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Posts Tagged ‘Chronic kidney disease’


Targeting Kidney Glomerulus

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Targeting the podocyte to treat glomerular kidney disease

Lal MA, Young KW, and Andag Uwe    1 CVMD iMED, AstraZeneca, Molndal, Sweden 2 Evotec AG, Germany
Drug Discovery Today  http://www.e-ditionsbyfry.com/olive/ODE/DDT/Default.aspx?href=DDT/2015/10/01

The number of randomized clinical trials addressing renal disease continues to be outpaced by most other specialties and the failure of a number of recent kidney clinical trials suggests a need for new thinking and new strategies to address CKD [5,58]. Delineation of the crucial molecular species and signaling pathways underlying podocyte function will undoubtedly lead to the identification of cell-specific targets that can be exploited for treating various glomerulopathies and CKD.

 

The majority of chronic kidney disease (CKD) cases have their origin in the glomerulus, the microvascular unit of the nephron that serves as a filter tasked with forming primary urine. This selective filtration process is determined to a large extent by the functional capacity of the podocyte, a highly differentiated cell type that enwraps the outer aspect of the glomerular capillary wall. In this short review, we describe the biology of the podocyte, its central role in the etiology of various glomerulopathies and highlight current and future opportunities to exploit the unique properties of this cell type for developing kidney-specific therapeutics.  

The kidneys   The kidneys are a remarkable set of paired organs that receive › 20% of cardiac output and that filter some 180 l blood daily, which is subsequently modified to give a final urine volume of approximately 1.5 l. This phenomenal physiological capacity is carried out by the concerted actions of the glomerular filter and renal tubular systems that together represent the single functional unit of the kidney known as the nephron. In performing this outstanding feat, the kidneys participate in establishing wholebody ion homeostasis, acid-base balance and blood pressure regulation. It should therefore come as no surprise that loss of kidney function and a compromised ability to carry out such fundamental processes is associated with considerable risks to overall health [1].  

Chronic kidney disease   Chronic kidney disease (CKD) is a progressive malady defined by reduced glomerular filtration rate, increased urinary albumin excretion or both, and is a major global public health concern with an extremely high unmet medical need. CKD is estimated to occur in 8–16% of the worldwide population and results in a substantially reduced life expectancy [1]. Diabetes itself is recognized as the primary cause of kidney failure in almost half of all new CKD cases and, given the current global diabetes pandemic, the prevalence of renal complications will continue to grow.  

There is a strong relationship between CKD and cardiovascular disease which itself is twice as common in CKD patients as compared with the general population [2]. Accordingly, the current clinical paradigm for treating CKD patients is primarily focused on reducing cardiovascular risk and is, not surprisingly, inadequate to prevent patients from continuing their inexorable loss of renal function because such strategies do not directly address the principal mechanisms accounting for renal disease itself. Despite overall advances in renal replacement therapy and dialysis for CKD patients ultimately succumbing to end-stage renal failure, these last resort options are insufficient in terms of limited organ availability and high mortality and/or cost. The potential identification and implementation of novel, kidney-specific treatment strategies therefore represents a significant untapped opportunity to improve the prognosis of these patients. The future development of such kidney-centric therapeutics will require the identification of suitable renal targets and can only be achieved through a comprehensive understanding of disease pathophysiology, the underlying molecular mechanisms of disease initiation and progression, and the implementation of translatable models and technologies [3].  

The glomerular filtration barrier   The vast majority of all cases of CKD have their origin in the glomerulus [4]. Clinically, although kidney disease progression shows the strongest correlation with the degree of fibrosis in the tubules and interstitium, it is lesions within the highly specialized microvascular unit that initiate disease. The primary function of the glomerulus is to filter blood selectively across the capillary wall and to elaborate an ultrafiltrate that is secondarily modified by the renal tubule system. The filtration capacity of the glomerulus is defined by the functional properties of the glomerular filtration barrier (GFB), a trilaminar molecular sieve comprising endothelial cells and visceral podocytes that medially elaborate the constituents of the glomerular basement membrane (GBM) that lies between them (Fig. 1). Although each individual component of the GFB is necessary for its concerted function, the podocyte is largely recognized as the final determinant of size-selective filtration and the ultimate barrier to albumin [4]. This is of particular clinical relevance because even small increases in albuminuria confer clinical risk and, at increasing levels of proteinuria, life expectancy is substantially reduced [5].  

FIGURE 1   Ultrastructural images of the glomerular capillary and filtration barrier. (a,b) Scanning electron micrographs of the exterior surface of the glomerular capillary depicting the intricate pattern in which podocyte foot processes interdigitate, surround and enwrap the outer capillary surface. (c) Transmission electron micrograph of the glomerular filtration barrier in cross section. The * indicates the slit diaphragm. Size marker 1 mm (a) and 200 nm (b,c). Abbreviations: FP, foot process; GBM, glomerular basement membrane; EC, endothelial cell; RBC, red blood cell; CL, capillary lumen; US, urinary space.

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The podocyte   The podocyte is a terminally differentiated, highly specialized cell type with a remarkable morphology exquisitely designed to match its function. The podocyte establishes the integrity and function of the GFB and slit diaphragm, maintains capillary structure and resists intraglomerular blood pressure, contributes to the formation and modulation of the glomerular basement membrane and also determines endothelial cell homeostasis [6]. Ultrastructurally, the podocyte is like no other cell. From its voluminous cell body exposed in the urinary space extend primary and secondary processes that arborize into slender foot processes that interdigitate with those of neighboring podocytes to enwrap the outer surface of the glomerular capillary firmly (Fig. 1). One of the most unique features of the podocytes and their foot processes is the slit diaphragm, a modified cell–cell adherens junction that spans the length of their interaction. The slit diaphragm, visible upon electron microscopy as a bridge connecting juxtaposed foot processes, comprises a host of unique structural proteins, the founding member of which is nephrin [4]. It is the initial identification of nephrin and genetic mutations therein as the cause of the massive proteinuria that characterizes patients with congenital nephrotic syndrome of the Finnish type (CNSF) that has placed the podocyte at the center of current activities aimed at understanding the molecular and cellular determinants of proteinuria. Since the discovery of nephrin, the identification of a host of additional human genetic mutations in various proteins implicit in podocyte function has highlighted the central involvement of this cell type in the etiology of various inherited diseases of the glomerulus [7] (Table 1). Collectively, glomerular diseases can be classified as a related spectrum of podocytopathies where abnormalities in podocyte biology (i.e. dysfunction, injury and loss) are shared among them. They encompass purely genetic forms of podocyte disease [such as CNSF, Alport syndrome and some forms of focal and segmental glomerular sclerosis (FSGS) and membranous nephropathy] and also include nonhereditary glomerular diseases that occur secondary to conditions such as hypertension and diabetic nephropathy [8]. Taken together, glomerular diseases account for the vast majority of all end-stage kidney disease cases with diabetic nephropathy representing the single largest cause.  

 

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One of the earliest cellular lesions observed in the various podocytopathies that lead to glomerulosclerosis is a loss of podocytes or, more specifically, a reduction in the number of podocytes per glomerulus. Because podocytes are terminally differentiated and have a limited capacity for repair or regeneration, glomerular function is particularly sensitive to situations in which there is a mismatch between podocyte number and the glomerular filtration surface area. Experimental animal models show that a loss of <20% of podocytes causes transient proteinuria and that further podocyte loss results in progressive proteinuria, glomerulosclerosis and eventually loss of renal function [9]. In a similar manner, if glomerular volume exceeds the threshold for podocytes to maintain a functional GFB, glomerular disease will ensue. Data obtained from humans with diabetic nephropathy corroborate these preclinical observations [10]. Based on this understanding of disease etiology, therapeutic strategies aimed specifically at improving the health and function of the podocyte represent a promising avenue to address CKD [11].  

