染料木黄酮在减轻肾间质纤维化中的作用

目的 探讨染料木黄酮(Genistein)在减轻单侧输尿管结扎模型(UUO)诱导肾间质纤维化中的作用及可能机制.方法 将30只SD大鼠随机分为5组,A组:假手术+DMSO 1 ml/d×14 d;B组:假手术+Genistein每日20 mg/ks体重×14 d;C组:UUO+DMSO 1 ml/d×14 d;D组:UUO+Genistein 5mg/kg体重每日×14d;E组:UUO+Genistein 20mg/kg体重每日×14d.术后14d比较各组大鼠左/右肾重量和长度的比值;酶联免疫吸附实验(ELISA)法检测血清INF-y和TGFa1表达;病理观察肾小管扩张和肾间质增生的程度,免疫组织化学观察肾脏á-SMA和ED-1表达.结果 C、D、E组左/右肾重量及长度比值均明显高于A、B组,但E组左/右肾重量及长度比值、血清TGF-a1及INF-y浓度、肾小管扩张分数和肾小管容量分数、á-SMA和ED-1的表达均低于C组.结论 染料木黄酮可明显降低由单侧输尿管结扎模型所导致的肾间质纤维化的病理改变,其可能机制是染料木黄酮抑制TGF-a1及INF-y的表达,从而抑制肾小管上皮细胞向成纤维细胞转化.     

Triptolide attenuates renal interstitial fibrosis in rats with unilateral ureteral obstruction

Aim: Extracts of Tripterygium wilfordii Hook F. have been used to treat glomerulonephritis for more than 30 years in China. Most of the anti‐inflammatory and immunosuppressive activities of these extracts can be attributed to triptolide (Trip). The present study was to investigate the effect of Trip on renal interstitial fibrosis in a model of unilateral ureteral obstruction (UUO). Methods: UUO or sham‐operated rats were randomly assigned to receive mycophenolate mofetil (MMF), Trip or vehicle and were killed on days 7 and 14 after UUO or sham operation. Kidney specimens were fixed for immunohistochemistry for myofibroblasts (α‐smooth muscle actin, α‐SMA), macrophages (ED‐1), monocyte chemoattractant protein‐1 (MCP‐1) and osteopontin. Interstitial collagen deposition and amounts of transforming growth factor‐β1 (TGF‐β1) were determined by Sirius red staining and enzyme‐linked immunosorbent assay, respectively. The mRNA expression of TGF‐β1, connective tissue growth factor (CTGF), MCP‐1 and osteopontin were measured by real‐time polymerase chain reaction analysis. Results: The scores for the density of α‐SMA‐ and ED‐1‐positive cells, the staining of MCP‐1 and osteopontin, interstitial collagen deposition and amounts of TGF‐β1 were significantly reduced by MMF or Trip. MMF or Trip significantly reduced the mRNA expression of TGF‐β1, CTGF, MCP‐1 and osteopontin. Conclusion: Trip significantly attenuated tubulointerstitial fibrosis in a rat UUO model and the effect of Trip on renal fibrosis was similar to that of MMF. Trip may be useful as a potential candidate in the treatment of renal fibrosis.     

