Involvement of pRB family in TGF beta-dependent epithelial cell hypertrophy.

Although renal hypertrophy is often associated with the progressive loss of renal function, the mechanism of hypertrophy is poorly understood. In both primary cultures of rabbit proximal tubules and NRK-52E cells (a renal epithelial cell line), transforming growth factor beta 1 (TGF beta) converted epidermal growth factor (EGF)-induced hyperplasia into hypertrophy. TGF beta did not affect EGF-induced increases in c-fos mRNA abundance or cyclin E protein abundance, but inhibited EGF-induced entry into S, G2, and M phases. EGF alone increased the amount of hyperphosphorylated (inactive) pRB; TGF beta blocked EGF-induced pRB phosphorylation, maintaining pRB in the active form. To determine the importance of active pRB in TGF beta-induced hypertrophy, NRK-52E cells were infected with SV40 large T antigen (which inactivates pRB and related proteins and p53), HPV16 E6 (which degrades p53), HPV16 E7 (which binds and inactivates pRB and related proteins), or both HPV16 E6 and E7. In SV40 large T antigen expressing clones, the magnitude of EGF + TGF beta-induced hypertrophy was inhibited and was inversely related to the magnitude of SV40 large T antigen expression. In the HPV16-infected cells, EGF + TGF beta-induced hypertrophy was inhibited in E7- and E6E7-expressing, but not E6-expressing cells. These results suggest a requirement for active pRB in the development of EGF + TGF beta-induced renal epithelial cell hypertrophy. We suggest a model of renal cell hypertrophy mediated by EGF-induced entry into the cell cycle with TGF beta-induced blockade at G1/S, the latter due to maintained activity of pRB or a related protein.

Apical membrane chloride/base exchange in the rat proximal convoluted tubule.

To examine whether Cl/base exchange is present on the apical membrane of the proximal convoluted tubule, cell pH was measured fluorometrically in the in vivo microperfused rat proximal tubule with (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. The effect of luminal chloride addition was examined in tubules perfused symmetrically with chloride-free solutions. In the absence of inhibitors, luminal chloride addition did not affect cell pH. However, after inhibition of basolateral membrane anion transport with peritubular 4-acetamido-4'-isothiocyano-(2,2')-disulfonic-stilbene (to amplify effects of apical membrane transport on cell pH), luminal chloride addition caused a small cell acidification (delta pHi = 0.02). When 1 mM formate was added to the solutions, luminal chloride addition caused a larger change in cell pH (delta pHi = 0.06) that was inhibited by (4,4')-diisothiocyano-(2,2')-disulfonic-stilbene. This stimulation of Cl/base exchange was not seen with 1 mM acetate addition. These results demonstrate apical membrane Cl/base exchange, a significant fraction of which is dependent on the presence of formate and probably represents Cl/formate exchange.

Cell pH in the rat proximal convoluted tubule. Regulation by luminal and peritubular pH and sodium concentration.

To examine the relative roles of apical and basolateral membrane transport mechanisms in the regulation of cell pH in the proximal convoluted tubule, cell pH was measured in the in vivo microperfused rat tubule using fluorescence. Decreasing luminal pH by 0.7 pH units caused cell pH to decrease by 0.08 pH units, whereas a similar decrease in peritubular pH caused cell pH to decrease by 0.32 pH units. Inhibition of basolateral membrane bicarbonate transport with peritubular 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) enhanced the response to luminal fluid acidification. Removal of luminal sodium caused a small transient acidification which was followed by a late alkalinization. Peritubular SITS increased the magnitude of the transient acidification, and eliminated the late alkalinization. The acidification was partially inhibited by luminal amiloride. The results demonstrate sodium-coupled processes on both the apical (Na/H antiport) and basolateral (Na/HCO3 symport) membranes. Basolateral membrane transporters are more important determinants of cell pH.

Chronic metabolic acidosis causes an adaptation in the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter in the rat proximal convoluted tubule.

