Norcantharidin inhibits renal interstitial fibrosis through inhibiting PP2Ac mediated the dephosphorylation of Smad3 linker region

Objective To investigate the inhibition of interstitial fibrosis by NCTD is related to the dephosphorylation of Smad3 linker region mediated by the inhibition of PP2Ac. Methods HK -2 cells were cultured and devided into 5 groups: (1) normal control group; (2) TGF-β1 group (5 μg/L); (3) TGF-β1+NCTD group (2.5 mg/L); (4) TGF-β1+PP2Ac shRNA group; (5)TGF-β1+PP2Ac shRNA+ NCTD group. Real-time PCR and Western blot were used to detect the expression of PP2Ac, FN, Col-I, α-SMA and E-cadherin. Additionally, the HK-2 cells were assigned to three groups:(1) normal control group; (2) TGF-β1 group; (3) TGF-β1+NCTD group. Immunofluorescence were used to analysis the distribution of pSmad3-L(Ser204) and pSmad3-L(Ser208). Western blot analysis were used to detect the protein expression of pSmad3-L(Ser204) and pSmad3-L(Ser208). Results (1) TGF-β1 stimulated the expression of PP2Ac in HK-2 cells, increased the expression of FN, Col-I and α-SMA, and decreased the expression of E - cadherin. Both NCTD and PP2Ac shRNA could inhibit PP2Ac expression accompanied with the downregulation of FN, Col - I and α - SMA, and upregulation of E - cadherin. However, compared with PP2Ac shRNA transfected group, cells transfecting with PP2Ac shRNA and incubated with NCTD showed no obvious differences on the relief of the above indicators induced by TGF-β1 in HK2 cells. (2)The expression of pSmad3-L(Ser204) and pSmad3-L(Ser208) in the nucleus of HK2 cells stimulated by TGF-β1 was significantly elevated. The expression of pSmad3-L(Ser204) and pSmad3 - L(Ser208) in the nucleus was further upregulated when treated with NCTD. Conclusions NCTD has anti - fibrosis effect and it may be due to the inhibition the dephosphorylation of Smad3 linker region mediated by the inhibition of PP2Ac.     

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.     

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.     

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.     

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.     

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.     

Effect of intermedin on microvascular injury of unilateral ureteral obstruction rats

Objective To observe the effect of intermedin(IMD) on microvascular injury of renal fibrosis in unilateral ureteral obstruction (UUO) rat model. Methods Seventy-two male Wistar rats were randomly divided into two groups: the sham - operation group (n=24) underwent the left ureteral dissection, the other 48 rats were made as unilateral ureteral obstruction models and sub - divided into model group(UUO, n=24) and IMD group (n=24). At the 7, 14, 21, 28 day after the operation, 6 randomly - selected rats from each of the three groups respectively were blooded by abdominal arotic and their obstructive kidneys were taken out. The renal histopathological changes were observed through HE and Masson staining, the contents of BUN, Scr and cystatin C (CysC) of the obstructive kidneys were determined, the expressions of transforming growth factor - β1 (TGF - β1), α-SMA, bone morphogenetic protein-7 (BMP-7), E-cadherin, thrombospondin 1 (TSP-1) and vascular endothelial growth factor (VEGF) were detected by RT - PCR and immunohistochemistry. Results Compared with the sham-operated group, the pathological changes of kidney in the model group showed that the degree of fibrosis was obvious, tubular interstitial damage aggravated, the levels of BUN, Scr, CysC in the model group increased (P＜0.05), the mRNA expression and protein content of TGF-β1, α-SMA, TSP-1 increased (P＜0.05), while the levels of BMP-7, E-cadherin and VEGF decreased (P＜0.05). Compared with the UUO group, renal tubular damage, interstitial fibrosis in the IMD group were lighter, the levels of BUN, Scr, CysC in the IMD group were lower (P＜0.05), the mRNA expression and protein content of TGF-β1, α-SMA,TSP-1 were down-regulated (P＜0.05), while the levels of BMP-7, E-cadherin and VEGF were up-regulated (P＜0.05). Conclusion IMD can ameliorate the renal interstitial fibrosis, and the mechanism may be related to the fact that VEGF mediated by IMD can reduce vascular injury.     

