Calcium-sensing receptor attenuates AVP-induced aquaporin-2 expression via a calmodulin-dependent mechanism

Abstract

Recent evidence suggests that arginine vasopressin (AVP)-dependent aquaporin-2 expression is modulated by the extracellular calcium-sensing receptor (CaSR) in principal cells of the collecting duct, but the signaling pathways mediating this effect are unknown. Using a mouse cortical collecting duct cell line (mpkCCD(cl4)), we found that increasing the concentration of apical extracellular calcium or treating with the CaSR agonists neomycin or Gd(3+) attenuated AVP-dependent accumulation of aquaporin-2 mRNA and protein; CaSR gene-silencing prevented this effect. Calcium reduced the AVP-induced accumulation of cAMP, but this did not occur by increased degradation of cAMP by phosphodiesterases or by direct inhibition of adenylate cyclase. Notably, the effect of extracellular calcium on AVP-dependent aquaporin-2 expression was prevented by inhibition of calmodulin. In summary, our results show that high concentrations of extracellular calcium attenuate AVP-induced aquaporin-2 expression by activating the CaSR and reducing coupling efficiency between V(2) receptor and adenylate cyclase via a calmodulin-dependent mechanism in cultured cortical collecting duct cells.

Figure 1.

Effect of high apical calcium on AVP-dependent AQP2 expression in mpkCCD_cl4 cells. Confluent mpkCCD_cl4 cells grown on filters in culture medium containing baseline (1 mM) calcium were preincubated at 37°C in the presence of 10⁻⁹ M AVP for 24 h to induce AQP2 expression. Cells were then incubated in the continuous presence of 10⁻⁹ M AVP for different lengths of time (3 to 24 h) with 1 or 5 mM apical calcium. (A and B) Total protein extracts (40 μg) were separated by 10% SDS-PAGE. AQP2 and the Na-K-ATPase α-subunit, used as a loading control, were detected by Western blotting. (A) A representative immunoblot is shown. (B) Densitometric quantification of AQP2 protein expressed as a percentage of control optical density values measured after 3 h of incubation in the presence of 1 mM extracellular calcium (100%). Bars are means ± SEM from 12 independent experiments. (C) RNA was extracted as described in the Concise Methods section. Real-time PCR was performed with primers specific for AQP2. Results are expressed as a percentage of control values determined after 24 h of incubation in the presence of 1 mM calcium (100%). Bars are means ± SEM from seven independent experiments. (D) Cells were preincubated in the continuous presence of AVP and 1 mM (○) or 5 mM (•) apical calcium for 24 h before addition of 5 × 10⁻⁶ M actinomycin D (ActD) for 3 or 5 h. RNA was then extracted and real-time PCR was performed. Results are expressed as a percentage of control values determined after 24 h of incubation in the presence of 1 mM calcium and without ActD (control [ctl], 100%). Circles are means ± SEM from four independent experiments. *P < 0.05.

Figure 2.

Effect of extracellular CaSR agonists on AQP2 expression. Confluent mpkCCD_cl4 cells grown on filters in culture medium containing baseline (1 mM) calcium were preincubated at 37°C with 10⁻⁹ M AVP for 24 h. Cells were then incubated in the continuous presence of AVP for another 24 h without (Ctl) or with increasing concentrations of neomycin (A and B) or gadolinium, two cationic CaSR agonists. Total protein extracts (40 μg) were separated by 10% SDS-PAGE and AQP2 and the Na-K-ATPase α-subunit, used as a loading control, were detected by Western blotting. (A and C) Representative immunoblot is shown. (B and D) Densitometric quantification of AQP2 protein expressed as a percentage of optical density values measured in the absence of drug (100%). Bars are means ± SEM from four to six independent experiments. *P < 0.05.

Figure 3.

Role of the CaSR in the calcium-induced attenuation of AVP-dependent AQP2 expression. (A) Confluent mpkCCD_cl4 cells grown on filters in culture medium containing baseline (1 mM) calcium were preincubated at 37°C with 10⁻⁹ M AVP for 24 h and then incubated in the continuous presence of AVP for another 24 h with 1 or 5 mM apical calcium. RNA or protein was then extracted and real-time PCR or Western blotting was performed as described in the Concise Methods section using primers or antibodies specific for CaSR, respectively. (B and C) Two days after transfection with either CaSR scramble RNAi (Scr) or CaSR RNAi, confluent mpkCCD_cl4 cells grown on filters in culture medium containing baseline (1 mM) calcium were preincubated at 37°C with 10⁻⁹ M AVP for 24 h and then incubated in the continuous presence of AVP for another 14 h with 1 or 5 mM apical calcium. RNA or protein was then extracted and real-time PCR or Western blotting was performed as described in the Concise Methods sections using primers or antibodies specific for CaSR, respectively (B), or primers specific for AQP2 (C). Na-K-ATPase α-subunit was used as a loading control for Western blotting, and representative immunoblots from two independent experiments are shown. CaSR and Na,K-ATPase α-subunit were detected as 140- and 110-kD bands, respectively. PCR results are expressed as a percentage of control values determined after incubation in the presence of 1 mM calcium (100%). Bars are means ± SEM from six independent experiments. *P < 0.05.

