Endothelin-1 stimulates the Na+/H+ and Na+/HCO3- transporters in rabbit renal cortex.

Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor identified to date, raising the strong possibility of its involvement in the pathogenesis of systemic hypertension. Whether ET-1 exerts a direct stimulating effect on sodium reabsorption in the renal proximal convoluted tubule, the dominant locus of sodium reabsorption in the nephron, is currently unknown. Such an effect would suggest yet another mechanism by which ET-1 might mediate systemic hypertension. In studies on membrane vesicles prepared from rabbit renal cortex, we show that ET-1 (10(-8) to 10(-11) M) exerts dose-dependent stimulation of the apical Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter; preincubation of vesicles with 10(-10) M ET-1 for five minutes enhanced the activity of each transporter by approximately 25%. This stimulation reflected an increase in the Vmax of each transporter but no change in the Km for sodium. The stimulatory effect of ET-1 was blocked in the presence of an ET-1 antiserum. Moreover, the stimulation of the apical Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter by ET-1 displayed specificity as indicated by the lack of effects on the activities of the apical Na(+)-glucose transporter and the basolateral Na(+)-succinate transporter. The data implicate ET-1 as a novel, direct and specific modulator of sodium reabsorption in the proximal tubule. As such, ET-1 might be a direct determinant of extracellular fluid volume under normal and pathophysiologic circumstances, including hypertensive disorders.     

Albumin regulates the Na+/H+ exchanger 3 in OKP cells

Albumin filtered by the glomerulus is reabsorbed in the proximal tubule. We have shown previously that proteinuria stimulates the proximal tubular Na(+)/H(+) exchanger 3 (NHE3) in rats. Activation of NHE3 may be a pathophysiologically important factor in the development of renal salt and water retention observed in the nephrotic syndrome. For examining whether albumin is a specific inducer of proximal tubular Na(+)/H(+) exchange and to determine the molecular mechanisms by which it regulates Na(+)/H(+) exchange, the effect of albumin on NHE3 in opossum kidney cells was studied. Albumin activated Na(+)/H(+) exchange in a time- and dose-dependent manner up to 100% in 48 h. In the early phase of stimulation (2 to 12 h), NHE3 activity was increased without changes in NHE3 protein and mRNA. At 24 h, increased NHE3 activity was accompanied by increase in cell surface NHE3 protein. The increase in surface NHE3 was associated with increased bidirectional trafficking of NHE3 protein between intracellular compartments and the cell surface. At 48 h, total cell NHE3 protein abundance and mRNA were increased as well. Whereas NHE3 translation was increased, NHE3 protein half-life remained unchanged. The effects of albumin on NHE3 protein abundance were modified by hydrocortisone in a complicated pattern. These results indicate that albumin directly regulates proximal tubular NHE3 at multiple levels.     

Red cell Na+/Li+ countertransport and Na+/H+ exchanger isoforms in human proximal tubules

BACKGROUND: Increased activity of the Na+/Li+ countertransporter (SLC) is a well-recognized intermediate phenotype of hypertension and diabetic nephropathy and may indicate a predisposition to hypertension. Previous work has attempted to link this membrane transport marker to altered Na+ reabsorption in the proximal tubule. Since the Na+/H+ exchanger (NHE) isoforms 1 and 3 are expressed in the basolateral and apical membranes of the proximal tubule, respectively, we investigated the relationship between these transport proteins and red cell SLC to examine whether the peripheral blood transport phenotype is associated with altered levels of transport proteins in the proximal tubule. METHODS: Proximal tubules were prepared from human nephrectomy specimens. NHE-1 and NHE-3 were detected on Western blots by specific antibodies. Red cell SLC was also measured. RESULTS: Both NHE-1 and NHE-3 proteins were demonstrated, with molecular weights of 97 and 85 kD, respectively. SLC was very strongly correlated with the level of NHE-3 protein (r = 0.78, P < 0.001) and was negatively related to NHE-1 protein (r = -0.32). In multiple regression analysis, only NHE-3 and NHE-1 protein levels were significant predictors of red cell SLC, accounting for up to about 70% of the variance of this parameter. CONCLUSIONS: We conclude that red cell SLC may be a marker of increased NHE-3 protein expression in the proximal tubule, which may account for the blunted pressure natriuresis and predisposition to hypertension.     

