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Bartter's syndrome represents a group of hereditary salt- and water-losing renal tubulopathies caused by loss-of-function mutations in proteins mediating or regulating salt transport in the thick ascending limb (TAL) of Henle's loop. Mutations in the ROMK channel cause type II antenatal Bartter's syndrome that presents with maternal polyhydramnios and postnatal life-threatening volume depletion. We have developed a colony of Romk null mice showing a Bartter-like phenotype and with increased survival to adulthood, suggesting the activation of compensatory mechanisms. To test the hypothesis that upregulation of [Na.sup.+]-transporting proteins in segments distal to the TAL contributes to compensation, we studied expression of salt-transporting proteins in ROMK-deficient ([Romk.sup.-/-]) mice. Plasma aldosterone was 40% higher and urinary [PGE.sub.2] excretion was 1.5-fold higher in [Romk.sup.-/-] compared with wild-type littermates. Semiquantitative immunoblotting of kidney homogenates revealed decreased abundances of proximal tubule [Na.sup.+]/[H.sup.+] exchanger (NHE3) and [Na.sup.+]-[P.sub.i] cotransporter (NaPi-IIa) and TAL-specific [Na.sup.+]-[K.sup.+]-2[Cl.sup.-]-cotransporter (NKCC2/BSC1) in [Romk.sup.-/-] mice, while the distal convoluted tubule (DCT)-specific [Na.sup.+]-[Cl.sup.-] cotransporter (NCC/TSC) was markedly increased. The abundance of the [alpha]-,[beta]-, and [gamma]-subunits of the epithelial [Na.sup.+] channel (ENaC) was slightly increased, although only differences for [gamma]-ENaC reached statistical significance. Morphometry revealed a fourfold increase in the fractional volume of DCT but not of connecting tubule (CNT) and collecting duct (CCD). Consistently, CNT and CD of [Romk.sup.-/-] mice revealed no apparent increase in the luminal abundance of the ENaC compared with those of wild-type mice. These data suggest that the loss of ROMK-dependent [Na.sup.+] absorption in the TAL is compensated predominately by upregulation of [Na.sup.+] transport in downstream DCT cells. These adaptive changes in [Romk.sup.-/-] mice may help to limit renal [Na.sup.+] loss, and thereby, contribute to survival of these mice. thick ascending limb of Henle's loop; distal convoluted tubule; thiazide-sensitive NaCl cotransporter; renal outer medulla [K.sup.+] channel; hyperprostaglandin E syndrome

Cantone A, Yang X, Yan Q, Giebisch G, Hebert SC, Wang T. Mouse model of type II Bartter's syndrome. I. Upregulation of thiazide-sensitive Na-Cl cotransport activity. Am J Physiol Renal Physiol 294: F1366-F1372, 2008. First published April 2, 2008; doi:10.1152/ajprenal.00608.2007.l-ROMK-deficient ([Romk.sup.-/-]) mice exhibit polyuria, natriuresis, and kaliuresis similar to individuals with type II Bartter's form of hyperprostaglandin E syndrome (HPS; antenatal Baartter's syndrome). In the present study, we utilized both metabolic and clearance studies to define the contributions of specific distal nephron segments to the renal salt wasting in these mice. The effects of furosemide, hydrochlorothiazide, and benzamil on urinary [Na.sup.+] and [K.sup.+] excretion in both wild-type ([Romk.sup.+/+]) and [Romk.sup.-/-] mice were used to assess and compare salt transport by the [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC2)-expressing thick ascending limb (TAL), the [Na.sup.+]-[Cl.sup.-] cotransporter (NCC)-expressing distal convoluted tubule (DCT1/DCT2), and the epithelial [Na.sup.+] channel (ENaC)-expressing connecting segment (CNT) and collecting duct (CD), respectively. Whole kidney glomerular filtration rate was reduced by 47% in [Romk.sup.-/-] mice. Furosemide-induced increments in the fractional excretion rate of [Na.sup.+] and [K.sup.+] and absolute excretion of [Na.sup.+] and [K.sup.+] were significantly blunted in [Romk.sup.-/-] mice, consistent with a major salt transport defect in the TAL. In contrast, hydrochlorothiazide produced an exaggerated natriuresis in [Romk.sup.-/-] mice, indicating upregulation of salt absorption by the DCT. Benzamil resulted in a similar increment in absolute Na excretion in both [Romk.sup.-/-] and [Romk.sup.+/+,] indicating no significant upregulation of [Na.sup.+] transport by ENaC in ROMK null mice. Moreover, hydrochlorothiazide increased the fractional [K.sup.+] excretion rate in [Romk.sup.-/-] mice, confirming our recent observation that maxi-K channels contribute to distal [K.sup.+] secretion in the absence of ROMK. ROMK knockout; furosemide; hydrochlorothiazide; benzamil

