INVITED REVIEWParadoxes of nitric oxide in the diabetic kidney - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 23 下一页

返回列表

查看: 354645\|回复: 228	go INVITED REVIEWParadoxes of nitric oxide in the diabetic kidney [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 13:38 |只看该作者 |倒序浏览 |打印

作者：RadkoKomers,  SharonAnderson,作者单位：1 Division of Nephrology and Hypertension,Department of Medicine, Oregon Health and Science University, and Portland Veterans Affairs Medical Center, Portland,Oregon 97201-2940; and Diabetes Center, Instituteof Clinical and Experimental Medicine, Prague, CzechRepublic
   ; ^4 g$ Q! ?2 U

      & T) C2 ]0 Q0 Y; I- b; G1 U
                     5 A$ Q' y2 @7 J+ Q. K; i

         ; }0 s5 o6 P; [6 N9 \






      【摘要】  V( W  F1 t5 I0 }
   As an important modulator of renalfunction and morphology, the nitric oxide (NO) system has beenextensively studied in the diabetic kidney. However, a number ofstudies in different experimental and clinical settings have producedoften confusing data and contradictory findings. We have reviewed awide spectrum of findings and issues that have amassed concerning thepathophysiology of the renal NO system in diabetes, pointed out thecontroversies, and attempted to find some explanation for thesediscrepancies. Severe diabetes with profound insulinopenia can beviewed as a state of generalized NO deficiency, including in thekidney. However, we have focused our hypotheses and conclusions on theevents occurring during moderate glycemic control with some degree oftreatment with exogenous insulin, representing more the clinicallyapplicable state of diabetic nephropathy. Available evidence suggeststhat diabetes triggers mechanisms that in parallel enhance and suppressNO bioavailability in the kidney. We hypothesize that during the earlyphases of nephropathy, the balance between these two opposing forces is shifted toward NO. This plays a role in the development ofcharacteristic hemodynamic changes and may contribute to consequentstructural alterations in glomeruli. Both endothelial (eNOS) andneuronal NO synthase can contribute to altered NO production. Theseenzymes, particularly eNOS, can be activated byCa 2  -independent and alternative routes of activation thatmay be elusive in traditional methods of investigation. As the duration of exposure to the diabetic milieu increases, factors that suppress NObioavailability gradually prevail. Increasing accumulations of advancedglycation end products may be one of the culprits in this process. Inaddition, this balance is continuously modified by actual metaboliccontrol and the degree of insulinopenia.
      【关键词】 reactive oxygen species endothelial function renal function signal transduction diabetic nephropathy
   INTRODUCTION
  _1 o$ P3 x2 N: N  r" p: g! T
NITRIC OXIDE (NO) IS A PARACRINE mediator with a wide spectrum of physiological actions, includingthe control of vascular tone, antithrombotic actions, cell cycleregulation, neurotransmission, signal transduction, and inflammation.NO is synthesized during conversion of its physiological precursor L -arginine ( L -Arg) to L -citrulline( 77 ). This reaction is catalyzed by a family of enzymesknown as NO synthases (NOS) ( 81 ). Three NOS isoforms [neuronal (nNOS, NOS1); inducible (iNOS, NOS2); and endothelial (eNOS,NOS3)] have been identified in mammalian tissues. nNOS and eNOS aretraditionally viewed as constitutive enzymes, with a limited tissuedistribution, exhibiting intracellular Ca 2  /calmodulindependency, although Ca 2  -independent activation hasbeen described ( 29, 34 ). nNOS and eNOS are responsible forproducing NO for a variety of physiological purposes( 68 ). In contrast, iNOS is an inducible enzyme, expressed in all nucleated cells that generate large bursts of NO inresponse to immunological and certain nonimmunological stimuli.iNOS is usually described as Ca 2  independent,although the presence of Ca 2  enhances iNOS activity( 142 ). To produce NO, NOS enzymes require molecular oxygenand a battery of cofactors and posttranslational modifications( 39, 68 ). Effects of NO are typically mediated byactivation of soluble guanylate cyclase, resulting in increased levelsof cGMP ( 85 ).( J5 s" O' u$ k( @

NO also acts as a potent modulator of renal function. A wealth ofinformation on renal actions of NO that has amassed during the past twodecades has been summarized in several excellent reviews ( 9, 68 ). All three NOS isoforms are found in the kidney. nNOSprotein and mRNA are found predominantly in the macula densa (MD)region of the distal tubule and renal nerves ( 7, 64, 127, 150 ). iNOS mRNA is detectable in most of tubular cells along thenephron ( 1, 84 ). eNOS is typically expressed inendothelial cells along the renal vascular tree ( 7 ). All three isoforms are expressed in the medulla ( 88, 104, 123 ), and medullary NO production exceeds that in the cortex( 154 ). Studies with nonspecific NOS inhibitors, such as L -Arg analogs, demonstrate that renal hemodynamics are verysensitive to NOS inhibition. NO controls both afferent and efferentvascular tone, the ultrafiltration coefficient ( 28, 159 ),and medullary blood flow ( 79 ), with preferential action onthe afferent arteriole ( 28 ). In addition, NO hasnatriuretic actions ( 4, 79 ).7 x6 ~+ h3 I  D1 E( m1 r

As an important modulator of renal function and morphology, the NOsystem has been extensively studied in the diabetic kidney. However, anumber of studies in different experimental settings have oftenproduced confusing data and contradictory findings. In this paper, wewill briefly review in vitro evidence on the renal NO system indiabetes; put more emphasis on in vivo evidence, in particular,pointing out the controversies in this literature; and attempt to findsome explanation for these discrepancies. Furthermore, based onprevailing evidence, we will propose a unifying hypothesis on thechanges in renal NO in diabetes and its role in the pathophysiology of nephropathy.

EFFECTS OF DIABETES ON THE RENAL NO SYSTEM IN VITRO8 H. k* I+ |5 Q+ q6 S. x
' g$ I* P  e: b" q9 l& E. u; z
Determinations of the effects of high glucose or glycosylationproducts on NO bioavailability in renal cell cultures( 143 ) and in isolated glomeruli ex vivo ( 22, 23, 112, 146 ) have been applied by several groups as an in vitroapproach to assess the effects of diabetes on the renal NO system.Considering the abundance of endothelial cells in the kidney, we alsomention several reports on this cell type. Although not performeddirectly in renal cells, we assume that the same processes as described by these authors could be active in the kidney and should be discussed in the context of this review ( 13, 30, 46 ). In general, invitro studies have provided important information on the mechanisms whereby high glucose or glycosylation products influence NObioavailability. These mechanisms include enhanced synthesis or actionof the prostanoid thromboxane A 2 and PKC activation( 22, 23, 46 ), NO quenching ( 14, 143 ),inhibition of both Ca 2  -dependent and -independent NOSactivities ( 30, 143 ), reactive oxygen species (ROS)production ( 46 ), and NO capture by glucose ( 13 ). Taken together, these studies have suggested adecrease in renal NO production, action, stability, or bioavailability in diabetes. In fact, in vitro studies have provided most of the experimentally available evidence implicating diabetes as a state ofrenal NO deficiency.
$ t' \( b/ K  U/ J8 D
Unlike the previously mentioned approaches, in vitro measurements ofNO-dependent renal vascular reactivity also provide functional aspectsof the renal NO system and are therefore discussed in more detail.Evidence discussed in this section encompasses studies using variousexperimental approaches and models, including the isolated kidney andisolated arteriolar and renal artery preparations (Table 1 )., \# _4 {+ p. [6 F/ ?

Table 1. Studies on effects of high glucose/diabetes on NO-dependent renalvascular reactivity in vitro

Studies in isolated rat kidneys reported increased ( 11 ) ornormal ( 10 ) vasodilator responses to ACh or metacholineand normal responses to nitroprusside ( 10, 11 ). However,other investigators have reported convincing evidence suggestingdeleterious effects of diabetes on renal NO-dependent vascularreactivity. Ohishi and Carmines ( 90 ) studied isolatedkidney preparations using videomicroscopy of in vitro blood-perfusedjuxtamedullary nephrons to directly assess renal afferent and efferentarteriolar diameters. Diabetic kidneys demonstrated greater baselineafferent diameters, whereas efferent diameters did not differ betweendiabetic and control rats. Apparently, this model reflects changes inarteriolar diameters in diabetic kidneys similar to those observed invivo in micropuncture studies ( 50, 160 ). Both afferent andefferent responses to the NOS inhibitor N G -nitro- L -arginine( L -NNA) were blunted in diabetic kidneys. The responses to NOS inhibition in diabetic rats were restored by treatmentwith SOD. These observations suggested decreased NO availability indiabetic glomerular arterioles, possibly due to accumulation ofNO-scavenging superoxide anions. Interestingly, this NO-deficientsituation was observed in kidneys with decreased afferent arteriolartone. Corresponding observations have been reported in alloxan-diabeticrabbits. ACh-induced vasodilator responses in preconstricted,microperfused afferent arterioles were impaired in diabetic rabbits,and the defect was corrected by using the SOD mimetic tempol( 109 ). However, using a similar model, Moore et al.( 86 ) reported somewhat different data compared with thetwo previously mentioned studies. The responses to ACh were unalteredin diabetic arterioles. Although the fractional N G -nitro- L -arginine methyl ester( L -NAME)-induced decrease in afferent diameter was lower indiabetic rats, absolute changes were greater in diabeticvessels. This discrepancy was attributable to differences inbaseline diameters between control and diabetic rats. Furthermore, theauthors performed direct intraluminal NO measurements and observed amarked increase in NO concentrations in diabetic vessels afterperfusion with a high-glucose perfusate. The authors also explored thevasoactive effects of advanced glycation end products (AGEs). TheAGE-containing perfusate induced no changes in diabetic vessels,although it blunted responses to ACh. Thus these findings are onlypartially consistent with previously mentioned studies by Bucala et al.( 14 ) and do not suggest a substantial role for AGE in NO quenching.
( k# _* R% Z7 y( b: ]; S- h
An interplay between NO and ANG II was addressed by Schoonmaker et al.( 111 ). Control afferent arterioles had enhancedvasoconstrictor responses to ANG II when pretreated with L -NNA. In contrast, L -NNA-treated and untreatedarteries harvested from diabetic rats demonstrated no differences inANG II-induced vasoconstriction. Moreover, responses of efferentarterioles were similar in control and diabetic rats ( 111 ). These observations corresponded to previousfindings in rabbit afferent arteriolar preparations microperfused witha normal and high-glucose (30 mM) perfusate ( 6 ) andsuggested attenuated NO action in counterbalancing the effects of ANGII specifically in afferent arterioles in diabetes and/or hyperglycemia." |  w" o0 b4 L2 l' B% f0 |5 u+ v

Diabetes-associated changes in the NO system have also beeninvestigated in isolated renal arteries. It is likely that observations in renal arteries reflect changes in the vascular system in general rather than contribute to an understanding of specific renal processes. However, controversial findings in this area may signal some specific features of the renal arteries compared with other large-conduit vessels that usually display diabetes-induced defects inendothelium-dependent vasodilation ( 99 ). Dai et al.( 25 ) reported that the defect in ACh-induced vasodilationin diabetic rats was ameliorated by pretreatment with a hydroxylradical scavenger or with a blockade of prostaglandinH 2 -thromboxane A 2 receptors. Corresponding to studies in other experimental settings ( 23, 90, 109 ),these data suggest an impairment of NO endothelial production or action in diabetic arteries, possibly mediated by the increased production ofROS and prostanoids that oppose the effects of NO. However, more recentstudies in that model found unchanged responses to ACh in renalarteries from insulin-treated diabetic rats preconstricted withserotonin and even enhanced responses to insulin compared with controls( 130, 131 ). In contrast to data by Dai et al. ( 25 ), enhanced responses to ACh and to NOS inhibition havealso been reported in renal arteries harvested from untreatedalloxan-diabetic rabbits ( 3 ).

Unlike the studies in cultured renal cells, evaluation of renalvascular reactivity in vitro has provided more controversial evidence,suggesting enhanced, normal, or decreased NO synthesis and activity inthe diabetic kidney (Table 1 ). These contrasting findings cannot beexplained by species differences, differences in duration of diabetes,metabolic control, or the presence or absence of insulin treatment.
! N6 E& n1 H2 N2 Z! G% I
One of the factors that could possibly explain differences among atleast some of these studies could relate to the method ofpreconstriction of isolated vessels. There is evidence suggesting thatdifferent vasoconstrictor stimuli may alter responses to ACh inafferent arterioles ( 43 ). Different constrictors may alsoexert variable effects on basal vascular tone and, as in the case ofserotonin, even activate eNOS ( 107 ).

Several caveats of in vitro approaches in evaluating the renal NOsystem in diabetes in general merit consideration. The in vitro studiesmay not precisely reflect the diabetic milieu, which is determined notonly by high glucose. For example, most of the in vitro evidenceregarding the effects of glucose on cellular NO systems has beenobtained by using media glucose concentrations from 25 to 33 mM. Theclinical correlate would be severely decompensated diabetes, which isknown to be associated with renal vasoconstriction as a result not onlyof specific effects of glucose on renal cells but also of physiologicalcompensation for volume depletion and electrolyte wasting( 50 ). However, as specifically discussed below, earlystages of nephropathy are characterized by glomerular hyperperfusion,which has important pathophysiological implications and is prominent inthe more clinically applicable model of moderate hyperglycemia(15-20 mM) ( 52, 162 ). Consequently, it is beneficial to use an in vitro approach to precisely model a particular condition and obtain answers to specific questions. However, the in vitro datashould be interpreted with caution, considering functional alterationstypical in the early stages of diabetic nephropathy.
+ A) l# e& x4 o- R8 L& s
Several in vitro studies also focused on the effects of hyperglycemiaon iNOS-derived NO in renal cell cultures ( 113 ).Considering the relatively weak in vivo evidence for the role of iNOSin diabetes-induced alterations in the renal NO system, these papersare not discussed in this review. Consequently, we also do not discussin vitro studies in mesangial cells. With respect to prevailingevidence, this cell type does not express constitutive NOS( 68 ).

