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ABSTRACT: Acid-secreting intercalated cells respond to changes in systemic pH through regulation of apical H(+) transporters. Little is known about the mechanism by which these cells sense changes in extracellular pH (pH(o)). Pyk2 is a non-receptor tyrosine kinase activated by auto-phosphorylation at Tyr402 by cell-specific stimuli, including decreased pH, and is involved in the regulation of MAPK signaling pathways and transporter activity. We examined whether the Pyk2 and MAPK signaling pathway mediates the response of transport proteins to decreased pH in outer medullary collecting duct cells. Immunoblot analysis of phosphorylated Pyk2 (Tyr402), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) was used to assay protein activation. To examine specificity of kinase activation and its effects, we used Pyk2 siRNA to knockdown Pyk2 expression levels, the Src kinase inhibitor PP 1 to inhibit Pyk2 phosphorylation, and the MEK inhibitor U0126 to inhibit ERK1/2 phosphorylation. The pH-sensitive fluorescent probe BCECF-AM was used to assay H(+) transporter activity. The activity of H(+) transporters was measured as the rate of intracellular pH recovery after an NH(4)Cl pre-pulse. We show that Pyk2 is endogenously expressed and activated by acid pH in mouse-derived outer medullary collecting duct (mOMCD1) cells. Incubation of mOMCD1 cells in acid media (pH(o) 6.7) increased the phosphorylation of Pyk2, ERK1/2 and p38. Reduction in pH(i) induced by an NH(4)Cl pre-pulse also increased the phosphorylation of Pyk2, ERK1/2 and p38. Consistent with our previous studies, we found that mOMCD1 cells exhibit H(+)-ATPase and H(+),K(+)-ATPase activity. Pyk2 inhibition by Pyk2 siRNA and PP 1 prevented Pyk2 phosphorylation as well as H(+)-ATPase-mediated recovery in mOMCD1 cells. In addition, ERK1/2 inhibition by U0126 prevented acid-induced ERK1/2 phosphorylation and H(+)-ATPase-mediated pHi recovery but not phosphorylation of p38. We conclude that Pyk2 and ERK1/2 are required for increasing H(+)-ATPase, but not H(+),K(+)-ATPase activity at decreased pH(i) in mOMCD1 cells.
AJP Renal Physiology 07/2012; · 4.42 Impact Factor
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ABSTRACT: The H(+)-K(+)-ATPase α-subunit (HKα(2)) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα(2) plasma membrane expression is regulated by PKA (Codina J, Liu J, Bleyer AJ, Penn RB, DuBose TD Jr. J Am Soc Nephrol 17: 1833-1840, 2006) and in a separate study demonstrated that genetic ablation of the proton-sensing G(s)-coupled receptor GPR4 results in spontaneous metabolic acidosis (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HKα(2) expression in HEK-293 cells. Chronic acidosis was modeled in vitro by using multiple methods: reducing media pH by adjusting bicarbonate concentration, adding HCl, or by increasing the ambient concentration of CO(2). PKA activity and HKα(2) protein were monitored by immunoblot analysis, and HKα(2) mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα(2) mRNA; however, PKA activity and HKα(2) protein abundance increased when media pH decreased from 7.4 to 6.8. Furthermore, this increase was independent of the method used to create chronic acidosis. Heterologous expression of GPR4 was sufficient to increase both basal and acid-stimulated PKA activity and similarly increase basal and acid-stimulated HKα(2) expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα(2) protein by increasing PKA activity without altering HKα(2) mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα(2) and acid-base balance.
