Effect of chronic renal failure on nitric oxide metabolism

Effect of Chronic Renal Failure on Nitric Oxide Metabolism N.D. Vaziri, MD, MACP ● Chronic renal failure (CRF) is associated with hypertension, endothelial dysfunction, and a strong propensity for arteriosclerotic cardiovascular disease. Nitric oxide (NO) is an endogenous modulator with diverse biological functions. Chronic inhibition of NO synthases (NOS) has been shown to cause hypertension and vasculopathy. In light of these considerations, numerous studies have explored the effect of CRF on NO metabolism with the assumption that NO deficiency may be involved in the pathogenesis of cardiovascular and other consequences of uremia. The purpose of this review is to provide a brief overview of the effect of CRF on (1) the bioavailability of NO substrate, L-arginine; (2) the expression of NOS isoforms in the relevant organs; (3) the interaction of NO with reactive oxygen species that are known to be increased in CRF, and (4) the accumulation of uremic inhibitors of NOS. © 2001 by the National Kidney Foundation, Inc. INDEX WORDS: Chronic renal failure (CRF); uremia; nitric oxide (NO); nitric oxide synthases (NOS); nitric oxide synthase (NOS) inhibitors; oxidative stress; reactive oxygen species (ROS); hypertension; cardiovascular disease (CVD); arteriosclerosis.

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NCE KNOWN AS a toxic industrial and environmental pollutant, nitric oxide (NO) has emerged as a major endogenous intercellular and intracellular signaling molecule with vast biological functions. For instance, NO serves as a neurotransmitter, a potent endogenous vasodilator, an inhibitor of renal tubular sodium reabsorption, a modulator of renal hemodynamics and tubuloglomerular feedback, and an innate immune affector. In addition, NO inhibits leukocyte adhesion; smooth muscle cell, fibroblast, and macrophage proliferation; and matrix protein accumulation.1 NO is produced by the stereo-specific oxidation of the terminal guanidino group of Larginine in the presence of oxygen and NADPH by a family of enzymes known as NO synthases (NOS). Three isoforms of NOS have thus far been identified: NOS I, also known as neuronal NOS (nNOS); NOS II, otherwise known as inducible NOS (iNOS); and NOS III, also known as endothelial NOS (eNOS). All three NOS isoforms are constitutively expressed at relatively low levels at basal condition. In addition, under certain pathological conditions, iNOS expres-

From the Division of Nephrology and Hypertension, Departments of Medicine, Physiology and Biophysics, University of California, Irvine, CA. Address reprints requests to N.D. Vaziri, MD, MACP, Division of Nephrology and Hypertension, UCI Medical Center, 101 The City Drive, Orange, CA 92868. E-mail: [email protected] © 2001 by the National Kidney Foundation, Inc. 0272-6386/01/3804-0114$35.00/0 doi:10.1053/ajkd.2001.27409 S74

sion can be induced in massive quantities in leukocytes, macrophages, endothelial cells, vascular smooth muscle cells, hepatocytes, and other cell types by endotoxin and various cytokines, such as interleukin-1 (IL-1), tumor necrosis factor, and interferons. This is best exemplified by septic shock, wherein production of massive quantities of iNOS-derived NO contributes to the decrease in peripheral vascular resistance, hemodynamic instability, and hypotension. The enzymatic activity of NOS is dependent on binding with calmodulin, which can only occur as a calcium-calmoduline complex with eNOS and nNOS. Thus, the enzymatic activity of eNOS and nNOS is regulated by changes in cytoplasmic [Ca2⫹]. In contrast, calmodulin binding to iNOS does not require Ca2⫹ and, as such, iNOS activity is calcium independent. Hypertension and atherosclerosis are among the most common complications of chronic renal failure (CRF). In fact, the risk of cardiovascular mortality in patients with end-stage renal disease (ESRD) is significantly greater than that in the general population. Several factors, including hypertension, dyslipidemia, hyperhomocysteinemia, oxidative stress, and vascular calcification, contribute to the pathogenesis of atherosclerotic cardiovascular disease in the ESRD population. Experimental NO deficiency, induced by the chronic administration of NOS inhibitors, has been shown to promote atherosclerosis and accelerate neointimal formation in hypercholesterolemic rabbits.2,3 Several factors contribute to the antiatherogenic actions of NO. These factors include NO-mediated inhibitions of fibroblast

