The phosphoinositide 3-kinase inhibitor alpelisib restores actin organization and improves proximal tubule dysfunction in vitro and in a mouse model of Lowe syndrome and Dent disease

Abstract

Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4,5-bisphosphate [PI(4,5)P₂] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI(4,5)P₂ and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI(3)P] has been recently identified as a coactivator with PI(4,5)P₂ in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K inhibitor copanlisib and class IA p110α PI3K inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3,4,5-trisphosphate, PI(4,5)P₂ and PI(3)P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110α phenocopied the actin phenotype. In a humanized Ocrl^Y/- mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2.

Keywords: cytoskeleton; endocytosis; lipids; proximal tubule; renal Fanconi syndrome.

Graphical abstract

Figure 1

PI3K inhibitors relieve aberrant actin assembly at endosomes in an OCRL-deficient human kidney (HK2) cell model. (a) Steps of phosphoinositide lipid conversion relevant for this study indicating the conversions between PI(4,5)P₂, PI(3,4,5)P₂, and PI(3)P with the most relevant enzymes in bold, most relevant conversions in solid lines, and others in dashed lines. PI(4,5)P₂ is elevated in Lowe syndrome due to the lack of 5-phosphatase activity from OCRL and is made from the phosphorylation of PI by phosphatidylinositol 4-kinases (PI4Ks) and PI(4)P 5-kinase (PIP5K). PI(4,5)P₂ is phosphorylated by class I PI3Ks to produce PI(3,4,5)P₃. PI(3,4,5)P₃ can be dephosphorylated to PI(4,5)P₂ by PTEN or to PI(3,4)P₂ by SH-2–containing inositol 5′ polyphosphatase (SHIP) 1 and 2, synaptojanin 1 and 2, and also OCRL, although this is thought to be minor. PI(3,4)P₂ is dephosphorylated to PI(3)P by inositol polyphosphate-4-phosphatase type I A (INPP4A) and B. PI(3)P is also made at endosomes via the phosphorylation of PI by class III PI3K, vacuolar sorting protein Vps34. (b) Western blots illustrating OCRL expression loss in the HK2 OCRL CRISPR knockout (KO) cell line compared with wild-type (WT) HK2 control cells with α-tubulin as loading control. (c–e) Representative Airyscan confocal micrographs and quantification of early endosome antigen 1 (EEA1)/actin overlap (expressed as a percentage of total detected EEA1+ vesicles) for HK2 WT or OCRL KO cells treated with either dimethylsulfoxide (DMSO) (d) or the indicated inhibitor and fixed using the 4% formaldehyde fix. In all cases, the images illustrate a single z-slice from an Airyscan-processed confocal stack of cells immunolabeled for EEA1 (yellow), phalloidin (actin, magenta), and 4′,6-diamidino-2-phenylindole (DAPI) (cyan). Bars = 5 μm. In the quantifications, the lines indicate the mean ± SEM and each data point is an individual cell. In all experiments, treatments were applied 16 hours before fixation. In all quantifications, statistical significance was assessed by a Kruskal-Wallis (K-W) analysis of variance with Dunn’s multiple comparisons test. (c) WT or KO cells treated with either DMSO or 100 nM of copanlisib, demonstrating rescue of the actin-endosomal overlap. K-W test: ∗∗∗P < 0.001, multiple comparisons; WT DMSO versus KO DMSO, KO DMSO versus KO copanlisib both ∗∗∗P < 0.001, WT copanlisib versus KO copanlisib P = 0.44 (not significant [ns]). N = 52, 85, 67, and 63 cells for WT DMSO, WT copanlisib, KO DMSO, and KO copanlisib, respectively. (d) WT or KO cells treated with either DMSO or 10 μM of alpelisib, demonstrating rescue of the actin-endosomal overlap. K-W test: ∗∗∗P < 0.001, multiple comparisons; WT DMSO versus KO DMSO, KO DMSO versus KO alpelisib both ∗∗∗P < 0.001, WT alpelisib versus KO alpelisib. P > 0.99 (ns). N = 31, 30, 43, and 41 cells for WT DMSO, WT alpelisib, KO DMSO, and KO alpelisib, respectively. (e) WT or KO cells treated with either DMSO, 10 μM of GSK2636771, or 10 μM of idelalisib, demonstrating that neither compound is able to significantly reduce the actin-endosomal overlap. K-W test: ∗∗∗P < 0.001, multiple comparisons; WT DMSO versus KO DMSO, ∗∗∗P < 0.001, KO DMSO versus KO GSK, ∗P = 0.04, KO DMSO versus KO idelalisib, P > 0.99 (ns). N = 159, 130, 203, 122, 131, and 145 cells for WT DMSO, WT GSK2636771, WT idelalisib, KO DMSO, KO GSK2636771, and KO idelalisib, respectively. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 2

