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Posts Tagged ‘endothelial cell’

Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases


Summary – Volume 4, Part 2:  Translational Medicine in Cardiovascular Diseases

Author and Curator: Larry H Bernstein, MD, FCAP

 

We have covered a large amount of material that involves

  • the development,
  • application, and
  • validation of outcomes of medical and surgical procedures

that are based on translation of science from the laboratory to the bedside, improving the standards of medical practice at an accelerated pace in the last quarter century, and in the last decade.  Encouraging enabling developments have been:

1. The establishment of national and international outcomes databases for procedures by specialist medical societies

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/06/stent-design-and-thrombosis-bifurcation-intervention-drug-eluting-stents-des-and-biodegrable-stents/

On Devices and On Algorithms: Prediction of Arrhythmia after Cardiac Surgery and ECG Prediction of an Onset of Paroxysmal Atrial Fibrillation
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
https://pharmaceuticalintelligence.com/2013/05/07/on-devices-and-on-algorithms-arrhythmia-after-cardiac-surgery-prediction-and-ecg-prediction-of-paroxysmal-atrial-fibrillation-onset/

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/11/04/mitral-valve-repair-who-is-a-candidate-for-a-non-ablative-fully-non-invasive-procedure/

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions
Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/07/23/cardiovascular-complications-of-multiple-etiologies-repeat-sternotomy-post-cabg-or-avr-post-pci-pad-endoscopy-andor-resultant-of-systemic-sepsis/

Survivals Comparison of Coronary Artery Bypass Graft (CABG) and Percutaneous Coronary Intervention (PCI) /Coronary Angioplasty
Larry H. Bernstein, MD, Writer And Aviva Lev-Ari, PhD, RN, Curator
https://pharmaceuticalintelligence.com/2013/06/23/comparison-of-cardiothoracic-bypass-and-percutaneous-interventional-catheterization-survivals/

Revascularization: PCI, Prior History of PCI vs CABG
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/04/25/revascularization-pci-prior-history-of-pci-vs-cabg/

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/08/27/outcomes-in-high-cardiovascular-risk-patients-prasugrel-effient-vs-clopidogrel-plavix-aliskiren-tekturna-added-to-ace-or-added-to-arb/

Endovascular Lower-extremity Revascularization Effectiveness: Vascular Surgeons (VSs), Interventional Cardiologists (ICs) and Interventional Radiologists (IRs)
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

and more

2. The identification of problem areas, particularly in activation of the prothrombotic pathways, infection control to an extent, and targeting of pathways leading to progression or to arrythmogenic complications.

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/07/23/cardiovascular-complications-of-multiple-etiologies-repeat-sternotomy-post-cabg-or-avr-post-pci-pad-endoscopy-andor-resultant-of-systemic-sepsis/

Anticoagulation genotype guided dosing
Larry H. Bernstein, MD, FCAP, Author and Curator
https://pharmaceuticalintelligence.com/2013/12/08/anticoagulation-genotype-guided-dosing/

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/06/stent-design-and-thrombosis-bifurcation-intervention-drug-eluting-stents-des-and-biodegrable-stents/

The Effects of Aprotinin on Endothelial Cell Coagulant Biology
Co-Author (Kamran Baig, MBBS, James Jaggers, MD, Jeffrey H. Lawson, MD, PhD) and Curator
https://pharmaceuticalintelligence.com/2013/07/20/the-effects-of-aprotinin-on-endothelial-cell-coagulant-biology/

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/08/27/outcomes-in-high-cardiovascular-risk-patients-prasugrel-effient-vs-clopidogrel-plavix-aliskiren-tekturna-added-to-ace-or-added-to-arb/

Pharmacogenomics – A New Method for Druggability  Author and Curator: Demet Sag, PhD
https://pharmaceuticalintelligence.com/2014/04/28/pharmacogenomics-a-new-method-for-druggability/

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage    Author: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-Sepsis-and-the-Cardiovascular-System-at-its-End-Stage/

3. Development of procedures that use a safer materials in vascular management.

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/06/stent-design-and-thrombosis-bifurcation-intervention-drug-eluting-stents-des-and-biodegrable-stents/

Biomaterials Technology: Models of Tissue Engineering for Reperfusion and Implantable Devices for Revascularization
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/05/05/bioengineering-of-vascular-and-tissue-models/

Vascular Repair: Stents and Biologically Active Implants
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, RN, PhD
https://pharmaceuticalintelligence.com/2013/05/04/stents-biologically-active-implants-and-vascular-repair/

Drug Eluting Stents: On MIT’s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES
Author: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN
http://PharmaceuticalIntelligence.com/2013/04/25/Contributions-to-vascular-biology/

MedTech & Medical Devices for Cardiovascular Repair – Curations by Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2014/04/17/medtech-medical-devices-for-cardiovascular-repair-curation-by-aviva-lev-ari-phd-rn/

4. Discrimination of cases presenting for treatment based on qualifications for medical versus surgical intervention.

Treatment Options for Left Ventricular Failure – Temporary Circulatory Support: Intra-aortic balloon pump (IABP) – Impella Recover LD/LP 5.0 and 2.5, Pump Catheters (Non-surgical) vs Bridge Therapy: Percutaneous Left Ventricular Assist Devices (pLVADs) and LVADs (Surgical)
Author: Larry H Bernstein, MD, FCAP And Curator: Justin D Pearlman, MD, PhD, FACC
https://pharmaceuticalintelligence.com/2013/07/17/treatment-options-for-left-ventricular-failure-temporary-circulatory-support-intra-aortic-balloon-pump-iabp-impella-recover-ldlp-5-0-and-2-5-pump-catheters-non-surgical-vs-bridge-therapy/

Coronary Reperfusion Therapies: CABG vs PCI – Mayo Clinic preprocedure Risk Score (MCRS) for Prediction of in-Hospital Mortality after CABG or PCI
Writer and Curator: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/06/30/mayo-risk-score-for-percutaneous-coronary-intervention/

ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery Reporter: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/11/05/accaha-guidelines-for-coronary-artery-bypass-graft-surgery/

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/11/04/mitral-valve-repair-who-is-a-candidate-for-a-non-ablative-fully-non-invasive-procedure/ 

5.  This has become possible because of the advances in our knowledge of key related pathogenetic mechanisms involving gene expression and cellular regulation of complex mechanisms.

What is the key method to harness Inflammation to close the doors for many complex diseases?
Author and Curator: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2014/03/21/what-is-the-key-method-to-harness-inflammation-to-close-the-doors-for-many-complex-diseases/

CVD Prevention and Evaluation of Cardiovascular Imaging Modalities: Coronary Calcium Score by CT Scan Screening to justify or not the Use of Statin
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2014/03/03/cvd-prevention-and-evaluation-of-cardiovascular-imaging-modalities-coronary-calcium-score-by-ct-scan-screening-to-justify-or-not-the-use-of-statin/

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2014/03/03/richard-lifton-md-phd-of-yale-university-and-howard-hughes-medical-institute-recipient-of-2014-breakthrough-prizes-awarded-in-life-sciences-for-the-discovery-of-genes-and-biochemical-mechanisms-tha/

Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)
Curator:  Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2014/01/15/pathophysiological-effects-of-diabetes-on-ischemic-cardiovascular-disease-and-on-chronic-obstructive-pulmonary-disease-copd/

Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Reviewer and Co-Curator: Larry H Bernstein, MD, CAP and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

Notable Contributions to Regenerative Cardiology  Author and Curator: Larry H Bernstein, MD, FCAP and Article Commissioner: Aviva Lev-Ari, PhD, RD
https://pharmaceuticalintelligence.com/2013/10/20/notable-contributions-to-regenerative-cardiology/

As noted in the introduction, any of the material can be found and reviewed by content, and the eTOC is identified in attached:

http://wp.me/p2xfv8-1W

 

This completes what has been presented in Part 2, Vol 4 , and supporting references for the main points that are found in the Leaders in Pharmaceutical Intelligence Cardiovascular book.  Part 1 was concerned with Posttranslational Modification of Proteins, vital for understanding cellular regulation and dysregulation.  Part 2 was concerned with Translational Medical Therapeutics, the efficacy of medical and surgical decisions based on bringing the knowledge gained from the laboratory, and from clinical trials into the realm opf best practice.  The time for this to occur in practice in the past has been through roughly a generation of physicians.  That was in part related to the busy workload of physicians, and inability to easily access specialty literature as the volume and complexity increased.  This had an effect of making access of a family to a primary care provider through a lifetime less likely than the period post WWII into the 1980s.

However, the growth of knowledge has accelerated in the specialties since the 1980’s so that the use of physician referral in time became a concern about the cost of medical care.  This is not the place for or a matter for discussion here.  It is also true that the scientific advances and improvements in available technology have had a great impact on medical outcomes.  The only unrelated issue is that of healthcare delivery, which is not up to the standard set by serial advances in therapeutics, accompanied by high cost due to development costs, marketing costs, and development of drug resistance.

I shall identify continuing developments in cardiovascular diagnostics, therapeutics, and bioengineering that is and has been emerging.

1. Mechanisms of disease

REPORT: Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness Signatures 

Science 11 April 2014:
Vol. 344 no. 6180 pp. 208-211
http://dx.doi.org/10.1126/science.1250217

Abstract: Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.

Yeasty HIPHOP

Laura Zahn
Sci. Signal. 15 April 2014; 7(321): ec103.   http://dx.doi.org/10.1126/scisignal.2005362

In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al.(p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions

Key Heart Failure Culprit Discovered

A team of cardiovascular researchers from the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai, Sanford-Burnham Medical Research Institute, and University of California, San Diego have identified a small, but powerful, new player in thIe onset and progression of heart failure. Their findings, published in the journal Nature  on March 12, also show how they successfully blocked the newly discovered culprit.
Investigators identified a tiny piece of RNA called miR-25 that blocks a gene known as SERCA2a, which regulates the flow of calcium within heart muscle cells. Decreased SERCA2a activity is one of the main causes of poor contraction of the heart and enlargement of heart muscle cells leading to heart failure.

Using a functional screening system developed by researchers at Sanford-Burnham, the research team discovered miR-25 acts pathologically in patients suffering from heart failure, delaying proper calcium uptake in heart muscle cells. According to co-lead study authors Christine Wahlquist and Dr. Agustin Rojas Muñoz, developers of the approach and researchers in Mercola’s lab at Sanford-Burnham, they used high-throughput robotics to sift through the entire genome for microRNAs involved in heart muscle dysfunction.

Subsequently, the researchers at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai found that injecting a small piece of RNA to inhibit the effects of miR-25 dramatically halted heart failure progression in mice. In addition, it also improved their cardiac function and survival.

“In this study, we have not only identified one of the key cellular processes leading to heart failure, but have also demonstrated the therapeutic potential of blocking this process,” says co-lead study author Dr. Dongtak Jeong, a post-doctoral fellow at the Cardiovascular Research Center at Icahn School of  Medicine at Mount Sinai in the laboratory of the study’s co-senior author Dr. Roger J. Hajjar.

Publication: Inhibition of miR-25 improves cardiac contractility in the failing heart.Christine Wahlquist, Dongtak Jeong, Agustin Rojas-Muñoz, Changwon Kho, Ahyoung Lee, Shinichi Mitsuyama, Alain Van Mil, Woo Jin Park, Joost P. G. Sluijter, Pieter A. F. Doevendans, Roger J. :  Hajjar & Mark Mercola.     Nature (March 2014)    http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13073.html

 

“Junk” DNA Tied to Heart Failure

Deep RNA Sequencing Reveals Dynamic Regulation of Myocardial Noncoding RNAs in Failing Human Heart and Remodeling With Mechanical Circulatory Support

Yang KC, Yamada KA, Patel AY, Topkara VK, George I, et al.
Circulation 2014;  129(9):1009-21.
http://dx.doi.org/10.1161/CIRCULATIONAHA.113.003863              http://circ.ahajournals.org/…/CIRCULATIONAHA.113.003863.full

The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

Junk DNA was long thought to have no important role in heredity or disease because it doesn’t code for proteins. But emerging research in recent years has revealed that many of these sections of the genome produce noncoding RNA molecules that still have important functions in the body. They come in a variety of forms, some more widely studied than others. Of these, about 90% are called long noncoding RNAs (lncRNAs), and exploration of their roles in health and disease is just beginning.

The Washington University group performed a comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

In their study, the researchers found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support,” wrote the researchers. “These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.”

‘Junk’ Genome Regions Linked to Heart Failure

In a recent issue of the journal Circulation, Washington University investigators report results from the first comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

“We took an unbiased approach to investigating which types of RNA might be linked to heart failure,” said senior author Jeanne Nerbonne, the Alumni Endowed Professor of Molecular Biology and Pharmacology. “We were surprised to find that long noncoding RNAs stood out.

In the new study, the investigators found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“We don’t know whether these changes in long noncoding RNAs are a cause or an effect of heart failure,” Nerbonne said. “But it seems likely they play some role in coordinating the regulation of multiple genes involved in heart function.”

Nerbonne pointed out that all types of RNA molecules they examined could make the obvious distinction: telling the difference between failing and nonfailing hearts. But only expression of the long noncoding RNAs was measurably different between heart failure associated with a heart attack (ischemic) and heart failure without the obvious trigger of blocked arteries (nonischemic). Similarly, only long noncoding RNAs significantly changed expression patterns after implantation of left ventricular assist devices.

Comment

Decoding the noncoding transcripts in human heart failure

Xiao XG, Touma M, Wang Y
Circulation. 2014; 129(9): 958960,  http://dx.doi.org/10.1161/CIRCULATIONAHA.114.007548 

Heart failure is a complex disease with a broad spectrum of pathological features. Despite significant advancement in clinical diagnosis through improved imaging modalities and hemodynamic approaches, reliable molecular signatures for better differential diagnosis and better monitoring of heart failure progression remain elusive. The few known clinical biomarkers for heart failure, such as plasma brain natriuretic peptide and troponin, have been shown to have limited use in defining the cause or prognosis of the disease.1,2 Consequently, current clinical identification and classification of heart failure remain descriptive, mostly based on functional and morphological parameters. Therefore, defining the pathogenic mechanisms for hypertrophic versus dilated or ischemic versus nonischemic cardiomyopathies in the failing heart remain a major challenge to both basic science and clinic researchers. In recent years, mechanical circulatory support using left ventricular assist devices (LVADs) has assumed a growing role in the care of patients with end-stage heart failure.3 During the earlier years of LVAD application as a bridge to transplant, it became evident that some patients exhibit substantial recovery of ventricular function, structure, and electric properties.4 This led to the recognition that reverse remodeling is potentially an achievable therapeutic goal using LVADs. However, the underlying mechanism for the reverse remodeling in the LVAD-treated hearts is unclear, and its discovery would likely hold great promise to halt or even reverse the progression of heart failure.

 

Efficacy and Safety of Dabigatran Compared With Warfarin in Relation to Baseline Renal Function in Patients With Atrial Fibrillation: A RE-LY (Randomized Evaluation of Long-term Anticoagulation Therapy) Trial Analysis

Circulation. 2014; 129: 951-952     http://dx.doi.org/10.1161/​CIR.0000000000000022

In patients with atrial fibrillation, impaired renal function is associated with a higher risk of thromboembolic events and major bleeding. Oral anticoagulation with vitamin K antagonists reduces thromboembolic events but raises the risk of bleeding. The new oral anticoagulant dabigatran has 80% renal elimination, and its efficacy and safety might, therefore, be related to renal function. In this prespecified analysis from the Randomized Evaluation of Long-Term Anticoagulant Therapy (RELY) trial, outcomes with dabigatran versus warfarin were evaluated in relation to 4 estimates of renal function, that is, equations based on creatinine levels (Cockcroft-Gault, Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI]) and cystatin C. The rates of stroke or systemic embolism were lower with dabigatran 150 mg and similar with 110 mg twice daily irrespective of renal function. Rates of major bleeding were lower with dabigatran 110 mg and similar with 150 mg twice daily across the entire range of renal function. However, when the CKD-EPI or MDRD equations were used, there was a significantly greater relative reduction in major bleeding with both doses of dabigatran than with warfarin in patients with estimated glomerular filtration rate ≥80 mL/min. These findings show that dabigatran can be used with the same efficacy and adequate safety in patients with a wide range of renal function and that a more accurate estimate of renal function might be useful for improved tailoring of anticoagulant treatment in patients with atrial fibrillation and an increased risk of stroke.

Aldosterone Regulates MicroRNAs in the Cortical Collecting Duct to Alter Sodium Transport.

Robert S Edinger, Claudia Coronnello, Andrew J Bodnar, William A Laframboise, Panayiotis V Benos, Jacqueline Ho, John P Johnson, Michael B Butterworth

Journal of the American Society of Nephrology (Impact Factor: 8.99). 04/2014;     http://dx. DO.org/I:10.1681/ASN.2013090931

Source: PubMed

ABSTRACT A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells.

Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport.

Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein.

A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3′ untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.

 

2. Diagnostic Biomarker Status

A prospective study of the impact of serial troponin measurements on the diagnosis of myocardial infarction and hospital and 6-month mortality in patients admitted to ICU with non-cardiac diagnoses.

Marlies Ostermann, Jessica Lo, Michael Toolan, Emma Tuddenham, Barnaby Sanderson, Katie Lei, John Smith, Anna Griffiths, Ian Webb, James Coutts, John hambers, Paul Collinson, Janet Peacock, David Bennett, David Treacher

Critical care (London, England) (Impact Factor: 4.72). 04/2014; 18(2):R62.   http://dx.doi.org/:10.1186/cc13818

Source: PubMed

ABSTRACT Troponin T (cTnT) elevation is common in patients in the Intensive Care Unit (ICU) and associated with morbidity and mortality. Our aim was to determine the epidemiology of raised cTnT levels and contemporaneous electrocardiogram (ECG) changes suggesting myocardial infarction (MI) in ICU patients admitted for non-cardiac reasons.
cTnT and ECGs were recorded daily during week 1 and on alternate days during week 2 until discharge from ICU or death. ECGs were interpreted independently for the presence of ischaemic changes. Patients were classified into 4 groups: (i) definite MI (cTnT >=15 ng/L and contemporaneous changes of MI on ECG), (ii) possible MI (cTnT >=15 ng/L and contemporaneous ischaemic changes on ECG), (iii) troponin rise alone (cTnT >=15 ng/L), or (iv) normal. Medical notes were screened independently by two ICU clinicians for evidence that the clinical teams had considered a cardiac event.
Data from 144 patients were analysed [42% female; mean age 61.9 (SD 16.9)]. 121 patients (84%) had at least one cTnT level >=15 ng/L. A total of 20 patients (14%) had a definite MI, 27% had a possible MI, 43% had a cTNT rise without contemporaneous ECG changes, and 16% had no cTNT rise. ICU, hospital and 180 day mortality were significantly higher in patients with a definite or possible MI.Only 20% of definite MIs were recognised by the clinical team. There was no significant difference in mortality between recognised and non-recognised events.At time of cTNT rise, 100 patients (70%) were septic and 58% were on vasopressors. Patients who were septic when cTNT was elevated had an ICU mortality of 28% compared to 9% in patients without sepsis. ICU mortality of patients who were on vasopressors at time of cTNT elevation was 37% compared to 1.7% in patients not on vasopressors.
The majority of critically ill patients (84%) had a cTnT rise and 41% met criteria for a possible or definite MI of whom only 20% were recognised clinically. Mortality up to 180 days was higher in patients with a cTnT rise.

