Triptolide inhibits the tubulointerstitial fibrosis in rats of unilateral ureteral obstruction by up-regulating Ski expression

Objective To investigate the effects of triptolide on tubulointerstitial fibrosis in rats kidneys with the unilateral ureteral obstruction (UUO) by examining the expression of collagen type Ι (Col-Ι), Ski, Smad3, TGF-β1. Methods Sixty male SD rats were divided into three groups: Sham operation group (Sham group), UUO group and triptolide (0.2 mg•kg-1•d-1) treatment group. The levels of blood urea nitrogen (BUN), serum creatinine (Scr), pathological changes were measured. Col-Ι, Ski and Smad3 expressions were assessed by immunohistochemistry. Protein and mRNA expressions of Ski, Smad3, TGF-β1 were assessed by Western blotting and real-time PCR. Results Compared with Sham group, Scr and BUN increased significantly in UUO group (P＜0.05). Interstitial fibrosis was prominent and renal interstitial injury score increased significantly in UUO group (P＜0.05). The expressions of Col-Ι and Smad3 were increased in UUO group (P＜0.05). Compared with Sham group, the protein expressions of TGF-β1 and Smad3 were increased, the Ski protein was decreased in UUO group (P＜0.05). In triptolide group, the morphological changes were notably reduced (P＜0.05). Comparison with UUO group, triptolide could increase the protein and mRNA expressions of Ski significantly, and decreased the protein and mRNA expressions of Smad3 and TGF-β1 (P＜0.05). Conclusion Triptolide can reduce the tubulointerstitial fibrosis by up-regulating Ski, and down-regulating TGF-β1 and Smad3.     

冬虫夏草菌粉对5/6肾大部切除大鼠肾脏纤维化的抑制作用及机制

目的 探讨冬虫夏草菌粉对5/6肾大部切除术大鼠肾脏纤维化的抑制作用及其可能机制.方法 30只雄性SD大鼠随机分为3组:假手术组(Sham组,n=10)、5/6肾大部切除模型组(SNx组,n=10)以及5/6肾大部切除+冬虫夏草菌粉干预组(CS组,n=10).术前及术后4、8、12周分别检测大鼠体质量、尿蛋白量变化,并于术后第12周末处死大鼠,检测血尿素氮、肌酐变化,取肾组织切片行HE、Masson染色观察肾脏病理变化,免疫组化观察转化生长因子β1(TGF-β1)及其Ⅰ型受体(TβR Ⅰ)、Ⅱ型受体(TβR Ⅱ)的表达,免疫荧光观察E-c adherin、α-SMA的表达,Western印迹法检测肾脏组织TGF-β1、TβR Ⅰ、TβR Ⅱ、磷酸化(p)Smad2/3、Smad7、E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)的表达.结果 术后CS组大鼠的体质量高于SNx组,尿蛋白量及血尿素氮、血肌酐低于SNx组.肾脏组织病理分析显示,CS组肾小球硬化、肾小管间质损伤程度均显著低于SNx组(均P＜0.01).CS组TGF-β1、TβR Ⅰ、TβR Ⅱ、p-Smad2/3蛋白表达量均显著低于SNx组(均P＜0.05),E-cadherin蛋白表达量显著高于SNx组(P＜0.05),α-SMA蛋白表达量显著低于SNx组(P＜0.05),Smad7蛋白表达量显著高于SNx组(P＜0.05).结论 冬虫夏草菌粉对5/6肾大部切除大鼠肾脏纤维化具有明显的抑制作用,其机制可能是与其抑制TGF-β1及其下游信号通路以及抑制EMT的发生有关.     

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.     

Astragaloside IV attenuates renal tubulointerstitial fibrosis by inhibiting p38 MAPK signaling

Objective To investigate the effect of astragaloside IV (AS-IV) on renal tubulointerstitial fibrosis and its regulation on p38 MAPK signaling. Methods In vivo, UUO model with renal tubulointerstitial injury was constructed. Mice in AS-IV group were orally administrated AS-IV 20 mg•kg-1•d-1 for 7 days after operation, and mice in other groups were administrated the equal volume vehicle. Bilateral kidneys were collected in 7 and 14 days after operation. Transverse kidney slices were stained with Masson trichrome to evaluate the severity of renal tubule injury. In vitro, normal human renal tubular epithelial cells (HK-2) were stimulated with recombinant TGF-β1 (10 ng/ml) and simultaneously treated with different concentrations of AS-IV (0, 50, 100, 200 μg/ml) for 24 h. SB203580 (10 μmol/L) was also ultilized to pre-treat HK-2 cells for 1 h to inhibit phosphorylation of p38 MAPK signaling. The expression of FN, Col IV, and α-SMA were investigated by western blotting and real-time PCR. The expression of p-p38 MAPKs were also observed by Western Blotting. Results Astragaloside IV morphologically ameliorated renal tubulointerstitial fibrosis. The proteins and mRNA expression of FN, Col IV, α-SMA, and TGF-β1 were also increased significantly in UUO kidney tissues (all P＜0.05), which could be reversed by AS-IV administration (all P＜0.05). In vitro, the expression of FN, Col IV, and α-SMA were up-regulated by TGF-β1 after stimulating for 24 h (all P＜0.05), which were decreased by AS-IV. The inhibition effect on FN and α-SMA were similar between AS-IV and MAPK inhibitor SB203580. AS-IV inhibited p-p38 MAPK signals both in vivo and in vitro. Conclusions AS-IV could attenuate renal tubulointerstitial fibrosis induced by UUO and TGF-β1 through reducing FN、Col IV、α-SMA expression in renal tubular cells. The mechanism of AS-IV protective effect might be associated with inhibition of p38 MAPK phosphorylation.     

