Objective To explore the relationship between resveratrol and Notch 1 signalling pathway in podocytes.Methods Interference RNA (RNAi) and doxycycline (Dox) were used to inhibit the Sirtuin (SIRT) 1 expression in the wild-type and inducible SIRT1 shRNA (CAGGS) podocytes respectively.Recombinant mouse delta-like ligand 4 (DLL4) was used to activate Notch1 signalling.The message RNA of SIRT1,Notch1 downstream gene Hes1 and Hey2,as well as the key enzymes of Notch1 signalling pathway were detected by using real-time PCR.Western blotting was used to detect intracellular domain of Notch 1 (ICD1),SIRT1,and metalloprotease (ADAM) 10 and components of γ-secretase complex protein expression.Results In WT murine podoytes,resveratrol up-regulated ICD1 protein production,as well as the mRNA of Hes1 and Hey2 in a dose-dependent manner.Treatment with resveratrol resulted Nicastrin mRNA and protein increase in podocytes (P ＜0.05),as well as inhibit ADAM10 expression (P ＜ 0.05),but all these changes were prevented after the use of SIRT1 RNAi(P ＜ 0.05).DLL4 up-regulated the expression of mRNA of Hes1 and Hey2,as well as ICD1 protein production in a dose-dependent manner.Treatment with doxycycline resulted decrease of SIRT1 gene and protein expression in CAGGS podocytes after 24 h and 48 h respectively(P ＜ 0.05),which weakend the role of DLL4 significantly(P ＜ 0.05).Conclusion Resveratrol induces Nicastrin expression,as well as activation of Notch1 signalling pathway in a SIRT1-dependent manner.

Objective To investigate the role of activated cylic AMP(cAMP) signaling in chemical-induced podocyte injury.Methods Eight-weeks-old male BalB/C mice were randomly divided into three groups:control group,Adriamycin (ADR) group and Forskolin+ADR group.ADR nephropathy models were established by tail intravenous injection,and part of them were injected Forskolin,an agonist of adenylate cyclase,intraperitoneally.Phosphorylation of cAMP response element binding protein (CREB) was detected by laser confocal microscopy,morphology of foot processes were determined with transmission electron microscope,and WT-1 expression in glomeruli were detected by immunohistochemistry.Conditionally immortalized podocytes were treated with puromycin aminonucleoside (PAN),Exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2-O-Me-cAMP (2Me),protein kinase A (PKA) antagonist H89 and its agonist pCPT-cAMP(pCPT).Western blot was used to detect the expression levels of Epac,caspase3 and cleaved caspase3.PKA activity was assayed using cAMP-dependent protein kinase detection system.Cell viability was determined by a cell count kit and podocyte apoptosis was estimated by TUNEL staining.Mitochondrial membrane potential was evaluated by JC-1 staining.Results (1)Compared with ADR group,the urine albumin decreased significantly (P ＜ 0.05) among Forskolin + ADR group and the WT-1 positive cells per glomerulus increased obviously (P ＜ 0.05).(2)PAN decreased podocyte number in a time-dependent manner (P ＜ 0.05),pre-treatment with pCPT obviously inhibited PAN induced podocyte decrease (P ＜0.05),but H89 prevented the effect of pCPT in a dose-dependent manner (P ＜ 0.05).(3)JC-1 staining showed that the percentage of podocyte with green fluorescence for control,PAN and pCPT+PAN group were (12.67±2.15)％,(31.35±4.60)％ and (16.96 ± 2.51)％ respectively (P ＜ 0.05),and pretreatment with H89 inhibited the effect of pCPT (P ＜ 0.05).(4) PAN promoted podocyte apoptosis and cleaved caspase3 expression (P ＜ 0.05),and pretreatment with pCPT significantly prevented PAN-induced podocyte apoptosis and cleaved caspase3 expression (P ＜ 0.05).Conclusions cAMP signaling activation ameliorated podocyte injury in ADR nice and PAN-induced podocyte apoptosis,and cAMP/ PKA pathway may mediate these processes.

