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

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

The criticality of renal function is easily overlooked until significant loss of nephron mass is overtly seen.  The kidneys become acutely and/or chronically dysfunctional in metabolic,  systemic inflammatory and immunological diseases of man.  We have described how the key role that nitric oxide and the NO synthases (eNOS and iNOS) play competing roles in reduction or in the genesis of reactive oxygen  species.  There is  a balance 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 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 37^oC. The NO donors

• sodium nitroprusside (SNP),
• glyceryl trinitrate (GTN), or
• 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.

Interferon a-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 overexpression 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 tubular 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

1.                   superoxide dismutase,
2.                   dimethylthiourea,
3.                   allopurinol, and
4.                    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 inducible nitric oxide synthase (iNOS) and production of high levels of nitric oxide (NO) also contribute to this injury. NO can combine 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, a stable biomarker of ONOO− formation.

1. The kidneys from I-R animals had increased levels of 3-nitrotyrosine-protein adducts compared with control animals
2. L-NIL-treated rats (3 mg/kg) subjected to I-R showed decreased levels of 3-nitrotyrosine-protein adducts.

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

In summary, 3-nitrotyrosine-protein adducts were detected in renal tubules after I-R injury. Selective inhibition of iNOS by L-NIL

• decreased injury,
• improved renal function, and
• decreased apparent ONOO− formation.

Reactive nitrogen species should be considered potential therapeutic targets in the prevention and treatment of renal I-R injury.

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.

Recent studies also suggest involvement of nitric oxide (NO), generated by inducible NO synthase (iNOS), in the pathogenesis of endotoxin-induced renal failure. TNF-a leads to a decrease in glomerular filtration rate (GFR). The present 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.

 

Effect of TNFsRp55 (10 mg/kg IP) on renal function in wild-type mice.

Mice were pretreated with TNFsRp55 for one hour before the administration of 5 mg/kg intraperitoneal endotoxin. GFR (A) and RPF (B) 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. In this review, we discuss the contribution of endothelial and epithelial cells and leukocytes to this inflammatory response. The roles of cytokines/chemokines in the injury and recovery phase are reviewed. The ability of the mouse kidney to be protected by prior exposure to ischemia or urinary tract obstruction is discussed as a potential model to emulate as we search for pharmacologic agents that will serve to protect the kidney against injury.

the 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 fibrosis by 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

• 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.

On the contrary we found 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, our 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 in diabetic nephropathy

Diabetic Nephropathy (DN) poses a major health problem. There is strong evidence for a potential role of the eNOS gene. The aim of this case control study was to investigate 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;

• 68 of these patients had no diabetic nephropathy (group 1) while
• 56 patients exhibited symptoms of diabetic nephropathy (group 2).
• 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).

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±0.220.001**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.

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).

 

Table 2: Tumour necrosis factior 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	17.60 ±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±0.950.02*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

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

1. decreased significantly NO, TNF α, BUN and CrCl while
2. 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 ARF

 

The 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.

RAS	Renal hemodynamics	Sodium balance
	Medullary perfusion
	Pressure-natriuresis
Salt intake	Tubulo-glomerular feedback	Blood pressure
	Tubular sodium reabsorption
Blood pressure	Renal sympathetic activity	Regulation
Extrarenal factors	Intrarenal functions	Physiological roles

 

Role of nitric oxide in renal physiology. RAS, renin-angiotensin system

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 dysoxia is 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. 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 (Figure 1).

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.

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.

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, rapid, 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.
• 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.

•  neither drugs with renal vasodilatory effects (i.e., dopamine, fenoldopam, endothelin receptors blockers, adenosine antagonists) or
• 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. 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.

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

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 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 dysfunction 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 on 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 has been long known, 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 evaulate complex and still incomplete and conflicting conclusions from many studies.  I thus broke the report into three major portions:

1 The kidney and its anatomy, physiology, and ontogeny.

2 The pathological disease variation affecting the kidney

a  a tie in to eNOS 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

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.  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 impofrtant 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 medularry circullation, and the renin-angiotensin-aldosterone system.  Substantial data and multiple patientys with many factors per patient would be need to extract the best model using a supercomputer.

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