Biomedical subjects

M Soleimani

Publications and source records attributed to M Soleimani.

At least 37 records · Page 2Linked to original sources

PubMed2000

Pathways for HCO3-reabsorption in mouse medullary collecting duct segments.

To determine pathways of HCO3- reabsorption in the collecting duct of the mouse kidney, the outer medullary collecting duct (OMCD) and the terminal inner medullary collecting duct (IMCDt) were dissected and perfused at 1 to 2 nL/min, and total CO2 was measured by microfluorometry. In the OMCD, net HCO3- flux (JtCO2) was 12.2 +/- 0.7 pmol/min/mm tubule length and decreased to 6.9 +/- 0.6 pmol/min/mm tubule length (n = 5) with 10 mol/L Schering 28080 (SCH) in perfusate (P < .001) and to 7.7 +/- 0.6 pmol/min/mm tubule length (P < .004; n = 4) with 50 micromol/L diethylstilbestrol (DES), an inhibitor of H+-adenosine triphosphatase; together they reduced JtCO2 to 3.7 +/- 0.2 pmol/min/mm tubule length (P = .0002; n = 4). In IMCDt, JtCO2 was 10.9 +/- 1.1 pmol/min/mm tubule length, and it decreased to 4.3 +/- 0.9 pmol/min/mm tubule length (n = 4) with 10 micromol/L SCH in perfusate (P < .05) and to 7.0 +/- 1.1 pmol/min/mm tubule length (P < .05; n = 4) with 50 micromol/L DES; together they decreased JtCO2 to 2.3 +/- 0.3 pmol/min/mm tubule length (P < .002; n = 4). Ouabain (1 mmol/L), an inhibitor of colonic H-K-adenosine triphosphatase (cHKA), in perfusate had no effect on JtCO2 in either segment. Northern hybridization studies showed a high level of expression of gastric HKA (gHKA) in outer medulla and a low level in inner medulla; cHKA expression was undetectable. Thus, in normal mouse OMCD and IMCDt, HCO3- reabsorption is predominantly mediated by gHKA and H+-adenosine triphosphatase and not cHKA. A third isoform of HKA could be present in mouse IMCDt.

Absorption↗

PubMed2000

CFTR induces the expression of DRA along with Cl(-)/HCO(3)(-) exchange activity in tracheal epithelial cells.

Thickening of airway mucus and lung dysfunction in cystic fibrosis (CF) results, at least in part, from abnormal secretion of Cl(-) and HCO(3)(-) across the tracheal epithelium. The mechanism of the defect in HCO(3)(-) secretion is ill defined; however, a lack of apical Cl(-)/HCO(3)(-) exchange may exist in CF. To test this hypothesis, we examined the expression of Cl(-)/HCO(3)(-) exchangers in tracheal epithelial cells exhibiting physiological features prototypical of cystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosis transmembrane conductance regulator (CFTR)] or normal trachea (CFT-1 cells transfected with functional wild-type CFTR, termed CFT-WT). Cells were grown on coverslips and were loaded with the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was monitored. Cl(-)/HCO(3)(-) exchange activity increased by approximately 300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1 cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited by DIDS. The mRNA expression of the ubiquitous basolateral AE-2 Cl(-)/HCO(3)(-) exchanger remained unchanged. However, mRNA encoding DRA, recently shown to be a Cl(-)/HCO(3)(-) exchanger (Melvin JE, Park K, Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274: 22855-22861, 1999.) was abundantly expressed in cells expressing functional CFTR but not in cells that lacked CFTR or that expressed mutant CFTR. In conclusion, CFTR induces the mRNA expression of "downregulated in adenoma" (DRA) and, as a result, upregulates the apical Cl(-)/HCO(3)(-) exchanger activity in tracheal cells. We propose that the tracheal HCO(3)(-) secretion defect in patients with CF is partly due to the downregulation of the apical Cl(-)/HCO(3)(-) exchange activity mediated by DRA.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Potassium deprivation upregulates expression of renal basolateral Na(+)-HCO(3)(-) cotransporter (NBC-1).