AJP Renal Physiology 06/2011; 301(3):F536-43. · 4.42 Impact Factor
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ABSTRACT: Proton receptors are G protein-coupled receptors that accept protons as ligands and function as pH sensors. One of the proton receptors, GPR4, is relatively abundant in the kidney, but its potential role in acid-base homeostasis is unknown. In this study, we examined the distribution of GPR4 in the kidney, its function in kidney epithelial cells, and the effects of its deletion on acid-base homeostasis. We observed GPR4 expression in the kidney cortex, in the outer and inner medulla, in isolated kidney collecting ducts, and in cultured outer and inner medullary collecting duct cells (mOMCD1 and mIMCD3). Cultured mOMCD1 cells exhibited pH-dependent accumulation of intracellular cAMP, characteristic of GPR4 activation; GPR4 knockdown attenuated this accumulation. In vivo, deletion of GPR4 decreased net acid secretion by the kidney and resulted in a nongap metabolic acidosis, indicating that GPR4 is required to maintain acid-base homeostasis. Collectively, these findings suggest that GPR4 is a pH sensor with an important role in regulating acid secretion in the kidney collecting duct.
Journal of the American Society of Nephrology 10/2010; 21(10):1745-55. · 9.66 Impact Factor
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ABSTRACT: The anion exchanger Pendrin, which is encoded by SLC26A4 (human)/Slc26a4 (mouse) gene, is localized on the apical membrane of non-acid-secreting intercalated (IC) cells in the kidney cortical collecting duct (CCD). To examine its role in the mediation of bicarbonate secretion in vivo and the apical Cl(-)/HCO(3)(-) 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 HCO(3)(-) 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(-)/HCO(3)(-) 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(-)/HCO(3)(-) 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 NH(4)(+) (ammonium) excretion increased significantly, consistent with increased trapping of NH(3) 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(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells in CCD. Additional apical Cl(-)/HCO(3)(-) exchanger(s) is (are) present in the CCD.
AJP Cell Physiology 04/2010; 299(1):C33-41. · 3.54 Impact Factor
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ABSTRACT: Slc26a7 is a member of a family of anion transport proteins, Solute-Linked Carrier 26 (Slc26). Slc26a7, which can mediate Cl-/HCO3- exchange, is expressed in the acid-secreting, A-intercalated cells of the kidney collecting duct. On the basolateral side of the A-intercalated cells, Slc26a7 co-localizes with the anion exchanger 1 (AE1), a Cl-/HCO3- exchanger that mediates bicarbonate reabsorption in the collecting duct.
To test if Slc26a7 is involved in acid-base regulation, as its localization and function suggest, we examined the effect of acid loading and deletion of AE1 on Slc26a7 expression with quantitative real-time RT-PCR and Western blotting.
Four days of acid loading increased Slc26a7 mRNA expression in the kidney inner medulla by 57% (n = 6 acid loaded vs. n = 6 control rats; p < 0.001), whereas mRNA expression in the outer medulla and the cortex did not change. Western blotting analysis demonstrated increased Slc26a7 protein expression in both outer (140%) and inner medulla (50%) in acid-loaded animals (n = 3) compared to controls (n = 3; p < 0.05). The expression of Slc26a7 mRNA was increased by 66% in the kidneys of AE1 knockout mice (n = 5) compared to the wild types (n = 5, p < 0.001). The increase in Slc26a7 mRNA correlated with a twofold increase in protein expression (p < 0.05).
We suggest that the increase in Slc26a7 expression caused by acid challenge and deletion of AE1 represents an adaptive response, indicating that Slc26a7 contributes to the regulation of acid-base balance by the kidney.
Nephron Physiology 02/2008; 109(3):p29-35. · 2.55 Impact Factor
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ABSTRACT: Intercalated cells (ICs) of the kidney collecting duct are rich in carbonic anhydrase II (CAII), which facilitates proton and bicarbonate transport. Bicarbonate secretion is mediated via Pendrin (Slc26a4), which is expressed on the apical membrane of B-ICs and nonA-nonB ICs in the cortical collecting ducts (CCD). Bicarbonate absorption is mediated via anion exchanger 1 (AE1-Slc4a1) in the CCD and via AE1 and possibly Slc26a7 in the OMCD. Both exchangers are expressed on the basolateral membrane of A-ICs. The aim of this study was to examine the expression of pendrin, Slc26a7, and AE1 in the kidneys of CAII-deficient (CAR2-null) mice.
For the expression studies, we used real-time RT-PCR, Northern hybridization, immunolabeling, and immunoblotting.
