Ammonium interaction with the epithelial sodium channel.

The purpose of this study was to investigate the direct effect of NH(3)/NH on mouse epithelial Na(+) channels (mENaC) expressed in Xenopus oocytes. Two-electrode voltage-clamp and ion-selective microelectrodes were used to measure the Na(+) current, intracellular pH (pH(i)), and ion activities in oocytes expressing mENaC. In oocytes expressing mENaC, removal of external Na(+) reversibly hyperpolarized membrane potential by 129 +/- 5.3 mV in the absence of 20 mM NH(4)Cl but only by 100 +/- 7.8 mV in its presence. Amiloride completely inhibited the changes in membrane potential. In oocytes expressing mENaC, butyrate (20 mM) caused a decrease in pH(i) (0.43 +/- 0.07) similar to the NH(4)Cl-induced pH(i) decrease (0.47 +/- 0.12). Removal of Na(+) in the presence of butyrate caused hyperpolarization that was not significantly different from that in the absence of butyrate at high pH(i) (in the absence of NH(4)Cl). Removal of external Na(+) resulted in an outward current of 3.7 +/- 0.8 microA (at -60 mV). The magnitude of this change in current was only 2.7 +/- 0.7 microA when Na(+) was removed in the presence of NH(4)Cl. In oocytes expressing mENaC, NH(4)Cl also caused a decrease in whole cell conductance at negative potential and an outward current at positive potential. In the presence of amiloride, steady-state current and the change in current caused by removal of Na(+) were not different from zero. These results indicate that NH(4)Cl inhibits Na(+) transport when mENaC is expressed in oocytes. The inhibition of voltage changes is not due to intracellular acidification caused by NH(4)Cl. Permeability and selectivity of ENaC to NH may play a role.

Transport of NH(3)/NH in oocytes expressing aquaporin-1.

The aim of this study was to determine whether expressing aquaporin (AQP)-1 could affect transport of NH(3). Using ion-selective microelectrodes, the experiments were conducted on frog oocytes (cells characterized by low NH(3) permeability) expressing AQP1. In H(2)O-injected oocytes, exposure to NH(3)/NH (20 mM, pH 7.5) caused a sustained cell acidification and no initial increase in pH(i) (as expected from NH(3) influx), and the cell depolarized to near 0 mV. The absence of Na(+), the presence of Ba(2+), or raising bath pH (pH(B)) did not inhibit the magnitude of the pH(i) decrease or result in an initial increase in pH(i) when NH(3)/NH was added. However, after the cell was acidified (because of NH(3)/NH), raising pH(B) to 8.0 caused a slow increase in pH(i) but had no effect on membrane potential. The changes in pH(i) with raising pH(B) did not occur in the absence of NH(3)/NH. In AQP1 oocytes, exposure to NH(3)/NH usually resulted in little or no change in pH(i), and in the absence of Na(+) there was a small increase in pH(i) (the cell still depolarized to near 0 mV). However, after exposure to NH(3)/NH, raising pH(B) to 8.0 caused pH(i) to increase more than two times faster than in control oocytes. This increase in pH(i) is likely the result of increased NH(3) entry and not the result of NH transport. These results indicate that 1) the oocyte membrane, although highly permeable to NH, has a significant NH(3) permeability and 2) NH(3) permeability is enhanced by AQP1.

Citrate and succinate transport in proximal tubule cells.

Urinary citrate, which inhibits calcium nephrolithiasis, is determined by proximal reabsorption via an apical dicarboxylate transporter. Citrate is predominantly trivalent at physiological pH, but citrate(-2) is transported at the apical membrane. We now demonstrate that low-Ca solutions induce transport of citrate(-2) and succinate in opossum kidney cells. With 1.2 mM extracellular Ca, citrate uptake was pH insensitive and not competed by succinate(-2). In contrast, with low extracellular Ca, citrate uptake increased twofold, was inhibited by succinate (and other dicarboxylates), was stimulated by lowering extracellular pH (consistent with citrate(-2) transport), and increased further by lowering extracellular Mg. The effect of Ca was incrementally concentration dependent, between 0 and 1.2 mM. The effect of Ca was not simply complexation with citrate because succinate (which is complexed significantly less) was affected by Ca similarly. Incubation of cells for 48 h in a low-pH media increased citrate transport (studied at control pH) more than twofold, suggesting induction of transporters.

Regulation of sodium transport in M-1 cells.

