Gastrin decreases Na⁺,K⁺-ATPase activity via a PI 3-kinase- and PKC-dependent pathway in human renal proximal tubule cells

Abstract

The natriuretic effect of gastrin suggests a role in the coordinated regulation of sodium balance by the gastrointestinal tract and the kidney. The renal molecular targets and signal transduction pathways for such an effect of gastrin are largely unknown. Recently, we reported that gastrin induces NHE3 phosphorylation and internalization via phosphatidylinositol (PI) 3-kinase and PKCα. In this study, we show that gastrin induced the phosphorylation of human Na⁺,K⁺-ATPase at serine 16, resulting in its endocytosis via Rab5 and Rab7 endosomes. The gastrin-stimulated phosphorylation of Na⁺,K⁺-ATPase was dependent on PI 3-kinase because the phosphorylation was blocked by the PI 3-kinase inhibitor wortmannin. The phosphorylation of Na⁺,K⁺-ATPase was also blocked by chelerythrine, a pan-PKC inhibitor, Gö-6976, a conventional PKC (cPKC) inhibitor, and BAPTA-AM, an intracellular calcium chelator, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The gastrin-mediated phosphorylation of Na⁺,K⁺-ATPase was also inhibited by U-73122, a phospholipase C (PLC) inhibitor. These results suggest that gastrin regulates sodium hydrogen exchanger and pump in renal proximal tubule cells at the apical and basolateral membranes.

cholecystokinin (CCK) and gastrin, which are synthesized and secreted by I cells in the upper intestine and G cells in the gastric antrum, respectively, regulate various functions in the gastrointestinal tract and central nervous system (5). These two hormones share the same receptors, the CCKA receptor (CCKAR) and CCKB receptor (CCKBR), that belong to the family of G protein-coupled receptors. Sulfated CCK has a 500- to 1,000-fold higher affinity than sulfated gastrin or nonsulfated CCK to CCKAR, whereas CCKBR binds and responds to gastrin or CCK with similar affinity, regardless of the state of sulfation. Since the levels of plasma gastrin are much higher than CCK, the CCKBR can be considered as the “gastrin receptor” in tissues other than the central nervous system (5).

CCKBR mRNA and protein have been shown to be expressed in the guinea pig and rat kidney; CCKAR mRNA has also been shown to be expressed in the murine and human kidney, but there is no information on the renal expression of CCKAR protein (5). In the kidney, CCKBR is expressed to a greater extent in proximal and distal tubules and collecting ducts and to a lesser extent in glomeruli (4, 27).

The existence of CCKBR in the gastrointestinal tract and the kidney suggests that gastrin, via CCKBR, may exert a coordinated regulation of sodium balance by regulating sodium transport in the gastrointestinal tract and kidney. Gastrin, via CCKBR, increases sodium excretion in the isolated perfused rat kidney (26). The intrarenal infusion of gastrin or CCK in rats induces a diuresis and natriuresis (1, 15). CCK also inhibits Na⁺,K⁺-ATPase activity in the intestinal mucosa (22). The inhibition of renal and intestinal sodium transport by gastrin is in contrast to its ability to increase H⁺,K⁺-ATPase activity in gastric parietal cells (14, 28) and the ability of CCK to increase Na⁺,K⁺-ATPase activity in pancreatic acinar cells (8–10), suggesting tissue specificity in the regulation of ion transport by gastrin and CCK. We have reported that the intrarenal infusion of gastrin increased sodium excretion and decreased Na⁺,K⁺-ATPase activity (1, 19). However, the cellular mechanisms and signal transduction pathways by which gastrin regulates Na⁺,K⁺-ATPase activity are not known.

We now report that in immortalized human renal proximal tubule cells, gastrin increased the phosphorylation of Na⁺,K⁺-ATPase at serine 16, caused its endocytosis, and decreased its expression at the cell surface. Such gastrin-mediated phosphorylation of Na⁺,K⁺-ATPase was phosphatidylinositol (PI) 3-kinase, PKC, and calcium dependent, which is similar to the effect of gastrin on sodium hydrogen exchanger type 3 (NHE3) (16).

METHODS

Chemicals and antibodies.

