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Potassium channels and transporters maintain potassium homeostasis and play significant roles in several different biological actions via potassium ion regulation. In previous decades, the key revelations that potassium channels and transporters are involved in the production of gastric acid and the regulation of secretion in the stomach have been recognized. Drugs used to treat peptic ulceration are often potassium transporter inhibitors. It has also been reported that potassium channels are involved in ulcerative colitis. Direct toxicity to the intestines from nonsteroidal anti-inflammatory drugs has been associated with altered potassium channel activities. Several reports have indicated that the long-term use of the antianginal drug Nicorandil, an adenosine triphosphate-sensitive potassium channel opener, increases the chances of ulceration and perforation from the oral to anal regions throughout the gastrointestinal (GI) tract. Several of these drug features provide further insights into the role of potassium channels in the occurrence of ulceration in the GI tract. The purpose of this review is to investigate whether potassium channelopathies are involved in the mechanisms responsible for ulceration that occurs throughout the GI tract.

Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk(-/-) mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1(-/-) mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1(+/+) and Romk1(-/-) mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1(+/+), but such regulation by high K intake was diminished with significant hyperkalemia in Romk1(-/-) mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K(+) secretion in the collecting tubule.

[][1] On April 25, 2018, Dr. Donald Seldin passed away at the age of 97, and the world lost a great treasure. Born in Coney Island, Brooklyn, Seldin attended James Madison High School and established his intellectual roots early on. He then attended New York University, where he was

The renal phenotype induced by loss-of-function mutations of inwardly rectifying potassium channel (Kir), Kcnj10 (Kir4.1), includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which Kir.4.1 mutations cause the tubulopathy is not completely understood. Here we demonstrate that Kcnj10 is a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and determines the expression of the apical Na-Cl cotransporter (NCC) in the DCT. Immunostaining demonstrated Kcnj10 and Kcnj16 were expressed in the basolateral membrane of DCT, and patch-clamp studies detected a 40-pS K channel in the basolateral membrane of the DCT1 of p8/p10 wild-type Kcnj10(+/+) mice (WT). This 40-pS K channel is absent in homozygous Kcnj10(-/-) (knockout) mice. The disruption of Kcnj10 almost completely eliminated the basolateral K conductance and decreased the negativity of the cell membrane potential in DCT1. Moreover, the lack of Kcnj10 decreased the basolateral Cl conductance, inhibited the expression of Ste20-related proline-alanine-rich kinase and diminished the apical NCC expression in DCT. We conclude that Kcnj10 plays a dominant role in determining the basolateral K conductance and membrane potential of DCT1 and that the basolateral K channel activity in the DCT determines the apical NCC expression possibly through a Ste20-related proline-alanine-rich kinase-dependent mechanism.

During the past century, investigators have increased our understanding of renal potassium excretion significantly using many techniques. Notable among these were renal clearance experiments, renal micropuncture, isolated tubule microperfusion, and electrophysiological and patch clamp analysis. These experiments have been made possible by technical advances that have allowed the measurement of potassium in progressively smaller quantities. Initially, the kidney was viewed as controlling potassium excretion by the regulated absorption of potassium from the glomerular filtrate, predominantly in the proximal tubule. This concept was supplanted when clearance experiments deduced and subsequent micropuncture studies directly identified the importance of the distal nephron and collecting duct as the principal site responsible for the regulation of potassium excretion. Additional micropuncture and microperfusion studies showed that a component of potassium secreted by the distal cortical nephron and cortical collecting duct is reabsorbed in the medullary collecting duct, which results in renal medullary potassium recycling. Studies have defined the cellular and molecular mechanisms responsible for potassium secretion and potassium reabsorption in the collecting duct. Further understanding of renal potassium handling will require integrated investigation of the renal and extrarenal signaling systems that control these transport mechanisms.

Intercalated cells in the collecting duct system express V-type H+-ATPases which participate in acid extrusion, bicarbonate secretion, and chloride absorption depending on the specific subtype. The activity of H+-ATPases is regulated by acid-base status and several hormones, including angiotensin II and aldosterone. Angiotensin II stimulates chloride absorption mediated by pendrin in type B intercalated cells and this process is energized by the activity of H+-ATPases. Moreover, angiotensin II stimulates bicarbonate secretion by the connecting tubule (CNT) and early cortical collecting duct (CCD). In the present study we examined the effect of angiotensin II (10 nM) on H+-ATPase activity and localization in isolated mouse connecting tubules and cortical collecting ducts. Angiotensin II stimulated Na+-independent intracellular pH recovery about 2-3 fold, and this was abolished by the specific H+-ATPase inhibitor concanamycin. The effect of angiotensin II was mediated through type 1 angiotensin II receptors (AT1-receptors) because it could be blocked by saralasin. Stimulation of H+-ATPase activity required an intact microtubular network - it was completely inhibited by colchicine. Immunocytochemistry of isolated CNT/CCDs incubated in vitro with angiotensin II suggests enhanced membrane associated staining of H+-ATPases in pendrin expressing intercalated cells. In summary, angiotensin II stimulates H+-ATPases in CNT/CCD intercalated cells, and may contribute to the regulation of chloride absorption and bicarbonate secretion in this nephron segment.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K + channel. However, the expression of CFTR in apical cell membranes or its function as a Cl − channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl − channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl − channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl − channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl − channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR inh -172 abolished apical Cl − channel activity in excised patches. Moreover, apical Cl − channel activity was completely absent in principal cells from transgenic mice expressing the ΔF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl − channels on salt handling by the collecting duct and on the functional CFTR–ROMK interactions in modulating the metabolic ATP-sensing of ROMK.

