Prostaglandin E2 receptor 1 antagonist attenuates mesangial cell lesion induced by TGF-β1 in mice through inhibiting ERK signal pathway

Objective To explore the effects and mechanisms of prostaglandin E2 (PGE2) receptor 1 antagonist (SC-19220) on proliferation, prostaglandin synthase and extracellular regulated protein kinases (ERK) signal pathway induced by transforming growth factor β1(TGF-β1) in glomerular mesangial cells. Methods Mouse glomerular mesangial cells (GMCs) were divided into 5 groups: control group, TGF-β1 (10 μg/L) group, TGF-β1 (10 μg/L) plus SC-19220 group (0.1, 0.5, 1.0 μmol/L). The proliferation of GMCs was measured by CCK-8. The PGE2 in supernatant was measured by ELISA. The expression of connective tissue growth factor (CTGF), laminin (LN), cyclooxygenase 2(COX2), membrane-bound prostaglandin E2 synthase 1 (mPGES1) protein and mRNA was examined by Western blotting and real-time quantitative PCR, ERK1/2 or phospho-ERK1/2 was measured by Western blotting as well. Results TGF-β1 induced the proliferation of GMCs and increased the secretion of PGE2. Besides, TGF-β1 significantly up-regulated the expression of CTGF, LN, COX2 and mPGES1 mRNA and protein (P﹤0.05), and increased the expression of phospho-ERK1/2 protein (P﹤0.05). However, SC-19220 significantly attenuated the changes of above-mentioned parameters and their activities (P﹤0.05). All the effects of SC-19220 were in dose-dependent manner. Conclusions SC-19220 may reduce TGF-β1-induced cell damage by suppressing the activity of ERK1/2, and feedback inhibition of COX2, mPGES1 and PGE2, thus decreases the expression of LN and CTGF.     

Protective effect ofEP2 agonists on mesangial cell lesion induced by TGF-β1 in mice

Objective To explore the effects and mechanism of Butaprost (PGE2 receptor 2 agonist) on proliferation and secretion of extracellular matrix (ECM) accumulation induced by TGF-β1 in mouse mesangial cell. Methods Mouse golmerular mesangial cell (GMC) were divided into 5 groups: control group; TGF-β1 group; TGF-β1 plus Butaprost group (10, 1, 0.1 μmol/L). The proliferation of GMCs was measured by CCK-8, the proportion of G2＋S phase in the cell cycle was measured by flow cytometry and cAMP, PGE2 secreted into media by ELISA assay. The expression of LN, FN, CTGF, COX1, COX2, p27 protein and mRNA was measured by Western blot and real time quantitative reverse PCR, phosphorylation of p38 MAPK was measured by Western blot as well. ResultsTGF β1 induced the proliferation of GMCs and increased the secretion of cAMP and PGE2 as well as the proportion of G2＋S phase. Besides, TGF β1 significantly upregulated the expression of FN, LN, CTGF, COX2 and phosphorylated p38 MAPK mRNA and protein while the expression ofp27Kip1 mRNA and protein was reduced (all P＜0.05). Butaprosteffectively reversed above changes and their activities (all P＜0.05). All the effects of Butaprost were dose-dependent. Conclusions Butaprost may inhibit TGF-β1-induced cell proliferation and ECM accumulation by upregulate the expression of cAMP and repress the activity of p38 MAP kinase thus decreased the expression of COX2, PGE2, FN, LN 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.     

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.     