Figure 4.

The effect of apical calcium on AVP-dependent AQP2 expression is independent of PKC and ERK activation. Confluent mpkCCD_cl4 cells grown on filters in culture medium containing baseline (1 mM) calcium were preincubated for 24 h at 37°C with 10⁻⁹ M AVP. Cells were then incubated for another 24 h in the continuous presence of AVP and in the presence of 1 or 5 mM apical calcium and treated or not (Ctl) with either 10⁻⁶ M of the GF109203X (GF), a PKC inhibitor, or 10⁻⁶ M UO126 (UO), a MEK-1 inhibitor. Total protein extracts (40 μg) were separated by 10% SDS-PAGE and AQP2 and the Na-K-ATPase α-subunit, used as a loading control, were detected by Western blotting. (A) A representative immunoblot is shown. (B) Densitometric quantification of AQP2 protein expressed as a percentage of OD values measured in the presence of 1 mM calcium and without drugs (100%). Bars are means ± SE from four independent experiments. *P < 0.05.

Figure 5.

Role of the cAMP-PKA pathway in the calcium-induced attenuation of AVP-dependent AQP2 expression. (A) Confluent mpkCCD_cl4 cells grown on filters in culture medium containing baseline (1 mM) calcium were preincubated for 24 h at 37°C with 10⁻⁹ M AVP. Cells were then incubated for another 24 h in the continuous presence of AVP and in the presence of 1 or 5 mM apical calcium and treated or not with 5 × 10⁻⁶ M myristoylated PKA inhibitor (PKI). After RNA extraction, real-time PCR was performed as described in the Concise Methods section using primers against AQP2. Results are expressed as a percentage of control values determined after 24 h of incubation in the presence of 1 mM calcium (100%). Bars are means ± SEM from four independent experiments. (B) Confluent mpkCCD_cl4 cells grown on filters were preincubated for 24 h at 37°C in the presence of 1 or 5 mM apical calcium and treated or not for 30 to 60 min with 10⁻⁹ M AVP. Cellular cAMP content was determined as described in the Concise Methods section. Results are expressed as pmol cAMP × 10⁶ cells⁻¹ and are means ± SEM from five independent experiments. (C) Cells were treated as in A before RNA extraction. Real-time PCR was performed as described in the Concise Methods section using primers against AQP2, the vasopressin V₂ receptor (V₂R), or type 6 AC (AC6). Results are expressed as a percentage of control values determined after 24 h of incubation in the presence of 1 mM calcium (100%). Bars are means ± SEM from four independent experiments. *P < 0.05.

Figure 6.

Role of phosphodiesterases and AC in the calcium-induced attenuation of AVP-dependent AQP2 expression. (A) Confluent mpkCCD_cl4 cells grown on filters were preincubated for 24 h at 37°C in the presence of 1 or 5 mM apical calcium before incubation for another 24 h with or without 10⁻³ M 8Br-cAMP, a cell-permeable cAMP analog. (B) Confluent mpkCCD_cl4 cells grown on filters were preincubated for 24 h at 37°C with 10⁻⁹ M AVP. Cells were then incubated for another 24 h in the continuous presence of AVP and in the presence of 1 or 5 mM apical calcium with or without 10⁻⁴ M IBMX, a phosphodiesterase inhibitor. (C) Confluent mpkCCD_cl4 cells grown on filters were preincubated for 24 h at 37°C in the presence of 1 or 5 mM apical calcium before incubation for another 24 h with or without 5 × 10⁻⁶ M forskolin, a direct activator of AC. After RNA extraction, real-time PCR was performed as described in the Concise Methods section using primers against AQP2. Results are expressed as a percentage of control values determined after 24 h of incubation in the presence of 1 mM calcium and in the absence of drugs (100%). Bars are means ± SEM from four to eight independent experiments. *P < 0.05.

Figure 7.

Role of calmodulin in the calcium-induced attenuation of AVP-dependent AQP2 expression. (A) Confluent mpkCCD_cl4 cells grown on filters were preincubated for 24 h at 37°C with 10⁻⁹ M AVP. Cells were then incubated for another 24 h in the continuous presence of AVP and in the presence of 1 or 5 mM apical calcium with or without 10⁻⁵ M W7 or trifluoperazine, two unrelated calmodulin inhibitors. After RNA extraction, real-time PCR was performed as described in the Concise Methods section using primers against AQP2. Results are expressed as a percentage of control values determined after 24 h of incubation in the presence of 1 mM calcium and in the absence of drugs (100%). Bars are means ± SEM from five to eight independent experiments. *P < 0.05.