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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 Article has an altmetric score of 43

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

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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 4

APOL1 G1 activates Gαq-PLC-IP3R/RYR signaling to liberate calcium from the ER into the cytosol of T-REx-293 cells.

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APOL1 G1 activates Gαq-PLC-IP3R/RYR signaling to liberate calcium from t...
(A) Fura-2 Ca2+ sensor shows that 8 hours of APOL1 G1 expression attenuates thapsigargin-induced surge of cytosolic Ca2+, which is rescued by VX-147 cotreatment (n = 8). (B) Thapsigargin-induced Ca2+ release plotted as bar graphs (n = 8). (C) Direct measurement of ER Ca2+ with fluorescent sensor (D1ER) shows that 8 hours of APOL1 G1 expression depletes basal ER Ca2+, which is restored by VX-147 cotreatment (n = 8–12). Addition of CPA, which further depletes ER Ca2+, has marginal effect on ER Ca2+ of APOL1 G1–expressing cells. (D) Basal ER Ca2+ level before CPA addition shown as bar graph (n = 12–30). (E) Real-time live-cell fluorescence shows increased DAG biosynthesis after 8 hours of G1 induction. G1-induced DAG synthesis is blocked by VX-147, U73122 (PLC inhibitor), and FR*359 (Gαq inhibitor) (n = 8). (F) Fluorescent Ca2+ sensor shows that combined inhibition of IP3R with XSpC and of RYR with JTV-519 rescues thapsigargin-induced Ca2+ response in G1-expressing T-REx-293 cells (12 hours of treatment), similar to the effect of VX-147 (n = 3–6). (G) Measurement of live-cell basal cytosolic Ca2+ levels shows that VX-147, XSpC, and JTV-519 cotreatment prevents G1-induced increase in cytosolic Ca2+ (n = 6–12). (H) Multitox assay shows that JTV-519, XSpC, and VX-147, but not SKF96365, rescue APOL1 G1–induced cytotoxicity in T-REx-293 after 24 hours of treatment (n = 4–12). (I) Immunoblot shows successful CRISPR/Cas9 knockout of IP3R1 and IP3R3 in T-REx-293 G1 cells (C1 and C10 are 2 independent clones). (J) Multitox assay shows loss of IP3R-mediated APOL1 G1–induced cytotoxicity in T-REx-293 after 24 hours of treatment (n = 5–7). (K) Schematic summary of Figure 4, showing how each step of this signaling cascade is reversible. All data are represented as mean ± SD. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001, ordinary 1-way ANOVA with Tukey’s multiple-comparison test.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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