Alpelisib effects on actin are dose-responsive and recapitulated by siRNA of PI3K p110α. (a) Representative Airyscan confocal micrographs (fixed using the 4% formaldehyde fix and immunolabeled for early endosome antigen 1 [EEA1], yellow; actin [phalloidin], magenta; and 4′,6-diamidino-2-phenylindole [DAPI], cyan; bars = 5 μm) and quantification of wild-type (WT) or knockout (KO) human kidney (HK2) cells treated with dimethylsulfoxide (DMSO) or the indicated doses of alpelisib for 16 hours, demonstrating dose-responsive rescue of the actin-endosomal overlap. In all cases, the lines indicate mean ± SEM and the points indicate individual cells. N = 31, 30, 71, 42, 90, 89, 41, 69, 66, and 67 cells for WT control, WT 10 μM, WT 50 μM, KO DMSO, and KO 2.5, 5, 10, 15, 25, and 50 μM alpelisib, respectively. Statistical significance assessed by the Kruskal-Wallis (K-W) test with Dunn’s multiple comparisons test: overall ∗∗∗P < 0.001, multiple comparisons; WT DMSO versus KO DMSO, KO DMSO versus KO 5, 15, 25, and 50 μM alpelisib all ∗∗∗P < 0.001, KO DMSO versus KO 2.5 μM alpelisib ∗P = 0.0131, KO DMSO versus KO 10 μM alpelisib ∗∗P = 0.0043, WT DMSO versus both WT 10 and 50 μM alpelisib P > 0.9999 (not significant [ns]). (b) Western blot for p110α and the loading control α-tubulin of WT or KO cells treated with either scramble (Scram.) or p110α siRNA. (c) Representative Airyscan confocal micrographs (fixed using the 4% formaldehyde fix and immunolabeled for EEA1, yellow; actin [phalloidin], magenta; and DAPI, cyan; bars = 5 μm) and quantification of WT or KO cells treated with either scramble (S) or p110α siRNA, demonstrating reduction of EEA1-actin overlap on p110α depletion. K-W test with Dunn’s multiple comparisons test: ∗∗∗P < 0.001, multiple comparisons; WT scramble versus KO scramble, KO scramble versus KO p110α siRNA both ∗∗∗P < 0.001. N = 79, 108, 93, and 131 cells, respectively. HK2, human kidney; PI3K, phosphatidylinositol-3′-kinase; siRNA, small, interfering RNA. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 3