Chemicals and antibodies were as follows: human gastrin (Genscript, Piscataway, NJ); Gö-6976 (LC Laboratories, Woburn, MA); bis(2-aminophenoxy)ethane-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM; Invitrogen, Grand Island, NY); phospho-Na⁺,K⁺-ATPase antibody (Abcam, Cambridge, MA); path scan antibody (Cell Signaling Technology, Danvers, MA); anti-Rab5, anti-Rab7, anti-phospho-S6, anti-eIF4E (eukaryotic initiation factor 4E), anti-Na⁺,K⁺-ATPase, antibody and secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); and U-73122, chelerythrine, and other chemicals, including protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO).

Cell culture.

Male human proximal tubule cells (NT16) that endogenously express the CCKBR receptor, but not CCKAR (16), were initially cultured as described (21) and then switched to DMEM medium with 4.5% FBS, antibiotics, and antimycotics (Invitrogen) for further growth. The cells were serum-starved for 3 h before treatment. For cells treated with gastrin and a particular inhibitor, the inhibitor was added 5 min before the addition of gastrin. Most inhibitors, including BAPTA-AM, U-73122, Gö-6976, and wortmannin, were dissolved in DMSO, whereas gastrin was dissolved in sterile deionized water. A corresponding amount of vehicle was added in the vehicle-treated (control) groups.

The average normal fasting blood gastrin level is 100 pg/ml. The meal-stimulated blood gastrin concentration is about two- to 4.5-fold higher and equivalent to a concentration of ∼200 pM. Gastrin is almost completely reabsorbed in the proximal tubule, particularly in the S1 segment, and the concentration in this segment may reach levels of ∼100- to 300-fold (17) in the filtrate, which gives a final concentration in the cells of 20–150 nM.

Based on these ranges of concentrations, we carried out an experiment to determine the optimum concentration of gastrin and incubation time on Na⁺,K⁺-ATPase phosphorylation. We treated NT16 cells with gastrin at concentrations of 100, 300, or 1,000 nM for 30 min, 1 h, 2 h, or 4 h and found that the effect of gastrin on Na⁺,K⁺-ATPase phosphorylation was highest at 100 nM gastrin treatment for 30 min and remained at the highest level for 2–3 h. Therefore, we chose 100 nM gastrin treatments for 3 h in our studies.

Immunoblot analyses.

The cells were collected into lysis buffer and then sonicated for 30 s. Equal amounts of protein were loaded onto 4–20 or 10–20% tricine gels (Invitrogen) for SDS-PAGE, transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA), and then probed with primary antibody (overnight at 4°C). The blots were then incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) (2 h at 22°C) and developed with enhanced chemiluminescence reagent (Santa Cruz Biotechnology). The autoradiographs were scanned, and the intensity of the signals (including a blank region) was quantified (NIH ImageJ). Control and treatment values were corrected for blank values and normalized to their respective GAPDH or tubulin bands. Quantifications were finally expressed as relative percentage changes over controls.

Endosome isolation.

Endosomes were fractionated on a flotation gradient, using essentially the technique described by others (2, 7). After treatment with gastrin the medium was removed, and the cells were scraped off in cold homogenization buffer containing 250 mM sucrose and 3 mM imidazole (pH 7.4). The cells were gently homogenized using a Dounce homogenizer, and the samples were centrifuged at 3,000 g for 5 min. The sucrose concentration of supernatants was adjusted to 40.6%; 1.5 ml was loaded into the bottom of a 5.0-ml centrifuge tube, and 1.5 ml of 16% sucrose in 3 mM imidazole and 0.5 mM EDTA was carefully added on top. After topping with 1 ml of 10% sucrose, 1 ml of homogenization buffer was added. The samples were centrifuged for 1 h at 110,000 g in a Beckman SW 41 rotor. Late endosomes were collected at the interface between the homogenization buffer and 10% sucrose, and early endosomes were collected at the interface between the 10 and 16% sucrose gradient.

Statistical analyses.

One-way ANOVA was used to compare more than two groups with one treatment, whereas two-way ANOVA was used to compare more than two groups with more than one treatment. Post hoc test utilized Tukey's multiple comparison tests, with a P value <0.05 deemed as significant.

