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Anion channels in the retinal pigment epithelium (RPE) play an essential role in the transport of Cl- between the outer retina and the choroidal blood to regulate the ionic composition and volume of the subretinal fluid that surrounds the photoreceptor outer segments. Recently, we reported that the anion conductance of the mouse RPE basolateral membrane is highly selective for the biologically active anion thiocyanate (SCN-), a property that does not correspond with any of the Cl- channels that have been found to be expressed in the RPE to date. The purpose of this study was to determine the extent to which SLC26A7, a SCN- permeable-anion exchanger/channel that was reported to be expressed in human RPE, contributes to the RPE basolateral anion conductance. We show by quantitative RT-PCR that Slc26a7 is highly expressed in mouse RPE compared with other members of the Slc26 gene family and Cl- channel genes known to be expressed in the RPE. By applying immunofluorescence microscopy to mouse retinal sections and isolated cells, we localized SLC26A7 to the RPE basolateral membrane. Finally, we performed whole cell and excised patch recordings from RPE cells acutely isolated from Slc26a7 knockout mice to show that the SCN- conductance and permeability of its basolateral membrane are dramatically smaller relative to wild-type mouse RPE cells. These findings establish SLC26A7 as the SCN--selective conductance of the RPE basolateral membrane and provide new insight into the physiology of an anion channel that may participate in anion transport and pH regulation by the RPE. [ABSTRACT FROM AUTHOR]

To define the stoichiometry and molecular identity of the Cl-/HCO3- exchanger in the apical membrane of pancreatic duct cells, changes in luminal pH and volume were measured simultaneously in interlobular pancreatic ducts isolated from wild-type and Slc26a6-null mice. Transepithelial fluxes of HCO3- and Cl- were measured in the presence of anion gradients favoring rapid exchange of intracellular HCO3- with luminal Cl- in cAMP-stimulated ducts. The flux ratio of Cl- absorption/ HCO3- secretion was∼0.7 in wild-type ducts and∼1.4 in Slc26a6-/- ducts where a different Cl-/HCO3 - exchanger, most likely SLC26A3, was found to be active. Interactions between Cl-/HCO3- exchange and cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated ducts were examined by measuring the recovery of intracellular pH after alkali-loading by acetate prepulse. Hyperpolarization induced by luminal application of CFTRinh-172 enhanced HCO3- efflux across the apical membrane via SLC26A6 in wild-type ducts but significantly reduced HCO3- efflux in Slc26a6-/- ducts. In microperfused wild-type ducts, removal of luminal Cl-, or luminal application of dihydro-4,4=-diisothiocyanatostilbene-2,2=-disulphonic acid to inhibit SLC26A6, caused membrane hyperpolarization, which was abolished in Slc26a6-/- ducts. In conclusion, we have demonstrated that deletion of Slc26a6 alters the apparent stoichiometry of apical Cl-/ HCO3 - exchange in native pancreatic duct. Our results are consistent with SLC26A6 mediating 1:2 Cl-/HCO3 - exchange, and the exchanger upregulated in its absence, most probably SLC26A3, mediating 2:1 exchange. [ABSTRACT FROM AUTHOR]

The NH2 terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH2-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH2 terminus of NBCe1A are important in its accurate targeting to the plasma membrane. [ABSTRACT FROM AUTHOR]

The expression of catabolic enzymes spermidine/spermine N¹-acetyltransferase (SSAT) and spermine oxidase (SMO) increases after ischemic reperfusion injury. We hypothesized that polyamine catabolism is upregulated and that this increase in catabolic response contributes to tissue damage in endotoxin-induced acute kidney injury (AKI). SSAT mRNA expression peaked at threefold 24 h following LPS injection and returned to background levels by 48 h. The activity of SSAT correlated with its mRNA levels. The expression of SMO also increased in the kidney after LPS administration. Serum creatinine levels increased significantly at ~ 15 h, peaking by 24 h, and returned to background levels by 72 h. To test the role of SSAT in endotoxin induced AKI, we injected wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice with LPS. Compared with SSAT-wt mice, the SSAT-ko mice subjected to endotoxic-AKI had less severe kidney damage as indicated by better preservation of kidney function. The role of polyamine oxidation in the mediation of kidney injury was examined by comparing the severity of renal damage in SSAT-wt mice treated with MDL72527, an inhibitor of both polyamine oxidase and SMO. Animals treated with MDL72527 showed significant protection against endotoxin-induced AKI. We conclude that increased polyamine catabolism through generation of by-products of polyamine oxidation contributes to kidney damage and that modulation of polyamine catabolism may be a viable approach for the treatment of endutoxin-induced AKI. [ABSTRACT FROM AUTHOR]

