Vacuolar H+-ATPase expression is increased in acid-secreting intercalated cells in kidneys of rats with hypercalcaemia-induced alkalosis.

AIMS: Hypercalcaemia is known to be associated with systemic metabolic alkalosis, although the underlying mechanism is uncertain. Therefore, we aimed to examine whether hypercalcaemia was associated with changes in the expression of acid-base transporters in the kidney. METHODS: Rats were infused with human parathyroid hormone (PTH, 15 microg kg(-1) day(-1)), or vehicle for 48 h using osmotic minipumps. RESULTS: The rats treated with PTH developed hypercalcaemia and exhibited metabolic alkalosis (arterial HCO: 31.1 +/- 0.8 vs. 28.1 +/- 0.8 mmol L(-1) in controls, P < 0.05, n = 6), whereas the urine pH of 6.85 +/- 0.1 was significantly decreased compared with the pH of 7.38 +/- 0.1 in controls (P < 0.05, n = 12). The observed alkalosis was associated with a significantly increased expression of the B1-subunit of the H(+)-ATPase in kidney inner medulla (IM, 233 +/- 45% of the control level). In contrast, electroneutral Na(+)-HCO cotransporter NBCn1 and Cl(-)/HCO anion exchanger AE2 expression was markedly reduced in the inner stripe of the outer medulla (to 26 +/- 9% and 65 +/- 6%, respectively). These findings were verified by immunohistochemistry. CONCLUSIONS: (1) hypercalcaemia-induced metabolic alkalosis was associated with increased urinary excretion of H(+); (2) the increased H(+)-ATPase expression in IM may partly explain the enhanced urinary acidification, which is speculated to prevent stone formation because of hypercalciuria and (3) the decreased expression of outer medullary AE2 suggests a compensatory reduction of the transepithelial bicarbonate transport.

Losartan decreases vasopressin-mediated cAMP accumulation in the thick ascending limb of the loop of Henle in rats with congestive heart failure.

INTRODUCTION: Vasopressin (AVP) stimulates sodium reabsorption and Na,K,2Cl-cotransporter (NKCC2) protein level in the thick ascending limb (TAL) of Henle's loop in rats. Rats with congestive heart failure (CHF) have increased protein level of NKCC2, which can be normalized by angiotensin II receptor type-1 (AT(1)) blockade with losartan. AIM: In this study, we investigated whether CHF rats displayed changes in AVP stimulated cAMP formation in the TAL and examined the role of AT(1) receptor blockade on this system. METHOD: CHF was induced by ligation of the left anterior descending coronary artery (LAD). SHAM-operated rats were used as controls. Half of the rats were treated with losartan (10 mg kg day(-1) i.p.). RESULTS: CHF rats were characterized by increased left ventricular end diastolic pressure. Measurement of cAMP in isolated outer medullary TAL showed that both basal and AVP (10(-6) m) stimulated cAMP levels were significantly increased in CHF rats (25.52 +/- 4.49 pmol cAMP microg(-1) protein, P < 0.05) compared to Sham rats (8.13 +/- 1.14 pmol cAMP microg(-1) protein), P < 0.05). Losartan significantly reduced the basal level of cAMP in CHF rats (CHF: 12.56 +/- 1.93 fmol microg(-1) protein vs. Los-CHF: 7.49 +/- 1.08, P < 0.05), but not in Sham rats (SHAM: 4.66 +/- 0.59 vs. Los-SHAM: 4.75 +/- 0.71). AVP-mediated cAMP accumulation was absent in both groups treated with losartan (Los-SHAM: 4.75 +/- 0.71 and Los-CHF: 7.49 +/- 1.08). CONCLUSION: The results indicate that the increased NKCC2 protein level in the mTAL from CHF rats is associated with increased cAMP accumulation in this segment. Furthermore, the finding that AT(1) receptor blockade prevents AVP-mediated cAMP accumulation in both SHAM and CHF rats suggests an interaction between angiotensin II and AVP in regulation of mTAL Na reabsorption.

A SCL4A10 gene product maps selectively to the basolateral plasma membrane of choroid plexus epithelial cells.

The choroid plexus epithelium of the brain ventricular system produces the majority of the cerebrospinal fluid and thereby defines the ionic composition of the interstitial fluid in the brain. The transepithelial movement of Na+ and water in the choroid plexus depend on a yet-unidentified basolateral stilbene-sensitive Na+-HCO3- uptake protein. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the expression in the choroid plexus of SLC4A10 mRNA, which encodes a stilbene-sensitive Na+-HCO3- transporter. Anti-COOH-terminal antibodies were developed to determine the specific expression and localization of this Na+-HCO3- transport protein. Immunoblotting demonstrated antibody binding to a 180-kDa protein band from mouse and rat brain preparations enriched with choroid plexus. The immunoreactive band migrated as a 140-kDa protein after N-deglycosylation, consistent with the predicted molecular size of the SLC4A10 gene product. Bright-field immunohistochemistry and immunoelectron microscopy demonstrated strong labeling confined to the basolateral plasma membrane domain of the choroid plexus epithelium. Furthermore, the stilbene-insensitive Na+-HCO3- cotransporter, NBCn1, was also localized to the basolateral plasma membrane domain of the choroid plexus epithelium. Hence, we propose that the SLC4A10 gene product and NBCn1 both function as basolateral HCO3- entry pathways and that the SLC4A10 gene product may be responsible for the stilbene-sensitive Na+-HCO3- uptake that is essential for cerebrospinal fluid production.

