Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

Massachusetts General Hospital Center for Systems Biology, Program in Membrane Biology and Nephrology Division, Massachusetts General Hospital, Boston, Massachusetts 02114-2790, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2008; 283(39):26643-61. DOI: 10.1074/jbc.M801071200
Source: PubMed


The unique phenotype of renal medullary cells allows them to survive and functionally adapt to changes of interstitial osmolality/tonicity. We investigated the effects of acute hypertonic challenge on AQP2 (aquaporin-2) water channel trafficking. In the absence of vasopressin, hypertonicity alone induced rapid (<10 min) plasma membrane accumulation of AQP2 in rat kidney collecting duct principal cells in situ, and in several kidney epithelial lines. Confocal microscopy revealed that AQP2 also accumulated in the trans-Golgi network (TGN) following hypertonic challenge. AQP2 mutants that mimic the Ser(256)-phosphorylated and -nonphosphorylated state accumulated at the cell surface and TGN, respectively. Hypertonicity did not induce a change in cytosolic cAMP concentration, but inhibition of either calmodulin or cAMP-dependent protein kinase A activity blunted the hypertonicity-induced increase of AQP2 cell surface expression. Hypertonicity increased p38, ERK1/2, and JNK MAPK activity. Inhibiting MAPK activity abolished hypertonicity-induced accumulation of AQP2 at the cell surface but did not affect either vasopressin-dependent AQP2 trafficking or hypertonicity-induced AQP2 accumulation in the TGN. Finally, increased AQP2 cell surface expression induced by hypertonicity largely resulted from a reduction in endocytosis but not from an increase in exocytosis. These data indicate that acute hypertonicity profoundly alters AQP2 trafficking and that hypertonicity-induced AQP2 accumulation at the cell surface depends on MAP kinase activity. This may have important implications on adaptational processes governing transcellular water flux and/or cell survival under extreme conditions of hypertonicity.

