Inhibition of vascular calcification by inositol phosphates derivatized with ethylene glycol oligomers.

Myo-inositol hexakisphosphate (IP6) is a natural product known to inhibit vascular calcification (VC), but with limited potency and low plasma exposure following bolus administration. Here we report the design of a series of inositol phosphate analogs as crystallization inhibitors, among which 4,6-di-O-(methoxy-diethyleneglycol)-myo-inositol-1,2,3,5-tetrakis(phosphate), (OEG2)2-IP4, displays increased in vitro activity, as well as more favorable pharmacokinetic and safety profiles than IP6 after subcutaneous injection. (OEG2)2-IP4 potently stabilizes calciprotein particle (CPP) growth, consistently demonstrates low micromolar activity in different in vitro models of VC (i.e., human serum, primary cell cultures, and tissue explants), and largely abolishes the development of VC in rodent models, while not causing toxicity related to serum calcium chelation. The data suggest a mechanism of action independent of the etiology of VC, whereby (OEG2)2-IP4 disrupts the nucleation and growth of pathological calcification.

Nephron-Specific Deletion of Circadian Clock Gene Bmal1 Alters the Plasma and Renal Metabolome and Impairs Drug Disposition.

The circadian clock controls a wide variety of metabolic and homeostatic processes in a number of tissues, including the kidney. However, the role of the renal circadian clocks remains largely unknown. To address this question, we performed a combined functional, transcriptomic, and metabolomic analysis in mice with inducible conditional knockout (cKO) of BMAL1, which is critically involved in the circadian clock system, in renal tubular cells (Bmal1lox/lox/Pax8-rtTA/LC1 mice). Induction of cKO in adult mice did not produce obvious abnormalities in renal sodium, potassium, or water handling. Deep sequencing of the renal transcriptome revealed significant changes in the expression of genes related to metabolic pathways and organic anion transport in cKO mice compared with control littermates. Furthermore, kidneys from cKO mice exhibited a significant decrease in the NAD+-to-NADH ratio, which reflects the oxidative phosphorylation-to-glycolysis ratio and/or the status of mitochondrial function. Metabolome profiling showed significant changes in plasma levels of amino acids, biogenic amines, acylcarnitines, and lipids. In-depth analysis of two selected pathways revealed a significant increase in plasma urea level correlating with increased renal Arginase II activity, hyperargininemia, and increased kidney arginine content as well as a significant increase in plasma creatinine concentration and a reduced capacity of the kidney to secrete anionic drugs (furosemide) paralleled by an approximate 80% decrease in the expression level of organic anion transporter 3 (SLC22a8). Collectively, these results indicate that the renal circadian clocks control a variety of metabolic/homeostatic processes at the intrarenal and systemic levels and are involved in drug disposition.

Mutation in the Monocarboxylate Transporter 12 Gene Affects Guanidinoacetate Excretion but Does Not Cause Glucosuria.

A heterozygous mutation (c.643C>A; p.Q215X) in the monocarboxylate transporter 12-encoding gene MCT12 (also known as SLC16A12) that mediates creatine transport was recently identified as the cause of a syndrome with juvenile cataracts, microcornea, and glucosuria in a single family. Whereas the MCT12 mutation cosegregated with the eye phenotype, poor correlation with the glucosuria phenotype did not support a pathogenic role of the mutation in the kidney. Here, we examined MCT12 in the kidney and found that it resides on basolateral membranes of proximal tubules. Patients with MCT12 mutation exhibited reduced plasma levels and increased fractional excretion of guanidinoacetate, but normal creatine levels, suggesting that MCT12 may function as a guanidinoacetate transporter in vivo However, functional studies in Xenopus oocytes revealed that MCT12 transports creatine but not its precursor, guanidinoacetate. Genetic analysis revealed a separate, undescribed heterozygous mutation (c.265G>A; p.A89T) in the sodium/glucose cotransporter 2-encoding gene SGLT2 (also known as SLC5A2) in the family that segregated with the renal glucosuria phenotype. When overexpressed in HEK293 cells, the mutant SGLT2 transporter did not efficiently translocate to the plasma membrane, and displayed greatly reduced transport activity. In summary, our data indicate that MCT12 functions as a basolateral exit pathway for creatine in the proximal tubule. Heterozygous mutation of MCT12 affects systemic levels and renal handling of guanidinoacetate, possibly through an indirect mechanism. Furthermore, our data reveal a digenic syndrome in the index family, with simultaneous MCT12 and SGLT2 mutation. Thus, glucosuria is not part of the MCT12 mutation syndrome.

