Active ion transport in the renal proximal tubule. II. Ionic dependence of the Na pump

The dependence of Na pump activity on intracellular and extracellular Na+ and K+ was investigated using a suspension of rabbit cortical tubules that contained mostly (86%) proximal tubules. The ouabain- sensitive rate of respiration (QO2) was used to measure the Na pump activity of intact tubules, and the Na,K-ATPase hydrolytic activity was measured using lysed proximal tubule membranes. The dependence (K0.5) of the Na pump on intracellular Na+ was affected by the relative intracellular concentration of K+, ranging from approximately 10 to 15 mM at low K+ and increasing to approximately 30 mM as the intracellular K+ was increased. The Na pump had a K0.5 for extracellular K+ of 1.3 mM in the presence of saturating concentrations of intracellular Na+. Measurements of the Na,K-ATPase activity under comparable conditions rendered similar values for the K0.5 of Na+ and K+. The Na pump activity in the intact tubules saturated as a function of extracellular Na at approximately 80 mM Na, with a K0.5 of 30 mM. Since Na pump activity under these conditions could be further stimulated by increasing Na+ entry with the cationophore nystatin, these values pertain to the Na+ entry step and not to the Na+ dependence of the intracellular Na+ site. When tubules were exposed to different extracellular K+ concentrations and the intracellular Na+ concentration was subsaturating, the Na pump had an apparent K0.5 of 0.4 mM for extracellular K. Under normal physiological conditions, the Na pump is unsaturated with respect to intracellular Na+, and indirect analysis suggests that the proximal cell may have an intracellular Na+ concentration of approximately 35 mM.     

Active ion transport in the renal proximal tubule. III. The ATP dependence of the Na pump

The dependence of the Na pump activity of intact renal tubules on the ATP concentration was investigated using a suspension of rabbit cortical tubules. Rotenone (an inhibitor of mitochondrial oxidative phosphorylation) was used in graded fashion to alter the cellular ATP, and the Na pump activity was measured when the pump was stimulated by adding KCl to tubules suspended in a K+-free medium. The K+ uptake into the tubule was measured using an extracellular K+ electrode, and the oxygen consumption (QO2) was measured using a Clark-type oxygen electrode. The Na pump activity was found to have a linear, nonsaturating dependence on the ATP concentration. However, the Na,K- ATPase hydrolytic activity (assayed biochemically) of lysed proximal tubule membranes demonstrated saturation and had a K0.5 value of 0.4 mM ATP. Presumably, unknown cytosolic factors present in the intact renal cell but not normally present in the biochemical assay accounted for the differences between the two measurements. The data suggest that an alteration in the intracellular ATP will result in a proportional change in active ion transport activity. Moreover, additional findings also suggest that the basal (non-transport-related) QO2 may be redirected to support the proximal Na pump activity when transport activity is stressed. Thus, basal respiration is not invariant under all conditions, and ion transport activity appears to be maintained foremost among cellular ATP-dependent processes.     

Rheogenic transport in the renal proximal tubule

The electrophysiology of the renal Na-K ATPase was studied in isolated perfused amphibian proximal tubules during alterations in bath (serosal) potassium. Intracellular and extracellular ionic activity measurements permitted continuous evaluation of the Nernst potentials for Na+, K+, and Cl- across the basolateral membrane. The cell membrane and transepithelial potential differences and resistances were also determined. Return of K to the basal (serosal) solution after a 20-min incubation in K-free solution hyperpolarized the basolateral membrane to an electrical potential that was more negative than the Nernst potential for either Na, Cl, or K. This constitutes strong evidence that at least under stimulated conditions the Na-K ATPase located at the basolateral membrane of the renal proximal tubule mediates a rheogenic process which directly transfers net charge across the cell membrane. Interpretation of these data in terms of an electrical equivalent circuit permitted calculation of both the rheogenic current and the Na/K coupling ratio of the basolateral pump. During the period between 1 and 3 min after pump reactivation by return of bath K, the basolateral rheogenic current was directly proportional to the intracellular Na activity, and the pump stoichiometry transiently exceeded the coupling ratio of 3Na to 2K reported in other preparations.     

