Regulation of ammonia production by mouse proximal tubules perfused in vitro. Effect of luminal perfusion.

To investigate factors regulating ammonia (NH3) production by isolated defined proximal tubule segments, we examined the rates of total NH3 (NH3 + NH+4) production by individual proximal tubule segments perfused in vitro under a variety of perfusion conditions. Segments consisting of late convoluted and early straight portions of superficial proximal tubules were incubated at 37 degrees C in Krebs-Ringer bicarbonate (KRB) buffer containing 0.5 mM L-glutamine and 1.0 mM sodium acetate, pH 7.4. The rate of total ammonia production was calculated from the rate of accumulation of total NH3 in the bath. The total ammonia production rate by unperfused proximal segments was 6.0 +/- 0.2 (+/- SE) pmol/mm per minute, which was significantly lower than segments perfused at a flow rate of 22.7 +/- 3.4 nl/min with KRB buffer (21.5 +/- 1.4 pmol/mm per minute; P less than 0.001) or with KRB buffer containing 0.5 mM L-glutamine (31.9 +/- 2.5; P less than 0.001). The rate of NH3 production was higher in segments perfused with glutamine than in segments perfused without glutamine (P less than 0.01). The perfusion-associated stimulation of NH3 production was characterized further. Analysis of collected luminal fluid samples revealed that the luminal fluid total NH3 leaving the distal end of the perfused proximal segment accounted for 91% of the increment in NH3 production observed with perfusion. Increasing the perfusion flow rate from 3.7 +/- 0.1 to 22.7 +/- 3.4 nl/min by raising the perfusion pressure resulted in an increased rate of total NH3 production in the presence or absence of perfusate glutamine (mean rise in rate of total NH3 production was 14.9 +/- 3.7 pmol/mm per minute in segments perfused with glutamine and 7.8 +/- 0.9 in those perfused without glutamine). In addition, increasing the perfusion flow rate at a constant perfusion pressure increased the rate of luminal output of NH3. Total NH3 production was not affected by reducing perfusate sodium concentration to 25 mM and adding 1.0 mM amiloride to the perfusate, a condition that was shown to inhibit proximal tubule fluid reabsorption. These observations demonstrate that the rate of total NH3 production by the mouse proximal tubule is accelerated by perfusion of the lumen of the segment, by the presence of glutamine in the perfusate, and by increased perfusion flow rates. The increased rate of NH3 production with perfusion seems not to depend upon normal rates of sodium reabsorption. The mechanism underlying the stimulation of NH3 production by luminal flow is unknown and requires further study.     

Effect of angiotensin II on ammonia production and secretion by mouse proximal tubules perfused in vitro.

The effects of angiotensin II on total ammonia (tNH3) production and net secretion were investigated using in vitro microperfused mouse S2 proximal tubule segments incubated in Krebs-Ringer bicarbonate buffer containing 0.5 mM L-glutamine. Basolateral exposure of mouse S2 segments to 10(-11), 10(-10), and 10(-9) M angiotensin II stimulated tNH3 production rates by 23, 52, and 49%, respectively. Addition of 10(-6) M angiotensin II inhibited the tNH3 production rate by 34%. 10(-10) M angiotensin II inhibited net luminal secretion of tNH3 in the presence of enhanced luminal acidification and in the absence of altered luminal tNH3 efflux rates. Measurements of intracellular pH (pHi) and intracellular calcium concentration [( Ca2+]i) suggested that the effects of angiotensin II on tNH3 production were not mediated by changes in pHi but by the stimulatory effect of angiotensin II correlated with increased [Ca2+]i. Inhibition of the calcium-calmodulin-dependent pathway with W-7 blocked the stimulatory effect of 10(-10) M angiotensin II on tNH3 production and luminal acidification. These results indicate that angiotensin II has concentration-dependent effects on tNH3 production; that its action to stimulate tNH3 production may be mediated by rises in [Ca2+]i and the calcium-calmodulin pathway; and that angiotensin II, at concentrations that stimulate tNH3 production, inhibits net luminal ammonia secretion by a mechanism that is not mediated by diminished luminal acidification or by changes in luminal ammonia efflux rates.     

Ammonia production by isolated mouse proximal tubules perfused in vitro. Effect of metabolic acidosis.