Na+/H+ exchangers in renal regulation of acid-base balance

The kidney plays key roles in extracellular fluid pH homeostasis by reclaiming bicarbonate (HCO(3)(-)) filtered at the glomerulus and generating the consumed HCO(3)(-) by secreting protons (H(+)) into the urine (renal acidification). Sodium-proton exchangers (NHEs) are ubiquitous transmembrane proteins mediating the countertransport of Na(+) and H(+) across lipid bilayers. In mammals, NHEs participate in the regulation of cell pH, volume, and intracellular sodium concentration, as well as in transepithelial ion transport. Five of the 10 isoforms (NHE1-4 and NHE8) are expressed at the plasma membrane of renal epithelial cells. The best-studied isoform for acid-base homeostasis is NHE3, which mediates both HCO(3)(-) absorption and H(+) excretion in the renal tubule. This article reviews some important aspects of NHEs in the kidney, with special emphasis on the role of renal NHE3 in the maintenance of acid-base balance.     

Bimodal acute effects of A1 adenosine receptor activation on Na+/H+ exchanger 3 in opossum kidney cells

Regulation of renal apical Na+/H+ exchanger 3 (NHE3) activity by adenosine has been suggested to contribute to acute control of mammalian Na(+) homeostasis. The mechanism by which adenosine controls NHE3 activity in a renal cell line was examined. The adenosine analog, N(6)-cyclopentyladenosine (CPA) exerts a bimodal effect on NHE3: CPA concentrations >10(-8) M inactivate NHE3, whereas concentrations <10(-8) M stimulate NHE3 activity. Acute CPA-induced control of NHE3 was blocked by antagonists of A1 adenosine receptors and inhibition of phospholipase C, pretreatment with BAPTA-AM (chelator of cellular calcium), and exposure to pertussis toxin. Stimulatory and to some extent also inhibitory CPA concentrations attenuated 8-bromo-cAMP and dopamine-mediated inhibition of NHE3. BAPTA eliminated the ability of a stimulatory dose of CPA to attenuate 8-bromo-cAMP-induced suppression of NHE3 activity. Upon inhibition of protein kinase C, CPA at an inhibitory dose provoked activation of NHE3, which is partially reverted by 8-bromo-cAMP and suppressed by pre-incubation with BAPTA-AM. Cytochalasin B, an actin-modifying agent, selectively prevented downregulation but did not affect upregulation of NHE3 activity by CPA. In conclusion, these observations demonstrate that (1) CPA modulates NHE3 activity by elevation of cellular Ca(2+) exerting a negative control on adenylate cyclase activity, (2) protein kinase C is the determining factor leading to CPA-induced downregulation of NHE3 activity, and (3) alterations of surface NHE3 abundance may contribute to A1 adenosine receptor-dependent inhibition of NHE3 activity.     

Subcellular distribution of Na+/H+ antiport activity in rat renal cortex

Phase partitioning analyses of a brush border membrane preparation obtained with a divalent cation precipitation procedure (Am J Physiol 246:F853-F858, 1984) confirmed that Na+/H+ antiport activity was localized primarily to the brush border membrane of the rabbit proximal tubular epithelial cell. This analysis also indicated that antiport activity was associated with membrane populations that appeared to be derived from cytoplasmic structures. However, since the starting point of the analysis was a partially-purified brush border sample rather than a total membrane sample, it was not possible to discern the magnitude of the potential cytoplasmic pool of antiport activity. We have now used a three dimensional analytical fractionation procedure, based on differential centrifugation, equilibrium density gradient centrifugation, and partitioning in an aqueous polymer 2-phase system, to survey the subcellular distribution of Na/H antiport activity in rat kidney cortex. Roughly 53% of the recovered antiport activity could be assigned to a population of brush border membrane vesicles characterized by a 15-fold enrichment of maltase. An additional 26% of the recovered activity could be assigned to a group of three membrane populations whose biochemical characteristics appeared equally consistent with origins in distinct microdomains of the brush border membrane and with origins in microdomains of the Golgi complex involved in the assembly or recycling of brush border membrane constituents. Therefore, depending on the identities of membranes which contained the secondary pool of Na+/H+ antiport activity, no more than one-third of the total recoverable Na+/H+ antiport activity could be assigned to cytoplasmic membranes of the proximal tubular epithelium.     