ObjectiveTo investigate whether advanced glycation end products (AGE) induce monocyte chemoattractant protein-1 (MCP-1) expression in mouse podocytes in vitro and the role of AGE receptor (RAGE). MethodsThe effects of AGE, CML, S100 protein and neutralizing antibody against RAGE on MCP-1 gene and protein expressions were examined by RT-PCR and ELISA in mouse podocytes. ResultsRAGE was detected in undifferentiated and differentiated podocytes. AGE and CML dose-dependently stimulated MCP-1 expression in podocytes. At 8 h of incubation, AGE-and CML-treated podocytes produced more MCP-1 than that treated with bovine serum albumin (BSA), the MCP-1 concentrations being (7.44?1.01 and 8.06?0.96)ng/L for AGE and CML respectively vs (3.77?0.39)ng/L for BSA (both P

Objective To investigate the signalling events follow the activation of RAGE in podocytes. Methods AGE and CML generated dichloroflurescin-sensitive intracellular ROS were measured by confocal microscopy. The activation of MAP kinases family were studied using Western blotting. MCP-1 mRNA expression was detected by semi-quantitative RT-PCR. Results Basal ROS was located in nucleus of starvation podocytes. AGE and CML rapidly generated intracellular ROS in podocytes. NAC pre-treated podocytes suppressed basal and inducible ROS generation and antibody for RAGE suppressed the inducible ROS. Blockage of ROS induced by NAC suppressed the expression of CML and H_2O_2-induced MCP-1. Phosphorylated extracellular signal-regulated kinase (ERK) was found in CML incubated podocytes at 10 min and was prevented by NAC or AFC. PD98058 Pre-treated podocytes partially inhibited the expression of CML-induced MCP-1 mRNA. No evidence were showed that p38 MAPK, SAPK/JNK, PI3K and PKC were involved in the signal transduction. Conclusion Activation of RAGE induces MCP-1 expression in podocytes via ROS-ERK signalling pathway.

OBJECTIVE: To study the expression of angiopoietin receptor Tie-2 in the renal tissue of diabetic rats and the effects of Astragalus. METHODS: SD rats were randomly divided into normal control group, diabetes group and Astragalus-treated group. The expression of receptor Tie-2 in the renal tissue was assessed by using real-time quantitative polymerase chain reaction and immunohistochemical method. RESULTS: Glomerule Tie-2 protein expression was significantly elevated in the diabetes group as compared with the normal control group (P<0.01). Glomerule Tie-2 protein expression in the Astragalus-treated group was decreased as compared with the diabetes group (P<0.01). CONCLUSION: Tie-2 may play an important role in the pathogenesis of the early stage diabetic renal injury. The reno-protection effect of Astragalus may be mediated by down-regulating the expression of Tie-2 in the kidney tissue of diabetic rats.

Objective To investigate the role of recombinant human interleukin 6 (rhlL-6) in calcification and osteogenic transition of cultured human umbilical artery smooth muscle cells (HUASMC), and the possible cell signal transduction way. Methods HUASMCs were isolated by the explant method. HUASMCs were treated with (treatment groups) or without (control group) rhIL-6. Alizarin Red S stain was applied for calcium deposition in extracellular matrix of control ceils and the cells treated with rhIL-6 50 μg/L at day 12. Calcium concentration in cell layer of control group and treatment group (treated with rhIL-6 10 μg/L and 50 μg/L, respectively) was determined calorimetrically by the o-cresolphthalein complexone method at day 3, 6, 9 and 12, and corrected by total cell proteins. The mRNA expressions of bone-specific alkaline phosphatase (BAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP2) and osteoprotegerin (OPG) were estimated by real-time PCR in 12, 24 and 72 hours. OPN, BMP2 and OPG expressions were assessed by Western blotting and the BAP concentration at the same time was checked by fluorometry method . Electrophoretie mobility shift assays (EMSA) was used to detect the binding activity of transcription factor Cbfα1 with or without inhibitors of p38-MAPK (SB203580) and PKC (DHC) after 6 hours stimulation by rhIL-6 10 μg/L. Results rhIL-6 induced a positive Alizarin Red S stain and a time-dose-dependent increasing of cell layer calcium deposition.Compared with control group, rhIL-6 10 μg/L enhanced gene expression and protein levels of BAP and BMP2 at the early time (12 and 24 hours), and of OPN and OPG at later hours (24 and 72 hours). RhIL-6 still induced an increasing of binding activity of Cbfα1, which could be partially blocked by DHC but not SB203580. Conclusions rhIL-6 induces HUASMCs calcification and osteogenie transition in vitro, which may be one of the mechanism involved in IL-6 associated vascular calcification as observed in clinical studies. The role of IL-6 in HUASMCs may partially achieved through the PKC cell signal transduction way.