ENaC activity in collecting ducts modulates NCC in cirrhotic mice.

Cirrhosis is a frequent and severe disease, complicated by renal sodium retention leading to ascites and oedema. A better understanding of the complex mechanisms responsible for renal sodium handling could improve clinical management of sodium retention. Our aim was to determine the importance of the amiloride-sensitive epithelial sodium channel (ENaC) in collecting ducts in compensate and decompensate cirrhosis. Bile duct ligation was performed in control mice (CTL) and collecting duct-specific αENaC knockout (KO) mice, and ascites development, aldosterone plasma concentration, urinary sodium/potassium ratio and sodium transporter expression were compared. Disruption of ENaC in collecting ducts (CDs) did not alter ascites development, urinary sodium/potassium ratio, plasma aldosterone concentrations or Na,K-ATPase abundance in CCDs. Total αENaC abundance in whole kidney increased in cirrhotic mice of both genotypes and cleaved forms of α and γ ENaC increased only in ascitic mice of both genotypes. The sodium chloride cotransporter (NCC) abundance was lower in non-ascitic KO, compared to non-ascitic CTL, and increased when ascites appeared. In ascitic mice, the lack of αENaC in CDs induced an upregulation of total ENaC and NCC and correlated with the cleavage of ENaC subunits. This revealed compensatory mechanisms which could also take place when treating the patients with diuretics. These compensatory mechanisms should be considered for future development of therapeutic strategies.

Regulation of 11ß-hydroxysteroid dehydrogenase type 2 by microRNA.

The enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) is selectively expressed in aldosterone target tissues, conferring aldosterone selectivity for the mineralocorticoid receptor. A diminished activity causes salt-sensitive hypertension. The mechanism of the variable and distinct 11ß-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) expression in the cortical collecting duct is poorly understood. Here, we analyzed for the first time whether the 11ß-HSD2 expression is modulated by microRNAs (miRNAs). In silico analysis revealed 53 and 27 miRNAs with potential binding sites on human or rat HSD11B2 3'-untranslated region. A reporter assay demonstrated 3'-untranslated region-dependent regulation of human and rodent HSD11B2. miRNAs were profiled from cortical collecting ducts and proximal convoluted tubules. Bioinformatic analyses showed a distinct clustering for cortical collecting ducts and proximal convoluted tubules with 53 of 375 miRNAs, where 13 were predicted to bind to the rat HSD11B2 3'-untranslated region. To gain insight into potentially relevant miRNAs in vivo, we investigated 2 models with differential 11ß-HSD2 activity linked with salt-sensitive hypertension. (1) Comparing Sprague-Dawley with low and Wistar rats with high 11ß-HSD2 activity revealed rno-miR-20a-5p, rno-miR-19b-3p, and rno-miR-190a-5p to be differentially expressed. (2) Uninephrectomy lowered 11ß-HSD2 activity in the residual kidney with differentially expressed rno-miR-19b-3p, rno-miR-29b-3p, and rno-miR-26-5p. In conclusion, miRNA-dependent mechanisms seem to modulate 11ß-HSD2 dosage in health and disease states.

Local renal circadian clocks control fluid-electrolyte homeostasis and BP.

