Electrophysiological analysis of rat renal sugar and amino acid transport

We have used electrophysiological techniques in rat kidney proximal tubule to study the transport of the followingl--amino acids: alanine, phenylalanine, glutamine, methionine, cysteine, cystine, proline, and hydroxyproline as well as the transport of glycine and -alanine. When applied in millimolar concentrations in the tubular lumen in the presence of Na⁺, all amino acids tested were found to depolarize the tubular cell membranes partially. This depolarization arises from current flow that is associated with the cotransport of Na⁺ and amino acids across the brushborder membrane (Frömter 1982 [9]). Peritubular application did not alter the membrane potentials in a conclusive way. The magnitude of the depolarization in response to luminal perfusion increased with increasing amino acid concentration and obeyed simple Michaelis-Menten kinetics, except for proline, hydroxyproline, and glycine, which exhibited double-site saturation kinetics. By analyzing the time course of the potential changes and distinguishing between initial and steady state depolarizations, it was possible to separate kinetic properties of the brushborder transport mechanisms from the lumped kinetic properties of the overall epithelium. Knowing the concentration dependence of the depolarizations, the competition of different amino acids for the same transport site was investigated: Two amino acids were applied in saturating concentrations in the tubular lumen, either singly or jointly, to determine whether the depolarizations were additive and whether the additivity exceeded the predictions from the kinetic experiments. From such studies the presence of three separate rheogenic transport systems is postulated for neutral amino acids: System I transports all neutrall--amino acids with the exception of cystine, system II transports proline, hydroxyproline and glycine, and system III transports -alanine or under physiological conditions taurine. Studies with the oxidant diamide suggest that cystine may be mostly reduced to cysteine and transported as cysteine, however it is likely that an extra transport system exists which transports cystine possibly in electroneutral fashion together with dibasic amino acids.     

Evidence for OH−/H+ permeation across the peritubular cell membrane of rat renal proximal tubule in HCO 3 − -free solutions

Membrane potentials and intracellular pH were measured on rat renal proximal tubular cells in vivo to test whether sodium-bicarbonate cotransport across the peritubular cell membrane accepts OH^– (or H⁺ in opposite direction) or whether it requires the CO₂, HCO ₃ ^– , CO ₃ ⁼ buffer to operate. It was found that step changes of peritubular pH in nominally HCO ₃ ^– -free and CO₂-free solutions produced qualitively similar initial potential responses and cell pH responses as changes in peritubular HCO ₃ ^– concentrations. These responses, however, were considerably smaller and they were neither reduced in Na⁺-free solutions nor inhibited by the stilbene derivative SITS which is known to block Na⁺ (HCO ₃ ^– ) _n cotransport completely. We conclude that the cotransporter requires the CO₂, HCO ₃ ^– , CO ₃ ⁼ buffer for its physiological operation but that high rates of OH^– or H⁺ can also be transferred across the peritubular cell membrane in HCO ₃ ^– -free solutions, probably through a separate transport system.     

The mechanism of action of harmaline on renal solute transport

Summary The effect of the hallucinogenic drug harmaline was tested on rat kidney proximal tubular solute and water transport, using in vivo micropuncture and electrophysiological techniques as well as in vitro biochemical techniques. During peritubular application harmaline (5 mmol/l) was found to block net tubular volume absorption reversibly (by 85%) through inhibition of active Na⁺ transport and possibly active HCO ₃ ^– transport. The inhibition was accompanied by a rapid strong depolarization of the tubular cell membranes. As a biochemical equivalent harmaline inhibited the Na⁺–K⁺-ATPase and the Mg²⁺-ATPase of peritubular cell membrane fractions as well as the HCO ₃ ^– -stimulated ATPase of a brush border membrane fraction with similar kinetics. By studying glucose tracer efflux and by measuring cell membrane potential and conductance changes in response to glucose perfusions, no evidence for a direct effect of harmaline on Na⁺-glucose (or amino acid) cotransport mechanisms in the brush border could be obtained. The data suggest that harmaline does not specifically compete with Na⁺ for transport sites. Neither are the cotransport systems in the brush border membrane specifically inhibited, nor could the inhibition of the Na⁺ pump in the peritubular cell membrane simply result from a competition between harmaline and Na⁺.     