Lipids and cellular Na+/H+ antiport activity in diabetic nephropathy.

Increased cellular Na+/H+ antiport activity has been documented in various cell types from hypertensive humans and rats. This membrane abnormality may be associated with the thickening of the vascular media of resistance vessels. Such an abnormality has also been demonstrated in cells from type I diabetic patients with nephropathy, and may indicate the predisposition to essential hypertension in such patients. We now demonstrate the importance of the rate-limiting enzyme for cholesterol and isoprenoid synthesis, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, in determining cellular Na+/H+ antiport activity. This finding may have application in the future treatment of diabetic patients with proteinuria.     

Ischemic preconditioning and Na+/H+ exchange inhibition improve reperfusion ion homeostasis

BACKGROUND: Intramyocyte sodium (Na+) increases during ischemia and reperfusion, which causes myocardial calcium (Ca2+) uptake and leads to myocyte injury or death. This study determines if ischemic preconditioning and myocyte sodium-hydrogen ion (Na+-H+) exchange (NHE) inhibition decreases Na+ gain that otherwise occurs with cardioplegic arrest and reperfusion. METHODS: Pigs had 1 hour of cardioplegic arrest followed by reperfusion. Group 1 had no intervention (controls). Group 2 received dimethyl amiloride (DMA, an NHE inhibitor), and group 3 had ischemic preconditioning before cardioplegic arrest. Precardioplegia to postreperfusion change in intramyocyte ion content was measured with atomic absorption spectrometry. The time to initial electrical activity and number of defibrillations needed to establish an organized rhythm postreperfusion were used as electrophysiologic variables to measure ischemia-reperfusion injury. RESULTS: Intramyocyte Na+ content for group 1 increased from 45.9+/-6.7 to 61.9+/-22.5 micromol/g (p = 0.02). Group 2 had an insignificant decrease in intramyocyte Na+ of 27.7+/-19.58 micromol/g (p = 0.06), and group 3 had an insignificant decrease of 10.8+/-46.33 micromol/g (p = 0.48). Interstitial water increased significantly in all groups, but there were no significant increases in intramyocyte water content. Electrophysiologic recovery was similar for all three groups. CONCLUSIONS: The NHE inhibition and ischemic preconditioning each eliminated the increase in intramyocyte Na+ content that otherwise occurred with cardioplegic arrest and reperfusion in this porcine model. Because their mechanisms are distinct, it is possible that an additive beneficial effect against ischemia-reperfusion injury can be achieved by using NHE inhibition together with a preconditioning stimulus as prereperfusion therapy.     

Cyclosporin A stimulates apical Na+/H+ exchange in LLC-PK1/PKE20 proximal tubular cells

