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S C Hebert

Publications and source records attributed to S C Hebert.

At least 37 records · Page 2Linked to original sources
PubMed1999

Molecular mechanisms.

The clinically useful and potent distal acting diuretics enhance urinary NaCl excretion by specific inhibition of distinct sodium transport processes in the loop of Henle and distal nephron. When these compounds were first used as diuretics little was known about their cellular mechanisms of action. Physiological investigations over the past 25 years, however, have shown that each class of diuretics inhibits a specific ion transport system in the kidney. Over the past few years, the molecular cloning of the distal diuretic-sensitive Na+ transporters has significantly enhanced our understanding of the mechanism of action of each class of diuretics and has clearly defined the specific protein (and its gene) that is the target for each of these diuretics. The identification of mutations in the genes encoding these transporters in inherited disorders characterized by alterations in salt balance has provided unequivocal evidence for roles of the cloned diuretic-sensitive transporters in sodium homeostasis. Many laboratories are actively engaged in defining the structural sites for ion transport and diuretic binding, and the molecular mechanisms of transport regulation. This information may enable the design of new diuretics and provide the basis for improved use of diuretics. This review will focus on this recent molecular information.

Animals↗
PubMed1998

Stimulation by in vivo and in vitro metabolic acidosis of expression of rBSC-1, the Na+-K+(NH4+)-2Cl- cotransporter of the rat medullary thick ascending limb.

To assess whether metabolic acidosis per se regulates rBSC-1, the rat medullary thick ascending limb (MTAL) apical Na+-K+(NH4+)-2Cl- cotransporter, rat MTALs were incubated for 16 h in an acid 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's medium. Cotransport activity was estimated in intact cells and membrane vesicles by intracellular pH and 22Na+ uptake measurements, respectively; rBSC-1 protein was quantified by immunoblotting analysis and mRNA by quantitative reverse transcription-polymerase chain reaction. As compared with incubation at pH approximately 7.35, acid incubation (pH approximately 7.10) up-regulated by 35-100% rBSC-1 transport activity in cells and membrane vesicles, and rBSC-1 protein and mRNA abundance. In contrast, acid incubation did not alter alkaline phosphatase and Na+/K+-ATPase enzyme activities or beta-actin protein abundance. After 3 h of in vivo chronic metabolic acidosis (CMA) rBSC-1 mRNA abundance increased in freshly harvested MTALs, which was accompanied after 1-6 days of CMA with enhanced rBSC-1 protein abundance. These results demonstrate that both in vivo and in vitro CMA stimulate rBSC-1 expression, which would contribute to the adaptive increase in MTAL absorption and urinary excretion of NH4+ in response to CMA.

Acidosis↗
PubMed1998

Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transports thyroid hormones and taurocholate and comparison with oatp2.

Two complementary DNAs for the organic anion transporter subtypes oatp2 and oatp3, which transport thyroid hormones as well as taurocholate, were isolated from a rat retina cDNA library. The sequence of oatp2 is identical to that recently reported (Noé, B., Hagenbuch, B., Stieger, B., and Meier, P. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10346-10350), whereas the sequence of oatp3 is novel. oatp3 consists of 670 amino acid residues and exhibits a structural architecture common to the organic anion transporter family, possessing the 12 putative membrane-spanning segments. Oocytes injected with oatp2 and oatp3 cRNAs showed taurocholate uptake in a saturable manner. The oatp2 and oatp3 cRNA-injected oocytes also showed significant uptake of both thyroxine and triiodothyronine. Northern blot and in situ analyses showed that the oatp2 mRNA was widely expressed in neuronal cells of the central nervous system, especially in the hippocampus, cerebellum, and choroid plexus as well as in the retina and liver. The oatp3 mRNA was highly expressed in the kidney and moderately abundant in the retina. This suggests that oatp2 and oatp3 are multifunctional transporters involved in the transport of thyroid hormones in the brain, retina, liver, and kidney.

Amino Acid Sequence↗
PubMed1998

The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels.