We examined the effects of metabolic acidosis in vivo and reduced bath and luminal pH in vitro on total NH3 (NH3 + NH+4) production rates by isolated mouse proximal tubule segments. Midproximal tubule segments were obtained from mice with NH4Cl-induced metabolic acidosis and from nonacidotic controls. The segments were perfused with modified Krebs-Ringer bicarbonate (KRB) buffer, incubated in KRB buffer containing 0.5 mM L-glutamine and 1.0 mM sodium acetate, and gassed with 95% O2 and 5% CO2. Isolated unperfused and perfused proximal tubules from acidotic mice produced total NH3 at higher rates than corresponding tubules from nonacidotic mice. Perfusion of the tubular lumen stimulated total NH3 production by tubules from both acidotic and nonacidotic mice. In contrast, lowering the bath pH to 7.0 by lowering the HCO3- concentration increased total NH3 production rates by tubules from nonacidotic mice but not by tubules from acidotic mice. Reducing the HCO3- concentration of the bath buffer to 10 mM while maintaining a pH of 7.4 had no significant effect on total NH3 production by tubules from nonacidotic mice. Lowering the luminal fluid pH by reducing the perfusate HCO-3 from 25 mM to 10, 5, or 1.2 mM while maintaining a bath pH of 7.4 lowered collected luminal fluid pH but had no effect on total NH3 production by proximal tubules from nonacidotic mice. These observations demonstrated that metabolic acidosis in vivo stimulated total NH3 production in isolated mouse proximal tubule segments and that low peritubular pH and HCO-3 stimulated total NH3 production by proximal tubule segments from nonacidotic mice in vitro.     

Luminal secretion of ammonia in the mouse proximal tubule perfused in vitro.

A major portion of the total ammonia (tNH3 = NH3 + NH+4) produced by the isolated perfused mouse proximal tubule is secreted into the luminal fluid. To assess the role of Na+-H+ exchange in net tNH3 secretion, rates of net tNH3 secretion and tNH3 production were measured in proximal tubule segments perfused with control pH 7.4 Krebs-Ringer bicarbonate (KRB) buffer or with modified KRB buffers containing 10 mM sodium and 0.1 mM amiloride. Net tNH3 secretion was inhibited by 90% in proximal tubule segments perfused with the pH 7.4 modified KRB buffer while tNH3 production remained unaffected. The inhibition of net tNH3 secretion by perfusion with the modified KRB buffer was only partially reversed by acidifying the modified KRB luminal perfusate from 7.4 to as low as 6.2. These data indicate that the Na+-H+ exchanger facilitates a major portion of net tNH3 secretion by the proximal tubule and that luminal acidification may play only a partial role in the mechanism by which the Na+-H+ exchanger mediates net tNH3 secretion.     

Spontaneous luminal disequilibrium pH in S3 proximal tubules. Role in ammonia and bicarbonate transport.

We determined whether a spontaneous luminal disequilibrium pH, pHdq (pH measured - pH equilibrium), was present in isolated perfused rabbit S2 and S3 proximal tubules. Luminal pH was measured by perfusing with the fluorescent pH probe 1,4-DHPN, and the equilibrium pH was calculated from the measured collected total CO2 and dissolved CO2 concentrations. S2 tubules failed to generate a spontaneous pHdq. S3 tubules generated a spontaneous acidic pHdq of -0.46 +/- 0.15 (P less than 0.05), which was obliterated following the addition of carbonic anhydrase (0.1 mg/ml) to the perfusate. In S3 tubules perfused and bathed in 4 mM total ammonia, luminal total ammonia rose from 4.08 +/- 0.05 mM (perfusate) to 4.95 +/- 0.20 mM (collected fluid) (P less than 0.02). Carbonic anhydrase added to the perfusate prevented the rise in the collected total ammonia concentration. We conclude that the rabbit S3 proximal tubule lacks functional luminal carbonic anhydrase. The acidic pHdq in the S3 segment enhances the diffusion of NH3 into the lumen. In contrast, the S2 segment has functional luminal carbonic anhydrase.     

Effect of luminal and peritubular HCO3(-) concentrations and PCO2 on HCO3(-) reabsorption in rabbit proximal convoluted tubules perfused in vitro.

