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Previously, we showed that global knockout (KO) of the circadian clock transcription factor PER1 in male, but not female, mice fed a high-salt diet plus mineralocorticoid treatment (HS/DOCP) resulted in nondipping hypertension and decreased night/day ratio of sodium (Na) excretion. Additionally, we have shown that the endothelin-1 (ET-1) gene is targeted by both PER1 and aldosterone. We hypothesized that ET-1 would exhibit a sex-specific response to HS/DOCP treatment in PER1 KO. Here we show that male, but not female, global PER1 KO mice exhibit a decreased night/day ratio of urinary ET-1. Gene expression analysis revealed significant genotype differences in ET-1 and endothelin A receptor ([ET.sub.A]) expression in male, but not female, mice in response to HS/DOCP. Additionally, both wild-type and global PER1 KO male mice significantly increase endothelin B receptor ([ET.sub.B]) expression in response to HS/DOCP, but female mice do not. Finally, siRNA-mediated knockdown of PER1 in mouse cortical collecting duct cells (mpkCCDc14) resulted in increased ET-1 mRNA expression and peptide secretion in response to aldosterone treatment. These data suggest that PER1 is a negative regulator of ET-1 expression in response to HS/DOCP, revealing a novel mechanism for the regulation of renal Na handling in response to HS/DOCP treatment. Key words: kidney, aldosterone, endothelin, nondipping hypertension, renal sodium handling. Precedemment, nous avons montre que le knock-out (KO) global du facteur de transcription PER1 de l'horloge circadienne entrainait une hypertension sans abaissement nocturne et une diminution du ratio nuit/jour de l'excretion de sodium (Na) chez des souris males auxquelles etait administre un regime alimentaire a teneur elevee en sel avec un mineralocorticoide (HS/DOCP), mais pas chez les femelles. De plus, nous avons montre que le gene de l'endotheline 1 (ET-1) est cible par le facteur PER1 comme l'aldosterone. Nous avons pose l'hypothese selon laquelle l'ET-1 entraine une reaction specifique au sexe a l'administration de HS/DOCP avec le KO de PER1. Nous montrons ici que, contrairement aux femelles, les souris KO PER1 males presentent une diminution du ratio nuit/jour de l'ET-1 urinaire. L'etude de l'expression genique a revele la presence de differences genotypiques notables dans l'expression de l'ET-1 et des recepteurs de l'endotheline de type A ([ET.sub.A]) en reaction a l'administration de HS/DOCP chez les souris males, mais pas chez les souris femelles. De plus, les souris males de type sauvage comme KO PER1 global presentaient une augmentation marquee de l'expression des recepteurs de l'endotheline de type B ([ET.sub.B]) en reaction a l'administration de HS/DOCP, ce qui n'etait pas le cas chez les souris femelles. Enfin, le << knock-down >> de PER1 medie par le siARN dans les cellules du tube collecteur cortical de souris (mpkCCDc14) entrainait une augmentation de l'expression en ARNm et de la secretion en peptides de l'ET-1 en reaction a l'administration d'aldosterone. Ces donnees laissent entendre que PER1 serait un regulateur inverse de l'expression de l'ET-1 en reaction a l'administration de HS/DOCP, ce qui revele un nouveau mode d'action pour la regulation renale du sodium en reaction a l'administration de HS/DOCP. [Traduit par la Redaction] Mots-cles: rein, aldosterone, endotheline, hypertension sans abaissement nocturne, regulation renale du sodium., Introduction Circadian clocks are physiological timekeepers that are built into nearly every living organism. These internal biological clocks regulate expression of thousands of genes in a tissue-specific manner, allowing for [...]