The M-1 cell line, derived from the mouse cortical collecting duct (CCD), is being used as a mammalian model of the CCD to study Na+ transport. The present studies aimed to further define the role of various hormones in affecting Na+ transport in M-1 cells grown in defined media. M-1 cells on permeable support, in serum-free media, developed amiloride-sensitive current 4-5 days after seeding. As expected for the involvement of epithelial Na+ channels, alpha-, beta-, and gamma-subunits of the epithelial Na+ channel were identified by RT-PCR. Either dexamethasone (Dex, 10-100 nM) or aldosterone (Aldo, 10(-6)-10(-7) M) for 24 h stimulated transport. Cells grown in the presence of Aldo and Dex had higher transport than with Dex alone. Spironolactone added to Dex media decreased transport. The acute effects of hormones reported to inhibit Na+ transport in CCD were also examined. Epidermal growth factor, phorbol esters, and increased intracellular Ca2+ with thapsigargin did not alter transport. Arginine vasopressin caused a transient increase in transport (probably Cl- secretion), which was not amiloride sensitive. Also, the protease inhibitor aprotinin decreased Na+ transport; in aprotinin-treated cells, trypsin stimulated transport. This study demonstrates that adrenal steroids (Dex > Aldo) stimulate Na+ transport in M-1 cells. At least part of this response may represent activation of mineralocorticoid receptors based on an additive effect of Dex and Aldo, as well as inhibition by spironolactone. Responses to immediate-acting hormones is limited. However, an endogenous protease activity, which activates Na+ transport, is present in these cells.

Metabolic support of collecting duct transport.

The present studies address the metabolic processes that support the reabsorption of sodium and the secretion of bicarbonate in the interspersed but distinct principal and intercalated cells of the cortical collecting duct (CCD). In microperfused rabbit CCD, sodium reabsorption was measured by lumen-to-bath 22Na flux, and bicarbonate transport was assayed by microcalorimetry. Flux measurements were made before and after metabolic substrate changes or application of metabolic inhibitors. Both sodium reabsorption and bicarbonate secretion were dependent on oxidative metabolism (inhibited by antimycin A) and appeared to have no special dependence on glycolysis or the hexose-monophosphate shunt pathways. Endogenous substrates (in the absence of exogenous metabolic substrates) supported a small component of sodium transport; in contrast, bicarbonate reabsorption in the outer medullary collecting duct, which was studied for comparison, was fully supported by endogenous substrates. In the CCD, sodium reabsorption was supported best by a mixture of basolateral metabolic substrates (glucose and acetate, as a fatty acid), whereas bicarbonate secretion was fully supported by either glucose or acetate. Alanine, as a representative amino acid, was not an effective metabolic substrate. Another contrasting feature of the two transport processes was that bicarbonate secretion, and not sodium transport, was supported to some extent by luminal glucose. In sum, principal cells and intercalated cells differ not only in their morphology and function, but also in their metabolism.

Citrate transport in proximal cell line.

Citrate uptake into kidney proximal tubules occurs via an apical dicarboxylate transporter and a poorly characterized process in the basolateral membrane. We used OK cells, a cell line derived from opossum kidney, to study citrate transport in proximal tubule-like cells. Citrate uptake into cell monolayers was studied using [14C]citrate with [3H]mannitol as a volume marker. Citrate uptake into these cells was sodium dependent and saturable with increasing concentrations of citrate. In contrast to previous models, citrate transport was altered minimally by changes in pH from 6.2 to 7.0 and increased at pH 7.4 to 7.8. A variety of di- and tricarboxylates were tested for interaction with citrate transport. The dicarboxylates succinate, malate, and oxaloacetate at 1 mM concentration inhibited citrate uptake minimally (uptake at least 80% of control); one dicarboxylate, alpha-ketoglutarate, did inhibit citrate uptake significantly. In contrast, the tricarboxylates isocitrate and tricarballylate inhibited citrate uptake significantly, indicating probable competitive inhibition with the transport process. These characteristics are distinctly different from those of the apical membrane dicarboxylate transporter. 1,2,3-Benzenetricarboxylic acid, an inhibitor of the mitochondrial tricarboxylate transporter, did not alter citrate uptake. In conclusion, the OK proximal cell line exhibits a novel citrate transport process compared with the apical transport of citrate described in most proximal systems. This transport process probably involves the trivalent species of citrate in contrast to the usual predominant transport of divalent citrate. This transport process may represent a process similar to that in the basolateral membrane of the proximal tubule.

Inner medullary collecting duct Na(+)-H+ exchanger.

Cells from the inner medullary collecting duct (IMCD) exhibit Na(+)-H+ exchange. The present studies were performed to address certain important characteristics of this process in cultured IMCD cells. First, Na(+)-H+ exchange was found to be present both at 37 degrees C and at 25 degrees C, in contrast to Na(+)-independent H+ extrusion, which was only observed in some cultures and only at 37 degrees C. Second, with the use of image analysis techniques, virtually all cells in IMCD cultures were demonstrated to possess Na(+)-H+ exchange, whether or not the cells exhibited Na(+)-independent intracellular pH recovery from acid loads. Also, Na(+)-H+ exchange was found to be expressed on the basolateral aspect of these cells, but not on the apical membrane. These properties of IMCD Na(+)-H+ exchange are consistent with a function to regulate intracellular pH rather than mediate transepithelial acid-base transport. Na(+)-H+ exchange in IMCD cells was also compared with that in cultured renal proximal tubule cells. Despite physiologically distinct roles in vivo, Na(+)-H+ exchange in these two cell types in culture was found to be similar with respect to the Km for Na+ and the Ki for 5-(N-ethyl-N-isopropyl)amiloride. These data are consistent with functionally similar (if not identical) processes mediating Na(+)-H+ exchange in these two cell types, but with opposite polarity.

Increased sodium transport by cortical collecting tubules from remnant kidneys.

To determine whether intrinsic changes in cortical collecting tubule (CCT) transport contribute to the maintenance of sodium and acid-base balance after loss of renal mass, we studied transport functions in isolated perfused CCT from rabbit remnant kidneys. The rabbits were sacrificed three weeks after surgical reduction of renal mass (by 3/4 to 7/8) at which time they were mildly azotemic but had no systemic electrolyte or acid-base disturbances. When perfused by standard methods in vitro, CCT from remnant kidneys exhibited sodium transport rates (lumen-to-bath 22Na-flux) approximately twice as high as those in CCT from control animals (111 +/- 19 vs. 54 +/- 7 pmol/min mm, P less than 0.02). A similar difference was present in the ouabain-sensitive sodium fluxes (81 +/- 16 vs. 39 +/- 8 pmol/min mm, P less than 0.05). In contrast, there were no significant differences in net bicarbonate transport. Significant hypertrophy of the remnant kidney CCT was reflected by 30 to 45% increases in tubule diameters. To examine the possible role of differences in food intake, we studied a separate group of weight-matched, pair-fed sham-operated and remnant kidney rabbits. Similar differences in total and ouabain-sensitive 22Na-flux, and in tubule size persisted in the pair-fed animals. A dissociation between active sodium transport and tubule hypertrophy was documented in the outer medullary collecting tubule: despite the lack of active sodium transport, hypertrophy was present. Our studies show that loss of renal mass results in a selective augmentation of certain transport processes in the CCT, implying selective or specific signals and mechanisms.

Effect of epidermal growth factor on sodium transport in the cortical collecting tubule.

Receptors for epidermal growth factor (EGF) have been demonstrated in the distal tubule. To determine whether there are acute functional correlates, we studied the effect of EGF on cortical collecting tubule (CCT) transepithelial voltage and transport of sodium and bicarbonate. Rabbit CCT were perfused in vitro and EGF was added to either the bathing medium or the luminal perfusate after base-line measurements of transport. Peritubular EGF in concentrations of 0.1 to 100 ng/ml (1.7 X 10(-11) to 1.7 X 10(-8) M) decreased sodium reabsorption, measured as 22Na absorption from the lumen, by 44-59%. There was a corresponding fall in the lumen-negative transepithelial voltage. Lower doses of EGF were without effect on transepithelial voltage or sodium transport. Pretreatment of the tubules with ouabain eliminated the effect of EGF on sodium transport. In contrast to peritubular EGF, luminal EGF (100 ng/ml) did not affect sodium transport. Peritubular EGF had no effect on either bicarbonate secretion or net bicarbonate transport in the CCT and no effect on net bicarbonate reabsorption in the medullary collecting tubule. Using the pH sensitive, fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, no change in principal cell intracellular pH was found after peritubular EGF. Two other growth factors, insulin and insulin-like growth factor I were without effect on sodium transport. We conclude that EGF inhibits active sodium absorption in CCT via receptors located at the basolateral membrane.

Control of bicarbonate transport in collecting tubules from normal and remnant kidneys.

Bicarbonate transport in the rabbit cortical collecting tubule (CCT) and outer medullary collecting tubule (MCT) in vitro was studied under two types of conditions that were anticipated to alter distal tubule bicarbonate transport: 1) reduction of renal mass, and 2) acid and base loading in vivo. Bicarbonate secretion (both total and acetazolamide sensitive) and bicarbonate reabsorption (studied separately) in CCT and bicarbonate reabsorption in the MCT were not different between tubules from normal and remnant kidneys. The control or conditioning of the separate processes of bicarbonate secretion and bicarbonate reabsorption was also studied in CCT from normal and remnant kidneys. Bicarbonate secretion was not increased by base-loading animals with either normal or remnant kidneys. In contrast, bicarbonate secretion was consistently decreased by acid loading (studied in CCT from remnant kidneys). Bicarbonate reabsorption in the CCT was not altered by acid or base loads given to animals with normal kidneys. And bicarbonate reabsorption in MCT was not increased by acid loading of animals with remnant kidneys. These studies demonstrate that bicarbonate transport (and its conditioning by acid or base loads in vivo) in both CCT and MCT in vitro is not altered by reduction of renal mass in rabbits. The predominant conditioning effect of acid or base loads in vivo is for acid loads to inhibit CCT bicarbonate secretion.