Role of nitric oxide in renal tubular apoptosis of unilateral ureteral obstruction

BACKGROUND: The obstructed kidney in unilateral ureteral obstruction (UUO) is characterized by renal atrophy and tissue loss, which is mediated by renal tubular apoptosis. We sought to determine whether NO is involved in renal tubular apoptosis in vitro and in vivo. METHODS: Rat renal tubular epithelial cells (NRK-52E) were subjected to mechanical stretch, and apoptosis and cell size were analyzed by flow cytometry. Furthermore, we studied UUO in mice lacking the gene for inducible nitric oxide synthase (iNOS-/-) and their wild-type littermates. Tubular apoptosis and proliferation were detected by immunostaining. NOS activity and NOS expression were assessed by a citrulline assay and Western blot, respectively. RESULTS: Stretching-induced apoptosis in NRK-52E, which was reduced when NO was increased; conversely, stretch-induced apoptosis was increased when a NOS inhibitor was added to the cells. Stretched cells are larger and more apoptotic than unstretched cells. In UUO, the obstructed kidney of iNOS-/- mice exhibited more apoptotic renal tubules than the wild-type mice through 14 days of UUO. The obstructed kidney of iNOS-/- mice at day 3 showed more proliferative tubules compared with wild type. The obstructed kidney of wild-type mice exhibited higher total NOS activity until day 7 after UUO compared with iNOS-/- mice. However, the obstructed kidney of day 14 wild-type mice exhibited significantly lower iNOS activity and protein compared with the day 0 kidney. CONCLUSION: These results suggest that mechanical stretch is related to renal tubular apoptosis and that NO plays a protective role in this system in UUO.     

Obstructive nephropathy and renal fibrosis: The role of bone morphogenic protein-7 and hepatocyte growth factor

BACKGROUND: The nephropathy induced by ureteral obstruction is associated with increased interstitial volume due to matrix deposition, fibroblast differentiation/proliferation, and monocyte infiltration. Recent studies indicate that transforming growth factor-beta (TGF-beta) is linked to renal fibrosis. Tumor necrosis factor (TNF-alpha) has a role in the recruitment of inflammatory cells. We found that infiltration of macrophages of the interstitium in unilateral ureteral obstruction (UUO) occurred as early as four hours after the onset of UUO. METHODS: Recent studies indicate that a renal tubular development morphogen, bone morphogenetic protein-7 (BMP-7), is effective in preventing the tubulointerstitial nephritis in the setting of obstructive nephropathy. The mechanism of action appears to be preservation of epithelial cell phenotype, inhibition of epithelial-mesenchymal transdifferentiation, and inhibition of injury-induced epithelial cell apoptosis. Hepatocyte growth factor (HGF) also inhibited tubulointerstitial fibrosis. RESULTS: In a treatment protocol in rats with ureteral ligation, BMP-7 restored renal function. The preservation of glomerular filtration rate (GFR) was accompanied by a significant decrease in cortical interstitial volume. In diabetic rats given BMP-7 proteinuria was normalized. In mice with ureteral obstruction, HGF suppressed the expression of TGF-beta and of platelet-derived growth factor. The onset of tubulointerstitial fibrosis was almost completely inhibited by HGF. CONCLUSION: Both BMP-7 and HGF attenuate the tubulointerstitial fibrosis due to ureteral obstruction. They also increase GFR and renal plasma flow.     

Unilateral ureteral obstruction in neonatal rats leads to renal insufficiency in adulthood

