Apolipoprotein L1 (APOL1)-miR193a axis has been reported to play a role in the maintenance of podocyte homeostasis. In the present study, we analyzed transcription factors relevant to miR193a in human podocytes and their effects on podocytes’ molecular phenotype. The motif scan of the miR193a gene provided information about transcription factors, including YY1, WT1, Sox2, and VDR-RXR heterodimer, which could potentially bind to the miR193a promoter region to regulate miR193a expression. All structure models of these transcription factors and the tertiary structures of the miR193a promoter region were generated and refined using computational tools. The DNA-protein complexes of the miR193a promoter region and transcription factors were created using a docking approach. To determine the modulatory role of miR193a on APOL1 mRNA, the structural components of APOL1 3’ UTR and miR193a-5p were studied. Molecular Dynamic (MD) simulations validated interactions between miR193a and YY1/WT1/Sox2/VDR/APOL1 3′ UTR region. Undifferentiated podocytes (UPDs) displayed enhanced miR193a, YY1, and Sox2 but attenuated WT1, VDR, and APOL1 expressions, whereas differentiated podocytes (DPDs) exhibited attenuated miR193a, YY1, and Sox2 but increased WT1, VDR, APOL1 expressions. Inhibition of miR193a in UPDs enhanced the expression of APOL1 as well as of podocyte molecular markers; on the other hand, DPD-transfected with miR193a plasmid showed downing of APOL1 as well as podocyte molecular markers suggesting a causal relationship between miR193a and podocyte molecular markers. Silencing of YY1 and Sox2 in UPDs decreased the expression of miR193a but increased the expression of VDR, and CD2AP (a marker of DPDs); in contrast, silencing of WT1 and VDR in DPDs enhanced the expression of miR193a, YY1, and Sox2. Since miR193a-downing by Vitamin D receptor (VDR) agonist not only enhanced the mRNA expression of APOL1 but also of podocyte differentiating markers, suggest that down-regulation of miR193a could be used to enhance the expression of podocyte differentiating markers as a therapeutic strategy.

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.