Current therapeutic options with unpredicted effects on podocyte biology  

The current state of the art for the treatment of CKD is focused on optimizing renal and cardiovascular risk factors and includes controlling blood pressure, albuminuria, blood glucose and blood lipids [5]. These current treatment options are based largely on repurposing of existing therapeutics and are not kidney-targetbased nor designed to address the underlying mechanisms of CKD progression. Nevertheless, there are now considerable amounts of data supporting the intriguing possibility that the beneficial effects on kidney function of a number of currently available drugs (including glucocorticoids, calcineurin inhibitors, rituximab, inhibitors of the renin–angiotensin–aldosterone system, among others) could be at least partially accounted for by their direct effects on the podocyte. Table 2 lists additional pathways and targets central to podocyte function for which therapeutic interventions have been described using preclinical models. Such examples will not be further described here so we invite the reader to consult some recent reviews on the subject [11–13].  

 

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The most intriguing and contentious clinical evidence available thus far supporting the possibility to improve renal function and prevent CKD progression by specifically targeting the podocyte comes from research examining the role of B7-1 (CD80) in this cell type [14–16]. Targeting of the podocyte at this molecular moiety with abatacept, an antibody-based inhibitor of B7-1, induced remission in five patients with FSGS [16]. Mechanistically, B7-1 overexpression in injured podocytes appears to interfere with b1-integrin-mediated cellular attachment to the glomerular basement membrane and abatacept exerts its protective effects by facilitating the stabilization of b1-integrin activation and podocyte adhesion. Although these results might be compelling, they are not without significant debate because the detection of B7-1 expression in podocytes as well as the efficacy of B7-1 blockers in treating FSGS patients are both highly controversial [17]. Future studies with sufficient sample size will hopefully clarify the situation.  

Focusing on the podocyte as a specific therapeutic  target   Based on the above genetic evidence and efficacy of the described pharmacological approaches to targeting the podocyte clinically, combined with the plethora of preclinical animal studies conclusively demonstrating that podocyte dysfunction per se results in foot process effacement, albuminuria and glomerular disease, there is tantalizing hope of a future where the pursuit of podocentric therapeutic strategies will be realized. Although the ability to alter the podocyte genetically in mice has led to an explosion in the number of gene products identified as requisite for podocyte biology and potentially amenable to pharmacological manipulation, the ubiquitous nature of many of these targets and their pathways in other cell types makes them difficult to exploit. However, by identifying the molecular components that uniquely define and contribute to podocyte signaling and function, it should be possible to identify and develop cell-specific targeting opportunities.  

One of the best-studied aspects of podocyte biology concerns that of the mechanisms and factors regulating its cytoskeleton. Indeed, podocyte effacement and loss of the slit diaphragm as seen in glomerular diseases are phenomena intricately tied to disrupted actin cytoskeletal homeostasis in this cell type [12,18–20]. Not surprisingly, podocyte-specific manipulation of the mouse genome clearly indicates that the RhoGTPase family of actin master regulators is intricately involved in determining podocyte function [21,22]. The ultimate question that remains to be addressed, however, is how to exploit such a ubiquitous cell biological process driven by canonical signaling pathways therapeutically. Perhaps, the best opportunity for such a scenario could come from further studies aimed at defining the unique, principal molecular components that impact podocyte actin cytoskeletal architecture. A number of actin cytoskeleton effectors with podocyte-enriched expression have been identified and they could provide novel entry points to facilitate the fine-tuning and manipulation of the actin cytoskeleton specifically in this cell type to promote the reestablishment of normal foot process architecture in disease [23–25].  

Defining the molecular footprint of the podocyte   The ability to isolate pure populations of glomeruli from the renal parenchyma and further selectively separate the podocyte from its neighboring endothelial and mesangial cells by lineage-tracking and fluorescently activated cell sorting combined with various omics technologies has allowed researchers to catalogue the podocyte transcriptome and proteome [26–28]. Further definition of the miRNA transcriptional profile, alternative gene splicing events, phosphoproteome and epigenome represent additional levels of molecular complexity that further enhance our understanding of the underpinnings of podocyte biology [26,29–31]. A computational, machine-learning-based approach has also been developed that is able to infer podocyte-specific expression from whole-tissue samples through iterative statistical analysis of shared gene expression patterns and will be particularly useful to elucidate the roles of individual cell-specific transcripts in human glomerular disease [32]. The daunting task remains to unravel the cellular role of these individual proteins in podocyte health and disease and to consider whether they represent viable therapeutic targets. Significant inroads have been made over the past two decades and it is not unreasonable to envision a not too distant future when the involvement of all individual genes in podocyte biology is achieved. Acquired glomerular diseases, such as diabetic nephropathy, are not due to genetic defects but rather can be seen as a consequence of crucial signaling pathways of the podocyte that have gone awry. Determining which of the proteins expressed in podocytes and their pathways that are most amenable to therapeutic intervention will require considerable efforts from the research community.  

Use of podocytes in phenotypic screens   The ability to propagate conditionally immortalized podocytes in culture combined with rapid advances in molecular biology has greatly complemented our knowledge of the molecular machinery that defines this cell type [33,34]. Although there are important limitations to consider when utilizing such podocyte cell lines, podocyte cell culture has been an instrumental tool for simulating various aspects of glomerular disease biology. From the drug discovery perspective, there remain significant opportunities to take advantage of this cellular system for target evaluation and discovery [13,35].  

Target-agnostic high-content or phenotypic screening approaches are a recent addition to the drug discovery process [36]. Here, hit compounds are identified as having a positive phenotypic readout in a cell-based assay. Key to this process is the disease relevance of the phenotype being investigated and ultimately the translatability of the assay to human disease states. In this regard, we have used human conditionally immortalized podocytes [34] treated with a combination of palmate and high glucose to mimic conditions observed in diabetic nephropathy. The podocyte response to this ‘disease-relevant’ stimulus can be measured using high-content imaging technology, such as the OperaTM confocal imaging platform, and manifests as changes in the actin cytoskeleton and overall cell morphology, followed by onset of cell apoptosis (Fig. 2).

 

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FIGURE 2   Podocyte high-content screening. Conditionally immortalized human podocytes were screened using a combination of palmitate and high glucose to mimic conditions observed in diabetic nephropathy. Small-molecule inhibitors of phenotypic changes were identified using the OperaTM high-content imaging platform. The primary screen was run using activation of caspase 3/7 as a marker of podocyte apoptosis. Hit compounds were also tested in an orthogonal assay which used Alexa-labeled phalloidin to detect changes in the actin cytoskeleton.

 

These events provide suitably robust outputs for compound screening. Evotec, in combination with AstraZeneca, has screened >120 000 compounds using protection from palmitate and highglucose-induced podocyte apoptosis as a primary readout. Protection against effects on the actin cytoskeleton was used as a secondary orthogonal assay. To this end, we have identified a number of targets, some previously recognized as important regulators of podocyte biology [e.g. mammalian target of rapamycin (mTOR), glucocorticoid receptor, all-trans retinoic acid, among others]. Interestingly , a number of compounds that had the capability to attenuate palmitate and high-glucose-driven podocyte apoptosis in the screen were identified as glycogen synthase kinase (GSK)3b inhibitors. 1-Azakenpaullone (1-AZK), a selective GSK3b inhibitor identified by the podocyte screening approach, was further analyzed for its potency to protect ex vivo isolated porcine glomeruli against palmitate and high-glucose injury. 1-AZK significantly increased cell viability of glomeruli exposed to palmitate and high glucose, highlighting again the protective effect of GSK3b inhibitors in this injury setting (Fig. 3). Recently, GSK3b inhibitors have been demonstrated to exert protective effects on injured podocytes in culture as well as in mouse models of kidney disease including diabetic nephropathy [37,38]. These findings suggest that pharmacologically targeting GSK3b could represent a therapeutic strategy to protect podocytes against injury.  