BACKGROUND: Although unilateral ureteropelvic junction obstruction is the most common cause of congenital obstructive nephropathy in infants and children, management remains controversial, and follow-up after pyeloplasty is generally limited to the pediatric ages. We have developed a model of temporary unilateral ureteral obstruction (UUO) in the neonatal rat: One month following the relief of five-day UUO, the glomerular filtration rate (GFR) of the postobstructed kidney was normal despite a 40% reduction in the number of glomeruli and residual vascular, glomerular, tubular, and interstitial injury. METHODS: To determine whether hyperfiltration and residual injury of remaining nephrons leads to progression of renal insufficiency in later life, 31 rats were sham operated or subjected to left UUO at one day of age, with relief of UUO five days later, and were studied at one year of age. GFR was measured by inulin clearance, and the number of glomeruli, tubular atrophy, glomerular sclerosis, and interstitial fibrosis were measured by histomorphometry in sham, obstructed (UUO), and intact opposite kidneys. Intrarenal macrophages and alpha-smooth muscle actin were identified by immunohistochemistry. RESULTS: Despite relief of UUO, ultimate growth of the postobstructed kidney was impaired. The number of glomeruli was reduced by 40%, and GFR was decreased by 80%. However, despite significant compensatory growth of the opposite kidney, there was no compensatory increase in GFR, and proteinuria was increased. Moreover, glomerular sclerosis, tubular atrophy, macrophage infiltration, and interstitial fibrosis were significantly increased not only in the postobstructed kidney, but also in the opposite kidney. CONCLUSIONS: Although GFR is initially maintained following relief of five-day UUO in the neonatal rat, there is eventual profound loss of function of the postobstructed and opposite kidneys because of progressive tubulointerstitial and glomerular damage. These findings suggest that despite normal postoperative GFR in infancy, children undergoing pyeloplasty for ureteropelvic junction obstruction should be followed into adulthood. Elucidation of the cellular response to temporary UUO may lead to improved methods to assess renal growth, injury, and functional reserve in patients with congenital obstructive nephropathy.     

Reimplantation of the ureter after unilateral ureteral obstruction provides a model that allows functional evaluation

Experimental unilateral ureteral obstruction (UUO) is widely used to study renal fibrosis; however, renal injury can only be scored semiobjectively by histology. We sought to improve the UUO model by reimplanting the obstructed ureter followed by removal of the contralateral kidney, thus allowing longitudinal measurements of renal function. Mice underwent UUO for different lengths of time before ureteral reimplantation and contralateral nephrectomy. Measurement of blood urea nitrogen (BUN) allows objective evaluation of residual renal function. Seven weeks after reimplantation and contralateral nephrectomy, mean BUN levels were increased with longer duration of UUO. Interstitial expansion, fibrosis, and T-cell and macrophage infiltration were similar in kidneys harvested after 10 days of UUO or following 10 weeks of ureter reimplantation, suggesting that the inflammatory process persisted despite relief of obstruction. Urinary protein excretion after reimplantation was significantly increased compared to control animals. Our study shows that functional assessment of the formerly obstructed kidney can be made after reimplantation and may provide a useful model to test therapeutic strategies for reversing renal fibrosis and preserving or restoring renal function.     

Effects of low molecular weight heparin in obstructed kidneys: decrease of collagen, fibronectin and TGF-beta, and increase of chondroitin/dermatan sulfate proteoglycans and macrophage infiltration.

BACKGROUND: Heparin exerts beneficial effects in different experimental models of nephropathy, as observed by the preservation of the structural morphology of the kidney after heparin therapy. Here we investigate molecular and cellular events involved in the protective effects of heparin in the progression of renal disease after unilateral ureteral obstruction. METHODS: Thirty-six rats were divided into six groups: group C (control) was not subjected to any surgical manipulation; group S (sham) was subjected to surgical manipulation but without ureteral ligation; group UUO was subjected to ureteral obstruction and received no treatment; group UUO + S was subjected to ureteral obstruction and received saline subcutaneously (s.c.) once daily; group UUO + H was subjected to ureteral obstruction and received low molecular weight heparin (LMW-Hep; 4 mg/kg) s.c. once daily; and group C + H was not subjected to any surgical manipulation and received LMW-Hep (4 mg/kg) s.c. once daily. After 14 days, the content of collagen, fibronectin, total glycosaminoglycans (GAGS), chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs), transforming growth factor-beta (TGF-beta) and cellular infiltration were determined in the kidneys by immunohistochemical and biochemical techniques. RESULTS: Collagen, fibronectin, total GAGS, CS/DSPGs, TGF-beta and cellular infiltration increased significantly in group UUO. LMW-Hep treatment reduced collagen, fibronectin and TGF-beta, but induced an increase in the content of total GAGS, CS/DSPGs and macrophage infiltration in group UUO + H when compared with group UUO. CONCLUSIONS: LMW-Hep diminishes fibrosis in obstructed kidneys by downregulating the synthesis of collagen, fibronectin and TGF-beta. The mechanisms underlying the overproduction of CS/DSPGs and the increase in cellular infiltration upon LMW-Hep administration remain to be elucidated.     

