Search

Ischemic acute kidney injury (AKI), a complication that frequently occurs in hospital settings, is often associated with hemodynamic compromise, sepsis, cardiac surgery, or exposure to nephrotoxins. Here, using a murine renal ischemia/ reperfusion injury (IRI) model, we show that intercalated cells (ICs) rapidly adopted a proinflammatory phenotype after IRI. Wwe demonstrate that during the early phase of AKI either blockade of the proinflammatory P2Y14 receptor located on the apical membrane of ICs or ablation of the gene encoding the P2Y14 receptor in ICs (a) inhibited IRI-induced increase of chemokine expression in ICs, (b) reduced neutrophil and monocyte renal infiltration, (c) reduced the extent of kidney dysfunction, and (d) attenuated proximal tubule damage. These observations indicate that the P2Y14 receptor participates in the very first inflammatory steps associated with ischemic AKI. In addition, we show that the concentration of the P2Y14 receptor ligand UDP-glucose (UDP-Glc) was higher in urine samples from intensive care unit patients who developed AKI compared with patients without AKI. In particular, we observed a strong correlation between UDP-Glc concentration and the development of AKI in cardiac surgery patients. Our study identifies the UDP-Glc/P2Y14 receptor axis as a potential target for the prevention and/or attenuation of ischemic AKI., Introduction Acute kidney injury (AKI) is a frequent medical complication seen in hospitalized patients. It is associated with an increased length of hospital stay, the development of chronic kidney disease, [...]

Pendrin is a Cl-/HCO3- exchanger expressed in the apical regions of renal intercalated cells. Following pendrin gene ablation, blood pressure falls, in part, from reduced renal NaCl absorption. We asked if pendrin is expressed in vascular tissue and if the lower blood pressure observed in pendrin null mice is accompanied by reduced vascular reactivity. Thus, the contractile responses to KCl and phenylephrine (PE) were examined in isometrically mounted thoracic aortas from wild-type and pendrin null mice. Although pendrin expression was not detected in the aorta, pendrin gene ablation changed contractile protein abundance and increased the maximal contractile response to PE when normalized to cross sectional area (CSA). However, the contractile sensitivity to this agent was unchanged. The increase in contractile force/cross sectional area observed in pendrin null mice was due to reduced cross sectional area of the aorta and not from increased contractile force per vessel. The pendrin-dependent increase in maximal contractile response was endothelium- and nitric oxide-independent and did not occur from changes in Ca2+ sensitivity or chronic changes in catecholamine production. However, application of 100 nM angiotensin II increased force/CSA more in aortas from pendrin null than from wild type mice. Moreover, angiotensin type 1 receptor inhibitor (candesartan) treatment in vivo eliminated the pendrin-dependent changes contractile protein abundance and changes in the contractile force/cross sectional area in response to PE. In conclusion, pendrin gene ablation increases aorta contractile force per cross sectional area in response to angiotensin II and PE due to stimulation of angiotensin type 1 receptor-dependent signaling. The angiotensin type 1 receptor-dependent increase in vascular reactivity may mitigate the fall in blood pressure observed with pendrin gene ablation.

Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl- absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice. NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure. [ABSTRACT FROM AUTHOR]

The urinary potassium (K+) excretion machinery is upregulated with increasing dietary K+, but the role of accompanying dietary anions remains inadequately characterized. Poorly absorbable anions, including HCO−3 , are thought to increase K+ secretion through a transepithelial voltage effect. Here, we tested if they also influence the K+ secretion machinery. Wild-type mice, aldosterone synthase (AS) knockout (KO) mice, or pendrin KO mice were randomized to control, high-KCl, or high-KHCO3 diets. The K+ secretory capacity was assessed in balance experiments. Protein abundance, modification, and localization of K+-secretory transporters were evaluated by Western blot analysis and confocal microscopy. Feeding the high-KHCO3 diet increased urinary K+ excretion and the transtubular K+ gradient significantly more than the high-KCl diet, coincident with more pronounced upregulation of epithelial Na+ channels (ENaC) and renal outer medullary K+ (ROMK) channels and apical localization in the distal nephron. Experiments in AS KO mice revealed that the enhanced effects of HCO−3 were aldosterone independent. The high-KHCO3 diet also uniquely increased the large-conductance Ca2+-activated K+ (BK) channel β4-subunit, stabilizing BKα on the apical membrane, the Cl−/HCO−3 exchanger, pendrin, and the apical KCl cotransporter (KCC3a), all of which are expressed specifically in pendrin-positive intercalated cells. Experiments in pendrin KO mice revealed that pendrin was required to increase K+ excretion with the high-KHCO3 diet. In summary, HCO−3 stimulates K+ excretion beyond a poorly absorbable anion effect, upregulating ENaC and ROMK in principal cells and BK, pendrin, and KCC3a in pendrin-positive intercalated cells. The adaptive mechanism prevents hyperkalemia and alkalosis with the consumption of alkaline ash-rich diets but may drive K+ wasting and hypokalemia in alkalosis. NEW & NOTEWORTHY Dietary anions profoundly impact K+ homeostasis. Here, we found that a K+-rich diet, containing HCO−3 as the counteranion, enhances the electrogenic K+ excretory machinery, epithelial Na+ channels, and renal outer medullary K+ channels, much more than a high-KCl diet. It also uniquely induces KCC3a and pendrin, in B-intercalated cells, providing an electroneutral KHCO3 secretion pathway. These findings reveal new K+ balance mechanisms that drive adaption to alkaline and K+-rich foods, which should guide new treatment strategies for K+ disorders. [ABSTRACT FROM AUTHOR]

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type [H.sup.+]-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/ NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting [alpha]-ICs with PP- ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an [alpha]- ketoglutarate ([alpha]-KG) paracrine signaling system. Coordinate regulation of a multigene [alpha]-KG synthesis and transport pathway resulted in [alpha]-KG secretion into pro-urine, as the [alpha]-KG-activated GPCR (Oxgrl) increased on the PP-IC apical surface, allowing paracrine delivery of [alpha]-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy., Introduction The sodium chloride cotransporter (NCC) of the SLC12A3 gene (1, 2) is the principal determinant of NaCl reabsorption in the distal convoluted tubule (DCT) (3) and the key target [...]

