Epithelial Na(+) channels are regulated by flow.

Na(+) absorption in the renal cortical collecting duct (CCD) is mediated by apical epithelial Na(+) channels (ENaCs). The CCD is subject to continuous variations in intraluminal flow rate that we speculate alters hydrostatic pressure, membrane stretch, and shear stress. Although ENaCs share limited sequence homology with putative mechanosensitive ion channels in Caenorhabditis elegans, controversy exists as to whether ENaCs are regulated by biomechanical forces. We examined the effect of varying the rate of fluid flow on whole cell Na(+) currents (I(Na)) in oocytes expressing mouse alpha,beta,gamma-ENaC (mENaC) and on net Na(+) absorption in microperfused rabbit CCDs. Oocytes injected with mENaC but not water responded to the initiation of superfusate flow (to 4-6 ml/min) with a reversible threefold stimulation of I(Na) without a change in reversal potential. The increase in I(Na) was variable among oocytes. CCDs responded to a threefold increase in rate of luminal flow with a twofold increase in the rate of net Na(+) absorption. An increase in luminal viscosity achieved by addition of 5% dextran to the luminal perfusate did not alter the rate of net Na(+) absorption, suggesting that shear stress does not influence Na(+) transport in the CCD. In sum, our data suggest that flow stimulation of ENaC activity and Na(+) absorption is mediated by an increase in hydrostatic pressure and/or membrane stretch. We propose that intraluminal flow rate may be an important regulator of channel activity in the CCD.

Flow-dependent K+ secretion in the cortical collecting duct is mediated by a maxi-K channel.

K+ secretion by the cortical collecting duct (CCD) is stimulated at high flow rates. Patch-clamp analysis has identified a small-conductance secretory K+ (SK) and a high-conductance Ca(2+)-activated K+ (maxi-K) channel in the apical membrane of the CCD. The SK channel, encoded by ROMK, is believed to mediate baseline K+ secretion. The role of the stretch- and Ca2+-activated maxi-K channel is still uncertain. The purpose of this study was to identify the K+ channel mediating flow-dependent K+ secretion in the CCD. Segments isolated from New Zealand White rabbits were microperfused in the absence and presence of luminal tetraethylammonium (TEA) or charybdotoxin, both inhibitors of maxi-K but not SK channels, or apamin, an inhibitor of small-conductance maxi-K+ channels. Net K+ secretion and Na+ absorption were measured at varying flow rates. In the absence of TEA, net K+ secretion increased from 8.3 +/- 1.0 to 23.4 +/- 4.7 pmol. min(-1). mm(-1) (P < 0.03) as the tubular flow rate was increased from 0.5 to 6 nl. min(-1). mm(-1). Flow stimulation of net K+ secretion was blocked by luminal TEA (8.2 +/- 1.2 vs. 9.9 +/- 2.7 pmol. min(-1). mm(-1) at 0.6 and 6 nl. min(-1). mm(-1) flow rates, respectively) or charybdotoxin (6.8 +/- 1.6 vs. 8.3 +/- 1.6 pmol. min(-1). mm(-1) at 1 and 4 nl. min(-1). mm(-1) flow rates, respectively) but not by apamin. These results suggest that flow-dependent K+ secretion is mediated by a maxi-K channel, whereas baseline K+ secretion occurs through a TEA- and charybdotoxin-insensitive SK (ROMK) channel.

Na(+)-K(+)-ATPase-mediated basolateral rubidium uptake in the maturing rabbit cortical collecting duct.

