Expression of Apolipoprotein L1 Risk Variants at the Plasma Membrane and Haplotype-Dependent Cytotoxicity

Despite apolipoprotein L1 (APOL1) risk variants being linked to various glomerular disorders, the pathophysiological mechanisms remain unclear. Recent studies suggest that APOL1 expression at the plasma membrane and its pore-forming activity may be proximal drivers of the mammalian risk variants' cytotoxicity. This has led to the development of Inaxaplin (VX-147), a specific APOL1 inhibitor. However, both the presence of APOL1 at the plasma membrane and the precise nature of the ion conductance involved in APOL1-mediated kidney disease (AMKD) remain subjects of ongoing debate,,; particularly, the Ca2+ permeability of the pore-forming APOL1 at the plasma membrane is unclear. Besides the pore-forming property, the APOL1 haplotype background is a significant contributor to AMKD. APOL1 risk variants expressed in the native African haplotype (E150, I228, and K255: African haplotype [EIK]) exhibit greater cytotoxicity than those in the reference (E150, M288, and R255: reference haplotype [EMR]) or world-wide common (K150, I228, and K255: world-wide common haplotype [KIK]) backgrounds, an effect mitigated by the M1 (N264K) variant. However, it remains unclear whether different haplotypes and their associated risk variants disproportionally dysregulate cytosolic Ca2+ concentration. To address these questions, we used HEK-293 cells transiently expressing APOL1 G0, G1, or G2 in EMR, KIK, and EIK haplotype backgrounds, with or without the M1 variant, to examine cation homeostasis, with particular focus on intracellular Ca2+ concentration [(Ca2+)i] alterations in relation to haplotype-dependent cytotoxicity. In parallel, we investigated the subcellular localization and near-plasma membrane dynamics of APOL1 variants. Key results were validated in human podocytes expressing different APOL1 risk variants and genotypes.

Full details are provided in the Supplemental Material. Site-Directed Mutagenesis APOL1 cDNA (splice isoform A) was cloned into the pCDNA3.1 vector with a C-terminal–enhanced green fluorescent protein (eGFP) tag. Mutations were introduced via standard overlap-extension PCR. Cell Lines and Cell Culture HEK-293 cells, inducible APOL1 podocyte lines, conditionally immortalized human podocytes endogenously expressing APOL1 G0/G0, G2/G2, and an APOL1 knockout with its isogenic wild-type control were used. Cells were cultured as previously described., APOL1 Transfection and Induction HEK-293 cells and human podocytes were transfected with APOL1 or empty vector plasmid using transfection reagents. APOL1 expression in inducible podocyte lines was stimulated with 500 ng/ml doxycycline, while endogenous expression in human podocytes was induced using 50 μg/ml polyinosinic:polycytidylic acid (poly[I:C])., Fluorescence-Activated Cell Sorting Transfection efficiency in HEK-293 cells was assessed by measuring eGFP fluorescence using flow cytometry. Cell Viability Cell viability was evaluated with the PrestoBlue high sensitivity cell viability reagent, as previously described. Real-Time Quantitative Polymerase Chain Reaction The total RNA used for Real-Time Quantitative Polymerase Chain Reaction was isolated using the RNeasy Micro Kit according to the manufacturer's instructions. Total RNA Sequencing Total RNA sequencing was performed on inducible stable podocyte lines treated with doxycycline for 72 hours by Macrogen Europe. Immunoblotting Cells used for immunoblotting were collected in phosphate buffer solution and lysed in radio-immunoprecipitation assay lysis buffer supplemented with protease and phosphatase inhibitors. Immunofluorescence and Confocal Microscopy Cells were fixed with 4% paraformaldehyde for 15 minutes prior to staining with antibodies corresponding to different subcellular organelles. The full list of the antibodies is provided in the Supplemental Table 1. Plasma Membrane Sheets Preparation and Image Acquisition Plasma membrane sheets were prepared using the plasma membrane isolation with colloidal silica technique, as previously described., Total Internal Reflection Fluorescence Microscopy Transfected cells seeded on 25-mm glass coverslips were placed in a custom-made chamber, and total internal reflection fluorescence (TIRF) imaging experiments were performed at 25°C, as previously described., Fluorimetric Intracellular Ca2+ Measurements Changes in (Ca2+)i were monitored 24 hours post-transfection using ratiometric FURA-2-based fluorimetry, as previously described. To determine whether Ca2+ is released from the endoplasmic reticulum (ER), transfected cells were stimulated for 2 minutes with 2 μg/ml trypsin or 500 nM thapsigargin. Whole-Cell Patch Clamping Whole-cell membrane currents were measured with an EPC-10 patch-clamp amplifier and PatchMasterPro Software (HEKA Elektronik), as previously described. Statistical Analysis Data were first tested for normality. Data are presented as mean±SEM or as median with 95% confidence intervals. P values ≤ 0.05 were considered significant.

APOL1 Colocalized with the ER and the Plasma Membrane in HEK-293 Cells To obtain a more comprehensive understanding of APOL1 subcellular localization, HEK-293 cells transiently overexpressing C-terminally eGFP-tagged African apolipoprotein L1 (APOL1-EIK) G0 and its corresponding risk variants were analyzed 24 hours post-transfection. Immunofluorescence analysis demonstrated colocalization between APOL1 and the ER-resident scaffolding protein, vesicle-associated membrane protein-associated protein B, indicating an association with the ER (Figure 1A). Furthermore, APOL1 expression at the plasma membrane was observed based on colocalization with an established plasma membrane marker, cholera toxin subunit B, using both colloidal silica–based plasma membrane isolation and immunofluorescence staining techniques (Figure 1B). However, no colocalization was observed between APOL1 and other organelles in HEK-293 cells (Supplemental Figure 1). Using TIRF microscopy, which selectively visualizes fluorophores within approximately 100–200 nm of the plasma membrane, we further confirmed the localization of APOL1 to the sub–plasma membrane region. At 18 hours post-transfection, APOL1-eGFP was consistently observed as static fluorescence that evenly covered the entire footprint of the cell at the glass coverslip. Additionally, motile punctate vesicle-like structures were regularly observed, with these structures being larger and less mobile in cells transfected with the risk variants (Figure 1C and Supplemental Videos 1–4). Characterization of the mobile APOL1-positive structures revealed that these vesicle-like compartments in the G1 and G2 variants colocalized with lysosome-associated membrane protein 1 (LAMP1)–positive vesicles (Figure 1D and Supplemental Videos 5–7). Total Internal Reflection Fluorescence