EP1 receptor mediates Adriamycin-induced podocyte injury through activating p38 MAPK

Objective To investigate the effect of prostaglandin E2 receptor 1 (EP1) on Adriamycin (ADR)-induced glomerular podocytes injury and its possible mechanism. Methods (1) In vivo experiments: 6-8 weeks old male Balb/c mice were randomly divided into four groups: Control group; ADR group; EP1 agonist 17-phenyl PGE2+ADR group; EP1 antagonist SC-19220+ADR group. The mouse model of nephrotic syndrome was induced by injection of ADR (10 mg/kg) into tail vein, and then EP1 agonist (1 μg/g) and antagonist (25 μg/g) were administered respectively. Six weeks later, all mice were sacrificed and urine, blood and kidney tissues were collected. Detecting urine protein, blood chemistry, changes of renal pathology and podocyte-related proteins, electron microscopy changes of podocytes. (2) In vitro experiments: Podocytes were cultured in vitro and divided into different groups: Control group; ADR group (0.2 μmol/L); EP1 agonist (0.1, 1, 10 μmol/L)+ADR (0.2 μmol/L) group; antagonist (0.1, 0.5, 1 μmol/L)+ADR (0.2 μmol/L) group. The proliferation of podocytes was measured by CCK-8. Expression of PGE2 in podocytes was detected by ELISA. Indirect immunofluorescence was used to determine the localization of podocyte-related proteins nephrin, podocin and CD2AP. Expression of nephrin, podocin, CD2AP, COX2 in podocytes was detected by Western blotting and Real-time quantitative PCR. p38 MAPK or phospho-p38 MAPK was measured by Western blotting as well. Flow cytometry was used to detect cell apoptosis. Results (1) In vivo experiments: Compared with control group, obvious proteinuria, blood biochemical changes and renal pathological changes were observed in ADR group, proteinuria, blood biochemical and renal pathological changes were more serious in mice dealt with agonist, while antagonist could reduce ADR-induced injury (all P＜0.05). Results of immunohistochemistry showed that the expression of podocyte-related proteins nephrin, podocin and CD2AP in ADR group were significantly lower than those in control group, and EP1 agonist could further inhibit expression of these proteins, while antagonist could reverse this inhibitory effects (P＜0.05). Electron microscopic results showed that mice in ADR group appeared foot enlargement and fusion, and the agonist group further aggravated the injury, while antagonist intervention could inhibit the injury of podocytes. (2) In vitro experiments: Compared with control group, expression of PGE2 and COX2 were increased; mRNA and protein expression of nephrin, podocin, CD2AP were decreased, p38 MAPK activity and podocytes apoptosis were increased in ADR group (P＜0.05); Agonist could aggravate podocytes damage (P＜0.05), while Antagonist could down-regulate the expression of PGE2 and COX2, promote the expression of nephrin, podocin and CD2AP, and inhibit the activity of p38 MAPK and podocytes apoptosis (P＜0.05). The addition of p38 MAPK inhibitor(10 μmol/L) could reduce the inhibitory effect of EP1 agonist on the expression of podocyte-related proteins nephrin, podocin and CD2AP (P＜0.05). Conclusions EP1 receptor may activate the p38 MAPK signaling pathway to inhibit podocytes-related proteins nephrin, podocin and CD2AP, as well as mediate the ADR induced podocyte injury. Inhibition of EP1 receptor however have a protective effect.     

Impact of STAT1-siRNA transfection on the expressions of STAT3 and transforming growth factor β1 in human glomerulur mesangial cells induced by high glucose

Objective To observe the cell proliferation and the protein expression of STAT1,phosphorylation of STAT1 (p-STAT1), STAT3, p-STAT3 and transforming growth factor β1 (TGF-β1) in human glomerulur mesangial cells (HMCs) induced by high glucose after STAT1-siRNA transfection. Methods Three STAT1-siRNA sequences were designed and synthetized. HMCs in 6-well plate were transiently transfected with STAT1-siRNA using Lipofectamine 2000. After transfection for 48 h or 72 h, STAT1 mRNA and protein expression were detected by real-time PCR and Western blotting, respectively, to choose the effective sequence in later experiments. After transfection for 24 h and stimulated with 25 mmol/L glucose for 24 h, 48 h, 72 h, cell proliferation was measured by MTT assays, the protein expressions of STAT1, p-STAT1, STAT3 and p-STAT3 were detected by Western blotting, the expression of TGF-β1 was detected by ELISA in each group. Results High glucose could stimulate HMCs proliferation. The protein expressions of p-STAT1, p-STAT3 and TGF-β1 were increased in the group stimulated by high glucose (P＜0.05). The protein expressions of p-STAT3 and TGF-β1 were further increased in HMCs induced by high glucose after STAT1-siRNA transfection (P＜0.05). Conclusions Under high glucose conditions, JAK-STAT signal transduction pathway of HMCs can be activated, then it is far greater when HMCs are induced by high glucose after STAT1-siRNA transfection. The secretion of TGF-β1 is increased in HMCs under the state of high glucose, and it is further increased after STAT1-siRNA transfection, which is related to the kidney fibrosis.     

