Rapamycin attenuates unilateral ureteral obstruction-induced renal fibrosis

Unilateral ureteral obstruction (UUO) is a well-characterized hydronephrosis model exhibiting interstitial inflammatory-cell infiltration and tubular dilatation followed by tubulointerstitial fibrosis of the obstructed kidney. Our recent report indicates that rapamycin is effective for 50% of transplant recipients with chronic allograft nephropathy. In this study, we investigate the effect of rapamycin on UUO-induced renal fibrosis. UUO or sham-operated rats were randomly assigned to rapamycin or vehicle and were killed on days 7 and 14 after UUO or sham operation. Rapamycin decreased cross-sectional and gross-morphology changes in the obstructed kidney significantly. Rapamycin markedly blunted the increase in weight of the obstructed kidney, obstructed kidney length, and the obstructed/non-obstructed kidney weight ratio (by 74.6, 42.8, and 61.6% on day 14, respectively, all P<0.01). The scores for tubular dilatation, interstitial volume, interstitial collagen deposition, and alpha-smooth muscle actin (alpha-SMA) after UUO were significantly reduced by rapamycin. Rapamycin also decreased the number of infiltrative anti-ED1-positive cells and the gene expression of transforming growth factor (TGF)-beta1 (84.8 and 80.2% on day 7) after UUO (both P<0.01). By double immunostaining and Western analysis, rapamycin blocked the TGF-beta1-induced loss of E-cadherin expression and de novo increase of the expression of alpha-SMA in a dose-dependent manner. In conclusion, rapamycin significantly attenuated tubulointerstitial damage in a UUO-induced rat model of renal fibrosis, suggesting that rapamycin may have the potential to delay the progression of tubulointerstitial renal fibrosis.     

Ureteral obstruction in neonatal mice elicits segment-specific tubular cell responses leading to nephron loss

BACKGROUND: To elucidate the sequence of renal responses leading to nephron loss in obstructive nephropathy, we examined the evolution of segmental nephron cellular changes consequent to chronic unilateral ureteral obstruction (UUO) in the neonatal mouse. METHODS: Neonatal mice were subjected to UUO or sham-operation, and kidneys were harvested 5, 12 or 19 days after surgery. Proximal tubules (PT), distal tubules (DT) and collecting ducts (CD) were identified with lectins. Histomorphometric quantitation was made for cellular necrosis, apoptosis, proliferation, tubular dilatation, tubular basement membrane (TBM) thickening, interstitial collagen, and glomerular maturation. The distribution of hypoxic tissue was determined using pimonidazole as a marker. Additional studies were performed by mechanically stretching monolayer cultures of mouse proximal tubular and collecting duct cells, and measuring apoptosis. RESULTS: Neonatal UUO induced an arrest of glomerular maturation throughout the period of study. Chronic UUO induced hypoxia, tubular necrosis, proliferation, and TBM thickening in the PT, but stimulated apoptosis in the DT and CD. Tubular dilation in the obstructed kidney was most severe in CD and least severe in PT. Tubular cell apoptosis closely paralleled tubular dilation (P < 0.05), and fibrosis surrounding individual tubules also correlated with tubular dilation (P < 0.001). Mechanical stretching of cultured mouse tubular cells induced apoptosis directly proportional to the magnitude of axial strain: apoptosis was consistently greater in CD than in PT cells (P < 0.05). CONCLUSIONS: Following UUO, the co-localization of hypoxia with cellular proliferation, necrosis, and TBM thickening of the PT is consistent with ischemic injury resulting from vasoconstriction. In contrast, a selective dilation of the distal portion of the nephron (DT and CD), which results from the greater tubular compliance there, leads to stretch-induced epithelial cell apoptosis, along with a progressive peritubular fibrosis. Nephron loss in the obstructed developing kidney likely results from complex, segment-specific cellular responses.     