Amino acid transport in the renal proximal tubule

Summary. In the kidney the proximal tubule is responsible for the uptake of amino acids. This occurs via a variety of functionally and structurally different amino acid transporters located in the luminal and basolateral membrane. Some of these transporters show an ion-dependence (e.g. Na⁺, Cl⁻ and K⁺) or use an H⁺-gradient to drive transport. Only a few amino acid transporters have been cloned or functionally characterized in detail so far and their structure is known, while little is known about a majority of amino acid transporters. Only few attempts have been untertaken looking at the regulation of amino acid transport. We summarized more recent information on amino acid transport in the renal proximal tubule emphasizing functional and regulatory aspects. Received August 8, 1999; Accepted April 20, 2000     

Sulphate and phosphate transport in the renal proximal tubule

Experiments performed on microperfused proximal tubules and brush-border membrane vesicles revealed that inorganic phosphate is actively reabsorbed in the proximal tubule involving a 2 Na+-HPO2-4 or H2PO-4 co-transport step in the brush-border membrane and a sodium-independent exit step in the basolateral cell membrane. Na+-phosphate co-transport is competitively inhibited by arsenate. The transtubular transport regulation is mirrored by the brush-border transport step: it is inhibited by parathyroid hormone intracellularly mediated by cyclic AMP. Transepithelial inorganic phosphate (Pi) transport and Na+-dependent Pi transport across the brush-border membrane correlates inversely with the Pi content of the diet. Intraluminal acidification as well as intracellular alkalinization led to a reduction of transepithelial Pi transport. Data from brush-border membrane vesicles indicate that high luminal H+ concentrations reduce the affinity for Na+ of the Na+-phosphate co-transport system, and that this mechanism might be responsible for the pH dependence of phosphate reabsorption. Contraluminal influx of Pi from the interstitium into the cell could be partly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). It is not, however, changed when dicarboxylic acids are present or when the pH of the perfusate is reduced to pH 6. Sulphate is actively reabsorbed, involving electroneutral 2 Na+-SO2-4 co-transport through the brush-border membrane. This transport step is inhibited by thiosulphate and molybdate, but not by phosphate or tungstate. The transtubular active sulphate reabsorption is not pH dependent, but is diminished by the absence of bicarbonate. The transport of sulphate through the contraluminal cell side is inhibited by DIDS and diminished when the capillary perfusate contains no bicarbonate or chloride. The latter data indicate the presence of an anion exchange system in the contraluminal cell membrane like that in the erythrocyte membrane.     

Ion exchangers mediating NaCl transport in the renal proximal tubule

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. Studies of isolated brush-border membrane vesicles confirmed the presence of Na+-H+ exchange and identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanisms of uphill Cl- entry. We found that formate and oxalate each stimulate NaCl absorption in microperfused proximal tubules. Stimulation of NaCl absorption by formate was blocked by the Na+-H+-exchange inhibitor EIPA, whereas stimulation by oxalate was blocked by omission of sulfate from the perfusion solutions. These observations were consistent with recycling of formate from lumen to cell by H+-coupled formate transport in parallel with Na+-H+ exchange and recycling of oxalate by oxalate-sulfate exchange in parallel with Na+-sulfate cotransport. Using isoform-specific antibodies, we found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the loop of Henle. The inhibitor sensitivity of Na+-H+ exchange in renal brush-border vesicles and of HCO3- absorption in microperfused tubules suggested that NHE3 is responsible for most, if not all, apical membrane Na+-H+ exchange in the proximal tubule. The role of NHE3 in mediating proximal tubule HCO3- absorption and formate-dependent Cl- absorption was confirmed by studies in NHE3 null mice. Finally, we cloned and functionally expressed CFEX, an anion transporter expressed on the apical surface of proximal tubule cells and capable of mediating Cl(-)-formate exchange.     

Angiotensin II and proximal tubule sodium transport.