 

Prognostic performance of high-sensitivity cardiac troponin T kinetic changes adjusted for elevated admission values and the GRACE score in an unselected emergency department population.

Moritz BienerMatthias MuellerMehrshad VafaieAllan S JaffeHugo A Katus,Evangelos Giannitsis

Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.54). 04/2014;   http://dx.doi.org/10.1016/j.cca.2014.04.007

Source: PubMed

ABSTRACT To test the prognostic performance of rising and falling kinetic changes of high-sensitivity cardiac troponin T (hs-cTnT) and the GRACE score.
Rising and falling hs-cTnT changes in an unselected emergency department population were compared.
635 patients with a hs-cTnT >99th percentile admission value were enrolled. Of these, 572 patients qualified for evaluation with rising patterns (n=254, 44.4%), falling patterns (n=224, 39.2%), or falling patterns following an initial rise (n=94, 16.4%). During 407days of follow-up, we observed 74 deaths, 17 recurrent AMI, and 79 subjects with a composite of death/AMI. Admission values >14ng/L were associated with a higher rate of adverse outcomes (OR, 95%CI:death:12.6, 1.8-92.1, p=0.01, death/AMI:6.7, 1.6-27.9, p=0.01). Neither rising nor falling changes increased the AUC of baseline values (AUC: rising 0.562 vs 0.561, p=ns, falling: 0.533 vs 0.575, p=ns). A GRACE score ≥140 points indicated a higher risk of death (OR, 95%CI: 3.14, 1.84-5.36), AMI (OR,95%CI: 1.56, 0.59-4.17), or death/AMI (OR, 95%CI: 2.49, 1.51-4.11). Hs-cTnT changes did not improve prognostic performance of a GRACE score ≥140 points (AUC, 95%CI: death: 0.635, 0.570-0.701 vs. 0.560, 0.470-0.649 p=ns, AMI: 0.555, 0.418-0.693 vs. 0.603, 0.424-0.782, p=ns, death/AMI: 0.610, 0.545-0.676 vs. 0.538, 0.454-0.622, p=ns). Coronary angiography was performed earlier in patients with rising than with falling kinetics (median, IQR [hours]:13.7, 5.5-28.0 vs. 20.8, 6.3-59.0, p=0.01).
Neither rising nor falling hs-cTnT changes improve prognostic performance of elevated hs-cTnT admission values or the GRACE score. However, rising values are more likely associated with the decision for earlier invasive strategy.

 

Troponin assays for the diagnosis of myocardial infarction and acute coronary syndrome: where do we stand?

Arie Eisenman

ABSTRACT: Under normal circumstances, most intracellular troponin is part of the muscle contractile apparatus, and only a small percentage (< 2-8%) is free in the cytoplasm. The presence of a cardiac-specific troponin in the circulation at levels above normal is good evidence of damage to cardiac muscle cells, such as myocardial infarction, myocarditis, trauma, unstable angina, cardiac surgery or other cardiac procedures. Troponins are released as complexes leading to various cut-off values depending on the assay used. This makes them very sensitive and specific indicators of cardiac injury. As with other cardiac markers, observation of a rise and fall in troponin levels in the appropriate time-frame increases the diagnostic specificity for acute myocardial infarction. They start to rise approximately 4-6 h after the onset of acute myocardial infarction and peak at approximately 24 h, as is the case with creatine kinase-MB. They remain elevated for 7-10 days giving a longer diagnostic window than creatine kinase. Although the diagnosis of various types of acute coronary syndrome remains a clinical-based diagnosis, the use of troponin levels contributes to their classification. This Editorial elaborates on the nature of troponin, its classification, clinical use and importance, as well as comparing it with other currently available cardiac markers.

Expert Review of Cardiovascular Therapy 07/2006; 4(4):509-14.   http://dx.doi.org/:10.1586/14779072.4.4.509 

 

Impact of redefining acute myocardial infarction on incidence, management and reimbursement rate of acute coronary syndromes.

Carísi A Polanczyk, Samir Schneid, Betina V Imhof, Mariana Furtado, Carolina Pithan, Luis E Rohde, Jorge P Ribeiro

ABSTRACT: Although redefinition for acute myocardial infarction (AMI) has been proposed few years ago, to date it has not been universally adopted by many institutions. The purpose of this study is to evaluate the diagnostic, prognostic and economical impact of the new diagnostic criteria for AMI. Patients consecutively admitted to the emergency department with suspected acute coronary syndromes were enrolled in this study. Troponin T (cTnT) was measured in samples collected for routine CK-MB analyses and results were not available to physicians. Patients without AMI by traditional criteria and cTnT > or = 0.035 ng/mL were coded as redefined AMI. Clinical outcomes were hospital death, major cardiac events and revascularization procedures. In-hospital management and reimbursement rates were also analyzed. Among 363 patients, 59 (16%) patients had AMI by conventional criteria, whereas additional 75 (21%) had redefined AMI, an increase of 127% in the incidence. Patients with redefined AMI were significantly older, more frequently male, with atypical chest pain and more risk factors. In multivariate analysis, redefined AMI was associated with 3.1 fold higher hospital death (95% CI: 0.6-14) and a 5.6 fold more cardiac events (95% CI: 2.1-15) compared to those without AMI. From hospital perspective, based on DRGs payment system, adoption of AMI redefinition would increase 12% the reimbursement rate [3552 Int dollars per 100 patients evaluated]. The redefined criteria result in a substantial increase in AMI cases, and allow identification of high-risk patients. Efforts should be made to reinforce the adoption of AMI redefinition, which may result in more qualified and efficient management of ACS.

International Journal of Cardiology 03/2006; 107(2):180-7. · 5.51 Impact Factor   http://www.sciencedirect.com/science/article/pii/S0167527305005279

 

3. Biomedical Engineerin3g

Safety and Efficacy of an Injectable Extracellular Matrix Hydrogel for Treating Myocardial Infarction 

Sonya B. Seif-Naraghi, Jennifer M. Singelyn, Michael A. Salvatore,  et al.
Sci Transl Med 20 February 2013 5:173ra25  http://dx.doi.org/10.1126/scitranslmed.3005503

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of application with substantial intrinsic hurdles, but where human translation is now occurring.

 Acellular Biomaterials: An Evolving Alternative to Cell-Based Therapies

J. A. Burdick, R. L. Mauck, J. H. Gorman, R. C. Gorman,
Sci. Transl. Med. 2013; 5, (176): 176 ps4    http://stm.sciencemag.org/content/5/176/176ps4

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of applications with substantial intrinsic hurdles, but where human translation is now occurring.


Instructive Nanofiber Scaffolds with VEGF Create a Microenvironment for Arteriogenesis and Cardiac Repair

Yi-Dong Lin, Chwan-Yau Luo, Yu-Ning Hu, Ming-Long Yeh, Ying-Chang Hsueh, Min-Yao Chang, et al.
Sci Transl Med 8 August 2012; 4(146):ra109.   http://dx.doi.org/ 10.1126/scitranslmed.3003841

Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation.

Manufacturing Challenges in Regenerative Medicine

I. Martin, P. J. Simmons, D. F. Williams.
Sci. Transl. Med. 2014; 6(232): fs16.   http://dx.doi.org/10.1126/scitranslmed.3008558

Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.

 

 

 

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Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine


Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine

Author and Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

This document is entirely devoted to medical and surgical therapies that have made huge strides in

  • simplification of interventional procedures,
  • reduced complexity, resulting in procedures previously requiring surgery are now done, circumstances permitting, by medical intervention.

This revolution in cardiovascular interventional therapy is regenerative medicine.  It is regenerative because it is largely driven by

  • the introduction into the impaired vasculature of an induced pleuripotent cell, called a stem cell, although
  • the level of differentiation may not be a most primitive cell line.

There is also a very closely aligned development in cell biology that extends beyond and including vascular regeneration that is called synthetic biology.  These developments have occurred at an accelerated rate in the last 15 years. The methods of interventional cardiology were already well developed in the mid 1980s.  This was at the peak of cardiothoracic bypass surgery.

Research on the endothelial cell,

  • endothelial cell proliferation,
  • shear flow in small arteries, especially at branch points, and
  • endothelial-platelet interactions

led to insights about plaque formation and vessel thrombosis.

Much was learned in biomechanics about the shear flow stresses on the luminal surface of the vasculature, and there was also

  • the concomitant discovery of nitric oxide,
  • oxidative stress, and
  • the isoenzymes of nitric oxide synthase (eNOS, iNOS, and nNOS).

It became a fundamental tenet of vascular biology that

  • atherogenesis is a maladjustment to oxidative stress not only through genetic, but also
  • non-genetic nutritional factors that could be related to the balance of omega (ω)-3 and omega (ω)-6 fatty acids,
  • a pro-inflammatory state that elicits inflammatory cytokines, such as, interleukin-6 (IL6) and c-reactive protein(CRP),
  • insulin resistance with excess carbohydrate associated with type 2 diabetes and beta (β) cell stress,
  • excess trans- and saturated fats, and perhaps
  • the now plausible colonic microbial population of the gastrointestinal tract (GIT).

There is also an association of abdominal adiposity,

  • including the visceral peritoneum, with both T2DM and with arteriosclerotic vessel disease,
  • which is presenting at a young age, and has ties to
  • the effects of an adipokine, adiponectin.

Much important work has already been discussed in the domain of cardiac catheterization and research done to

  • prevent atheroembolization.and beyond that,
  • research done to implant an endothelial growth matrix.

Even then, dramatic work had already been done on

  • the platelet structure and metabolism, and
  • this has transformed our knowledge of platelet biology.

The coagulation process has been discussed in detailed in a previous document.  The result was the development of a

  • new class of platelet aggregation inhibitors designed to block the activation of protein on the platelet surface that
  • is critical in the coagulation cascade.

In addition, the term long used to describe atherosclerosis, atheroma notwithstanding, is “hardening of the arteries”.  This is particularly notable with respect to mid-size arteries and arterioles that feed the heart and kidneys. Whether it is preceded by or develops concurrently with chronic renal insufficiency and lowered glomerular filtration rate is perhaps arguable.  However, there is now a body of evidence that points to

  • a change in the vascular muscularis and vessel stiffness, in addition to the endothelial features already mentioned.

This has provided a basis for

  • targeted pharmaceutical intervention, and
  • reduction in salt intake.

So we have a  group of metabolic disorders, which may alone or in combination,

  • lead to and be associated with the long term effects of cardiovascular disease, including
  • congestive heart failure.

This has been classically broken down into forward and backward failure,

  • depending on decrease outflow through the aorta (ejection fraction), or
  • decreased venous return through the vena cava,

which involves increased pulmonary vascular resistance and decreased return into the left atrium.

This also has ties to several causes, which may be cardiac or vascular. This document, as the previous, has four pats.  They are broadly:

  1. Stem Cells in Cardiovascular Diseases
  2. Regenerative Cell and Molecular Biology
  3. Therapeutics Levels In Molecular Cardiology
  4. Research Proposals for Endogenous Augmentation of circulating Endothelial Progenitor Cells (cEPCs)

As in the previous section, we start with the biology of the stem cell and the degeneration in cardiovascular diseases, then proceed to regeneration, then therapeutics, and finally – proposals for augmenting therapy with circulating endogenous endothelial progenitor cells (cEPCs).

 

context

stem cells

 

theme

regeneration

 

 

 

 

theme

Therapeutics

 

theme

augmentation

 

 

 

 

 

 

 

 

 

 

Key pathways involving NO

Key pathways involving NO

 

 

 

 

stem cell lin28

stem cellLlin28

1479-5876-10-175-1-l  translational research with feedback loops

Tranlational Research -Lab to Bedside

 

 

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Circulating Endothelial Progenitors Cells (cEPCs) as Biomarkers

Article Curator: Larry H. Bernstein, MD, FCAP

and

Topic Curator: Aviva Lev-Ari, PhD, RN

Circulating progenitor cells have gained much interest rapidly in the past year primarily in identification of damaged tissue that has turnover of cells that are identifiable in the circulation.  This has to require a sensitivity for identification at one or two logs lower than circulating hematopoietic cells.  I mention this untested view only because cells of the circulation are detected routinely by automated hematology instruments like those of Beckman-Coulter and Siemens, with graphical presentation of results.  The Sysmex also reports immature granulocytes that are a small percent of the neutrophil count.  In the evaluation of leukemias, flow cytometry has been used for years, but require a preparative step.  Cell types have been identified by acidic and basic dye stains to identify basophilic, acidophilic and neutrophilic granulocyte series, and by size of the cell population, and nuclear features, differentiating mature and nucleated red cells, the granulocyte series, monocytes and lymphocytes, as well as platelets (aggregation gives an underestimate of platelet count).  But to detect cancer cells or damaged endothelial cells, the number of cells in the circulation requires and antibody to the surface with a visualizable ligand attached to an antibody for identification.  Visualization could be by a fluorophor, or perhaps a luciferase reaction.  Here are two articles that identify circulating endothelial cells, making them suitable for biomarkers of cardiovascular injury.  Whether they can detect early predictive ischemia, or frank AMI needs investigation.  The concept of piecemeal necrosis in the heart may be applicable to cardiomyocyte injury that is found unexpectedly at autopsy as “silent infarct”.

Circulating endothelial progenitors–cells as biomarkers

Rosenzweig, Anthony
N Engl J Med. 2005 Sep 8;353(10):1055-7

Comment on

Circulating endothelial progenitor cells and cardiovascular outcomes

[N Engl J Med. 2005]  PMID: 16148292 [PubMed – indexed for MEDLINE]

Endothelial injury and dysfunction are thought to be critical events in the  pathogenesis of atherosclerosis. Thus,

  • understanding the mechanisms that  maintain and restore endothelial function
    • may have important clinical  implications.

A series of clinical and basic studies prompted by the discovery 

  • of bone marrow derived endothelial progenitor cells1 have
  • provided insights into these processes and
    • opened a door to the development of new therapeutic approaches.

Growing evidence suggests that bone marrow derived endothelial progenitor cells circulate in the blood and

  • play an important role in the formation of new blood vessels as well as
  • contribute to vascular homeostasis in the adult.

Circulating endothelial progenitor cells were initially identified

  • through their expression of CD34
    (a surface marker common to hematopoietic stem cells and mature endothelial cells)
  • and vascular endothelial cell growth-factor receptor 2
    (VEGFR2 or kinase-domain related [KDR] receptor),

but not of other markers seen on fully differentiated endothelial cells.1

Subsequent studies have also used other identifiers, such as

  • the stem-cell marker CD133, and
  • functional assays, including
    • the ability to form endothelial colonies.

Endothelial progenitor cells defined in these ways probably represent

  • a heterogeneous population, which,
  • in combination with the lack of a consensual definition,

complicates the interpretation of work in this field.

Nevertheless, numerous studies in animals have shown that endothelial  progenitor cells can integrate into new and existing blood vessels.2,3,4
Intravenous injection of cytokine-mobilized human endothelial progenitor cells

  • improved myocardial neoangiogenesis and
  • the recovery of functioning in a rat model of infarction.3

Repeated injection of bone marrow derived cells in a mouse model of atherosclerosis

  • reduced the rate of plaque formation without altering serum lipids levels, and
  • donor endothelial progenitor cells could subsequently be identified in the recipient’s blood vessels.4

Previous clinical studies have shown that

  • traditional risk factors for coronary atherosclerosis
  • are associated with low levels of circulating endothelial progenitor cells,5 whereas
  • protective interventions, including statin therapy6 and exercise,7
    • appear to increase the supply of these cells.

Hill et al. found that even in healthy volunteers,

  • levels of endothelial progenitor cells were inversely correlated with the Framingham risk score and
  • actually appeared to predict vascular function better than the Framingham risk score.5

Together, these data suggest that circulating endothelial progenitor cells may participate

  • not only in forming new blood vessels
  • but also in maintaining the integrity and function of vascular  endothelium,

thereby mitigating disease processes such as atherosclerosis.

In this issue of the Journal, Werner and colleagues have further advanced our understanding of the clinical implications of endothelial progenitor cells.8 Endothelial progenitor cells were quantitated in 519 patients with coronary artery disease who

  • were followed for one year after undergoing catheterization.

Patients with higher levels of endothelial progenitor cells had

  • a reduced risk of death from cardiovascular causes and of
  • the composite end point of major cardiovascular events.

These relationships were preserved even

  • after adjustment for traditional risk factors and prognostic variables.

A similar relationship was seen

  • whether endothelial progenitor cells were  identified by virtue of expression
    either of CD34 and KDR or of CD133 or
  • because of their ability to form endothelial colonies,

further strengthening the authors’ conclusions. Repeated catheterization was not performed in this  cohort, so

  • we do not know whether the reduction in clinical events reflected a slowed progression of atherosclerosis or some other clinical effect.

A  dissociation between anatomical measures of atherosclerosis and clinical events has been well documented in other settings.

Although this study is consistent with prior work suggesting that circulating endothelial progenitor cells may play a protective role in vascular homeostasis, other explanations

  • for the association between endothelial progenitor number and outcome remain possible.

Changes in the number of endothelial progenitor cells and

  • in clinical events might reflect a common underlying etiology,
      • rather than a causal relation.

For example, a defect in the production of nitric oxide, which plays an important role

  • in both the mobilization of endothelial progenitor cells9 and blood-vessel function, might account for both observations.

Similarly, the number of endothelial progenitor cells

  • may mirror a person’s regenerative capacity more broadly and
  • predict clinical events on that basis.

Even if endothelial progenitor cells are mechanistically linked to clinical cardiovascular events,

  • such clinical studies do not distinguish between the possibility
  • that the protection is mediated through the integration of endothelial  progenitor cells into blood vessels and

its possible mediation by other  mechanisms, such as the

  • paracrine benefits of endothelial progenitor  cell secreted products.

Although such questions will undoubtedly continue to provide fertile ground  for fundamental investigation,

  • the report by Werner and colleagues has more  immediate clinical implications.

First, it suggests that circulating cell  populations may represent a new class of biomarkers

  • that naturally integrate  diverse genetic and environmental effects,
  • thereby providing robust  physiological and prognostic insights.

Second, in the context of coronary  disease, the study shows that

  • the number of endothelial progenitor cells is an independent predictor of hard clinical outcomes.

As with other biomarkers, a demonstration of clinical usefulness will ultimately require

  • the examination of other patient populations, as well as
  • a demonstration that clinical therapy can be guided and enhanced by this information.

Finally, the increased risk associated with reduced levels of endothelial progenitor cells

  • supports the growing interest in the therapeutic potential of enhancing the level of these cells.

The most dramatic extension of this line of reasoning involves

transferring  bone marrow or peripheral blood cells that are likely to include endothelial  progenitor cells to patients with coronary artery disease. Although it would be premature to judge the clinical success of these strategies, early trials, including one randomized (though incompletely blinded) trial, have suggested

  • at least short-term functional benefits of intracoronary infusion of bone marrow cells after acute infarction.10

Trials are planned to address more definitively the potential benefits of such cells

  • in the settings of acute infarction and chronic ischemic cardiomyopathy.