Norcantharidin inhibits renal interstitial fibrosis through down-regulating protein phosphatase-2Ac

Objective To observe the relationship between protein phosphatase-2Ac (PP2Ac) and renal interstitial fibrosis, and to investigate the effects and the underlyingmechanism of norcantharidin (NCTD) on renal fibrosis in unilateral ureteral obstruction (UUO) rats. Methods Sixteen male Sprague-Dawley rats were randomly divided into four groups: Sham operation group, UUO day 3 group, UUO day 7 group and UUO day 14 group. The rats were sacrificed at day 1, 3, 7, 14 respectively and kidneys were harvested. Immunohistochemical staining were used to detect the expression of fibronectin (FN), type I collagen (Col-I) and PP2Ac. Another twenty male Sprague-Dawley rats were also randomly divided into four groups: Sham operation group, UUO group, low dose NCTD treatment group (0.05mg·kg-1·d-1) and high dose NCTD treatment group (0.1 mg·kg-1·d-1). The rats in NCTD treatment groups were injected with norcantharidin into abdominal cavity 1 day before operation, while the Sham and UUO group were injected with equal normal saline. Rats were sacrificed at day 14 after surgery and the kidneys were harvested. Immunohistochemical staining, Western blotting and real-time PCR were used to detect the expression of FN, Col-I, α-SMA, E-cadherin and PP2Ac. Results (1)Compared with sham group, the expression of PP2Ac, FN and Col-I increased with the development of ureteral obstruction (all P＜0.05). The expression of PP2Ac was positively correlated with the expression of FN and Col-I (r＝0.894 and 0.887, all P＜0.05). (2)Compared with sham group, the expression of FN, Col-I, α-SMA increased and the E-cadherin decreased in UUO group, the expression of PP2Ac also increased (all P＜0.05). After the treatment of NCTD, the above changes were all alleviated in a dose dependent manner, and the expression of PP2Ac was down-regulated (all P＜0.05). Conclusion NCTD can ameliorate tubulointerstitial fibrosis and its anti-fibrosis effect may be related to its inhibition to PP2Ac.     

Effects of irbesartan on the expression of hypoxia-induced factor 1α and connective tissue growth factor in the kidney of unilateral ureteral ligation operation model rats

Objective To investigate the expression of hypoxia-induced factor 1α (HIF-1α)and connective tissue growth factor (CTGF) in the kidneys of unilateral ureteral ligation operation (UUO)model rats and the effect of irbesartan on the expression. Methods Thirty healthy adult male SD rats were randomly divided into 3 groups: sham operation group (n＝10), UUO group (n＝10) and irbesartan group (n＝10, UUO rats treated with irbesartan by lavage 2 days before operation). The rats in sham group and UUO group were treated with equal normal saline by lavage. Renal function, histopathological changes, urinary protein of 24 hours in rats at week 2 were measured. In situ hybridization and Western blotting were applied to measure the expression of HIF-1α and CTGF. Results At week 2, the levels of BUN, Scr and the expressions of HIF-1α and CTGF were significantly increased in UUO group compared with those in sham group (all P＜0.01). There was significant positive correlation between HIF-1α mRNA and CTGF mRNA (r＝0.697, P＜0.01). Compared with UUO group, the levels of urine protein and Scr were significantly decreased [(103.44±8.76) mg/24 h vs (278.23±26.15) mg/24 h, P＜0.01; (109.15±3.93) μmol/L vs (185.04±13.45) μmol/L P＜0.01], and renal tubulointerstitial lesion area became smaller (0.28±0.02 vs 0.51±0.05, P＜0.01) in irbesartan group. The expression of HIF-1α mRNA and protein was also significantly decreased after the treatment of irbesartan (all P＜0.01). Conclusions The expressions of HIF-1α and CTGF in UUO rats increase significantly. Irbesartan can improve renal fibrosis through down-regulating the expression of HIF-1α and CTGF.     