Pendrin mRNA expression was reduced 63% along with decreased pendrin immunolabeling in the cortex of CAR2-null mice present predominantly in nonA-nonB ICs. Slc26a7 mRNA expression was decreases by 73% and Slc26a7 immunolabeling, present in A-ICs, severely reduced in the outer medulla of CAR2-null mice. AE1 mRNA expression was decreased to a similar degree (62%) along with reduced AE1 immunolabeling. The expression of aquaporin 2 (AQP2) water channel, exclusively present in principal cells of the collecting duct, was comparable in the wild type and CAR2-null mice.
CAII deficiency results in a significant decrease in the gene and protein expression of bicarbonate transport proteins from Slc26 gene family - Slc26a4 (pendrin) and Slc26a7. These results emphasize the critical role of CAII for the maintenance of the intercalated cell phenotype.
Cellular Physiology and Biochemistry 02/2008; 21(1-3):95-108. · 2.86 Impact Factor
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ABSTRACT: Slc26a6 (PAT1, CFEX) is a major chloride/base exchanger located on the apical membrane of the kidney proximal tubule. The purpose of the present study was to examine the effect of Slc26a6 deletion on the apical Na+/H+ exchanger 3 (NHE3) in the straight segment (S3) of the proximal tubule, which is the major site for the reabsorption of filtered chloride in the kidney.
The proximal tubule S3 segment was perfused and the intracellular pH and apical Na+/H+ exchanger activity and expression were measured.
In the proximal tubule straight segments that were microperfused in vitro, baseline intracellular pH, measured by BCPCF-AM, was 7.10 +/- 0.02 in Slc26a6-/- and 7.33 +/- 0.02 in Slc26a6+/+ animals, a significant reduction in Slc26a6 mutant mice (p < 0.00001). The activity of the apical Na+/H+ exchanger was 0.49 +/- 0.02 pH units/min in Slc26a6+/+ and 0.26 +/- 0.03 pH units/min in Slc26a6-/- animals, a significant reduction in Slc26a6-/- mice (p < 0.0001). Formate-induced intracellular alkalinization, which is mediated via NHE3, was significantly blunted in Slc26a6-/- animals, with an alkalinization magnitude of 0.16 pH unit in Slc26a6-/- versus 0.37 in Slc26a6+/+ animals (p < 0.00001, n = 5 separate animals). Angiotensin II stimulation of NHE3 activity was intact in Slc26a6-/- animals. Buffering capacity was comparable in Slc26a6+/+ and Slc26a6-/- mice. Immunoblotting and immunofluorescent labeling demonstrated comparable NHE3 abundance and distribution in kidney proximal tubules of Slc26a6+/+ and Slc26a6-/- mice.
In conclusion, Slc26a6 deletion downregulates the apical Na+/H+ exchanger activity in the straight segment of the proximal tubule. The absence of a significant renal sodium loss in Slc26a6-null mice, despite NHE3 downregulation in the in vitro perfused tubules, points to possible activation of signaling pathways that can stimulate the apical Na+/H+ exchanger in vivo.
American Journal of Nephrology 01/2008; 28(2):330-8. · 2.54 Impact Factor
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ABSTRACT: SLC26A7 is a newly identified basolateral Cl(-)/HCO(3)(-) exchanger specific to alpha-intercalated cells of the outer medullary collecting duct (OMCD). The purpose of the present experiments was to examine the expression of SLC26A7 in kidneys of vasopressin-deficient Brattleboro rats before and after treatment with desamino-Cys(1),d-Arg(8)-vasopressin (dDAVP). Brattleboro rats were treated with dDAVP, a vasopressin analog, for 8 days, and their kidneys were examined for the expression of SLC26A7. The expression of SLC26A7 protein, as examined by immunofluorescence, was undetectable in kidneys of Brattleboro rats. However, treatment with dDAVP induced expression of SLC26A7 protein, restoring it to levels observed in normal rats. These results were verified by Western blot analysis. The mRNA expression of SLC26A7 remained unchanged in response to dDAVP. Immunofluorescent labeling demonstrated abundant levels of anion exchanger type 1 in the OMCD of Brattleboro rats and a mild reduction in response to dDAVP. The abundance of H(+)-ATPase was not affected by dDAVP. The increased SLC26A7 expression directly correlated with enhanced aquaporin-2 expression, which is proportional to increased interstitial osmolarity in the medulla. In conclusion, vasopressin increases the expression of SLC26A7 protein through posttranscriptional mechanisms in the OMCD. The induction of SLC26A7 by vasopressin in OMCD cells of Brattleboro rats is likely an attempt by cells to regulate their cell volume and maintain HCO(3)(-) absorption in a state associated with increased interstitial medullary tonicity.
