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Objective:To explore the isolation and culture methods of mouse parietal epithelial cells (PECs) of Bowman′s capsule, so as to provide a cell tool for further study.Methods:Mouse renal corpuscles were isolated by cell sieving combined with magnetic separation. After primary culture, identified parietal epithelial cells were induced to differentiate into podocytes. Immunofluorescence staining, real-time quantitative PCR and Western blotting were used to detect specific markers of parietal epithelial cells and podocytes.Results:Primary cultured PECs grew like paving stone and expressed Claudin-1 (PECs specific marker), CD133 (stem cell marker) and CD24 (stem cell marker), without the expression of tubular epithelial cell proteins, mesangial cell and podocyte specific proteins. Cultured to 6 generations in vitro, the PECs still expressed Claudin-1, CD133 and CD24. After incubated with differentiation medium, PECs were able to express podocyte markers WT-1 and Synaptopodin. Conclusion:The renal corpuscles are extracted by cell sieving combined with magnetic separation, and the mouse PECs successfully cultured in vitro can be induced to express podocytes′ markers.
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Objective@#To investigate the incidence and prognosis of cognitive impairment and to find out the risk factors associated with the outcome for better understanding and preventing cognitive impairment in maintenance hemodialysis (MHD) patients.@*Methods@#The patients who met the criteria as below: MHD patients (≥3 months) in Renji Hospital, Shanghai Jiao Tong University School of Medicine from January 2000 to July 2014, ≥18 years old were enrolled and could carry on the montreal cognitive assessment (MoCA) of voluntary cooperation. According to the score of MoCA, all enrolled patients were divided into two groups: cognitive impairment (MoCA<26) group and non-cognitive impairment (MoCA≥26) group. The follow-up period was 3 years. There were 130 males, and the incidence, demography data, medical history, hemodialysis data, laboratory examination and prognosis of cognitive impairment in hemodialysis patients were prospectively compared and analyzed. Logistic regression analysis was used to investigate the risk factors of cognitive impairment. Kaplan-Meier survival curve and Cox regression model were used for prognostic analysis.@*Results@#A total of 219 MHD patients were enrolled. The incidence of cognitive impairment in MHD patients was 51.6%. There were 130 males, and the ratio of male to female was 1.46∶1. Age was (60.07±12.44) years old and dialysis vintage was (100.79±70.23) months. Compared with non-cognitive impairment group (n=106), patients in cognitive impairment group (n=113) were older, and had higher proportion of education status<12 years, history of diabetes and anuria (all P<0.05); however, the post-dialysis systolic pressure, pre-dialysis diastolic pressure, post-dialysis diastolic pressure, platelet and spKt/V were lower (all P<0.05). Multivariate logistic regression analysis showed that education status<12 years (OR=3.428, 95%CI 1.919-6.125, P<0.001), post-dialysis diastolic pressure<73 mmHg (OR=2.234, 95%CI 1.253-3.984, P=0.006) and spKt/V<1.72(OR=1.982, 95%CI 1.102-3.564, P=0.022) were the independent risk factors for cognitive impairment in MHD patients. The Kaplan-Meier survival curve analysis showed that the survival rate of patients with cognitive impairment was lower than that of non-cognitive impairment group in MHD patients during 3 years follow-up (χ2=3.977, P=0.046). Multivariate Cox regression analysis showed that cognitive impairment was an independent risk factor for death in MHD patients (RR=2.661, 95%CI 0.967-7.321, P=0.058).@*Conclusions@#Cognitive impairment is one of the common complications and an independent risk factor for death in MHD patients. The mortality is high in patients who suffer cognitive impairment. Education status<12 years, post-dialysis diastolic pressure<73 mmHg and spKt/V<1.72 are the independent risk factors for cognitive impairment in MHD patients.
