miR-192 Mediates TGF- /Smad3-Driven Renal Fibrosis

TGF/Smad3 promotes renal fibrosis, but the mechanisms that regulate profibrotic genes remain unclear. We hypothesized that miR-192, a microRNA expressed in the kidney may mediate renal fibrosis in a Smad3-dependent manner. Microarray and real-time PCR demonstrated a tight association between upregulation of miR-192 in the fibrotic kidney and activation of TGF/Smad signaling. Deletion of Smad7 promoted miR-192 expression and enhanced Smad signaling and fibrosis in obstructive kidney disease. In contrast, overexpression of Smad7 to block TGF/Smad signaling inhibited miR-192 expression and renal fibrosis in the rat 5/6 nephrectomy model; in vitro, overexpression of Smad7 in tubular epithelial cells abolished TGF1–induced miR-192 expression. Furthermore, Smad3 but not Smad2 mediated TGF1–induced miR-192 expression by binding to the miR-192 promoter. Last, overexpression of a miR-192 mimic promoted and addition of a miR-192 inhibitor blocked TGF1–induced collagen matrix expression. Taken together, miR-192 may be a critical downstream mediator of TGF/Smad3 signaling in the development of renal fibrosis. J Am Soc Nephrol 21: 1317–1325, 2010. doi: 10.1681/ASN.2010020134 TGFis a profibrogenic cytokine that mediates renal fibrosis positively by activating its downstream mediators called Smad2 and Smad3 but negatively by its inhibitory factor Smad71– 4; however, the mechanisms as to how the Smads regulate the fibrogenic genes during renal fibrosis remain unclear. MicroRNAs (miRNAs) are small, noncoding RNAs with approximately 22 nucleotides. The mature miRNAs can complementarily bind to the mRNA 3 untranslated region to regulate the gene expression by translational repression or induction of mRNA degradation.5 Increasing evidence shows that TGFmay act by regulating miRNAs to exhibit its biological effects such as epithelial-to-mesenchymal transition (EMT),5 suggesting that TGFregulates the expression of these miRNAs to promote EMT. Several miRNAs, including miR-192, -194, -204, -215, and -216, are highly expressed in the kidney, as compared with other organs.6,7 In the context of renal fibrosis, expression levels of miR-192 increased significantly in glomeruli isolated from diabetic mice.8 In vitro, miR-192 is induced by TGF1 and mediates TGF–induced collagen expression in mesangial cells (MCs) by downregulating ZEB2 expression.8 In contrast, a recent study also found that TGF1 suppresses miR-192 expression in human tubular epithelial cells (TEC) and loss of miR-192 promotes fibrogenesis in diabetic nephropathy.9 The discrepancy in these two studies with opposite findings and understanding of miR-192 in diabetic nephropathy necessitates further investigation of the potential role of miR192 and the mechanisms that regulate miR-192 expression during renal fibrosis under various disease conditions. Thus, this study tested the hypothesis that TGF1 may act by stimulating Smad3 to regulate miR-192 expression during renal fibrosis. This was Received February 2, 2010. Accepted March 24, 2010. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Hui Y. Lan, Department of Medicine and Therapeutics and Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong, China. Phone: 852-3763-6077; Fax: 852-2145-7190; E-mail: [email protected] Copyright © 2010 by the American Society of Nephrology BASIC RESEARCH www.jasn.org J Am Soc Nephrol 21: 1317–1325, 2010 ISSN : 1046-6673/2108-1317 1317 tested in rodent models of obstructive and remnant kidney diseases induced in mice that lacked Smad3 or Smad7, had conditional knockout (KO) for Smad2, or overexpressed renal Smad7. In addition, TGF–induced miR-192 expression via the Smad3dependent mechanism was determined in TECs overexpressing Smad7 or knocking down for Smad2 or Smad3 and in Smad2 or Smad3 KO mouse embryonic fibroblasts (MEFs).