David Mordasini

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.

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 and Potassium Balance Depends on αENaC Expression in Connecting Tubule

Mutations in α, β, 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.

Short-Term Aldosterone Action on Na,K-ATPase Surface Expression

Abstract: Aldosterone controls extracellular volume and blood pressure by regulating Na+ reabsorption across epithelial cells of the aldosterone‐sensitive distal nephron (ASDN). This effect is mediated by a coordinate action on the luminal channel ENaC (generally rate limiting) and the basolateral Na,K‐ATPase. Long‐term effects of aldosterone (starting within 3 to 6 hours and increasing over days) are mediated by the direct and indirect induction of stable elements of the Na+ transport machinery (e.g., Na,K‐ATPase α subunit), whereas short‐term effects appear to be mediated by the upregulation of short‐lived elements of the machinery (e.g., ENaC α subunit) and of regulatory proteins, such as the serum‐ and glucocorticoid‐regulated kinase SGK1. We have recently shown that in cortical collecting duct (CCD) from adrenalectomized (ADX) rats, the increase in Na,K‐ATPase activity (approximately threefold in 3 h), induced by a single aldosterone injection, can be fully accounted for by the increase in Na,K‐ATPase cell‐surface expression. Using the model cell line mpkCCDcl4, we showed that the parallel increase in Na,K‐ATPase function [assessed by Na+ pump current (Ip) measurements] and cell‐surface expression depends on transcription and translation, and that it is not secondary to a change in apical Na+ influx. As a first approach to address the question whether the aldosterone‐induced regulatory protein SGK1 might play a role in mediating Na,K‐ATPase translocation, we have used the Xenopus laevis expression system. SGK1 coexpression indeed increased both the Na+ pump current and the surface expression of pumps containing the rat α1 subunits. In summary, aldosterone controls Na+ reabsorption in the short term not only by regulating the apical cell‐surface expression of ENaC but also by coordinately acting on the basolateral cell‐surface expression of the Na,K‐ATPase. Results obtained in the Xenopus oocyte expression system suggest the possibility that this effect could be mediated in part by the aldosterone‐induced kinase SGK1.

Mechanism of Control of Na,K‐ATPase in Principal Cells of the Mammalian Collecting Duct

Abstract: The collecting duct is the site of final Na reabsorption according to Na balance requirements. Using isolated rat cortical collecting ducts (CCD) and mpkCCDcl4 cells, a mouse cortical collecting duct cell line, we have studied the physiological control of Na,K‐ATPase, the key enzyme that energizes Na reabsorption. Aldosterone, a major regulator of Na transport by the collecting duct, stimulates Na,K‐ATPase activity through both recruitment of intracellular pumps and increased total amounts of Na pump subunits. This effect is observed after a lag time of 1 hour and is independent of Na entry through ENaC, but requires de novo transcription and translation. Vasopressin and cAMP, its second messenger, stimulate Na,K‐ATPase activity within minutes through translocation of Na pumps from a brefeldin A‐sensitive intracellular pool to the plasma membrane. Dysregulation of collecting duct Na,K‐ATPase activity is at least in part responsible of the Na retention observed in nephritic syndrome. In this setting, Na,K‐ATPase activity and subunit synthesis are specifically increased in CCD. In conclusion, aldosterone, vasopressin, and intracellular Na control the cell surface expression of Na,K‐ATPase and translocation from intracellular stores is a major mechanism of regulation of Na,K‐ATPase activity in collecting duct principal cells.

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 Henles 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.

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.

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 1081LLV1083 as an ER exit signal necessary for maturation of NKCC2. Mutation of 1081LLV1083 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.

Cytokines and Sodium Induce Protein Kinase A–Dependent Cell-Surface Na,K-ATPase Recruitment via Dissociation of NF-κB/IκB/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.

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.