The circadian clock protein basic helix-loop-helix aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a transcription factor that impacts kidney function, including blood pressure (BP) control. Previously, we have shown that male, but not female, kidney-specific cadherin Cre-positive BMAL1 knockout (KS-BMAL1 KO) mice exhibit lower BP compared with littermate controls. The goal of this study was to determine the BP phenotype and immune response in male KS-BMAL1 KO mice in response to a low-K+ high-salt (LKHS) diet. BP, renal inflammatory markers, and immune cells were measured in male mice following an LKHS diet. Male KS-BMAL1 KO mice had lower BP following the LKHS diet compared with control mice, yet their circadian rhythm in pressure remained unchanged. Additionally, KS-BMAL1 KO mice exhibited lower levels of renal proinflammatory cytokines and immune cells following the LKHS diet compared with control mice. KS-BMAL1 KO mice were protected from the salt-sensitive hypertension observed in control mice and displayed an attenuated immune response following the LKHS diet. These data suggest that BMAL1 plays a role in driving the BP increase and proinflammatory environment that occurs in response to an LKHS diet. [ABSTRACT FROM AUTHOR]

In the collecting duct (CD), H-K-ATPases function in cation reabsorption and H secretion. This study evaluated H-K-ATPase-mediated H secretion along the mouse CD, measured as EIPA- and luminal bafilomycin [A.sub.1]-insensitive intracellular pH ([pH.sub.1]) recovery from acute H loading (NH4) using BCECF. pHi recovery was measured in 1) microperfused cortical, outer medullary, and inner medullary CDs (CCD, OMCD, and IMCD) from C57BL/6J mice fed a normal diet and 2) common murine CD cell lines. H-K-ATPase activity along the native, microperfused CD was greatest in the CCD, less in the OMCD, and least in the IMCD (0.10 [+ or -] 0.02, 0.04 [+ or -] 0.01, and 0.01 + 0.002 U/min, respectively). H-K-ATPase activity was 0.30 [+ or -] 0.03 and 0.26 [+ or -] 0.03 in A- and B-type ICs, respectively, and was sensitive to Sch-28080 or ouabain, phi recovery was greatest in the [OMCD.sub.1] cell line (0.25 [+ or -] 0.01) and less in [mpkCCD.sub.c14] (0.17 [+ or -] 0.01), mIMCD-K2 (0.12 [+ or -] 0.01), and mIMCD-3 (0.05 [+ or -] 0.01) cells. EIPA inhibited the majority of pHi recovery in these cells (100%, 64%, 75%, and 80% in [mpkCCD.sub.c14], [OMCD.sub.1], mIMCD-K2, and mIMCD-3, respectively). In [OMCD.sub.1] cells, where EIPA-insensitive [pH.sub.i] recovery was greatest, H-K-ATPase activity was 0.10 [+ or -] 0.01 and was significantly inhibited (80%) by Sch-28080. We conclude that I) H-K-ATPase-mediated H secretion in the native mouse CD is greatest in the ICs of the CCD, 2) A- and B-type ICs possess [HK[alpha].sub.1] and [HK[alpha].sub.2] H-K-ATPase activity, and 3) the [OMCD.sub.1] cell line best exhibits H-K-ATPase. microperfusion; intracellular pH; acid-base balance; intercalated cell; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein doi: 10.1152/ajprenal.00333.2009

Am J Physiol Renal Physiol 298: F12-F21, 2010. First published July 29, 2009; doi: 10.1152/ajprenal.90723.2008.--The [H.sup.+]-[K.sup.+]-ATPases are ion pumps that use the energy of ATP hydrolysis to transport protons ([H.sup.+]) in exchange for potassium ions ([K.sup.+]). These enzymes consist of a catalytic [alpha]-subunit and a regulatory [beta]-subunit. There are two catalytic subunits present in the kidney, the gastric or HK[[alpha].sub.j] isoform and the colonic or HK[[alpha].sub.2] isoform. In this review we discuss new information on the physiological function, regulation, and structure of the renal [H.sup.+]-[K.sup.+]-ATPases. Evaluation of enzymatic functions along the nephron and collecting duct and studies in HK[[alpha].sub.1] and HK[[alpha].sub.2] knockout mice suggest that the [H.sup.+]-[K.sup.+]-ATPases may function to transport ions other than protons and potassium. These reports and recent studies in mice lacking both HK[[alpha].sub.1] and HK[[alpha].sub.2] suggest important roles for the renal [H.sup.+]-[K.sup.+]-ATPases in acid/base balance as well as potassium and sodium homeostasis. Molecular modeling studies based on the crystal structure of a related enzyme have made it possible to evaluate the structures of HK[[alpha].sub.1] and HK[[alpha].sub.2] and provide a means to study the specific cation transport properties of [H.sup.+]-[K.sup.+]-ATPases. Studies to characterize the cation specificity of these enzymes under different physiological conditions are necessary to fully understand the role of the [H.sup.+]-[K.sup.+] ATPases in renal physiology. adenosine 5'-triphosphatase; potassium