RESULTS

Gastrin stimulates the phosphorylation of Na⁺,K⁺-ATPase at serine 16. To determine whether gastrin causes phosphorylation of Na⁺,K⁺-ATPase at serine 16, we treated immortalized human renal proximal tubule NT16 cells with gastrin and quantified the levels of phospho-Na⁺,K⁺-ATPase with a phospho-specific Na⁺,K⁺-ATPase antibody that recognizes Na⁺,K⁺-ATPase only when it is phosphorylated at serine 16. As shown in Fig. 1, A, top, and B, gastrin treatment for 3 h at the concentration of 100 or 300 nM increased sevenfold the phosphorylation of Na⁺,K⁺-ATPase at serine 16. Immunoblotting using an antibody that recognizes total Na⁺,K⁺-ATPase (Fig. 1, A and C) shows that gastrin treatment actually decreased threefold the total Na⁺,K⁺-ATPase protein. These results show that the gastrin treatment probably stimulates the degradation of the Na⁺,K⁺-ATPase in the renal proximal tubule cells via phosphorylation at serine 16. The changes in the levels of phosphorylated and total Na⁺,K⁺-ATPase in response to gastrin were in opposite directions. Thus, the increase in the ratio of phosphorylated and total Na⁺,K⁺-ATPase showed an even more impressive increase in response to gastrin treatment (Fig. 1D).

Fig. 1.

Gastrin treatment increases human Na⁺,K⁺-ATPase phosphorylation at serine 16. NT16 cells were treated with gastrin at the indicated concentrations for 3 h. Samples were then subjected to immunoblotting with phospho-Na⁺,K⁺-ATPase S16 antibody (A, top), total Na⁺,K⁺-ATPase antibody (A, middle), or GAPDH antibodies (A, bottom). The set of blots is 1 of 3 independent experiments. B: quantification of phospho-Na⁺,K⁺-ATPase levels in NT16 cells without and with treatment with gastrin at the indicated concentrations for 3 h. C: quantification of total Na⁺,K⁺-ATPase levels in NT16 cells without and with treatment with gastrin at the indicated concentrations for 3 h. D: ratio of phospho-Na⁺,K⁺-ATPase and total Na⁺,K⁺-ATPase levels in NT16 cells without and with treatment with gastrin at the indicated concentrations for 3 h. *P < 0.05 vs. gastrin 0 nM; n = 3.

Gastrin treatment induces the endocytosis of phospho-Na⁺,K⁺-ATPase. The phosphorylation of Na⁺,K⁺-ATPase is known to be a mechanism in the acute regulation of the subcellular distribution and activity of this protein (2, 3). To determine the subcellular distribution of Na⁺,K⁺-ATPase as a consequence of its phosphorylation, we isolated both early and late endosomes from NT16 cells treated with gastrin. Endosomes from the same amount of cells were isolated by sucrose gradient ultracentrifugation and fractionation. Enrichment of early and late endosome fractions by sucrose gradient centrifugation was evaluated by immunoblotting for Rab 5 and Rab 7 (7), markers for early and late endosomes, respectively (Fig. 2A). The samples were subjected to immunoblotting with phospho-Na⁺,K⁺-ATPase antibody. As shown in Fig. 2B, in the untreated group, no signal was observed in either early or late endosomes. However, gastrin induced a concentration-dependent increase in amount of phospho-Na⁺,K⁺-ATPase in early endosomes (Fig. 2, B and C). A similar pattern was observed in late endosomes (Fig. 2, B and D). However, phospho-Na⁺,K⁺-ATPase was much less in late than in early endosomes (Fig. 2B).

Fig. 2.

Gastrin treatment increases the endocytosis of phosphorylated (serine 16) human Na⁺,K⁺-ATPase. NT16 cells were treated with gastrin at the indicated concentrations for 3 h. A and B: early and late endosomes were isolated according to the protocol described in methods. Samples were then subjected to immunoblotting with Rab5 or Rab7 antibody for early and late endosome markers, respectively (A), and phospho-Na⁺,K⁺-ATPase S16 antibody (B); 1 set of blots from 3 independent experiments is shown. C: quantification of phospho-Na⁺,K⁺-ATPase levels in early endosomes of NT16 cells after treatment with gastrin at the indicated concentrations for 3 h as in A (*P < 0.05 vs. gastrin 0 nM; n = 3). D: quantification of phospho-Na⁺,K⁺-ATPase levels in late endosomes of NT16 cells after treatment with gastrin at the indicated concentrations for 3 h as in A (each group is assigned a letter or the combination of 2 letters, and groups without shared letters are significantly different from each other, P < 0.05; n = 3).