EFFECTS OF DIABETES ON THE RENAL NO SYSTEM IN VIVO

Expression of NOS Isoforms in the Diabetic Kidney In Vivo

A number of studies have examined renal NOS expression in diabetesin vivo (Table 2 ). Some of the studiesmentioned in this section combined measurements of NOS expression withrenal hemodynamics and will be discussed further in the followingsections. Choi et al. ( 19 ) found markedly increased renalcortical expression of all NOS isoforms in streptozotocin(STZ)-diabetic rats; medullary expression of NOS was not different fromcontrols. Another group ( 118 ) found enhancedimmunohistochemical expression and NADPH diaphorase staining,reflecting constitutive NOS activity, in endothelia of afferent but notefferent arterioles of STZ-diabetic rats. Enhanced NADPH diaphorasestaining was associated with increased afferent diameter, increasedglomerular filtration rate (GFR) assessed by creatinine clearance, andglomerular hypertrophy. These changes were corrected by insulintreatment or by treatment with L -NAME. Inaccordance with the previous report, Veelken et al. ( 141 )reported increased cortical eNOS expression in untreated hyperfilteringdiabetic rats. In addition, increased medullary expressions of nNOS andeNOS were found in the medulla of diabetic rats treated with insulin( 115 ).) x! `9 F* O# X0 T( X& Y
4 {. z9 M4 G) @0 Q  q6 b2 o  c
Table 2. Studies on effects of diabetes on NOS renal expression in vivo6 f; {' ^' A: f1 o, a

Early studies that focused on nNOS immunoreactivity in MD cellsreported less intense immunohistochemical staining for constitutive NOS(presumably nNOS) in MD and glomerular arterioles in rats with diabetesfor various durations ( 157 ). In concert with those findings, Keynan et al. ( 60 ) reported decreased nNOS mRNAand diaphorase MD staining in rats at 7 days of diabetes. Furthermore, they found no differences in cortical eNOS between control and diabeticrats. Unlike most of the studies dealing with this issue, they founddecreased urinary nitrite/nitrate excretion (U NOx V) indiabetic rats compared with controls.: a8 N) I/ D% j* ?) ]+ r6 {

Most recent analyses also do not provide unequivocal evidence. Ishii etal. ( 54 ) found no differences in cortical expression ofNOS isoforms between diabetic animals and controls. Diaphorase stainingalso did not differ among groups. Another study ( 92 ), using whole kidney samples, found no change in nNOS and increased eNOSin female rats after 4 wk of diabetes. In addition, whole kidney eNOSand iNOS mRNAs evaluated by RT-PCR were unchanged in diabetic rats( 112 ). Finally, increased eNOS expression was found inpurified renal vascular trees of diabetic rats ( 26 ).* w0 f6 d1 d" }( d7 m

Thus it appears that consensus can be reached with respect to eNOSexpression. Most of the available evidence suggests increased expression of this isoform in the diabetic renal cortex. There aredisparate findings concerning nNOS, suggesting increased, normal, ordecreased expression (Table 2 ). It should be noted that a decrease innNOS in the diabetic renal cortex has been observed only in ratswithout insulin treatment. With the exception of one study( 19 ), iNOS cortical protein and mRNA expression have beenfound to be unchanged ( 54, 112 ) or barely detectable ( 119, 141 ) during the hyperfiltering stage. Twoobservations suggest that iNOS is detectable in the renal cortex ofrats with long-term diabetes. iNOS was immunohistochemically detectedin glomeruli of rats after 1 yr of diabetes but not in controlage-matched animals ( 119 ). Another long-term study (32 wk)demonstrated weak glomerular expression in both control and diabeticrats ( 116 ) but no differences between the groups.

Disparate findings have also been reported in studies that determinedrenal NOS activity by a citrulline generation assay. Keynan et al.( 60 ) found decreased cortical NOS activity. However, onemight question the validity of the assay in this particular studybecause NOS activity was not detectable in the medulla, which shouldhave much higher NOS activity than the cortex ( 154 ). Incontrast, Omer et al. ( 91 ) and Ishii et al.( 54 ) found increased cortical NOS activity in diabeticrats. Importantly, these studies differed in the absence( 60 ) or presence ( 54, 91 ) of insulin treatment.

ROLE OF NO IN RENAL HEMODYNAMIC ALTERATIONS IN DIABETES1 M0 h1 {# l8 w/ e
* z5 A( S1 q: X0 l
Early stages of diabetic nephropathy are associated with increasesin GFR and variable increases in renal plasma flow (RPF) and filtrationfraction, both clinically and experimentally ( 5 ). This"diabetic hyperfiltration" has been implicated in the pathogenesis of diabetic nephropathy both in humans as well as in animal models ofdiabetes ( 49, 83, 105, 160, 161 ). At the single-nephron level, diabetic hyperfiltration is characterized by disproportionately decreased afferent arteriolar resistance, resulting in elevated glomerular capillary pressure (P GC ) ( 50, 90, 160 ). Considering the renal hemodynamic actions of NO, thissubstance is a good candidate for mediating diabetic hyperfiltration.In this section, we will discuss studies exploring acutehemodynamic effects of modulation of NO synthesis in vivo (Table 3 ).
8 z6 \' N+ V' ?# w
Table 3. Studies on effects of NOS modulation on renal hemodynamics in models ofdiabetes8 P5 r. F4 e2 r6 q0 k/ f
3 G+ u7 v% h6 a) {0 k! [& ^6 m
Most of the evidence concerning the role of NO in control of renalhemodynamics in diabetes was obtained using nonspecific NOS inhibitors.More recently, several studies have attempted to test the activity ofindividual NOS isoforms with newly available isoform-specificinhibitors. In a number of these studies, GFR and RPF were measured asclearances of inulin and PAH using constant-infusion techniques.Despite the explosive developments in molecular biology, we considersuch evidence as the gold standard when evaluating the role of aparticular substance in the control of renal hemodynamics in vivo. Inaddition to measurements of renal hemodynamics, these studies have alsooften relied on measurements of U NOx V and urinary cGMP asmarkers of renal NO production.

To our knowledge, the first in vivo evidence indicating that modulationof NO synthesis could influence renal hemodynamics in diabetes wasreported in an abstract by King et al. ( 62 ). Theirpreliminary data showed a major renal vasoconstrictor effect of apressor dose of NOS inhibitor, suggesting greater NO dependence ofrenal hemodynamics in diabetic compared with control rats. Full papersby other groups addressing this issue followed shortly. Bank andAynedjian ( 8 ) found an increase in U NOx V inhyperfiltering diabetic rats, consistent with increased NO production.However, when administered increasing doses of NLA, both diabetic andnormal rats had similar decreases in GFR and RPF. Despite thesedisparate findings, the authors concluded that NO synthesis wasincreased in hyperfiltering diabetic rats and that excessive NOsynthesis contributed to hyperfiltration. Tolins et al.( 128 ) also found increased U NOx V inhyperfiltering diabetic rats. However, NOS inhibition with L -NAME normalized GFR in diabetic rats and induced greatervasoconstrictor responses under various perfusion pressures. Importantly, perfusion pressure was modified independently of thesystemic blood pressure changes that would usually be associated withsystemic NOS inhibition. Thus both excretion of stable metabolites ofNO and renal hemodynamic responses suggested enhanced renal generationof NO and its contribution to the pathogenesis of diabetic hyperfiltration.6 e5 y3 @1 q* {  {$ u3 |5 S' b
: }- @- S2 i% E. f
We evaluated the effects of L -NAME in conscious control andhyperfiltering diabetic rats ( 63 ). At a low dose of L -NAME, diabetic rats demonstrated a blunted mean arterialpressure (MAP) response and a proportionately significant reduction inGFR and RPF compared with controls. At a supramaximal dose, the L -NAME-induced rise in MAP was similar indiabetic and control rats. However, renal vasoconstriction was greaterin the diabetic animals. U NOx V was increased in diabeticrats and significantly reduced by low-dose L -NAME.Furthermore, diabetic rats demonstrated no response to the NO donorglyceryl trinitrate (GTN), which induced significant renal vasodilationin control rats. We interpreted the lack of a GTN effect in thediabetic kidney as a further indication of enhanced NO productionand/or signaling that cannot be significantly altered by additional NO.Interestingly, this phenomenon was later quoted in support of impairedNO actions in the diabetic kidney ( 96 ). These results, inaccordance with the previous study, suggested a role for NO in thepathogenesis of hyperfiltration and increased renal NO generation indiabetes. Similar data, i.e., significant reduction in hyperfiltrationin anesthetized diabetic rats in response to L -NAME, werethen reported by Mattar et al. ( 78 ) and in several studiesthat focused on other issues ( 118, 141 ). Furthermore,extensive studies by Omer et al. ( 91 ) confirmed ourfindings in virgin and pregnant normal and diabetic rats. Both groupsdemonstrated marked hyperfiltration that was nearly normalized by L -NNA. Moreover, substantial increases in RPF in diabeticanimals, further enhanced by pregnancy, also showed marked sensitivityto NOS inhibition. Similar to our studies ( 63 ), the MAPresponse was attenuated in virgin diabetic animals compared withcontrols. These hemodynamic observations were associated with parallelchanges in U NOx V and increased baseline NO renal generationas assessed by the citrulline generation assay. Interestingly, thisgroup also found an enhanced response to ACh in aortic rings fromdiabetic rats ( 91 ).

Most recently, the effects of nonspecific NOS inhibition were evaluatedin hydronephrotic kidneys of control and moderately hyperglycemicfemale rats at 6 wk of diabetes ( 26 ). This model was usedto allow intravital microscopy for evaluations of changes in renalvascular diameters. Increased vasoconstrictor responses to L -NAME in diabetic rats, suggesting local hyperproductionand activity of NO, were apparent in all segments of the renalvasculature. One might argue that using the hydronephrotic kidney modelinvolves factors other than the diabetic milieu that would alter local NO production and therefore complicate interpretation of the results. However, renal vascular responses were in good accord with eNOS proteinexpression determined in vascular trees of intact kidneys.% u: O* q, N* `
7 [" i) C. u5 w6 a- U
Not all authors have found enhanced whole kidney hemodynamic responsesto NO inhibition. Using Doppler probes, Kiff et al. ( 61 )found similar responses to L -NAME in conscious control anddiabetic rats. Another study ( 37 ) reported rathercontroversial observations. In basal conditions, diabetic ratsdemonstrated marked increases in creatinine clearance (C Cr )and U NOx V compared with controls. L -Argincreased U NOx V in controls but not in diabetic rats. L -NAME had no effect in controls but decreasedU NOx V in diabetic animals. In further studies,uninephrectomized control and diabetic rats were evaluated after 60 minof renal ischemia. Control rats responded with less severedecreases in C Cr and higher U NOx V, whereasdiabetic animals demonstrated an opposite trend that was furtheraggravated by L -NAME. Despite the finding in basalconditions, the authors concluded that renal NO production is defectivein diabetes ( 37 ). Given the pathophysiological complexityof ischemic renal failure, it is also unclear whether kidneysafter 60 min of total occlusion of the renal artery represent anappropriate model for studies of the NO system in diabetes.

Pflueger et al. ( 96 ) applied videomicroscopy in controland STZ-diabetic rats to assess cortical and medullary blood flow insuperficial cortical capillaries and papillary vasa recta. Diabeticrats demonstrated basal increases in these parameters, as well asincreases in GFR (measured as inulin clearance in the contralateralkidney) and plasma NOx levels. U NOx V did not differ betweencontrol and diabetic rats. Despite increased blood flow and a higherGFR, diabetic rats showed attenuated responses of the cortical andpapillary microcirculations to both systemic NOS inhibition with L -NMMA and stimulation of NO synthesis with L -Arg. The unresponsiveness of NO synthesis inhibition wasgreatest in cortical capillaries. Thus, despite baselinehyperperfusion, these data suggest a defect in renal, and particularlyrenal cortical, NO production or action in diabetes in vivo. In aparallel study, the same group reported increased sensitivity ofdiabetic renal vasculature to adenosine due to defectivecounterregulatory NO production ( 97 ). Of note, increasesin GFR observed in contralateral kidneys in diabetic rats werenormalized by NOS inhibition. It is not clear how measurements of asingle capillary diameter and red blood cell velocity relate to thetraditional methods for determining renal function, because the changesin GFR were not reflected by the effects of NOS inhibition on corticalcapillaries in the contralateral kidney.: P& G1 t  C5 G6 k2 k/ d

Whereas the preceding studies relied on nonspecific NOS inhibitors, thecontribution of individual NOS isoforms to NO production in thediabetic kidney has only recently been evaluated. Research in thisdirection has been accelerated by the availability of new inhibitorsthat selectively block NOS isoforms.; m9 q( |) `/ C% d( b
" G' v: {! ?( ]3 Q- v8 ~
Studies by Wang et al. ( 146 ) represent a less frequentlyused approach to investigate in vivo whole kidney responses to NO modulation. In these studies, the authors evaluated responses tointravenous infusion of ACh. Therefore, these studies could beinterpreted as exploring isoform-specific NO production (eNOS). Therenal vasodilator response to ACh was diminished in diabetic rats butnot in normoglycemic diabetic rats. Acute treatment with insulin didnot restore the response to ACh, although the blood glucose level wasnormalized. These in vivo experiments further supported the in vitrodata by this group ( 146 ), suggesting reduced NObioavailability and/or impaired signaling in the renal vascular endothelium and the previously discussed possible defects inreceptor-mediated eNOS stimulation.. O; Q6 d. v& i  Q
3 B3 _7 R: _7 l) T7 x3 r+ r$ _2 V
There is abundant literature suggesting defective endothelium-dependent(i.e., eNOS dependent) NO production or function in diabetes, inapparent contrast to observations of substantial NO dependency of renalhemodynamics in hyperfiltering rats, as revealed with nonspecific NOSinhibitors. We hypothesized that increased renal nNOS activity couldexplain this paradox. This isoform is expressed in MD cells ( 7, 150 ), and nNOS-derived NO in MD decreases predominantly afferentarteriolar tone ( 51, 150 ), contributes to control ofintraglomerular pressure ( 124, 150 ), and counteractsafferent vasoconstriction induced by activation of tubuloglomerularfeedback (TGF) ( 51 ). We explored the acute effects ofsystemic nNOS inhibition with the specific nNOS inhibitor S -methyl- L -thiocitrulline (SMTC)( 89 ) in diabetic rats ( 66 ). SMTC inducedstronger renal vasoconstriction responses in diabetic compared withcontrol animals. Renal vasoconstriction was partly opposed by ANG IIAT 1 receptor blockade, suggesting an interaction ofnNOS-derived NO with ANG II. Further studies were designed to diminishthe possible systemic effects of SMTC ( 64 ). When administered directly into the abdominal aorta above the left renalartery, SMTC did not influence MAP but nearly normalized GFR inhyperfiltering diabetic rats. These observations indicate that nNOScontributes to altered renal NO production and hemodynamics inexperimental diabetes. Renal hemodynamic effects of SMTC were attenuated in normoglycemic diabetic rats, suggesting that alterations in nNOS activity are related to the level of metabolic control. Moreover, the nonspecific NOS inhibitor L -NAME did notinfluence GFR but further decreased RPF in diabetic rats pretreatedwith SMTC. These data suggest that nNOS is the major isoformresponsible for hyperfiltration, although other isoforms act in concertwith nNOS in the control of renal perfusion.