Antibody to transforming growth factor-beta ameliorates tubular apoptosis in unilateral ureteral obstruction

BACKGROUND: Unilateral ureteral obstruction (UUO) is characterized by progressive renal atrophy, renal interstitial fibrosis, an increase in renal transforming growth factor-beta (TGF-beta), and renal tubular apoptosis. The present study was undertaken to determine the effect of a monoclonal antibody to TGF-beta (1D11) in UUO. METHODS: Mechanical stretch was applied to tubular epithelial cells (NRK-52E) by a computer-assisted system. Three doses of 1D11 (either 0.5, 2, or 4 mg/rat) were administered to rats one day prior to UUO and every two days thereafter, and kidneys were harvested at day 13. Fibrosis was assessed by measuring tissue hydroxyproline and mRNA for collagen and fibronectin. Apoptosis was assessed with the terminal deoxy transferase uridine triphosphate nick end-labeling assay. TGF-beta levels were determined by bioassay. Western blot and immunostaining were used to identify proliferating cell nuclear antigen (PCNA), p53, bcl-2, and inducible nitric oxide synthase (iNOS). RESULTS: Stretch significantly induced apoptosis in NRK-52E cells, which was accompanied by an increased release of TGF-beta; 1D11 (10 microg/mL) totally inhibited stretch-induced apoptosis. Control obstructed kidney contained 20-fold higher TGF-beta as compared with its unobstructed kidney; 1D11 neutralized tissue TGF-beta of the obstructed kidney. Control obstructed kidney exhibited significantly more fibrosis and tubular apoptosis than its unobstructed counterpart, which was blunted by 1D11. In contrast, 1D11 significantly increased tubular proliferation. p53 immunostaining was localized to renal tubular nuclei of control obstructed kidney and was diminished by 1D11. In contrast, bcl-2 was up-regulated in the 1D11-treated obstructed kidney. Total NOS activity and iNOS activity of the obstructed kidney were increased by 1D11 treatment. CONCLUSION: The present study strongly suggests that an antibody to TGF-beta is a promising agent to prevent renal tubular fibrosis and apoptosis in UUO.     

Is enalapril adequate for the prevention of renal tissue damage caused by unilateral ureteral obstruction and/or hyperoxaluria?

Unilateral ureteral obstruction (UUO) and hyperoxaluria (HOX) can lead to end-stage renal disease with tubulointerstitial fibrosis. We investigated the effects of enalapril (E), an ACE-inhibitor, on rat kidneys with either UUO or HOX. Sham-operated, UUO, HOX, UUO+HOX, UUO+E and HOX+E rats were killed 14 days after UUO and/or HOX was initiated. Rat kidney sections were histologically scored for tissue damage and monocyte/macrophage infiltration was demonstrated with ED1 antibody and measured by computer image analysis software. Serious glomerular and tubulointerstitial damage was found for UUO and HOX, consisting of glomerular basement membrane thickening, tubular dilatation/collapse, tubular basement membrane thickening and the infiltration of mononuclear leucocytes (mainly macrophages). For HOX, calcium oxalate crystals were visible. Neither the scored histological parameters nor monocyte/macrophage infiltration was significantly decreased when E-treated were compared with untreated groups. We conclude that E did not ameliorate the parameters scored in either UUO or HOX. This being contrary to findings by other research groups, we hypothesize that E may be effective only in short-term UUO/HOX, with transforming growth factor, TGF-beta1, formation becoming partly independent of Ang II in late-stage UUO/HOX, or other fibrogenic cytokines than TGF-beta1 becoming predominant.     

