Shank2 redistributes with NaPilla during regulated endocytosis

Abstract

Serum phosphate levels are acutely impacted by the abundance of sodium-phosphate cotransporter IIa (NaPiIIa) in the apical membrane of renal proximal tubule cells. PSD-95/Disks Large/Zonula Occludens (PDZ) domain-containing proteins bind NaPiIIa and likely contribute to the delivery, retention, recovery, and trafficking of NaPiIIa. Shank2 is a distinctive PDZ domain protein that binds NaPiIIa. Its role in regulating NaPiIIa activity, distribution, and abundance is unknown. In the present in vivo study, rats were maintained on a low-phosphate diet, and then plasma phosphate levels were acutely elevated by high-phosphate feeding to induce the recovery, endocytosis, and degradation of NaPiIIa. Western blot analysis of renal cortical tissue from rats given high-phosphate feed showed NaPiIIa and Shank2 underwent degradation. Quantitative immunofluorescence analyses, including microvillar versus intracellular intensity ratios and intensity correlation quotients, showed that Shank2 redistributed with NaPiIIa during the time course of NaPiIIa endocytosis. Furthermore, NaPiIIa and Shank2 trafficked through distinct endosomal compartments (clathrin, early endosomes, lysosomes) with the same temporal pattern. These in vivo findings indicate that Shank2 is positioned to coordinate the regulated endocytic retrieval and downregulation of NaPiIIa in rat renal proximal tubule cells.

Fig. 1.

Plasma phosphate levels following high Pi feeding. In rats acutely subjected to the high-phosphate diet (1.2% Pi; n = 3; closed circles), plasma phosphate levels were essentially doubled after 1, 2, and 4 h. In rats maintained on the low-phosphate diet (0.1% Pi; n = 3; open circles), plasma phosphate levels decreased modestly over the 4-h study period. *P < 0.05 vs. 0 h levels.

Fig. 2.

Time course of NaPiIIa and Shank2 degradation following high Pi feeding. A: Western blot analysis of kidney cortex lysates from rats that received a high-phosphate diet showed NaPiIIa levels were diminished after 1 h and markedly decreased after 2 and 4 h. Shank2 levels appeared lower after 2 and 4 h. In contrast, EBP50 and PDZK1 levels were unchanged during the entire time course. Actin served as a loading control. B: densitometry of the high-phosphate feeding time course studies (n = 3) measured a significant decrease in NaPiIIa/Actin levels after 1, 2, and 4 h and a significant decrease in Shank2/actin after 4 h. C: in contrast, there was no change in EBP50/actin or PDZK1/actin levels at any time point. *P < 0.05 vs. 0 h levels.

Fig. 3.

Microvillar distribution of EBP50 following high Pi feeding. Immunofluorescence imaging of EBP50 (green) and F-actin (red) in rat renal PT cells following high-phosphate feeding showed that EBP50 remained largely colocalized with the microvillar F-actin (colocalized: yellow) after 0, 1, 2, and 4 h following high-phosphate feeding. Bar = 10 μm.

Fig. 4.

Coincident redistribution of NaPiIIa and Shank2 following high Pi feeding. A: rat kidney proximal tubule (PT) cells were stained for F-actin (blue), NaPiIIa (green), and Shank2 (red). F-actin is used to demarcate both the apical microvillar region and the basal membrane of the PTs. Merge shows all three proteins within a single image. At 0 h, NaPiIIa and Shank2 were largely constrained within the apical microvillar region. After 1 h, NaPiIIa and Shank2 broaden their distributions. While still present within both the microvillar region, both proteins now also appear within the cell interior. After 2 and 4 h, a greater percentage of NaPiIIa and Shank2 distributed within the cell interior. Bar = 10 μm. B: images are from the boxed areas in the Merge panels from above. The apical membranes are facing right; the basal membranes are facing left. In contrast to the tightly constrained distribution of NaPiIIa (green) and Shank2 (red) within the microvillar region at 0 h, these proteins display a temporal exodus from the apical microvillar region after 1, 2, and 4 h after high-phosphate feeding. This includes the appearance of NaPiIIa and Shank2 in intracellular areas below the microvillar region and increasing amounts of the microvillar regions that are largely devoid of NaPiIIa and Shank2, as seen by increasing areas of blue versus yellow microvillar regions. Bar = 5 μm.

Fig. 5.