Evidence for a Na+/Ca2+ exchange mechanism in rat peritoneal mast cells.

Mast cells lose their ability to secrete when incubated in nominally Ca2+-free medium, but the Na+/K+ pump inhibitor ouabain prevents this loss, suggesting a Na+ dependence of the Ca2+ gradient in rat mast cells. The present study includes measurements of histamine release from cell suspensions, and fura-2/AM and current-clamp experiments on single cells. KB-R7943, an inhibitor of the reverse mode of the Na+/Ca2+ exchanger, 2,4-dichlorobenzamil and La3+ counteracted the increase in histamine release induced by ouabain in a dose-dependent manner. The Ca2+ response to compound 48/80 was reduced by preincubation of the mast cells for 30 min in nominally Ca2+-free medium. This reduction was partly prevented by ouabain or by a low extracellular Na+ concentration. Superfusion of cells with a medium containing a low Na+concentration resulted in a hyperpolarization of the cells of 38.6+/-8.6 mV, n=8, followed by a repolarization after the superfusion had ceased (45.7+/-5.9 mV, n=4). KB-R7943 reduced the hyperpolarization and repolarization induced by a low extracellular Na+ concentration to 15.5+/-2.9 mV (n=7) and 0.2+/-3.4 mV (n=3), respectively. These results are consistent with the presence of a Na+/Ca2+ exchanger in rat peritoneal mast cells.

Sodium coupled bicarbonate transporters in the kidney, an update.

Recently five genes have been cloned, which code for sodium dependent bicarbonate transport proteins. These genes belong to the SLC4A gene family. This short review summarizes our knowledge of these gene products with respect to their renal distribution and function. The best characterized members are the SLC4A4 and SLC4A7. SLC4A4 codes for an electrogenic Na(+), HCO(3) (-)-cotransporter (NBCe1), which is present in the basolateral membranes of proximal tubules and is responsible for the bicarbonate efflux here, and thus about 80% of the renal bicarbonate reabsorption. SLC4A7 codes for an electroneutral NBC (called NBC3 and NBCn1), which is present basolaterally in the thick ascending limb and the distal part of the collecting ducts and in intercalated cells (either apically or basolaterally) in the connecting and collecting tubules. In the thick ascending limb NBCn1 may be important for NH(4) (+) reabsorption. SLCA5 codes for an electrogenic NBC (called NBC4 and NBCe2), which based on RT-PCR is located to the kidney but the exact localization awaits a good antibody. This is also the case for the SLC4A8 and SLC4A10 gene products, which are sodium dependent Cl(-), HCO(3) (-) exchangers. The recent development in this field substantially increases our understanding of the complex renal regulation of acid base status.

Specific lectin binding to beta1 integrin and fibronectin on the apical membrane of madin-darby canine kidney cells.

Although lectins have previously been used to identify specific cell types in the kidney and various other tissues, the proteins labeled were not identified. We hypothesized that fluorescently labeled lectins could provide a useful tool for direct labeling of membrane-associated glycoproteins. Protein fractions from Madin-Darby canine kidney (MDCK) cells were exposed to a panel of 16 fluorescently labeled lectins to identify suitable lectin-protein pairs. Peanut agglutinin (PNA) selectively bound a 220-240 kDa O-linked glycoprotein with a slightly acidic isoelectric point, while Sambucus nigra agglutinin (SNA) labeled a 130 kDa glycoprotein with a highly acidic isoelectric point. Both proteins were readily labeled by lectins applied to the apical surface of living confluent cells. The proteins were isolated by lectin affinity columns and identified by mass spectrometry. Peptides from the PNA-binding protein shared molecular weight and amino acid composition with fibronectin. Fragments of the SNA-binding protein showed amino-acid identity with peptides from beta1 integrin. The identities of these proteins were validated by Western blotting. Binding of PNA to a 220 kDa protein was inhibited by an anti-fibronectin antibody, and binding of a 130 kDa protein by SNA was diminished by an anti-beta1 integrin antibody. We conclude that PNA and SNA can be used as specific markers for fibronectin and beta1 integrin, respectively, in MDCK cells.

Beta1-integrins in the primary cilium of MDCK cells potentiate fibronectin-induced Ca2+ signaling.

