Hui John Zhao*,
Suwan Wang*,
Huifang Cheng*,
Ming-zhi Zhang*,
Takamune Takahashi*,
Agnes B. Fogo*,,
Matthew D. Breyer* and
Raymond C. Harris*
* Division of Nephrology and Hypertension, Department of Medicine, and Department of Pathology, Vanderbilt University, Nashville, Tennessee
Address correspondence to: Dr. Raymond C. Harris, Department of Medicine, Division of Nephrology and Hypertension, Vanderbilt University Medical Center, C-3121 MCN, Nashville, TN 37232. Phone: 615-343-0030; Fax: 615-343-2675; E-mail: ray.harris{at}vanderbilt.edu
Functionally significant polymorphisms in endothelial nitricoxide synthase (eNOS) and reduced vascular eNOS activity havebeen associated with increased human diabetic nephropathy (DN),but the pathogenic role of eNOS deficiency in the developmentof DN has not yet been confirmed. This study characterizes theseverity of DN in eNOS–/– mice that were backcrossedto C57BLKS/J db/db mice. Although the severity of hyperglycemiawas similar to C57BLKS/J db/db mice, by 26 wk, eNOS–/–C57BLKS/J db/db mice exhibited dramatic albuminuria, arteriolarhyalinosis, increased glomerular basement membrane thickness,mesangial expansion, mesangiolysis, and focal segmental andearly nodular glomerulosclerosis. Even more remarkable, eNOS–/–C57BLKS db/db exhibited decreases in GFR to levels <50% ofthat in eNOS+/+ C57BLKS db/db, as confirmed by increased serumcreatinine. In summary, eNOS–/–db/db mice providethe most robust model of type II DN that has been describedto date and support a role for deficient eNOS-derived NO productionin the pathogenesis of DN.
Diabetic hyperglycemia causes microvascular dysfunction, whichcontributes to the development of ESRD (1–3). In recentyears, studies have focused first on the mesangial cell andmore recently on the podocyte as both initiators and targetsof diabetic nephropathy (DN) (4–6). Although these cellsundoubtedly are involved in the development of DN, there isequally compelling reason to consider a role for endothelialcell dysfunction in the initiation and propagation of the characteristicglomerular lesions that are seen in DN.
Endothelial cell–derived vasodilators, such as nitricoxide (NO), seem to be important modulators of permeabilityin the vasculature. Inhibition or genetic deletion of endothelialNO synthase (eNOS, NOS III) induces opening of the interendothelialjunctions and increases vascular permeability in microvascularbeds, suggesting that NO production may play an important rolein regulating the endothelial barrier function (7,8).
Functionally significant polymorphisms in eNOS have been recognizedin human DN (9). In addition, vascular eNOS activity is alteredin diabetes. NO products have been reported to be increasedearly after the onset of diabetes and may be involved in mediatingrenal vasodilation and hyperfiltration; however, with more prolongeddiabetes, renal eNOS production decreases (10). The exact functionof eNOS in the development of DN remains undetermined. Thesestudies were designed not only to understand better the roleof eNOS-mediated events in the development and progression ofDN but also to determine whether eNOS deficiency results ina mouse model of DN that more closely approximates the functionaland structural changes that are seen in human DN.
Animals
eNOS+/– mice initially on the C57/B6 background and dbheterozygous mice on the C57BLKS/J (BKS) background were purchasedfrom The Jackson Laboratory (Bar Harbor, ME). The eNOS+/–mice were backcrossed for 10 generations to the C57BLKS/J backgroundand then crossed with db heterozygous mice. Genotyping was performedby PCR. Whenever possible, the same mice underwent all physiologicassessments (BP measurement, GFR, and albumin/creatinine ratio[ACR] measurement) and procurement of kidney tissue for histologicanalysis.
BP Measurement
Systolic BP (SBP) was measured in conscious, trained mice atroom temperature using a tail-cuff monitor (BP-2000 BP Analysissystem, Visitech System, NC).
Determination of Blood Glucose and Creatinine
Blood glucose was determined using the OneTouch glucometer andtest strips (LifeScan, Milpitas, CA). Serum creatinine was measuredby a previously described HPLC method (11).
