Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

Abstract

The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34-deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux.

Keywords: glomerulus; podocyte; proteinuria.

Figure 1.

Conditional PIKfyve deletion in mouse podocytes results in normal phenotype. (A, top panel) Hematoxylin and eosin staining of paraffin–embedded mouse kidney sections from PIKfyve^{fl/fl,Nphs2-Cre−} (control) and PIKfyve^{fl/fl,Nphs2-Cre+} (PIKfyve^podko) mice. (A, middle and bottom panels) Transmission electron micrographs of kidneys from control and PIKfyve^podko mice show a normal podocyte foot process architecture and the absence of vacuoles in the PIKfyve^podko podocytes. (B) Number of junctions per micrometer of glomerular basement membrane (GBM) surface as seen by transmission EM. Data are means±SEMs.(C) Urine collected from PIKfyve^podkomice and their age–matched littermate controls was resolved using SDS-PAGE. BSA standards were run for comparison. Results shown are representative of five or more 12-month-old mice per group. (D) Urine albumin-to-creatinine ratio in PIKfyve^podko and age–matched littermate control mice as measured by ELISA. Error bars are SEMs.

Figure 2.

Deletion of PIKfyve in mouse podocytes. (A) Immunofluorescence images of frozen kidney section showing green fluorescence exclusively in the glomerulus in a podocyte-specific distribution when the mTmG Cre-reporter mouse was bred with the Nphs2-Cre mouse. (B) Deletion of the floxed segment was confirmed by reverse transcription of mRNA extracted from podocytes isolated by flow cytometry. PCR amplification with primers (arrows) flanking the floxed segment shows a change in the size of the PCR product by the predicted 172 bp. Depicted is the position of the primers along the PIKfyve gene (arrows). The box shows the amplified section of the gene. The length of the sequence between the loxP sites is 150 bp. (C) In situ hybridization using RNA scope probes against exon 6 of PIKfyve. Each individual dot indicates an mRNA strand. (C, upper panel) There are fluorescent dots in the cells on the periphery of the glomerular tuft in wild-type mouse (arrowheads). C, lower panel shows absence of dots in the cell periphery. Podocin was used to label the podocytes. (D) Lysates from wild-type podocytes (lane 1) and podocytes lacking PIKfyve (lane 2) were resolved using SDS-PAGE and blotted with antibody against the C terminus of PIKfyve. Lysates from HEK293 cells transfected with GFP-tagged PIKfyve were used as positive control. Quantification of band densities using Unscan presented next to the blot. Error bars are SEMs (n=4). Twelve-month-old mice were used in these experiments.

Figure 3.

PIKfyve-deleted podocytes develop vacuoles in vitro. (A) Glomeruli isolated from 6-month-old mTmG/PIKfyve^{fl/fl,Nphs2-Cre} mouse kidneys develop large vacuoles by days 6–8 in culture. (B) Quantitation by HPLC from four independent experiments calculated as a percentage of the total PI radioactivity and presented as means±SEMs. The total PI radioactivity was obtained by summing the counts within the elution times corresponding to the individual [³H]GroPIns peaks as detailed in Concise Methods. The changes in PtdIns(3,5)P₂, PtdIns(5)P, and PtdIns(3)P in PIKfyve^fl/fl,Cre− versus PIKfyve^fl/fl,Cre+ were statistically significant. *P<0.01 using paired t test analysis.

Figure 4.

PIKfyve deletion in mouse proximal tubular epithelial cells triggers formation of large cytoplasmic vacuoles. (A, top panel) Immunofluorescence images of frozen kidney sections showing expression of green fluorescence in proximal tubules when the mTmG^fl/fl mouse was bred with the PEPCK-Cre mouse. There is a mosaic pattern of GFP expression in the proximal tubules (arrowheads). (A, middle panel) Numerous vacuoles are seen in the cytoplasm of proximal tubular cells expressing GFP. Frozen kidney sections of a 2-month-old mTmG/PIKfyve^fl/fl mouse carrying both Nphs2-Cre and PEPCK-Cre (double knockout) show expression of GFP in both podocytes and proximal tubular cells. (A, bottom panel) There are numerous vacuoles in the proximal epithelial cells adjacent to a glomerulus with normal-appearing podocytes. (B, top panel) Hematoxylin and eosin–stained paraffin–embedded kidney section showing presence of large vacuoles in the PIKfyve–deleted proximal epithelial cells. (B, middle and bottom panels) Transmission EM images confirm the presence of large vacuoles in the cytoplasm of the proximal epithelial cells deleted of PIKfyve.

Figure 5.