The anion exchanger Pendrin, which is encoded by SLC26A4 (human)/ Slc26a4 (mouse) gene, is localized on the apical membrane of nonacid-secreting intercalated (IC) cells in the kidney cortical collecting duct (CCD). To examine its role in the mediation of bicarbonate secretion in viva and the apical Cl-/HCO3- exchanger in the kidney CCD, mice with genetic deletion of pendrin were generated. The mutant mice show the complete absence of pendrin expression in their kidneys as assessed by Northern blot hybridization. Western blot, and immunofluorescence labeling. Pendrin knockout (KO) mice display significantly acidic urine at baseline [pH 5.20 in KO vs. 6.01 in wild type (WT); P < 0.0001] along with elevated serum HCO3- concentration (27.4 vs. 24 meq/l in KO vs. WT, respectively: P < 0.02), consistent with decreased bicarbonate secretion in vivo. The urine chloride excretion was comparable in WT and KO mice. For functional studies, CCDs were microperfused and IC cells were identified by their ability to trap the pH fluorescent dye BCECF. The apical Cl-/HCO3- exchanger activity in B-IC and non-A, non-B-IC cells, as assessed by intracellular pH monitoring, was significantly reduced in pendrin-null mice. The basolateral Cl-/HCO3- exchanger activity in A-IC cells and in non-A, non-B-IC cells, was not different in pendrin KO mice relative to WT animals. Urine NH4+ (ammonium) excretion increased significantly, consistent with increased trapping of NH3 in the collecting duct in pendrin KO mice. We conclude that Slc26a4 (pendrin) deletion impairs the secretion of bicarbonate in vivo and reduces apical Cl-/HCO3- exchanger activity in B-IC and non-A, non-B-IC cells in CCD. Additional apical Cl-/HCO3- exchanger(s) is (are) present in the CCD. [ABSTRACT FROM AUTHOR]

CFTR has been recognized to function as both an anion channel and a key regulator of Slc26 anion transporters in heterologous expression systems. Whether this regulatory relationship between CFTR and Slc26 transporters is seen in native intestine, and whether this effect is coupled to CFTR transport function or other features of this protein, has not been studied. The duodena of anesthetized CFTR-, NHE3-, Slc26a6-, and Scl26a3-deficient mice and wild-type (WT) littermates were perfused, and duodenal bicarbonate (HCO3-) secretion (DBS) and fluid absorptive or secretory rates were measured. The selective NHE3 inhibitor S1611 or genetic ablation of NHE3 significantly reduced fluid absorptive rates and increased DBS. Slc26a6 (PATI) or Slc26a3 (DRA) ablation reduced the S1611-induced DBS increase and reduced fluid absorptive rates, suggesting that the effect of S1611 or NHE3 ablation on HCO secretion may be an unmasking of Slc26a6- and Slc26a3- mediated CI-/HCO3- exchange activity. In the absence of CFTR expression or after application of the CFTR(inh)-172, fluid absorptive rates were similar to those of WT, but S161 1 induced virtually no increase in DBS, demonstrating that CFTR transport activity, and not just its presence, is required for Slc26-mediated duodenal HCO3- secretion. A functionally active CFTR is an absolute requirement for Slc26-mediated duodenal HCO3- secretion, but not for Slc26-mediated fluid absorption, in which these transporters operate in conjunction with the Na+/H+ exchanger NHE3. This suggests that Slc26a6 and Slc26a3 need proton recycling via NHE3 to operate in the Cl- absorptive mode and CI- exit via CFTR to operate in the HCO3- secretory mode. [ABSTRACT FROM AUTHOR]