Effect of telmisartan on the expression of urate transporter protein in the renal tubule epithelial cells

Objective To explore the protective effect and underlying mechanism of telmisartan on hyperuricemic nephropathy. Methods (1)High level of uric acid (600 μmol/L) and telmisartan in different concentrations (10nmol/L, 100 nmol/L, 1000 nmol/L, 10000 nmol/L) were added to renal tubule epithelial cells and cultured for 48 h, the expression of UAT, TGF-β1 and α-SMA were detected by Real-time PCR, RT-PCR, Western blotting or cell immunofluorescence. (2) Wister rats were randomly divided into normal control group(Con), high uric acid group (HU), and telmisartan treatment group (Tel). Four weeks later, Scr, BUN and serum uric acid of the rats were detected. The expression of UAT in rat kidney was detected by Western blotting. Results (1)In vitro, compared to control group, high uric acid (600 μmol/L) inhibited the expression of UAT (P＜0.01), and the inhibition could be alleviated by telmisartan; Telmisartan inhibited the upregulation of TGF-β1 and α-SMA induced by high uric acid(all P＜0.05); (2)In vivo, compared to high uric acid group rats, telmisartan group rats had significantly reduced serum uric acid levels (189.9 μmol/L vs 204.5 μmol/L, P＜0.05), upregulated UAT and downregulated TGF-β1 expression in rat kidney (all P＜0.05). Conclusion Telmisartan significantly inhibits the upregulation of TGF-β1 and α-SMA induced by uricemia, which may prevent kidney from fibrosis. The protect effect of telmisartan may be related to the upregulation of UAT.     

支架蛋白JLP在单侧输尿管梗阻小鼠肾间质纤维化中的作用及其机制探讨

Objective To observe the effect of JLP deficiency on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction (UUO), and to investigate the role and underlying mechanism of JLP in the development of renal fibrosis in obstructive nephropathy. Methods jlp Wild type (jlp+/+) and jlp deficient (jlp-/-) mice were divided into four groups: jlp+/+- and jlp-/--sham-operated groups(jlp-/--sham group and jlp+/+-sham group), jlp+/+- and jlp-/--unilateral ureteral obstruction (UUO)-operated groups (jlp-/--UUO group and jlp+/+-UUO group). Mice were sacrificed at 7 days and 14 days after the operation respectively to evaluate the fibrosis by Masson staining.The expression of JLP in jlp+/+renal tissue was assayed by immunohistochemistry staining, immunofluorescence and Western blotting. Immunohistochemical staining was used to detect the expression of α-smooth muscle actin (α-SMA), collagen Ⅰ(COL-Ⅰ), collagen Ⅲ(COL-Ⅲ) and transforming growth factor-β1 (TGF-β1) in sham and UUO groups. Besides, the α-SMA, COL-Ⅰ, COL-Ⅲ, TGF-β1, p-Smad2 and p-Smad3 protein levels were also analyzed by Western blotting in four groups. Results The expression of JLP was mainly demonstrated in the renal tubules of mice. A large amount of collagen deposition was observed in the renal interstitial area in jlp-/--UUO group compared to jlp+/+-UUO group. Similarly, the expression of α-SMA, COL-Ⅰ, COL-Ⅲ and TGF-β1 was significantly increased in the kidney cortices in jlp-/-- UUO-operated groups. Meanwhile, Western blotting showed that the expression of α-SMA, COL-Ⅰ, COL-Ⅲ, and TGF-β1 protein was obviously higher in jlp-/--UUO group. Moreover, the expression of p-Smad2 and p-Smad3 protein was markedly higher in jlp-/--UUO group. Conclusion Scaffolding protein JLP is critical in preventing renal fibrosis through the inhibition of TGF-β1 expression and myo-fibroblast production.     

Protective effect of emodin on renal interstitial fibrosis in mice of unilateral ureteral obstruction via PI3K/Akt/mTOR signaling pathway

Objective To investigate the effect and mechanism of emodin (EM) in renal interstitial fibrosis of unilateral ureteral obstruction (UUO) mice. Methods Male C57BL/6J mice were randomly divided into 4 groups, including sham operation group (n=8), UUO operation group (n=8), UUO operation+losartan (LST) group (n=8) and UUO operation+EM group (n=8). The mice in each group were ingested the suspensions by gavage for 14 days after surgery. Mice in UUO+LST and UUO+EM groups were given 10 mg?kg-1?d-1 LST and 20 mg?kg-1?d-1 EM, respectively. LST and EM were mixed with 0.5% sodium carboxymethyl cellulose. Mice in sham group and UUO group were given 0.5% sodium carboxymethyl cellulose. The mice were sacrificed at the 14th day. Interstitial fibrosis was observed by HE, Masson and PAS stain. Real-time PCR was used to detect LC3, Beclin-1 and mTOR mRNA. Protein expressions of TGF-β1, α-SMA, E-cadherin, LC3, Beclin-1, PI3K, p-Akt and mTOR were detected by Western blotting. The autophagy was observed with transmission electron microscopy in the renal tissue. Results Compared with sham mice, UUO mice at the 14th day displayed obvious renal fibrosis. Meanwhile, UUO mice had increased expressions of TGF-β1 and α-SMA (all P＜0.01), and decreased expressions of E-cadherin (P＜0.01). Their renal expressions of PI3K, p-Akt and mTOR were also raised (all P＜0.01). Compared with those in UUO group, in UUO+LST group and UUO+EM group, expressions of autophagy protein LC3 and Beclin-1 were increased (all P＜0.01), and the number of autophagic was increased. Additionally, expressions of TGF-β1 and α-SMA were reduced in UUO+LST group and UUO+EM group (all P＜0.01), while the expression of E-cadherin was increased by emodin treatment (P＜0.05). And expressions of PI3K, p-Akt and mTOR were decreased in UUO+LST group and UUO+EM group (all P＜0.05), meanwhile renal tissue fibrosis significantly reduced. Conclusions Emodin can promote autophagy, ameliorate renal interstitial fibrosis and protect renal function through PI3K/Akt/mTOR signaling pathway.     

