NADPH oxidase 4 deficiency increases tubular cell death during acute ischemic reperfusion injury.

NADPH oxidase 4 (NOX4) is highly expressed in kidney proximal tubular cells. NOX4 constitutively produces hydrogen peroxide, which may regulate important pro-survival pathways. Renal ischemia reperfusion injury (IRI) is a classical model mimicking human ischemic acute tubular necrosis. We hypothesized that NOX4 plays a protective role in kidney IRI. In wild type (WT) animals subjected to IRI, NOX4 protein expression increased after 24 hours. NOX4 KO (knock-out) and WT littermates mice were subjected to IRI. NOX4 KO mice displayed decreased renal function and more severe tubular apoptosis, decreased Bcl-2 expression and higher histologic damage scores compared to WT. Activation of NRF2 was decreased in NOX4 KO mice in response to IRI. This was related to decreased KEAP1 oxidation leading to decreased NRF2 stabilization. This resulted in decreased glutathione levels. In vitro silencing of NOX4 in cells showed an enhanced propensity to apoptosis, with reduced expression of NRF2, glutathione content and Bcl-2 expression, similar to cells derived from NOX4 KO mice. Overexpression of a constitutively active form of NRF2 (caNRF2) in NOX4 depleted cells rescued most of this phenotype in cultured cells, implying that NRF2 regulation by ROS issued from NOX4 may play an important role in its anti-apoptotic property.

Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress.

Cell volume homeostasis is vital for the maintenance of optimal protein density and cellular function. Numerous mammalian cell types are routinely exposed to acute hypertonic challenge and shrink. Molecular crowding modifies biochemical reaction rates and decreases macromolecule diffusion. Cell volume is restored rapidly by ion influx but at the expense of elevated intracellular sodium and chloride levels that persist long after challenge. Although recent studies have highlighted the role of molecular crowding on the effects of hypertonicity, the effects of ionic imbalance on cellular trafficking dynamics in living cells are largely unexplored. By tracking distinct fluorescently labeled endosome/vesicle populations by live-cell imaging, we show that vesicle motility is reduced dramatically in a variety of cell types at the onset of hypertonic challenge. Live-cell imaging of actin and tubulin revealed similar arrested microfilament motility upon challenge. Vesicle motility recovered long after cell volume, a process that required functional regulatory volume increase and was accelerated by a return of extracellular osmolality to isosmotic levels. This delay suggests that, although volume-induced molecular crowding contributes to trafficking defects, it alone cannot explain the observed effects. Using fluorescent indicators and FRET-based probes, we found that intracellular ATP abundance and mitochondrial potential were reduced by hypertonicity and recovered after longer periods of time. Similar to the effects of osmotic challenge, isovolumetric elevation of intracellular chloride concentration by ionophores transiently decreased ATP production by mitochondria and abated microfilament and vesicle motility. These data illustrate how perturbed ionic balance, in addition to molecular crowding, affects membrane trafficking.

Proteinuria Increases Plasma Phosphate by Altering Its Tubular Handling.

Proteinuria and hyperphosphatemia are cardiovascular risk factors independent of GFR. We hypothesized that proteinuria induces relative phosphate retention via increased proximal tubule phosphate reabsorption. To test the clinical relevance of this hypothesis, we studied phosphate handling in nephrotic children and patients with CKD. Plasma fibroblast growth factor 23 (FGF-23) concentration, plasma phosphate concentration, and tubular reabsorption of phosphate increased during the proteinuric phase compared with the remission phase in nephrotic children. Cross-sectional analysis of a cohort of 1738 patients with CKD showed that albuminuria≥300 mg/24 hours is predictive of higher phosphate levels, independent of GFR and other confounding factors. Albuminuric patients also displayed higher plasma FGF-23 and parathyroid hormone levels. To understand the molecular mechanisms underlying these observations, we induced glomerular proteinuria in two animal models. Rats with puromycin-aminonucleoside-induced nephrotic proteinuria displayed higher renal protein expression of the sodium-phosphate co-transporter NaPi-IIa, lower renal Klotho protein expression, and decreased phosphorylation of FGF receptor substrate 2α, a major FGF-23 receptor substrate. These findings were confirmed in transgenic mice that develop nephrotic-range proteinuria resulting from podocyte depletion. In vitro, albumin did not directly alter phosphate uptake in cultured proximal tubule OK cells. In conclusion, we show that proteinuria increases plasma phosphate concentration independent of GFR. This effect relies on increased proximal tubule NaPi-IIa expression secondary to decreased FGF-23 biologic activity. Proteinuria induces elevation of both plasma phosphate and FGF-23 concentrations, potentially contributing to cardiovascular disease.

Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion.

Coordinated cell proliferation and ability to form intercellular seals are essential features of epithelial tissue function. Tight junctions (TJs) classically act as paracellular diffusion barriers. More recently, their role in regulating epithelial cell proliferation in conjunction with scaffolding zonula occludens (ZO) proteins has come to light. The kidney collecting duct (CD) is a model of tight epithelium that displays intense proliferation during embryogenesis followed by very low cell turnover in the adult kidney. Here, we examined the influence of each ZO protein (ZO-1, -2 and -3) on CD cell proliferation. We show that all 3 ZO proteins are strongly expressed in native CD and are present at both intercellular junctions and nuclei of cultured CD principal cells (mCCDcl1). Suppression of either ZO-1 or ZO-2 resulted in increased G0/G1 retention in mCCDcl1 cells. ZO-2 suppression decreased cyclin D1 abundance while ZO-1 suppression was accompanied by increased nuclear p21 localization, the depletion of which restored cell cycle progression. Contrary to ZO-1 and ZO-2, ZO-3 expression at intercellular junctions dramatically increased with cell density and relied on the presence of ZO-1. ZO-3 depletion did not affect cell cycle progression but increased cell detachment. This latter event partly relied on increased nuclear cyclin D1 abundance and was associated with altered ß1-integrin subcellular distribution and decreased occludin expression at intercellular junctions. These data reveal diverging, but interconnected, roles for each ZO protein in mCCDcl1 proliferation. While ZO-1 and ZO-2 participate in cell cycle progression, ZO-3 is an important component of cell adhesion.

Epithelial sodium channel abundance is decreased by an unfolded protein response induced by hyperosmolality.

Large shifts of osmolality occur in the kidney medulla as part of the urine concentrating mechanism. Hyperosmotic stress profoundly challenges cellular homeostasis and induces endoplasmic reticulum (ER) stress. Here, we examined the unfolded protein response (UPR) in hyperosmotically-challenged principal cells of the kidney collecting duct (CD) and show its relevance in controlling epithelial sodium channel (ENaC) abundance, responsible for the final adjustment of Na(+) excretion. Dehydration increases medullary but not cortical osmolality. Q-PCR analysis of microdissected CD of water-deprived mice revealed increased aquaporin-2 (AQP2) expression in outer medullary and cortical CD while ENaC abundance decreased in outer medullary but not cortical CD. Immunoblotting, Q-PCR and immunofluorescence revealed that hyperosmolality induced a transient ER stress-like response both ex vivo and in cultured CD principal cells and increased activity of the canonical UPR mediators PERK and ATF6. Both hyperosmolality and chemical induction of ER stress decreased ENaC expression in vitro. ENaC depletion by either stimulus was abolished by transcriptional inhibition and by the chemical chaperone 4-phenylbutyric acid and was partly abrogated by either PERK or ATF6 silencing. Our data suggest that induction of the UPR by hyperosmolality may help preserve body fluid homeostasis under conditions of dehydration by uncoupling AQP2 and ENaC abundance in outer medullary CD.

NADPH oxidase 4 deficiency reduces aquaporin-2 mRNA expression in cultured renal collecting duct principal cells via increased PDE3 and PDE4 activity.

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.

Sodium transport is modulated by p38 kinase-dependent cross-talk between ENaC and Na,K-ATPase in collecting duct principal cells.

In relation to dietary Na(+) intake and aldosterone levels, collecting duct principal cells are exposed to large variations in Na(+) transport. In these cells, Na(+) crosses the apical membrane via epithelial Na(+) channels (ENaC) and is extruded into the interstitium by Na,K-ATPase. The activity of ENaC and Na,K-ATPase must be highly coordinated to accommodate variations in Na(+) transport and minimize fluctuations in intracellular Na(+) concentration. We hypothesized that, independent of hormonal stimulus, cross-talk between ENaC and Na,K-ATPase coordinates Na(+) transport across apical and basolateral membranes. By varying Na(+) intake in aldosterone-clamped rats and overexpressing γ-ENaC or modulating apical Na(+) availability in cultured mouse collecting duct cells, enhanced apical Na(+) entry invariably led to increased basolateral Na,K-ATPase expression and activity. In cultured collecting duct cells, enhanced apical Na(+) entry increased the basolateral cell surface expression of Na,K-ATPase by inhibiting p38 kinase-mediated endocytosis of Na,K-ATPase. Our results reveal a new role for p38 kinase in mediating cross-talk between apical Na(+) entry via ENaC and its basolateral exit via Na,K-ATPase, which may allow principal cells to maintain intracellular Na(+) concentrations within narrow limits.

Albuminuria induces a proinflammatory and profibrotic response in cortical collecting ducts via the 24p3 receptor.

Albuminuria is strongly associated with progressive kidney tubulo-interstitial damage and chronic kidney disease (CKD) progression. In proteinuric nephropathies, albumin reabsorption by the proximal tubule is saturated and the distal nephron is exposed to high concentrations of luminal albumin that may produce adverse effects. Since proximal tubular cells exposed to albuminuria exhibit a proinflammatory and profibrotic response, we assessed the effect of albuminuria in the collecting duct (CD). With the use of kidney sections and isolated cortical CDs (CCDs) from puromycin-aminonucleoside-induced nephrotic rats (PAN rats) exhibiting proteinuria, immunofluorescence microscopy revealed internalized albumin in CD cells. In these proteinuric rats, increased expression levels of cytokines and profibrotic signaling markers were detected in isolated CCDs and bands of inflammatory fibrosis could be observed around CDs. Albumin endocytosis was confirmed by FITC-albumin uptake in cultured murine CCD (mCCDcl1) cells. Exposure of mCCDcl1 cells to albumin induced NF-κB activation as assessed by luciferase reporter gene assay, nuclear translocation of NF-κB p65 subunit, and increased NF-κB target gene expression. Moreover, albuminuria-like condition results in transforming growth factor-ß1 (TGF-ß1) overexpression and the upregulation of profibrotic signaling markers such as Snail or vimentin via an autocrine mechanism. In mCCDcl1 cells, neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin-2/24p3 receptor (24p3R) mediates albumin endocytosis as well as activation of NF-κB and TGF-ß1 signaling pathways. Therefore, CD may play a key role in initiation and/or progression of inflammation and fibrosis in response to proteinuria.

Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters.

Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. Here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, LC3-II generation and Ptdlns3K-dependent formation of LC3- and ATG12-positive puncta. Lysosomotropic agents chloroquine and bafilomycin A 1, but not nutrient deprivation or rapamycin treatment, further increased LC3-II generation, as well as ATG12-positive puncta, indicating that hypertonic stress increases autophagic flux. Autophagy induction upon hypertonic stress enhanced cell survival since cell death was increased by ATG12 siRNA-mediated knockdown and reduced by rapamycin. We additionally showed that hypertonicity induces fast reorganization of microtubule networks, which is associated with strong reorganization of microtubules at centrosomes and fragmentation of Golgi ribbons. Microtubule remodeling was associated with pericentrosomal clustering of ATG12-positive autolysosomes that colocalized with SQSTM1/p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is at least partly selective. Efficient autophagy by hypertonic stress required microtubule remodeling and was DYNC/dynein-dependent as autophagosome clustering was enhanced by paclitaxel-induced microtubule stabilization and was reduced by nocodazole-induced tubulin depolymerization as well as chemical (EHNA) or genetic [DCTN2/dynactin 2 (p50) overexpression] interference of DYNC activity. The data document a general and hitherto overlooked mechanism, where autophagy and microtubule remodeling play prominent roles in the osmoprotective response.

STIM1 juxtaposes ER to phagosomes, generating Ca²âº hotspots that boost phagocytosis.

BACKGROUND: Endoplasmic reticulum (ER) membranes are recruited to phagosomes, but the mechanism and functional significance of this ER recruitment is not known. Here, we show that the ER Ca(2+) sensor stromal interaction molecule 1 (STIM1) sustains high-efficiency phagocytosis by recruiting thin ER cisternae that interact productively but do not fuse with phagosomes. RESULTS: Endogenous STIM1 was recruited to phagosomes upon ER Ca(2+) depletion in mouse neutrophils, and exogenous YFP-STIM1 puncta coincided with localized Ca(2+) elevations around phagosomes in fibroblasts expressing phagocytic receptors. STIM1 ablation decreased phagocytosis, ER-phagosome contacts, and periphagosomal Ca(2+) elevations in both neutrophils and fibroblasts, whereas STIM1 re-expression in Stim1(-/-) fibroblasts rescued these defects, promoted the formation and elongation of tight ER-phagosome contacts upon ER Ca(2+) depletion and increased the shedding of periphagosomal actin rings. Re-expression of a signaling-deficient STIM1 mutant unable to open Ca(2+) channels recruited ER cisternae to the vicinity of phagosomes but failed to rescue phagocytosis, actin shedding, and periphagosomal Ca(2+) elevations. The periphagosomal Ca(2+) hotspots were decreased by extracellular Ca(2+) chelation and by Ca(2+) channels inhibitors, revealing that the Ca(2+) ions originate at least in part from phagosomes. CONCLUSIONS: Our findings indicate that STIM1 recruits ER cisternae near phagosomes for signaling purposes and that the opening of phagosomal Ca(2+) channels generates localized Ca(2+) elevations that promote high-efficiency phagocytosis.

Simvastatin enhances aquaporin-2 surface expression and urinary concentration in vasopressin-deficient Brattleboro rats through modulation of Rho GTPase.

Statins are 3-hydroxyl-3-methyglutaryl-CoA reductase inhibitors that are commonly used to inhibit cholesterol biosynthesis. Emerging data have suggested that they also have "pleotropic effects," including modulating actin cytoskeleton reorganization. Here, we report an effect of simvastatin on the trafficking of aquaporin-2 (AQP2). Specifically, simvastatin induced the membrane accumulation of AQP2 in cell cultures and kidneys in situ. The effect of simvastatin was independent of protein kinase A activation and phosphorylation at AQP2-Ser(256), a critical event involved in vasopressin (VP)-regulated AQP2 trafficking. Further investigation showed that simvastatin inhibited endocytosis in parallel with downregulation of RhoA activity. Overexpression of active RhoA attenuated simvastatin's effect, suggesting the involvement of this small GTPase in simvastatin-mediated AQP2 trafficking. Finally, the effect of simvastatin on urinary concentration was investigated in VP-deficient Brattleboro rats. Simvastatin acutely (3-6 h) increased urinary concentration and decreased urine output in these animals. In summary, simvastatin regulates AQP2 trafficking in vitro and urinary concentration in vivo via events involving downregulation of Rho GTPase activity and inhibition of endocytosis. Our study provides an alternative mechanism to regulate AQP2 trafficking, bypassing the VP-vasopressin receptor signaling pathway.

Osmoprotective transcription factor NFAT5/TonEBP modulates nuclear factor-kappaB activity.

Tonicity-responsive binding-protein (TonEBP or NFAT5) is a widely expressed transcription factor whose activity is regulated by extracellular tonicity. TonEBP plays a key role in osmoprotection by binding to osmotic response element/TonE elements of genes that counteract the deleterious effects of cell shrinkage. Here, we show that in addition to this "classical" stimulation, TonEBP protects cells against hypertonicity by enhancing nuclear factor-κB (NF-κB) activity. We show that hypertonicity enhances NF-κB stimulation by lipopolysaccharide but not tumor necrosis factor-α, and we demonstrate overlapping protein kinase B (Akt)-dependent signal transduction pathways elicited by hypertonicity and transforming growth factor-α. Activation of p38 kinase by hypertonicity and downstream activation of Akt play key roles in TonEBP activity, IκBα degradation, and p65 nuclear translocation. TonEBP affects neither of these latter events and is itself insensitive to NF-κB signaling. Rather, we reveal a tonicity-dependent interaction between TonEBP and p65 and show that NF-κB activity is considerably enhanced after binding of NF-κB-TonEBP complexes to κB elements of NF-κB-responsive genes. We demonstrate the key roles of TonEBP and Akt in renal collecting duct epithelial cells and in macrophages. These findings reveal a novel role for TonEBP and Akt in NF-κB activation on the onset of hypertonic challenge.

Controlled aquaporin-2 expression in the hypertonic environment.

The corticomedullary osmolality gradient is the driving force for water reabsorption occurring in the kidney. In the collecting duct, this gradient allows luminal water to move across aquaporin (AQP) water channels, thereby increasing urine concentration. However, this same gradient exposes renal cells to great osmotic challenges. These cells must constantly adapt to fluctuations of environmental osmolality that challenge cell volume and incite functional change. This implies profound alterations of cell phenotype regarding water permeability. AQP2 is an essential component of the urine concentration mechanism whose controlled expression dictates apical water permeability of collecting duct principal cells. This review focuses on changes of AQP2 abundance and trafficking in hypertonicity-challenged cells. Intracellular mechanisms governing these events are discussed and the biological relevance of altered AQP2 expression by hypertonicity is outlined.

Aquaporin-2 abundance in the renal collecting duct: new insights from cultured cell models.

The renal cortico-papillary osmotic gradient is generated by sodium reabsorption in the thick ascending limb. The antidiuretic hormone arginine vasopressin (AVP) increases collecting duct water permeability by enhancing aquaporin-2 (AQP2) water channel insertion in the apical membrane of principal cells, allowing water to passively flow along the osmotic gradient from the tubule lumen to the interstitium. In addition to short-term AQP2 redistribution between intracellular compartments and the cell surface, AQP2 whole cell abundance is tightly regulated. AVP is a major transcriptional activator of the AQP2 gene, and stimulation of insulin- and calcium-sensing receptors respectively potentiate and reduce its action. Extracellular tonicity is another key factor that determines the levels of AQP2 abundance. Its effect is dependent on activation of the tonicity-responsive enhancer binding protein that reinforces AVP-induced AQP2 transcriptional activation. Conversely, activation of the NF-kappaB transcriptional factor by proinflammatory factors reduces AQP2 gene transcription. Aldosterone additionally regulates AQP2 whole cell abundance by simultaneously reducing AQP2 gene transcription and stimulating AQP2 mRNA translation. These examples illustrate how cross talk between various stimuli regulates AQP2 abundance in collecting duct principal cells and consequently contributes to maintenance of body water homeostasis.

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.

Effects of the renal medullary pH and ionic environment on vasopressin binding and signaling.

The kidney has a cortico-medullary interstitial gradient of decreasing pH and increasing concentrations of sodium chloride and urea, but the influence of these gradients on receptor signaling is largely unknown. Here, we measured G-protein coupled receptor function in LLC-PK1 cells acutely exposed to conditions mimicking different kidney regions. Signaling through the parathyroid hormone receptor, normally expressed in the cortex, was greatly reduced at an acidic pH similar to that of the inner medulla. Parathyroid hormone receptor, tagged with green fluorescent protein, showed no ligand-induced internalization. In contrast, under both acidic and hyperosmotic conditions, vasopressin increased intracellular cAMP, and upon binding to its type 2 receptor (V2R) was internalized and degraded. Dose-displacement binding assays with selective vasopressin/oxytocin receptor ligands under inner medullary conditions indicated a shift in the V2R pharmacological profile. Oxytocin did not bind to the V2R, as it does under normal conditions and the vasopressin type 1 receptor (V1R) had reduced affinity for vasopressin compared to the V2R in low pH and high osmolality. We suggest that the cortico-medullary gradient causes a receptor-specific selectivity in ligand binding that is of functional significance to the kidney. While the gradient is important for urinary concentration, it may also play a substantial role in fine-tuning of the vasopressin response through the V2R.

A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK1 cells expressing aquaporin-2.

Vasopressin (VP)-induced exocytosis was dissected in native and aquaporin-2 (AQP2)-expressing renal LLC-PK(1) cells by a fluorimetric exocytosis assay based on soluble secreted yellow fluorescent protein (ssYFP). YFP was targeted to the secretory pathway by addition of an 18-amino acid signal peptide from hen egg white lysozyme. Immunofluorescence labeling, together with analysis of Alexa 555-dextran internalization, revealed that ssYFP is exclusively located in the secretory pathway. Immunofluorescence and immunogold electron microscopy showed significant colocalization of ssYFP and AQP2. Fluorimetry and Western blot analysis demonstrated similar constitutive ssYFP secretion in native LLC-PK(1) and AQP2-expressing cells. In AQP2-expressing cells, a twofold increase in ssYFP secretion was observed within 15 min of VP stimulation. This transient burst of ssYFP secretion was abolished by the PKA inhibitor H-89 and was not observed in native cells. The endocytotic inhibitor methyl-beta-cyclodextrin, which also promotes membrane accumulation of AQP2, had no effect on ssYFP secretion. Although cells expressing phosphorylation-deficient AQP2-S256A showed significantly lower baseline levels of constitutive secretion, VP induced a significant increase in exocytosis. Our data indicate that 1) this assay can monitor exocytosis in cultured epithelial cells, 2) VP has an acute stimulatory effect on ssYFP secretion in AQP2-expressing, but not native, cells, and 3) phosphorylation of AQP2 at S256 may be involved in the regulation of constitutive AQP2 exocytosis and play only a minor role in the VP-induced burst. These results support the idea that, in addition to its role in reducing AQP2 endocytosis, VP increases AQP2 exocytosis.

NF-kappaB modulates aquaporin-2 transcription in renal collecting duct principal cells.

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.

Phosphorylation events and the modulation of aquaporin 2 cell surface expression.

PURPOSE OF REVIEW: This review highlights the role of phosphorylation in the trafficking and targeting of aquaporin 2. Current knowledge will be put into the context of modulating the cell surface expression of aquaporin 2 by vasopressin in renal epithelial cells, which is critical for regulation of urinary concentration and control of fluid and electrolyte homeostasis. RECENT FINDINGS: In addition to previously identified phosphorylation sites on aquaporin 2, new data have revealed three other serine residues in the C-terminus whose phosphorylation is altered by vasopressin. Several steps in aquaporin 2 recycling, including exocytosis and endocytosis, are coordinated by phosphorylation and dephosphorylation to regulate cell surface accumulation. Aquaporin 2 phosphorylation on serine 256 regulates aquaporin 2 association with proteins that are involved in trafficking, including hsc/hsp70 and myelin and lymphocyte-associated protein. SUMMARY: Aquaporin 2 trafficking is regulated by phosphorylation of serine 256 and other amino acid residues in its cytoplasmic domain. These events increase or decrease interaction of aquaporin 2 with key regulatory proteins to determine the cellular distribution and fate of aquaporin 2, both after vasopressin addition and under baseline conditions. Better understanding of these mechanisms may provide new therapeutic avenues for patients with X-linked nephrogenic diabetes insipidus, as well as providing basic cell biological information relevant to membrane trafficking processes in general.