Such efforts would be aided substantially by the identification of specific markers as well as

  • an improved understanding of the role of subtypes of endothelial progenitor cells and
  • of the mechanisms by which they work.

Ironically, the data presented by Werner and colleagues in combination with work showing

  • the impaired functioning of endothelial progenitor cells in high-risk patients5 suggest
  • that the patients most in need of endothelial progenitor cells may be
      • those who are least able to donate them for autologous transplantation.

Whether these limitations can be overcome through

  • ex vivo expansion or  genetic modification of endothelial progenitor cells is unclear.

In addition to possible cell-based therapies, work on endothelial progenitor cells provides yet another rationale

  • for redoubling efforts to comply with established therapeutic guidelines,
  • including lifestyle modifications and the use of statin therapy,
      • both of which appear to enhance the number of circulating endothelial progenitor cells.

Whether there will be a downside to enhancing the number and function of  endothelial progenitor cells remains unclear,

  • although obvious concerns  include exacerbating conditions that are characterized by adverse vessel  formation,
    • such as diabetic retinopathy and tumor angiogenesis.

Small studies have suggested an association between high levels of circulating endothelial progenitor cells and the risk of certain cancers, such as multiple myeloma.11 Moreover, studies in animals show that

  • bone marrow derived endothelial progenitors participate in tumor angiogenesis, thereby
      • enhancing tumor growth.12

In the study by Werner and colleagues,

  • the number of deaths from cardiovascular causes among patients with high levels of endothelial progenitor cells
  • was substantially lower than that among patients with lower levels of these cells,
  • without a reduction in the risk of death overall.8

Although this finding could raise the specter of a counterbalancing adverse effect of endothelial progenitor cells,

  • there was no apparent pattern in the deaths due to other causes,
  • and no deaths from cancer were noted in this population.

It is possible that as we learn more about the biology of endothelial progenitor cells, there may be opportunities

  • to target vessel formation more specifically.

In addition, therapeutic strategies

  • tailored to individualized risk will undoubtedly help in practice.

For example, in the study by Werner et al.,

  • patients in the group with the lowest baseline levels of endothelial progenitor cells
  • had a risk of death from cardiovascular causes of 8.3 percent during one year of follow-up,
  • suggesting that the benefits of enhancing the function and number of endothelial progenitor cells
      • may well outweigh the risks in such high-risk populations.

Additional studies will be necessary to address these questions definitively. Larger studies

  • of longer duration performed in different cohorts will be required to determine fully
    • the clinical usefulness of endothelial progenitor cells as a biomarker.

Rigorous interventional studies will indicate

  • whether levels of endothelial progenitor cells can be used to guide therapy and
  • whether cell transfer has a role in augmenting the levels of these cells.

Basic-science studies should help guide these clinical efforts by

  • further defining the desirable subpopulations of endothelial progenitor cells and
  • the mechanisms by which they mediate their effects.

By establishing a connection between circulating endothelial progenitor cells and hard clinical end points, Werner and colleagues

  • provide a potent stimulus for clinical and basic studies to address these important issues.

Source Information

From the Program in Cardiovascular Gene Therapy, Massachusetts General  Hospital, and Harvard Medical School ― both in Boston.

References

Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor  endothelial cells for angiogenesis. Science 1997;275:964-967.

Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced  mobilization of bone marrow-derived endothelial progenitor cells for  neovascularization. Nat Med 1999;5:434-438.

Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001; 7: 430-436.

Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, et al. Aging, progenitor cell exhaustion, and atherosclerosis. Circulation 2003; 108: 457-463.

Hill JM, Zalos G, Halcox JPJ, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348:593-600.

Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial  progenitor cells by statin therapy in patients with stable coronary artery  disease. Circulation 2001; 103: 2885-2890.

Laufs U, Werner N, Link A, et al. Physical training increases endothelial  progenitor cells, inhibits neointima formation, and enhances angiogenesis.  Circulation 2004; 109: 220-226.

Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005; 353: 999-1007.

Aicher A, Heeschen C, Mildner-Rihm C, et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med  2003; 9: 1370-1376.

Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow  cell transfer after myocardial infarction: the BOOST randomised controlled  clinical trial. Lancet 2004; 364: 141-148.

Zhang H, Vakil V, Braunstein M, et al. Circulating endothelial progenitor cells in multiple myeloma: implications and significance. Blood 2005; 105: 3286-3294.

Lyden D, Hattori K, Dias S, et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 2001;7:1194-1201.

Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction.

Kelly Bethel, Madelyn S Luttgen, Samir Damani, Anand Kolatkar, Rachelle Lamy, Mohsen Sabouri-Ghomi, Sarah Topol, Eric J Topol, Peter Kuhn

Physical Biology (Impact Factor: 2.62). 01/2014; 11(1):016002. http://dx.doi.org/10.1088/1478-3975/11/1/016002
Source: PubMed

Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted

  • a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay),
  • to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs.

Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined

  • by positive staining for DAPI, CD146 and von Willebrand Factor
  • and negative staining for CD45.

In addition, CECs exhibited distinct morphological features that

  • enable differentiation from surrounding white blood cells.
  1. CECs were found both as individual cells and as aggregates.
  2. CEC numbers were higher in MI patients compared with healthy controls.
  3. VS patients had lower CEC counts when compared with MI patients

but were not different from healthy controls.

Both HD-CEC and CellSearch® assays could discriminate

  • MI patients from healthy controls with comparable accuracy

but the HD-CEC assay exhibited

  • higher specificity while maintaining high sensitivity.

Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients.

MicroRNA function in endothelial cells

Solving the mystery of an unknown target gene using microRNA Target Site Blockers
Dr. Virginie Mattot
Dr. Virgine Mattot works in the team “Angiogenesis, endothelium activation and Cancer” directed by Dr. Fabrice Soncin at the Institut de Biologie de Lille in France where she studies the roles played by microRNAs in endothelial cells during physiological and pathological processes such as angiogenesis or endothelium activation. She has been using Target Site Blockers to investigate the role of microRNAs on putative targets which functions are yet unknown.
What is the main focus of the research conducted in your lab?
We are studying endothelial cell functions with a particular interest
  • in angiogenesis and endothelium activation during physiological and tumoral vascular development.
How did your research lead to the study of microRNAs?
A few years ago, we identified in my team
  • a new endothelial cell-specific gene which harbors a microRNA in its intronic sequence.

We have since been working on understanding

  • the functions of both this new gene and
  • its intronic microRNA in endothelial cells

What is the aim of your current project?

While we were searching for the functions of the intronic microRNA,
  • we identified an unknown gene as a putative target.
The aim of my project was to investigate if this unknown gene was actually a genuine target and
  • if regulation of this gene by the microRNA was involved in endothelial cell function.
We had already characterized the endothelial cell phenotype associated with the inhibition of our intronic microRNA.
We then used miRCURY LNA™ Target Site Blockers to demonstrate
  • that the expression of this unknown gene is actually controlled by this microRNA.
Further, we also demonstrated that the microRNA regulates
  • specific endothelial cell properties through regulation of this unknown gene.
How did you perform the experiments and analyze the results?
LNA™ enhanced target site blockers (TSB) for our microRNA were designed by Exiqon.
We transfected the TSBs into endothelial cells using our standard procedure and
  • analysed the induced phenotype.
As a control for these experiments, a mutated version of the TSB was designed by Exiqon and
  • transfected into endothelial cells.
We first verified that this TSB was functional by
  • analyzing the expression of the miRNA target
      • against which the TSB was directed in transfected cells.
Finally, we showed that the TSB induced similar phenotypes as those found when we inhibited the microRNA in the same cells. 
What were some specific challenges in your experiments and how did you overcome them?
The fact that the target gene for our microRNA was unknown was a major challenge. Without specific available tools, like antibodies,
  • it becomes difficult to demonstrate the effect of the microRNA on the gene in question and
  • to show that the unknown gene is indeed responsible for the functions of the microRNA.
However through the use of specific target site blockers, we were able to demonstrate
  • that this unknown gene was associated with the phenotype observed
    • when the microRNA was inhibited in endothelial cells.
How do you feel about your results so far?
We are very pleased with the results of the TSB experiments and
  • altogether these results demonstrate that our miRNA of interest
  • is functional in endothelial cells
    • through the regulation of a target gene with a previously unknown role.
What do you find to be the main benefits/advantage of the LNA™ microRNA target site blockers from Exiqon?
Target Site Blockers are efficient tools to demonstrate the

  • specific involvement of putative microRNA targets
  • in the function played by this microRNA.
The use of LNA™ allows the design of short oligonucleotides that are very specific and easy to work with. 
What would be your advice to colleagues about getting started with microRNA functional analysis?
In order to address the role played by a microRNA,
  • it is essential to perform both gain and loss of functions experiments.
What are the next steps in the current project and how do you plan to perform them?
We plan to use microRNA inhibitor libraries to identify
  • more microRNAs specifically involved in the processes that we currently study.
When and where will be hear /read more about your studies?
We are currently in the process of submitting a manuscript regarding the function of my microRNA of interest.

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Endothelial Dysfunction (release into the circulation of damaged endothelial cells) as A Risk Marker for Ischemia and MI

Reporter and Curator: Larry H Bernstein, MD, FCAP

Endothelial Dysfunction: An Early Cardiovascular Risk Marker in Asymptomatic Obese Individuals with Prediabete

AK Gupta, E Ravussin, DL Johannsen, AJ Stull,WT.Cefalu and WD Johnson at Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA Brit J Med & Med Res 2012; 2(3):413-423 [www.ScienceDomain.org]

provides an exceedingly interesting insight into the relationship between type 2 diabetes mellitus, obesity and risk for cardiovascular disease in patients who are asymptomatic prediabetics, defined as a fasting blood glucose between 1000 and 1240 mg/L, or a Hb A1c (may not accurate for African Americans) between 5.6 and 6.5.  They would be expected to show an abnormal 5-hr GTT.

Obesity is associated with the release from adipocytes of adiponectin, which it has been reported is countered by resistin.  We might also have the effect of the insulin secreting beta cell, that releases insulin without a relationship to an anabolic function, through IGF-1 related to feedback to the pituitary GH, which takes a dominant catabolic role. Thus, insulin resistance. This is an oversimplification, and far greater depth is found elsewhere.

This study is consistent with another study on  Metabolism Influences Cancer

Reuben Shaw, Ph.D., a geneticist and researcher at the Salk Institute: Metabolism Influences Cancer

Recent development on Human Stem Cell Therapies for comorbidity and Cardiovascular disease

Human Stem Cell Therapies: UCSD New Discovery addressing the Limiting Factor and Providing the Solution

https://pharmaceuticalintelligence.com/2014/01/06/human-stem-cell-therapies-ucsd-new-discovery-addressing-the-limiting-factor-and-providing-the-solution/

This study reported a potential early marker of myocardial infarction by the release into the circulation of damaged endothelial cells that are to be measured in patients suspected of severe ischemia in a clinical trial.  The question that I raised in my comment was whether this would have to be a special immunochemical assay of tagged cells, and if that were the case, would it be measured on an automated flow-based hemocytometer, which can differentiate several populations of cells – granulocytes, lymphocytes, red cells, platelets, immature granuloytes, BLASTS.  That would be a very practical extension of the technology for labs worldwide.

Abstract

Aims: To elucidate if endothelial dysfunction is an early CV risk marker in obese men and women with prediabetes.
Study Design: Cross-sectional study.

Place and Duration of Study: Clinical Research Unit, Pennington Biomedical Research Center, Baton Rouge, LA. United States.

Background: Overweight and obese status denotes an increasing adipose tissue burden which spills over into ectopic locations, including the visceral compartment, muscle and liver. Associated co-morbidities enhance cardiovascular (CV) risk. Endothelium which is the largest receptor-effector end-organ in our bodies, while responding to numerous physical and chemical stimuli maintains vascular homeostasis. Endothelial dysfunction (ED) is the initial perturbation, which precedes fatty streak known to initiate atherosclerosis: insidious process which often culminates as sudden catastrophic CV adverse event.

Methodology:  Asymptomatic men and women; [n=42] coming in after an overnight fast had demographic, anthropometric, clinical chemistry and

  • resting endothelial function (EF)
  • increased test finger peripheral arterial tone (PAT) relative to control;
    • expressed as relative hyperemia index (RHI)] assessments.

Results: Adults with desirable weight [n=12] and overweight [n=8] state, had normal fasting plasma glucose [Mean(SD)]: FPG [91.1(4.5), 94.8(5.8) mg/dL], insulin [INS, 2.3(4.4), 3.1(4.8) µU/ml], insulin sensitivity by homeostasis model assessment [HOMA-IR, 0.62(1.2), 0.80(1.2)] and desirable resting clinic blood pressure [SBP/DBP, 118(12)/74(5), 118(13)/76(8) mmHg].

Obese adults [n=22] had

  • prediabetes [FPG, 106.5(3.5) g/dL],
  • hyperinsulinemia [INS 18.0(5.2) µU/ml],
  • insulin resistance [HOMA-IR .59(2.3)],
  • prehypertension [PreHTN; SBP/DBP 127(13)/81(7) mmHg] and
  • endothelial dysfunction [ED;
  • reduced RHI 1.7(0.3) vs. 2.4(0.3); all p<0.05].

Age-adjusted RHI correlated with BMI [r=-0.53; p<0.001]; however,

    • BMI-adjusted RHI was not correlated with age [r=-0.01; p=0.89].

Conclusion: Endothelial dysfunction reflective of cardiometabolic changes in obese adults can be an early risk marker for catastrophic CV events.

Keywords: Fasting plasma glucose; healthy adults; reverse cholesterol transport pathway; insulin resistance; body weight; relative hyperemia index.

ABBREVIATIONS

ADA: American Diabetes association; BMI: body mass index; CVD: cardiovascular disease; CV: cardiovascular; DBP: diastolic blood pressure; ED: endothelial dysfunction; EF: resting endothelial function; FPG: fasting plasma glucose; HOMA-IR: homeostasis model assessment; INS: insulin; JNC 7: Joint National Commission 7; LDL-C/HDL-C: low density lipoprotein cholesterol to high density lipoprotein; NCEP ATP III: National Cholesterol Education Program Adult Treatment Panel III; PAT: peripheral arterial tone; PreDM: prediabetes; PreHTN: prehypertension; PBRC: Pennington Biomedical Research Center; RHI: relative hyperemia index; SBP: systolic blood pressure; Total-C/HDL-C: total cholesterol to high density lipoprotein cholestrol; TG/HDL-C: triglycerides to high density lipoprotein cholesterol; WC: waist circumference.

Introduction

Healthy adults with no chronic medical conditions, on no prescription medications (n=24) and with low cardiovascular risk, in a randomized-order, cross-over clinical trial, with a 2 week washout period, exhibitd improved endothelial function (measured with flow mediated dilatation) with a diet rich in antioxidants (Franzini et al., 2012). Healthy over weight and obese volunteers with normal glucose appear to attenuate flow mediated dilation after high
glycemic index carbohydrate meals (Suessenbacher et al., 2011). In matched (age, work place, physical activity, tobacco use, blood pressure, serum lipids and family history of premature coronary artery disease) male shift and no shift workers, peripheral endothelial function (peripheral arterial tone (PAT) index obtained with the EndoPAT technique) was impaired in shift workers, suggesting elevated cardiovascular risk (Lavi et al., 2009).

Endothelial function thus appears to be an exquisitely sensitive marker for a variety of populations, under various conditions. Although endothelial function has been evaluated in numerous disease conditions and perturbed with a variety of agents, there has, to our knowledge, not been a comparison of resting endothelial function in free living healthy lean, overweight and obese subjects. Using a noninvasive assessment for resting endothelial function (by measuring the peripheral arterial tone, Bonetti et al., 2004), we tested the hypothesis that fasting glucose escalation in otherwise asymptomatic obese men and women is functionally reflected as endothelial dysfunction.

Endothelial Function

Assessment of resting endothelial function was done with the participant in fasting state, after having avoided stimulants (caffeine, tobacco, alcohol, exercise) for 12 hours, at the same fixed clock hour (range 8-10 AM), using the EndoPAT 2000 device manufactured by ITAMAR Medical®. This assessment technique has been previously validated (Bonetti et al., 2004), has been used in numerous (>250) peer reviewed publications (Carty et al., 2012; Kuvin et al., 2003) and has been in routine use in our clinical core. Briefly: subjects coming
in from home, after an overnight fast and having avoided stimulants for 12-hours, were placed in a supine position for 20 minutes in a quiet room before the test. A patented single use finger sleeve was then placed on the index finger of each hand to continuously measure peripheral arterial tone. A blood pressure cuff applied to the upper arm of the non-dominant arm (test arm) was then used to occlude the brachial artery for 5 minutes. This was followed by a rapid release. The dominant arm without any manipulation served as the control. The
built in, validated software integrated the data gathered from the finger sleeves of the control (undisturbed) and the test arms (during the baseline, occlusion and release phases), thus providing the relative hyperemia index (RHI) for the test arm. This flow mediated dilatation induced change in the test arm, relative to the control arm, served as the measure for endothelial function (RHI).

The subjects with desirable and overweight body weight were significantly younger [36.7(19.1) and 27.4(3.9) years, respectively], than those who were obese [53.2(11.6) years]. We performed correlations between the measure for endothelial function (RHI) and confounding factors like BMI, age and gender. Age-adjusted RHI correlated with BMI [r=- 0.53, p<0.001]; however, BMI-adjusted RHI was not associated with age [r=-0.01, p=0.89]. Fig. 1 depicts panels for the regression line for RHI as a function of age, (and BMI, glucose
and HOMA-IR, respectively) superimposed on a scatter plot. No correlation was observed between endothelial function and age (r²=0.07), while endothelial function was highly correlated with body mass index, glucose and insulin sensitivity (r²=0.3).

DISCUSSION

Asymptomatic obese adults with prediabetes (when compared to asymptomatic desirable weight and overweight adults with normal glucose), exhibit above the upper limits for desirable fasting plasma total cholesterol (>200mg/dL) and triglycerides (>150 mg/dL), but due to a relatively lower HDL-C display higher cardiac risk ratios (Total-C/HDL-C; p=0.05 and TG/HDL-C; p=0.02). A lower HDL-C and the elevated cardiac risk ratios are early clinical indicators for an impaired reverse cholesterol transport (RCT) pathway, a process by which cholesterol from the periphery is transported to the liver (Tall, 1998). The RCT pathway has been shown to be a sensitive indicator of the net flux (deposition vs. removal) of cholesterol homeostasis at the endothelium (Gupta et al., 1993; Tall et al., 2000). It is at the endothelium that the first fatty streaks, which over time deteriorate into atherosclerosis, have been shown to develop (Rosenfeld et al., 2000).

Impaired endothelial dysfunction is the first step in the process of atherosclerosis, even before the development of the fatty streak (Davignon, 2004; Ross 1999). These healthy obese men and women with prediabetes, prehypertension and impaired reverse cholesterol transport pathway were assessed to have impaired resting endothelial function, which is consistent with latent early onset cardiovascular disease.