Global adiponectin suppress the high expression of monocyte chemotactic protein-1 induced by high glucose in NRK52E cells

Objective To investigate the effect of globular adiponectin on the high expression of monocyte chemotactic protein-1 (MCP-1) induced by high glucose in rat renal tubular epithelial cells(NRK52E), and its relationship with adiponectin receptors and p38MAPK. Methods NRK52E cells were cultured in vitro and divided into six groups: normal glucose group (NG, 5.6 mmol/L glucose), high glucose group(HG, 25 mmol/L glucose), gAd group1 (HG+gAd 2 mg/L), gAd group2 (HG+gAd 5 mg/L), gAd group3 (HG+gAd 10 mg/L), p38MAPK antagonist group：(SB, HG+SB203580 10 μmol/L). The protein expression of phosphorylated p38MAPK (p-p38MAPK), total p38MAPK (t-p38MAPK), MCP-1 and AdipoR1/AdipoR2 were examined by western blotting. The mRNA expression of MCP-1 and AdipoR1/AdipoR2 were detected by RT-PCR and real-time PCR respectively. Results Compared with NG group, the mRNA and protein expression of MCP-1 increased significantly in HG group (all P＜0.05). The phosphorylation of p38MAPK increased (P＜0.05) with no change in t-p38MAPK protein. The addition of gAd or SB203580 inhibited the unregulation of MCP-1 and p-p38MAPK induced by HG. Two kinds of adipoR,adipoR1 and adipoR2,were all detectable in NG group, and mRNA and protein expression of adipoR1 was higher than that of adipoR2 (P＜0.01). Compared with NG group, the expression of adipoR decreased in HG group, but the difference had no statistical significance(P＞0.05). Compared to HG group, the mRNA and protein expression of adipoR1 increased in gAd groups (all P＜0.01). Conclusion The gAd can dose-dependently attenuate the overexpression of MCP-1 induced by high glucose, and this protective effect may be mediated by adipoR1 and p38MAPK.     

Roles of prostaglandin E receptors in mesangial cells under high-glucose conditions.

BACKGROUND: High glucose reportedly stimulates prostaglandin (PG) E2 production and DNA synthesis in mesangial cells (MCs). However, the pathophysiological significance of PGE2 in MCs has remained unclear. METHODS: The effects of prostanoids on [3H]-thymidine uptake and cAMP production in rat MCs cultured with 5.6 mM glucose, 25 mM glucose, or 5.6 mM glucose supplemented with 19.4 mM mannitol were examined. The gene expression of PGE2 receptor (EP) subtypes in MCs was analyzed with Northern blotting techniques. RESULTS: Northern blotting indicated EP1 and EP4 gene expression in MCs. EP1 agonists and PGE2 stimulated [3H]-thymidine uptake in MCs. EP1 antagonists dose dependently attenuated high-glucose-induced [3H]-thymidine uptake, which suggests EP1 involvement, by an increase in intracellular Ca2+, in DNA synthesis of MCs. On the other hand, forskolin, db-cAMP, and 11-deoxy-PGE1, an EP4/EP3/EP2 agonist, significantly decreased DNA synthesis in MCs. These inhibitory effects are thought to be mediated via EP4 as a result of an increase in cAMP synthesis. The effects via EP4 seem to be particularly important because PGE2-induced cAMP synthesis was significantly attenuated in the high-glucose group compared with the mannitol group, in which [3H]-thymidine uptake did not increase in spite of augmented PGE2 production. CONCLUSION: The increase in DNA synthesis in MCs under high-glucose conditions can be explained, at least in part, by the high-glucose-induced inhibition of cAMP production via EP4, which augments EP1 function in conjunction with the overproduction of PGE2.     