Levels of phosphatidylinositol (PI) 4,5-bisphosphate [PI(4,5)P₂] are elevated with OCRL knockout (KO), and PI(3,4,5)P₃, PI(4,5)P₂, and PI(3)P are suppressed by alpelisib treatment. In all experiments, the indicated treatments were applied 16 hours before fixation. In the quantifications, the lines indicate the mean ± SEM and each data point results from an individual cell. In all quantifications, statistical significance was assessed by a Kruskal-Wallis (K-W) analysis of variance with Dunn’s multiple comparisons test. Bars = 20 μm in all images. (a) Representative widefield micrographs of wild-type (WT) or KO human kidney (HK2) cells treated with dimethylsulfoxide (DMSO) or 10 μM of alpelisib and then fixed with the plasma membrane fix and immunolabeled for PI(3,4,5)P₃ (red) and 4′,6-diamidino-2-phenylindole (DAPI) (cyan), with quantification of the mean cellular PI(3,4,5)P₃ labeling intensity, showing the effectiveness of alpelisib treatment on PI(3,4,5)P₃ synthesis. N = 96, 128, and 109 cells for WT DMSO, WT alpelisib, KO DMSO, and KO alpelisib, respectively. K-W test: overall ∗∗∗P < 0.001, multiple comparisons; WT DMSO versus KO DMSO, WT DMSO versus WT alpelisib, and KO DMSO versus KO alpelisib all ∗∗∗P < 0.001. (b) Representative confocal micrographs of WT or KO HK2 cells treated with either DMSO, 10 μM-, or 50-μM alpelisib for 16 hours and then fixed with the Golgi fix and labeled using the mCh-2xFYVE PI(3)P probe (magenta) and DAPI (cyan), with quantification of the number of PI(3)P-positive puncta detected in cells treated with a range of alpelisib concentrations, as indicated, showing PI(3)P-positive punctae are reduced in a dose-responsive fashion. N = 280, 254, 210, 287, 324, 239, 340, 250, 240, and 215 cells for WT control, WT 10 μM, WT 50 μM, KO DMSO, and KO 2.5, 5, 10, 25, and 50 μM alpelisib, respectively. K-W test: overall ∗∗∗P < 0.001, multiple comparisons; WT DMSO versus KO DMSO, WT DMSO versus WT 10 and 50 μM alpelisib, KO DMSO versus KO 50 μM alpelisib all ∗∗∗P < 0.001; KO DMSO versus KO 2.5 and 5 μM alpelisib both P > 0.9999 (not significant [ns]); KO DMSO versus KO 10 μM alpelisib ∗∗P = 0.0049; KO DMSO versus KO 25 μM alpelisib ∗∗∗P = 0.0002. (c) Representative widefield micrographs of WT or KO HK2 cells treated with DMSO or 10 μM of alpelisib and then fixed with the plasma membrane fix and immunolabeled for PI(4,5)P₂ (yellow) and DAPI (cyan), with quantification of the mean cellular PI(4,5)P₂ labeling intensity, showing increased plasma membrane PI(4,5)P₂ in KO cells, which is reduced by alpelisib treatment, specifically in KO cells. N = 126, 112, 85, and 116 cells for WT DMSO, WT alpelisib, KO DMSO, and KO alpelisib, respectively. K-W test: overall ∗∗∗P < 0.001, multiple comparisons; WT DMSO versus KO DMSO and KO DMSO versus KO alpelisib both ∗∗∗P < 0.001; WT DMSO versus WT alpelisib P = 0.4752 (ns). (d) Representative widefield micrographs of WT or KO HK2 cells treated with DMSO or 10 μM of alpelisib and then fixed with the 4% formaldehyde fix and immunolabeled for PI(4,5)P₂ (yellow) and DAPI (cyan), with quantification of the number of PI(4,5)P₂-positive puncta, showing increased PI(4,5)P₂ puncta in KO cells, which is reduced by alpelisib treatment, specifically in KO cells. N = 75, 65, 52, and 69 cells for WT DMSO, WT alpelisib, KO DMSO, and KO alpelisib, respectively. K-W test: overall ∗∗∗P < 0.001, multiple comparisons; WT DMSO versus KO DMSO and KO DMSO versus KO alpelisib both ∗∗∗P < 0.001; WT DMSO versus WT alpelisib P > 0.99 (ns). a.u., arbitrary units. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 4