American Journal of Kidney Diseases, Vol 38, No 4, Suppl 1 (October), 2001: pp S74-S79

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and vascular smooth muscle cell proliferation, matrix protein accumulation, leukocyte adhesion, platelet adhesion and activation, and reduced endothelial hyperpermeability—events that are central to the atherogenic process. The potential role of NO deficiency in the pathogenesis of atherosclerosis is evidence that proatherogenic conditions, such as diabetes, hypertension, hyperlipidemia, chronic tobacco use, ESRD, and so forth, are accompanied by endothelial dysfunction and depressed basal and stimulated NO release in atherosclerotic vessel.4 Total body NO production, as discerned from the urinary excretion of stable NO metabolites (nitrate ⫹ nitrite ⫽ NO␹), is significantly reduced in the animals with CRF.5-8 Similarly, the total body production of NO␹ and, hence, NO is significantly reduced in ESRD patients.9-11 CRF can adversely affect NO metabolism by several mechanisms. The present article is intended to provide an overview of the known effects of CRF on NO metabolism.

In addition to the alterations in renal arginine biosynthesis and catabolism noted above, a reduction in protein intake associated with uremiainduced anorexia can diminish exogenous supply of L-arginine. Finally, losses of amino acids, including L-arginine during dialysis, can further impact the available L-arginine supply. Thus, CRF affects several aspects of arginine metabolism. In fact, plasma L-arginine concentration has been variably reported as being either normal15 or reduced16 in dialysis-dependent ESRD patients.

EFFECT OF CRF ON L-ARGININE AVAILABILITY

Kidney Using an immunohistochemical method, Aiello et al8 were the first to demonstrate a significant and progressive decline in immunodetectable iNOS but not in eNOS in the remnant kidneys of rats with renal mass reduction accomplished by subtotal renal infarction technique. The downregulation of iNOS observed by Aiello et al8 was subsequently confirmed by Vaziri et al5 using Western blot in rats with renal mass reduction accomplished by excisional 5/6 nephrectomy. In addition, the latter study showed significant downregulation of eNOS in the remnant kidney.5 Downregulations of both eNOS and iNOS expression in the remnant kidneys of CRF rats shown by Vaziri et al5 was recently confirmed by Kim et al7 in this model. Moreover, Roczniak et al17 have recently shown marked downregulation of nNOS mRNA and protein expressions in the remnant kidneys of rats with CRF produced by 5/6 nephrectomy. Thus, the available data from several groups point to marked downregulation of all NOS isoforms in the remnant kidneys of animals with CRF. The reduction in renal tissue NOS isoforms in animals with CRF may contribute to the pathogenesis of the associated hypertension, altered renal tubular transport, and progressive loss of renal

The available pool of L-arginine is primarily derived from its dietary intake and endogenous biosynthesis. Normally filtered citruline is converted to arginine in the proximal tubular epithelial cells that contain arginine synthase complex.12 Thus, the kidney is a major source of endogenous arginine production. Advanced CRF can theoretically affect both dietary intake (anorexia) and endogenous production of arginine by the kidney (reduction in renal mass). In fact, arginine synthase complex activity is reportedly reduced in rats with advanced CRF.13 It should be noted that production of arginine by remnant kidney may be preserved for extended periods.14 This is accomplished by a combination of increased single nephron GFR, proximal tubular hypertrophy, and increased plasma citruline concentration, which helps to maintain the filtered load of citruline despite a marked reduction of the glomerular filtration rate. However, renal biosynthesis of arginine is inevitably curtailed when ESRD is reached. The reduction of renal arginine biosynthesis with progressive loss of renal function may be, in part, offset by diminished arginine conversion to ornithine by arginase, which is abundantly present in the kidney.

EFFECT OF CRF ON EXPRESSION OF NOS ISOFORMS

In an attempt to discern the mechanism of altered NO metabolism and endothelial dysfunction, several groups of investigators have examined expression of various NOS isoforms in different organs of animals with experimental CRF. The results of these studies for each of the tested organs are summarized below.

