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

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.

Intracellular Na+ controls cell surface expression of Na,K-ATPase via a cAMP-independent PKA pathway in mammalian kidney collecting duct cells.

In the mammalian kidney the fine control of Na+ reabsorption takes place in collecting duct principal cells where basolateral Na,K-ATPase provides the driving force for vectorial Na+ transport. In the cortical collecting duct (CCD), a rise in intracellular Na+ concentration ([Na+]i) was shown to increase Na,K-ATPase activity and the number of ouabain binding sites, but the mechanism responsible for this event has not yet been elucidated. A rise in [Na+]i caused by incubation with the Na+ ionophore nystatin, increased Na,K-ATPase activity and cell surface expression to the same extent in isolated rat CCD. In cultured mouse mpkCCDcl4 collecting duct cells, increasing [Na+]i either by cell membrane permeabilization with amphotericin B or nystatin, or by incubating cells in a K(+)-free medium, also increased Na,K-ATPase cell surface expression. The [Na+]i-dependent increase in Na,K-ATPase cell-surface expression was prevented by PKA inhibitors H89 and PKI. Moreover, the effects of [Na+]i and cAMP were not additive. However, [Na+]i-dependent activation of PKA was not associated with an increase in cellular cAMP but was prevented by inhibiting the proteasome. These findings suggest that Na,K-ATPase may be recruited to the cell membrane following an increase in [Na+]i through cAMP-independent PKA activation that is itself dependent on proteasomal activity.

NF-kappaB inhibits sodium transport via down-regulation of SGK1 in renal collecting duct principal cells.

Tubulointerstitial inflammation is a common feature of renal diseases. We have investigated the relationship between inflammation and Na(+) transport in the collecting duct (CD) using the mCCD(cl1) and mpkCDD(cl4) principal cell models. Lipopolysaccharide (LPS) decreased basal and aldosterone-stimulated amiloride-sensitive transepithelial current in a time-dependent manner. This effect was associated with a decrease in serum and glucocorticoid-regulated kinase 1 (SGK1) mRNA and protein levels followed by a decrease in epithelial sodium channel (ENaC) alpha-subunit mRNA levels. The LPS-induced decrease in SGK1 expression was confirmed in isolated rat CD. This decreased expression of either SGK1 or the ENaC alpha-subunit was not due to enhanced degradation of mRNA. In contrast, LPS inhibited transcriptional activity of the SGK1 promoter measured by luciferase-reporter gene assay. The effect of LPS was not mediated by inhibition of mineralocorticoid or glucocorticoid receptor, because expression of both receptors was unchanged and blockade of either receptor by spironolactone or RU486, respectively, did not prevent the down-regulation of SGK1. The effect of LPS was mediated by the canonical NF-kappaB pathway, as overexpression of a constitutively active mutant, IKKbeta (inhibitor of nuclear factor kappaB kinase-beta) decreased SGK1 mRNA levels, and knockdown of p65 NF-kappaB subunit by small interfering RNA increased SGK1 mRNA levels. Chromatin immunoprecipitation showed that LPS increased p65 binding to two NF-kappaB sites along the SGK1 promoter. In conclusion, we show that activation of the NF-kappaB pathway down-regulates SGK1 expression, which might lead to decreased ENaC alpha-subunit expression, ultimately resulting in decreased Na(+) transport.

Stimulation of Na+ transport by AVP is independent of PKA phosphorylation of the Na-K-ATPase in collecting duct principal cells.

Arginine-vasopressin (AVP) stimulates Na(+) transport and Na-K-ATPase activity via cAMP-dependent PKA activation in the renal cortical collecting duct (CCD). We investigated the role of the Na-K-ATPase in the AVP-induced stimulation of transepithelial Na(+) transport using the mpkCCD(c14) cell model of mammalian collecting duct principal cells. AVP (10(-9) M) stimulated both the amiloride-sensitive transepithelial Na(+) transport measured in intact cells and the maximal Na pump current measured by the ouabain-sensitive short-circuit current in apically permeabilized cells. These effects were associated with increased Na-K-ATPase cell surface expression, measured by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. The effects of AVP on Na pump current and Na-K-ATPase cell surface expression were dependent on PKA activity but independent of increased apical Na(+) entry. Time course experiments revealed that in response to AVP, the cell surface expression of both endogenous Na-K-ATPase and hybrid Na pumps containing a c-myc-tagged wild-type human alpha(1)-subunit increased transiently. Na-K-ATPase cell surface expression was maximal after 30 min and then declined toward baseline after 60 min. Immunoprecipitation experiments showed that PKA activation did not alter total phosphorylation levels of the endogenous Na-K-ATPase alpha-subunit. In addition, mutation of the PKA phosphorylation site (S943A or S943D) did not alter the time course of increased cell surface expression of c-myc-tagged Na-K-ATPase in response to AVP or to dibutyryl-cAMP. Therefore, stimulation of Na-K-ATPase cell surface expression by AVP is dependent on PKA but does not rely on alpha(1)-subunit phosphorylation on serine 943 in the collecting duct principal cells.

Insulin potentiates AVP-induced AQP2 expression in cultured renal collecting duct principal cells.