Effects of triptolide on proliferation and apoptosis in rat mesangial cells induced by transforming growth factor β1 and related mechanisms

Objective To investigate the effects of triptolide on proliferation, apoptosis and the changes of Ski, Smad3, Smad7 and collagen type Ι (ColΙ) in cultured rat mesangial cells induced by transforming growth factor (TGF)?β1. Methods Cultured HBZY?1 rat mesangial cells were divided into 5 groups: (1)normal control group; (2)TGF?β1 group (10 μg/L); (3)-(5)triptolide (0.4, 2, 10 μg/L)+TGF?β1 (10 μg/L) groups. The cell proliferation was detected by MTT. Apoptosis of mesangial cells was detected by TUNEL assay. The expressions of Ski, Smad3, Smad7 mRNA were examined by real?time quantitative PCR. The expressions of Ski, Smad3, Smad7 and ColΙ protein were detected by Western blotting. The localizations of Ski and Smad3 protein were detected by laser confocal fluorescence microscope. Results Compared with the normal control, TGF?β1 (10 μg/L) significantly stimulated mesangial cells proliferation, while decreased apoptosis. The mRNA and protein expressions of Ski, Smad7, Smad3 and ColΙ protein expression in TGF?β1 group were increased (P＞0.05). In comparison with TGF?β1 group, triptolide could significantly inhibit TGF?β1?induced mesangial cells proliferation in dose?dependent manner, and promote the apoptosis of mesangial cells. In TGF?β1 group, mRNA and protein expresscons of Ski and Smad7 were increased (P<0.05), Smad3 mRNA and protein were decreased (P＞0.05), and ColΙ protein was decreased (P<0.01). In comparison with TGF?β1 group, fluorescence intensity of Ski, Smad3 proteins was significantly increased in cytoplasm, while decreased in nucleus. Conclusions Triptolide can inhibit TGF?β1?induced mesangial cells proliferation through regulating the expressions of Ski, Smad7 mRNA and protein, inhibiting Ski. Smad7 translocation to the nucleus, and down?regulating Smad3 mRNA and protein expression. Triptolide can promote apoptosis of mesangial cells.     

移植肾IgA肾病临床病理及预后分析

目的探讨移植肾IgA肾病(IgAN)复发或新发的诱因及移植肾生存的危险因素。方法选取2012年11月至2018年12月浙江大学医学院附属第一医院经肾活检确诊为移植肾IgAN的患者,按照血肌酐(Scr)增高水平、估算肾小球滤过率(eGFR)下降率分为稳定组(Scr升高值<20μmol/L,eGFR下降率<10%)和进展组(Scr增高但未达翻倍值,30%相似文献   

Effects of metformin on expression of AMP - activated protein kinase in rat glomerular mesangial cells

ObjectiveTo observe the effects of metformin on expression of Adenosine 5’- monophosphate (AMP)-activated protein kinase (AMPK), nuclear factor-κB (NF-κB) and transforming growth factor β1 (TGF - β1) in cultured rat glomerular mesangial cells (MCs), and explore its reno - protective mechanisms. Methods MCs were cultured in the medium with normal glucose (group NG, 5.6 mmol/L), high glucose (group HG, 25mmol/L) and different concentrations of metformin (group M1, M2, M3). After 48 h exposure, the supernatants and MCs were collected. The expression of NF-κB and TGF-β1 mRNA was analyzed by real time-PCR. Total-AMPK, phospho-Thr-172 AMPK (p-AMPK), NF -κB p65 and TGF-β1 were visualized by Western blot. ResultsThe real time-PCR and Western blot result showed MCs could express AMPK, NF-κB and TGF-β1 mRNA and protein. After stimulated by HG, the levels of intracellular NF - κB and TGF - β1 expressions were significantly increased compared with group NG (P＜0.05); The levels of NF-κB and TGF-β1 were significantly decreased in group M1, M2 and group M3 compared with group HG in a dose-dependent manner. After stimulated by HG, the level of intracellular p-AMPK were down-regulated compared with group NG(all P＜0.05); The expression of p-AMPK increased with the rising of metformin concentration, presenting the opposite trend (P＜0.05), while the level of total-AMPK protein was unchanged with exposure to HG or different concentrations of metformin(P＞0.05). ConclusionMetformin can suppress the expression of NF- κB and TGF-β1 of glomerular MCs induced by HG via AMPK activation, which may partly contribute to its reno-protection.     