We have demonstrated a high prevalence of isolated prediabetes or prehypertension and co-existing prediabetes and prehypertension, among the otherwise healthy US adults (Gupta et al., 2011). We have also elucidated that asymptomatic obese adults with overly heightened systemic inflammation, tend to have prediabetes and prehypertension (Gupta et al., 2010a). These individuals by various conventional measures (larger waist circumference, exacerbated systemic inflammation, higher insulin resistance, elevated triglycerides, lower high-density lipoprotein cholesterol, above average cardiac risk ratios and a significant co-existence of two or three concomitant metabolic risk factors) appear to be on an accelerated pathway towards early adverse cardiovascular events (Gupta et al., 2010a, 2010b). With this study we provide a dynamic, non-invasive, functional correlate: significant resting endothelial dysfunction, as an early biomarker for pre-atherosclerosis in obese adults with prediabetes.

Increased organ ectopic adipose burden especially in the muscle and liver appears to drive clinically recognizable adverse cardio metabolic changes (Hamdy et al., 2006). Increased inflammation (local and systemic) along with enhanced insulin resistance (liver, muscle) manifests as dysglycemia, dyslipidemia, excess reactive oxygen species, hyper-coagulablility and loss of blood pressure control (Gastaldelli et al., 2010).

We demonstrate an early impairment in the reverse cholesterol transport pathway, indicating a net deposition versus removal of cholesterol at the endothelium. In asymptomatic obese men and women with predisease  conditions (prediabetes and prehypertension) when contrasted with ideal bodyweight or overweight adults with normoglycemia and normal blood pressure, resting endothelial dysfunction can be an early warning sign for future catastrophic cardiovascular adverse events.

© 2012 Gupta et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

REFERENCES on circulating Endothelial Progenitor Cells as Biomarkers for Cardiovascular Disease and their Angiogenesis Potential.

Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964-967.

Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999;5:434-438.

Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001;7:430-436.

Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, et al. Aging, progenitor cell exhaustion, and atherosclerosis. Circulation 2003;108:457-463.

Hill JM, Zalos G, Halcox JPJ, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348:593-600.

Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation 2001;103:2885-2890

Laufs U, Werner N, Link A, et al. Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis. Circulation 2004;109:220-226.

Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005;353:999-1007.

Aicher A, Heeschen C, Mildner-Rihm C, et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med 2003;9:1370-1376.

Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004;364:141-148.

Zhang H, Vakil V, Braunstein M, et al. Circulating endothelial progenitor cells in multiple myeloma: implications and significance. Blood 2005;105:3286-3294

Lyden D, Hattori K, Dias S, et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 2001;7:1194-1201.

Other related articles that were published in this Open Access Online Scientific Journal include the following:

Lev-Ari, A. 2/28/2013 The Heart: Vasculature Protection – A Concept-based Pharmacological Therapy including THYMOSIN

https://pharmaceuticalintelligence.com/2013/02/28/the-heart-vasculature-protection-a-concept-based-pharmacological-therapy-including-thymosin/

Lev-Ari, A. 2/27/2013 Arteriogenesis and Cardiac Repair: Two Biomaterials – Injectable Thymosin beta4 and Myocardial Matrix Hydrogel

https://pharmaceuticalintelligence.com/2013/02/27/arteriogenesis-and-cardiac-repair-two-biomaterials-injectable-thymosin-beta4-and-myocardial-matrix-hydrogel/

Lev-Ari, A. 11/13/2012 Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes

https://pharmaceuticalintelligence.com/2012/11/13/peroxisome-proliferator-activated-receptor-ppar-gamma-receptors-activation-pparγ-transrepression-for-angiogenesis-in-cardiovascular-disease-and-pparγ-transactivation-for-treatment-of-dia/

Lev-Ari, A. 8/29/2012 Positioning a Therapeutic Concept for Endogenous Augmentation of cEPCs — Therapeutic Indications for Macrovascular Disease: Coronary, Cerebrovascular and Peripheral

https://pharmaceuticalintelligence.com/2012/08/29/positioning-a-therapeutic-concept-for-endogenous-augmentation-of-cepcs-therapeutic-indications-for-macrovascular-disease-coronary-cerebrovascular-and-peripheral/ 

Lev-Ari, A. 8/28/2012 Cardiovascular Outcomes: Function of circulating Endothelial Progenitor Cells (cEPCs): Exploring Pharmaco-therapy targeted at Endogenous Augmentation of cEPCs

https://pharmaceuticalintelligence.com/2012/08/28/cardiovascular-outcomes-function-of-circulating-endothelial-progenitor-cells-cepcs-exploring-pharmaco-therapy-targeted-at-endogenous-augmentation-of-cepcs/

Lev-Ari, A. 8/27/2012 Endothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs

https://pharmaceuticalintelligence.com/2012/08/27/endothelial-dysfunction-diminished-availability-of-cepcs-increasing-cvd-risk-for-macrovascular-disease-therapeutic-potential-of-cepcs/

Lev-Ari, A. 8/24/2012 Vascular Medicine and Biology: CLASSIFICATION OF FAST ACTING THERAPY FOR PATIENTS AT HIGH RISK FOR MACROVASCULAR EVENTS Macrovascular Disease – Therapeutic Potential of cEPCs

https://pharmaceuticalintelligence.com/2012/08/24/vascular-medicine-and-biology-classification-of-fast-acting-therapy-for-patients-at-high-risk-for-macrovascular-events-macrovascular-disease-therapeutic-potential-of-cepcs/

Lev-Ari, A. 7/19/2012 Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Lev-Ari, A. 4/30/2012 Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair

https://pharmaceuticalintelligence.com/2012/04/30/93/

Lev-Ari, A. 7/2/2012 Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

https://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

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Summary of Genomics and Medicine: Role in Cardiovascular Diseases


Summary of Genomics and Medicine: Role in Cardiovascular Diseases

Author: Larry H. Bernstein, MD, FCAP

The articles within Chapters and Subchapters you have just read have been organized into four interconnected parts.
  1. Genomics and Medicine
  2. Epigenetics – Modifyable Factors Causing CVD
  3. Determinants of CVD – Genetics, Heredity and Genomics Discoveries
  4. Individualized Medicine Guided by Genetics and Genomics Discoveries
The first part established the
  • rapidly evolving science of genomics
  • aided by analytical and computational tools for the identification of nucleotide substitutions, or combinations of them
that have a significant association with the development of
  • cardiovascular diseases,
  • hypercoagulable state,
  • atherosclerosis,
  • microvascular disease,
  • endothelial disruption, and
  • type-2DM, to name a few.
These may well be associated with increased risk for stroke and/or peripheral vascular disease in some cases,
  • essentially because the involvement of the circulation is systemic in nature.

Part 1

establishes an important connection between RNA and disease expression.  This development has led to
  • the necessity of a patient-centric approach to patient-care.
When I entered medical school, it was eight years after Watson and Crick proposed the double helix.  It was also
  • the height of a series of discoveries elucidating key metabolic pathways.
In the period since then there have been treatments for some of the important well established metabolic diseases of
  • carbohydrate,
  • protein, and
  • lipid metabolism,
such as –  glycogen storage disease, and some are immense challenges, such as
  • Tay Sachs, or
  • Transthyretin-Associated amyloidosis.
But we have crossed a line delineating classical Mendelian genetics to
  • multifactorial non-linear traits of great complexity and
involving combinatorial program analyses to resolve.
The Human Genome Project was completed in 2001, and it has opened the floodgates of genomic discovery.  This resulted in the identification of
genomic alterations in
  • cardiovascular disease,
  • cancer,
  • microbial,
  • plant,
  • prion, and
  • metabolic diseases.
This has also led to
  • the identification of genomic targets
  • that are either involved in transcription or
  • are involved with cellular control mechanisms for targeted pharmaceutical development.
In addition, there is great pressure on the science of laboratory analytics to
  • codevelop with new drugs,
  • biomarkers that are indicators of toxicity or
  • of drug effectiveness.
I have not mentioned the dark matter of the genome. It has been substantially reduced, and has been termed dark because
  • this portion of the genome is not identified in transcription of proteins.
However, it has become a lightning rod to ongoing genomic investigation because of
  • an essential role in the regulation of nuclear and cytoplasmic activities.
Changes in the three dimensional structure of these genes due to
  • changes in Van der Waal forces and internucleotide distances lead to
  • conformational changes that could have an effect on cell activity.

Part 2

is an exploration of epigenetics in cardiovascular diseases.  Epigenetics is
  • the post-genomic modification of genetic expression
  • by the substitution of nucleotides or by the attachment of carbohydrate residues, or
  • by alterations in the hydrophobic forces between sequences that weaken or strengthen their expression.
This could operate in a manner similar to the conformational changes just described.  These changes
  • may be modifiable, and they
  • may be highly influenced by environmental factors, such as
    1. smoking and environmental toxins,
    2. diet,
    3. physical activity, and
    4. neutraceuticals.
While neutraceuticals is a black box industry that evolved from
  • the extraction of ancient herbal remedies of agricultural derivation
    (which could be extended to digitalis and Foxglove; or to coumadin; and to penecillin, and to other drugs that are not neutraceuticals).

The best examples are the importance of

  • n-3 fatty acids, and
  • fiber
  • dietary sulfur (in the source of methionine), folic acid, vitamin B12
  • arginine combined with citrulline to drive eNOS
  • and of iodine, which can’t be understated.
In addition, meat consumption involves the intake of fat that contains

  • the proinflammatory n-6 fatty acid.

The importance of the ratio of n-3/n-6 fatty acids in diet is not seriously discussed when

  • we look at the association of fat intake and disease etiology.
Part 2 then leads into signaling pathways and proteomics. The signaling pathways are
  • critical to understanding the inflammatory process, just as
  • dietary factors tie in with a balance that is maintained by dietary intake,
    • possibly gut bacteria utilization of delivered substrate, and
    • proinflammatory factors in disaease.
These are being explored by microfluidic proteomic and metabolomic technologies that were inconceivable a half century ago.
This portion extended into the diagnosis of cardiovascular disease, and
  • elucidated the relationship between platelet-endothelial interaction in the formation of vascular plaque.
It explored protein, proteomic, and genomic markers
  1. for identifying and classifying types of disease pathobiology, and
  2. for following treatment measures.

Part 3

connected with genetics and genomic discoveries in cardiovascular diseases.

Part 4

is the tie between life style habits and disease etiology, going forward with
  • the pursuit of cardiovascular disease prevention.
The presentation of walking and running, and of bariatric surgery (type 2DM) are fine examples.
It further discussed gene therapy and congenital heart disease.  But the most interesting presentations are
  • in the pharmacogenomics for cardiovascular diseases, with
    1. volyage-gated calcium-channels, and
    2. ApoE in the statin response.

This volume is a splendid example representative of the entire collection on cardiovascular diseases.

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Diagnostic Value of Cardiac Biomarkers


Diagnostic Value of Cardiac Biomarkers

Author and Curator: Larry H Bernstein, MD, FCAP 

These presentations covered several views of the utilization of cardiac markers that have evolved for over 60 years.  The first stage was the introduction of enzymatic assays and isoenzyme measurements to distinguish acute hepatitis and acute myocardial infarction, which included lactate dehydrogenase (LD isoenzymes 1, 2) at a time that late presentation of the patient in the emergency rooms were not uncommon, with the creatine kinase isoenzyme MB declining or disappeared from the circulation.  The world health organization (WHO) standard definition then was the presence of two of three:

1. Typical or atypical precordial pressure in the chest, usually with radiation to the left arm

2. Electrocardiographic changes of Q-wave, not previously seen, definitive; ST- elevation of acute myocardial injury with repolarization;
T-wave inversion.

3. The release into the circulation of myocardial derived enzymes –
creatine kinase – MB (which was adapted to measure infarct size), LD-1,
both of which were replaced with troponins T and I, which are part of the actomyosin contractile apparatus.

The research on infarct size elicited a major research goal for early diagnosis and reduction of infarct size, first with fibrinolysis of a ruptured plaque, and this proceeded into the full development of a rapidly evolving interventional cardiology as well as cardiothoracic surgery, in both cases, aimed at removal of plaque or replacement of vessel.  Surgery became more imperative for multivessel disease, even if only one vessel was severely affected.

So we have clinical history, physical examination, and emerging biomarkers playing a large role for more than half a century.  However, the role of biomarkers broadened.  Patients were treated with antiplatelet agents, and a hypercoagulable state coexisted with myocardial ischemic injury.  This made the management of the patient reliant on long term followup for Warfarin with the international normalized ratio (INR) for a standardized prothrombin time (PT), and reversal of the PT required transfusion with thawed fresh frozen plasma (FFP).  The partial thromboplastin test (PPT) was necessary in hospitalization to monitor the heparin effect.

Thus, we have identified the use of traditional cardiac biomarkers for:

1. Diagnosis
2. Therapeutic monitoring

The story is only the beginning.  Many patients who were atypical in presentation, or had cardiovascular ischemia without plaque rupture were problematic.  This led to a concerted effort to redesign the troponin assays for high sensitivity with the concern that the circulation should normally be free of a leaked structural marker of myocardial damage. But of course, there can be a slow leak or a decreased rate of removal of such protein from the circulation, and the best example of this would be the patient with significant renal insufficiency, as TnT is clear only through the kidney, and TNI is clear both by the kidney and by vascular endothelium.  The introduction of the high sensitivity assay has been met with considerable confusion, and highlights the complexity of diagnosis in heart disease.  Another test that is used for the diagnosis of heart failure is in the class of natriuretic peptides (BNP, pro NT-BNP, and ANP), the last of which has been under development.

While there is an exponential increase in the improvement of cardiac devices and discovery of pharmaceutical targets, the laboratory support for clinical management is not mature.  There are miRNAs that may prove valuable, matrix metalloprotein(s), and potential endothelial and blood cell surface markers, they require

1. codevelopment with new medications
2. standardization across the IVD industry
3. proficiency testing applied to all laboratories that provide testing
4. the measurement  on multitest automated analyzers with high capability in proteomic measurement  (MS, time of flight, MS-MS)

nejmra1216063_f1   Atherosclerotic Plaques Associated with Various Presentations               nejmra1216063_f2     Inflammatory Pathways Predisposing Coronary Arteries to Rupture and Thrombosis.        atherosclerosis progression

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Curator & Author: Larry H. Bernstein, MD, FCAP

Leaders in Pharmaceutical Intelligence

Subtitle: Nitric Oxide, Peroxinitrite, and NO donors in Renal Function Loss

Summary: The criticality of renal function is traced to the emergence of animal forms from the sea to land. It also becomes acutely and/or chronically dysfunctional in metabolic, systemic inflammatory and immunological diseases of man. We have already described the key role that nitric oxide and the NO synthases play in reduction of oxidative stress, and we have seen that a balance has to be struck between pro- and anti-oxidative as well as inflammatory elements for avoidance of diseases, specifically involving the circulation, but effectively not limited to any organ system. In this discussion we shall look at kidney function, NO and NO donors. This is an extension of a series of posts on NO and NO related disorders.

__________________________________________________________________________________________________________________________________________________________

Part I. The evolution of kidney structure and Function Evolution of kidney function

In fish the nerves that activate breathing take a short journey from an ancient part of the brain, the brain stem, to the throat and gills. For the ancient tadpole, the nerve controlling a reflex related to hiccup in man served a useful purpose, allowing the entrance to the lung to remain open when breathing air but closing it off when gulping water – which would then be directed only to the gills.

For humans and other mammals it provides a bit of evidence of our common ancestry. DNA evidence has pinned iguanas and chameleons as the closest relatives to snakes. In utero, we develop three separate kidneys in succession, absorbing the first two before we wind up with the embryonic kidney that will become our adult kidney. The first two of these reprise embryonic kidneys of ancestral forms, and in the proper evolutionary order.

The pronephric kidney does not function in human and other mammalian embryos. It disappears and gives rise to the Mesonephric kidney. This kidney filters wastes from the blood and excretes them to the outside of the body via a pair of tubes called the mesonephric ducts (also “Wolffian ducts”). The mesonephric kidney goes on to develop into the adult kidney of fish and amphibians.

This kidney does function for a few weeks in the human embryo, but then disappears as our final kidney forms, which is the Metanephric kidney. This begins developing about five weeks into gestation, and consists of an organ that filters wastes from the blood and excretes them to the outside through a pair ureters. In the embryo, the wastes are excreted directly into the amniotic fluid. The metanephric kidney is the final adult kidney of reptiles, birds, and mammals.

The first two kidneys resemble, in order, those of primitive aquatic vertebrates (lampreys and hagfish) and aquatic or semiaquatic vertebrates (fish and amphibians): an evolutionary order.

The explanation, then, is that we go through developmental stages that show organs resembling those of our ancestors. Take a step back and we see that fresh water fish have glomerular filtration. Cardiac contraction provides the pressure to force the water, small molecules, and ions into the glomerulus as nephric filtrate. The essential ingredients are then reclaimed by the tubules, returning to the blood in the capillaries surrounding the tubules. The amphibian kidney also functions chiefly as a device for excreting excess water.

But the problem is to conserve water, not eliminate it. The frog adjusts to the varying water content of its surroundings by adjusting the rate of filtration at the glomerulus. When blood flow through the glomerulus is restricted, a renal portal system is present to carry away materials reabsorbed through the tubules. Bird kidneys function like those of reptiles (from which they are descended). Uric acid is also their chief nitrogenous waste. All mammals share our use of urea as their chief nitrogenous waste. Urea requires much more water to be excreted than does uric acid. Mammals produce large amounts of nephric filtrate but are able to reabsorb most of this in the tubules. But even so, humans lose several hundred ml each day in flushing urea out of the body.

In his hypothesis of the evolution of renal function Homer Smith proposed that the formation of glomerular nephron and body armor had been adequate for the appearance of primitive vertebrates in fresh water and that the adaptation of homoiotherms to terrestrial life was accompanied by the appearance of the loop of Henle.

In the current paper, the increase in the arterial blood supply and glomerular filtration rate and the sharp elevation of the proximal reabsorption are viewed as important mechanisms in the evolution of the kidney. The presence of glomeruli in myxines and of nephron loops in lampreys suggests that fresh water animals used the preformed glomerular apparatus of early vertebrates, while mechanisms of urinary concentration was associated with the subdivision of the kidney into the renal cortex and medulla. The principles of evolution of renal functions can be observed at several levels of organizations in the kidney.

Natochin YV. Evolutionary aspects of renal function. Kidney International 1996; 49: 1539–1542; doi:10.1038/ki.1996.220. Smith HW: From Fish to Philosopher. Boston, Little, Brown, 1953.