The mineralocorticoid aldosterone is a major regulator of sodium transport in target epithelia and contributes to the control of blood pressure and cardiac function. It specifically functions to increase renal absorption of sodium from tubular fluid via regulation of the [alpha] subunit of the epithelial sodium channel ([alpha]ENaC). We previously used microarray technology to identify the immediate transcriptional targets of aldosterone in a mouse inner medullary collecting duct cell line and found that the transcript induced to the greatest extent was the circadian clock gene Period 1. Here, we investigated the role of Period 1 in mediating the downstream effects of aldosterone in renal cells. Aldosterone treatment stimulated expression of Period 1 (Per1) mRNA in renal collecting duct cell lines and in the rodent kidney. RNA silencing of Period 1 dramatically decreased expression of mRNA encoding [alpha]ENaC in the presence or absence of aldosterone. Furthermore, expression of [alpha]ENaC-encoding mRNA was attenuated in the renal medulla of mice with disruption of the Per1 gene, and these mice exhibited increased urinary sodium excretion. Renal [alpha]ENaC-encoding mRNA was expressed in an apparent circadian pattern, and this pattern was dramatically altered in mice lacking functional Period genes. These results suggest a role for Period 1 in the regulation of the renal epithelial sodium channel and more broadly implicate the circadian clock in control of sodium balance., Introduction Sodium (Na) is the principal extracellular cation and plays a vital role in maintaining extracellular fluid volume and therefore blood pressure. The mineralocorticoid hormone aldosterone acts directly on the [...]

Two classes of H pumps, H-K-ATPase and H-ATPase, contribute to luminal acidification and HC[O.sub.3] transport in the collecting duct (CD). At least two H-K-ATPase [alpha]-subunits are expressed in the CD: HK[[alpha].sub.1] and HK[[alpha].sub.2]. Both exhibit K dependence but have different inhibitor sensitivities. The HK[[alpha].sub.1] H-K-ATPase is Sch-28080 sensitive, whereas the pharmacological profile of the HK[[alpha].sub.2] H-K-ATPase is not completely understood. The present study used a nonpharmacological, genetic approach to determine the contribution of HK[[alpha].sub.1] and HK[[alpha].sub.2] to cortical CO (CCD) intercalated cell (IC) proton transport in mice fed a normal diet. Intracellular pH ([pH.sub.i]) recovery was determined in ICs using in vitro microperfusion of CCD after an acute intracellular acid load in wild-type mice and mice of the same strain lacking expression of HK[[alpha].sub.1], HK[[alpha].sub.2], or both H-K-ATPases (HK[[alpha].sub.1,2]). A-type and B-type ICs were differentiated by luminal loading with BCECF-AM and peritubular chloride removal from C[O.sub.2]/HC[O.sub.3]-buffered solutions to identify the membrane locations of Cl/HC[O.sub.3] exchange activity. H-ATPase--and Na/H exchange-mediated H transport were inhibited with bafilomycin At (100 nM) and EIPA (10 [micro]M), respectively. Here, we report 1) initial [pH.sub.i] and buffering capacity were not significantly altered in the ICs of HK[alpha]-deficient mice, 2) either HK[[alpha].sub.1] or HK[[alpha].sub.2] deficiency resulted in slower acid extrusion, and 3) A-type ICs from HK[[alpha].sub.1,2]-deficient mice had significantly slower acid extrusion compared with A-type ICs from HK[[alpha].sub.1]-deficient mice alone. These studies are the first nonpharmacological demonstration that both HK[[alpha].sub.1] and HK[[alpha].sub.2] contribute to H secretion in both A-type and B-type ICs in animals fed a normal diet. potassium; microperfusion; pH; acid-base balance; P-type ATPase