Selectins mediate macrophage infiltration in obstructive nephropathy in newborn mice

BACKGROUND: Urinary tract obstruction during development leads to tubular atrophy and causes interstitial fibrosis. Macrophage infiltration into the interstitium plays a central role in this process. Selectins, a family of three adhesion molecules, are involved in leukocyte recruitment to sites of inflammation and immune activity. We investigated the role of selectins in obstructive nephropathy in newborn mice. METHODS: Triple selectin-deficient mice (EPL-/-), L-selectin deficient mice (L-/-) and wild type mice (WT) were subjected to complete unilateral ureteral obstruction (UUO) or sham operation within the first 48 hours of life, and were sacrificed 5 and 12 days later. Kidneys were removed, and sections were stained for macrophage infiltration (mAb F4/80), apoptosis (TUNEL), tubular atrophy (periodic acid-Schiff) and interstitial fibrosis (Masson trichrome). RESULTS: Selectin deficient mice showed a marked reduction in macrophage infiltration into the obstructed kidney compared to WT at day 5 and day 12 after UUO. Tubular apoptosis was strongly reduced in EPL-/- at day 5 after UUO, and in EPL-/- and L-/- at day 12 after UUO when compared to WT. The number of apoptotic tubular cells was correlated with macrophage infiltration, suggesting that macrophages stimulate tubular apoptosis in obstructive nephropathy. In addition, tubular atrophy and interstitial fibrosis were significantly diminished in EPL-/- and L-/- compared to WT at day 12 after UUO. CONCLUSION: Following UUO, selectins mediate macrophage infiltration into the obstructed kidney, which in turn may induce tubular apoptosis, tubular atrophy and interstitial fibrosis.     

Locally activated renin-angiotensin system associated with TGF-beta1 as a major factor for renal injury induced by chronic inhibition of nitric oxide synthase in rats

Chronic inhibition of nitric oxide synthase (NOS) is known to cause renal parenchymal injury with systemic hypertension. To elucidate the pathogenetic mechanism in renal damage induced by NOS inhibition, N(omega)-nitro-L-arginine methyl ester (L-NAME) was given orally for 12 wk in Wistar rats, and the roles of tissue renin-angiotensin system and transforming growth factor-beta1 (TGF-beta1) were investigated. BP and urinary protein excretion increased significantly in L-NAME rats compared with control rats, and glomerulosclerosis and interstitial fibrosis developed. In L-NAME rats, the cortical tissue levels of angiotensin-converting enzyme activity and angiotensin II were significantly higher than those in control rats. The cortical mRNA expressions of both TGF-beta1 and fibronectin were significantly elevated in L-NAME rats. Immunohistochemically, increased expressions of both fibronectin and alpha-smooth muscle actin were also revealed in L-NAME rats. In L-NAME rats, these histologic injuries and the increased expression of TGF-beta1 were equally ameliorated by either angiotensin-converting enzyme inhibitor or angiotensin II type 1 receptor antagonist, but not by hydralazine. In conclusion, the locally activated renin-angiotensin system in connection with the increased TGF-beta1 expression is a major pathogenetic feature of renal injury in chronically NOS-inhibited rats.     

Angiotensin II and prostaglandin interactions on systemic and renal effects of L-NAME in humans.

For investigation of whether interactions between prostaglandins and angiotensin II modulate renal response to acute nitric oxide synthesis inhibition in humans, seven young volunteers who were kept on a 240-mM Na diet underwent four experiments with 90 min of infusion of 3.0 microg/kg.min(-1) NG-nitro-L-arginine methyl ester (L-NAME), each preceded by a 3-d treatment with placebo (PL), 50 mg of losartan (LOS), 75 to 125 mg of indomethacin (IND), or both drugs. Mean arterial pressure (MAP), GFR, effective renal plasma flow (ERPF), and Na excretion rate (UNaV) were measured at baseline and from 0 to 45 min and 45 to 90 min of L-NAME infusion. After PL, L-NAME reduced GFR by 5% at 45 min (P < 0.05) and by 9% at 90 min (P < 0.001), ERPF by 11 to 17% (P < 0.001), and UNaV by 28 to 45% (P < 0.001). MAP, unchanged at 45 min, rose by 5% (P < 0.001) at 90 min. LOS prevented pressor but not renal effects of L-NAME. With L-NAME+IND, MAP rose even at 45 min (+5%; P < 0.001 versus baseline) with a 10% rise at 90 min (P < 0.001). Changes in GFR (-13 to -20%), ERPF (-19 to -26%), and UNaV (-51 to -70%) were greater than those with L-NAME+PL or L-NAME+LOS (P < 0.05 to 0.001). With L-NAME+IND+LOS, MAP did not increase, and GFR, ERPF, and UNaV fell much less than with L-NAME+IND alone (P < 0.02 to 0.001) with no differences versus PL or LOS alone. Angiotensin II blockade does not affect renal changes caused by L-NAME but prevents their potentiation by prostaglandin inhibition. Thus, endogenous prostaglandins counteract renal actions of endogenous angiotensin II in Na-repleted humans even when nitric oxide synthesis is inhibited.     

