SNAP-23 in rat kidney: colocalization with aquaporin-2 in collecting duct vesicles.

Vesicle targeting proteins ("SNAREs") have been proposed to direct vasopressin-induced trafficking of aquaporin-2 water channels in kidney collecting ducts. A newly identified SNARE protein, SNAP-23, is proposed to mediate vesicle targeting to the plasma membrane in diverse tissues. The current studies were done to determine whether SNAP-23 is expressed in collecting ducts with an intracellular distribution compatible with a role in aquaporin-2 trafficking. RT-PCR demonstrated SNAP-23 mRNA in microdissected collecting ducts and other tubular segments including the proximal tubule and thick ascending limb. Immunoblotting using a polyclonal antibody raised against a COOH-terminal peptide revealed a solitary band at an apparent molecular mass of 30 kDa in renal medullary membrane fractions and inner medullary collecting duct suspensions. Differential centrifugation revealed that SNAP-23 is present in membrane fractions including the low-density fraction enriched in intracellular vesicles. Immunocytochemistry revealed SNAP-23 labeling at both the apex and the cytoplasm of collecting duct principal cells. Immunoblotting of intracellular vesicles immunoisolated using an aquaporin-2 antibody revealed the presence of both SNAP-23 and synaptobrevin-2 (VAMP-2) in aquaporin-2-bearing vesicles. We conclude that SNAP-23 is strongly expressed in collecting duct principal cells, consistent with a role in vasopressin-regulated trafficking of aquaporin-2. However, localization of SNAP-23 in both intracytoplasmic vesicles and plasma membranes suggests a function different from that originally proposed for SNAP-25 in synaptic vesicle targeting.

Expression of synaptotagmin VIII in rat kidney.

The synaptotagmins are a family of integral membrane proteins proposed to function as regulators of both exocytosis and endocytosis. Here, we have used immunochemical techniques and RT-PCR to assess sites of renal expression of synaptotagmin VIII. A polyclonal antibody was raised to a synthetic peptide corresponding to the carboxy-terminal 21 amino acids of mouse synaptotagmin VIII. On immunoblots of membrane fractions from renal cortex and medulla (and in several other tissues), the antibody labeled a 52-kDa band (absent with preimmune IgG). Immunofluorescence localization was carried out in tissue sections from rat kidney. The synaptotagmin VIII antibody labeled early proximal tubules, thin ascending limbs, thick ascending limbs, connecting tubules, and collecting ducts. In collecting ducts, both type A and B intercalated cells exhibited basolateral labeling, whereas principal cells were labeled chiefly in the apical and subapical portion of the cells. Thick ascending limbs were labeled in both the basolateral and apical regions. RT-PCR experiments using total RNA extracted from cortex and medulla or microdissected inner medullary collecting ducts gave a single band of appropriate size. Sequencing of the PCR product confirmed that the amplified target is synaptotagmin VIII. We conclude that synaptotagmin VIII is broadly expressed among renal tubule epithelia, raising the possibility that it is involved in regulation of transport and/or cell remodeling at several sites in the nephron and collecting duct.

Expression of syntaxins in rat kidney.

Previously, we demonstrated that a putative vesicle-targeting protein, syntaxin-4, is expressed in renal collecting duct principal cells and is localized to the apical plasma membrane, suggesting a role in targeting aquaporin-2-containing vesicles to the apical plasma membrane. To investigate whether other syntaxin isoforms are present in the renal collecting duct, we determined the intrarenal localization of syntaxin-2 and -3. Reverse transcription-polymerase chain reaction (RT-PCR) experiments using total RNA extracted from kidney and various organs revealed that both syntaxin-2 and -3 are expressed in kidney cortex and medulla. RT-PCR experiments using microdissected tubules and vascular structures from the kidney revealed that syntaxin-3 mRNA, but not syntaxin-2, is expressed in collecting duct cells. Syntaxin-3 mRNA was also relatively abundant in the thick ascending limb of Henle's loop and in vasa recta. Syntaxin-2 mRNA was found chiefly in glomeruli. To investigate the localization of syntaxin-3 protein, a peptide-derived polyclonal antibody was raised in rabbits. In immunoblotting experiments, this antibody labeled a 37-kDa protein in inner medulla that was most abundant in plasma membrane-enriched subcellular fractions. Immunoperoxidase labeling of thin cryosections combined with immunogold electron microscopy showed that, in contrast to the labeling seen for syntaxin-4, syntaxin-3 labeling in medullary collecting duct was predominantly in the basolateral plasma membrane of intercalated cells. These results suggest the possibility that syntaxin-3 may be involved in selective targeting of acid-base transporters and/or in basolateral membrane remodeling in response to systemic acid-base perturbations.