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function. In support of the latter proposition, several studies have shown significant benefit with long-term administration of low-dose Larginine in CRF rats. Vascular Tissue Using immunohistochemical studies, Aiello et al8 have shown a significant increase in NOS activity and protein expression in the aortas of rats rendered uremic using the subtotal renal infarction method. In contrast, using a CRF model produced by 5/6 excisional nephrectomy, Vaziri et al5 showed significant downregulations of eNOS and iNOS using a Western blot in the thoracic aorta of CRF rats. Downregulations of aorta eNOS and iNOS shown by Vaziri et al5 were recently confirmed by Kim et al7 in 5/6 nephrectomized rats. The reason for the disparity between the results of the earlier study by Aiello et al8 and those of Vaziri et al5 and Kim et al7 is not entirely clear. It should be noted, however, that renal mass reduction by ligation of branches of renal artery in uninephrectomized animals causes CRF, which is associated with severe hypertension. In contrast, CRF induced by surgical resection of the upper and lower thirds of the kidney is associated with mild to moderate hypertension. Given the known stimulatory effect of increased pressure and shear stress on eNOS expression,18,19 the observed upregulation of vascular eNOS isoform found in the former study may have been due to the more severe hypertension overriding the inhibitory effect of CRF in the renal infarction model. In an attempt to test this hypothesis, we recently modified our resectional nephrectomy model by producing punctuated scars on the surface of the remnant kidney using electrocautery. This technique produced a CRF model with severe hypertension and a compensatory upregulation of eNOS in the aorta and left ventricle (unpublished data), thus replicating the findings of Aiello et al.8 Brain Ye et al20 have demonstrated a significant increase in norepinephrine turnover in the posterior hypothalamic nuclei, locus coeruleus, paraventricular nuclei, and the rostral ventral medulla in CRF rats. This finding was coupled with a significant increase in NO␹ content and nNOS, mRNA abundance in all tested brain nuclei of

N.D. VAZIRI

CRF rats. Given the fact that nNOS-derived NO in the brain exerts an inhibitory action on central sympathetic outflow, the authors concluded that upregulation NO production in the brain of CRF animals may mitigate the severity of hypertension.20 Possible Mechanisms of Altered NOS Expression in CRF The mechanism of CRF-associated downregulation of NOS isoforms in the remnant kidney is uncertain. However, upregulations of transforming growth factor-␤ (TGF-␤) and platelet-derived growth factor (PDGF) in the damaged kidney have been considered as a potential reason for depressed iNOS, because TGF-␤ and PDGF are known to inhibit cytokine-mediated induction of iNOS in cultured rat mesangial cells. In addition, increased endothelin-1 (ET-1) has also been proposed as a possible mediator of the observed downregulation of iNOS in the remnant kidney, based on the observation that ET-1 can inhibit cytokine-mediated induction of iNOS in both cultured mesangial and epithelial cells. The potential role of ET-1 is supported by the following observations. (1) Local production of ET-1 by proximal tubules increases significantly coincident with development of proteinuria in animals with renal mass reduction. (2) The addition of specific ETA receptor blocker reverses the ET-1–mediated inhibition of iNOS induction by cytokines in cultured mesangial and epithelial cells.21 However, it is not clear whether or not the constitutively expressed iNOS in the renal tubular epithelial cells and vascular smooth muscle is regulated by cytokines. If not, the inhibitory action of TGF-␤, PDGF, and ET-1 on the cytokine-mediated classic pathway of iNOS induction may be of less relevance in this case. In search for a possible alternative explanation for suppressed renal and vascular tissue NOS expression in CRF, we explored the role of excess parahormone (PTH) and the resultant elevation of resting cytoplasmic [Ca2⫹].5 This was based on the observation that excess PTH downregulates expression of several other genes encoding for a number of enzymes and receptors in CRF animals. To test this possibility, we compared kidney and aorta eNOS and iNOS protein expressions in CRF rats, parathyroidectomized CRF rats (supplemented with calcium