The Cl-/HCO3- exchanger pendrin in the kidney maintains acid-base balance and intravascular volume. Pendrin is upregulated in models associated with high circulating aldosterone concentration, such as dietary NaCl restriction or an aldosterone infusion. However, it has not been established if pendrin is similarly regulated by aldosterone with a high-K+ diet because the effects of accompanying anions have not been considered. Here, we explored how pendrin is modulated by different dietary potassium salts. Wild-type (WT) and aldosterone synthase (AS) knockout (KO) mice were randomized to control, high-KHCO3, or high-KCl diets. Dietary KCl and KHCO3 loading increased aldosterone in WT mice to the same extent but had opposite effects on pendrin abundance. KHCO3 loading increased pendrin protein and transcript abundance. Conversely, high-KCl diet feeding caused pendrin to decrease within 8 h of switching from the high-KHCO3 diet, coincident with an increase in plasma Cl- and a decrease in HCO3-. In contrast, switching the high-KCl diet to the high-KHCO3 diet caused pendrin to increase in WT mice. Experiments in AS KO mice revealed that aldosterone is necessary to optimally upregulate pendrin protein in response to the high-KHCO3 diet but not to increase pendrin mRNA. We conclude that pendrin is differentially regulated by different dietary potassium salts and that its regulation is prioritized by the dietary anion, providing a mechanism to prevent metabolic alkalosis with high-K+ base diets and safeguard against hyperchloremic acidosis with consumption of high-KCl diets. NEW & NOTEWORTHY Regulation of the Cl-/HCO3- exchanger pendrin has been suggested to explain the aldosterone paradox. A high-K+ diet has been proposed to downregulate a pendrin-mediated K+-sparing NaCl reabsorption pathway to maximize urinary K+ excretion. Here, we challenged the hypothesis, revealing that the accompanying anion, not K+, drives pendrin expression. Pendrin is downregulated with a high-KCl diet, preventing acidosis, and upregulated with an alkaline-rich high-K+ diet, preventing metabolic alkalosis. Pendrin regulation is prioritized for acid-base balance. [ABSTRACT FROM AUTHOR]

The renal response to acid-base disturbances involves phenotypic and remodeling changes in the collecting duct. This study examines whether the proximal tubule controls these responses. We examined mice with genetic deletion of proteins present only in the proximal tubule, either the A variant or both A and B variants of isoform 1 of the electrogenic Naþ-bicarbonate cotransporter (NBCe1). Both knockout (KO) mice have spontaneous metabolic acidosis. We then determined the collecting duct phenotypic responses to this acidosis and the remodeling responses to exogenous acid loading. Despite the spontaneous acidosis in NBCe1-A KO mice, type A intercalated cells in the inner stripe of the outer medullary collecting duct (OMCDis) exhibited decreased height and reduced expression of Hþ-ATPase, anion exchanger 1, Rhesus B glycoprotein, and Rhesus C glycoprotein. Combined kidney-specific NBCe1-A/B deletion induced similar changes. Ultrastructural imaging showed decreased apical plasma membrane and increased vesicular Hþ-ATPase in OMCDis type A intercalated cell in NBCe1-A KO mice. Next, we examined the collecting duct remodeling response to acidosis. In wild-type mice, acid loading increased the proportion of type A intercalated cells in the connecting tubule (CNT) and OMCDis, and it decreased the proportion of non-A, non-B intercalated cells in the connecting tubule, and type B intercalated cells in the cortical collecting duct (CCD). These changes were absent in NBCe1-A KO mice. We conclude that the collecting duct phenotypic and remodeling responses depend on proximal tubule-dependent signaling mechanisms blocked by constitutive deletion of proximal tubule NBCe1 proteins. [ABSTRACT FROM AUTHOR]

Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloride-sensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of [Na.sup.+] and Cl in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the [Na.sup.+]-driven [Cl.sup.-]/[HCO.sub.3.sup.-] exchanger (NDCBE/SLC4A8) and the [Na.sup.+]-independent [Cl.sup.-]/[HCO.sub.3.sup.-] exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial [Na.sup.+] reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice., Introduction Sodium chloride is the main extracellular osmotic constituent and thereby determines extracellular volume and blood pressure. To maintain a constant extracellular volume, the kidney has to match sodium excretion [...]