Calcitonin stimulates H+ secretion in rat kidney intercalated cells.

Calcitonin (CT) modulates rat intercalated cell (IC) functions of the rat cortical collecting duct (CCD) [E. Siga, B. Mandon, N. Roinel, and C. de Rouffignac. Am.J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F221-F227, 1993]. To characterize the specific function regulated by CT, rat CCDs were perfused in vitro. Total CO2 net fluxes (JtCO2, pmol.mm-1.min-1) and transepithelial voltage (Vt) were measured. Bath CT induced a significant tCO2 reabsorption. This effect was higher on CCDs harvested from acid-loaded than from control rats. When HCO3- secretion was blocked, CT also raised JtCO2 and Vt. When H+ secretion was blocked, CT was ineffective on JtCO2 and Vt. When HCO3- secretion was increased and H+ secretion was inhibited, CT did not change JtCO2, whereas isoproterenol (ISO) increased tCO2 secretion from -13.5 +/- 2.0 (control) to -19.0 +/- 2.4 (ISO). In rat CCD studied under these same preceding conditions plus luminal amiloride to block the Na(+)-dependent Vt, CT did not alter Vt, whereas ISO increased it by 4.5 +/- 0.7 mV. We conclude from these data that, in the rat CCD, calcitonin stimulates H+ secretion, likely by so-called alpha-intercalated (alpha-IC) cells, whereas ISO stimulates HCO3- secretion, likely by so-called beta-IC cells.

Syntaxin-4 is localized to the apical plasma membrane of rat renal collecting duct cells: possible role in aquaporin-2 trafficking.

To evaluate the possible role of a putative vesicle-targeting protein, syntaxin-4, in vasopressin-regulated trafficking of aquaporin-2 water channel vesicles to the apical plasma membrane of renal collecting duct cells, we have carried out immunoblotting, immunocytochemistry, and reverse transcription (RT)-PCR experiments in rat kidney. Immunochemical studies used an affinity-purified, peptide-directed polyclonal antibody to rat syntaxin-4. Immunoblots using membrane fractions from inner medullary collecting duct (IMCD) cell suspensions revealed a solitary protein of 36 kD, the expected molecular mass of syntaxin-4. This protein was enriched in a plasma membrane-enriched membrane fraction from IMCD cells. Immunoperoxidase immunocytochemistry in 0.85-microm cryosections from rat inner medulla revealed discrete labeling of the apical plasma membrane of IMCD cells. RT-PCR demonstrated the presence of syntaxin-4 mRNA in microdissected IMCD segments, confirmed by direct sequencing of the PCR product. In addition, RT-PCR experiments demonstrated syntaxin-4 mRNA in glomeruli, vasa recta, connecting tubules, and thin descending limbs of Henle's loops. The demonstrated localization of syntaxin-4 in the apical plasma membrane of collecting duct principal cells, coupled with previous demonstration of syntaxin-4's putative cognate receptor VAMP2 in aquaporin-2-containing vesicles, supports the view that these proteins could play a role of aquaporin-2 vesicle targeting to the apical plasma membrane.

Rat renal arcade segment expresses vasopressin-regulated water channel and vasopressin V2 receptor.