Histone methyltransferase MLL1 accelerates the development of renal fibrosis via promoting the epithelial-mesenchymal transition of HK-2 cells

Objective To investigate the possible effects of histone methyltransferase MLL1 on renal interstitial fibrosis and epithelial-mesenchymal transition (EMT). Methods Forty-two male SD rats were randomly divided into normal group, sham operation group and unilateral ureteral occlusion (UUO) group, and then UUO group was further divided into group 1 d, 1 week, 2 week, 3 week and 4 week after operation. The expression of MLL1, E-cadherin, α-SMA, Vimentin and Col3α1 in UUO rat kidney tissue as well as TGF-β1 stimulated HK-2 cells were detected by real-time PCR and Western blotting. siRNA-MLL1 plasmids was used to inhibit the expression of MLL1 and the protein levels of MLL1, α-SMA, Vimentin, E-cadherin, Col3α1 and H3K4me3 induced by TGF-β1 stimulation were detected by Western blotting. The level of H3K4me3 in promoter region of EMT related genes was observed by chromatin immunoprecipitation (CHIP). Results Compared with normal and sham operated groups, the loss of renal function in UUO group was more obvious with the obstruction time (P＜0.05). The renal fibrosis was most obvious 1 week and 2 weeks after the rats underwent the UUO operation (all P＜0.05), with the highest protein expressions of MLL1, E-cadherin, α-SMA, Vimentin and Col3α1 (all P＜0.05). Compared with the control group, 3 ng/ml TGF-β1 induced the highest expression of MLL1 and the most obvious EMT in HK-2 cells (all P＜0.05). Moreover, the EMT and the high level of H3K4me3 in HK-2 triggered by TGF-β1 were all inhibited by siRNA-MLL1 plasmids transfection (all P＜0.05). Conclusions MLL1 can enhance the occurrence of EMT induced by TGF-β1 in HK-2 cells by increasing the level of H3K4me3 in the promoter region of α-SMA and Vimentin.     

洋川芎内酯A 对单侧输尿管梗阻大鼠肾间质纤维化的作用及机制研究

目的:探讨洋川芎内酯A(SenA)对单侧输尿管梗阻(UUO)大鼠肾脏病变的保护作用。方法:50只雄性SD大鼠随机分为假手术组、模型组、SenA低剂量(20 mg/kg)组、SenA高剂量(40 mg/kg)组及阳性对照(厄贝沙坦,20 mg/kg)组,行UUO术制备单侧输尿管模型,并于术后21天处死大鼠。根据试剂盒说明书检测血清尿素氮(BUN)、肌酐(Scr)的含量,HE染色法观察肾脏病理改变,免疫组化法检测肾组织α-SMA及CollagenⅠ蛋白的表达,Western blotting检测各组大鼠肾皮质Wnt4、β-catenin、p-GSK-3β、E-cadherin的表达水平。结果:与模型组比较,SenA低、高剂量组及阳性对照组大鼠血清中Scr、BUN含量明显降低(P<0.05),肾脏病变情况得到不同程度的改善。同时,SenA可明显减少大鼠肾组织中α-SMA和CollagenⅠ蛋白的分泌(P<0.05),下调Wnt4、β-catenin及p-GSK-3β的表达水平(P<0.05),并伴有E-cadherin蛋白表达显著上调(P<0.01)。结论:SenA可通过下调Wnt4/β-catenin信号通路,抑制肾组织细胞外基质沉积并改善肾功能,最终改善UUO模型大鼠肾间质纤维化的病变进程。     

