Abstract: TH-PO1098
Functional APOL1-miR193a Axis (AMA) Prevents Podocyte Injury
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Kumar, Vinod, Feinstein Institute for Medical Research, Manhasset, New York, United States
- Vashistha, Himanshu, Ochsner Health System, New Orleans, Louisiana, United States
- Lan, Xiqian, Feinstein Institute for Medical Research, Manhasset, New York, United States
- Adnani, Harsha, Feinstein Institute of Research, Manhasset, New York, United States
- Qayyum, Maleeha, Northwell health , Jamaica, New York, United States
- Chinnapaka, Sushma, Northwell health , Jamaica, New York, United States
- Ayasolla, Kamesh R., Feinstein Institute for Medical Research, Manhasset, New York, United States
- Malhotra, Ashwani, Feinstein Institute Medical 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-miR193a axis plays a vital role in the maintenance of podocyte molecular phenotype. It is bifunctional in podocytes expressing APOL1G0 (wild-type). APOL1 inversely regulates miR193a expression and vice versa in podocytes. Both Puromycin aminonucleoside (PAN) or Adriamycin (ADR) are known to induce apoptosis in human podocytes. However, the role of APOL1-miR193a axis remain to be elucidated. We asked whether modulation of this axis carries a potential to prevent PAN- and ADR-induced podocyte injury.
Methods
Human podocytes (immortalized) stably expressing either vector (V-podocytes) or APOL1G0 (G0-podocytes) were differentiated (incubating in special media for ten days, V-D or G0-D). V-Dpodocytes and G0-Dpodocytes were incubated in media containing different concentrations of Adriamycin (5, 15, 30, and 35 nM) or PAN (0, 5, 10, 25, 50, and 100 nM) for 48 hours (n=6); in other sets, VD-podocytes and G0-Dpodocytes were treated with Adriamycin (30 mM) or PAN (50 nM), with/without miR193a inhibitor (25 nM) for 48 hours (n=6). Cells were evaluated for reactive oxygen species (ROS) generation (DCF detection assay), caspase-3 cleavage, and apoptosis (TUNEL assay). Proteins and RNAs were extracted from cells treated under similar conditions (n=6). Protein blots were probed for APOL1 and caspase-3; RNAs were assayed for mIR193a. Differentiated podocytes (DPDs) were transduced with either empty vector or miR193a plasmid and evaluated for APOL1 and caspase-3 expression.
Results
G0-Dpodocytes showed a two-fold increase in expression of APOL1, but a 2.8-fold decrease in miR193a levels when compared to V-Dpodocytes. V-Dpodocytes showed increased (P<0.01) ROS generation and a higher (P<0.01) percentage of TUNEL +ve cells when compared to G0D-podocytes in Adriamycin and PAN milieus. Both Adriamycin and PAN displayed an upregulation in miR193a and caspase-3 expression in both V-D and G0-Dpodocytes from their baseline. MicroRNA193a inhibitor decreased miR193a levels, increased APOL1 expression, and attenuated number of TUNEL +ve cells in both Adriamycin and PAN milieus. DPDs overexpressing miR193a displayed reduced expression of APOL1 and enhanced cleavage of caspase-3.
Conclusion
Functional AMA prevents podocyte injury in adverse milieus through down-regulation of miR193.
Funding
- NIDDK Support