The arcades are long, branched renal tubules which connect deep and mid-cortical nephrons to cortical collecting ducts in the renal cortex. Because they are inaccessible by standard physiological techniques, their functions are poorly understood. In this paper, we demonstrate that the arcades are a site of expression of two proteins, aquaporin-2 (the vasopressin-regulated water channel) and the V2 vasopressin receptor, that are important to regulated water transport in the kidney. Using a peptide-derived polyclonal antibody to aquaporin-2, quantitative ELISA in microdissected segments showed that aquaporin-2 is highly expressed in arcades and that the expression is increased in response to restriction of fluid intake. Immunocytochemistry revealed abundant aquaporin-2 labeling of structures in the cortical labyrinth in a pattern similar to that of the Na(+)-Ca2+ exchanger and kallikrein, marker proteins expressed in arcades but not in cortical collecting ducts. RT-PCR experiments demonstrated substantial aquaporin-2 and V2 receptor mRNA in microdissected arcades. In situ hybridization, using 35S-labeled antisense cRNA probes for the V2 receptor demonstrated strong labeling of both arcades and cortical collecting ducts. Thus, these results indicate that the arcades contain the specific proteins associated with vasopressin-regulated water transport, and may be a heretofore unrecognized site of free water absorption.

Renal aquaporins.

Aquaporins (AQPs) are a newly recognized family of transmembrane proteins that function as molecular water channels. At least four aquaporins are expressed in the kidney where they mediate rapid water transport across water-permeable epithelia and play critical roles in urinary concentrating and diluting processes. AQP1 is constitutively expressed at extremely high levels in the proximal tubule and descending limb of Henle's loop. AQP2, -3 and -4 are expressed predominantly in the collecting duct system. AQP2 is the predominant water channel in the apical plasma membrane and AQP3 and -4 are found in the basolateral plasma membrane. Short-term regulation of collecting duct water permeability by vasopressin is largely a consequence of regulated trafficking of AQP2-containing vesicles to and from the apical plasma membrane.

Molecular analysis of beta-adrenergic receptor subtypes in rat collecting duct: effects on cell cAMP and Ca2+ levels.

Expression and functional properties of beta-adrenergic receptors (beta-ARs) were studied in rat collecting tubules isolated by microdissection. Reverse transcription-polymerase chain reaction experiments demonstrated that the beta 1- and beta 2-AR mRNAs, but not the beta 3-subtype, are expressed in the cortical collecting duct (CCD). Quantitation of mRNAs, carried out using mutant RNAs as internal standards, further showed that beta 1- and beta 2-ARs transcripts are present at comparable amounts in CCD (3,000-4,000 copies/mm of tubular length), but reach 6-8 times lower levels in the outer medullary collecting duct (OMCD: beta 1, 480 +/- 180; beta 2, 590 +/- 110 copies/mm of tubular length). Functional studies, carried out in CCD, corroborated the expression of these two receptor subtypes. The rank order of potency of beta-agonists for stimulating adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was isoproterenol > norepinephrine = epinephrine, and similar efficiencies were found for a beta 1- and a beta 2-antagonist to inhibit isoproterenol-dependent cAMP formation. Fura 2 fluorescence measurements revealed that isoproterenol (10 microM) induces a biphasic rise of intracellular free Ca2+ concentration ([Ca2+]i), consisting of an initial fast increase (delta [Ca2+]i = 122 nM) followed by a plateau phase (delta [Ca2+]i = 58 nM). In the absence of basolateral Ca2+, the initial peak was still observed, suggesting intracellular Ca2+ release. Norepinephrine and epinephrine, as well as selective beta 1- and beta 2-agonists, also increased [Ca2+]i in CCD. Only slight [Ca2+]i variations were produced by isoproterenol in the OMCD (delta [Ca2+]i = 21 nM) and the cortical thick ascending limb (delta [Ca2+]i = 25 nM). These results show that both beta 1- and beta 2-ARs are expressed in the collecting tubule and that they predominate in the CCD. The two receptor subtypes contribute to cAMP accumulation induced by beta-agonists. They also trigger [Ca2+]i variations, indicating their possible coupling to several transduction pathways in the rat CCD.