The purpose of the present experiments was to examine the effect of potassium deprivation on the expression of the renal basolateral Na(+)-HCO(3)(-) cotransporter (NBC-1). Rats were placed on a K(+)-free diet for various time intervals and examined. NBC-1 mRNA levels increased by about threefold in the cortex (P < 0.04) at 72 h of K(+) deprivation and remained elevated at 21 days. NBC activity increased by approximately 110% in proximal tubule suspensions, with the activity increasing from 0.091 in control to 0.205 pH/min in the K(+)-deprived group (P < 0.005). The inner stripe of outer medulla and cells of medullary thick ascending limb of Henle (mTAL) showed induction of NBC-1 mRNA and activity in K(+)-deprived rats, with the activity in mTAL increasing from 0.010 in control to 0.133 pH/min in the K(+)-deprived group (P < 0.004). K(+) deprivation also increased NBC-1 mRNA levels in the renal papilla (P < 0.02). We conclude that 1) K(+) deprivation increases NBC-1 expression and activity in proximal tubule and 2) K(+) deprivation causes induction of NBC-1 expression and activity in mTAL tubule and inner medulla. We propose that NBC-1 likely mediates enhanced HCO(3)(-) reabsorption in proximal tubule, mTAL, and inner medullary collecting duct in K(+) deprivation and contributes to the maintenance of metabolic alkalosis in this condition.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Early polyuria and urinary concentrating defect in potassium deprivation.

The time course of the onset of nephrogenic diabetes insipidus and its relationship to aquaporin-2 (AQP2) expression in K(+) deprivation (KD) remains unknown. Rats were fed a K(+)-free diet and killed after 12 h, 1, 2, 3, 6, or 21 days. Serum K(+) concentration was decreased only after, but not before, 3 days of a K(+)-free diet. Urine osmolality, however, decreased as early as 12 h of KD (1,061 +/- 26 vs. 1,487 +/- 102 mosmol/kgH(2)O in control, P < 0.01). It decreased further at 24 h (to 858 +/- 162 mosmol/kgH(2)O in KD, P < 0.004) and remained low at 21 days of KD (436 +/- 58 mosmol/kgH(2)O, P < 0.0001 compared with baseline). Water intake decreased at 12 h (P < 0.002) but increased at 24 h (P < 0.05) and remained elevated at 21 days of KD. Urine volume increased at 24 h of KD (8 +/- 2 to 15 +/- 2 ml/24 h, P < 0.05) and remained elevated at 21 days. Immunoblot analysis demonstrated that AQP2 protein abundance in the outer medulla remained unchanged at 12 h (P > 0.05), decreased at 24 h ( approximately 44%, P < 0.001), and remained suppressed ( approximately 52%, P < 0.03) at 21 days of KD. In the inner medulla the AQP2 protein abundance remained unchanged at both 12 and 24 h of KD. AQP2 protein abundance in the cortex, however, decreased at 12 h ( approximately 47%, P < 0.01) and remained suppressed at 24 h ( approximately 77%, P < 0.001) of KD. Northern blot analysis showed that AQP2 mRNA decreased as early as 12 h of KD in both cortex (P < 0.02) and outer medulla (P < 0.01) and remained suppressed afterward. In conclusion, the urinary concentrating defect in KD is an early event and precedes the onset of hypokalemia. These studies further suggest that the very early urinary concentrating defect in KD (after 12 but before 24 h) results primarily from the suppression of cortical AQP2, whereas the later onset of a urinary concentrating defect (after 24 h) also involves a downregulation of medullary AQP2.

Animals↗

PubMed1999

Differential expression of ileal Na(+)/H(+) exchanger isoforms after enterectomy.

