Abstract: FR-PO918
APOL1 Renal Risk Variants (RRVs)-Induced Translocation of miR193a and Podocyte mRNAs to P-Bodies Prevents Their Degradation and Facilitates Translation
Session Information
- Glomerular Diseases: Podocyte Biology - II
November 08, 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
- 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
- Singhal, Pravin C., North Shore LIJ Health System, Great Neck, New York, United States
Background
A disruption of APOL1-miR193a axis (AMA) has been reported in podocytes expressing APOL1 renal risk variants (RRVs, G1, and G2). However, the involved mechanism of disruption of AMA is not understood. We hypothesize that miR193a binding with APOL1G0 mRNA directs it for degradation, whereas, non-complementary binding with APOL1RRV mRNAs would facilitate their translocation to P-bodies to prevent their degradation as well as facilitate translation. To validate this hypothesis, we have evaluated the binding of APOL1G0 and APOL1RRVs mRNAs with miR193a and PKR in cultured podocytes. Additionally, we have examined the role of miR193a and involved mechanism of PKR activation in dual transgenic mice expressing miR193a and APOL1G0/APOL1RRVs.
Methods
RNAs of podocytes stably expressing G0s, G1s, and G2s were immunoprecipitated with anti-PKR antibody, and IP fractions were evaluated for PKR and downstream signaling (total and phospho--PKR, total and phospho-eIF2a, and GAPDH) and assayed for miR193a (n=6). Co-labeling of PKR and LSM14A/HEDLs (to localize PKR in P-bodies) were conducted. Protein blots of renal tissues of 10-weeks old control, single and dual transgenic (APOL1G0/G1/G2 and miR193a transgenic) mice (n=6; started on doxycycline [to induce miR193a and APOL1] feed at age 4 weeks) were probed for total and phospho--PKR, total and phospho-eIF2a and GAPDH. RNAs from renal tissues of single and dual transgenics were immunoprecipitated with anti-PKR antibody, and IP fractions were evaluated for PKR activation.
Results
PKR IP fractions from G1- and G2-podocytes displayed enhanced binding of APOL1mRNA; similarly, PKR IP fractions of renal tissues of dual G1- and G2 as well as single G1- and G2 transgenic mice displayed enhanced expression of miR193a. Immunolabeling studies showed enhanced translocation of PKR into P-bodies in G1- and G2-podocytes vs. G0 podocytes. Cellular lysates of G1- and G2-podocytes, renal tissues of both single and dual G1- and G2- transgenic mice, and miR193a mice displayed activation of PKR and associated downstream signaling.
Conclusion
APOL1RRVs facilitates translocation of miR193a and other podocyte mRNAs to P-bodies. It prevents their degradation and facilitates their translation.
Funding
- NIDDK Support