SAT-372 Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

Abstract

Optimal expression of the Actin Cytoskeleton (AC) proteins is considered to be an integral part of podocyte health. Nephrin is one of the most important constituents of the ACs and is transcribed by Wilms tumor type (WT) 1 transcription factor. Since miR193a inversely regulates the expression of WT1, it also inversely regulates the transcription of nephrin in podocytes. A decrease in nephrin expression disintegrated the ACs and resulted in nuclear import of dendrin, followed by the activation of a pro-apoptotic pathway. In the present study, we have examined the role of APOL1– miR193a axis in the maintenance of stability of the ACs and the integrity of actin cytoskeleton in human differentiated podocytes. Also, we have evaluated the effects of disruption of APOL1-mIR193a axis on the stability of the ACs and the organization of actin filaments in podocytes expressing APOL1 risk alleles. We used interventions, which resolved the defective signaling in adverse milieus, and podocytes expressing APOL1 risk alleles. Human Podocytes were conditionally immortalized and stable cell lines with overexpression of APOL1 G0, G1 and G2 were generated (DPDG0s/G1s/G2). Invivo studies were conducted on transgenic mice expressing APOL1 non risk and risk variants. Transient podocyte cell were exposed to Puromycin Aminonucleoside (PAN) and analyzed for for miR193a and AC complex (CD2AP, Nephrin, Dynamic and Dendrin) at protein and mRNA expression. The change in the expression of ACs complex molecules was also analyzed with APOL1 transgenic mice and the finding were further validated by using invitro stable podocyte cell lines. Protein protein interaction studies done by using immunoprecipitation and Insilco PPI methods. DPDG1s/G2s exhibited an increase in miR193a and down-regulation of the expression of adherens complex’s constituents (CD2AP, nephrin, and dendrin). DPDG0s showed decreased Cathepsin L, enhanced dynamin expressions, and the intact actin cytoskeleton. On the contrary, DPDG1s/G2s displayed an increase in Cathepsin L, but down-regulation of dynamin expressions and disorganization of the actin cytoskeleton. APOL1 silencing enhanced miR193a and Cathepsin L, but down-regulated dynamin expressions. DPDG1s/G2s displayed nuclear import of dendrin, indicating an occurrence of destabilization of adherens complexes in APOL1 risk milieu. These findings suggest that DPDG1s and DPDG2s developed disorganized actin cytoskeleton as a consequence of disrupted APOL1-miR193a axis. Interestingly, docking and co-labeling studies suggested an interaction between APOL1 and CD2AP. APOL1G1/G1 and APOL1G1/G2 transgenic mice displayed nuclear import of dendrin indicating destabilization of adherens complexes in podocytes; moreover, these mice showed a four-fold increase in urinary albumin to creatinine ratio and development of focal segmental glomerular lesions. We conclude that an intact APOL1-miR193a axis plays an important role in the maintenance of the integrity of actin cytoskeleton, whereas, its disruption in podocytes expressing APOL1 risk alleles make them susceptible to disorganization of actin flaments.