Eur J Clin Pharmacol (2006) 62: 123–130DOI 10.1007/s00228-005-0003-0

REVIEW ARTICLE

Chris Baylis

Nitric oxide deficiency in chronic renal disease

Published online: 26 October 2005# Springer-Verlag 2005

Abstract There is clear evidence that chronic inhibition ofnitric oxide synthase (NOS) in animals causes hypertensionand also leads to progressive kidney damage. There is alsoevidence that nitric oxide (NO) deficiency occurs in manwith chronic kidney disease (CRD) and this may contributeto further progression of CRD, to hypertension, and to othercardiovascular complications. There are multiple ways inwhich NO deficiency develops in CRD. At end stage thereare uremic factors in plasma that inhibit L-arginine transportinto cells and this may cause a “net” substrate deficiency.Also, increases occur in endogenous NOS inhibitors, inparticular asymmetric dimethylarginine (ADMA). The in-creased oxidative stress of CRD is likely to be a primarycause of the increased plasma ADMA since the catabolicenzyme, dimethylarginine dimethylaminohydrolase (DDAH)is extremely sensitive to inhibition by oxidants. Animal stud-ies demonstrate a decrease in abundance of the neuronal NOSwithin the injured kidney that correlates with extent of injury.Overall, there is substantial clinical, “in vitro,” and animaldata to suggest that systemic, endothelial, and renal NOdeficiency is a common feature of CRD irrespective of theprimary genesis of the disease. This NO deficiency, which ismultifactorial, contributes to the progressive nature of theCRD and the endothelial dysfunction and associated risk forcardiovascular events. Strategies that reverse NOS inhibitionand/or can boost the ability of the damaged kidney to produceNO might help preserve residual renal function and/or slowdown the rate of progression to end stage.

Keywords Asymmetric dimethylarginine (ADMA) .Renal neuronal nitric oxide synthase . L-arginine transport

Introduction

There is clear evidence from animal studies that chronicnitric oxide synthase (NOS) inhibition leads to hyperten-sion and progressive kidney damage. A moderate level ofchronic NOS inhibition over 2 months in the rat led tomoderate systemic and glomerular hypertension, mild pro-teinuria, and moderate focal and segmental glomerularsclerosis [1]. More complete NOS inhibition led to anaccelerated model of kidney damage with malignant hy-pertension appearing at very high levels of NOS inhibition.The increasing systemic hypertension was associated withglomerular hypertension, which contributed to the in-creased severity of kidney damage at high levels of NOSinhibition [2]. In addition, the potent vasoconstrictor effectof nitric oxide (NO)-deficiency caused declines in renalfunction [2]. Nonhemodynamic actions of NO includegrowth inhibition on mesangial cell growth and extracel-lular matrix production; thus, NO deficiency would alsolikely lead to a state of unrestrained mesangial growth and/or matrix overproduction [2].

The clinically important issue is whether NO deficiencycan occur in man in situations where chronic kidney dis-ease (CRD) occurs, since, if present, NO deficiency couldcontribute to further progression. Evidence to suggest thatNO deficiency does occur in end-stage renal disease(ESRD) and in CRD is presented below, as well as con-sideration of some potential mechanisms whereby NOdeficiency might develop in renal disease.

As shown in the simplified version of the NO synthesispathway in Fig. 1, there aremultiple ways in which a net NOdeficiency could develop. Reduction in substrate avail-ability (L-arginine) could lead to NO deficiency. This couldreflect decreases in either ingested or endogenous arginine(synthesized mainly in kidney), or limitations on L-argininetransport to the NOS. Accumulation of endogenous NOSinhibitors such as the methylated arginine analogs, in par-ticular asymmetric dimethylarginine (ADMA), could re-duce NOS activity. A decrease in abundance or activity ofthe NOS isozymes, due to reduction in protein content,inhibitory signals, or reduced availability of essential co-

C. Baylis (*)Department of Physiology and Functional Genomics,University of Florida,P.O. Box 100274 Gainesville, FL 32667, USAe-mail: baylisc@ufl.eduTel.: +1-352-3927869Fax: +1-352-3927935

factors would again lead to NO deficiency. Any of theseevents, either alone or acting together, would lead to re-duction in the total amount of NO generated, and this wouldbe reflected by a reduced production rate of nitrite andnitrate, the stable oxidation products of NO. Other, moredownstream events could also reduce NO-dependent ac-tivity; for example by increased NO inactivation with su-peroxide anion, by decreasing the access of NO to tissue (asoccurs with a deposition of advanced glycosylated endproducts), and/or by alteration in the NO target proteins/enzymes and their transduction mechanisms. There is ev-idence both of increased oxidant stress and formation ofadvanced glycosylated end products in patients with renaldisease, and these mechanisms must contribute to a net NO-deficient state. However, this review will focus on the firststeps of the L-arginine/NO pathway, which control the pro-duction rate of NO.