The circadian timing system is critically involved in the maintenance of fluid and electrolyte balance and BP control. However, the role of peripheral circadian clocks in these homeostatic mechanisms remains unknown. We addressed this question in a mouse model carrying a conditional allele of the circadian clock gene Bmal1 and expressing Cre recombinase under the endogenous Renin promoter (Bmal1(lox/lox)/Ren1(d)Cre mice). Analysis of Bmal1(lox/lox)/Ren1(d)Cre mice showed that the floxed Bmal1 allele was excised in the kidney. In the kidney, BMAL1 protein expression was absent in the renin-secreting granular cells of the juxtaglomerular apparatus and the collecting duct. A partial reduction of BMAL1 expression was observed in the medullary thick ascending limb. Functional analyses showed that Bmal1(lox/lox)/Ren1(d)Cre mice exhibited multiple abnormalities, including increased urine volume, changes in the circadian rhythm of urinary sodium excretion, increased GFR, and significantly reduced plasma aldosterone levels. These changes were accompanied by a reduction in BP. These results show that local renal circadian clocks control body fluid and BP homeostasis.

α-Ketoglutarate regulates acid-base balance through an intrarenal paracrine mechanism.

Paracrine communication between different parts of the renal tubule is increasingly recognized as an important determinant of renal function. Previous studies have shown that changes in dietary acid-base load can reverse the direction of apical α-ketoglutarate (αKG) transport in the proximal tubule and Henle's loop from reabsorption (acid load) to secretion (base load). Here we show that the resulting changes in the luminal concentrations of αKG are sensed by the αKG receptor OXGR1 expressed in the type B and non-A-non-B intercalated cells of the connecting tubule (CNT) and the cortical collecting duct (CCD). The addition of 1 mM αKG to the tubular lumen strongly stimulated Cl(-)-dependent HCO(3)(-) secretion and electroneutral transepithelial NaCl reabsorption in microperfused CCDs of wild-type mice but not Oxgr1(-/-) mice. Analysis of alkali-loaded mice revealed a significantly reduced ability of Oxgr1(-/-) mice to maintain acid-base balance. Collectively, these results demonstrate that OXGR1 is involved in the adaptive regulation of HCO(3)(-) secretion and NaCl reabsorption in the CNT/CCD under acid-base stress and establish αKG as a paracrine mediator involved in the functional coordination of the proximal and the distal parts of the renal tubule.

PA21, a new iron-based noncalcium phosphate binder, prevents vascular calcification in chronic renal failure rats.

Chronic renal failure (CRF) is associated with the development of secondary hyperparathyroidism and vascular calcifications. We evaluated the efficacy of PA21, a new iron-based noncalcium phosphate binder, in controlling phosphocalcic disorders and preventing vascular calcifications in uremic rats. Rats with adenine-diet-induced CRF were randomized to receive either PA21 0.5, 1.5, or 5% or CaCO3 3% in the diet for 4 weeks, and were compared with uremic and nonuremic control groups. After 4 weeks of phosphate binder treatment, serum calcium, creatinine, and body weight were similar between all CRF groups. Serum phosphorus was reduced with CaCO3 3% (2.06 mM; P ≤ 0.001), PA21 1.5% (2.29 mM; P < 0.05), and PA21 5% (2.21 mM; P ≤ 0.001) versus CRF controls (2.91 mM). Intact parathyroid hormone was strongly reduced in the PA21 5% and CaCO3 3% CRF groups to a similar extent (1138 and 1299 pg/ml, respectively) versus CRF controls (3261 pg/ml; both P ≤ 0.001). A lower serum fibroblast growth factor 23 concentration was observed in the PA21 5%, compared with CaCO3 3% and CRF, control groups. PA21 5% CRF rats had a lower vascular calcification score compared with CaCO3 3% CRF rats and CRF controls. In conclusion, PA21 was as effective as CaCO3 at controlling phosphocalcic disorders but superior in preventing the development of vascular calcifications in uremic rats. Thus, PA21 represents a possible alternative to calcium-based phosphate binders in CRF patients.