Pendrin is expressed in the apical regions of type B and non-A, non-B intercalated cells, where it mediates [Cl.sup.-] absorption and HC[O.sup.-.sub.3] secretion through apical [Cl.sup.-]/HC[O.sup.-.sub.3] exchange. Since pendrin is a robust [I.sup.-] transporter, we asked whether pendrin is upregulated with dietary [I.sup.-] restriction and whether it modulates [I.sup.-] balance. Thus [I.sup.-] balance was determined in pendrin null and in wild-type mice. Pendrin abundance was evaluated with immunoblots, immunohistochemistry, and immunogold cytochemistry with morphometric analysis. While pendrin abundance was unchanged when dietary [I.sup.-] intake was varied over the physiological range, [I.sup.-] balance differed in pendrin null and in wild-type mice. Serum [I.sup.-] was lower, while [I.sup.-] excretion was higher in pendrin null relative to wild-type mice, consistent with a role of pendrin in renal [I.sup.-] absorption. Increased [H.sub.2]O intake enhanced differences between wild-type and pendrin null mice in [I.sup.-] balance, suggesting that [H.sub.2]O intake modulates pendrin abundance. Raising water intake from ~4 to ~11 ml/day increased the ratio of B cell apical plasma membrane to cytoplasm pendrin label by 75%, although circulating renin, aldosterone, and serum osmolality were unchanged. Further studies asked whether [H.sub.2]O intake modulates pendrin through the action of AVP. We observed that [H.sub.2]O intake modulated pendrin abundance even when circulating vasopressin levels were clamped. We conclude that [H.sub.2]O intake modulates pendrin abundance, although not likely through a direct, type 2 vasopressin receptor-dependent mechanism. As water intake rises, pendrin becomes increasingly critical in the maintenance of [Cl.sup.-] and [I.sup.-] balance. chloride; apical anion exchange; vasopressin; intercalated cells; vasopressin escape doi: 10.1152/ajprenal.90581.2008.

NKCC1 is a widely expressed isoform of the Na-2Cl-K cotransporter that mediates several direct and indirect vascular effects and regulates expression and release of renin. In this study, we used NKCC1-deficient ([NKCC1.sup.-/-]) and wild-type (WT) mice to assess day/night differences of blood pressure (BP), locomotor activity, and renin release and to study the effects of high (8%) or low (0.03%) dietary NaCl intake on BP, activity, and the renin/aldosterone system. On a standard diet, 24-h mean arterial blood pressure (MAP) and heart rate determined by radiotelemetry, and their day/night differences, were not different in [NKCC1.sup.-/-] and WT mice. Spontaneous and wheel-running activities in the active night phase were lower in [NKCC1.sup.-/-] than WT mice. In [NKCC1.sup.-/-] mice on a high-NaCl diet, MAP increased by 10 mmHg in the night without changes in heart rate. In contrast, there was no salt-dependent blood pressure change in WT mice. MAP reductions by hydralazine (1 mg/kg) or isoproterenol (10 [micro]g/mouse) were significantly greater in [NKCC1.sup.-/-] than WT mice. Plasma renin (PRC; ng ANG I x [ml.sup.-1] x [h.sup.i]) and aldosterone (aldo; pg/ml) concentrations were higher in [NKCC1.sup.-/-] than WT mice (PRC: 3,745 [+ or -] 377 vs. 1,245 [+ or -] 364; aldo: 763 [+ or -] 136 vs. 327 [+ or -] 98). Hyperreninism and hyperaldosteronism were found in [NKCC1.sup.-/-] mice during both day and night. High Na suppressed PRC and aldosterone in both [NKCC1.sup.-/-] and WT mice, whereas a low-Na diet increased PRC and aldosterone in WT but not [NKCC1.sup.-/-] mice. We conclude that 24-h MAP and MAP circadian rhythms do not differ between [NKCCI.sup.-/-] and WT mice on a standard diet, probably reflecting a balance between anti- and prohypertensive factors, but that blood pressure of [NKCC1.sup.-/-] mice is more sensitive to increases and decreases of Na intake. radiotelemetry; running wheels; renin; aldosterone; vasodilators; sodium-2 chloride-potassium cotransporter-deficient mice

Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct. AM J Physiol Renal Physiol 292: F192-F201, 2008. Fist published April 16, 2008: doi:10.1152/ajprenal.00011-2008.--Collecting duct cells swell when exposed to argininge vasopressin (AVP) in the presence of a transepithelial osmolality gradient. We investigated the mechanisms of AVP-induced cell swelling in isolated , perfused rate inner medullary collecting ducts (IMCDs) using quantitative video microscopy and fluorescence-based measurements of transepithelial water transport. We tested the roles of transepithelial water flow, basolateral solute entry, and the cytoskeleton (actomysosin). Whe a transepithelial osmolality gradient was imposed by addition of NaCl to the bath, AVP significantly increased both water flux and cell height. When the osmolality gradient was imposed by addition of mannitol, AVP increased water flux but not cell height, suggesting that AVP-induced cell swelling requires a NaCl gradien and is not merely dependent on the associated water flux. Bumetanide (Na-K-2Cl contransporter inhibitor) added to the bath markedly diminished the AVP-induced cell height increase. AVP-induced cell swelling was absent in IMCDs form NKCC1-knockout mice. In rat IMCDs, replacement of Na, K, or Cl in the peritubular bath cased significant cell shrinkage, consistent with a basolateral solute transport pathway dependent on all three ions. Immunocytochemistry using an antibody to NKCC1 confirmed basolateral expression in IMCD cells. The conventional nonmuscle myosin II inhibitor blebbistatin also diminished the AVP-induced cell height increase and cell shape change, consistent with a role for the actin cytoskeleton and myosin II. We conclude that the AVP-induced cell height increase is dependent on basolateral solute uptake via NKCC1 and changes in actin organization via myosin II, but is not dependent specifically on increased apical water entry. cell volume; blebbistatin; cytoskeleton; kidney; inner medulla