Urine Albumin and Creatinine
Spot urine was collected from individually caged mice usingpolycarbonate metabolic cages. Urinary albumin and creatinineexcretion was determined using Albuwell-M kits (Exocell Inc.,Philadelphia, PA).
Measurement of GFR
GFR was measured by a single-bolus FITC-inulin injection method,as described previously (12).
Histologic Analysis
Renal histology was assessed in mice that were killed at 24to 26 wk of age. The perfused kidneys were removed and fixedovernight in 10% formalin at 4°C, and 3-µm-thick sectionswere stained with periodic acid-Schiff. Histologic evaluationwas performed without knowledge of the identity of the variousgroups. A semiquantitative index was used to evaluate the degreeof glomerular mesangial expansion and sclerosis. Each glomeruluson a single section was graded from 0 to 4, where 0 representsno lesion, and 1, 2, 3, and 4 represent mesangial matrix expansionor sclerosis, involving 25, 25 to 50, 50 to 75, or >75% ofthe glomerular tuft area, respectively.
Immunohistochemistry
Immunohistochemical detection of fibronectin staining was performedusing an anti-fibronectin antibody (Sigma, St. Louis, MO). Thesections then were incubated using the avidin-biotin-horseradishperoxidase technique (Elite Vectastain ABC kit; Vector Laboratories,Burlingame, CA), and staining was visualized using 3,3'-diaminobenzidine.
Statistical Analyses
All values are presented as means ± SEM. Bonferroni ttest corrected for multiple comparisons was used for statisticalanalysis, and differences were considered significant at P <0.05.
Development of Type 2 Diabetes in eNOS–/– C57BLKS db/db Mice
We first investigated the timing of development of diabetesin this model. Hyperglycemia was evident by approximately 6to 8 wk of age. The fasting blood glucose level in db/db micewas significantly higher than that in the lean controls at 26wk of age (P < 0.05; Figure 1A). eNOS deletion did not alterfasting blood glucose in either db/db or control mice. As wasdescribed previously (13,14), moderate hypertension was observedin lean mice with genetic deletion of eNOS by age 24 to 28 wk(Figure 1B). SBP was significantly greater in eNOS–/–C57BLKS/J db/db mice (eNOS–/–db/db) than in db/dbC57BLKS/J mice (db/db; 158 ± 10 versus 110 ± 3;n = 4; P < 0.05) but was not significantly higher than innondiabetic eNOS–/– C57BLKS/J mice (eNOS–/–;143 ± 7 mmHg; n = 5; NS).
Figure 1. Metabolic and physiologic parameters. (A) Fasting blood glucose at 24 to 26 wk in db/db mice and age-matched controls ± eNOS–/–. *P < 0.05 versus control and eNOS–/–. Values are means ± SE of at least eight mice. (B) Systolic BP. *P < 0.05 eNOS–/– or eNOS–/–db/db versus control or db/db. Values are means ± SE of at least four mice.
Albuminuria in eNOS–/– C57BLKS/J db/db Mice
Albuminuria is a hallmark of DN. At 26 wk of age, moderate albuminuriawas observed in db/db (262 ± 24 µg albumin/mg creatinine;n = 10) and eNOS–/– (387 ± 27; n = 19), comparedwith control mice (43 ± 9; n = 8; P < 0.05; Figure 2A).In contrast, eNOS–/–db/db exhibited a marked increasein spot urine ACR (1503 ± 176; n = 12; P < 0.001 comparedwith their age-matched controls, db/db, or eNOS–/–).Urine ACR was elevated by age 8 wk in eNOS–/–db/db(data not shown).
Figure 2. Functional renal parameters. (A) Urinary albumin/creatinine ratio (ACR) at 24 to 26 wk of age. Values are means ± SE of at least eight mice. *P < 0.05 versus respective controls, db/db, and eNOS–/–. (B) GFR in diabetic mice. *P < 0.05 versus control, db/db, and eNOS–/–. Values are means ± SE of at least eight mice. (C) Correlation of serum creatinine with GFR.