上调肾组织intermedin表达抑制单侧输尿管梗阻大鼠肾间质纤维化

目的观察上调肾组织intermedin(IMD)表达对单侧输尿管梗阻(UUO)大鼠肾间质纤维化的影响。 方法健康雄性Wistar大鼠随机分为假手术组、UUO组、IMD＋UUO组、空质粒＋UUO组。IMD＋UUO组和空质粒＋UUO组在输尿管结扎前分别将IMD-pcDNA3.1真核表达质粒和空质粒转入肾组织,real-time RT-PCR及免疫组化法检测转染效率。各组分别于术后7 d、14 d留取梗阻侧肾组织。HE、Masson染色观察肾组织病理变化;real-time RT-PCR检测肾组织中转化生长因子-β1(TGF-β1)、纤连蛋白(Fn1)的mRNA表达;Western印迹法检测Fn1的蛋白表达;免疫组化法检测TGF-β1的蛋白表达。 结果与假手术组相比,UUO组肾脏出现明显的病理改变,肾间质纤维化程度随梗阻时间延长加重(与假手术组比较,7 d, t＝11.927,P＝0.0003;14 d, t＝8.891,P＝0.0009);IMD＋UUO组肾脏病理改变及肾间质纤维化程度较同时间点UUO组明显减轻(7 d, t＝3.892,P＝0.018;14 d, t＝4.047,P＝0.016),而空质粒＋UUO组与UUO组无显著差别(7 d, t＝0.562,P＝0.604;14 d, t＝0.035,P＝0.974)。与同时间点假手术组相比,UUO组TGF-β1、Fn1的表达明显升高(TGF-β1 mRNA水平7 d, t＝4.432,P＝0.011;14 d, t＝4.873,P＝0.006;蛋白质水平7 d, t＝5.312,P＝0.006;14 d, t＝4.482,P＝0.011;Fn1 mRNA水平7 d, t＝6.053,P＝0.004;14 d, t＝7.345,P＝0.002;蛋白质水平7 d, t＝8.791,P＝0.009;14 d t＝8.027,P＝0.001);转染IMD质粒后Fn1的表达较同时间点UUO组明显下降(mRNA水平7 d, t＝3.103,P＝0.036;14 d, t＝2.913,P＝0.044;蛋白质水平7 d, t＝2.955,P＝0.042;14 d, t＝2.991,P＝0.040);而转染空质粒后Fn1的表达无明显变化(mRNA水平7 d, t＝0.095,P＝0.929;14 d, t＝0.158,P＝0.882;蛋白质水平7 d, t＝0.159,P＝0.881;14 d, t＝0.170,P＝0.874)。转染IMD和空质粒对TGF-β1的表达均无明显影响(转染IMD质粒mRNA水平7 d, t＝0.176,P＝0.869;14 d, t＝0.126,P＝0.906;蛋白质水平7 d, t＝0.198,P＝0.853;14 d, t＝0.196,P＝0.854;转染空质粒mRNA水平7 d, t＝0.100,P＝0.925;14 d, t＝0.097,P＝0.928;蛋白质水平7 d, t＝0.042,P＝0.968; 14 d, t＝0.060,P＝0.955)。 结论上调肾组织IMD的表达能明显减轻肾间质纤维化,但其作用不是通过直接抑制TGF-β1的表达实现的。     

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.     

Role of apoptosis and Bcl-2/Bax in the development of tubulointerstitial fibrosis during experimental obstructive nephropathy

BACKGROUND/AIMS: To examine the role of apoptosis in experimental unilateral ureteral obstruction (UUO). METHODS: Rat kidneys were examined 3, 7 and 11 days following UUO or sham operation (SO). Tissue was immunohistochemically stained for alpha-smooth muscle actin (alpha-SMA), proliferating cell nuclear antigen (PCNA), Bcl-2 and Bax proteins. Apoptotic analysis was carried out in kidney sections using in situ end labelling of endonuclease cleaved DNA. RESULTS: The relative volume (Vv) of cortical interstitium and interstitial alpha-SMA increased progressively following UUO. ED1-positive monocytes/macrophages peaked at day 7 and significantly decreased at day 11. PCNA-positive cells in tubulointerstitium were significantly increased on day 3. Staining returned to the level of the SO group by day 11, meanwhile those in the interstitium remained much higher than baseline. TUNEL-positive cells were persistently raised following UUO. Transient tubular cell proliferation seemed unable to counteract the apoptosis since tubular atrophy was apparently present by day 11 of UUO. However, interstitial cell proliferation was high enough to overwhelm apoptosis, particularly with respect ot myofibroblasts, since alpha-SMA immunostaining and Vv remained elevated. The ratio of the number of PCNA-positive cells to apoptotic cells formed a predictive pattern for the staining score of interstitial alpha-SMA (R(2) = 47.23%, p < 0.05) and Vv (R(2) = 49.93%, p < 0.05). Tubular Bcl-2 immunostaining peaked on day 3, and then gradually decreased to baseline by day 11. The expression of Bax protein was inhibited on day 3 when compared with that of the SO group, but increased with time following UUO. CONCLUSION: These findings suggest an important role for apoptosis and its regulatory proteins in the processes of tubular atrophy and fibrogenesis following UUO.     