NO METABOLISM IN CHRONIC RENAL FAILURE

citrate to maintain normal plasma Ca concentration), and sham-operated control animals. Parathyroidectomy reversed CRF-induced elevation of resting cytoplasmic [Ca2⫹] and normalized eNOS and iNOS protein expression in the remnant kidney and thoracic aorta. In an attempt to dissect the effect of excess PTH from that of the associated increase in cytoplasmic [Ca2⫹], we included a group of CRF animals that were treated with a long-acting dihydropyridine calcium channel blocker. As with parathyroidectomy, calcium channel blockade improved urinary NO␹ excretion and increased both vascular and renal tissue iNOS and eNOS expression.5 These observations provided strong evidence for contribution of chronic PTH-mediated elevation of cytoplasmic [Ca2⫹] to the pathogenesis of the associated downregulation of renal and vascular eNOS and iNOS expression. With regards to brain nNOS, in a recent study, Ye et al22 showed that upregulation of the brain nNOS is accompanied by upregulation of IL-1␤ mRNA. They further showed that administration of specific antibody to IL-1␤ into the lateral ventricle raised blood pressure, increased norepinephrine secretion, and suppressed nNOS mRNA expression in both CRF and normal control rats. They, therefore, concluded that CRF-associated upregulation of nNOS in the brain nuclei is mediated by IL-1␤.22 ACCUMULATION OF NATURALLY OCCURRING NOS INHIBITORS IN CRF

Advanced CRF results in accumulation of naturally occurring compounds that are capable of inhibiting NOS activity and NO production. The functional relevance of this phenomenon is shown by the recent findings by Xiao et al23 that the addition of 20% plasma from hemodialysisdependent and peritoneal dialysis patients to the culture medium significantly inhibits NO production by cultured human and bovine endothelial cells. The inhibitory action of uremic plasma could be overcome by the addition of excess L-arginine. Interestingly, the NOS inhibitory action of uremic plasma persisted after hemodialysis in this study. Several naturally occurring compounds, such as asymmetrical dimethyl Larginine (ADMA), the L-arginine analogue, NGmonomethyl-L-arginine (LNMMA), methylguanidine, guanidine, and urea, which accumulate in

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the uremic milieu have been shown to inhibit all or some of the NOS isoforms.15,16,24 It should be noted that some investigators have suggested that the magnitude of the increase in plasma concentration of the uremic NOS inhibitors, such as ADMA, may be insufficient to significantly affect NO production in CRF patients. However, a low to normal L-arginine level, together with a low L-arginine to NOS inhibitor ratio, can work in concert to limit NOS activity in CRF. Consequently, the accumulation of various uremic NOS inhibitors can potentially contribute to uremic hypertension, atherogenic diathesis, impaired immune response, etc. It is of note that, although ADMA, LNMMA, and methylguanidine can nonspecifically inhibit all NOS isoforms, aminoguanidine-related compounds and urea specifically inhibit iNOS. Effect of CRF-Associated Oxidative Stress Oxidative stress occurs when generation of the reactive oxygen species (ROS) exceeds the natural antioxidant capacity of the organism. In the presence of oxidative stress, uncontained ROS are free to attack, denature, or modify structural and functional molecules, such as lipids, proteins, DNA, and various other compounds. Oxidative stress is a well-recognized metabolic consequence of CRF. The presence of oxidative stress in CRF is based on the elevation of the plasma concentration of the lipid peroxidation product melonedialdehyde, depressed antioxidant capacity, and impaired antioxidant enzymes in CRF patients. Increased ROS activity plays an important role in the nonenzymatic production of reactive carbonyl compounds and lipoperoxides, which can, in turn, react with and modify structural and functional proteins, leading to the formation of advanced glycation end products (AGE) and advanced lipoxidation end products (ALE).25 The accumulation of these products undoubtedly contributes to the longterm complications of CRF. ROS avidly react with and inactivate NO and, in the process, produce highly reactive and cytotoxic products, such as peroxynitrite (ONOO) and peroxynitrous acid (ONOOH). Peroxynitrite, in turn, reacts with and modifies various molecules, such as lipids, DNA, and proteins. For instance, peroxynitrite reacts with the tyrosine and cystein residues in protein molecules