The effect of chronic dietary acid on the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter was examined in the in vivo microperfused rat proximal tubule. Transporter activity was assayed with the epifluorescent measurement of cell pH using the intracellular, pH-sensitive fluorescent dye, (2'7')-bis(carboxyethyl)-(5,6)-carboxy-fluorescein (BCECF). BCECF was calibrated intracellularly, demonstrating similar pH-sensitivity of the dye in control and acidotic animals. In subsequent studies, lumen and peritubular capillaries were perfused to examine Na/H and Na(HCO3)3 transporter activity in the absence of contact with native fluid. The initial rate of change in cell pH (dpHi/dt) was 97, 50, and 44% faster in tubules from acidotic animals when peritubular [HCO3] was changed from 25 to 10 mM in the presence or absence of chloride, or peritubular [Na] was changed from 147 to 50 mM, respectively. dpHi/dt was 57% faster in tubules from acidotic animals when luminal [Na] was changed from 152 to 0 mM. Buffer capacities, measured using NH3/NH+4 addition, were similar in the two groups. The results demonstrate that chronic metabolic acidosis causes an adaptation in the intrinsic properties of both the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter.

Contributions of cellular leak pathways to net NaHCO3 and NaCl absorption.

Proton and formic acid permeabilities were measured in the in vivo microperfused rat proximal convoluted tubule by examining the effect on intracellular pH when [H] and/or [formic acid] were rapidly changed in the luminal or peritubular fluids. Apical and basolateral membrane H permeabilities were 0.52 +/- 0.07 and 0.67 +/- 0.18 cm/s, respectively. Using these permeabilities we calculate that proton backleak from the luminal fluid to cell does not contribute significantly to net proton secretion in the early proximal tubule, but may contribute in the late proximal tubule. Apical and basolateral membrane formic acid permeabilities measured at extracellular pH 6.62 were 4.6 +/- 0.5 X 10(-2) and 6.8 +/- 1.5 X 10(-2) cm/s, respectively. Control studies demonstrated that the formic acid permeabilities were not underestimated by either the simultaneous movement of formate into the cell or the efflux of formic acid across the opposite membrane. The measured apical membrane formic acid permeability is too small to support all of transcellular NaCl absorption in the rat by a mechanism that involves Na/H-Cl/formate transporters operating in parallel with formic acid nonionic diffusion.

Cyclic adenosine monophosphate acutely inhibits and chronically stimulates Na/H antiporter in OKP cells.

Parathyroid hormone, dopamine, alpha-adrenergic catecholamines, and angiotensin II regulate renal Na excretion, at least in part through modulation of acute cyclic (c)AMP-induced proximal tubule Na/H antiporter inhibition. The present studies examined the effect of chronic increases in cell cAMP on Na/H antiporter activity in OKP cells. Whereas 8-bromo cAMP acutely inhibited Na/H antiporter activity, chronic application for 6 h led to a 24% increase in Na/H antiporter activity measured 16-20 h after cAMP removal. This chronic persistent activation of the Na/H antiporter required > 2 h exposure. This effect was not a nonspecific effect of 8-bromo cAMP, in that addition of forskolin or forskolin + 3-isobutyl-1-methylxanthine for 6 h also led to a chronic persistent increase in Na/H antiporter activity. Inhibition of protein synthesis with cycloheximide prevented 8-bromo cAMP-induced Na/H antiporter stimulation. Although 8-bromo cAMP addition decreased cell pH by 0.15-0.20 pH U, Na/H antiporter stimulation could be dissociated from cell acidification. In summary, while cAMP acutely inhibits Na/H antiporter activity, it chronically increases antiporter activity. This chronic activation occurs with exogenous addition or endogenous generation of cAMP. These results imply that for hormones that modulate renal Na excretion and proximal tubule Na/H antiporter activity via cAMP and protein kinase A, acute effects may not predict chronic effects.

Active and passive components of chloride transport in the rat proximal convoluted tubule.