As a target site for angiotensin II (A-II), renal proximal tubule is unique in that it may be equipped with a local A-II generating system and that both basolateral and apical membranes may be accessible for A-II's action. We have recently conducted studies to examine these possibilities. With in vitro cultured proximal tubular cells, we have demonstrated de novo synthesis of angiotensinogen and renin. With isolated renal brush border membrane (BBM), we have confirmed the presence of A-II receptors and found that A-II directly stimulated BBM Na(+)-H+ exchange. In search of the signal transduction mechanism, we have found that A-II also activated BBM phospholipase A2 (PLA) and that BBM contained a pertussis toxin-sensitive guanine nucleotide binding protein (G-protein) which mediates the effects of A-II. Further studies showed that prevention of PLA activation abolished A-II's effect on Na(+)-H+ exchange, and that activation of PLA by mellitin and addition of arachidonic acid similarly enhanced Na(+)-H+ exchange activity, suggesting that PLA activation may mediate the stimulatory effect of A-II on Na(+)-H+ exchange. These results thus indicate that a local signal transduction mechanism involving G-protein mediated PLA activation exists in renal BBM which mediates A-II's effect on Na(+)-H+ exchange. Taken together, we propose that, independent of A-II in the circulation, local luminal A-II may serve as an important regulatory system on sodium transport in renal proximal tubule.     

Kinetic transport model for cellular regulation of pH and solute concentration in the renal proximal tubule.

An open circuit kinetic model was developed to calculate the time course of proximal tubule cell pH, solute concentrations, and volume in response to induced perturbations in luminal or peritubular fluid composition. Solute fluxes were calculated from electrokinetic equations containing terms for known carrier saturabilities, allosteric dependences, and ion coupling ratios. Apical and basolateral membrane potentials were determined iteratively from the requirements of cell electroneutrality and equal opposing transcellular and paracellular currents. The model converged to membrane potentials accurate to 0.05% in one to four iterations. Model variables included cell concentrations of Na, K, HCO3, glucose, pH (uniform CO2), volume, and apical and basolateral membrane potentials. The basic model contained passive apical membrane transport of Na/H, Na/glucose, H and K, basolateral transport of Na/3HCO3, K, H, and glucose, and paracellular transport of Na, K, Cl, and HCO3; apical H and basolateral 3Na/2K-ATPases were present. Apical Na/H and basolateral K transport were regulated allosterically by pH. Apical Na/H transport, basolateral Na/3HCO3 transport, and the 3Na/2K-ATPase were saturable. Model parameters were chosen from data in the rat proximal tubule. Model predictions for the magnitude and time course of cell pH, Na, and membrane potential in response to rapid changes in apical and peritubular Na and HCO3 were in excellent agreement with experiment. In addition, the model requires that there exist an apical H-ATPase, basolateral Na/3HCO3 transport saturable with HCO3, and electroneutral basolateral K transport.     

Transepithelial sulfate transport by avian renal proximal tubule epithelium in primary culture

The mechanisms and control of transepithelial inorganic sulfate (Si) transport by primary cultures of chick renal proximal tubule monolayers in Ussing chambers were determined. The competitive anion, S2 O 3 2- (5 mM), reduced both unidirectional reabsorptive and secretory fluxes and net Si reabsorption with no effect on electrophysiological properties. The carbonic anhydrase (CA) inhibitor ethoxzolamide decreased net Si reabsorption approximately 45%. CAII protein and activity were detected in isolated chick proximal tubules by immunoblots and biochemical assay, respectively. Cortisol reduced net Si reabsorption up to approximately 50% in a concentration-dependent manner. Thyroid hormone increased net Si reabsorption threefold in 24 h, and parathyroid hormone (PTH) acutely stimulated net Si reabsorption approximately 45%. These data indicate that CA participates in avian proximal tubule active transepithelial Si reabsorption, which cortisol directly inhibits and T3 and PTH directly stimulate.     

Alpha 2-adrenoceptor control of ion and water transport in the newt renal distal tubule.

To study the nature of adrenergic stimulation of ions and water reabsorption in the newt renal distal tubule, stationary microperfusion of the nephron and electron probe analysis were used. After application of norepinephrine (NE 10(-6) M) to the tubule surface, the fractional reabsorption of fluid increased from 15.0 +/- 3.1 to 41.30 +/- 10.4% (n = 7, p < 0.01), of Na+ from 69.30 +/- 6.6 to 79.10 +/- 7.5% (p < 0.05), Cl- from 63.30 +/- 7.6 to 72.40 +/- 7.9% (p < 0.05). Instead of secretion (control), there was reabsorption of K+. Fractional reabsorption of Ca2+ decreased from 51.00 +/- 6.0 to 43.00 +/- 7.0% (p < 0.05). The nonspecific alpha-adrenergic antagonist dibenamine 10(-6) M completely inhibited the effect of NE while, under the action of propranolol (2 x 10(-6) M) NE increased ion and water reabsorption significantly. When applied alone, or with NE, the specific alpha 2-adrenoblocker idazoxan, 2 x 10(-6) M, did not interfere with reabsorption in the distal tubule. At the same time, under the action of alpha 1-adrenoblocker prazosin 2 x 10(-6) M NE, increased the fractional reabsorption of fluid from 24.10 +/- 3.4 to 44.40 +/- 4.0% (n = 6, p < 0.001). These results serve as evidence that there exist specific alpha 2-adrenoceptors in the newt distal tubule the stimulation of which increases membrane permeability of the distal tubule to water, Na+, K+, Cl-, but not to Ca2+.     