Stimulation of cAMP production and cyclooxygenase-2 by prostaglandin E(2) and selective prostaglandin receptor agonists in murine osteoblastic cells

Prostaglandins (PGs), particularly PGE(2), can stimulate bone resorption and formation and auto-amplify their effects by inducing cyclooxygenase (COX)-2. We examined the role of different PG receptors in stimulating cAMP production and COX-2 expression in murine calvarial osteoblasts. Cells were obtained from PGE(2) receptor (EP2R and EP4R) wild-type and knockout (KO) mice and from mice transgenic for the COX-2 promoter fused to a luciferase reporter. We analyzed effects of selective agonists, EP2A and EP4A, for EP2R and EP4R, which mediate the increase in cAMP in response to PGE(2). We also tested agonists for other PGE(2) receptors (EP1A and EP3A) and for prostacyclin (IPA), prostaglandin D(2) (DPA), thromboxane (TPA), and prostaglandin F(2alpha) (FPA) receptors. PGE(2) and EP2A were the most effective stimulators of cAMP production. EP4A, IPA, and DPA produced smaller responses, and EP1A, EP3A, FPA, and TPA were ineffective. In EP2R KO cells, cAMP responses to PGE(2) were reduced by 80%, and responses to EP2A were abrogated. In EP4R KO cells, cAMP responses to PGE(2) and EP2A showed a small reduction, while the response to EP4A was abrogated. Pretreatment with PGE(2), EP2A, or EP4A down-regulated the subsequent response to the respective ligands. COX-2 induction was measured by increased luciferase activity and mRNA expression. PGE(2) was the most effective agonist; EP2A and another selective EP2R agonist, butaprost, showed similar efficacy, and EP4A was less effective. EP2A and EP4A effects on luciferase activity were additive, and effects of the combination were similar to PGE(2) itself. IPA, TPA, and DPA produced 2- to 6-fold increases in COX-2 expression. FPA was a weak agonist, while EP1A and EP3A were inactive. Treatment with specific inhibitors indicated that PGE(2), EP2A, and EP4A induced COX-2 expression largely through protein kinase A (PKA). We conclude that the PG induction of COX-2 in this system generally paralleled effects on cAMP production and was mediated predominantly via the PKA pathway.     

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.     

Effect of MG132 on the expression of ERK1/2 and connective tissue growth factor in rat peritoneal mesothelial cells induced by high glucose

Objective To observe the effect of MG132 on the expression of extracellular regulated kinase 1/2 (ERK1/2) and connective tissue growth factor (CTGF) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose. Methods RPMCs were isolated, cultured and passaged by trypsin, then identified. The second generation of cultured RPMCs were used in the experiment. RPMCs were divided into normal control group, high glucose (1.5%, 2.5%, 4.25%) for 24 hours, high glucose (2.5%) for 0, 12, 24, 48 hours,incubated with MG132 (0.5, 1, 2 μmol/L) for half an hour and then with high glucose (2.5%) for 24 hours. ERK1/2 protein was detected by Western blotting, and CTGF protein in supernatant was detected by ELISA. Results Compared with the control group, the expression of p-ERK1/2 was significantly increased in the groups stimulated by high glucose (P＜0.01), reached the peak at 24th hour (P＜0.01), and then the expression decreased at 48th hour, but still was higher than that in the normal control group (P＜0.01). CTGF protein expression of RPMCs induced by high glucose increased, in time- and dose-dependent manner (P＜0.05). MG132 could significantly decrease the expression of ERK1/2 and CTGF induced by high glucose (P＜0.05). Conclusions MG132 can decrease the expression of p-ERK1/2 and CTGF in RPMCs induced by high glucose. The ubiquitin proteasome pathway participates in the development of peritoneal fibrosis, and blocking the way may contribute to the prevention of peritoneal fibrosis.     