Rat proximal convoluted tubules were perfused in vivo to examine the active and passive components of chloride absorption. Chloride flux was a linear function of the transepithelial electrochemical driving force, yielding a permeability coefficient of 20.6 X 10(-5) cm/s. In the absence of an electrochemical driving force, chloride absorption persisted at the rate of 131 peq/mm X min, thus demonstrating active absorption of chloride. Addition of luminal cyanide to tubules absorbing chloride inhibited net chloride absorption. In tubules perfused with a low luminal chloride concentration in which there was net chloride secretion, addition of luminal cyanide increased the magnitude of net chloride secretion. These studies demonstrate that transepithelial chloride transport involves two components: a passive paracellular flux and an active transcellular flux. Cyanide affects net chloride flux by inhibiting active transcellular chloride absorption.

Effects of extracellular fluid volume and plasma bicarbonate concentration on proximal acidification in the rat.

The effects of systemic bicarbonate concentration and extracellular fluid volume status on proximal tubular bicarbonate absorption, independent of changes in luminal composition and flow rate, were examined with in vivo luminal microperfusion of rat superficial proximal convoluted tubules. Net bicarbonate absorption and bicarbonate permeability were measured using microcalorimetry. From these data, net bicarbonate absorption was divided into two parallel components: proton secretion and passive bicarbonate diffusion. The rate of net bicarbonate absorption was similar in hydropenic and volume-expanded rats when tubules were perfused with 24 mM bicarbonate, but was inhibited in volume-expanded rats when tubules were perfused with 5 mM bicarbonate. Volume expansion caused a 50% increase in bicarbonate permeability, which totally accounted for the above inhibition. The rate of proton secretion was unaffected by volume expansion in both studies. The rate of net bicarbonate absorption was markedly inhibited in alkalotic expansion as compared with isohydric expansion. Bicarbonate permeabilities were not different in these two conditions, and the calculated rates of proton secretion were decreased by greater than 50% in alkalosis. Net bicarbonate absorption was stimulated in acidotic rats compared to hydropenic rats. This stimulation was attributable to a 25% increase in the rate of proton secretion. We conclude that (a) proton secretion is stimulated in acidosis, inhibited in alkalosis, and is not altered by volume status; (b) bicarbonate permeability is increased by volume expansion but is not altered by increases in plasma bicarbonate concentration; (c) when luminal bicarbonate concentrations are similar to those of plasma, net bicarbonate absorption is dominated by proton secretion and is thus sensitive to peritubular bicarbonate concentrations, and insensitive to extracellular fluid volume; (d) when luminal bicarbonate concentrations are low and proton secretion is slowed, bicarbonate permeability and thus extracellular fluid volume have a greater influence on net bicarbonate absorption.

Long-term activation of protein kinase c causes chronic Na/H antiporter stimulation in cultured proximal tubule cells.

To examine the role of protein kinase C as a chronic regulator of proximal tubule Na/H antiporter activity, the effect of phorbol 12-myristate 13-acetate (PMA) on the Na/H antiporter was studied in cultured proximal tubule cells. Short-term activation of protein kinase C by 5 min exposure to PMA caused an acute increase in Na/H antiporter activity that was not prevented by cycloheximide or actinomycin D and did not persist 24 h later. Long-term activation of protein kinase C by 2 h exposure to PMA caused a dose-dependent increase in Na/H antiporter activity 24 h later. This latter effect was due to protein kinase C activation in that it was inhibited by sphingosine and was not seen with 4 alpha-PMA, an inactive analogue. The chronic effect of PMA was inhibited by 10 nM actinomycin D or 7 microM cycloheximide. Proximal tubule cells exposed to PMA for 2 h demonstrated a two- to threefold increase in Na/H antiporter mRNA (mRNANa/H) abundance 4 h later. In conclusion, short-term activation of protein kinase C leads to a transient increase in Na/H antiporter activity that is independent of transcription and translation, whereas long-term activation of protein kinase C causes a persistent increase in antiporter activity that is dependent on transcription and translation and is associated with increased mRNANa/H abundance. This latter effect may mediate increased Na/H antiporter activity in a number of chronic conditions.