BACKGROUND: Na(+)/H(+) exchangers (NHE) are transporters involved in the absorption of NaCl along the gastrointestinal tract. The aim of this study was to determine the expression pattern of the intestinal brush border NHE isoforms 2 and 3 following massive small bowel resection (SBR). Additionally, the effect of epidermal growth factor (EGF) and salivarectomy (removal of the primary source of EGF) on the expression pattern was studied. MATERIALS AND METHODS: ICR mice underwent a proximal SBR or sham surgery and then received either orogastric saline or EGF (50 microg/kg/day). In separate experiments mice underwent salivarectomy followed by SBR or sham. Postoperatively the remaining ileum was isolated and levels of NHE-2 and NHE-3 mRNA and protein were resolved. RESULTS: Following SBR, the expression of both mRNA and protein for NHE-3 increased by approximately 2.5-fold. Treatment with EGF enhanced NHE-3 mRNA in sham animals with further elevation following SBR. The expression of NHE-2 mRNA demonstrated minimal change while protein marginally increased (40%) following SBR. EGF did not affect the expression of NHE-2 mRNA. Salivarectomy did not influence NHE-2 protein expression and inhibited the increased NHE-3 protein expression following SBR. CONCLUSIONS: Following SBR, the expression pattern for brush border NHE isoforms is distinctive. Increased expression of NHE-3 secondary to SBR and/or EGF treatment with loss of this increase following salivarectomy implies a common mechanism to enhance enterocyte proliferation and luminal absorption of NaCl and water. These results suggest that NHE-3 is an important ileal exchanger following SBR.

Animals↗

PubMed1999

NH4+ secretion in inner medullary collecting duct in potassium deprivation: role of colonic H+-K+-ATPase.

UNLABELLED: NH4+ secretion in inner medullary collecting duct in potassium deprivation: Role of colonic H+-K+-ATPase. BACKGROUND: In K+ deprivation (KD), gastric (g) H+-K+-ATPase (HKA) is suppressed, whereas colonic (c) HKA is induced in the terminal inner medullary collecting duct (IMCD). We hypothesized that in KD, cHKA is induced and can mediate the secretion of NH4+. METHODS: Rats were sacrificed after 2, 3, 6, or 14 days on regular (NML) or K+-free (KD) diet. mRNA expression of HKA isoforms in terminal inner medulla was examined and correlated with NH4+ secretion in perfused IMCD in vitro. RESULTS: Urinary NH4+ excretion increased after K+-free diet for six days. In terminal inner medulla, cHKA expression was strongly induced, whereas gHKA expression was decreased. NH4+ secretion increased by 62% in KD (JtNH4+ 0.57 vs. 0.92 pmol/min/mm tubule length, P < 0.001). Ouabain (1 mM) in perfusate inhibited NH4+ secretion in KD by 45% (P < 0.002) but not in NML. At luminal pH 7.7, which inhibits NH3 diffusion, NH4+ secretion in IMCD was 140% higher in KD (0.36 vs. 0.15, P < 0.03) and was sensitive to ouabain. ROMK-1 mRNA expression was induced in parallel with cHKA in inner medulla. CONCLUSIONS: These data suggest that in KD, cHKA replaces gHKA and mediates enhanced secretion of NH4+ (and H+) into the lumen facilitated by K+ recycling through ROMK-1.

Acid-Base Equilibrium↗

PubMed1999

CFTR drives Na+-nHCO-3 cotransport in pancreatic duct cells: a basis for defective HCO-3 secretion in CF.

Pancreatic dysfunction in patients with cystic fibrosis (CF) is felt to result primarily from impairment of ductal HCO-3 secretion. We provide molecular evidence for the expression of NBC-1, an electrogenic Na+-HCO-3 cotransporter (NBC) in cultured human pancreatic duct cells exhibiting physiological features prototypical of CF duct fragments (CFPAC-1 cells) or normal duct fragments [CAPAN-1 cells and CFPAC-1 cells transfected with wild-type CF transmembrane conductance regulator (CFTR)]. We further demonstrate that 1) HCO-3 uptake across the basolateral membranes of pancreatic duct cells is mediated via NBC and 2) cAMP potentiates NBC activity through activation of CFTR-mediated Cl- secretion. We propose that the defect in agonist-stimulated ductal HCO-3 secretion in patients with CF is predominantly due to decreased NBC-driven HCO-3 entry at the basolateral membrane, secondary to the lack of sufficient electrogenic driving force in the absence of functional CFTR.