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to produce nitrotyrosine and nitrocystein, which are considered to be a footprint of NO interaction with ROS. In addition to these and other harmful biochemical reactions, the oxidation of NO by ROS inevitably results in functional NO deficiency, which can contribute to the pathogenesis and maintenance of hypertension and its longterm consequences. In fact, Vaziri et al26 have demonstrated the role of oxidative stress in the pathogenesis of various forms of hypertension including uremic hypertension, lead-induced hypertension, diet-induced hypertension, aorta coarctation-induced hypertension, and genetic hypertension. In all instances, depressed NO availability was coupled with widespread accumulation of nitrotyrosine in all tested tissues, denoting NO inactivation and sequestration by ROS. Moreover, NO sequestration, hypertension, and reduced NO availability improved with antioxidant therapy or irradication of the source of oxidative stress in these models. In fact, Deng et al27 recently demonstrated heavy accumulation of nitrotyrosine in the brain of CRF rats. Antioxidant therapy ameliorated hypertension and reduced brain nitrotyrosine abundance in these animals. The role of oxidative stress, per se, in the pathogenesis of hypertension was recently proven convincingly by the demonstration that induction of oxidative stress by glutathione depletion can cause severe antioxidant-remediable hypertension in genetically normal, otherwise intact animals.28 Thus, the effects of CRF-associated downregulations of renal and vascular NOS isoforms and accumulation of uremic inhibitors of NOS are compounded by ROS-mediated inactivation of NO. The resultant NO deficiency can contribute to the pathogenesis of hypertension, arteriosclerotic cardiovascular disease, and numerous other long-term complications of CRF. ACKNOWLEDGMENT The author is grateful to Ms Carmen Eaton for her technical assistance with this paper. Because of the mandatory restrictions in the length of the “Reference” section, many important references were regrettably deleted.

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regulation of neuronal nitric oxide synthase in the rat remnant kidney. J Am Soc Nephrol 10:594-560, 1999 18. Vaziri ND, Ni Z, Oveisi F: Upregulation of renal and vascular nitric oxide synthase in young spontaneously hypertensive rats. Hypertension 31:1248-1254, 1998 19. Barton CH, Ni Z, Vaziri ND: Effect of severe aortic banding above the renal arteries on nitric oxide synthase isotype expression. Kidney Int 59:654-661, 2001 20. Ye S, Nosrati S, Campese VM: Nitric oxide modulates the neurogenic control of blood pressure in rats with chronic renal failure. J Clin Invest 99:540-548, 1997 21. Markewitz BA, Michael JR, Kohan DE: Endothelin-1 inhibits the expression of inducible nitric oxide sythase. Am J Physiol 272:L1078-L1083, 1997 22. Ye S, Mozayeni P, Gamburd M, Zhang H, Campese VM: Interleukin-1 beta and neurogenic control of blood pressure in normal rats and rats with chronic renal failure. Am J Physiol (Renal Physiol) 279:F671-F678, 2000 23. Xiao S, Schmidt RJ, Baylis C: Plasma from ESRD patients inhibits nitric oxide synthase activity in cultured

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human and bovine endothelial cells. Miner Electrolyte Metab 25:384-390, 1999 24. Vallance P, Leone A, Calver A, Collier J, Moncada S: Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 339:572-575, 1992 25. Miyata T, Kurokawa K, Van Ypersele De Strihou C: Advanced glycation and lipoxidation end products: Role of reactive carbonyl compounds generated during carbohydrate and lipid metabolism. J Am Soc Nephrol 11:1744-1752, 2000 26. Vaziri ND, Oveisi F, Ding Y: Role of increased oxygen free radical activity in the pathogenesis of uremic hypertension. Kidney Int 53:1748-1754, 1998 27. Deng G, Vaziri ND, Jabbari B, Yan XX: Increasd tyrosine nitration of the brain in chronic renal insufficiency: Reversal by antioxidant therapy. J Am Soc Nephrol (in press) 28. Vaziri ND, Wang XQ, Oveisi F, Rad B: Induction of oxidative stress by glutathione depletion causes severe hypertension in normal rats. Hypertension 36:142-146, 2000