In the present study, we have used the two-electrode voltage-clamp and patch-clamp techniques to study the effects of forskolin and cAMP on the ROMK1 channels, which are believed to be the native K+ secretory channels in the kidney. Addition of 1 microM forskolin or 100 microM 8-bromo-cAMP, within 10 min, has no significant effect on the current of ROMK1 channels expressed in Xenopus oocytes. In contrast, application of 1 microM forskolin, within 3 min, significantly increased whole-cell K+ current by 35%, when ROMK1 channels were coexpressed with the A kinase anchoring protein AKAP79, which was cloned from neuronal tissue. Two lines of evidence indicate that the effect of forskolin is mediated by a cAMP-dependent pathway: (i) Addition of 100 microM 8-bromo-cAMP mimics the effect of forskolin and (ii) the effect of forskolin and cAMP is not additive. That AKAP is required for the effect of cAMP is further supported by experiments in which addition of ATP (100 microM) and cAMP (100 microM) restored the activity of run-down ROMK1 channels in inside-out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in those that expressed ROMK1 alone. Moreover, when we used RII, the regulatory subunit of type II protein kinase A, in an overlay assay, we identified a RII-binding protein in membranes obtained from the kidney cortex but not in membranes from oocytes. This suggests that the insensitivity of ROMK1 channels to forskolin and cAMP is due to the absence of AKAPs. We conclude that AKAP may be a critical component that mediates the effect of protein kinase A on the ROMK channels in the kidney.

Adenosine Triphosphate↗
PubMed1998

Estradiol enhances thiazide-sensitive NaCl cotransporter density in the apical plasma membrane of the distal convoluted tubule in ovariectomized rats.

Recent data suggest that sex hormones affect the thiazide-sensitive NaCl cotransporter (TSC) density or binding capacity (Chen, Z., D.A. Vaughn, and D.D. Fanestil. 1994. J. Am. Soc. Nephrol. 5:1112-1119). Thus, we determined the effect of ovariectomy (OVX) and estrogen replacement on the ultrastructural localization of TSC in rat kidney using immunocytochemistry. Kidneys of intact female (CON) and OVX rats fed ad libitum for 6 and 9 wk or pair-fed for 9 wk were processed for transmission electron microscopy. Immunogold localization of rat TSC (rTSC1) demonstrated intense label in the apical plasma membrane of CON distal convoluted tubule (DCT). In OVX DCT, rTSC1 label and apical plasma membrane microprojections were decreased. Western blots of renal membrane protein from pair-fed CON and OVX revealed bands at 129-135 kD, but the OVX signal was reduced. Morphometric analyses demonstrated that injecting 10 microg/ kg body weight 17beta-estradiol subcutaneously 4x/wk in OVX rats restored DCT apical microprojections and label density for rTSC1. Thus, in OVX rats (a) rTSC1 immunoreactive renal membrane protein is reduced; (b) apical plasma membrane complexity and immunogold label for rTSC1 in DCT is decreased; and (c) estradiol replacement restores DCT ultrastructure and rTSC1 label to normal. We conclude that estrogen enhances the density of rTSC1 in the DCT, and may alter renal Na transport by this mechanism.

Animals↗
PubMed1998

Novel molecular variants of the Na-K-2Cl cotransporter gene are responsible for antenatal Bartter syndrome.

Antenatal Bartter syndrome is a variant of inherited renal-tubular disorders associated with hypokalemic alkalosis. This disorder typically presents as a life-threatening condition beginning in utero, with marked fetal polyuria that leads to polyhydramnios and premature delivery. Another hallmark of this variant is a marked hypercalciuria and, as a secondary consequence, the development of nephrocalcinosis and osteopenia. We have analyzed 15 probands belonging to 13 families and have performed SSCP analysis of the coding sequence and the exon-intron boundaries of the NKCC2 gene; and we report 14 novel mutations in patients with antenatal Bartter syndrome, as well as the identification of three isoforms of human NKCC2 that arise from alternative splicing.

Amino Acid Sequence↗
PubMed1998

The extracellular calcium-sensing receptor: its role in health and disease.

