Eric Féraille

Phosphorylation of the catalyic alpha-subunit constitutes a triggering signal for Na+,K+-ATPase endocytosis.

Inhibition of Na+,K+-ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na+,K+-ATPase activity in response to dopamine is associated with endocytosis of its α- and β-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na+,K+-ATPase and inhibition of its activity were accompanied by phosphorylation of the α-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the α-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent α-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the α-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent α-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the α-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.

Tonicity-Responsive Enhancer Binding Protein Is an Essential Regulator of Aquaporin-2 Expression in Renal Collecting Duct Principal Cells

Tonicity-responsive enhancer binding protein (TonEBP) plays a key role in protecting renal cells from hypertonic stress by stimulating transcription of specific genes. Under hypertonic conditions, TonEBP activity is enhanced via increased nuclear translocation, transactivation, and abundance. It was reported previously that hypertonicity exerted a dual, time-dependent effect on vasopressin-inducible aquaporin-2 (AQP2) expression in immortalized mouse collecting duct principal cells (mpkCCDcl4). Whereas AQP2 abundance decreased after 3 h of hyperosmotic challenge, it increased after 24 h of hypertonic challenge. This study investigated the role that TonEBP may play in these events by subjecting mpkCCDcl4 cells to 3 or 24 h of hypertonic challenge. Hypertonic challenge increased TonEBP mRNA and protein content and enhanced TonEBP activity as illustrated by both increased TonEBP-dependent luciferase activity and mRNA expression of several genes that are targeted by TonEBP. Irrespective of the absence or presence of vasopressin, decreased TonEBP activity in cells that were transfected with either TonEBP small interfering RNA or an inhibitory form of TonEBP strongly reduced AQP2 mRNA and protein content under iso-osmotic conditions and blunted the increase of AQP2 abundance that was induced after 24 h of hypertonic challenge. Conversely, decreased TonEBP activity did not significantly alter reduced expression of AQP2 mRNA that was induced by 3 h of hypertonic challenge. Mutation of a TonE enhancer element located 489 bp upstream of the AQP2 transcriptional start site abolished the hypertonicity-induced increase of luciferase activity in cells that expressed AQP2 promoter-luciferase plasmid constructs, indicating that TonEBP influences AQP2 transcriptional activity at least partially by acting directly on the AQP2 promoter. These findings demonstrate that in collecting duct principal cells, TonEBP plays a central role in regulating AQP2 expression by enhancing AQP2 gene transcription.

Cell shrinkage triggers the activation of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending limb of the Henle's loop. Requirement of p38 kinase for the regulatory volume increase response.

The kidney medulla is exposed to very high interstitial osmolarity leading to the activation of mitogen-activated protein kinases (MAPK). However, the respective roles of increased intracellular osmolality and of cell shrinkage in MAPK activation are not known. Similarly, the participation of MAPK in the regulatory volume increase (RVI) following cell shrinkage remains to be investigated. In the rat medullary thick ascending limb of Henle (MTAL), extracellular hypertonicity produced by addition of NaCl or sucrose increased the phosphorylation level of extracellular signal-regulated kinase (ERK) and p38 kinase and to a lesser extent c-Jun NH2-terminal kinase with sucrose only. Both hypertonic solutions decreased the MTAL cellular volume in a dose- and time-dependent manner. In contrast, hypertonic urea had no effect. The extent of MAPK activation was correlated with the extent of MTAL cellular volume decrease. Increasing intracellular osmolality without modifying cellular volume did not activate MAPK, whereas cell shrinkage without variation in osmolality activated both ERK and p38. In the presence of 600 mosmol/liter NaCl, the maximal cell shrinkage was observed after 10 min at 37 °C and the MTAL cellular volume was reduced to 70% of its initial value. Then, RVI occurred and the cellular volume progressively recovered to reach about 90% of its initial value after 30 min. SB203580, a specific inhibitor of p38, almost completely inhibited the cellular volume recovery, whereas inhibition of ERK did not alter RVI. In conclusion, in rat MTAL: 1) cell shrinkage, but not intracellular hyperosmolality, triggers the activation of both ERK and p38 kinase in response to extracellular hypertonicity; and 2) RVI is dependent on p38 kinase activation.

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.