ObjectiveTo investigate the effects of pravastation intervention on tumor necrosis factor (TNF)-α-indueed ossifie calcification in human umbilical artery smooth muscle cells (hUASMCs). MethodshUASMCs were cultured by tissue explant in vitro, hUASMC were treated with TNF-α 50 μg/L and pravastatin of three different concentrations. The calcium deposition was determined by O-cresolphthalein eomplexone method. The mRNA expression of BAP and OPN was determined by real time-PCR. The protein expression of BAP, OPN and BMP-2 was determined by Western blotting. ResultsPravastatin inhibited the proliferation of hUASMC (r=-0.946, P＜0.01) and decreased the cell calcium deposition (r=-0.973, P＜0.01) in a dosedependent manner. Pravastatin down-regulated the expression of BAP, OPN and BMP-2 induced by TNF-α in a dose-dependent manner (mRNA, r=-0.972, P＜0.01;BAP protein, r=-0.820, P＜0.01;OPN protein, r=-0.972, P＜0.01;BMP-2 protein, r=-0.928, P＜0.01). ConclusionPravastatin can inhibit the proliferation of hUASMC, decrease the cell calcium deposition and inhibit the ossifie calcification of hUASMC induced by TNF-α.

Objective To study the effect of uremic serum on the calcification and osteogenic transition of cultured human umhilical artery smooth muscle cells(HUASMC).Methods Sera from 40 healthy controls(control group),40 nondialysis uremic patients(nondialysis group)and 45 uremic patients on dialysis(dialysis group)were detected fi)r biochemical indexes concerned and used to treat the cultured HUASMC.Alizarin red S stain was applied to examined calcium deposition in the cell layer.Calcium concentration was determined calorimetrically by the Ocresolphtha]ein complexone method,and corrected by total cell proteins.The mRNA expression of bone specific alkaline phosphatase(BAP),osteopontin(OPN)and bone morphogenelic protein 2(BMP2)was estimated by realtime PCR.OPN and BMP2 protein expression was assessed by Western blotting and fluorometry method was used to check the BAP concentration. Results Serum biochemical detection revealed thai both uremic groups had higher levels of phosphate,triglyseride,iPTH,C-reactive protein(CRP)and IL-6,and lower level of fetuin-A than healthy control(P＜0.05).Furthermore,dialysis serum had higher levels of triglyseride,CRP and IL-6 than nondialysis serum(P＜0.05).Compared with control group,both uremic scra induced more cell layer calcium deposition and higher mRNA and protein expression levels of BMP2,BAP and OPN(P＜0.05).Higher mRNA and protein expression levels of above factors were found in dialysis group as compared to nondialysis group(P＜0.05). Conclusions Uremic serum can induce HUASMC calcification and osteogenic transition in vitro,which may be one of the mechanisms involved in vascular calcification of ESRD patients.Microinflammatory state may promote the osteogenic transition and vascular calcification in dialysis patients.