Earlier renal fibrosis aggravates acute kidney injury induced by ischemia reperfusion in mice

Objective To investigate the influence of earlier renal fibrosis on ischemia and reperfusion induced acute kidney injury. Methods Male C57BL/6 mice at eight to twelve weeks old age were divided into 4 groups randomly: (1)Sham (n=3); (2)Unilateral ureter obstruction (UUO, n=6): UUO for 3 days (UUO3d, n=3) and UUO for 5 days (UUO5d, n=3)；(3)Ischemia and reperfusion (IR, n=7): bilateral kidney ischemia for 40 minutes followed by 24 hours of reperfusion; (4)UUO for 3 days plus IR (UUO3d+IR, n=6): bilateral kidney ischemia after UUO 2 days for 40 minutes followed by 24 hours of reperfusion, and the real time for UUO was 3 days. Pathologic analysis for acute or chronic injury was performed on paraffin embedded kidney sections with hematoxylin and eosin (HE) or Masson staining. Apoptosis was detected by immunohistochemistry(IHC) and Western blotting with anti-caspase-3 antibody, and proliferation was observed by IHC with anti-ki67 antibody. Results On kidney sections with HE or Masson staining, it showed that the chronic kidney lesions and fibrosis got more severe as time of UUO prolonged from 3 days to 5 days; the area of matrix deposition increased in UUO5d and UUO3d mice significantly compared to Sham mice (P＜0.05) and was smaller in UUO3d mice compared with UUO5d mice obviously (P＜0.05). Acute kidney injury could be observed in UUO3d+IR mice, such as massive inflammatory cells infiltration, tubules dilation, brush border disappearance, tubular epithelial cells vacuolar degeneration, necrosis, casting formation, coexisting with chronic lesions: thinner cortex, broadened interstitial space, and increased blue stained matrix. Acute kidney injury score in UUO3d+IR mice was higher than that in IR mice significantly (P＜0.05), and serum creatinine level increased significantly in UUO3d+IR mice compared to Sham mice (P＜0.05). Caspase-3 expression increased and ki67 positive tubular cells decreased in UUO3d+IR mice than those in IR mice obviously (P＜0.05). Conclusion Earlier renal fibrosis aggravates acute kidney injury induced by ischemia reperfusion in mice through increasing apoptosis and decreasing proliferation of tubular epithelial cells.     

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.     

Renal tubulointerstitial injury from ureteral obstruction in the neonatal rat is attenuated by IGF-1

BACKGROUND: The administration of insulin-like growth factor-1 (IGF-1) has been shown to ameliorate the renal injury resulting from ischemic acute renal failure. As there are a number of similarities between acute renal failure and obstructive nephropathy, we examined the effects of IGF-1 on the renal cellular response to unilateral ureteral obstruction (UUO) in the neonatal rat. METHODS: Forty-five rats were subjected to UUO or sham operation within the first 48 hours of life and received IGF-1 (2 mg/kg/day) or saline for the following three or seven days, after which kidneys were removed for study by morphometry and immunohistochemistry. To determine the effects of UUO on endogenous expression of IGF-1 and its receptor, six additional rats were subjected to UUO or sham operation, and mRNA was measured by solution hybridization. RESULTS: There was no effect of seven days of UUO on the renal expression of endogenous IGF-1 or its receptor. Moreover, seven days of exogenous IGF-1 did not improve the suppression of nephrogenesis, the delay in glomerular maturation, or the reduction in tubular proliferation induced by ipsilateral UUO. However, in the obstructed kidney, IGF-1 reduced tubular expression of vimentin, apoptosis, and tubular atrophy by 38 to 50% (P < 0.05). In addition, IGF-1 also decreased renal interstitial collagen deposition in the obstructed kidney by 44% (P < 0.05). Following three days of UUO, the administration of IGF-1 also reduced tubular apoptosis (P < 0.05), but did not alter tubular proliferation. CONCLUSIONS: IGF-1 has a profound salutary effect on the tubular and interstitial response to UUO in early development, without affecting glomerular injury or development. These results suggest that IGF-1 may have therapeutic potential in the management of congenital obstructive nephropathy.     