Effects of nitric oxide on gentamicin toxicity in isolated perfused rat kidneys

BACKGROUND: There have been many studies in recent years concerning the role of nitric oxide (NO) in acute renal failure (ARF). In this study, the effects of the inhibition or the induction of NO synthase (NOS) on gentamicin-induced ARF was investigated in isolated perfused rat kidneys. METHODS: Kidneys from male Sprague-Dawley rats were perfused in situ for 90 min. Perfusion was conducted in the presence of inulin (60 mg/dL in perfusion buffer) as a glomerular filtration rate (GFR) marker. Six groups (total: 42 rats) were studied: group 1, controls with no treatment; group 2, L-arginine (2 mM in perfusate); group 3, L-nitro-arginine-methyl ester (L-NAME, 0.1 mM in perfusate); group 4, gentamicin (GM, 0.5 mg/mL in perfusate); group 5, GM + L-arginine (same dose as groups 2 and 4) and; group 6, GM + L-NAME (same dose as groups 3 and 4). Cell injury was assessed by measuring N-acetyl-beta-D-glucosaminidase (NAG), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity in urine. RESULTS: L-arginine prevented, whereas L-NAME enhanced, GM-induced enzyme release and GFR reduction. Histological studies showed that GM-treated kidneys had clear signs of tubular damage and this damage was increased by simultaneous L-NAME and GM administration. CONCLUSION: This study suggests that NO formation could prevent the GM-induced nephrotoxicity in this ARF model.     

Renal structural and functional repair in a mouse model of reversal of ureteral obstruction

The end point of immune and nonimmune renal injury typically involves glomerular and tubulointerstitial fibrosis. Although numerous studies have focused on the events that lead to renal fibrosis, less is known about the mechanisms that promote cellular repair and tissue remodeling. Described is a model of renal injury and repair after the reversal of unilateral ureteral obstruction (UUO) in male C57bl/6J mice. Male mice (20 to 25 g) underwent 10 d of UUO with or without 1, 2, 4, or 6 wk of reversal of UUO (R-UUO). UUO resulted in cortical tubular cell atrophy and tubular dilation in conjunction with an almost complete ablation of the outer medulla. This was associated with interstitial macrophage infiltration; increased hydroxyproline content; and upregulated type I, III, IV, and V collagen expression. The volume density of kidney occupied by renal tubules that exhibited a brush border was measured as an assessment of the degree of repair after R-UUO. After 6 wk of R-UUO, there was an increase in the area of kidney occupied by repaired tubules (83.7 +/- 5.9%), compared with 10 d UUO kidneys (32.6 +/- 7.3%). This coincided with reduced macrophage numbers, decreased hydroxyproline content, and reduced collagen accumulation and interstitial matrix expansion, compared with obstructed kidneys from UUO mice. GFR in the 6-wk R-UUO kidneys was restored to 43 to 88% of the GFR in the contralateral unobstructed kidneys. This study describes the regenerative potential of the kidney after the established interstitial matrix expansion and medullary ablation associated with UUO in the adult mouse.     

