Kidney Week

Abstract: SA-PO357

Modulation of APOL1-miR193a Axis Protects Against Apoptosis in Adverse Milieus

Session Information

• Glomerular Diseases: Immunology and Inflammation - III
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1202 Glomerular Diseases: Immunology and Inflammation

Authors

• Kumar, Vinod, Fienstine Institute for Medical Research, NEW YORK, New York, United States

Vinod Kumar,

• Paliwal, Nitpriya, Feinstein Institute for Medical Research, Manhasset, New York, United States

Nitpriya Paliwal,

• Lan, Xiqian, Feinstein Institute for Medical Research, Manhasset, New York, United States

Xiqian Lan,

• Ayasolla, Kamesh R., Feinstein Institute for Medical Research, Manhasset, New York, United States

Kamesh R. Ayasolla,

• Chowdhary, Sheetal, Feinstein Institute of Medical Research, New Hyde Park, New York, United States

Sheetal Chowdhary,

• Marashi Shoshtari, Seyedeh Shadafarin, The Feinstein Institute for Medical Research, Manhasset, New York, United States

Seyedeh Shadafarin Marashi Shoshtari,

• Malhotra, Ashwani, Feinstein Institute Medical Research and NSLIJ, MANHASSET, New York, United States

Ashwani Malhotra,

• Skorecki, Karl, Rambam Health Care Campus, Haifa, Israel

Karl Skorecki,

• Singhal, Pravin C., Feinstein Institute for Medical Research, Manhasset, New York, United States

Pravin C. Singhal,

Background

Both Puromycin aminonucleoside (PAN) and adriamycin are known to promote podocyte (PD) apoptosis in both in vitro and in vivo studies. We have recently reported that APOL1 wild-type (G0) has a potential to preserve podocyte molecular phenotype in high glucose milieu. APOL1 inversely regulates PD expression of miR193a and forms reciprocally linked feedback relationship. We now hypothesize that upregulation of APOL1-miR193a axis has a potential to prevent PD apoptosis in PAN and adriamycin milieus.

Methods

To aim differentiation, immortalized human podocytes (PDs) stably expressing vector (PDV) or APOL1G0 (wild- type, PDG0) were incubated in media for 10 days. Differentiated (D) PDVs and PDG0 were treated with different concentrations of Adriamycin (0,5, 15, 30, 50, and 100 nM) or PAN (0, 5, 10, 25, 50, and 100 nM) for 48 hours (n=4); in other sets, DPDVs were incubated in media containing Adriamycin (30 mM) , PAN (50 nM), with/without miR193a inhibitor (25 nM) for 48 hours (n=4). 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=4). Protein blots were probed for APOL1 and caspase-3; RNAs were assayed for miR193a. DPDs were transduced with either empty vector and miR193a plasmid and evaluated for APOL1 and caspase-3 expressions.

Results

PDVs displayed higher (P<0.01 vs. respective controls) generation of ROS and a greater (P<0.01 vs. respective controls) percentage of apoptosis when compared to PDG0, both in adriamycin and PAN milieus. PDG0 displayed enhanced (two-fold) expression of APOL1, but decreased (2.8-fold) miR193a levels when compared to PDVs. Both Adriamycin and PAN exhibited enhanced PD expression of miR193a and caspase-3. MicroRNA193a inhibitor decreased (3-fold) miR193a levels, increased APOL1 (1.8-fold) expression, and attenuated (P<0.5 vs. adriamycin and PAN) apoptosis in both adriamycin and PAN milieus. DPDs overexpressing miR193a displayed decreased expression of APOL1 and enhanced cleavage of caspase-3.

Conclusion

APOL1Go provides protection against apoptosis in adverse milieus through down-regulation of miR193a and attenuated generation of ROS.

Funding

• NIDDK Support