Resistance to cadmium (Cd)-induced testicular necrosis is an autosornal recessive trait defined as the Cdm locus. Using positional cloning, we previously identified the Slc39a8 (encoding an apical-surface ZIP8 transporter protein) as the gene most likely responsible for the phenotype. In situ hybridization revealed that endothelial cells of the testis vasculature express high ZIP8 levels in two sensitive inbred mouse strains and negligible amounts in two resistant strains. In the present study, we isolated a 168.7-kb bacterial artificial chromosome (BAC), carrying only the Slc39a8 gene, from a Cd-sensitive 129/SvJ BAC library and generated BAC-transgenic mice. The BTZIP8-3 line, having three copies of the I 29/SvJ Slc39a8 gene inserted into the Cd-resistant C57BL/6J genome (having its normal two copies of the Slc39a8 gene), showed tissue-specific ZIP8 mRNA expression similar to wild-type mice, mainly in lung, testis, and kidney. The ~2.5-fold greater expression paralleled the fact that the BTZIP8-3 line has five copies, whereas wild-type mice have two copies, of the Slc39a8 gene. The ZIP8 mRNA and protein localized especially to endothelial cells of the testis vasculature in BTZIP8-3 mice. Cd treatment reversed Cd resistance (seen in nontransgenic littermates) to Cd sensitivity in BTZIP8-3 mice; reversal of the testicular necrosis phenotype confirms that Slc39a8 is unequivocally the Cdm locus. ZIP8 also localized specifically to the apical surface of proximal tubule cells in the BTZIP8-3 kidney. Cd treatment caused acute renal failure and signs of proximal tubular damage in the BTZIP8-3 but not nontransgenic littermates. BTZIP8-3 mice should be a useful model for studying Cd-induced disease in kidney. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion injury (IRI) in liver and other organs is manifested as an injury phase followed by recovery and resolution. Control of cell growth and proliferation is essential for recovery from the injury. We examined the expression of three related regulators of cell cycle progression in liver IRI: spermidine/spermine N-acetyltransferase (SSAT), p21 (a cyclin-dependent kinase inhibitor), and stathmin. Mice were subjected to hepatic IRI, and liver tissues were harvested at timed intervals. The expression of SSAT, the rate-limiting enzyme in the polyamine catabolic pathway, had increased fivefold 6 h after IRI and correlated with increased putrescine levels in the liver, consistent with increased SSAT enzymatic activity in IRI. The expression of p21, which is transactivated by p53, was undetectable in sham-operated animals but was heavily induced at 12 and 24 h of reperfusion and declined to undetectable baseline levels at 72 h of reperfusion. The interaction of the polyamine pathway with the p53-p21 pathway was shown in vitro, where activation of SSAT with polyamine analog or the addition of putrescine to cultured hepatocytes induced the expression of p53 and p21 and decreased cell viability. The expression of stathmin, which is under negative transcriptional regulation by p21 and controls cell proliferation and progression through mitosis, remained undetectable at 6, 12, and 24 h of reperfusion and was progressively and heavily induced at 48 and 72 h of reperfusion. Double-immunofluorescence labeling with antibodies against stathmin and PCNA, a marker of cell proliferation, demonstrated colocalization of stathmin and PCNA at 48 and 72 h of reperfusion in hepatocytes, indicating the initiation of cell proliferation. The distinct and sequential upregulation of SSAT, p21, and stathmin, along with biochemical activation of the polyamine catabolic pathway in IRI in vivo and the demonstration of p53-p21 upregulation by SSAT and putrescine in vitro, points to the important role of regulators of cell growth and cell cycle progression in the pathophysiology and/or recovery in liver IRI. The data further suggest that SSAT may play a role in the initiation of injury, whereas p21 and stathmin may be involved in the resolution and recovery after liver IRI. [ABSTRACT FROM AUTHOR]

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylon) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9. [ABSTRACT FROM AUTHOR]

SLC26A6 (PAT1, CFEX) is an anion exchanger that is expressed on the apical membrane of the kidney proximal tubule and the small intestine. Modes of transport mediated by SLC26A6 include Cl-/formate exchange, Cl-/HCO3- exchange, and Cl-/oxalate exchange. To study its role in kidney and intestinal physiology, gene targeting was used to prepare mice lacking Slc26a6. Homozygous mutant Slc26a6-/- mice appeared healthy and exhibited a normal blood pressure, kidney function, and plasma electrolyte profile. In proximal tubules microperfused with a low-HCO3-/highCl- solution, the baseline rate of fluid absorption (Jv), an index of NaCl transport under these conditions, was the same in wild-type and null mice. However, the stimulation of Jv by oxalate observed in wild-type mice was completely abolished in Slc26a6-null mice (P < 0.05). Formate stimulation of Jv was partially reduced in null mice, but the difference from the response in wild-type mice did not reach statistical significance. Apical membrane Cl-/base exchange activity, assayed with the pH-sensitive dye BCPCF in microperfused proximal tubules, was decreased by 58% in Slc26a6-/- animals (P < 0.001 vs. wild types). In the duodenum, the baseline rate of HCO3- secretion measured in mucosal tissue mounted in Ussing chambers was decreased by ∼30% (P < 0.03), whereas the forskolin-stimulated component of HCO3- secretion was the same in wild-type and Slc26a6-/- mice. We conclude that Slc26a6 mediates oxalate-stimulated NaCl absorption, contributes to apical membrane Cl-/base exchange in the kidney proximal tubule, and also plays an important role in HCO3- secretion in the duodenum. [ABSTRACT FROM AUTHOR]