POSH (plenty of SH3) is a scaffold protein that has been shown to act as an E3 ubiquitin ligase. Here we report that POSH stimulates the ubiquitination of Kir1.1 (ROMK) and enhances the internalization of this potassium channel. Immunostaining reveals the expression of POSH in the renal cortical collecting duct. Immunoprecipitation of renal tissue lysate with ROMK antibody and glutathione S-transferase pulldown experiments demonstrated the association between ROMK and POSH. Moreover, immunoprecipitation of lysates of HEK293T cells transfected with ROMK1 or with constructs encoding the ROMK-N terminus or ROMK1-C-Terminus demonstrated that POSH binds to ROMK1 on its N terminus. To study the effect of POSH on ROMK1 channels, we measured potassium currents with electrophysiological methods in HEK293T cells and in oocytes transfected or injected with ROMK1 and POSH. POSH decreased potassium currents, and the inhibitory effect of POSH on ROMK channels was dose-dependent. Biotinylation assay further showed that POSH decreased surface expression of ROMK channels in HEK293T cells transfected with ROMK1 and POSH. The effect of POSH on ROMK1 channels was specific because POSH did not inhibit sodium current in oocytes injected with ENaC-alpha, beta, and gamma subunits. Moreover, POSH still decreased the potassium current in oocytes injected with a ROMK1 mutant (R1Delta373-378), in which a clathrin-dependent tyrosine-based internalization signal residing between amino acid residues 373 and 378 is deleted. However, the inhibitory effect of POSH on ROMK channels was absent in cells expressing with dominant negative dynamin and POSHDeltaRING, in which the RING domain was deleted. Expression of POSH also increased the ubiquitination of ROMK1, whereas expression of POSHDeltaRING diminished its ubiquitination in HEK293T cells. The notion that POSH may serve as an E3 ubiquitin ligase is also supported by in vitro ubiquitination assays in which adding POSH increased the ROMK ubiquitination. We conclude that POSH inhibits ROMK channels by enhancing dynamin-dependent and clathrin-independent endocytosis and by stimulating ubiquitination of ROMK channels.

Stimulation of the basolateral Na(+)/K(+)-ATPase in the isolated perfused rabbit cortical collecting duct by raising either bath potassium or lumen sodium increases potassium secretion, sodium absorption and their apical conductances. Here we determined the effect of stimulating Na(+)/K(+)-ATPase on potassium secretion without luminal sodium transport. Acutely raising bath potassium concentrations from 2.5 to 8.5 mM, without luminal sodium, depolarized the basolateral membrane and transepithelial voltages while increasing the transepithelial, basolateral and apical membrane conductances of principal cells. Fractional apical membrane resistance and cell pH were elevated. Net potassium secretion was maintained albeit diminished and was still enhanced by raising bath potassium, but was reduced by basolateral ethylisopropylamiloride, an inhibitor of Na(+)/H(+) exchange. Luminal iberitoxin, a specific inhibitor of the calcium-activated big-conductance potassium (BK) channel, impaired potassium secretion both in the presence and absence of luminal sodium. In contrast, iberitoxin did not affect luminal sodium transport. We conclude that basolateral Na(+)/H(+) exchange in the cortical collecting duct plays an important role in maintaining potassium secretion during compromised sodium supplies and that BK channels contribute to potassium secretion.

This review provides an overview of the molecular mechanisms of K transport in the mammalian connecting tubule (CNT) and cortical collecting duct (CCD), both nephron segments responsible for the regulation of renal K secretion. Aldosterone and dietary K intake are two of the most important factors regulating K secretion in the CNT and CCD. Recently, angiotensin II (AngII) has also been shown to play a role in the regulation of K secretion. In addition, genetic and molecular biological approaches have further identified new mechanisms by which aldosterone and dietary K intake regulate K transport. Thus, the interaction between serum-glucocorticoid-induced kinase 1 (SGK1) and with-no-lysine kinase 4 (WNK4) plays a significant role in mediating the effect of aldosterone on ROMK (Kir1.1), an important apical K channel modulating K secretion. Recent evidence suggests that WNK1, mitogen-activated protein kinases such as P38, ERK, and Src family protein tyrosine kinase are involved in mediating the effect of low K intake on apical K secretory channels.

ROMK null mice with a high survival rate and varying severity of hydronephrosis provide a good model to study type II Bartter syndrome pathophysiology ( 26 ). During the development of such a colony, we found that more male than female null mice survived, 58.7% vs. 33.3%. To investigate the possible mechanism of this difference, we compared the survival rates, renal functions, degree of hydronephrosis, as well as PGE2 and TXB2 production between male and female ROMK wild-type and null mice. We observed that female ROMK Bartter's mice exhibited lower GFR (0.37 vs. 0.54 ml·min−1·100 g BW−1, P < 0.05) and higher fractional Na+ excretion (0.66% vs. 0.48%, P < 0.05) than male Bartter's. No significant differences in acid-base parameters, urinary K+ excretion, and plasma electrolyte concentrations were observed between sexes. In addition, we assessed the liquid retention rate in the kidney to evaluate the extent of hydronephrosis and observed that 67% of male and 90% of female ROMK null mice were hydronephrotic mice. Urinary PGE2 excretion was higher in both sexes of ROMK null mice: 1.35 vs. 1.10 ng/24 h in males and 2.90 vs. 0.87 ng/24 h in females. TXB2 excretion was higher in female mice in both wild-type and ROMK null mice. The increments of urinary PGE2 and TXB2 were significantly higher in female null mice than males, 233.33% vs. 22.74% of PGE2 and 85.67% vs. 20.36% of TXB2. These data demonstrate a more severe Bartter phenotype in female ROMK null mice, and higher PGE2 and TXB2 production may be one of the mechanisms of this manifestation.

Dietary K intake plays an important role in the regulation of renal K secretion: a high K intake stimulates whereas low K intake suppresses renal K secretion. Our previous studies demonstrated that the Src family protein-tyrosine kinase and mitogen-activated protein kinase (MAPK) are involved in mediating the effect of low K intake on renal K channels and K secretion. However, the molecular mechanism by which low K intake stimulates MAPK is not completely understood. Here we show that inhibitor of growth 4 (ING4), a protein with a highly conserved plant homeodomain finger motif, is involved in mediating the effect of low K intake on MAPK. K restriction stimulates the expression of ING4 in the kidney and superoxide anions, and its related products are involved in mediating the effect of low K intake on ING4 expression. We used HEK293 cells to express ING4 and observed that expression of ING4 increased the phosphorylation of p38 and ERK MAPK, whereas down-regulation of ING4 with small interfering RNA decreased the phosphorylation of p38 and ERK. Immunocytochemistry showed that ING4 was expressed in the renal outer medullary potassium (ROMK)-positive tubules. Moreover, ING4 decreased K currents in Xenopus oocytes injected with ROMK channel cRNA. This inhibitory effect was reversed by blocking p38 and ERK MAPK. These data provide evidence for the role of ING4 in mediating the effect of low K intake on ROMK channel activity by stimulation of p38 and ERK MAPK.

With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr(1092), Tyr(1094), and Tyr(1143), and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr(1092) or Tyr(1143) to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4(Y1092/1094F). Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser(1196), an effect that was abrogated in the double mutant. WNK4(Y1143F) inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4(Y1143F) could not be reversed by SGK1. The failure to reverse WNK4(Y1143F)-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4-PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4(Y1143F)-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr(1092) and Tyr(1143) plays an important role in modulating the inhibitory effect of WNK4 on ROMK.