Curcumin mediates repulsive guidance molecule B (RGMb) in the treatment mechanism of renal fibrosis induced by unilateral ureteral obstruction

In this study, we explored the role and mechanism of repulsive guidance molecule B (RGMb, also known as Dragon) in the protective effects of curcumin against renal fibrosis and verified Dragon’s effect on renal tubular epithelial cell apoptosis and cell programmability. Unilateral ureteral obstruction (UUO) was surgically induced in rats to establish a model of renal interstitial fibrosis (RIF). The rats were then treated with curcumin. Curcumin prominently decreased the serum creatinine (SCr) and blood urea nitrogen (BUN) levels, and also improved the tubular injury in the UUO-induced rats. Curcumin significantly downregulated the TGF-β1, P-Smad2/3, cleaved caspase-3, cleaved caspase-8 and Dragon levels. Dragon knockdown also markedly reduced the TGF-β1, P-Smad2/3, Smad2/3, cleaved caspase-3, cleaved caspase-8, fibronectin, collagen I, collagen IV, vimentin, and α-SMA expression levels. Conversely, Dragon overexpression caused higher expression levels of these proteins, and curcumin reversed this effect. Furthermore, Dragon knockdown increased the E-cadherin levels, whereas Dragon overexpression decreased these levels. Overexpressing Dragon significantly decreased the cell viability, and curcumin reversed this effect. In conclusion, curcumin acted on Dragon and attenuated RIF in UUO rat models. Curcumin downregulated the TGF-β1/Smad signaling pathway and inhibited Dragon and fibrogenic molecules in both rats and HK-2 cells.     

Variable chronic partial ureteral obstruction in the neonatal rat: a new model of ureteropelvic junction obstruction

BACKGROUND: Congenital ureteropelvic junction (UPJ) obstruction is a common developmental anomaly. To elucidate the mechanisms underlying the renal consequences of congenital UPJ obstruction, we have developed a new model of variable partial unilateral ureteral obstruction (UUO) in the neonatal rat. METHODS: Rat pups were subjected to sham-operation, complete UUO, or variable partial UUO within the first day of life. After 14 or 28 days, the relative number of glomeruli, cell proliferation, tubular apoptosis, tubular atrophy, and interstitial fibrosis were quantitated in histologic sections. Glomerular filtration rate (GFR) was determined after 28 days of partial or complete UUO. RESULTS: Following 70% to 75% reduction in ureteral diameter, renal growth from 14 to 28 days was reduced by 60%, and the number of glomeruli decreased by 50%. Renal pelvic diameter increased in proportion to the severity of obstruction following 14 days of partial UUO, and by 28 days, was maximally dilated regardless of the luminal diameter. Renal proliferation was increased, while tubular apoptosis, tubular atrophy, and interstitial fibrosis were less severe 14 days following partial UUO than in complete UUO. GFR was reduced by 80%, and proteinuria developed following 28 days of partial UUO. CONCLUSION: Renal function is impaired by chronic ipsilateral partial UUO, which reduces the number of nephrons, and leads to progressive renal pelvic dilatation. Tubular atrophy and interstitial fibrosis develop prior to significant renal pelvic dilatation. Correlation of clinically measurable parameters with renal morphometry or imaging studies in this model may lead to new approaches to the management of congenital UPJ obstruction.     

Protein restriction ameliorates renal tubulointerstitial nephritis and reduces renal transforming growth factor-beta expression in unilateral ureteral obstruction.