Pendrin (encoded by Pds, Slc26a4) is a [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger expressed in the apical regions of type B and non-A, non-B intercalated cells of kidney and mediates renal [Cl.sup.-] absorption, particularly when upregulated. Aldosterone increases blood pressure by increasing absorption of both [Na.sup.+] and [Cl.sup.-] through increased protein abundance and function of [Na.sup.+] transporters, such as the epithelial [Na.sup.+] channel (ENaC) and the [Na.sup.+]-[Cl.sup.-] cotransporter (NCC), as well as [Cl.sup.-] transporters, such as pendrin. Because aldosterone analogs do not increase blood pressure in [Slc26a4.sup.-/-] mice, we asked whether [Na.sup.+] excretion and [Na.sup.+] transporter protein abundance are altered in kidneys from these mutant mice. Thus wild-type and Slc26a4-null mice were given a NaCl-replete, a NaCl-restricted, or NaCl-replete diet and aldosterone or aldosterone analogs. Abundance of the major renal [Na.sup.+] transporters was examined with immunoblots and immunohistochemistry. Slc26a4-null mice showed an impaired ability to conserve [Na.sup.+] during dietary NaCl restriction. Under treatment conditions in which circulating aldosterone is increased, [alpha]-, [beta]-, and 85-kDa [gamma]-ENaC subunit protein abundances were reduced 15-35%, whereas abundance of the 70-kDa fragment of [gamma]-ENaC was reduced ~70% in Slc26a4-null relative to wild-type mice. Moreover, ENaC-dependent changes in transepithelial voltage were much lower in cortical collecting ducts from Slc26a4-null than from wild-type mice. Thus, in kidney, ENaC protein abundance and function are modulated by pendrin or through a pendrin-dependent downstream event. The reduced ENaC protein abundance and function observed in Slc26a4-null mice contribute to their lower blood pressure and reduced ability to conserve [Na.sup.+] during NaCl restriction. intercalated cell; Slc26a4; apical [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger; epithelial sodium channel; aldosterone

The low [Ca.sup.2+] concentration ([[Ca.sup.2+]]) of mammalian endolymph in the inner ear is required for normal hearing and balance. We reported (Yamauchi et al., Biochem Biophys Res Commun 331: 1353-1357, 2005) that the epithelial [Ca.sup.2+] channels TRPV5 and TRPV6 (transient receptor potential types 5 and 6) are expressed in the vestibular system and that TRPV5 expression is stimulated by 1,25-dihydroxyvitamin [D.sub.3], as also reported in kidney. TRPV5/6 channels are known to be inhibited by extracellular acidic pH. Endolymphatic pH, [[Ca.sup.2+]], and transepithelial potential of the utricle were measured in [Cl.sup.-]/H[C.sup.-.sub.3] exchanger pendrin (SLC26A4) knockout mice in vivo. Slc26a[4.sup.-/-] mice exhibit reduced pH and utricular endolymphatic potential and increased [[Ca.sup.2+]]. Monolayers of primary cultures of rat semicircular canal duct cells were grown on permeable supports, and cellular uptake of [sup.45][Ca.sup.2+] was measured individually from the apical and basolateral sides. Net uptake of [sup.45][Ca.sup.2+] was greater after incubation with 1,25-dihydroxyvitamin [D.sub.3]. Net [sup.45][Ca.sup.2+] absorption was dramatically inhibited by low apical pH and was stimulated by apical alkaline pH. Gadolinium, lanthanum, and ruthenium red reduced apical uptake. These observations support the notion that one aspect of vestibular dysfunction in Pendred syndrome is a pathological elevation of endolymphatic [[Ca.sup.2+]] due to luminal acidification and consequent inhibition of TRPV5/6-mediated [Ca.sup.2+] absorption. epithelial calcium channel; vitamin D; SLC26a4; HC[O.sup.-.sub.3] secretion doi:10.1152/ajprenal.00432.2006

Studies in rat aorta have shown that the Na-K-2Cl cotransporter NKCC1 is activated by vasoconstrictors and inhibited by nitrovasodilators, contributes to smooth muscle tone in vitro, and is upregulated in hypertension. To determine the role of NKCC1 in systemic vascular resistance and hypertension, blood pressure was measured in rats before and after inhibition of NKCC1 with bumetanide. Intravenous infusion of bumetanide sufficient to yield a free plasma concentration above the I[C.sub.50] for NKCC1 produced an immediate drop in blood pressure of 5.2% (P < 0.001). The reduction was not prevented when the renal arteries were clamped, indicating that it was not due to a renal effect of bumetanide. Bumetanide did not alter blood pressure in NKCC1-null mice, demonstrating that it was acting specifically through NKCC1. In third-order mesenteric arteries, bumetanide-inhibitable efflux of [sup.86]Rb was acutely stimulated 133% by phenylephrine, and bumetanide reduced the contractile response to phenylephrine, indicating that NKCC1 influences tone in resistance vessels. The hypotensive effect of bumetanide was proportionately greater in rats made hypertensive by a 7-day infusion of norepinephrine (12.7%, P < 0.001 vs. normotensive rats) but much less so when hypertension was produced by a fixed aortic coarctation (8.0%), again consistent with an effect of bumetanide on resistance vessels rather than other determinants of blood pressure. We conclude that NKCC1 influences blood pressure through effects on smooth muscle tone in resistance vessels and that this effect is augmented in hypertension. resistance arteries; hypertension; bumetanide doi:10.1152/ajpheart.01402.2006