Ischemic renal injury can be classified into the initiation and extension phase followed by the recovery phase. The recovery phase is characterized by increased dedifferentiated and mitotic cells in the damaged tubules. Suppression subtractive hybridization was performed by using RNA from normal and ischemic kidneys to identify the genes involved in the physiological response to ischemia-reperfusion injury (IRI). The expression of stathmin mRNA increased by fourfold at 24 h of reperfusion. The stathmin mRNA did not increase in sodium-depleted animals or in animals with active, persistent injury secondary to cis-platinum. Immunofluorescent labeling demonstrated that the expression of stathmin increased dramatically at 48 h of reperfusion. Labeling with antibodies to stathmin and proliferating cell nuclear antigen (PCNA) indicates that the expression of stathmin was induced before the upregulation of PCNA and that all PCNA-positive cells expressed stathmin. Double immunofluorescent labeling demonstrated the colocalization of stathmin with vimentin, a marker of dedifferentiated cells. Stathmin expression was also significantly enhanced in acute tubular necrosis in humans. On the basis of its induction profile in IRI, the data indicating its enhanced expression in proliferating cells and regenerating organs, we propose that stathmin is a marker of dedifferentiated, mitotically active epithelial cells that may contribute to tubular regeneration and could prove useful in distinguishing the injury phase from recovery phase in IRI. [ABSTRACT FROM AUTHOR]

Metabolic acidosis is associated with alteration in fluid and electrolyte reabsorption in a number of nephron segments. However, the effects of metabolic acidosis on urine osmolality and aquaporin-2 (AQP-2) remain poorly understood. In these studies, we examined the effects of chronic metabolic acidosis on water handling by the kidney. Rats were placed in metabolic cages and subjected to water (control) or 280 mM NH4CI loading for 120 h to induce metabolic acidosis. The results indicated a significant increase in urine osmolality with no change in urine volume or urinary Na+ excretion in acid-loaded animals. This effect was independent of alteration in fluid intake or salt/C1- loading. Immunoblotting and Northern hybridization studies indicated that AQP-2 protein abundance and mRNA expression levels increased significantly along the collecting duct system of NH4CI- but not NaC1-loaded animals. RIA results indicated that metabolic acidosis was associated with a fourfold increase in circulating levels of vasopressin (AVP) and a significant increase in brain AVP mRNA expression levels. In conclusion, metabolic acidosis upregulates the expression levels of AQP-2 and increases urine osmolality, suggesting an adaptive increase in water reabsorption in the collecting duct. A concomitant increase in AVP synthesis and secretion likely plays an essential role in the adaptation of AQP-2 in metabolic acidosis. [ABSTRACT FROM AUTHOR]

SLC26A6 (or putative anion transporter 1, PAT1) is located on the apical membrane of mouse kidney proximal tubule and mediates Cl[sup -]/HCO[sup -, sub 3] exchange in in vitro expression systems. We hypothesized that PAT1 along with a Cl[sup -]/HCO[sup -, sub 3] exchange is present in apical membranes of rat kidney proximal tubules. Northern hybridizations indicated the exclusive expression of SLC26A6 (PAT1 or CFEX) in rat kidney cortex, and immunocytochemical staining localized SLC26A6 on the apical membrane of proximal tubules, with complete prevention of the labeling with the pre-adsorbed serum. To examine the functional presence of apical Cl[sup -]/HCO[sup -, sub 3] exchanger, proximal tubules were isolated, microperfused, loaded with the pH-sensitive dye BCPCF-AM, and examined by digital ratiometric imaging. The pH of the perfusate and bath was kept at 7.4. Buffering capacity was measured, and transport rates were calculated as equivalent base flux. The results showed that in the presence of basolateral DIDS (to inhibit Na[sup +]HCO[sup -, sub 3] cotransporter 1) and apical EIPA (to inhibit Na[sup +]/H[sup +] exchanger 3), the magnitude of cell acidification in response to addition of luminal Cl[sup -] was ∼5.0-fold higher in the presence than in the absence of CO[sub 2]/HCO[sup -, sub 3]. The Cl[sup -]-dependent base transport was inhibited by ∼61% in the presence of 0.5 mM luminal DIDS. The presence of physiological concentrations of oxalate in the lumen (200 µM) did not affect the Cl[sup -]/HCO[sup -, sub 3] exchange activity. These results are consistent with the presence of SLC26A6 (PAT1) and Cl[sup -]/HCO[sup -, sub 3] exchanger activity in the apical membrane of rat kidney proximal tubule. We propose that SLC26A6 is likely responsible for the apical Cl[sup -]/HCO[sup -, sub 3] (and Cl[sup -]/OH[sup -]) exchanger activities in kidney proximal tubule. [ABSTRACT FROM AUTHOR]

Studies the role of cystic fibrosis transmembrane conductance regulator (CFTR) in the expression of basolateral cotransporter in cultured pancreatic duct cells. Use of the Northern blot hybridization; Cloning of cotransporters; Enhancement of the expression of the cotransporters due to CFTR.

Examines the effects of angiotensin II (ANG II) on sodium cotranmsport system. Concentration-dependent effects of ANG II; Mediation of the arachidonic acid pathway of the ANG II inhibitory effect; Role of protein kinase C in the stimulatory effect of ANG II.

Presents a letter to the editor replying to a commentary on an article about the regulation of aquaporin-2 by metabolic acidosis.

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylon) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9. [ABSTRACT FROM AUTHOR]