Alpelisib alleviates actin defects of Ocrl in cultured humanized Ocrl^Y/−mouse PTCs (mPTCs). (a) Representative maximum intensity Z-projection confocal micrographs of Ocrl^Y/+ or Ocrl^Y/− mPTCs treated with dimethylsulfoxide (DMSO) or 10 μM of alpelisib for 16 hours and then fixed with the 4% formaldehyde fix and immunolabeled for actin (phalloidin) (white) and 4′,6-diamidino-2-phenylindole (DAPI) (blue), with quantification of the degree to which stress fibers are present in each condition, showing that stress fibers lost in Ocrl^Y/− mPTCs are rescued by alpelisib treatment. The lines indicate means ± SEM, and the data points indicate each imaging region: N = 5 imaging regions per condition (each containing approximately 15–20 cells). Significance was assessed by ordinary 1-way analysis of variance (ANOVA) with Holm-Sidak’s multiple comparison test, ∗∗P = 0.001, multiple comparisons; Ocrl^Y/+ DMSO versus Ocrl^Y/− DMSO ∗∗P = 0.002, Ocrl^Y/− DMSO versus Ocrl^Y/− alpelisib ∗∗P = 0.004, Ocrl^Y/+ DMSO versus Ocrl^Y/− alpelisib P = 0.50 (not significant [ns]). Bars = 20 μm. (b) High-magnification representative 3D surface renderings of Ocrl mPTCs treated with DMSO or 10 μM of alpelisib for 16 hours and then fixed with the 4% formaldehyde fix and immunolabeled for early endosome antigen 1 (EEA1) (purple), actin (phalloidin, yellow), and DAPI (blue), and quantification illustrating rescue of the actin-endosomal overlap by alpelisib. Lower-magnification views indicating overviews of these regions are shown in Supplementary Figure S4B and Supplementary Movies S1–S3. Lines indicate mean ± SEM. N = 42, 47, and 45 randomly selected fields for Ocrl^Y/+ + DMSO, Ocrl^Y/− + DMSO, and Ocrl^Y/− + alpelisib conditions, respectively, in each case pooled from 4 mouse kidneys per condition. Significance was tested by Kruskal-Wallis (K-W) ANOVA with Dunn’s multiple comparisons test: overall ∗∗∗P < 0.001, multiple comparisons; Ocrl^Y/+ DMSO versus Ocrl^Y/− DMSO and Ocrl^Y/− DMSO versus Ocrl^Y/− alpelisib ∗∗∗P < 0.001; Ocrl^Y/+ DMSO versus Ocrl^Y/− alpelisib P > 0.99 (ns). Bars = 1 μm. (c) Representative confocal micrographs of Ocrl^Y/+ or Ocrl^Y/− mPTCs treated with DMSO or 10-μM alpelisib and then fixed with the Golgi fix and labeled using the mCh-2xFYVE PI(3)P probe (magenta) and DAPI (cyan), with quantification of the number of PI(3)P-positive puncta detected in cells. N = 188, 177, 246, and 206 cells from Ocrl^Y/+ + DMSO, Ocrl^Y/+ + alpelisib, Ocrl^Y/− + DMSO, and Ocrl^Y/− + alpelisib, respectively; cells were pooled from 3 Ocrl kidneys per group. K-W test: overall ∗∗∗P < 0.001, multiple comparisons; Ocrl^Y/+ DMSO versus Ocrl^Y/− DMSO, Ocrl^Y/+ DMSO versus Ocrl^Y/+ alpelisib, and Ocrl^Y/− DMSO versus Ocrl^Y/− alpelisib, all ∗∗∗P < 0.001. Bars = 20 μm. (d) Representative confocal micrographs of Ocrl^Y/+ or Ocrl^Y/− mPTCs treated with DMSO or 10 μM of alpelisib for 16 hours then fixed with the 4% formaldehyde fix and immunolabeled for phosphatidylinositol (PI) 4,5-bisphosphate [PI(4,5)P₂] (yellow) and DAPI (cyan), with quantification of the number of PI(4,5)P₂-positive puncta, showing increased PI(4,5)P₂ puncta in Ocrl^Y/− cells, which is reduced by alpelisib treatment. N = 477, 444, 423, and 494 from Ocrl^Y/+ + DMSO, Ocrl^Y/+ + alpelisib, Ocrl^Y/− + DMSO, and Ocrl^Y/− + alpelisib, respectively; cells were pooled from 3 Ocrl kidneys per group. K-W test: overall ∗∗∗P < 0.001, multiple comparisons; Ocrl^Y/+ DMSO versus Ocrl^Y/− DMSO, Ocrl^Y/+ DMSO versus Ocrl^Y/+ alpelisib, and Ocrl^Y/− DMSO versus Ocrl^Y/− alpelisib, all ∗∗∗P < 0.001. Bars = 20 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 5