Similar results regarding the role of nNOS were more recently reportedby Ito et al. ( 55 ), using a different nNOS inhibitor [7-nitro indazole (7-NI)]. Furthermore, data by Schwartz et al. ( 112 ), showing enhanced renal hemodynamic response to anonspecific NOS inhibitor, in conjunction with an absence of responseto the iNOS blocker L - N 6 -(1-iminoethyl)lysine( L -NIL) and unchanged renal iNOS and eNOS mRNA expressions,indirectly point to nNOS. Further indirect support for the role of nNOSin diabetic alterations in glomerular hemodynamics can be derived fromobservations that the activity of TGF is blunted in diabetes( 135 ). The physiological roles of nNOS-derived NO ( 51 ) and its suggested higher production by MD cells indiabetes are consistent with this abnormality. In addition, Veelken et al. ( 141 ) reported the lack of effect of a selective iNOSinhibitor, L -NIL, in contrast to the effect of L -NAME, on renal hemodynamics in conscious STZ-diabeticrats. Because cortical eNOS expression was increased in diabetic rats,the authors suggested the role of eNOS in the pathogenesis ofhyperfiltration. However, these findings do not exclude the possibilitythat nNOS is involved in the process, because the reduction of GFR wasachieved with a nonspecific NOS inhibitor.' l6 h# A+ b+ d" Q- ^0 d. b  C

Unlike in vitro and in vivo studies focusing on renal NOS expression,we are able to identify some differences in in vivo hemodynamicstudies, suggesting enhanced renal NO production/activity in diabetesand those studies reporting opposite data. There is a clear distinctionbetween the two lines of evidence with respect to methods ofmeasurements of renal function. In those studies that used formalclearance techniques to determine GFR and RPF, diabetic ratsdemonstrated hyperfiltration and enhanced responses to NOS inhibition,suggesting increased renal production and/or activity. In contrast,studies with contrary evidence used either C Cr ortechniques requiring extensive instrumentation ( 96, 97 ).
7 m) [4 L9 F6 x. e) I- Y) _5 ]* l
Another factor that could influence the outcome of these studies is thepresence or absence of insulin treatment. Those investigators who usedinsulin treatment to achieve moderate hyperglycemia have generallyfound hyperfiltration and enhanced hemodynamic responses to NOSinhibition. One might argue that decreased renal hemodynamic responsiveness to NOS inhibitors has also been observed in moderately hyperglycemic rats. However, in those studies, despite an absence ofexogenous insulin treatment, residual endogenous insulin secretion prevented severe hyperglycemia ( 96, 97 ). As discussedbelow, the combination of moderate hyperglycemia and exogenous insulin treatment may be a crucial factor creating the milieu for NO-dependent diabetic hyperfiltration. This interpretation is in accord with theavailable micropuncture data ( 110 ). An exception to this rule may be the elegant studies of Carmines et al. ( 90, 111 ). However, the technique used by that group (evaluation ofarteriolar diameters in isolated blood-perfused nephrons) is still anin vitro technique. Consequently, arterioles and glomeruli in diabetic animals are not exposed to the same metabolic milieu as occurs in vivo.* W' J6 F1 P- [  C% Y9 J. ~: F- N
+ g& [8 K4 z' K; `
Many in vivo studies have buttressed their conclusions of enhancedrenal NO production by demonstration of increased U NOx V. However, these measurements should be interpreted with caution. Considering present knowledge of the distribution of NOS activities inthe kidney ( 154 ), and studies by Suto et al.( 120 ) that directly addressed this issue, it is clear thatmeasurements of urinary NOx can hardly reflect NO changes in thecortex. Although some attempts to directly determine renal NOproduction in vivo have been communicated, our present knowledge stillrelies mostly on indirect indicators of renal NOS activity. Directmeasurements of NO in the kidney represent some of the challenges forfuture research.2 Y/ F2 e& h/ b: d

Pieper ( 98 ) has suggested that the duration of diabetes isan important determinant for NO production in the vascular system, evolving from an increase during the early stages, followed by a periodof normal production, and then to a decrease in production as theduration of the disease increases. However, this construct does notreally explain the discrepancies in published data regarding eitherrenal NOS expression or in vivo studies of hemodynamics. The evidenceremains controversial irrespective of the duration of diabetes.; v6 F2 Y; A$ y- o4 c3 M
! V% G3 E( t2 K1 b7 F/ H$ ~
Effects of Long-Term Modulation of NO Synthesis in the DiabeticKidney  ~/ Q4 V& g& l- O- N3 P
. D, O+ Z" s) V: W
One would expect that long-term modulation of NO would ultimatelyanswer many questions regarding the role of NO in the development ofdiabetic nephropathy. However, even these types of studies have notprovided unequivocal data. Several studies have suggested anephroprotective role of NO in the development of nephropathy indiabetic rats. Reyes et al. ( 103 ) found lower proteinuria and amelioration of hyperfiltration in STZ-diabetic rats after 14 wk of L -Arg treatment. However, this study reported several findings that are difficult to interpret. Administration of L -Arg did not lead to increased cGMP and U NOx V;in fact, these parameters were lower in treated rats. Thus the studyfailed to provide proof that the decrease in proteinuria was mediatedvia increased renal NO production. Paradoxically, in both vehicle- and L -Arg-treated diabetic rats, urinary protein excretion washigher at baseline than after treatment, and the achieved valuesexceeded levels seen in animals with severe glomerulosclerosis.Furthermore, histological evaluation did not reveal any differences inglomerulosclerosis scores between L -Arg-treated andvehicle-treated rats.0 ~+ X: |, a! v; S- h8 |4 d

Further evidence for a possible renoprotective role of NO indiabetes was reported by Craven et al. ( 21 ).STZ-diabetic rats received nonpressor doses of L -NAME for 4 wk. At the end of the study, L -NAME-treated rats had slightly but significantly higher albuminuria, although still in the microgram range. Importantly, renaltransforming growth factor- (TGF- ) was increased in treated rats.However, the study was too short to determine whether chronic nonpressor L -NAME could accelerate the course ofnephropathy. This issue was addressed by Soulis et al.( 116 ), who found no effect of chronic (6 mo) treatmentwith a nonpressor dose of L -NAME on the progression ofnephropathy in STZ-diabetic rats. In addition, corresponding to ouracute studies ( 64 ), our preliminary data suggest thatlong-term nNOS inhibition with a nonpressor dose of SMTC modestlyretards development of renal injury in diabetes ( 67 ).

POSTRANSLATIONAL MODULATION OF NOS ACTIVITY: IMPACT OF DIABETES
9 E# n  E7 w. S! T! a8 {# F, x
In the previous sections, we focused on individual studies,pointed out some differences, and attempted to find some explanation for disparate findings in studies with generally similar designs. Inthe following paragraphs, we will discuss additional controversial issues that arise when one analyzes particular aspects of renal NOphysiology and pathophysiology with respect to the pathogenesis ofdiabetic nephropathy. To shed light on the issues, we will include someevidence from the extrarenal literature. Although we will discuss thefollowing issues separately, it is apparent that they represent aconglomerate of interacting mechanisms.

Changes in eNOS Conformation and Generation of ROS9 {; Z) s2 ^7 x; i" `
0 y( t- }$ e8 T0 a$ X4 |" |
To function as NO-producing enzymes, NOS requires a battery ofcofactors, conformational changes, and fatty acid acylations responsible for membrane targeting of the enzyme (reviewed in Refs. 39 and 81 ). A traditional pathway of eNOS activation involves receptor stimulation by agonists such as ACh or bradykinin, resulting in a mobilization of intracellular Ca 2  andconsequent interaction of calmodulin and eNOS, releasing the enzymefrom an inhibitory complex with caveolin-1 ( 58 ). On moreprolonged agonist stimulation, eNOS may be released from its membranelocation and translocated to other subcellular compartments. Thisreversible translocation may represent a mechanism to downregulate ordisconnect the enzyme from receptor occupancy ( 76 ).8 u/ [4 l' j% e- d( L* _# M

In addition to synthesizing NO, purified NOS catalyzesO 2 − formation under certain conditions, such astetrahydrobiopterin (BH4) or L -Arg deficiency ( 102, 138, 139, 155, 156 ). In this "uncoupled state," electronsflowing from the NOS reductase domain to the oxygenase domain arediverted to molecular oxygen rather than to L -Arg( 139, 148, 155 ). Thus NOS acts as an enzyme with dualfunctions, capable of producing both NO and ROS. It has been proposedthat formation of a dimer is crucial for NO production by NOS( 45 ). BH4 and calmodulin have been suggested as moleculesessential for dimer formation, although there the evidence for BH4 iscontroversial ( 45 ).

Enhanced production of ROS appears to be an important mechanism in thepathophysiology of diabetic complications, including nephropathy. Theirrole has been validated in long-term studies ( 71, 80 ). ROSmay be involved in diabetes-induced alterations in lipids and proteins,cellular signaling ( 40 ), as well as inactivation of NO.After generation by these enzymes, O 2 − may undergoreaction with NO, resulting in formation of cytotoxic ONOO, or may be transformed into the more stable radicalH 2 O 2. This reaction is catalyzed by SOD.H 2 O 2 is further metabolized by catalase. Twostudies determined nitrosylated protein (NT) expression in diabetickidneys by Western blotting as a measure of ONOO formation ( 54, 92 ). Both studies found increases in this parameter in diabetic kidneys, indicating enhanced interaction betweenNO and O 2 −. However, those studies did not provideinformation about localization of NT in the kidney. Furthermore, in thelatter study ( 93 ), renal tissue was analyzed as a wholewithout separation of cortex and medulla. Considering high NOSactivities in the medulla, it is not clear how much ONOO was formed in that compartment. Postmortem human data show increased NTformation in proximal tubules of patients with long-standing diabetescompared with kidneys from patients with glomerulonephritis or normalkidneys ( 125 ). Thus far, then, there is no proof of enhanced NT formation in the renal vascular tree in diabetes.

There are several sources of ROS ( 108 ). Some investigatorshave addressed the question of whether eNOS could be an important source of ROS in diabetes. Cosentino et al. ( 20 ) reportedincreased eNOS expression associated with parallel increases in NO andO 2 − in human aortic endothelial cells exposed to highglucose (22.2 mM) for 5 days. Hink et al. ( 46 ) found thatimpaired endothelium-dependent vasodilation in STZ-diabetic rats wasassociated with increased vascular eNOS expression and ROS production.Because the increase in ROS production by diabetic vessels wascorrected by NOS inhibition, the results suggested uncoupled eNOS as animportant source of ROS in diabetic arteries. In addition, the authorsshowed that PKC inhibition might prevent eNOS uncoupling. Themechanisms described in aortic tissue could also be active in thekidney. Indeed, Ishii et al. ( 54 ) have recentlydemonstrated increased NOS activity and larger quantities ofO 2 − produced in the diabetic rat kidney cortex.Further evidence suggests that eNOS uncoupling could be prevented byBH4, identifying diabetes as a state of BH4 depletion( 99 ).
: s# x& ]5 }9 f  @
In an aforementioned in vitro report, L -Arg had improvedbasal, but not ACh-stimulated, cGMP production by isolated glomeruli ( 23 ). This phenomenon suggests a specificglucose-induced defect in receptor-mediated eNOS activation,mediated by factors already mentioned ( 23 ) or, forexample, by ROS-induced alterations in eNOS-caveolae interactions, eNOScellular localization, and cofactor integrity ( 56, 95 ).1 Y2 `+ w, F2 L5 X3 ?; J# Q( B
: s$ [: {& }1 G+ G! _" @
The fact that eNOS undergoes important posttranslational changes andprotein-protein interactions documents the importance of more detailedexperimental approaches in evaluating NOS expression in the diabetickidney. For example, the changes in renal expression of NOS mRNA ortotal protein in the diabetic kidney may not reflect the functionalstatus of the enzyme. Because most of the evidence concerning eNOSexpression in the diabetic kidney has not taken into account thequaternary structure of NOS and other posttranslational modifications,it is difficult to draw links between most of the available knowledgeconcerning renal NOS expression and its function. Considering thedisparate findings in some studies focusing on eNOS-mediated vascularreactivity in diabetes, it is possible that the results depend on theactual balance between NO and O 2 −. This balance couldbe influenced by a number of factors, including the experimental technique.; R  {* O* ~, x3 }4 c$ ]9 {
& ?! C9 n1 W$ y; [* m3 ^) K
We have recently attempted to address some of these issues. Ourpreliminary data suggest alterations in some of those posttranslational modifications and other functionally important characteristics, such asdecreased formation of the NO-producing eNOS dimer, alterations inmembrane eNOS targeting, and eNOS- caveolin-1 interaction in thediabetic renal cortex ( 65 ). Further documenting the need for more detailed analytic approaches to assess renal NOS, apreliminary report by Carmines et al. ( 15 ) demonstratedreduced renal cortical expression and function of heat shock protein 90 in diabetes, another protein cofactor facilitating eNOS catalytic activities.