Pendrin (Slc26a4) localizes to type B and non-A, non-B intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (CCD), where it mediates apical [Cl.sup.-]/HC[O.sup.-.sub.3] exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, [J.sub.Cl] in mouse CCD through a pendrin-dependent mechanism. [J.sub.Cl] and transepithelial voltage, [V.sub.T], were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice ingesting a NaCl-replete diet or a NaCl-replete diet and furosemide. In CCDs from wild-type mice ingesting a NaClreplete diet, [V.sub.T] and [J.sub.Cl] were not different from zero either in the presence or absence of angiotensin II ([10.sup.-8] M) in the bath. Thus further experiments employed mice given the high-NaCl diet and furosemide to upregulate renal pendrin expression. CCDs from furosemide-treated wild-type mice had a lumen-negative [V.sub.T] and absorbed [Cl.sup.-]. With angiotensin II in the bath, [Cl.sup.-] absorption doubled although [V.sub.T] did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, [Cl.sup.-] secretion and a [V.sub.T] of ~0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished [V.sub.T] although [J.sub.Cl] fell only ~50%. Thus substantial [Cl.sup.-] absorption is observed in the absence of an electromotive force. Attenuating apical anion exchange with the peritubular application of the [H.sup.+]-ATPase inhibitor bafilomycin abolished benzamil-insensitive [Cl.sup.-] absorption. In conclusion, angiotensin II increases transcellular [Cl.sup.-] absorption in the CCD through a pendrin- and [H.sup.+]-ATPase-dependent process. transepithelial voltage; bafilomycin; [H.sup.+]-ATPase; knockout mice; Slc26a4; intercalated cell

Mechanisms of [K.sup.+] secretion and absorption along the collecting duct are not understood fully. Because KCNQ1 participates in [K.sup.+] secretion within the inner ear and stomach, distribution of KCNQ1 in mouse kidney was studied using Northern and Western analyses, RT-PCR of isolated tubules, and immunohistochemistry. Northern blots demonstrated KCNQ1 transcripts in whole kidney. RT-PCR showed KCNQ1 mRNA in isolated distal convoluted tubule (DCT), connecting segment (CNT), collecting ducts (CD), and glomeruli. Immunoblots of kidney and stomach revealed a ~75-kDa protein, the expected mobility for KCNQ1. KCNQ1 was detected by immunohistochemistry throughout the distal nephron and CD. Thick ascending limbs exhibited weak basolateral immunolabel. In DCT and CNT cells, immunolabel was intense and basolateral, although KCNQ1 label was stronger in late than in early DCT. Initial collecting tubule and cortical CD KCNQ1 immunolabel was predominantly diffuse, but many cells exhibited discrete apical label. Double-labeling experiments demonstrated that principal cells, type B intercalated cells, and a few type A intercalated cells exhibited distinct apical KCNQ1 immunolabel. In inner medullary CD, principal cells exhibited distinct basolateral KCNQ1 immunolabel, whereas intercalated cells showed diffuse cytoplasmic staining. Thus KCNQ1 protein is widely distributed in mouse distal nephron and CD, with significant axial and cellular heterogeneity in location and intensity. These findings suggest that KCNQ 1 has cell-specific roles in renal ion transport and may participate in [K.sup.+] secretion and/or absorption along the thick ascending limb, DCT, connecting tubule, and CD. KvLQT1; Kv7.1 ; slowly activating potassium channel

Pendrin, encoded by Slc26a4, is a [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger expressed in the apical region of type B and non-A, non-B intercalated cells, which regulates renal NaCl excretion. Dietary C1 restriction upregulates total pendrin protein expression. Whether the subcellular expression of pendrin and whether the apparent vascular volume contraction observed in Slc26a4 null mice are [Cl.sup.-] dependent, but [Na.sup.+] independent, is unknown. Thus the subcellular distribution of pendrin and its role in acid-base and fluid balance were explored using immunogold cytochemistry and balance studies of mice ingesting a NaCl-replete or a [Na.sup.+]-replete, [Cl.sup.-]-restricted diet, achieved through substitution of NaCl with NaHCO3. Boundary length and apical plasma membrane pendrin label density each increased by ~60-70% in type B intercalated cells, but not in non-A, non-B cells, whereas cytoplasmic pendrin immunolabel increased ~60% in non-A, non-B intercalated cells, but not in type B cells. Following either NaC1 restriction or [Cl.sup.-] restriction alone, S1c26a4 null mice excreted more [Cl.sup.-] and had a higher arterial pH than pair-fed wild-type mice. In conclusion, 1) following dietary [Cl.sup.-] restriction, apical plasma membrane pendrin immunolabel increases in type B intercalated cells, but not in non-A, non-B intercalated cells; and 2) pendrin participates in the regulation of renal [C1.sup.-] excretion and arterial pH during dietary [C1.sup.-] restriction. acid-base and fluid balance; mammalian collecting duct