Amino Acid Sequence↗

PubMed1999

CFTR upregulates the expression of the basolateral Na(+)-K(+)-2Cl(-) cotransporter in cultured pancreatic duct cells.

The purpose of the current experiments was 1) to assess basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) expression and 2) to ascertain the role of cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of this transporter in a prototypical pancreatic duct epithelial cell line. Previously validated human pancreatic duct cell lines (CFPAC-1), which exhibit physiological features prototypical of cystic fibrosis, and normal pancreatic duct epithelia (stable recombinant CFTR-bearing CFPAC-1 cells, termed CFPAC-WT) were grown to confluence before molecular and functional studies. High-stringency Northern blot hybridization, utilizing specific cDNA probes, confirmed that NKCC1 was expressed in both cell lines and its mRNA levels were twofold higher in CFPAC-WT cells than in CFPAC-1 cells (P < 0.01, n = 3). Na(+)-K(+)-2Cl(-) cotransporter activity, assayed as the bumetanide-sensitive, Na(+)- and Cl(-)-dependent NH(+)(4) entry into the cell (with NH(+)(4) acting as a substitute for K(+)), increased by approximately 115% in CFPAC-WT cells compared with CFPAC-1 cells (P < 0.01, n = 6). Reducing the intracellular Cl(-) by incubating the cells in a Cl(-)-free medium increased Na(+)-K(+)-2Cl(-) cotransporter activity by twofold (P < 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cells and mediates the entry of Cl(-). NKCC1 activity is enhanced in the presence of an inward Cl(-) gradient. The results further indicate that the presence of functional CFTR enhances the expression of NKCC1. We speculate that CFTR regulates this process in a Cl(-)-dependent manner.

Biological Transport↗

PubMed1999

H+-K+-ATPases: regulation and role in pathophysiological states.

Molecular cloning experiments have identified the existence of two H+-K+-ATPases (HKAs), colonic and gastric. Recent functional and molecular studies indicate the presence of both transporters in the kidney, which are presumed to mediate the exchange of intracellular H+ for extracellular K+. On the basis of these studies, a picture is evolving that indicates differential regulation of HKAs at the molecular level in acid-base and electrolyte disorders. Of the two transporters, gastric HKA is expressed constitutively along the length of the collecting duct and is responsible for H+ secretion and K+ reabsorption under normal conditions and may be stimulated with acid-base perturbations and/or K+ depletion. This regulation may be species specific. To date there are no data to indicate that the colonic HKA (HKAc) plays a role in H+ secretion or K+ reabsorption under normal conditions. However, HKAc shows adaptive regulation in pathophysiological conditions such as K+ depletion, NaCl deficiency, and proximal renal tubular acidosis, suggesting an important role for this exchanger in potassium, HCO-3, and sodium (or chloride) reabsorption in disease states. The purpose of this review is to summarize recent functional and molecular studies on the regulation of HKAs in physiological and pathophysiological states. Possible signals responsible for regulation of HKAs in these conditions will be discussed. Furthermore, the role of these transporters in acid-base and electrolyte homeostasis will be evaluated in the context of genetically altered animals deficient in HKAc.

Acidosis↗

PubMed1999

Characterization of Na+/HCO-3 cotransporter isoform NBC-3.