NO production in man

We conducted a series of clinical studies to measure nitriteand nitrate in patients with end-stage renal disease. Inaddition to NO-derived nitrite and nitrate (NOX), theseanions can also enter the body fluid in the drinking waterand the diet; therefore, strict control over the dietary NOX

intake is essential. All of our studies were conducted undercontrolled low-NOX intake diet. As shown in Fig. 2, des-pite similar NOX intake, the total amount of NOX excretedin a 24-h period was significantly decreased in peritonealdialysis and hemodialysis patients at end stage [3, 4], aswell as in CRD patients with ∼25% residual renal function[5]. Although this is a useful qualitative index of NOproduction, it cannot be used quantitatively nor does itgive information on regional NO production, for reasonsdiscussed elsewhere [6]. Nevertheless, the lower NOX

excretion demonstrates that total body NO production issubstantially reduced in ESRD. Similar conclusions werereached by Blum et al. [7] and by Wever et al. [8] using15N2-labeled L-arginine-to-15N-labeled L-citrulline con-version in man with CRD, although not by Lau et al. [9]

who reported increased arginine-to-citrulline conversion inESRD. In general, the evidence suggests decreased NOproduction in man with CRD, and the animal data arealsoconvincing for multiple models of CRD (Fig. 3) [10–14];see also below.

The urinary excretion of the second messenger cGMPhas also been used as a surrogate marker for NOS activity,

endogenous NOS inhibitors

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Fig. 1 Simplified scheme of theNO biosynthesis pathway and anumber of possible mechanismsthat could cause a state ofrelative NO deficiency. NOSnitric oxide synthase, ADMAasymmetric dimethylarginine,O2

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Fig. 2 Upper panel: NOX (nitrite+nitrate) output, measured either asurinary excretion or output in dialysate, in subjects with normalrenal function (Control) and in end-stage renal disease (ESRD)patients on either peritoneal dialysis (PD) or hemodialysis (HD).Lower panel: the cGMP output is given for the same four groups.All subjects were on a low,constant dietary NOX intake (~300 μmol/24 h). * denotes a significant difference versus controls. CRDchronic renal disease. Data are from references [3–5]

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but, as shown in Fig. 2, urinary cGMP excretion does notalways correlate with the urinary NOX excretion, beingsimilar to control values in both CRD and hemodialysispatients [3–5]. Since cGMP signals for other agonists inaddition to NO, this variability is not surprising. Further,plasma and urine cGMP levels do not accurately reflect theactivity of the NO system during graded reduction in NOSactivity by administration of increasing doses of NOSinhibitor. As shown by Arnal and colleagues [15], vesselwall cGMP but not plasma or urinary cGMP variedinversely with blood pressure in rats chronically treatedwith NOS inhibitor. Thus, urine and plasma cGMP datashould be interpreted cautiously.

L-Arginine availability

The kidney is a major site of the endogenous synthesisof arginine, and arginine made in the kidney is exportedthroughout the circulation and utilized in various metabolicprocesses, including generation of NO [16]. It is possiblethat in patients with severe CRD or ESRD, where there islittle or no normal functional renal mass, this source of L-arginine might become deficient and could lead to a re-duction in substrate availability. When plasma L-argininelevels are measured in patients with renal disease, they tendto be at the low end of normal range, but, of importance,even when moderately reduced, plasma L-arginine levelsremain well in excess of the KM of the various NOS iso-zymes [3, 4]. Net endogenous arginine synthesis has beenreported to be intact in ESRD patients [9], and, at least at themRNA level, all key enzymes are preserved in the old malerat kidney with severe CRD [17]. This could be interpretedto indicate that there is no L-arginine deficiency in renaldisease; however, the intracellular L-arginine concentration

in immediate proximity to the NOS enzyme is the criticalelement in determining availability of substrate. Thus, therate of transport of L-arginine into endothelial cells may bean important determinant of endothelial NOS (eNOS)activity.