Recovery After Photobleaching Revealed Distinct Modes of APOL1 Variants Recovery at the Plasma Membrane Although previous studies have validated the translocation of APOL1 to the plasma membrane,, there is no knowledge available on the molecular dynamics of APOL1 near the plasma membrane. Therefore, we used total internal reflection fluorescence recovery after photobleaching (TIR-FRAP) to selectively monitor the kinetics of repopulation of the membrane and the subcellular structures involved in the trafficking toward the plasma membrane (Figure 2A). Following the fluorescent bleaching step, we analyzed the recovery of global eGFP fluorescence in the TIRF field as an index of the rate of movement of APOL1-EIK proteins from non-bleached parts of the cells toward the bleached peri-membrane area (Figure 2B and Supplemental Videos 8–10). In cells overexpressing APOL1 G0-eGFP, recovery of global fluorescence in the TIRF field could be well described using a single exponential time constant (τ) 49.1±4.8 s, reaching a maximal recovery of 83%±8% of the global eGFP of the normalized pre-bleach fluorescence. On the other hand, the APOL1 G1-eGFP– (τ=47.3±6.2 s) and APOL1 G2-eGFP– (τ=57.3±6.6) expressing cells recovered at a similar speed, but the global eGFP recovery was significantly lower (63.0%±2.0% of the normalized pre-bleach fluorescence) compared to the G0 variant (Figure 2, C–E). Next, we investigated the cytoskeletal tracks mediating the trafficking of dynamic APOL1-containing structures toward the plasma membrane. To this end, transfected cells were treated for 1 hour prior to TIR-FRAP analysis with either the microtubule-depolymerizing agent nocodazole or the actin-depolymerizing agent cytochalasin D. Cytochalasin D incubation resulted in a significant decrease in the fluorescence recovery rate of APOL1-EIK G0 transfected cells (Figure 2, F–H), without a significant decrease in the speed of recovery (τ; Figure 2I). In contrast, the fluorescence recovery rate of APOL1-EIK G0 was not affected in cells treated with nocodazole (Figure 2, F–H, and Supplemental Videos 11–13). Cytochalasin D or nocodazole pretreatment of cells expressing APOL1-EIK G1 or G2 risk variants did not affect the fluorescence recovery rate (Supplemental Figure 2). APOL1 Expression Affected Basal Cytosolic Ca2+ Levels in a Haplotype-Dependent Manner Next, cytosolic (Ca2+)i levels in APOL1-transfected HEK-293 cells were investigated via microfluorimetric calcium measurements to examine the contribution of APOL1 in calcium homeostasis. Basal Ca2+ levels were significantly elevated in cells overexpressing APOL1-EIK G0 compared to empty vector and non-transfected cells (Figure 3, A and B), an effect also observed in the KIK and EMR haplotypes (Supplemental Figure 4, B and C). However, direct comparison of G0 across haplotypes revealed significantly higher basal (Ca2+)i levels in APOL1 G0-EIK than in the other backgrounds (Figure 3, C and D). Interestingly, expression of APOL1 risk variants in the EIK background resulted in an approximately three-fold increase in basal Ca2+ levels relative to KIK and EMR (Supplemental Figure 3, A, B, D, and E). Importantly, this haplotype-dependent increased (Ca2+)i was independent of APOL1 expression levels, measured as the eGFP intensity (Figure 3E and Supplemental Figure 3, C and F). Besides, eGFP tagging did not affect APOL1 function, as untagged APOL1 construct similarly resulted in a robust increase in cytosolic Ca2+ levels (Supplemental Figure 3, G and H). These data suggested that elevated cytosolic (Ca2+)i levels depended on the APOL1 haplotype background. APOL1 Modulated Extracellular Ca2+ Influx in a Risk Variant–Dependent Manner Next, we compared the cytosolic (Ca2+)i levels between the risk variants and their G0 within each haplotype. Within each haplotype, the basal (Ca2+)i conferred by G1 and G2 was significantly higher compared to G0-expressing cells (Figure 4, A and B, and Supplemental Figure 4, B and C). Moreover, this risk variant–dependent increased (Ca2+)i was independent of the eGFP intensity (Supplemental Figure 4A), excluding the hypothesis of increased expression levels of the risk variants. Patients with AMKD may be compound heterozygous for the risk variants, and disease risk can also be conferred by a single risk allele. To model these genetic scenarios, HEK-293 cells were transfected with 1:1 mixture of cDNAs encoding G0, G1, or G2. Under these conditions, cells expressing G1/G2, G1/G0, or G2/G0 exhibited significantly elevated basal cytosolic Ca2+ levels compared with cells expressing G0 alone (Figure 4, A and B, and Supplemental Figure 4, B–D). Taken together, these data demonstrated that APOL1 risk variants showed elevated cytosolic (Ca2+)i levels independent of their expression levels at the plasma membrane. To determine whether the elevated cytosolic (Ca2+)i reflects ER Ca2+ store depletion or extracellular Ca2+ influx, a series of calcium imaging experiments were performed. Stimulation of APOL1-EIK–transfected cells with trypsin (2 μg/ml) resulted in a minimal increase in cytosolic (Ca2+)i (Supplemental Figure 4E). Consistently, perfusion with Ca2+-free extracellular solution (0 mM Ca2+) significantly reduced cytosolic (Ca2+)i compared to 2 mM Ca2+ conditions in APOL1-EIK transfected cells (Figure 4, C and D), indicating that the (Ca2+)i increase originated predominantly from the extracellular compartment. To further exclude ER-derived Ca2+ release, cells co-transfected with C-terminally red fluorescent protein-tagged APOL1 variants and the endoplasmic reticulum-targeted GCaMP6f (ER-GCaMP6f, Ca2+ sensor), were loaded with fura-2-acetoxymethyl ester and perfused with thapsigargin, a sarco/ER Ca2+-ATPase inhibitor, in Ca2+-free solution. Although thapsigargin induced an increase in cytosolic Ca2+, no differences were observed between APOL1-transfected and non-transfected cells. Consistently, ER-GCaMP6f imaging provided no evidence for APOL1 variant–specific ER Ca2+ release, suggesting a minimal contribution of ER Ca2+ release (Supplemental Figure 4, F–I). Supporting an extracellular origin of Ca2+ influx, and consistent with TIR-FRAP data indicating that actin filaments are required for APOL1 trafficking to the plasma membrane, treatment of APOL1-EIK G0 transfected cells with cytochalasin D for 3 hours significantly reduced cytosolic (Ca2+)i levels compared with untreated transfected cells (Supplemental Figure 4, H and K). Together, these results suggested that elevated cytosolic (Ca2+)i in APOL1-expressing cells is most probably driven by extracellular Ca2+ influx rather than ER store release. Next, the APOL1 inhibitor, VX-147 (1 μM), was assessed for its effect on the elevated cytosolic (Ca2+)i in APOL1-transfected cells. This concentration was selected based on an exploratory dose-response analysis of cell viability (Supplemental Figure 6H). VX-147 significantly