The renal-specific [Na.sup.+]-[K.sup.+]-[2Cl.sup.-] cotransporter NKCC2 belongs to the SLC12 gene family; it is the target for loop diuretics and the cause of type 1 Bartter's syndrome. Because the NKCC2 sequence contains two putative Nlinked glycosylation sites, one of which is conserved with the renal [Na.sup.+]-[Cl.sup.-] cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild-type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by [sup.86Rb.sup.+] influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442A52Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced ~20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in [Cl.sup.-] affinity by N442Q and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting insertion into the plasma membrane and the intrinsic activity of the cotransporter. bumetanide; thick ascending limb; isoforms; salt reabsorption

Gastric acid secretion is activated by two distinct pathways: a neuronal pathway via the vagus nerve and release of acetylcholine and an endocrine pathway involving gastrin and histamine. Recently, we demonstrated that activation of [H.sup.+]-[K.sup.+]-ATPase activity in parietal cells in freshly isolated rat gastric glands is modulated by the calcium-sensing receptor (CaSR). Here, we investigated if the CaSR is functionally expressed in freshly isolated gastric glands from human patients undergoing surgery and if the CaSR is influencing histamine-induced activation of [H.sup.+]-[K.sup.+]ATPase activity. In tissue samples obtained from patients, immunohistochemistry demonstrated the expression in parietal cells of both subunits of gastric [H.sup.+]-[K.sup.+]-ATPase and the CaSR. Functional experiments using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein and measurement of intracellular pH changes allowed us to estimate the activity of [H.sup.+]-[K.sup.+]-ATPase in single freshly isolated human gastric glands. Under control conditions, [H.sup.+]-[K.sup.+]ATPase activity was stimulated by histamine (100 [micro]M) and inhibited by omeprazole (100 [micro]M). Reduction of the extracellular divalent cation concentration (0 [Mg.sup.2+], 100 [micro]M [Ca.sup.2+]) inactivated the CaSR and reduced histamine-induced activation of [H.sup.+]-[K.sup.+]-ATPase activity. In contrast, activation of the CaSR with the trivalent cation [Gd.sup.3+] caused activation of omeprazole-sensitive [H.sup.+]-[K.sup.+]-ATPase activity even in the absence of histamine and under conditions of low extracellular divalent cations. This stimulation was not due to release of histamine from neighbouring enterochromaffin-like cells as the stimulation persisted in the presence of the [H.sub.2] receptor antagonist cimetidine (100 [micro]M). Furthermore, intracellular calcium measurements with fura-2 and fluo-4 showed that activation of the CaSR by [Gd.sup.3+] led to a sustained increase in intracellular [Ca.sup.2+] even under conditions of low extracellular divalent cations. These experiments demonstrate the presence of a functional CaSR in the human stomach and show that this receptor may modulate the activity of acid-secreting [H.sup.+]-[K.sup.+]ATPase in parietal cells. Furthermore, our results show the viability of freshly isolated human gastric glands and may allow the use of this preparation for experiments investigating the physiological regulation and properties of human gastric glands in vitro. stomach; [H.sup.+]-[K.sup.+]-ATPase; parietal cells