Considering the data from long-term studies ( 71, 80 ), onewould not question the contribution of ROS to the development ofnephropathy, although beneficial effects of antioxidant treatment remain to be established in large clinical trials. However, the datasuggesting the contribution of NO-ROS interactions to alterations inrenal vasomotor function in diabetes raise several questions. First,this evidence suggests that O 2 −, a renalvasoconstrictor, decreases bioavailability of NO or limits thebuffering capacity of NO against vasoconstrictors ( 111 ). Therefore, one would expect that the net effect of this imbalance wouldresult in renal vasoconstriction. However, this is difficult toreconcile with the fact of diabetic hyperfiltration. Unlike most of theother groups, Carmines et al. ( 16 ) have offered anexplanation for this contradiction. They suggested that a decrease inafferent arteriolar tone in diabetes is a result of a functional defectin afferent arteriolar voltage-gated Ca 2  channels, leadingto impaired vasoconstriction. In addition, studies in isolated nephronsand arterioles ( 90, 109 ) that provided the most compellingevidence for the ROS-NO interaction in the control of renalhemodynamics in diabetes did not take into account NO fromnonendothelial sources, such as MD.
& U6 ~* }% l: w( H; x9 K
Second, we are not aware of any evidence demonstrating the effect ofO 2 − scavenging with SOD or a SOD mimetic on basalarteriolar tone in diabetic rats. Considering theNO-O 2 − interaction, one would expect enhancedSOD-induced afferent vasodilator responses not only during AChstimulation ( 109 ) but also in the basal, unstimulatedstate. However, the SOD mimetic tempol did not alter basal afferenttone in diabetic rabbits ( 109 ). Corresponding to vascularreactivity studies, antioxidant treatment did not alter basal mesangialcell NO production in high-glucose media ( 132 ).

Third, O 2 − dismutation by SOD results in formation ofH 2 O 2. Unlike O 2 −, H 2 O 2 acts as a renal vasodilator ( 108, 137 ). Provided that renal arteriolar NO and O 2 − are produced in large quantities, vascular responses to SOD aredetermined by protection of NO from quenching, by the elimination ofthe vasoconstrictor actions of O 2 −, and by furthervasodilator effects of H 2 O 2. It has beensuggested that H 2 O 2 could be one of putativeendothelial vasodilators often referred to as endothelium-derived hyperpolarizing factor ( 137 ). Therefore, the effects ofSOD on ACh-stimulated vascular reactivity may be attributable, in part, to H 2 O 2. However, it should be noted that therole of H 2 O 2 is not supported by data obtainedwith tempol. This substance has similar effects to those of SOD onNO-dependent responses in diabetes, but it also enhancesH 2 O 2 dismutation ( 72 ), presumablyeliminating the contribution of H 2 O 2. Fourth,the most persuasive evidence for potentiation of NO-dependentvasodilation by O 2 − quenching was obtained in vitro.In vitro systems may be predisposed to the formation of ROS. One ofthose mechanisms could be a lack of L -Arg in cultivationmedia or perfusion solutions. As already mentioned, eNOS uncouplingcould be enhanced in situations with reduced substrate availability( 139 ). In contrast, in vivo studies demonstrated thatantioxidant treatment may normalize hyperfiltration in diabetes( 71 ). Considering the decrease in the filtration fractionin diabetic rats treated with antioxidants observed in that study, onewould expect efferent arteriolar effects of antioxidant treatment.These data suggest that these effects in the renal microvasculature areattributable not only to protection of NO from quenching but also toother mechanisms. One of the mechanisms compatible with the long-termglomerular hemodynamic action of antioxidants could be, for example,inhibition of ANG II signaling via ROS ( 40 ).& Z! c* {% L# b+ c9 g' s6 O
2 V: O# K7 l) I4 W5 P7 d
Effects of Diabetes on Signaling Pathways That Modulate RenalActivity and/or Expression of NOS

In vitro evidence has demonstrated that eNOS is regulated bycoordinated signaling via phosphorylation and dephosphorylation oftyrosine, serine, and threonine amino acid residues ( 32 ). For example, phosphorylation of Ser1177/1179 leads to activation of theenzyme ( 42 ), whereas phosphorylation of Thr495 has an opposite effect ( 33 ). Diabetes, by altering varioussignaling pathways, may theoretically influence the phosphorylationstatus of eNOS and presumably also of other NOS isoenzymes.

The impact of PKC activation in the pathophysiology of nephropathy hasbeen well established ( 70 ). Phosphorylation of eNOS by PKCinhibits its catalytic activity ( 47 ), and thisinhibition is accomplished by phosphorylation of Thr495 anddephosphorylation of Ser1177 ( 82 ). Specifically, thePKC- isoform has been implicated in the pathophysiology ofcomplications ( 69 ). This isoform inactivates eNOS inendothelial cells and microvessels of insulin-resistant Zucker rats( 75 ). Identification of the link between PKC and eNOSactivity provides strong support for the concept of NO insufficiency inthe diabetic kidney (Fig. 1 ).0 k$ E/ Q, m; p5 ?

Fig. 1. Schematic presentation of mechanisms associated withmetabolic milieu that can result in both activation as well assuppression of the renal nitric oxide (NO) system. eNOS and nNOS,endothelial and neuronal nitric oxide synthase, respectively; TGF-,transforming growth factor-.

More recently, other signaling pathways have been identified asmodulators of NOS activity along with evidence suggesting alterationsof these pathways in diabetes. Akt kinase (PKB), a downstream effectorof phosphatidylinositol 3-kinase, has been identified as the kinaseresponsible for Ca 2  -independent activation of eNOS( 29, 34 ). Akt activates eNOS by Ser1177/1179phosphorylation. This kinase has been implicated in cellular signalingof factors relevant to diabetic complications, such as insulin( 162, 163 ), VEGF ( 34 ), ANG II ( 38, 121, 134 ), leptin ( 140, 152 ), TGF- ( 17 ),and shear stress ( 42 ) (Fig. 1 ). Recently, Feliers et al.( 31 ) demonstrated an increase in Akt expression andactivity in the renal cortex of db/db mice, a genetic modelof type 2 diabetes.

We have repeatedly emphasized the presence or absence of insulintreatment preventing severe hyperglycemia as a possible contributor todisparate findings in a number of the preceding studies. In ouropinion, the crucial role of exogenous insulin is not merely inmodulating blood glucose levels but also in its ability to influence NOsynthesis. Indeed, early after induction of diabetes, residualsecretion of insulin may be sufficient to achieve moderate hyperglycemia. However, to achieve given levels of blood glucose, theamount of exogenous insulin could be substantially greater comparedwith the amount of endogenously secreted hormone. Thus, acting via Akt,insulin could be an independent activator of eNOS in various tissuesincluding the kidney. This pathway would not be reflected, for example,in studies using stimulation with ACh, a widely accepted approach fortesting eNOS function. We cannot exclude that impaired agonist-inducedNO production, as observed in a number of studies, could becounterbalanced by alternative signaling pathways, such as Akt. Localactivation of Akt by hyperinsulinemia may be an example of such asituation (Fig. 1 ).; j8 R- i" j* j* n" s/ z+ G

Some investigators have suggested a defect in Akt signaling as one ofthe sites of insulin resistance ( 18, 73 ), a hallmark feature of type 2 diabetes. An important issue is whether resistance tometabolic actions of insulin affects not only metabolic but alsovasoactive or renal actions of the hormone. An increase in Aktexpression in the kidney in insulin-resistant mice suggests that thesetwo processes may be separated ( 31 ). However, opposite evidence suggesting resistance to insulin-induced NOS activation alsoexists ( 75 ). Furthermore, another line of in vitroevidence suggests that in high glucose, the Akt-dependentphosphorylation site responsible for eNOS activation can be modified by N -acetyl glucosamine ( 30 ), in a process linkedto mitochondrial O 2 − production. There is alsoconflicting evidence with respect to the possible impact of PKC on Aktsignaling, resulting in eNOS activation ( 75, 133 ).2 A) [0 z+ W8 Y- k& a
- R/ b8 _$ q6 W
Unlike eNOS, there is much less evidence on the effects of insulin anddiabetes-induced signaling pathways on nNOS activity. However, there isindirect evidence from other tissues suggesting that insulin couldactivate nNOS. For example, pial arteriolar vasodilation associatedwith insulin-induced hypoglycemia is mediated by nNOS-derived NO( 106 ). Similar to eNOS, rat nNOS contains an Akt-dependentphosphorylation motif ( 34 ). However, phosphorylation ofthis motif has not been shown to significantly modulate NO productionby the enzyme ( 34 ).
. g3 z5 P( V9 z' [7 f9 u4 B
Although cellular signaling pathways possibly modulating nNOS activityin the diabetic kidney have not been elucidated, other mechanismsinvolved in renal nNOS regulation have been identified. Importantly,some of these mechanisms may be active in the diabetic kidney. Underphysiological conditions, sodium delivery to the distal tubule is themajor acute determinant of nNOS activity in the MD cells ( 51, 149 ). However, based on micropuncture studies by Vallon et al.( 135 ) showing a decrease in solute content in early distaltubular fluid in diabetic rats, stimulation of nNOS by increased solutedelivery to the MD in diabetes is unlikely. Welch and Wilcox( 147 ) reported that blunting of TGF responses by NO couldbe limited by L -Arg availability in the tubular lumen andby its uptake via the y transport system. To ourknowledge, the availability of L -Arg in diabetic comparedwith normal kidneys remains unknown. Indirect clues that couldhelp elucidate this issue are rather conflicting. Plasma L -Arg levels are decreased in diabetes ( 100 ),but urinary L -Arg excretion has been reported to bemarkedly increased ( 103 ). Renal nNOS is chronicallyactivated in parallel with the renin-angiotensin system (RAS) in suchpathophysiological states as two-kidney, one-clip hypertension,furosemide treatment, or dietary sodium restriction ( 12 ).Assuming that a low-sodium diet decreases distal sodium delivery, thismechanism corresponds to the situation in the distal tubule asdescribed by Vallon et al. ( 135 ) in diabetic rats and thusrepresents a possible pathway resulting in activation of nNOS.

NO AND RENAL STRUCTURAL CHANGES IN DIABETES$ `7 W- K. i- y1 w3 ?9 U' u) G; r

In the diabetic kidney, most cells undergo hypertrophy. Thisprocess, together with accumulation of extracellular matrix, underliesrenal structural changes in diabetes that are characterized bymesangial expansion ( 93 ), later development ofglomerulosclerosis, and by tubular hypertrophy and later interstitialfibrosis ( 36 ). Data reported by several groups haveprovided evidence that this hypertrophy is, at least in part,attributable to altered cell cycle regulation. This complex process,reviewed elsewhere ( 151 ), is associated with increasedexpression of cyclin-dependent kinase (CDK) inhibitors, such asp21 Cip1 and p27 Kip1 ( 74, 153 ),resulting in G 1 -phase arrest ( 151 ). Thesemolecules can be induced by glucose and other mediators of the diabetic milieu, such as glucose-TGF- or RAS-TGF- pathways( 151 ), molecules whose importance in the pathophysiologyof diabetic nephropathy is well documented ( 2 ).
# Y8 r7 ?+ @+ ?( \' L
Antiproliferative actions of NO on mesangial cells or vascular smoothmuscle cells, and its tendency to shift cells into the hypertrophicphenotype, have been recognized for over a decade ( 35, 57, 144 ). Later studies have provided evidence for mechanisms responsible for these actions ( 41, 53, 114, 122 ). NOdirectly modulates smooth muscle cell cycle progression by upregulation of p21 Cip1 and inhibition of cyclin-dependent kinase 2 activity. Thus it appears that NO growth actions in the kidney displaysubstantial similarities to the diabetic milieu. Importantly, theseeffects of NO are independent of cGMP generation ( 122 ).Furthermore, TGF- can induce eNOS in vitro ( 52 ) as wellas in vivo in both diabetic ( 94 ) and nondiabetic tissues( 101, 158 ). TGF- -induced activation of eNOS may involveAkt kinase signaling ( 17, 94 ) (Fig. 1 ).) R; Q3 [+ v) E9 `3 e9 P
0 E( I6 j$ X" \
Another factor implicated in the pathogenesis of diabetic nephropathyand signaling via Akt is VEGF ( 27 ). Reviewing the data onVEGF well illustrates the controversial nature of evidence discussed inthis review. There is abundant evidence suggesting NO as a mediator ofVEGF actions ( 44, 48, 136 ) (Fig. 1 ). However, some actionsof VEGF presumably mediated by NO, such as endothelial proliferation( 87 ), are not typically observed in the diabetic kidneyand are not in accordance with the effects of NO on cellular trophicstatus discussed above. In contrast, other effects, such as localNO-dependent increases in vascular permeability, vasodilation, orglomerular hypertrophy, are well consistent with diabetic renalpathophysiology ( 27, 126 ). In any case, provided that thehypothesis about the role of VEGF is valid, it is hard to reconcilewith those findings suggesting a NO deficit in the diabetic kidney.$ [4 p; V# l, l2 Q. b8 k7 u4 c( C

As with other issues discussed in this review, convincing contradictoryevidence has also been reported in this area, suggesting that a NOdeficit may underlie the development of renal structural changes indiabetes. Several in vitro reports have indicated that NO can interferewith pathways, resulting in TGF- induction by high glucose or othermechanisms active in the diabetic kidney ( 24, 117 ). It hasrecently been reported that TGF- suppression by NO in mesangialcells cultured in high glucose is mediated by downregulation ofthrombospondin-1 ( 145 ). This view was further supported byobservations that the RAS-TGF- axis is involved in mediating organinjury during chronic NOS inhibition in diabetic ( 21 ) andnondiabetic models ( 59, 129 ). Thus mechanisms discussed inthis section suggest that both increased as well as decreased renal NOactivity may result in characteristic changes in diabetic renal morphology.  m+ B7 ~7 ~1 j) O$ x" w7 R