The tetrameric K channel ROMK provides an important pathway for K secretion by the mammalian kidney, and the gating of this channel is highly sensitive to changes in cytosolic pH. Although charge–charge interactions have been implicated in pH sensing by this K channel tetramer, the molecular mechanism linking pH sensing and the gating of ion channels is poorly understood. The x-ray crystal structure KirBac1.1, a prokaryotic ortholog of ROMK, has suggested that channel gating involves intermolecular interactions of the N- and C-terminal domains of adjacent subunits. Here we studied channel gating behavior to changes in pH using giant patch clamping of Xenopus laevis oocytes expressing WT or mutant ROMK, and we present evidence that no single charged residue provides the pH sensor. Instead, we show that N–C- and C–C-terminal subunit–subunit interactions form salt bridges, which function to stabilize ROMK in the open state and which are modified by protons. We identify a highly conserved C–C-terminal arginine–glutamate (R-E) ion pair that forms an intermolecular salt bridge and responds to changes in proton concentration. Our results support the intermolecular model for pH gating of inward rectifier K channels.

The multisubunit vacuolar-type H + ATPases mediate acidification of various intracellular organelles and in some tissues mediate H + secretion across the plasma membrane. Mutations in the B1-subunit of the apical H + ATPase that secretes protons in the distal nephron cause distal renal tubular acidosis in humans, a condition characterized by metabolic acidosis with an inappropriately alkaline urine. To examine the detailed cellular and organismal physiology resulting from this mutation, we have generated mice deficient in the B1-subunit ( Atp6v1b1 -/- mice). Urine pH is more alkaline and metabolic acidosis is more severe in Atp6v1b1 -/- mice after oral acid challenge, demonstrating a failure of normal urinary acidification. In Atp6v1b1 -/- mice, the normal urinary acidification induced by a lumen-negative potential in response to furosemide infusion is abolished. After an acute intracellular acidification, Na + -independent pH recovery rates of individual Atp6v1b1 -/- intercalated cells of the cortical collecting duct are markedly reduced and show no further decrease after treatment with the selective H + ATPase inhibitor concanamycin. Apical expression of the alternative B-subunit isoform, B2, is increased in Atp6v1b1 -/- medulla and colocalizes with the H + ATPase E-subunit; however, the greater severity of metabolic acidosis in Atp6v1b1 -/- mice after oral acid challenge indicates that the B2-subunit cannot fully functionally compensate for the loss of B1. Our results indicate that the B1 isoform is the major B-subunit isoform that incorporates into functional, plasma membrane H + ATPases in intercalated cells of the cortical collecting duct and is required for maximal urinary acidification.

NHE3−/− mice display a profound defect in proximal tubule bicarbonate reabsorption but are only mildly acidotic owing to reduced glomerular filtration rate and enhanced H+ secretion in distal nephron segments. In vivo microperfusion of rat distal tubules suggests that a significant fraction of bicarbonate reabsorption in this nephron segment is mediated by NHE2. Two approaches were used to evaluate the role of distal tubule NHE2 in compensating for the proximal defect of H+ secretion in NHE3−/− mice. First, renal clearance experiments were used to assess the impact of HOE694, an inhibitor with significant affinity for NHE2, on excretion of bicarbonate in NHE3−/− and NHE2−/− mice. Second, in vivo micropuncture and microperfusion were employed to measure the concentration of bicarbonate in early distal tubule fluid and to measure distal bicarbonate reabsorption during a constant bicarbonate load. Our data show that HOE694 had no effect on urinary bicarbonate excretion in NHE3+/+ mice, whereas bicarbonate excretion was higher in NHE3−/− mice receiving HOE694. HOE694 induced a significant increase in bicarbonate excretion in mice given an acute bicarbonate load, but there was no effect during metabolic acidosis. Bicarbonate excretion was not affected by HOE694 in bicarbonate-loaded NHE2−/− mice. In vivo micropuncture revealed that early distal bicarbonate concentration was elevated in both bicarbonate-loaded and NHE3−/− mice. Further, microperfusion experiments showed that HOE694-sensitive bicarbonate reabsorption capacity was higher in acidotic and NHE3 null animals. We conclude that NHE2 contributes importantly to acidification in the distal tubule, and that it plays a major role in limiting urinary bicarbonate losses in states in which a high luminal bicarbonate load is presented to the distal tubule, such as in NHE3 null mice.

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.

A key question in systems biology is how diverse physiologic processes are integrated to produce global homeostasis1. Genetic analysis can contribute by identifying genes that perturb this integration. One system orchestrates renal NaCl and K+ flux to achieve homeostasis of blood pressure and serum K+ concentration (refs. 2,3). Positional cloning implicated the serine-threonine kinase WNK4 in this process4; clustered mutations in PRKWNK4, encoding WNK4, cause hypertension and hyperkalemia (pseudohypoaldosteronism type II, PHAII5) by altering renal NaCl and K+ handling. Wild-type WNK4 inhibits the renal Na-Cl cotransporter (NCCT); mutations that cause PHAII relieve this inhibition6. This explains the hypertension of PHAII but does not account for the hyperkalemia. By expression in Xenopus laevis oocytes, we show that WNK4 also inhibits the renal K+ channel ROMK. This inhibition is independent of WNK4 kinase activity and is mediated by clathrin-dependent endocytosis of ROMK, mechanisms distinct from those that characterize WNK4 inhibition of NCCT. Most notably, the same mutations in PRKWNK4 that relieve NCCT inhibition markedly increase inhibition of ROMK. These findings establish WNK4 as a multifunctional regulator of diverse ion transporters; moreover, they explain the pathophysiology of PHAII. They also identify WNK4 as a molecular switch that can vary the balance between NaCl reabsorption and K+ secretion to maintain integrated homeostasis.