In the renal collecting duct (CD), water reabsorption depends on the presence of aquaporin-2 (AQP2) in the apical membrane of principal cells. AQP2 expression and subcellular repartition are under the control of AVP. Some pieces of experimental evidence indicate that additional hormonal factors, including insulin, may also control AQP2 expression and thereby CD water permeability. We have previously shown that AVP induces endogenous AQP2 expression in cultured mouse mpkCCD(cl4) CD principal cells (23). In the present study, we investigated the effect of insulin on AQP2 expression in mpkCCD(cl4) cells. Addition of insulin to the basal medium of cells grown on filters slightly increased AQP2 mRNA and protein expression, whereas insulin potentiated the effect of AVP. The potentiation of AVP-induced AQP2 expression by insulin was abolished by actinomycin D, a transcriptional inhibitor. Analysis of AQP2 protein expression under conditions of AVP washout and/or in the presence of chloroquine, a lysosomal degradation inhibitor, revealed that insulin did not significantly alter AQP2 protein degradation. Inhibition of ERK, p38 kinase, and phosphatidylinositol 3'-kinase (PI 3-kinase) activities prevented the insulin-induced stimulation of AQP2 expression, whereas inhibition of PKC has no effect. Taken together, our results indicate that insulin increased AQP2 protein expression mostly through increased AQP2 mRNA levels in cultured mpkCCD(cl4) cells. This effect most likely relies on increased AQP2 gene transcription in response to MAPK and PI 3-kinase activation.

Extracellular hypotonicity increases Na,K-ATPase cell surface expression via enhanced Na+ influx in cultured renal collecting duct cells.

In the renal collecting duct (CD), the Na,K-ATPase, which provides the driving force for Na+ absorption, is under tight multifactorial control. Because CD cells are physiologically exposed to variations of interstitial and tubular fluid osmolarities, the effects of extracellular anisotonicity on Na,K-ATPase cell surface expression were studied. Results show that hypotonic conditions increased, whereas hypertonic conditions had no effect on Na,K-ATPase cell surface expression in confluent mpkCCDcl4 cells. Incubating cells with amphotericin B, which increases [Na+]i, under isotonic or anisotonic conditions, revealed that Na,K-ATPase recruitment to the cell surface was not directly related to variations of cell volume and osmolarity. The effects of amphotericin B and extracellular hypotonicity were not additive, and both were prevented by protein kinase A and proteasome inhibitors, suggesting a common mechanism of action. In line with this hypothesis, extracellular hypotonicity induced a sustained stimulation of the amiloride-sensitive short-circuit current, indicating increased Na+ influx through the apical epithelial Na+ channel. Moreover, inhibiting apical Na+ entry by amiloride, a blocker of epithelial Na+ channel, or incubating cells in Na+ -free medium prevented the cell surface recruitment of Na,K-ATPase in response to extracellular hypotonicity. Altogether, these findings strongly suggest that extracellular hypotonicity stimulates apical Na+ influx leading to increased [Na+]i, protein kinase A activation, and recruitment of Na,K-ATPase units to the cell surface of mpkCCDcl4 cells.

Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells.

Fine regulation of water reabsorption by the antidiuretic hormone [8-arginine]vasopressin (AVP) occurs in principal cells of the collecting duct and is largely dependent on regulation of the aquaporin-2 (AQP2) water channel. AVP-inducible long term AQP2 expression was investigated in immortalized mouse cortical collecting duct principal cells. Combined RNase protection assay, Western blot, and immunofluorescence analyses revealed that physiological concentrations of AVP added to the basal side, but not to the apical side, of cells grown on filters induced both AQP2 mRNA and apical protein expression. The stimulatory effect of AVP on AQP2 expression followed a V(2) receptor-dependent pathway because [deamino-8-d-arginine]vasopressin (dDAVP), a specific V(2) receptor agonist, produced the same effect as AVP, whereas the V(2) antagonist SR121463B antagonized action of both AVP and dDAVP. Moreover, forskolin and cyclic 8-bromo-AMP fully reproduced the effects of AVP on AQP2 expression. Analysis of protein degradation pathways showed that inhibition of proteasomal activity prevented synthesis of AVP-inducible AQP2 mRNA and protein. Once synthesized, AQP2 protein was quickly degraded, a process that involves both the proteasomal and lysosomal pathways. This is the first study that delineates induction and degradation mechanisms of AQP2 endogenously expressed by a renal collecting duct principal cell line.

Increased synthesis and avp unresponsiveness of Na,K-ATPase in collecting duct from nephrotic rats.

Renal sodium retention is responsible for ascites and edema in nephrotic syndrome. In puromycin aminonucleoside (PAN)-induced nephrosis, sodium retention originates in part from the collecting duct, and it is associated with increased Na,K-ATPase activity in the cortical collecting duct (CCD). The aims of this study were to evaluate whether the outer medullary collecting duct (OMCD) also participates to sodium retention and to determine the mechanisms responsible for stimulation of Na,K-ATPase in CCD. PAN nephrosis increased Na,K-ATPase activity in the CCD but not in OMCD. The two-fold increase of Na,K-ATPase activity in CCD was associated with two-fold increases in the number of alpha and beta Na,K-ATPase subunits mRNA determined by quantitative RT-PCR and of the total amount of Na,K-ATPase alpha subunits estimated by Western blotting. PAN nephrosis also increased two-fold the amount of Na,K-ATPase alpha subunit at the basolateral membrane of CCD principal cells, as determined by Western blotting after biotinylation and streptavidin precipitation and by immunofluorescence. The intracellular pool of latent Na,K-ATPase units also increased in size and was no longer recruitable by vasopressin and cAMP. This unresponsiveness of the intracellular pool of Na,K-ATPase to vasopressin was not the result of any alteration of the molecular and functional expression of the vasopressin V(2) receptor/adenylyl cyclase (AC) complex. It is concluded that PAN nephrosis (1) does not alter sodium reabsorption in OMCD, (2) is associated with increased synthesis and membrane expression of Na,K-ATPase in the CCD, and (3) alters the normal trafficking of intracellular Na,K-ATPase units to the basolateral membrane.