American journal of physiology. Renal physiology 06/2006; 290(5):F1194-201. · 3.68 Impact Factor
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ABSTRACT: SLC26A7 is a Cl(-)/HCO(3)(-) exchanger that is expressed on the basolateral membrane and in the cytoplasm of two distinct acid-secreting epithelial cells: The A-intercalated cells in the kidney outer medullary collecting duct and the gastric parietal cells. The intracellular localization of SLC26A7 suggests the possibility of trafficking between cell membrane and intracellular compartments. For testing this hypothesis, full-length human SLC26A7 cDNA was fused with green fluorescence protein and transiently expressed in MDCK epithelial cells. In monolayer cells in isotonic medium, SLC26A7 showed punctate distribution throughout the cytoplasm. However, in medium that was made hypertonic for 16 h, SLC26A7 was detected predominantly in the plasma membrane. The presence of mitogen-activated protein kinase inhibitors blocked the trafficking of SLC26A7 to the plasma membrane. Double-labeling studies demonstrated the localization of SLC26A7 to the transferrin receptor-positive endosomes. A chimera that was composed of the amino terminal fragment of SLC26A7 and the carboxyl terminal fragment of SLC26A1, and a C-terminal-truncated SLC26A7 were retained in the cytoplasm in hypertonicity. In separate studies, SLC26A7 showed predominant localization in plasma membrane in potassium-depleted isotonic medium (0.5 or 2 mEq/L KCl) versus cytoplasmic distribution in normal potassium isotonic medium (4 mEq/L). It is concluded that SLC26A7 is present in endosomes, and its targeting to the basolateral membrane is increased in hypertonicity and potassium depletion. The trafficking to the cell surface suggests novel functional upregulation of SLC26A7 in states that are associated with hypokalemia or increased medullary tonicity. Additional studies are needed to ascertain the role of SLC26A7 in enhanced bicarbonate absorption in outer medullary collecting duct in hypokalemia and in acid-base regulation in conditions that are associated with increased medullary tonicity.
Journal of the American Society of Nephrology 05/2006; 17(4):956-67. · 9.66 Impact Factor
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ABSTRACT: The objective of these studies was to examine the effects of long-term vasopressin treatment on acid-base transporters in the collecting duct of rat kidney.
Brattleboro rats were placed in metabolic cages and treated with daily injections of 1-desamino-8-D-arginine vasopressin (dDAVP), a selective V2-receptor agonist, or its vehicle (control) for up to 8 days.
dDAVP treatment resulted in a significant reduction in serum bicarbonate concentration, and caused the upregulation of key ammoniagenesis enzymes, along with increased urinary NH4+ excretion. Northern hybridization and immunofluorescence labeling indicated a significant increase (+80%) in mRNA expression of the apical Cl-/HCO3- exchanger pendrin (PDS), along with a sharp increase in its protein abundance in B-type intercalated cells in the cortical collecting duct in dDAVP-treated rats. In the inner medullary collecting duct, the abundance of basolateral Cl-/HCO3- exchanger (AE1) and apical H+-ATPase was significantly reduced in dDAVP-treated rats. Kidney renin mRNA increased significantly and correlated with an increase in serum aldosterone levels in dDAVP-injected rats. Serum corticosterone levels were, however, reduced and correlated with increased mRNA levels of renal 11beta-hydroxysteroid dehydrogenase-2 (11beta-HSD2) and decreased mRNA expression of 11beta-hydroxylase in the adrenal gland of dDAVP-injected rats.