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The Kidney: Anatomy and Physiology

The kidney lies in the lower abdomen capped by the adrenal glands. It has an outer cortex and an inner medulla. The basic unit is the nephron, which filters blood at the glomerulus, and not only filters urine eliminating mainly urea, also uric acid, and other nitrogenous waste, but also reabsorbs Na+ in exchange for H+/(reciprocal K+) through the carbonic anhydrase of the epithelium. In addition, it serves as a endocrine organ and receptor through the renin-angiotensin/aldosterone system, sensitivity to water loss controlled by antidiuretic hormone, and is sensitive to the natriuretic peptides of the heart. The kidney is an elegant structure with a high concentration of glomeruli in the cortex, and in the medulla one finds a U-shaped tube that is critical in a countercurrent multiplier system with a descending limb, Loop of Henley, and ascending limb.

As the filtrate flows through the glomerulus into the descending limb, there is reabsorption of glucose and of H+ by the carbonic anhydrase conversion to water and CO2, except with serious acidemia, in which K+ is reabsorbed with H+ loss to the filtrate, resulting in a hyperkalemia. In the descending limb Na+ is absorbed into the interstitium, and the hypertonic interstitium draws water back for circulation, regulated by the action of ADH on the epithelium of the ascending limb. The result in terms of basic urinary clearance, the volume of urine loss is moderated by the amount needed for circulation (10 units of whole blood) without dehydration, and an amount sufficient for metabolite loss (including drug metabolites). The urine flows into the kidney pelvis and flow down the ureters.

The renal blood flow needs mention. The blood reaches the glomerulus by way of the afferent arteriole and leaves by way of the efferent arteriole. In a book by the Harvard Pathologist Shields Warren on diabetes he made a distinction between hypertension and diabetes in that efferent arteriolar sclerosis is present in both, but diabetes is uniquely identified by afferent arteriolar sclerosis. In diabetes you also have a typical glomerulosclerosis, which might be related to the same hyalinization found in the pancreatic islets – a secondary amyloidosis.

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English: Nephron, Diagram of the urine formati...

English: Nephron, Diagram of the urine formation. The number inside tubular urin concentration in mOsm/l – when ADH acts Polski: Nefron, Schemat tworzenia moczu. Cyfry wewnątrz kanalików oznaczają lokalne stężenie w mOsm/l – gdy działa ADH (dochodzi do zagęszczania moczu). (Photo credit: Wikipedia)

Loop of Henle (Grey's Anatomy book)

Loop of Henle (Grey’s Anatomy book) (Photo credit: Wikipedia)

Frontal section through the kidney

Frontal section through the kidney (Photo credit: Wikipedia)

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_ Part IIa. Nitric Oxide role in renal tubular epithelial cell function Tubulointerstitial Nephritides

As part of the exponential growth in our understanding of nitric oxide (NO) in health and disease over the past 2 decades, the kidney has become appreciated as a major site where NO may play a number of important roles. Although earlier work on the kidney focused more on effects of NO at the level of larger blood vessels and glomeruli, there has been a rapidly growing body of work showing critical roles for NO in tubulointerstitial disease. In this review we discuss some of the recent contributions to this important field.

Mattana J, Adamidis A, Singhal PC. Nitric oxide and tubulointerstitial nephritides. Seminars in Nephrology 2004; 24(4):345-353.

Nitric oxide donors and renal tubular (subepithelial) matrix

Nitric oxide (NO) and its metabolite, peroxynitrite (ONOO-), are involved in renal tubular cell injury. If NO/ONOO- has an effect to reduce cell adhesion to the basement membrane, does this effect contribute to tubular obstruction and would it be partially responsible for the harmful effect of NO on the tubular epithelium during acute renal failure (ARF)?

Wangsiripaisan A, et al. examined the effect of the NO donors

  • [1] (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (DETA/NO),
  • [2] spermine NONOate (SpNO), and
  • [3] the ONOO- donor 3-morpholinosydnonimine (SIN-1) on

cell-matrix adhesion to collagen types I and IV, and also fibronectin using three renal tubular epithelial cell lines:

  • [1] LLC-PK1,
  • [2] BSC-1, and
  • [3] OK.

It was only the exposure to SIN-1 that caused a dose-dependent impairment in cell-matrix adhesion.

Similar results were obtained in the different cell types and matrix proteins. The effect of SIN-1 (500 microM) on LLC-PK1 cell adhesion was not associated with either cell death or alteration of matrix protein and was attenuated by either

  • [1] the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide,
  • [2] the superoxide scavenger superoxide dismutase, or
  • [3] the ONOO- scavenger uric acid in a dose-dependent manner.

These investigators concluded in this seminal paper that ONOO- generated in the tubular epithelium during ischemia/reperfusion has the potential to impair the adhesion properties of tubular cells, which then may contribute to the tubular obstruction in ARF.

Wangsiripaisan A, Gengaro PE, Nemenoff RA, Ling H, et al. Effect of nitric oxide donors on renal tubular epithelial cell-matrix adhesion. Kidney Int 1999; 55(6):2281-8.

Coexpressed Nitric Oxide Synthase and Apical β1 Integrins

In sepsis-induced acute renal failure, actin cytoskeletal alterations result in shedding of proximal tubule epithelial cells (PTEC) and tubular obstruction.

This study examined the hypothesis that inflammatory cytokines, released early in sepsis, cause PTEC cytoskeletal damage and alter integrin-dependent cell-matrix adhesion. The question of whether the intermediate nitric oxide (NO) modulates these cytokine effects was also examined. After exposure of human PTEC to tumor necrosis factor-α, interleukin-1α, and interferon-γ, the actin cytoskeleton was disrupted and cells became elongated, with extension of long filopodial processes.

Cytokines induced shedding of viable, apoptotic, and necrotic PTEC, which was dependent on NO synthesized by inducible NO synthase (iNOS) produced as a result of cytokine actions on PTEC. Basolateral exposure of polarized PTEC monolayers to cytokines induced maximal NO-dependent cell shedding, mediated in part through NO effects on cGMP. Cell shedding was accompanied by dispersal of basolateral β1 integrins and E-cadherin, with corresponding upregulation of integrin expression in clusters of cells elevated above the epithelial monolayer.

These cells demonstrated coexpression of iNOS and apically redistributed β1 integrins. These authors point out that the major ligand involved in cell anchorage was laminin, probably through interactions with the integrin α3β1.

This interaction was downregulated by cytokines but was not dependent on NO. They posulate a mechanism by which inflammatory cytokines induce PTEC damage in sepsis, in the absence of hypotension and ischemia.

Glynne PA, Picot J and Evans TJ. Coexpressed Nitric Oxide Synthase and Apical β1 Integrins Influence Tubule Cell Adhesion after Cytokine-Induced Injury. JASN 2001; 12(11): 2370-2383.

Potentiation by Nitric Oxide of Apoptosis in Renal Proximal Tubule Cells

Proximal tubular epithelial cells (PTEC) exhibit a high sensitivity to undergo apoptosis in response to proinflammatory stimuli and immunosuppressors and participate in the onset of several renal diseases. This study examined the expression of inducible nitric oxide (NO) synthase after challenge of PTEC with bacterial cell wall molecules and inflammatory cytokines and analyzed the pathways that lead to apoptosis in these cells by measuring changes in the mitochondrial transmembrane potential and caspase activation.

The data show that the apoptotic effects of proinflammatory stimuli mainly were due to the expression of inducible NO synthase. Cyclosporin A and FK506 inhibited partially NO synthesis.

However, both NO and immunosuppressors induced apoptosis, probably through a common mechanism that involved the irreversible opening of the mitochondrial permeability transition pore. Activation of caspases 3 and 7 was observed in cells treated with high doses of NO and with moderate concentrations of immunosuppressors.

The conclusion is that the cooperation between NO and immunosuppressors that induce apoptosis in PTEC might contribute to the renal toxicity observed in the course of immunosuppressive therapy.

Hortelano S, Castilla M, Torres AM, Tejedor A, and Bosca L.  Potentiation by Nitric Oxide of Cyclosporin A and FK506- Induced Apoptosis in Renal Proximal Tubule Cells. J Am Soc Nephrol 2000; 11: 2315–2323.

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Part IIb. Related studies with ROS and/or RNS on nonrenal epithelial cells

Reactive nitrogen species block cell cycle re-entry Endogenous sources of reactive nitrogen species (RNS) act as second messengers in a variety of cell signaling events, whereas environmental sources of RNS like nitrogen dioxide (NO2) inhibit cell survival and growth through covalent modification of cellular macromolecules. Murine type II alveolar cells arrested in G0 by serum deprivation were exposed to either NO2 or SIN-1, a generator of RNS, during cell cycle re-entry.

In serum-stimulated cells, RNS blocked cyclin D1 gene expression, resulting in cell cycle arrest at the boundary between G0 and G1. Dichlorofluorescin diacetate (DCF) fluorescence indicated that RNS induced sustained production of intracellular hydrogen peroxide (H2O2), which normally is produced only transiently in response to serum growth factors.

Loading cells with catalase prevented enhanced DCF fluorescence and rescued cyclin D1 expression and S phase entry.

These studies indicate environmental RNS interfere with cell cycle re-entry through an H2O2-dependent mechanism that influences expression of cyclin D1 and progression from G0 to the G1 phase of the cell cycle.

Yuan Z, Schellekens H, Warner L, Janssen-Heininger Y, Burch P, Heintz NH. Reactive nitrogen species block cell cycle re-entry through sustained production of hydrogen peroxide. Am J Respir Cell Mol Biol. 2003;28(6):705-12. Epub 2003 Jan 10.

Peroxynitrite modulates MnSOD gene expression

Peroxynitrite (ONOO-) is a strong oxidant derived from nitric oxide (‘NO) and superoxide (O2.-), reactive nitrogen (RNS) and oxygen species (ROS) present in inflamed tissue. Other oxidant stresses, e.g., TNF-alpha and hyperoxia,   induce mitochondrial, manganese-containing superoxide dismutase (MnSOD) gene expression.   3-morpholinosydnonimine HCI (SIN-1) (10 or 1000 microM) increased MnSOD mRNA, but did not change hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA.   Authentic peroxynitrite (ONOO ) (100-500 microM) also increased MnSOD mRNA but did not change constitutive HPRT mRNA expression.   ONOO stimulated luciferase gene expression driven by a 2.5 kb fragment of the rat MnSOD gene 5′ promoter region.

MnSOD gene induction due to ONOO- was

  • [1] inhibited effectively by L-cysteine (10 mM) and
  • [2] partially inhibited by N-acetyl cysteine (NAC)(50 mM) or
  • [3] pyrrole dithiocarbamate (10 mM).

.NO from 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) (100 or 1000 microM) did not change MnSOD or HPRT mRNA, nor did either H202 or NO2-, breakdown products of SIN-1 and ONOO, have any effect on MnSOD mRNA expression; ONOO- and SIN-1 also did not increase detectable MnSOD protein content or increase MnSOD enzymatic activity.

Nevertheless, increased steady state [O2.-] in the presence of .NO yields ONOO , and ONOO has direct, stimulatory effects on MnSOD transcript expression driven at the MnSOD gene 5′ promoter region inhibited completely by L-cysteine and partly by N-acetyl cysteine in lung epithelial cells. This raises a question of whether the same effect is seen in renal tubular epithelium.

Jackson RM, Parish G, Helton ES. Peroxynitrite modulates MnSOD gene expression in lung epithelial cells. Free Radic Biol Med. 1998; 25(4-5):463-72.

Comparative impacts of glutathione peroxidase-1 gene knockout on oxidative stress

Selenium-dependent glutathione peroxidase-1 (GPX1) protects against reactive-oxygen-species (ROS)-induced oxidative stress in vivo, but its role in coping with reactive nitrogen species (RNS) is unclear. Primary hepatocytes were isolated from GPX1-knockout (KO) and wild-type (WT) mice to test protection of GPX1 against cytotoxicity of

  • [1] superoxide generator diquat (DQ),
  • [2]NO donor S-nitroso-N-acetyl-penicillamine (SNAP) and
  • [3] peroxynitrite generator 3-morpholinosydnonimine (SIN-1).

Treating cells with SNAP in addition to DQ produced synergistic cytotoxicity that minimized differences in apoptotic cell death and oxidative injuries between the KO and WT cells. Less protein nitrotyrosine was induced by 0.05-0.5 mM DQ+0.25 mM SNAP in the KO than in the WT cells.

Total GPX activity in the WT cells was reduced by 65 and 25% by 0.5 mM DQ+0.1 mM SNAP and 0.5 mM DQ, respectively. Decreases in Cu,Zn-superoxide dismutase (SOD) activity and increases in Mn-SOD activity in response to DQ or DQ+SNAP were greater in the KO cells than in the WT cells.

The study indicates GPX1 was more effective in protecting hepatocytes against oxidative injuries mediated by ROS alone than by ROS and RNS together, and knockout of GPX1 did not enhance cell susceptibility to RNS-associated cytotoxicity. Instead, it attenuated protein nitration induced by DQ+SNAP.

To better understand the mechanism(s) underlying nitric oxide (. NO)-mediated toxicity, in the presence and absence of concomitant oxidant exposure, postmitotic terminally differentiated NT2N cells (which are incapable of producing . NO) were exposed to [1]PAPA-NONOate (PAPA/NO) and [2] 3-morpholinosydnonimine (SIN-1).

Exposure to SIN-1, which generated peroxynitrite (ONOO) in the range of 25-750 nM/min, produced a concentration- and time-dependent delayed cell death.   In contrast, a critical threshold concentration (>440 nM/min) was required for . NO to produce significant cell injury.   There is a largely necrotic lesion after ONOO exposure and an apoptotic-like morphology after . NO exposure.

Cellular levels of reduced thiols correlated with cell death, and pretreatment with N-acetylcysteine (NAC) fully protected from cell death in either PAPA/NO or SIN-1 exposure. NAC given within the first 3 h posttreatment further delayed cell death and increased the intracellular thiol level in SIN-1 but not . NO-exposed cells.

Cell injury from . NO was independent of cGMP, caspases, and superoxide or peroxynitrite formation.   Overall, exposure of non-. NO-producing cells to . NO or peroxynitrite results in delayed cell death, which, although occurring by different mechanisms,   appears to be mediated by the loss of intracellular redox balance.

Gow AJ, Chen Q, Gole M, Themistocleous M, Lee VM, Ischiropoulos H. Two distinct mechanisms of nitric oxide-mediated neuronal cell death show thiol dependency. Am J Physiol Cell Physiol. 2000; 278(6):C1099-107.

NO2 effect on phosphatidyl choline   Nitrogen dioxide (NO2) inhalation affects the extracellular surfactant as well as the structure and function of type II pneumocytes.

The studies had differences in oxidant concentration, duration of exposure, and mode of NO2 application. This study evaluated the influence of the NO2 application mode on the phospholipid metabolism of type II pneumocytes. Rats were exposed to identical NO2 body doses (720 ppm x h), which were applied continuously (10 ppm for 3 d), intermittently (10 ppm for 8 h per day, for 9 d), and repeatedly (10 ppm for 3 d, 28 d rest, and then 10 ppm for 3 d). Immediately after exposure, type II cells were isolated and evaluated for cell yield, vitality, phosphatidylcholine (PC) synthesis, and secretion.

Type II pneumocyte cell yield was only increased from animals that had been continuously exposed to NO2, but vitality of the isolated type II pneumocytes was not affected by the NO2 exposure modes. Continuous application of 720 ppm x h NO2 resulted in increased activity of the cytidine-5-diphosphate (CDP)-choline pathway.   After continuous NO2 application,

  • [1] specific activity of choline kinase,
  • [2] cytidine triphosphate (CTP):cholinephosphate cytidylyltransferase,
  • [3] uptake of choline, and
  • [4] pool sizes of CDP-choline and PC   were significantly increased over those of controls.

Intermittent application of this NO2 body dose provoked less increase in PC synthesis and the synthesis parameters were comparable to those for cells from control animals after repeated exposure. Whereas PC synthesis in type II cells was stimulated by NO2, their secretory activity was reduced.   Continuous exposure reduced the secretory activity most, whereas intermittent exposure nonsignificantly reduced this activity as compared with that of controls. The repeated application of NO2 produced no differences.

The authors conclude that…. type II pneumocytes adapt to NO2 atmospheres depending on the mode of its application, at least for the metabolism of PC and its secretion from isolated type II pneumocytes.

The reader asks whether this effect could also be found in renal epithelial cells, for which PC is not considered vital as for type II pneumocytes and possibly related to surfactant activity in the lung.

Müller B, Seifart C, von Wichert P, Barth PJ. Adaptation of rat type II pneumocytes to NO2: effects of NO2 application mode on phosphatidylcholine metabolism. Am J Respir Cell Mol Biol. 1998; 18(5): 712-20.

iNOS involved in immediate response to anaphylaxis

The generation of large quantities of nitric oxide (NO) is implicated in the pathogenesis of anaphylactic shock. The source of NO, however, has not been established and conflicting results have been obtained when investigators have tried to inhibit its production in anaphylaxis.

This study analyzed the expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in a mouse model of anaphylaxis.   BALB/c mice were sensitized and challenged with ovalbumin to induce anaphylaxis. Tissues were removed from the heart and lungs, and blood was drawn at different time points during the first 48 hours after induction of anaphylaxis. The Griess assay was used to measure nitric oxide generation.

Nitric oxide synthase expression was examined by reverse transcriptase polymerase chain reaction and immunohistochemistry. A significant increase in iNOS mRNA expression and nitric oxide production was evident as early as 10 to 30 minutes after allergen challenge in both heart and lungs.

In contrast, expression of eNOS mRNA was not altered during the course of the experiment. The results support involvement of iNOS in the immediate physiological response of anaphylaxis.

Sade K, Schwartz IF, Etkin S, Schwartzenberg S, et al. Expression of Inducible Nitric Oxide Synthase in a Mouse Model of Anaphylaxis. J Investig Allergol Clin Immunol 2007; 17(6):379-385.

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Part IIc. Additional Nonrenal Related NO References

1. Nitrogen dioxide induces death in lung epithelial cells in a density-dependent manner. Persinger RL, Blay WM, Heintz NH, Hemenway DR, Janssen-Heininger YM. Am J Respir Cell Mol Biol. 2001 May;24(5):583-90. PMID: 11350828 [PubMed – indexed for MEDLINE] Free Article

2. Molecular mechanisms of nitrogen dioxide induced epithelial injury in the lung. Persinger RL, Poynter ME, Ckless K, Janssen-Heininger YM. Mol Cell Biochem. 2002 May-Jun;234-235(1-2):71-80. Review. PMID: 12162462 [PubMed – indexed for MEDLINE]

3. Nitric oxide and peroxynitrite-mediated pulmonary cell death. Gow AJ, Thom SR, Ischiropoulos H. Am J Physiol. 1998 Jan;274(1 Pt 1):L112-8. PMID: 9458808 [PubMed – indexed for MEDLINE] Free Article

4. Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells. Nabeyrat E, Jones GE, Fenwick PS, Barnes PJ, Donnelly LE. Am J Physiol Lung Cell Mol Physiol. 2003 Jun;284(6):L1112-20. Epub 2003 Feb 21. PMID: 12598225 [PubMed – indexed for MEDLINE] Free Article

5. Peroxynitrite inhibits inducible (type 2) nitric oxide synthase in murine lung epithelial cells in vitro. Robinson VK, Sato E, Nelson DK, Camhi SL, Robbins RA, Hoyt JC. Free Radic Biol Med. 2001 May 1;30(9):986-91. PMID: 11316578 [PubMed – indexed for MEDLINE]

6. Nitric oxide-mediated chondrocyte cell death requires the generation of additional reactive oxygen species. Del Carlo M Jr, Loeser RF. Arthritis Rheum. 2002 Feb;46(2):394-403. PMID: 11840442 [PubMed – indexed for MEDLINE]

7. Colon epithelial cell death in 2,4,6-trinitrobenzenesulfonic acid-induced colitis is associated with increased inducible nitric-oxide synthase expression and peroxynitrite production.

