Disruption of the exocyst induces podocyte loss and dysfunction

Abstract

Although the slit diaphragm proteins in podocytes are uniquely organized to maintain glomerular filtration assembly and function, little is known about the underlying mechanisms that participate in trafficking these proteins to the correct location for development and homeostasis. Identifying these mechanisms will likely provide novel targets for therapeutic intervention to preserve podocyte function following glomerular injury. Analysis of structural variation in cases of human nephrotic syndrome identified rare heterozygous deletions of EXOC4 in two patients. This suggested that disruption of the highly-conserved eight-protein exocyst trafficking complex could have a role in podocyte dysfunction. Indeed, mRNA profiling of injured podocytes identified significant exocyst down-regulation. To test the hypothesis that the exocyst is centrally involved in podocyte development/function, we generated homozygous podocyte-specific Exoc5 (a central exocyst component that interacts with Exoc4) knockout mice that showed massive proteinuria and died within 4 weeks of birth. Histological and ultrastructural analysis of these mice showed severe glomerular defects with increased fibrosis, proteinaceous casts, effaced podocytes, and loss of the slit diaphragm. Immunofluorescence analysis revealed that Neph1 and Nephrin, major slit diaphragm constituents, were mislocalized and/or lost. mRNA profiling of Exoc5 knockdown podocytes showed that vesicular trafficking was the most affected cellular event. Mapping of signaling pathways and Western blot analysis revealed significant up-regulation of the mitogen-activated protein kinase and transforming growth factor-β pathways in Exoc5 knockdown podocytes and in the glomeruli of podocyte-specific Exoc5 KO mice. Based on these data, we propose that exocyst-based mechanisms regulate Neph1 and Nephrin signaling and trafficking, and thus podocyte development and function.

Keywords: cilia; ciliopathy; connecting cilium; endocytosis; exocyst; photoreceptor; podocyte; protein trafficking; trafficking.

Figure 1.

Intensities of the CNV region using exome chip genotypes acquired from the patients. A–C, log R ratio from exome chip data at the Chr. 7: locus is presented. Black dots, SNPs within the CNV region. Smaller values on y axis, lower intensity is supportive of a deletion. D, expression level of exocyst complex members decreases following injury with PAN. mRNA profiling of podocytes injured with PAN showed down-regulation of various components of the exocyst complex and its regulator CDC42. (*, p < 0.05, paired Student's t test).

Figure 2.

Podocyte-specific knockout of Exoc5. A, podocyte-specific Exoc5 KO mice on tdTomato background (tdTom–Exoc5fl/fl;Pod–Cre/+) showed tdTomato expression (red color without staining) in the glomerular podocytes (black arrows). There was no red Tomato expression in tdTom–Exoc5fl/fl glomeruli, where Cre had not been expressed. Bar, 100 μm. B, kidney sections from podocyte-specific Exoc5 KO mice, on a tdTomato background, stained with Exoc5 antibody (green) showed tdTomato expression (red) in podocytes (arrows), where Exoc5 was inactivated by Cre. Expression of Exoc5 (green) in glomerular cells other than podocytes was readily visible. Bar, 50 μm. C, RT-PCR confirming the loss of Exoc5 expression in podocytes isolated by FACS sorting from the glomeruli of podocyte-specific Exoc5 KO mice. (*, p < 0.01, paired t test.) D, podocyte-specific KO of Exoc5 (tdTom–Exoc5fl/fl;Pod- Cre/+) mice leads to stunted growth compared with littermate control (tdTom–Exoc5fl/fl) mice. Survival curve showed that all podocyte-specific Exoc5 knockout mice die within 30 days after birth (E), SDS-PAGE (F), and albumin/creatinine ratio (μg/μg) (G) showed massive proteinuria in podocyte-specific Exoc5 KO mice (tdTom–Exocfl/fl;Pod Cre/+) compared with control (tdTom–Exoc5fl/fl) mice.

Figure 3.

Histological and ultrastructural analyses. A, hematoxylin and eosin (H&E), periodic acid Schiff (PAS), and Masson's trichrome stains of kidney sections from podocyte-specific Exoc5 KO (Exoc5fl/fl;Pod–Cre/+) mice show proteinaceous casts in tubules (white arrows), altered glomerular morphology (black arrow), and increased fibrosis (blue color with Masson's trichrome) compared with the heterozygous podocyte-specific Exoc5 KO (Exoc5fl/+;Pod–Cre/+) and control (Exoc5fl/fl) mice. Higher magnification images to the right of each panel are 6×. The blue color depicts areas of fibrosis. Bar, 100 μm. B and C, ultrastructural (SEM) analysis of podocyte-specific Exoc5 KO glomeruli showed significant loss of foot processes (white arrow), and the TEM showed foot process fusion and loss of slit diaphragm (black arrow) when compared with the littermate controls. Bars, 1 μm.

Figure 4.

