aPKCλ maintains the integrity of the glomerular slit diaphragm through trafficking of nephrin to the cell surface

Abstract

The slit diaphragm (SD), the specialized intercellular junction between renal glomerular epithelial cells (podocytes), provides a selective-filtration barrier in renal glomeruli. Dysfunction of the SD results in glomerular diseases that are characterized by disappearance of SD components, such as nephrin, from the cell surface. Although the importance of endocytosis and degradation of SD components for the maintenance of SD integrity has been suggested, the dynamic nature of the turnover of intact cell-surface SD components remained unclear. Using isolated rat glomeruli we show that the turnover rates of cell-surface SD components are relatively high; they almost completely disappear from the cell surface within minutes. The exocytosis, but not endocytosis, of heterologously expressed nephrin requires the kinase activity of the cell polarity regulator atypical protein kinase C (aPKC). Consistently, we demonstrate that podocyte-specific deletion of aPKCλ resulted in a decrease of cell-surface localization of SD components, causing massive proteinuria. In conclusion, the regulation of SD turnover by aPKC is crucial for the maintenance of SD integrity and defects in aPKC signalling can lead to proteinuria. These findings not only reveal the pivotal importance of the dynamic turnover of cell-surface SD components but also suggest a novel pathophysiological basis in glomerular disease.

Keywords: aPKC; cell-surface localization; glomerular disease; nephrin; slit diaphragm.

Fig. 1

SD integrity is maintained by rapid turnover of cell-surface SD components. (A) Schematic representation of the cell-surface biotinylation assay. (B) Isolated glomeruli were subjected to the cell-surface biotinylation assay as in (A). Biotinylated SD components were isolated with streptavidin sepharose and isolated proteins detected by immunoblot. (C) Quantification of the results in (B). The total protein level and cell-surface localization of SD components were normalized to those at the start of labelling. (D) Schematic representation of the endocytosis assay. (E) Isolated glomeruli were subjected to the endocytosis assay and analysed by immunoblot. (F) Quantification of the results in (E). The endocytosed proteins were expressed as the percentage of to those at the start of labelling (see ‘Materials and Methods’ section). (G) Schematic representation of the biotinylation degradation assay. (H) Isolated glomeruli were subjected to biotinylation degradation and analysed by immunoblot. (I) Quantification of the results in (H). The biotinylated SD components were normalized to those at the start of labelling. The data shown in C, F and I are the mean ± SD of three independent experiments.

Fig. 2

aPKC is required for the cell-surface localization of SD components, including nephrin. (A) Isolated rat glomeruli were treated with 10 µM aPKC pseudosubstrate (PS) or SC for 30 min at 37°C in HBSS(+), then subjected to the cell-surface biotinylation assay. (B) Quantification of the results in (A). (C) HCT116-nephrin cells were treated with 20 µM of aPKC-PS or SC for 2 h at 37°C and subjected to the cell-surface biotinylation assay. (D) Quantification of the results in (C). (E) HCT116-nephrin cells were transiently transfected with aPKC WT or KN cDNA and incubated for 48 h and then subjected to the cell-surface biotinylation assay. (F) Quantification of the results in (E). (G) HCT116-nephrin cells were transiently transfected with aPKCλ/ζ siRNA and incubated for 70 h. Both isotypes of aPKC are expressed in HCT116 cells (data not shown). After incubation, the cells were subjected to the cell-surface biotinylation assay. (H) Quantification of the results in (G). The values shown in B, D, F and H were normalized to the appropriate control and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t-test.

Fig. 3

aPKC does not suppress the endocytosis of nephrin. (A) HCT116-nephrin cells were treated with 20 µM aPKC-PS or SC for 2 h at 37°C and then subjected to the endocytosis assay as in Fig. 1D. (B) Quantification of the results in (A). (C) HCT116-nephrin cells were transiently transfected with aPKC WT or KN cDNA and incubate for 48 h and then subjected to the endocytosis assay. (D) Quantification of the results in (C). The amount of endocytosed nephrin shown in B and D was expressed as the percentage of those at the start of labelling and are the mean ± SD of three independent experiments. (E) HCT116-nephrin cells were treated with 20 µM aPKC PS with or without 10 µM chlorpromazine or 10 mM MβCD for 30 min at 37°C and then subjected to the cell-surface biotinylation assay.

Fig. 4

aPKC is required for exocytosis of newly synthesized nephrin. (A) HeLa Tet-On Advanced cells were transiently transfected with nephrin cDNA and incubated for 48 h. After incubation, cells were treated with 20 µM of aPKC-PS or SC for 2 h, and then incubated with 100 ng/ml doxycycline for the indicated times to induce the expression of nephrin. After doxycycline treatment, the cells were subjected to the cell-surface biotinylation assay. (B) Quantification of the results in (A). (C) HeLa Tet-On Advanced cells were transiently transfected with nephrin and aPKC WT or KN cDNA, and incubated for 48 h. After incubation, the cells were incubated with 100 ng/ml doxycycline for the indicated times to induce the expression of nephrin. After doxycycline treatment, the cells were subjected to the cell-surface biotinylation assay. (D) Quantification of the results in (C). The values shown in B and D were normalized to those at the start of doxycycline treatment and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t-test.

Fig. 5

aPKC is required for the cell-surface localization of SD components in vivo. (A) aPKC cKO and control mice at P10 or P11 were transcardially perfused with 2 mg/ml sulfo-NHS-SS-biotin/PBSCM for 5 min. Then, the kidneys were lysed and biotinylated proteins isolated with streptavidin sepharose and detected by immunoblot. The white arrowhead represents the mature-glycosylated, cell-surface form, and the black arrowhead represents the N-glycosylated, ER-form of nephrin. (B) Quantification of the results in (A). The values were normalized to control mice and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t-test. (C) The cell-surface biotinylated aPKC cKO and control kidney were immunostained with nephrin, neph1 or podocin and biotin (Supplementary Fig. S5), and the colocalization coefficient was calculated with LAS-AF software provided by Leica. The P values were determined by two-tailed Student’s t-test. (D) The ultrathin cryosections of aPKC cKO and control kidney were labelled with anti-nephrin or anti-podocin antibodies followed by 10 nm gold particle-conjugated secondary antibody. Black arrowheads represent nephrin localized to the intracellular region, and white arrowheads represent nephrin localized to the rough ER. FP, foot process; GBM, glomerular basement membrane. (E) The distance of gold particle-labelled nephrin or podocin from the plasma membrane in aPKC cKO podocytes was compared with that of the control kidney. The P values were determined by two-tailed Mann–Whitney U-test.