To investigate the impact of uremia on L-arginine trans-port capacity, we used a range of cultured vascular endo-thelial cells from different species and regions of thecirculation. Cells were preincubated with diluted plasma(20 vol%) from normal people or ESRD patients, and alsosome synthetic media, and then the rate of L-argininetransport into the cell was measured [18]. As shown inFig. 4, uremic plasma from both peritoneal dialysis and pre-hemodialysis patients inhibited L-arginine transport. He-modialysis did clear some of the inhibiting factors fromplasma, but there was residual inhibition of L-argininetransport by plasma obtained immediately post-hemodial-ysis [18]. We conducted experiments with syntheticsolutions to try and determine what the factors might be,and discovered that urea in uremic concentrations (presentat >15 mmol/l) was capable of inhibiting L-arginine trans-port in all endothelial cell types [18]. This effect was notosmotically mediated, required preincubation, was notacutely reversible with excess L-arginine, and exhibited an“all or nothing” response, switching on when the concen-tration in the medium reached 15 mmol/l [18]. We sub-sequently showed that the urea has to enter the endothelialcell since in the presence of phloretin, a urea transport in-hibitor, the inhibitory effect of urea on L-arginine transportwas abolished [13]. We, therefore, propose the followingscheme, which might provide a mechanism of local en-dothelial intracellular arginine deficiency despite appar-ently adequate circulating levels in uremia. The eNOS isanchored to the inside of the endothelial cell membrane inthe caveolae in close proximity to the L-arginine transport-er. There is also evidence that even with normal plasma L-arginine concentrations, L-arginine may be rate limiting insome circumstances, raising the possibility that it is the L-arginine delivered into the microenvironment immediatelyaround the eNOS that determines eNOS activity [19]. Wesuggest that normally urea is transported out of endothelialcells (where it is made by resident arginases), probably bythe B-type urea transporter. In uremic conditions, the ureaconcentration gradient is reversed and urea enters theendothelial cells, where it inhibits L-arginine transport by acurrently unknown mechanism. Of note, there have beenreports that high levels of urea can inhibit other membranetransporters (see ref. [18]), which raises the possibility of awidespread transport inhibition in uremia.

Endogenous inhibitors of NO synthesis

In addition to a possible local substrate deficiency, NOdeficiency could result from increased levels of circulatingor local NOS inhibitors. Vallance and colleagues were thefirst to report that circulating endogenous NOS inhibitorsaccumulate in patients with renal failure leading to falls inthe ratio of plasma arginine to methylated arginine [20].

Animal models of CRD

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Fig. 3 NOX (nitrite+nitrate) output, measured as urinary excretionin male Sprague-Dawley rats studied in control, 5/6th renal ablation/infarction (A/I), both at 11 weeks after the start of the observationperiod; with chronic glomerulonephritis (GN) after 20 weeks ofobservation; with chronic puromycin aminonucleoside nephrosis(PAN) after 15 weeks of observation and in rats at 22 months ofage (Aging). All rats were maintained on a similar low NOX diet.* denotes a significant difference versus controls. Data are fromreferences [10–14]

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There have been many subsequent studies, all reportingincreased methylated arginine levels in patients with renalfailure, although the baseline value and magnitude of theincrease vary [21–24]. Our findings in both peritoneal di-alysis and hemodialysis patients agree quantitatively withthe observations of Vallance et al. [20] with markedincreases in the potent NOS inhibitor ADMA, as well as inthe inactive symmetric dimethylarginine (SDMA) [3, 4].Studies by Vallance and colleagues [25, 26] suggest thatthese levels of ADMA (2–5 μmol/l) can functionally in-hibit eNOS activity in vivo. Our in vitro studies supportthis, since NOS activity in cultured endothelial cells in-cubated with plasma from ESRD patients was inhibited byabout 30% compared to cells incubated with plasma fromcontrols with normal renal function [14] (Fig. 5). There issignificant clearance of both methylated arginine speciesacutely during hemodialysis; however, plasma L-arginineis also cleared so that the arginine-to-ADMA molar ratioremains low. This may account for our finding that im-mediately after hemodialysis the NOS inhibitory activity ofESRD plasma was still fully present [14]. Using syntheticsolutions, concentrations of ADMA similar to those seen inESRD (>1.5 μmol/l) have a functionally significant in-hibitory effect on NOS activity [27]. Of note, however, ourcell culture studies using ESRD patient plasma used a 1 in5 dilution; thus the ADMA concentration to which the cellswere exposed was always less that 1.5 μmol/l since nopatient had plasma ADMA in excess of 7.5 μmol/l. Despitethis, significant inhibition of NOS activity was seen in cellsexposed to ESRD plasma, strongly suggesting the presenceof additional NOS inhibitors. Given the marked effectsseen in vitro with a ESRD plasma dilution of 1:5 (v/v), theimpact in vivo on endothelial cells exposed to undilutedESRD plasma is likely to be very significant.