Alpelisib improves endocytic uptake of humanized Ocrl^Y/−mouse PTCs (mPTCs). (a) Schematic illustrating the pulse-chase experiment used to examine the binding and internalization of Alexa 488-bovine serum albumin (BSA) into mPTCs. Cells were exposed to Alexa 488-BSA (0.2 mg/ml) for 1 hour at 4 °C to allow BSA to bind to cell surface receptors (pulse) and then warmed to 37 °C in cell medium for 20 minutes before fixation to allow ligand internalization (chase). (b) Representative confocal micrographs of Ocrl^Y/+ or Ocrl^Y/− mPTCs treated with dimethylsulfoxide (DMSO) or 10 μM of alpelisib for 16 hours and subjected to the Alexa 488-BSA (green) pulse-chase experiment, before being fixed and labeled for 4′,6-diamidino-2-phenylindole (DAPI) (blue). The pulse phase of the experiment is shown in the top panel for each condition, with the chase below. Bars = 20 μm. (c) Quantification of cell surface Alexa 488-BSA (i) and internalized Alexa 488-BSA (ii), evaluated as mean fluorescence intensities per cell. Alpelisib rescues the rescued BSA binding and internalization observed in Ocrl^Y/−cells. N = 209, 228, 186, and 196 cells for Ocrl^Y/+ DMSO, Ocrl^Y/+ alpelisib, Ocrl^Y/− DMSO, and Ocrl^Y/− alpelisib conditions, respectively in (i) and N = 203, 183, 199, and 233 cells for Ocrl^Y/+ DMSO, Ocrl^Y/+ alpelisib, Ocrl^Y/− DMSO, and Ocrl^Y/− alpelisib conditions, respectively in (ii), in each case pooled from 2 mouse kidneys per condition; each data point represents the mean fluorescence intensity in an individual cell. Significance was tested by Kruskal-Wallis (K-W) analysis of variance (ANOVA) with Dunn’s multiple comparisons test: for (i) cell surface BSA, overall ∗∗∗P < 0.001, multiple comparisons: Ocrl^Y/+ DMSO versus Ocrl^Y/− DMSO and Ocrl^Y/− DMSO versus Ocrl^Y/− alpelisib ∗∗∗P < 0.001; Ocrl^Y/+ DMSO versus Ocrl^Y/+ alpelisib P = 0.60 (not significant [ns]); for (ii) internalized BSA, overall ∗∗∗P < 0.001, multiple comparisons: Ocrl^Y/+ DMSO versus Ocrl^Y/− DMSO and Ocrl^Y/− DMSO versus Ocrl^Y/− alpelisib ∗∗∗P < 0.001; Ocrl^Y/+ DMSO versus Ocrl^Y/+ alpelisib P = 0.66 (ns). (d) Quantification of the ratio between the cell surface Alexa 488-BSA and the internalized Alexa 488–BSA fluorescence intensities, showing no significant differences in ratios between each condition. Each point represents the average of the ratio in a field containing approximately 15–20 cells (N = 13, 13, 10, and 11 randomly selected fields for Ocrl^Y/+ DMSO, Ocrl^Y/+ alpelisib, Ocrl^Y/− DMSO, and Ocrl^Y/− alpelisib conditions, respectively, in each case pooled from 2 mouse kidneys per condition). Significance was tested by K-W ANOVA with Dunn’s multiple comparisons test: overall P = 0.46 (ns), multiple comparisons: Ocrl^Y/+ DMSO versus Ocrl^Y/− DMSO and Ocrl^Y/− DMSO versus Ocrl^Y/− alpelisib P > 0.99 (ns); Ocrl^Y/+ DMSO versus Ocrl^Y/+ alpelisib P = 0.84 (ns). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 6