Inverse PCR-mediated cloning of the promoter for the rat vasopressin V2 receptor gene.

The adenylyl cyclase-coupled vasopressin V2 receptor has been cloned recently and shown, in rats, to be produced from the predominant form of two alternate spliced variants. To begin to unravel the transcriptional regulation of this receptor, we have isolated the 5' flanking region of the rat vasopressin V2 receptor gene and characterized its promoter sequence. The method of inverse polymerase chain reaction (PCR), which allows the amplification of DNA fragments adjacent to a segment of known sequence, was used as an alternative approach to genomic DNA library screening. Using a probe encompassing part of the coding region, first we identified by Southern blot analysis, a single BstX I hybridizing fragment of 2.3 kilobases (kb). This size predicted a BstX I restriction site 1.5 kb upstream to the gene coding region. Cloning of this fragment was accomplished through circularization of BstX I restriction digests and inverse PCR-mediated amplification. Sequence analysis of the gene 5' flanking domain enabled the design of oligonucleotide primers with the usual forward/reverse orientation, and additional clones were generated from native genomic DNA using a high fidelity thermoresistant DNA polymerase. Reverse transcription-PCR (RT-PCR) and primer extension analysis mapped the major transcription start site 422 nucleotides upstream to the translation initiation codon. The promoter region lacks a TATA box but contains a CAAT box and a consensus binding site for transcription factor Sp1. Multiple potential binding sites for the transcription factor PEA3 are clustered in two DNA portions located 0.6 kb and 1 kb upstream to the coding region. In addition, sequences homologous to glucocorticoid response elements are present and might be responsible for the regulation by adrenal steroids of vasopressin-dependent adenylyl cyclase activity in the kidney.

Arachidonic acid inhibits hormone-stimulated cAMP accumulation in the medullary thick ascending limb of the rat kidney by a mechanism sensitive to pertussis toxin.

The possible regulation of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by arachidonic acid (AA) was studied in segments, microdissected from the rat kidney, which are sensitive to arginine vasopressin (AVP). In the presence of 5 microM indomethacin, the addition of 5 microM AA did not impair AVP-dependent cAMP accumulation (measured during 4 min at 35 degrees C) in the cortical or outer medullary collecting tubule, but decreased this response in the thick ascending limb with an inhibition much more pronounced in the medullary portion (MTAL) than in the cortical portion. In MTAL, the response to 10 nM AVP was inhibited by 34.4 +/- 9.6% (SEM) and 65.8 +/- 5.4% with 1 microM and 5 microM AA, respectively, N = 5 experiments. AVP-, glucagon- and calcitonin-sensitive cAMP levels in MTAL were inhibited by 5 microM AA to a similar extent. AA-induced inhibition was unaffected by the presence of inhibitors of AA metabolism: (1) either 10 microM indomethacin or 50 microM ibuprofen added to all media; (2) a 10-min pre-incubation and a 4-min incubation of MTAL samples with 10 microM eicosa-5,8,11,14-tetrayonic acid, (3) a 1-h preincubation with either 30 microM SKF-525A, 20 microM ketoconazole, or 20 microM nordihydroguariaretic acid. In contrast to AA, 11 other saturated or unsaturated fatty acids had no inhibitory effect on the AVP-dependent cAMP level. In fura-2-loaded MTAL samples, AA induced a slow increase of the intracellular calcium concentration ([Ca2+]i) which reached 21.0 +/- 3.8 nM and 92.9 +/- 21.4 nM over basal values (n = 11) at 2 min and 4 min, respectively, after the beginning of the superfusion of 5 microM AA. AA-induced inhibition of AVP-dependent cAMP accumulation was due neither to the increase in [Ca2+]i elicited by AA, nor to an activation of protein kinase C because this inhibition: (1) was not blocked when MTAL samples were incubated either in zero Ca2+ medium, or in the presence of 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid (BAPTA) to chelate [Ca2+]i, and (2) it was not reproduced by a pre-treatment of MTAL segments with a phorbol ester. Pre-incubation of MTAL (6 h at 35 degrees C) with 500 ng/ml pertussis toxin (PTX) prevented AA-induced inhibition: in the presence of PTX inhibition was 24.7 +/- 6.6% vs 10 nM AVP, as compared to 81.6 +/- 4.0% in control groups, i.e in the absence of PTX, N = 6.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular analysis of vasopressin receptors in the rat nephron. Evidence for alternative splicing of the V2 receptor.

