miR-200 family promotes podocyte differentiation through repression of RSAD2

The mature podocytes, also known as glomerular epithelial cells, are highly differentiated cells that reside on the glomerular basement membrane (GBM). During glomerulogenesis, podocytes develop from precursor cells, which arise from induced mesenchymal renal stem cells, into their adult phenotype, which is characterized by a complex pattern of processes1. The function of podocytes is mainly based on their special structure and includes regulation of the glomerular filter. Recently, several studies have indicated that the ectopic development of podocytes may lead to abnormal glomerulogenesis and subsequent kidney diseases. Kidneys of Lmx1b mutant mice exhibit pathological changes, which places podocytes at the center of the pathomechanism leading to proteinuria, hematuria, and chronic renal disease2,3. Concordantly, The recent discovery of several novel podocyte proteins and their mutation analysis, including the Nephrin homologue Neph14, Nephrin5, CD2-associated protein(CD2AP)6, Podocin7, and transient receptor potential cation channel 6 (TRPC6)8,9, have indicated the critical role of the structural integrity of podocytes in maintaining the normal function of the glomerular filtration barrier. MicroRNAs (miRNAs) are single-stranded, noncoding RNA molecules that are thought to modulate gene expression by translational inhibition and destabilization of messenger RNAs (mRNAs)10,11. Since the first miRNA, the lin-4, was found in the nematode worm Caenorhabditis elegans12, miRNAs are present in multiple species, including plants, animals and even an algae13, implying that the regulation of genes by RNA silencing is an ancient mechanism. In fact, some new emerging studies have reported that podocyte-specific deletion of Dicer or Drosha result in proteinuria and glomerulosclerosis, which suggests the important role of miRNAs in podocytes for renal function. The miR-200 family of miRNAs comprises five members organized as two clusters, miRs-200b/a/429 and miRs-200c/141, on chromosomes 1 and 12 in humans and 4 and 6 in mice. Although abundant studies of the role of miR-200 family in the oncology field have been accumulated, the contribution of the family in nephrogenesis, especially in specific cell types during differentiation, is still largely unknown. Raman Agrawal et al., showed that miR-200 family was one of the most abundant miRNAs in kidney14. miR-200 was expressed in the developing pronephros, the skin and part of the somites15. However, the accurate role of miR-200 family in podocyte differentiation is not clear. Therefore, a study in our laboratory has identified the miRNAs and mRNA expression profiles in undifferentiated and differentiated podocytes using miRNA and mRNA microarray16. These works lay the groundwork for our further molecular mechanism study in podocyte differentiation. RSAD2, also known as Viperin or Cig5, plays a key role in the innate immune response system. RSAD2 is rapidly induced by both type I and II interferons and has a wide variety of antiviral activity17. RSAD2 subsequently localizes to the endoplasmic reticulum (ER) via its N-terminal amphipathic α-helix18, the process of which appears to result in the disruption of lipid raft microdomains and the prevention of influenza virus from budding of the plasma membrane19. Although increasing amounts of researchers have reported on the antiviral effect of RSAD2 in various organs and cells, very few studies have been conducted on the expression or function of system during cell development. One recent study from Steinbusch et al. has shown that RSAD2 knockdown induces ER stress and skews the chondrocyte phenotype towards hypertrophy in developing chondrocytes in vivo20. However, whether and how the RSAD2 is regulated by miRNA, especially by miR200 family during podocyte differentiation is still unclear. Herein, we hypothesize that miR-200 family promotes podocyte differentiation through repression of RSAD2 expression. Our results indicate that the expression of miR-200 family is significantly up-regulated in differentiated podocytes comparing with the undifferentiated one, and the inhibition of miR-200 family can arrest cell differentiation. Furthermore, bioinformatic analysis and Luciferase reporter assay revealed that RASD2 was directly negatively regulated by miR-200 family. Together, we show that miR-200 family and RASD2 cooperate to assure a normal differentiation of podocytes.