Chronic administration of dDAVP to Brattleboro rats is associated with the upregulation of PDS and downregulation of H+-ATPase and AE1 in the collecting duct, along with increased ammoniagenesis. Stimulation of the renin-angiotensin-aldosterone system and/or decreased glucocorticoid levels likely plays a role in the transduction of these effects.
American Journal of Nephrology 02/2006; 26(2):194-205. · 2.54 Impact Factor
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ABSTRACT: 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.
AJP Cell Physiology 11/2005; 289(4):C826-35. · 3.54 Impact Factor
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ABSTRACT: 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-/high-Cl- 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 approximately 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.
AJP Cell Physiology 05/2005; 288(4):C957-65. · 3.54 Impact Factor
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ABSTRACT: The Na+-HCO3- cotransporter NBC1 is located exclusively on the basolateral membrane and mediates vectorial transport of bicarbonate in a number of epithelia, including kidney and pancreas. To identify the motifs that direct the targeting of kidney NBC1 to basolateral membrane, wild type and various carboxyl-terminally truncated kidney NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in kidney epithelial cells. GFP was linked to the NH2 terminus of NBC1, and labeling was examined by confocal microscopy. Full-length (1035 aa) and mutants with the deletion of 3 or 20 amino acids from the COOH-terminal end of NBC1 (lengths 1032 and 1015 aa, respectively) showed strong and exclusive targeting on the basolateral membrane. However, the deletion of 26 amino acid residues from the COOH-terminal end (length 1010 aa) resulted in retargeting of NBC1 to the apical membrane. Expression studies in oocytes demonstrated that the NBC1 mutant with the deletion of 26 amino acid residues from the COOH-terminal end is functional. Additionally, the deletion of the last 23 amino acids or mutation in the conserved residue Phe at position 1013 on the COOH-terminal end demonstrated retargeting to the apical membrane. We propose that a carboxyl-terminal motif with the sequence QQPFLS, which spans amino acid residues 1010-1015, and specifically the amino acid residue Phe (position 1013) are essential for the exclusive targeting of NBC1 to the basolateral membrane.
Journal of Biological Chemistry 11/2004; 279(41):43190-7. · 4.77 Impact Factor
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ABSTRACT: SLC26A7 is a recently identified Cl(-)/HCO(3)(-) exchanger that co-localizes with AE1 on the basolateral membrane of Alpha intercalated cells (A-IC) in outer medullary collecting duct (OMCD). The purpose of these studies was to determine whether AE1 and SLC26A7 are differentially regulated in OMCD in pathophysiologic states. Toward this end, the expression and regulation of AE1 and SLC26A7 was examined in water deprivation, a condition known to increase the osmolality of the medulla. Rats were subjected to 3 d of water deprivation while having free access to food. Northern hybridizations demonstrated that in the outer medulla, the mRNA expression of SLC26A7 increased by approximately 300% (P < 0.01 versus control; n = 3), whereas the expression of AE1 decreased by approximately 50% (P < 0.05 versus control, n = 3) in water-deprived rats. Immunoblot analysis studies demonstrated that in the outer medulla, SLC26A7 abundance increased by approximately 3.5-fold (P < 0.02 versus control; n = 3), whereas the AE1 abundance decreased by approximately 55% (P < 0.05 versus control) in water deprivation. The expression of SLC26A7 remained unchanged in the kidney cortex and stomach in water deprivation, indicating the specificity of SLC26A7 upregulation in outer medulla. In situ hybridization indicated the exclusive expression of SLC26A7 in the outer medulla and double immunofluorescence labeling confirmed the co-localization of AE1 and SLC26A7 on the basolateral membrane of A-IC cells in OMCD. It is concluded that AE1 and SLC26A7 are differentially regulated in OMCD in water deprivation. On the basis of these results and previous functional studies indicating the activation of SLC26A7 activity by high osmolality, it is proposed that SLC26A7 may play an important role in bicarbonate reabsorption and or cell volume regulation in OMCD (specifically under hypertonic conditions).