Expression and regulation of vasopressin V2 and V1a receptors were studied at the mRNA level in the rat kidney. Two V2 mRNA variants were identified and shown to arise from a single gene by alternative splicing using one donor and two different acceptor sites. The long (V2L) form encodes the adenylyl cyclase-coupled receptor. The short (V2S) form lacks the nucleotide sequence encoding the putative seventh transmembrane domain and undergoes a frame shift in its 3'end coding region; it is inactive on the cyclase pathway in transfected cells. Measurement of mRNAs, carried out by quantitative reverse transcription-polymerase chain reaction (RT-PCR) on microdissected nephrons, demonstrated that neither V2L, V2S nor V1A mRNAs are expressed in glomeruli and proximal tubules (< 100 mRNA copies/glomerulus or mm of tubular length), whereas they are present in the ascending limb of Henle's loop and in the collecting tubule. The V2L mRNA, which is always predominant in these structures, is expressed throughout the collecting tubule at 10 times higher levels (30,000 copies/mm) than in the thin and thick ascending limbs. The ratio of the V2S over V2L mRNA is constant (15%) in all nephron segments; hence high V2S levels are only observed in the collecting tubule. The V1A mRNA is slightly expressed in the thin ascending limb, absent in the thick ascending limb and reaches its maximum in the cortical collecting duct (4,000 copies/mm), before gradually decreasing to undetectable levels in the terminal collecting duct. Finally, in vivo administration of a vasopressin V2 agonist decreased by 50% V2L and V2S mRNAs, but did not alter the V1A mRNA level. We conclude that this study provides the quantitation, on a molar basis, of vasopressin receptor mRNAs in kidney tubules and demonstrates the occurrence of two V2 mRNA spliced variants which are similarly down-regulated.

Insulin stimulates Na+, Cl-, Ca2+, and Mg2+ transports in TAL of mouse nephron: cross-potentiation with AVP.

Insulin (Ins) decreases Na+ delivery in the final urine. To determine whether the loop of Henle participates in this reduction, the effects of Ins were tested on cortical (CTAL) and medullary thick ascending limbs (MTAL) of the mouse nephron, microperfused in vitro. In the MTAL, Ins increased the transepithelial potential difference (Vt) and the Na+ and Cl- net reabsorption fluxes (JNa and JCl, respectively) in a dose-dependent manner, the threshold being below 10(-9) M. At 10(-7) M, Ins reversibly increased JNa and JCl, leaving Mg2+ and Ca2+ fluxes (JMg and JCa, respectively) close to zero. In the CTAL, 10(-7) M Ins reversibly increased Vt, JNa, JCl, JMg, and JCa. In CTAL segments perfused under asymmetrical conditions, with a bath-to-lumen-directed NaCl gradient (lumen 50 mM NaCl, bath 150 mM NaCl), addition of 10(-7) M Ins to the bath resulted in a large increase in JMg and JCa. Thus the responses of CTAL and MTAL to Ins are in all ways similar to those already reported for the adenosine 3',5'-cyclic monophosphate (cAMP)-generating hormones acting on these nephron segments. When 10(-10) M arginine vasopressin (AVP) and 10(-7) M Ins were used in combination, previous addition of one hormone to the bath potentiated the response to the second hormone. In cAMP accumulation experiments, performed in the presence of a phosphodiesterase inhibitor, the amounts of cAMP formed with 10(-7) M Ins and 10(-10) M AVP (which elicit maximal physiological responses in these segments) were in the same range.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+, Mg2+ and K+ transport in the cortical and medullary thick ascending limb of the rat nephron: influence of transepithelial voltage.