 

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FIGURE 3   Glycogen synthase kinase (GSK)3b inhibition protects against podocyte injury. Representative images of the degree of podocyte apoptosis measured as caspase 3/7 activation (green) under control (a) and 200 mM palmitate with 25 mM glucose (b) conditions. Podocyte nuclei are counterstained red. (c) 1-Azakenpaullone (1-AZK) dose-dependently reduces caspase 3/7 activation in human podocytes challenged with 200 mM palmitate and 25 mM glucose for 48 h. Podocyte apoptosis was quantified and normalized to confluence level by measuring caspase 3/7 activity and confluence using IncuCyte live cell imaging (n = 4). (d) Overall cell viability, quantified by AlamarBlueWassay (n = 3), of glomeruli freshly isolated from Gottingen minipig via graded sieving technique and cultured in 11 mM glucose ( › ) or 400 mM palmitate with 25 mM glucose (+) with indicated 1-AZK concentrations for three days.

 

The second, often more complex phase, of phenotypic screening is the need to identify the target or targets of hits found in the screening phase. Here, the process involves expansion of the chemical matter around the initial hit chemotype to understand the basic structure-related properties for the molecule. Next, chemical proteomics is employed to identify cellular binding partners for the hits and ultimately these targets need to be confirmed using the screening assay set up [39,40]. Together, Evotec and AstraZeneca have successfully progressed podocyte hit compounds to target identification and are currently developing compounds around these novel targets.  

Tools to evaluate podocyte function   Although the ability to culture podocytes has tremendously enhanced our understanding of the function of individual proteins in this cell type at a biochemical level, such methods and the conclusions drawn from their results need to be integrated within the complex framework of podocyte function in vivo as a requisite component of the GFB. This is important to consider because the podocyte in vitro is an imperfect representation of its in vivo counterpart at a molecular and functional level [25–27,41]. The isolated podocyte certainly mimics certain facets of podocyte biology observed in vivo, but there is a need for additional unbiased and integrated strategies that can facilitate our understanding of the mechanistic determinants of podocyte function in its native microenvironment. A number of novel and exciting strategies have been developed to fill the void between cell culture and whole-animal, mammalian studies of podocyte biology.  

The concept of the podocyte as a dynamic, motile cell type in vitro has been around for some time, but the corollary of this phenomenon in vivo and its putative physiological significance in human health and disease is a subject of active debate. Through the use of transgenic animals (i.e. mice and zebrafish) expressing fluorescently labeled podocytes and advanced microscopic imaging techniques it is possible to study and interrogate the occurrence of podocyte motility in vivo and its relevance to glomerular disease [42,43]. The conclusions drawn from such studies are not in complete harmony and continue to fuel the debate over podocyte plasticity and the cellular nature of their origin. Regardless, such strategies are valuable tools that will be important to evaluating the efficacy of novel therapeutic approaches designed to exploit podocyte dynamics and the potential to control podocyte regeneration from progenitor cell niches.  

Another novel and very interesting imaging-based approach for the study of glomerular function is a recently published technique based on repetitive noninvasive in vivo imaging of GFB integrity in isolated glomeruli transplanted into the anterior chamber of the mouse eye [44]. Podocytes from such glomeruli maintained their differentiation and displayed interdigitating podocyte foot processes for up to six months after transplantation and are amenable to repeated imaging. Transplanting healthy or diseased human glomeruli into mouse eyes might provide a means to image functioning human glomeruli and acutely examine the effect of therapeutics on glomerular function.  

The fruit fly has also recently seen its debut as a model organism to study podocyte biology [45]. Despite gross anatomical differences in the excretory system of humans and fruit flies, the Drosophila pericardial nephrocyte bears a remarkable evolutionary conservation of molecular components and functional properties with the mammalian podocyte. To exploit this observation, and to harness the power of Drosophila genetics, a reporter system of nephrocyte filtration function has been combined with a large RNA interference genetic screen that enables the scanning of the entire genome for genes specifically required for pericardial nephrocyte function [46]. An initial genetic screen of about 1000 genes resulted in the identification of about 7% that were essential for nephrocyte function, notable among them the mammalian homologs of nephrin and podocin [46]. In the near future, the complete repertoire of genes required for nephrocyte-specific function will be defined and this will be an invaluable source of unbiased information ready to be translated to higher animal species.  

The zebrafish, as mentioned above, has taken its place as an exceptional model for studying podocyte biology and glomerular function. The ability to knockdown individual genes and subsequently to follow functional parameters of filtration capacity has been an important tool in the armamentarium of the podocyte biologist to accelerate the study of podocyte gene function [47]. Reverse genetic screens can be readily used to investigate the potential role of novel podocyte proteins. Additionally, novel transgenic zebrafish models that incorporate inducible podocyte injury with a fluorescent tracer for proteinuria make it possible to use this functional model for the screening of therapeutic candidates that could improve overall podocyte health and GFB integrity [48].  

Another strategy not yet fully realized for the kidney, but demonstrated in principal for other organ systems including that of the lung and liver, is the microfluidic organ-on-a-chip which aims to facilitate the in vitro organomimetic modeling of complex human physiology [49,50]. Similar to recapitulating the alveolarcapillary interface of the lung in a mechanical environment that mirrors physiological function, the GFB of the kidney would appear to be a particularly well-suited functional unit for such a strategy. One can envisage the culturing of human podocytes and glomerular endothelial cells on either side of a structural support in a self-contained unit where separated perfusion chambers bath cells in appropriate media and biomechanical properties such as hydrostatic pressure, fluid flow and shear stress can be modulated to simulate in vivo properties. Such a biomimetic device could be what is required to promote the differentiation of cultured podocytes to develop foot processes and slit diaphragms as is functionally required for GFB function. Interestingly, it has also been demonstrated through the cellular repopulation of decellularized kidneys that the native kidney extracellular matrix, including that of the GBM, provides cellular cues that might promote the potential differentiation of podocytes in the glomerulus [51]. The future development of a human GFB biomimetic represents a significant hurdle but holds great promise as a means to study podocyte function and to evaluate drug pharmacology and toxicity.  

Podocyte delivery and targeting   Briefly, cell-specific targeting of therapeutic agents to podocytes of the glomerulus might potentially be an attractive method to increase their efficacy and/or minimize their side-effects. Size and charge characteristics could be exploited to facilitate the capability of drug–carrier conjugates to reach and interact specifically with the correct target cells. Within the kidney, the podocyte is an attractive target cell because of its unique properties. Large molecules that accumulate in the GBM might not need to be filtered to reach the podocyte because of the robust endocytic capacity of this cell type [52]. This raises the possibility of using tissue-specific homing strategies to deliver large proteins, antibodies and siRNAs into cells in a biologically active form [53,54]. Ultrasound-microbubble-mediated delivery of gene therapies to the kidney is an interesting approach that has been used to achieve drug targeting to the kidney [55]. Efficacy of ultrasound-microbubble-mediated gene delivery has been demonstrated in rat models of renal fibrosis but upregulation of the transgene of interest was noted in all kidney tissues [56]. The ability to combine such ultrasound microbubble technology with cell-specific targeting should be possible for the podocyte as has been demonstrated for achieving endothelium-specific transgene expression of a shRNA [57].  

Concluding remarks   The number of randomized clinical trials addressing renal disease continues to be outpaced by most other specialties and the failure of a number of recent kidney clinical trials suggests a need for new thinking and new strategies to address CKD [5,58]. Delineation of the crucial molecular species and signaling pathways underlying podocyte function will undoubtedly lead to the identification of cell-specific targets that can be exploited for treating various glomerulopathies and CKD. Realizing this goal will require a holistic approach where the short-comings of any single strategy in isolation will be overcome only by integrating their individual strengths. With this in mind, the future for developing podocyte specific therapeutics looks bright.