Effect of parathyroid hormone on acid/base transport in rabbit renal proximal tubule S3 segment

The effect of parathyroid hormone (PTH) on acid/base transport in isolated rabbit renal proximal tubule S3 segment was investigated with double-barreled and conventional microelectrodes. PTH (10 nM) induced a small depolarization and enhanced the initial rates of cell pH (pH_i) increase and cell Cl^– ([Cl^–]_i) decrease in response to bath Cl^– removal by 28.0±2.1% and 31.0±6.4% respectively. The calculated initial HCO₃ ^– influx to bath Cl^– removal was also enhanced by 28%. On the other hand, PTH reduced the initial rate of pH_i decrease to luminal Na⁺ removal in the absence of HCO₃ ^–/CO₂ by 20.4±3.9%. The PTH-induced depolarization was not accompanied with changes in steadystate pH_i or [Cl^–]_i levels, but was greatly attenuated in the presence of ouabain (0.1 mM). Either dibutyrylcAMP (0.1 mM) plus theophylline (1 mM) or forskolin (10 M) alone could reproduce all the effects of PTH. These results indicate that (a) PTH inhibits the luminal Na⁺/H⁺ exchanger but stimulates the basolateral Cl^–/HCO₃ ^– exchanger in the S3 segment; (b) the PTH-induced depolarization largely results from inhibition of Na⁺/K⁺-ATPase and (c) all these effects are at least partly mediated by a cAMP-dependent mechanism.     

Phosphate transport by isolated renal brush border vesicles

Summary A sodium dependent specific transport system for phosphate is present in the brush border microvilli but absent from the basal-lateral plasma membranes. The apparent affinity of this transport system for phosphate is 0.08 mM at 100 mM sodium and pH 7.4. It is inhibited competitively by arsenate with an apparent inhibitor constant of 1.1 mM (100 mM sodium, pH 7.4). Sodium dependent phosphate uptake is two times higher at pH 8 compared to the uptake observed at pH 6. The apparent affinity of the transport system for sodium is also pH-dependent, half-maximal stimulation of uptake is found at pH 6 with 129 mM sodium, at pH 7.4 with 60 mM sodium and at pH 8 with 50 mM sodium. Under all conditions a nonhyperbolic dependence of phosphate uptake on the sodium concentration is observed. The uptake of phosphate by brush border microvilli vesicles shows a typical overshoot phenomenon in the presence of sodium gradient across the membrane {\text{ }}C_{Na_i } )$$ " align="middle" border="0"> . The amount of phosphate taken up after 2 min is about twice the equilibrium value reached after 2 h of incubation. At pH 7.4 the initial rate of uptake is increased only slightly (12%) by inside negative membrane diffusion potentials and inhibited to the same extent by inside positive membrane diffusion potentials.These results indicate that the entry of phosphate across the brush border membrane into the epithelial cell of the proximal tubule is coupled to the entry of sodium. The transfer of phosphate is dependent on its concentration gradient and on the concentration difference of sodium. The data are best explained by the following hypothesis: Both the primary phosphate as well as the secondary phosphate are transported in cotransport with sodium. The divalent form however seems to be transported preferentially. Its transport occurs electroneutral with 2 sodium ions; the monovalent phosphate also enters the cell together with 2 sodium ions but as a positively charged complex.The exit of phosphate across the contraluminal cell border is sodium independent and is favoured by the high intracellular phosphate concentration and the inside negative membrane potential.Part of these data have been presented at the Spring Meeting of the German Physiological Society at Bochum 1975 (Pflügers Arch.355, R 98, 1975).     

Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane

In order to study the mechanism of pancreatic HCO ₃ ^– transport, a perfused preparation of isolated intra-and interlobular ducts (i.d. 20–40 m) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PD_te) in ducts bathed in HCO ₃ ^– -free and HCO ₃ ^– -containing solution was –0.8 and –2.6 mV, respectively. The equivalent short circuit current (I_sc) under similar conditions was 26 and 50 A·cm^–2. The specific transepithelial resistance (R_te) was 88 cm². In control solutions the PD across the basolateral membrane (PD_bl) was –63±1 mV (n=314). Ouabain (3 mmol/l) depolarized PD_bl by 4.8±1.1 mV (n=6) within less than 10 s. When the bath K⁺ concentration was increased from 5 to 20 mmol/l, PD_bl depolarized by 15.9±0.9 mV (n=50). The same K⁺ concentration step had no effect on PD_bl if the ducts were exposed to Ba²⁺, a K⁺ channel blocker. Application of Ba²⁺ (1 mmol/l) alone depolarized PD_bl by 26.4±1.4 mV (n=19), while another K⁺ channel blocker TEA⁺ (50 mmol/l) depolarized PD_bl only by 7.7±2.0 mV (n=9). Addition of amiloride (1 mmol/l) to the bath caused 3–4 mV depolarization of PD_bl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PD_bl. An increase in the bath HCO ₃ ^– concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PD_bl by 8.5±1.0 mV (n=149). It was investigated whether the effect of HCO ₃ ^– was due to a Na⁺⁺-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K⁺ conductance caused by lowered intracellular pH. Experiments showed that the HCO ₃ ^– effect was present even when the bath Na⁺ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO ₃ ^– effect remained unchanged after Ba²⁺ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na⁺+K⁺)-ATPase, a Ba²⁺ sensitive K⁺ conductance and an amiloride sensitive Na⁺/H⁺ antiport. The HCO ₃ ^– effect on PD_bl is most likely due to rheogenic anion exit across the luminal membrane.     

Voltage-clamp studies of the Na+/glucose cotransporter cloned from rabbit small intestine

Inward Na⁺ currents associated with the cloned intestinal Na⁺/glucose cotransporter expressed in Xenopus oocytes have been studied using the two-microelectrode voltage-clamp method. The steady-state current/voltage relations showed voltage-dependent (V _m from +20 to –75 mV) and relatively voltage-independent (V _m from –75 to –150 mV) regions. The apparent I _max for Na⁺ and glucose increased with negative membrane potentials, and the apparent K _0.5 for glucose (K _0.5 ^Glc ) depended on V _m and [Na]_o. Increasing [Na]_o from 7 to 110 mmol/l had the same effect in decreasing K _0.5 ^Glc from 0.44 to 0.03 mmol/l as increasing the V _m from –40 to –150 mV. The I/V curves under saturating conditions (20 mmol/l external sugars and 110 mmol/l [Na]_o) were identical for d-glucose, d-galactose, -methyl d-glucopyranoside and 3-O-methyl d-glucoside. The specificity of the cotransporter for sugars was: d-glucose, d-galactose, -methyl d-glucopyranoside > 3-O-methyl d-glucoside d-xylose > d-allose d-mannose. K _i for phlorizin ( 10 mol/l) was independent of V _m at saturating [Na]_o. We conclude that a variety of sugars are transported by the cloned Na⁺/glucose cotransporter at the same maximal rate and that membrane potential affects both the maximal current and the apparent K _0.5 of the cotransporter for Na⁺ and glucose.     

Effect of organic acid absorption on bicarbonate transport in rat colon

The absorption of organic anions and the influence of these anions on the movement of HCO ₃ ^– were studied in vivo in rat colon using a perfusion technique. The absorption of short chain fatty acids (SCFA's) such as acetate, propionate, and butyrate was much greater than that of succinate or lactate. With increasing initial concentration of SCFA up to 100 mmol · l^–1, SCFA absorption increased linearly in correspondence with HCO ₃ ^– appearance. FinalpCO₂ level of the perfusion solution with SCFA was the same as the plasma level. Among the SCFA's, no significant differences in absorption or their effects on HCO ₃ ^– appearance were observed. The presence of Na⁺ stimulated SCFA absorption, and the maximum value was obtained at more than 100 mmol · l^–1 of Na⁺.These results suggest that a specific system for HCO ₃ ^– secretion activated by SCFA exists in the colon, and that this system may control the intraluminal pH by the alkalization of intestinal contents.     