血小板反应蛋白-1及其受体CD47在肾间质纤维化血管病变中的表达

目的观察血小板反应蛋白-1(TSP-1)及其受体CD47在单侧输尿管梗阻(UUO)大鼠模型中的表达,探讨TSP-1-CD47在肾周毛细血管(PTC)病变中的作用。 方法将60只SD大鼠随机分为2组：UUO组45只和假手术组(SOR) 15只。UUO组随机选取15只分别于术后3 d、7 d、14 d处死;SOR组随机选取5只于相同时间点处死。检测24 h尿蛋白定量,采用全自动生化分析仪检测各组大鼠的肾功能,通过Masson染色观察肾间质病理改变;免疫荧光双染色观察TSP-1及其受体CD47在UUO模型中是否存在共表达。Western印迹检测毛细血管病变指标CD34和血管内皮生长因子(VEGF)及TSP-1的表达;多组间均数比较使用方差分析;TSP-1与CD34、VEGF的相关性进行Pearson相关分析。 结果与SOR组相比,UUO各组大鼠血清肌酐、尿素氮、24 h尿蛋白定量随梗阻时间延长无明显变化,但肾间质纤维化逐渐加重,肾小管损伤评分增加。免疫荧光双染色显示TSP-1及其受体CD47在肾小管间质存在共表达。Western印迹检测显示随UUO时间延长,TSP-1蛋白水平逐渐增加,而CD34及VEGF蛋白表达随梗阻时间延长逐渐下降。相关分析显示TSP-1蛋白水平与CD34及VEGF的表达呈负相关(r＝－0.931, P<0.01;r＝－0.953, P<0.01)。 结论TSP-1及其受体CD47在UUO大鼠模型肾间质存在共表达,且与PTC呈负相关。TSP-1可能通过CD47参与UUO肾脏PTC病变的发生发展。     

Effect of NADPH oxidases inhibition on the development of interstitial fibrosis in unilateral ureter obstruction rats

ObjectiveTo clarify whether the NADPH oxidases (NOXs) family contributed to the reactive oxygen species (ROS) production and subsequent interstitial fibrosis in unilateral ureter obstruction (UUO) rats. MethodsMale Wistar rats were randomly divided into sham operation group (n＝8), sham operation + apocynin treatment group (n＝8), UUO operation group (n＝8) and UUO operation+apocynin treatment group (n＝8). Either vehicle or apocynin (100 mg/kg per day) were given by gavage for 7 days after surgery. Rats were sacrificed at 7th day. ELISA was used to detect the activity of superoxide dismutase (SOD) and catalase (CAT), and the level of 8-iso-prostaglandin F2alpha (8-iso- PGF2α) in renal tissue. Western blotting was used to detect the protein expressions of NADPH oxidase subunit NOX2 and NOX4, α- smooth muscle actin(α-SMA), collagen I (COL-I) and the level of ERK1/ 2 phosphorylation (p-ERK1/2). ResultsUUO rats with vehicle displayed increased oxidative stress, as measured by renal tissue 8-iso-PGF2α, accompanied with increased renal expression of NADPH oxidases (NOX2, 1.5-fold and NOX4, 1.7-fold, respectively), compared with sham-operated rats (P＜ 0.05). Furthermore, vehicle treated UUO rats showed increased renal COL - I and α - SMA levels, compared with sham-operated rats (P＜0.05). ERK1/2 was also activated as detected by p-ERK1/2 expression in UUO rats with vehicle (P＜0.05). Apocynin treatment significantly decreased renal tissue 8-iso-PGF2α level and expressions of NOX2 (-28.7%) and NOX4 (-31.0%) in UUO rats, respectively, compared with vehicle treated rats (P＜0.05). And significant decrease of COL-I (-26.4%) and α-SMA expression (-80.0%) were also observed (P＜0.05). The activation of ERK1/2 in UUO rats was greatly inhibited by apocynin treatment (P＜0.01). Despite the pronounced dysregulation of pro – oxidative NOXs family, no compensatory increase of antioxidative enzyme activities occurred. ConclusionThe NOXs family contributes largely to the production of ROS and subsequent interstitial fibrosis after ureter ligation, and inhibition of the NOXs family may be a choice for preventing interstitial fibrosis.     

干细胞因子联合粒细胞集落刺激因子动员骨髓干细胞促进单侧输尿管梗阻大鼠肾脏损伤的修复

目的 研究干细胞因子（SCF）联合粒细胞集落刺激因子（G-CSF）动员单侧输尿管梗阻（UUO）大鼠骨髓干细胞对肾间质中微血管、纤维化程度和肾功能的影响,并探讨其对微血管影响的可能机制。 方法 128只大鼠按数字随机法分为假手术组（Sham组）、SCF联合G-CSF动员组（SCF-G组）、UUO组、UUO+SCF-G组。于实验第 5、14、21、28天每组各随机抽取8只处死,检测Scr、肾间质CD34阳性表达细胞数目和Ⅷ因子阳性表达细胞数目、肾间质纤维化和间质病理损害积分、肾皮质血管内皮生长因子（VEGF）mRNA和血小板反应蛋白1（TSP-1）mRNA的表达。 结果 (1)UUO组2周时可见到肾间质纤维化伴肾小管周微血管的丢失。（2）UUO+SCF-G组肾间质干细胞归巢数目明显高于UUO组和Sham组（P < 0.05）。（3）UUO+SCF-G组肾小管周微血管指数减少出现的时间晚于UUO组（P < 0.05）。（4）第14、21、28天UUO+SCF-G组间质化纤维程度和肾小管损伤程度均轻于UUO组（P < 0.05）。（5）UUO+SCF-G组术后VEGF mRNA表达下调出现的时间晚于UUO组,且表达均高于同期UUO组 （P < 0.05)。（6）UUO+SCF-G组术后TSP-1 mRNA表达增高出现的时间晚于UUO组,且表达均低于同期UUO组（P < 0.05）。（7）在UUO组和UUO+SCF-G组中,肾小管周微血管指数与Scr、间质纤维化积分和肾小管间质病理积分均呈负相关;肾皮质VEGF mRNA表达与肾小管周微血管指数呈正相关;肾皮质TSP-1 mRNA表达与肾小管周微血管指数呈负相关。 结论 （1）UUO大鼠存在肾小管周微血管丢失,并与肾间质纤维化及间质病理损伤相关。（2）联合应用SCF和G-CSF动员骨髓干细胞可以归巢至受损的肾脏,有助于减少肾小管周微血管丢失,并进而减轻肾间质纤维化和间质损害,保护肾功能。（3）联合应用SCF和G-CSF可以上调肾皮质VEGF mRNA水平和下调TSP-1 mRNA水平,这可能是其促进内皮细胞修复及保护肾间质微血管损伤的机制之一。     

