Differential modulation of intracellular Ca2+ responses associated with calcium-sensing receptor activation in renal collecting duct cells.

In this work, we studied G protein-coupled Extracellular Calcium Sensing Receptor (CaR) signaling in mouse cortical collecting duct cells (MCD4) expressing endogenous CaR. Intracellular [Ca2+] measurements performed with real time video imaging revealed that CaR stimulation with 5mM Ca2+, 300µM Gd3+ and with 10µM of specific allosteric modulator NPS-R 568, all resulted in an increase in [Ca2+]i although displaying different features. Specifically, Ca2+ as well as stimulation with NPS-R 568 induced a rapid peak of [Ca2+]i while stimulation with Gd3+ induced transient intracellular Ca2+ oscillations. PLC inhibition completely abolished any [Ca2+]i increase after stimulation with CaR agonists. Inhibition of Rho or Rho kinase (ROK) abolished [Ca2+]i oscillations induced by Gd3+, while the peak induced by high Ca2+ was similar to control. Conversely, emptying the intracellular calcium stores abolished the response to Gd3+. On the other hand, the inhibition of calcium influx did not alter calcium changes. We conclude that in our cell model, CaR stimulation with distinct agonists activates two distinct transduction pathways, both PLC-dependent. The transient cytosolic Ca2+ oscillations produced by Gd3+ are modulated by Rho-Rho kinase signaling, whereas the rapid peak of intracellular Ca2+ in response to 5mM [Ca2+]o is mainly due to PLC/IP3 pathway activation

Integrin signaling modulates AQP2 trafficking via Arg-Gly-Asp (RGD) motif.

Aquaporin-2 (AQP2) increases the water permeability of renal collecting ducts in response to vasopressin. Vasopressin stimulation is accompanied by a profound remodeling of actin cytoskeleton whose dynamics are regulated by crosstalk between intracellular and extracellular signals. Here, we report that AQP2 contains a conserved RGD domain in its external C-loop. Co-immunoprecipitation experiments demonstrated that AQP2 binds integrin β1 in renal tissue and in MCD4 cells. To investigate the role of this interaction on AQP2 trafficking, cells were exposed to synthetic RGD-containing peptides, GRGDNP or GRGDSP, able to bind certain integrins. Incubation with these peptides increased the membrane expression of AQP2 in the absence of hormonal stimulation as assessed by confocal analysis and cell surface biotinylation. To identify the signals underlying the effects of peptides on AQP2 trafficking, some possible intracellular messengers were evaluated. Exposure of MCD4 cells to GRGDNP increased intracellular cAMP as assessed by FRET studies while GRGDSP increased intracellular calcium concentration. Taken together, these data propose integrins as new players controlling the cellular localization of AQP2, via two distinct signal transduction pathways dependent on cAMP and calcium respectively

Calcium-Sensing Receptor and Aquaporin 2 Interplay in Hypercalciuria-Associated Renal Concentrating Defect in Humans. An In Vivo and In Vitro Study

One mechanism proposed for reducing the risk of calcium renal stones is activation of the calcium-sensing receptor (CaR) on the apical membranes of collecting duct principal cells by high luminal calcium. This would reduce the abundance of aquaporin-2 (AQP2) and in turn the rate of water reabsorption. While evidence in cells and in hypercalciuric animal models supports this hypothesis, the relevance of the interplay between the CaR and AQP2 in humans is not clear. This paper reports for the first time a detailed correlation between urinary AQP2 excretion under acute vasopressin action (DDAVP treatment) in hypercalciuric subjects and in parallel analyzes AQP2-CaR crosstalk in a mouse collecting duct cell line (MCD4) expressing endogenous and functional CaR. In normocalciurics, DDAVP administration resulted in a significant increase in AQP2 excretion paralleled by an increase in urinary osmolality indicating a physiological response to DDAVP. In contrast, in hypercalciurics, baseline AQP2 excretion was high and did not significantly increase after DDAVP. Moreover DDAVP treatment was accompanied by a less pronounced increase in urinary osmolality. These data indicate reduced urinary concentrating ability in response to vasopressin in hypercalciurics. Consistent with these results, biotinylation experiments in MCD4 cells revealed that membrane AQP2 expression in unstimulated cells exposed to CaR agonists was higher than in control cells and did not increase significantly in response to short term exposure to forskolin (FK). Interestingly, we found that CaR activation by specific agonists reduced the increase in cAMP and prevented any reduction in Rho activity in response to FK, two crucial pathways for AQP2 translocation. These data support the hypothesis that CaR–AQP2 interplay represents an internal renal defense to mitigate the effects of hypercalciuria on the risk of calcium precipitation during antidiuresis. This mechanism and possibly reduced medulla tonicity may explain the lower concentrating ability observed in hypercalciuric patients

A Protein Kinase A-Independent Pathway Controlling Aquaporin 2 Trafficking as a Possible Cause for the Syndrome of Inappropriate Antidiuresis Associated with Polycystic Kidney Disease 1 Haploinsufficiency