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Fig. 4 Effects of diluted humanplasma (20 vol%) from subjectswith normal renal function(Control), patients on peritonealdialysis (PD), and both pre- andpost-hemodialysis (HD), on L-arginine transport in culturedvascular endothelial cells after6 h incubation. Studies were inhuman dermal microvascularcells (HDMEC), human glo-merular capillary endothelialcells (HGEC), and bovine aorticendothelial cells (BAEC)studied between passage 4–7.Measurements were made inthe baseline state (solidhistograms) and with 5 mmol/lNG-monomethyl-L-arginine(L-NMMA) (open histograms),which competitively inhibits themajor endothelial cell argininetransporter. *P<0.01 comparedto control. #P<0.05 comparedto pre-HD and control.Reproduced with permissionfrom reference [18]

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Fig. 5 Effects of diluted human plasma (20 vol%) (from controls,peritoneal dialysis, PD, and pre-hemodialysis, HD, patients) on theNOS activity in human dermal microvascular endothelial cells(HDMEC, top panel), human glomerular capillary endothelial cells(HGEC, middle panel), and bovine thoracic aortic endothelial cells(BAEC, bottom panel). After 6 h incubation, NOS activity wasdetermined by measuring the conversion rate of L-arginine to L-citrulline (solid histograms). The NOS inhibitor L-NMMA (5 mmol/l) was added to some wells (open histograms) to confirm that the L-arginine-to-L-citrulline conversion reflected specific NOS activity.Results are mean±SEM of three separate experiments, each per-formed in triplicate. *P<0.05 compared to control. PD peritonealdialysis, pre-HD pre-hemodialysis. Reproduced with permissionfrom reference [14]

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We have also studied patients with CRD where residualrenal function exists [5]. As shown in Fig. 2, the CRDpatients with ∼20% residual renal function all exhibitedreduced total NO production (from the 24-h urinary NOX

excretion rate). In the plasma of these patients, there were noinhibitory factors of transport of L-arginine, presumablyreflecting the lesser degree of hemoconcentration of uremicfactors versus ESRD. However, a NOS inhibitory factor waspresent in the plasma of some, but not all of the CRD patients[27]. There are a number of different primary diseases, butthe presence or absence of NOS inhibitory activity in CRDpatient plasma was not predicted either by disease or bymedication. For example, four patients had diabetic neph-ropathy, twowith normal and two with NOS inhibitory factor(s) in their plasma. As shown in Fig. 6, the plasma ADMAconcentration predicted the in vitro NOS inhibitory poten-tial of the plasma from CRD patients [27]. Those patientswith normal plasma eNOS activity had low (normal) plasmaADMA concentration, whereas those, in whom eNOS activ-ity was reduced, showed elevated plasma levels of ADMA.This variability was not due to differences in renal clearancebetween the groups, since plasma creatinine and blood ureanitrogen (BUN) were uniformly elevated (Fig. 6).

In contrast to the variable ADMA plasma levels, plasmaSDMA levels were elevated in all CRD patients, and this