SUMMARY

We have reviewed a wide spectrum of findings and issues that haveamassed concerning the pathophysiology of the renal NO system indiabetes. It is apparent that it might be practically impossible toreconcile such complex and controversial evidence and find a unifyingscenario characterizing these processes. However, we believe that somepatterns are emerging. One of major general controversies existsbetween the in vitro findings, which generally suggest decreasedbioavailability of NO in the diabetic kidney, and in vivo observations,which tend to suggest enhanced renal NO production and/or activity indiabetes, at least in the early stages. We believe that the diabeticmilieu is complex and that in vitro approaches may miss some importantmechanisms that comodulate NO activity in a particular system. On theother hand, these studies are indispensable in the identification ofprecise mechanisms resulting in alterations of the NO system indiabetes. Most importantly, in vitro studies describe processes wherebyhigh glucose levels, a hallmark metabolic feature of diabetes, exertdeleterious effects on NO bioavailability.& d! P& a( z9 z4 ^4 O4 W0 k8 t

There is little doubt that severe diabetes with profound insulinopeniacan be viewed as a state of general NO deficiency, including in thekidney. However, it is important to note that we focus our hypothesesand conclusions on the events occurring during moderate glycemiccontrol, with some degree of treatment with exogenous insulin (Fig. 1 ).This situation represents more the clinically applicable model mostclosely resembling the situation in patients destined for developmentof nephropathy.( p7 E& t0 v; F& y5 T
+ [2 l+ y, |  j# |+ _/ @1 y
Diabetes triggers mechanisms that in parallel enhance and suppress NObioavailability in the kidney (Fig. 1 ). We hypothesize that during theearly phases of nephropathy, the balance between these two opposingforces is shifted toward NO. This plays a role in the development ofcharacteristic hemodynamic changes and may contribute to consequentstructural alterations in glomeruli. Both eNOS and nNOS can contributeto altered NO production. These enzymes, in particular eNOS, can beactivated by Ca 2  -independent and alternative routes ofactivation that may be elusive in traditional methods of investigation.As the duration of exposure to the diabetic milieu increases, factorsthat suppress NO bioavailability gradually prevail. Increasingaccumulation of AGE may be one of the culprits in this process. Inaddition, this balance is continuously modified by actual metaboliccontrol and the degree of insulinopenia.
! o% Q9 d( @" e! V! J3 U: M
Alterations of the NO system in the diabetic kidney and their role inthe pathophysiology of diabetic nephropathy still represent a greatchallenge for future research. There are a number of topics in thisarea that warrant further investigation. Future investigations in thisarea may focus on, among other topics, direct in vivo measurements ofNO production in different compartments of the diabetic kidney andtheir changes in response to various stimuli; the biochemistry of eNOSand nNOS with respect to the changes in quaternary structure andcellular localization and posttranslational changes; and activity ofsignaling pathways, leading to modulation of NOS activities, as well ason possible alterations in NO signaling. Considering the epidemicincrease in type 2 diabetes-associated nephropathy, studies shouldfocus on evaluating these systems in models of type 2 diabetes., g- X) e4 C; q# r2 t, C  p
, _  I" e! v7 s
ACKNOWLEDGEMENTS# L& q' a4 F5 w  U/ m3 b$ c
. Y1 x+ v" \1 i9 F% z
This work was supported, in part, by National Institute on AgingGrant AG-14699, the American Diabetes Association, and the JuvenileDiabetes Research Foundation. R. Komers is also supported by GrantVZ/CEZ L17/98:00023001 from the Ministry of Healthcare, Czech Republic.0 O0 t5 \$ i$ [5 q/ f/ E
      【参考文献】
1. Ahn, KY,Mohaupt MG,Madsen KM,andKone BC. In situ hybridization localization of mRNA encoding inducible nitric oxide synthase in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 267:F748-F757,1994 .: G( d8 @( x1 S; F6 g. e

3 u" D8 _% Y& O6 b. \
2 ]* Y1 }% e2 z" T
2. Al-Douahji, M,Brugarolas J,Brown PA,Stehman-Breen CO,Alpers CE,andShankland SJ. The cyclin kinase inhibitor p21WAF1/CIP1 is required for glomerular hypertrophy in experimental diabetic nephropathy. Kidney Int 56:1691-1699,1999  .9 o  `1 U- Y3 v7 j- m: }1 ?: @

9 ]4 [# }  g9 v% @. p3 t

3. Alabadi, JA,Miranda FJ,Llorens S,Ruiz de Apodaca RF,Centeno JM,andAlborch E. Diabetes potentiates acetylcholine-induced relaxation in rabbit renal arteries. Eur J Pharmacol 415:225-232,2001  .) H8 }9 k- q9 B% j+ d; s

8 b! ~& k8 M( Q' _& e' P

4. Alberola, A,Pinilla JM,Quesada T,Romero JC,Salom MG,andSalazar FJ. Role of nitric oxide in mediating renal response to volume expansion. Hypertension 9:780-784,1992.

5 A. b) i7 f. G0 A
5. Anderson, S,andKomers R. Pathogenesis of diabetic glomerulopathy: the role of glomerular hemodynamic factors.In: The Kidney and Hypertension in Diabetes Mellitus, edited by Mogensen CE.. Boston, MA: Kluwer, 2000, p. 281-294./ K# e5 n% u8 y' D6 V/ a% _
( i6 a+ y; s7 t* }: G* e

6 Z; a$ w8 v6 V( a9 q. L* L
6. Arima, S,Ito S,Omata K,Takeuchi K,andAbe K. High glucose augments angiotensin II action by inhibiting NO synthesis in in vitro microperfused rabbit afferent arterioles. Kidney Int 48:683-689,1995  .+ u* Z( A0 {' @; i% |+ c9 G

, b1 W' t# W$ B1 G4 ^! X

7. Bachmann, S,Bosse HM,andMundel P. Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney. Am J Physiol Renal Fluid Electrolyte Physiol 268:F885-F898,1995 .

+ @1 P+ J7 W; z$ @3 k) ]9 t

8. Bank, N,andAynedjian HS. Role of EDRF (nitric oxide) in diabetic renal hyperfiltration. Kidney Int 43:1306-1312,1993  .* T, }% k* i! l; O5 ~# Z8 w. i

9. Baylis, C,andQiu C. Importance of nitric oxide in the control of renal hemodynamics. Kidney Int 49:1727-1731,1996  .
7 o6 V( U. ]7 A* X( z

10. Beenen, OH,Mathy MJ,Pfaffendorf M,andvan Zwieten PA. Vascular responsiveness in isolated perfused kidneys of diabetic hypertensive rats. J Hypertens 14:1125-1130,1996  .2 I" h7 `+ l1 f& K2 I4 W( a

# V! s+ ^0 v9 r+ @9 h' s
9 ^2 g* [9 G9 o' S
11. Bhardwaj, R,andMoore PK. Increased vasodilator response to acetylcholine of renal blood vessels from diabetic rats. J Pharm Pharmacol 40:739-742,1988  .
$ V+ o. _! H2 P! I& D

12. Bosse, HM,Bohm R,Resch S,andBachmann S. Parallel regulation of constitutive NO synthase and renin at JGA of rat kidney under various stimuli. Am J Physiol Renal Fluid Electrolyte Physiol 269:F793-F805,1995 .
# o; x) K$ L" a) H$ P
7 B% Y3 {8 s7 V" S: ^( c) Z

13. Brodsky, SV,Morrishow AM,Dharia N,Gross SS,andGoligorsky MS. Glucose scavenging of nitric oxide. Am J Physiol Renal Physiol 280:F480-F486,2001 .# N9 n% B8 z! [  G& n; Y! `# Y* e

# \* ]7 Y  ^0 b2 Z! ]$ i; N- \
14. Bucala, R,Tracey KJ,andCerami A. Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes. J Clin Invest 87:432-438,1991  .3 _6 C4 z& N! ]( ?% z, A

' e1 T' j$ V& {/ ^/ c  C
15. Carmines, PK,Fallet RW,Roscow J,Sasser JM,andPollock JS. Renal cortical Hsp90 and its impact on NOS activity and afferent arteriolar tone are diminished in diabetes mellitus (Abstract). J Am Soc Nephrol 13:532A,2002.8 R4 V3 x7 J' a+ y

" D+ |$ Y9 a% r  r2 s3 ?
16. Carmines, PK,Ohishi K,andIkenaga H. Functional impairment of renal afferent arteriolar voltage-gated calcium channels in rats with diabetes mellitus. J Clin Invest 98:2564-2571,1996  .
0 c- Z9 J1 i, [& q* r  C6 l: K6 v

2 j& D9 B& h5 L! l5 W, n8 i7 Q/ M
17. Chen, H,Li D,Saldeen T,andMehta JL. TGF- 1 modulates NOS expression and phosphorylation of Akt/PKB in rat myocytes exposed to hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol 281:H1035-H1039,2001 .
9 `% }3 o) G4 Z  w6 [  v/ q

18. Cho, H,Mu J,Kim JK,Thorvaldsen JL,Chu Q,Crenshaw EB, III,Kaestner KH,Bartolomei MS,Shulman GI,andBirnbaum MJ. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB ). Science 292:1728-1731,2001 .! d! o8 ], B0 y" W$ C

+ m3 W5 G7 w7 @7 C; m5 G5 \
- o7 A( o( x( C5 Y, L. $ o( F4 e
19. Choi, KC,Kim NH,An MR,Kang DG,Kim SW,andLee J. Alterations of intrarenal renin-angiotensin and nitric oxide systems in streptozotocin-induced diabetic rats. Kidney Int 52, Suppl 60:S23-S27,1997 .) U2 N; T( M- I& $ M' s6 ^

- ~9 O/ |* {& A8 O- s, W3 g8 u

20. Cosentino, F,Hishikawa K,Katusic ZS,andLuscher TF. High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation 96:25-28,1997 .6 [  O# H1 ], r+ G8 U

21. Craven, PA,DeRubertis FR,andMelhem M. Nitric oxide in diabetic nephropathy. Kidney Int 52, Suppl 60:S46-S53,1997.! T) X8 }- q4 e5 \. w7 v

" l  s+ M( W/ N2 x% G) @; R
22. Craven, PA,Studer RK,andDeRubertis FR. Impaired nitric oxide release by glomeruli from diabetic rats. Metabolism 44:695-698,1995  .

$ U, A8 ~1 ?9 F2 G9 v4 s! i! H/ m
23. Craven, PA,Studer RK,andDeRubertis FR. Impaired nitric oxide-dependent cyclic guanosine monophosphate generation in glomeruli from diabetic rats. Evidence for protein kinase C-mediated supression of cholinergic response. J Clin Invest 93:311-320,1994  .! q1 ?- Y* U  z4 @

( a6 j, E8 d! d* Y/ t# ^( f
5 {* L1 d9 N- s, W" S  ]$ o& F  O
24. Craven, PA,Studer RK,Felder J,Phillips S,andDeRubertis FR. Nitric oxide inhibition of transforming growth factor-beta and collagen synthesis in mesangial cells. Diabetes 46:671-681,1997 .

25. Dai, FX,Diederich A,Skopec J,andDiederich D. Diabetes-induced endothelial dysfunction in streptozotocin-treated rats: role of prostaglandin endoperoxides and free radicals. J Am Soc Nephrol 4:1327-1336,1993 ." s* b( V* U7 x% A

26. De Vriese, AS,Stoenoiu MS,Elger M,Devuyst O,Vanholder R,Kriz W,andLameire NH. Diabetes-induced microvascular dysfunction in the hydronephrotic kidney: role of nitric oxide. Kidney Int 60:202-210,2001  .' y5 j) c) v0 v8 @
# n) a6 z- L) U
( z8 R) l6 o8 k! x

27. De Vriese, AS,Tilton RG,Elger M,Stephan CC,Kriz W,andLameire NH. Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. J Am Soc Nephrol 12:993-1000,2001 ." t5 D3 Y; \5 k; `0 S, y
9 ]. ~* L& ]. c0 l! Z- r3 l+ O" m
8 W! v; C$ R$ V9 {
: A8 S" c" b4 U+ o
28. Deng, A,andBaylis C. Locally produced EDRF controls preglomerular resistance and ultrafiltration coefficient. Am J Physiol Renal Fluid Electrolyte Physiol 264:F212-F215,1993 .* S1 E% U7 }$ Y8 a' I  p; Q

( B9 y8 H+ \8 U# y

29. Dimmeler, S,Fleming I,Fisslthaler B,Hermann C,Busse R,andZeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399:601-605,1999 .% J7 W# r; k' r( R
+ t/ ^# T8 |7 `, M  W( w0 l: b9 T
) [/ ^4 c7 r8 {4 K1 Q! m
& G8 r3 d6 P. d% k$ N( h
30. Du, XL,Edelstein D,Dimmeler S,Ju Q,Sui C,andBrownlee M. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest 108:1341-1348,2001  .- a' m+ {% o' j

' h1 K1 O& R) [7 G" p4 y$ |
31. Feliers, D,Duraisamy S,Faulkner JL,Duch J,Lee AV,Abboud HE,Choudhury GG,andKasinath BS. Activation of renal signaling pathways in db/db mice with type 2 diabetes. Kidney Int 60:495-504,2001  .
7 H! i) v1 E; Y- m& w1 I4 h
( [' v) K7 R% g! _- {# t
, W! h0 X( @( w: t, X9 X
32. Fleming, I,Bauersachs J,Fisslthaler B,andBusse R. Ca 2  -independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress. Circ Res 82:686-695,1998 .