内质网应激相关凋亡途径参与单侧输尿管梗阻大鼠肾间质纤维化

Objective To investigate the effect of endoplasmic reticulum stress (ERS)-associated apoptosis on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO) in rats. Methods Eighteen healthy male Wistar rats undergoing UUO were sacrificed at 3,7,14 days after operation. Additional seven rats underwent sham operation. Histological changes were observed by HE and Masson staining. Immunohistochemistry was performed on renal tissue for α-smooth muscle actin (α-SMA). Chromatometry was used to detect the content of hydroxyproline. Apoptosis cells were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and the electrophoresis analysis of genome DNA. The mRNA expression of glucose-regulated protein 78 (GRP78), which are important markers of ERS, were detected by RT-PCR. Western blotting was used to assess the protein level of GRP78 and cysteinyl aspartate specific proteinase-3 (caspase-3). Results Compared with sham operation group, the degree of renal interstitial and the level of hydroxyproline content of UUO group increased significantly (P<0.05). Immunohistochemistry staining indicated that a-SMA extensively expressed in renal tubular and interstitial cells. The apoptotic cells in the renal tubular and interstitium were continuously increased from day 3 to the end of experiment of UUO group. As early as 3 days after surgery, the mRNA level of GRP78 in UUO group increased compared with sham operation group (P<0.01), while the protein expression increased on day 7 after surgery (P<0.01). Prolonged ERS triggered apoptosis, the protein expression of caspase-3 increased significantly on day 3 after surgery (P< 0.05), and the expression sustained high level during the experiment afterwards. There was a positive correlation between GRP78 protein expression and hydroxyproline content (r =0.657, P< 0.01) as well as caspase-3 protein expression (r=0.714, P<0.01). Conclusions UUO induces a significant up-regulation in endoplasmic reticulum molecular chaperones at early stage, indicating that ERS response is activated in the rat kidney. Prolonged ERS can lead to renal tubular and interstitial cell apoptosis, and caspase-3-mediated ERS associated apoptosis may contribute to the fibrosis.     

内质网应激相关凋亡途径参与单侧输尿管梗阻大鼠肾间质纤维化

Objective To investigate the effect of endoplasmic reticulum stress (ERS)-associated apoptosis on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO) in rats. Methods Eighteen healthy male Wistar rats undergoing UUO were sacrificed at 3,7,14 days after operation. Additional seven rats underwent sham operation. Histological changes were observed by HE and Masson staining. Immunohistochemistry was performed on renal tissue for α-smooth muscle actin (α-SMA). Chromatometry was used to detect the content of hydroxyproline. Apoptosis cells were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and the electrophoresis analysis of genome DNA. The mRNA expression of glucose-regulated protein 78 (GRP78), which are important markers of ERS, were detected by RT-PCR. Western blotting was used to assess the protein level of GRP78 and cysteinyl aspartate specific proteinase-3 (caspase-3). Results Compared with sham operation group, the degree of renal interstitial and the level of hydroxyproline content of UUO group increased significantly (P<0.05). Immunohistochemistry staining indicated that a-SMA extensively expressed in renal tubular and interstitial cells. The apoptotic cells in the renal tubular and interstitium were continuously increased from day 3 to the end of experiment of UUO group. As early as 3 days after surgery, the mRNA level of GRP78 in UUO group increased compared with sham operation group (P<0.01), while the protein expression increased on day 7 after surgery (P<0.01). Prolonged ERS triggered apoptosis, the protein expression of caspase-3 increased significantly on day 3 after surgery (P< 0.05), and the expression sustained high level during the experiment afterwards. There was a positive correlation between GRP78 protein expression and hydroxyproline content (r =0.657, P< 0.01) as well as caspase-3 protein expression (r=0.714, P<0.01). Conclusions UUO induces a significant up-regulation in endoplasmic reticulum molecular chaperones at early stage, indicating that ERS response is activated in the rat kidney. Prolonged ERS can lead to renal tubular and interstitial cell apoptosis, and caspase-3-mediated ERS associated apoptosis may contribute to the fibrosis.     

