Abstract: FR-PO917
Podocytes Expressing APOL1 Non-Risk and Renal Risk Variants Carry Potential to Secrete APOL1 Through Exosomal Pathway
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 of Research, Manhasset, New York, United States
- Adnani, Harsha, Feinstein Institute of Research, Manhasset, New York, United States
- Lan, Xiqian, Feinstein Institute for Medical 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., North Shore LIJ Health System, Great Neck, New York, United States
Background
Recent reports indicate a vital role of APOL1-miR193a axis in the development of focal segmental glomerulosclerosis. Circulatory APOL1 is bound to HDL complex and unlikely to pass through glomerular filtration barrier. Although APOL1 has been considered to be a secretory protein but its secretion by podocytes remains controversial. However, the realese of APOL1 from podocytes throuhg the exosomal pathway has not been studied to date. We asked whether cultured podocytes have potential to release APOL1 in exosomes. To confirm exosomal secretion, we validated this hypothesis in human embryonic kidney cells (HEKs) expressing vector, APOL1G0, APOL1G1, and APOL1G2. Additionally, we evaluated exosomal contents in the urine of the APOL1G0, APOLG1, and APOL1G2 transgenic mice.
Methods
Immortalized human podocytes and HEKs stably expressing vector (V), APOL1G0 (G0), APOL1G1 (G1), APOL1G2 (G2) were incubated with exosome-free media for 48 hours (n=6). Subsequently, media were collected and ultra-centrifuged for exosome isolation; Western blotting and FACS analysis were carried out for the expression of exosomal markers (CD81, HSP70), and cytosolic marker (Calnexin). The isolated exosomes were analyzed for their size by using Nano site system. To determine the presence of other relevant proteins, the single exo RNAseq and proteomic analysis was carried out. To validate these findings in vivo, the exosomes were harvested from the urine of the APOL1G0, APOLG1, and APOL1G2 transgenic mice.
Results
Exosomes harvested from both podocytes and HEKs expressing APOL1G0 and G1/G2 displayed the presence of APOL1. Nano Site analysis characterized the isolated exosomes in the range from 90-125 nM in size. Western blotting and FACS analysis displayed the exosomal expression of CD81 and HSP70 but Calnexin was not expressed. Release of exosome amounts differed in incubation med harvested from G0-, G1-, and G2-podocytes and HEKs. However, exosomes harvested from podocytes and HEKs expressing APOL1 risk variants showed a decrease in APOL1 expression. Data from urinary exosomes from APOL1 transgenic mice were consistent with in vitro findings.
Conclusion
Podocytes expressing APOL1G0, APOL1G1, and APOL1G2 carry potential to secrete APOL1 through the exosomal pathway.
Funding
- NIDDK Support