In contrast to the substantial evidence for attenuation of the glomerular lesions by a low-protein (LP) diet, it remains to be determined whether and how such a diet lessens the progression of tubulointerstitial lesions, which show the strongest correlation with renal function. Chronic unilateral ureteral obstruction (UUO) results in interstitial fibrosis of the affected kidney. We investigated the therapeutic effects of an LP diet on the progression of interstitial fibrosis in UUO mice. Sixty ICR mice underwent UUO or sham operation; half of these mice were fed a normal-protein (NP) and the other half a LP diet. They were sacrificed at 3, 7 and 14 days postoperatively. The degree of tubular lesion, the distribution of transforming growth factor-beta (TGF-beta), alpha-smooth muscle actin and fibronectin and the activated TGF-beta 1 level were determined. The LP diet significantly reduced the progression of tubular injury, depositions of fibronectin, tubulointerstitial myofibroblast formation, the interstitial expression of TGF-beta-positive cells (at 14 days; NP = 6.91 +/- 3.35 vs. LP = 1.67 +/- 0.41; p < 0.005), and renal active TGF-beta 1 concentration (at 14 days; NP = 5.72 +/- 2.03 vs. LP = 2.96 +/- 0.72; p < 0.01). We conclude that protein restriction may aid the attenuation of progression of tubulointerstitial fibrosis through the reduction in tubulointerstitial expression of TGF-beta.     

单侧输尿管梗阻大鼠肾脏转化生长因子β受体亚型表达的变化

目的 探讨转化生长因子β（TGF-β）Ⅰ型受体（RⅠ）、Ⅱ型（RⅡ）受体以及下游Smad蛋白在单侧输尿管梗阻（UUO）大鼠模型肾脏中表达及意义。 方法 90只雌性Wistar大鼠随机分为正常对照组（CON组）、假手术组（SOR组）和单侧输尿管梗阻组（UUO组）,分别于术后1、3、7、14、21 d处死,检测各组大鼠肾功能;PAS与Masson染色观察大鼠肾间质病理形态改变;实时定量PCR基因芯片分析正常大鼠和肾间质纤维化大鼠肾组织TGF-βⅠ、Ⅱ、Ⅲ型受体及Smad蛋白家族表达。筛选出差异表达的受体亚型,进一步应用实时荧光定量PCR、蛋白免疫印迹法、免疫荧光法检测和验证筛选出的差异受体亚型在不同分期肾间质纤维化大鼠肾组织的分布和表达。 结果 与CON组相比,UUO组大鼠的Scr及BUN于术后3 d开始升高（P < 0.05）,第21天达峰值（P < 0.01）;UUO组术后3 d肾间质可见明显炎性细胞浸润;14 d后出现明显肾小管萎缩;21 d可见明显肾间质纤维化。UUO组肾组织TGF-βⅠ型受体ALK-5、ALK-7和TGF-βRⅡ的mRNA表达于术后3 d上升并随梗阻时间延长逐渐增加（P < 0.05）,于14 d达到峰值（均P < 0.01）;ALK-6的mRNA表达于术后3 d下降（P < 0.05）并随梗阻时间延长逐渐减少,于14 d达谷值（P < 0.01）。ALK-5、ALK-6、ALK-7和TGF-βRⅡ蛋白表达与基因表达一致。Smad2/3及磷酸化（p）-Smad2/3的蛋白表达于术后3 d上升（均P < 0.05）并随梗阻时间延长逐渐增加,于14 d达到峰值（均P < 0.01）。 结论 在肾间质纤维化进展中不同TGF-β受体亚型存在不同的变化规律并与肾间质纤维化进展密切关联。     

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.     

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.     

Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis

Connective tissue growth factor (CTGF/CCN2) is one of the candidate factors mediating fibrogenic activity of TGF-beta. It was shown previously that the blockade of CTGF by antisense oligonucleotide (ODN) inhibits TGF-beta-induced production of fibronectin and type I collagen in cultured renal fibroblasts. The in vivo contribution of CTGF in renal interstitial fibrosis, however, remains to be clarified. With the use of a hydrodynamics-based gene transfer technique, the effects of CTGF antisense ODN are investigated in rat kidneys with unilateral ureteral obstruction (UUO). FITC-labeled ODN injection via the renal vein showed that the ODN was specifically introduced into the interstitium. At day 7 after UUO, the gene expression of CTGF, fibronectin, fibronectin ED-A, and alpha1(I) collagen in untreated or control ODN-treated obstructed kidneys was prominently upregulated. CTGF antisense ODN treatment, by contrast, markedly attenuated the induction of CTGF, fibronectin, fibronectin ED-A, and alpha1(I) collagen genes, whereas TGF-beta gene upregulation was not affected. The antisense treatment also reduced interstitial deposition of CTGF, fibronectin ED-A, and type I collagen and the interstitial fibrotic areas. The number of myofibroblasts determined by the expression of alpha-smooth muscle actin was significantly decreased as well. Proliferation of tubular and interstitial cells was not altered with the treatment. These findings indicate that CTGF expression in the interstitium plays a crucial role in the progression of interstitial fibrosis but not in the proliferation of tubular and interstitial cells during UUO. CTGF may become a potential therapeutic target against tubulointerstitial fibrosis.     