WNK1 and WNK4 [WNK, with no lysine (K)] are serine--threonine kinases that function as molecular switches, eliciting coordinated effects on diverse ion transport pathways to maintain homeostasis during physiological perturbation. Gain-of-function mutations in either of these genes cause an inherited syndrome featuring hypertension and hyperkalemia due to increased renal NaCl reabsorption and decreased [K.sup.+] secretion. Here, we reveal unique biochemical and functional properties of WNK3, a related member of the WNK kinase family. Unlike WNK1 and WNK4, WNK3 is expressed throughout the nephron, predominantly at intercellular junctions. Because WNK4 is a potent inhibitor of members of the cation-cotransporter SLC12A family, we used coexpression studies in Xenopus oocytes to investigate the effect of WNK3 on NCC and NKCC2, related kidney-specific transporters that mediate apical NaCl reabsorption in the thick ascending limb and distal convoluted tubule, respectively. In contrast to WNK4's inhibitory activity, kinase-active WNK3 is a potent activator of both NKCC2 and NCC-mediated transport. Conversely, in its kinase-inactive state, WNK3 is a potent inhibitor of NKCC2 and NCC activity. WNK3 regulates the activity of these transporters by altering their expression at the plasma membrane. Wild-type WNK3 increases and kinase-inactive WNK3 decreases NKCC2 phosphorylation at Thr-184 and Thr-189, sites required for the vasopressin-mediated plasmalemmal translocation and activation of NKCC2 in vivo. The effects of WNK3 on these transporters and their coexpression in renal epithelia implicate WNK3 in NaCl, water, and blood pressure homeostasis, perhaps via signaling downstream of vasopressin. hypertension | ion transport | protein serine--threonine kinases

I. Introduction A. Physiological roles for [K.sup.+] channels in kidney B. Overview of renal [K.sup.+] channel diversity and structure II. Function and Regulation of Potassium Channels in the Kidney A. [...]

The distal nephron plays a capital role in the fine regulation of sodium reabsorption. Compared with the cortical collecting duct, much less information is available on the hormonal regulation of sodium transporter genes in the distal convoluted tubule (DCT), where the thiazide-sensitive [Na.sup.+]-[Cl.sup.-] cotransporter (NCC) is the major entry pathway for [Na.sup.+]. The purpose of this study was to characterize the in vitro effects of aldosterone (Aldo; 1 [micro]M) and cAMP (8-BrcAMP; 0.5 mM) on mouse DCT (mDCT) by using an immortalized mDCT cell line. Western blot analysis and semiquantitative RT-PCR were performed to analyze the expression of genes involved in sodium transport. The mDCTcell line expressed the 11[beta]-hydroxysteroid dehydrogenase type 2 gene and both the mineralocorticold and glucocorticoid receptor genes, suggesting Aldo responsiveness. In this sense, we found that mDCT cells expressed the anfilofide-sensitive [Na.sup.+] channel (ENaC) and responded to Aldo by upregulating the [alpha]-subunit protein. Similarly, [[alpha].sub.1] [Na.sup.+]-[K.sup.+]-ATPase protein was upregulated by Aldo and 8-BrcAMP. In addition, the Aldo intermediate gene sgk1 mRNA was increased in response to both Aldo and 8-BrcAMP, and the transcription factor HNF-3[alpha] mRNA was induced by 8-BrcAMP. With respect to NCC regulation, although Aldo induced NCC protein levels in mice in vivo, neither Aldo nor 8-BreAMP significantly induced the NCC mRNA or protein levels in mDCT cells. These results suggest that in mDCT, Aldo and cAMP modulate some downstream mediators and effectors in vitro but do not influence the expression of NCC in this cell model. sodium reabsoption; gene expression; mineralocorticoids; cell culture

We previously demonstrated that dietary K intake regulates the expression of Src family PTK, which plays an important role in controlling the expression of ROMK1 in plasma membrane (Wei Y, Bloom P, Lin D-H, Gu RM, and Wang WH. Am J Physiol Renal Physiol 281: F206-F212, 2001). In the present study, we used the immunofluorescence staining technique to demonstrate the presence of c-Src, a member of Src family PTK, in the thick ascending limb (TAL) and collecting duct. Confocal microscopy shows that c-Src is highly expressed in the renal cortex and outer medulla. Moreover, c-Src and ROMK are coexpressed in the same nephron segment. Also, the positive staining of c-Src is visible in tubules stained with Tamm-Horsfall glycoprotein or aquaporin-2. This suggests that c-Src is present in the TAL, cortical collecting duct (CCD), and outer medullary collecting duct (OMCD). To study the role of PTK in the regulation of ROMK membrane expression in the TAL and CCD, we carried out immunocytochemical staining with ROMK antibody in the CCD or TAL from rats on either a high-K (HK) or K-deficient (KD) diet. A sharp membrane staining of ROMK can be observed in the TAL from rats on both HK and KD diets. However, a clear plasma membrane staining can be observed only in the CCD from rats on a HK diet but not from those on a KD diet. Treatment of the CCD from rats on a HK diet with phenylarsine oxide (PAO) decreases the positive staining in the plasma/subapical membrane and increases the ROMK staining in the intracellular compartment. However, PAO treatment did not significantly alter the staining pattern of ROMK in the TAL. Moreover, the biotinylation technique has also confirmed that neither herbimycin A nor PAO has significantly changed the biotin-labeled ROMK2 in HEK293 cells transfected with ROMK2 and c-Src. We conclude that c-Src is expressed in the TAL, CCD, and OMCD and that stimulation of PTK increases the ROMK channels in the intracellular compartment but decreases them in the apical/subapical membrane in the CCD. potassium secretion; endocytosis; exocytosis; dietary potassium intake; c-Src

