MiRNA-30 Family And Podocyte Injury

The visceral glomerular epithelial cell,also known as the podocyte,is a terminally differentiated,highly specialized cell.The function of the glomerular filtration barrier depends on the integrity of the podocyte.Podocyte loss and injury have been observed in human and experimental models of glomerular diseases,Recently,extensive studies have been performed to understand the relationship of miRNA and podocyte injury.In the animal model of Dicer selective deletion in podocytes that leads to proteinruia and glomerulosclerosis,miR-30 predicted targets were enriched in the upregulated genes in the glomeruli of the mice,suggesting miR-30 may play a role in the homeostasis of podocytes,and its loss by Dicer deficiency might contribute to the phenotypes observed.Based on these previous studies,we performed further studies to investigate the function of miR-30s in podocytesPart One:MicroRNA-30 and Podocyte InjuryObjective:miRNAs are essential for glomerular podocyte homeostasis as shown by selective Dicer deletion in mouse podocytes,and miR-30 has been suggested to be a player as evidenced by its anti-apoptotic effect in podocytes.Here We hypothesized that loss of miR-30 as a novel and specific mechanism induced podocyte apoptosis in glomerulosclerosis and may participate in the Pathogenesis of FSGS.Methods:Glomeruli were microdissected from renal biopsies,and subjected to RNA preparation and qRT-PCR of miR-30.Conditionally-immortalized human podocyte cell line was used for in vitro studies.Podocyte injury was evaluated by apoptosis,cytoskeletal alteration and injury markers.The expressions of miR-30 targets were examined by western blotting.Results:miR-30 members were abundantly expressed in podocytes of glomeruli,but all downregulated in patients of FSGS.In vitro,human podocytes were treated with TGF-?,LPS and PAN,and miR-30 members were found all downregulated.When exogenous miR-30 was expressed to sustain miR-30 level of the podocytes in the treatment,both apoptosis and cytoskeletal damage were signifiantly ameliorated.We further show that miR-30 exerted its protective role,at least partly,through inhibition of its targets,Notchl and p53.Lastly,we show glucocorticosteroid prevented TGF-? and LPS-induced miR-30 downregulation in podocytes,accompanied with prevention of upregulation of proapoptotic p53,Bax and Notchl,and restoration of anti-apoptotic Bcl2 and CD2AP expression.Conclusions:miR-30 protects podocytes,at least partly,through inhibiting its targets,Notchl and p53,two known culprits in podocyte injury.Downregulation of miR-30 causes podocytes vunerable to injury.miR-30 expression appears to have a signfianl clinical relevance as shown by its downregulation in podocytes of patients,its correlation with proteinuria,its restoration by glucocorticosteroid in vitro.Thus,miR-30 family is a promising therapeutic target for glomerular disease.Part Two:Regulation of Calcium-Calcineurin Signaling by miR-30s in PodocytesObjective:Calcium-calcineurin signaling activation induces podocyte injury,but the mechanism underlying its regulation is largely unknown.Many components of the signaling are predicted to be miR-30 targets and they are upregulated in injured podocytes in which miR-30s are downregulated,we therefore hypothesize that miR-30s control calcium-calcineurin signaling by inhibiting the expression of its multiple components and miR-30 downregulation leads to the activation of the signaling.Methods:Studies were performed with human podocyte cell line,podocyte-selective miR-30s sponge transgenic mice,puromycin aminonucleoside(PAN)-treated rats,miR-30a gene transferring in vivo,and FSGS patients' renal biopsies.Results:We found that calcium-calcineurin signaling components,TRPC6,PPP3ca,PPP3cb,PPP3r1,and NFATc3,have abundant mRNA but not protein in normal podocytes,and that their protein but not mRNA levels are greatly elevated in the podocytes of FSGS patients.We confirmed by luciferase reporter assays that these genes are miR-30 targets and showed that.miR-30 overexpression reduced their expressions,as well as calcium influx,calcineurin activity and NFAT3c nuclear translocation in PAN-treated podocytes.In vivo,podocyte-selective miR-30s sponge transgenic mice displayed increased expressions of the five genes in the podocytes and the mice developed significantly higher levels of proteinuria than control mice when treated with PAN.The expressions of the five genes were also upregulated in the podocytes of PAN-treated rats,in which miR-30s were downregulated.As expected,the upregulation of these genes in the podocytes of PAN-treated rats was prevented by miR-30a gene transferring to the podocytes,accompanied by alleviation of proteinuria and podocyte injury.Conclusions:miR-30s tightly control calcium-calcineurin signaling by inhibiting the expression of multiple components of the signaling,and miR-30 downregulation results in their upregulation and thus the signaling activation.