We previously demonstrated that carbon monoxide (CO) stimulates the apical 70-pS K+ channel in the thick ascending limb (TAL) of the rat kidney (Liu HJ, Mount DB, Nasjletti A, and Wang WH. J Clin Invest 103: 963-970, 1999). Because the apical K+ channel plays a key role in K+ recycling, we tested the hypothesis that heme oxygenase (HO)-dependent metabolites of heme may affect Na+ transport in the TAL. We used in vivo microperfusion to study the effect of chromium mesoporphyrin (CrMP), an inhibitor of HO, on fluid absorption ( Jv) and Na+ absorption ( JNa) in the loop of Henle and renal clearance methods to examine the effect of CrMP on renal sodium excretion. Microperfusion experiments demonstrated that addition of CrMP to the loop of Henle decreased Jv by 13% and JNa by 20% in animals on normal rat chow and caused a decrease in Jv (39%) and JNa (40%) in rats on a high-K+ (HK) diet. The effect of CrMP is the result of inhibition of HO because addition of MgPP, an analog of CrMP that does not inhibit HO, had no effect on Jv. Western blot analysis showed that HO-2 is expressed in the kidney and that the level of HO-2 was significantly elevated in animals on a HK diet. Renal clearance studies demonstrated that the infusion of CrMP increased the excretion of urinary Na+ (ENa) and volume (UV) without changes in glomerular filtration rate. The effect of CrMP on ENa and UV was larger in HK rats than those kept on normal chow. We conclude that HK intake increases HO-2 expression in the kidney and that HO-dependent metabolites of heme, presumably CO, play a significant role in the regulation of Na+ transport in the loop of Henle.

Proximal solute and fluid absorption is greatly reduced in mice in which the gene encoding the Na/H exchanger isoform 3 has been ablated (NHE3-/-). To obtain information on the intracellular functional consequences of such selective NHE3 deficiency, Na, Cl and K concentrations and cell Rb uptake were measured using electron microprobe analysis after a 30-s infusion of Rb (an index of basolateral Na/K-ATPase activity) in proximal convoluted tubule (PCT) cells of NHE3-/- and wild-type (NHE3+/+) mice. In addition, the relative abundance of the alpha1-subunit of the Na/K-ATPase in the outer cortex was determined by Western blot analysis. PCT cell Na concentration in NHE3-/- mice was slightly but significantly lower than in NHE3+/+ [13.1+/-0.6 ( n=64) vs. 14.9+/-0.6 ( n=62) mmol/kg wet wt.; means +/-SEM]. The lower intracellular Na concentration was associated with significantly reduced Rb uptake rates [9.7+/-0.6 ( n=59) vs. 14.8+/-0.8 ( n=50) mmol/kg wet wt./30 s], but the abundance of the alpha1-subunit of the Na/K-ATPase was not different between NHE3-/- and NHE3+/+ mice. Intracellular Cl concentration was higher (14.2+/-0.4 vs. 12.8+/-0.4 mmol/kg wet wt.) and K concentration unchanged (122.7+/-2.7 vs. 121.6+/-2.5 mmol/kg wet wt.) in PCT cells in NHE3-/- compared with NHE3+/+ mice. These findings suggest that the elimination of apical NHE3 in PCT cells of NHE3-/- mice reduces apical Na entry and, due to lower cell Na concentrations, Na/K-ATPase activity. The observed changes in intracellular Na concentration did not affect the expression of Na/K-ATPase in the renal cortex of NHE3-/- mice. There were no significant changes of cell Na concentration and Rb uptake in distal convoluted tubule, connecting tubule, principal and intercalated cells.

Potassium secretory flux ( JK) by the distal nephron is regulated by systemic and luminal factors. In the present investigation, JKwas measured with a double-barreled K+electrode during paired microperfusion of superficial segments of the rat distal nephron. We used control solutions (100 mM NaCl, pH 7.0) and experimental solutions in which Cl−had been replaced with a less permeant anion and/or pH had been increased to 8.0. JKincreased when Cl−was replaced by either acetate (∼37%), sulfate (∼32%), or bicarbonate (∼62%), and also when the pH of the control perfusate was increased (∼26%). The majority (80%) of acetate-stimulated JKwas Ba2+sensitive, but furosemide (1 mM) further reduced secretion (∼10% of total), suggesting that K+-Cl−cotransport was operative. Progressive reduction in luminal Cl−concentration from 100 to 20 to 2 mM caused increments in JKthat were abolished by inhibitors of K+-Cl−cortransport, i.e., furosemide and [(dihydroindenyl)oxy]alkanoic acid. Increasing the pH of the luminal perfusion fluid also increased JKeven in the presence of Ba2+, suggesting that this effect cannot be accounted for only by K+channel modulation of K+secretion in the distal nephron of the rat. Collectively, these data suggest a role for K+-Cl−cotransport in distal nephron K+secretion.

A significant fraction of active chloride reabsorption across the apical membrane of the proximal tubule is mediated by a chloride/formate exchange process, whereby intracellular formate drives the transport of chloride into the cell. When chloride/formate exchange operates in parallel with Na+/H+exchange and H+-coupled recycling of formate, the net result is electroneutral NaCl reabsorption. Pendrin is the protein product of the PDS gene (SLC26A4) and functions in several different anion exchange modes, including chloride/formate exchange. Pendrin is expressed in the kidney and may serve as the transporter responsible for formate-dependent NaCl reabsorption. In the present study, Pds-knockout mice were used to determine the role of pendrin in proximal tubule chloride reabsorption. We show that formate-dependent NaCl absorption in microperfused proximal tubules is similar between wild-type and pendrin-deficient mice. In addition, there is no difference in the rate of formate-mediated chloride transport in brush-border membrane vesicles isolated from wild-type and pendrin-deficient mice. These studies demonstrate that pendrin is not responsible for formate-dependent NaCl reabsorption in the proximal tubule.

A complex pump-leak system involving both active and passive transport mechanisms is responsible for the appropriate distribution of potassium (K) between the intra- and extracellular fluid compartments. In addition, the kidneys, and to a lesser extent the colon, safeguard maintenance of the narrow range of low K concentrations in the extracellular fluid. Early renal clearance studies showed that K is normally both reabsorbed and secreted by renal tubules, and that regulated secretion is the major source of K excretion. Studies at the tubule and cell level have localized secretion and reabsorption of K to principal and intercalated cells in the collecting ducts. Measurements of the electrochemical driving forces across individual cell membranes have permitted the characterization of specific ATPases, K channels and K cotransporters and also provided insights into the molecular structure of individual transporters that regulate K excretion.

