MiR-30d Improves Glucocorticoid Sensitivity Through Regulating Glucocorticoid Receptor α In Murine Podocyte

Many of the major clinical manifestations of the disease,such as membranous nephropathy(MN),focal stage glomerular sclerosis(FSGS)and so on,thoes patients of primary podocytopathy,all have a various degrees of the structural and functional abnormalities of glomerular epithelial cells which also called podocyte.Glucocorticoid(GC)is still the first-line drugs for treatment of primary podocytopathy,but the therapeutic efficacy of hormone is uncertain,part of patients with steroid resistant effect the clinical remission.Factors affecting hormone sensitivity is not completely determined.The expression of GRα gene in peripheral blood mononuclear cells(PBMC)of patients with primary podocytopathy was analyzed in this study,at the same time,the model of injury and repair of podocyte was constructed in basic research,and with the application of mi RNA interference technology to explore whether the mi R-30 d have a targeted regulatory effect on GRα in the podocyte and affect the sensitivity of the hormone,revealing the essence of podocytopathy from a different point of view and exploringa new therapeutic method and interventing targets.Part Ⅰ Analysis of the expression of GRα in mononuclear cells of peripheral blood in patients with podocytopathyObjective: Analysis of the expression of GRα in mononuclear cells of peripheral blood in patients with podocytopathy.Methods:Retrospective randomly selected 9 cases patients of podocytopathy diagnosed by renal biopsyin from 2013 March to February 2016 in Department of Nephrology,the First Affiliated Hospital of Dalian Medical University,Including minute lesions(3 cases),focal segmental glomerular sclerosis(3 cases),membranous nephropathy(3cases).The baseline data were recorded in groups of patients,and also record blood pressure 、 medication 、 24 hour urinary protein 、 serum albumin 、 creatinine 、cholesteroland other clinical indicators before hormonal therapy,take the morning fasting peripheral blood of patients ahead of one week before hormonal therapy and pick up the peripheral blood mononuclear cells.q RT-PCR were used to detect GRαgene expression in PBMC of patients.At the same time,choose 3 cases healthy adult that age and weight matches with patients’ as normal control group.Result: Randomly collected data of 9 patients in accordance with the inclusion criteria.With 6 cases of male patients(60%)and 4(40%)cases of female,male to female ratio was 1.5:1,there was no significant difference between different age(P > 0.05);The blood pressure are the number of 126/76 mm Hg,weight is 71.5Kg;onset time is 4.25 months.q RT-PCR results showed that,compared with the Control group,GRα gene expression in PBMC of minute lesions(3 cases),focal segmental glomerular sclerosis(3 cases),membranous nephropathy(3 cases)were all decreased(P<0.05).Conclusions:Decreased expression of GRα in PBMC of patients with podocytopathy may be related to hormone resistance.Part Ⅱ mi R-30 d improves glucocorticoid sensitivity through regulating glucocorticoid receptor α in murine podocyteObjective: To investigate the molecular mechanism of mi RNA-30 d improves glucocorticoid sensitivity through regulation of glucocorticoid receptor α in podocyte.Methods:The conditionally immortalized mouse podocyte cell line was cultured,under the differentiation and maturational condition,with the podocyte injury and repair model-induced by puromycin(Pan)(4.5 ug / ml,48h),after transfecting the podocyte with mi R-30 d mimics 、mimics negative control and inhibitor 、inhibitor negative control,dexamethasone(DEX)were used to stimulate the podocyte with 0h,6h,12 h,24h.Then,QRT-PCR and Western blot were used to detect the GR alpha gene and protein expression levels.Semi quantitative analysis of protein were detected by immunofluorescence microscopy,with the laser scanning confocal microscope detect ing the nuclear translocation of GR alpha.dual luciferase reporter assay system test the effect of mi R-30 d on GRα m RNA full-length luciferase reporter gene fusion expression.testing whether the mi R-30 d has directly targeted regulation.on the GRα with Further research.Results: Under the normal differentiation and injury-repair conditions in podocyte the expression of GR was down regulated to the same.Transfection of mi R-30 d Mimics and Inhibitor to podocytes induced by PAN,the differences of expression of GR gene and protein level were not statistically significant;Transfecting of mi R-30 d Mimics and inhibitor to podocytes induced by PAN and DEX pretreatment,the difference of expression level of GR gene and protein alpha showed no statistical significance.Dual luciferase reporter assay results also showed that,the mi R-30 d had no obvious effect on expression of dual luciferase reporter gene with the fusion predicted target gene of GR alpha m RNA full-length sequences.Conclusions:Glucocorticoid can reduce podocyte apoptosis induced by PAN,but mi R-30 d does not exist on GRα targeting mechanism directly.