Isolated segments of rat cortical (cTAL) and medullary (mTAL) thick ascending limbs were microperfused and the transepithelial net fluxes (JX) were determined by measuring the composition of the collected fluid with an electron microprobe. When perfused with symmetrical solutions both segments showed similar JNa and JCl and lumen-positive transepithelial voltage (Vte = 7-8 mV). JMg, JCa and JK were not significantly different from zero. When perfused with asymmetrical solutions (lumen 50 mM, bath 150 mM NaCl), the mean Vte were 23 mV and 17 mV in the cTAL and mTAL respectively; this rise was accompanied by significant increases in JMg and JCa in the cTAL, but not in the mTAL, and a marked increase in JK in both segments. It is concluded that, in the rat, divalent cations can be reabsorbed in the cTAL, and K+ can be reabsorbed in the cTAL and mTAL. The transport is voltage-dependent. The mTAL can reabsorb neither Mg2+ nor Ca2+, whatever Vte.

Hormonal stimulation of Ca2+ and Mg2+ transport in the cortical thick ascending limb of Henle's loop of the mouse: evidence for a change in the paracellular pathway permeability.

Recent studies from our laboratory have shown that in the cortical thick ascending limb of Henle's loop of the mouse (cTAL) Ca2+ and Mg2+ are reabsorbed passively, via the paracellular shunt pathway. In the present study, cellular mechanisms responsible for the hormone-stimulated Ca2+ and Mg2+ transport were investigated. Transepithelial voltages (PDte) and transepithelial ion net fluxes (JNa, JCl, JK, JCa, JMg) were measured in isolated perfused mouse cTAL segments. Whether parathyroid hormone (PTH) is able to stimulate Ca2+ and Mg2+ reabsorption when active NaCl reabsorption and thus PDte, is abolished by luminal furosemide was first tested. With symmetrical lumen and bath Ringer's solutions, no Ca2+ and Mg2+ net transport was detectable, either in the absence or in the presence of PTH. In the presence of luminal furosemide and a chemically imposed lumen-to-bath directed NaCl gradient, which generates a lumen-negative PDte, PTH slightly but significantly increased Ca2+ and Mg2+ net secretion. In the presence of luminal furosemide and a chemically imposed bath-to-lumen-directed NaCl gradient, which generates a lumen-positive PDte, PTH slightly but significantly increased Ca2+ and Mg2+ net reabsorption. In view of the observed small effect of PTH on passive Ca2+ and Mg2+ movement, a possible interference of furosemide with the hormonal response was considered. To investigate this possibility, Ca2+ and Mg2+ transport was first stimulated with PTH in tubules under control conditions. Then active NaCl reabsorption was abolished by furosemide and the effect of PTH on JCa and JMg measured. In the absence of PDte and under symmetrical conditions, no Ca2+ and Mg2+ transport was detectable, either in the presence or absence of PTH. In the presence of a bath-to-lumen-directed NaCl gradient, Ca2+ and Mg2+ reabsorption was significantly higher in the presence than in the absence of PTH. Finally, when active NaCl transport was not inhibited by furosemide, but reduced by a bath-to-lumen-directed NaCl gradient, PTH strongly increased JCa and JMg, whereas only a small increase in PDte was noted. In conclusion, these data suggest that PTH exerts a dual action on Ca2+ and Mg2+ transport in the mouse cTAL by increasing the transepithelial driving force for Ca2+ and Mg2+ reabsorption through hormone-mediated PDte alterations and by modifying the passive permeability for Ca2+ and Mg2+ of the epithelium, very probably at the level of the paracellular shunt pathway.

Effects of calcitonin on function of intercalated cells of rat cortical collecting duct.