Heterogeneity in the processing of CLCN5 mutants related to Dent disease.

Mutations in the electrogenic Cl(-)/H(+) exchanger ClC-5 gene CLCN5 are frequently associated with Dent disease, an X-linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in Xenopus laevis oocytes and in HEK293 cells of nine previously reported, pathogenic, missense mutations of ClC-5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild-type ClC-5, but exhibited reduced currents. Three mutants displayed improper N-glycosylation, and were nonfunctional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC-5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing, and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC-5 loss of function in Dent disease.

Regulation of pendrin by pH: dependence on glycosylation.

Mutations in the anion exchanger pendrin are responsible for Pendred syndrome, an autosomal recessive disease characterized by deafness and goitre. Pendrin is highly expressed in kidney collecting ducts, where it acts as a chloride/bicarbonate exchanger and thereby contributes to the regulation of acid-base homoeostasis and blood pressure. The present study aimed to characterize the intrinsic properties of pendrin. Mouse pendrin was transfected in HEK (human embryonic kidney) 293 and OKP (opossum kidney proximal tubule) cells and its activity was determined by monitoring changes in the intracellular pH induced by variations of transmembrane anion gradients. Combining measurements of pendrin activity with mathematical modelling we found that its affinity for Cl-, HCO3- and OH- varies with intracellular pH, with increased activity at low intracellular pH. Maximal pendrin activity was also stimulated at low extracellular pH, suggesting the presence of both intracellular and extracellular proton regulatory sites. We identified five putative pendrin glycosylation sites, only two of which are used. Mutagenesis-induced disruption of pendrin glycosylation did not alter its cell-surface expression or polarized targeting to the apical membrane and basal activity, but fully abrogated its sensitivity to extracellular pH. The hither to unknown regulation of pendrin by external pH may constitute a key mechanism in controlling ionic exchanges across the collecting duct and inner ear.

Sodium and potassium balance depends on αENaC expression in connecting tubule.

Mutations in α, ß, or γ subunits of the epithelial sodium channel (ENaC) can downregulate ENaC activity and cause a severe salt-losing syndrome with hyperkalemia and metabolic acidosis, designated pseudohypoaldosteronism type 1 in humans. In contrast, mice with selective inactivation of αENaC in the collecting duct (CD) maintain sodium and potassium balance, suggesting that the late distal convoluted tubule (DCT2) and/or the connecting tubule (CNT) participates in sodium homeostasis. To investigate the relative importance of ENaC-mediated sodium absorption in the CNT, we used Cre-lox technology to generate mice lacking αENaC in the aquaporin 2-expressing CNT and CD. Western blot analysis of microdissected cortical CD (CCD) and CNT revealed absence of αENaC in the CCD and weak αENaC expression in the CNT. These mice exhibited a significantly higher urinary sodium excretion, a lower urine osmolality, and an increased urine volume compared with control mice. Furthermore, serum sodium was lower and potassium levels were higher in the genetically modified mice. With dietary sodium restriction, these mice experienced significant weight loss, increased urinary sodium excretion, and hyperkalemia. Plasma aldosterone levels were significantly elevated under both standard and sodium-restricted diets. In summary, αENaC expression within the CNT/CD is crucial for sodium and potassium homeostasis and causes signs and symptoms of pseudohypoaldosteronism type 1 if missing.

Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing.