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Objective To investigate the incidence and prognosis of cognitive impairment and to find out the risk factors associated with the outcome for better understanding and preventing cognitive impairment in maintenance hemodialysis (MHD) patients. Methods The patients who met the criteria as below: MHD patients (≥3 months) in Renji Hospital, Shanghai Jiao Tong University School of Medicine from January 2000 to July 2014, ≥18 years old were enrolled and could carry on the montreal cognitive assessment (MoCA) of voluntary cooperation. According to the score of MoCA, all enrolled patients were divided into two groups: cognitive impairment (MoCA<26) group and non-cognitive impairment (MoCA≥26) group. The follow-up period was 3 years. There were 130 males, and the incidence, demography data, medical history, hemodialysis data, laboratory examination and prognosis of cognitive impairment in hemodialysis patients were prospectively compared and analyzed. Logistic regression analysis was used to investigate the risk factors of cognitive impairment. Kaplan-Meier survival curve and Cox regression model were used for prognostic analysis. Results A total of 219 MHD patients were enrolled. The incidence of cognitive impairment in MHD patients was 51.6％. There were 130 males, and the ratio of male to female was 1.46:1. Age was (60.07 ± 12.44) years old and dialysis vintage was (100.79 ± 70.23) months. Compared with non-cognitive impairment group (n=106), patients in cognitive impairment group (n=113) were older, and had higher proportion of education status<12 years, history of diabetes and anuria (all P<0.05); however, the post-dialysis systolic pressure, pre-dialysis diastolic pressure, post-dialysis diastolic pressure, platelet and spKt/V were lower (all P<0.05). Multivariate logistic regression analysis showed that education status<12 years (OR=3.428, 95％CI 1.919-6.125, P<0.001), post-dialysis diastolic pressure<73 mmHg (OR=2.234, 95％CI 1.253-3.984, P=0.006) and spKt/V<1.72(OR=1.982, 95％CI 1.102-3.564, P=0.022) were the independent risk factors for cognitive impairment in MHD patients. The Kaplan-Meier survival curve analysis showed that the survival rate of patients with cognitive impairment was lower than that of non-cognitive impairment group in MHD patients during 3 years follow-up (χ2=3.977, P=0.046). Multivariate Cox regression analysis showed that cognitive impairment was an independent risk factor for death in MHD patients (RR=2.661, 95％CI 0.967-7.321, P=0.058). Conclusions Cognitive impairment is one of the common complications and an independent risk factor for death in MHD patients. The mortality is high in patients who suffer cognitive impairment. Education status<12 years, post-dialysis diastolic pressure<73 mmHg and spKt/V<1.72 are the independent risk factors for cognitive impairment in MHD patients.
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Objective·To explore effects of pCPT-cAMP on proteinuria and dedifferentiation of podocytes in adriamycin (ADR)-induced nephropathy mice. Methods·Male BALB/c mice were divided into three groups. The control group did not make any intervention, and the other mice were administrated with ADR in a dose of 10 mg/kg by intravenous injection (ADR group). Some ADR-injected mice were treated with pCPT-cAMP [50 mg/(kg·d)] by intraperitoneal injection everyday (A+P group). Albumin urine was detected by Coomassie blue stain. Urine creatinine concentration was estimated by ELISA. The expression of WT-1 was detected by immunohistochemical staining. Immunofluorescence staining and Western blotting were used to evulate the dedifferentiation of podocytes. Results·Compared with the control group, the ratio of urinary albumin/creatinine in ADR nephropathy mice was significantly increased. WT-1 immunohistochemical staining showed that the number of podocytes was significantly decreased, while immunofluorescence double staining of podocyte-specific protein synaptopodin and podocalyxin remarkably reduced, and the expression of desmin was increased. pCPT-cAMP intervention decreased the ratio of albumin/creatinine in ADR mice, elevated the quantity of WT-1 positive cells and the expression of synaptopodin and podocalyxin, while desmin expression decreased. Conclusion·pCPT-cAMP activates the PKA signaling and protects ADR nephropathy mice by preventing the loss of podocytes and ameliorating the urine albumin/creatinine ratio, which may be mediated by pCPT-cAMP-prevented dedifferentiation of podocytes.
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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.