Within the renal cortical collecting duct (CCD), transepithelial Na(+) absorption and K(+) secretion are linked to basolateral Na(+)-K(+)-ATPase activity. Our purpose was to examine the developmental changes in basolateral Na(+)-K(+)-ATPase-mediated (86)rubidium (Rb) uptake, its inhibitor sensitivity and relationship to pump hydrolytic activity and Na(+) transport. Multiple CCDs ( approximately 6 mm) from maturing rabbits were affixed to coverslips, preincubated at 37 degrees C for 10 min (+/-1-2.5 mM ouabain or 10 or 100 micro M Schering-28080, an inhibitor of H(+)-K(+)-ATPase), and then transferred to prewarmed incubation solution containing tracer amounts of (86)Rb (+/-inhibitors). After 1 min at 37 degrees C, tubular samples were rinsed and permeabilized and isotope counts were measured to calculate basolateral Rb uptake. Ouabain-inhibitable Rb uptake, an index of basolateral Na(+)-K(+) pump activity, increased approximately 3-fold during the 1st 8 wk of postnatal life (P < 0.03). The approximately 2-fold increase in absolute rate of Rb uptake between 1 and 6 wk (2.64 +/- 0.45 to 5.02 +/- 0.32 pmol. min(-1). mm(-1)) did not reach statistical significance. The rate of basolateral Rb uptake increased further after the 6th wk of life to 7.29 +/- 0.53 pmol. min(-1). mm(-1) in adult animals (P < 0.03 vs. 6 wk). Schering-28080 failed to inhibit Rb uptake, implying that functional H(+)-K(+)-ATPase is absent at the basolateral membrane. Na(+)-K(+)-ATPase hydrolytic activity, determined by using a microassay that measured inorganic phosphate release from [gamma-(32)P]ATP under maximum velocity (V(max)) conditions, also increased in the differentiating CCD (from 316.2 +/- 44.4 pmol. h(-1). mm(-1) at 2 wk to 555.9 +/- 105.1 at 4 wk to 789.7 +/- 145.0 at 6 wk; r = 1.0 by linear regression analysis; P < 0.005). The parallel approximately 2.5-fold increases in Na(+)-K(+)-ATPase activity and ouabain-sensitive Rb uptake between 2- and 6-wk postnatal age suggest that the developmental increase in basolateral transport capacity is due predominantly to an increase in enzyme abundance. The signals mediating the developmental increase in Na(+)-K(+)-ATPase activity in the CCD remain to be defined.

Developmental expression of ROMK mRNA in rabbit cortical collecting duct.

The cortical collecting duct (CCD) is a major site of regulation of K+ homeostasis in the fully differentiated mammalian kidney. CCDs isolated from adult rabbits and microperfused in vitro secrete K+ into the tubular fluid at high rates. However, CCDs dissected from newborn animals show no significant net K+ secretion until the 3rd wk of life, at least in part because of a paucity of conducting apical secretory K+ (SK) channels. To determine whether the abundance of genes encoding the SK channel is developmentally regulated, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis to test for the presence of mRNA encoding rat outer medullary K+ channel (ROMK), considered to be a major subunit of the SK channel, in kidney and single CCDs isolated from maturing rabbits. Using rat ROMK-specific primers, RT-PCR of rabbit kidney yielded an amplification product of expected size and sequence. Northern blot analysis identified a single band at approximately 2.9 kb in kidney at all ages. Densitometric analysis revealed a progressive increase in steady state expression of ROMK message in kidney after birth. RT-PCR of individual CCDs yielded a single band of predicted size for ROMK in all segments isolated from animals > or =3 wk old. In contrast, transcripts were not detected in any CCD samples obtained from 1-wk-old animals and were identified in only 30% of CCD samples isolated from 2-wk-old rabbits. In all of the latter tubular samples, a specific PCR product of correct size for beta-actin mRNA was detected. These results suggest that an increase in steady state expression of ROMK mRNA contributes to the developmental appearance of conducting secretory K+ channels in the CCD.

Developmental expression of ROMK in rat kidney.