 

   1 Eckardt, K.U. et al. (2013) Evolving importance of kidney disease: from subspecialty to global health burden. Lancet 382, 158–169

2 de Zeeuw, D. et al. (2004) Proteinuria, a target for renoprotection in patients with type 2 diabetic nephropathy: lessons from RENAAL. Kidney Int. 65, 2309–2320

3 Gashaw, I. et al. (2011) What makes a good drug target? Drug Discov. Today 16, 1037–1043

4 Tryggvason, K. et al. (2006) Hereditary proteinuria syndromes and mechanisms of proteinuria. N. Engl. J. Med. 354, 1387–1401

5 Lambers Heerspink, H.J. and de Zeeuw, D. (2013) Novel drugs and intervention strategies for the treatment of chronic kidney disease. Br. J. Clin. Pharmacol. 76, 536–550

6 Pavenstadt, H. et al. (2003) Cell biology of the glomerular podocyte. Physiol. Rev. 83, 253–307

7 Fogo, A.B. (2014) Causes and pathogenesis of focal segmental glomerulosclerosis. Nat. Rev. Nephrol. 11, 76–87

8 Wiggins, R.C. (2007) The spectrum of podocytopathies: a unifying view of glomerular diseases. Kidney Int. 71, 1205–1214

9 Wharram, B.L. et al. (2005) Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene. J. Am. Soc. Nephrol. 16, 2941–2952

10 Pagtalunan, M.E. et al. (1997) Podocyte loss and progressive glomerular injury in type II diabetes. J. Clin. Invest. 99, 342–348

11 Mathieson, P.W. (2012) The podocyte as a target for therapies—new and old. Nat. Rev. Nephrol. 8, 52–56

12 Brinkkoetter, P.T. et al. (2013) The role of the podocyte in albumin filtration. Nat. Rev. Nephrol. 9, 328–336

13 Reiser, J. et al. (2010) Toward the development of podocyte-specific drugs. Kidney Int. 77, 662–668

14 Fiorina, P. et al. (2014) Role of podocyte B7-1 in diabetic nephropathy. J. Am. Soc. Nephrol. 25, 1415–1429

15 Reiser, J. et al. (2004) Induction of B7-1 in podocytes is associated with nephrotic syndrome. J. Clin. Invest. 113, 1390–1397

16 Yu, C.C. et al. (2013) Abatacept in B7-1-positive proteinuric kidney disease. N. Engl. J. Med. 369, 2416–2423

17 Benigni, A. et al. (2014) Abatacept in B7-1-positive proteinuric kidney disease. N. Engl. J. Med. 370, 1261–1263

18 Kriz, W. (2002) Podocyte is the major culprit accounting for the progression of chronic renal disease. Microsc. Res. Tech. 57, 189–195

19 Kriz, W. et al. (2013) The podocyte’s response to stress: the enigma of foot process effacement. Am. J. Physiol. Renal. Physiol. 304, F333–F347

20 Welsh, G.I. and Saleem, M.A. (2012) The podocyte cytoskeleton—key to a functioning glomerulus in health and disease. Nat. Rev. Nephrol. 8, 14–21

21 Lal, M.A. and Tryggvason, K. (2012) Knocking out podocyte rho GTPases: and the winner is. J. Am. Soc. Nephrol. 23, 1128–1129

22 Mouawad, F. et al. (2013) Role of Rho-GTPases and their regulatory proteins in glomerular podocyte function. Can. J. Physiol. Pharmacol. 91, 773–782

23 Akilesh, S. et al. (2011) Arhgap24 inactivates Rac1 in mouse podocytes, and a mutant form is associated with familial focal segmental glomerulosclerosis. J. Clin. Invest. 121, 4127–4137

24 Asanuma, K. et al. (2006) Synaptopodin orchestrates actin organization and cell motility via regulation of RhoA signalling. Nat. Cell Biol. 8, 485–491

25 Lal, M.A. et al. (2015) Rhophilin-1 is a key regulator of the podocyte cytoskeleton and is essential for glomerular filtration. J. Am. Soc. Nephrol. 26, 647–662

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Investigational Bioengineered Blood Vessel: Humacyte Presents Interim First-in-Human Data at the American Heart Association (AHA) Scientific Sessions 2013

Reporter: Aviva Lev-Ari, PhD, RN

The investigational bioengineered blood vessels represent a research and development milestone in vascular tissue engineering, as this technology could have the potential to help reduce or avoid surgical interventions and hospitalizations for patients with end-stage renal disease.

The Humacyte investigational bioengineered blood vessels are manufactured in a novel bioreactor system. The investigational bioengineered vessels go through a process of decellularization, which is designed to render them potentially non-immunogenic and implantable into any patient. These investigational bioengineered vessels are designed to be stored off-the-shelf for up to 12 months under standard refrigerated conditions, including, if successfully developedand approved,  on-site in hospitals.

 

Gail Thornton
Media Relations, Humacyte
1 908 392 3420 MOBILE
gail@westmillconsulting.com

Jim Modica

West Mill Consulting

908-872-4919

Jim@westmillconsulting.com

Humacyte Presents Interim First-in-Human Data

For Investigational Bioengineered Blood Vessel at the American Heart Association (AHA) Scientific Sessions 2013

  • The Humacyte investigational bioengineered blood vessel technology represents a research and development milestone in vascular tissue engineering.
  • Interim data from 28 patients in a three-center, first-in-human study in Poland indicate that all of the investigational blood vessels to date remain open to blood flow (patent), with no indication of an immune response in recipients, no aneurysms, and flow rates and durability suitable for dialysis.
  • The interim data suggest that the Humacyte investigational technology may have the potential to have high patency rates.
  • Longer follow-up and additional clinical studies will be required to confirm these preliminary observations.

 

RESEARCH TRIANGLE PARK, N.C., November 20, 2013 –Humacyte, Inc., a pioneer in regenerative medicine, today announced the presentation of interim, first-in-human data from an ongoing, multi-center study in Poland, evaluating the company’s investigational bioengineered blood vessel in hemodialysis patients with End-Stage Renal Disease (ESRD). The data were presented by Dr. Jeffrey H. Lawson, M.D., Ph.D., at the American Heart Association Scientific Sessions 2013 in Dallas, Texas (abstract). Dr. Lawson is Professor of Surgery and Pathology with tenure at Duke University Medical Center (Durham, North Carolina, USA), and Director of the Vascular Research Laboratory and Director of Clinical Trials for the Department of Surgery. He is also Clinical Consultant to Humacyte.

This is the first time surgical data from patients have been reported for the Humacyte investigational bioengineered vessel; the interim data come from a cohort of 28 study participants out of a total of 30 that will ultimately be enrolled in the three-site study in Poland (http://clinicaltrials.gov/show/NCT01744418%20CLN-PRO-V001%20NCT01744418). The first patients were implanted with the investigational vessels in December, 2012, and the vessels were first used for hemodialysis in February, 2013. The primary endpoints of the study in Poland are safety, tolerability, and patency to be examined at each visit within the first six months after graft implantation. Patients will be followed for an additional six months.

The interim patient data suggest that the Humacyte investigational bioengineered vessel may potentially be associated with low rates of vessel clotting, low infection rates, and low rates of surgical interventions. Low rates of clotting and intervention are consistent with preclinical data from animal testing that indicated little intimal hyperplasia. Preclinical data also indicated that, in animals, investigational vessels were remodeled to become living and more similar to native tissue. To date in the Polish study, the investigational vessel has remained open to blood flow (patent), with no indication of an immune response in recipients, no aneurysms (abnormal widening or ballooning of part of an artery due to weakness in the blood vessel wall), and flow rates and durability suitable for dialysis. Longer follow-up and additional clinical studies will be required to confirm these preliminary observations.

Co-authors on the presentation were: Drs. Marek Iłżecki, Tomasz Jakimowicz, Alison Pilgrim, Stanisław Przywara, Jacek Szmidt, Jakub Turek, Wojciech Witkiewicz, Norbert Zapotoczny, Tomasz Zubilewicz, and Laura Niklason.

Described by Investigator as “Breakthrough Investigational Technology”

“Based on our experience to date, this is breakthrough investigational technology,” said Principal Investigator Prof. Tomasz Zubilewicz, M.D., Ph.D., head, Department of Vascular Surgery and Angiology, Medical University of Lublin, Poland. “The investigational bioengineered vessel seems like it could have the potential to be shown to be superior to synthetic grafts in vascular access for hemodialysis in all aspects. This technology also has potential for other areas of vascular surgery, including replacement of infected synthetic grafts.”