The recent cloning of an extracellular calcium (Ca2+o)-sensing receptor (CaR) from parathyroid, kidney and other cell types has clarified the mechanisms through which Ca2+o exerts its direct actions on various cells and tissues. In the parathyroid, the CaR mediates the inhibitory effects of Ca2+o on parathyroid hormone (PTH) secretion and likely on expression of the PTH gene and parathyroid cellular proliferation. In the kidney, the receptor mediates direct inhibition of the reabsorption of divalent cations in the cortical thick ascending limb, and it likely underlies the inhibitory actions of hypercalcemia on the urinary-concentrating mechanism in the medullary thick ascending limb and inner medullary collecting duct. The identification of inherited diseases of Ca2+o-sensing that arise from mutations in the CaR gene has proven, by genetic means, the central role of the CaR in mineral ion homeostasis and the importance of the receptor in regulating the parathyroid and kidney. An allosteric CaR agonist ("calcimimetic") is currently being tested for the treatment of primary hyperparathyroidism, and CaR-based therapeutics will likely be applicable to other disorders in which CaRs are under- or overactive. Thus the discovery of the CaR and its associated diseases has documented that Ca2+o plays an essential role as an extracellular first messenger, in addition to serving its better recognized role as an intracellular second messenger.

Absorption↗
PubMed1998

Functional demonstration of Na+-K+-2Cl- cotransporter activity in isolated, polarized choroid plexus cells.

The function of the apical Na+-K+-2Cl- cotransporter in mammalian choroid plexus (CP) is uncertain and controversial. To investigate cotransporter function, we developed a novel dissociated rat CP cell preparation in which single, isolated cells maintain normal polarized morphology. Immunofluorescence demonstrated that in isolated cells the Na+-K+-ATPase, Na+-K+-2Cl- cotransporter, and aquaporin 1 water channel remained localized to the brush border, whereas the Cl-/HCO-3 (anion) exchanger type 2 was confined to the basolateral membrane. We utilized video-enhanced microscopy and cell volume measurement techniques to investigate cotransporter function. Application of 100 microM bumetanide caused CP cells to shrink rapidly. Elevation of extracellular K+ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling was blocked completely by Na+ removal or by addition of 100 microM bumetanide. Exposure of CP cells to 5 mM BaCl2 induced rapid swelling that was inhibited by 100 microM bumetanide. We conclude that the CP cotransporter is constitutively active and propose that it functions in series with Ba2+-sensitive K+ channels to reabsorb K+ from cerebrospinal fluid to blood.

Absorption↗
PubMed1998

Identification and localization of extracellular Ca(2+)-sensing receptor in rat intestine.

The extracellular calcium (Ca2+o)-sensing receptor (CaR) plays vital roles in Ca2+o homeostasis, but no data are available on its expression in small and large intestine. Polymerase chain reaction products amplified from reverse-transcribed duodenal RNA using CaR-specific primers showed > 99% homology with the rat kidney CaR. Northern analysis with a CaR-specific cRNA probe demonstrated 4.1- and 7.5-kb transcripts in all intestinal segments. Immunohistochemistry with CaR-specific antisera showed clear basal staining of epithelial cells of small intestinal villi and crypts and modest apical staining of the former, whereas there was both basal and apical staining of colonic crypt epithelial cells. In situ hybridization and immunohistochemistry also demonstrated CaR expression in Auerbach's myenteric plexus of small and large intestines and in the submucosa in the region of Meissner's plexus. Our results reveal CaR expression in several cell types of small and large intestine, in which it may modulate absorptive and/or secretomotor functions.

Animals↗
PubMed1998

Role of the NH2 terminus of the cloned renal K+ channel, ROMK1, in arachidonic acid-mediated inhibition.

We have previously demonstrated that the ROMK channel maintains the property of arachidonic acid (AA) sensitivity observed originally in the native ATP-sensitive K+ channel of the rat cortical collecting duct (16). We used the patch-clamp technique to extend these studies to other NH2-terminal splice variants of the ROMK channel family, ROMK2 and ROMK3, expressed in Xenopus oocytes to determine the mechanism by which AA inhibits channel activity. Although the conductance, channel open probability, and open/closed times of the three homologs were determined to be similar, addition of 5-10 microM AA caused only a moderate inhibition of ROMK2 (15 +/- 8%) and ROMK3 (13 +/- 9%) activity, indicating that differences in the NH2 termini of ROMK channels strongly influence the AA action. We consequently examined the effect of AA on a ROMK1 variant, R1ND37, in which the NH2 terminal amino acids 2-37 were deleted, and on a mutant ROMK1, R1S4A, in which the serine-4 residue was mutated to alanine. Like ROMK2 and ROMK3, AA had a diminished effect on these variants. Addition of 1 nM exogenous protein kinase C (PKC) inhibited ROMK1 but not the mutant, R1S4A. However, the effect of AA is not a result of stimulation of a membrane bound PKC, since PKC inhibitors, calphostin C and chelerythrine, failed to abolish the AA-induced inhibition. In contrast, application of 5 microM staurosporine, a nonspecific protein kinase inhibitor at high concentration, abolished the effect of AA. We conclude that phosphorylation of serine-4 residue in the NH2 terminus plays a key role in determination of AA effect on ROMK channels.