reduced cytosolic (Ca2+)i in cells overexpressing APOL1-EIK variants (Figure 4, E and F), indicating inhibition of APOL1-induced Ca2+ entry. This effect was comparable in G1- and G2-expressing cells, suggesting independence from the specific risk variant (Supplemental Figure 4L). VX-147 had no effect on empty vector controls (Figure 4E), and the presence of a C-terminal eGFP tag did not alter the inhibitory action (Supplemental Figure 3G). Based on the hypothesis that APOL1 forms a functional ion-conducting pore capable of mediating Ca2+ influx from the extracellular medium, we investigated the functional properties of APOL1 at the plasma membrane. Specifically, we examined two potential modulators of channel activity: extracellular pH and hypotonic cell swelling., The application of an acidic solution (pH 5.4) to cells overexpressing APOL1-EIK variants resulted in a significant inhibition of cytosolic (Ca2+)i levels (Figure 4, G and H). Notably, this inhibitory effect by protons was abolished upon the application of a neutral extracellular bath solution (pH 7.4) and even more significantly with a further increase toward pH 8.4 (Figure 4, G and I). Conversely, the application of a hypotonic solution to APOL1-EIK–overexpressing cells did not cause any significant change in cytosolic (Ca2+)i (Supplemental Figure 4M). In summary, these findings provided evidence that the elevated cytosolic Ca2+ levels observed in APOL1-transfected cells resulted from Ca2+-influx from the extracellular medium, which can be blocked by the APOL1 inhibitor, VX-147, as well as by extracellular protons. In addition, cytosolic (Ca2+)i levels were further increased in cells expressing the G1 and G2 variants, suggesting that both variants represented gain-of-function proteins characterized by enhanced basal APOL1 activity. APOL1-Transfected HEK-293 Cells Showed Increased Current Density Amplitudes Compared to Non-transfected Cells To further investigate the effect of APOL1 expression at the plasma membrane, whole-cell patch-clamp recordings were performed on HEK-293 cells, transiently overexpressing different APOL1-EIK variants. The basal APOL1-EIK G0 activity was monitored by application of voltage ramps ranging from −120 mV toward +120 mV. At indicated time points, the extracellular cations were all replaced by the larger cation N-methyl-d-glucamine (NMDG+) to evaluate for leak detection. Basal recordings resulted in a linear current (I)—voltage (V) relationship with a reversal potential around 0 mV. The mean current amplitudes in APOL1-EIK G0–transfected cells were significantly larger compared to non-transfected cells (Figure 5, A–D, and G). Application of the APOL1 blocker, VX-147 (1 μM), evoked robust voltage independent inhibition (82.4%±6.0% block at −80 mV) of the in- and outward currents in APOL1-EIK G0–expressing cells, while VX-147 was without effect on non-transfected cells (3.5%±1.0% block at −80 mV; Figure 5, A–D, and H). Interestingly, cells overexpressing the APOL1-EIK G2 variant showed a similar linear I–V relationship with a reversal potential around 0 mV and a strong block (64.5%±7.0% at −80 mV) by VX-147 (Figure 5, E, F, and H). Moreover, the mean current amplitudes in APOL1-EIK G2–transfected cells were increased compared to APOL1-EIK G0–expressing cells (Figure 5G). Subsequently, increasing the amount of protons in the extracellular medium by lowering the pH toward pH 5.4 significantly reduced the current densities in APOL1-EIK G2–transfected cells. Shifting the pH of the extracellular bath solution toward pH 8.4 induced an increase in current densities at +80 mV and −80 mV (Supplemental Figure 5, D–F). In contrast, lowering the extracellular pH toward 5.4 in non-transfected cells (Supplemental Figure 5, A–C) resulted in an increase in current amplitude at +80 mV by activation of the endogenously expressed proton-activated outwardly rectifying anion channel in HEK-293 cells. Taken together, these results indicated that APOL1 overexpression induced increased current-density amplitudes with a linear voltage-dependence, which could be inhibited by VX-147 and by extracellular protons. These key biophysical properties were also evident in APOL1-EIK G2–transfected cells; however, current density amplitudes were higher in G2- than in G0-transfected cells, consistent with enhanced basal activity of the APOL1 G2 variant. Reduction of the Extracellular Ca2+ Levels or Pharmacologic Blockade of APOL1 Pore Activity Attenuated APOL1-Induced Cytotoxicity Next, we investigated whether the elevated cytosolic Ca2+ levels affected the viability of APOL1-transfected cells, and whether we could modulate the effect by either blocking the channel activity with VX-147 or by lowering the extracellular Ca2+-levels. Overexpression of APOL1 G0 haplotypes in HEK-293 cells for 24 hours resulted in a haplotype-dependent reduction in cell viability, with APOL1-EIK G0 exhibiting the highest cytotoxicity compared to EMR and KIK haplotypes (Figure 6A). While APOL1-EMR G0 did not induce detectable toxicity at 24 hours, a modest but significant effect was observed after 48 hours (Supplemental Figure 6A). Because G1 and G2 occur naturally in the EIK haplotype, we compared their cytotoxicity across haplotypes and with APOL1-EIK G0 variant. Overexpression of APOL1-EIK variants resulted in risk variant–dependent cytotoxicity (Figure 6B), which increased over time (Supplemental Figure 6B). Remarkedly, G1 and G2 toxicity was dramatically reduced when expressed outside the EIK background (Supplemental Figure 6, C and D). Additionally, the presence of a single risk variant resulted in decreased cell viability (Supplemental Figure 6E). Importantly, these effects were independent of APOL1 expression levels at both the mRNA (Figure 6, C and D, and Supplemental Figure 6F) and protein levels (Figure 6, E and F, and Supplemental Figure 6G). Given our finding that APOL1 localized to the plasma membrane and mediated extracellular Ca2+ influx, we next examined whether APOL1-induced Ca2+ entry is the principal driver of cytotoxicity. Reducing extracellular Ca2+ concentrations from 2 mM to 0.5 mM or 0.2 mM significantly improved the viability of APOL1-overexpressing cells, whereas increasing extracellular Ca2+ to 8 mM further exacerbated cell death (Figure 6G). Next, we evaluated the effect of VX-147 in APOL1-EIK–transfected cells. VX-147 conferred a significant, concentration-dependent protective effect on APOL1-EIK–expressing cells' viability, with no effect observed in empty vector (Supplemental Figure 6H). Notably, the calculated IC50 of approximately 1 μM demonstrated comparable efficacy across all APOL1 variants, restoring cell viability to levels similar to empty vector-transfected HEK-293 cells (Figure 6H). Importantly, this protective effect was not attributed to a reduced APOL1 transfection efficiency (Figure 6I). The APOL1 M1 Variant