Renal water reabsorption is controlled by arginine vasopressin (AVP), which binds to V2 receptors, resulting in protein kinase A (PKA) activation, phosphorylation of aquaporin 2 (AQP2) at serine 256, and translocation of AQP2 to the plasma membrane. However, AVP also causes dephosphorylation of AQP2 at S261. Recent studies showed that cyclin-dependent kinases (cdks) can phosphorylate AQP2 peptides at S261 in vitro. We investigated the possible role of cdks in the phosphorylation of AQP2 and identified a new PKA-independent pathway regulating AQP2 trafficking. In ex vivo kidney slices and MDCK-AQP2 cells, R-roscovitine, a specific inhibitor of cdks, increased pS256 levels and decreased pS261 levels. The changes in AQP2 phosphorylation status were paralleled by increases in cell surface expression of AQP2 and osmotic water permeability in the absence of forskolin stimulation. R-Roscovitine did not alter cAMP-dependent PKA activity but specifically reduced protein phosphatase 2A (PP2A) expression and activity in MDCK cells. Notably, we found reduced PP2A expression and activity and reduced pS261 levels in Pkd1(+/-) mice displaying a syndrome of inappropriate antidiuresis with high levels of pS256, despite unchanged AVP and cAMP. Similar to previous findings in Pkd1(+/-) mice, R-roscovitine treatment caused a significant decrease in intracellular calcium in MDCK cells. Our data indicate that reduced activity of PP2A, secondary to reduced intracellular Ca(2+) levels, promotes AQP2 trafficking independent of the AVP-PKA axis. This pathway may be relevant for explaining pathologic states characterized by inappropriate AVP secretion and positive water balance

Patient characteristics and baseline urinary parameters.

<p><u>Abbreviations</u>: M = male; F = female; SDS = standard deviation score; UOsm = urine osmolarity, UCreat = urine creatinine, UUN = urine urea nitrogen; FeNa = fractional excretion of sodium; FeK = fractional excretion of potassium; FeCl = fractional excretion of chloride; FeCa = fractional excretion of calcium; UCa/UCreat = urinary calcium:creatinine ratio; UMg/UCreat = urinary magnesium:creatinine ratio; TRP = tubular reabsorption of phosphorus; FeUA = fractional excretion of uric acid; UGluc/UCreat = urinary glucose:creatinine ratio; UProt/UCreat = urinary protein:creatinine ratio.</p>(*)<p>numbers indicate the presenting symptom or finding.</p

AQP2 excretion during urinary concentration test.

<p>For the urinary concentration test, urinary AQP2 excretion and osmolality were measured on samples obtained hourly after 20 µg intranasal DDAVP administration. AQP2 urinary excretion. (A) In normocalciuric children, DDAVP administration resulted in a significant increase in urinary AQP2 excretion. Hypercalciuric children had a high basal AQP2 excretion and DDAVP administration did not result in a significant increase in urinary AQP2 excretion. Data were analyzed by Wilcoxon Signed Rank ANOVA test for paired non parametric data (*p = 0.0023). (B) Comparison of basal AQP2 excretion in hypercalciurics and normocalciurics. patients. In hypercalciurics a significant higher AQP2 excretion was observed (p = 0.036 ANOVA test).</p

Effect of CaR signaling on AQP2 trafficking in MCD4 cells.

<p><b>Apical surface biotinylation.</b> (A) MCD4 cells were preincubated with 5 mM Ca²⁺, 300 µM Gd³⁺ or 10 µM NPS-R 568 then exposed to FK10⁻⁴ M or left under control conditions. Apical membrane-expressed AQP2 was quantitated by apical surface biotinylation. FK-induced AQP2 membrane accumulation was significantly reduced in the presence of CaR agonists. CaR agonists induced a mild increase in AQP2 membrane expression even in the absence of FK stimulation. The total amount of AQP2 in the starting preparation was comparable in each experimental condition (total AQP2). (B) Densitometric analysis of the 29 kDa biotinylated AQP2 band. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033145#s2" target="_blank">Results</a> are expressed as mean values ± SEM. The values obtained in five independent experiments are expressed as percentages of the basal condition. Data were compared by one-way Anova and Tukey's multiple comparison test (* P<0.05 relative to ctr, # P<0.05 relative to FK).</p

RhoA activity in cells exposed to CaR agonists.

<p><b>A. Scheme of the Raichu-RBD probe mechanism.</b> Raichu-RBD contains YFP and CFP separated by rhotekin-RBD (RBD). Active RhoGTP binds RBD, separating the donor (CFP) from the acceptor (YFP) thus reducing FRET. As internal control MCD4 cells were incubated for 3 hours with C3 toxin (1 µg/ml), which inactivates Rho proteins, and FRET signals was recorded. As shown in the figure, C3 toxin (1 µg/ml for 3 hours) (n = 124) leaded to a ‘closed’ conformation increasing FRET compared to control cells (n = 71). <b>B. Rho activity during CaR activation.</b> RhoA activity was evaluated in MCD4 cells exposed to CaR agonists. MCD4 cells were preincubated with 5 mM Ca²⁺, 300 µM Gd³⁺ or 10 µM NPS-R 568 for 30 min and then stimulated with 10⁻⁴ M FK or analyzed at rest. The amount of active RhoA was evaluated by FRET using a probe consisting of a Rho-binding domain (RBD) of Rhotekin sandwiched by YFP and CFP (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033145#s4" target="_blank">Methods</a>). In this system, any increase in RhoA activity results in a decrease in FRET efficiency. In non-treated cells, FK stimulation (n = 59) caused a significant decrease in the amount of active RhoA compared to control conditions (n = 51). In cells pretreated with 300 µM Gd³⁺, RhoA activity was significantly increased (decreased FRET signal, n = 50) compared to control untreated cells. The decrease in RhoA activity in response to FK was prevented in cells preincubated either with Gd³⁺ (n = 66) or NPS-R 568 (n = 60). Values are expressed as mean ± SEM Data were compared by one-way Anova and Tukey's multiple comparison test (*P<0.05 relative to control).</p