NKCC1 null mice are hypotensive, in part, from the absence of NKCC1-mediated vasoconstriction. Whether these mice have renal defects in NaCl and water handling which r contribute to the hypotension is unexplored. Therefore, we asked 1) whether NKCC1 (-/-) mice have a defect in the regulation of NaCl and water balance, which might contribute to the observed hypotension and 2) whether the hypotension observed in these mice is accompanied by endocrine abnormalities and/or downregulation of renal [Na.sup.+] transporter expression. Thus we performed balance studies, semi-quantitative immunoblotting, and immunohistochemistry of kidney tissue from NKCC1 (+/+) and NKCC1 (-/-) mice which consumed either a high (2.8% NACl)- or a low-NaCl (0.01% NaCl) diet for 7 days. Blood pressure was lower in NKCC1 (-/-) than NKCC1 (+/+) mice following either high or low dietary NaCl intake. Relative to wild-type mice, NKCC1 null mice had a lower plasma ANP concentration, a higher plasma renin and a higher serum [K.sup.+] concentration with inappropriately low urinary [K.sup.+] excretion, although serum aldosterone was either the same or only slightly increased in the mutant mice. Expression of NHE3, the a-subunit of the Na-K-ATPase, NCC, and NKCC2 were higher in NKCC1 null than in wild-type mice, although differences were generally greater during NaCl restriction. NKCC1 null mice had a reduced capacity to excrete free water than wild-type mice, which resulted in hypochloremia following the NaCl-deficient diet. Hypochloremia did not occur from increased aquaporin-1 (AQP1) or 2 protein expression or from redistribution of AQP2 to the apical regions of principal cells. Instead, NKCC1 null mice had a blunted increase in urinary osmolality following vasopressin administration, which should increase free water excretion and attenuate the hypochloremia. In conclusion, aldosterone release is inappropriately low in NKCC1 null mice. Moreover, the action of aldosterone and vasopressin is altered within kidneys of NKCC1 null mice, which likely contributes to their hypotension. Increased [Na.sup.+] transporter expression, increased plasma renin, and reduced plasma ANP, as observed in NKCC1 null mice, should increase vascular volume and blood pressure, thus minimizing hypotension. [Na.sup.+] transporters; chloride; water; renin; aldosterone

Slc26a4 (Pds) encodes pendrin, a C1 /HCO3 exchanger expressed in the apical region of type B and non-A, non-B cells, which mediates secretion of OH equivalents. Thus genetic disruption of S1c26a4 leads to systemic alkalosis in some treatment models. However, humans and mice with genetic disruption of Slc26a4 have normal acid-base balance under basal conditions. Thus we asked: 1) Is net acid excretion altered in S1c26a4 (-/-) mice under basal conditions? 2) In the absence of pendrin-mediated OH secretion, are increases in intracellular and systemic pH minimized through changes in intercalated cell subtype abundance or intercalated cell [H.sup.+]/O[H.sup.-] transporter expression? To answer these questions, net acid excretion and [H.sup.+]/O[H.sup.-] transporter expression were examined in Slc26a4 (-/-) and Slc26a4 (+/+) mice using balance studies, immunolocalization, and immunoblotting. Excretion of ammonium, titratable acid, and citrate were the same in Slc26a4 null and wild-type mice. However, urinary pH and P[CO.sub.2] were much lower in Sic26a4 null relative to wild-type mice due to reduced urinary buffering of secreted [H.sup.+] by HC[O.sup.-.sub.3]. Abundance of non-A, but not type A intercalated cells, was reduced within the cortical collecting ducts of S1c26a4 null mice. Moreover, kidneys from Slc26a4 null mice had reduced [H.sup.+]-ATPase, NBC3 and RhBG total protein expression, particularly within type B and non-A, non-B intercalated cells, although RhCG protein expression was unchanged. Reduced intercalated cell [H.sup.+]/O[H.sup.-] transporter expression is observed in S1c26a4 null mice, which likely attenuates the rise in intracellular and systemic pH expected with genetic disruption of Slc26a4. NBC3; AEl; acid-base balance

In normal rats, vasopressin and hyperosmolality enhance urea permeability ([P.sub.urea]) in the terminal, but not in the initial inner medullary collecting duct (IMCD), a process thought to occur through the UT-A1 urea transporter. In the terminal IMCD, UT-A1 is detected as 97- and 117-kDa glycoproteins. However, in the initial IMCD, only the 97-kDa form is detected. During streptozotocin-induced diabetes mellitus, UT-A1 protein abundance is increased, and the 117-kDa UT-A 1 glycoprotein appears in the initial IMCD. We hypothesize that the 117-kDa glycoprotein mediates the vasopressin- and osmolality-induced changes in [P.sub.urea]. Thus, in the present study, we measured [P.sub.urea] in in vitro perfused initial IMCDs from diabetic rats by imposing a 5 mM bath-to-lumen urea gradient without any osmotic gradient. Basal [P.sub.urea] was similar in control vs. diabetic rats (3 [+ or -] 1 vs. 5 [+ or -] 1 x [10.sup.-5] cm/s, n = 4, P = not significant). Vasopressin (10 [micro]M) significantly increased [P.sub.urea] to 16 [+ or -] 5 x [10.sup.-5] cm/s (n = 4, P < 0.05) in diabetic but not in control rats. Forskolin (10 [micro]M, adenylyl cyclase activator) also significantly increased [P.sub.urea] in diabetic rats. In contrast, increasing osmolality to 690 mosmol/kg[H.sub.2]O did not change [P.sub.urea] in diabetic rats. We conclude that initial IMCDs from diabetic rats have vasopressin- and forskolin-, but not hyperosmolality-stimulated [P.sub.urea]. The appearance of vasopressin-stimulated [P.sub.urea] in initial IMCDs correlates with an increase in UT-A1 protein abundance and the appearance of the 117-kDa UT-A1 glycoprotein in this region during diabetes. This suggests that the 117-kDa UT-A1 glycoprotein is necessary for vasopressin-stimulated urea transport. diabetes mellitus; inner medullary collecting duct; hyperosmolality

