Inducible Deletion of MicroRNA Activity in Kidney Mesenchymal Cells Exacerbates Kidney Fibrosis

Background: Renal interstitial mesenchymal cell-specific deletion of Dicer results in hypoplastic kidneys with abnormal differentiation of nephron tubules and vasculature (Kidney International, 2015); however, the role of Dicer in these cells in adult kidney diseases remains unexplored. We developed a new mouse model and investigated the role of Dicer and Dicer-dependent miRNA activity in renal interstitial mesenchymal cells. Methods: We generated Dicer conditional knockout (cKO) mice by crossing hemizygous Pdgfr-β-CreERT2 mice with homozygous Dicer floxed (DicerFL/FL) mice. DicerFL/FL mice without CreERT2 were used as controls. After administering oral tamoxifen, these mice underwent unilateral ureter obstruction (UUO) or intraperitoneal injection of folic acid (FA, 250 mg/kg). Fibrosis in the renal interstitium was assessed using immunofluorescent staining and real-time PCR (RT-PCR). RNA sequencing was performed using total RNA from Sham- or UUO-kidneys. Primary renal fibroblasts were transfected with miR-9-5p inhibitors (20 nM) and treated with 5 ng/mL TGF-β1 for 48 hours. RT-PCR was performed to evaluate Pdgfrb and Acta2 expression in vitro assay. Results: Compared with control mice, cKO mice significantly increased expression of PDGFR-β in injured kidneys by immunofluorescence staining and RT-PCR. The percentage of Sirius red staining in injured kidneys was also significantly elevated in cKO mice compared with that in control mice. Microarray analysis indicated decreased expression of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice. The major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, actin cytoskeleton, and fibroblast proliferation. In vitro assays demonstrated significant upregulation of Pdgfrb and Acta2 in primary renal fibroblasts transfected with miR-9-5p inhibitor following TGF-β1 treatment. Conclusion: This study revealed that renal interstitial mesenchymal cell-specific deletion of Dicer increased activation of PDGFR-β and exacerbated renal fibrosis by decreasing levels of miR-9-5p, miR-344g-3p, and miR-7074-3p. Activation of PDGFR-β was sufficient to drive renal fibrosis, and these miRNAs are potential therapeutic targets for renal fibrosis. Funding: Government Support – Non-U.S.