Apical potassium re cycling is crucial for salt transport by the thick ascending limb (TAL) Loss-of-function mutations in the K channel, ROMK (Kir1.1; KCNJ1) cause Bartter syndrome, a genetically heterogeneous disorder character ized by severe reduction in salt absorption by the TAL, Na wasting polyuria, and hypokalemic alkalosis. ROMK(-/-) null mice exhibit Bartter phenotype and lack the small-conductance (30-pS) apical K channel (SK) in the TAL. However, a distinct 70-pS K channel can also significantly contribute to the apical conductance of TAL. We now examine the effect of ROMK deletion on the functional expression of the 70-pS K channel in the TAL. Functional expression of the 70-pS K channel was low [average channel acitivty (N[P.sub.O] = 0.02] in ROMK(+/+) mice on a control K diet but increased to 0.27 by high-K intake for 2 wk. In contrast, the high-K diet decreased N[P.sub.O] of SK by ~30%, from 2.04 to 1.44. In ROMK heterozygous (+/-) mice on control K diet, SK activity was about one-half of that observed in ROMK(+/+) mice (0.95 vs. 2.04). The high-K diet also reduced SK activity in ROMK(+/-) mice by -40% (from 0.95 to 0.55) but in creased NPo of the 70-pS K channel from 0 to 0.09 in ROMK (+/-) mice. This corresponds to ~30% of channel activity (N[P.sub.O] = 0.27 observed in ROMK(+/+) mice. Neither the 70-pS nor the 30-pS K channels were observed in TAL cells from ROMK(-/-) mice on either the normal or high-K diets. Thus functional expression of the 70-pS K channel is enhanced by increasing dietary K and requires expression of ROMK. It is likely that ROMK forms a critical subunit of the 70-pS K channel, accounting for the loss of apical K secretory channel activity in ROMK Bartter syndrome. small-conductance potassium channel; intermediate-conductance potassium channel; Kit 1.1; Bartter syndrome

We recently cloned a homologue of the bovine parathyroid calcium receptor from the kidney of a spiny dogfish (Squalus acanthias) and termed this new protein SKCaR. SKCaR senses alterations in extracellular [Mg.sup.2+] after its expression in human embryonic kidney cells (Nearing J, Betka M, Quinn S, Hentschel H, Elger M, Baum M, Bai M, Chattopadyhay N, Brown E, Hebert S, and Harris HW. Proc Natl Acad. Sci USA 99: 9231-9236, 2002). In this report, we used light and electron microscopic immunocytochemical techniques to study the distribution of SKCaR in dogfish kidney. SKCaR antiserum bound to the apical membranes of shark kidney epithelial cells in the following tubular segments: proximal tubules (PIa and PIIb), late distal tubule, and collecting tubule/collecting duct as well as diffusely labeled cells of early distal tubule. The highly specific distribution of SKCaR in mesial tissue as well as lateral countercurrent bundles of dogfish kidney is compatible with a role for SKCaR to sense local tubular [Mg.sup.2+] concentrations. This highly specific distribution of SKCaR protein in dogfish kidney could possibly work in concert with the powerful [Mg.sup.2+] secretory system present in the PIIa segment of elasmobranch fish kidney to affect recycling of [Mg.sup.2+] from putative [Mg.sup.2+]-sensing/[Mg.sup.2+]-reabsorbing segments. These data provide support for the possible existence of [Mg.sup.2+] cycling in elasmobranch kidney in a manner analogous to that described for mammals. renal handling of magnesium; transmembrane receptor protein; immunohistochemistry