The apical secretory K+ (SK) channel in the principal cell represents the rate-limiting step for K+ secretion across the cortical collecting duct (CCD). Patch clamp analysis of maturing rabbit principal cells identifies an increase in number of conducting SK channels after the 2nd week of life [L. M. Satlin and L. G. Palmer. Am. J. Physiol. 272 (Renal Physiol. 41): F397-F404, 1997], approximately 1 wk after an increase in activity of the amiloride-sensitive epithelial Na+ channel (ENaC) is detected. To correlate the postnatal increase in channel activity with developmental expression of ROMK, the molecular correlate of the SK channel, we used gene-specific probes to show a developmental increase in abundance of renal ROMK mRNA and a ROMK-specific antibody to examine the nephron distribution, localization, and abundance of this protein in developing rat kidney. Using antibodies directed against aquaporin-3 (AQP-3) and Tamm-Horsfall protein (THP), we confirmed that ROMK was expressed along the apical membranes of principal cells and thick ascending limbs of Henle (TALH) in adult kidney. Within the midcortex of the neonatal kidney, ROMK-positive segments revealed weak coincident staining with the TALH-specific antibody but did not colabel with an antibody directed against distal and connecting tubule (CNT)-specific kallikrein or the lectin Dolichos biflorus agglutinin (DBA), which labels proximal tubules and collecting ducts. In inner cortex and outer medulla of kidneys from 1-wk-old animals, ROMK protein was identified in medullary TALH (MTALH) and DBA-positive collecting ducts. By 3 wk of age, coincident ROMK and DBA expression was detected in midcortical and outer cortical CNTs and CCDs. Immunoblot analysis of plasma membrane-enriched fractions of maturing rat kidney revealed a developmental increase in a approximately 40-kDa band, the expected size for ROMK. Immunolocalization of alpha-ENaC showed apical staining of a majority of cells in distal nephron segments after the 1st week of postnatal life. The beta- and gamma-ENaC subunit expression was routinely detected in a mostly cytoplasmic distribution immediately after birth, albeit in low abundance; gamma-ENaC showed some apical polarization. These results suggest that the postnatal increases in a principal cell apical SK and Na+ channel activity are mediated, at least in part, by increases in abundance of ROMK message and protein and ENaC subunit proteins.

Regulation of potassium transport in the maturing kidney.

Kidneys of full-term newborn humans and animals conserve potassium (K+), a condition essential for growth. The cortical collecting duct (CCD) is uniquely adapted to accomplish this task early in life. CCDs isolated from newborn rabbits and microperfused in vitro show no net K+ secretion until after the third week of life; in contrast, segments isolated from adult animals secrete net K+ at high rates. The magnitude and direction of net K+ transport in the CCD reflect the balance of opposing fluxes of K+ secretion and K+ absorption mediated by principal and intercalated cells, respectively. The absence of net K+ secretion in the CCD early in life may thus be caused by a limited capacity of principal cells for K+ secretion and/or an excess of K+ absorption by intercalated cells. Recent studies provide data to support both possibilities. Patch-clamp analysis detects few conducting apical K+-secretory channels in neonatal principal cells, whereas fluorescent functional assays identify significant activity of the apical hydrogen, potassium adenosine triphosphatase (H+,K+-ATPase), a pump that reabsorbs K+ in exchange for H+s, in adjacent intercalated cells. Under conditions prevailing in vivo, the sum of the fluxes mediated by these two cell types likely contributes to the relative K+ retention characteristic of the neonatal kidney.

Characterization and regulation of H-K-ATPase in intercalated cells of rabbit cortical collecting duct.

K-dependent H+ extrusion was investigated using fluorescence techniques in rabbit cortical collecting tubules (CCTs). Experiments were performed in split-open tubules from normal animals exposed to the intracellular pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). This preparation permitted the study of individual intercalated cells (ICs). In the ICs, partial recovery of pH(i) was observed in response to an acute acid load upon readdition of 5 mM K to the superfusate. This recovery was SCH 28080-inhibitable (10(-5) M) and ouabain-insensitive suggesting the process is mediated by a gastric-type H-K ATPase. To see if H-K ATPase plays a role in acid secretion its function was evaluated under chronic metabolic acidosis (CMA) conditions. CMA was induced by replacing drinking water with 75 mM NH4Cl in 5% sucrose for 10-14 days. The SCH 28080-inhibitable K-dependent pH(i) recovery rate was three-fold higher in CMA ICs compared to controls. To determine the location of the H-K ATPase, CCTs were microperfused and individual peanut lectin binding (PNA) ICs studied. K-dependent pH(i) recovery was measured in response to an NH4Cl pulse. An apical SCH 28080-inhibited K-dependent pH(i) recovery process was observed in control and CMA ICs. Taken together these data confirm the existence of a gastric-type H-K ATPase in ICs of rabbit CCT. Based on our findings the H-K ATPase is found on the apical side of the cell and is stimulated under conditions of CMA.