三七总皂苷对单侧输尿管梗阻大鼠肾组织BMP-7及TGF-β1/samds信号传导通路的影响

目的:观察三七总皂苷(PNS)对单侧输尿管梗阻(UUO)大鼠肾组织骨形成蛋白-7(BMP-7)及TGF-β1/Smads信号传导通路的影响。方法:将50只雄性Wistar大鼠随机分为5组:假手术组、模型组、肾康注射液组(对照组)、PNS低剂量组(低剂量组)、PNS高剂量组(高剂量组),每组10只,除假手术组,其余各组用UUO法建立肾间质纤维化动物模型,术前1d起各组给予相应浓度和剂量的药物,术后第14天处死所有大鼠,检测大鼠血肌酐(Scr)和尿素氮(BUN)变化,梗阻侧肾组织行HE染色、PAS染色和Masson染色,观察肾组织病理变化并半定量计算肾小管损伤指数(TII),免疫组化法检测BMP-7、TGF-β1、Smad2、Smad7在肾组织的表达。结果:与模型组相比,PNS和肾康注射液能明显降低UUO大鼠术后14dScr、BUN(P<0.05);对梗阻侧肾组织HE染色、PAS染色进行TII评分发现,2个试验组大鼠肾小管损伤指数显著低于模型组(P<0.01);Masson染色观察发现,2个实验组大鼠肾小管间质病变明显轻于模型组;免疫组化法染色并对其灰度值测定发现,与模型组相比,2个试验组大鼠TGF-β1、Smad2在肾组织的表达下调(P<0.01),BMP-7、Smad7在肾脏组织的表达上调(P<0.01)。结论:PNS可能通过干预BMP-7/Smads/TGF-β1信号转导通路抑制了TGF-β1信号的细胞内转导,而起到抗肾间质纤维化的作用。     

骨形态发生蛋白-7在幼年大鼠肾小管间质损伤中的表达及意义

目的：探讨单侧输尿管梗阻（UUO）幼年大鼠肾间质纤维化形成过程中骨形态发生蛋白-7（BMP-7）的表达趋势及其与转化生长因子-β1（TGF-β1）、α-平滑肌肌动蛋白（α-SMA）的相关关系;观察血管紧张素转换酶抑制剂（ACEI）和血管紧张素受体拮抗剂（ARB）的干预作用。方法：采用单侧输尿管结扎制备UUO模型。3～4周龄幼年Wistar雄性大鼠随机分为对照组、模型组和干预组。于实验第3、7、14、28天取大鼠8只处死。HE及Masson染色观察肾组织的病理改变;免疫组化半定量检测各组大鼠肾组织BMP-7、TGF-β1及α-SMA蛋白表达。了解BMP-7与TGF-β1、α-SMA、肾间质纤维化程度的关系。结果：随梗阻时间延长,模型组BMP-7表达逐渐下降,TGF-β1、α-SMA表达进行性增高,干预组BMP-7表达较模型组显著增加（P〈0.05）;TGF-β1、α-SMA表达较模型组明显减少（P〈0.05）。模型组肾小管间质TGF-β1、α-SMA表达明显增高（P〈0.05）,BMP-7表达显著减少（P〈0.05）。与模型组相比,干预组肾小管间质TGF-β1、α-SMA表达显著减少（P〈0.05）,而BMP-7表达显著增多（P〈0.05）。BMP-7与TGF-β1、α-SMA、肾间质纤维化程度成负相关（r分别为-0.844、-0.787、-0.952,P均〈0.01）。结论：BMP-7表达减少伴随着肾小管上皮细胞转分化出现,提示BMP-7可能具有维持小管上皮细胞表型作用。苯那普利联合氯沙坦可能通过下调TGF-β1、α-SMA蛋白的异常高表达,上调BMP-7蛋白的异常低表达,直接或间接负性调控肾小管上皮细胞转分化,阻止肾间质纤维化进展。     