Na+-HCO-3 cotransporters mediate the transport of HCO-3 into or out of the cell. Two Na+-HCO-3 cotransporters (NBC) have been identified previously, which are referred to as NBC-1 and NBC-2. A cDNA library from uninduced human NT-2 cells was screened with an NBC-2 cDNA probe. Several clones were identified and isolated. Sequence analysis of these clones identified a partial coding region (2 kb) of a novel NBC (called here NBC-3), which showed 53% and 72% identity with NBC-1 and NBC-2, respectively. Northern blot analysis revealed that NBC-3 encodes a 4.4-kb mRNA with a tissue distribution pattern distinct from NBC-1 and NBC-2. NBC-3 is highly expressed in brain and spinal column, with moderate levels in trachea, thyroid, and kidney. In contrast with NBC-1, NBC-3 shows low levels of expression in pancreas and kidney cortex. In the kidney, NBC-3 expression is predominantly limited to the medulla. Cultured mouse inner medullary collecting duct (mIMCD-3) cells showed high levels of NBC-1 and low levels of NBC-3 mRNA expression. Subjecting the mutagenized mIMCD-3 cells to sublethal acid stress decreased the mRNA expression of NBC-1 by approximately 90% but increased the Na+-dependent HCO-3 cotransport activity by approximately 7-fold (as assayed by DIDS-sensitive, Na+-dependent, HCO-3-mediated intracellular pH recovery). This increase was associated with approximately 5.5-fold enhancement of NBC-3 mRNA levels. NBC showed significant affinity for Li+ in the mutant but not the parent mIMCD-3 cells. On the basis of the widespread distribution of NBC-3, we propose that this isoform is likely involved in cell pH regulation by transporting HCO-3 from blood to the cell. We further propose that enhanced expression of NBC-3 in severe acid stress could play an important role in cell survival by mediating the influx of HCO-3 into the cells.

Acids↗

PubMed1999

HCO-3 reabsorption in renal collecting duct of NHE-3-deficient mouse: a compensatory response.

Mice with a targeted disruption of Na+/H+ exchanger NHE-3 gene show significant reduction in HCO-3 reabsorption in proximal tubule, consistent with the absence of NHE-3. Serum HCO-3, however, is only mildly decreased (P. Schulties, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282-285, 1998), indicating possible adaptive upregulation of HCO-3-absorbing transporters in collecting duct of NHE-3-deficient (NHE-3 -/-) mice. Cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) were perfused, and total CO2 (net HCO-3 flux, JtCO2) was measured in the presence of 10 microM Schering 28080 (SCH, inhibitor of gastric H+-K+-ATPase) or 50 microM diethylestilbestrol (DES, inhibitor of H+-ATPase) in both mutant and wild-type (WT) animals. In CCD, JtCO2 increased in NHE-3 mutant mice (3.42 +/- 0.28 in WT to 5.71 +/- 0.39 pmol. min-1. mm tubule-1 in mutants, P < 0.001). The SCH-sensitive net HCO-3 flux remained unchanged, whereas the DES-sensitive HCO-3 flux increased in the CCD of NHE-3 mutant animals. In OMCD, JtCO2 increased in NHE-3 mutant mice (8.8 +/- 0.7 in WT to 14.2 +/- 0.6 pmol. min-1. mm tubule-1 in mutants, P < 0.001). Both the SCH-sensitive and the DES-sensitive HCO-3 fluxes increased in the OMCD of NHE-3 mutant animals. Northern hybridizations demonstrated enhanced expression of the basolateral Cl-/HCO-3 exchanger (AE-1) mRNA in the cortex. The gastric H+-K+-ATPase mRNA showed upregulation in the medulla but not the cortex of NHE-3 mutant mice. Our results indicate that HCO-3 reabsorption is enhanced in CCD and OMCD of NHE-3-deficient mice. In CCD, H+-ATPase, and in the OMCD, both H+-ATPase and gastric H+-K+-ATPase contribute to the enhanced compensatory HCO-3 reabsorption in NHE-3-deficient animals.

Absorption↗

PubMed1998

NHE-1 is the sodium-hydrogen exchanger isoform present in erythroid cells.

Erythrocyte sodium hydrogen exchanger (NHE) represents one of a limited number of sodium entry pathway in erythrocytes. At least five NHE isoforms have been identified, differing in tissue specificity, regulatory characteristics, and pharmacological sensitivities. Although physiological characteristics of erythrocyte NHE suggest that the widely expressed NHE-1 isoform may be present, evidence is not conclusive and does not exclude the existence of other isoforms. In this study, Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses were used to test for five NHE isoforms in erythroid cells. Blood from patients with sickle cell disease was depleted of white blood cells (WBC) by passage through leukocyte filters and cellulose column. RT-PCR performed on WBC depleted reticulocyte RNA using a NHE-1 primer set yielded product a of expected size, the sequence of which was identical to the published human NHE-1 sequence. Northern blot analysis of the reticulocyte RNA using a 1.6 kb probe revealed a message of approximately 5.0 kb in size. RT-PCR analysis of rat kidney RNA using primers specific for NHE isoforms -2, -3, -4 and rat brain RNA using primer specific for NHE-5 isoform yielded products of expected size, whereas WBC depleted RNA under identical conditions yielded no products. These results identify the erythroid isoform of the sodium hydrogen exchanger as NHE-1.