雌激素对单侧输尿管梗阻大鼠肾间质纤维化的保护作用

目的探讨雌激素对单侧输尿管梗阻（UUO）大鼠肾间质纤维化的作用。方法雌性SD大鼠30只，随机分为4组：Ⅰ组对照组；Ⅱ组生理雌激素组；Ⅲ组低雌激素组；Ⅳ组高雌激素组。UUO术后21d处死各组大鼠，光镜观察梗阻肾组织病理变化，并分别用免疫组化和RT-PCR方法检测各组肾组织α-平滑肌肌动蛋白（α-SMA）和金属蛋白酶1组织抑制剂（TIMP-1）的表达。结果低雌激素组间质纤维化病变最明显，高雌激素组病变显著减轻（P〈0．01）。与生理雌激素组相比，低雌激素组α—SMA和TIMP-1蛋白和基因的表达增加（P〈0．05）；高雌激素组上述物质表达则减少（P〈0．05）。结论雌激素可能通过抑制α-SMA和TIMP-1的表达进而减少细胞外基质的沉积而发挥肾保护作用。     

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.     

Effect of SOST gene deletion on the progression of renal interstitial fibrosis in obstructive kidney injury

Abstract

Objectives: The role of SOST/sclerostin in mediating tissue fibrogenic response to injury/inflammation remains largely unknown. Thus, we conducted this study to determine whether SOST/sclerostin plays a role in renal interstitial fibrosis (RIF) for the first time. Methods: Unilateral ureteral obstruction (UUO) was performed to create obstructive kidney injury model. Twelvemale SOST knockout (SOST KO) mice and 12 age-matched wild-type (WT) mice were divided into three groups: sham surgery, UUO 3?d and UUO 7?d. The mice were sacrificed at each time point and kidney tissues were collected. Histopathological changes were evaluated by hematoxylin and eosin and Masson staining, while α-smooth muscle actin (α-SMA), type I collagen (Col-I) and fibronectin (FN) expression levels were detected by RT-PCR and western-blot. Results: In sham control group, neither WT nor SOST KO exhibited fibrotic change. On 3 days after UUO, total renal histopathological score and fibrotic area were aggravated and α-SMA, Col-I and FN expressions were upregulated, but no difference was observed between WT and SOST KO. On 7 days after UUO, compared with WT, SOST KO mice showed higher total renal histopathological score and fibrotic area percentage, as well as a higher level of fibrogenic marker mRNA/protein expression (except for α-SMA mRNA and FN mRNA). Conclusion: It is supposed that SOST gene is involved in the regulation of RIF progression. In obstructive kidney injury, SOST gene deletion would probably enhance renal fibrogenic response and promote the progression of RIF. But more evidences are needed to further identify the role of SOST/sclerostin in mediating RIF progression.     

γ-干扰素对梗阻性积水肾间质纤维化的治疗作用

目的 探讨γ干扰素(IFN-γ)对梗阻性肾积水肾间质纤维化的抑制作用及其可能的机制.方法 将65只雄性SD大鼠随机分成4组:治疗组(20只)、模型组(20只)、药物对照组(20只)、假手术组(5只).在建模和给药后的第3、7、14、21、28天,每组各随机处死动物4只(假手术组1只).采用苏木素-伊红(HE)和Masson染色观察病变肾脏间质纤维化的情况;采用聚合酶链反应(RT-PCR)技术检测肾组织中转化生长因子-β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原(Col-Ⅰ)的mRNA表达情况;免疫组织化学法观察以上三种蛋白的变化.结果 模型组大鼠术后肾间质逐渐出现纤维化改变,并随梗阻时间的延长逐渐加重,TGF-β1、α-SMA和Col-Ⅰ的mRNA和蛋白表达亦逐渐升高.第14天,模型组TGF-β1和α-SMA的表达量达到高峰,28 d时各指标的表达均达到最高,TGF-β1、α-SMA和Col-Ⅰ分别为51.84%、72.59%和68.73%.治疗组各指标在不同时间点均低于模型组,第28天时,三项指标依次为33.84%、32.59%和48.73%,与模型组比较P＜0.05.Banff评分显示治疗组间质纤维化程度明显减轻(P＜0.01).其余两组未见肾间质纤维化改变.结论 IFN-γ具有减轻积水后肾间质纤维化程度的作用,该作用与其下调TGF-β1的表达、抑制肌成纤维细胞(MyoF)激活和减少Col-Ⅰ生成有关.     