Intracellular ion activities in Necturus proximal tubule

Ion-sensitive microelectrodes were used to measure the intracellular activities of Na, K, and Cl in proximal tubules of the perfused Necturus kidney. Cell Cl was 2-3 times higher than the value predicted for passive distribution during perfusion with normal Ringer; intracellular Na was far below the level for passive distribution. Cell Na and Cl fell to very low values when the lumen was NaCl-free. Cl entry into the tubule cell from the lumen required luminal Na. Na entered the cell across the luminal membrane both by diffusion and by coupled movement with Cl.     

Axial heterogeneity of organic cation transport along the rabbit renal proximal tubule: studies with brush-border membrane vesicles

Brush-border membrane vesicles prepared from rabbit kidney outer cortex (rich in S1 and S2) and outer medulla (rich in S3) were used to evaluate the axial heterogeneity of tetraethylammonium transport in the proximal tubule. The vesicle preparations had similar Km values but the Vmax values differed, suggesting that axial heterogeneity of tetraethylammonium secretion may be due to differences in transport across the brush-border membrane.     

Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3- transport

We have used pH-, Na-, and Cl-sensitive microelectrodes to study basolateral HCO3- transport in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum. In one series of experiments, we lowered basolateral pH (pHb) from 7.5 to 6.8 by reducing [HCO3-]b from 10 to 2 mM at a constant pCO2. This reduction of pHb and [HCO3-]b causes a large (approximately 0.35), rapid fall in pHi as well as a transient depolarization of the basolateral membrane. Returning pHb and [HCO3-]b to normal has the opposite effects. Similar reductions of luminal pH (pHl) and [HCO3-]l have only minor effects. The reduction of [HCO3-]b and pHb also produces a reversible fall in aiNa. In a second series of experiments, we reduced [Na+]b at constant [HCO3-]b and pHb, and also observed a rapid fall in pHi and a transient basolateral depolarization. These changes are reversed by returning [Na+]b to normal. The effects of altering [Na+]l in the presence of HCO3-, or of altering [Na+]b in the nominal absence of HCO3-, are substantially less. Although the effects on pHi and basolateral membrane potential of altering either [HCO3-]b or [Na+]b are largely blocked by 4-acetamido-4- isothiocyanostilbene-2,2'-disulfonate (SITS), they are not affected by removal of Cl-, nor are there accompanying changes in aiCl consistent with a tight linkage between Cl- fluxes and those of Na+ and HCO3-. The aforementioned changes are apparently mediated by a single transport system, not involving Cl-. We conclude that HCO3- transport is restricted to the basolateral membrane, and that HCO3- fluxes are linked to those of Na+. The data are compatible with an electrogenic Na/HCO3 transporter that carries Na+, HCO3-, and net negative charge in the same direction.     

Angiotensin II actions in the rabbit proximal tubule. Angiotensin II mediated signaling mechanisms and electrolyte transport in the rabbit proximal tubule.

Angiotensin II (AngII) is a potent regulator of electrolyte transport with biphasic effects on salt and HCO3-resorption in proximal tubule epithelia (PCT). In cultured PCT cells, pM to nM AngII activates a GTP-binding protein to inhibit cAMP formation and thus releases inhibition of apical Na/H exchange. Phospholipase A2 is activated by nM to microM AngII releasing arachidonate which is metabolized by a novel P450 epoxygenase to form 5,6-epoxy-eicosatrienoic acid (5,6-EET). 5,6-EET and nM apical AngII cause dihydropyridine-sensitive Ca2+ influx from the extracellular space, inhibition of apical-to-basolateral Na flux, and decrease in epithelial monolayer short circuit current. 5,6-EET also inhibits Na/K-ATPase by 50%. This P450 epoxygenase is physiologically important in the AngII-signaling system because the P450 inhibitor ketoconazole blocks AngII effects while potentiating exogenous 5,6-EET effects. Finally, these AngII-mediated signaling systems are polarized in the PCT with pM basolateral AngII inhibiting adenylate cyclase and nM apical AngII activating PLA2 and subsequent generation of 5,6-EET.     