This study determined whether nucleotides that bind to purinergic receptors (P2R) regulate the expression or function of serum- and glucocorticoid-inducible kinase-1 (SGK1) in mouse renal inner medullar collecting duct cells (mIMCD-3). The SGK1 protein was detected by Western blotting. A significant reduction of cytosolic SGK1 expression was observed in the cells pretreated with P2R agonist adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma]S), and the reduction could be reversed by P2R antagonists. This reduction was also observed in cells that were pretreated with agonists for P2R subtypes. Using ELISA, we observed a reduced SGK1 kinase activity in ATP[gamma]S-pretreated cells. This effect was reversed by P2R antagonists. Furthermore, an increase of SGK1 kinase activity in aldosterone-pretreated cells was suppressed by ATP[gamma]S. These studies demonstrate for the first time that SGK1 can be downregulated by nucleotides in renal collecting duct epithelial cells, likely via the activation of P2R, and suggest that activation of renal purinergic signaling regulates a SGK1-dependent pathway that is known to modulate ion transport in the renal collecting duct. purinergic signaling; phosphatidylinositol 4,5-bisphosphate; epithelial sodium channels; mineralocorticoid receptor; renal ion transport; serum- and glucocorticoid-inducible kinase-1

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

A putative [H.sup.+]-[K.sup.+]-ATPase in the Atlantic stingray, Dasyatis sabina: primary sequence and expression in gills. Am J Physiol Regul Integr Comp Physiol 287: R981-R991, 2004. First published June 24, 2004; 10.1152/ajpregu.00513.2003.--In mammals, the gastric [H.sup.+]-[K.sup.+]-ATPase (HK[alpha]1)mediates acid secrete acid from their stomachs, but unlike mammals they primarily use their gills for systemic acid excretion instead of their kidneys. The purpose of this study was to determine in an HK[alpha]orthologue exists in an elasmo-branch (Atlantic stingray, Dasyatis sabina), to determine if it is expressed in gills and, if so, to localize its expression and determine if its expression is regulated during hypercapnia or freshwater acclimation. A polyclonal antibody made against HK[alpha]1 peptide detected HK[alpha]1 immunoreactivity was present in a subpopulation of epithelial cells in both organs. Double-labeling experiments in the gills showed that HK[alpha]1 immunoreactivity occurred in [NA.sup.+]-[K.sup.+]-ATPase-rich cells and not in V-type [H.sup.+]-ATPase-rich cells. RT-PCRs were used to deduce the primary sequence of putative [H.sup.+]-[K.sup.+]-ATPase from the stomach of Atlantic stingrays. The 3,421-base pair cDNA includes a coding region for a 1,025-amino acid protein that is over 80% identical to HK[alpha]1 of mammals. RT-PCRs were then used to demonstrate that this transcript is also expressed in the gills. To our knowledge, this is the first [H.sub.+]-[K.sup.+]-ATPase sequence reported for any elasmobranch and the first full-length sequence for any fish. We also provide the first evidence for its expression in the gills of any fish and demonstrate that its expression increased during freshwater acclimation but no exposure to hypercapnia. elasmobranch; proton pump; gastric; stomach

Kidney tubules are targets for the activation of locally released nucleotides through multiple P2 receptor types. Activation of these P2 receptors modulates cellular [Ca.sup.2+] signaling and downstream cellular function. The purpose of this study was to determine whether P2 receptors were present in mIMCD-3 cells, a mouse inner medullary collecting duct cell line, and if so, to examine their link with intracellular [Ca.sup.2+] homeostasis. To monitor intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]), experiments were conducted using the fluorescent dye fura 2. ATP (0.1-100 [micro]M) produced a dose-dependent increase in [[[Ca.sup.2+]].sub.i] in a physiological [Ca.sup.2+]-containing solution, with an E[C.sub.50] of 2.5 [micro]M. The P2-receptor antagonist FPADS reduced the effect of ATP on [[[Ca.sup.2+]].sub.i], and the P1-receptor agonist adenosine caused only a small increase in [[[Ca.sup.2+]].sub.i]. Preincubation of cells with the phospholipase C antagonist U-73122 blocked the ATP-induced increase in [[[Ca.sup.2+]].sub.i], indicating P2Y receptors were involved in this process. In a [Ca.sup.2+]-free bath solution, thapsigargin and ATP induced intracellular [Ca.sup.2+] release from an identical pool. Nucleotides caused an increase in [[[Ca.sup.2+]].sub.i] in the potency order of UTP = ATP > ATP[gamma]S > ADP > UDP that is best fitted with the P2[Y.sub.2] subtype profile. Although the P2Y agonist UTP induced a similar large transient increase in [[[Ca.sup.2+]].sub.i] as did ATP, a small but sustained increase in [[[Ca.sup.2+]].sub.i] occurred only in ATP-stimulated cells, suggesting the role of P2X receptors in [Ca.sup.2+] influx. The sustained increase in [[[Ca.sup.2+]].sub.i] could be blocked by either nonselective cation channel blockers [Gd.sup.3+] or P2X antagonists PPADS and PPNDS. Furthermore, when either [Gd.sup.3+] or PPNDS was applied to the bath solution before ATP application, the ATP-induced increase in [[[Ca.sup.2+]].sub.i] was significantly reduced. Both RT-PCR and Western blotting corroborated the presence of P2[X.sub.1] and P2[Y.sub.2] receptors. These studies demonstrate that mIMCD-3 cells have both P2X and P2Y subtype receptors and that the activation of both P2X and P2Y receptors by extracellular ATP appears to be required to regulate intracellular [Ca.sup.2+] signaling. epithelia; purinergic receptors; collecting duct; calcium channel; kidney

