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Kidney Week

Abstract: FR-PO1027

Role of Epigenetics in Parietal Epithelial Cell Transition

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Kumar, Vinod, Fienstine Institute for Medical Research, NEW YORK, New York, United States
  • Ayasolla, Kamesh R., Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Lan, Xiqian, Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Paliwal, Nitpriya, Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Chowdhary, Sheetal, Feinstein Institute of Medical Research, New Hyde Park, New York, United States
  • Marashi Shoshtari, Seyedeh Shadafarin, The Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Malhotra, Ashwani, Feinstein Institute Medical Research and NSLIJ, MANHASSET, New York, United States
  • Meyer-Schwesinger, Catherine, University of Hamburg, Hamburg, Germany
  • 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 a minor component of circulating lipid-rich multiprotein complexes in certain primate species including humans. It is expressed in liver, pancreas, kidney, brain, macrophages, and endothelial cells. In kidneys, APOL1 protein is expressed in podocytes, tubular, and vascular smooth muscle cells. Parietal epithelial cells (PECs) do not express APOL1 under a normal physiologic state. We hypothesize that epigenetic factors have a potential to induce APOL1 expression in PECs for their participation in PDs renewal in adverse milieus,

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, a 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 β-actin. cDNAs were amplified for DNMT1-4, HDAC1-4, and APOL1 with specific primers. 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; as expected, inhibition of miR193a in PECs also resulted in the induction of APOL1expression. The treatment of PECs with either azacytidine or SAHA induced the expression of APOL1 as well as decreased (3-fold) miR193a levels. HIV and IFN-γ enhanced the expression of DNMT3b, HDAC4, HK4me3, and H3K27me3, but down-regulated the expression of H3K8/9ac. ChIP assay revealed histone methylation at 27 lysine residues. RIP-ChIP assay confirmed binding of miR193a on APOL1 gene promoter. Bisulphite-sequencing displayed enhanced methylation of CpG islands at miR193a gene in PECHIVs.

Conclusion

Epigenetic factors play a role in the induction of PECs expression of APOL1 through modulation of miR193a expression in adverse milieus.

Funding

  • NIDDK Support