Abstract: FR-PO675

APOL1-B3 Isoform Is Involved in the Processing of the IL-1β Production

Session Information

Category: Glomerular

  • 1001 Glomerular: Basic/Experimental Immunology and Inflammation

Authors

  • Wakashin, Hidefumi, Dokkyo Medical University, Shimotsuga, Japan
  • Kopp, Jeffrey B., NIDDK, NIH, Bethesda, Maryland, United States
Background

APOL1 genetic variants G1 and G2 increase risk for glomerular disease. Previously, we identified an intracellular splice isoform, APOL1-B3, that is expressed in glomerular cells and tubular cells in vivo (ms submitted). In transgenic mice, APOL1-B3-G2 expression enhanced podocyte injury, increased pro-IL-1β mRNA from isolated glomeruli, and increased renal IL-1β protein production. APOL1-B3 interacted with NLRP12, a negative regulator of Toll-like receptor signaling, potentially explaining elevation of pro-IL-1β mRNA in glomeruli. Here we examined the role of APOL1-B3 in inflammasome activation and production of mature IL-1β.

Methods

We generated stable THP-1 monocytic cell lines expressing APOL1 under the control of the chicken actin promoter, CAG-APOL1-B3-FLAG-G0 (common variant) or -G2 (renal risk variant); G1 variant cells were not viable. THP-1 cells were treated with phorbol myristate acetate to induce differentiation into macrophages, which were stimulated with lipopolysaccharide (LPS) and nigericin to activate inflammasomes. We generated CAG-APOL1-B3 transgenic mice, using linear DNA encoding CAG promoter-APOL1-B3 (G0 or G2), with FLAG-SV40 polyA signal sequence. Mice were studied at 6-8 w of age. Random urine was obtained prior to and 3 days after uninephrectomy.

Results

In THP-1 derived macrophages, over expression of APOL1-B3-G0 and –G2 significantly enhanced both LPS-stimulated IL-1β production (mean±SD: control =193±3.8, G0=585±33, G2=669±79) (P<0.05) and release of caspase-1 into supernatant (control =3393±520, G0=9828±1541, G2= 6783±608) (P<0.05). Following uninephrectomy, increased urinary caspase-1 was seen in both APOL-B3 mice (G0, G2) and wild type mice, with no difference among groups. The ratio of IL-1β to pro-IL-1β in the kidney, assessed by Western blot, increased following uninephrectomy, and was numerically greater in APOL1-B3-G2 mice. These findings suggest enhanced processing of pro-IL-1β by the APOL1 risk variant . By Western blot, both APOL1-B3-G0- and -G2 interacted with NLRP3.

Conclusion

These findings suggest that under the conditions studied, the APOL1-B3 isoform affects processing of IL-1b and add a further dimension to the role of APOL1-B3 in modulating NLRP3 pathway signaling.