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Abstract: FR-PO953

APOL1 Renal Risk Variants Induce Disruption in APOL1-miR193a Axis Through Downregulation of Vitamin D Receptor

Session Information

Category: Pathology and Lab Medicine

  • 1601 Pathology and Lab Medicine: Basic

Authors

  • Kumar, Vinod, Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Jha, Alok, Feinstein Institute for Medical Research, Manhasset, New York, 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 , Hicksville, New York, United States
  • Chinnapaka, Sushma, Northwell health , Hicksville, New York, United States
  • Malhotra, Ashwani, Feinstein Institute Medical Research and NSLIJ, Manhasset, New York, United States
  • Singhal, Pravin C., Feinstein Institute for Medical Research, Manhasset, New York, United States
Background

APOL1-miR193a Axis has been demonstrated to play a vital role in the maintenance of podocyte molecular phenotype. Both APOL1G0 (wild-type) and miR193a inversely regulate each other. However, APOL1 renal risk variants ([RRVs], G1 and G2) upregulated instead of downregulation of miR193a expression in podocytes. Recent reports indicate that Vitamin D Receptor agonist (VDA) downregulates miR193a expression in podocytes. We hypothesize that APOL1RRVs enhance degradation of Vitamin D Receptor (VDR) and that would enhance the expression of miR193a in podocytes. VDR heterodimerizes with RXR and VDR-RXR binds on the gene promoter.

Methods

Immortalized human podocytes stably expressing vector (V-podocytes), APOL1G0 (G0-podocytes), APOL1G1 (G1-podocytes), and APOL1G2 (G2-podocytes) were differentiated. Proteins and RNAs were extracted. Protein blots of V-podocytes, G0-, G1-, G2-podocytes were probed for APOL1, VDR, and GAPDH (n=6); cDNAs were amplified for VDR; RNAs were assayed for miR193a. Bioinformatics studies suggested that VDR binds at miR193a gene. To validate the binding of VDR and RXR on miR193 promoter, ChiP assay was carried out. To determine the role of proteasomal degradation, V-, G0-, G1-, and G2-podocytes were incubated in media containing either vehicle or MG132 (10 nM) for 48 hours (n=4); protein blots were probed for APOL1, VDR, and GAPDH. Podocytes were transfected with either scrambled or VDR siRNA; protein blots were probed for VDR and GAPDH; RNAs were assayed for miR193a.

Results

G0-podocytes displayed enhanced (P<0.05) APOL1 and VDR but attenuated (P<0.05) expression of miR193 when compared to V-podocytes. In contrasts, G1- and G2-podocytes showed decreased (P<0.05) VDR but enhanced (P<0.01) expression of miR193a when compared to V- and G0 podocytes. MG132-treated G1- and G2-podocytes showed an increased expression of VDR vs. only vehicle treated G1-and G2-podocytes. Podocytes silenced for VDR displayed enhanced miR193a expression. ChiP assay revealed binding of VDR and RXR at miR193a promoter.

Conclusion

APOL1RRVs enhance miR193a expression in podocytes through downregulation VDR.

Funding

  • NIDDK Support