APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease
Somenath Datta, … , Christopher B. Newgard, Opeyemi A. Olabisi
Somenath Datta, … , Christopher B. Newgard, Opeyemi A. Olabisi
Published January 16, 2024
Citation Information: J Clin Invest. 2024;134(5):e172262. https://doi.org/10.1172/JCI172262.
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Research Article Nephrology

APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease

Abstract

Two coding variants of apolipoprotein L1 (APOL1), called G1 and G2, explain much of the excess risk of kidney disease in African Americans. While various cytotoxic phenotypes have been reported in experimental models, the proximal mechanism by which G1 and G2 cause kidney disease is poorly understood. Here, we leveraged 3 experimental models and a recently reported small molecule blocker of APOL1 protein, VX-147, to identify the upstream mechanism of G1-induced cytotoxicity. In HEK293 cells, we demonstrated that G1-mediated Na+ import/K+ efflux triggered activation of GPCR/IP3–mediated calcium release from the ER, impaired mitochondrial ATP production, and impaired translation, which were all reversed by VX-147. In human urine-derived podocyte-like epithelial cells (HUPECs), we demonstrated that G1 caused cytotoxicity that was again reversible by VX-147. Finally, in podocytes isolated from APOL1 G1 transgenic mice, we showed that IFN-γ–mediated induction of G1 caused K+ efflux, activation of GPCR/IP3 signaling, and inhibition of translation, podocyte injury, and proteinuria, all reversed by VX-147. Together, these results establish APOL1-mediated Na+/K+ transport as the proximal driver of APOL1-mediated kidney disease.

Authors

Somenath Datta, Brett M. Antonio, Nathan H. Zahler, Jonathan W. Theile, Doug Krafte, Hengtao Zhang, Paul B. Rosenberg, Alec B. Chaves, Deborah M. Muoio, Guofang Zhang, Daniel Silas, Guojie Li, Karen Soldano, Sarah Nystrom, Davis Ferreira, Sara E. Miller, James R. Bain, Michael J. Muehlbauer, Olga Ilkayeva, Thomas C. Becker, Hans-Ewald Hohmeier, Christopher B. Newgard, Opeyemi A. Olabisi

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Figure 1

VX-147 protects T-REx-293 cells from APOL1 G1– and G2–induced cellular swelling and cell death.

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VX-147 protects T-REx-293 cells from APOL1 G1– and G2–induced cellular s...
(A) Illustration of a T-REx-293 cell with Tet-inducible APOL1 G0, G1, or G2 expression construct. Tet-induced G1 or G2 but not G0 causes cytotoxicity at 24 hours that is completely prevented by VX-147 (n = 8). (B) Immunoblot of T-REx-293 cell lysates shows that Tet induces similar expression of G0, G1, and G2 proteins at 8 hours and VX-147 further increases G1 and G2 levels. (C) Live-cell fluorescence microscopy of T-REx-293 G1 cells shows that 8 hours of G1 expression causes cell swelling, pushing the PM (red) away from the nuclei (green), quantified in lower panel. Scale bars: 36.8 μm; zoom in scale bars: 311 μm. (D) Multitox assay shows that APOL1 G1 expression causes 100% cytotoxicity in T-REx-293 by 24 hours (topmost bar), an effect completely reversed by VX-147 within 12 hours and partially reversed at 16 hours following Tet induction (n = 6). (E) Live-cell fluorescence microscopy of T-REx-293 G1 cells shows that cell swelling caused by 12 hours of Tet-induced APOL1 G1 (top row) is reversed by a subsequent 12 hours of VX-147 treatment (middle row). Untreated T-REx-293 G1 cells served as controls. Scale bars: 311 μm. (F) Clonogenic survival assay (cell nuclei stained blue) performed after 10 to 12 days of continuous treatment shows that G0 expression does not affect the survival of T-REx-293 cells (top row), whereas G1 and G2 cause complete loss of cell survival that is rescued by VX-147. Scale bars: 10,000 μm (n = 3). Quantification of cells or colony counts (right panel). All data are represented as mean ± SD. ****P ≤ 0.0001, ordinary 1-way ANOVA with Tukey’s multiple-comparison test.