Because beta(1)-integrin is involved in sensing of fluid flow rate in endothelial cells, a function that in Madin-Darby canine kidney (MDCK) cells is confined to the primary cilium, we hypothesized beta(1)-integrin to be an important part of the primary ciliary mechanosensory apparatus in MDCK cells. We observed that beta(1)-integrin, alpha(3)-integrin, and perhaps alpha(5)-integrin were localized to the primary cilium of MDCK cells by combining lectin and immunofluorescence confocal microscopy. beta(1)-Integrin was also colocalized with tubulin to the primary cilia of the rat renal collecting ducts, as well as to the cilia of proximal tubules and thick ascending limbs. Immunogold-electron microscopy confirmed the presence of beta(1)-integrin on primary cilia of MDCK cells and rat collecting ducts. Intracellular Ca(2+) levels, monitored by fluorescence microscopy on fluo 4-loaded MDCK cells, significantly increased on addition of fibronectin, a beta(1)-integrin ligand, to mature MDCK cells with an IC(50) of 0.02 mg/l. In immature, nonciliated cells or in deciliated mature cells, the IC(50) was 0.40 mg/l. Blocking the fibronectin-binding sites of beta(1)-integrin with RGD peptide prevented the Ca(2+) signal. Cross-linking of beta(1)-integrins by Sambucus nigra agglutinin produced a Ca(2+) response similar to the addition of fibronectin. Furthermore, the fibronectin-induced response was not dependent on flow or a flow-induced Ca(2+) response. Finally, the flow-induced Ca(2+) response was not prevented by the fibronectin-induced signal. Although beta(1)-integrin on the primary cilium greatly potentiates the fibronectin-induced Ca(2+) signaling in MDCK cells, the flow-dependent Ca(2+) signal is not mediated through activation of beta(1)-integrin.

The cystic fibrosis transmembrane conductance regulator is not a base transporter in isolated duodenal epithelial cells.

Duodenal epithelial bicarbonate secretion has previously been shown to be greatly impaired in mice deficient of the cystic fibrosis transmembrane conductance regulator (CFTR). It has been proposed that transmembranal bicarbonate transport occurs through the CFTR channel itself. In the present study, the transport of acid/base equivalents across the plasma membrane of proximal duodenal epithelial cells from CFTR deficient mice was compared with that of cells from normal littermates. Mixed epithelial cells from both villi and crypts were isolated from proximal duodenum and intracellular pH was assessed by cuvette-based fluorescence spectrometry using the pH sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. The steady state intracellular pH, the acid extrusion rate and the alkaline extrusion rate were unaffected by CFTR deficiency in the presence of CO(2)/HCO(-)(3). Forskolin had no effect on acid extrusion or alkaline extrusion rates. In control experiments without CO(2)/HCO(-)(3), the intrinsic buffering capacities, the steady state intracellular pH and the acid extrusion rates were equivalent in the cells from CFTR deficient mice and normal littermates. The results are consistent with a model where acid/base transport is almost exclusively mediated by the previously described transporters in the murine duodenum (i.e. Na+/H+ exchange, Cl(-)/HCO(-)(3). exchange and Na+:HCO(-)(3). cotransport). There were no evidence for significant CFTR dependent HCO(-)(3). transport in proximal duodenal epithelial cells of mixed villus and crypt origin.

Molecular and functional evidence for electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters in murine duodenum.

Inward Na(+)-HCO(3)(-) cotransport has previously been demonstrated in acidified duodenal epithelial cells, but the identity and localization of the mRNAs and proteins involved have not been determined. The molecular expression and localization of Na(+)-HCO(3)(-) cotransporters (NBCs) were studied by RT-PCR, sequence analysis, and immunohistochemistry. By fluorescence spectroscopy, the intracellular pH (pH(i)) was recorded in suspensions of isolated murine duodenal epithelial cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Proximal duodenal epithelial cells expressed mRNA encoding two electrogenic NBC1 isoforms and the electroneutral NBCn1. Both NBC1 and NBCn1 were localized to the basolateral membrane of proximal duodenal villus cells, whereas the crypt cells did not label with the anti-NBC antibodies. DIDS or removal of extracellular Cl(-) increased pH(i), whereas an acidification was observed on removal of Na(+) or both Na(+) and Cl(-). The effects of inhibitors and ionic dependence of acid/base transporters were consistent with both inward and outward Na(+)-HCO(3)(-) cotransport. Hence, we propose that NBCs are involved in both basolateral electroneutral HCO(3)(-) transport as well as basolateral electrogenic HCO(3)(-) transport in proximal duodenal villus cells.

NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells.

Na(+)/H(+)-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na(+)-dependent processes to acid extrusion, 2) sensitivity to Na(+)/H(+) exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pH(i)) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na(+) concentration ([Na(+)](o)) during pH(i) recovery decreased H(+) efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na(+). The Na(+)/H(+) exchange inhibitors ethylisopropylamiloride and amiloride inhibited H(+) efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na(+)](o) and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na(+)/H(+) exchange by isoforms NHE1, NHE2, and NHE3.