AE2 isoforms in rat kidney: immunohistochemical localization and regulation in response to chronic N[H.sub.4]Cl loading. Am J Physiol Renal Physiol 286:F1163--F1170, 2004. First published January 28, 2004; 10.1152/ajprenal.00409.2003.--Three splice variants of anion exchanger (AE)2 (AE2a, b, and c) have been described in the rat, but their relative distribution in rat kidney is not known. The purpose of this study was to describe the segmental and cellular distribution of the AE2 isoforms in the rat kidney and to evaluate whether the expression levels of these AE2 isoforms are regulated independently in response to chronic N[H.sub.4]Cl loading. Two polyclonal antibodies were generated, respectively, recognizing a N[H.sub.2]-terminal peptide unique to AE2a and an amino acid sequence common to AE2a and AE2b. Antibody specificities were tested using cells transfected separately with the AE2a, AE2b, and AE2c isoforms, Immunohistochemistry on sections of paraffin-embedded rat kidneys showed a distribution of AE2a/AE2b labeling in the kidney similar to the distribution of AE2 in the rat kidney reported previously. AE2 is highly expressed in the medullary thick ascending limb, cortical thick ascending limb (cTAL), and macula densa. The pattern of AE2a-specific labeling differed from the pattern of AE2a/AE2b labeling in that relatively more of the total immunolabel was observed in the terminal inner medullary collecting duct. N[H.sub.4]Cl loading (0.033 mrnol N[H.sub.4]Cl/g body wt for 7 days) did not change the labeling of AE2 isoforms in the medulla, whereas the labeling in the cortex was intensified and included more distal parts of the cTAL. Immunoblotting confirmed upregulation of AE2a/b expression in the cortex. These results indicate that AE2a and AE2b are differentially expressed and regulated in the rat kidney. The regulation following N[H.sub.4]Cl loading of AE2b in the cTAL suggests a role for AE2 in transepithelial bicarbonate reabsorption in this segment. immunohistochemistry; collecting duct; nephron segments

Prolonged lithium treatment of humans and rodents often results in hyperchloremic metabolic acidosis. This is thought to be caused by diminished net [H.sup.+] secretion and/or excessive back-diffusion of acid equivalents. To explore whether lithium treatment is associated with changes in the expression of key renal acid-base transporters, semiquantitative immunoblotting and immunocytochemistry were performed using kidneys from lithium-treated (n = 6) and control (n = 6) rats. Rats treated with lithium for 28 days showed decreased urine pH, whereas no significant differences in blood pH and plasma HC[O.sup.-.sub.3] levels were observed. Immunoblot analysis revealed that lithium treatment induced a significant increase in the expression of the [H.sup.+]-ATPase ([B.sub.1]-subunit) in cortex (190 [+ or -] 18%) and inner stripe of the outer medulla (190 [+ or -] 9%), and a dramatic increase in inner medulla (900 [+ or -] 104%) in parallel to an increase in the expression of type 1 anion exchanger (400 [+ or -] 40%). This was confirmed by immunocytochemistry and immunoelectron microscopy, which also revealed increased density of intercalated cells. Moreover, immunoblotting and immunocytochemistry revealed a significant increase in the expression of the type 1 electrogenic [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter (NBC) in cortex (200 [+ or -] 23%) and of the electroneutral NBCn1 in inner stripe of the outer medulla (250 [+ or -] 54%). In contrast, there were no changes in the expression of [Na.sup.+]/[H.sup.+] exchanger-3 or of the [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger pendrin. These results demonstrate that the expression of specific renal acid-base transporters is markedly altered in response to long-term lithium treatment. This is likely to represent direct or compensatory effects to increase the capacity for HC[O.sup.-.sub.3] reabsorption, N[H.sup.+.sub.4] reabsorption, and proton secretion to prevent the development of systemic metabolic acidosis. acid-base balance; distal tubular acidosis; hydrogen ion-adenosinetriphosphatase; electroneutral sodium-HC[O.sup.-.sub.3] cotransporter; pendrin

Wall, Susan M., Michael P. Fischer, Dawn M. Glapion, and Mae De La Calzada. ANG II reduces net acid secretion in rat outer medullary collecting duct. Am J Physiol Renal Physiol 285: F930-F937, 2003. First published July 8, 2003; 10.1152/ajprenal.00400.2002.-In rat outer medullary collecting duct (OMCD), the mechanism(s) and regulation of [H.sup.+] secretion are not understood fully. The effect of changes in acid-base balance and the renin-angiotensin system on net [H.sup.+] secretion was explored. Rats received NaCl, NaHC[O.sub.3], N[H.sub.4]Cl, or nothing in their drinking water for 7 days. Total ammonia and total C[O.sub.2] ([J.sub.tco2) fluxes were measured in OMCD tubules perfused in vitro from rats in each treatment group. [J.sub.tCO2] was reduced in tubules from rats drinking N[H.sub.4]Cl relative to those drinking NaHC[O.sub.3]. Because N[H.sub.4]CI intake increases plasma renin and aldosterone, we asked if upregulation of the renin-angiotensin system reduces net [H.sup.+] secretion. Deoxycorticosterone pivalate administered in vivo did not affect J[tCO.sub.2]. However, ANG II given in vivo at 0.1 ng/min reduced J[tCO.sub.2] by 35%. To determine if ANG II has a direct effect on acid secretion, J[tCO.sub.2] was measured with ANG II applied in vitro. ANG II ([10.sup.8] M) present in the bath solution reduced J[tCO.sub.2] by 35%. This ANG II effect was not observed in the presence of the AT[sub.1] receptor blocker candesartan. In conclusion, in rat OMCD, J[tCO.sub.2] is paradoxically reduced with N[H.sub.4]Cl ingestion. Increased circulating ANG II, as occurs during metabolic acidosis, reduces J[tCO.sub.2]. ammonium; acidification; angiotensin 2; aldosterone; metabolic acidosis; metabolic alkalosis