The small-conductance K+ channel (SK) in the apical membrane of the cortical-collecting duct (CCD) is regulated by adenosine triphosphate (ATP) and phosphorylation-dephosphorylation processes. When expressed in Xenopus oocytes, ROMK, a cloned K+ channel similar to the native SK channel, can be stimulated by phosphatidylinositol bisphosphate (PIP2), which is produced by phosphoinositide kinases from phosphatidylinositol. However, the effects of PIP2 on SK channel activity are not known. In the present study, we investigated the mechanism by which hydrolyzable ATP prevented run-down of SK channel activity in excised apical patches of principal cells from rat CCD. Channel run-down was significantly delayed by pretreatment with hydrolyzable Mg-ATP, but ATPγS and AMP-PNP had no effect. Addition of alkaline phosphatase also resulted in loss of channel activity. After run-down, SK channel activity rapidly increased upon addition of PIP2. Exposure of inside-out patches to phosphoinositide kinase inhibitors (LY294002, quercetin or wortmannin) decreased channel activity by 74% in the presence of Mg-ATP. PIP2 added to excised patches reactivated SK channels in the presence of these phosphoinositide kinase inhibitors. The protein kinase A inhibitor, PKI, reduced channel activity by 36% in the presence of Mg-ATP. PIP2 was also shown to modulate the inhibitory effects of extracellular and cytosolic ATP. We conclude that both ATP-dependent formation of PIP2 through membrane-bound phosphoinositide kinases and phosphorylation of SK by PKA play important roles in modulating SK channel activity.

The ROMK (Kir1.1; Kcnj1) gene is believed to encode the apical small conductance K(+) channels (SK) of the thick ascending limb (TAL) and cortical collecting duct (CCD). Loss-of-function mutations in the human ROMK gene cause Bartter's syndrome with renal Na(+) wasting, consistent with the role of this channel in apical K(+) recycling in the TAL that is crucial for NaCl reabsorption. However, the mechanism of renal K(+) wasting and hypokalemia that develop in individuals with ROMK Bartter's syndrome is not apparent given the proposed loss of the collecting duct SK channel. Thus, we generated a colony of ROMK null mice with approximately 25% survival to adulthood that provides a good model for ROMK Bartter's syndrome. The remaining 75% of null mice die in less than 14 days after birth. The surviving ROMK null mice have normal gross renal morphology with no evidence of significant hydronephrosis, whereas non-surviving null mice exhibit marked hydronephrosis. ROMK protein expression was absent in TAL and CCD from null mice but exhibited normal abundance and localization in wild-type littermates. ROMK null mice were polyuric and natriuretic with an elevated hematocrit consistent with mild extracellular volume depletion. SK channel activity in TAL and CCD was assessed by patch clamp analysis in ROMK wild-type ROMK(+/+), heterozygous ROMK(+/-), and null ROMK(-/-) mice. In 313 patches with successful seals from the three ROMK genotypes, SK channel activity in ROMK (+/+ and +/-) exhibited normal single channel kinetics. The expression frequencies are as follows: 67 (TAL) and 58% (CCD) in ROMK(+/+); about half that of the wild-type in ROMK(+/-), being 38 (TAL) and 25% (CCD); absent in both TAL or CCD in ROMK(-/-) between 2 and 5 weeks in 15 mice (61 and 66 patches, respectively). The absence of SK channel activity in ROMK null mice demonstrates that ROMK is essential for functional expression of SK channels in both TAL and CCD. Despite loss of ROMK expression, the normokalemic null mice exhibited significantly increased kaliuresis, indicating alternative mechanisms for K(+) absorption/secretion in the nephron.

ATP-sensitive potassium (K(ATP)) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of K(ATP) channel activity may involve direct binding of this nucleotide to the pore-forming inward rectifier (Kir) subunit despite the lack of known nucleotide-binding motifs. To examine this possibility, we assessed the binding of the fluorescent ATP analogue, 2',3'-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)adenosine 5'-triphosphate (TNP-ATP) to maltose-binding fusion proteins of the NH(2)- and COOH-terminal cytosolic regions of the three known K(ATP) channels (Kir1.1, Kir6.1, and Kir6.2) as well as to the COOH-terminal region of an ATP-insensitive inward rectifier K(+) channel (Kir2.1). We show direct binding of TNP-ATP to the COOH termini of all three known K(ATP) channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of K(ATP) channels because this nucleotide did not bind to the NH(2) termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to K(ATP) COOH termini of Kir1.1, Kir6.1, and Kir6.2 were similar. Binding was abolished by denaturing with 4 m urea or SDS and enhanced by reduction in pH. TNP-ATP to protein stoichiometries were similar for all K(ATP) COOH-terminal proteins with 1 mol of TNP-ATP binding/mole of protein. Competition of TNP-ATP binding to the Kir1.1 COOH terminus by MgATP was complex with both Mg(2+) and MgATP effects. Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of K(ATP) tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer.

Inwardly rectifying, ATP-sensitive K + channels (K ATP ) couple metabolism to either cell excitability (Kir6.x) or potassium secretion (Kir1.1). Phosphatidylinositol phospholipids, like PI(4,5)P 2 , antagonize nucleotide inhibition of K ATP channels enhancing the coupling of metabolic events to cell electrical or transport activity. The mechanism by which phospholipids relieve ATP block is unclear. We have shown that maltose-binding fusion proteins (MBP) containing the COOH termini of K ATP channels (Kir1.1, Kir6.1, and Kir6.2) form functional tetramers that directly bind at least two ATP molecules with negative cooperativity. Here we show that purified phosphatidylinositol phospholipids compete for 2,4,6,-trinitrophenyl (TNP)-ATP binding to the COOH termini of K ATP channels with EC 50 values for PIP 2 between 6–8 μM. The phospholipid potency profile was PIP 3 > PIP 2 = PIP > PI, suggesting that net phospholipid charge was important. A role for head group charge was supported by polycations (neomycin, spermine, and polylysine) reversing the effect of PIP 2 on TNP-ATP binding to the Kir1.1 channel COOH terminal fusion protein. In contrast, the water-soluble charged hydrolytic product of PIP 2 , inositol(1,4,5)P 3 (IP 3 ), had no effect on TNP-ATP binding, suggesting that the acyl chain of PIP 2 was also necessary for its effect on TNP-ATP binding. Indeed, neutral and charged lipids had weak, but significant, effects on TNP-ATP binding. Whereas μM concentrations of PIP 2 could compete with TNP-ATP, we found that mM concentrations of MgATP were required to compete with PIP 2 for binding to these K ATP channel COOH termini. Thus the COOH termini of K ATP channels form a nucleotide- and phospholipid-modulated channel gate on which ATP and phospholipids compete for binding.

