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.

The calcium-sensing receptor (CaSR) is a member of class C of the G protein–coupled receptors (GPCR) and its role is to constantly monitor Ca²⁺ in the extracellular environment.¹ In the kidney, CaSR is essential for sensing Ca²⁺ in both the urinary filtrate and interstitial fluid to adequately modulate calcium excretion. To achieve this, CaSR is expressed all along the nephron.^2–5

Ca²⁺ and salt (NaCl) handling in the kidney are particularly integrated in two segments of the nephron: the thick ascending limb of Henle’s loop (TALH) and the distal convoluted tubule (DCT). In the TALH Ca²⁺ is reabsorbed by a paracellular route, a process which is largely dependent on NaCl reabsorption.^6,7 Apical NaCl influx via the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) is accompanied by potassium recycling to the lumen, through the apical renal outer medullary K⁺ channel (ROMK, KCNJ1), and by the basolateral extrusion of NaCl by the Na⁺/K⁺-ATPase and the chloride channel Kb (CLCNKB).⁸ The apical recycling of K⁺ generates a transepithelial voltage difference providing a driving force that drags paracellular reabsorption of cations, among them, Ca²⁺.⁹ Consequently, a positive correlation exists between NaCl and Ca²⁺ reabsorption in this nephron segment. For instance, patients with Bartter syndrome exhibit a salt-losing nephropathy and hypercalciuria.¹⁰ Likewise, clinicians have taken advantage of this positive correlation phenomenon by using loop diuretics to treat hypercalcemia.¹¹

In the TALH, CaSR is expressed in the basolateral membrane^2,5 where it senses increased interstitial Ca²⁺ and promotes its urinary excretion by halting NKCC2 and ROMK activity.^4,12–15 In this manner, the increase in Ca²⁺ excretion is due to decreased NaCl reabsorption in the TALH that must be reabsorbed beyond the macula densa. Indeed, gain-of-function mutations of CaSR have been reported to produce a Bartter-like syndrome.^16,17

The DCT reabsorbs approximately 5%–10% of the filtered NaCl and Ca²⁺.^6,7,18 Its effect on BP and Ca²⁺ excretion is prominent because NaCl reabsorption beyond the macula densa is not regulated by tubuloglomerular feedback and no specific Ca²⁺ reabsorption pathways are present beyond this point.⁷ In the DCT, reabsorption of NaCl occurs through the thiazide-sensitive Na⁺-Cl⁻ cotransporter (NCC), whereas that of Ca²⁺ occurs through the apical transient receptor potential cation channel subfamily V (TRPV5).¹⁸ In this part of the nephron, NaCl and Ca²⁺ transport occurs in opposite directions; increased NaCl reabsorption is associated with decreased Ca²⁺ reabsorption.¹⁹ For instance, patients with Gitelman syndrome present a salt-losing nephropathy accompanied by hypocalciuria.²⁰ Clinicians have taken advantage of this inverse reabsorption by using thiazide diuretics to promote Ca²⁺ reabsorption in patients with urolithiasis.²¹ The exact mechanism for this inverse relationship is still unclear. CaSR is expressed both in the basolateral and apical membranes of DCT cells.^4,5,22 However, the role CaSR might play in regulating NaCl reabsorption in this nephron segment is not known.

The activity of NCC is modulated by a kinase pathway consisting of the with-no-lysine-kinases (WNKs) acting upon the Ste20-related proline alanine–rich kinase (SPAK).²³ Active WNK kinases phosphorylate SPAK,²⁴ which subsequently phosphorylates and activates NCC.²⁵ Two proteins, Cullin 3 (CUL3) and Kelch-like 3 (KLHL3), are part of an E3-RING ubiquitin ligase complex that in turn regulates WNK kinases. KLHL3 specifically binds to WNKs marking them for degradation.^26,27 Disease-causing mutations in WNK4, KLHL3, or CUL3 result in impaired degradation of WNK kinases leading to increased NCC activity that results in a syndrome called pseudohypoaldosteronism type II.^28,29

Hormones that regulate NaCl reabsorption in the DCT do so by affecting the KLHL3-WNK-SPAK-NCC pathway. Angiotensin II (AngII) regulates NCC activity in a WNK4-dependent manner.^30,31 This regulation occurs via protein kinase C (PKC), which directly phosphorylates WNK4 in two main sites, S64 and S1196, increasing WNK4 activity.³² PKC also promotes phosphorylation of KLHL3 in a serine residue (S433) that lays in the WNK4-binding domain preventing degradation of WNK4.³³ The effects of AngII in the DCT are mediated by the AT1 receptor, a pleiotropic GPCR whose intracellular signaling mechanisms are similar to that of CaSR.³⁴ Both receptors are preferentially coupled to Gαq and thus activate PLC transduction pathway, increasing intracellular Ca²⁺ and activating PKC.^14,35 In this work, using a combination of in vitro and in vivo approaches, we sought to test the hypothesis that activation of CaSR modulates NCC activity through the KLHL3-WNK4-SPAK pathway.