Vesicular localization of nephrin in Vps34–deleted mouse podocytes. (A) Indirect immunofluorescence staining of paraffin–embedded 4-week-old mouse kidney sections from Vps34^podko shows that nephrin is present largely in vesicles (arrowheads). Nephrin staining in PIKfyve^podko mice is similar to that of the littermate controls. Staining for synaptopodin and ZO-1 is similar in both Vps34^podko and PIKfyve^podko and their littermate control kidneys. Scale bars, 50μm. (B) Immunogold transmission EM shows presence of nephrin in vesicles (arrowheads) in Vps34^podko mouse podocytes. Images were photographed at ×5800 and cropped to emphasize the presence of immunogold particles in vesicles.

Figure 6.

Membrane trafficking and endocytic flux from Golgi is impaired in Vps34–deleted mouse podocytes. Immunofluorescence staining of kidney sections from 4-week-old Vps34–deleted and 6-month-old PIKfyve–deleted mouse podocytes for various vesicular markers. There is increased staining for Lamp1, APPL1 Rab5, Rab11, and Syntaxin-6. Rab11 and Syntaxin-6are markers of vesicles that originate from the Golgi. Staining in PIKfyve-deleted podocytes for these markers is similar to that in wild-type podocytes. Rows 4–6 are enlarged images of the boxed areas in rows 1–3. Arrowheads point to the podocytes at the periphery of the glomerulus. Scale bars, 20 μmin rows 1–3; 5 μm in rows 4–6.

Figure 7.

Aberrant accumulation of endosomal markers in PIKfyve–deleted proximal tubular cells. (A) PIKfyve–deleted proximal tubular cells show increased staining for vesicular markers APPL1, EEA1, Rab5, Rab11, and Lamp1. Lamp1 staining is used to identify cells that have undergone Cre–mediated PIKfyve deletion because of the mosaic pattern of PEPCK promoter–driven Creexpression. Two-month-old mice were used for these experiments. Scale bars, 20 μm. (B) Quantification of the immunofluorescence in PIKfyve–deleted proximal tubular cells processed by ImageJ software. Data show significant accumulation of Lamp1 in proximal tubular epithelial cells. Lamp1 staining was used to double-stain cells that have deletion of PIKfyve. There is accumulation of Clathrin, EEA1, Rab5, and Rab11 in PIKfyve–deleted proximal tubular epithelial cells. Results are expressed as integrated density and corrected total cell fluorescence. Error bars are SEMs. WT, wild type. Scale bars, 20 μm. **P<0.01; ***P<0.001.

Figure 8.

Deletion of PIKfyve in mouse podocytes and tubular cells does not affect autophagy markers LC3 and p62. (A) There is accumulation of autophagy marker LC3 in Vps34–deleted mouse podocytes indicative of altered autophagic flux. (B) Quantification of LC3 staining using ImageJ software in Vps34- and PIKfyve-deleted podocytes. (C) p62 Staining in wild-type podocytes, Vps34-deleted podocytes, PIKfyve-deleted podocytes, and PIKfyve–deleted proximal tubular cells. Note the increase of the p62 staining in Vps34-deleted podocytes (arrowheads in row 5). (D) Quantification of p62 staining using ImageJ software. Results are expressed as integrated density and corrected total cell fluorescence; 4-week-old Vps34 knockout mice and 3-month-old PIKfyve knockout mice were used in these experiments. Error bars are SEMs. KO, knockout; WT, wild type. Scale bars, 20 μm in A, upper panel and B; 5 μm in A, lower panel. ***P<0.001.

Figure 9.

Podocytes fail to accumulate FITC-halb. (A) Perfusion of 3-week-old kidneys of Vps34^{fl/fl,Nphs2-Cre+} mice and their littermate controls shows leak of FITC albumin in a few severely affected glomeruli (arrowheads). Accumulation of FITC-halb is not seen in wild–type or Vps34–deleted mouse podocytes. Even at 6 weeks of age, the Vps34–deleted mouse podocytes show only minimal accumulation of FITC-halb in rare glomeruli (row 4). Scale bars, 20 μm. (B) Perfusion time course of 12-month-old kidneys of PIKfyve^{fl/fl,Nphs2-Cre+}mice and their littermate controls shows absence of FITC-halb in podocytes. B, right panel shows enlarged images of boxed area in B, left panel. Scale bars, 20 μm in B, left panel; 5 μm in B, right panel.

Figure 10.

Occasional vacuole formation in PIKfyve–deleted mouse podocytes after unilateral nephrectomy. (A) Immunofluorescence images showing increased expression of Lamp1 (arrowheads) in PIKfyve-deleted podocytes 5 days after unilateral nephrectomy (A, top panel). Transmission EM images show vacuoles in PIKfyve-deleted podocytes. Large vacuoles were evident only in a few podocytes. The adjoining podocyte in the image does not have any vacuoles. (B) Quantification of immunofluorescence using ImageJ software showing significantly elevated Lamp1 expression in PIKfyve-deleted podocytes. Scale bars, 20 μm. *P<0.01.