Yue G, Lai PS, Yin K, Sun FF, Nagele RG, Liu X, Linask KK, Wang C, Lin KT, Wong PY. J Pharmacol Exp Ther. 2001 Jun;297(3):915-25. PMID: 11356911 [PubMed – indexed for MEDLINE] Free Article

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Part IIIa. Acute renal failure   Acute renal failure (ARF), characterized by sudden loss of the ability of the kidneys to [1] excrete wastes, [2] concentrate urine, [3] conserve electrolytes, and [4] maintain fluid balance, is a frequent clinical problem, particularly in the intensive care unit, where it is associated with a mortality of between 50% and 80%.

This clinical entity was described as an acute loss of kidney function that occurred in severely injured crush victims because of histological evidence for patchy necrosis of renal tubules at autopsy. In the clinical setting, the terms ATN and acute renal failure (ARF) are frequently used interchangeably. However, ARF does not include increases in blood urea due to [1] reversible renal vasoconstriction (prerenal azotemia) or [2] urinary tract obstruction (postrenal azotemia). Acute hemodialysis was first used clinically during the Korean War in 1950 to treat military casualties, and this led to a decrease in mortality of the ARF clinical syndrome from about 90% to about 50%.   In the half century that has since passed, much has been learned about the pathogenesis of ischemic and nephrotoxic ARF in experimental models, but there has been very little improvement in mortality. This may be explained by changing demographics: [1] the age of patients with ARF continues to rise, and [2] comorbid diseases are increasingly common in this population. Both factors may obscure any increased survival related to improved critical care. Examining the incidence of ARF in several military conflicts does, however, provide some optimism. The incidence of ARF in seriously injured casualties decreased between World War II and the Korean War, and again between that war and the Vietnam War, despite the lack of any obvious difference in the severity of the injuries. What was different was the rapidity of the fluid resuscitation of the patients? Fluid resuscitation on the battlefield with the rapid evacuation of the casualties to hospitals by helicopter began during the Korean War and was optimized further during the Vietnam War. For seriously injured casualties the incidence of ischemic ARF was one in 200 in the Korean War and one in 600 in the Vietnam War. This historical sequence of events suggests that early intervention could prevent the occurrence of ARF, at least in military casualties.   In experimental studies it has been shown that progression from an azotemic state associated with renal vasoconstriction and intact tubular function (prerenal azotemia) to established ARF with tubular dysfunction occurs if the renal ischemia is prolonged. Moreover, early intervention with fluid resuscitation was shown to prevent the progression from prerenal azotemia to established ARF. Diagnostic evaluation of ARF One important question, therefore, is how to assure that an early diagnosis of acute renal vasoconstriction can be made prior to the occurrence of tubular dysfunction, thus providing the potential to prevent progression to established ARF. In this regard, past diagnostics relied on observation of the patient response to a fluid challenge: [1] decreasing levels of blood urea nitrogen (BUN) indicated the presence of reversible vasoconstriction, [2] while uncontrolled accumulation of nitrogenous waste products, i.e., BUN and serum creatinine, indicated established ARF.

This approach, however, frequently led to massive fluid overload in the ARF patient with resultant

  • [1] pulmonary congestion,
  • [2] hypoxia, and
  • [3] premature need for mechanical ventilatory support and/or hemodialysis.

On this background the focus turned to an evaluation of urine sediment and urine chemistries to differentiate between renal vasoconstriction with intact tubular function and established ARF.

It was well established that if tubular function was intact, renal vasoconstriction was associated with enhanced tubular sodium reabsorption. Specifically, the fraction of filtered sodium that is rapidly reabsorbed by normal tubules of the vasoconstricted kidney is greater than 99%.

Thus, when nitrogenous wastes, such as creatinine and urea, accumulate in the blood due to a fall in glomerular filtration rate (GFR) secondary to renal vasoconstriction with intact tubular function, the fractional excretion of filtered sodium (FENa = [(urine sodium × plasma creatinine) / (plasma sodium × urine creatinine)]) is less than 1%. An exception to this physiological response of the normal kidney to vasoconstriction is when the patient is receiving a diuretic, including mannitol, or has glucosuria, which decreases tubular sodium reabsorption and increases FENa.

It has recently been shown in the presence of diuretics that a rate of fractional excretion of urea (FEurea) of less than 35 indicates intact tubular function, thus favoring renal vasoconstriction rather than established ARF as a cause of the azotemia.

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English: Physiology of Nephron

English: Physiology of Nephron (Photo credit: Wikipedia)

Structures of the kidney: 1.Renal pyramid 2.In...

Structures of the kidney: 1.Renal pyramid 2.Interlobar artery 3.Renal artery 4.Renal vein 5.Renal hilum 6.Renal pelvis 7.Ureter 8.Minor calyx 9.Renal capsule 10.Inferior renal capsule 11.Superior renal capsule 12.Interlobar vein 13.Nephron 14.Minor calyx 15.Major calyx 16.Renal papilla 17.Renal column (no distinction for red/blue (oxygenated or not) blood, arteriole is between capilaries and larger vessels (Photo credit: Wikipedia)

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Mechanisms of ARF

Based on the foregoing comments, this discussion of mechanisms of ARF will not include nitrogenous-waste accumulation due to renal vasoconstriction with intact tubular function (prerenal azotemia) or urinary tract obstruction (postrenal azotemia). The mechanisms of ARF involve both vascular and tubular factors. An ischemic insult to the kidney will in general be the cause of the ARF. While a decrease in renal blood flow with diminished oxygen and substrate delivery to the tubule cells is an important ischemic factor, it must be remembered that a relative increase in oxygen demand by the tubule is also a factor in renal ischemia.

Approximately 30–70% of these shed epithelial tubule cells in the urine are viable and can be grown in culture. Recent studies using cellular and molecular techniques have provided information relating to the structural abnormalities of injured renal tubules that occur both in vitro and in vivo. In vitro studies using chemical anoxia have revealed abnormalities in the proximal tubule cytoskeleton that are associated with translocation of Na+/K+-ATPase from the basolateral to the apical membrane.

A comparison of cadaveric transplanted kidneys with delayed versus prompt graft function has also provided important results regarding the role of Na+/K+-ATPase in ischemic renal injury. This study demonstrated that, compared with kidneys with prompt graft function, those with delayed graft function had a significantly greater cytoplasmic concentration of Na+/K+-ATPase and actin-binding proteins — spectrin (also known as fodrin) and ankyrin — that had translocated from the basolateral membrane to the cytoplasm.

Such a translocation of Na+/K+-ATPase from the basolateral membrane to the cytoplasm could explain the decrease in tubular sodium reabsorption that occurs with ARF. An important focus of research is the mechanisms whereby the critical residence of Na+/K+-ATPase in the basolateral membrane (which facilitates vectorial sodium transport) is uncoupled by hypoxia or ischemia.  The actin-binding proteins,

  • spectrin and
  • ankyrin,

serve as substrates for the calcium-activated cysteine protease calpain.

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In vitro studies in proximal tubules have shown a rapid rise in cytosolic calcium concentration during acute hypoxia, which antedates the evidence of tubular injury as assessed by lactic dehydrogenase (LDH) release. There is further evidence to support the importance of the translocation of Na+/K+-ATPase from the basolateral membrane to the cytoplasm during renal ischemia/reperfusion.

Specifically, calpain-mediated breakdown products of the actin-binding protein spectrin occur with renal ischemia. Calpain activity was demonstrated to increase during hypoxia in isolated proximal tubules. Measurement of LDH release following calpain inhibition indicated attenuation of hypoxic damage to proximal tubules. There was no evidence of an increase in cathepsin, a (cysteine protease) in proximal tubules during hypoxia , but there is a calcium-independent pathway for calpain activation during hypoxia.

Calpastatin, an endogenous cellular inhibitor of calpain activation, was shown to be diminished during hypoxia in association with the rise in another cysteine protease, caspase.

This effect of diminished calpastatin activity could be reversed by caspase inhibition. Proteolytic pathways appear to be involved in calpain-mediated proximal tubule cell injury during hypoxia. Calcium activation of phospholipase A has also been shown to contribute to renal tubular injury during ischemia.

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Tubular obstruction during ARF

The existence of proteolytic pathways involving cysteine proteases, namely calpain and caspases, may therefore explain

  • the decrease in proximal tubule sodium reabsorption and
  • increased FENa

secondary to proteolytic uncoupling of Na+/K+-ATPase from its basolateral membrane anchoring proteins.

This tubular perturbation alone, however, does not explain the fall in GFR that leads to nitrogenous-waste retention and thus the rise in BUN and serum creatinine.   Decreased proximal tubule sodium reabsorption may lead to a decreased GFR during ARF. First of all, brush border membranes and cellular debris could provide the substrate for intraluminal obstruction in the highly resistant portion of distal nephrons.

In fact, microdissection of individual nephrons of kidneys from patients with ARF demonstrated obstructing casts in distal tubules and collecting ducts. This observation could explain the dilated proximal tubules that are observed upon renal biopsy of ARF kidneys. The intraluminal casts in ARF kidneys stain prominently for Tamm-Horsfall protein (THP), which is produced in the thick ascending limb. Importantly, THP is secreted into tubular fluid as a monomer but subsequently may become a polymer that forms a gel-like material in the presence of increased luminal Na+ concentration, as occurs in the distal nephron during clinical ARF with the decrease in tubular sodium reabsorption.

Thus, the THP polymeric gel in the distal nephron provides an intraluminal environment for distal cast formation involving viable, apoptotic, and necrotic cells.

The loss of the tubular epithelial cell barrier and/or the tight junctions between viable cells during acute renal ischemia could lead to a leak of glomerular filtrate back into the circulation. (If this occurs and normally non-reabsorbable substances, such as inulin, leak back into the circulation, then a falsely low GFR will be measured as inulin clearance. It should be noted, however, that the degree of extensive tubular damage observed in experimental studies demonstrating tubular fluid backleak is rarely observed with clinical ARF in humans). Moreover, dextran sieving studies in patients with ARF demonstrated that, at best, only a 10% decrease in GFR could be explained by backleak of filtrate. Cadaveric transplanted kidneys with delayed graft function, however, may have severe tubular necrosis, and thus backleak of glomerular filtration may be more important in this setting.

Inflammation and NO

There is now substantial evidence for the involvement of inflammation in the pathogenesis of the decreased GFR associated with acute renal ischemic injury. In this regard, there is experimental evidence that iNOS may contribute to tubular injury during ARF. Hypoxia in isolated proximal tubules has been shown to increase NO release, and there is increased iNOS protein expression in ischemic kidney homogenates. An antisense oligonucleotide was shown to block the upregulation of iNOS and afford functional protection against acute renal ischemia. Moreover, when isolated proximal tubules from iNOS, eNOS, and neuronal NO synthase (nNOS) knockout mice were exposed to hypoxia, only the tubules from the iNOS knockout mice were protected against hypoxia, as assessed by LDH release. The iNOS knockout mice were also shown to have lower mortality during ischemia/reperfusion than wild-type mice.  The scavenging of NO by oxygen radicals produces peroxynitrite causing tubule damage during ischemia. While iNOS may contribute to ischemic injury of renal tubules,  the vascular effect of eNOS in the glomerular afferent arteriole is protective against ischemic injury. In this regard, eNOS knockout mice are more sensitive to endotoxin-related injury than normal mice.

Moreover, the protective role of vascular eNOS may be more important than the deleterious effect of iNOS at the tubule level during renal ischemia.   This is because treatment of mice with the nonspecific NO synthase (NOS) inhibitor L-NAME, which blocks both iNOS and eNOS, worsens renal ischemic injury. NO may downregulate eNOS and is a potent inducer of heme oxygenase-1, which has been shown to be cytoprotective against renal injury. The MAPK pathway also appears to be involved in renal oxidant injury. Activation of extracellular signal–regulated kinase (ERK) or inhibition of JNK ameliorates oxidant injury–induced necrosis in mouse renal proximal tubule cells in vitro. Upregulation of ERK may also be important in the effect of preconditioning whereby early ischemia affords protection against a subsequent ischemia/reperfusion insult. Alterations in cell cycling are also involved in renal ischemic injury. Upregulation of p21, which inhibits cell cycling, appears to allow cellular repair and regeneration, whereas homozygous p21 knockout mice demonstrate enhanced cell necrosis in response to an ischemic insult.

Prolonged duration of the ARF clinical course and the need for dialysis are major factors projecting a poor prognosis. Patients with ARF who require dialysis have a 50–70% mortality rate. Infection and cardiopulmonary complications are the major causes of death in patients with ARF. Excessive fluid administration in patients with established ARF may lead to pulmonary congestion, hypoxia, the need for ventilatory support, pneumonia, and multiorgan dysfunction syndrome, which has an 80–90% mortality rate. Until means to reverse the diminished host defense mechanisms in azotemic patients with clinical ARF are available, every effort should be made to avoid invasive procedures such as the placement of bladder catheters, intravenous lines, and mechanical ventilation. Over and above such supportive care, it may be that combination therapy will be necessary to prevent or attenuate the course of ARF. Such combination therapy must involve agents with potential beneficial effects on vascular tone, tubular obstruction, and inflammation.

Schrier RW, Wang W, Poole B, and Mitra A. Acute renal failure: definitions, diagnosis, pathogenesis, and therapy. The Journal of Clinical Investigation 2004; 114(1):5-14. http://www.jci.org

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Part IIIb. Additional Related References on NO, oxidative stress and Kidney

Shelgikar PJ, Deshpande KH, Sardeshmukh AS, Katkam RV, Suryakarl AN. Role of oxidants and antioxidants in ARF patients undergoing hemodialysis. Indian J Nephrol 2005;15: 73-76.

Lee JW. Renal Dysfunction in Patients with Chronic Liver Disease. Electrolytes Blood Press 7:42-50, 2009ㆍdoi: 10.5049/EBP.2009.7.2.42.

Saadat H, et al. Endothelial Nitric Oxide Function and Tubular Injury in Premature Infants. Int J App Sci and Technol 2012; 7(6): 77-81. http://www.ijastnet.com.

Amerisan MS. Cardiovascular disease in chronic kidney disease. Indian J Nephrol 2005;15: 1-7.

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Traditional risk factors for CVD in CKD

  • Hypertension
  • Older Age
  • Diabetes Mellitus
  • Male gender
  • High LDL
  • White Race
  • Low HDL
  • Physical inactivity
  • Smoking
  • Menopause
  • LVH

CKD Related CV Risk Factors

  • Blood Pressure
  • ? Homocysteinemia
  • Anemia
  • ? Inflammation
  •   Ca++ x P++
  • ? NO synthesis
  • Na+ Retention
  • ? Lp (a)
  • Hypervolemia
  • ? Insulin Resistance
  • Proteinuria & Hypoalbuminemia
  • Iron over load
  • ? Adeponectin
  • ??Vit. C or E
  • ? 5 Lipoxygenase
  • ROS
  • Genetic factors
  • ADMA (Asymmetric Dimethyl Arginine)

S Vikrant, SC Tiwari. Essential Hypertension – Pathogenesis and Pathophysiology. J Indian Acad Clinical Medicine 2001; 2(3):141-161. Scheme for pathogenesis of salt dependent hypertension.

The hypothesis proposes that early hypertension is episodic and is mediated by a hyperactive sympathetic nervous system or activated renin-angiotensin system.

Cell membrane alterations

Hypotheses linking abnormal ionic fluxes to increased peripheral resistance through increase in cell sodium, calcium, or pH.   The hypertension that is more common in obese people may arise in large part from the insulin resistance and resultant hyperinsulinaemia that results from the increased mass of fat. However, rather unexpectedly, insulin resistance may also be involved in hypertension in non-obese people.

Overall scheme for the mechanisms by which obesity, if predominantly upper body or visceral in location, could promote

________________________________________________________________________________________________________________________________________________________

  • diabetes,
  • dyslipidemia and
  • hypertension via hyperinsulinemia.

The explanation for insulin resistance found in as many as half of nonobese hypertensive is not obvious and may involve one or more aspects of insulin’s action

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Proposed mechanisms by which insulin resistance and/or hyperinsulinemia may lead to increased blood pressure.

  1. Enhanced renal sodium and water reabsorption.
  2. Increased blood pressure sensitivity to dietary salt intake
  3. Augmentation of the pressure and
  4. aldosterone responses to AII
  5. Changes in transmembrane electrolyte transport
  • a. Increased intracellular sodium
  • b. Decreased Na+/K+ – ATPase activity
  • c. Increased intracellular Ca2+ pump activity
  • d. Increased intracellular Ca2+ accumulation
  • e. Stimulation of growth factors

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Part IV. New Insights on NO donors

This study investigated the involvement of nitric oxide (NO) into the irradiation-induced increase of cell attachment. These experiments explored the cellular mechanisms of low-power laser therapy. HeLa cells were irradiated with a monochromatic visible-tonear infrared radiation (600–860 nm, 52 J/m2) or with a diode laser (820 nm, 8–120 J/m2) and the number of cells attached to a glass matrix was counted after 30 minute incubation at 37oC. The NO donors

  1. sodium nitroprusside (SNP),
  2. glyceryl trinitrate (GTN), or
  3. sodium nitrite (NaNO2)

were added to the cellular suspension before or after irradiation. The action spectra and the concentration and fluence dependencies obtained were compared and analyzed.

The well-structured action spectrum for the increase of the adhesion of the cells, with maxima at 619, 657, 675, 740, 760, and 820 nm, points to the existence of a photoacceptor responsible for the enhancement of this property (supposedly cytochrome c oxidase, the terminal respiratory chain enzyme), as well as signaling pathways between the cell mitochondria, plasma membrane, and nucleus.

Treating the cellular suspension with SNP before irradiation significantly modifies the action spectrum for the enhancement of the cell attachment property (band maxima at 642, 685, 700, 742, 842, and 856 nm). The action of SNP, GTN, andNaNO2 added before or after irradiation depends on their concentration and radiation fluence.

The NO donors added to the cellular suspension before irradiation eliminate the radiation induced increase in the number of cells attached to the glass matrix, supposedly by way of binding NO to cytochrome c oxidase. NO added to the suspension after irradiation can also inhibit the light-induced signal downstream. Both effects of NO depend on the concentration of the NO donors added.

The results indicate that NO can control the irradiation-activated reactions that increase the attachment of cells.

Karu TI, Pyatibrat LV, and Afanasyeva NI. Cellular Effects of Low Power Laser Therapy Can be Mediated by Nitric Oxide. Lasers Surg. Med 2005; 36:307–314.