Acid activation of immediate early genes in renal epithelial cells.

These studies examined the effect of acidosis on immediate early (IE) gene expression in renal tubule cells. In MCT cells, an SV40 transformed mouse proximal tubule cell line, incubation in acid media led to transient increases in c-fos, c-jun, junB, and egr-1 mRNA abundance, peaking at 30 min to 1 h. In vivo metabolic acidosis caused more prolonged increases in these mRNA species in renal cortex. Nuclear runon studies demonstrated increased rates of transcription for these IE genes. In addition, pretreatment of cells with cycloheximide caused superinduction of these mRNA by acid incubation. These responses are similar to those elicited by growth factors. Inhibition of tyrosine kinase pathways prevented IE gene activation by acid, while inhibition of protein kinase C and/or increases in cell calcium had no effect. In 3T3 cells, acid activated IE genes by a different mechanism in that the increase in mRNA did not include c-jun, was more prolonged, and was blocked by cycloheximide. In summary, incubation of renal cells in acid media leads to activation of IE genes that is similar to growth factor-induced IE gene activation, and is likely mediated by tyrosine kinase pathways.

Electrogenic sodium/bicarbonate cotransport in rabbit renal cortical basolateral membrane vesicles.

The present studies examined the mechanism of bicarbonate transport across basolateral membrane vesicles prepared from rabbit renal cortex. Isotopic sodium uptake was stimulated by bicarbonate when compared with gluconate (2.5 nmol/mg protein per 5 s versus 1.4 nmol/mg protein per 5 s), and this process was inhibited by disulfonic stilbenes. Imposition of an interior-positive potassium diffusion potential further stimulated isotopic sodium uptake to 3.4 nmol/mg protein per 5 s, an effect that occurred only in the presence of bicarbonate and was blocked by disulfonic stilbenes. Kinetic analysis of the rate of bicarbonate-dependent sodium uptake as a function of sodium concentration revealed saturable stimulation with a Vmax of 2.7 nmol/mg protein per 2 s and a Km of 10.4 mM. The effect of bicarbonate concentration on bicarbonate-dependent sodium uptake was more complex. The present results demonstrate an electrogenic (negatively charged) sodium/bicarbonate cotransporter in basolateral membrane vesicles from the rabbit renal cortex. The electrogenicity implies a stoichiometry of at least two bicarbonate ions for each sodium ion.

Endothelin(B) receptor activates NHE-3 by a Ca2+-dependent pathway in OKP cells.

To examine the mechanisms by which endothelin (ET) regulates the Na/H antiporter isoform, NHE-3, OKP cells were stably transfected with ET(A) and ET(B) receptor cDNA. In cells overexpressing ET(B), but not ET(A) receptors, ET-1 increased Na/H antiporter activity (JNa/H). This effect was inhibited by a nonselective endothelin receptor blocker and by a selective ET(B) receptor blocker but was not inhibited by an ET(A) selective receptor blocker. In ET(B)-overexpressing cells, 10(-8) M ET-1 inhibited adenylyl cyclase, but protein kinase A inhibition and pertussis toxin pretreatment did not affect Na/H antiporter activation by ET-1. ET-1 caused a transient increase in cell [Ca2+], followed by a sustained increase. Increases in cell [Ca2+] were partially inhibited by pertussis toxin. ET-1-induced increases in J(Na/H) were 50% inhibited by clamping cell [Ca2+] low with BAPTA, and by KN62, a Ca-calmodulin kinase inhibitor. Inhibitors of protein kinase C, cyclooxygenase, lipoxygenase, and cytochrome P450 and cyclic GMP were without effect. In ET(A)-overexpressing cells, ET-1 increased cell [Ca2+] but did not increase JNa/H. In summary, binding of ET-1 to ET(B) receptors increases Na/H antiporter activity in OKP cells, an effect mediated in part by increases in cell [Ca2+] and Ca-calmodulin kinase. Increases in cell [Ca2+] are not sufficient for Na/H antiporter activation.