GFR in eNOS–/– C57BLKS/J db/db Mice
HPLC serum creatinine determined at 26 wk was 0.10 ±0.001 (n = 5) in the control lean mice, 0.11 ± 0.01 (n= 7) in db/db, 0.115 ± 0.001 (n = 9) in eNOS–/–,and 0.17 ± 0.02 (n = 5) in eNOS–/–db/db (P< 0.05). GFR was examined in diabetic mice and age-matchedcontrols using FITC-inulin clearance. Given the disparity inbody weights between the control (lean) and diabetic (obese)mice, GFR was calculated per mouse as well as per gram of bodyweight. At 26 wk of age, GFR determined per mouse was numericallyincreased in db/db (366 ± 39 µl/min per mouse;n = 8) but was not statistically significant compared with age-matchedcontrols (331 ± 25; n = 10). In contrast, the GFR ofeNOS–/–db/db (164 ± 27; n = 10) was significantlydecreased compared with db/db (P < 0.001) and eNOS–/–(265 ± 21; n = 12; P < 0.05; Figure 2B). At 26 wk,the body weights of the obese mice were more than double thoseof the aged-matched controls (lean controls 23.4 ± 0.9g; db/db 59.4 ± 2.4; eNOS–/– 26.5 ±1.5; eNOS–/–db/db 58.1 ± 2.3). GFR per gramof body weight were as follows: lean controls 14.1 ±1.1 µl/min per g; db/db 6.2 ± 0.6; eNOS–/–9.9 ± 0.6; and eNOS–/–db/db 2.5 ±0.4. Given that body fat represents approximately 50% of totalbody weight in db/db mice (15), determination of GFR/body weightin the obese mice will underestimate the true GFR. Serum creatininescorrelated with the GFR measurements in mice from all groupsin which both measurements were obtained (n = 23; r2 = 0.699,P < 0.0001; Figure 2C).
Histologic Analysis
Kidneys were assessed by light and electron microscopy (Figure 3).Compared with control (Figure 3A, a), moderate mesangial expansionwas observed in glomeruli of db/db (Figure 3A, b and e) andeNOS–/– (Figure 3A, c) at 26 wk of age. In contrast,marked mesangial expansion, focal nodular sclerosis, and mesangiolysis(Figure 3A, d and f) as well as arteriolar hyalinosis (Figure 3A,f) were noted in eNOS–/–db/db glomeruli at 26 wkof age. There was only minimal tubulointerstitial fibrosis ineNOS–/–db/db kidneys. Immunohistochemical examinationalso revealed minimal accumulation of fibronectin in the mesangialregions of control (Figure 3B, a), db/db (Figure 3B, b), andeNOS–/– (Figure 3B, c), compared with the strikingfibronectin accumulation that was observed in the eNOS–/–db/dbglomeruli (Figure 3B, d).
Figure 3. Glomerular histopathology. (A) Representative glomerular lesions of diabetic mouse kidneys at 24 to 26 wk: control (a); db/db (b and e); eNOS–/– (c); eNOS–/–db/db (d and f). (f) Arteriolar hyalinosis (big arrow) and early nodular glomerulosclerosis (small arrow) in eNOS–/–db/db mice (periodic acid-Schiff). (B) Glomerular fibronectin expression in control (a), db/db (b), eNOS–/– (c), and eNOS–/–db/db mice (d). (C) Glomerular injury scores in diabetic mice. Mesangial expansion and sclerosis were scored on a scale from 0 to 4+ as described in The Materials and Methods section. *P < 0.05, eNOS–/–db/db (24 to 26 wk) mice versus age-matched controls, db/db, and eNOS–/– mice. (D) Correlation of glomerular injury index with albuminuria. (E) Electron micrographs of glomeruli (16 wk). Opposed arrows indicate GBM. GBM thickening was notable in eNOS–/–db/db mice (c) compared with control (a) and db/db mice (b). Magnifications: x400 in A, a through d, and B; x200 in A, e and f.