AMPK attenuates inflammation to reduce fibrosis induced by acute ischemia reperfusion injury in mice

Objective To observe the effect of adenosine monophosphate activated protein kinase (AMPK) on attenuating inflammation in fibrosis induced by acute ischemia reperfusion injury (IRI) in mice. Methods Forty eight male C57BL/6 mice were randomly divided into four groups: sham operation group (sham group), IRI group, AMPK inhibitor+IRI group (AMPK/IRI group) and normal saline+IRI group (NS/IRI group), 12 mice each group. The mice with renal IRI were occluded for 30 min through clipping bilateral renal pedicle, then released renal perfusion. Mice in sham group were performed the separation of renal pedicle without clipping. Mice in AMPK/IRI group and NS/IRI group were respectively intraperitoneal injected AMPK inhibitor and normal saline before IRI. At the 2 d after operation, 6 randomly-selected mice from each group were blooded by extraction eyeball to detect BUN and Scr. The renal histopathological changes were observed through HE staining. The mRNA expression of IL-1β, IL-6 and TNF-α was detected by real time PCR, and the level of AMPK phosphorylation was detected by Western blotting. At the 14 d after operation, Collagen 1 (COL1), α-SMA and fibronectin (FN) were detected by immunofluorescence and Western blotting in 6 remained mice from each group. The degree of kidney fibrosis was observed through sirus red staining. Results Compared with those in sham group, tubular interstitial damage was aggravated (P＜0.05), BUN and Scr were increased (P＜0.05), the mRNA expression of IL-1β, IL-6 and TNF-α was increased at the 2 d after operation (all P＜0.05), and the level of AMPK phosphorylation was activated in IRI group and NS/IRI group (all P＜0.05); the degree of kidney fibrosis and the expression of COL1, α-SMA and FN were increased obviously at the 14 d (all P＜0.05). Compared with those in IRI group, in AMPK/IRI group tubular interstitial damage was aggravated (P＜0.05), BUN and Scr were increased (all P＜0.05), the mRNA expression of IL-1β, IL-6 and TNF-α was increased at the 2 d (all P＜0.05), and the level of AMPK phosphorylation was decreased (P＜0.05). Moreover, the degree of kidney fibrosis and the expression of COL1, α-SMA and FN were increased obviously at the 14 d in AMPK/IRI group (all P＜0.05). Conclusions AMPK can ameliorate the acute renal ischemia reperfusion injury induce fibrosis in mice, and the mechanism may be related to the decrease of inflammatory reaction.     

Effect of bone morphogenetic protein-7 transfected bone marrow mesenchymal stem cells on renal injury in rats with chronic renal failure

Objective To observe the bone marrow mesenchymal stem cells (BMSCs) modified by bone morphogenetic protein-7 (BMP-7) gene on the expression of renal BMP-7, transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF), further to explore its protective mechanism on renal injury in rats with chronic renal failure (CRF). Methods BMSCs with high expression of BMP-7 gene (BMSCs-BMP-7) and empty vector-BMSCs (BMSCs-EV) were obtained by lentiviral-mediated gene transfection. Thirty male Sprague-Dawley (SD) rats were randomly divided into 5 groups, 6 in each group: normal control (CON) group; PBS intervention (CRF with PBS infusion, CRF+PBS) group; BMSCs intervention (CRF with BMSCs infusion, CRF+BMSCs) group; BMSCs-EV intervention (CRF with BMSCs-empty vector infusion, CRF+BMSCs-EV) group and BMSCs-BMP-7 intervention (CRF with BMSCs-BMP-7 infusion, CRF+BMSCs-BMP-7) group. The CRF model was established by 5/6 nephrectomy. The CON group was a sham operation group. The corresponding 12-weeks interventions of each experimental group were performed after 2 weeks of modeling, the rats in the CON group and the CRF+PBS group were injected with 1 ml of PBS through the tail vein, and the other three groups were injected with 1 ml of the corresponding cell suspension once a week. At the time of sacrifice, blood and renal tissue samples were reserved. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by routine biochemical methods, and the expression of BMP-7, VEGF, TGF-β1 in kidney was assayed by Western blotting. Results At the time of sacrifice, the levels of Scr and BUN in the CRF+PBS group were significantly higher than those in the CON group (all P＜0.01); Compared with the CRF+PBS group, the Scr and BUN of the CRF+BMSCs group, CRF+BMSCs-EV group and CRF+BMSCs-BMP-7 group were decreased to different extents, the differences were statistically significant (all P＜0.01); the Scr and BUN of the CRF+BMSCs-BMP-7 group were significantly lower than CRF+BMSCs group and CRF+BMSCs-EV group (all P＜0.05). The expression of BMP-7 and VEGF were the lowest in the CRF+PBS group. Compared with the CRF+PBS group, the expression of BMP-7 and VEGF in the CRF+BMSCs group, CRF+BMSCs-EV group and CRF+BMSCs-BMP-7 group were significantly increased respectively (all P＜0.05). The expression of the BMP-7 and VEGF in the CRF+BMSCs-BMP-7 group were higher than those in the CRF+BMSCs group and CRF+BMSCs-EV group (P＜0.01). Compared with the CON group, the expression of TGF-β1 in the CRF+PBS group was significantly increased (P＜0.01); compared with the CRF+PBS group, the expression of TGF-β1 in the CRF+BMSCs group, CRF+BMSCs-EV and CRF+BMSCs-BMP-7 group was significantly decreased (all P＜0.01); the expression of TGF-β1 in the CRF+BMSCs-BMP-7 group was lower than the CRF+BMSCs and CRF+BMSCs-EV group (both P＜0.01). Conclusions BMSCs modified by BMP-7 has a protective effect on CRF rats; its protective mechanism may be related to antagonizing TGF-β1 and up-regulation of renal VEGF expression.     