内质网应激相关凋亡途径参与单侧输尿管梗阻大鼠肾间质纤维化

目的 探讨内质网应激（ERS）相关凋亡途径在单侧输尿管梗阻（UUO）大鼠肾间质纤维化发生、发展中的作用。 方法 健康雄性Wistar大鼠25只,按随机数字表法分为UUO模型组（n=18）和假手术组（n=7）,UUO模型组行左侧输尿管结扎术,假手术组仅分离输尿管不结扎,分别于术后3 d、7 d、14 d处死各组大鼠,行HE和Masson染色,观察肾脏病理变化;比色法测定肾组织羟脯氨酸（HYP）含量;免疫组化法检测α平滑肌肌动蛋白（α-SMA）;原位末端标记法（TUNEL）与DNA电泳观察肾小管间质细胞凋亡情况;RT-PCR法检测梗阻侧肾组织ERS相关分子葡萄糖调节蛋白78（GRP78）mRNA表达变化;Western印迹法分析凋亡相关蛋白半胱氨酸天门冬氨酸蛋白酶3（caspase-3）和GRP78的蛋白表达变化。 结果 与假手术组比较,UUO模型组肾脏病理改变加重,肾间质纤维化程度随梗阻时间延长逐渐加重,肾组织HYP含量显著升高（P < 0.05）,肾组织α-SMA也在肾小管间质细胞广泛表达,TUNEL染色及DNA琼脂糖凝胶电泳提示大量的肾小管间质细胞凋亡。UUO模型组GRP78 mRNA表达于术后3 d即发生显著上调,而蛋白表达在术后7 d开始出现显著变化,与假手术组差异均有统计学意义（均P < 0.01）;在此后观察期间内GRP78 mRNA和蛋白均持续高水平表达。模型组大鼠肾组织caspase-3的蛋白表达在UUO术后3 d即有显著上调（P < 0.05）,且随着梗阻时间延长进行性升高,于术后7 d、14 d增多更为显著,与假手术组差异均有统计学意义（P < 0.05）。相关分析显示GRP78蛋白表达与肾组织HYP含量和caspase-3蛋白表达均呈正相关（r = 0.657,P < 0.01;r = 0.714,P < 0.01）。 结论 UUO早期即可诱导ERS标志蛋白表达变化,触发ERS。长期ERS可诱导肾小管间质细胞凋亡;caspase-3介导的ERS相关凋亡途径可能参与了肾间质纤维化过程。     

Smad3 deficiency attenuates renal fibrosis, inflammation,and apoptosis after unilateral ureteral obstruction

BACKGROUND: Transforming growth factor-beta (TGF-beta) has been implicated in the development of renal fibrosis induced by unilateral ureteral obstruction (UUO). However, there is little information on signaling pathways mediating TGF-beta activity involved in molecular and cellular events leading to renal fibrosis induced by UUO. In this study, we sought to determine whether Smad3, a major signaling component of TGF-beta, mediated renal fibrosis induced by UUO. METHODS: Renal fibrosis, inflammation, and apoptosis induced by UUO were macroscopically and histologically compared between wild-type mice and Smad3 null mice. RESULTS: Gross appearance of the kidney after UUO showed relatively intact kidney in Smad3 null mice [Smad3(-/-) mice] when compared with that of wild-type mice [Smad3(+/+) mice]. Renal interstitial fibrosis based on the interstitial area stained with Aniline-blue or Sirius red solution was significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Deposition of type I and type III collagens were also significantly reduced in the obstructed kidney of Smad3(-/-) mice. In addition, the numbers of myofibroblasts, macrophages, and CD4/CD8 T cells infiltrated into the kidney after UUO were significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Furthermore, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining after UUO showed significantly reduced number of tubular apoptotic cells in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Endogenous Smad pathway was activated in the obstructed kidney after UUO in wild-type mice as judged by the increase of phosphorylated Smad2 or phosphorylated Smad2/3-positive cells in renal interstitial area. CONCLUSION: Smad3 deficiency attenuated renal fibrosis, inflammation, and apoptosis after UUO, suggesting that Smad3 was a key molecule mediating TGF-beta activity leading to real fibrosis after UUO.     