& O+ B$ D; \' e$ C

33. Fleming, I,Fisslthaler B,Dimmeler S,Kemp BE,andBusse R. Phosphorylation of Thr 495 regulates Ca 2  /calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res 88:E68-E75,2001  .
* u( ^- p" f% H  m
( Y5 M5 u: j  I1 J! R
- b; H* ]" u* A
34. Fulton, D,Gratton JP,McCabe TJ,Fontana J,Fujio Y,Walsh K,Franke TF,Papapetropoulos A,andSessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399:597-601,1999 .) ^- i8 ^3 |% ?- c% \9 {! v4 l" q

* d# B3 V+ B. M) i) V
35. Garg, UC,andHassid A. Inhibition of rat mesangial cell mitogenesis by nitric oxide generating vasodilators. Am J Physiol Renal Fluid Electrolyte Physiol 257:F60-F66,1989 .4 B$ S% K" |  S. q2 m9 l4 o! Z, ]
* H% T4 o4 ^( k

* H% R. k2 C- N$ G6 ^' @
36. Gilbert, RE,andCooper ME. The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury? Kidney Int 56:1627-1637,1999  .7 V; k2 m' |2 U: @0 W2 d, }! Q

37. Goor, Y,Peer G,Iaina A,Blum M,Wollman Y,Chernihovsky T,Silverberg D,andCabili S. Nitric oxide in ischaemic acute renal failure of streptozotocin diabetic rats. Diabetologia 39:1036-1040,1996  .) _# A: l) R8 O3 w
6 I3 N/ y! V$ U* h. j# S. i9 T
8 \( f' u) _! C$ D' U: {2 b' M" O
$ w6 ^0 B5 F1 F$ }; h
38. Gorin, Y,Kim NH,Feliers D,Bhandari B,Choudhury GG,andAbboud HE. Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase. FASEB J 15:1909-1920,2001 .1 S, S/ c$ I* K3 c1 a
" a: m3 `6 p6 M  N; D; f; g2 }

. I. f! U/ I* Z0 a3 x0 F, e, ?
39. Govers, R,andRabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol 280:F193-F206,2001 .& G1 }! A8 J6 h% q% c
, w2 q& U; Q; g# N/ v" M4 d

6 T0 h/ k$ H" [1 _, X
40. Griendling, KK,andUshio-Fukai M. Reactive oxygen species as mediators of angiotensin II signaling. Regul Pept 91:21-27,2000  .7 z1 Z) V9 k! O: R  u( @
6 n9 T4 ^$ B* V+ x+ T% u' Y

8 N/ E2 n4 `4 m- \
41. Guo, K,Andres V,andWalsh K. Nitric oxide-induced downregulation of Cdk2 activity and cyclin A gene transcription in vascular smooth muscle cells. Circulation 97:2066-2072,1998 .

0 y, d; V& E2 E+ ~9 g8 f+ D
- v; J2 f; z5 p
42. Harris, MB,Ju H,Venema VJ,Liang H,Zou R,Michell BJ,Chen ZP,Kemp BE,andVenema RC. Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation. J Biol Chem 276:16587-16591,2001 .6 h+ H7 x5 f0 n5 n1 E' k
' R% Z5 d$ Q; l9 M- I
/ A) Y. _5 N6 T- ?. Y2 `

43. Hayashi, K,Loutzenhiser R,Epstein M,Suzuki H,andSaruta T. Multiple factors contribute to acetylcholine-induced renal afferent arteriolar vasodilation during myogenic and norepinephrine- and KCl-induced vasoconstriction. Studies in the isolated perfused hydronephrotic kidney. Circ Res 75:821-828,1994 .
# z0 i9 c! k3 R4 ]- A
. I, P( _9 x9 R
2 o' E+ Q( H: H$ ?7 u/ Y
44. He, H,Venema VJ,Gu X,Venema RC,Marrero MB,andCaldwell RB. Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through flk-1/KDR activation of c-Src. J Biol Chem 274:25130-25135,1999 .1 s* D) }- ^8 O7 A3 W

& J9 n. X, K( D# M/ P: E% e" l

45. Hellermann, GR,andSolomonson LP. Calmodulin promotes dimerization of the oxygenase domain of human endothelial nitric-oxide synthase. J Biol Chem 272:12030-12034,1997 .9 ^4 Q7 u# Y5 N' x- O8 y
8 A8 X3 Y! }4 P! x! ~7 U$ \, p* l
. V, ]$ u. I9 C, d  `

46. Hink, U,Li H,Mollnau H,Oelze M,Matheis E,Hartmann M,Skatchkov M,Thaiss F,Stahl RA,Warnholtz A,Meinertz T,Griendling K,Harrison DG,Forstermann U,andMunzel T. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res 88:E14-E22,2001  .
) C0 ]9 v" P7 V( n

( l" x7 A- U0 D& J( C2 O+ t
47. Hirata, K,Kuroda R,Sakoda T,Katayama M,Inoue N,Suematsu M,Kawashima S,andYokoyama M. Inhibition of endothelial nitric oxide synthase activity by protein kinase C. Hypertension 25:180-185,1995 .7 q* }% G8 i1 N/ l( P

. a5 \1 s3 ]7 Y7 F0 \

48. Horowitz, JR,Rivard A,van der Zee R,Hariawala M,Sheriff DD,Esakof DD,Chaudhry GM,Symes JF,andIsner JM. Vascular endothelial growth factor/vascular permeability factor produces nitric oxide-dependent hypotension. Evidence for a maintenance role in quiescent adult endothelium. Arterioscler Thromb Vasc Biol 17:2793-2800,1997 .0 l& X$ r$ O/ B

) n6 M# }0 h2 R" ]0 s
3 A( M; `# m% @9 t$ R2 y! k
49. Hostetter, TH,Rennke HG,andBrenner BM. The case for intrarenal hypertension in the initiation and progression of diabetic and other glomerulopathies. Am J Med 80:443-453,1982.' ^. E0 b, U6 ?+ q

50. Hostetter, TH,Troy JL,andBrenner BM. Glomerular hemodynamics in experimental diabetes mellitus. Kidney Int 19:410-415,1981  .) }9 @8 q8 M. i) M2 [" t5 m
' x. p; K! y/ \

51. Ichihara, A,Inscho EW,Imig JD,andNavar LG. Neuronal nitric oxide synthase modulates rat renal microvacular function. Am J Physiol Renal Physiol 274:F516-F524,1998 .7 z) s7 h* g& k$ y4 ~
2 o% a. b. h; R, p2 ~& n
3 E: J+ i4 X) C
& j3 a* a/ P. v* ~: |9 j+ L9 S2 H/ h
52. Inoue, N,Venema RC,Sayegh HS,Ohara Y,Murphy TJ,andHarrison DG. Molecular regulation of the bovine endothelial cell nitric oxide synthase by transforming growth factor-beta 1. Arterioscler Thromb Vasc Biol 15:1255-1261,1995 .1 J5 p4 H4 S$ q7 L8 m
& B3 H& [4 T( V6 q( o- e

53. Ishida, A,Sasaguri T,Kosaka C,Nojima H,andOgata J. Induction of the cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1) by nitric oxide-generating vasodilator in vascular smooth muscle cells. J Biol Chem 272:10050-10057,1997 .
% `0 T  e9 r0 O

54. Ishii, N,Patel KP,Lane PH,Taylor T,Bian K,Murad F,Pollock JS,andCarmines PK. Nitric oxide synthesis and oxidative stress in the renal cortex of rats with diabetes mellitus. J Am Soc Nephrol 12:1630-1639,2001 .; n9 W7 ^  s( d2 @9 s+ @8 \
% Z1 F$ L# I) E! M+ y+ L  C% O

55. Ito, A,Uriu K,Inada Y,Qie YL,Takagi I,Ikeda M,Hashimoto O,Suzuka K,Eto S,Tanaka Y,andKaizu K. Inhibition of neuronal nitric oxide synthase ameliorates renal hyperfiltration in streptozotocin-induced diabetic rat. J Lab Clin Med 138:177-185,2001  .) V" d7 M9 m, j' H
4 C/ Y% E  K8 O' [( ?; E
. B1 s. C; g2 G1 f! Q

56. Jaimes, EA,Sweeney C,andRaij L. Effects of the reactive oxygen species hydrogen peroxide and hypochlorite on endothelial nitric oxide production. Hypertension 38:877-883,2001 .; p4 M) G0 k+ r$ `+ Z7 X

& J/ V7 a! ]% Z$ m. q0 d3 v- x$ }  X
57. Janssens, S,Flaherty D,Nong Z,Varenne O,van Pelt N,Haustermans C,Zoldhelyi P,Gerard R,andCollen D. Human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation after balloon injury in rats. Circulation 97:1274-1281,1998 .4 x! B# e: E3 @* J5 J/ |
/ D3 Q, v3 ]1 R' F* r2 w$ X
/ Z3 b0 ^( ~" m- G
' N3 _# A3 g# T! h, x7 p
58. Ju, H,Zou R,Venema VJ,andVenema RC. Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem 272:18522-18525,1997 .

  n3 f' b) K" W! [% R( r# D
1 W; T9 \& H9 @" k% J
59. Kashiwagi, M,Shinozaki M,Hirakata H,Tamaki K,Hirano T,Tokumoto M,Goto H,Okuda S,andFujishima M. Locally activated renin-angiotensin system associated with TGF- 1 as a major factor for renal injury induced by chronic inhibition of nitric oxide synthase in rats. J Am Soc Nephrol 11:616-624,2000 .7 y0 P# W0 `4 k

$ c+ _4 e, s$ a& y& J+ b3 F3 }
' T( e/ w+ h3 i- d1 O3 J
60. Keynan, S,Hirshberg B,Levin-Iaina N,Wexler ID,Dahan R,Reinhartz E,Ovadia H,Wollman Y,Chernihovskey T,Iaina A,andRaz I. Renal nitric oxide production during the early phase of experimental diabetes mellitus. Kidney Int 58:740-747,2000  .2 O7 W+ _6 u  G5 v' v- y& S

61. Kiff, RJ,Gardiner SM,Compton AM,andBennett T. The effects of endothelin-1 and N G -nitro- L -arginine methyl ester on regional haemodynamics in conscious rats with streptozotocin-induced diabetes mellitus. Br J Pharmacol 103:1321-1326,1991  ." @) c3 B) M2 Z! s

! [4 p/ q) S7 D* v1 e8 f7 s
4 Z0 O  ~( o' F" O
62. King, AJ,Zayas MA,Troy JL,Downes SJ,andBrenner BM. Inhibition of diabetes-induced hyperfiltration and hyperemia by N -monomethyl- L -arginine ( L -NMMA) (Abstract). J Am Soc Nephrol 1:666,1990.  C6 ^7 g& Z  b& K
  `% \5 T' H) g% Y: ~1 U0 z
% @9 c$ C1 Z. N7 X
: @+ E- F" o# S/ |2 h" Y& Y* o
63. Komers, R,Allen TJ,andCooper ME. Role of endothelium-derived nitric oxide in the pathogenesis of the renal hemodynamic changes of experimental diabetes. Diabetes 43:1190-1197,1994 .

8 K# X" s! X5 y7 r$ @
64. Komers, R,Lindsley JN,Oyama TT,Allison KM,andAnderson S. Role of neuronal NOS (NOS1) in the pathogenesis of renal hemodynamic changes in diabetes. Am J Physiol Renal Physiol 279:F573-F583,2000 .$ ^- ]$ T# y1 A+ v# t

9 [3 I8 s& R: v  V
# ~1 s5 W! f' w: W  u
65. Komers, R,Lindsley JN,Oyama TT,andAnderson S. Functional expression of endothelial nitric oxide synthase and caveolin-1 in diabetic kidney cortex (Abstract). J Am Soc Nephrol 13:532A,2002.
" E  T  ~/ P" ^
" C, g/ N4 N) M4 g
, U  P. l1 e" J) d% D
66. Komers, R,Oyama TT,Chapman JG,Allison KM,andAnderson S. Effects of systemic inhibition of neuronal nitric oxide synthase in diabetic rats. Hypertension 34:655-661,2000.

+ c3 E+ x0 m. B( ~/ e& L
, r( M# ^) \* A% g8 r& ^, G
67. Komers, R,Oyama TT,Lindsley JN,andAnderson S. Long-term inhibition of neuronal nitric oxide (NO) synthase (NOS1) postpones development of proteinuria in uninephrectomized diabetic rats (Abstract). J Am Soc Nephrol 12:A4391,2001.. i% J, L7 s* O8 ]) ?
7 X4 F, G7 @4 A5 j& G$ r2 t/ ?3 R  R
0 D0 J( V. ^: p4 M7 Y
8 p$ p! L$ N. u7 ]! p2 S) `+ [
68. Kone, BC,andBaylis C. Biosynthesis and homeostatic roles of nitric oxide in the normal kidney. Am J Physiol Renal Physiol 272:F561-F578,1997 ." I! l1 O1 L. [# |+ R5 Z
6 W; X  N2 @* w* B" H( N7 J: V
4 @# \( E5 Y2 w+ ]9 Z4 K' g

69. Koya, D,Jirousek MR,Lin YW,Ishii H,Kuboki K,andKing GL. Characterization of protein kinase C- isoform activation on the gene expression of transforming factor-, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats. J Clin Invest 100:115-126,1997  .1 P  G9 y, v; e" n7 \

70. Koya, D,andKing GL. Protein kinase C activation and the development of diabetic complications. Diabetes 47:859-866,1998 .

71. Koya, D,Lee IK,Ishii H,Kanoh H,andKing GL. Prevention of glomerular dysfunction in diabetic rats by treatment with D - -tocopherol. J Am Soc Nephrol 8:426-435,1997 .

6 J4 r) V& U* `+ |$ b

72. Krishna, MC,Samuni A,Taira J,Goldstein S,Mitchell JB,andRusso A. Stimulation by nitroxides of catalase-like activity of hemeproteins. Kinetics and mechanism. J Biol Chem 271:26018-26025,1996 .% G, ]5 _: ~$ @
" k+ |" a* s  i3 N; c& p6 d3 W# I

73. Krook, A,Roth RA,Jiang XJ,Zierath JR,andWallberg-Henriksson H. Insulin-stimulated Akt kinase activity is reduced in skeletal muscle from NIDDM subjects. Diabetes 47:1281-1286,1998 .