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。     

依贝沙坦对单侧输尿管梗阻小鼠肾脏整合素连接激酶表达的影响及意义

目的研究依贝沙坦（Irb）对单侧输尿管梗阻（UUO）小鼠肾脏整合素连接激酶（ILK）表达的影响,并探讨其与肾小管上皮间充质转化的关系。方法将雄性CD-1小鼠随机分为假手术对照组（C,n=20）、UUO组（UUO,n=40）和Irb治疗组（UUO＋Irb,n=40）,分别于术后1、3、7和14d处死小鼠。Masson染色观察肾间质纤维化。免疫组化检测小鼠肾组织ILK、上皮细胞钙黏蛋白（E-cadherin）和α平滑肌肌动蛋白（α-SMA）表达。Western印迹观察小鼠肾组织ILK蛋白表达。荧光实时定量PCR（real-time PCR）检测ILK、E-cadherin、α-SMA和纤连蛋白（FN）mRNA表达。结果与C组相比,UUO组术后1d ILK mRNA及蛋白表达均增高;术后3d FN mRNA表达上调,E-cadherin mRNA及蛋白表达明显减少,α-SMA mRNA及蛋白表达显著增加。与UUO组相同时间点比较,Irb治疗组ILK、FN及α-SMA表达均被显著抑制（P均＜0．05）;E-cadherin表达则增高（P＜0．01）。ILK蛋白表达与α-SMA蛋白表达呈正相关（r =0．707,P＜0．01）,与E-cadherin蛋白表达呈负相关（r=-0．919,P＜0．01）。结论Irb能减轻肾脏纤维化,抑制肾小管上皮细胞转分化,这可能与其抑制肾小管细胞ILK表达有关。     

肝细胞生长因子负性调控细胞转分化在肾间质纤维化中的作用

目的 研究肝细胞生长因子(HGF)对单侧输尿管梗阻(UUO)大鼠肾间质纤维化的保护作用及其可能机制&#65377;方法 大鼠随机分为UUO组&#65380;HGF治疗组和假手术组&#65377;用实时荧光定量RT-PCR&#65380;Western杂交和免疫组化检测术后大鼠肾组织结缔组织生长因子(CTGF)和骨形成蛋白7(BMP7)表达量&#65377;免疫组化检测大鼠肾组织TGF-β1&#65380;FN及α-SMA表达&#65377;结果 与假手术组相比,UUO组及HGF治疗组CTGF mRNA&#65380;TGF-β1&#65380;α-SMA&#65380;FN&#65380;CTGF蛋白表达均增高,且UUO组明显高于治疗组;UUO组及HGF治疗组BMP7 mRNA和蛋白表达均减少,且UUO组显著低于治疗组&#65377;结论 HGF能减轻肾间质纤维化,负性调控肾小管上皮细胞-肌成纤维细胞转分化,调节CTGF及BMP7表达可能是其作用途径&#65377;     

去甲斑蝥素对TGF-β_1诱导的人近端肾小管细胞上皮间质转分化以及转录因子Snail1表达的影响

目的：探讨去甲斑蝥素对体外人源性肾小管细胞株（HK-2）上皮间质转分化以及转录因子Snail1的影响。方法：常规培养HK-2细胞,分为对照组、TGF-β1组、去甲斑蝥素组（NCTD组）。对照组为无血清DMEM培养,TGF-β1组为TGF-β15ng/ml诱导,NCTD组为不同浓度NCTD（0.5、1.0、2.5μg/ml）与TGF-β1（5ng/ml）共同作用,各组作用时间48h。采用RT-PCR、Western blot分别检测α-SMA、E-cadherin与Snail1表达水平的变化。结果：与对照组相比,TGF-β1组α-SMA mRNA和蛋白表达上调（P〈0.05）,E-cadherin mRNA和蛋白表达下调（P〈0.05）;与TGF-β1组相比,NCTD干预组α-SMA mRNA和蛋白表达下调（P〈0.05）,而E-cadherin mRNA和蛋白表达上调（P〈0.05）,且均呈剂量依赖性。与对照组相比,TGF-β1组Snail1 mRNA和蛋白表达上调（P〈0.05）;与TGF-β1组比较,NCTD干预组Snail1 mRNA和蛋白表达下调（P〈0.05）,具有一定的剂量依赖关系。结论：去甲斑蝥素具有抑制肾小管上皮细胞EMT的作用,该作用可能与下调转录因子Snail1的表达有关。     

