Abstract: SA-PO580
Epigenetic Factors Regulate APOL1-miR193a Axis in Parietal Epithelial Cells
Session Information
- Glomerular Diseases: Fibrosis, Extracellular Matrix
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix
Authors
- Kumar, Vinod, Feinstein Institutes for Medical Research, Manhasset, New York, United States
- Vashistha, Himanshu, Ochsner Health System, New Orleans, Louisiana, United States
- Ayasolla, Kamesh R., Feinstein Institutes for Medical Research, Manhasset, New York, United States
- Adnani, Harsha, Feinstein Institutes for Medical Research, Manhasset, New York, United States
- Malhotra, Ashwani, Feinstein Institutes for Medical Research and NSLIJ, Manhasset, New York, United States
- Singhal, Pravin C., Feinstein Institute of Research, Manhasset, New York, United States
Background
Bifunctional APOL1-mIR193 axis plays a vital role in the podocyte renewal during normal physiology and pathological states. Parietal epithelial cells (PECs) serve as progenitor cells for podocytes in juvenile. We have recently demonstrated the role of APOL1 wild-type (G0) in the transition of PECs. Since PECs have a higher expression of miR193a, they do not express APOL1. However, downregulation of miR193a induces APOL1 expression in PECs. We hypothesize that epigenetic factors regulate APOL1-miR193 axis through modulation of miR193a expression in PECs.
Methods
Immortalized human PECs underwent several experimental designs: PECs were transduced with either vector (V) or HIV (NL4 (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 empty vector (EV) of a specific miR193 inhibitor (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. ChiP assay was carried out to evaluate histone acetylation at miR193a promoter. The RIP-ChIP assay was performed to examine the binding of miR193a at APOL1 gene promoter. In PECVs and PECHIVs, methylation of CpG islands at miR193a gene was detected by Bisulphite sequencing.
Results
HIV, as well as IFN-γ, induced APOL1 expression in PECs. PECHIV and IFN-γ-treated PECs showed 2.0-2.5-fold decrease in miR193a expressions, respectively; inhibition of miR193a in PECs by a specific miR193a inhibitor also resulted in the induction of APOL1 expression. The treatment of PECs with either azacytidine or SAHA induced the expression of APOL1 as well as decreased (three-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 the binding of miR193a on APOL1 gene promoter. Bisulfite sequencing displayed enhanced methylation of CpG islands at miR193a gene in PECHIV.
Conclusion
Epigenetic factors regulate APOL1-miR193a axis in PECs.
Funding
- NIDDK Support