may implicate differences in the activity of the enzymedimethylarginine dimethylaminohydrolase (DDAH) whichhydrolyzes ADMA but not SDMA. DDAH is widely dis-tributed in the tissues and the vasculature, and co-segregateswith the NOS enzymes in the kidney [28]. Inhibition ofDDAH (that allows accumulation of ADMA) inhibitsendothelium-dependent relaxation [29]. Thus, a change inthe ADMA to SDMAmolar ratio as seen in some, but not allCRD patients, suggests an altered DDAH activity. Wespeculate that loss of renal clearance is responsible for theincreased plasma ADMA in some CRD patients, but that inothers, who have amore active DDAH enzyme, ADMA canbe metabolized by this mechanism, preventing the earlyincrease in this endogenous NOS inhibitor. Several poly-morphisms have recently been reported in the endothelialisoform of the DDAH gene [30], which may impact plasmaADMA levels. There are also many factors that can influ-ence the expression and/or activity of the DDAH isozymes.Oxidative stress inhibits DDAH activity by oxidation of asulfhydryl group in the active site of the enzyme causingADMA levels to increase [31, 32]. This is particularlyrelevant to the renal disease population where oxidativestress appears early and persists throughout the course of theCRD [33]. The importance of the activity of DDAH isclearly demonstrated by the prospective study of Zoccaliand colleagues [23], who found that plasma ADMA con-centration was a strong and independent predictor of overallmortality and cardiovascular outcome in ESRD patients.There are also local inhibitory influences that could impacton NO production in various locations. ADMA is syn-thesized in the endothelial cell (as well as in many otherlocations) and is capable of influencing NO productionlocally and in adjacent cells [34]. In addition, the NOSisozymes have regulatory interactions with a number ofproteins [35]. Of note, one of these, the protein inhibitor ofneuronal NOS (nNOS), i.e. PIN, has been shown to besignificantly upregulated in the rat kidney following 5/6th-reduction of renal mass, a model for rapidly progressingCRD [36].

NOS protein abundance in the kidney

In addition to reduction in the arginine to ADMA molarratio, falls in either abundance or activity of the NOSenzymes will cause NO deficiency. We have investigatedthe renal NOS protein abundance and in vitro NOS activityin several rat models of renal disease. The most widelystudied, experimentally induced model of CRD is 5/6thablation or combined ablation/infarction of kidney mass,and several groups have reported that total NO production,and in vitro renal NOS activity declines [37, 38]. We havemade similar observations of reduction in total NO gen-eration, in vitro renal NOS activity, and abundance of thenNOS proteins in the remnant kidney in Sprague-Dawleyrats [19, 39]. In fact, as shown in Fig. 7, there is a linearrelationship between degree of glomerular damage anddecline in renal cortical and medullary nNOS protein, whena threshold of ∼20% glomerular injury is exceeded [39].

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Fig. 6 NOS activity of plasma on cultured human dermal micro-vascular endothelial cells, and plasma concentrations of ADMA(asymmetric dimethylarginine), SDMA (symmetric dimethylargi-nine), creatinine (Cr), and BUN (blood urea nitrogen), in subjectswith normal renal function (Control), the entire group of patientswith chronic renal disease (CRD), a subgroup of CRD patientswhose plasma had no inhibitory effect on eNOS activity (CRD I),and a subgroup of CRD patients whose plasma had an inhibitoryeffect on eNOS activity (CRD II). *P<0.05 compared to control;#P<0.05 compared to CRD I patients with normal NOS activity inplasma. Reproduced with permission from reference [27]

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Most of the animal studies on the NO system in CRD hasbeen on the model of renal ablation or ablation/infarction,which is not an optimal model for CRD in man. To de-termine whether renal NO deficiency occurs in all forms ofCRD, we also investigated other rat models and as shownin Fig. 8, in every case we saw reduction of renal corticalnNOS abundance: in the Sprague-Dawley rat, when the5/6th ablation infarction injury was accelerated with highsodium and protein intake (AA/I) [39]; in a chronic glo-merular nephritis model (GN) [40]; with chronic puromy-cin aminonucleoside nephrosis (PAN); and in the male withadvancing age (aging), we found declines in nNOS proteinabundance and in the in vitro NOS activity in the solublefraction of renal cortex (location of the nNOS) [10, 11]. Inaddition, we have seen a similar result in other strains; inthe Zucker obese inbred hyperglycemic rat which developsa severe focal and segmental glomerular damage as well asinterstitial injury (unpublished data), and also in the chron-ic allograft rejection model of the Fisher-344 to Lewis rat[41].

In contrast to the uniform changes in the renal nNOS, therenal eNOS isoform abundance is highly variable, showingincreases in the 5/6th renal ablation/infarction model [39,41], no change in the GN and PAN models [10, 40] and theobese hyperglycemic Zucker rat (unpublished data), anddeclines in aging and the renal transplant rejection model[11, 41]. Based on this we have concentrated our inves-tigations into the renal nNOS and begun to address thequestion of whether the nNOS decline is functionallysignificant.

