Apolipoprotein L1 Dynamics in Human Parietal Epithelial Cell Molecular Phenotype Kinetics

Abstract

. CC-BY-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/259267 doi: bioRxiv preprint first posted online Feb. 4, 2018; 3 APOL1 (G0) is a minor component of circulating lipid-rich trypanolytic multiprotein complexes in certain primate species including humans (1). It is expressed in liver, pancreas, kidney, brain, macrophages, and endothelial cells (1). Approximately 34% of African Americans carry one of the two risk variants (G1 and G2) and 13% have both variants (2, 3). Increased expression of G1 and G2 risk variant APOL1, has been shown to induce cellular injury, including podocytes (PDs), both in vitro and in vivo (4-13).Parietal epithelial cells (PECs) and PDs are derived from the same mesenchymal cells during embryogenesis (14-16); however, the expression level of miR193a determines the net phenotype-PECs vs. PDs-in these cells (17). We have recently demonstrated that APOL1-miR193a axis preserves podocyte molecular phenotype in adverse milieus (18). We now hypothesize that APOL1 and miR193a forms a reciprocally linked feedback loop to regulate PECs' phenotype in humans. In this loop, lack of APOL1 assures PECs' phenotype while its presence initiates their transition to PDs. APOL1 and miR193a are reciprocally linked with a feedback loop in PECsPECs have not been shown to display expression of APOL1 both in vitro and in vivo studies (1,19). To examine the expression of APOL1, lysates of cultured PECs, differentiated PDs and HepG2 (positive control) and HEKs (negative control) were probed for APOL1 and GAPDH. HepG2s and PDs displayed robust expression of APOL1, but PECs and HEKs did not display any expression of APOL1 protein (Fig. 1A).. CC-BY-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/259267 doi: bioRxiv preprint first posted online Feb. 4, 2018; 4 Since APOL1 is expressed by PDs (1, 4, 19), we asked whether PECs transiting to PDs' phenotype would also display APOL1. PECs were incubated in media for different time intervals followed by RNA and protein extractions. Protein blots were probed for APOL1 and GAPDH. PECs did not show expression of APOL1 protein on day 0, however, APOL1 expression emerged on day 4 and increased further during transition (Fig.1B). RNAs were assayed for miR193a and cDNAs were amplified with an APOL1 specific primer. APOL1 mRNA expression increased (Fig. 1C) but miR193a levels decreased (Fig. 1D) during transition.To evaluate a possible causal relationship, PECs were transfected with either scrambled or APOL1 siRNAs. cDNAs were amplified for APOL1 and RNAs were quantified for miR193. Down regulation of APOL1 mRNA (Lower panel, Fig. 1E) was associated with an up regulation of miR193a (Upper panel, Fig. 1E). These results suggest that PECs have a functional APOL1 protein which is not detectable by available tools; nonethele...