Cyclosporin A (CyA) causes renal Na⁺ retention which may lead to arterial hypertension. The apical Na⁺/H⁺ exchanger (NHE3) is responsible for bulk proximal tubular Na⁺ reabsorption. The aim of this study was to investigate the effects of CyA on the NHE3 of polarized proximal tubular cells to evaluate cellular mechanisms of CyA-associated arterial hypertension. The change of the intracellular pH (Δ-[pH]_i/min) was determined as a measure of the activity of the NHE in LLC-PK₁/PKE₂₀ cells using 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The NHE activity was identified as the apical NHE3 since it could be inhibited by the inhibitor S3226, but not by inhibitors of the basolateral isoform (NHE1) amiloride or HOE 694. CyA stimulated the NHE3 activity dose dependently. The mean increase stimulated by relevant CyA concentrations was 61±11%. A 24-h application of CyA also stimulated an increase of NHE3 activity which did not seem to be mediated by an increase of NHE3 RNA expression. The less immunosuppressive derivatives cyclosporin H and cyclosporin G caused NHE3 activation as well. Carbachol and ATP, which both induce a Ca²⁺ release from internal Ca²⁺ stores, also increased the NHE3 activity. The Ca²⁺ chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,-N′,N′-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) abolished the CyA-associated NHE3 stimulation, whereas low extracellular Ca²⁺ had no effect. CyA-associated effects did not seem to be mediated via inhibition of protein kinase C (PKC). CyA had no additive effects on the angiotensin II-associated NHE3 stimulation. Concurrent application of losartan did not impair the CyA-induced NHE3 stimulation. In conclusion CyA stimulates the apical NHE3 in proximal tubular cells. This is mediated by Ca²⁺ release from intracellular stores but is independent of the action of angiotensin II or PKC.     

Na+/H+ exchangers: physiology and link to hypertension and organ ischemia

PURPOSE OF REVIEW: Na/H exchangers (NHEs) are ubiquitous proteins with a very wide array of physiological functions, and they are summarized in this paper in view of the most recent advances. Hypertension and organ ischemia are two disease states of paramount importance in which NHEs have been implicated. The involvement of NHEs in the pathophysiology of these disorders is incompletely understood. This paper reviews the principal findings and current hypotheses linking NHE dysfunction to hypertension and ischemia. RECENT FINDINGS: With the advent of large-scale sequencing projects and powerful in-silico analyses, we have come to know what is most likely the entire mammalian NHE gene family. Recent advances have detailed the roles of NHE proteins, exploring new functions such as anchoring, scaffolding and pH regulation of intracellular compartments. Studies of NHEs in disease models, even though not conclusive to date, have contributed new evidence on the interplay of ion transporters and the delicate ion balances that may become disrupted. SUMMARY: This paper provides the interested reader with a concise overview of NHE physiology, and aims to address the implication of NHEs in the pathophysiology of hypertension and organ ischemia in light of the most recent literature.     

Luminal regulation of Na+/H+ exchanger gene expression in rat ileal mucosa

It is well recognized that ileostomy patients suffer from chronic depletion of Na⁺ through the stoma effluent. In this study we evaluated the effects of ileostomy on messenger RNA levels that encode different Na⁺/H⁺ exchanger isoforms (NHE-2 and NHE-3). Loop ileostomies were created in Sprague-Dawley rats. Segments of diverted ileum were harvested for quantitation of mRNA levels encoding these isoforms and the Na⁺/K⁺ ATPase in mucosal scrapings and for immunofluorescence microscopy, specifically of the NHE-3 protein. Our studies indicate that as early as 8 days after diversion, NHE-3 gene expression is selectively attenuated in poststomal ileal mucosa. Mucosal morphology remains undisturbed, and the distribution of protein expression along the crypt/villus axis is not altered. Infusion of Na⁺ or the enterocyte nutrient, glutamine, into the lumen of the diverted segment restores or even augments mRNA levels for NHE-3, again without altering the histologic appearance or distribution of the protein along the crypt/ villus axis. These effects are specific because nonpolar osmolytes (mannitol) and related organic nutrients not specific for the enterocyte (i.e., butyrate) have no effect on mRNA levels of NHE-3. Further work is required to understand how the early changes in mRNA contribute to mucosal function and response to luminal diversion. Present at the Forty-Second Annual Meeting of The Society for Surgery of the Alimentary Tract in Atlanta, Georgia, May 20–23, 2001 (poster presentation). Supported by grant RO1 DK 44571-11 from the National Institutes of Health and by funds from the Brigham Surgical Group Foundation.     