N-硝基-L-精氨酸甲酯对大鼠隐睾生殖细胞凋亡的保护作用

目的探讨一氧化氮合酶(NOS)抑制剂N-硝基-L-精氨酸甲酯(L-NAME)对大鼠隐睾生殖细胞凋亡的保护作用.方法用22 d SD雄性大鼠复制单侧隐睾模型.实验分假手术组、隐睾组、隐睾+L-NAME组[术后腹腔注射L-NAME,10 mg/(kg·d)],每组大鼠各10只.术后7 d,用生物素-dUTP/酶标亲和素测定法检测睾丸生殖细胞凋亡,用硝酸还原酶法测定睾丸组织中NO含量,用化学比色法测定睾丸组织中NOS活性.结果术后第7 d,与假手术组睾丸相比,隐睾组睾丸发生凋亡的生殖细胞数显著增加,隐睾+L-NAME组睾丸发生凋亡的生殖细胞数比隐睾组显著减少(P＜0.01),隐睾+L-NAME组睾丸组织中NO含量及NOS活性与隐睾组相比显著降低(P＜0.01).结论隐睾组织中NO和NOS升高是隐睾生殖细胞凋亡增加的病理机制之一,L-NAME通过抑制NOS活性、减少NO的产生来降低睾丸组织生殖细胞的凋亡发挥其保护作用.     

Hepatocyte growth factor suppresses interstitial fibrosis in a mouse model of obstructive nephropathy

BACKGROUND: As tubulointerstitial fibrosis (TIF) reflects the prognosis of patients with various chronic renal diseases, the pathogenesis of TIF has to be clarified. Transforming growth factor-beta (TGF-beta) is a key mediator for renal fibrosis. We reported that hepatocyte growth factor (HGF) prevents renal fibrosis in nephrotic mice. However, the function of HGF in chronic renal failure, except for nephrotic syndrome, remains to be determined. METHODS: Using mice subjected to unilateral ureter-ligated obstruction (UUO), we investigated the roles of HGF in TIF, as induced by obstructive nephropathy. Pathophysiological changes in the kidney after UUO treatment were analyzed focusing on expressions of renal HGF and TGF-beta, TIF, tubular proliferation, and apoptosis. Neutralizing antibody against rodent HGF, or recombinant human HGF (rhHGF), was administrated to the UUO mice, and pathophysiological changes after neutralization or supplements of HGF were analyzed. RESULTS: In this UUO model, TIF with tubular apoptosis became evident, and it was accompanied by a decrease in renal HGF expression and an increase in renal TGF-beta expression. Neutralization of endogenous HGF accelerated the progression of TIF, accompanied by increases in TGF-beta expression and tubular apoptosis as well as by decreases in tubular proliferation. In contrast, rhHGF attenuated TIF progression, and there were decreases in TGF-beta expression and tubular apoptosis, and an increase in tubular proliferation. CONCLUSIONS: Endogenous as well as exogenous HGF attenuated the progression of the fibrosis caused by obstructive nephropathy in these mice. Thus, local reduction in HGF levels may account for TIF in chronic renal diseases.     

Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction

BACKGROUND: The small GTPase Rho is involved in cell-to-substratum adhesion and cell contraction. These actions of Rho mediated by downstream Rho effectors such as Rho-associated coiled-coil forming protein kinase (ROCK) may be partly responsible for the progression of renal interstitial fibrosis. METHODS: The anti-fibrosis effects of Y-27632, a specific ROCK inhibitor, were studied both in vivo (unilateral ureteral obstruction; UUO) and in vitro. To investigate the therapeutic efficacy of Y-27632 in UUO kidneys, smooth muscle alpha actin (SMalphaA) expression, macrophage infiltration and fibrosis in the obstructed kidneys were studied. SMalphaA, transforming growth factor beta (TGF-beta), alpha1 (I) collagen, osteopontin, macrophage chemoattractant peptide-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) gene expression were examined by Northern blotting. To elucidate the mechanism linking the Rho-ROCK pathway with renal fibrosis, the effects of Y-27632 on in vitro cell proliferation and cell migration were studied. RESULTS: In vivo analysis showed that Y-27632 suppressed SMalphaA expression, macrophage infiltration and interstitial fibrosis, and that Y-27632 suppressed SMalphaA, TGF-beta and alpha1 (I) collagen mRNA expression. In vitro analysis showed that Y-27632 did not suppress proliferation of renal fibroblasts but suppressed migration of macrophages. CONCLUSIONS: The Rho-ROCK system may play an important role in the development of tissue fibrosis, and the Rho-ROCK signaling pathway may be a new therapeutic target for preventing interstitial fibrosis in progressive renal disease.     

