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Abstract: SA-PO360

Disrupted APOL1-miRNA Axis Induces an Imbalance Between Autophagic Load and Handling in Podocytes Expressing APOL1 Risk Alleles

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Mishra, Abheepsa, The Feinstein Institute for Medical Research, Northwell Health, MANHASSET, New York, United States
  • Ayasolla, Kamesh R., Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Paliwal, Nitpriya, Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Marashi Shoshtari, Seyedeh Shadafarin, The Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Kumar, Vinod, Fienstine Institute for Medical Research, NEW YORK, 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


MicroRNA193a has been reported to enhance autophagy in several cell types. A recent report suggests that human podocytes (PDs) expressing APOL1 risk alleles (G1 and G2) have an altered autophagic reflux. We hypothesize that disruption of APOL1-miR193a axis would contribute to an imbalance between autophagic load and handling in PDs expressing APOL1 risk alleles.

Methods

Immortalized human podocytes (PDs) stably expressing vector, APOL1G0/G1/G2 were differentiated and evaluated for APOL1 (protein and mRNA) and miR193a expressions. To determine the effect of miR193a on autophagy, PDs were transfected with empty vector or different concentrations of miR193a plasmid (0, 50, 100 nM), or an inhibitor of miR193a (0, 25, 50, and 100 nM), followed by an evaluation for autophagy markers (P62, LC3II, and beclin 1). To determine the role of the mTOR pathway, PDs were transfected with either empty vector, miR193a, or a miR193a inhibitor (plasmids) followed by analysis for phos-mTOR and GAPDH by Western blotting. To examine the role of miR193a-APOL1 axis, PDs were transfected with either scrambled or APOL1 siRNA and then evaluated for miR193a expression and autophagy markers. To examine autophagic flux, PDs expressing vector (PDV), APOL1G0/G1/G2 were transfected with GFP-tagged LC3II plasmid and treated with Bafilomycin (100 nm) and examined at 0, 2, and 4 hours under a confocal microscope.

Results

APOL1G0 down-regulated but APOL1G1 and APOL1G2 upregulated miR193a expression in PDs. Overexpression of miR193a down-regulated but a miR193a inhibitor enhanced the expression of phos-mTOR. APOL1-silenced PDs showed enhanced expression of miR193a as well as of autophagy markers. PDs expressing APOL1G1 and G2 displayed a higher expression of LC3II in a time course manner in Bafilomycin blockade experiments, suggesting an increased load or sluggish autophagic flux. Since APOL1 risk alleles displayed 2.5- fold higher expression of miR193a when compared to PDs expressing APOL1G0, it appears that they carried an enhanced autophagic load, in addition to sluggish autophagic flux.

Conclusion

Disrupted APOL1-miR193a axis induces an imbalance between autophagic load and handing in podocytes expressing APOL1 risk alleles.

Funding

  • NIDDK Support