Alanine↗
PubMed1998

Localization of the extracellular Ca2+/polyvalent cation-sensing protein in rat kidney.

We previously identified transcripts encoding a G protein-coupled, extracellular calcium/polyvalent cation-sensing receptor, RaKCaR, in rat kidney (D. Riccardi, J. Park, W.-S. Lee, G. Gamba, E. M. Brown, and S. C. Hebert. Proc. Natl. Acad. Sci. USA 92:131-135, 1994), which was proposed to provide the mechanism for modulating a variety of renal functions in response to changes in extracellular Ca2+ (E. M. Brown. In: Handbook of Physiology. Bethesda, MD: Am. Physiol. Soc., 1992, sect. 8, vol. 2, chapt. 39, p. 1841-1916; and S. C. Hebert. Kidney Int. 50: 2129-2139, 1996). Here, we examine the cellular and regional distribution of receptor protein by immunofluorescence microscopy using a polyclonal antibody raised against a 22 amino acid region of the NH2 terminus of the receptor. The most intense fluorescence was seen at the basolateral border of cortical thick ascending limb cells. Basolateral staining for the receptor was also detected in medullary thick ascending limbs, in macula densa cells identified by costaining with antibody to brain nitric oxide synthase, NOS-B1, and in distal convoluted tubule cells distinguished by costaining for the apical thiazide-sensitive Na(+)-Cl- cotransporter. Apical anti-RaKCaR staining was detected at the base of the brush border of proximal tubules with decreasing intensity from S1 to S3 segments. In cortical collecting ducts, anti-RaKCaR staining was detected in some, but not all, type A intercalated cells identified by costaining with anti-H(+)-ATPase and anti-AE1 Cl-/HCO3- exchanger antibodies. The present study demonstrates that RaKCaR protein is expressed in many different nephron segments and that the polarity of receptor expression varies with cell type along the nephron. These results suggest potential roles for the extracellular Ca2+/ polyvalent cation-sensing receptor in responding to both circulating and urinary concentrations of divalent minerals and potentially other polyvalent cations (e.g., aminoglycoside antibiotics) to modulate nephron function.

Animals↗
PubMed1998

Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats.

To investigate how hypercalcemia blunts renal concentrating ability, alterations in basal and arginine vasopressin (AVP)-elicited osmotic water (Pf) and urea (Purea) permeabilities were measured in isolated perfused terminal inner medullary collecting ducts (IMCD) from control and chronically hypercalcemic rats after dihydrotachysterol (DHT) (M. Levi, L. Peterson, and T. Berl. Kidney Int. 23: 489-497, 1983) treatment. The IMCD Pf of DHT-treated rats did not increase significantly after AVP and was accompanied by a significant 87 +/- 4% reduction in aquaporin-2 (AQP-2) protein but not mRNA. In contrast, both basal and AVP-elicited IMCD Purea from DHT rats were significantly increased and accompanied by a significant 41 +/- 11% increase in AVP-regulated urea transporter protein content. Immunoblotting with anti-calcium/polyvalent cation-sensing receptor protein (CaR) antiserum revealed specific alterations in CaR bands in endosomes purified from the apical membranes of inner medulla of DHT rats. These data are the first detailed analyses of hypercalcemia-induced alterations in AVP-regulated permeabilities and membrane transporters in IMCD. We conclude that selective alterations in IMCD transport occur in hypercalcemia, permitting the body to dispose of excess calcium without forming calcium-containing renal stones.

Animals↗
PubMed1998

Roles of Na-K-2Cl and Na-Cl cotransporters and ROMK potassium channels in urinary concentrating mechanism.

The cloning of the apical transport proteins involved in NaCl absorption in the thick ascending limb of Henle and the distal convoluted tubule has ushered in a new era that promises to provide molecular details of the urinary concentrating and diluting processes. We now have at hand tools to examine the regulation of these proteins that can affect the concentrating and diluting capacity.

Animals↗
PubMed1998

Partially active channels produced by PKA site mutation of the cloned renal K+ channel, ROMK2 (kir1.2).