“We are very encouraged by the Humacyte investigational bioengineered vessel’s performance in end-stage renal disease patients,” said Dr. Lawson. “Tremendous medical need exists for vascular access grafts in patients with ESRD who require dialysis. Based on this interim data and other ongoing research, we believe that the investigational bioengineered vessel has potential to meet this significant need.”

Need to Overcome Limitations of PTFE Grafts

Currently available synthetic vessels made from polytetrafluoroethylene (PTFE) are subject to many complications and about half fail within a year, requiring replacement surgery. PTFE vessels tend to become blocked (have low patency rates), have high rates of stenosis (an abnormal narrowing in a blood vessel that can be associated with hemodialysis), and high intervention rates.

“We continue to make significant progress in our research and development program with the Humacyte investigational bioengineered blood vessel,” said Laura E. Niklason, M.D., Ph.D., professor and vice chair of Anesthesia, professor of Biomedical Engineering, Yale University, and founder, Humacyte. “With our current interim study data, all of the Humacyte vessels have remained open to blood flow, with 20 out of the 28 implants requiring no intervention to date. We are grateful to patients, investigators, regulators and the broader vascular community for their ongoing collaboration and support in advancing this science.”

Unmet Medical Need in Chronic Kidney Disease

The Humacyte investigational technology is being developed with the goal of pursuing approval for use in patients with chronic kidney disease, a major global health problem affecting 26 million Americans[i] and around 40 million people in the European Union (EU).[ii] Individuals who progress to end-stage renal disease (ESRD) require renal replacement therapy (hemodialysis or kidney transplant); more than 380,000 patients currently require hemodialysis in the U.S.[iii] and some 250,000 patients require hemodialysis or have had kidney transplants in the EU.[iv] The investigational bioengineered vessels, if successfully developed and approved for use in ESRD by regulatory authorities, could offer the potential for significant cost savings to the healthcare system. These investigational bioengineered vessels represent a research and development milestone in vascular tissue engineering, as this technology could have the potential to help reduce or avoid surgical interventions and hospitalizations for patients with ESRD.

Investigators Highlight Preliminary Experiences In Patients

The investigators involved with the study in Poland cited their clinical observations in connection with the release of the preliminary patient data obtained for the Humacyte investigational technology.

“It was an exciting experience to be involved with this study, and to participate in this potential breakthrough in vascular surgery. This investigational bioengineered vein is a promising development for vascular surgeons,” said Principal Investigator Prof. Jacek Szmidt, head of the Department of General, Vascular and Transplant Surgery, Medical University of Warsaw, Poland.

“The Humacyte investigational bioengineered vessel was very easy to handle during implantation in this study. The graft maintained excellent mechanical properties, and based on our team’s experience, the complication rate to date has been very low compared with synthetic grafts,” said Investigator Stanisław Przywara, M.D., Ph.D., senior assistant, Department of Vascular Surgery and Angiology, Medical University of Lublin, Poland.

“During implantation in this study, the Humacyte investigational vessel behaved very much like a native vein.  Anastomotic hemostasis was achieved almost immediately. Insertion of needles to perform hemodialysis was easy and as reported by our nephrologists, provides very good adequacy of hemodialysis,” said Investigator Marek Iłżecki, M.D., Ph.D., senior resident, Department of Vascular Surgery and Angiology, Medical University of Lublin, Poland.

U.S. Clinical Trial Started in May, 2013

A multi-center U.S. clinical trial began in May, 2013 under a U.S. Investigational New Drug (IND) application. The U.S. trial will involve up to 20 patients across three sites to assess safety and performance of the innovative, investigational bioengineered blood vessels to provide vascular access for hemodialysis in ESRD patients.

About the Investigational Bioengineered Blood Vessels

The Humacyte investigational bioengineered blood vessels are manufactured in a novel bioreactor system. The investigational bioengineered vessels go through a process of decellularization, which is designed to render them potentially non-immunogenic and implantable into any patient. These investigational bioengineered vessels are designed to be stored off-the-shelf for up to 12 months under standard refrigerated conditions, including, if successfully developed and approved,  on-site in hospitals. Subject to receipt of regulatory approval, these properties could make the investigational bioengineered vessels readily available to surgeons and patients, and could eliminate the wait for vessel production or shipping. Data from studies of the investigational bioengineered vessels in large animal models reflect resistance to thickening for up to one year, and the early human studies that are now underway will provide safety and performance data in patients to support a future application for regulatory approval.

About Humacyte

Humacyte, Inc., a privately held company founded in 2005, is a medical research, discovery and development company with clinical and pre-clinical stage investigational products. Humacyte is primarily focused on developing and commercializing a proprietary novel technology based on human tissue-based products for key applications in regenerative medicine and vascular surgery. The company uses its innovative, proprietary platform technology to engineer human, extracellular matrix-based tissues that are designed be shaped into tubes, sheets, or particulate conformations, with properties similar to native tissues. These are being developed for potential use in many specific applications, with the goal to significantly improve treatment outcomes for a variety of patients, including those with vascular disease and those requiring hemodialysis. The company’s proprietary technologies are designed to result in off-the-shelf products that, once approved, can be utilized in any patient. The company web site is www.humacyte.com.

Forward-Looking Statement

Information in this news release contains “forward-looking statements” about Humacyte. These statements, including statements regarding management’s projections relating to future results and operations, are based on, among other things, management’s views, assumptions and estimates, developed in good faith, all of which are subject to known and unknown factors that may cause actual results, performance or achievements, or industry results, to differ materially from those expressed or implied by such forward-looking statements.

 

References


[iv]http://www.ekha.eu/usr_img/info/factsheet.pdf

SOURCE

From: Gail Thornton <gail@westmillconsulting.com>
Reply-To: Gail Thornton <gail@westmillconsulting.com>
Date: Wed, 20 Nov 2013 09:24:32 -0800 (PST)
To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>
Subject: Re: American Heart Association: Humacyte Investigational Bioengineered Blood Vessels

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Humacyte, Inc., a pioneer in regenerative medicine, presented the results of foundational U.S. preclinical studies of its investigational bioengineered blood vessel at the American Society of Nephrology’s ‘Kidney Week 2013’ Annual Meeting in Atlanta, GA.

Reporter: Aviva Lev-Ari, PhD, RN

HUMACYTE

Media Contacts:

Gail Thornton

West Mill Consulting

908-392-3420

Gail@westmillconsulting.com

Jim Modica

West Mill Consulting

908-872-4919

Jim@westmillconsulting.com

Humacyte Highlights Preclinical Data

Of Its Investigational Bioengineered Blood Vessel

 

  • Humacyte investigational bioengineered blood vessel technology represents a research and development milestone in the field of vascular tissue engineering.
  • Preclinical data on the investigational bioengineered blood vessel were presented at the American Society for Nephrology ‘Kidney Week’ meeting.
  • The pre-clinical data suggest that the Humacyte technology may have the potential to be associated with lowered vessel clotting and incorporation with animal model tissues.

RESEARCH TRIANGLE PARK, N.C., November 13, 2013Humacyte, Inc., a pioneer in regenerative medicine, presented the results of foundational U.S. preclinical studies of its investigational bioengineered blood vessel at the American Society of Nephrology’s ‘Kidney Week 2013’ Annual Meeting in Atlanta, GA.

The scientific presentation – by Shannon L. M. Dahl, Ph.D., co-founder and vice president, Technology and Pipeline Development, Humacyte – summarized U.S. preclinical data of the company’s investigational bioengineered vessel technology, which is being developed for use as the first off-the-shelf, human-derived, artificial blood vessel. The presentation’s title was ‘Preclinical Dataset Supports Initiation of Clinical Studies for Bioengineered Vascular Access Grafts.’ Co-authors were: Jeffrey H. Lawson, M.D., Ph.D.; Heather L. Prichard, Ph.D.; Roberto J. Manson, M.D.; William E.Tente, M.S.; Alan P. Kypson, M.D.; Juliana L. Blum, Ph.D.; and Laura E. Niklason, M.D., Ph.D.

