The Calcium-Sensing Receptor Increases Activity of the Renal NCC through the WNK4-SPAK Pathway

Abstract

Background Hypercalciuria can result from activation of the basolateral calcium-sensing receptor (CaSR), which in the thick ascending limb of Henle's loop controls Ca²⁺ excretion and NaCl reabsorption in response to extracellular Ca²⁺ However, the function of CaSR in the regulation of NaCl reabsorption in the distal convoluted tubule (DCT) is unknown. We hypothesized that CaSR in this location is involved in activating the thiazide-sensitive NaCl cotransporter (NCC) to prevent NaCl loss.Methods We used a combination of in vitro and in vivo models to examine the effects of CaSR on NCC activity. Because the KLHL3-WNK4-SPAK pathway is involved in regulating NaCl reabsorption in the DCT, we assessed the involvement of this pathway as well.Results Thiazide-sensitive ²²Na⁺ uptake assays in Xenopus laevis oocytes revealed that NCC activity increased in a WNK4-dependent manner upon activation of CaSR with Gd³⁺ In HEK293 cells, treatment with the calcimimetic R-568 stimulated SPAK phosphorylation only in the presence of WNK4. The WNK4 inhibitor WNK463 also prevented this effect. Furthermore, CaSR activation in HEK293 cells led to phosphorylation of KLHL3 and WNK4 and increased WNK4 abundance and activity. Finally, acute oral administration of R-568 in mice led to the phosphorylation of NCC.Conclusions Activation of CaSR can increase NCC activity via the WNK4-SPAK pathway. It is possible that activation of CaSR by Ca²⁺ in the apical membrane of the DCT increases NaCl reabsorption by NCC, with the consequent, well known decrease of Ca²⁺ reabsorption, further promoting hypercalciuria.

Keywords: Na transport; distal tubule; diuretics; hypertension.

Graphical abstract

Figure 1.

CaSR activates NCC in a WNK4-dependent manner in X. laevis oocytes. (A) The presence of non-activated CaSR has no effect on WNK4- or WNK1-induced activation of NCC. Functional expression assay shows the thiazide-sensitive Na⁺ uptake in groups of oocytes injected with NCC, NCC+hWNK4, and NCC+WNK1D11 cRNA (black bars), or together with CaSR cRNA (gray bars), as stated. Uptake in oocytes injected with NCC cRNA alone was arbitrarily set to 100% and the corresponding groups were normalized accordingly. **P<0.01 versus NCC. (B) Activation of CaSR with GdCl₃ increased the activity of NCC only in the presence of WNK4. Uptake was performed in control conditions (black bars) or after stimulation with GdCl₃ 80 µM for 15 minutes. Each group in control conditions (black bars) was arbitrarily set to 100% and the corresponding group with GdCl₃ was normalized accordingly (gray bars). ***P<0.001 versus its own control. Supplemental Figure 1 shows the same experiments but with data expressed as picomoles per oocyte per hour. cRNA, complementary RNA.

Figure 2.

CaSR phosphorylates SPAK in a WNK4-dependent manner in HEK-293 cells. (A) Representative immunoblot of cells transfected with hSPAK-GFP-HA, mWNK4-HA, and hCaSR in different combinations, as stated. The day before the experiment, cells were serum-starved in the normal growth medium and left overnight. The next day, cells were stimulated with R-568 (200 nM) for 30 minutes. (B) Densitometric analysis of (A). SPAK transfection alone in control conditions was arbitrarily set to 1 and the corresponding groups were normalized accordingly. Bars represent mean±SEM of at least three independent experiments. *P<0.05 versus control. (C) Representative immunoblot showing two experiments of cells transfected with empty vector (Empty), hSPAK-GFP-HA, mWNK4-HA, and hCaSR and treated as in (A). The WNK inhibitor WNK463 was added to the medium for 2 hours on the day of the experiment to a final concentration of 4 µM. (D) Densitometric analysis of (C). SPAK in control conditions was arbitrarily set to 1 and the corresponding groups were normalized accordingly. Bars represent mean±SEM of at least three independent experiments. *P<0.05 versus control (no stimulation with R-568 and no WNK463). ***P<0.01 versus R-568.

Figure 3.

An activating mutation of CaSR increases WNK4 abundance. (A) Representative immunoblot of HEK-293 cells transfected with mWNK4-HA, hCaSR WT, and CaSR mutants with or without KLHL3 DNA (40 ng). For this set of experiments, cells were maintained in normal growth medium after transfection. (B) Densitometric analysis of (A), where the expression of WNK4 alone (WNK4) was set to 1 and the rest of the groups were normalized accordingly. Bars represent mean±SEM of at least three independent experiments. ***P<0.001 and **P<0.05 versus WNK4. (C and D) Densitometric analysis where WNK4 (Control) (C) without KLHL3 cotransfection or (D) with KLHL3 were set to 1 and the rest of the groups were normalized accordingly. Bars depict mean±SEM of at least three independent experiments. **P<0.001 versus WNK4+KLHL3 (Control of [D]).

