Objective To examine the expression of TGF-?1/Smads signaling pathway in renal tubulointerstitial fibrosis.Methods Male SD rats were divided into control,sham operation and tubulointerstitial fibrosis groups.They were sacrificed at day 3,7,14,28 after operation.Use masson coloration to examine the area of pathological changes.The level of TGF?1,p-Smad2/3 and smad7 protein was examined by immunohistochemistry staining and Western blot.The level of TGF-?1 mRNA and smad7 mRNA was analyzed by RTPCR.Results Compared with sham operation group,TGF-?1 and p-Smad2/3 were significantly increased in UUO rats,while smad7 protein reduced in it.The mRNA of TGF-?1 and smad 7 increased from day 3 to day 28.Smads protein plays an important role in the synthesis and accumulation of extracellular matrix in tubulointerstitial area.The reduction of smad 7 protein may be a major cause of the interstitial fibrosis in this model.Conclusion TGF-? /Smads protein pathway perhaps is essential in the development of tubulointerstitial fibrosis.

Objective To study the expression and significance of the p38 mitogen-activated protein kinase (MAPK) and cAMP-responsive element binding protein (CREB 1) in experimental diabetic mellitus rats, and their relationship with extracllular matrix (ECM) restructuration. Methods Sixty male Wistar rats, with their kidneys removed surgically, were divided in two groups: control group (n=30) and diabetic group (n=30). Diabetic model was reproduced by intraperitoneal injection of Streptozocin (STZ, 65mg/kg). Six rats of each group were sacrificed at 1, 2, 4, 8 and 12weeks after STZ injection. Immunohistochemistry was used to assess the expression of p-p38 MAPK and p-CREB 1 in both control and diabetic groups. Western blot was performed to determine the activity of p-p38 MAPK and p-CREB 1 in both groups. Semiquantitative competitive reverse transcription polymerase chain reaction (PCR) was performed to detect the expression of mRNA of p38 MAPK, CREB 1 and fibronectin (FN). Results Compared with the control group, expression of p-p38 MAPK in diabetic glomeruli was significantly enhanced at 1, 2, 4, and 8 weeks, then it lowered at 12 weeks. P-CREB 1 was increased at 2, 4, 8 weeks, also decreased at 12 weeks. Elevation of FN mRNA began after 4 weeks in diabetic rats. Conclusions Glomerular p38 MAPK activity was increased in early stage of diabetes. This activated p38 MAPK pathway in diabetic glomeruli may in part play a role in the pathogenesis of extracellular matrix restructuring.

AIM: To investigate the effect of activation of JAK/STAT signaling pathway on the transdifferentiation induced by high concentration of glucose in renal proximal tubular epithelial cells(HKCs).METHODS: Cultured HKCs cells were divided into four groups: low glucose group(LG),high glucose group(HG),high mannitol group(LG+M),and HG+AG490 group(AG).Immunoprecipitation and Western blotting analysis were used to determine the expression of tryosine phosphorylated Janus kinase 2(p-JAK2).The protein expressions of STAT1,STAT3,p-STAT1 and p-STAT3 and expressions of ?-SMA,E-cadherin were observed by Western blotting.The contents of TGF-?1 and type I collagen in the supernatants of the cultured HKCs were detected by enzyme-linked immunoadsorbent assay(ELISA).TGF-?1 mRNA were measured by reverse transcription and polymerase chain reaction(RT-PCR).RESULTS: Compared with low glucose control group,the expression of JAK2,p-STAT1,p-STAT3 and TGF-?1 mRNA was significantly increased in HG group at different stimulated time from 6 h to 72 h.Meanwhile,the contents of TGF-?1 and collagen I in the supernatants and the expression of ?-SMA were increased,the expression of E-cadherin were decreased.The expression of JAK2,p-STAT1,p-STAT3 and TGF-?1 mRNA and the levels of TGF-?1,collagen I in the supernatants in HG+AG490 group were obviously lower than those in HG group.The expressions of ?-SMA and E-cadherin were also decreased in HG+AG490 group.CONCLUSION: Activation of JAK/STAT signaling pathway may be involved in the high glucose-induced transdifferentiation and overproduction of TGF?1 and ECM proteins in HKCs.

Objective To investigate the effects of angiotensin II type I receptor antagonist losartan on apoptosis and the expression of Fas and Fas-L in the kidney of diabetic rats. Methods Uninephrectomized, STZ(65 mg/kg) -induced diabetic male SD rats were used. Losartan(40 mg/kg) was delivered daily by gavage from the next day of the induction to diabetes for 12 weeks. Apoptosis was examined by means of terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling(TUNEL) . Flow cytometry and immunohistochemistry were used to detect the expression of Fas and Fas-L. Results Compared with those in the kidneys of control group, apoptotic cells were more in number and the expression of Fas and Fas-L was higher in the diabetic kidneys( P