Effect of suramin on the epithelial-mesenchymal transition in peritoneal mesothelial cells induced by high concentration glucose

Objective To explore the effect of suramin on the epithelial-mesenchymal transition (EMT) and the excretion of transforming growth factor-β1 (TGF-β1) in peritoneal mesothelial cells (PMCs) induced by high concentrations of glucose solution (GS). Methods Cultured PMCs were divided into three groups: (1) normal control group; (2) GS-treated group: cells were treated with 1.5%, 2.5%, 4.25% GS for 12 h, 24 h, 48 h, respectively; (3) Suramin-treated group: PMCs cultured with 4.25% GS were exposed to different doses of suramin (25, 50, 100 μmol/L) for 48 h. Expression levels of α-smooth muscle actin (α-SMA) and E-cadherin were detected by Western blotting and the concentration of TGF-β1 in the culture supernatant was determined by ELISA. Results Compared with normal control group, GS-treated PMCs exhibited a time-dependent increase in the expression of α-SMA, and decrease in the expression of E-cadherin. GS also stimulated PMCs to secrete TGF-β1. In the presence of suramin, GS-induced α-SMA expression and TGF-β1 production were reduced, E-cadherin expression was increased. Conclusions Suramin can inhibit high glucose-induced EMT of PMCs by down-regulating the expression of TGF-β1. Suramin may be a novel therapeutic agent for the treatment of peritoneal fibrosis.     

Effect of vitamin K2 on β-glycerophosphate-induced calcification in rat vascular smooth muscle cells and the mechanism

Objective To explore the effect of vitamin K2 on β-glycerophosphate(β-GP)-induced rat vascular smooth muscle cells (VSMCs) calcification and and the mechanism. Methods VSMCs were obtained from rat aortic, and identified by immunocytochemistry, then randomly divided into control group, high phosphorus group, vitamin K2 group (the group was settled three subgroups according to the concentration of vitamin K2 based on the high phosphorus medium, namely 10 μmol/L, 25 μmol/L, 50 μmol/L) and noggin (bone morphogenetic protein pathway inhibitor) group. Calcification was visualized by Alizarin red staining, calcium load in cells was quantified by o-cresolphthalein complexone method and alkaline phosphatase (ALP) activity was measured after stimulating 14 days, gene expressions of bone morphogenetic protein - 2 (BMP-2), SMAD1, SMAD7 and Runx2 mRNA were detected by RT-PCR, Runx2 protein levels was detected by Western blotting after stimulating 3 days. Results Compared with the cells in control group, high phosphorus induced cell calcification, increased ALP activity, up-regulated the expression of BMP-2, SMAD1, Runx2 mRNA (P＜0.05) and down-regulated the expression of SMAD7 (P＜0.01),while compared with high phosphorus group, the calcium deposition, ALP activity and the expression of BMP-2, SMAD1, Runx2 mRNA were remarkably reduced in a dose-dependent manner by treatment with vitamin K2 (P＜0.05) and the expression of SMAD7 was increased (P＜0.01). Compared with high phosphorus group, SMAD1 and Runx2 expression in noggin group were remarkably reduced(P＜0.01). Conclusion Vitamin K2 inhibits β-glycerophosphate-induced VSMCs calcification which correlates with the suppression of the expression of osteoblast markers through the down-regulation of bone morphogenetic protein pathway.     