' F- ~( t/ O$ f2 S/ s- g
74. Kuan, CJ,al-Douahji M,andShankland SJ. The cyclin kinase inhibitor p21WAF1, CIP1 is increased in experimental diabetic nephropathy: potential role in glomerular hypertrophy. J Am Soc Nephrol 9:986-993,1998 .
, m& I; [. F; O

75. Kuboki, K,Jiang ZY,Takahara N,Ha SW,Igarashi M,Yamauchi T,Feener EP,Herbert TP,Rhodes CJ,andKing GL. Regulation of endothelial constitutive nitric oxide synthase gene expression in endothelial cells and in vivo: a specific vascular action of insulin. Circulation 101:676-681,2000 .' ^' G/ K; |; h7 P; V9 ^2 B* h

/ x9 A5 T- q: e/ T' u4 Q9 g
! @) [! r( z' H% y3 m" P
76. Malinski, T,Taha Z,Grunfeld S,Patton S,Kapturczak M,andTomboulian P. Diffusion of nitric oxide in the aorta wall monitored in situ by porphyrinic microsensors. Biochem Biophys Res Commun 193:1076-1082,1993  .
4 s8 K- N4 J7 W$ p# H* n9 B$ O% L
5 _& P( V9 G9 B
# e8 F( a" c, D; x
77. Marletta, MA. Nitric oxide: biosynthesis and biological significance. Trends Pharmacol Sci 158:348-352,1989.. ?$ j" ^2 w# E7 ]: [. b& s1 ^
% z1 `5 Q( S0 @2 J  u) \0 z) R

$ S- I7 ]0 i& C
78. Mattar, AL,Fujihara CK,Ribeiro MO,de Nucci G,andZatz R. Renal effects of acute and chronic nitric oxide inhibition in experimental diabetes. Nephron 74:136-143,1996  .

$ J" u9 ^% Q3 B. ^* @
: \8 T3 m( C4 x8 F/ Q
79. Mattson, DL,Roman RJ,andCowley AW. Role of nitric oxide in renal papillary blood flow and sodium excretion. Hypertension 19:766-769,1992 .

7 ~5 e$ D. s: V5 N5 ^! d
5 u* w5 o/ s0 u4 {$ i
80. Melhem, MF,Craven PA,andDerubertis FR. Effects of dietary supplementation of -lipoic acid on early glomerular injury in diabetes mellitus. J Am Soc Nephrol 12:124-133,2001 .0 O; b! y5 d+ L: u% C$ D( q6 X

. g7 e: C4 ~0 a( e- z' y
81. Michel, T,andFeron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest 100:2146-2152,1997  .
6 S" |9 u3 y4 e8 j) _+ J
  ~! j" ~$ g: [9 U! ?: I7 _

82. Michell, BJ,Chen Z,Tiganis T,Stapleton D,Katsis F,Power DA,Sim AT,andKemp BE. Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. J Biol Chem 276:17625-17628,2001 .
7 l. g1 c$ Z6 \% M" i- y2 z

83. Mogensen, CE. Early glomerular hyperfiltration in insulin-dependent diabetics and late nephropathy. Scand J Clin Invest 1986:201-206,1986.

! h( q& D: X4 w! w9 L# o* O' g

84. Mohaupt, MG,Elzie JL,Ahn KY,Clapp WL,Wilcox CS,andKone BC. Differential expression and induction of mRNAs encoding two inducible nitric oxide synthases in rat kidney. Kidney Int 46:653-665,1994  .$ f, n9 `/ _- W& X4 T" v/ Q0 O
8 K* B4 k3 p* y' x+ |

85. Moncada, S,andHiggs EA. The L -arginine-nitric oxide pathway. N Engl J Med 329:2002-2012,1993 .
# o6 i: R# j3 |

' a3 u- v' x; n0 i/ x
86. Moore, LC,Thorup C,Ellinger A,Paccione J,Casellas D,andKaskel FJ. Advanced glycosylation end-products and NO-dependent vasodilation in renal afferent arterioles from diabetic rats. Acta Physiol Scand 168:101-106,2000  .
) M. W& M; ]9 d1 A

87. Morbidelli, L,Chang CH,Douglas JG,Granger HJ,Ledda F,andZiche M. Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium. Am J Physiol Heart Circ Physiol 270:H411-H415,1996 .0 Y0 N* {9 H( h$ }1 ~
9 B" ?7 C+ j" A+ Q) H5 G
8 c, f9 M7 F0 Z0 c# r

88. Morrisey, JJ,McCracken R,Kaneto H,Vehaskari M,Montani D,andKlahr S. Location of an inducible nitric oxide synthase mRNA in the normal kidney. Kidney Int 45:998-1005,1994  .

89. Narayanan, K,Spack L,McMillan K,Kilbourn RG,Hayward MA,Masters BSS,andGriffith OW. Salkyl- L -thiocitrullines. J Biol Chem 270:11103-11110,1995 .% I  i# ]$ i+ d+ u) O

5 J2 k  ]- U% k
90. Ohishi, K,andCarmines PK. Superoxide dismutase restores the influence of nitric oxide on renal arterioles in diabetes mellitus. J Am Soc Nephrol 5:1559-1566,1995 .

91. Omer, S,Shan J,Varma DR,andMulay S. Augmentation of diabetes-associated renal hyperfiltration and nitric oxide production by pregnancy in rats. J Endocrinol 161:15-23,1999 .+ S1 Z. w( s' t1 Q" [& q* Y

# p* D: m0 K7 G. m9 W4 C4 f6 V

92. Onozato, ML,Tojo A,Goto A,Fujita T,andWilcox CS. Oxidative stress and nitric oxide synthase in rat diabetic nephropathy: effects of ACEI and ARB. Kidney Int 61:186-194,2002  .& U1 q* n0 H1 y! }

; n" l1 p: e: g4 G1 I, c
93. Osterby, R. Glomerular structural changes in type 1 (insulin-dependent) diabetes mellitus: causes, consequences, and prevention. Diabetologia 35:803-812,1992  .
, _* Q3 l7 U" e# a9 u
' x! {2 M6 p$ ]) X' G! T" t

94. Oyadomari, S,Gotoh T,Aoyagi K,Araki E,Shichiri M,andMori M. Coinduction of endothelial nitric oxide synthase and arginine recycling enzymes in aorta of diabetic rats. Nitric Oxide 5:252-260,2001  ., M* S, ~. ?8 e) M2 c

4 H/ G' Q* G0 [3 x( U; g1 n3 ~
+ {8 H7 ^5 K$ }, J9 N0 t
95. Peterson, TE,Poppa V,Ueba H,Wu A,Yan C,andBerk BC. Opposing effects of reactive oxygen species and cholesterol on endothelial nitric oxide synthase and endothelial cell caveolae. Circ Res 85:29-37,1999 .
4 H( j# U8 q: p# `3 z" w, A
$ v# v& J4 |6 m

96. Pflueger, AC,Larson TS,Hagl S,andKnox F. Role of nitric oxide in intrarenal hemodynamics in experimental diabetes mellitus in rats. Am J Physiol Regul Integr Comp Physiol 277:R725-R733,1999 .

97. Pflueger, AC,Osswald H,andKnox F. Adenosine-induced renal vasoconstriction in diabetes mellitus. Am J Physiol Renal Physiol 276:F340-F346,1999 .- M. h% C0 x4 h& a5 \5 X, H: [3 R
# e; c" _, w9 G1 P# l; f
8 C& i7 l5 b( Y" Y+ W) }' [* Q
# Q0 v' M8 T+ ?$ S! {) _
98. Pieper, GM. Enhanced, unaltered and impaired nitric oxide-mediated endothelium-dependent relaxation in experimental diabetes mellitus: importance of disease duration. Diabetologia 42:204-213,1999  .
1 w6 x! z2 f8 E9 f

/ j1 c# s' x/ m, c: V) U  i3 Q% R
99. Pieper, GM. Review of alterations in endothelial nitric oxide production in diabetes. Protective role of arginine on endothelial dysfunction. Hypertension 31:1047-1060,1998 .7 ]7 E8 `5 ~7 |/ x2 B
& Y7 h. d/ V3 @

& P" T: O  I7 F4 ]
100. Pieper, GM,Jordan M,Adams MB,andRoza AM. Syngeneic pancreatic islet transplantation reverses endothelial dysfunction in experimental diabetes. Diabetes 33:1106-1113,1995.
6 a( z0 b4 v, }: R+ {" z: Y

101. Poppa, V,Miyashiro JK,Corson MA,andBerk BC. Endothelial NO synthase is increased in regenerating endothelium after denuding injury of the rat aorta. Arterioscler Thromb Vasc Biol 18:1312-1321,1998 .

3 H4 h2 {/ G7 M* L, V5 N" R
) `! R0 ?# \' B7 V% _2 W0 h% P# @
102. Pou, S,Pou WS,Bredt DS,Snyder SH,andRosen GM. Generation of superoxide by purified brain nitric oxide synthase. J Biol Chem 267:24173-24176,1992 .- k  ^6 ^1 n% k$ T) P
0 W3 J  [. g! ?

103. Reyes, AA,Karl IE,Kissane J,andKlahr S. L -arginine administration prevents glomerular hyperfiltration and decreases proteinuria in diabetic rats. J Am Soc Nephrol 4:1039-1045,1993 .5 y# d' `0 A" }. |: m# ~( ~4 I$ ]; \

7 T9 q" p* B" v  v8 t. B5 X0 h1 m
104. Roczniak, A,Zimpelmann J,andBurns KD. Effect of dietary salt on neuronal nitric oxide synthase in inner medullary collecting duct. Am J Physiol Renal Physiol 275:F46-F54,1998 .
; W# R* k' d5 z0 v& v, z

105. Rudberg, S,Persson B,andDahlquist G. Increased glomerular filtration rate as a predictor of diabetic nephropathy-an 8-year prospective study. Kidney Int 41:822-828,1992  .

106. Santizo, RA,Koenig HM,andPelligrino DA. -Adrenoceptor and nNOS-derived NO interactions modulate hypoglycemic pial arteriolar dilation in rats. Am J Physiol Heart Circ Physiol 280:H562-H568,2001 .
0 d5 D5 m! ]5 w. O
$ w5 H# J9 h6 s9 H+ {& E0 H( M

107. Schini-Kerth, VB,andVanhoutte PM. Nitric oxide synthases in vascular cells. Exp Physiol 80:885-905,1995  .! F3 ^- K) J# |8 y+ t

% a2 ]7 r& ]# E. U8 U  {- i
' Q9 p( \4 F7 M9 n$ r! G
108. Schnackenberg, CG. Physiological and pathophysiological roles of oxygen radicals in the renal microvasculature. Am J Physiol Regul Integr Comp Physiol 282:R335-R342,2002 .# w' a3 a. |: x3 q3 B. p6 O
* A# L7 }: g$ X

109. Schnackenberg, CG,andWilcox CS. The SOD mimetic tempol restores vasodilation in afferent arterioles of experimental diabetes. Kidney Int 59:1859-1864,2001  .' n0 I* S1 d; B9 m) L" x% o

110. Scholey, JW,andMeyer TW. Control of glomerular hypertension by insulin administration in diabetic rats. J Clin Invest 83:1384-1389,1989  .

111. Schoonmaker, GC,Fallet RW,andCarmines PK. Superoxide anion curbs nitric oxide modulation of afferent arteriolar ANG II responsiveness in diabetes mellitus. Am J Physiol Renal Physiol 278:F302-F309,2000 .
* J$ s" H2 k* k5 |* {
4 Q; G$ _; X: c+ {. C  R3 W

112. Schwartz, D,Schwartz IF,andBlantz RC. An analysis of renal nitric oxide contribution to hyperfiltration in diabetic rats. J Lab Clin Med 137:107-114,2001  .

4 [3 Z0 z/ O& i

113. Sharma, K,Danoff TM,DePiero A,andZiyadeh FN. Enhanced expression of inducible nitric oxide synthase in murine macrophages and glomerular mesangial cells by elevated glucose levels: possible mediation via protein kinase C. Biochem Biophys Res Commun 207:80-88,1995  .. {4 R$ Q1 [4 V2 }, e. L; y
: [* r% n) t/ ~% T/ Q. t& L
& ~1 Z/ W; c5 V* l7 e: [, c

114. Sharma, RV,Tan E,Fang S,Gurjar MV,andBhalla RC. NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 276:H1450-H1459,1999 .

6 o* o1 b8 @) {0 D% L0 j
1 K) {& G7 e* E% D' T6 M# }3 N: Z3 k
115. Shin, SJ,Lai FJ,Wen JD,Hsiao PJ,Hsieh MC,Tzeng TF,Chen HC,Guh JY,andTsai JH. Neuronal and endothelial nitric oxide synthase expression in outer medulla of streptozotocin-induced diabetic rat kidney. Diabetologia 43:649-659,2000  .( Z5 c5 P. b' X6 ~  b4 E

2 O- W6 C$ d4 w) M( a! k- ?
116. Soulis, T,Cooper ME,Sastra S,Thallas V,Panagiotopoulos S,Bjerrum OJ,andJerums G. Relative contributions of advanced glycation and nitric oxide synthase inhibition to aminoguanidine-mediated renoprotection in diabetic rats. Diabetologia 40:1141-1151,1997  .( w  ^( {/ V' l
: J! b' e; o9 ~- u1 {" \$ m
/ |# u( {" @/ b2 z  k2 x
& `1 @0 H6 C' Z* a) c
117. Studer, RK,DeRubertis FR,andCraven PA. Nitric oxide suppresses increases in mesangial cell protein kinase C, transforming growth factor beta, and fibronectin synthesis induced by thromboxane. J Am Soc Nephrol 7:999-1005,1996 .  _7 R1 D3 _% Y4 e/ H
+ a4 D3 i2 V8 H
- Y, w* Y! N5 a* s* D
  n- a. H* b5 n8 X5 F* u
118. Sugimoto, H,Shikata K,Matsuda M,Kushiro M,Hayashi Y,Hiragushi K,Wada J,andMakino H. Increased expression of endothelial cell nitric oxide synthase (ecNOS) in afferent and glomerular endothelial cells is involved in glomerular hyperfiltration of diabetic nephropathy. Diabetologia 41:1426-1434,1998  .

+ A# ^& D/ p! s* k
119. Sugimoto, H,Shikata K,Wada J,Horiuchi S,andMakino H. Advanced glycation end products-cytokine-nitric oxide sequence pathway in the development of diabetic nephropathy: aminoguanidine ameliorates the overexpression of tumour necrosis factor-alpha and inducible nitric oxide synthase in diabetic rat glomeruli. Diabetologia 42:878-886,1999  .
) }) o9 L0 Q+ A3 |  V6 U# d9 D
& o& A1 h; b9 L; R

120. Suto, T,Losonczy G,Qiu C,Hill C,Samsell L,Ruby J,Charon N,Venuto R,andBaylis C. Acute changes in urinary excretion of nitrite nitrate do not necessarily predict renal vascular NO production. Kidney Int 48:1272-1277,1995  .