钙三醇抑制肾间质成纤维细胞的活化

目的 通过观察钙三醇对肾间质成纤维细胞增殖和凋亡的影响，及其对转化生长因子（TGF）β1诱导的成纤维细胞转分化和细胞外基质(ECM)合成的干预作用，旨在探讨钙三醇抗肾间质纤维化的潜在效应及相关机制。 方法 体外培养大鼠肾间质成纤维细胞NRK-49F，分别以不同浓度TGF-β1（1、2、5和10 μg/L）和（或）不同浓度钙三醇（10－4、10－5、10－6、10－7、和10－8 mmol/L）进行干扰。MTT法观察细胞增殖情况；流式细胞术分析细胞周期和细胞凋亡改变；实时定量PCR和Western印迹法分别检测细胞内α平滑肌肌动蛋白（α-SMA）、结缔组织生成因子（CTGF）及纤连蛋白（FN）的mRNA和蛋白表达。 结果 钙三醇能明显抑制正常和TGF-β1（5 μg/L）诱导的NRK-49F细胞增殖（P < 0.01）。经不同浓度钙三醇（10－4、10－5、10－6、10－7、和10－8 mmol/L）作用48 h后，NRK-49F细胞G2/S期细胞比例较TGF-β1刺激组均明显减少（分别为25.88%、21.81%、21.73%、23.28%、23.61%比27.42%，均P < 0.05），但对细胞凋亡无明显影响。NRK-49F细胞经TGF-β1刺激后，其α-SMA、CTGF、FN mRNA表达量显著上升，并在TGF-β1 1~5 μg/L的范围内呈剂量依赖效应。钙三醇能明显抑制TGF-β1诱导的α-SMA、CTGF、FN mRNA表达上调（均P < 0.05），而不同钙三醇浓度干预组之间差异无统计学意义。钙三醇能显著降低TGF-β1诱导的α-SMA和FN蛋白表达（P < 0.05）。 结论 钙三醇可通过G1期停滞抑制大鼠肾间质成纤维细胞增殖，但不影响凋亡。钙三醇具有拮抗 TGF-β1致肾间质纤维化的潜在作用。     

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。     

JAK-STAT通路在小鼠单侧输尿管梗阻模型肾间质纤维化中的作用

目的 探讨Janus蛋白酪氨酸激酶-信号转导子和转录激活子(JAK-STAT)通路在小鼠单侧输尿管梗阻(UUO)模型.肾间质纤维化过程中的作用.方法 选用30只雄性Balb/c小鼠建立小鼠UUO模型(n=24)和假手术小鼠(n=6),术后第1、4、7和14天检测JAK-STAT磷酸化情况.另把18只雄性Balb/c小鼠随机分为假手术组、UUO模型组和治疗组,每组各6只.治疗组在建模前2 h开始给予选择性JAK2抑制剂AG490治疗,每天1次;模型组仅注射溶媒.术后第14天处死动物.组织学评估肾小管损伤和.肾间质纤维化程度;免疫组化检测肾脏巨噬细胞浸润和α-SMA表达;RT-PCR检测Ⅲ型胶原和单核细胞趋化蛋白(MCP)1 mRNA表达;Western印迹检测JAK2和STATl磷酸化.结果 JAK2-STAT1在UUO模型中被激活,其磷酸化水平与病情、肾小管组织学损害以及.肾间质纤维化相一致.AG490能显著抑制JAK2和STAT1的磷酸化(P＜0.01).AG490治疗显著减轻肾小管损害[(21.7±1.7)%比(49.4±1.0)%]和肾间质纤维化(1.0±0.1比2.3±0.2)、α-SMA表达(0.9±0.1比2.1±0.2)和巨噬细胞积聚[(13.3±1.6)细胞/HPF比(34.4±1.0)细胞/HPF](均P＜0.01).AG490治疗显著抑制Ⅲ型胶原和MCP-1 mRNA表达.结论 JAK-STAT信号通路在肾小管间质炎性反应和纤维化中发挥重要作用.     

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.