Methods

In Vitro Experiments

To test the effects of CaSR on NCC activity in vitro we assessed NCC activity in Xenopus laevis oocytes by measuring tracer ²²Na⁺ uptake when CaSR was stimulated with gadolinium chloride (GdCl₃), as described in the complete methods (Supplemental Material). In mammalian cells, the effect of CaSR activation was assessed in HEK-293 cells transiently transfected with CaSR wild type (WT), CaSR mutants, mWNK4-HA, and hSPAK-GFP-HA, with/without KLHL3 and mWNK4-5A-HA mutant. Cells were stimulated with the calcimimetic NPS R-568 (R-568) (Tocris Biosciences) and SPAK phosphorylation, and WNK4 abundance and phosphorylation were assessed by western blot analysis (complete methods, Supplemental Material).

In Vivo Experiments

To test the effect of activating CaSR on the WNK4-SPAK-NCC pathway in vivo we used C57BL/6 male mice, 12–16 weeks old, exposed to vehicle or R-568 (3.0 µg/g of weight) by oral gavage,^36,37 or a single furosemide (Sigma) ip dose of 15 mg/kg⁵⁸. Three hours later, kidneys were extracted and proteins were prepared for western blot (complete methods, Supplemental Information). We also used ex vivo kidney preparations such as the Langendorff system, as previously described.^38,39 Kidneys were perfused with vehicle or the calcimimetic, R-568, at a rate of 0.60 µg/ml per minute for 30 minutes.

Statistical Analyses

Unpaired t test (two tailed) was used for comparison between two groups. One-way ANOVA with Dunnett’s multiple comparison test was performed for comparison between multiple groups. P<0.05 was considered significant. Values are reported as mean±SEM.

Results

CaSR Activates NCC in a WNK4-Dependent Manner in X. laevis Oocytes

Xenopus oocytes were coinjected with WT CaSR and NCC cRNA, with or without WNK4 or WNK1 cRNA, and subjected to thiazide-sensitive tracer ²²Na⁺ transport assays as previously reported.⁴⁰ Coexpressing NCC with WNK4 or WNK1 promoted marked increases in basal NCC activity of two- and four-fold (P<0.01), respectively (Figure 1A), as previously described.^41,42 However, this increase was not affected by the presence of CaSR (Figure 1A). Thus, unstimulated CaSR by itself had no effect on NCC activity. We then tested the effect of CaSR stimulation in the absence or presence of WNK1 or WNK4 kinases. As Figure 1B shows, after exposing oocytes to the type 1 CaSR agonist, Gd³⁺, NCC uptake increased two-fold (P<0.001) only in oocytes coexpressing both CaSR and WNK4 (Figure 1B). We observed no effect of Gd³⁺ in oocytes injected with NCC+CaSR or NCC+WNK1D11+CaSR. These results suggest that, similar to the effects of AngII,^30,31 WNK4 is required for the activation of CaSR to have an effect on NCC.

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.

CaSR Phosphorylates SPAK in a WNK4-Dependent Manner in HEK-293 Cells

To test whether the CaSR-NCC effect could also be observed in a human cell model, we analyzed the effects of activating CaSR on SPAK phosphorylation (pSPAK), as a surrogate of SPAK-NCC activation by WNKs in HEK-293 cells.²⁴ Cells were transiently transfected with SPAK-GFP-HA, WNK4-HA, and CaSR and then treated with the calcimimetic R-568.^43–45 Results show that R-568 induced a time- and dose- dependent pSPAK increase in cells fasted in a serum-free medium (Supplemental Figure 2, A and B). We next evaluated the role of WNK4 on SPAK phosphorylation by CaSR. HEK-293 cells were transfected with SPAK-GFP-HA, CaSR, and/or WNK4-HA. In cells transfected with CaSR alone, pSPAK did not increase after treatment with the calcimimetic. Only in the presence of CaSR and WNK4 together did the calcimimetic promote a significant increase in pSPAK (P<0.05) (Figure 2, A and B). To further test that WNK4 is required for translating CaSR activation to SPAK phosphorylation, we assessed the effect of the highly specific WNK inhibitor, WNK463,⁴⁶ on CaSR-induced SPAK phosphorylation. As shown in Figure 2, C and D, the positive effect of R-568 on pSPAK was completely prevented by the presence of WNK463 inhibitor, confirming that in mammalian cells the effect of CaSR is WNK4-dependent. It is known that CaSR activation leads to activation by phosphorylation of the mitogen-activated protein kinase ERK1,2.⁴⁷ Therefore, we analyzed ERK1,2 phosphorylation to verify CaSR activation in these experiments. As shown in Figure 2A, a clear functional activation of CaSR was achieved with R-568 in CaSR-transfected cells, as demonstrated by increased ERK1,2 phosphorylation, but SPAK phosphorylation by CaSR only increases in the presence of WNK4.

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.

An Activating Mutation of CaSR Increases WNK4 Abundance

Mutations in the CASR gene result in Mendelian disorders characterized by altered Ca²⁺ homeostasis.⁴⁸ Activating mutations of the receptor cause autosomal dominant hypocalcemia, whereas inactivating mutations cause dominant familial hypocalciuric hypercalcemia or recessive neonatal severe hyperparathyroidism.^15,49,50 We used two reported mutations, one activating, CaSR-E228K, and one inactivating, CaSR-R185Q, to assess their effects on the WNK4-SPAK-NCC pathway.^51–53 We transfected HEK-293 cells with the WT CaSR or the mutants with WNK4 and observed that CaSR-E228K increased WNK4 abundance (Figure 3, A and C). We reasoned that if CaSR was acting by the same signal transduction pathway as the AT1 receptor, the presence of KLHL3 would enhance this effect on WNK4. As expected, cotransfection of KLHL3 induced a significant decrease of WNK4 abundance (Figure 3, A and B) that was prevented by CaSR-E228K, but not by CaSR-R185Q, establishing a significant KLHL3-dependent increase in WNK4 total protein levels only in the presence of the active mutant CaSR-E228K (Figure 3D). These results are consistent with the proposal that active CaSR may elicit the same signal transduction pathway as that of AT1 receptor, resulting in decreased degradation of WNK4, likely due to inhibition of KLHL3.³³

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]).

CaSR Promotes KLHL3 and WNK4 Phosphorylation by PKC

Two previous studies have demonstrated that AngII effects on WNK4 are due to a Gαq-PKC signaling transduction pathway.^32,33 To further determine whether CaSR activation elicited similar effects, we assessed if PKC-mediated phosphorylation of KLHL3 and WNK4 occurred after CaSR activation. KLHL3-Flag was immunoprecipitated from lysates of HEK-293 cells cotransfected with CaSR WT or CaSR mutants and subjected to immunoblotting with an mAb that recognizes PKC phosphorylation site, pRRXS.^32,33,54 In the presence of the active mutant CaSR-E228K, KLHL3 pRRXS phosphorylation remarkably increased (P<0.01), whereas this was not observed with the inactive mutant CaSR-R185Q (Figure 4A). If PKC was responsible for these effects, we would expect that inhibition of PKC would prevent CaSR-induced pRRXS increase in KLHL3. As shown in Figure 4B, bisindolylmaleimide I, used at a concentration considered to be an inhibitor of PKC,⁵⁵ significantly reduced KLHL3 pRRXS phosphorylation.

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.

We next evaluated if CaSR-induced activation of PKC also promoted WNK4 phosphorylation. To this end we analyzed whether activating CaSR in HEK-293 cells with R-568 promoted phosphorylation of a key WNK4 PKC phosphorylation site, serine residue S1196.³² After transfection of WNK4-HA, SPAK-GFP-HA, and CaSR, incubation with the calcimimetic resulted in a clear increase in S1196 phosphorylation P<0.05 (Figure 4C). Because the experiment was done with an acute CaSR activation of 30 minutes, no changes were seen in total WNK4 abundance; however, activation by phosphorylation of this site has been previously established³², partially explaining why we can see an effect before WNK4 abundance increases. Furthermore, we used a WNK4 mutant that has all five serines of the PKC consensus sites (RRXS sites) mutated to alanines (WNK4–5A), which prevents PKC-induced phosphorylation.³² The 5A mutation did not alter WNK4 abundance but remarkably reduced the CaSR effect on SPAK phosphorylation (Figure 4D), suggesting that phosphorylation of these sites, and the consequent activation of WNK4 by PKC, is necessary for the complete effect of CaSR on the WNK4-SPAK pathway.

CaSR Promotes NCC Phosphorylation In Vivo

To define whether the CaSR effect on NCC occurred in vivo, we administered C57BL/6 male WT mice with an acute oral treatment of R-568 (3 µg/g body wt)^36,37 and, 3 hours later, mice were euthanized to investigate the effects on NCC phosphorylation by immunoblotting. Calcimimetics directly target the TALH CaSR function,¹² thereby decreasing NKCC2 activity (hence, phosphorylation) and promoting increased luminal Ca²⁺ and NaCl delivery to the distal nephron.¹² To test if this effect occurred in our in vivo model we assessed NKCC2 phosphorylation after the administration of the calcimimetic. Figure 5, A and B, shows that mice treated with the calcimimetic exhibited a significant decrease of NKCC2 phosphorylation P<0.05.

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.

Activation of NCC is associated with increased phosphorylation of three residues, T55, T60, and S73, in human NCC^25,56; therefore, phosphorylation of any of these residues has been extensively used as surrogate of NCC activation.⁵⁶ As expected, treatment with the calcimimetic induced a 1.5-fold increase in NCC phosphorylation (P<0.05) (Figure 5, C and D), without promoting changes in total NCC (NCC/β-actin 1.00 versus 0.96531, P=NS). Moreover, in concordance with our in vitro data, activation of CaSR resulted in a significant increase in total WNK4 protein (1.7-fold increase, P<0.05) (Figure 5, C and D). To evaluate if the increased WNK4 protein was activated by PKC, we analyzed the phosphorylation of residue S64, as previously reported.³² We found that most of the WNK4 protein in the calcimimetic-administered group was phosphorylated in S64 (Figure 5C). However, the pS64/WNK4 ratio between vehicle and R-568 groups remained similar (pS64/WNK4 1.00 versus 1.3050, P=NS). The absence of significance between the vehicle- and R-568–administered groups could be due to the concurrent increase in WNK4 protein. Additionally, immunofluorescence microscopy of kidneys extracted from WT mice showed increased membrane abundance after an acute dose of the calcimimetic (Figure 5E). Interestingly, the increase in NCC phosphorylation was not present in knock-in mice in which SPAK cannot be activated by WNKs (mutation T243A)⁵⁷ (pNCC/NCC 1.00 versus 0.99, P=NS) (Figure 5, F and G).

CaSR is expressed at both the apical and basolateral membranes of DCT cells. To investigate if increasing Ca²⁺ delivery to the DCT, and therefore, only activation of the apical CaSR is sufficient to elicit NCC phosphorylation, we administered C57BL/6 male WT mice with an acute treatment of furosemide (15 mg/kg over 3 hours), as previously described.⁵⁸ This specific dosage and short time of treatment has been described to increase Ca²⁺ and NaCl delivery to the DCT, without promoting dehydration.⁵⁸ No changes in plasma potassium after 3 hours were observed (vehicle 4.3±73 versus furosemide 4.3±0.25, P=NS). As expected, furosemide administration increased NCC phosphorylation four-fold (P<0.05) while not increasing total NCC (NCC/β-actin 1.00 versus 0.9456, P=NS) (Figure 6, A and B). In addition, furosemide administration was associated with increased WNK4 total protein and increased phosphorylation of WNK4 at S64 (Figure 6, C and D). Taken together, these results suggest that the acute inhibition of NKCC2 is associated with increased WNK4-NCC phosphorylation that was probably triggered by increased luminal Ca²⁺.

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).

CaSR Promotes NCC Phosphorylation Ex Vivo

The administration of the calcimimetic in the previous experiments could have promoted NCC activation either by a direct effect on the kidney, through the mechanism proposed in our hypothesis, or by a secondary effect due to activation/modification of any of the multiple hormonal systems that can activate NCC.⁵⁹ Acute calcimimetic administration is associated with decreased activity of the renin-AngII system,^60,61 making this possibility unlikely. Nevertheless, we studied NCC phosphorylation using an ex vivo system where intervention of the central nervous system and other extra renal hormonal systems are not expected to be present. Kidneys of WT male Wistar rats were perfused with physiologic saline with vehicle or with R-568 (0.60 µg/ml per minute). The concentration of R-568 used in these experiments did not change the perfusion pressure, arguing against the presence of an intrarenal AngII effect. As shown in Figure 7, A and B, NCC and SPAK phosphorylation levels were significantly higher in kidneys perfused with the calcimimetic.

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.