Attenuation of ureteral obstruction-induced renal injury by polyenylphosphatidylcholine

BACKGROUND: The cytoprotective, antioxidant and antifibrotic effects of polyenylphosphatidylcholine (lecithin, PPC) have been demonstrated both experimentally and clinically. The present study investigated whether PPC treatment has any beneficial effect on renal injury in unilateral partial ureteral obstruction (UUO) in rats. METHODS: Forty Wistar-Albino rats were split into three groups (sham-operated controls, untreated and treated rats). Rats of the untreated and treated groups (n = 15) underwent UUO with two-thirds of the left ureter embedded in the psoas muscle. In group 3, PPC was given orally at a dose of 100 mg/day for 30 days. At the end of the 30th day of the experimental period, obstructed kidneys and blood samples were harvested. To investigate the therapeutic efficacy of PPC treatment in UUO kidneys, oxidant and antioxidant enzyme levels, lipid peroxidation, proinflammatory cytokines (interleukin-1, interleukin-6, tumor necrosis factor alpha), transforming growth factor beta-1 (TGFbeta-1), alpha smooth muscle actin (alpha-SMA) and nuclear factor kappa beta (NF-kappabeta) expression, leukocyte infiltration (ED1, ED2, CD4 and CD8 immunohistochemistry), and tubulointerstitial damage in the obstructed kidneys were studied. RESULTS: Oxidative stress, neutrophil infiltration, release of cytotoxic mediators, TGFbeta-1 levels, tubulointerstitial damage, alpha-SMA and NF-KB expressions in kidney tissue were significantly increased in the UUO rats. PPC treatment attenuated oxidative stress, leukocyte infiltration, cytotoxic mediator, and TGFbeta-1 levels and also decreased expressions of alpha-SMA and NF-kappabeta. It was associated with decreased tubulointerstitial damage, compared with UUO alone. CONCLUSIONS: These results indicate that PPC treatment protects against UUO-induced renal injury in rats possibly through its antioxidant, anti-inflammatory and antifibrotic actions.     

COMP-angiopoietin-1 ameliorates renal fibrosis in a unilateral ureteral obstruction model

Injury to the renal microvasculature may be a major factor in the progression of renal disease; therefore, protection of endothelial cells (EC) in renal vasculature may have a therapeutic role in renal fibrosis. Recently, a soluble, stable, and potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, was developed. The contribution of COMP-Ang1 in renal interstitial fibrosis, however, remains to be clarified. This study investigated the effects of COMP-Ang1 on peritubular capillary EC in the renal cortex and the renal fibrogenic process that is triggered by unilateral ureteral obstruction. COMP-Ang1 preserved renal platelet-EC adhesion molecule-1-and Tie2-positive EC. Morphologic examination indicated less tubular injury and tubulointerstitial fibrosis in mice that received COMP-Ang1 than vehicle-treated mice. Interstitial type I collagen and myofibroblast accumulation were significantly suppressed by COMP-Ang1 treatment. COMP-Ang1 increased Tie2 and Akt phosphorylation in ureteral obstructed kidneys. Renal surface microvasculature and renal blood flow were higher after treatment with COMP-Ang1 than with vehicle. COMP-Ang1 treatment decreased monocyte/macrophage infiltration, tissue levels of TGF-beta1, and Smad 2/3 phosphorylation and increased Smad 7 in the obstructed kidney. These results demonstrate that COMP-Ang1 treatment can decrease the progression of renal fibrosis in unilateral ureteral obstruction. COMP-Ang1 may be an endothelium-specific therapeutic modality in fibrotic renal disease.     