Dent's disease is an X-linked recessive disorder affecting the proximal tubules and is frequently associated with mutations in CLCN5, which encodes the electrogenic chloride-proton exchanger ClC-5. To better understand the functional consequences of CLCN5 mutations in this disease, we screened four newly identified missense mutations (G179D, S203L, G212A, L469P), one new nonsense mutation (R718X), and three known mutations (L200R, C219R, and C221R), in Xenopus laevis oocytes and HEK293 cells expressing either wild-type or mutant exchanger. A type-I mutant (G212A) trafficked normally to the cell surface and to early endosomes, underwent complex glycosylation at the cell surface like wild-type ClC-5, but exhibited significant reductions in outwardly rectifying ion currents. The type-II mutants (G179D, L200R, S203L, C219R, C221R, L469P, and R718X) were improperly N-glycosylated and were non-functional due to retention in the endoplasmic reticulum. Thus these mutations have distinct mechanisms by which they could impair ClC-5 function in Dent's disease.

A highly conserved motif at the COOH terminus dictates endoplasmic reticulum exit and cell surface expression of NKCC2.

Mutations in the apically located Na(+)-K(+)-2Cl(-) co-transporter, NKCC2, lead to type I Bartter syndrome, a life-threatening kidney disorder, yet the mechanisms underlying the regulation of mutated NKCC2 proteins in renal cells have not been investigated. Here, we identified a trihydrophobic motif in the distal COOH terminus of NKCC2 that was required for endoplasmic reticulum (ER) exit and surface expression of the co-transporter. Indeed, microscopic confocal imaging showed that a naturally occurring mutation depriving NKCC2 of its distal COOH-terminal region results in the absence of cell surface expression. Biotinylation assays revealed that lack of cell surface expression was associated with abolition of mature complex-glycosylated NKCC2. Pulse-chase analysis demonstrated that the absence of mature protein was not caused by reduced synthesis or increased rates of degradation of mutant co-transporters. Co-immunolocalization experiments revealed that these mutants co-localized with the ER marker protein-disulfide isomerase, demonstrating that they are retained in the ER. Cell treatment with proteasome or lysosome inhibitors failed to restore the loss of complex-glycosylated NKCC2, further eliminating the possibility that mutant co-transporters were processed by the Golgi apparatus. Serial truncation of the NKCC2 COOH terminus, followed by site-directed mutagenesis, identified hydrophobic residues (1081)LLV(1083) as an ER exit signal necessary for maturation of NKCC2. Mutation of (1081)LLV(1083) to AAA within the context of the full-length protein prevented NKCC2 ER exit independently of the expression system. This trihydrophobic motif is highly conserved in the COOH-terminal tails of all members of the cation-chloride co-transporter family, and thus may function as a common motif mediating their transport from the ER to the cell surface. Taken together, these data are consistent with a model whereby naturally occurring premature terminations that interfere with the LLV motif compromise co-transporter surface delivery through defective trafficking.

Proteinase-activated receptor 2 stimulates Na,K-ATPase and sodium reabsorption in native kidney epithelium.

Proteinase-activated receptors 2 (PAR2) are expressed in kidney, but their function is mostly unknown. Since PAR2 control ion transport in several epithelia, we searched for an effect on sodium transport in the cortical thick ascending limb of Henle's loop, a nephron segment that avidly reabsorbs NaCl, and for its signaling. Activation of PAR2, by either trypsin or a specific agonist peptide, increased the maximal activity of Na,K-ATPase, its apparent affinity for sodium, the sodium permeability of the paracellular pathway, and the lumen-positive transepithelial voltage, featuring increased NaCl reabsorption. PAR2 activation induced calcium signaling and phosphorylation of ERK1,2. PAR2-induced stimulation of Na,K-ATPase Vmax was fully prevented by inhibition of phospholipase C, of changes in intracellular concentration of calcium, of classical protein kinases C, and of ERK1,2 phosphorylation. PAR2-induced increase in paracellular sodium permeability was mediated by the same signaling cascade. In contrast, increase in the apparent affinity of Na,K-ATPase for sodium, although dependent on phospholipase C, was independent of calcium signaling, was insensitive to inhibitors of classical protein kinases C and of ERK1,2 phosphorylation, but was fully prevented by the nonspecific protein kinase inhibitor staurosporine, as was the increase in transepithelial voltage. In conclusion, PAR2 increases sodium reabsorption in rat thick ascending limb of Henle's loop along both the transcellular and the paracellular pathway. PAR2 effects are mediated in part by a phospholipase C/protein kinase C/ERK1,2 cascade, which increases Na,K-ATPase maximal activity and the paracellular sodium permeability, and by a different phospholipase C-dependent, staurosporine-sensitive cascade that controls the sodium affinity of Na,K-ATPase.

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.

Sodium retention and ascites formation in a cholestatic mice model: role of aldosterone and mineralocorticoid receptor?

UNLABELLED: Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone-sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenal-intact or corticosteroid-clamped mice. Corticosteroid-clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24-hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodium- and potassium-dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4-5 weeks, cirrhosis induced sodium retention in adrenal-intact mice and formation of ascites in 50% of mice. At that time, sodium- and potassium-dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum- and glucocorticoid-regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid-clamp prevented induction of sgk1 but not cirrhosis-induced sodium retention, formation of ascites and stimulation of sodium- and potassium-dependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. CONCLUSION: Bile duct ligation in mice induces cirrhosis which, within 4-5 weeks, leads to the induction of sodium- and potassium-dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium- and potassium-dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids.

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.

Hormonal and nonhormonal mechanisms of regulation of the NA,K-pump in collecting duct principal cells.

In the kidney, the collecting duct (CD) is the site of final Na+ reabsorption, according to Na+ balance requirements. In this segment of the renal tubule, principal cells may reabsorb up to 5% of the filtered sodium. The driving force for this process is provided by the basolateral Na,K-adenosine triphosphatase (ATPase) (sodium pump). Na,K-ATPase activity and expression in the CD are modulated physiologically by hormones (aldosterone, vasopressin, and insulin) and nonhormonal factors including intracellular [Na+] and extracellular osmolality. In this article, we review the short- and long-term hormonal regulation of Na,K-ATPase in CD principal cells, and we analyze the integrated network of implicated signaling pathways with an emphasis on the latest findings.

Cytokines and sodium induce protein kinase A-dependent cell-surface Na,K-ATPase recruitment via dissociation of NF-kappaB/IkappaB/protein kinase A catalytic subunit complex in collecting duct principal cells.

Collecting duct (CD) principal cells are exposed to large physiologic variations of apical Na+ influx as a result of variations of Na(+) intake and extrarenal losses. It was shown previously that increasing intracellular [Na+] induces recruitment of Na,K-ATPase to the cell surface in a protein kinase A (PKA)-dependent manner in both native and cultured renal CD principal cells. As described previously in response to cytokines in nonrenal cells, PKA activation in response to increased intracellular [Na+] was independent of cAMP and required proteasomal activity. With the use of cultured mpkCCD(cL4) cells as a model of CD principal cells, whether cytokines and increased intracellular [Na+] share a common signaling pathway leading to cell-surface Na,K-ATPase recruitment was investigated. Results showed that two potent inducers of NF-kappaB, LPS and TNF-alpha, enhance Na+ transport and induce cell-surface Na,K-ATPase recruitment in mpkCCD(cL4) cells via cAMP-independent PKA activation. In addition, increased intracellular [Na+] after selective plasma membrane permeabilization by a low concentration of the Na+ ionophore amphotericin B (1 microg/ml) induced dissociation of the PKA catalytic subunit from p65-NF-kappaB and IkappaBalpha. Moreover, inhibitors of NF-kappaB/IkappaB dissociation prevented both Na+-dependent stimulation of PKA activity and cell-surface Na,K-ATPase recruitment. Altogether, these results revealed the presence of a novel Na+-dependent intracellular signaling pathway leading to Na,K-ATPase cell-surface recruitment via dissociation of the PKA catalytic subunit from a macromolecular complex that contains NF-kappaB and IkappaBalpha in CD epithelial cells.

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.