# W; F9 {$ N) J

121. Takahashi, T,Taniguchi T,Konishi H,Kikkawa U,Ishikawa Y,andYokoyama M. Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 276:H1927-H1934,1999 .

122. Tanner, FC,Meier P,Greutert H,Champion C,Nabel EG,andLuscher TF. Nitric oxide modulates expression of cell cycle regulatory proteins: a cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation. Circulation 101:1982-1989,2000 .
' [, R+ T2 e* F2 S/ z8 N4 S, M
& W0 J# M: q! [8 _' h4 d5 G
: m! _2 R/ j3 `3 W" x! ^9 l
123. Terada, Y,Tomita K,Nonoguchi H,andMarumo F. Polymerase chain reaction localization of constitutive nitric oxide synthase and soluble guanylate cyclase messenger RNAs in microdissected rat nephron segments. J Clin Invest 90:659-665,1992  .
' n; L/ m- t/ \( L/ e1 P

! e8 L. m, o' |4 Q! f: {, b
124. Thorup, C,Erik A,andPersson G. Macula densa derived nitric oxide in regulation of glomerular capillary pressure. Kidney Int 49:430-436,1996  .3 ]0 a8 |. s- J% r  t! S+ P: A

125. Thuraisingham, RC,Nott CA,Dodd SM,andYaqoob MM. Increased nitrotyrosine staining in kidneys from patients with diabetic nephropathy. Kidney Int 57:1968-1972,2000  .
; h8 k" o- Q) u! }9 S" @
& M% e  ?- c0 Y% p1 |4 K

126. Tilton, RG,Kawamura T,Chang KC,Ido Y,Bjercke RJ,Stephan CC,Brock TA,andWilliamson JR. Vascular dysfunction induced by elevated glucose levels in rats is mediated by vascular endothelial growth factor. J Clin Invest 99:2192-2202,1997  .
; q6 q% H8 D$ i+ k+ j0 r: e

127. Tojo, A,Gross SS,Zhang L,Tisher CC,Schmidt HHHW,Wilcox CS,andMadsen KM. Immunohistochemical localization of distinct isoforms of nitric oxide synthase in the juxtaglomerular apparatus of normal rat kidney. J Am Soc Nephrol 4:1438-1447,1994 .
0 z7 z/ k& Y5 |6 k

& g' h. P# S/ M! n; _- f, [, b( e4 {8 y
128. Tolins, JP,Shultz PJ,Raij L,Brown DM,andMauer SM. Abnormal renal hemodynamic response to reduced renal perfusion pressure in diabetic rats: role of NO. Am J Physiol Renal Fluid Electrolyte Physiol 265:F886-F895,1993 .& V% ?8 F* K- h2 Z% Z8 G
5 w* o/ s$ c2 h

$ w' K8 o* D7 o$ Q6 u1 k! g3 }! Y
129. Tomita, H,Egashira K,Ohara Y,Takemoto M,Koyanagi M,Katoh M,Yamamoto H,Tamaki K,Shimokawa H,andTakeshita A. Early induction of transforming growth factor-beta via angiotensin II type 1 receptors contributes to cardiac fibrosis induced by long-term blockade of nitric oxide synthesis in rats. Hypertension 32:273-279,1998 .

, K8 j9 O1 p, {6 ~
130. Torffvit, O,andEdvinsson L. Blockade of nitric oxide decreases the renal vasodilatory effect of neuropeptide Y in the insulin-treated diabetic rat. Pflügers Arch 434:445-450,1997  .

! X8 u4 m' ]7 ~3 {1 U" G

131. Torffvit, O,andEdvinsson L. Relaxing effect of insulin in renal arteries from diabetic rats. Regul Pept 79:147-152,1999  .3 C, H, R: Y8 f$ n& v+ H- A% m" p
2 O) H; t" v) P4 r  }8 y' k7 ]
/ R8 N9 S- z  F" k* h1 [

132. Trachtman H, Futterweit S, and Crimmins DL. High glucose inhibitsnitric oxide production in cultured rat mesangial cells. JAm Soc Nephrol : 1276-1282, 1997.

$ v; }& v! J* m1 \. c* ^3 [
% }; ~7 P. |/ L) y! K9 Q
133. Uematsu, M,Ohara Y,Navas JP,Nishida K,Murphy TJ,Alexander RW,Nerem RM,andHarrison DG. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol Cell Physiol 269:C1371-C1378,1995 ." @7 E7 j) |, l- L, C" w* h

134. Ushio-Fukai, M,Alexander RW,Akers M,Yin Q,Fujio Y,Walsh K,andGriendling KK. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem 274:22699-22704,1999 .3 F0 G3 D( L4 _' `- s: y
( f0 ?! l, x3 \7 g
& V' q* |  z5 M5 G7 M
6 I$ I8 L+ F, H
135. Vallon, V,Blantz RC,andThomson S. Homeostatic efficiency of tubuloglomerular feedback is reduced in established diabetes mellitus in rats. Am J Physiol Renal Fluid Electrolyte Physiol 269:F876-F883,1995 .1 K3 O6 W; @' n- I9 ^
8 s5 R9 `( L% G3 ]% e3 D, D! @

136. Van der Zee, R,Murohara T,Luo Z,Zollmann F,Passeri J,Lekutat C,andIsner JM. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation 95:1030-1037,1997 ., N* S9 |, D" s4 v

5 t& j/ V2 F# ]: g/ g
137. Vanhoutte, PM. Endothelium-derived free radicals: for worse and for better. J Clin Invest 107:23-25,2001  .8 G5 u2 s; A% w6 s; k: b1 y2 O

/ O: f/ I, r: m# _8 g$ Z- U, z  @- R; C
138. Vasquez-Vivar, J,Hogg N,Martasek P,Karoui H,Pritchard KA, Jr,andKalyanaraman B. Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase. J Biol Chem 274:26736-26742,1999 .9 r; g) ~, v, g$ u
+ _( T+ C6 q* A
! f8 }: A4 T& p6 ]9 G) Z' K

139. Vasquez-Vivar, J,Kalyanaraman B,Martasek P,Hogg N,Masters BS,Karoui H,Tordo P,andPritchard KA, Jr. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci USA 95:9220-9225,1998 .
1 X2 r4 ~2 V; v5 `
9 K9 g9 P! T$ }" h- `- B  Q: Q" L
& L3 O! B0 T5 R( B
140. Vecchione, C,Maffei A,Colella S,Aretini A,Poulet R,Frati G,Gentile MT,Fratta L,Trimarco V,Trimarco B,andLembo G. Leptin effect on endothelial nitric oxide is mediated through Akt-endothelial nitric oxide synthase phosphorylation pathway. Diabetes 51:168-173,2002 .* b0 C1 B! k4 G, W3 n$ B2 D

5 l+ H0 J) x( W1 B# z6 E; v1 O

141. Veelken, R,Hilgers KF,Hartner A,Haas A,Bohmer K,andSterzel RB. Nitric oxide synthase isoforms and glomerular hyperfiltration in early diabetic nephropathy. J Am Soc Nephrol 11:71-79,2000 .3 q4 k- d9 r& w5 W6 ]) Q; R
; O6 y9 \  Z8 r; t3 E: m
0 G5 j+ b8 n3 }! k& p  N% R
% Y: D; v( o$ {0 ?2 Y+ d
142. Venema, RC,Sayegh HS,Kent JD,andHarrison DG. Identification, characterization, and comparison of calmodulin binding domains of the endothelial and inducible nitric oxide synthases. J Biol Chem 271:6435-6440,1996 .8 f3 ~% E" T, o& z( m
0 I0 O, j- J6 N& |2 m1 Z
, d; X5 b7 |1 f: y9 o9 \: r. Q. \
0 p  P$ ^* n; p. }2 p# ^
143. Verbeke, P,Perichon M,Friguet B,andBakala H. Inhibition of nitric oxide synthase activity by early and advanced glycation end products in cultured rabbit proximal tubular epithelial cells. Biochim Biophys Acta 1502:481-494,2000 .9 Q0 L2 f( D' P: y# V
8 h7 Y6 F* r$ ?- Z+ H  H6 V

144. Von der Leyen, HE,Gibbons GH,Morishita R,Lewis NP,Zhang L,Nakajima M,Kaneda Y,Cooke JP,andDzau VJ. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci USA 92:1137-1141,1995 .' a. l. d/ P5 y8 Y% u9 L

145. Wang, S,Shiva S,Poczatek MH,Darley-Usmar V,andMurphy-Ullrich JE. Nitric oxide and cGMP-dependent protein kinase regulation of glucose- mediated thrombospondin1-dependent TGF- activation in mesangial cells. J Biol Chem 277:9880-9888,2002 .7 ^% }- P* [2 G3 W0 `
( O  \! j9 r" n* J# F  e
; y& s  G2 \  M2 g. F- Y3 i4 ^

146. Wang, YX,Brooks DP,andEdwards RM. Attenuated glomerular cGMP production and renal vasodilation in streptozotocin-induced diabetic rats. Am J Physiol Regul Integr Comp Physiol 264:R952-R956,1993 .

1 I/ _/ a1 h+ q* F9 C& w
  Q# n+ E/ U1 z4 _  D7 k
147. Welch, WJ,andWilcox CS. Macula densa arginine delivery and uptake in the rat regulates glomerular capillary pressure. J Clin Invest 100:2235-2242,1997  ., D# W5 Q& v. r' ]+ N1 j6 n8 f# L

# ?- \0 J+ m: j7 |" [/ p2 I, k

148. Wever, RM,van Dam T,van Rijn HJ,de Groot F,andRabelink TJ. Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase. Biochem Biophys Res Commun 237:340-344,1997  .; }. M! Z; e  b2 x( U
1 z5 d6 ?4 i9 c- j9 l1 K' j7 _0 B( ^

149. Wilcox, CS,andWelch WJ. Macula densa nitric oxide synthase: expression, regulation, and function. Kidney Int 54:S53-S57,1998.
" {0 s6 M( }3 e5 ^

1 ?4 o2 p% L( b2 ?6 B
150. Wilcox, CS,Welch WJ,Murad F,Gross SS,Taylor G,Levi R,andSchmidt HH. Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc Natl Acad Sci USA 89:11993-11997,1992 .3 y/ \! x+ H6 c

151. Wolf, G. Cell cycle regulation in diabetic nephropathy. Kidney Int 58, Suppl 77:S-59-S-66,2000.# V  u% n, \3 l; D# c. B

- B8 p6 l1 `( E# e& r
: [2 j) q4 r1 j! k" R
152. Wolf, G,Hamann A,Han DC,Helmchen U,Thaiss F,Ziyadeh FN,andStahl RA. Leptin stimulates proliferation and TGF- expression in renal glomerular endothelial cells: potential role in glomerulosclerosis. Kidney Int 56:860-872,1999  .4 I0 c9 x) E' i

( ]* L) j3 Z" n* y
# a& s8 |$ R+ h5 `! O3 U
153. Wolf, G,Schroeder R,Thaiss F,Ziyadeh FN,Helmchen U,andStahl RA. Glomerular expression of p27Kip1 in diabetic db/db mouse: role of hyperglycemia. Kidney Int 53:869-879,1998  .2 [, L# A4 h4 ~% x3 }( y

2 s& p( t  Z3 \

154. Wu, F,Park F,Cowley AW,andMattson DL. Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney. Am J Physiol Renal Physiol 276:F874-F881,1999 ." G8 |/ ?; R- O& y9 ?
# G0 J* b. }; b$ d
8 `5 P1 N8 u% b
& }- p; d' W( ~  w6 ^* }
155. Xia, Y,Tsai AL,Berka V,andZweier JL. Superoxide generation from endothelial nitric-oxide synthase. A Ca 2  /calmodulin-dependent and tetrahydrobiopterin regulatory process. J Biol Chem 273:25804-25808,1998 .
% G! F* E/ m% X0 M' T$ }7 v) a
0 G' P2 n: {, A$ Y7 V+ f

156. Xia, Y,andZweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci USA 94:6954-6958,1997 .

- ^, G  I- o2 k8 M, W7 s! T6 c

157. Yagihashi, N,Nishida N,Seo HG,Taniguchi N,andYagihashi S. Expression of nitric oxide synthase in macula densa in streptozotocin diabetic rats. Diabetologia 39:793-799,1996  .

' ]( x3 m) E3 Z. _0 [5 Q
$ P/ M, k/ Q' f0 K& E
158. Ying, WZ,andSanders PW. Dietary salt enhances glomerular endothelial nitric oxide synthase through TGF- 1. Am J Physiol Renal Physiol 275:F18-F24,1998 .

6 ^; z# }5 ^- i- \4 J
159. Zatz, R,andde Nucci G. Effects of acute nitric oxide inhibition on rat glomerular microcirculation. Am J Physiol Renal Fluid Electrolyte Physiol 261:F360-F363,1991 .
+ Y) p+ z0 k* R# O6 U
% B1 _0 m/ Y4 p, Q1 K) ~$ p

160. Zatz, R,Dunn BR,Meyer TW,Anderson S,Rennke HG,andBrenner BM. Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. J Clin Invest 77:1925-1930,1986  .5 z) a0 i" e! P. {4 _
0 W1 G3 _# n0 g6 L  |# d8 A4 [

161. Zatz, R,Meyer TW,Rennke HG,andBrenner BM. Predominance of hemodynamic rather than metabolic factors in the pathogenesis of diabetic nephropathy. Proc Natl Acad Sci USA 82:5963-5967,1985 .. o* C4 v0 N, Q/ b$ v9 b% g

5 ^' I* V3 x: A$ \, a1 n, G! r
9 I  w& `5 F1 I) L$ N
162. Zeng, G,Nystrom FH,Ravichandran LV,Cong LN,Kirby M,Mostowski H,andQuon MJ. Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation 101:1539-1545,2000 .

163. Zeng, G,andQuon MJ. Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells. J Clin Invest 98:894-898,1996  .

收藏0 分享0 顶0 踩0