APOL1 kidney disease risk variants cause cytotoxicity by depleting cellular potassium and inducing stress-activated protein kinases

Abstract

Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K(+) efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K(+) efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K(+) and subsequent induction of stress-activated protein kinase pathways.

Keywords: African-American; apolipoprotein L1; genetics; kidney; protein kinase.

Fig. 1.

Tetracycline-induced comparable expression levels of G0, G1, and G2 APOL1 in T-REx-293 cells. Stably transfected T-REx-293 cells were treated with the indicated concentrations of tetracycline for 12 h. Immunoblot analyses of ApoL1 and GAPDH in the indicated stable APOL1–T-REx-293 cell lines were performed using anti-Flag or anti-GAPDH antibodies.

Fig. 2.

Expression of G1 or G2 APOL1 in T-REx-293 cells induces cytotoxicity and apparent cell swelling. (A) Quantitation of serial cytotoxicity:viability ratio of G0, G1, or G2 APOL1–T-REx-293 cells after induction with tetracycline (50 ng/mL) at time point zero. Each data point represents mean ± SD of three separate experiments. (B) Immunoblot of APOL1, Flag, and GAPDH 24 h after T-REx cells were tet-induced or not to express G0, G1, and G2 APOL1. (C) Representative phase contrast micrographs of transgenic T-REx-293 cells 9 h postinduction of G0, G1, and G2 APOL1. Nuclei were counterstained with Hoechst 33342. Arrows indicate swollen cytoplasm. The region of the 20× micrograph delineated by dotted rectangle is amplified at 40× magnification. More G1 or G2 APOL1-expressing cells exhibit swollen cytoplasm compared with G0-expressing cells. (Scale bars: 200 or 100 μm for 20× and 40×, respectively.)

Fig. S1.

Schematic representation of tetracycline-inducible APOL1 expression vector stably transfected into T-REx-293 cells. The * represents mutations that constitute G1 (S342 and I 384M) and G2 (deletion of N388 and Y389).

Fig. S2.

Chloroquine increased G1 APOL1-induced LC3-II accumulation. G0 or G1 APOL1–T-REx-293 cells were tet-induced (50 ng/mL) in the presence or absence of chloroquine (5 μM) for 9 h. Autophagy flux was quantitated at 9 h by measuring LC3-II/LC3-I in cell lysates.

Fig. S3.

Autophagy inhibitors do not reduce G1 APOL1 cytotoxicity in T-REx-293 cells. Cytotoxicity: viability ratio of G1 APOL1–T-REx-293 cells after tet induction (50 ng/mL) in the presence or absence of autophagy inhibitors chloroquine (CQ) or wortmannin. All treatments lasted 24 h. Each bar represents mean ± SD of experimental triplicates.

Fig. S4.

Expression of G1 or G2 APOL1 does not increase apoptosis in T-REx-293 cells. Quantitation of apoptosis (caspase-3/7 cleavage) after tet-induction of G0, G1, or G2 APOL1 expression for 24 h. Each bar represents mean ± SD.

Fig. 3.

Expression of G1 or G2 APOL1 inhibits STAT3 phosphorylation. (A) Quantitation of p-STAT3 (Y705) in T-REx-293 cell lysates after 9-h tet induction of G0, G1, and G2 APOL1. (B) Immunoblot of p-STAT3, total STAT3, Flag-tagged APOL1, and beta-actin at the specified time points postinduction of APOL1. Cells were cultured in 10% (vol/vol) FBS until the last 1 h, when they were transitioned to serum-free media for an additional 1 h before treatment with oncostatin M (OSM) for 10 min to induce STAT3 phosphorylation. (C) Immunoblot of basal p-STAT3, p-STAT3 (S727), total STAT3, APOL1 (Flag), and GAPDH after 9 h induction with the indicated tet concentration. Cells remained in 10% (vol/vol) FBS for the entire 9 h and were not treated with OSM. (D) Immunoblot of p-STAT3 and total STAT3 after induction with tet for 9 h [first 8 h in 10% (vol/vol) FBS, last 1 h in serum-free DMEM]. Cells were treated with or without IL-6 (10 ng/mL) for 30 min followed by IL-6 withdrawal for the specified time. (E) Immunoblot of p-STAT3 and p-STAT1 (Y701) after 9-h induction of APOL1. Cells were serum starved for the last 1 h before 10-min treatments with IL-6 (10 ng/mL), OSM (10 ng/mL), or INFγ (10 ng/mL). *, difference is statistically significant compared with EV.

Fig. S5.

Representative confocal micrographs of maximum intensity projection (15 optical Z-stacked images) of STAT3 nuclear localization G0, G1, or G2 T-REx-293 cells that were tet-induced for 9 h, followed by treatment or not with OSM (10 ng/mL, 10 min). EV are empty vector control cells. All images were collected under identical settings and were processed similarly. (Scale bars: 100 μm.)

Fig. 4.

G1 or G2 APOL1 reduces GP130 protein but not mRNA level in T-REx-293 cells. (A) Immunoblot of GP130, p-GP130, beta-actin, and Flag-tagged G0, G1, and G2 APOL1 at the specified time points after induction with tet (50 ng/mL). (B) Immunoblot of GP130, Flag-tagged APOL1, EGFR, and Na⁺/K⁺-ATPase in biotin pull-down (biotinylated membrane proteins) and in total lysate (4%) after T-REx-293 cell induction with tet (50 ng/mL) for 9 h. (C) Quantitative PCR of GP130 and beta-actin after induction of T-REx-293 cells with tet (50 ng/mL) for the specified time periods.