Dual Effects of Hypertonicity on Aquaporin-2 Expression in Cultured Renal Collecting Duct Principal Cells

The driving force for renal water reabsorption is provided by the osmolarity gradient between the interstitium and the tubular lumen, which is subject to rapid physiologic variations as a consequence of water intake fluctuations. The effect of increased extracellular tonicity/osmolarity on vasopressin-inducible aquaporin-2 (AQP2) expression in immortalized mouse collecting duct principal cells (mpkCCD(cl4)) is investigated in this report. Increasing the osmolarity of the medium either by the addition of NaCl, sucrose, or urea first decreased AQP2 expression after 3 h. AQP2 expression then increased in cells exposed to NaCl- or sucrose-supplemented hypertonic medium after longer periods of time (24 h), while urea-supplemented hyperosmotic medium had no effect. Altered AQP2 expression induced by both short-term (3 h) and long-term (24 h) exposure of cells to hypertonicity arose from changes in AQP2 gene transcription because hypertonicity did not modify AQP2 mRNA stability nor AQP2 protein turnover. On the long-term, vasopressin (AVP) and hypertonicity increased AQP2 expression in a synergistic manner. Hypertonicity altered neither the dose-responsiveness of AVP-induced AQP2 expression nor cAMP-protein kinase (PKA) activity, while PKA inhibition did not reduce the extent of the hypertonicity-induced increase of AQP2 expression. These results indicate that in collecting duct principal cells: (1) a short-term increase of extracellular osmolarity decreases AQP2 expression through inhibition of AQP2 gene transcription; (2) a long-term increase of extracellular tonicity, but not osmolarity, enhances AQP2 expression via stimulation of AQP2 gene transcription; and (3) long-term hypertonicity and PKA increases AQP2 expression through synergistic but independent mechanisms.

Protein kinase A induces recruitment of active Na+,K+‐ATPase units to the plasma membrane of rat proximal convoluted tubule cells

1 The aim of this study was to investigate the mechanism of control of Na+,K+‐ATPase activity by the cAMP‐protein kinase A (PKA) pathway in rat proximal convoluted tubules. For this purpose, we studied the in vitro action of exogenous cAMP (10−3 M dibutyryl‐cAMP (db‐cAMP) or 8‐bromo‐cAMP) and endogenous cAMP (direct activation of adenylyl cyclases by 10−5 M forskolin) on Na+,K+‐ATPase activity and membrane trafficking. 2 PKA activation stimulated both the cation transport and hydrolytic activity of Na+,K+‐ATPase by about 40 %. Transport activity stimulation was specific to the PKA signalling pathway since (1) db‐cAMP stimulated the ouabain‐sensitive 86Rb+ uptake in a time‐ and dose‐dependent fashion; (2) this effect was abolished by addition of H‐89 or Rp‐cAMPS, two structurally different PKA inhibitors; and (3) this stimulation was not affected by inhibition of protein kinase C (PKC) by GF109203X. The stimulatory effect of db‐cAMP on the hydrolytic activity of Na+,K+‐ATPase was accounted for by an increased maximal ATPase rate (Vmax) without alteration of the efficiency of the pump, suggesting that cAMP‐PKA pathway was implicated in membrane redistribution control. 3 To test this hypothesis, we used two different approaches: (1) cell surface protein biotinylation and (2) subcellular fractionation. Both approaches confirmed that the cAMP‐PKA pathway was implicated in membrane trafficking regulation. The stimulation of Na+,K+‐ATPase activity by db‐cAMP was associated with an increase (+40 %) in Na+,K+‐ATPase units expressed at the cell surface which was assessed by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. Subcellular fractionation confirmed the increased expression in pump units at the cell surface which was accompanied by a decrease (‐30 %) in pump units located in the subcellular fraction corresponding to early endosomes. 4 In conclusion, PKA stimulates Na+,K+‐ATPase activity, at least in part, by increasing the number of Na+‐K+ pumps in the plasma membrane in proximal convoluted tubule cells.