Suppressed APOL1-Mediated Ca2+ Influx To elucidate the protective effect of the M1 variant in individuals with the APOL1 G2/G2 genotype, we characterized its functional properties. Immunofluorescence and TIRF microscopy analysis demonstrated ER association and plasma membrane localization of the M1 variant (Figure 7, A–C). Similar to APOL1-EIK variants, motile punctate vesicle-like structures were frequently detected; however, these structures exhibited increased mobility in cells overexpressing APOL1-EIK G2-N264K variant compared with APOL1-EIK G2 (Supplemental Videos 14–17). Consistently, TIR-FRAP analysis revealed comparable global eGFP recovery following photobleaching in APOL1 G0-eGFP cells with or without the N264K substitution. In contrast, in APOL1 G2-eGFP cells, the presence of N264K was associated with a significantly enhanced global eGFP recovery (Figure 7, D and E, and Supplemental Figure 7A, and Supplemental Videos 18–21). To assess the contribution of the M1 variant to Ca2+ homeostasis, basal cytosolic Ca2+ levels were measured in APOL1-transfected HEK-293 cells. Interestingly, cells expressing the N264K variant in both G0 and G2 backgrounds exhibited significantly lower basal cytosolic Ca2+ levels compared with their respective non-N264K counterparts, reaching values comparable to those observed in empty vector-transfected cells (Figure 7, F and G). Consistent with these findings, application of an alkaline extracellular solution (pH 8.4) failed to modulate pore-forming activity in cells expressing the M1 variant (Supplemental Figure 7, B and C), suggesting a loss-of-function phenotype. Next, we examined the effect of M1 variant on APOL1-induced cytotoxicity. Expression of the N264K variant conferred a significant protective effect on cell viability in APOL1-EIK–transfected cells, independent of APOL1 expression levels (Figure 7H and Supplemental Figure 7D). Collectively, these findings indicated that the M1 variant suppressed APOL1-mediated Ca2+ influx, providing a mechanistic explanation for its protective effect in individuals with G2/G2 genotype. Validation of Key Findings in Human Podocyte Lines Podocytopathy is a defining feature of AMKD. Accordingly, we examined whether findings from HEK-293 cells could be recapitulated in human podocytes. Consistent with the findings from HEK-293 cells, immunofluorescence analysis of undifferentiated human podocytes transiently expressing C-terminally eGFP-tagged APOL1-EIK variants demonstrated APOL1 colocalization exclusively with the ER and the plasma membrane (Figure 8, A and B, and Supplemental Figure 8). Moreover, TIRF and TIR-FRAP analyses of plasma membrane dynamics in differentiated human podocytes (cultured for 14 days at 37°C) revealed the presence of motile punctate vesicle-like structures, which were larger and less mobile in podocytes expressing the risk variants, compared to the G0 variant (Figure 8C and Supplemental Videos 22–25). Accordingly, TIR-FRAP analysis demonstrated significantly decreased eGFP recovery in G1- and G2-overexpressing podocytes compared to APOL1 G0 cells (Figure 8, D–F, and Supplemental Videos 26–28). Next, we investigated whether the observed elevated cytosolic Ca2+ levels associated with APOL1 risk variants in HEK-293 cells represented a translatable phenotype in human podocytes. APOL1 overexpression in inducible stable podocyte lines resulted in risk variant–dependent and significantly increased basal cytosolic Ca2+ levels across multiple time points (Figure 9, A–C). Consistently, upregulation of endogenous APOL1 expression by poly(I:C) (Supplemental Figure 9A) resulted in a significantly increased cytosolic Ca2+ levels. Under both basal and poly(I:C)-stimulated conditions, podocytes expressing APOL1 G2/G2 exhibited significantly higher intracellular Ca2+ levels compared with APOL1 G0/G0 podocytes (Supplemental Figure 9, B–E). Next, we examined whether the elevated basal cytosolic Ca2+ levels affected podocyte viability. Induced overexpression of APOL1 in stable podocyte lines resulted in a risk variant–dependent reduction in viability after 48 hours, which was markedly attenuated by VX-147 (Figure 9, D–F). Similarly, podocytes endogenously expressing APOL1 G2/G2 showed significantly reduced metabolic activity under basal conditions compared with G0/G0 podocytes (Supplemental Figure 9F). Moreover, endogenous APOL1 expression induced by 24-hour poly(I:C) treatment led to a pronounced decrease in cell viability (Supplemental Figure 9G). These effects were independent of APOL1 expression levels (Figure 9, G and H, and Supplemental Figure 9, H and I). Collectively, these findings in human podocytes closely mirrored those observed in HEK-293 cells.

The mechanisms by which APOL1 risk variants induce kidney disease in people with African ancestry remain incompletely understood. Using complementary overexpression models in HEK-293 cells and human podocytes, combined with genetic, electrophysiological, and advanced imaging approaches, we provide three key insights into AMKD pathogenesis. First, APOL1 variants are trafficked to the plasma membrane via actin-dependent transport, where they display distinct dynamic properties. Second, APOL1 is functionally expressed at the plasma membrane as a voltage-dependent non-selective cation-permeable pore that mediates haplotype-dependent cytotoxicity. Third, APOL1 variants expressed in the African haplotype background promote increased Ca2+ influx into the cytoplasm, enhancing cytotoxicity, an effect attenuated by the N264K (M1) variant. Previous studies have reported variability in the subcellular localization of APOL1, attributable in part to the lack of selective antibodies for APOL1 and to differences in haplotype background. To overcome these limitations, we transiently transfected undifferentiated human podocytes and HEK-293 cells with C-terminally eGFP-tagged APOL1-EIK variants. Consistent with Scales et al., plasma membrane isolation using colloidal silica and immunofluorescence analyses revealed that APOL1 predominantly colocalizes with the ER and the plasma membrane in both models. Via live-cell TIR-FRAP microscopy, we were able to delineate, for the first time, the trafficking pathways and plasma membrane–proximal dynamics of APOL1 variants. Our results indicate that APOL1 is transported to the plasma membrane via actin-dependent mechanisms, where it exhibits variant-specific dynamic behavior and functional expression. In differentiated human podocytes and HEK-293 cells expressing the APOL1 risk variants G1 and G2, we observed larger, less mobile vesicle-like structures, accompanied by reduced global fluorescence recovery following photobleaching. Interestingly, introduction of the N264K substitution in the APOL1-EIK G2 variant reversed these observations, restoring increased vesicular mobility and significantly enhancing global eGFP recovery. These