Endothelin-1/endothelin-B receptor-mediated increases in NHE3 activity in chronic metabolic acidosis.

Decreases in blood pH activate NHE3, the proximal tubular apical membrane Na/H antiporter. In cultured renal epithelial cells, activation of the endothelin-B (ET(B)) receptor increases NHE3 activity. To examine the role of the ET(B) receptor in the response to acidosis in vivo, the present studies examined ET(B) receptor-deficient mice, rescued from neonatal lethality by expression of a dopamine beta-hydroxylase promoter/ET(B) receptor transgene (Tg/Tg:ET(B)(-/-) mice). In proximal tubule suspensions from Tg/Tg:ET(B)(+/-) mice, 10(-8) M endothelin-1 (ET-1) increased NHE3 activity, but this treatment had no effect on tubules from Tg/Tg:ET(B)(-/-) mice. Acid ingestion for 7 days caused a greater decrease in blood HCO(3)(-) concentration in Tg/Tg:ET(B)(-/-) mice compared with Tg/Tg:ET(B)(+/+) and Tg/Tg:ET(B)(+/-) mice. Whereas acid ingestion increased apical membrane NHE3 by 42-46% in Tg/Tg:ET(B)(+/+) and Tg/Tg:ET(B)(+/-) mice, it had no effect on NHE3 in Tg/Tg:ET(B)(-/-) mice. In C57BL/6 mice, excess acid ingestion increased renal cortical preproET-1 mRNA expression 2.4-fold and decreased preproET-3 mRNA expression by 37%. On a control diet, Tg/Tg:ET(B)(-/-) mice had low rates of ammonium excretion, which could not be attributed to an inability to acidify the urine, as well as hypercitraturia, with increased titratable acid excretion. Acid ingestion increased ammonium excretion, citrate absorption, and titratable acid excretion to the same levels in Tg/Tg:ET(B)(-/-) and Tg/Tg:ET(B)(+/+) mice. In conclusion, metabolic acidosis increases ET-1 expression, which increases NHE3 activity via the ET(B) receptor.

Chronic hyperosmolality increases NHE3 activity in OKP cells.

This study investigated the effect of chronic hypertonicity on the OKP cell Na/H antiporter, encoded by Na/H exchanger 3 (NHE3). Chronic (48 h) increases in extracellular glucose, mannitol, or raffinose concentration caused a significant increase in Na/H antiporter activity, while increases in urea concentration were without effect. This effect was seen with changes in osmolality of only 20 mOsm/liter, a magnitude that is observed clinically in poorly controlled diabetes mellitus. Increases in mannitol concentration acutely inhibited and chronically stimulated Na/H antiporter activity. The increase in Na/H antiporter activity induced by hypertonic incubation was resistant to 10(-7) and 5 x 10(-6) M but inhibited by 10(-4) M ethylisopropyl amiloride, consistent with regulation of NHE3. In addition, hypertonicity increased total cellular and plasma membrane NHE3 protein abundance twofold, with only a small increase in NHE3 mRNA abundance. We conclude that chronic pathophysiologically relevant increases in tonicity lead to increases in NHE3 protein abundance and activity. This may be responsible for increased proximal tubule apical membrane Na/H antiporter activity in poorly controlled diabetes mellitus, which could then contribute to hypertension, glomerular hyperfiltration and diabetic nephropathy.

Regulation of cell pH by ambient bicarbonate, carbon dioxide tension, and pH in the rabbit proximal convoluted tubule.

UNLABELLED: To study the regulation of cell pH by ambient pH, carbon dioxide tension (PCO2), and bicarbonate (HCO3), cell pH was measured in the isolated, in vitro microperfused rabbit proximal convoluted tubule using the fluorescent dye (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein. For the same changes in external pH, changes in [HCO3] and PCO2 affected cell pH similarly ([HCO3]: pHi/pHe = 0.67, PCO2: pHi/pHe = 0.64, NS). Isohydric changes in extracellular [HCO3] and PCO2 did not change cell pH significantly. Changes in peritubular [HCO3] elicited larger changes in cell pH than changes in luminal [HCO3], which were enhanced by peritubular 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). The cell pH defense against acute increases and decreases in PCO2 was inhibited by sodium, but not by chloride removal. Peritubular SITS inhibited the cell pH defense against increases and decreases of PCO2, whereas luminal amiloride inhibited cell pH defense against increases in PCO2. CONCLUSIONS: (a) Steady-state cell pH changes in response to changes in extracellular [HCO3] and PCO2 are quantitatively similar for a given change in extracellular pH; (b) the rate of the basolateral Na/(HCO3)3 cotransporter is a more important determinant of cell pH than the rate of the apical membrane mechanism(s); (c) cell pH defense against acute changes in PCO2 depends on the basolateral Na/(HCO3)3 cotransporter (acid and alkaline loads) and the luminal Na/H antiporter (acid loads).

Differential regulation of Na/H antiporter by acid in renal epithelial cells and fibroblasts.

Increased Na/H antiporter activity has been demonstrated after in vivo chronic metabolic acidosis as well as in vitro acid preincubation of cultured rabbit renal tubule cells. To study the underlying molecular mechanisms of this adaptive increase in Na/H antiporter activity, the present studies examined the effect of low pH media on Na/H antiporter activity and mRNA abundance in cultured renal tubule cells. Na/H antiporter activity was increased by 60% in a mouse renal cortical tubule cell line (MCT), and by 90% in an opossum kidney cell line (OKP) after 24 h of preincubation in acid (low [HCO3]) media. The ethylisopropylamiloride sensitivity of the Na/H antiporters were different in these two cell lines (MCT IC50 = 65 nM; OKP IC50 = 4.5 microM). In MCT cells, Na/H antiporter mRNA abundance measured by RNA blots increased by two- to fivefold after 24 h in low [HCO3] media. Na/H antiporter mRNA abundance was also increased in MCT cells with high CO2 preincubation as well as in rat renal cortex with in vivo chronic acid feeding. In contrast to renal epithelia, acid preincubation of NIH 3T3 fibroblasts led to suppression of Na/H antiporter activity. RNA blots of 3T3 fibroblasts revealed the same size Na/H antiporter transcript as in MCT cells. However, Na/H antiporter mRNA levels were suppressed by acid preincubation. These studies demonstrate differential regulation of Na/H antiporter activity and mRNA abundance in renal epithelial cells and fibroblasts in response to an acidotic environment.

Glucocorticoids enhance acid activation of the Na+/H+ exchanger 3 (NHE3).

In the absence of exogenous glucocorticoids, decreasing media pH (from 7.4 to 6.8) for 24 hours increased the Na+/H+ exchanger 3 (NHE3) activity in opossum kidney (OKP) cells. 10(-7) M and 10(-8) M hydrocortisone increased NHE3 activity, and in their presence, acid incubation further increased NHE3 activity. Hydrocortisone (10(-9) M) had no effect on NHE3 activity, but in its presence, the effect of acid incubation on NHE3 activity increased twofold. Aldosterone (10(-8) M) had no effect. In the absence of hydrocortisone, acid incubation increased NHE3 protein abundance by 47%; in the presence of 10(-9) M hydrocortisone, acid incubation increased NHE3 protein abundance by 132%. The increase in NHE3 protein abundance was dependent on protein synthesis. However, 10(-9) M hydrocortisone did not modify the effect of acid incubation to cause a twofold increase in NHE3 mRNA abundance. In the absence of protein synthesis, 10(-9) M hydrocortisone did potentiate an effect of acid on NHE3 activity, which was due to trafficking of NHE3 to the apical membrane. These results suggest that glucocorticoids and acid interact synergistically at the level of NHE3 translation and trafficking.