Organic anion transporting polypeptide mediates organic anion/HCO3- exchange.

Organic anion transporting polypeptide (oatp) is an integral membrane protein cloned from rat liver that mediates Na+-independent transport of organic anions such as sulfobromophthalein and taurocholic acid. Previous studies in rat hepatocytes suggested that organic anion uptake is associated with base exchange. To better characterize the mechanism of oatp-mediated organic anion uptake, we examined transport of taurocholate in a HeLa cell line stably transfected with oatp under the regulation of a zinc-inducible promoter (Shi, X., Bai, S., Ford, A. C., Burk, R. D., Jacquemin, E., Hagenbuch, B., Meier, P. J., and Wolkoff, A. W. (1995) J. Biol. Chem. 270, 25591-25595). Whereas noninduced transfected cells showed virtually no uptake of [3H]taurocholate, taurocholate uptake by induced cells was Na+-independent and saturable (Km = 19.4 +/- 3.3 microM; Vmax = 62.2 +/- 1.4 pmol/min/mg protein; n = 3). To test whether organic anion transport is coupled to HCO3- extrusion, we compared the rates of taurocholate-dependent HCO3- efflux from alkali-loaded noninduced and induced cells. Monolayers grown on glass coverslips were loaded with the pH-sensitive dye 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; intracellular pH (pHi) was measured by excitation ratio fluorometry. Noninduced and induced cells were alkalinized to an equivalent pHi ( approximately 7.7) by transient exposure to a 50 mM HCO3-, Cl--free solution. In the absence of extracellular Cl- and taurocholate, isohydric reduction of superfusate HCO3- concentration from 50 to 25 mM resulted in an insignificant change in pHi over time (dpHi/dt) in both groups. Addition of 25 microM taurocholate to the superfusate led to a rapid fall in pHi in induced (-0.037 +/- 0.011 pH units/min to pHi of 7.41 +/- 0.14) but not in noninduced (0.003 +/- 0.006 pH units/min to pHi of 7.61 +/- 0.08) cells (p < 0.03). These data indicate that oatp-mediated taurocholate transport is Na+-independent, saturable, and accompanied by HCO3- exchange. We conclude that organic anion/base exchange is an important, potentially regulatable component of oatp function.

Expression of carbonic anhydrase IV in carbonic anhydrase II-deficient mice.

Chronic metabolic acidosis (CMA) in the rabbit upregulates carbonic anhydrase (CA) IV in the proximal convoluted tubule (PCT). This study was designed to assess CA IV expression in a model of CMA in the mouse, i.e., congenital deficiency in CA II [CA(II)D]. In female CA(II)D mice, CA IV specific activity but not CA IV immunoreactivity was upregulated in the renal cortex, specifically in microdissected PCTs. Western blot analysis showed higher expression of CA IV immunoreactive protein in renal membranes from males than in those from females.

Apical K+ conductance in maturing rabbit principal cell.

Net K+ secretion is not detected in cortical collecting ducts (CCDs) isolated from newborn rabbits and perfused in vitro. To establish whether a low apical K+ permeability of the neonatal principal cell limits K+ secretion early in life, we used the patch-clamp technique in split-open CCDs isolated from maturing rabbits to study the properties and density of conducting K+ channels in principal cells. With KCl in the pipette and a NaCl solution warmed to 37 degrees C in the bath, inward currents with a conductance of approximately 42 pS were observed in 0% (0 out of 13 or 0/13), 10% (2/21), 18% (5/28), 29% (4/14), and 56% (10/18) of cell-attached patches obtained in 1-, 2-, 3-, 4-, and 5-wk-old animals, respectively. The conductance and reversal potential of this channel led us to suspect that it represented the low-conductance K+ channel previously described in the rat CCD by L. G. Palmer, L. Antonian, and G. Frindt (J. Gen. Physiol. 104: 693-710, 1994). The mean number of open channels per patch (NPo) increased progressively (P < 0.05) after birth, from 0 at 1 wk, to 0.06 +/- 0.04 at 2 wk, to 0.40 +/- 0.18 at 3 wk, to 0.74 +/- 0.41 at 4 wk, and to 1.06 +/- 0.28 at 5 wk. The increase in NPo appeared to be due primarily to a developmental increase in N, which is the number of channels; open probability, Po, remained constant at approximately 0.5 for all channels identified after the 2nd wk of life. The increase in number of conducting K+ channels during postnatal life is likely to contribute to the maturational increase in net K+ secretion in the CCDs.

H-K-ATPase activity in PNA-binding intercalated cells of newborn rabbit cortical collecting duct.

Functional and immunocytochemical studies indicate that intercalated cells in the adult rabbit cortical collecting duct (CCD) possess an H-K-adenosinetriphosphatase (H-K-ATPase). Because growing subjects must retain K+ and excrete H+, we sought to determine whether H-K-ATPase is present in the CCD early in life and, if so, to assess its activity and polarity. H-K-ATPase activity was defined as the initial rate of Sch-28080-inhibitable K+-dependent cell pH (pHi) recovery observed, in the absence of Na+, in response to an in vitro acid load. Transporter activity was assayed in intercalated cells labeled with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and apical cell surface marker rhodamine peanut lectin (PNA) in split-open CCDs isolated from neonatal and adult New Zealand White rabbits. In Na+-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions (nominal absence of CO2/HCO3-), the rate of K+-dependent pH(i) recovery from a NH4Cl-induced acid load was similar in newborn (0.056 +/- 0.015 pH U/min, n = 9) and adult (0.060 +/- 0.019 pH U/min; n = 9, P = not significant) cells. This rate of K+-dependent pH(i) recovery was significantly reduced by 10-20 pM Sch-28080, an inhibitor of gastric H-K-ATPase, in both newborns (0.009 +/- 0.003 pH U/min, n = 7) and adults (0.013 +/- 0.007 pH U/min, n = 9) (P < 0.05 compared with rates in absence of inhibitor). To determine whether the location of the transporter is consistent with a role in K+ absorption and H+ secretion, pH(i) recovery of acutely acid-loaded intercalated cells in neonatal CCDs (n = 7) microperfused and bathed in the absence of Na+ and K+ was monitored after selective addition of K+ to either the luminal or basolateral membrane. Addition of 5 mM K+ led to a significantly greater rate of pH(i) recovery when it was added to the luminal rather than the peritubular solution (0.049 +/- 0.005 vs. 0.018 +/- 0.005 pH U/min, P < 0.05). We conclude that PNA-binding intercalated cells of the neonatal CCD possess H-K-ATPase activity, predominantly located in the apical membrane. This provides a mechanism for H secretion and K+ retention, processes required for growth.

Apical Na+ conductance in maturing rabbit principal cell.

Net Na+ absorption in microperfused rabbit cortical collecting ducts (CCDs) is low during the 1st wk of postnatal life, increasing substantially thereafter [L. M. Satlin. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F57-F65, 1994]. To establish whether the low rate of Na+ absorption observed immediately after birth is due to a low apical Na+ permeability of the neonatal principal cell, we used the patch-clamp technique in split-open CCDs isolated from maturing rabbits to estimate conductance, number (N), and open probability (Po) of apical Na+ channels in principal cells. With LiCl in the pipette and a NaCl or potassium gluconate solution, warmed to 37 degrees C, in the bath, inward currents with a conductance of approximately 11 pS (n = 23) were observed in 17% of cell-attached patches at 1 wk, 41% of patches at 2 wk, and 43% of patches at 5 wk. The mean N per patch in the 1st wk (0.22 +/- 0.09; n = 36) was significantly less than that observed in the 2nd (1.38 +/- 0.39; n = 34) and 5th (1.24 +/- 0.37; n = 21) wk of life. Po, studied at positive pipette voltages, was significantly lower in the 1st wk (0.085 +/- 0.035; n = 5) than in the 2nd wk (0.345 +/- 0.063; n = 9) and 5th wk (0.291 +/- 0.058; n = 4). To confirm that the 11-pS channel represented the amiloride-sensitive apical Na+ channel, cell-attached patches in CCDs isolated from 2-wk-old rabbits were studied with 0.5 microM amiloride added to the LiCl pipette solution. Amiloride led to > 90% reductions in mean open and closed times of the 11-pS conductance, consistent with blockade of the channel. These data indicate that N and Po of apical amiloride-sensitive Na+ channels in principal cells increase significantly after birth.