Objective To investigate the incidence of colorectal disease in patients with chronic kidney disease (CKD) and analyze the risk factor of colorectal disease in patients with CKD.Methods The clinical data of 719 patients with CKD underwent colonoscopy examination and 404 patients without CKD underwent colonoscopy examination were collected.The incidence of colorectal disease was compared between patients of the two groups.According to the results of colonoscopy examination,the patients with CKD were divided into colonoscopy positive group and negative group,and clinical biochemical indexes of the two groups were analyzed.The rank-sum test or t-test was used to compare the measurement data.Rates were compared by Chi-square test.The risk factors of colorectal disease in patients with CKD were evaluated by logistic regression.Results The positive rate of colonoscopy examination in 719 patients with CKD was 21.28％ (153/719),which was higher than that of patients without CKD (12.62 ％,51/404; x2 =13.036,P＜0.01).The positive rate of colonoscopy in patients with CKD at stage 1 was 17.50％ (56/320),at stage 2 or 3 was 22.68％(66/291),at stage 4 or 5 was 28.70％ (31/108).There were significant differences among the three groups (x2-6.623,P＜0.05).The incidence of colorectal cancer in patients with CKD was 3.89 ％ (28/719),which was higher than that of patients without CKD (1.73％,7/404; x2 =4.003,P＜0.05).The incidence of colorectal polyps in CKD group was 8.34％(60/719),which was higher than that of non-CKD group (5.20％,21/404; x2 =3.827,P＜0.05).The incidence of inflammatory bowel disease in CKD group was 9.04％(65/719),which was higher than that of non-CKD group (5.69 ％,23/404; x2 =4.013,P＜0.05).The incidence of colorectal cancer and colorectal polyps in patients with CKD at stage Ⅰ was 2.50％(8/320) and 6.25％(20/320),at stage 2 or 3 was 3.78％(11/291) and 8.59％(25/291),at stage 4 or 5 was 8.33％(9/108) and 13.89％ (15/108).There were significant differences among the three groups (x2-7.359 and 6.199,both P＜ 0.05).The age of colonoscopy positive group was older than that of colonoscopy negative group (t=-3.821,P＜0.01); there were lower hemoglobin (t=3.541,P＜0.01),increased erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) (Z=-4.996 and-7.493,both P＜0.01),higher cholesterol and low density lipoprotein (t=-2.659 and-3.248,both P＜0.01),increased serum creatinine (Z=-3.683,P＜0.01) and declined glomerular filtration rate (Z=-6.227,P＜0.01) in colonoscopy positive group than in colonoscopy negative group; the differences were statistically significant.Logistic regression analysis indicated that age (β=0.981,95％ CI 0.965 to 0.998,P =0.032),serum creatinine (β=1.006,95％CI 1.002 to 1.009,P=0.001) and ESR (β=1.029,95％CI 1.018 to 1.040,P＜0.01) were risk factors of colorectal disease in patients with CKD.Conclusions The incidence of colorectal disease in patients with CKD is high,and it increases along with the declined glomerular filtration rate.The colorectal disease in patients with CKD patients may be associated with age,anemia,lipid metabolism,inflammation and impaired renal function.

Objective To evaluate the value of immunofecal occult blood test (IFOBT) as a prognostic indicator in CKD patients with colorectal impairment.Methods A total of 176CKD patients and 180 healthy adults as control were enrolled.Serum biochemistry was measured at baseline and gastrointestinal bleeding was determined by IFOBT.All the CKD patients were followed up for 4.5 years.Renal replacement therapy or death was defined as end-point event.The Logistic regression analysis was used for risk factors.Kaplan-Meier analysis and COX regression model were used for survival analysis.Results The positive rate of IFOBT in CKD patients was significantly higher than healthy control (17％ vs 5.3％,χ2=13.236,P＜0.01).When comparing with IFOBT negitive patients,IFOBT positive patients were older [(62.030±15.544) years old vs (48.660±19.018)years old,P＜0.01],had higher ESR [(71.800±31.657) mu/h vs (57.210±32.712) mm/h,P＜0.05],C-reactive protein [6.230 (3.000～14.148) mg/L vs 3.000 (3.000～6.833)mg/L,P＜0.05],serum creatinine [419.100 (103.200～546.625) μmol/L vs 175.100 (68.150～462.950) μmol/L,P＜0.05],and had lower hemoglobin level [(97.970±20.590) g/L vs (107.170±27.988) g/L,P＜0.05] and eGFR [11.400 (8.671～53.544) ml·min1·(1.73 m2)1 vs 35.274(10.961～82.145) ml·min-1·(1.73 m2)-1,P＜0.01].There was a negative correlation between IFOBT value and eGFR in CKD patients (r=-0.20,P＜0.01).Positive correlations of IFOBT value with age (r=0.175,P＜0.05) and serum creatinine (r=0.171,P＜0.05) were found.Logistic regression and COX regression analysis showed that IFOBT value,eGFR and ESR were important factors that influenced the prognosis of CKD patients.Kaplan-Meier analysis revealed that IFOBT value ＞100μg/L predicted progression of renal function.Conclusions The prevalence of gastrointestinal bleeding disorder is high in patients with CKD.Value of IFOBT independently predicts decline in renal function of CKD patients.