Abstract: TH-PO083

Epigenetics-Induced Down-Regulation of MicroRNA (miR)193a Determines APOL1 Expression in Parietal Epithelial Cells

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Kumar, Vinod, Fienstine Institute for Medical Research, New York, New York, United States
  • Lan, Xiqian, Feinstein Institute for Medical Research, Great Neck, New York, United States
  • Marashi Shoshtari, Seyedeh Shadafarin, The Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Chowdhary, Sheetal, Feinstein Institute of Medical Research, New Hyde Park, New York, United States
  • Bhooplapur, Manali, Feinstein Institute for medical research, Dix Hills, New York, United States
  • Meyer-Schwesinger, Catherine, University of Hamburg, Hamburg, Germany
  • Malhotra, Ashwani, Feinstein Inst.Med research and NSLIJ, Manhasset, New York, United States
  • Skorecki, Karl, Rambam Health Care Campus, Haifa, Israel
  • Singhal, Pravin C., North Shore LIJ Health System, Great Neck, New York, United States
Background

APOL1 is expressed in kidneys of some primate species, including humans. Its trypanolytic effect has been well studied. Podocytes express APOL1 and overexpression of APOL1G1 and G2 has been demonstrated to be cytotoxic to podocytes, both in vitro and in vivo studies. Parietal epithelial cells (PECs) do not express APOL1; however, HIV has the potential to induce APOL1 expression in PECs (submitted abstract ASN, 2017). We hypothesize an epigenetic mechanism for the induction of APOL1 in PECs.

Methods

Immortalized human PECs (at 33°C) were transduced with either vector (V) or HIV (NL4-3) (n=4); PECs were incubated in media containing variable concentration of IFN-γ (0, 5, 10, and 20 nM) for 48 hours (n=4); PECs were treated with either buffer, azacytidine (5 µM, a demethylating agent), or SAHA (10µM, an histone deacetylation inhibitor) for 48 Hours (n=4). Protein blots were probed for APOL1, DNMT 1-3, HDAC 1-4, H3K27me3, H3K4me3, H3K8/9ac, and reprobed for actin. cDNAs were amplified for DNMT1-4, HDAC1-4, and APOL1. RNAs were assayed for miR193a. To confirm histone acetylation at miR193a gene promoter, ChiP assay was carried out. To confirm binding of miR193a to APOL1 gene promoter, RIP-ChIP assay was performed. To measure methylation of CpG islands at miR193a gene, Bisulphite sequencing was carried out in PECVs and PECHIVs.

Results

Both HIV and IFN-γ induced APOL1 expression in PECs. PECHIV and IFN-γ-treated PECs showed 2 to 2.5-fold decrease in miR193a expressions, respectively; on the other hand, miR193a knockout PECs displayed induction of APOL1expression in PECs. Both azacytidine and SAHA not only induced APOL1 expression but also decreased (3-fold) miR193a levels in PECs. Both HIV and IFN-γ-treated PECs displayed enhanced DNMT3b, HDAC4, HK4me3, and H3K27me3 but down-regulation of H3K8/9ac expressions. RIP-ChIP assay confirmed binding of miR193a to the APOL1 gene promoter. Bisulphite sequencing displayed enhanced methylation of CpG islands at miR193a gene in PECHIV. These findings suggest that HIV down regulates miR93a through methylation at CpG islands and at histone 3 lysine 27 residues.

Conclusion

HIV induces APOL1 expression in PECs through down-regulation of miR193a via epigenetic mechanisms.

Funding

  • NIDDK Support