This study assessed the functional role of Na+/H+exchanger (NHE) isoforms NHE3 and NHE2 in the proximal tubule, loop of Henle, and distal convoluted tubule of the rat kidney by comparing sensitivity of transport to inhibition by Hoe-694 (an agent known to inhibit NHE2 but not NHE3) and S-3226 (an agent with much higher affinity for NHE3 than NHE2). Rates of transport of fluid ( Jv) and HCO[Formula: see text]( JHCO3) were studied by in situ microperfusion. In the proximal tubule, addition of ethylisopropylamiloride or S-3226 significantly reduced Jvand JHCO3, but addition of Hoe-694 caused no significant inhibition. In the loop of Henle, JHCO3was also inhibited by S-3226 and not by Hoe-694, although much higher concentrations of S-3226 were required than what was necessary to inhibit transport in the proximal tubule. In contrast, in the distal convoluted tubule, JHCO3was inhibited by Hoe-694 but not by S-3226. These results are consistent with the conclusion that NHE2 rather than NHE3 is the predominant isoform responsible for apical membrane Na+/H+exchange in the distal convoluted tubule, whereas NHE3 is the predominant apical isoform in the proximal tubule and possibly also in the loop of Henle.

ATP-regulated (K(ATP)) channels are formed by an inward rectifier pore-forming subunit (Kir) and a sulfonylurea (glibenclamide)-binding protein, a member of the ATP binding cassette family (sulfonylurea receptor (SUR) or cystic fibrosis transmembrane conductance regulator). The latter is required to confer glibenclamide sensitivity to K(ATP) channels. In the mammalian kidney ROMK1-3 are components of K(ATP) channels that mediate K(+) secretion into urine. ROMK1 and ROMK3 splice variants share the core polypeptide of ROMK2 but also have distinct NH(2)-terminal extensions of 19 and 26 amino acids, respectively. The SUR2B is also expressed in rat kidney tubules and may combine with Kir.1 to form renal K(ATP) channels. Our previous studies showed that co-expression of ROMK2, but not ROMK1 or ROMK3, with rat SUR2B in oocytes generated glibenclamide-sensitive K(+) currents. These data suggest that the NH(2)-terminal extensions in both ROMK1 and ROMK3 block ROMK-SUR2B interaction. Seven amino acids in the NH(2)-terminal extensions of ROMK1 and ROMK3 are identical (amino acids 13-19 in ROMK1 and 20-26 in ROMK3) and may determine ROMK-SUR2B interaction. We constructed a series of hemagglutinin-tagged ROMK1 NH(2)-terminal deletion and substitution mutants and examined glibenclamide-sensitive K(+) currents in oocytes when co-expressed with SUR2B. These studies identified an amino acid triplet "IRA" within the conserved segment in the NH(2) terminus of ROMK1 and ROMK3 that blocks the ability of SUR2B to confer glibenclamide sensitivity to the expressed K(+) currents. The position of this triplet in the ROMK1 NH(2)-terminal extension is also important for the ROMK-SUR2B interactions. In vitro co-translation and immunoprecipitation studies with hemagglutinin-tagged ROMK mutants and SUR2B indicted that direct interaction between these two proteins is required for glibenclamide sensitivity of induced K(+) currents in oocytes. These results suggest that the IRA triplet in the NH(2)-terminal extensions of both ROMK1 and ROMK3 plays a key role in subunit assembly of the renal secretary K(ATP) channel.

The absorption of NaCl in the proximal tubule is markedly stimulated by formate. This stimulation of NaCl transport is consistent with a cell model involving Cl−-formate exchange in parallel with pH-coupled formate recycling due to nonionic diffusion of formic acid or H+-formate cotransport. The formate recycling process requires H+ secretion. Although Na+-H+ exchanger isoform NHE3 accounts for the largest component of H+ secretion in the proximal tubule, 40–50% of the rates of HCO[Formula: see text] absorption or cellular H+ extrusion persist in NHE3 null mice. The purpose of the present investigation is to use NHE3 null mice to directly test the role of apical membrane NHE3 in mediating NaCl absorption stimulated by formate. We demonstrate that formate stimulates NaCl absorption in the mouse proximal tubule microperfused in vivo, but the component of NaCl absorption stimulated by formate is absent in NHE3 null mice. In contrast, stimulation of NaCl absorption by oxalate is preserved in NHE3 null mice, indicating that oxalate-stimulated NaCl absorption is independent of Na+-H+ exchange. The virtually complete dependence of formate-induced NaCl absorption on NHE3 activity raises the possibility that NHE3 and the formate transporters are functionally coupled in the brush border membrane.

Pathophysiological stimuli, including hypoxia, lead to K+efflux from the intracellular lumen to the extracellular space, thereby increasing local tissue K+concentrations and depolarizing resident cells. In this study, we investigated the effects of increased extracellular K+concentrations ([K+]e) on heat shock protein (HSP) expression in the porcine proximal tubule epithelial cell line LLC-PK1. We analyzed HSP-25, HSP-72, HSC-73, and HSP-90 protein expression by Western blot analyses and HSP-72 promoter activity by luciferase reporter gene assays using the proximal 1,440 bp of the HSP-72 promoter. Elevating [K+]efrom 20 to 50 mM increased HSP-72 protein expression and promoter activity but did not affect HSP-25, HSC-73, or HSP-90 levels. Addition of identical concentrations of sodium chloride did not increase HSP-72 expression to a similar amount. The Ca2+channel blocker diltiazem and the Ca2+-specific chelator EGTA-AM abolished high [K+]e-induced HSP-72 expression by 69.7 and 75.2%, respectively, indicating that the transcriptional induction of HSP-72 involves Ca2+influx. As measured by confocal microscopy using the Ca2+dye fluo 3-AM, we also observed a rapid increase of intracellular Ca2+concentration as early as 30 s after placing LLC-PK1cells in high [K+]e. We further analyzed whether Ca2+influx was necessary for induction of HSP-72 expression by high [K+]eusing Ca2+-free medium. Here, induction of HSP-72 in response to high [K+]ewas completely abolished. Our data thus demonstrate activation of a protective cellular response to ionic stress, e.g., elevated K+concentrations, by specifically increasing protein levels of HSP-72.

A key function of the proximal tubule is retrieval of most of the vast quantities of NaCl and water filtered by the kidney. Physiological studies using brush border vesicles and perfused tubules have indicated that a major fraction of Cl − reabsorption across the apical membrane of proximal tubule cells occurs via Cl − -formate exchange. The molecular identity of the transporter responsible for renal brush border Cl − -formate exchange has yet to be elucidated. As a strategy to identify one or more anion exchangers responsible for mediating Cl − reabsorption in the proximal tubule, we screened the expressed sequence tag database for homologs of pendrin, a transporter previously shown to mediate Cl − -formate exchange. We now report the cDNA cloning of CFEX, a mouse pendrin homolog with expression in the kidney by Northern analysis. Sequence analysis indicated that CFEX very likely represents the mouse ortholog of human SLC26A6. Immunolocalization studies detected expression of CFEX, but not pendrin, on the brush border membrane of proximal tubule cells. Functional expression studies in Xenopus oocytes demonstrated that CFEX mediates Cl − -formate exchange. Taken together, these observations identify CFEX as a prime candidate to mediate Cl − -formate exchange in the proximal tubule and thereby to contribute importantly to renal NaCl reabsorption. Given its wide tissue distribution, CFEX also may contribute to transcellular Cl − transport in additional epithelia such as the pancreas and contribute to transmembrane Cl − transport in nonepithelial tissues such as the heart.