findings contrast with Gupta et al. who reported accelerated plasma membrane trafficking of risk variants. Given that recovery kinetics were comparable among all variants in our study, our data instead suggest that trafficking dynamics of APOL1 risk variants at or near the plasma membrane are altered, rather than accelerated. To further examine this possibility, cells overexpressing APOL1-EIK risk variants were pretreated with cytochalasin D for 1 hour prior to analysis. Although a trend toward reduced fluorescence recovery was observed in APOL1-EIK G1–transfected cells, this effect was not statistically significant, potentially reflecting pre-existing disruption of the actin cytoskeleton. Notably, APOL1-positive vesicle-like structures in G1- and G2-expressing cells colocalized with lysosome-associated membrane protein 1-positive compartments, suggesting that these punctate structures may traffic through the late endosomal or lysosomal pathway. Nevertheless, plasma membrane isolation assays confirmed the presence of APOL1 at the plasma membrane. Together, these findings indicate that although actin-dependent processes contribute to APOL1 trafficking, the precise mechanisms regulating APOL1 membrane trafficking remain incompletely defined and warrant further investigation including functional characterization of the APOL1-positive vesicle-like structures. Plasma membrane localization of APOL1 has been postulated as an essential mechanism in AMKD., Here, we identify APOL1 as a pore-forming channel that conducts Ca2+ and Na+ ions in both a heterologous expression system and in human podocytes. This conclusion is supported by several observations: (1) APOL1-transfected cells exhibited elevated basal cytosolic Ca2+ levels compared with non-transfected and empty vector transfected controls, an effect that was inhibited by VX-147 or by introduction of the N264K variant; (2) increased cytosolic Ca2+ levels were observed in differentiated human podocytes expressing APOL1; (3) APOL1 expression was associated with increased basal whole-cell current densities relative to non-transfected cells; (4) APOL1-mediated currents displayed a linear current–voltage relationship with a reversal potential near 0 mV, consistent with a non-selective cation conductance; and (5) basal inward currents were selectively inhibited by VX-147 and protons in APOL1-overexpressing cells. Although APOL1-mediated Na+ and Ca2+ conductance has been reported,,, its ion selectivity remains debated. Earlier studies have proposed that APOL1 promotes K+ efflux,,, whereas others have suggested anion permeability, particularly Cl− selectivity., Alternatively, APOL1-induced cation flux has been proposed to occur indirectly through activation of endogenous plasma membrane ion channels such as the transient receptor potential Ca2+ channel 6 (TRPC6). However, RNA sequencing was performed in stable podocyte lines expressing APOL1 following doxycycline treatment for 72 hours to determine whether the calcineurin (Cn)/nuclear factor of activated T cells (NFAT)/TRPC6 signaling pathway is concomitantly activated. Transcriptomic analysis revealed no significant differential expression of genes within the Cn/NFAT/TRPC6 pathway (Supplemental Figures 10–11). These findings indicate that, under our experimental conditions, the Cn/NFAT/TRPC6 pathway is not activated and therefore is unlikely to play a parallel role in APOL1-mediated podocytopathy. APOL1 channel activity is modulated by multiple factors., We demonstrate that APOL1 exhibits basal activity at the plasma membrane, in the absence of exogenous ligands, and that this APOL1 activity is modulated by extracellular protons, as shown by both Ca2+ microfluorimetric and patch-clamp analysis. This observed proton inhibition result aligns with other studies.,, Although APOL1 risk variants expression has been associated with cell swelling,,, presumably as a consequence of dysregulated cation influx,, under our experimental conditions, we did not observe an effect of hypotonic-induced swelling on cytosolic (Ca2+)i levels in APOL1-expressing HEK-293 cells. Furthermore, we identified the extracellular milieu as the primary source of the APOL1-induced Ca2+ influx, representing an upstream event that drives cellular cytotoxicity. This conclusion is consistent with findings by Giovinazzo et al., but contrasts with those reported by Datta et al. Although the ER plays a central role in Ca2+ storage, sequestration, and release,, several lines of evidence support an extracellular origin of the increased cytosolic Ca2+ observed in our study. First, depletion of ER Ca2+ stores using thapsigargin failed to produce a significant increase in cytosolic Ca2+ levels or a concomitant decrease in ER-GCaMP6f fluorescence. Notably, no significant differences were detected in ER Ca2+ content between APOL1-G0 and -G1/G2–expressing cells. Second, removal of extracellular Ca2+ resulted in a marked reduction of basal cytosolic Ca2+ levels in APOL1-transfected cells. Third, increasing extracellular Ca2+ concentrations caused a dose-dependent exacerbation of cytotoxicity in APOL1-overexpressing cells. Finally, pharmacologic inhibition of APOL1 pore activity with VX-147 induced an immediate reduction in cytosolic Ca2+ levels. Moreover, further characterization of the N264K variant demonstrates that this protective variant has preserved plasma membrane expression but does not result in elevated cytosolic Ca2+ levels. Collectively, these findings underscore a critical role for extracellular Ca2+ influx in AMKD and highlight the need for future studies to further elucidate the role of Ca2+ dysregulation in the pathogenesis of AMKD. In the present study, we provide evidence for basal APOL1 channel activity under standard conditions, as mean basal current amplitudes were significantly higher in cells overexpressing the APOL1 G2 variant compared with G0. In addition, cells expressing APOL1 risk variants exhibited a higher increase in (Ca2+)i influx than those expressing the non-risk APOL1 G0 variant. Similar phenotypes were observed in differentiated human podocyte lines. Notably, expression of APOL1 risk variants in the EIK haplotype background resulted in an approximately threefold increase in cytosolic Ca2+ levels compared with other haplotypes, whereas introduction of the N264K variant did not result in increased cytosolic Ca2+ levels. These findings are consistent with previous reports and support a model in which haplotype-dependent APOL1 Ca2+ channel activity drives risk variant cytotoxicity.,, Although the mechanisms linking these variants to cytotoxicity remain incompletely understood, dysregulated intracellular K+ efflux and elevations in intracellular Ca2+ and Na+ levels have been associated with AMKD., Accordingly, if APOL1 channel activity contributes directly to cell death, cytotoxicity would be expected to parallel changes