Electrophysiological characterization of rabbit distal convoluted tubule cell

The distal convoluted tubule (DCT) from rabbit kidney were perfused in vitro to study the conductive properties of the cell membranes by using electrophysiological methods. When the lumen and the bath were perfused with a biearbonate free solution buffered with HEPES, the transepithelial voltage (V _T) averaged –2.8±0.6 mV (n=20), lumen negative. The basolateral membrane voltage (V _B) averaged –77.8±1.1 mV (n=33) obtained by intracellular impalement of microelectrodes. Cable analysis performed by injecting a current from perfusion pipette revealed that the transepithelial resistance was 21.8±1.7 ·cm² and the fractional resistance of the luminal membrane was 0.78±0.03 (n=8), indicating the existence of ionic conductances in the luminal membrane. Addition of amiloride (10^–5 mol/l) to the luminal perfusate or Na⁺ removal from the lumen abolished the lumen negativeV _T and hyperpolarized the apical membrane. An increase in luminal K⁺ concentration from 5 to 50 mmol/l reduced the apical membrane potential (V _A) by 37.5±2.6 mV (n=7), whereas a reduction of Cl^– in the luminal perfusate did not changeV _A significantly (0.5±0.5 mV,n=4). Addition of Ba²⁺ to the lumen reducedV _A by 42.6±1.0 mV (n=4). When the bathing fluid was perfused with 50 mmol/l K⁺ solution, the basolateral membrane voltage (V _B) fell from –76.8±1.5 to –31.0±1.3 mV (n=18), and addition of Ba²⁺ to the bath reducedV _B by 18.3±4.8 mV (n=7). Although a reduction of Cl^– in the bathing fluid from 143 to 5 mmol/l did not cause any significant fast initial depolarization (1.8±1.7 mV,n=8), a spike like depolarization (14.0±2.5 mV,n=4) was observed, upon Cl^– reduction in the presence of Ba²⁺ in the bath. From these results, we conclude that the apical membrane of DCT has both K⁺ and Na⁺ conductances and the basolateral membrane has a K⁺ conductance and a small Cl^– conductance.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule

The mechanism of bicarbonate transport across the peritubular cell membrane was investigated in rat kidney proximal tubules in situ by measuring cell pH and cell Na⁺ activity in response to sudden reduction of peritubular Na⁺ and/or HCO ₃ ^– . The following observations were made: 1. sudden peritubular reduction of either ion concentration produced the same transient depolarizing potential response; 2. bicarbonate efflux in response to peritubular reduction of bicarbonate was accompanied by sodium efflux; 3. sodium efflux in response to peritubular sodium removal was accompanied by cell acidification indicating bicarbonate efflux; 4. all aforementioned phenomena were inhibited by SITS (10^–3 mol/l) except for a small SITS-independent sodium efflux and depolarization which occurred in response to peritubular sodium removal and was not accompanied by cell pH changes; 5. bicarbonate efflux and accompanying potential changes in response to reduction of peritubular bicarbonate virtually vanished in sodium-free solutions. From these observations we conclude that bicarbonate efflux proceeds as rheogenic sodium-bicarbonate cotransport with a stoichiometry of bicarbonate to sodium greater than 1. The question which of the charged species of the bicarbonate buffer system moves cannot yet be decided. Attempts to determine the stoichiometry from the SITS-inhibitable initial cell depolarization and from the SITS-inhibitable initial fluxes suggest a stoichiometry of 3 HCO ₃ ^– : 1 Na⁺. In addition to sodium-dependent bicarbonate flux, evidence was obtained for a sodium-independent transport system of acids or bases which is able to regulate cell pH even in sodium-free solutions.     