Fig. 5.

G1 and G2 APOL1 down-regulate GP130-STAT3 signaling and induce cytotoxicity via time-dependent activation of p38 MAPK. (A) Immunoblot of p-MKK3 (S189), p-MKK6 (S207), p-P38 (T180/Y182), p-JNK (T183/Y185), p-ERK (T202/Y204) MAPKs, beta-actin, Flag-tagged APOL1, GP130, and p-STAT3 (Y705) in whole cell lysates after treatment with tet (50 ng/mL) with/without p-38 inhibitor, SB202190, 10 μM for 9 h. (B) Immunoblot of p-P38 (T180/Y182) MAPK and GAPDH in whole cell lysates after treatment with tet (50 ng/mL) for the specified time periods. (C) Cell cytotoxicity/viability after 24-h treatment with or without tet (50 ng/mL) in the presence or absence of inhibitors of p38 MAPK (SB202190, 10 μM), JNK (JNK-IN-8, 1 μM), MEK (PD0325901, 10 μM), or a combination of p38 and JNK inhibitors. *, compared with tet-treated cells, reduction in cytotoxicity is statistically significant.

Fig. S6.

Expression of G1 or G2 APOL1 in T-REx-293 cells activates MAPK signaling pathways. After inducing expression of G0, G1, or G2 APOL1 in T-REx cells for 9 h using tetracycline, cell lysates were immunoblotted for the p38, JNK, and ERK and their substrates. Note that ATF2 is a substrate of both p38 MAPK and JNK.

Fig. S7.

MEK and JNK are activated after 6 h of expression of G1 or G2 APOL1 in T-REx-293 cells. After tetracycline-induced expression of G0, G1, or G2 APOL1 for the specified time points, cell lysates were immunoblotted for phospho-MEK (S217/221), MEK (total), and phospho-JNK.

Fig. 6.

Several p38 MAPK inhibitors reduce G1 APOL1-induced cytotoxicity. T-REx-293 cell cytotoxicity/viability after 24-h treatment with or without tet (50 ng/mL) in the presence or absence of inhibitors of p38 MAPK: SB202190, (1–10 μM), Losmapimod (1–100 μM), VX-702 (1–10 μM), and LY2228820 (0.5–1 μM). *, reduction in cytotoxicity/viability is statistically significant compared with tet-treated cells.

Fig. 7.

Cytotoxicity of G1 and G2 APOL1 is mediated by depletion of intracellular potassium and activation of SAPKs in T-REx-293 cells. (A and B) Intracellular K contents were measured using XRpro X-ray fluorescence after inducing G0, G1, or G2 APOL1 expression with tet (50 ng/mL) in DMEM for the specified time periods in A and for 9 h in DMEM or high-K⁺ media, CKCM in B. (C) Phase contrast micrograph of T-REx-293 cells 9 h postinduction with tet (50 ng/mL) in DMEM or CKCM. (Magnification: 40×.) (Scale bars: 100 μm.) (D) T-REx-293 cells cytotoxicity/viability ratio after 24-h treatment with or without tet (50 ng/mL) in DMEM or CKCM, in the presence or absence of p38 inhibitor, SB202190 (10 μM). (E) Immunoblot of whole cell lysates for p-p38 (T180/Y182), total p38, Flag-tagged APOL1, and GAPDH after 9-h induction (with specified concentrations of tet) in DMEM or CKCM. (F) Immunoblot of Flag, p-p38(T180/Y182), GP130, pSTAT3 (Y705), total STAT3, and GAPDH in whole cell lysates from T-REx-293 cells in which expression of G2 APOL1 was induced for 9 h with tet (50 ng/mL) either in DMEM, CKCM, or NMDG. (G) Comparison of G2 APOL1-induced cytotoxicity in T-REx-293 cells after 24-h induction in DMEM, CKCM or NMDG. *, differences are statistically significant.

Fig. S8.

Inhibition of p38 MAPK does not reduce G2 APOL1-induced autophagy. G2 APOL1–T-REx-293 cells were induced or not with tet (50 ng/mL) for 9 h (total). Cells were exposed to SB202190 (10 μM) for 3, 6, or 9 h. Autophagy was quantitated in all cells after 9 h.

Fig. S9.

Elevated extracellular [K⁺] does not reduce G1 or G2 APOL1-induced autophagy. G0, G1, or G2 APOL1–T-REx-293 cells were induced with tet for 9 h in DMEM or CKCM. Autophagy was quantitated in all cells after 9 h.

Fig. 8.

A model of G1 or G2 APOL1-induced cytotoxicity mediated by K⁺ efflux and activation of SAPK signaling. APOL1 proteins form K⁺-permeable cation-selective pores in the plasma membrane. Pores formed by G1 or G2 mediate increased efflux of intracellular K⁺, leading to depletion of intracellular K⁺ and resulting in activation of p38, JNK, and ERK MAPKs. The aberrantly activated SAPKs (p38 and JNK) cause cell toxicity and death likely via their downstream effectors. Down-regulation of GP130-STAT3 signaling is a downstream consequence of activated p38 MAPK. However, the direct contribution of the GP130-STAT pathway in the pathogenesis of G1 or G2 APOL1 cytotoxicity is yet to be determined. Apparent cytoplasmic swelling results from influx of Na⁺ (likely G1 or G1 APOL1-related), with accompanying Cl⁻ and H₂0. G1 or G2 APOL1-induced autophagy occurs independently of K⁺ efflux and does not seem to contribute to G1 or G2 APOL1-induced death of HEK293 cells.