If the decline in renal nNOS contributes importantly tothe progression of the CRD, we would expect to see main-tained renal nNOS in situations where progression did notoccur after the initial insult. In contrast to Sprague-Dawleyand Wistar rats, which are very sensitive to ablation/infarction-induced progressive renal disease, the Wistar

Furth rat is remarkably resistant [42]. We have confirmedthis observation [12] and observed that the renal nNOSabundance and activity are significantly preserved after5/6th renal ablation/infarction in comparison to theSprague-Dawley rat. Moreover, a very low level of chronicNOS inhibition that has no further impact on the progres-sion of CRD in the Sprague-Dawley rat converts the Wistar

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Fig. 7 a Correlation between glomerular damage and relativenNOS abundance expressed as % of the appropriate sham mean inrenal cortex for all rats studied. Open boxes at 100% cortical nNOSdenote sham groups. The solid symbols denote data from rats studiedat 1–11 weeks after surgery. r=0.678, P<0.0001. b Correlation be-tween glomerular damage and relative nNOS abundance expressed

as % of the appropriate sham mean in renal medulla for all ratsstudied. Open circles denote shams. The solid symbols denote datafrom rats studied at 1–11 weeks after surgery. r=0.722, P<0.0001.nNOS neuronal nitric oxide synthase. Reproduced with permissionfrom reference [39]

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Fig. 8 Abundance of the neuronal NOS isoform (given asintegrated optical density, IOD, factored for the internal standard,Int Std=cerebellar lysate, and factored for the total protein loadedper lane given by the ponceau red staining) in homogenates of renalcortex in sham controls (solid histograms) and experimental modelsof chronic renal failure (CRD; open histograms) in male rats sub-jected to 11 weeks 5/6th renal mass ablation/infarction (A/I);accelerated A/I (A/II)=3 weeks post A/I with high sodium and pro-tein intake; 20 weeks after induction of glomerulonephritis (GN); 15weeks after induction of puromycin aminonucleoside nephrosis(PAN); in the 22-month old Sprague-Dawley; in the 10-month oldobese and lean Zucker rat with diabetic nephropathy (DN); 22weeks after transplantation (Tx) of a Fisher-344 rat kidney into aLewis host. * denotes a significant difference versus controls. Dataare collated from references [10–12, 39–41]

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Furth rat into a model of rapidly progressive renal disease[12]. We have made similar observations in the chronicPAN model, with the Sprague-Dawley rats developingESRD after 15 weeks of PAN while the Wistar Furth ratsmaintain normal kidney function and structure, havingrecovered completely from the acute insult of PAN ad-ministration [10]. This recovery (failure to progress) isagain associated with maintained renal nNOS abundanceand activity. In ongoing studies in the C57Bl6 mouse, astrain highly resistant to development of renal mass re-duction-induced injury, we have found that both chronicnonselective NOS inhibition and selective nNOS inhibitiongreatly accelerate the course of the renal damage [43].Future studies are planned in nNOS (and other NOS iso-form) knockout mice to test the possibility that the nNOSknockout mice will be particularly vulnerable to developCRD following renal mass reduction. Finally, while in-trarenal NOS activity (soluble fraction) and the abundanceof renal nNOS protein declines in the male Sprague-Dawley rat with advancing age, as CRD develops, theaging female is protected from functional declines andstructural damage and shows preserved renal nNOS proteinand NOS activity [11]. Taken together, these data suggestthat NO deficiency within the kidney may play a primaryrole in progression of CRD.

Conclusion

There are substantial clinical, in vitro, and in vivo data tosuggest that systemic, endothelial, and renal NO deficiencyis a common feature of CRD irrespective of the primarygenesis of the disease. Some evidence suggests that thisNO deficiency contributes importantly to the progressivenature of the CRD, and the endothelial NO deficiencycertainly contributes to the cardiovascular damage in renalfailure patients. Strategies that reverse eNOS inhibitionand/or can boost the ability of the damaged kidney toproduce NO might help preserve residual renal functionand/or reduce the rate of progression to end stage.

Acknowledgements I thank past and present members of mylaboratory who have contributed to various aspects of this work. Thiswork has been funded by NIH grants HL 31933, DK 45517 and DK56843

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