Glomerular injury that was assessed semiquantitatively was significantlyincreased in eNOS–/–db/db at 24 to 26 wk of age,as compared with other groups (n = 6 to 9/group; P < 0.05;Figure 3C). The amount of albuminuria correlated with the glomerularinjury index in mice from all groups in which both measureswere obtained (n = 19; r2 = 0.58, P < 0.0002; Figure 3D).
Glomerular ultrastructure was examined by electron microscopyat age 16 wk (Figure 3E). Compared with control mice (270 ±47 nm; Figure 3E, a), at this age, there was no thickening ofglomerular basement membrane (GBM) in db/db mice (240 ±76 nm; Figure 3E, b), in contrast to the markedly thickenedGBM that was seen in eNOS–/–db/db mice (322 ±57 nm; Figure 3E, c). No electron-dense deposits were present.
Human DN is a characteristic clinical syndrome that consistsof albuminuria, progressively declining GFR, and defined histopathologicfeatures that include thickening of the GBM and mesangial expansion,often with nodular glomerulosclerosis, arteriolar hyalinosis,and tubulointerstitial fibrosis (16,17). These features wouldbe important features of a robust animal model of DN (18).
Previous animal models of diabetic kidney disease have manifestedalbuminuria and early renal pathologic changes such as GBM thickeningand mesangial expansion but with only minimal or inconsistentexpression of other characteristic histopathologic featuressuch as arteriolar hyalinosis and nodular glomerulosclerosis.Furthermore, the failure of previous models to manifest a declinein renal function has called into question their utility asanalogues of human DN (16,18,19).
eNOS–/– C57BLKS db/db mice not only developed strikingalbuminuria and characteristic pathologic changes of DN butalso exhibited remarkably decreased GFR on the basis of inulinclearance and serum creatinine. Furthermore, the onset and theprogression of the pathologic features in eNOS–/–db/dbmice were rapid, which makes this DN model attractive as a potentialplatform for testing efficacy of new therapeutic agents.
The eNOS–/–db/db mice had significant obesity andhypertension, which typically is seen in humans with type 2diabetes (20,21). The mutated leptin receptor in db/db miceleads to defective signaling of leptin in the hypothalamus andresults in persistent hyperphagia and obesity and developmentof peripheral insulin resistance (19). In addition, when thedb/db mutation is on the C57BLKS background, mice develop alate (>4 mo) insulitis so that insulin levels are inappropriatelylow for the level of hyperglycemia (22). eNOS–/–mice were reported previously to develop moderate systemic hypertension(13,14). This increased SBP was slightly greater in diabeticeNOS–/–db/db mice, although the results did notreach statistical significance.
There is growing evidence that endothelial cell dysfunctioncontributes to hypertension and microvascular complicationsof diabetes (2,23). A major defense of endothelial cells againstvascular injury is eNOS, which generates NO in the presenceof the substrate L-arginine, and the co-factor (6R)-5,6,7,8-tetrahydro-L-biopterin(BH4). NADPH oxidases are major sources of reactive oxygen speciesin endothelium and are activated in animal models of hypertensionand diabetes. Superoxide reacts avidly with vascular NO·to form peroxynitrite, leading to BH4 oxidation and subsequentpromotion of superoxide production by eNOS itself, so-called"eNOS uncoupling" (24,25). Uncoupled eNOS is detected in conditionsthat are associated with oxidant stress, hypertension, and diabetes(23).
Human DN progresses through several pathophysiologic stages,initially characterized by early hyperfiltration and hypertrophyfollowed by microalbuminuria and mesangial expansion, and thenovert proteinuria, sclerosis, and a progressive decline of GFR(16,17). Early in diabetes, increased endothelial NO productionmay contribute to the observed hyperfiltration and microalbuminuria(10), but with advancing nephropathy, there is progressive NOdeficiency. Hyperglycemia, advanced glycosylation end products,increased oxidant stress, endogenous inhibitors of NO such asasymmetric dimethylarginine, activation of protein kinase C,and TGF- all are potential contributors to decreased NO productionand/or eNOS uncoupling (26,27).