Figure 4.

CaSR promotes KLHL3 and WNK4 phosphorylation by PKC. (A) Representative immunoblot of immunopurified KLHL3-Flag from HEK-293 cells transfected with KLHL3, WT hCaSR, and CaSR mutants. Cells were maintained in normal growth medium after transfection. Graph depicts densitometric analysis of at least three independent experiments. KLHL3 immunopurified from transfection alone (Control) was set as 1 and the rest of the groups were normalized accordingly. Bars represent mean±SEM. **P<0.01 versus Control. (B) Representative image of immunopurified KLHL3-Flag from HEK-293 cells transfected with KLHL3, CaSR-E228K, and treated with a PKC inhibitor (bisindolylmaleimide I [BIM]). BIM (4 µM) was added to the normal growth medium and left overnight. The next day, cells were lysed and immunoblotted. Graph shows densitometric analysis of at least three independent experiments. Bars represent mean±SEM. *P<0.05 versus KLHL3 CaSR-E228K without BIM. (C) Representative immunoblot of cells transfected with SPAK-GFP-HA, mWNK4-HA, and WT hCaSR, serum-starved and stimulated with R-568 (200 nM) for 30 minutes. Lysates were blotted with the indicated antibodies. The graph depicts densitometric analysis. *P<0.05 versus Control (no stimulation with R-568). (D) Cells were transfected with SPAK-GFP-HA, mWNK4-HA, and WT hCaSR or the mutant mWNK4-5A, which has all PKC-phosphorylation sites mutated to alanines, and then stimulated as in (C). The graph represents densitometric analysis of at least three independent experiments for the mWNK45A mutant. Bars are mean±SEM. ***P<0.001 versus its own control (data for SPAK-mWNK4-CaSR are shared with Figure 2D). IP, immunoprecipitation.

Figure 5.

CaSR promotes NCC phosphorylation in vivo. Animals were administered with vehicle or with R-568, 3 µg/g body wt through oral gavage. Three hours later, kidneys were harvested and processed for immunoblot. Each column of the representative immunoblot represents the kidneys from one animal. (A and C) Representative immunoblot of the effect of oral R-568 administration on NCC and NKCC2 phosphorylation, WNK4 abundance, and phosphorylation in S64 in WT mice (upper image). pS64/WNK4 1.00 versus 1.3050, P=NS. (E) Immunofluorescent staining of kidney sections from WT mice treated with Vehicle or R-568. Scale bars, 20 µm. (F) Representative immunoblot of the effect of R-568 on NCC phosphorylation in SPAK knock-in mice (SPAK^243A/243A). (B, D, and G) Densitometric analysis of representative immunoblots. Bars represent mean±SEM. *P<0.05 versus Vehicle.

Figure 6.

An acute furosemide treatment promotes NCC phosphorylation in vivo. Animals were administered with vehicle or with furosemide, 15 mg/kg body wt through ip injection. Three hours later, kidneys were harvested and processed for immunoblot. Each column of the representative immunoblot represents the kidney from one animal. (A and C) Representative immunoblots of the effect of the acute administration of furosemide on NCC phosphorylation, WNK4 abundance, and phosphorylation in S64 in WT mice. pS64/WNK4 1.00 versus 1.53, P=NS. (B and D) Densitometric analysis of n=8 controls and n=7 furosemide-administered mice. Bars represent mean±SEM. *P<0.05 versus Vehicle (B was analyzed with Mann-Whitney U test).

Figure 7.

CaSR promotes NCC phosphorylation ex vivo. (A) Representative immunoblot of protein extracts from ex vivo perfused rat kidneys. The kidneys were perfused with physiologic saline with vehicle or with R-568 at a rate of 0.60 µg/ml per minute. Each column of the immunoblot represents one kidney. (B) Bars represent mean±SEM of the densitometric analysis of (A). n=6 vehicles and n=7 R-568. **P<0.01 versus vehicle. *P<0.05 versus vehicle.

Figure 8.

Proposed model for CaSR activation of NCC through a PKC-WNK4-SPAK pathway. Increased extracellular Ca²⁺ leads to CaSR-mediated inhibition of NKCC2 and ROMK, halting the transepithelial voltage difference that drags paracellular reabsorption of Ca²⁺ ions. Reduction in Ca²⁺ reabsorption in the TALH causes increased NaCl and Ca²⁺ delivery to the distal nephron. In the DCT, integration of calcium and NaCl homeostasis by the CaSR must respond to prevent unwanted NaCl loss. We propose the existence of a mechanism in the DCT where apically expressed CaSR responds to increased intratubular Ca²⁺ concentration, evoking a CaSR-Gαq-PKC-WNK4 signaling transduction pathway that promotes NCC activation. Cln-14/16, Claudin 14 and 16 heterodimers.