Gastrin-induced phosphorylation of Na⁺,K⁺-ATPase is PI 3-kinase dependent. As shown in Fig. 3, A and B, gastrin induced the phosphorylation of Na⁺,K⁺-ATPase as well as S6 and eIF4E. These effects were blocked by pretreatment with wortmannin, suggesting that PI 3-kinase was involved in the phosphorylation of Na⁺,K⁺-ATPase and S6.

Fig. 3.

Gastrin-induced Na⁺,K⁺-ATPase phosphorylation is phosphatidylinositol (PI) 3-kinase dependent. NT16 cells were treated with gastrin at the indicated concentrations alone or in combination with wortmannin (1 μM) for 3 h. A: samples were then subjected to immunoblotting with phospho-Na⁺,K⁺-ATPase (S16), phospho-S6, eukaryotic initiation factor 4E (eIF4E), and GAPDH antibodies. One set of blots from 3 independent experiments is shown. B: quantification of phospho-Na⁺,K⁺-ATPase levels in NT16 cells after treatment with gastrin at the indicated concentrations alone or in combination with wortmannin (1 μM) for 3 h as in A (n = 3). Each group is assigned a letter or a combination of letters. Groups without shared letters indicate significant difference with P < 0.05. Thus, a is different from bc and d but not different from ab; bc is different from a and d but not different from ab, and d is different from all other groups.

The ability of gastrin to phosphorylate Na⁺,K⁺-ATPase was biphasic (Fig. 3); the phosphorylation of both Na⁺,K⁺-ATPase and S6 increased after 3 h with 100 to 300 nM gastrin (Fig. 1–3) but was blunted with 1,000 nM (Fig. 3).

Gastrin-induced Na⁺,K⁺-ATPase phosphorylation is phospholipase C (PLC) dependent. As shown in Fig. 4,A and B, gastrin treatment increased the phosphorylation of Na⁺,K⁺-ATPase, in agreement with the data shown in Figs. 1–3. Although there was some variability in the relative change induced by 300 nM gastrin, the PLC inhibitor U-73122 reduced both basal and gastrin-induced phosphorylation of Na⁺,K⁺-ATPase, suggesting that PLC is involved in the gastrin signal transduction pathway. We then used a PLC isoform antibody kit to determine the PLC isoform involved in the gastrin signaling pathway. PLCγ2 protein was not found in renal proximal tubule cells, in agreement with previous studies showing that this PLCγ isoform is expressed mainly in hematopoietic cells (29). By contrast, gastrin treatment increased the phosphorylation of PLCγ1 (data not shown). This is reminiscent of gastrin-induced IP3 formation through PLCγ1 and pp60c-src kinase in rat colonic epithelial cells (30).

Fig. 4.

Gastrin-induced phosphorylation of Na⁺,K⁺-ATPase is blocked by U-73122, a phospholipase C (PLC) inhibitor. NT16 cells were treated with gastrin at the indicated concentrations alone or in combination with PLC inhibitor U-73122 (10 μM) for 3 h. A: samples were subjected to immunoblotting with phospho-Na⁺,K⁺-ATPase antibody; 1 set of blots from 3 independent experiments is shown. B: quantification of phospho-Na⁺,K⁺-ATPase levels in NT16 cells after treatment with gastrin at the indicated concentrations alone or in combination with the PLC inhibitor U-73122 (10 μM) for 3 h as in A (n = 3). Each group is assigned a letter, and different letters indicate significant difference with P < 0.05. Thus, a is different from b, c, and d; b is different from a, c, and d; c is different from a, b, and d; and d is different from a, b, and c.

Gastrin-induced Na⁺,K⁺-ATPase phosphorylation is conventionally PKC dependent. Gastrin treatment increased the phosphorylation of Na⁺,K⁺-ATPase, S6, and eIF4E, as shown in Figs. 3A and 5A; gastrin cotreatment with the conventional PKC (cPKC) inhibitor Gö-6976 (Fig. 5, A and B) blocked the basal and gastrin-induced phosphorylation, suggesting that the signaling pathway activated by gastrin included cPKC. Similar results were obtained with the pan-PKC inhibitor chelerythrine (data not shown). In a separate study, we reported that gastrin treatment increased the phosphorylation of PKCα (16). These data suggest that PKCα is involved in the signal transduction of gastrin.

Fig. 5.