Effect of heat shock protein 47 on epithelial-mesenchymal transdifferentiation induced by transforming growth factor β1 in the renal tubular epithelial cells

Objective To detect the expression of heat shock protein 47(HSP47) in renal proximal epithelial cell lines (HK-2) and to investigate the role of HSP47 in the progress of transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transdifferentiation (EMT) in HK-2 cells. Methods HK-2 cells were exposed to TGF-β1 (0, 2.5, 5, 10 μg/L) for different time (0, 12, 24, 48 h). The expression of HSP47 was examined by Western blotting. Then HK-2 cells were exposed to 10 μg/L TGF-β1, the expressions of vimentin, zona occludens-1 (ZO-1) were examined by Western blotting and real-time PCR. Furthermore, the expressions of p-Smad3 and Smad3 were examined by Western blotting. HK-2 cells were transfected with HSP47 siRNA and siRNA negative control before exposing to TGF-β1. Then the expressions of vimentin, ZO-1 were detected by Western blotting and real-time PCR, meanwhile Western blotting for HSP47, p-Smad3 and Smad3. Results Stimulating HK-2 with TGF-β1resulted in a significant increased expression of HSP47 in time-and concentration-dependent manner (P＜0.05). Meanwhile, TGF-β1up-regulated the protein and mRNA expression of vimentin (P＜0.05), and down-regulated the protein and mRNA expression of ZO-1 (P＜0.05), all in time-dependent manner. Stimulating HK-2 with TGF-β1 resulted in phosphorylation of Smad3, which was peaked at 30 min, slightly decreased at 1 h, and then increased again between 24 and 48 h (P＜0.05). Compared to the TGF-β1group, inhibition of HSP47 expression in HK-2 up-regulated the protein and mRNA expression of ZO-1, down-regulated the protein and mRNA expression ofvimentin (P＜0.05) and down-regulated the ratio of p-Smad3/Smad3. HSP47 siRNA negative control had no significant effect on the expressions of ZO-1, vimentin and p-Smad3/Smad3 (P＞0.05). Conclusion HSP47 can promote the EMT of renal tubular epithelial cell which is possibly via the TGF-β1-Smad3 pathway.     

Protective effect of resveratrol on 5/6 nephrectomized rats and its mechanism

Objective To investigate the protective effect of resveratrol (RSV) on 5/6 nephrectomized rats and its mechanism. Methods Fifty male SD rats were randomly divided into three groups: sham operated (Sham, n＝10）, 5/6 nephrectomy (Nx, n＝20), and 5/6 nephrectomy＋RSV 20 mg/kg (Nx＋RSV, n＝20). RSV or normal saline was administered one week after 5/6 nephrectomy. Proteinuria was detected every 4 weeks. Serum creatinine and the renal pathological changes were measured after 12 weeks. Immunohistochemisty staining of fibronectin (FN), collagenⅠ, transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) were used to analyze the changes of renal fibrosis. Western blotting was used to measure the expression of Smad3, phospho-Smad3, and acety-Smad3. Immunoprecipitation was used to detect the interaction between Sirt1 and Smad3. Results Compared with the sham operated rats, subtotal nephrectomy significantly increased proteinuria [(152.14±30.49) mg/24 h vs (25.34±7.54) mg/24 h], serum creatinine[(111.60±21.50) μmol/L vs (53.90±11.59) μmol/L], glomerular sclerosis index (1.56±0.34 vs 0.35±0.08) and the expressions of fibronectin, collagenⅠ, TGF-β and CTGF in renal tissue at 12 weeks after operation (all P＜0.01), and RSV treatment significantly inhibited the above up-regulations (all P＜0.01). Compared with the sham operated rats, subtotal nephrectomy increased the expression of phospholylation and acetylation of Smad3. RSV treatment significantly reduced the expression of acety-Smad3, but had no effect on the phospho-Smad3. Immunoprecipitation revealed a binding effect of Smad3 with Sirt1. Conclusions RSV treatment can attenuate proteinuria, protect renal function and inhibit renal fibrosis in 5/6 nephrectomized rats. This renal protective effect is associated with reduced Smad3 acetylation and activation of Sirt1, which suggesting that Sirt1 may be a potential therapeutic target of CKD.     

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.