Potential Of Investigational Bioengineered Vessels Explored In Pre-Clinical Studies

These U.S. preclinical data suggest that the investigational bioengineered vessel may be associated with lowered vessel clotting and incorporation with animal model tissues. This investigational technology is being developed with the goal of pursuing approval for use in patients with chronic kidney disease, a major global health problem affecting 26 million Americans[i] and around 40 million people in the European Union (EU).[ii] Individuals who progress to end-stage renal disease (ESRD) require renal replacement therapy (hemodialysis or kidney transplant); more than 380,000 patients currently require hemodialysis in the U.S.,[iii] and some 250,000 patients require hemodialysis or have had kidney transplants in the EU.[iv]

In ESRD patients, synthetic vascular grafts are prone to wall thickening, which results in graft clotting. Such clotting is the major cause of graft failures. As a result, ESRD patients experience frequent hospitalization and re-operation. The investigational bioengineered vessels, if successfully developed and approved by regulatory authorities, could offer the potential for significant cost savings to the healthcare system if approved for use in patients who require vascular access for ESRD. These investigational bioengineered vessels represent a research and development milestone in the field of vascular tissue engineering, as this technology could have the potential to help reduce or avoid surgical interventions and hospitalizations for patients with ESRD.

First Off-the-Shelf Investigational Bioengineered Vessel In Clinical Studies

“In the preclinical studies described, our investigational bioengineered vessels were repopulated with cells and remodeled like living tissue in the animal model,” said Dr. Dahl. “These investigational bioengineered vessels are produced using donated human vascular cells and then go through a process that is intended to decellularize the investigational vessels to remove the donor identity from the newly created vessels. This process is designed to produce investigational human grafts with the potential to be implanted into any patient at the time of medical need, enabling our investigational product to become the first truly off-the-shelf engineered graft to have moved into clinical evaluation. Demonstrating safety and performance in patients with end-stage renal disease could set the stage for follow-on development of our technology in other vascular procedures, such as replacement or bypass of diseased vessels, of vessels damaged by trauma, or for other vascular procedures.”

In 2012, Humacyte submitted an Investigational New Drug (IND) application to the U.S. Food and Drug Administration to conduct a multi-center U.S. clinical trial, involving up to 20 patients across three sites. In this trial, which will assess safety and performance of the investigational bioengineered vessels to provide vascular access for hemodialysis in ESRD patients, the first investigational bioengineered vessel was implanted in the arm of a kidney dialysis patient at Duke University Hospital in June, 2013.

European studies are already underway; as part of a multi-center study in Poland, the first patients were implanted with the investigational vessels in December 2012 and the vessels were first used for hemodialysis in February 2013. The primary endpoints of the study in Poland are safety, tolerability, and patency, to be examined at each visit within the first six months after graft implantation (see clinicaltrials.gov).

Studies Planned in Additional Patient Populations

Humacyte also will carry out a study in Poland to test safety and performance of the investigational bioengineered vessel as an above-knee bypass graft in patients with peripheral arterial disease (PAD). The study began in October of this year.

First-in-human interim study results for the investigational bioengineered vessel technology from Humacyte will be presented on Wednesday, November 20, 2013, at the American Heart Association Scientific Sessions (abstract) in Dallas, TX.

About Investigational Bioengineered Blood Vessels

The Humacyte investigational bioengineered blood vessels are manufactured in a novel bioreactor system. The investigational bioengineered vessels go through a process of decellularization, which is designed to render vessels potentially non-immunogenic and implantable into any patient. These investigational bioengineered vessels are designed to be stored for up to 12 months under standard refrigerated conditions, including, if successfully developed and approved, on-site in hospitals. Subject to receipt of regulatory approval, these properties could make the investigational bioengineered vessels readily available to surgeons and patients, and could eliminate the wait for vessel production or shipping. Data from studies of the investigational bioengineered vessels in large animal models reflect resistance to thickening for up to one year, and the early human studies that are now underway will provide safety and performance  data in patients to support a future application for regulatory approval.

About Humacyte

Humacyte, Inc., a privately held company founded in 2005, is a medical research, discovery and development company with clinical and pre-clinical stage investigational products. Humacyte is primarily focused on developing and commercializing a proprietary novel technology based on human tissue-based products for key applications in regenerative medicine and vascular surgery.  The company uses its innovative and proprietary platform technology to engineer human, extracellular matrix-based tissues that are designed be shaped into tubes, sheets, or particulate conformations, with properties similar to native tissues. These are being developed for potential use in many specific applications, with the goal to significantly improve treatment outcomes for a variety of patients, including those with vascular disease and those requiring hemodialysis. The company’s proprietary technologies are designed to result in off-the-shelf products that, once approved, can be utilized in any patient. The company web site is www.humacyte.com.

Forward-Looking Statement

Information in this news release contains “forward-looking statements” about Humacyte. These statements, including statements regarding management’s projections relating to future results and operations, are based on, among other things, management’s views, assumptions and estimates, developed in good faith, all of which are subject to known and unknown factors that may cause actual results, performance or achievements, or industry results, to differ materially from those expressed or implied by such forward-looking statements.

# # #


[iv]http://www.ekha.eu/usr_img/info/factsheet.pdf

SOURCE

From: Gail Thornton <gail@westmillconsulting.com>
Reply-To: Gail Thornton <gail@westmillconsulting.com>
Date: Wed, 13 Nov 2013 16:47:00 -0800 (PST)
To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>
Subject: American Society of Nephrology Kidney Week – Humacyte Press Release

 

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Subtitle: The Balance of Nitric Oxide, Peroxinitrite, and NO donors in Maintenance of Renal Function

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

The Nitric Oxide and Renal is presented in FOUR parts:

Part I: The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide

Part II: Nitric Oxide and iNOS have Key Roles in Kidney Diseases

Part III: The Molecular Biology of Renal Disorders: Nitric Oxide

Part IV: New Insights on Nitric Oxide donors 

Conclusion to this series is presented in

The Essential Role of Nitric Oxide and Therapeutic NO Donor Targets in Renal Pharmacotherapy

Evolution of kidney function

The Emergence of  a Mammalian Kidney as Subterranian Life Crawls from Sea to Land
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.

The Metanephric kidney 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. Thus, mammals produce large amounts of nephric filtrate but are able to reabsorb most of this in the tubules. 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 homoisotherms 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.

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 distal acting 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 functioning of a countercurrent multiplier system with a descending limb, Loop of Henley, and ascending limb.

The glomerulus is a dense ball of capillaries (glomerular capillaries) that branches from the afferent arteriole that enters the nephron. Because blood in the glomerular capillaries is under high pressure, substances in the blood that are small enough to pass through the pores (fenestrae, or endothelial fenestrations) in the capillary walls are forced out and into the encircling glomerular capsule. The glomerular capsule is a cup-shaped body that encircles the glomerular capillaries and collects the material (filtrate) that is forced out of the glomerular capillaries. The filtrate collects in the interior of the glomerular capsule, the capsular space, which is an area bounded by an inner visceral layer (that faces the glomerular capillaries) and an outer parietal layer.

The glomerular filtrate passes into the proximal convoluted tubule (PCT),  a winding tube in the renal cortex.  The PCT is mitochondria roch and has a high-energy yield.  The large surface area of these cells support their functions of reabsorption and secretion. The filtrate passes down the descending tubule and reaches the Loop of Henle. The  loop is shaped like a hairpin and consists of a descending limb that drops into the renal medulla and an ascending limb that rises back into the renal cortex.  The distal convoluted tubule (DCT) coils within the renal cortex and empties into the collecting duct.   In the final portions of the DCT and the collecting duct, there are cells that respond to the hormones aldosterone and antidiuretic hormone (ADH), and there are cells that secrete H+ in an effort to maintain proper pH.