Regulation of NaCl transport in the renal collecting duct: lessons from cultured cells

The fine control of NaCl absorption regulated by hormones takes place in the distal nephron of the kidney. In collecting duct principal cells, the epithelial sodium channel (ENaC) mediates the apical entry of Na⁺, which is extruded by the basolateral Na⁺,K⁺-ATPase. Simian virus 40-transformed and “transimmortalized” collecting duct cell lines, derived from transgenic mice carrying a constitutive, conditionally, or tissue-specific promoter-regulated large T antigen, have been proven to be valuable tools for studying the mechanisms controlling the cell surface expression and trafficking of ENaC and Na⁺,K⁺-ATPase. These cell lines have made it possible to identify sets of aldosterone- and vasopressin-stimulated proteins, and have provided new insights into the concerted mechanism of action of serum- and glucocorticoid-inducible kinase 1 (Sgk1), ubiquitin ligase Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), and 14-3-3 regulatory proteins in modulating ENaC-mediated Na⁺ currents. Epidermal growth factor and induced leucine zipper protein have also been shown to repress and stimulate ENaC-dependent Na⁺ absorption, respectively, by activating or repressing the mitogen-activated protein kinase externally regulated kinase_1/2. Overall, these findings have provided evidence suggesting that multiple pathways are involved in regulating NaCl absorption in the distal nephron.     

Tetraethylammonium transport by snake renal brush-border membrane vesicles

Transport of tetraethylammonium (TEA) by snake (Thamnophis spp.) renal brush-border membrane vesicles (BBMV) was studied. An outwardly directed proton gradient (pH 6.0 in, pH 7.5 out) stimulated uptake of TEA into BBMV and supported concentrative accumulation. Uptake of radioactively labeled TEA was also stimulated by outwardly directed gradients of unlabeled TEA and choline. The initial rate of TEA uptake was a saturable process that was adequately described by Michaelis-Menten kinetics. TEA uptake was not influenced by changes in the electrical potential difference across the membranes. Although uptake of TEA was stimulated by an outwardly directed Na⁺ gradient and inhibited by an inwardly directed Na⁺ gradient, these effects were probably secondary to the generation of proton gradients via a Na⁺/H⁺ exchanger demonstrated in these same BBMV. In agreement with previous studies with intact snake renal tubules, the present results indicate that TEA transport across the brush-border membrane involves electroneutral countertransport for protons or organic cations.     

Erythrocyte sodium content and transport in borderline and mild hypertension

Summary Sodium content and transport of red blood cells were examined in 98 male blood donors. Regarding their blood pressure they were classified into the following groups: (a) 57 normotensives, (RR<140/90 mm Hg); (b) 24 borderline hypertensives (140/90RR<160/95 mm Hg); and (c) 17 hypertensives (RR>160/100 mm Hg). Compared with the normotensives the borderline hypertensives have significantly reduced red cell sodium content. The ouabain-resistant net Na⁺ uptake and the relative Na⁺ uptake, as a measure of the Na⁺/K⁺ pump, were significantly increased. With rising blood pressures the measured values turn to normal, so that no difference exists between the normotensive and hypertensive groups. It is supposed that in the initial or even prehypertensive state a considerable enhancement of the pump activity occurs, simultaneously accompanied by less marked increases in sodium influx, leading to a reduced intracellular sodium content. In the course of hypertension, possibly caused by the formation of a pump inhibitor, the sodium content of red cells turns to normal or supernormal values.Abbreviations BMI body mass index - BHT borderline hypertensive - Ca _ion ²⁺ ionized plasma calcium - HT hypertensive - k relative OR net Na⁺ uptake - [Na⁺]_i, [K⁺]_i intracellular sodium and potassium content in RBCs - NT normotensive - OR ouabain-resistant - RBCs red blood cells - Na OR net Na⁺ uptake     