In humans, the eNOS gene is found on chromosome 7q. Genome-widescans have indicated that regions on 7q, 18q, and 22q may influenceproteinuria and/or development of DN in type 2 diabetes (27).Of note, evidence for a region on 7q overlapped all studies(28). Recent studies have reported an association between eNOSpolymorphisms that lead to decreased eNOS expression and thedevelopment of advanced DN in both patients with type 1 (29–31)and with type 2 diabetes (32,33). Other studies also have foundan association of these polymorphisms with nondiabetic causesof ESRD (9,34). However, not all studies have detected an associationof disease with these eNOS polymorphisms (35–39).
Although short-term studies have examined the effect of administrationof NOS inhibitors in experimental models of diabetes (10,40),we are not aware of any published studies to date that haveexamined long-term chronic administration (4 to 6 mo). Whereasadministration of NOS inhibitors alone produces renal lesionsthat are consistent with ischemic nephropathy with minimal glomerulosclerosis(41), chronic administration of nonspecific NOS inhibitors doesaccelerate glomerulosclerosis in the remnant model of rat glomerulopathy(42), although similar studies are yet to be performed in mousemodels. Therefore, it is possible that NOS inhibitors may imparta similar acceleration of injury in experimental diabetic models,although a potential advantage of the current genetic modelin dissecting pathophysiologic mechanisms of DN is that eNOSis selectively deleted while other NOS isoforms remain active.
Our results demonstrate that eNOS–/–db/db mice exhibitsignificant albuminuria and glomerular pathology that parallelthe later phase of DN in patients with type 2 diabetes and includearteriolar hyalinosis, mesangial expansion, thickening of GBM,and focal segmental and early nodular glomerulosclerosis. Thismodel should prove useful for studying the role of endothelialdysfunction in development of DN and in facilitating the developmentof new diagnostic and therapeutic interventions.
Acknowledgments
This work was supported by National Institutes of Health grantsDK061018 (Mouse Models of Diabetic Complications Consortium),DK39261 (Vanderbilt George O'Brien Kidney and Urologic DiseasesCenter), and DK59637 (Vanderbilt Mice Metabolic Phenotype Center)and funds from the Department of Veterans Affairs.
We thank Dr. Stephen Dunn for measuring plasma creatinine levelsand Xiaofeng Fan for technical support.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Futrakul N, Butthep P, Vongthavarawat V, Futrakul P, Sirisalipoch S, Chaivatanarat T, Suwanwalaikorn S: Early detection of endothelial injury and dysfunction in conjunction with correction of hemodynamic maladjustment can effectively restore renal function in type 2 diabetic nephropathy.
Clin Hemorheol Microcirc 34
: 373
–381, 2006[Medline]
Jawa A, Nachimuthu S, Pendergrass M, Asnani S, Fonseca V: Impaired vascular reactivity in African-American patients with type 2 diabetes mellitus and microalbuminuria or proteinuria despite angiotensin-converting enzyme inhibitor therapy.
J Clin Endocrinol Metab 91
: 31
–35, 2006[Abstract/Free Full Text]
Wautier J-L, Schmidt AM: Protein glycation: A firm link to endothelial cell dysfunction.
Circ Res 95
: 233
–238, 2004[Abstract/Free Full Text]
Mason RM, Wahab NA: Extracellular matrix metabolism in diabetic nephropathy.
J Am Soc Nephrol 14
: 1358
–1373, 2003[Abstract/Free Full Text]
Wendt TM, Tanji N, Guo J, Kislinger TR, Qu W, Lu Y, Bucciarelli LG, Rong LL, Moser B, Markowitz GS, Stein G, Bierhaus A, Liliensiek B, Arnold B, Nawroth PP, Stern DM, D’Agati VD, Schmidt AM: RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy.
Am J Pathol 162
: 1123
–1137, 2003[Abstract/Free Full Text]
White KE, Bilous RW: Structural alterations to the podocyte are related to proteinuria in type 2 diabetic patients.