IFNa-2b (IFN-a) effect on barrier function of renal tubular epithelium

IFNa treatment can be accompanied by impaired renal function and capillary leak. This study shows IFNa produced dose-dependent and time-dependent decrease in transepithelial resistance (TER) ameliorated by tyrphostin, an inhibitor of phosphotyrosine kinase with increased expression of occludin and E-cadherin. In conclusion, IFNa can directly affect barrier function in renal epithelial cells via ovewrexpression or missorting of the junctional proteins occludin and E-cadherin.

Lechner J, Krall M, Netzer A, Radmayr C, et al. Effects of interferon a-2b on barrier function and junctional complexes of renal proximal tubulat LLC-pK1 cells. Kidney Int 1999; 55:2178-2191.

Ischemia-reperfusion injury

The pathophysiology of acute renal failure (ARF) is complex and not well understood. Numerous models of ARF suggest that oxygen-derived reactive species are important in renal ischemia-reperfusion (I-R) injury, but the nature of the mediators is still controversial. Treatment with oxygen radical scavengers, antioxidants, and iron chelators such as

  • superoxide dismutase,
  • dimethylthiourea,
  • allopurinol, and
  • deferoxamine

are protective in some models, and suggest a role for the hydroxyl radical formation. However, these compounds are not protective in all models of I-R injury, and direct evidence for the generation of hydroxyl radical is absent. Furthermore, these inhibitors have another property in common.

They all directly scavenge or inhibit the formation of peroxynitrite (ONOO−), a highly toxic species derived from nitric oxide (NO) and superoxide. Thus, the protective effects seen with these inhibitors may be due in part to their ability to inhibit ONOO− formation. Even though reactive oxygen species are thought to participate in ischemia-reperfusion (I-R) injury, induction of and production of high levels of  inducible nitric oxide (NO)  also contribute to this injury.

NO combines with superoxide to form the potent oxidant peroxynitrite (ONOO−). NO and ONOO− were investigated in a rat model of renal I-R injury using the selective iNOS inhibitor L-N6-(1-iminoethyl)lysine (L-NIL).

I-R surgery significantly increased plasma creatinine levels to 1.9 ± 0.3 mg/dl (P < .05) and caused renal cortical necrosis. L-NIL administration (3 mg/kg) in animals subjected to I-R significantly decreased plasma creatinine levels to 1.2 ± 0.10 mg/dl (P < .05 compared with I-R) and reduced tubular damage.

ONOO− formation was evaluated by detecting 3-nitrotyrosine-protein adducts (3NTyPAs), a stable biomarker of ONOO− formation.   The kidneys from I-R animals had increased levels of 3NTyPAs compared with control animals   L-NIL-treated rats (3 mg/kg) subjected to I-R showed decreased levels of 3NTyPAs.

These results suggests that iNOS-generated NO mediates damage in I-R injury possibly through ONOO− formation.

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In summary,

  1. 3-nitrotyrosine-protein adducts were detected in renal tubules after I-R injury.
  2. Selective inhibition of iNOS by L-NIL decreased injury, improved renal function, and decreased apparent ONOO− formation.
  3. Reactive nitrogen species should be considered potential therapeutic targets in the prevention and treatment of renal I-R injury.

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Walker LM, Walker PD, Imam SZ, et al. Evidence for Peroxynitrite Formation in Renal Ischemia-Reperfusion Injury: Studies with the Inducible Nitric Oxide Synthase InhibitorL-N6-(1-Iminoethyl)lysine1. 2000.

Role of TNFa independent of iNOS Renal failure is a frequent complication of sepsis, mediated by renal vasoconstrictors and vasodilators. Endotoxin induces several proinflammatory cytokines, among which tumor necrosis factor (TNF) is thought to be of major importance. Tumor necrosis factor (TNF) has been suggested to be a factor in the acute renal failure in sepsis or endotoxemia. Passive immunization by anti-TNFa prevented development of septic shock in animal experiments.The development of ARF involves excessive intrarenal vasoconstriction. Involvement of nitric oxide (NO), generated by inducible NO synthase (iNOS), is still a factor in the pathogenesis of endotoxin-induced renal failure. TNF-a leads to a decrease in glomerular filtration rate (GFR).

This study tested the hypothesis that the role of TNF-a in endotoxic shock related ARF is mediated by iNOS-derived NO.   An injection of lipopolysaccharide (LPS) constituent of gram-negative bacteria to wild-type mice resulted in a 70% decrease in glomerular filtration rate (GFR) and in a 40% reduction in renal plasma flow (RPF) 16 hours after the injection.   The results occurred independent of hypotension, morphological changes, apoptosis, and leukocyte accumulation. In mice pretreated with TNFsRp55, only a 30% decrease in GFR was observed without a significant change in RPF as compared with controls. Pretreatment with TNKsRp55 on renal function Wild-type mice were pretreated with TNFsRp55(10 mg/kg IP)  for one hour before the administration of 5 mg/kg intraperitoneal endotoxin. GFR and RPF were determined 16 hours thereafter. Data are expressed as mean 6, SEM, N 5 6. *P , 0.05 vs. Control; §P , 0.05 vs. LPS, by ANOVA.

The serum NO concentration was significantly lower in endotoxemic wild-type mice pretreated with TNFsRp55, as compared with untreated endotoxemic wild-type mice. In LPS-injected iNOS knockout mice and wild-type mice treated with a selective iNOS inhibitor, 1400W, the development of renal failure was similar to that in wild-type mice. As in wild-type mice,TNFsRp55 significantly attenuated the decrease in GFR (a 33% decline, as compared with 75% without TNFsRp55) without a significant change in RPF in iNOS knockout mice given LPS. These results demonstrate a role of TNF in the early renal dysfunction (16 h) in a septic mouse model independent of iNOS,

  • hypotension,
  • apoptosis,
  • leukocyte accumulation,and
  • morphological alterations,

thus suggesting renal hypoperfusion secondary to an imbalance between, as yet to be defined renal vasoconstrictors and vasodilators.

Knotek M, Rogachev B, Wang W,….., Edelstein CL, Dinarello CA, and Schrier RW. Endotoxemic renal failure in mice: Role of tumor necrosis factor independent of inducible nitric oxide synthase. Kidney International 2001; 59:2243–2249

Ischemic acute renal failure

Inflammation plays a major role in the pathophysiology of acute renal failure resulting from ischemia. This review discusses the contribution of

  • endothelial
  • epithelial cells and
  • leukocytes

to this inflammatory response. The roles of cytokines/chemokines in the injury and recovery phase are reviewed. The protection of mouse kidney prior to exposure to ischemia or urinary tract obstruction is  a potential model to  search for pharmacologic agents to protect the kidney against injury by inflammatory mediators produced by tubular epithelial cells and activated leukocytes in renal ischemia/reperfusion (I/R) injury. Tubular epithelia produce

  • TNF-a,
  • IL-1,
  • IL-6,
  • IL-8,
  • TGF-b,
  • MCP-1,
  • ENA-78,
  • RANTES, and
  • fractalkines,

whereas leukocytes produce

  • TNF-a,
  • IL-1,
  • IL-8,
  • MCP-1,
  • ROS, and
  • eicosanoids.

The release of these chemokines and cytokines serve as effectors for a positive feedback pathway enhancing inflammation and cell injury, the cycle of tubular epithelial cell injury and repair following renal ischemia/reperfusion.   Tubular epithelia are typically cuboidal in shape and apically-basally polarized; the Na+/K+-ATPase localizes to basolateral plasma membranes, whereas cell adhesion molecules, such as integrins localize basally. In response to ischemia reperfusion,

  • the Na+/K+-ATPase appears apically, and
  • integrins are detected on lateral and basal plasma membranes.

Some of the injured epithelial cells undergo necrosis and/or apoptosis detaching from the underlying basement membrane into the tubular space where they contribute to tubular occlusion. Viable cells that remain attached, dedifferentiate, spread, and migrate to repopulate the denuded basement membrane. With cell proliferation, cell-cell and cell-matrix contacts are restored, and the epithelium redifferentiates and repolarizes, forming a functional, normal epithelium Inflammation is a significant component of renal I/R injury, playing a considerable role in its pathophysiology.

Although significant progress has been made in defining the major components of this process, the complex cross-talk between endothelial cells, inflammatory cells, and the injured epithelium with each generating and often responding to cytokines and chemokines is not well understood. In addition, we have not yet taken full advantage of the large body of data on inflammation in other organ systems.

Furthermore, preconditioning the kidney to afford protection to subsequent bouts of ischemia may serve as a useful model challenging us to therapeutically mimic endogenous mechanisms of protection.

Understanding the inflammatory response prevalent in ischemic kidney injury will facilitate identification of molecular targets for therapeutic intervention.

Bonventre JV and Zuk A. Ischemic acute renal failure: An inflammatory disease? Forefronts in Nephrology 2002;.. :480-485

Gene expression profiles in renal proximal tubules In kidney disease renal proximal tubular epithelial cells (RPTEC) actively contribute to the progression of tubulointerstitial fibrosisby mediating both

  • an inflammatory response and
  • via epithelial-to-mesenchymal transition.

Using laser capture microdissection we specifically isolated RPTEC from cryosections of the healthy parts of kidneys removed owing to renal cell carcinoma and from kidney biopsies from patients with proteinuric nephropathies. RNA was extracted and hybridized to complementary DNA microarrays after linear RNA amplification. Statistical analysis identified 168 unique genes with known gene ontology association, which separated patients from controls. Besides distinct alterations in signal-transduction pathways (e.g. Wnt signalling), functional annotation revealed a significant upregulation of genes involved in

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  • cell proliferation and cell cycle control (like insulin-like growth factor 1 or cell division cycle 34),
  • cell differentiation (e.g. bone morphogenetic protein 7),
  • immune response,
  • intracellular transport and
  • metabolism

__________________________________________________________________________________________________________________________________________________________

in RPTEC from patients.

The study also revealed differential expression of a number of genes responsible for cell adhesion (like BH-protocadherin) with a marked downregulation of most of these transcripts. In summary, the results obtained from RPTEC revealed a differential regulation of genes, which are likely to be involved in either pro-fibrotic or tubulo-protective mechanisms in proteinuric patients at an early stage of kidney disease.

Rudnicki M, Eder S, Perco P, Enrich J, et al. Gene expression profiles of human proximal tubular epithelial cells in proteinuric nephropathies. Kidney International 2006; xx:1-11. Kidney International advance online publication, 20 December 2006; doi:10.1038/sj.ki.5002043. http://www.kidney-international.org

Oxidative stress involved with diabetic nephropathy

Diabetic Nephropathy (DN) poses a major health problem. There is strong evidence for a potential role of the eNOS gene. This case control study investigated the possible role of genetic variants of the endothelial Nitric Oxide Synthase (eNOS) gene and oxidative stress in the pathogenesis of nephropathy in patients with diabetes mellitus. The study included 124 diabetic patients;

  1. 68 of these patients had no diabetic nephropathy (group 1) while
  2. 56 patients exhibited symptoms of diabetic nephropathy (group 2).
  3. Sixty two healthy non-diabetic individuals were also included as a control group.

Blood samples from subjects and controls were analyzed to investigate the eNOS genotypes and to estimate

  • the lipid profile and
  • markers of oxidative stress such as malondialdehyde (MDA) and nitric oxide (NO).

No significant differences were found in the frequency of eNOS genotypes between diabetic patients (either in group 1 or group 2) and controls (p >0.05). Also, no significant differences were found in the frequency of eNOS genotypes between group 1 and group 2 (p >0.05). Both group 1 and group 2 had significantly higher levels of nitrite and MDA when compared with controls (all p = 0.0001). Also group 2 patients had significantly higher levels of nitrite and MDA when compared with group 1 (p = 0.02, p = 0.001 respectively).

The higher serum level of the markers of oxidative stress in diabetic patients particularly those with diabetic nephropathy suggest that oxidative stress and not the eNOS gene polymorphism is involved in the pathogenesis of the diabetic nephropathy in this subset of patients

Badawy A, Elbaz R, Abbas AM, Ahmed Elgendy A, et al. Oxidative stress and not endothelial Nitric Oxide Synthase gene polymorphism involved in diabetic nephropathy. Journal of Diabetes and Endocrinology 2011; 2(3): 29-35.

Metformin in renal ischemia reperfusion

Renal ischemia plays an important role in renal impairment and transplantation. Metformin is a biguanide used in type 2 diabetes, it inhibits hepatic glucose production and increases peripheral insulin sensitivity. While the mode of action of metformin is incompletely understood, it appears to have anti-inflammatory and antioxidant effects involved in its beneficial effects on insulin resistance.   Control, Sham, ischemia/reperfusion (I/R) and Metformin treated I /R groups   A renal I/R injury was done by a left renal pedicle occlusion to induce ischemia for 45 min followed by 60 min of reperfusion with contralateral nephrectomy. Metformin pretreated I/R rats in a dose of 200 mg/kg/day for three weeks before ischemia induction.

  • Nitric oxide (NO),
  • tumor necrosis factor alpha (TNF α) ,
  • catalase (CAT) and
  • reduced glutathione (GSH) activities

were determined in renal tissue, while

  • creatinine clearance (CrCl) ,
  • blood urea nitrogen (BUN) were measured and

5 hour urinary volume and electrolytes were estimated . BUN and CrCl levels in the I/R group were significantly higher than in control rats (p<0.05) table (1).

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Table 1: Creatinine clearance (Cr Cl) and blood urea nitrogen (BUN) levels in control and test groups.
(Mean ± SD)

Groups CrCl   (ml/min) BUN (mg/dl)
Control group 1.30 ±0.11 14.30±0.25
Sham group+ metformin 1.27±0.09 15.70±0.19
I/R group (P1) 1.85±0.25 (<0.001 ) 28.00±0.62 (<0.001)
I/R+ metformin group (P2,P3) 1.55±0.22 (0.001, 0.028) 18.10±1.00 (<0.001, <0.001)
  • P1: Statistical significance between control
    group and saline treated I/R group.
  • P2 Statistical significance between control
    group and Metformin treated I/R group.
  • P3 Statistical significance between saline treated
    I/R group and Metformin treated I/R group

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When metformin was administered before I/R, BUN and CrCl levels were still significantly higher than control group but their elevation were significantly lower in comparison to I/R group alone (P<0.05).   TNF α and NO levels were significantly higher in the I/R group than those of the control group (Table 2). Pre-treatment with metformin significantly lowered their levels in comparison to I/R group (P<0.05).

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Table 2: Tumor necrosis factor α (TNF α) and inducible nitric oxide (iNO) levels in control and test groups.
(Mean ± SD)

Groups TNF α (pmol/mg tissue) iNO (nmol/ mg tissue)
Control group 1 7.60 ±5.98 2.54 ± 0.82
Sham group+ metformin 16.70 ±5.50 2.35 ±0.80
I/R group (P1) 54. 00±6.02 (<0.001) 4.50±0.89 (<0.001)
I/R+metformin group (P2,P3) 39 ± 14.01 (<0.001, 0.006) 3.53±0.95 (0.02, 0.03)

 

  • P1: Statistical significance between control group
    and saline treated I/R group.
  • P2 Statistical significance between control group
    and Metformin treated I/R group.
  • P3 Statistical significance between saline treated
    I/R group and Metformin treated I/R group

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These results showed significant increase in NO,TNF α, BUN , CrCl and significant decrease in urinary volume , electrolytes, CAT and GSH activities in the I/R group than those in the control group. Metformin decreased significantly NO, TNF α, BUN and CrCl while increased urinary volume, electrolytes, CAT and GSH activities.   Lipid peroxidation is related to I/R induced tissue injury. Production of inducible NO synthase (NOS) under lipid peroxidation and inflammatory conditions results in the induction of NO which react with O2 liberating peroxynitrite (OONO-). NO itself inactivates the antioxidant enzyme system CAT and GSH. Alteration in NO synthesis have been observed in other kidney injuries as nephrotoxicity and acute renal failure induced by endotoxins.

Treatment with iNOS inhibitors improved renal function and decreased peroxynitrite radical which is believed to be responsible for the shedding of proximal convoluted tubules in I/R.   Metformin produced anti-inflammatory renoprotective effect on CrCl and diuresis in renal I/R injury.

Malek HA. The possible mechanism of action of metformin in renal ischemia reperfusion in rats. The Pharma Research Journal 2011; 6(1):42-49.

Possible role of NO donors in ARFThe L-arginine-nitric oxide (NO) pathway has been implicated in many physiological functions in the kidney, including

  • regulation of glomerular hemodynamics,
  • mediation of pressure-natriuresis,
  • maintenance of medullary perfusion,
  • blunting of tubuloglomerular feedback (TGF),
  • inhibition of tubular sodium reabsorption and
  • modulation of renal sympathetic nerve activity

Its net effect in the kidney is to promote natriuresis and diuresis, contributing to adaptation to variations of dietary salt intake and maintenance of normal blood pressure. Nitric oxide has been implicated in many physiologic processes that influence both acute and long-term control of kidney function. Its net effect in the kidney is to promote natriuresis and diuresis, contributing to adaptation to variations of dietary salt intake and maintenance of normal blood pressure. A pretreatment with nitric oxide donors or L-arginine may prevent the ischemic acute renal injury. In chronic kidney diseases, the systolic blood pressure is correlated with the plasma level of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase. A reduced production and biological action of nitric oxide is associated with an elevation of arterial pressure, and conversely, an exaggerated activity may represent a compensatory mechanism to mitigate the hypertension.

JongUn Lee. Nitric Oxide in the Kidney : Its Physiological Role and Pathophysiological Implications. Electrolyte & Blood Pressure 2008; 6:27-34.

Renal Hypoxia and Dysoxia following Reperfusion

Acute renal failure (ARF) is a common condition which develops in 5% of hospitalized patients. Of the patients who develop ARF, ~10% eventually require renal replacement therapy. Among critical care patients who have acute renal failure and survive, 2%-10% develop terminal renal failure and require long-term dialysis.   The kidneys are particularly susceptible to ischemic injury in many clinical conditions such as renal transplantation, treatment of suprarenal aneurysms, renal artery reconstructions, contrast-agent induced nephropathy, cardiac arrest, and shock. One reason for renal sensitivity to ischemia is that the kidney microvasculature is highly complex and must meet a high energy demand.

Under normal, steady state conditions, the oxygen (O2) supply to the renal tissues is well in excess of oxygen demand.   Under pathological conditions, the delicate balance of oxygen supply versus demand is easily disturbed due to the unique arrangement of the renal microvasculature and its increasing numbers of diffusive shunting pathways.  

The renal microvasculature is serially organized, with almost all descending vasa recta emerging from the efferent arterioles of the juxtamedullary glomeruli. Adequate tissue oxygenation is thus partially dependent on the maintenance of medullary perfusion by adequate cortical perfusion. This, combined with the low amount of medullary blood flow (~10% of total renal blood flow) in the U-shaped microvasculature of the medulla allows O2 shunting between the descending and ascending vasa recta and contributes to the high sensitivity of the medulla and cortico-medullary junction to decreased O2 supply.