Coupling between proximal tubular transport processes

Summary The rate of active transport by the proximal renal tubule of amino acid (l-histidine), sugar (-methyl-d-glycoside), H⁺ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (c) of these substances. In the case of calcium the electrochemical potential differencec +zFc_i /RT) was the criterion employed. The rate of isotonic Na⁺-absorption (J_Na) was measured with the shrinking droplet method. The effect of ouabain on the transport of these substances was tested in the golden hamster and the effect of SITS (4-acetamido-4isothiocyanatostilbene 2,2-disulfonic acid) was observed in rats.Ouabain (1 mM) applied peritubularly incompletely inhibited J_Na (80%), but in combination with acetazolamide (0.2 mM) the inhibition was almost complete (93%). In addition, ouabain inhibited the sodium coupled (secondary active) transport processes ofl-histidine, -methyl-d-glycoside, calcium and phosphate by more than 75%. It did not affect H⁺ (glycodiazine) transport and PAH transport was only slightly affected.When SITS (1 mM) was applied from both sides of the cell it inhibited H⁺ (glycodiazine) transport by 72% and reduced J_Na by 38% when given from only the peritubular cell side. SITS (1 mM), however, had no significant affect on H⁺ secretion and sodium reabsorption if it was applied from only the luminal side. Furthermore it had no affect on the other transport processes tested, regardless of the cell side to which it was applied.When the HCO ₃ ^– buffer or physically related buffers were omitted from the perfusate the absorption of Na⁺ was reduced by 66%, phosphate by 44%, andl-histidine by 15%. All the other transport processes tested were not significantly affected.The data are consistent with the hypothesis that the active transport processes of histidine, -methyl-d-glycoside and phosphate, which are located in the brush border, are driven by a sodium gradient which is abolished by ouabain. This may also apply to the Na⁺-Ca²⁺ countertransport located at the contraluminal cell side. The residual Na⁺ transport remaining in the presence of ouabain is likely to be passively driven by the continuing H⁺ transport which probably is driven directly by ATP. SITS seems to inhibit the exit step of HCO ₃ ^– from the cell and secondary to that, the luminal H⁺-Na⁺ exchange and consequently the Na⁺ reabsorption. In the absence of HCO ₃ ^– buffer in the perfusates the luminal H⁺-Na⁺ exchange seems to be affected and the pattern of inhibition of the other transport processes is almost the same as with SITS. The different effects onP _i reabsorption observed under these conditions might be explained by possible variations in intracellular pH.     

Na/Ca exchangers in collecting cells of rat kidney

Single pieces of fura-2-loaded cortical collecting tubule (CCT) isolated either from normal or adrenalectomized (ADX) rats were superfused in vitro, and the cytosolic calcium concentration ([Ca²⁺]_i) was calculated from fluorescence recordings. The effects of altering the sodium gradient across cell membranes were investigated. Switching external sodium from 164 mM to 27 mM (low [Na⁺]_o) had little effect on [Ca²⁺]_i in normal tubules (106±9 versus 101±9 nM, n=15) whereas it resulted in a large peak of [Ca²⁺]_i in CCT from ADX-rats (270±32 versus 135±11 nM, n=21). Since CCT from ADX rats are known to have a reduced Na-pump activity, the effect of ouabain treatment on CCT from normal rats was also tested. When CCT from normal rats were exposed to 1 mM of ouabain in the presence of 164 mM of [Na⁺]_o, [Ca²⁺]_i increased only moderately (123±15 versus 111±11 nM, n=13); when the low [Na⁺]_o solution was applied to these ouabain-treated tubules, a large and transient increase in [Ca²⁺]_i was obtained (287±38 versus 123±15 nM, n=13). This response was absent with [Ca²⁺]_o=0. The data suggest the presence of 3 Na⁺/1 Ca²⁺ exchangers in cell membranes of rat CCT. The calcium flux equation derived by Läuger for the exchanger indicates a non-linear relationship between net calcium flux and driving force which could account for the difference observed here between the poor effect of applying either low [Na⁺]_o or ouabain alone and the large peak of [Ca²⁺]_i induced by combining these two conditions.     

Bidirectional active transport of thiosulfate in the proximal convolution of the rat kidney