Roles of nitric oxide and oxidative stress in the regulation of blood pressure and renal function in prehypertensive Ren-2 transgenic rats

AIMS: The present study was performed to evaluate the role of nitric oxide (NO) and its interaction with superoxide anion (O2-) in the regulation of blood pressure (BP) and renal function during the developmental phase of hypertension in Ren-2 transgenic rats (TGR). The first aim was to compare BP and renal functional responses to acute NO synthase (NOS) inhibition achieved by intravenous (i.v.) infusion of Nomega-nitro-L-arginine-methyl ester (L-NAME) in prehypertensive heterozygous TGR and in transgene-negative Hannover Sprague-Dawley (HanSD) rats. The second aim was to evaluate whether scavenging of O2- by infusion of the superoxide dismutase mimetic tempol increases NO bioavailability which therefore should augment BP and renal functional responses to L-NAME. Methods: Rats were anesthetized, prepared for clearance experiments and BP and renal functional responses were evaluated in response to i.v. L-NAME administration (20 microg.100 g(-1).min(-1)) without or with tempol pretreatment (i.v., 300 microg.100 g(-1).min(-1)). In renal cortical tissue, nitrotyrosine protein expression was assessed by immunoblotting as marker of O2- production and urinary 8-epi-PGF(2alpha) excretion as marker of intrarenal oxidative stress was assessed by enzyme immunoassay. Results: BP, glomerular filtration rate (GFR), renal plasma flow (RPF) and sodium excretion were similar in TGR and HanSD. L-NAME infusion induced greater increases in BP in TGR than in HanSD (+42 +/- 4 vs. +25 +/- 3 mmHg, p < 0.05). In the absence of a significant change in GFR, L-NAME caused similar decreases in RPF (-32 +/- 6 and -25 +/- 4%, p < 0.05) in TGR and HanSD. Despite significantly higher renocortical expression of nitrotyrosine and urinary 8-epi-PGF2alpha excretion in TGR than in HanSD, pretreatment with tempol did not augment the rise in BP and the decrease in RPF induced by L-NAME. CONCLUSIONS: The greater BP response to L-NAME in TGR suggests that prehypertensive TGR exhibit an enhanced NO activity in the systemic vasculature as compared with HanSD. Despite increased intrarenal oxidative stress in TGR, the dependency of the intrarenal vascular tone on NO appears to be similar in TGR and HanSD. The lack of a compensatory increase in renal NO activity may partially account for the enhanced renal vascular response to ANG II present in TGR.     

黄芪当归合剂对单侧输尿管梗阻大鼠肾脏血管活性物质变化的影响

目的 本研究通过观察黄芪当归合剂(A&A)对单侧输尿管梗阻(UUO)模型肾组织血管紧张素Ⅱ(Ang-Ⅱ)、内皮素-1(ET-1)、一氧化氮(NO)水平及一氧化氮合酶(NOS)的影响，以进一步揭示A&A抑制肾纤维化机制。方法 Wistar大鼠随机分为假手术(Sham)、UUO和UAA(UUO+A&A)组，造模后0、3、7、10 d分析各组肾脏中NO、Ang-Ⅱ、ET-1水平和NOS活性及3种NOS的表达。结果 (1)造模后UUO组的Ang-Ⅱ和ET-1水平持续增高；A&A仅在第3天时降低Ang-Ⅱ水平(P < 0.05)。(2)造模后UUO组的NO浓度在第10天时才明显增高；而UAA组中NO浓度逐渐增高，第3天时明显高于UUO组(P < 0.05)。(3)UUO组内皮型eNOS在髓质血管内表达增高，且UAA组与UUO组间没有显著性差异。第3天时UAA组神经型nNOS表达低于UUO组(P < 0.05)。UAA组cNOS的活性明显高于Sham和UUO组。诱导型iNOS表达和活性3组间差异均无统计学意义。结论 梗阻性肾病中，A&A在早期降低肾内Ang-Ⅱ水平、且持续增强eNOS活性使NO产生增加，从而可能降低血管张力、改善肾脏的缺血及缺氧状态，减轻肾间质纤维化。     

The outcome of non-selective vs selective nitric oxide synthase inhibition in lipopolysaccharide treated rats.