Microvillar versus cell interior distribution of NaPiIIa and Shank2. With the use of the apical F-actin staining to demarcate the microvillar domain, the relative intensities of NaPiIIa (A) and Shank2 (B) were measured within the microvilli (shaded bars) and cell interiors (solid bars). At 0 h, both proteins were predominantly distributed within the microvilli and progressively diminished from the microvillar domain at 1, 2, and 4 h. This was accompanied by a marked increase in NaPiIIa and Shank2 within the cell interior after 1 h. The comparative intensities within the cell interior decreased after 2 and 4 h, likely reflecting the net loss of NaPiIIa and Shank2 during this period. *P < 0.05.

Fig. 6.

NaPiIIa and Shank2 colocalization following high Pi feeding. The degree of NaPiIIa and Shank2 colocalization was qualitatively and quantitatively evaluated. A: with the use of a “colocalization highlighter,” pixels with both NaPiIIa (green) and Shank2 (red) appear white. Despite the redistribution of these proteins into the cell interior during the later time points, the proteins were largely colocalized at 0, 1, 2, and 4 h after high-phosphate feeding. Bar = 10 μm. B: intensity correlation quotient values for NaPiIIa and Shank2 were markedly positive under low-phosphate conditions (0 h) and remained positive 1, 2, and 4 h after high-phosphate feeding. *P < 0.05 vs. 0 h levels.

Fig. 7.

Temporal distribution of NaPiIIa in distinct endosomal compartments. At 0 h, NaPiIIa (green) distributes with F-actin (red) within the apical microvilli while the endosomal markers (blue; Clathrin, EEA1, LAMP1, rab11) distribute within the cell interior. At 1, 2, and 4 h after high-phosphate feeding, NaPiIIa redistributes into the cell interior and partially colocalizes (light blue) with Clathrin, EEA1, and LAMP1. Little or no colocalization was observed between NaPiIIa and rab11 at any time point. Bar = 5 μm.

Fig. 8.

Temporal distribution of Shank2 in distinct endosomal compartments. At 0 h, Shank2 (green) distributes with F-actin (red) within the apical microvilli, while the endosomal markers (blue; Clathrin, EEA1, LAMP1, rab11) distribute within the cell interior. At 1, 2, and 4 h after high-phosphate feeding, Shank2 redistributes into the cell interior and partially colocalizes (light blue) with Clathrin, EEA1, and LAMP1. Little or no colocalization was observed between Shank2 and rab11 at any time point. Bar = 5 μm.

Fig. 9.

Intensity correlation quotient (ICQ) analysis of NaPiIIa and Shank2 endosomal redistribution. The temporal ICQ patterns were markedly similar for NaPiIIa (A) and Shank2 (B) with different endosomal markers. At 0 h, ICQ values for both NaPiIIa and Shank2 with all four endosomal markers (clathrin, EEA1, LAMP1, and rab11) were markedly negative. ICQ values for both NaPiIIa and Shank2 with clathrin and EEA1 were similarly increased at 1 h and remained positive after 2 and 4 h. With an apparent modest delay, ICQ values for both NaPiIIa and Shank2 with LAMP1 were also increased at 1 h and remained positive after 2 and 4 h. No significant increases in ICQ values were measured for either NaPiIIa or Shank2 with rab11 at any time point. *P < 0.05 for NaPiIIa and Shank2 against clathrin, EEA1 and LAMP1 at 1, 2, and 4 h compared with 0 h values. ICQ values for NaPiIIa and Shank2 with F-actin were strongly positive at all time points.

Fig. 10.

NaPiIIa and Shank2 abundance in lysosome preparations. In lysosomal fractions from renal cortex tissues collected from rats following 0, 1, 2, and 4 h of high-phosphate feeding, LAMP1 was markedly enriched compared with the initial homogenates. The homogenate and lysosome LAMP1 data shown were from the same blot. NaPiIIa and Shank2 were also present in each of the lysosomal fractions at all four time points. Interestingly, relative levels of both these proteins appeared to be increased after 1 and 2 h of high-phosphate feeding. The blots shown are representative of three separate time course studies.

Fig. 11.

Presence of F-actin on NaPiIIa- and Shank2-containing endosomes. Images are from rat PTs after 1 h following high phosphate feeding. In the subapical domain of these tubules, endosomes co-label with F-actin (left) and NaPiIIa (middle; top row) or F-actin (left panel) and Shank2 (middle; bottom row). Merged images show F-actin (red) was colocalized with NaPiIIa (green) and Shank2 (green). Arrows point to examples of dual-labeled endosomes. Bar = 5 μm.