Nephrol Dial Transplant 19
: 1437
–1440, 2004[Abstract/Free Full Text]
Predescu D, Predescu S, Shimizu J, Miyawaki-Shimizu K, Malik AB: Constitutive eNOS-derived nitric oxide is a determinant of endothelial junctional integrity.
Am J Physiol Lung Cell Mol Physiol 289
: L371
–L381, 2005[Abstract/Free Full Text]
Sessa WC: eNOS at a glance.
J Cell Sci 117
: 2427
–2429, 2004[Free Full Text]
Noiri E, Satoh H, Taguchi J, Brodsky SV, Nakao A, Ogawa Y, Nishijima S, Yokomizo T, Tokunaga K, Fujita T: Association of eNOS Glu298Asp polymorphism with end-stage renal disease.
Hypertension 40
: 535
–540, 2002[Abstract/Free Full Text]
Prabhakar SS: Role of nitric oxide in diabetic nephropathy.
Semin Nephrol 24
: 333
–344, 2004[CrossRef][Medline]
Yuen PST, Dunn SR, Miyaji T, Yasuda H, Sharma K, Star RA: A simplified method for HPLC determination of creatinine in mouse serum.
Am J Physiol Renal Physiol 286
: F1116
–F1119, 2004[Abstract/Free Full Text]
Qi Z, Whitt I, Mehta A, Jin J, Zhao M, Harris RC, Fogo AB, Breyer MD: Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance.
Am J Physiol Renal Physiol 286
: F590
–F596, 2004[Abstract/Free Full Text]
Shesely EG, Maeda N, Kim HS, Desai KM, Krege JH, Laubach VE, Sherman PA, Sessa WC, Smithies O: Elevated blood pressures in mice lacking endothelial nitric oxide synthase.
Proc Natl Acad Sci U S A 93
: 13176
–13181, 1996[Abstract/Free Full Text]
Van Vliet BN, Chafe LL, Montani JP: Characteristics of 24 h telemetered blood pressure in eNOS-knockout and C57Bl/6J control mice.
J Physiol 549
: 313
–325, 2003[Abstract/Free Full Text]
Cox JE, Powley TL: Development of obesity in diabetic mice pair-fed with lean siblings.
J Comp Physiol Psychol 91
: 347
–358, 1977[CrossRef][Medline]
Breyer MD, Bottinger E, Brosius FC 3rd, Coffman TM, Harris RC, Heilig CW, Sharma K: Mouse models of diabetic nephropathy.
J Am Soc Nephrol 16
: 27
–45, 2005[Abstract/Free Full Text]
Parving H-H, Mauer M, Ritz E: Diabetic nephropathy. In:
The Kidney, 7th Ed., edited by Brenner BM, Philadelphia, WB Saunders, 2004
, pp 1777
–1818
Breyer MD, Bottinger E, Brosius FC, Coffman TM, Fogo A, Harris RC, Heilig CW, Sharma K: Diabetic nephropathy: Of mice and men.
Adv Chronic Kidney Dis 12
: 128
–145, 2005[CrossRef][Medline]
Sharma K, McCue P, Dunn SR: Diabetic kidney disease in the db/db mouse.
Am J Physiol Renal Physiol 284
: F1138
–F1144, 2003[Abstract/Free Full Text]
Sarafidis PA, Ruilope LM: Insulin resistance, hyperinsulinemia, and renal injury: Mechanisms and implications.
Am J Nephrol 26
: 232
–244, 2006[CrossRef][Medline]
Dobesh PP: Managing hypertension in patients with type 2 diabetes mellitus.
Am J Health Syst Pharm 63
: 1140
–1149, 2006[Abstract/Free Full Text]
Coleman DL, Leiter EH, Schwizer RW: Therapeutic effects of dehydroepiandrosterone (DHEA) in diabetic mice.
Diabetes 31
: 830
–833, 1982[Abstract]
Forstermann U, Munzel T: Endothelial nitric oxide synthase in vascular disease: From marvel to menace.
Circulation 113
: 1708
–1714, 2006[Abstract/Free Full Text]
Chalupsky K, Cai H: Endothelial dihydrofolate reductase: Critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase.