The murine apical bumetanide-sensitive [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter gene (mBSC1) exhibits two spliced isoform products that differ at the COOH-terminal domain. A long COOH-terminal isoform (L-mBSC1) encodes the [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter, and a short isoform (S-mBSC1) exerts a dominant-negative effect on L-mBSC1 cotransporter activity that is abrogated by cAMP. However, the mechanism of this dominant-negative effect was not clear. In this study, we used confocal microscopic analysis of an enhanced green fluorescent protein (EGFP) fusion construct (L-mBSC1-EGFP) expressed to characterize the surface expression of the L-BSC1 isoform in Xenopus laevis oocytes. Functional expression was also assessed in L-mBSC1-injected oocytes by measuring the bumetanide-sensitive [sup.86][Rb.sup.+] uptake. Oocytes injected with L-mBSC1-EGFP cRNA developed a distinct plasma membrane-associated fluorescence that colocalized with the fluorescent membrane dye FM 4-64. The fluorescence intensity in L-mBSC1-EGFP oocytes did not change after cAMP was added to the extracellular medium. In contrast, L-mBSC1-EGFP fluorescence intensity was reduced in a dose-dependent manner, with coexpression of S-mBSC1. The inhibitory effect of S-mBSC1 was abrogated by cAMP. Finally, the exocytosis inhibitor colchicine blocked the effect of cAMP on the L-mBSC1-EGFP/S-mBSC1-coinjected oocytes. All changes in L-mBSC1 surface expression correlated with modification of bumetanide-sensitive [sup.86][Rb.sup.+] uptake. Our data suggest that the dominant-negative effect of S-mBSC1 on L-mBSC1 transport function is due to the effects of the cotransporter on trafficking. kidney; thick ascending limb; Xenopus laevis; oocytes; green fluorescent protein; NKCC2

Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kg[H.sub.2]O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA (P < 0.04) and protein (P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA (P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-D-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium (P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with dexamethasone compared with ADX rats (P < 0.02). These results establish glucocorticoids and medium NaCl concentration as direct regulators of MTAL ROMK mRNA and protein expression, which may be modulated by cAMP-dependent factors. regulation of gene expression; ROMK channel; immunoblotting analysis; quantitative reverse transcriptase-polymerase chain reaction; isolated tubules

Expression of calcium-sensing receptor in rat colonic epithelium: evidence for modulation of fluid secretion. Am J Physiol Gastrointest Liver Physiol 283: G240-G250, 2002. First published March 20, 2002; 10.1152/ajpgi.00500.2001.--The calcium-sensing receptor (CaSR) is activated by extracellular calcium ([Ca.sup.2+.sub.o]) and mediates many of the known effects of extracellular divalent minerals on body cells. Both surface and crypt cells express CaSR transcripts and protein on both apical and basolateral surfaces. Raising [Ca.sup.2+.sub.o] elicited increases in intracellular calcium ([Ca.sup+.sub.1]) in both surface and crypt cells with an E[C.sub.50] of 2 mM. The [Ca.sup.2+.sub.o]-induced increase in [Ca.sup.2+.sub.1] was associated with increases in inositol 1,4,5-trisphosphate and eliminated by U-73129, an inhibitor of phosphatidylinositolphospholipase C, as well as by thapsigargin. Other CaSR agonists, [Gd.sup.3+] and neomycin, mimicked these [Ca.sup.2+.sub.o]-induced responses. Both luminal and bath [Ca.sup.2+.sub.o], [Gd.sup.3+], and neomycin induced increases in [Ca.sup.2+] in isolated perfused crypts. The stimulatory effect of forskolin on net fluid secretion in perfused crypts was abolished by increasing [Ca.sup.2+.sub.o] in either luminal or bath perfusates. Thus both apical and basolateral CaSR on crypt cells are functional and provide pathways modulating net intestinal fluid transport that may have important implications for the prevention and treatment of certain diarrheal diseases associated with elevated cAMP. calcium-sensing receptor; intracellular calcium; net fluid movement; isolated perfused colonic crypt