intermedin对单侧输尿管梗阻大鼠肾间质纤维化的影响

目的 观察intermedin( IMD)对单侧输尿管梗阻(UUO)大鼠肾间质纤维化的影响.方法 雄性Wistar大鼠40只,按随机数字表法分为假手术组(n=10,行左输尿管分离术)、模型组(UUO,n=10)、氯沙坦组(n=10)和IMD组(n=10),后3组行左输尿管结扎术.各组大鼠分别于术后第14、21天随机选取5只,腹主动脉采血并留取梗阻侧肾组织之后处死.HE、Masson染色观察肾组织病理变化;比色法测定血尿素氮(BUN)、血肌酐(Scr)及新鲜肾组织羟脯氨酸(Pro)含量;免疫组化方法检测肾组织中转化生长因子β1 (TGF-β1)、IMD的表达水平,并进行半定量分析.结果 与假手术组相比,不同时间点UUO组血BUN、Scr、肾组织Pro含量及TGF-β1、IMD的阳性表达均显著升高(P＜0.05);与UUO组相比,氯沙坦组血BUN、Scr、肾组织Pro含量及TGF-β1、IMD表达均降低(P＜0.05),IMD组除IMD表达增加外,其余均降低(P＜0.05).结论 IMD可减轻UUO肾间质纤维化,改善肾功能,其机制可能与拮抗纤维化炎性介质TGF-β1有关.     

Losartan attenuates renal vasoconstriction in response to acute unilateral ureteral occlusion in pigs

Unilateral ureteral obstruction in pigs is associated with an enhanced, de novo generation of angiotensin II from the ipsilateral kidney. In order to further investigate the role of this system during unilateral ureter obstruction, the renal hemodynamic response to the non-peptide angiotensin II antagonist losartan was investigated. Danish land race pigs were operated on under general anesthesia. Catheters were placed in both renal veins by x-ray and ultrasonic flow probes were mounted on the renal arteries. Losartan (2 mg/kg/h) was administered intravenously to an experimental group ( n=9) continuously over 8 h of unilateral ureteral occlusion. This group was then compared to a matched control group which received only saline ( n=6). Ipsilateral pelvic pressure, renal blood flow using ultrasound transit time, glomerular filtration rate, mean arterial pressure and heart rate were measured. Renal handling of angiotensin II was examined by determining the renal extraction and secretion rates of immunoreactive angiotensin II. The anticipated reduction in ipsilateral renal blood flow after the onset of obstruction was attenuated in the losartan treated pigs, but the ipsilateral glomerular filtration rate was unaffected as compared with the controls. In the losartan group, the increase in renal vascular resistance was significantly reduced compared with un-treated controls (141+/-25% vs 217+/-24%, P<0.05). Plasma immunoreactive angiotensin II increased significantly from all three sample locations in both groups after the onset of obstruction, being more pronounced in the losartan treated group in which immunoreactive angiotensin II from the ipsilateral renal vein increased from 5.1+/-0.5 pmol/l to 41.6+/-19.6 pmol/l, P=0.027. In the controls immunoreactive angiotensin II increased from 2.7+/-0.3 pmol/l to 24.8+/-10.2 pmol/l. Furthermore, plasma aldosterone was significantly reduced after losartan administration (from 80.4 pmol/l to 36.0 pmol/l, P=0.005), indicating effective blockade of the angiotensin II type-1 receptor. The results from the present study suggest that continuous intravenous administration of losartan blocks the angiotensin II receptor mediated effects in the pig. Losartan is able to reduce ipsilateral vasoconstriction in the obstructed kidney during unilateral ureter obstruction supporting the view that angiotensin II is an important mediator of vasoconstriction during unilateral ureter obstruction in the pig model with acute unilateral occlusion of the ureter.     

双环醇对单侧输尿管梗阻大鼠肾间质纤维化的影响及作用机制

肾间质纤维化是反映肾功能下降严重程度和判断预 后最重要的指标[1],寻求抗肾间质纤维化的新药物已成为当前肾脏病研究的热点之一.双环醇是在联苯双酯的基础上,经结构改造和筛选得到的国家Ⅰ类抗肝炎新药.近来研究发现,双环醇对肝纤维化具有明显的抑制作用[2].本研究应用双环醇动态观察单侧输尿管梗阻(UUO)术后肾组织NF-κB的表达水平,探讨双环醇对UUO大鼠肾间质纤维化的影响及其可能机制.     