Journal of the American Society of Nephrology 08/2004; 15(8):2002-11. · 9.66 Impact Factor
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ABSTRACT: Formate stimulates sodium chloride and fluid reabsorption in kidney proximal tubule; however, the exact cellular mechanism of this effect remains unknown. We hypothesized that the primary target of formate is the apical Na(+)/H(+) exchanger. Here, we demonstrate that formate directly enhances the apical Na(+)/H(+) exchanger (NHE3) activity in mouse kidney proximal tubule. In the absence of CO(2)/HCO(3)(-), addition of formate (500 microM) to the bath and lumen of microperfused mouse kidney proximal tubule caused significant intracellular alkalinization, with intracellular pH (pH(i)) increasing from baseline levels 7.17 +/- 0.01 to 7.55 +/- 0.01 (P < 0.001, n = 14), with a Delta pH of 0.38 +/- 0.02. Removal of luminal chloride did not block cell pH alkalinization by formate (baseline pH of 7.26 +/- 0.01 to 7.53 +/- 0.01 with formate, P < 0.001, n = 10), indicating that the apical Cl(-)/OH(-) exchanger was not the primary mediator of the effect of formate on cell pH. However, removal of sodium from the lumen or addition of EIPA completely prevented cell pH alkalinization. Addition of formate to the lumen and bath in the outer medullary collecting duct, which does not express any apical Na(+)/H(+) exchanger, did not cause any cell pH alkalinization. At lower concentrations (50 microM), formate caused significant pH(i) alkalinization in proximal tubule cells, with pH(i) increasing from baseline levels 7.15 +/- 0.02 to 7.36 +/- 0.02 (P < 0.02, n = 11). Acetate, at 50 microM, had no effect on pH(i). Formate's effect was observed both in the absence and presence of CO(2)/HCO(3)(-) in the media. We conclude that formate stimulates the apical Na(+)/H(+) exchanger NHE3 in the kidney proximal tubule. We propose that formate stimulation of chloride reabsorption in the proximal tubule is indirect and is secondary to the activation of apical Na(+)/H(+) exchanger NHE3, which then leads to the stimulation of the apical chloride/base exchanger.
American journal of physiology. Renal physiology 08/2004; 287(2):F336-46. · 3.68 Impact Factor
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ABSTRACT: 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.
AJP Cell Physiology 06/2004; 286(5):C1203-11. · 3.54 Impact Factor
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ABSTRACT: The outer medullary collecting duct (OMCD) plays an important role in bicarbonate reabsorption and acid-base regulation. An apical V-type H+-ATPase and a basolateral Cl-/HCO3- exchanger, located in intercalated cells of OMCD, mediate the bicarbonate reabsorption. Here we report the identification of a new basolateral Cl-/HCO3- exchanger in OMCD intercalated cells in rat kidney. Northern hybridizations demonstrated the predominant expression of this transporter, also known as SLC26A7, in the outer medulla, with lower expression levels in the inner medulla. SLC26A7 was recognized as a approximately 90-kDa band in the outer medulla by immunoblot analysis and was localized on the basolateral membrane of a subset of OMCD cells by immunocytochemical staining. No labeling was detected in the cortex. Double-immunofluorescence labeling with the aquaporin-2 and SLC26A7 antibodies or anion exchanger-1 and SLC26A7 antibodies identified the SLC26A7-expressing cells as alpha-intercalated cells. Functional studies in oocytes demonstrated that increasing the osmolality of the media (to simulate the physiological milieu in the medulla) increased the Cl-/HCO3- exchanger activity mediated via SLC26A7 by about threefold (P < 0.02 vs. normal condition). We propose that SLC26A7 is a basolateral Cl-/HCO3- exchanger in intercalated cells of the OMCD and may play an important role in bicarbonate reabsorption in medullary collecting duct.