in intracellular Ca2+ levels. Consistent with this hypothesis, cell viability decreased in a variant- and haplotype-dependent manner, with maximal cytotoxicity observed for the risk variants in the EIK haplotype background. This effect was attenuated by the N264K variant, in agreement with previous findings of Lannon et al. The enhancement of pore-forming activity conferred by the EIK haplotype, together with its attenuation by the M1 variant, supports a gain-of-function mechanism for G1 and G2 risk variants and a rather loss-of-function effect for M1 variant in the Ca2+ permeability. However, we cannot exclude the possibility that the N264K variant retains additional functional properties, including potential conductance of other ions such as Na+, or exerts context-dependent effects not captured in the present assays. Nevertheless, although these findings implicate APOL1 channel activity in cytotoxicity, a direct causal relationship between risk variant channel function and the development of AMKD has yet to be fully established. Accordingly, the precise molecular mechanisms underlying these effects warrant further investigation. This study has several limitations. While we focused on the pore-forming activity of APOL1, we did not evaluate the contribution of other cations such as K+ or Mg2+, nor did we assess the potential contribution of mitochondrial calcium in the pathogenesis of AMKD. Additionally, possible interactions between APOL1 and APOL3, as well as other plasma membrane ion channels that may modulate APOL1 channel function, were not investigated. In conclusion, we demonstrated that APOL1 variants traffic to the plasma membrane through actin-dependent mechanisms, where they form non-selective pores permeable to Na+ and Ca2+ ions, an effect attenuated by the M1 variant and VX-147. We further identified the extracellular medium as the primary origin of cytosolic Ca2+ influx driving AMKD. Collectively, these findings support a gain-of-function model in which APOL1 risk variants promote haplotype-dependent cytotoxicity and highlight both the protective M1 variant pathway and pharmacologic inhibition with VX-147 as potential therapeutic strategies for AMKD. Further investigation of APOL1 trafficking and plasma membrane dynamics may facilitate the development of more and effective interventions for this disorder. Disclosures Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/F894. Author Contributions Conceptualization: Oyindamola Christiana Adebayo, Veerle Labarque, Elena N. Levtchenko, Joris Vriens, Lambertus van den Heuvel. Data curation: Oyindamola Christiana Adebayo, Tjessa Bondue, Nikky Corthout, Charlotte Cresens, Ilhem Dallali, Sergio Gimeno-Rocafort, Sara Kerselaers, Sandra Van Aerschot. Formal analysis: Oyindamola Christiana Adebayo, Nnamdi Joseph Asouzu, Tjessa Bondue, Charlotte Cresens, Ilhem Dallali, Sara Kerselaers, Joris Vriens. Funding acquisition: Oyindamola Christiana Adebayo, Veerle Labarque. Investigation: Oyindamola Christiana Adebayo, Tjessa Bondue, Nikky Corthout, Charlotte Cresens, Ilhem Dallali, Sergio Gimeno-Rocafort, Sara Kerselaers, Sandra Van Aerschot. Methodology: Oyindamola Christiana Adebayo, Nikky Corthout, Charlotte Cresens, Ilhem Dallali, Rik Gijsbers, Annelies Janssens, Sara Kerselaers, Joris Vriens. Project administration: Oyindamola Christiana Adebayo, Veerle Labarque, Joris Vriens. Resources: Veerle Labarque, Joris Vriens. Software: Nnamdi Joseph Asouzu, Joris Vriens. Supervision: Veerle Labarque, Elena N. Levtchenko, Lambertus van den Heuvel, Joris Vriens. Validation: Oyindamola Christiana Adebayo, Joris Vriens. Visualization: Oyindamola Christiana Adebayo, Joris Vriens. Writing – original draft: Oyindamola Christiana Adebayo, Joris Vriens. Writing – review & editing: Oyindamola Christiana Adebayo, Veerle Labarque, Elena N. Levtchenko, Lambertus van den Heuvel, Joris Vriens. Funding O.C. Adebayo: Fonds Wetenschappelijk Onderzoek (11A5621N and 11A5623N). T. Bondue: Fonds Wetenschappelijk Onderzoek (11A7821N and 11A7823N). J. Vriens: Fonds Wetenschappelijk Onderzoek (G0D1417N, G084515N, and G0A6719N) and KU Leuven (C14/24/152). V. Labarque: CSL Behring, Abacus. Acknowledgments The authors gratefully acknowledge Evelien van Hoeymissen, Alex Englezakis, and Melissa Benoit (Laboratory of Ion Channel Research, KU Leuven, Belgium) and Inge Bongaers, Henry Okpoli, and Sara Akalay (Laboratory of Pediatric Nephrology, KU Leuven, Belgium) for their technical assistance. Further gratitude is extended to Marleen Derweduwe and Prof. Frederik De Smet (Laboratory for Precision Cancer Medicine, KU Leuven, Belgium) for their support and assistance with the usage of the microplate reader. Prof. Pieter Vanden Berghe (Confocal Imaging Cluster [CIC], KU Leuven, Belgium) is acknowledged for allowing the usage of the confocal microscope (supported by Hercules AKUL/15/37_GOH1816N and FWO G.0929.15 to Pieter Vanden Berghe, KU Leuven). We appreciate Prof. Llew Roderick (Laboratory of Experimental Cardiology, KU Leuven, Belgium) and Tom Leroy (Laboratory of Membrane Trafficking, KU Leuven, Belgium) for providing the authors with the calreticulin plasmid and VAPB antibody, respectively. Maxime Smits and Joris Van Asselberghs (Leuven Viral Vector Core, KU Leuven, Belgium) are acknowledged for expert technical support in generating the lentiviral vectors. We are grateful to Dr. Enrico Skoruppa (School of Physics and Astronomy, The University of Edinburgh, Scotland) for his help with analyzing the ER-GCaMP6f data. Abstracts have been presented at the Annual Meeting of the Belgian Society of Nephrology, May 22, 2025, Vilvoorde, Belgium, and at the 15th International Podocyte Conference, June 11–June 13, 2025, Hamburg, Germany. Data Availability Statements Original data generated for the study are available in a public access repository. Original data generated for the study will be made available upon reasonable request to the corresponding author. Data Type: Image Data; Software Executable Code; Raw Data/Source Data. Repository Name: GitHub. The code used for the differential expression analysis of the total RNA sequencing data is publicly available at https://github.com/asouzujoseph/APOL1-at-the-plasma-membrane-and-cytotoxicity. Supplemental videos associated with this study can be accessed at https://doi.org/10.48804/DTBMHW. All plasmids and cell lines are available from the corresponding author upon request, in accordance with institutional material transfer agreements and third-party agreements. The values for all data points in the graphs are available upon request from the corresponding author. Supplemental Material This article contains supplemental material online, published as provided by the authors, at http://links.lww.com/JSN/F895. Supplemental Video 1. TIRF video of HEK-293 cell transfected with empty vector. Video is related to Figure 