Proc Natl Acad Sci U S A 102
: 9056
–9061, 2005[Abstract/Free Full Text]
Prabhakar SS: Tetrahydrobiopterin reverses the inhibition of nitric oxide by high glucose in cultured murine mesangial cells.
Am J Physiol Renal Physiol 281
: F179
–F188, 2001[Abstract/Free Full Text]
Saran R, Novak JE, Desai A, Abdulhayoglu E, Warren JS, Bustami R, Handelman GJ, Barbato D, Weitzel W, D’Alecy LG, Rajagopalan S: Impact of vitamin E on plasma asymmetric dimethylarginine (ADMA) in chronic kidney disease (CKD): A pilot study.
Nephrol Dial Transplant 18
: 2415
–2420, 2003[Abstract/Free Full Text]
Tarnow L, Hovind P, Teerlink T, Stehouwer CD, Parving HH: Elevated plasma asymmetric dimethylarginine as a marker of cardiovascular morbidity in early diabetic nephropathy in type 1 diabetes.
Diabetes Care 27
: 765
–769, 2004[Abstract/Free Full Text]
Ng DP, Krolewski AS: Molecular genetic approaches for studying the etiology of diabetic nephropathy.
Curr Mol Med 5
: 509
–525, 2005[CrossRef][Medline]
Zanchi A, Moczulski DK, Hanna LS, Wantman M, Warram JH, Krolewski AS: Risk of advanced diabetic nephropathy in type 1 diabetes is associated with endothelial nitric oxide synthase gene polymorphism.
Kidney Int 57
: 405
–413, 2000[Medline]
Li C, Dong Y, Lu W: The association between polymorphism of endothelial nitric oxide synthase gene and diabetic nephropathy [in Chinese].
Zhonghua Nei Ke Za Zhi 40
: 729
–732, 2001[Medline]
Neugebauer S, Baba T, Watanabe T: Association of the nitric oxide synthase gene polymorphism with an increased risk for progression to diabetic nephropathy in type 2 diabetes.
Diabetes 49
: 500
–503, 2000[Abstract]
Asakimori Y, Yorioka N, Yamamoto I, Okumoto S, Doi S, Hirai T, Taniguchi Y: Endothelial nitric oxide synthase intron 4 polymorphism influences the progression of renal disease.
Nephron 89
: 219
, 2001[CrossRef][Medline]
Degen B, Schmidt S, Ritz E: A polymorphism in the gene for the endothelial nitric oxide synthase and diabetic nephropathy.
Nephrol Dial Transplant 16
: 185
–223, 2001[Free Full Text]
Freedman BI, Yu H, Anderson PJ, Roh BH, Rich SS, Bowden DW: Genetic analysis of nitric oxide and endothelin in end-stage renal disease.
Nephrol Dial Transplant 15
: 1794
–1800, 2000[Abstract/Free Full Text]
Lin S, Qu H, Qiu M: Allele A in intron 4 of ecNOS gene will not increase the risk of diabetic nephropathy in type 2 diabetes of Chinese population.
Nephron 91
: 768
, 2002[CrossRef][Medline]
Shimizu T, Onuma T, Kawamori R, Makita Y, Tomino Y: Endothelial nitric oxide synthase gene and the development of diabetic nephropathy.
Diabetes Res Clin Pract 58
: 179
–185, 2002[CrossRef][Medline]
Komers R, Anderson S: Paradoxes of nitric oxide in the diabetic kidney.
Am J Renal Physiol 284
: F1121
–F1137, 2003[Abstract/Free Full Text]
Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R: Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension.
Hypertension 20
: 298
–303, 1992[Abstract/Free Full Text]
Fujihara CK, De Nucci G, Zatz R: Chronic nitric oxide synthase inhibition aggravates glomerular injury in rats with subtotal nephrectomy.
J Am Soc Nephrol 5
: 1498
–1507, 1995[Abstract]
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Top
Abstract
Introduction
25, 25 to 50, 50 to 75, or >75% of the glomerular tuft area, respectively. 
all are potential contributors to decreased NO production and/or eNOS uncoupling (26,27). 