Pendrin is an anion exchanger expressed in type B intercalated cells of the cortical collecting duct (CCD). Whether pendrin localizes to other nephron segments with intercalated cells is unknown. Moreover, whether pendrin is expressed in proximal tubule is debated. Thus the distribution of pendrin mRNA and protein expression in mouse kidney was investigated by using light and electron microscopic immunohistochemistry and quantitative real-time PCR. We observed that pendrin mRNA is expressed mainly in cortex. Within cortex, pendrin mRNA is at least fivefold higher in CCD and the connecting tubule (CNT) than in the other segments. Pendrin protein was observed in a subset of cells within the distal convoluted tubule as well as in type B and in non-A-non-B intercalated cells of the CNT and CCD. In type B intercalated cells, pendrin immunoreactivity was highest in apical cytoplasmic vesicles with little immunolabel along the apical plasma membrane. In non-A-non-B intercalated cells, intense pendrin immunoreactivity was detected along the apical plasma membrane. These differences in the subcellular distribution of pendrin immunolabel were confirmed by morphometric analysis. In conclusion, pendrin is expressed in the mouse distal convoluted tubule, CCD, and CNT along the apical plasma membrane of non-A-non-B intercalated cells and in subapical cytoplasmic vesicles of type B intercalated cells. intercalated cell; distal convoluted tubule; cortical collecting duct; connecting tubule; anion exchange

Pendrin is an intercalated cell Cl−/HCO−3 exchanger thought to participate in K+-sparing NaCl absorption. However, its role in K+ homeostasis has not been clearly defined. We hypothesized that pendrin-null mice will develop hypokalemia with dietary K+ restriction. We further hypothesized that pendrin knockout (KO) mice mitigate urinary K+ loss by downregulating the epithelial Na+ channel (ENaC). Thus, we examined the role of ENaC in Na+ and K+ balance in pendrin KO and wild-type mice following dietary K+ restriction. To do so, we examined the relationship between Na+ and K+ balance and ENaC subunit abundance in K+-restricted pendrin-null and wild-type mice that were NaCl restricted or replete. Following a NaCl-replete, K+-restricted diet, K+ balance and serum K+ were similar in both groups. However, following a Na+, K+, and Cl−-deficient diet, pendrin KO mice developed hypokalemia from increased K+ excretion. The fall in serum K+ observed in K+-restricted pendrin KO mice was enhanced with ENaC stimulation but eliminated with ENaC inhibition. The fall in serum K+ observed in K+-restricted pendrin KO mice was enhanced with ENaC stimulation but eliminated with ENaC inhibition. However, reducing ENaC activity also reduced blood pressure and increased apparent intravascular volume contraction, since KO mice had lower serum Na+, higher blood urea nitrogen and hemoglobin, greater weight loss, greater metabolic alkalosis, and greater NaCl excretion. We conclude that dietary Na+ and K+ restriction induces hypokalemia in pendrin KO mice. Pendrin-null mice limit renal K+ loss by downregulating ENaC. However, this ENaC downregulation occurs at the expense of intravascular volume. NEW & NOTEWORTHY Pendrin is an apical Cl−/HCO−3 exchanger that provides renal K+-sparing NaCl absorption. The pendrin-null kidney has an inability to fully conserve K+ and limits renal K+ loss by downregulating the epithelial Na+ channel (ENaC). However, with Na+ restriction, the need to reduce ENaC for K+ balance conflicts with the need to stimulate ENaC for intravascular volume. Therefore, NaCl restriction stimulates ENaC less in pendrin-null mice than in wild-type mice, which mitigates their kaliuresis and hypokalemia but exacerbates volume contraction. [ABSTRACT FROM AUTHOR]

In rat inner medullary collecting duct, N[H.sup.+.sub.4] uptake by the Na,K-ATPase is increased during hypokalemia. Am J Physiol Renal Physiol 282: F91-F102, 2002. First published August 21, 2001; 10.1152/ajprenal.00141.2002.-- In rat terminal inner medullary collecting duct (tIMCD), the Na,K-ATPase mediates N[H.sup.+.sub.4] uptake, which increases secretion of net [H.sup.+] equivalents. [K.sup.+] and N[H.sup.+.sub.4] compete for a common binding site on the Na,K-ATPase. Therefore, N[H.sup.+.sub.4] uptake should increase during hypokalemia because interstitial [K.sup.+] concentration is reduced. We asked whether upregulation of the Na,K-ATPase during hypokalemia also increases basolateral N[H.sup.+.sub.4] uptake. To induce hypokalemia, rats ate a diet with a low [K.sup.+] content. In tIMCD tubules from rats given 3 days of dietary [K.sup.+] restriction, Na,K-ATPase [[beta].sub.1]-subunit (NK-[[beta].sub.1]) protein expression increased although NK-[[alpha].sub.1] protein expression and Na,K-ATPase activity were unchanged relative to [K.sup.+]-replete controls. However, after 7 days of [K.sup.+] restriction, both NK-[[alpha].sub.1] and NK-[[beta].sub.1] subunit protein expression and Na,K-ATPase activity increased. The magnitude of Na,K-ATPase-mediated N[H.sup.+.sub.4] uptake across the basolateral membrane ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]) was determined in tIMCD tubules perfused in vitro from rats after 3 days of a normal or a [K.sup.+]-restricted diet. [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] was the; same in tubules from rats on either diet when measured at the same extracellular [K.sup.+] concentration. However, in either treatment group, increasing [K.sup.+] concentration from 10 to 30 mM reduced [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] >60%. In conclusion, with 3 days of [K.sup.+] restriction, N[H.sup.+.sub.4] uptake by Na,K-ATPase is increased in the tIMCD primarily from the reduced interstitial [K.sup.+] concentration. sodium, hydrogen-adenosinetriphosphatase; terminal inner medullary collecting duct; potassium; ammonium