Alpelisib improves the proximal tubule (PT) function of Ocrl^Y/−mice, restoring ligand uptake and megalin expression. (a) Experimental setup. Ocrl mice were treated for 6 weeks with a daily oral dose of either carboxymethylcellulose 1% (vehicle) or alpelisib (50 mg/kg body weight). On the last day of the treatment, mice were injected with Cy5-labeled β-lactoglobulin (N = 6 Ocrl^Y/−, or 8 Ocrl^Y/− mice per group). (b–d) The Δ values shown indicate the mean change from BL to D42 for the condition ± SEM. (b) Clara cell protein 16 (CC16), (c) albumin urinary output (both within 15 hours), and (d) body mass was measured in Ocrl^Y/− mice treated with either vehicle or alpelisib at the indicated time point. Each dot represents 1 mouse. Significance was assessed by 2-tailed paired Student’s t test; in (b) CC16 output change relative to baseline; + vehicle, P = 0.9802 (not significant [ns]), + alpelisib, ∗P = 0.0334; in (c) albumin output change relative to baseline; + vehicle, P = 0.0502 (ns), + alpelisib, ∗∗∗P = 0.0006; in (d) body mass change relative to baseline; + vehicle, ∗∗∗P < 0.0001, + alpelisib, ∗∗P = 0.0083. (e) Representative confocal micrographs showing Cy5-labeled β-lactoglobulin (magenta) 15 minutes after tail vein injection and labeled for 4′,6-diamidino-2-phenylindole (DAPI) (cyan), plus quantification of the corresponding fluorescent signals from Ocrl mouse kidneys (N = 412, 500, and 588 tubules, respectively, for Ocrl^Y/++ vehicle, Ocrl^Y/− + vehicle, and Ocrl^Y/− + alpelisib) for 3 mice per treatment group. β-Lactoglobulin uptake is rescued by alpelisib treatment. Bars = 20 μm. In the quantifications, each dot represents fluorescence intensity normalized by tubule area; plotted data indicate the mean ± SEM. Significance was assessed by the Kruskal-Wallis (K-W) test followed by Dunn’s multiple comparison test; ∗∗∗P < 0.001, multiple comparisons; Ocrl^Y/++ vehicle versus Ocrl^Y/− + vehicle and Ocrl^Y/− + vehicle versus Ocrl^Y/− + alpelisib, both ∗∗∗P < 0.001. (f) Western blotting and densitometry analysis of megalin levels in whole kidney lysates from Ocrl mice. α-Tubulin was used as a loading control. The reduced megalin expression in Ocrl^Y/− is rescued by alpelisib. In the quantification of the densitometry analysis, each dot represents 1 mouse (N = 4 Ocrl^Y/++ vehicle and Ocrl^Y/− + vehicle and N = 3 Ocrl^Y/− + alpelisib mice); lines indicate mean ± SEM. Significance was assessed by 2-tailed unpaired Student’s t tests: Ocrl^Y/++ vehicle versus Ocrl^Y/− + vehicle; ∗∗P = 0.0057, Ocrl^Y/− + vehicle versus Ocrl^Y/− + alpelisib, ∗P = 0.0246. (g) Representative confocal micrographs with high-magnification insets and quantification of megalin (yellow) intensity in AQP1⁺ PTs (magenta) from Ocrl kidneys also labeled for DAPI (cyan) illustrating rescue of megalin levels after alpelisib treatment. Bars = 20 μm. In the quantifications, each dot represents fluorescence intensity normalized by tubule area; plotted data indicate the mean ± SEM; N = 142, 191, and 266 tubules, respectively, for Ocrl^Y/++ vehicle, Ocrl^Y/− + vehicle, and Ocrl^Y/− + alpelisib for 3 mice per treatment group. Significance was assessed by K-W analysis of variance followed by Dunn’s multiple comparison test; overall ∗∗∗P < 0.001, multiple comparisons; Ocrl^Y/++ vehicle versus Ocrl^Y/− + vehicle and Ocrl^Y/− + vehicle versus Ocrl^Y/− + alpelisib, both ∗∗∗P < 0.001. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 7

Proposed model depicting the therapeutic effect of alpelisib on proximal tubule cells in Lowe syndrome. Proximal tubule cells reabsorb urinary ligands (e.g., albumin and low-molecular-weight proteins) through megalin-mediated endocytosis. The 5-phosphatase activity of OCRL regulates the transition from high phosphatidylinositol (PI) 4,5-bisphosphate [PI(4,5)P₂] at the plasma membrane to low levels at the early endosome with a transient coincidence of PI(4,5)P₂ and PI(3)P in the vesicles. Once in the early endocytic compartment, the ligands dissociate from the receptors and are delivered to the lysosome for degradation, whereas the receptors recycle back to the plasma membrane for a new cycle of cargo binding. The loss of OCRL leads to an ectopic accumulation of PI(4,5)P₂ at the endosomal compartment, which results in a persistent coincidence with PI(3)P. We suggest that this event is responsible for aberrant F-actin polymerization blocking endocytic recycling and preventing ligand reabsorption (middle panel). Alpelisib rebalances the levels of  PI(4,5)P₂ and PI(3)P, which results in decreased actin polymerization and improvement of the endocytic machinery and absorptive capacity of proximal tubule cells (right panel).