Effects of renal sympathetic denervation on renal interstitial fibrosis in rats with unilateral ureteral obstruction

Objective To observe the influence of renal sympathetic denervation (RSD) on renal interstitial fibrosis and transforming growth factor beta 1(TGF-β1) and microRNA-21 (miR-21) in rats with unilateral ureteral obstruction(UUO). Methods 40 male Wistar rats were randomly divided into UUO group (A group, n=10), sham UUO group (B group, n=10), RSD+UUO group (C group, n=10) and RSD+sham UUO group (D group, n=10). Rats in A group and C group underwent unilateral ureteral ligation, while those in B group and D group underwent sham operation. Rats in C group and D group were followed by RSD. Rats were sacrificed at 21 days after the operation to evaluate the fibrosis by Masson staining. Immunohistochemical staining and Western blotting were used to detect the expressions of collagen I (COL-I), collagen Ⅲ(COL-Ⅲ) and TGF-β1 in four groups. The expression of miR-21 was detected by fluorescence in situ hybridization (FISH) and quantitative real-time PCR (RT-qPCR). Results A large amount of collagen deposition was observed in the renal interstitial area in A and C group compared to either B or D group (P＜0.05), but the change in C group was decreased significantly than that in A group (P＜0.05). Similarly, the expressions of COL-I, COL-Ⅲ, TGF-β1 and miR-21 were obviously higher in A and C group compared to either B or D group (P＜0.05), but those change in C group were decreased significantly than those in A group (P＜0.05). The above indexes were not significantly different between B group and D group (P＞0.05). Conclusion RSD may relieve the renal interstitial fibrosis in UUO rats, and down-regulate the expression of TGF-β1 and miR-21.     

Dietary arginine supplementation attenuates renal damage after relief of unilateral ureteral obstruction in rats

BACKGROUND: Progression of renal injury after relief of unilateral ureteral obstruction (UUO) has been demonstrated. Nitric oxide (NO) may be an effective intervention due to its vasodilatory, antifibrotic, and anti-apoptotic effects. Herein, we used dietary L-arginine (ARG) supplementation in a UUO relief model. METHODS: This study comprised group 1, control (no treatment). All other rats were subject to 3-day UUO, which was then relieved, and the rats maintained for 7 additional days. Group 2, no additional treatment; group 3, L-ARG; group 4, L-NAME, NO synthase inhibitor; group 5, ARG and L-NAME. Urinary NO(2/3) was quantified. GFR and ERPF were measured at day 10. Interstitial fibrosis and fibroblast expression, macrophage infiltration, tubular apoptosis, and proliferation, NOS expression, and the levels of tissue TGF-beta were evaluated. RESULTS: Urinary NO(2/3) was significantly increased by ARG treatment and decreased by L-NAME. GFR and ERPF measured 7 days following relief were not significantly different in the previously obstructed kidneys (POK) of groups 2 and 3. L-NAME significantly reduced GFR and ERPF in the POK. ARG significantly reduced apoptosis, macrophage infiltration, and fibroblast expression in the POK. L-NAME exacerbated the effects on apoptosis and fibroblasts. Fibrosis was minimal in groups 1 through 3, but was significantly increased by L-NAME. ARG did not affect renal NOS expression and tissue TGF-beta1 levels. CONCLUSION: Dietary ARG supplementation during UUO relief did not improve ERPF or GFR. However, renal damage, including fibrosis, apoptosis, and macrophage infiltration was significantly improved by ARG treatment. This suggests that increasing NO availability could be beneficial in the setting of UUO relief.     

红细胞生成素对单侧输尿管梗阻大鼠肾间质纤维化的保护作用

肾间质纤维化是各种肾脏疾病进展到终末期肾病过程的共同途径.转化生长因子β1(TGF-β1)-Smads信号传导通路在肾间质纤维化的发生发展过程中起重要作用[1].