Discussion

In this study, we show that CaSR activation is associated with increased NCC activity in vitro and in vivo. This increase involves PKC activation of the WNK4-SPAK pathway, supporting the hypothesis that CaSR modulates NCC activity. As previously shown for AngII, modulation of NCC via WNK4-SPAK occurs by two different pathways—phosphorylation and concurrent activation of WNK4, and prevention of WNK4 degradation by KLHL3 phosphorylation. CaSR-induced activation of NCC has an implication in the physiologic response to increased extracellular Ca²⁺, which requires the kidney to promote its excretion at the apparent expense of reducing NaCl reabsorption in the TALH, thus increasing the delivery of NaCl and Ca²⁺ to the distal nephron.¹⁴ Integration of NaCl and Ca²⁺ homeostasis by CaSR in the DCT could prevent unwanted NaCl loss, while further permitting Ca²⁺ excretion. In this regard, CaSR expression in the apical membrane of the DCT has been clearly established by many groups and recent studies colocalize CaSR with NCC in human and mouse kidneys.^5,22 Taking together the observations in this study, we propose the existence of a mechanism in the DCT, where apical CaSR responds to increased intratubular Ca²⁺ concentration evoking a CaSR-Gαq-PKC-WNK4-SPAK signaling transduction pathway that promotes NCC activation to recover the NaCl that was not reabsorbed in the TALH, due to NKCC2 and ROMK inhibition (Figure 8). Because it is known that increased NaCl reabsorption in the DCT is associated with decreased Ca²⁺ absorption,¹⁴ this mechanism not only claims the NaCl, but also further promotes hypercalciuria. The controversy of whether the thiazide effect on Ca²⁺ excretion occurs directly in the DCT or elsewhere^62,63 does not contradict our findings.

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.

We are aware of the possibility that the basolateral CaSR in DCT may also elicit a response to activate NCC, and our results do not rule out this possibility. In this scenario, increased extracellular Ca²⁺ could simultaneously reduce NKCC2 activity in the TALH but increase NCC activity in the DCT, by activating the basolateral receptor in both segments. However, because of the presence of CaSR and NCC in the apical membrane, is it likely that luminal Ca²⁺ is also involved in this response. NCC activation elicited by a single acute dose of furosemide, known to promote increased Ca²⁺ delivery to DCT, supports the fact that activation of apical CaSR is enough to provoke the proposed response. It is also worth mentioning that patients with autosomal dominant hypocalcemia (due to CaSR activating mutations) may exhibit a Bartter-like syndrome (also known as Bartter syndrome type V) that has been described as mild in most patients.^16,17 Perhaps CaSR activation in the DCT helps to reduce natriuresis, as compared with other types of Bartter syndrome.

A similar mechanism prompted by CaSR in the nephron has been described before. It has been clinically recognized for many years that hypercalcemia induces polyuria.^64,65 Increasing urinary Ca²⁺ to the distal nephron could also promote precipitation of Ca²⁺ and phosphate salts. Sands et al.⁶⁶ elegantly demonstrated that apical CaSR in the collecting duct responds to increased luminal Ca²⁺ to blunt vasopressin-induced insertion of AQP-2 water channels into the apical membrane. The latter would prevent water reabsorption in the collecting duct, allowing the urine to be diluted and thus preventing Ca²⁺ precipitation and formation of renal stones. The authors also demonstrated that the signaling pathway and molecular mechanisms initiated by CaSR was by Gαq and PKC proteins.⁶⁶ More recently, other groups have further established the association of active apical CaSR with decreased AQP2 abundance.^67–69

The observation that CaSR activation modulates NCC activity via WNK4-SPAK pathway may have further implications beyond the physiologic mechanism of how NaCl is recovered in DCT when TALH NaCl reabsorption is decreased by Ca²⁺. First, it is known that arterial hypertension is highly prevalent in primary hyperparathyroidism, ranging from 40% to 65%, which is much higher than the expected 25%–30% of hypertension in the general adult population.⁷⁰ Given our observations, a possible mechanism could be that increased Ca²⁺ in the tubular fluid, as occurs in hypercalcemia, stimulates the activity of NCC promoting NaCl reabsorption and, hence, the development of hypertension. Second, it has been recently demonstrated that glucose and other sugars act as type II calcimimetics, enhancing CaSR affinity for Ca²⁺.⁷¹ This could be relevant in the apical membrane of the DCT because all of the filtered glucose is reabsorbed in the proximal tubule and therefore these cells are not continuously exposed to glucose. In patients with diabetes, the excess filtered glucose often escapes reabsorption in the proximal tubule, allowing a significant amount of glucose in the tubular fluid that reaches the DCT. It is possible that the presence of glucose acting as a calcimimetic increases apical CaSR sensibility, enhancing NCC activity of thus NaCl reabsorption, which could help to explain the higher prevalence of hypertension in patients with diabetes.⁷² These possibilities are speculative but can certainly be explored in future studies.

Disclosures

None.