American journal of physiology. Renal physiology 02/2004; 286(1):F161-9. · 3.68 Impact Factor
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ABSTRACT: The molecular identity of the apical HCO3(-)-secreting transporter in gastric mucous cells remains unknown despite its essential role in preventing injury and ulcer by gastric acid. Here we report the identification of a Cl-/HCO3- exchanger that is located on apical membranes of gastric surface epithelial cells. RT-PCR studies of mouse gastrointestinal tract mRNAs demonstrated that this transporter, known as anion exchanger isoform 4 (AE4), is expressed in both stomach and duodenum. Northern blot analysis of RNA from purified stomach epithelial cells indicated that AE4 is expressed at higher levels in mucous cells than in parietal cells. Immunoblotting experiments identified AE4 as a approximately 110- to 120-kDa protein in membranes from stomach epithelium and apical membranes from duodenum. Immunocytochemical staining demonstrated that AE4 is expressed in apical membranes of surface cells in both mouse and rabbit stomach and duodenum. Functional studies in oocytes indicated that AE4 functions as a Cl-/HCO3- exchanger. These data show that AE4 is an apical Cl-/HCO3- exchanger in gastric mucous cells and duodenal villus cells. On the basis of its function and location, we propose that AE4 may play an important role in mucosal protection.
AJP Gastrointestinal and Liver Physiology 01/2004; 285(6):G1225-34. · 3.43 Impact Factor
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ABSTRACT: SLC26A6 (or putative anion transporter 1, PAT1) is located on the apical membrane of mouse kidney proximal tubule and mediates Cl-/HCO3- exchange in in vitro expression systems. We hypothesized that PAT1 along with a Cl-/HCO3- 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 preadsorbed serum. To examine the functional presence of apical Cl-/HCO3- 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+-HCO3- cotransporter 1) and apical EIPA (to inhibit Na+/H+ exchanger 3), the magnitude of cell acidification in response to addition of luminal Cl- was approximately 5.0-fold higher in the presence than in the absence of CO2/HCO3-. The Cl--dependent base transport was inhibited by approximately 61% in the presence of 0.5 mM luminal DIDS. The presence of physiological concentrations of oxalate in the lumen (200 microM) did not affect the Cl-/HCO3- exchange activity. These results are consistent with the presence of SLC26A6 (PAT1) and Cl-/HCO3- exchanger activity in the apical membrane of rat kidney proximal tubule. We propose that SLC26A6 is likely responsible for the apical Cl-/HCO3- (and Cl-/OH-) exchanger activities in kidney proximal tubule.
AJP Cell Physiology 10/2003; 285(3):C608-17. · 3.54 Impact Factor
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ABSTRACT: The basolateral Cl(-)/HCO(3)(-) exchanger in parietal cells plays an essential role in gastric acid secretion mediated via the apical gastric H(+)-K(+)-ATPase. Here, we report the identification of a new Cl(-)/HCO(3)(-) exchanger, which shows exclusive expression in mouse stomach and kidney, with expression in the stomach limited to the basolateral membrane of gastric parietal cells. Tissue distribution studies by RT-PCR and Northern hybridizations demonstrated the exclusive expression of this transporter, also known as SLC26A7, to stomach and kidney, with the stomach expression significantly more abundant. No expression was detected in the intestine. Cellular distribution studies by RT-PCR and Northern hybridizations demonstrated predominant localization of SLC26A7 in gastric parietal cells. Immunofluorescence labeling localized this exchanger exclusively to the basolateral membrane of gastric parietal cells, and functional studies in oocytes indicated that SLC26A7 is a DIDS-sensitive Cl(-)/HCO(3)(-) exchanger that is active in both acidic and alkaline pH(i). On the basis of its unique expression pattern and function, we propose that SLC26A7 is a basolateral Cl(-)/HCO(3)(-) exchanger in gastric parietal cells and plays a major role in gastric acid secretion.
AJP Gastrointestinal and Liver Physiology 07/2003; 284(6):G1093-103. · 3.43 Impact Factor