Hepatocyte growth factor gene therapy retards the progression of chronic obstructive nephropathy

BACKGROUND: Unilateral ureteral obstruction (UUO) is characterized by progressive tubular atrophy and interstitial fibrosis. Rupture of the balance between cell proliferation and apoptosis plays a critical role in renal atrophy. Hepatocyte growth factor (HGF) is a cytokine function on cell survival and tissue regeneration. We studied the effects and possible mechanisms of HGF gene therapy on tubular cell survival and anti-fibrosis in chronic obstructed nephropathy. METHODS: An in vivo transfection procedure of repeatedly transducing skeletal muscles with the HGF gene using liposomes containing the hemagglutinating virus of Japan (HVJ liposome) was tested on UUO rats. Expression of HGF and c-Met were examined by in situ hybridization, ELISA, or immunohistochemical staining. Interstitial fibrosis and macrophage infiltration were evaluated by Masson's Trichrome staining, alpha-smooth muscle actin and ED-1 immunostaining. Cell survival indices including proliferating cell nuclear antigen (PCNA), Bcl-2, Bcl-xL and Bax were measured by immunohistochemistry and Western blots. Apoptosis was determined by the TUNEL method. RESULTS: After HVJ-HGF gene transfer, endogenous HGF and c-Met were up-regulated in UUO kidneys. Renal fibrosis, macrophage infiltration and tubular atrophy were suppressed both at day 14 and 28 after UUO (P < 0.05 or 0.01). Tubular cell proliferation was activated while apoptosis was inhibited, especially at the late stage of UUO. Bcl-2 was enhanced in the HGF-transfected UUO rats, while no changes of Bcl-xL and Bax were found. CONCLUSIONS: In vivo HGF gene transfection retards the progression of chronic obstructed nephropathy and protects tubular cell survival in the long-term UUO model. Bcl-2 rather than Bcl-xL or Bax may contribute to the anti-apoptotic function of HGF.     

Complement C5 mediates experimental tubulointerstitial fibrosis

Renal fibrosis is the final common pathway of most progressive renal diseases. C5 was recently identified as a risk factor for liver fibrosis. This study investigated the role of C5 in the development of renal tubulointerstitial fibrosis by (1) induction of renal fibrosis in wild-type and C5(-/-) mice by unilateral ureteral ligation (UUO) and (2) investigation of the effects of a C5a receptor antagonist (C5aRA) in UUO. In C5(-/-) mice, when compared with wild-type controls, markers of renal fibrosis (Sirius Red, type I collagen, fibronectin, alpha-smooth muscle actin, vimentin, and infiltrating macrophages) were significantly reduced on day 5 of UUO. On day 10, fibronectin mRNA and protein expression were still reduced in the C5(-/-) mice. Cortical mRNA of all PDGF isoforms and of TGF-beta(1) (i.e., central mediators of renal disease) were significantly reduced in C5(-/-) mice when compared with controls. Renal tubular cell expression of the C5aR was sparse in normal cortex but markedly upregulated after UUO. Treatment of wild-type UUO mice with C5aRA also led to a significant reduction of cortical Sirius Red staining, fibronectin protein expression, and PDGF-B mRNA expression on day 5. Neither genetic C5 deficiency nor C5aRA treatment caused any histologic changes in the nonobstructed kidneys. In cultured murine cortical tubular cells, C5a stimulated production of TGF-beta(1), and this was inhibited by C5aRA. Using a combined genetic and pharmacologic approach, C5, in particular C5a, is identified as a novel profibrotic factor in renal disease and as a potential new therapeutic target.