Whereas past investigations have focused mainly on tubular injury as the main cause of ischemia-related acute renal failure, increasing evidence implicates alterations in the intra-renal microcirculation pathway and in the O2 handling. Indeed, although acute tubular necrosis (ATN) has classically been believed to be the leading cause of ARF, data from biopsies in patients with ATN have shown few or no changes consistent with tubular necrosis.

The role played by microvascular dysfunction, however, has generated increasing interest. The complex pathophysiology of ischemic ARF includes the inevitable

  • reperfusion phase associated with oxidative stress,
  • cellular dysfunction and
  • altered signal transduction.

During this process, alterations in oxygen transport pathways can result in cellular hypoxia and/or dysoxia. In this context, the distinction between hypoxia and dysoxiais that

  • cellular hypoxia refers to the condition of decreased availability of oxygen due to inadequate convective delivery from the microcirculation.
  • Cellular dysoxia, in contrast, refers to a pathological condition where the ability of mitochondria to perform oxidative phosphorylation is limited, regardless of the amount of available oxygen.

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The latter condition is associated with mitochondrial failure and/or activation of alternative pathways for oxygen consumption. Thus, we would expect that an optimal balance between oxygen supply and demand is essential to reducing damage from renal ischemia-reperfusion (I/R) injury. Complex interactions exist between

  • tubular injury,
  • microvascular injury, and
  • inflammation after renal I/R.

On the one hand, insults to the tubule cells promotes the liberation of a number of inflammatory mediators, such as TNF-á, IL-6, TGF-â, and chemotactic cytokines(RANTES, monocyte chemotactic protein-1, ENA-78, Gro-á, and IL-8). On the other hand, chemokine production can promote

  • leukocyte-endothelium interactions and
  • leukocyte activation,

resulting in…..

  • renal blood flow impairment and
  • the expansion of tubular damage
  • impaired renal hemodynamics and
  • electrolyte reabsorption

Adequate medullary tissue oxygenation, in terms of balanced oxygen supply and demand, is dependent on the maintenance of medullary perfusion by adequate cortical perfusion and also on the high rate of O2 consumption required for active electrolyte transport. Furthermore, renal blood flow is closely associated with renal sodium transport, mitochondrial activity and NO-mediated O2 consumption In addition to having a limited O2 supply due to the anatomy of the microcirculation anatomy, the sensitivity of the medulla to hypoxic conditions results from this high O2 consumption.

Renal sodium transport is the main O2-consuming function of the kidney and is closely linked to renal blood flow for sodium transport, particularly in the thick ascending limbs of the loop of Henle and the S3 segments of the proximal tubules. Medullary renal blood flow is also highly dependent on cortical perfusion, with almost all descending vasa recta emerging from the efferent arteriole of juxta medullary glomeruli. A profound reduction in cortical perfusion can disrupt medullary blood flow and lead to an imbalance between O2 supply and O2 consumption. On theother hand, inhibition of tubular reabsorption by diuretics increases medullary pO2 by decreasing the activity of Na+/K+-ATPases and local O2 consumption.

Mitochondrial activity and NO-mediated O2 consumption

The medulla has been found to be the main site of production of NO in the kidney. In addition to the actions described above, NO appears to be a key regulator of renal tubule cell metabolism by inhibiting the activity of the Na+-K+-2Cl- cotransporter and reducing Na+/H+ exchange. Since superoxide (O2-) is required to inhibit solute transport activity, it was assumed that these effects were mediated by peroxynitrite (OONO-). Indeed, mitochondrial nNOS upregulation, together with an increase in NO production, has been shown to increase mitochondrial peroxynitrite generation, which in turn, can induce cytochrome c release and promote apoptosis. NO has also been shown to directly compete with O2 at the mitochondrial level. These findings support the idea that NO acts as an endogenous regulator to match O2 supply to O2 consumption, especially in the renal medulla.   NO reversibly binds to the O2 binding site of cytochrome oxidase, and acts as a potent, rapidMitochondrial activity and NO-mediated O2 consumption, and reversible inhibitor of cytochrome oxidase in competition with molecular O2. This inhibition could be dependent on the O2 level, since the IC50 (the concentration of NO that reduces the specified response by half) decreases with reduction in O2 concentration. The inhibition of electron flux at the cytochrome oxidase level switches the electron transport chain to a reduced state, and consequently leads to depolarization of the mitochondrial membrane potential and electron leakage.

To summarize, while the NO/O2 ratio can act as a regulator of cellular O2 consumption by matching decreases in O2 delivery to decreases in cellular O2 cellular, the inhibitory effect of NO on mitochondrial respiration under hypoxic conditions further impairs cellular aerobic metabolism. This leads to a state of “cytopathic hypoxia,” as described in the sepsis literature.   Only cell-secreted NO competes with O2 and to regulate mitochondrial respiration. In addition to the 3 isoforms (eNOS, iNOS, cnNOS), an α-isoform of neuronal NOS, the mitochondrial isoform (mNOS) located in the inner mitochondrial membrane, has also been shown to regulate mitochondrial respiration. These data support a role for NO in the balanced regulation of renal O2 supply and O2 consumption after renal I/R However, the relationships between the determinants of O2 supply, O2 consumption, and renal function, and their relation to renal damage remain largely unknown.

Sustained endothelial activation Ischemic renal failure leads to persistent endothelial activation, mainly in the form of endothelium-leukocyte interactions and the activation of adhesion molecules. This persistent activation can compromise renal blood flow, prevent the recovery of adequate tissue oxygenation, and jeopardize tubular cell survival despite the initial recovery of renal tubular function. A 30-50% reduction in microvascular density was seen 40 weeks after renal ischemic injury in a rat model. Vascular rarefaction has been proposed to induce chronic hypoxia resulting in tubulointerstitial fibrosis via the molecular activation of fibrogenic factors such as transforming growth factor (TGF)-β, collagen, and fibronectin, all of which may play an important role in the progression of chronic renal disease.

Adaptation to hypoxia Over the last decade, the role of hypoxia-inducible factors (HIFs) in O2 supply and adaptation to hypoxic conditions has found increasing support. HIFs are O2-sensitive transcription factors involved in O2-dependent gene regulation that mediate cellular adaptation to O2 deprivation and tissue protection under hypoxic conditions in the kidney.   NO generation can promote HIF-1α accumulation in a cGMP-independent manner. However, Hagen et al. (2003) showed that NO may reduce the activation of HIF in hypoxia via the inhibitory effect of NO on cytochrome oxidase.

Therefore, it seems that NO has pleiotropic effects on HIF expression, with various responses related to different pathways. HIF-1α upregulates a number of factors implicated in cytoprotection, including angiogenic growth factors, such as vascular endothelial growth factors (VEGF), endothelial progenitor cell recruitment via the endothelial expression of SDF-1, heme-oxygenase-1 (HO-1), and erythropoietin (EPO), and vasomotor regulation.

HO-1 produces carbon monoxide (a potent vasodilator) while degrading heme, which may preserve tissue blood flow during reperfusion. Thus, it has been suggested that the induction of HO-1 can protect the kidney from ischemic damage by decreasing oxidative damage and NO generation.

Finally, in addition to its anti-apoptotic properties, EPO may protect the kidney from ischemic damage by restoring the renal microcirculation by stimulating the mobilization and differentiation of progenitor cells toward an endothelial phenotype and by inducing NO release from eNOS.

Pharmacological interventions

Use of pharmacological interventions which act at the microcirculatory level may be a successful strategy to overcome ischemia-induced vascular damage and prevent ARF. Activated protein C (APC), an endogenous vitamin K-dependent serine protease with multiple biological activities, may meet these criteria. Along with antithrombotic and profibrinolytic properties, APC can reduce the chemotaxis and interactions of leukocytes with activated endothelium.

However, renal dysfunction was not improved in the largest study published so far. In addition, APC has been discontinued by Lilly for the use intended in severe sepsis. Moreover, neither drugs with renal vasodilatory effects (i.e., dopamine, fenoldopam, endothelin receptors blockers, adenosine antagonists) nor agents that decrease renal oxygen consumption (i.e., loop diuretics) have been shown to protect the kidney from ischemic damage. We have to bear in mind that a magic bullet to treat the highly complex condition of which is renal I/R is not in sight.

We can expect that understanding the balance between O2 delivery and O2 consumption, as well as the function of O2-consuming pathways (i.e., mitochondrial function, reactive oxygen species generation) will be central to this treatment strategy.

Take home point

The deleterious effects of NO are thought to be associated with the NO generated by the induction of iNOS and its contribution to oxidative stress both resulting in vascular dysfunction and tissue damage. Ischemic injury also leads to structural damage to the endothelium and leukocyte infiltration. Consequently, renal tissue hypoxia is proposed to promote the initial tubular damage, leading to acute organ dysfunction.   Comment: I express great appreciation for refeering to this work, which does provide enormous new insights into hypoxia-induced acute renal failure, and ties together the anatomy, physiology, and gene regulation through signaling pathways.

Ince C, Legrand M, Mik E , Johannes T, Payen D. Renal Hypoxia and Dysoxia following Reperfusion of the Ischemic Kidney. Molecular Medicine (Proof) 2008; pp36. http://www.molmed.org

Nitric oxide and non-hemodynamic functions of the kidney

One of the major scientific advances in the past decade in understanding of the renal function and disease is the prolific growth of literature incriminating nitric oxide (NO) in renal physiology and pathophysiology. NO was first shown to be identical with endothelial derived relaxing factor (EDRF) in 1987 and this was followed by a rapid flurry of information defining the significance of NO in not only vascular physiology and hemodynamics but also in neurotransmission, inflammation and immune defense systems. Although most actions of NO are mediated by cyclic guanosine monophosphate (cGMP) signaling, S-nitrosylation of cysteine residues in target proteins constitutes another well defined non-cGMP dependent mechanism of NO effects. Recent years have witnessed a phenomenal scientific interest in the vascular biology, particularly the relevance of nitric oxide (NO) in cardiovascular and renal physiology and pathophysiology. Although hemodynamic actions of NO received initial attention, a variety of non-hemodynamic actions are now known to be mediated by NO in the normal kidney, which include

  • tubular transport of electrolyte and water,
  • maintenance of acid-base homeostasis,
  • modulation of glomerular and interstitial functions,
  • renin-angiotensin activation and
  • regulation of immune defense mechanism in the kidney.

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Table 1 : Functions of NO in the kidney

  • 1. Renal macrovascular and microvascular dilatation (afferent > efferent)
  • 2. Regulation of mitochondrial respiration.
  • 3. Modulation renal medullary blood flow
  • 4. Stimulation of fluid, sodium and HCO3 – reabsorption in the proximal tubule
  • 5. Stimulation of renal acidification in proximal tubule by stimulation of NHE activity
  • 6. Inhibition of Na+, Cl- and HCO3 – reabsorption in the mTALH
  • 7. Inhibition of Na+ conductance in the CCD
  • 8. Inhibition of H+-ATPase in CCD

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One of the renal regulatory mechanisms related to maintenance of arterial blood pressure involves the phenomenon of pressure-natriuresis in response to elevation of arterial pressure. This effect implies inhibition of tubular sodium reabsorption resulting in natriuresis, in an effort to lower arterial pressure. Experimental evidence from indicates that intra-renal NO modulates pressure natriuresis.

Furthermore many studies have confirmed the role of intra renal NO in mediating tubulo-glomerular feedback (TGF). In vivo micropuncture studies have shown that NO derived from nNOS in macula densa specifically inhibits the TGF responses leading to renal afferent arteriolar vasoconstriction in response to sodium reabsorption in the distal tubule. Other recent studies support the inhibitory role of NO from eNOS and iNOS in mTALH segment on TGF effects.

Recent observations in vascular biology have yielded new information that endothelial dysfunction early in the course might contribute to the pathophysiology of acute renal failure.  Structural and functional changes in the vascular endothelium are demonstrable in early ischemic renal failure. Altered NO production and /or decreased bioavailability of NO comprise the endothelial function in acute renal failure.

Several studies have indicated imbalance of NOS activity with enhanced expression and activity of iNOS and decreased eNOS in ischemic kidneys.

The imbalance results from enhanced iNOS activity and attenuated eNOS activity in the kidney.  

Many experimental studies support a contributory role for NO in glomerulonephritis (GN). Evidence from recent studies pointed out that NO may be involved in peroxynitrite formation, pro-inflammatory chemokines and signaling pathways in addition to direct glomerular effects that promote albumin permeability in GN. Although originally macrophages and other leukocytes were first considered as the source renal NO production in GN, it is now clear iNOS derived NO from glomerular mesangial cells are the primary source of NO in GN.

In most pathological states, the role of NO is dependent by the stage of the disease, the nitric oxide synthase (NOS) isoform involved and the presence or absence of other modifying intrarenal factors. Additionally NO may have a dual role in several disease states of the kidney such as acute renal failure, inflammatory nephritides, diabetic nephropathy and transplant rejection.

A rapidly growing body of evidence supports a critical role for NO in tubulointerstitial nephritis (TIN). In the rat model of autoimmune TIN, Gabbai et al. demonstrated increased iNOS expression in the kidney and NO metabolites in urine and plasma. However the effects of iNOS on renal damage in TIN seem to have a biphasic effect- since iNOS specific inhibitors (eg. L-Nil) are renoprotective in the acute phase while they actually accelerated the renal damage in the chronic phase.

Thus chronic NOS inhibition is used to induce chronic tubulointerstitial injury and fibrosis along with mild glomerulosclerosis and hypertension.

Major pathways of L-arginine metabolism.

L-arginine may be metabolized by the urea cycle enzyme arginase to L-ornithine and urea by arginine decarboxylase to agmatine and CO2 or by NOS to nitric oxide (NO) and L-citrulline.

Adapted from Klahr S: Can L-arginine manipulation reduce renal disease? Semin Nephrol 1999; 61:304-309.

It is obvious that kidney is not only a major source of arginine and nitric oxide but NO plays an important role in the water and electrolyte balance and acid-base physiology and many other homeostatic functions in the kidney. Unfortunately we are far from a precise understanding of the significance of NO alterations in various disease states primarily due to conflicting data from the existing literature.

Therapeutic potential for manipulation of L-arginine- nitric oxide axis in renal disease states has been discussed. More studies are required to elucidate the abnormalities in NO metabolism in renal diseases and to confirm the therapeutic potential of L-arginine.

Sharma SP. Nitric oxide and the kidney. Indian J Nephrol 2004;14: 77-84

Inhibition of Constitutive Nitric Oxide Synthase

Excess NO generation plays a major role in the hypotension and systemic vasodilatation characteristic of sepsis. Yet the kidney response to sepsis is characterized by vasoconstriction resulting in renal dysfunction. We have examined the roles of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) on the renal effects of lipopolysaccharide administration by comparing the effects of specific iNOS inhibition, L-N6-(1-iminoethyl)lysine (L-NIL), and 2,4-diamino-6-hydroxy-pyrimidine vs. nonspecific NOS inhibitors (nitro-L-arginine-methylester). cGMP responses to carbamylcholine (CCh) (stimulated, basal) and sodium nitroprusside in isolated glomeruli were used as indices of eNOS and guanylate cyclase (GC) activity, respectively. LPS significantly decreased blood pressure and GFR (P =0.05) and inhibited the cGMP response to CCh.

GC activity was reciprocally increased. L-NIL and 2,4-diamino-6-hydroxy-pyrimidine administration prevented the decrease in GFR, restored the normal response to CCh, and GC activity was normalized. In vitro application of L-NIL also restored CCh responses in LPS glomeruli. Neuronal NOS inhibitors verified that CCh responses reflected eNOS activity.

L-NAME, a nonspecific inhibitor, worsened GFR, a reduction that was functional and not related to glomerular thrombosis, and eliminated the CCh response. No differences were observed in eNOS mRNA expression among the experimental groups. Selective iNOS inhibition prevents reductions in GFR, whereas nonselective inhibition of NOS further decreases GFR.

These findings suggest that the decrease in GFR after LPS is due to local inhibition of eNOS by iNOS, possibly via NO autoinhibition.

Schwartz D, Mendonca M, Schwartz I, Xia Y, et al. Inhibition of Constitutive Nitric Oxide Synthase (NOS) by Nitric Oxide Generated by Inducible NOS after Lipopolysaccharide Administration Provokes Renal Dysfunction in Rats. J. Clin. Invest. 1997; 100:439–448.

Salt-Sensitivity and Hypertension Renin-angiotensin system (RAS) plays a key role in the regulation of renal function, volume of extracellular fluid and blood pressure. The activation of RAS also induces oxidative stress, particularly superoxide anion (O2-) formation.

Although the involvement of O2 – production in the pathology of many diseases is known for long, recent studies also strongly suggest its physiological regulatory function of many organs including the kidney. However, a marked accumulation of O2- in the kidney alters normal regulation of renal function and may contribute to the development of salt-sensitivity and hypertension.

In the kidney, O2- acts as vasoconstrictor and enhances tubular sodium reabsoption. Nitric oxide (NO), another important radical that exhibits opposite effects than O2 -, is also involved in the regulation of kidney function. O2- rapidly interacts with NO and thus, when O2- production increases, it diminishes the bioavailability of NO leading to the impairment of organ function. As the activation of RAS, particularly the enhanced production of angiotensin II, can induce both O2- and NO generation, it has been suggested that physiological interactions of

  • RAS,
  • NO and
  • O2-

provide a coordinated regulation of kidney function.   The imbalance of these interactions is critically linked to the pathophysiology of salt-sensitivity and hypertension.

Kopkan L, Červenka L. Renal Interactions of Renin-Angiotensin System, Nitric Oxide and Superoxide Anion: Implications in the Pathophysiology of Salt-Sensitivity and Hypertension. Physiol. Res. 2009; 58 (Suppl. 2): S55-S67.

Epicrisis

In this review I attempted to evaluate complex and still incomplete and conflicting conclusions from many studies. I thus broke the report into three major portions:

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  • 1 The kidney and its anatomy, physiology, and ontogeny.
  • 2 The pathological disease variation affecting the kidney
  • a: a tie in to eNON and iNos, nitric oxide, cGMP and glutaminase – in acute renal failure, hypertension, chronic renal failure, dialysis the pathology of acute tubular necrosis, glomerular function, efferent arteriolar and kidney medullary circulatory impairment, and cast formation related to Tamm Horsfall protein
  • b :The role of NO, eNOS and iNOS in disorders of the lund alveolar cell and subendothelial matrix, and of liver disease also affecting the kidney, and the heart. c Additional references
  • 3.     a Acute renal failure, oxidate stress, ischemia-reperfusion injury, tubulointerstitial chronic inflammation
  • 3       b Additional references 4. Nitric oxide donors – opportunities for therapeutic targeting? As we see this in as full a context as possible, it is hard to distinguish the cart from the horse.

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We know that there is an unquestionable role of NO, and a competing balance to be achieved between eNOS, iNOS, an effect on tubular water and ion-cation reabsorptrion, a role of TNFa, and consequently an important role in essential/malignant hypertension, with the size of the effect related to the stage of disorder, the amount of interstitial fibrosis, the remaining nephron population, the hypertonicity of the medulla, the vasodilation of the medullary circulation, and the renin-angiotensin-aldosterone system. Substantial data and multiple patients with many factors per patient would be need to extract the best model using a supercomputer.

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