In the rat cortical collecting duct (CCD), the presence of highly specific receptors to calcitonin (CT) coupled to a sensitive adenylate cyclase system suggests that this segment is a target site for CT. Our aim was to explore the effects of CT on the rat CCD microperfused in vitro. The hormone failed to alter the osmotic water permeability and did not affect net Na+ transport but generated a lumen-positive transepithelial potential difference (PDte), which under control conditions was close to zero. This response was dose dependent and was still observed in the presence of luminal amiloride, despite the luminal positivity generated by the Na+ channel blocker (PDte increased from 4.0 +/- 0.8 to 9.5 +/- 1.1 mV). In contrast, the nominal absence of CO2/HCO3- or the use of a low-Cl- solution totally prevented the PDte changes caused by CT. The CT-induced lumen-positive PDte was reduced by 2.3 +/- 0.8 mV after the basolateral addition of the Cl- channel inhibitor diphenylamine-2-carboxylate. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and acetazolamide, which inhibit Cl-/HCO3- exchangers and carbonic anhydrase activities, respectively, also inhibited the CT-induced PDte by 4.6 +/- 0.5 and 5.0 +/- 0.9 mV. To test whether the acid-base status of the animals influences the response to CT, rats underwent an acid or alkali load. CCD dissected from acid-loaded rats responded to CT to the same extent as control animals, but the hormonal action was significantly attenuated when the CCD was harvested from alkali-loaded rats (PDte increases: acid 4.0 +/- 0.3 vs. alkali 1.6 +/- 0.6 mV, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal control of renal magnesium handling.

In the kidney, the main site of magnesium transport is the thick ascending limb of Henle's loop which reabsorbs about 70% of filtered magnesium. In the mouse and rat, this reabsorption takes place essentially in the cortical portion of the thick ascending limb (cTAL). In the medullary portion (mTAL) the transport is not significantly different from zero, irrespective of the voltage, whereas in the cTAL, it is exclusively voltage-dependent. In the cTAL, up to six hormones (including PTH) or agonists can stimulate Mg transport. They are totally inactive in the mTAL. The data obtained in the mouse cTAL indicate that the hormone-dependent increases in magnesium transport result from two synergistic effects: (1) a rise in the transepithelial voltage and (2) an increase in the permeability to magnesium of the paracellular shunt pathway.

Stimulation of NaCl reabsorption by antidiuretic hormone in the cortical thick ascending limb of Henle's loop of the mouse.

The effect of antidiuretic hormone (ADH) on transepithelial Na+ Cl-, Ca2+ and Mg2+ net fluxes (JNa, JCl, JMg, JCa) was investigated in isolated perfused cortical thick ascending limb segments (cTAL) of the mouse nephron, using the microperfusion technique and the electron microprobe analysis to determine the ionic composition of the collected tubular fluid. Simultaneously, the transepithelial potential difference (PDte) and the transepithelial resistance (Rte) were recorded. Prior to the flux measurements cTAL segments were perfused for one hour. During this equilibration period PDte decreased significantly from +19.9 +/- 1.6 to +14.9 +/- 1.1 mV and Rte increased from 30.6 +/- 3.5 omega cm2 to 38.8 +/- 2.4 omega cm2 (n = 7), reflecting a decline in NaCl transport. After ADH was added to the bath solution at 10(-10) mol.l-1, PDte increased from +14.4 +/- 1.1 to +18.0 +/- 1.5 mV, accompanied by a rise in JNa and JCl from 205 +/- 11 to 273 +/- 19 and from 216 +/- 12 to 283 +/- 21 pmol.min-1.mm-1 (n = 7), respectively. JCa and JMg also increased from 0.81 +/- 0.07 to 1.50 +/- 0.12 and from 0.43 +/- 0.11 to 0.76 +/- 0.08 pmol.min-1.mm-1 (n = 7), respectively. All these effects were fully reversible after withdrawal of the hormone. In conclusion our data indicate that ADH stimulates divalent cation transport and NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse.