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A study was conducted to describe the mechanism of outer medullary collecting duct (OMCD(sub is)) proton secretion. Using in vitro microperfusion, it was revealed that the principal cell of (OMCD(sub is)) is responsible for luminal potassium reabsorption and urinary acidification. Findings also show that the proton transporters of the intercalated cells of the (OMCD(sub is)) have similar functions as those found in the (OMCD(sub is)) principal cell.

The full-length coding sequence of two hydrogen-potassium-adenine triphosphatase (H-K-ATPase) subunits from the rabbit kidney and thin expression in renal cortex and RCCT-28A cells was examined. The mechanism of intracellular pH recovery from an intracellular acid load were also investigated. The findings demonstrated the existence of three distant H-K-ATPases in the rabbit kidney and RCCT-28A cells. These data also provided insights into the regulation of potassium homeostasis.

The molecular mechanisms regulating ammonia metabolism are fundamental to acid-base homeostasis. Deletion of the A splice variant of Naþ-bicarbonate cotransporter, electrogenic, isoform 1 (NBCe1-A) partially blocks the effect of acidosis to increase urinary ammonia excretion, and this appears to involve the dysregulated expression of ammoniagenic enzymes in the proximal tubule (PT) in the cortex but not in the outer medulla (OM). A second NBCe1 splice variant, NBCe1-B, is present throughout the PT, including the OM, where NBCe1-A is not present. The purpose of the present study was to determine the effect of combined renal deletion of NBCe1-A and NBCe1-B on systemic and PT ammonia metabolism. We generated NBCe1-A/B deletion using CreloxP techniques and used Cre-negative mice as controls. As renal NBCe1-A and NBCe1-B expression is limited to the PT, Crepositive mice had PT NBCe1-A/B deletion [PT-NBCe1-A/B knockout (KO)]. Although on a basal diet, PT-NBCe1-A/B KO mice had severe metabolic acidosis, yet urinary ammonia excretion was not changed significantly. PT-NBCe1-A/B KO decreased the expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase and increased the expression of glutamine synthetase, an ammonia-recycling enzyme, in PTs in both the cortex and OM. Exogenous acid loading increased ammonia excretion in control mice, but PT-NBCe1-A/B KO prevented any increase. PT-NBCe1-A/B KO significantly blunted acid loading-induced changes in phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase expression in PTs in both the cortex and OM. We conclude that NBCe1-B, at least in the presence of NBCe1-A deletion, contributes to PT ammonia metabolism in the OM and thereby to systemic acid-base regulation. [ABSTRACT FROM AUTHOR]

Several experiments were undertaken to identify the B cell apical anion exchanger through the examination of its regulation. The stimulus selected for the studies was CO2/HCO(super -)(sub 3) since it amplified the cortical collecting duct HCO(super -)(sub 3) secretion. Results revealed that exogenous CO2/HCO(super -)(sub 3) induced B cell apical anion exchange activity. The anion exchanger identified in the studies was a Cl-/base exchanger which transported bases other than bicarbonate.

In vitro microperfusion was used to examine the role of proton transporters in the A-type intercalated cell (A cell) of the cortical collecting duct (CCD) in rabbit kidneys. Intracellular hydrogen-ion concentration (pHi) was monitored to determine the effects of proton transporters on basal pHi regulation and recovery. Results indicate that basolateral sodium/hydrogen ion exchange is predominantly the mechanism in pHi recovery while apical hydrogen adenosine triphosphatase is the primary transporter in contributing to pHi regulation.

The role of the enzyme H+-K+-adenosinetriphosphatase (ATPase) in the renal cortical collecting duct (CCD) was investigated by examining the binding of tetramethylrhodamine isothiocyanate-labeled tomato lectin to rabbit CCD B-type intercalated cells after the cells were acid loaded. The normal pH recovery of cells was slightly altered by inhibitors such as N-ethylmaleimide. In contrast, peritubular Sch-28080 and peritubular potassium removal had no effect on pH regulation. Tomato lectin was shown to bind to the gastric H+-K+-ATPase beta-subunit, indicating that H+-K+-ATPase activity is localized at the B cell apical membrane.

There are sex differences in renal ammonia metabolism and structure, many of which are mediated by testosterone. The goal of the present study was to determine the role of renal expression of testosterone's canonical receptor, androgen receptor (AR), in these sexual dimorphisms. We studied mice with kidney-specific AR deletion [KS-AR-knockout (KO)] generated using Cre/loxP techniques; control mice were Cre-negative littermates (wild type). In male but not female mice, KS-AR-KO increased ammonia excretion, which eliminated sex differences. Although renal structural size typically parallel ammonia excretion, KS-AR-KO decreased kidney size, cortical proximal tubule volume density, and cortical proximal tubule cell height in males--neither were altered in females and collecting duct volume density was unaltered in both sexes. Analysis of key protein involved in ammonia handling showed in male mice that KS-AR-KO increased both phosphoenolpyruvate carboxykinase (PEPCK) and Na+-K+-2Cl- cotransporter (NKCC2) expression and decreased Na+/H+ exchanger isoform 3 (NHE3) and electrogenic Na+-bicarbonate cotransporter 1 (NBCe1)-A expression. In female mice, KS-AR-KO did not alter these parameters. These effects occurred even though KS-AR-KO did not alter plasma testosterone, food intake, or serum Na+, K+, or HCO-3 significantly in either sex. In conclusion, AR-dependent signaling pathways in male, but not female, kidneys regulate PEPCK and NKCC2 expression and lead to the sexual differences in ammonia excretion. Opposing effects on NHE3 and NBCe1-A expression likely limit the magnitude of ammonia excretion changes. As AR is not present in the thick ascending limb, the effect of KS-AR-KO on NKCC2 expression is indirect. Finally, AR mediates the greater kidney size and proximal tubule volume density in male compared with female mice. [ABSTRACT FROM AUTHOR]

Angiotensin II regulates ion transport in the distal portions of the kidney by stimulating B-type intercalated cells to secrete bicarbonate ions and create an alkaline environment in the collecting duct. Angiotensin II introduced into the cortical collecting duct caused an increase in the pH of the luminal fluid, an effect that was reversed by the addition of losartan since losartan is an angiotensin-receptor antagonist.

A study of in vitro microperfusion in solutions containing CO2/HCO3 to examine the location of intercalated cell Cl-/base exchange activity and distribution of cortical collecting duct (CCD) intercalated cells confirms that basolateral Cl-/HCO3 exchange activity is present in all CCD intercalated cells. The activity, however, differs in A-type and B-type intercalated cells. Both intercalated cell type and function exhibit axial heterogeneity.

Study of cultured rabbit inner medullary collecting duct cells suggests that a band 3 protein negative Cl-/HCO3- exchanger helps bicarbonate transport in these cells. The mechanism is independent of Na+ levels. There is no distinct sodium-bicarbonate cotransport activity. The mechanism may be involved in urinary acidification and bicarbonate reabsorption.

Studies were conducted to examine intracellular pH (pHi) control in two cell populations of the outer medullary collecting duct (OMCDis) and to investigate the functions and place of activity of Na+/H+ exchange, Cl-/HCO(sub 3)(super -) exchange and Na(+)-independent H+ transfer. The results reveal that quantitatively identical cells which are in various conditions of functional working constitute OMCDis. A few reveal more H+ (independent of Na+) discharge and reabsorb more HCO(sub 3)(super -) than other cells, although they have almost identical properties.

Rhesus C glycoprotein (Rhcg), an ammonia transporter, is a key molecule in urinary acid excretion and is expressed mainly in the intercalated cells (ICs) of the renal collecting duct. In the present study we investigated the role of aldosterone in the regulation of Rhcg expression. In in vivo experiments using C57BL/6J mice, Western blot analysis showed that continuous subcutaneous administration of aldosterone increased the expression of Rhcg in membrane fraction of the kidney. Supplementation of potassium inhibited the effect of aldosterone on the Rhcg. Next, mice were subjected to adrenalectomy with or without administration of aldosterone, and then ad libitum 0.14 M NH4Cl containing water was given. NH4Cl load increased the expression of Rhcg in membrane fraction. Adrenalectomy decreased NH4Cl-induced Rhcg expression, which was restored by administration of aldosterone. Immunohistochemical studies revealed that NH4Cl load induced the localization of Rhcg at the apical membrane of ICs in the outer medullary collecting duct. Adrenalectomy decreased NH4Cl-induced membrane localization of Rhcg, which was restored by administration of aldosterone. For in vitro experiments, IN-IC cells, an immortalized cell line stably expressing Flag-tagged Rhcg (Rhcg-Flag), were used. Western blot analysis showed that aldosterone increased the expression of Rhcg-Flag in membrane fraction, while the increase in extracellular potassium level inhibited the effect of aldosterone. Both spiron olactone and GÓ§6983, a PKC inhibitor, inhibited the expression of Rhcg-Flag in the membrane fraction. These results suggest that aldosterone regulates the membrane expression of Rhcg through the mineralocorticoid receptor and PKC pathways, which is modulated by extracellular potassium level. [ABSTRACT FROM AUTHOR]

Sexual dimorphic variations are present in many aspects of biology and involve the structure and/or function of nearly every organ system. Acid-base homeostasis is critical for optimal health, and renal ammonia metabolism has a major role in the maintenance of acid-base homeostasis. Recent studies have shown sex-dependent differences in renal ammonia metabolism with regard to both basal ammonia excretion and the response to an exogenous acid load. These sexual dimorphisms are associated with structural changes in the proximal tubule and the collecting duct and variations in the expression of multiple proteins involved in ammonia metabolism and transport. Studies using orchiectomy-induced testosterone deficiency and physiological testosterone replacement have shown that testosterone underlies much of the sex-dependent differences in the proximal tubule. This parallels the finding that the canonical testosterone target receptor, androgen receptor (AR), is present exclusively in the proximal tubule. Thus testosterone, possibly acting through AR activation, regulates multiple components of renal structure and ammonia metabolism. The lack of detectable AR in the remainder of the nephron and collecting duct suggests that some dimorphisms in renal structure and ammonia transporter expression are mediated through mechanisms other than direct testosteronedependent AR activation. A better understanding of the mechanism and biological implications of sex's effect on renal structure and ammonia metabolism is critical for optimizing our ability to care for both men and women with acid-base disturbances. [ABSTRACT FROM AUTHOR]

There are substantial sex differences in renal structure and ammonia metabolism that correlate with differences in expression of proteins involved in ammonia generation and transport. This study determined the role of testis-derived testosterone in these differences. We studied 4-mo-old male C57BL/6 mice 4 and 8 wk after either bilateral orchiectomy (ORCH) or sham-operated control surgery and determined the effect of testosterone replacement to reverse the effects of ORCH. Finally, we determined the cellular expression of androgen receptor (AR), testosterone's canonical target receptor. ORCH decreased kidney and proximal tubule size, and testosterone replacement reversed this effect. ORCH increased ammonia excretion in a testosterone-dependent fashion; this occurred despite similar food intake, which is the primary component of endogenous acid production. ORCH increased expression of both phosphoenolpyruvate, a major ammonia-generating protein, and Na+- K+-2Cl- cotransporter, which mediates thick ascending limb ammonia reabsorption; these changes were reversed with testosterone replacement. Orchiectomy also decreased expression of Na+/H+ exchanger isoform 3, which mediates proximal tubule ammonia secretion, in a testosterone-dependent pattern. Finally, ARs are expressed throughout the proximal tubule in both the male and female kidney. Testosterone, possibly acting through ARs, has dramatic effects on kidney and proximal tubule size and decreases ammonia excretion through its effects on several key proteins involved in ammonia metabolism. [ABSTRACT FROM AUTHOR]

Frank, Amy E., Charles S. Wingo, and I. David Weiner. Effects of ammonia on bicarbonate transport in the cortical collecting duct. Am. J. Physiol. Renal Physiol. 278: F219-F226, 2000.--Both acidosis and hypokalemia stimulate renal ammoniagenesis, and both regulate urinary proton and potassium excretion. We hypothesized that ammonia might play an important role in this processing by stimulating [H.sup.+]-[K.sup.+]-ATPase-mediated ion transport. Rabbit cortical collecting ducts (CCD) were studied using in vitro microperfusion, bicarbonate reabsorption was measured using microcalorimetry, and intracellular pH (p[H.sub.i]) was measured using the fluorescent, pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Ammonia caused a concentration-dependent increase in net bicarbonate reabsorption that was inhibited by luminal addition of either of the [H.sup.+]-[K.sup.+]-ATPase inhibitors, Sch-28080 or ouabain. The stimulation of net bicarbonate reabsorption was not mediated through apical [H.sup.+]-ATPase, basolateral [H.sup.+]-[K.sup.+]-ATPase, or luminal electronegativity. Although ammonia caused intracellular acidification, similar changes in phi induced by inhibiting basolateral [Na.sup.+]/[H.sup.+] exchange did not alter net bicarbonate reabsorption. We conclude that ammonia regulates CCD proton and potassium transport, at least in part, by stimulating apical [H.sup.+]-[K.sup.+]-ATPase. proton-potassium-exchanging ATPase; kidney tubules; collecting ducts; hydrogen ion concentration; proton-transporting ATP synthase; rabbits