Using the standing droplet method in the late proximal convolution and simultaneous microperfusion of the peritubular capillaries, the zero net flux transtubular concentration difference of thiosulfate at 45 s was determined, the latter being taken as a measure of active thiosulfate transport. Under control conditions, in the presence of Na⁺, near zero c values were observed. When 1 mmol/l carinamide or paraaminohippurate (PAH) were added to the perfusates significant reabsorptive c arose. However, when 7.5 mmol/l sulfate was added to the Na⁺-free secretory c values were observed. Tested under Na⁺-free conditions, the secretory c was not influenced by simultaneously present 5 mmol/l of SO ₄ ^2– but was diminished by 50 mmol/l SO ₄ ^2– . PAH (1 mmol/l), carinamide (0.2 mmol/l) and probenecid (1 mmol/l) decreased the secretory c by 48, 65 and 48%, respectively. The PAH secretion was not influenced, when thiosulfate or sulfate up to 50 mmol/l was added to both perfusates. Under Na⁺-free conditions the c of thiosulfate in early loops of the proximal convolution is higher than in late loops, while for PAH this pattern is reversed. Taken together with the previously published inhibition of sulfate reabsorption by thiosulfate the data indicate 1. thiosulfate is reabsorved by the Na⁺-dependent sulfate transport system and 2. thiosulfate is simultaneously secreted by a carinamide-, probenecid-and PAH-sensitive secretory system. The secretory system might also be shared by sulfate. The thiosulfate net flux is the result of the difference in the activity of the counteracting transporters, located at the luminal and contraluminal cell side. Is is possible that the higher activity of the transporter at one cell side leads to a reversal of the flux through the transporter at the other cell side.     

Chloride transport in the renal proximal tubule

The renal proximal tubule is responsible for most of the renal sodium, chloride, and bicarbonate reabsorption. Micropuncture studies and electrophysiological techniques have furnished the bulk of our knowledge about the physiology of this tubular segment. As a consequence of the leakiness of this epithelium, paracellular ionic transport--in particular that of Cl(-)--is of considerable importance in this first part of the nephron. It was long accepted that proximal Cl(-) reabsorption proceeds solely paracellularly, but it is now known that transcellular Cl(-) transport also exists. Cl(-) channels and Cl(-)-coupled transporters are involved in transcellular Cl(-) transport. In the apical membrane, Cl(-)/anion (formate, oxalate and bicarbonate) exchangers represent the first step in transcellular Cl(-) reabsorption. A basolateral Cl(-)/HCO(3)(-) exchanger, involved in HCO(3)(-) reclamation, participates in the rise of intracellular Cl(-) activity above its equilibrium value, and thus also contributes to the creation of an outwardly directed electrochemical Cl(-) gradient across the cell membranes. This driving force favours Cl(-) diffusion from the cell to the lumen and to the interstitium. In the basolateral membrane, the main mechanism for transcellular Cl(-) reabsorption is a Cl(-) conductance, but a Na(+)-driven Cl(-)/HCO(3)(-) exchanger may also participate in Cl(-) reabsorption.     

Energy source for transepithelial sodium transport in rat renal proximal tubules

Summary The effects of various metabolic inhibitors on isotonic fluid absorption (J _V ) in rat proximal tubules and on the Na⁺–K⁺-ATPase of isolated cell membranes of rat kidney cortex were investigated by the shrinking split oil droplet technique and biochemical methods respectively.Both Oligomycin (5×10^–5 M, 10^–4 M) and Antimycin A (10^–5 M, 10^–4 M) inhibited isotonic fluid absorption by 80% when applied intratubularly but only in conjunction with bovine serum albumin. At these concentrations they inhibited a Na⁺–K⁺ activated adenosine triphosphate phosphohydrolase (Na⁺–K⁺ ATPase E.C. 3.6.1.3.) of cell membranes isolated from rat kidney cortex by 77%, 82% and 55%, 95%, respectively.Sodium phosphoenolpyruvate (PEP) 5×10^–3 M could partially reverse the inhibition of the isotonic fluid absorption but only with 10^–5 M Antimycin A when the Na⁺–K⁺ ATPase inhibition was apparently small.The uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (10^–3 M), as well as sodium cyanide (5×10^–3 M) inhibitedJ _V 100%, but only when applied through peritubular blood capillary perfusion.From these findings it was concluded thatall proximal tubular isotonic fluid absorption is supported by energy fromoxidative processes, and that in a least 80% of this sodium reabsorption, ATP from oxidative phosphorylation is directly involved, while, for the remaining 20% non ATP energy is responsible.C. J. Martin Fellow of the National Health and Medical Research Council of Australia.