Abstract
Patients carrying APOL1 risk alleles (G1 and G2) have a higher risk of developing Focal Segmental Glomerulosclerosis (FSGS); we hypothesized that escalated levels of miR193a contribute to kidney injury by activating renin-angiotensin system (RAS) in the APOL1 milieus. Differentiated podocytes (DPDs) stably expressing vector (V/DPD), G0 (G0/DPDs), G1 (G1/DPDs), and G2 (G2/DPDs) were evaluated for renin, Vitamin D receptor (VDR), and podocyte molecular markers (PDMMs, including WT1, Podocalyxin, Nephrin, and Cluster of Differentiation [CD]2 associated protein [AP]). G0/DPDs displayed attenuated renin but an enhanced expression of VDR and Wilms Tumor [WT]1, including other PDMMs; in contrast, G1/DPDs and G2/DPDs exhibited enhanced expression of renin but decreased expression of VDR and WT1, as well as other PDMMs (at both the protein and mRNA levels). G1/DPDs and G2/DPDs also showed increased mRNA expression for Angiotensinogen and Angiotensin II Type 1 (AT1R) and 2 (AT2R) receptors. Protein concentrations of Brain Acid-Soluble Protein [BASP]1, Enhancer of Zeste Homolog [EZH]2, Histone Deacetylase [HDAC]1, and Histone 3 Lysine27 trimethylated [H3K27me3] in WT1-IP (immunoprecipitated proteins with WT1 antibody) fractions were significantly higher in G0/DPDs vs. G1/DPD and G2/DPDs. Moreover, DPD-silenced BASP1 displayed an increased expression of renin. Notably, VDR agonist-treated DPDs showed escalated levels of VDR and a higher expression of PDMMs, but an attenuated expression of renin. Human Embryonic Kidney (HEK) cells transfected with increasing APOL1(G0) plasmid concentrations showed a corresponding reduction in renin mRNA expression. Bioinformatics studies predicted the miR193a target sites in the VDR 3'UTR (untranslated region), and the luciferase assay confirmed the predicted sites. As expected, podocytes transfected with miR193a plasmid displayed a reduced VDR and an enhanced expression of renin. Renal cortical section immunolabeling in miR193a transgenic (Tr) mice showed renin-expressing podocytes. Kidney tissue extracts from miR193aTr mice also showed reduced expression of VDR and PDMMs, but enhanced expression of Renin. Blood Ang II levels were higher in miR193aTr, APOLG1, and APOL1G1/G2 mice when compared to control mice. Based on these findings, miR193a regulates the activation of RAS and podocyte molecular markers through modulation of VDR and WT1 in the APOL1 milieu.