Adenosine triphosphate citrate lyase mediates hypocitraturia in rats.

Chronic metabolic acidosis increases proximal tubular citrate uptake and metabolism. The present study addressed the effect of chronic metabolic acidosis on a cytosolic enzyme of citrate metabolism, ATP citrate lyase. Chronic metabolic acidosis caused hypocitraturia in rats and increased renal cortical ATP citrate lyase activity by 67% after 7 d. Renal cortical ATP citrate lyase protein abundance increased by 29% after 3 d and by 141% after 7 d of acid diet. No significant change in mRNA abundance could be detected. Hypokalemia, which causes only intracellular acidosis, caused hypocitraturia and increased renal cortical ATP citrate lyase activity by 28%. Conversely, the hypercitraturia of chronic alkali feeding was associated with no change in ATP citrate lyase activity. Inhibition of ATP citrate lyase with the competitive inhibitor, 4S-hydroxycitrate, significantly abated hypocitraturia and increased urinary citrate excretion fourfold in chronic metabolic acidosis and threefold in K+-depletion. In summary, the hypocitraturia of chronic metabolic acidosis is associated with an increase in ATP citrate lyase activity and protein abundance, and is partly reversed by inhibition of this enzyme. These results suggest an important role for ATP citrate lyase in proximal tubular citrate metabolism.

Chronic metabolic acidosis enhances NHE-3 protein abundance and transport activity in the rat thick ascending limb by increasing NHE-3 mRNA.

Chronic metabolic acidosis (CMA) is associated with an adaptive increase in the bicarbonate absorptive capacity of the rat medullary thick ascending limb (MTAL). To specify whether NHE-3, the apical MTAL Na/H exchanger, is involved in this adaptation, NHE-3 mRNA was quantified by a competitive RT-PCR using an internal standard which differed from the wild-type NHE-3 mRNA by an 80-bp deletion. CMA increased NHE-3 mRNA from 0.025+/-0.003 to 0.042+/-0.009 amol/ng total RNA (P < 0.005). NHE-3 transport activity was measured as the initial proton flux rate calculated from the Na-dependent cell pH recovery of Na-depleted acidified MTAL cells in the presence of 50 microM HOE694 which specifically blocks NHE-1, the basolateral MTAL NHE isoform. CMA caused a 68% increase in NHE-3 transport activity (P < 0.001). In addition, CMA was associated with a 71% increase in NHE-3 protein abundance (P < 0.05) as determined by Western blot analysis on MTAL membranes using a polyclonal antiserum directed against a cytoplasmic epitope of rat NHE-3. Thus, NHE-3 adapts to CMA in the rat MTAL via an increase in the mRNA transcript that enhances NHE-3 protein abundance and transport activity.

Role of the Na+/H+ antiporter in rat proximal tubule bicarbonate absorption.

Amiloride and the more potent amiloride analog, 5-(N-t-butyl) amiloride (t-butylamiloride), were used to examine the role of the Na+/H+ antiporter in bicarbonate absorption in the in vivo microperfused rat proximal convoluted tubule. Bicarbonate absorption was inhibited 29, 46, and 47% by 0.9 mM or 4.3 mM amiloride, or 1 mM t-butylamiloride, respectively. Sensitivity of the Na+/H+ antiporter to these compounds in vivo was examined using fluorescent measurements of intracellular pH with (2', 7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein (BCECF). Amiloride and t-butylamiloride were shown to be as potent against the antiporter in vivo as in brush border membrane vesicles. A model of proximal tubule bicarbonate absorption was used to correct for changes in the luminal profiles for pH and inhibitor concentration, and for changes in luminal flow rate in the various series. We conclude that the majority of apical membrane proton secretion involved in transepithelial bicarbonate absorption is mediated by the Na+-dependent, amiloride-sensitive Na+H+ antiporter. However, a second mechanism of proton secretion contributes significantly to bicarbonate absorption. This mechanism is Na+-independent and amiloride-insensitive.