Stimulation of apical H-K-ATPase in intercalated cells of cortical collecting duct with chronic metabolic acidosis.

This study evaluated the role of H-K-adenosinetriphosphatase (H-K-ATPase) with chronic metabolic acidosis (CMA) in intercalated cells (ICs) of rabbit cortical collecting duct (CCD). CMA was induced by replacing drinking water with 75 mM NH4Cl in 5% sucrose for 10-14 days. CCDs isolated from CMA and control rabbits were split open and exposed to the intracellular pH (pHi) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions, the resting pHi in ICs was similar for both groups. K-dependent pHi recovery (5 mM K, 140 mM N-methyl-D-glucamine) was monitored in response to a pulse of NH4Cl (10 mM). The K-dependent pHi recovery rate was threefold higher in CMAICs compared with controls and was abolished with the gastric H-K-ATPase inhibitor, Sch-28080 (10(-5) M). Polarity of the H-K-ATPase was studied in microperfused CMA and control CCDs. Luminal K-dependent pHi recovery was monitored in response to an acute pulse of NH4Cl in individual peanut lectin agglutinin (PNA)-binding ICs. The apical Sch-28080-inhibitable K-dependent pHi recovery rate was significantly greater in CMA ICs than control ICs. In summary, CMA enhances functional activity of an apical H-K-ATPase in PNA-binding ICs of rabbit CCD.

H+ secretion in rabbit mesonephric collecting tubule.

The mesonephros, the precursor of the metanephros, the definitive kidney, is the functional excretory organ in the 12- to 20-day-old rabbit fetus. It is believed to acidify allantoic fluid. To determine whether H+ excretion occurs in the distal nephron, we examined isolated perfused mesonephric collecting tubules by microcalorimetry and pH-sensitive fluorescent dyes. Collecting tubules secreted H+ (absorbed HCO3-) at rates twice those observed in the mature outer medullary collecting duct (OMCD) of the metanephric kidney. H+ secretion was not inhibited by ouabain (18.5 +/- 2.2 vs. 16.7 +/- 4.0 pmol.min-1.mm-1; n = 7, P = NS) but was reversibly inhibited by removing Cl- from the bathing solution (15.1 +/- 2.3 to -0.6 +/- 3.7 to 15.5 +/- 1.1 pmol.min-1.mm-1; n = 5, P < 0.05); luminal application of N-ethylmaleimide (NEM), an inhibitor of the H(+)-ATPase, also inhibited H+ secretion (n = 2). These results suggested that H+ secretion in the mesonephric collecting tubule is mediated by transporters similar to those of the OMCD. To test this hypothesis, we stained collecting tubules from 15-20 day embryos with 6-carboxyfluorescein (6-CF) diacetate to identify intercalated-like cells or perfused them with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) to measure intracellular pH (pHi). We found that 139 +/- 15 cells/mm concentrated 6-CF or BCECF, consistent with the phenotype of metanephric intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of bicarbonate transport in peanut lectin-positive intercalated cells by a monoclonal antibody.

Intercalated cells (ICC) of the collecting duct are believed to secrete acid (alpha-type) or HCO3 (beta-type). Although these two types of ICC are functionally mirror images of each other, several components in their cell membranes are clearly unique. As a first step in defining the molecular composition of beta-ICC membranes, we raised cell-specific monoclonal antibodies (MAb) against surface antigens. One of these MAb, designated B63, reacts with the apical membrane of peanut lectin agglutinin (PNA)-positive cells of the kidney cortex. B63-positive cells do not react with antibodies against band 3 (the basolateral C1/HCO3 exchanger) or ST.48, markers for alpha-ICC and principal cells, respectively. Despite a significant positive correlation between PNA and B63 staining intensities, determined by flow cytometry, these markers react with separate antigens, as indicated by competition studies and the different distribution of the two antigens. In addition to renal beta-ICC, B63 antigen is present in tissues that are involved in HCO3 secretion, such as the pancreas, salivary glands, and the small intestine, suggesting that it might play a role in HCO3 secretion. To test this hypothesis more directly, we tested the effect of MAb B63 on HCO3 secretion and on changes in intracellular pH (pHi) in isolated perfused cortical collecting ducts. Luminal Cl removal in the presence of luminal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid resulted in a reversible increase in pHi. Luminal application of MAb B63 prevented this change in pHi. MAb B63 also significantly inhibited (by 37.7 +/- 7.3%) HCO3 secretion by isolated perfused tubules, whereas another MAb (MAb 601), which reacts with a separate antigen on beta-ICC, did not alter HCO3 secretion or pHi. These results indicate that B63 antigen plays an important role in HCO3 secretion: it might be either the apical anion exchanger of beta-ICC or an associated regulatory protein.

Postnatal maturation of potassium transport in rabbit cortical collecting duct.

Clearance studies in newborns demonstrate low rates of urinary excretion of potassium, suggesting that the neonatal kidney contributes to the conservation of potassium necessary for growth. Because the cortical collecting duct (CCD) is a primary site for potassium secretion in the adult, we sought to examine the transport capacity of this segment for potassium during postnatal maturation. CCDs isolated from rabbits of various ages (5-6 animals/age group) were microperfused in vitro with solutions simulating plasma. The concentrations of potassium in samples of collected fluid, measured by helium glow photometry, were used to calculate net transport. At a flow rate of approximately 1.6 nl.min-1 x mm-1 net potassium secretion was absent at birth, first became evident at 4 wk of age (-11.08 +/- 2.39 pmol.min-1 x mm-1), and increased sharply thereafter to reach mature rates (-23.08 +/- 3.47 pmol.min-1 x mm-1; P < 0.05) by 6 wk of age. To determine whether low distal tubular flow rates limit net potassium secretion in the neonate, we perfused CCDs at two or more flow rates in the 0.5-5 nl.min-1 x mm-1 range. In CCDs taken from animals > or = 6 wk of age, potassium secretion showed a significant linear correlation with flow rate (y = -10.0x - 7.45; r = 0.87; n = 12).(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation of rabbit cortical collecting duct to in vitro acid incubation.

Cortical collecting ducts (CCDs) isolated from acid-loaded rabbits and perfused in vitro absorb HCO3-, whereas CCDs from normal animals secrete HCO3-. We have previously shown that CCDs incubated in vitro for 3 h at pH 6.9 show a reduction in net (baseline and stimulated) HCO3- secretion. In this study we ascertained the minimum duration of an acidic stimulus necessary to induce adaptive changes in stimulated HCO3- secretion (determined in the absence of basolateral Cl-) and the roles of protein synthesis and cytoskeletal function in this process. CCDs incubated in acid (pH 6.8, HCO3- 6 mM) for 1 h followed by incubation at pH 7.4 (HCO3- 25 mM) for 2 h showed a 41% reduction in stimulated HCO3- secretion (P < 0.001), similar to that observed after 3 h of incubation at pH 6.8. However, this incubation protocol failed to enhance stimulated HCO3- absorption (determined in the absence of luminal Cl-). Addition of 10 microM anisomycin, a reversible inhibitor of protein synthesis, throughout the entire period of incubation (1 h at pH 6.8 plus 2 h at pH 7.4) blocked adaptive reduction in HCO3- secretion, as did exposure to anisomycin only during the initial 1 h of acid incubation. In contrast, anisomycin application during the 2-h incubation at pH 7.4 failed to block this adaptation of HCO3- secretion. Application of 4 microM actinomycin D, an inhibitor of DNA transcription, during the acid incubation also prevented the adaptive response, as did application during the total or during the 2-h pH 7.4 incubation period of 0.2 microM cytochalasin D, an inhibitor of actin filament function.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal maturation of rabbit renal collecting duct. III. Peanut lectin-binding intercalated cells.

Measurements of transepithelial HCO3 transport in the rabbit cortical collecting duct (CCD) indicate that net HCO3 secretion becomes apparent only after the first month of life [F. M. Mehrgut, L. M. Satlin, and G. J. Schwartz, Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F801-F808, 1990]. We used fluorescent probes and immunocytochemistry to trace the postnatal functional development of the beta-intercalated cell, the HCO3-secreting cell of the fully differentiated CCD. Throughout maturation, the beta-intercalated cell was empirically identified by its selective uptake of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, an alkaline cell pH, apical binding to peanut agglutinin (PNA) and monoclonal antibody B63, and by its functional capacity for apical Cl-HCO3 exchange as manifested by Cl-dependent extrusion of an intracellular alkali load. Compared with the mature segment, the neonatal mid-CCD exhibited fewer intercalated cells, which were characterized by a less alkaline cell pH, reduced apical Cl-HCO3 exchange activity, and shorter apical binding profiles for PNA. There was evidence for basolateral Cl conductance and similar buffering capacity at all ages. In the neonatal outer cortex there was little or no binding to PNA or to B63. As soon as cell surface antigens characteristic of the fully differentiated beta-cell were detected, functional studies indicated the presence, albeit reduced, of apical Cl-HCO3 exchange. Thus there is postnatal proliferation and maturation of HCO3-secreting intercalated cells in the rabbit kidney; the origin of these cells remains to be determined.

Postnatal maturation of rabbit renal collecting duct. II. Morphological observations.

The renal collecting duct exhibits cellular heterogeneity. An ultrastructural analysis of this nephron segment during maturation has been performed to determine morphological characteristics of developing principal and intercalated cells (IC). Apical surface features and cytoplasmic ultrastructure were best revealed by scanning electron microscopy and by transmission electron microscopy, respectively. Morphological maturity was assessed by comparing measurements of apical perimeter, vesicular profiles, and mitochondrial volume percent. In mature cortical collecting duct (CCD), approximately 30% of total cells were rich in mitochondria and generally dark in matrix staining, i.e., IC. Most IC in outer cortex were microvillated, whereas in inner cortex most IC were microplicated with interspersed microvilli. Only approximately 16% of cells in outer medullary collecting duct (OMCD, outer stripe) were IC, with light cytoplasm and large protruding apical surfaces covered exclusively with microplicae. Neonatal CCDs showed no IC in outer cortex; a single type of principal-like cell was found in outer cortical region. Neonatal IC in both CCDs and OMCDs showed smaller apical perimeters, vesicular volumes, and mitochondrial volume percents than did mature IC. Whereas the number of cortical IC was reduced in the newborn, IC were present in mature numbers in OMCD and appeared relatively more mature than did IC of CCD. Some (approximately 10%) of IC in neonatal CCD did not show an identifiable apical surface pattern. Principal cells from both newborn CCD and OMCD were also morphometrically immature. It is clear that the neonatal collecting duct undergoes postnatal proliferation and differentiation.

Maturation of renal potassium transport.

The studies outlined in this review suggest that the immaturity of distal nephron segments may hinder urinary excretion of potassium early in life. Among the factors that may limit potassium secretion by principal cells in the neonatal cortical collecting duct are an unfavorable electrochemical gradient (reduced Ki, Na(+)-K(+)-ATPase activity and/or Vte), limited membrane permeability to potassium and sodium, low tubular fluid flow rate, reduced luminal sodium concentration, or increased paracellular backleak. Alternatively, enhanced potassium absorption by other relatively well-differentiated distal nephron segments may contribute in part to a reduced net potassium excretory rate in the newborn. It should be kept in mind, however, that the limited potassium secretory capacity of the immature kidney becomes clinically relevant only under conditions of potassium excess. Under normal circumstances, the tendency of the newborn to retain potassium is an appropriate and necessary condition for growth.(ABSTRACT TRUNCATED AT 250 WORDS)