Podocyte-specific Ift88 knockout mice are not proteinuric and are not more susceptible to glomerular injury. A, podocyte-specific Ift88 knockout mice were generated (Ift88fl/fl;Pod–Cre/+). Immunoblot and RT-PCR analysis of glomeruli isolated from Ift88 KO and control mice. ***, p < 0.001. B, SDS-PAGE of the urine from Ift88 podocyte-specific KO mice showed no evidence of proteinuria. C and D, podocyte-specific Ift88 knockout and control mice (Ift88fl/fl;Pod–Cre/+) were subjected to acute (NTS) (C) and chronic (adriamycin) (D) glomerular injury, and the extent of albuminuria was assessed. No changes in albuminuria, compared with controls, were noted in either of these models.

Figure 5.

Exoc5 KD in cultured podocytes and RNA-Seq. A, immunoblot analysis of Exoc5 protein levels in various Exoc5 KD clones. B, volcano plot of differentially expressed genes from control and Exoc5 knockdown cells. C, heat map of hierarchical clustering from RNA-Seq data showed differentially regulated genes in Exoc5 knockdown and control podocytes. The adjusted p values (≤0.05) using Benjamin and Hochberg's approach that were reported by DESeq2 and assigned to the differentially regulated genes are shown. n = 3 experimental replicates. D, GO enrichment analysis showed multiple cellular trafficking events (presented as histograms) that were enriched in response to Exoc5 knockdown.

Figure 6.

Podocyte-specific Exoc5 deletion alters Nephrin and Neph1 expression/localization. A, there was significant mislocalization of Nephrin and Neph1 (green) in podocyte-specific Exoc5 KO (Exoc5fl/fl;Pod-Cre/+), compared with littermate control (tdTom–Exoc5fl/fl), glomeruli. Nephrin and Neph1 normally colocalize (merged images) with synaptopodin (red). Blue color represents 4′,6-diamidino-2-phenylindole–stained nuclei. Bar, 10 μm. B, expression analysis for various exocyst components in human Exoc5 KD podocytes using RT-PCR. C, Western blot analysis of Exoc5 KD podocytes showed increased ERK, SMAD3, and Neph1 phosphorylation along with concomitant loss of Exoc4 expression. D, relative protein quantification from C. **, p < 0.01, paired Student's t test.

Figure 7.

Slit diaphragm proteins Nephrin and Neph1 are mislocalized in Exoc5 KD podocytes. A and B, membrane localization of endogenous Neph1 at the cell–cell junctions was reduced in Exoc5 KD podocytes. Bar, 5 μm. 4′,6-Diamidino-2-phenylindole -stained nuclei (DNA) are shown in blue (n = 10). C, schematic of the chimeric Neph1 and Nephrin constructs, where FLAG-tag was inserted following the signal peptide sequence. D–I, chimeric Nephrin and Neph1 were expressed in cultured podocytes (D and G), and stable cell lines were created. Nephrin and Neph1 in these cells were extracellularly labeled with FLAG antibody and live-cell staining was performed. The surface localization of Neph1 and Nephrin was analyzed by immunofluorescence using confocal microscopy (E and H), and the images were quantified using ImageJ, which showed significant loss of surface Neph1 and Nephrin in Exoc5 knockdown podocytes (n = 10) (F and I). Bar, 25 μm. In each experiment, 2 million cells were plated on each coverslip, and 10 or more cells were analyzed for quantification and statistical analysis. **, p < 0.001; *, p < 0.01, paired Student's t test.

Figure 8.

Overexpression of Exoc5 rescued loss of Nephrin and Neph1 localization in Exoc5 KD podocytes. A lentiviral expression construct of mouse Exoc5 was used to rescue Exoc5 KD in cultured human podocytes. A, Western blot analysis of Exoc5 overexpression in Exoc5 KD podocytes. B and C, cultured stable podocytes overexpressing FLAG-tagged Nephrin (B) and Neph1 (C) in Exoc5 KD podocytes were treated with lentiviral particles expressing mouse Exoc5, and surface staining was performed using anti-FLAG antibody. The localization of Nephrin (B) and Neph1 (C) to the podocyte cell membrane was restored in podocytes following exogenous expression of mouse Exoc5. D, quantification of fluorescence in B and C is shown (n = 10). Bar, 25 μm. (**, p < 0.001, paired Student's t test.) In each experiment, 2 million cells were plated on each coverslip, and 10 or more cells were analyzed for quantification and statistical analysis.

Figure 9.

Exoc5 KD podocytes display reduced movement of cherry-Neph1 vesicles. A, podocytes expressing mCherry–Neph1 on a WT or Exoc5 KD background were plated on a glass-bottom cell culture plate, and the movement of Neph1-containing vesicles was analyzed using time-lapse live imaging. The vesicular movement was plotted as displacement (micrometers, y axis) versus time (seconds, x axis). B, decreased displacement (micrometers) and speed (micrometers per second) of mCherry–Neph1 vesicles were noted in the Exoc5 KD podocytes (p < 0.0001). Data are presented as means ± S.E.

Figure 10.

Exoc5 deletion induces apoptosis. A and B, significant amounts of TUNEL-positive nuclei were seen in the glomeruli of podocyte-specific Exoc5 knockout mice, but they were absent in the control mice. Bar, 20 μm (n = 10, ***, p < 0.001, paired Student's t test).