Abstract: SA-PO304
Expression of APOL1 Risk Alleles (G1 and G2) Compromises Spreading of Podocytes by Down Regulation of β3 Integrin
Session Information
- Extracellular Matrix Biology, Fibrosis, Cell Adhesion
November 04, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Cell Biology
- 204 Extracellular Matrix Biology, Fibrosis, Cell Adhesion
Authors
- Hussain, Ali, Feinstein Institute of Medical Research, New York, New York, United States
- Aslam, Rukhsana, Feinstein Institute for medical research, Glenoaks, New York, United States
- Kumar, Vinod, Feinstein Inst.Med research and NSLIJ, Manhasset, New York, United States
- Malhotra, Ashwani, Feinstein Inst.Med research and NSLIJ, Manhasset, New York, United States
- Skorecki, Karl, Rambam Health Care Campus, Haifa, Israel
- Singhal, Pravin C., North Shore LIJ Health System, Great Neck, New York, United States
Background
Genetic epidemiology indicated that Africans Americans are at four fold higher risk for the development of focal segmental glomerulosclerosis when compared to European Americans. This disparity amongst African American has been attributed to carrying APOL1 risk alleles (G1 and G2). Expression of APOL1 G1 and G2 by podocytes has been reported to promote cell death both in vitro and in vivo studies. However, the role of APOL1G0 (wild- type) in podocytes is far from clear. Podocytes are normally detached and excreted in the urine in a normal physiological state. However, adjacent podocytes are able to maintain the integrity of the glomerular filtration barrier by spreading over the naked basement membrane caused by detachment of podocytes. We hypothesize that APOL1 risk alleles (G1 and G2) could be affecting the spread of podocytes compromising integrity of glomerular filtration barrier.
Methods
Cultured immortalized human podocytes proliferate at 33°C and differentiate at 37°C. Podocytes stably expressing vector, APOL1G0, APOL1G1, and APOL1G2 grown on coverslips (collagen coated) were incubated in media for 12 and 24 hours at 37○C (n=4). Subsequently, podocytes were labeled with α-actinin antibody and examined under a confocal microscope. Average size of podocytes determined at 8 random fields using J Image program. Proteins extracted from the cells treated under similar conditions, were probed for β3 integrin and α-actinin expession and reprobed for actin.
Results
Podocytes expressing APOL1 G1 and G2 displayed decreased (P<0.01) spreading when compared to podocytes expressing vector (V) or APOLO1G0 (Mean podocyte size at 12 hours: V, 74.3 ± 18.6; APOLO1G0, 87.8 ± 15.3; APOL1G1, 42.5 ± 12.2; APOL1G2, 35.2 ± 9.7 μM and at 24 hours: V, 118.1 ± 15.7; APOL1G0, 130.6 ± 17.5; APOL1G1, 65.1 ± 18.0; APOL1G2, 79.7 ± 16.4 μM) both at 12 h and 24 h. Podocytes expressing APOL1G0 displayed increased (P<0.05) spreading when compared to Vector. Western blot analysis demonstrated 2- 5 fold decreased expression of β3 integrin and α-actinin in podoctyes expressing APOL1G1 and G2 when compared to podocytes expressing vector and APOL1G0.
Conclusion
Expression of APOL1 risk alleles (G1 and G2) compromised podocyte spreading by down regulation of β3 intergrin α-actinin expressions.
Funding
- NIDDK Support