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    • "This view is corroborated by a recent study of the role of NOX4 in murine models of kidney disease [50]. In the present study, we show that AQP2 mRNA abundance is decreased by siNOX4 in mpkCCDcl4 cells challenged with DDAVP, which increases cAMP concentration, but not hypertonicity, which increases AQP2 expression independently of a rise in cAMP levels [51], [52]. This repressive effect was found to be associated with increased PDE3 and PDE4 activity and depressed PKA and CREB activity. "
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    ABSTRACT: The final control of renal water reabsorption occurs in the collecting duct (CD) and relies on regulated expression of aquaporin-2 (AQP2) in principal CD cells. AQP2 transcription is primarily induced by type 2 vasopressin receptor (V2R)-cAMP-protein kinase A (PKA) signaling but also by other factors, including TonEBP and NF-κB. NAPDH oxidase 4 (NOX4) represents a major source of reactive oxygen species (ROS) in the kidney. Because NOX-derived ROS may alter PKA, TonEBP and NF-κB activity, we examined the effects of NOX4 depletion on AQP2 expression. Depleted NOX4 expression by siRNA (siNOX4) in mpkCCDcl4 cells attenuated increased AQP2 mRNA expression by arginine vasopressin (AVP) but not by hypertonicity, which induces both TonEBP and NF-κB activity. AVP-induced AQP2 expression was similarly decreased by the flavoprotein inhibitor diphenyleneiodonium. siNOX4 altered neither TonEBP nor NF-κB activity but attenuated AVP-inducible cellular cAMP concentration, PKA activity and CREB phosphorylation as well as AQP2 mRNA expression induced by forskolin, a potent activator of adenylate cyclase. The repressive effect of siNOX4 on AVP-induced AQP2 mRNA expression was abolished by the non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and was significantly decreased by selective PDE antagonists cilostamide and rolipram, but not vinpocetine, which respectively target PDE3, PDE4 and PDE1. Thus, by inhibiting PDE3 and PDE4 activity NOX4-derived ROS may contribute to V2R-cAMP-PKA signaling and enhance AQP2 transcription.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "Prior to the increase in u-AQP2, there was an abrupt rise in p-osm and p-AVP induced by the hypertonic saline infusion. Animal studies have shown that hypertonicity can cause an up regulation of AQP2 expression in the apical membrane comparable with that achieved by AVP alone [23,24]. It cannot be excluded that this might play an active part in the increased excretion of u-AQP2. "
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    ABSTRACT: The renal distal nephron plays an important role in the maintenance of sodium balance, extra cellular volume and blood pressure. The degree of water transport, via aquaporin2 water channels (AQP2), and sodium transport, via epithelial sodium channels (ENaC) in renal collecting duct principal cells are reflected by the level of urinary excretion of AQP2 (u-AQP2) and the gamma-fraction of ENaC (u-ENaCgamma). The effects of an acute intravenous volume load with isotonic saline, hypertonic saline and glucose on u-AQP2, u-ENaCgamma and underlying mechanisms have never been studied in a randomized, placebo-controlled trial in healthy humans. We studied the effects of 0.9% saline (23 ml/kg), 3% saline (7 ml/ kg) and 5% glucose (23 ml /kg) on u-AQP2 and u-ENaCgamma, fractional sodium excretion (FENa), free water clearance (CH2O), and plasma concentrations of vasopressin (AVP), renin (PRC), angiotensin II (ANG II) and aldosterone (Aldo) in a randomized, crossover study of 23 healthy subjects, who consumed a standardized diet, regarding calories, sodium and fluid for 4 days before each examination day. After isotonic saline infusion, u-AQP2 increased (27%). CH2O and u-ENaCgamma were unchanged, whereas FENa increased (123%). After hypertonic saline infusion, there was an increase in u-AQP2 (25%), u-ENaCgamma (19%) and FENa (96%), whereas CH2O decreased (-153%). After isotonic glucose infusion, there was a decrease in u-AQP2 (-16%), ENaCgamma (-10 %) and FENa (-44%) whereas CH2O increased (164%). AVP remained unchanged after isotonic saline and glucose, but increased after hypertonic saline (139%). PRC, AngII and p-Aldo decreased after isotonic and hypertonic saline infusion, but not after glucose infusion. Volume expansion with 3% and 0.9% saline increased u-AQP2, while isotonic glucose decreased u-AQP2. Infusion of hypertonic saline increased u-ENaCgamma, whereas u-ENaCgamma was not significantly changed after isotonic saline and tended to decrease after glucose. Thus, the transport of water and sodium is changed both via the aquaporin 2 water channels and the epithelial sodium channels during all three types of volume expansion to regulate and maintain water- and sodium homeostasis in the body.Trial registrationClinical Trial no: NCT01414088.
    Full-text · Article · Sep 2013 · BMC Nephrology
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    ABSTRACT: Renal tubulo-interstitial inflammation is frequently associated with polyuria and urine concentration defects. This led us to investigate the effects of the major pro-inflammatory nuclear factor kappaB (NF-kappaB) pathway on aquaporin 2 (AQP2) expression by the collecting duct. Using immortalized collecting duct principal cells (mpkCCDcl4), we found that, acting independently of vasopressin, activation of NF-kappaB by lipopolysaccharide (LPS) decreased AQP2 mRNA and protein levels in a time- and dose-dependent manner but did not decrease AQP2 mRNA stability. Consistently, constitutively active IkappaB kinase beta decreased AQP2 expression. The LPS-induced decrease in AQP2 mRNA levels was confirmed in rat kidney slices and was reproduced both under conditions of elevated cAMP concentration and V(2) receptor antagonism. Computer analysis of the AQP2 promoter revealed two putative kappaB elements. Mutation of either kappaB element abolished the LPS-induced decrease of luciferase activity in cells expressing AQP2 promoter-luciferase plasmid constructs. Chromatin immunoprecipitation revealed that LPS challenge decreased p65, increased p50 and p52, and had no effect on RelB and c-Rel binding to kappaB elements of the AQP2 promoter. RNA-mediated interference silencing of p65, p50, and p52 confirmed controlled AQP2 transcription by these NF-kappaB subunits. We additionally found that hypertonicity activated NF-kappaB in mpkCCDcl4 cells, an effect that may counteract the Tonicity-responsive enhancer binding protein (TonEBP)-dependent increase in AQP2 gene transcription. Taken together, these findings indicate that NF-kappaB is an important physiological regulator of AQP2 transcription.
    Preview · Article · Sep 2008 · Journal of Biological Chemistry
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