The juxtaglomerular apparatus (JGA) is an area of the nephron where the afferent arteriole and the initial portion of the distal convoluted tubule are in close contact. Here, specialized smooth muscle cells of the afferent arteriole, called granular juxtaglomerular (JG) cells, act as mechanoreceptors that monitor blood pressure in the afferent arteriole. In the adjacent distal convoluted tubule, specialized cells, called macula densa, act as chemoreceptors that monitor the concentration of Na+ and Cl in the urine inside the tubule. Together, these cells help regulate blood pressure and the production of urine in the nephron.

The operation of the human nephron consists of three processes:

  • Glomerular filtration
  • Tubular reabsorption
  • Tubular secretion

The net filtration pressure (NFP) determines the quantity of filtrate that is forced into the glomerular capsule. The NFP, estimated at about 10 mm Hg, is the sum of pressures that promote filtration less the sum of those that oppose filtration. The following contribute to the NFP:

  • The glomerular hydrostatic pressure (blood pressure in the glomerulus) promotes filtration.
  • The glomerular osmotic pressure  is created as a result of the movement of water and solutes out of the glomerular capillaries. The increase in the concentration of solutes in the glomerular capillaries draws into the glomerular capillaries.
  • The capsular hydrostatic pressure inhibits filtration. This pressure develops as water collects in the glomerular capsule.

As the filtrate flows through the glomerulus into the descending limb, glucose is reabsorbed  to a threshhold maximum, and H+ is converted by the carbonic anhydrase  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 reabsorption of most substances from the tubule to the interstitial fluids requires a membrane-bound transport protein that carries these substances across the tubule cell membrane by active transport. When all of the available transport proteins are being used, the rate of reabsorption reaches a transport maximum (Tm), and substances that cannot be transported are lost in the urine.

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.

The renal artery for each kidney enters the renal hilus and successively branches into segmental arteries and then into interlobar arteries, which pass between the renal pyramids toward the renal cortex. The interlobar arteries then branch into the arcuate arteries, which curve as they pass along the junction of the renal medulla and cortex. Branches of the arcuate arteries, called interlobular arteries, penetrate the renal cortex, where they again branch into afferent arterioles, which enter the filtering mechanisms, or glomeruli, of the nephrons.

Summary

The criticality of renal function is traced to the emergence of animal forms from the sea to land, and its evolutionary change is recapitulated in the embryo.  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 addition, we have noted the importance of oxidative stress and modifications in mitochondrial function in oncogenesis related to a reliance on aerobic glycolysis to support both energy and synthetic activities in growth and proliferation of the “cancer” cell, that becomes more like a cancer “prototype” than its forebears.  In this discussion we pay attention to kidney function, and what follows is the adaptive role of NO and NO donors. This is an extension of a series of posts on NO and NO related disorders.

Frontal section through the kidney

Frontal section through the kidney (Photo credit: Wikipedia)

Structures of the kidney: Renal pyramid Interl...

Structures of the kidney: Renal pyramid Interlobar artery Renal artery Renal vein Renal hylum Renal pelvis Ureter Minor calyx Renal capsule Inferior extremity Superior extremity Interlobar vein Nephron Renal sinus Major calyx Renal papilla Renal column (no distinction for red/blue (oxygenated or not) blood, arteriole is between capilaries and larger vessels) (Photo credit: Wikipedia)

Distribution of blood vessels in cortex of kid...

Distribution of blood vessels in cortex of kidney. (Although the figure labels the efferent vessel as a vein, it is actually an arteriole.) (Photo credit: Wikipedia)

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

English: Nephron, Diagram of the urine formation. The number inside tubular urine concentration in mOsm/l – when ADH acts  (Photo credit: Wikipedia)

Anatomy of the Kidneys. CliffsNotes. www.cliffsnotes.com/…/Anatomy-of-the-Kidneys

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Reporter: Aviva Lev-Ari, PhD, RN

 

Stroke and Bleeding in Atrial Fibrillation with Chronic Kidney Disease

Original Article

Stroke and Bleeding in Atrial Fibrillation with Chronic Kidney Disease

J.B. Olesen and Others

The prevalence of both atrial fibrillation and chronic kidney disease increases with age. The prevalence of atrial fibrillation is 2.3% among persons 40 years of age or older and 5.9% among those 65 years of age or older, and the prevalence of end-stage renal disease increases from approximately 3.5% among persons 45 to 64 years of age to nearly 6% among those 75 years of age or older. Many patients have both disorders, and the number of such patients is increasing, owing in part to the aging population and the improved survival in both diseases.

Clinical Pearls

  What is the effect of chronic kidney disease on the risk of stroke?

The U.S.-based Renal Data System has reported that chronic kidney disease increases the risk of stroke by a factor of 3.7, and end-stage renal disease (i.e., disease requiring renal-replacement therapy) increases the risk by a factor of 5.8. Atrial fibrillation increases the risk of stroke by a factor of 5, and chronic kidney disease increases the risk of stroke among patients without atrial fibrillation.

  What is a CHA2DS2-VASc score?

This study evaluated the risk of stroke or systemic thromboembolism, with adjustment for CHA2DS2-VASc risk factors. The CHA2DS2-VASc score extends the CHADS2 algorithm to include additional nonmajor risk factors for stroke, including vascular disease (V), age between 65 to 74 years (A), and female gender (sex category or Sc).

Morning Report Questions

Q. What were the results of this study of patients with atrial fibrillation and chronic kidney disease with respect to risk of stroke or systemic embolism? 

A. This study demonstrated that warfarin treatment was associated with a significantly decreased risk of stroke or systemic thromboembolism overall and among patients requiring renal-replacement therapy, and with a nonsignificantly decreased risk among patients with non–end-stage chronic kidney disease. In an analysis that compared all patients who had any renal disease with those who had no renal disease, warfarin decreased the risk of stroke or systemic thromboembolism (hazard ratio, 0.76; 95% CI, 0.64 to 0.91; P=0.003), as did warfarin plus aspirin (hazard ratio, 0.74; 95% CI, 0.56 to 0.98; P=0.04). Aspirin alone was associated with an increased risk of stroke or systemic thromboembolism overall and among patients who had any renal disease, as compared with those who had no renal disease (hazard ratio, 1.17; 95% CI, 1.01 to 1.35; P=0.04).

Table 3. Hazard Ratios for Stroke or Systemic Thromboembolism.

Q. How did the risk of bleeding differ among patients with and without kidney disease? 

A. The risk of bleeding was higher among patients with non–end-stage chronic kidney disease and among patients requiring renal-replacement therapy as compared to patients without renal disease, and treatment with warfarin, aspirin, or both incrementally increased this risk. Among all patients who had any renal disease, as compared with those who had no renal disease, there was an increased risk of bleeding with warfarin (hazard ratio, 1.33; 95% CI, 1.16 to 1.53; P<0.001), aspirin (hazard ratio, 1.17; 95% CI, 1.02 to 1.34; P=0.03), and warfarin plus aspirin (hazard ratio, 1.61; 95% CI, 1.32 to 1.96; P<0.001). Among patients with non–end-stage chronic kidney disease, the risk of bleeding increased with a higher dose of loop diuretics. The risk of bleeding was highest among patients with chronic glomerulonephritis and lowest among those with chronic tubulointerstitial nephropathy.

Table 4. Hazard Ratios for Bleeding.

 Original article

Stroke and Bleeding in Atrial Fibrillation with Chronic Kidney Disease

Jonas Bjerring Olesen, M.D., Gregory Y.H. Lip, M.D., Anne-Lise Kamper, M.D., D.M.Sc., Kristine Hommel, M.D., Lars Køber, M.D., D.M.Sc., Deirdre A. Lane, Ph.D., Jesper Lindhardsen, M.D., Gunnar Hilmar Gislason, M.D., Ph.D., and Christian Torp-Pedersen, M.D., D.M.Sc.