The apical small conductance (SK) channel plays a key role in K secretion in the cortical collecting duct (CCD). A high-K intake stimulates renal K secretion and involves a significant increase in the number of SK channels in the apical membrane of the CCD. We used the patch-clamp technique to examine the role of protein tyrosine kinase (PTK) in regulating the activity of SK channels in the CCD. The application of 100 μM genistein stimulated SK channels in 11 of 12 patches in CCDs from rats on a K-deficient diet, and the mean increase in NPo, a product of channel number ( N) and open probability ( Po), was 2.5. In contrast, inhibition of PTK had no effect in tubules from animals on a high-K diet in all 10 experiments. Western blot analysis further shows that the level of cSrc, a nonreceptor type of PTK, is 261% higher in the kidneys from rats on a K-deficient diet than those on a high-K diet. However, the effect of cSrc was not the result of direct inhibition of channel itself, because addition of exogenous cSrc had no effect on SK channels in inside-out patches. In cell-attached patches, application of herbimycin A increased channel activity in 14 of 16 patches, and the mean increase in NPowas 2.4 in tubules from rats on a K-deficient diet. In contrast, herbimycin A had no effect on channel activity in any of 15 tubules from rats on a high-K diet. Furthermore, herbimycin A pretreatment increased NPoper patch from the control value (0.4) to 2.25 in CCDs from rats on a K-deficient diet, whereas herbimycin failed to increase channel activity ( NPo: control, 3.10; herbimycin A, 3.25) in the CCDs from animals on a high-K diet. We conclude that PTK is involved in regulating the number of apical SK channels in the kidney.

We have used the patch-clamp technique to study the effects of changing extracellular ATP concentration on the activity of the small-conductance potassium channel (SK) on the apical membrane of the mouse cortical collecting duct. In cell-attached patches, the channel conductance and kinetics were similar to its rat homologue. Addition of ATP to the bathing solution of split-open single cortical collecting ducts inhibited SK activity. The inhibition of the channel by ATP was reversible, concentration dependent (K(i) = 64 microM), and could be completely prevented by pretreatment with suramin, a specific purinergic receptor (P(2)) blocker. Ranking of the inhibitory potency of several nucleotides showed strong inhibition by ATP, UTP, and ATP-gamma-S, whereas alpha, beta-Me ATP, and 2-Mes ATP failed to affect channel activity. This nucleotide sensitivity is consistent with P(2)Y(2) purinergic receptors mediating the inhibition of SK by ATP. Single channel analysis further demonstrated that the inhibitory effects of ATP could be elicited through activation of apical receptors. Moreover, the observation that fluoride mimicked the inhibitory action of ATP suggests the activation of G proteins during purinergic receptor stimulation. Channel inhibition by ATP was not affected by blocking phospholipase C and protein kinase C. However, whereas cAMP prevented channel blocking by ATP, blocking protein kinase A failed to abolish the inhibitory effects of ATP. The reduction of K channel activity by ATP could be prevented by okadaic acid, an inhibitor of protein phosphatases, and KT5823, an agent that blocks protein kinase G. Moreover, the effect of ATP was mimicked by cGMP and blocked by L-NAME (N(G)-nitro-l-arginine methyl ester). We conclude that the inhibitory effect of ATP on the apical K channel is mediated by stimulation of P(2)Y(2) receptors and results from increasing dephosphorylation by enhancing PKG-sensitive phosphatase activity.

In 1992, the defmition of erectile dysfunction (ED) replaced the old term of impotence that had been used for many centuries, in order to avoid the general confusion existing until today in this field. In the past, the origin of impotence had been attributed to different causes such as psychological diseases, or witches and demons. In 1764, Fusanacci described impotence that can occur in the male as well as in the female, as a defect in reproduction. The main causes responsible for impotence were identified as inability to achieve erection, a very fluid seminal ejaculation, a defective organ, dryness of testicles, and lack of copulative power after many sexual intercourses with healthy women. Some decades later in 1847, Beatty affirmed that one of the most remarkable changes that can be observed in the passage from childhood to maturity is the development of the sexual organs, followed by the appearanee of new sensations. He dis1inguished impotenee that oeeurs only in men from sterility that for him affeets women, and divided the eauses of impotenee into 3 classes: organie, functional and moral (today defmed as psyehologieal). For Beatty, the . progress of knowledge had taken away magie and soreery from the human mind and now it was widespread only ," among the lowest and most ignorant classes of humble people. In this way, he shows the evolution ofmedieal scienee d~ in XIX eentury in aceepting only the rational explanation of the human diseases.

Research in renal physiology began later in the USA than in Europe. Initial studies in the early 1900s dealt with the pathophysiology of renal failure. Micropuncture study of single nephron function was initiated in the laboratory of A.N. Richards in the early 1920s and with the later addition of microperfusion provided important insights into the site and mechanisms of solute transport. In parallel, with the leadership of Homer Smith, the development of noninvasive clearance methods to measure glomerular filtration rate and renal blood flow initiated a fruitful period of quantification of renal hemodynamics and of transport by the tubules. The steady progress of renal physiology in the USA owes much to the generous support of basic reasearch by the National Institutes of Health. It is also worthy of mention that progress in renal physiology in the US owes much to work carried out in clinical departments and to the type of comparative studies exemplified by work at the Mt. Desert Island Biological Laboratory.