Gastrin-induced phosphorylation of Na⁺,K⁺-ATPase is blocked by the conventional PKC (cPKC) inhibitor Gö-6976. NT16 cells were treated with gastrin alone or in combination with either Gö-6976 (10 μM) for 3 h. A: samples were subjected to immunoblotting with phospho-Na⁺,K⁺-ATPase, phospho-S6, eIF4E, and GAPDH antibodies; 1 set of blots from 3 independent experiments is shown. B: quantification of phospho-Na⁺,K⁺-ATPase in NT16 cells after treatment with gastrin at the indicated concentrations as in A (n = 3). Each group is assigned a letter, and different letters indicate significant difference with P < 0.05. Thus, a is different from b, c, and d; b is different from a, c, and d; c is different from a, b, and d; and d is different from a, b, and c.

Gastrin-induced Na⁺,K⁺-ATPase phosphorylation is intracellular calcium dependent. We found in another study that the gastrin-mediated phosphorylation of NHE3 in human renal proximal tubule cells was dependent on an increase in intracellular calcium (16). In the current study, we also found that the intracellular chelator BAPTA-AM blocked the basal and gastrin-induced phosphorylation of Na⁺,K⁺-ATPase (Fig. 6). The gastrin-mediated phosphorylation of Na⁺,K⁺-ATPase in BAPTA-AM-treated cells was not detectable by immunoblot, and therefore, it could not be quantified.

Fig. 6.

Gastrin-induced phosphorylation of Na⁺,K⁺-ATPase is dependent on intracellular calcium. NT16 cells were treated with gastrin at the indicated concentrations alone or in combination with intracellular calcium chelator bis(2-aminophenoxy)ethane-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM; 25 μM) for 3 h, and samples were subjected to immunoblotting with phospho-Na⁺,K⁺-ATPase and α-tubulin antibodies; 1 set of blots from 3 independent experiments is shown.

DISCUSSION

Under normal circumstances, ingested sodium is absorbed in the gastrointestinal track and excreted by the kidney. The balance between intestinal absorption and urinary excretion of sodium is essential for the maintenance of sodium balance in the body. Some hormones secreted by the gastrointestinal track can act on the kidney to influence sodium transport. Gastrin is one such hormone, the secretion of which is increased after food intake and is natriuretic (6, 11, 26).

The natriuretic effect of gastrin was reported more than 10 years ago, but its mechanism of action is not known (19, 20, 26). The receptor of gastrin, CCKBR, has been shown to be present in the kidney to a greater extent in proximal and distal tubules and collecting ducts and to a lesser extent in glomeruli (19, 27). The molecular target, the signal transduction pathway, and the mechanism of the natriuretic effect of gastrin in the kidney are not well known. We have reported that gastrin through its receptor, CCKBR, stimulates the NHE3 phosphorylation and internalization that are associated with a decrease in its activity. This process involved both the PI 3-kinase and PKC pathways (16). Sodium reabsorption by the renal proximal tubule depends on multiple sodium transporters and exchangers and pumps, of which the major sodium exchanger is NHE3 located at the apical membrane, whereas sodium inside the cell is pumped out by Na⁺,K⁺-ATPase at the basolateral membrane (31).

Gastrin has been reported to inhibit Na⁺,K⁺-ATPase in the kidney of normotensive rats (1, 20). These studies were designed to determine how gastrin inhibits Na⁺,K⁺-ATPase and the signal transduction pathway involved in human renal proximal tubule cells. We first treated human renal proximal tubule cells with gastrin and found that gastrin increased the phosphorylation of Na⁺,K⁺-ATPase that was blocked by wortmannin, suggesting that PI 3-kinase is needed for such phosphorylation. We have reported that gastrin also increased S6 phosphorylation along with NHE3 via a similar manner (16).

The phosphorylation of Na⁺,K⁺-ATPase caused by parathyroid hormone is associated with its endocytosis through the endosome (12, 13). One week of NaCl (4%) load was also shown to cause Na⁺,K⁺-ATPase endocytosis (18). To determine whether gastrin also causes endocytosis of Na⁺,K⁺-ATPase, we treated NT16 cells with gastrin and isolated both early and late endosomes and measured the level of phospho-Na⁺,K⁺-ATPase in those fractions. As shown in Fig. 2, phospho-Na⁺,K⁺-ATPase was not detected in early endosomes from cells treated with vehicle. However, the amount of phospho-Na⁺,K⁺-ATPase in early endosomes was increased by gastrin in a concentration-dependent manner.