Primary cultures of rabbit renal proximal tubule cells: I. Growth and biochemical characteristics

Summay Before the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures within 7 d after plating. Multicellular done formation, an indicator of transepithelial solute transport, was expressed after confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme,N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture. respectively. In contrast, the activity of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers into the culture medium, which are routinely used to measure cytoxicity, stabilized after 8 d in culture. The ratio of cellular protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/μg DNA in preconfluent cultures to 0.059 mg protein/μg DNA in confluent cultures. These results document the characteristics of a primary proximal tubule cell culture system for future studies in in vitro toxicology. This paper was resented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by grants GM 07145, The Johns Hopkins Center for Alternatives to Animal Testing, and a Sigma Xi Grants-in-Aid of Research Award.     

Transport of chlorine in the proximal tubule. Its effects on water-electrolyte absorption

Several studies in rat kidney have established that an appreciable fraction of proximal absorption is passive in nature and occurs across the highly conductive paracellular pathway. Passive absorption is generally ascribed to the transepithelial Cl- distribution, luminal Cl- activity (alpha lCl) being higher than plasma Cl- activity (alpha pCl). The inequality alpha lCl greater than alpha pCl generates a transepithelial diffusion potential, lumen positive, which taken together with the chemical potential differences of Cl- and Na+ across the epithelium gives rise to transepithelial electrochemical potential differences for Cl- and Na+ favoring their absorption. The alpha lCl greater than alpha pCl distribution is traditionally ascribed to preferential bicarbonate absorption. We argue that HCO3- absorption alone cannot generate a non equilibrium transepithelial Cl- distribution. Other mechanisms are necessary. Our measurements in amphibian proximal tubule demonstrate that the intracellular Cl- activity, alpha cCl, is higher than the theoretical value predicted for equilibrium. This distribution is the result of two basolateral coupled transport processes (Cl-/HCO3- exchange and Cl-/Na+ cotransport). It contributes to the exit of Cl- from cell to lumen (by passive diffusion and K+/Cl- cotransport), yielding alpha lCl values higher than the theoretical value for equilibrium with regard to plasma. Thus, a small transcellular flux of Cl- (without solvent) proceeds from interstitium to lumen. It compensates the dissipative tendency of a much higher paracellular Cl- absorptive flux (in association with water) on the transepithelial Cl- gradient. The result is a steady-state luminal Cl- distribution above equilibrium, along the major part of the proximal tubule.     

Effect of stevioside on PAH transport by isolated perfused rabbit renal proximal tubule

Stevioside, a non-caloric sweetening agent, is used as a sugar substitute. An influence of stevioside on renal function has been suggested, but little is known about its effect on tubular function. Therefore, the present study was designed to explore the direct effect of stevioside on transepithelial transport of p-aminohippurate (PAH) in isolated S2 segments of rabbit proximal renal tubules using in vitro microperfusion. Addition of stevioside at a concentration of 0.45 mM to either the tubular lumen, bathing medium, or both at the same time had no effect on transepithelial transport of PAH. Similarly, a concentration of 0.70 mM (maximum solubility in the buffer) when present in the lumen, had no effect on PAH transport. However, this concentration in the bathing medium inhibited PAH transport significantly by about 25-35%. The inhibitory effect of stevioside was gradually abolished after it was removed from the bath. Addition of 0.70 mM stevioside to both lumen and bathing medium at the same time produced no added inhibitory effect. Stevioside at this concentration has no effect on Na+/K+-ATPase activity as well as cell ATP content. These findings suggest that stevioside, at a pharmacological concentration of 0.70 mM, inhibits transepithelial transport of PAH by interfering with the basolateral entry step, the rate-limiting step for transepithelial transport. The lack of effect of stevioside on transepithelial transport of PAH on the luminal side and its reversible inhibitory effect on the basolateral side indicate that stevioside does not permanently change PAH transport and should not harm renal tubular function at normal human intake levels.