Inhibition of Src kinase can ameliorate renal interstitial fibrosis in unilateral ureteral obstruction mice

Objective To investigate the effect and mechanism of Src kinase in renal interstitial fibrosis of unilateral ureteral obstruction (UUO) mice. Methods Male C57BL/6J mice were randomly divided into 4 groups, including sham operation group (n=8), sham operation+PP2 group (n=8), UUO operation group (n=8) and UUO operation+PP2 group (n=8). The mice were injected 2 mg/kg PP2 by intraperitoneal everyday after surgery in sham+PP2 group and UUO+PP2 group. PP2 dissolved in 1% DMSO (formulated with normal saline). Sham and UUO group were given equal 1% DMSO. The mice were sacrificed at 7th day. Renal collagen was observed with Sirius red stain. The activities of Src, protein kinase B (PKB, AKT), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and the protein expressions of α-smooth muscle actin (α-SMA) and fibronectin (FN) were detected by Western blotting. The expression of collagen I (COLⅠ) was detected by immunohistochemistry and the expressions of matrix metalloprotein 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6) were measured by ELISA. Results Compared with sham mice, UUO mice on 7th day displayed obvious renal fibrosis. Meanwhile, UUO mice had increased expressions of COLⅠ and FN, and activities of AKT, ERK and p38 MAPK (all P＜0.05). Their renal expressions of α-SMA, TGF-β1, MMP-9, TIMP-1, MCP-1 and IL-6 were also raised (all P＜0.05). Compared with those in UUO group, in UUO+PP2 group the activities of Src, AKT, p38 MAPK and ERK, and expressions of TGF-β1, MCP-1 and IL-6 decreased (all P＜0.05). Additionally, expressions of COLⅠ, FN and α-SMA, collagen deposition and renal fibrosis receded in UUO+PP2 group (all P＜0.05). However, the expressions of MMP-9 and TIMP-1 were not influenced by PP2 treatment. Conclusions Src kinase promotes myofibroblasts accumulation and inflammatory reaction through activating its downstream signaling pathway in the progressing of renal interstitial fibrosis.     

intermedin预处理对大鼠肾脏缺血再灌注损伤修复和再生过程的作用

目的 探讨intermedin （IMD）预处理对大鼠肾脏缺血再灌注（IR）损伤修复和再生过程的作用。 方法 将Wistar大鼠按随机数字表法分为4组：假手术组（sham）、IR组、转空质粒组和转IMD组。在切除右肾后,转IMD组用超声微泡造影剂介导的基因转染方法将IMD真核质粒转染到大鼠肾组织,用RT-PCR和Western印迹法检测转染效率。转染成功后,制作肾脏IR损伤模型,分别于再灌注后1 d、2 d、3 d、4 d、7 d和14 d 6个时间点各取6只大鼠,留取血清及肾组织标本,常规检测血清BUN和Scr;HE和PAS染色观察肾组织的病理变化;免疫组化法观察肾小管上皮细胞的增殖程度。 结果 （1）转IMD组比转空质粒组的IMD蛋白和mRNA表达均增多（均P < 0.05）,且转IMD组7 d时表达最多,与转IMD组4 d时差异无统计学意义;（2）与sham组相比,IR组1 d和2 d时Scr和BUN均显著增高（P < 0.05）;与IR组相比,转IMD组显著下降（P < 0.05）;转空质粒组与IR组相比差异无统计学意义（P > 0.05）。（3）IR组、转空质粒组和转IMD组大鼠的肾小管均受损,但转IMD组的损伤较轻,均以2 d时病理损伤最重。（4）sham组肾小管和肾小球内几乎没有增殖细胞核抗原（PCNA）阳性细胞的表达;IR组和转空质粒组的PCNA阳性数在IR损伤1 d时开始增加,7 d时最多;转IMD组的PCNA阳性细胞数在IR损伤1 d时开始增加,3 d时最多。与IR组1~4 d相比,转IMD组的PCNA阳性细胞数显著增加（P < 0.05）;与IR组7 d相比,转IMD组7 d的PCNA阳性细胞数显著减少（P < 0.05）。 结论 IMD预处理可以促进肾小管上皮细胞增殖,加速肾脏IR损伤修复和再生。