Effects of Na(+)-HCO3- symport on ischemia/reperfusion injury; Na+/H+ exchanger inhibitor and buffer composition

We studied the impact of perfusate buffer composition on the relative degree of protection afforded by Na+/H+ exchanger (NHE) inhibition during ischemia as opposed to during reperfusion. Isolated rat hearts were perfused with bicarbonate- or HEPES-buffered medium. There was infusion of HOE 694 immediately before ischemia, during initial reperfusion, or during both of these periods. With bicarbonate-buffered medium, HOE 694 improved the post-ischemic recovery of left ventricular developed pressure (LVDP) when given before ischemia and before ischemia plus during reperfusion. In the presence of HEPES-buffered medium, however, HOE 694 significantly improved recovery of LVDP in all protocols. HOE 694 also provided an almost complete recovery of LVDP (88 +/- 9% vs 30 +/- 7% in controls) when given before ischemia plus during reperfusion. In conclusion, our results suggest that the influence of NHE activity during reperfusion on the extent of functional recovery is modulated significantly by perfusate buffer composition.     

Long-term myocardial preservation: beneficial and additive effects of polarized arrest (Na+-channel blockade), Na+/H+-exchange inhibition, and Na+/K+/2Cl- -cotransport inhibition combined with calcium desensitization

BACKGROUND: Polarized arrest, induced by tetrodotoxin (TTX) at an optimal concentration of 22 micromol/L, has been shown to reduce ionic imbalance and improve myocardial preservation compared with hyperkalemic (depolarized) arrest. Additional pharmacologic manipulation of ionic changes (involving inhibition of Na+ influx by the Na+/H+ exchanger [HOE694] and Na+/K+/2Cl- cotransporter [furosemide], and calcium desensitization [BDM]) may further improve long-term preservation. In this study, we (i) established optimal concentrations of each drug, (ii) determined additive effects of optimal concentrations of each drug and (iii) compared our optimal preservation solution to an established depolarizing cardioplegia (St Thomas' Hospital solution No 2: STH2) used during long-term hypothermic storage for clinical transplantation. METHODS: The isolated working rat heart, perfused with Krebs Henseleit (KH) buffer was used; cardiac function was measured after 20 min aerobic working mode perfusion. The hearts (n=6/group) were arrested with a 2 ml infusion (for 30 sec) of the polarizing (control) solution (22 micromol/L TTX in KH) or control+drug and subjected to 5 hr or 8 hr of storage at 7.5 degrees C in the arresting solution. Postischemic function during reperfusion was measured (expressed as percentage of preischemic function). RESULTS: Dose-response studies established optimal concentrations of HOE694 (10 micromol/L), furosemide (1.0 micromol/L) and BDM (30 mmol/L) in the polarizing (control) solution. Sequential addition to the control solution (Group I) of optimal concentrations of HOE694 (Group II), furosemide (Group III), and BDM (Group IV) were compared with STH2 (Group V); postischemic recovery of aortic flow was 29+/-7%, 49+/-6%*, 56+/-2%*, 76+/-3%*, and 25+/-6%, respectively (*P<0.05 vs. I and V). Creatine kinase leakage was lowest, and myocardial ATP content was highest in Group IV. CONCLUSIONS: A polarizing preservation solution (KH+TTX) containing HOE694, furosemide, and BDM significantly enhanced long-term preservation compared with an optimized depolarizing solution (STH2) used clinically for long-term donor heart preservation.     

Role of glucocorticoids in the maturation of the rat renal Na+/H+ antiporter (NHE3).

