ABSTRACT
Objective To investigate the role of vitamin D receptor (VDR) in the protection of bufalin on podocyte injury induced by adriamycin (ADR).Methods (1) In vitro:the toxic effect of different concentrations of bufalin (10-9,10-8,10-7,104 mol/L) on podocyte was evaluated by lactate dehydrogenase (LDH) test;Annexin V-FITC and RT-PCR were utilized for podocyte apoptosis and VDR mRNA level respectively.Western blotting was used to analyze the protein expression of VDR and nephrin.SiRNA intervene was also applied to evaluate the role of VDR in bufalin's protective effect on podocyte injury induced by ADR.(2) In vitro:24 SD rats were randomly divided into three groups:control group,ADR group and ADR+bufalin group.TUNEL assay was applied to detect the apoptosis of podocytes in the kidney.Immunofluorescence and transmission electron microscope (TEM) were applied to analyze the expression of VDR and the ultrastructure of the glomerulus.Results Bufalin concentration lower than 10-7 mol/L had no toxicity on normal podocyte.Bufalin reduced the urinary protein excretion (P ＜ 0.05),alleviated the removal of podocyte foot processes and attenuated the changes in nephrin expression in the glomerulus of the adriamycin (ADR) rats (P ＜ 0.05).Bufalin notably inhibited the down-regulation of VDR in protein levels on the glomerulus of the ADR rats.Additionally,bufalin inhibited the down-regulation of VDR in both mRNA levels and protein levels (P ＜ 0.05),nephrin protein expression (P＜ 0.05),and apoptosis induced by ADR in cultured podocytes.Additionally,VDR specific siRNA intervene abolished the protective effect of bufalin in ADR-induced podocyte injury.Conclusion Bufalin can alleviate ADR-induced podocyte injury via enhancing VDR expression.
ABSTRACT
Objective Long noncoding RNA (lncRNA) has been identified to regulate DNA methylation, histone acetylation, and gene post-transcriptional regulation in kinds of diseases, including tumorigenesis, obesity and so on.Therefore, lncRNAs might be the potential targets of mesangial cells proliferation.Methods Mesangial cells were exposed to suitable concentration of TGF-β through cell proliferation assay;then the lncRNAs expression levels were detected by microarray in experimental group and control group separately;finally the differentially expressed lncRNAs were identified by RT-PCR;meanwhile, and the expression levels of target genes were also detected by RT-PCR.Results Cell viability assay confirmed that 10 ng/ml TGF-β could promote mesangial cell proliferation significantly.Totally, over 30 000 lncRNAs were detected in TGF-β treated MCs and control group cells separately.Compared to the control group, 5550 lncRNAs differentially expressed in TGF-β treated MCs, including 119 up-regulated and 147 down-regulated over 2 fold.RT-PCR results appeared that uc.60, MRAK079149, MRAK029456, XR_005507, XR_007641, uc.14, and uc.412 were significantly up-regulated in TGF-β treated MCs, and BC088254, DQ402472, BC098733, BC158832,BC098746 were stably down-regulated.Compared to the control group, the mRNA expression levels of AATF and NEK were increased in the TGF-β treated mesangial cells (P ＜ 0.05).AATF and NEK were downstream target genes of uc.412 and MRAK079149 respectively.Conclusion The differential expression of long noncoding RNAs presents in the experimental mesangial cells proliferation induced by TGF-β.
ABSTRACT
Objective To investigate the effect of insulin (INS) on nuclear factor-kappa B (NF-KB) activation in glomerular mesangial cells(GMC) of the Zucker rats,and the correlation between the activity of NF-kB induced by insulin to the ages and genotype of Zucker rats. Methods (1) Four groups of cultured GMCs(O3m,O10m,L3m and L10m) from the Zucker obese rats(3 months old and 10 months old) and Zucker lean rats(3 months old and 10 months old) were stimulated by insulin. (2) Electrophoretic mobility shift assay (EMSA) was used to detect the activity of NF-KB. Gel supershift assay was used to detect the subunit of NF-KB dimer. (3) The protein of NF-KB p65 in cytoplasm and cytoblast was analysed by Western Blot. Results (1) NF-KB activity in 4 groups GMCs was significantly higher than that in control group after induced of INS ( F=219. 65 P