pH is an important modulator of the low-conductance ATP-sensitive K+ channel of the distal nephron. To examine the mechanism of interaction of protons with the channel-forming protein, we expressed the cloned renal K channel, ROMK (Kir1.x), in Xenopus oocytes and examined the response to varied concentrations of protons both in the presence and in the absence of ATP. Initial experiments were performed on inside-out patches in the absence of ATP in Mg2+-free solution, which prevents channel rundown. A steep sigmoidal relationship was shown between bath pH and ROMK1 or ROMK2 channel function with intracellular acidification reducing channel activity. We calculated values for p K = 7.18 and 7.04 and Hill coefficients = 3.1 and 3.3, for ROMK1 and ROMK2, respectively. Intracellular acidification (pH 7.2) also increased the Mg-ATP binding affinity of ROMK2, resulting in a leftward shift of the relationship between ATP concentration and the reduction in channel activity. The K 1/2 for Mg-ATP decreased from 2.4 mM at pH 7.4 to ∼0.5 mM at pH 7.2. Mutation of lysine-61 to methionine in ROMK2, which abolishes pH sensitivity, modulated but did not eliminate the effect of pH on ATP inhibition of channel activity. We previously demonstrated that the putative phosphate loop in the carboxy terminus of ROMK2 is involved in ATP binding and channel inhibition [C. M. McNicholas, Y. Yang, G. Giebisch, and S. C. Hebert. Am. J. Physiol. 271 ( Renal Fluid Electrolyte Physiol. 40): F275–F285, 1996]. Conceivably, therefore, protonation of the histidine residue within this region could alter net charge (i.e., positive shift) and increase affinity for the negatively charged nucleotide.

Isoforms of the H-K-ATPase participate in active K resorption in the renal collecting tubule. The cytoplasmic tail of the β-subunit of the gastric H-K-ATPase includes a 4 amino acid motif which is highly homologous to tyrosine-based endocytosis signals. We have generated transgenic mice expressing an H-K-ATPase β-subunit in which the tyrosine residue in this sequence has been mutated to alanine. Mice expressing the mutated protein manifest constitutive hypersecretion of gastric acid, demonstrating that the β-subunit tyrosine-based motif is required for the regulated endocytosis of the H-K pump and hence the cessation of gastric acid output. To test the possibility that the tyrosine-based sequence in the tail of the H-K-ATPase β-subunit plays a role in regulating the function of renal H-K-ATPases, we examined renal K clearance in normal and in transgenic mice. Blood pressure, urine volume, glomerular filtration rate (GFR), plasma Na, and Na excretion are similar in control and transgenic mice. However, plasma K concentrations are significantly higher in transgenic mice (4.76 ± 0.13 meq/l in transgenic and 4.12 ± 0.04 meq/l in control; n = 9, P < 0.05) and K excretion is lower in the transgenic animals (fractional excretion of K was 26.2 ± 3.62% in transgenic and 50.1 ± 4.78% in control; n = 9, P < 0.01). These data suggest that the tyrosine-based signal in the cytoplasmic tail of the H-K-ATPase β-subunit functions in the kidney as it does in the stomach to internalize H-K pump and thus inactivate pump function. Its elimination may result in the constitutive presence of the pump at the cell surface and lead to excessive urinary K reabsorption.

1. The contribution of apical Na+-H+ exchange to sodium reabsorption in the thick ascending limb of the loop of Henle (TALH) in vivo was examined in anaesthetized rats by perfusing loops of Henle of superficial nephrons with solutions containing the Na+-H+ exchange inhibitor, ethyl isopropyl amiloride (EIPA). 2. Using a standard perfusate, no statistically significant effect of EIPA on net sodium reabsorption (JNa) was detected. However, when sodium reabsorption in the pars recta of the proximal tubule was minimized by using a low-sodium perfusate, EIPA reduced JNa from 828 +/- 41 to 726 +/- 37 pmol min-1 (P0.05), indicating that apical Na+-H+ exchange can make a small contribution to net sodium reabsorption in the TALH in vivo. This contribution appears to be dependent on the bicarbonate load, since an increase in the latter led to an enhancement of EIPA-sensitive sodium transport. 3. Addition of the Na+-K+-2Cl- cotransport inhibitor, bumetanide, to the low-sodium perfusate reduced baseline JNa to 86 +/- 27 pmol min-1. In this setting, EIPA reduced JNa further, to -24 +/- 18 pmol min-1 (P0.05), an effect similar to that seen in the absence of bumetanide. This finding argues against previous suggestions (based on in vitro evidence) that inhibition of the Na+-K+-2Cl- cotransporter leads to an increase in apical Na+-H+ exchange in the TALH.

Two mechanisms of H + ion secretion in the proximal tubule that mediate bicarbonate reabsorption have been identified: the brush border Na/H exchanger and electrogenic H + ion secretion. Angiotensin II (AII) has been shown to be a regulator of the luminal Na + /H + exchanger and the basolateral Na + /HCO 3 − cotransporter. In the present study, we examined the effects of AII on H + -ATPase activity in isolated proximal tubule fragments. H + -ATPase activity was assessed by monitoring intracellular pH after Na + removal from the bath. In addition, we investigated the effects on pH recovery of the proton pump inhibitor bafilomycin A 1 , removal of Cl − , and of colchicine. pH was continuously measured with the pH-sensitive fluorescent dye 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recovery of cell pH was observed in the absence of external Na + and was significantly accelerated by AII. The AII-stimulated pH recovery was completely abolished by bafilomycin A 1 , by removal of Cl − , by NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate; a potent Cl − channel blocker], and by colchicine. We conclude from these studies that AII stimulates proton extrusion via H + -ATPase by a Cl − -dependent process involving brush border insertion of vesicles. This process may contribute to up-regulation of HCO 3 − reabsorption along the proximal tubule when tubules are exposed to AII.

NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

The regulation of potassium metabolism involves mechanisms for the appropriate distribution between the intra- and extracellular fluid compartments and for the excretion by the kidney. Clearance and single-nephron studies show that renal excretion is determined by regulated potassium secretion and potassium reabsorption, respectively, in principal and intercalated cells of the distal nephron. Measurement of the electrochemical driving forces acting on potassium transport across individual cell membranes and characterization of several ATPases and potassium channels provide insights into the transport and regulation of renal potassium excretion.

We have used the patch clamp technique to study the effects of inhibiting the apical Na+ transport on the basolateral small-conductance K+ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 microM amiloride decreased the activity of SK, defined as nPo (a product of channel open probability and channel number), to 61% of the control value. Application of 1 microM benzamil, a specific Na+ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na+ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 microM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca2+ because removal of Ca2+ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca2+, we observed that amiloride and benzamil significantly decreased intracellular Ca2+ in the Ca2+-containing solution but had no effect in a Ca2+-free bath. Furthermore, raising intracellular Ca2+ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca2+ are responsible for the effects on SK activity of inhibition of the Na+ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca2+ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP-dependent pathways. Addition of 10 microM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 microM 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca2+-free bath solution. We conclude that Ca2+-dependent NO generation mediates the effect of inhibiting the apical Na+ transport on the basolateral SK in the rat CCD.