Animals↗

PubMed1998

Functional characterization of a cloned human kidney Na+:HCO3- cotransporter.

Functional properties of a cloned human kidney Na+:HCO3- cotransporter (NBC-1) were studied in cultured HEK-293 cells that were transiently transfected with NBC-1 cDNA. The Na+:HCO3- cotransporter activity was assayed as the Na+ and HCO3-dependent pHi recovery from intracellular acidosis with the use of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In acid-loaded cells and in the presence of amiloride (to block Na+/H+ exchange), switching to a Na+-containing solution (115 mM) resulted in rapid pHi recovery only in the presence of HCO3-. This recovery was completely abolished by 300 microM 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid. Replacing the Na+ with Li+ (115 mM) caused significant HCO3--dependent, DIDS-sensitive pHi recovery from intracellular acidosis, with Li+ showing lower affinity than Na+. Potassium (K+) had no affinity for the Na+:HCO3- cotransporter. The Na+-dependent HCO3- cotransport was abolished in the presence of 0.2 mM harmaline. The Na+:HCO3- cotransporter could also function in Na+:OH- cotransport mode, although only at high external pH (7.8). Based on functional similarities with the mammalian kidney experiments, we propose that NBC-1 is the proximal tubule Na+:HCO3- cotransporter.

Bicarbonates↗

PubMed1998

Potassium depletion downregulates chloride-absorbing transporters in rat kidney.

Potassium depletion (KD) causes renal chloride wasting, suggesting defect(s) in Cl- reabsorption in renal tubules. To determine whether alterations in expression of the major Cl- transporter genes might contribute to the chloride wasting, we analyzed their expression in renal cortex and medulla of animals placed on KD diet. Feeding KD diet to rats resulted in significant hypokalemia at 14 d but not at 6 d. Northern hybridization revealed that mRNA levels for the apical Na-K-2Cl cotransporter in the medulla decreased by 56 and 51% at 6 and 14 d of KD diet, respectively. Functional studies in tubular suspensions from medullary thick ascending limb demonstrated that the Na-K-2Cl cotransporter activity decreased by approximately 45 and approximately 37% at 6 and 14 d of KD diet, respectively. mRNA levels for the thiazide-sensitive Na-Cl cotransporter decreased by 57 and 64% at 6 and 14 d of KD diet. Decreased expression of the apical Na-Cl and the Na-K-2Cl cotransporters became evident at 48 and 72 h of KD, respectively. Urinary chloride excretion increased at 48 h and further increased at 72 h of KD, correlating with suppression of the Na-Cl and the Na-K-2Cl transporters. Our results indicate that increased urinary chloride loss in KD results from suppression of the chloride-absorbing transporters. Downregulation of chloride transporters in KD is an early event and can lead to hypochloremia and subsequently hypovolemia and decreased glomerular filtration rate.

Animals↗

PubMed1998

Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.

NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

Animals↗

PubMed1998

Colonic H+-K+-ATPase is induced and mediates increased HCO3- reabsorption in inner medullary collecting duct in potassium depletion.