NADPH-Oxidase 4 Protects against Kidney Fibrosis during Chronic Renal Injury

NADPH oxidases synthesize reactive oxygen species that may participate in fibrosis progression. NOX4 and NOX2 are NADPH oxidases expressed in the kidneys, with the former being the major renal isoform, but their contribution to renal disease is not well understood. Here, we used the unilateral urinary obstruction model of chronic renal injury to decipher the role of these enzymes using wild-type, NOX4-, NOX2-, and NOX4/NOX2-deficient mice. Compared with wild-type mice, NOX4-deficient mice exhibited more interstitial fibrosis and tubular apoptosis after obstruction, with lower interstitial capillary density and reduced expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in obstructed kidneys. Furthermore, NOX4-deficient kidneys exhibited increased oxidative stress. With NOX4 deficiency, renal expression of other NOX isoforms was not altered but NRF2 protein expression was reduced under both basal and obstructed conditions. Concomitant deficiency of NOX2 did not modify the phenotype exhibited by NOX4-deficient mice after obstruction. NOX4 silencing in a mouse collecting duct (mCCD(cl1)) cell line increased TGF-β1-induced apoptosis and decreased NRF2 protein along with expression of its target genes. In addition, NOX4 silencing decreased hypoxia-inducible factor-1α and expression of its target genes in response to hypoxia. In summary, these results demonstrate that the absence of NOX4 promotes kidney fibrosis, independent of NOX2, through enhanced tubular cell apoptosis, decreased microvascularization, and enhanced oxidative stress. Thus, NOX4 is crucial for the survival of kidney tubular cells under injurious conditions.

Osmoprotective Transcription Factor NFAT5/TonEBP Modulates Nuclear Factor-κB Activity

Tonicity responsive binding protein (TonEBP) is a transcription factor that plays a key role in osmoprotection. Here, we demonstrate enhanced activity of prosurvival NF-κB—at the onset of hypertonic challenge that depends on p38 kinase—and Akt-dependent formation of p65-TonEBP complexes that bind to elements of NF-κB-responsive genes.

Effect of insulin on Na+,K(+)-ATPase in rat collecting duct.

1. The collecting duct is involved in the whole antinatriuretic effect of insulin, as indicated in vitro by the stimulatory effect of the hormone on ouabain‐sensitive 86Rb+ uptake. Since Na+,K(+)‐ATPase drives Na+ reabsorption, the contribution of the Na+ pump to the effect of insulin was investigated in rat isolated cortical and outer medullary collecting duct. 2. Insulin enhanced ouabain‐sensitive 86Rb+ uptake in the absence, as well as in the presence, of either 5 x 10(‐4) M amiloride or 10(‐3) M hydrochlorothiazide (HCT). Maximal ouabain‐sensitive 86Rb+ uptake, measured in Na(+)‐loaded tubules, was also enhanced by insulin. The insulin effect persisted both in the absence of external Na+, when the Na+,K(+)‐ATPase operates in a Rb(+)‐Rb+ exchange mode, and in tubules depolarized by a high external concentration (20 mM) of Rb+ or by addition of 3 mM Ba2+. 3. Insulin treatment did not alter the intracellular Na and K concentrations, the specific binding of [3H]ouabain measured in intact tubules, or the hydrolytic activity of Na+,K(+)‐ATPase measured after permeabilization of the tubule cells. 4. In conclusion, in the rat collecting duct, insulin increased Na+,K(+)‐ATPase‐mediated cation transport independently of Na+ availability, membrane potential and recruitment of pump units. The effect of insulin was lost after cell permeabilization, suggesting the presence of a cytosolic factor which controls the turnover of Na+,K(+)‐ATPase.

Aldosterone activates NF-kappaB in the collecting duct.

Besides its classical effects on salt homeostasis in renal epithelial cells, aldosterone promotes inflammation and fibrosis and modulates cell proliferation. The proinflammatory transcription factor NF-kappaB has been implicated in cell proliferation, apoptosis, and regulation of transepithelial sodium transport. The effect of aldosterone on the NF-kappaB pathway in principal cells of the cortical collecting duct, a major physiologic target of aldosterone, is unknown. Here, in both cultured cells and freshly isolated rat cortical collecting duct, aldosterone activated the canonical NF-kappaB signaling pathway, leading to increased expression of several NF-kappaB-targeted genes (IkappaBalpha, plasminogen activator inhibitor 1, monocyte chemoattractant protein 1, IL-1beta, and IL-6). Small interfering RNA-mediated knockdown of the serum and glucocorticoid-inducible kinase SGK1, a gene induced early in the response to aldosterone, but not pharmacologic inhibition of extracellular signal-regulated kinase and p38 kinase, attenuated aldosterone-induced NF-kappaB activation. Pharmacologic antagonism or knockdown of the mineralocorticoid receptor prevented aldosterone-induced NF-kappaB activity. In addition, activation of the glucocorticoid receptor inhibited the transactivation of NF-kappaB by aldosterone. In agreement with these in vitro findings, spironolactone prevented NF-kappaB-induced transcriptional activation observed in cortical collecting ducts of salt-restricted rats. In summary, aldosterone activates the canonical NF-kappaB pathway in principal cells of the cortical collecting duct by activating the mineralocorticoid receptor and by inducing SGK1.