N Engl J Med 2012; 367:625-635  August 16, 2012

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Reporter: Aviva Lev-Ari, PhD, RN

The combined creatinine–cystatin C equation performed better than equations based on either of these markers alone and may be useful as a confirmatory test for chronic kidney disease. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.)

Estimating Glomerular Filtration Rate from Serum Creatinine and Cystatin C

Lesley A. Inker, M.D., Christopher H. Schmid, Ph.D., Hocine Tighiouart, M.S., John H. Eckfeldt, M.D., Ph.D., Harold I. Feldman, M.D., Tom Greene, Ph.D., John W. Kusek, Ph.D., Jane Manzi, Ph.D., Frederick Van Lente, Ph.D., Yaping Lucy Zhang, M.S., Josef Coresh, M.D., Ph.D., and Andrew S. Levey, M.D. for the CKD-EPI Investigators

N Engl J Med 2012; 367:20-29  July 5, 2012

BACKGROUND

Estimates of glomerular filtration rate (GFR) that are based on serum creatinine are routinely used; however, they are imprecise, potentially leading to the overdiagnosis of chronic kidney disease. Cystatin C is an alternative filtration marker for estimating GFR.

METHODS

Using cross-sectional analyses, we developed estimating equations based on cystatin C alone and in combination with creatinine in diverse populations totaling 5352 participants from 13 studies. These equations were then validated in 1119 participants from 5 different studies in which GFR had been measured. Cystatin and creatinine assays were traceable to primary reference materials.

RESULTS

Mean measured GFRs were 68 and 70 ml per minute per 1.73 m2 of body-surface area in the development and validation data sets, respectively. In the validation data set, the creatinine–cystatin C equation performed better than equations that used creatinine or cystatin C alone. Bias was similar among the three equations, with a median difference between measured and estimated GFR of 3.9 ml per minute per 1.73 m2 with the combined equation, as compared with 3.7 and 3.4 ml per minute per 1.73 m2 with the creatinine equation and the cystatin C equation (P=0.07 and P=0.05), respectively. Precision was improved with the combined equation (interquartile range of the difference, 13.4 vs. 15.4 and 16.4 ml per minute per 1.73 m2, respectively [P=0.001 and P<0.001]), and the results were more accurate (percentage of estimates that were >30% of measured GFR, 8.5 vs. 12.8 and 14.1, respectively [P<0.001 for both comparisons]). In participants whose estimated GFR based on creatinine was 45 to 74 ml per minute per 1.73 m2, the combined equation improved the classification of measured GFR as either less than 60 ml per minute per 1.73 m2 or greater than or equal to 60 ml per minute per 1.73 m2 (net reclassification index, 19.4% [P<0.001]) and correctly reclassified 16.9% of those with an estimated GFR of 45 to 59 ml per minute per 1.73 m2 as having a GFR of 60 ml or higher per minute per 1.73 m2.

CONCLUSIONS

The combined creatinine–cystatin C equation performed better than equations based on either of these markers alone and may be useful as a confirmatory test for chronic kidney disease. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.)

Supported by grants (UO1 DK 053869, UO1 DK 067651, and UO1 DK 35073) from the National Institute of Diabetes and Digestive and Kidney Diseases.

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

This article was updated on July 5, 2012, at NEJM.org.

We thank Dr. Aghogho Okparavero for providing assistance with communications and manuscript preparation. (Additional acknowledgments are provided in the Supplementary Appendix.)

SOURCE INFORMATION

From Tufts Medical Center, Boston (L.A.I., C.H.S., H.T., Y.L.Z., A.S.L.); the University of Minnesota, Minneapolis (J.H.E.); the University of Pennsylvania School of Medicine, Philadelphia (H.I.F.); the University of Utah, Salt Lake City (T.G.); National Institutes of Health, Bethesda, MD (J.W.K.); Johns Hopkins University, Baltimore (J.M., J.C.); and Cleveland Clinic Foundation, Cleveland (F.V.L.).

Address reprint requests to Dr. Inker at the Division of Nephrology, Tufts Medical Center, 800 Washington St., Box 391, Boston, MA 02111, or at linker@tuftsmedicalcenter.org.

Additional investigators in the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) are listed in the Supplementary Appendix, available at NEJM.org.

N Engl J Med 2012; 367:20-29

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Risks of Hypoglycemia in Diabetics with Chronic Kidney Disease (CKD)

Reporter: Aviva Lev-Ari, PhD, RN

Risks of Hypoglycemia in Diabetics with CKD

By Mark Abrahams, MD

Reviewed by Loren Wissner Greene, MD, MA (Bioethics), Clinical Associate Professor of Medicine, NYU School of Medicine, New York, NY

Published: 03/13/2012

 http://www.medpagetoday.com/resource-center/diabetes/Risks-Hypoglycemia-Diabetics-CKD/a/31634

According to the National Institutes of Health (NIH), approximately 40% of adults with diabetes have some degree of chronic kidney disease (CKD).1 That’s a lot of patients—perhaps more than one might think.

What should we be doing differently for these patients? Sure, they should be getting an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin receptor blocker (ARB) for renoprotection, and blood pressure and lipids should be aggressively managed, but how does (or should) our approach to managing their antidiabetic therapy change?

We might consider taking a more aggressive approach to their glycemic control. In clinical trials, tight glycemic control has been shown to be the primary determinant of decreased microvascular complications.1 However, once we’ve decided how aggressively to manage glycemia, the choice of which antidiabetic to use (and how to dose it) is especially important in these patients.

Unfortunately, when the therapeutic strategy is to maximize glycemic control, the risk of hypoglycemia also increases – in both frequency and severity.2 Patients taking oral antidiabetics that are primarily eliminated by the kidneys are particularly susceptible.1 Furthermore, it should be noted that older patients are also at higher risk.3

Dosing errors are common in CKD patients and can cause poor outcomes.3 Drugs cleared renally should be dose-adjusted based on creatinine clearance or estimated glomerular filtration rate (eGFR). Dose reductions, lengthening of the dosing interval, or both may be required.3

As metformin is nearly 100% renally excreted, it is contraindicated in a number of patients: when serum creatinine is higher than 1.5 mg/dL in men or 1.4 mg/dL in women, in patients older than 80 years, or in patients with chronic heart failure. The primary concern here is that other hypoxic conditions (e.g., acute myocardial infarction, severe infection, respiratory disease, liver disease) may increase the risk of lactic acidosis. Because of this danger, and despite the fact that metformin is usually the recommended first-line treatment for type 2 diabetes, one should use caution when considering metformin in patients with renal impairment.3

Similarly, sulfonylureas should be used with care in diabetics with CKD. The clearance of both sulfonylureas and their metabolites is highly dependent on kidney function. As such, severe and sustained episodes of hypoglycemia due to sulfonylurea use have been described in dialysis patients.2

Regardless of which antidiabetic agent is selected, HbA1c and kidney function should be regularly monitored and the antidiabetic regimen appropriately adjusted. As patients with type 2 diabetes tend to progress over time, most will require a combination of agents to achieve desired glycemic control. These combinations should be chosen carefully in patients with CKD.1

Finally, awareness of and screening for renal impairment in diabetics is a necessary precursor to successful intervention. In these patients, CKD is underdiagnosed and undertreated, and awareness of the disease is low among providers and patients alike.1

Early detection of disease via eGFR or urinary albumin excretion can lead to timely, evidence-based intervention and help prevent or delay progression of CKD. The benefit? Improved kidney and cardiovascular outcomes, and lower associated costs.1

References:

  1. Bakris GL. Recognition, Pathogenesis, and Treatment of Different Stages of Nephropathy in Patients With Type 2 Diabetes MellitusMayo Clin Proc. 2011;86:444-456.
  2. Cavanaugh KL. Diabetes Management Issues for Patients With Chronic Kidney DiseaseClin Diab. 2007;25:90-97.
  3. Munar MY, et al. Drug Dosing Adjustments in Patients With Chronic Kidney Disease. Am Fam Physician. 2007;75:1487-1496.

 

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