PKC-mediated phosphorylation and endocytosis of Na⁺,K⁺-ATPase in renal proximal tubule cells have already been reported (2, 3, 24). Therefore, to identify further the signaling pathway involved in ligand-induced Na⁺,K⁺-ATPase phosphorylation, we tested the effects of the PLC inhibitor U-73122 and the cPKC inhibitor Gö-6976. We found that U-73122 and Gö-6976 reduced basal and gastrin-induced Na⁺,K⁺-ATPase phosphorylation. BAPTA-AM, an intracellular calcium chelator, not only reduced but actually completely blocked the basal and gastrin-induced phosphorylation of Na⁺,K⁺-ATPase. These studies suggest that cPKC is involved, but an increase in intracellular calcium is even more important. This could be taken to indicate that gastrin may increase intracellular calcium by additional mechanisms. In a separate report, we showed that gastrin-phosphorylated NHE3 also involved PLC, PKCα, and intracellular calcium (16). The gastrin-induced phosphorylation of S6 and elF4A always paralleled that of Na⁺,K⁺-ATPase, suggesting that they share the same signaling pathway. Parathyroid hormone also inhibits Na⁺,K⁺-ATPase activity via PKCα in opossum kidney cells that have both proximal (13) and distal tubule characteristics (25), although the role of calcium in the process was not tested. In COS-7 cells, a fibroblast-like cell line derived from the African green monkey, phorbol 12,13-dibutyrate, decreased Na⁺,K⁺-ATPase activity as well as its cell surface expression in cells incubated at 37°C but increased its activity in cells incubated at 18°C. In rat parotid acinar cells, ouabain-initiated serine 16 phosphorylation of Na⁺,K⁺-ATPase is PKC independent (23). Therefore, the inhibition of Na⁺,K⁺-ATPase activity is probably cell type and condition specific.

NHE3 is located at the apical and Na⁺,K⁺-ATPase at the basolateral membrane of the renal proximal tubule and thick ascending limb (31). We now report that Na⁺,K⁺-ATPase is regulated by gastrin in a fashion similar to NHE3, sharing the same pathway, involving PI 3-kinase, PLC, cPKC, and intracellular calcium. In addition, we show that the endocytosis of Na⁺,K⁺-ATPase occurs via Rab 5- and Rab 7-related endosomes. Based on the findings described in this and our previous report (16), we propose the following model of gastrin regulation of sodium transport in human renal proximal tubule cells (Fig. 7). Gastrin induces the phosphorylation of NHE3 and Na⁺,K⁺-ATPase located at the apical and basolateral membranes of renal proximal tubule cells, respectively. Such phosphorylation is PI 3-kinase, PLC, PKC, and calcium dependent. The phosphorylation of Na⁺,K⁺-ATPase and NHE3 causes their dislocation away from the plasma membrane into endosomes, causing an inhibition of sodium transport.

Fig. 7.

Hypothetical gastrin-cholecystokinin B receptor (CCKBR) signal transduction pathway. Gastrin signaling through CCKBR causes PI 3-kinase activation and the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which in turn increases the binding of PLC to the plasma membrane. PLC then activates PKCα through both diacylglycerol (DAG) and calcium, and PKCα, but not Akt, causes the phosphorylation of Na⁺,K⁺-ATPase, sodium hydrogen exchanger type 3 (NHE3), and S6. Phosphorylation of Na⁺,K⁺-ATPase and NHE3 increases their internalization and decreases renal sodium transport.

GRANTS

This study was supported National Institutes of Health Grants DK-039308 and HL-092196 to P. A. Jose.

DISCLOSURES

The authors declare no competing or financial interests.

AUTHOR CONTRIBUTIONS

P.A.J. conception and design of research; T.L., P.R.K., and Y.Y. performed experiments; P.A.J., T.L., P.R.K., and Y.Y analyzed data; P.A.J., T.L., and P.R.K. interpreted results of experiments; T.L., P.R.K., and Y.Y. prepared figures; P.A.J., T.L., and P.R.K. drafted manuscript; P.A.J., T.L., P.R.K., and Y.Y. edited and revised manuscript; P.A.J., T.L., P.R.K., and Y.Y approved final version of manuscript.