BACKGROUND: Potassium depletion increases HCO3- reabsorption in outer medullary collecting duct (OMCD) by activation of colonic (c) H-K-ATPase (HKA). The purpose of the current experiments was to examine the role of the isoforms of HKA in HCO3- reabsorption by terminal inner medullary collecting duct (IMCD) cells in potassium depletion. METHODS: Sprague-Dawley rats were fed a potassium-free diet and studied after 8 to 10 days. mRNA expression of HKA isoforms in terminal portion of inner medulla was examined and correlated with HCO3- reabsorption in the terminal IMCD. RESULTS: Gastric (g) HKA mRNA decreased whereas colonic (c) HKA mRNA expression was heavily induced in terminal portion of inner medulla in potassium depleted rats. Net HCO3- flux (JtCO2) in terminal IMCD increased in potassium depletion (4.56 to 7.06 pmol/min/mm tubule length, P < 0.001). In normal rats, 1 mM ouabain in perfusate had no effect on JtCO2, whereas 10 microM Schering 28080 (SCH) decreased JtCO2 to 2.4 (P < 0.002). In KD rats, 1 mM ouabain decreased JtCO2 to 4.9 (P < 0.005) and 10 microM SCH decreased JtCO2 to 3.3 (P < 0.001). However, the inhibitory effects of SCH and ouabain on JtCO2 in potassium depleted animals were not additive. CONCLUSIONS: The data indicate that gHKA is suppressed whereas cHKA is induced in potassium depletion and mediates increased HCO3- reabsorption in terminal IMCD. The results further indicate that cHKA in vivo is sensitive to both SCH and ouabain.

Animals↗

PubMed1998

Effect of long-term hyperosmolality on the Na+/H+ exchanger isoform NHE-3 in LLC-PK1 cells.

The effects of long-term exposure to hyperosmotic medium on the Na+/H+ exchanger isoform NHE-3 were examined in cultured renal epithelial cells (LLC-PK1). LLC-PK1 cells were grown to confluence in control medium (310 mOsm/kg H2O) and then either switched to a hyperosmotic medium (510 mOsm/kg H2O; addition of NaCl or mannitol) or maintained in the control medium for 48 hours. The Na+/H+ exchanger activity was then assessed in isosmotic solutions by measurement of amiloride-sensitive acid-stimulated 22Na+ influx or Na+-dependent acid extrusion. Acid-stimulated 22Na+ influx was decreased significantly in cells incubated in hyperosmotic medium (10.5 +/- 0.9 nmol/mg protein, control vs. 5.8 +/- 0.6, hyperosmotic; P < 0.01). Incubation in hyperosmotic medium also decreased the initial rate of Na+-dependent acid extrusion by approximately 60% over the intracellular pH range 6.9 to 7.3. Intracellular buffering power did not differ in the control and hyperosmotic groups. The Na+/H+ exchanger isoform NHE-3 mRNA and protein, assessed by Northern hybridization and immunoblot analysis, respectively, were unchanged in LLC-PK1 cells incubated in hyperosmotic medium compared with controls, suggesting post-translational regulation by high osmolality. These results demonstrate that long-term exposure to hyperosmotic medium causes an adaptive decrease in Na+/H+ exchange (NHE-3) activity in LLC-PK1 cells, and that this effect is unlikely to involve antiporter gene regulation or a change in protein abundance.

Acids↗

PubMed1998

ANG II controls Na(+)-K+(NH4+)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb.

Cell pH was monitored in medullary thick ascending limbs to determine effects of ANG II on Na(+)-K+(NH4+)-2Cl- cotransport. ANG II at 10(-16) to 10(-12) M inhibited 30-50% (P < 0.005), but higher ANG II concentrations were stimulatory compared with the 10(-12) M ANG II level cotransport activity; eventually, 10(-6) M ANG II stimulated 34% cotransport activity (P < 0.003). Inhibition by 10(-12) M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase, or cytochrome P-450-dependent monooxygenase blockade; 10(-12) M ANG II had no effect additive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE). Stimulation by 10(-6) M ANG II was abolished by PLC and protein kinase C (PKC) blockade and was partially suppressed when the rise in cytosolic Ca2+ was prevented. All ANG II effects were abolished by DUP-753 (losartan) but not by PD-123319. Thus < or = 10(-12) M ANG II inhibits via 20-HETE, whereas > or = 5 x 10(-11) M ANG II stimulates via PKC Na(+)-K+(NH4+)-2Cl- cotransport; all ANG II effects involve AT1 receptors and PLC activation.

Angiotensin II↗