The mineralocorticoid aldosterone is a major regulator of [Na.sup.+] and acid-base balance and control of blood pressure. Although the long-term effects of aldosterone have been extensively studied, the early aldosterone-responsive genes remain largely unknown. Using DNA array technology, we have characterized changes in gene expression after 1 h of exposure to aldosterone in a mouse inner medullary collecting duct cell line, mIMCD-3. Results from three independent microarray experiments revealed that the expression of many transcripts was affected by aldosterone treatment. Northern blot analysis confirmed the upregulation of four distinct transcripts identified by the microarray analysis, namely, the serum and glucose-regulated kinase sgk, connective tissue growth factor, period homolog, and preproendothelin. Immunoblot analysis for preproendothelin demonstrated increased protein expression. Following the levels of the four transcripts over time showed that each had a unique pattern of expression, suggesting that the cellular response to aldosterone is comp]ex. The results presented here represent a novel list of early aldosterone-responsive transcripts and provide new avenues for elucidating the mechanism of acute aldosterone action in the kidney. kidney; sgk; period homolog; connective tissue growth factor; endothelin-1

Research has been conducted on the M1-M2 segment of the rectifying K (super)+ channel. The single and double mutations in this segment have been characterized via the use of the patch-clamp technique and the details are presented.

Ammonia stimulates cortical collecting duct (CCD) net bicarbonate reabsorption by activating an apical [H.sup.+]-[K.sup.+]-ATPase through mechanisms that are independent of ammonia's known effects on intracellular pH and active sodium transport. The present studies examined whether this stimulation occurs through soluble N-ethylmaleimide-sensitive fusion attachment receptor (SNARE) protein-mediated vesicle fusion. Rabbit CCD segments were studied using in vitro microperfusion, and transepithelial bicarbonate transport was measured using microcalorimetry. Ammonia's stimulation of bicarbonate reabsorption was blocked by either chelating intracellular calcium with 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester or by inhibiting microtubule polymerization with colchicine compared with parallel studies performed in the absence of these inhibitors. An inactive structural analog of colchicine, lumicolchicine, did not alter ammonia's stimulation of bicarbonate reabsorption. Tetanus toxin, a zinc endopeptidase specific for vesicle-associated SNARE (v-SNARE) proteins, prevented ammonia from stimulating net bicarbonate reabsorption. Consistent with the functional evidence for v-SNARE involvement, antibodies directed against a conserved region of isoforms 1-3 of the tetanus toxin-sensitive, vesicle-associated membrane protein (VAMP) members of v-SNARE proteins labeled the apical and subapical region of collecting duct intercalated cells. Similarly, antibodies to NSF protein, a protein involved in activation of SNARE proteins for subsequent vesicle fusion, localized to the apical and subapical region of collecting duct intercalated cells. These results indicate that ammonia stimulates CCD bicarbonate reabsorption through an intracellular calcium-dependent, microtubule-dependent, and v-SNARE-dependent mechanism that appears to involve insertion of cytoplasmic vesicles into the apical plasma membrane of CCD intercalated cells. intracellular calcium; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; microtubule; soluble N-ethylmaleimide-sensitive fusion attachment receptor protein; hydrogen-potassium-adenosine triphosphatase