The activity of the cloned renal K+ channel (ROMK2) is dependent on a balance between phosphorylation and dephosphorylation. There are only three protein kinase A (PKA) sites on ROMK2, with the phosphorylated residues being serine-25 (S25), serine-200 (S200), and serine-294 (S294) (Z.-C. Xu, Y. Yang, and S. C. Hebert. J. Biol. Chem. 271: 9313-9319, 1996). We previously mutated these sites from serine to alanine to study the contribution of each site to overall channel function. Here we have studied each of these single PKA site mutants using the single-channel configuration of the patch-clamp technique. Both COOH-terminal mutations at sites S200A and S294A showed a decreased open channel probability (Po), whereas the NH2-terminal mutation at site S25A showed no change in Po compared with wild-type ROMK2. The decrease in Po for the S200A and S294A mutants was caused by the additional presence of a long closed state. In contrast, the occurrence of the S25A channel was approximately 66% less, suggesting fewer active channels at the membrane. The S200A and S294A channels had different kinetics compared with wild-type ROMK2 channels, showing an increased occurrence of sublevels. Similar kinetics were observed when wild-type ROMK2 was excised and exposed to dephosphorylating conditions, indicating that these effects are specifically a property of the partially phosphorylated channel and not due to an unrelated effect of the mutation.

Animals↗
PubMed1998

Intrarenal and subcellular localization of rat CLC5.

Dent's disease, an inherited disorder characterized by hypercalciuria, nephrolithiasis, nephrocalcinosis, rickets, low-molecular-weight proteinuria, Fanconi's syndrome, and renal failure, is caused by mutations in the renal chloride channel, CLC5. The normal role of CLC5 is unknown. We have investigated the intrarenal and subcellular localization of CLC5 in rat kidney by in situ hybridization and immunohistochemistry. By in situ hybridization, CLC5 mRNA was detected predominantly in cortical medullary ray and outer medullary tubule epithelial cells. Polyclonal antiserum was generated against a CLC5 fusion protein, affinity purified, and immunoadsorbed against CLC3 and CLC4 to yield a CLC5 isoform-specific antiserum. By immunohistochemistry, CLC5 protein was localized to the intracellular domain of tubular epithelial cells in the S3 segment of the proximal tubule and the medullary thick ascending limb. By subcellular membrane fractionation and flow cytometry, CLC5 expression was found in outer medullary endosomes. These findings are consistent with a model in which CLC5 encodes an endosomal chloride channel that facilitates acidification and trafficking of renal epithelial endosomes.

Animals↗
PubMed1998

pH-dependent modulation of the cloned renal K+ channel, ROMK.

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 pK = 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 K1/2 for Mg-ATP decreased from 2.4 mM at pH 7.4 to approximately 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.

Acids↗
PubMed1998

Electroneutral Na-coupled cotransporter expression in the kidney during variations of NaCl and water metabolism.

The purpose of the present study was to analyze the long-term regulation of renal bumetanide-sensitive Na+-K+-2Cl- cotransporter and thiazide-sensitive Na+-Cl- cotransporter gene expression during changes in NaCl and water metabolism. Male Wistar rats exposed to high or low NaCl intake, saline loading, dehydration, water loading, and furosemide administration during 7 days were studied. Control groups had access to regular food and tap water. Rats were kept in metabolic cages for 4 days before and during the experiment to determine daily urinary electrolyte excretion and osmolarity. At the end of the experiment, creatinine clearance and serum electrolyte levels were also measured. Kidneys were excised and macroscopically subdivided into cortex and outer and inner medulla. Total RNA was extracted from each individual cortex or outer medulla by use of the guanidine/cesium chloride method. The Na+-K+-2Cl- cotransporter expression in outer medulla total RNA was assessed by nonradioactive Northern blot analysis and the Na+-Cl- cotransporter expression in renal cortex total RNA was assessed by semiquantitative polymerase chain reaction. Experimental maneuvers were adequately tolerated, and all groups developed the appropriate renal response to each challenge. However, the level of expression of both cotransporters did not change in any model, except for a 2.8-fold increase in the Na+-Cl- cotransporter expression during dehydration. We conclude that nephron adaptation to 7-day modifications in NaCl and water metabolism does not include changes in the amount of electroneutral sodium-coupled cotransporter gene expression at the mRNA level.

Animals↗