Nitric oxide (NO), generated by inducible nitric oxide synthase (NOS) following lipopolysaccharide (LPS) administration, produces renal failure through autoinhibition of glomerular endothelial NOS activity. Preadministration of selective iNOS inhibitors abolishes this effect. Although nonselective NOS inhibitors further decrease GFR, current clinical trials investigate the effect of nonselective NOS inhibition in septic patients. The goals of our study were to determine whether treatment with selective NOS inhibitors can reverse the decrease in GFR in LPS treated rats with already established renal failure and to define the outcome of LPS treated rats following nonselective NOS inhibition. Four hours following the administration of LPS (4 mg/kg), we measured creatinine clearance (CrCl) before and after the administration of either L-NIL (selective iNOS inhibitor, 3 mg every 20 minutes) or saline. Selective iNOS inhibition attenuated the decrease in blood pressure [Controls: 105 +/- 6 to 98 +/- 5, LPS: 92 +/- 5* to 83 +/- 4*, LPS + L-NIL: 88 +/- 6* to 94 +/- 6 mm Hg; *p < 0.05, vs controls (n = 6)], and reversed the decrease in GFR after LPS [Controls: 2.21 +/- 0.13 to 2.07 +/- 0.11, LPS: 0.82 +/- 0.18* to 0.66 +/- 0.22*, LPS + L-NIL: 0.76 +/- 0.15* to 1.86 +/- 0.15 ml/min; *p < 0.05 vs controls (n = 6)]. We next studied the effect of complete non-selective NOS inhibition (L-NAME 200 mg, 2 hours after LPS) on LPS treated rats. All (6/6) animals treated with both LPS and L-NAME died within 2 hours following LPS, while rats treated with either LPS, L-NAME, or LPS + L-NIL survived. Histologic studies performed in all experimental groups were unremarkable. Overnight mortality was studied using smaller doses of L-NAME. All LPS + L-NAME (10/10) and 1/10 LPS treated rats died. L-NAME, control, and LPS + L-NIL animals survived. The characteristic histologic findings in LPS + L-NAME rats were diffuse ischemic changes, most importantly acute myocardial infarction. In conclusion: Selective iN-OS inhibition might prove to have clinical application as it prevents the decrease in GFR following LPS, even after renal failure is established. Treatment with a non selective NOS inhibitor in septic patients should be reconsidered.     

IN-1130, a novel transforming growth factor-beta type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy

The transforming growth factor-beta (TGF-beta) plays a central role in the progression of renal fibrosis. TGF-beta transduces its signal through the activin receptor-like kinase (ALK)5. IN-1130, a novel small molecule ALK5 inhibitor, inhibited the purified kinase domain of ALK5-mediated Smad3 phosphorylation with an IC(50) value of 5.3 nM. IN-1130 proved to be highly selective in a panel of 27 serine/threonine and tyrosine kinases including p38alpha mitogen-activated protein kinase. We evaluated the efficacy of IN-1130 to block renal fibrogenesis induced by unilateral ureteral obstruction (UUO) in rats. Either vehicle (saline) or IN-1130 (10 and 20 mg/kg/day) was intraperitoneally administered to UUO rats for 7 and 14 days. Phosphorylated Smad2 (pSmad2) and markers of fibrosis were analyzed in kidney tissues. In UUO control kidneys, interstitial fibrosis including tubular atrophy, loss and dilation, inflammatory cell infiltration, and fibroblast cell proliferation was prominent. These morphological changes were notably reduced by IN-1130 treatment. IN-1130 decreased levels of TGF-beta1 messenger RNA (mRNA), type I collagen mRNA, and pSmad2, compared to UUO control rats. As determined by measuring the hydroxyproline content, total kidney collagen amount was increased in UUO control kidneys, but significantly reduced by IN-1130 treatment, which was comparable to results of histochemical staining for collagen. IN-1130 also suppressed the expression of alpha-smooth muscle actin (alpha-SMA) and fibronectin in UUO kidneys. Our results show that IN-1130 suppressed the fibrogenic process of UUO, further underscoring the potential clinical benefits of IN-1130 in the treatment of renal fibrosis.