1C. https://doi.org/10.48804/DTBMHW. Supplemental Video 2. TIRF video of HEK-293 cell transfected with APOL1-EIK G0. Video is related to Figure 1C. https://doi.org/10.48804/DTBMHW. Supplemental Video 3. TIRF video of HEK-293 cell transfected with APOL1-EIK G1. Video is related to Figure 1C. https://doi.org/10.48804/DTBMHW. Supplemental Video 4. TIRF video of HEK-293 cell transfected with APOL1-EIK G2. Video is related to Figure 1C. https://doi.org/10.48804/DTBMHW. Supplemental Video 5. TIRF video of HEK-293 cell co-transfected with APOL1-EIK G0 and mCherry-LAMP1. Video is related to Figure 1D. https://doi.org/10.48804/DTBMHW. Supplemental Video 6. TIRF video of HEK-293 cell co-transfected with APOL1-EIK G1 and mCherry-LAMP1. Video is related to Figure 1D. https://doi.org/10.48804/DTBMHW. Supplemental Video 7. TIRF video of HEK-293 cell co-transfected with APOL1-EIK G2 and mCherry-LAMP1. Video is related to Figure 1D. https://doi.org/10.48804/DTBMHW. Supplemental Video 8. Baseline TIR-FRAP video of HEK-293 cell transfected with APOL1-EIK G0. Video is related to Figure 2B. https://doi.org/10.48804/DTBMHW. Supplemental Video 9. Baseline TIR-FRAP video of HEK-293 cell transfected with APOL1-EIK G1. Video is related to Figure 2B. https://doi.org/10.48804/DTBMHW. Supplemental Video 10. Baseline TIR-FRAP video of HEK-293 cell transfected with APOL1-EIK G2. Video is related to Figure 2B. https://doi.org/10.48804/DTBMHW. Supplemental Video 11. TIR-FRAP video of untreated APOL1-EIK G0–transfected HEK-293 cell. Video is related to Figure 2F. https://doi.org/10.48804/DTBMHW. Supplemental Video 12. TIR-FRAP video of APOL1-EIK G0–transfected HEK-293 cell treated with Cytochalasin D. Video is related to Figure 2F. https://doi.org/10.48804/DTBMHW. Supplemental Video 13. TIR-FRAP video of APOL1-EIK G0–transfected HEK-293 cell treated with Nocodazole. Video is related to Figure 2F. https://doi.org/10.48804/DTBMHW. Supplemental Video 14. TIRF video of HEK-293 cell transfected with APOL1-EIK G0. Video is related to Figure 7C. https://doi.org/10.48804/DTBMHW. Supplemental Video 15. TIRF video of HEK-293 cell transfected with APOL1-EIK G0-N264K. Video is related to Figure 7C. https://doi.org/10.48804/DTBMHW. Supplemental Video 16. TIRF video of HEK-293 cell transfected with APOL1-EIK G2. Video is related to Figure 7C. https://doi.org/10.48804/DTBMHW. Supplemental Video 17. TIRF video of HEK-293 cell transfected with APOL1-EIK G2-N264K. Video is related to Figure 7C. https://doi.org/10.48804/DTBMHW. Supplemental Video 18. Baseline TIR-FRAP video of HEK-293 cell transfected with APOL1-EIK G0. Video is related to Supplemental Figure 7A. https://doi.org/10.48804/DTBMHW. Supplemental Video 19. Baseline TIR-FRAP video of HEK-293 cell transfected with APOL1-EIK G0-N264K. Video is related to Supplemental Figure 7A. https://doi.org/10.48804/DTBMHW. Supplemental Video 20. Baseline TIR-FRAP video of HEK-293 cell transfected with APOL1-EIK G2. Video is related to Supplemental Figure 7A. https://doi.org/10.48804/DTBMHW. Supplemental Video 21. Baseline TIR-FRAP video of HEK-293 cell transfected with APOL1-EIK G2-N264K. Video is related to Supplemental Figure 7A. https://doi.org/10.48804/DTBMHW. Supplemental Video 22. TIRF video of differentiated human podocyte transfected with empty vector. Video is related to Figure 8C. https://doi.org/10.48804/DTBMHW. Supplemental Video 23. TIRF video of differentiated human podocyte transfected with APOL1-EIK G0. Video is related to Figure 8C. https://doi.org/10.48804/DTBMHW. Supplemental Video 24. TIRF video of differentiated human podocyte transfected with APOL1-EIK G1. Video is related to Figure 8C. https://doi.org/10.48804/DTBMHW. Supplemental Video 25. TIRF video of differentiated human podocyte transfected with APOL1-EIK G2. Video is related to Figure 8C. https://doi.org/10.48804/DTBMHW. Supplemental Video 26. Baseline TIR-FRAP video of differentiated human podocyte transfected with APOL1-EIK G0. Video is related to Figure 8D. https://doi.org/10.48804/DTBMHW. Supplemental Video 27. Baseline TIR-FRAP video of differentiated human podocyte transfected with APOL1-EIK G1. Video is related to Figure 8D. https://doi.org/10.48804/DTBMHW. Supplemental Video 28. Baseline TIR-FRAP video of differentiated human podocyte transfected with APOL1-EIK G2. Video is related to Figure 8D. https://doi.org/10.48804/DTBMHW. Supplemental Table 1. List of reagents. Supplemental Figure 1. Subcellular localization of APOL1 in HEK-293 cells. Supplemental Figure 2. TIR-FRAP revealed distinct modes of APOL1 variants recovery at the plasma membrane. Supplemental Figure 3. APOL1 expression affected basal cytosolic Ca2+ levels in a haplotype-dependent manner. Supplemental Figure 4. APOL1 modulated extracellular Ca2+ influx in a risk variant–dependent manner. Supplemental Figure 5. Electrophysiological characterization of HEK-293 cells overexpressing different APOL1-EIK variants. Supplemental Figure 6. APOL1-induced cytotoxicity was rescued by extracellular Ca2+ reduction or pharmacologic pore inhibition. Supplemental Figure 7. The APOL1 M1 variant was associated with a loss-of-function in pore-forming activity. Supplemental Figure 8. Subcellular localization of APOL1 in human podocytes. Supplemental Figure 9. Differentiated human podocyte lines endogenously expressing different APOL1 genotypes exhibited altered cytosolic Ca2+ levels and cellular viability. Supplemental Figure 10. Total RNA sequencing of differentiated human podocyte lines stably expressing APOL1 variants. Supplemental Figure 11. Transcriptomic profiling of differentiated human podocyte lines stably expressing APOL1 variants.

1. Ekulu PM, Nkoy AB, Betukumesu DK, et al. APOL1 risk genotypes are associated with early kidney damage in children in sub-Saharan Africa. Kidney Int Rep. 2019;4(7):930–938. doi:10.1016/j.ekir.2019.04.0022. Ekulu PM, Nkoy AB, Adebayo OC, et al. A focus on the association of Apol1 with kidney disease in children. Pediatr Nephrol. 2021;36(4):777–788. doi:10.1007/s00467-020-04553-z3. Adebayo OC, Betukumesu DK, Nkoy AB, et al. Clinical and genetic factors are associated with kidney complications in African children with sickle cell anaemia. Br J Haematol. 2022;196(1):204–214. doi:10.1111/bjh.178324. Friedman DJ, Pollak MR. Genetics of kidney failure and the evolving story of APOL1. J Clin Invest. 2011;121(9):3367–3374. doi:10.1172/JCI462635. Adebayo OC, Nkoy AB, van den Heuvel LP, et al. Glomerular hyperfiltration: part 2-clinical significance in children. Pediatr Nephrol. 2023;38(8):2529–2547. doi:10.1007/s00467-022-05826-56. Datta S, Antonio BM, Zahler NH, et al. APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease. J Clin Invest. 