BACKGROUND: Neonates have a lower Na+/H+ antiporter activity on the apical membrane of proximal tubule than that of adults. The maturational increase in Na+/H+ antiporter activity occurs at the time when there is a rise in serum glucocorticoid levels in rats. The purpose of the present study was to examine whether glucocorticoids are responsible for the postnatal increase in Na+/H+ antiporter activity. METHODS: Nine-day-old Sprague-Dawley rats were compared with rats studied at 30 days of age who had either a sham operation or adrenalectomy (ADX) at nine days of age and with rats that had an adrenalectomy and physiologic corticosterone replacement (ADX-Cort) to determine whether glucocorticoid deficiency prevented the maturational increase in Na+/H+ antiporter activity. Na+/H+ antiporter activity was measured in proximal convoluted tubules perfused in vitro by the change in cell pH (pHi) following luminal sodium removal. NHE3 mRNA abundance was measured using Northern blot analysis, and NHE3 protein abundance was measured by immunoblot. RESULTS: Na+/H+ antiporter activity was 93.8 +/- 17.7, 157.0 +/- 18.0, 356.7 +/- 29.9, and 402.5 +/- 14.5 pmol/mm. min in nine-day-old, ADX, ADX-Cort, and sham control groups, respectively. The ADX-Cort and sham control were higher than the 9-day-old and the 30-day-old ADX group (P < 0.05). Brush-border membrane NHE3 protein abundance in the nine-day-old and ADX groups were sixfold less than ADX-Cort and sham control groups (P < 0.001). Nine-day-old neonates had fivefold less renal cortical NHE3 mRNA than the ADX, ADX-Cort, and sham-operated control groups (P < 0.01). CONCLUSIONS: These data demonstrate that glucocorticoids play a role in the postnatal maturation of the proximal tubule Na+/H+ antiporter activity and brush-border membrane NHE3 protein abundance. Glucocorticoid deficiency does not completely prevent the maturational increase in Na+/H+ antiporter activity and does not affect NHE3 mRNA abundance.     

Luminal osmolarity downregulates gene expression of Na+/H+ exchanger (NHE3) in rat colon mucosa

Water-coupled Na⁺ absorption in the colon is mediated principally by Na⁺/H⁺ exchange (isoforms NHE2 and NHE3). To determine whether luminal ion composition or osmolarity influences NHE expression in colon mucosa, two groups (n = 6 in each) of adult male Sprague-Dawley rats underwent sham laparotomy or loop ileostomy. In these studies, diversion did not markedly alter mRNA levels for NTHE2, NHE3, or Na⁺/K⁺, at 8 or 21 days, indicating that loss of luminal volume does not alter NHE gene expression. To evaluate the effects of specific luminal components, we infused equal volumes of half-normal (154 mOsm) or iso-osmolar (308 mOsm) solutions of saline and mannitol into the diverted colon. All solutions elicited significant (45% to 60%; P <0.05) decreases in mRNA levels for NHE3, with iso-osmolar mannitol eliciting the greatest changes. Decreases in NHE2 and Na⁺/K⁺ mRNA levels were observed following these infusions but were not as marked as the changes for NHE3. These findings suggest that (1) loss of luminal Na⁺ is not, in itself, a signal that regulates NHE expression and (2) infusion of any solute, including Na⁺ itself, provides a signal to downregulate expression of NHE3 in colon mucosa. Supported by the Brigham Surgical Group Foundation and National Institutes of Health Award RO1-DK44571 (D.I.S.).     

The Na+/H+ exchange inhibitor: a new therapeutic approach for hepatic ischemia injury in rats

BACKGROUND: Na+/H+ exchanger (NHE) is one of the major mechanisms for restoring pH after ischemia-induced intracellular acidosis. However, activation of NHE during ischemia and reperfusion (I/R) is also involved in the paradoxical induction of cell injury. This study was designed to evaluate the effects of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), an NHE inhibitor, on hepatic I/R injury. METHODS: Partial hepatic ischemia was induced in male Wistar rats by cross-clamping the hepatic arterial and portal venous branches to the left lateral and median lobes of the liver for 120 minutes. The caudate and right lateral lobes were removed immediately after reperfusion. The concentrations of serum enzymes and ATP levels and energy charge in the live tissue were examined after 1-hour reperfusion. RESULT: EIPA afforded considerable protection against I/R injury, as demonstrated by decreased transaminase release and reduced histologic hepatocyte damage and increased energy charge. The 7-day survival rate was significantly improved from 15.4% to 55.6% (P <.05). CONCLUSIONS: This study shows for the first time that NHE may play an important role in the hepatic I/R injury and that EIPA should be considered as a new therapeutic approach to prevent hepatic I/R injury.