Objective To investigate the effect of high glucose on the expression of transforming growth factor-?1(TGF-?1),?-SMA,E-Cadherin in renal proximal tubular epithelial cells(HKCs)and role of AG490,an antagonist of Janus kinase.Methods Cultured HKCs cells were divided into four groups:low glucose group(LG),high glucose group(HG),high mannitol group(LG+M),and HG+AG490 group(AG).Immunoprecipitation and Western blot analysis were used to measure the expressions of tryosine phosphorylated Janus kinase 2(p-JAK2),STAT1,STAT3,p-STAT1,p-STAT3 and ?-SMA,E-Cadherin.The contents of TGF-?1 and type I collagen in the supernatants of the cultured HKCs were detected by ELISA.TGF-?1 mRNA were measured by RT-PCR.ResultsCompared with low glucose control group,the expressions of JAK2,p-STAT1,p-STAT3 and TGF-?1 mRNA were significantly increased in HG group from 6 to 72 h.Meanwhile,the contents of TGF-?1 and collagen I in the supernatants and the expression of ?-SMA were increased,the expression of E-Cadherin was decreased.The expression of JAK2,p-STAT1,p-STAT3 and TGF-?1 mRNA and the level of TGF-?1,collagen I in the supernatant s in HG+AG490 group were obviously lower than those of the HG group.The expression of ?-SMA and E-Cadherinwas also decreased in HG+AG490 group.Conclusion Activation of JAK/STAT signaling pathway seems to be involved in the high glucos induced overproduction of TGF-?1 and ECM proteins in HKCs.

AIM: To study the effect of high fat diet on the expression of sterol regulatory element biding protein-1 (SREBP-1) and transforming growth factor β_1 (TGF-β_1) in renal tubular cells and rosiglitazone intervention. METHODS: Wistar rats were treated with high fat diet and rosiglitazone for 3 months. The serum glucose, serum insulin and serum triglyceride were detected. Oil Red O staining was used to observe the renal lipid deposit and Masson staining was for the detection of ECM accumulation. SREBP-1, TGF-β_1 and FN protein were determined by the methods of immunohistochemistry and Western blotting. SREBP-1 mRNA was detected by in situ hybridization. RESULTS: Rosiglitazone prevented effectively the increase in serum glucose, serum insulin and serum triglyceride resulted from high fat diet. High fat diet led to lipid droplet formation in renal tubular cells and interstitial ECM accumulation, which was decreased by rosiglitazone treatment. Compared to normal rats, SREBP-1 protein and SREBP-1 mRNA showed high expressions in high fat diet rats that were lowered by rosiglitazone. The precursor segment and mature segment of SREBP-1 protein were decreased by 27.39% and 27.32%. Similarly, the high expressions of TGF-β_1 and FN protein in kidney of high fat diet rats were also prevented by rosiglitazone intervention. Compared to high fat diet rats, the expression of TGF-β_1 in rosiglitazone treatment rats was lowered by 19.14%. CONCLUSION: Rosiglitazone prevents effectively the over-expression of SREBP-1 and TGF-β_1 in renal tubular cells, and decreases lipid accumulation and ECM production in rats fed with high fat diet.

significantly increased (all P < 0.01). Following SREBP-1 was down-regulated by siRNA, high glucose-stimulated TGF-β1 and FN protein expressions were decreased by 17.9% and 24.6% ,respectively(all P<0.01).

Aim To construct eukaryotic expression vector of shRNA(small hairpin RNA)for human SREBP-1(sterol regulation element binding protein-1)gene and explore its effects on lipid droplet formation in human renal proximal tubular epithelial cell line(HKC)under the stimulation of high glucose.Methods Two eukaryotic expression vectors of shRNA were constructed for human SREBP-1 gene.The HKC cells were transfected with negative control plasmid(pGenesil-1-HK)and two recombinant vectors(pGenesil-1-SREBP1-1 and pGenesil-1-SREBP1-2)and then were cultured under the stimulation of high glucose for about 48 h.The expression of SREBP-1 mRNA and FAS mRNA was detected by RT-PCR and SREBP-1 protein expression was investigated by Western blot.Lipid droplets were detected by Oil Red O staining.Results DNA sequencing showed that the target segments were successfully cloned into pGenesil-1 vector respectively.RT-PCR indicated that two recombinant vectors could inhibit the expression of SREBP-1 mRNA and FAS mRNA in HKC cells under the stimulation of high glucose.Similarly,SREBP-1 protein was also inhibited by the transfection with recombinant vectors.Oil Red O staining found that silencing of SREBP-1 gene resulted in lipid droplets decrease.Conclusions The eukaryotic expression vector of shRNA for human SREBP-1 gene was successfully constructed,and the expression of SREBP-1 was inhibited effectively by the expressed siRNA in HKC cells that resulted in lipid droplets decrease through FAS mRNA transcription inhibition.

Aim To investigate the effects of Ang Ⅱ receptor antagonist irbesartan on the expressions of connective tissue growth factor(CTGF) and membrane-type 1 matrix metalloproteinase(MT1-MMP) in high glucose-cultured rat glomerular mesangial cells(GMCs).Methods High concentration glucose and irbesartan were used to stimulate the cultured rat GMCs in vitro.The mRNA and protein expressions of CTGF and MT1-MMP were detected with semi-quantitative RT-PCR and Western blot.The secreted collagen Ⅳ in the supernatants of the GMCs was detected by enzyme-linked immunoadsorbent assay(ELISA).Results Compared with control group,the expressions of CTGF were continuously increased in GMCs under high concentration glucose medium;otherwise the mRNA and protein levels of MT1-MMP in GMCs were decreased in a time-dependent manner at the same time.These changes were accompanied by increased secretion of collagen Ⅳ.Irbesartan could inhibit those changes induced by high glucose.Conclusions High glucosecould induce the expression of CTGF and inhibit the expression of MT1-MMP in GMCs.Irbesartan could inhibit the secretion of ECM in GMCs under high concentration glucose medium,partly by regulating the expressions of CTGF and MT1-MMP.