2024;134(5):e172262. doi:10.1172/JCI1722627. Olabisi OA, Zhang J-Y, VerPlank L, et al. APOL1 kidney disease risk variants cause cytotoxicity by depleting cellular potassium and inducing stress-activated protein kinases. Proc Natl Acad Sci U S A. 2016;113(4):830–837. doi:10.1073/pnas.15229131138. Gupta N, Waas B, Austin D, et al. Apolipoprotein L1 (APOL1) renal risk variant-mediated podocyte cytotoxicity depends on African haplotype and surface expression. Sci Rep. 2024;14(1):3765. doi:10.1038/s41598-024-53298-49. Egbuna O, Zimmerman B, Manos G, et al. Inaxaplin for proteinuric kidney disease in persons with two APOL1 variants. N Engl J Med. 2023;388(11):969–979. doi:10.1056/NEJMoa220239610. Thomson R, Finkelstein A. Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: relevance to trypanosome lysis. Proc Natl Acad Sci U S A. 2015;112(9):2894–2899. doi:10.1073/pnas.142195311211. Giovinazzo JA, Thomson RP, Khalizova N, et al. Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity. eLife. 2020;9:e51185. doi:10.7554/eLife.5118512. Heneghan JF, Vandorpe DH, Shmukler BE, et al. BH3 domain-independent apolipoprotein L1 toxicity rescued by BCL2 prosurvival proteins. Am J Physiol Cell Physiol. 2015;309(5):C332–C347. doi:10.1152/ajpcell.00142.201513. Pays E. The mechanism of kidney disease due to APOL1 risk variants: involvement of two distinct processes. J Am Soc Nephrol. 2024;35(6):818–821. doi:10.1681/ASN.000000000000036714. Friedman DJ, Pollak MR. In search of the mechanism of APOL1 kidney disease. J Am Soc Nephrol. 2024;35(6):815–817. doi:10.1681/ASN.000000000000036815. Adebayo OC, Okpoli HC, Vriens J, Levtchenko E, Heuvel LVD, Labarque V. APOL1-mediated kidney disease: a narrative review of the lessons learnt from the past 15 years. BMC Nephrol. 2025;27(1):60. doi:10.1186/s12882-025-04687-y16. Lannon H, Shah SS, Dias L, et al. Apolipoprotein L1 (APOL1) risk variant toxicity depends on the haplotype background. Kidney Int. 2019;96(6):1303–1307. doi:10.1016/j.kint.2019.07.01017. Gupta Y, Friedman DJ, McNulty MT, et al. Strong protective effect of the APOL1 p.N264K variant against G2-associated focal segmental glomerulosclerosis and kidney disease. Nat Commun. 2023;14(1):7836. doi:10.1038/s41467-023-43020-918. Hung AM, Assimon VA, Chen HC, et al. Genetic inhibition of APOL1 pore-forming function prevents APOL1-mediated kidney disease. J Am Soc Nephrol. 2023;34(11):1889–1899. doi:10.1681/ASN.000000000000021919. Martinelli E, Ke J, Khan A, et al. Precision diagnosis in APOL1 kidney disease with the p.N264K M1 protective variant. JAMA Netw Open. 2026;9(3):e261452. doi:10.1001/jamanetworkopen.2026.145220. Graham FL, Russell WC, Smiley J, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977;36(1):59–72. doi:10.1099/0022-1317-36-1-5921. Saleem MA, O'Hare MJ, Reiser J, et al. A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression. J Am Soc Nephrol. 2002;13(3):630–638. doi:10.1681/ASN.V13363022. Ekulu PM, Adebayo OC, Decuypere JP, et al. Novel human podocyte cell model carrying G2/G2 APOL1 high-risk genotype. Cells. 2021;10(8):1914. doi:10.3390/cells1008191423. Uzureau S, Lecordier L, Uzureau P, et al. APOL1 C-terminal variants may trigger kidney disease through interference with APOL3 control of actomyosin. Cell Rep. 2020;30(11):3821–3836.e13. doi:10.1016/j.celrep.2020.02.06424. Vriens J, Owsianik G, Janssens A, Voets T, Nilius B. Determinants of 4 alpha-phorbol sensitivity in transmembrane domains 3 and 4 of the cation channel TRPV4. J Biol Chem. 2007;282(17):12796–12803. doi:10.1074/jbc.M61048520025. Ramazani Y, Knops N, Berlingerio SP, et al. Therapeutic concentrations of calcineurin inhibitors do not deregulate glutathione redox balance in human renal proximal tubule cells. PLoS One. 2021;16(4):e0250996. doi:10.1371/journal.pone.025099626. Chaney LK, Jacobson BS. Coating cells with colloidal silica for high yield isolation of plasma membrane sheets and identification of transmembrane proteins. J Biol Chem. 1983;258(16):10062–10072. doi:10.1016/s0021-9258(17)44606-027. Paparelli L, Corthout N, Pavie B, et al. Inhomogeneity based characterization of distribution patterns on the plasma membrane. PLoS Comput Biol. 2016;12(9):e1005095. doi:10.1371/journal.pcbi.100509528. Ghosh D, Pinto S, Danglot L, et al. VAMP7 regulates constitutive membrane incorporation of the cold-activated channel TRPM8. Nat Commun. 2016;7(1):10489. doi:10.1038/ncomms1048929. Ghosh D, Segal A, Voets T. Distinct modes of perimembrane TRP channel turnover revealed by TIR-FRAP. Sci Rep. 2014;4(1):7111. doi:10.1038/srep0711130. Van Hoeymissen E, Held K, Nogueira Freitas AC, Janssens A, Voets T, Vriens J. Gain of channel function and modified gating properties in TRPM3 mutants causing intellectual disability and epilepsy. eLife. 2020;9:e57190. doi:10.7554/eLife.5719031. Scales SJ, Gupta N, De Maziere AM, et al. Apolipoprotein L1-specific antibodies detect endogenous APOL1 inside the endoplasmic reticulum and on the plasma membrane of podocytes. J Am Soc Nephrol. 2020;31(9):2044–2064. doi:10.1681/ASN.201908082932. Gbadegesin RA, Ulasi I, Ajayi S, et al. APOL1 Bi- and monoallelic variants and chronic kidney disease in West Africans. N Engl J Med 2025;392(3):228–238. doi:10.1056/NEJMoa240421133. Aryal SP, Xia M, Adindu E, Davis C, Ortinski PI, Richards CI. ER-GCaMP6f: an endoplasmic reticulum-targeted genetic probe to measure calcium activity in astrocytic processes. Anal Chem. 2022;94(4):2099–2108. doi:10.1021/acs.analchem.1c0432134. Vriens J, Watanabe H, Janssens A, Droogmans G, Voets T, Nilius B. Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4. Proc Natl Acad Sci U S A. 2004;101(1):396–401. doi:10.1073/pnas.030332910135. O'Toole JF, Schilling W, Kunze D, et al. ApoL1 overexpression drives variant-independent cytotoxicity. J Am Soc Nephrol. 2018;29(3):869–879. doi:10.1681/ASN.201612132236. Lambert S, Oberwinkler J. Characterization of a proton-activated, outwardly rectifying anion channel. J Physiol. 2005;567(1):191–213. doi:10.1113/jphysiol.2005.08988837. Winkler RL, Bruno J, Buchanan P, Edwards JC. Cation channel activity of apolipoprotein L1 is modulated by haplotype background. J Am Soc Nephrol. 2022;33(9):1673–1675. doi:10.1681/ASN.202202021338. Perez-Morga D, Vanhollebeke B, Paturiaux-Hanocq F, et al. Apolipoprotein L-I promotes trypanosome lysis by forming pores in lysosomal membranes. Science. 2005;309(5733):469–472. doi:10.1126/science.111456639. Bruno J, Pozzi N, Oliva J, Edwards JC. Apolipoprotein L1 confers pH-switchable ion permeability to phospholipid vesicles. J Biol Chem. 2017;292(44):18344–18353. doi:10.1074/jbc.M117.81344440. Vandorpe DH, Heneghan JF, Waitzman JS, et al. Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes. Pflügers Archiv. 2023;475(3):323–341. doi:10.1007/s00424-022-02767-841. Zhivotovsky B, Orrenius S. Calcium and cell death mechanisms: a perspective from the cell death community. Cell Calcium. 2011;50(3):211–221. doi:10.1016/j.ceca.2011.03.00342. Berridge MJ. The endoplasmic reticulum: a multifunctional signaling organelle. Cell Calcium. 2002;32(5-6):235–249. doi:10.1016/s0143416002001823