Aim To investigate the effects of fluvastatin on the cellular proliferation and the expression of connective tissue growth factor(CTGF)and type Ⅳ collagen(Col Ⅳ)in rat mesangial cells(MCs)induced by transforming growth factor-?1(TGF-?1).Methods MCs are divided into the following groups according to different factors:control group,TGF-?1 group,TGF-?1 plus fluvastatin(Flu,different concentrations)groups.The influence of Flu on rat proliferation of MCs was detected by MTT.The expression of CTGF was measured with RT-PCR and Western blot respectively.The secretion of Col Ⅳ protein was quantitated by ELISA.Results 5 ?g?L-1 TGF-?1 could stimulate proliferation of MCs and the expression of CTGF.Col Ⅳ in MCs significantly.Flu could inhibit proliferation of MCs and the expression of CTGF,Col Ⅳ in MCs induced by TGF-?1 in a dose-dependent manner:1 ?mol?L-1 Flu could suppress proliferation of MCs and downregulate the expression of CTGF,Col Ⅳ significantly,while 10 ?mol?L-1 Flu could just suppress the expression of CTGF protein significantly.Simultaneously exogenous CTGF could promote the expression of Col Ⅳ in a dose-dependent manner:2.5 ?g?L-1 CTGF could just upregulate the expression of Col Ⅳ.Conclusion Fluvastatin could inhibit proliferation of MCs and expression of CTGF-mediated Col Ⅳ in MCs.

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.

Objective To observe the effects of bone marrow?derived mesenchymal stem cells (BMSC) on glomerular podocyte injured by lipopolysaccharide (LPS) and the expression of related protein. Methods Podocytes are divided into control group, BMSC group, LPS group and LPS plus BMSC group. After 24 hours of intervention, observing each experimental group podocyte form under inverted phase contrast microscope;detecting the expressions of mRNA and protein of nephrin, CD2AP, synaptopodin, and TRPC6 by RT?PCR and Western?blot. Results Compared with control group, expressions of nephrin, CD2AP, and synaptopodin in LPS group decreased (P<0.05) while that of TRPC6 increased (P<0.05); compared with LPS group, expressions of nephrin, CD2AP, and synaptopodin in LPS+MSC group increased (P<0.05) while that of TRPC6 decreased (P<0.05). Conclusion BMSC may relieve LPS?induced podocyte injury.

Objective To investigate the effects of erythropoietin (EPO) on complement 3a (C3a)-induced renal tubular epithelial to mesenchymal transition. Methods The HK-2 cells were divided into 6 groups namely control group,EPO group,TGF-β group,TGF-β+EPO group,C3a group and EPO+C3a group.The mRNA and protein expressions of α-SMA,E-cadherin and C3 were investigated by RT-PCR,Western blot and immunofluorescence respectively. Results Compared with control group and EPO group,the mRNA and protein expressions of α-SMA in HK-2 cells were up-regulated after the intervention of C3a or TGF-β (all P＜0.05).On the contrast,the mRNA and protein expressions of E-cadherin were down-regulated(P＜0.05),the mRNA and protein expressions of C3 were enhanced (all P＜0.05).However,all those above effects of C3a or TGF-β were inhibited after the intervention of EPO (all P＜0.05). Conclusion EPO is capable of suppressing the epithelial to mesenchymal transition induced by C3a.

Objective: To investigate the effect and mechanism of renal fibrosis after macrophage depletion in C3-deficient unilateral ureteral obstruction mice. Methods: Renal interstitial fibrosis model was established by unilateral ureteral obstruction (UUO) in male C3-deficient mice and age-matched C57BL/6 WT mice (8-12 weeks of age). Mice were randomly divided into 4 groups, including sham operation in wild type group (WT/sham) (n=18), UUO operation in wild type group (WT/UUO) (n=18), sham operation in C3-deficient group (C3KO/sham) (n=18), and UUO operation in C3-deficient group (C3KO/UUO) (n=18). The expression of complement C3 was detected by immunohistochemical staining and renal interstitial macrophages were assessed by immunofluorescence staining. Tubulointerstitial fibrosis was observed by both HE staining and Masson staining after 14 days of UUO. Collagen accumulation and score of tubulointerstitial injury were obtained. Wild type and C3-deficient UUO mice were treated by liposome clodronate in early or late stage respectively and then interstitially infiltrated macrophages and renal fibrosis were analysed. Mice were sacrificed randomly at 3,7,14 days after UUO and obstructed kidneys were collected. Macrophage phenotype was detected by double-labeling immunofluorescence with F4/80 and iNOS for the M1, F4/80 and CD206 for the M2 macrophage subpopulation. iNOS, Arg-1 and CD206 were also detected by western blot. Results: C3 deficient mice exhibited attenuated renal fibrosis, reduced collagen accumulation and tubulointerstitial injury score compared with WT mice (P<0.01). Meanwhile, macrophage depletion in early or late stage of UUO reduced renal fibrosis in WT mice, but had no effect on C3-deficient UUO mice. Decreased accumulation of M1 macrophages and expression of iNOS, increased accumulation of M2 macrophages and expression of Arg-1, CD206 were found in C3 deficient mice compared with WT mice in early stage of UUO (P<0.01). Conclusion Renal fibrosis is not reduced after depletion of macrophages in C3 deficient UUO mice due to the altered macrophage polarization.