The effect of luminal and peritubular HCO3(-) concentrations and PCO2 on HCO3(-) reabsorption was examined in rabbit proximal convoluted tubules perfused in vitro. Increasing luminal HCO3(-) concentration from 25 to 40 mM without changing either peritubular HCO3(-) concentration or PCO2, stimulated HCO3(-) reabsorption by 41%. When luminal HCO3(-) concentration was constant at 40 mM and peritubular HCO3(-) concentration was increased from 25 to 40 mM without changing peritubular PCO2, a 45% reduction in HCO3(-) reabsorption was observed. This inhibitory effect of increasing peritubular HCO3(-) concentration was reversed when peritubular pH was normalized by increasing PCO2. Passive permeability for HCO3(-) was also measured and found to be 1.09 +/- 0.17 X 10(-7) cm2 s-1. Using this value, the passive flux of HCO3(-) could be calculated. Only a small portion (less than 23%) of the observed changes in net HCO3(-) reabsorption can be explained by the passive HCO3(-) flux. We conclude that luminal and peritubular HCO3(-) concentrations after HCO3(-) reabsorption by changing the active H+ secretion rate. Analysis of these data suggest that both luminal and peritubular pH are major determinants of HCO3(-) reabsorption.     

Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit.

Overall characteristics and kinetics of tubular absorption of albumin (Alb) were studied in isolated perfused proximal convoluted tubules of the rabbit. The fate of absorbed Alb was determined in tubules perfused with low [Alb]. Alb was labeled with tritium by reductive methylation ( [3H3C]Alb). At [Alb] = 0.03 mg/ml, approximately 80% of the absorbed [3H3C]Alb was released to the peritubular bathing solution as catabolic products. Transcellular transport of intact [3H3C]Alb was negligible. Iodoacetate (IAA, 4 mM) inhibited albumin absorption (JAlb) by greater than 95% and fluid reabsorption (JV) by 55%. At [Alb] = 0.1 mg/ml the absorption rate of a derivatized cationic Alb (pI = 8.4) was fivefold greater (P less than 0.01) than that of anionic Alb. Higher cationic [Alb] had deleterious effects on tubular functions. Overall Alb absorption was of high capacity and low affinity (JmaxAlb = 3.7 ng/min per mm tubule length, apparent Michaelis constant (Km) = 1.2 mg/ml). A low capacity system that saturates at near physiological loads was also detected (JmaxAlb = 0.064 ng/min per mm, apparent Km = 0.031 mg/ml). High [Alb] did not alter the rate of endocytic vesicle formation as determined by the tubular uptake of [14C]inulin. Results show that Alb absorption is a saturable process that is inhibited by high IAA concentrations and is affected by the charge of the protein. Absorbed Alb is hydrolyzed by tubular cells and catabolic products are readily released to the peritubular side. The dual kinetics of Alb absorption may be due to a combination of adsorptive endocytosis (low capacity system) and fluid endocytosis of albumin aggregates (high capacity system). Results indicate that albuminuria occurs much before albumin absorption is saturated. The kinetic characteristics of the process of tubular absorption of albumin helps to explain the concomitance of albuminuria, increased renal catabolic rates of albumin, and renal cell deposition of protein absorption droplets in severe glomerular proteinurias.     

Effect of inhibition of gluconeogenesis on ammonia production in the perfused rat kidney.

1. Gluconeogenesis from lactate or from glutamine is inhibited by 90-100% by sodium quinolinate (1 mmol/l) or 3-mercaptopicolinate (150 nmol/l) in the perfused rat kidney. L-Tryptophan is not metabolized and is without effect. 2. Lactate uptake and glucose production are inhibited to the same degree by 3-mercaptopicolinate in the kidneys of well-fed or starved rats. 3. Inhibition of gluconeogenesis from glutamine (1 mmol/l) by 3-mercaptopicolinate is accompanied by 50% inhibition of ammonia production, and 34% inhibition of glutamine uptake, in the kidneys of acidotic rats. Ammonia production from glutamine was not inhibited in kidneys from non-acidotic rats. 4. It is concluded that the increased rate of gluconeogenesis from glutamine which occurs in acidotic rats is an essential and primary event regulating all of the increase in ammonia formation.     

Activation of potassium channels contributes to hypoxic injury in proximal tubules.

The mechanisms responsible for the loss of cell potassium during renal ischemia are poorly understood. The present studies examined the hypothesis that potassium channels are activated as an early response to hypoxia and contribute to potassium loss independent from an inhibition of active K+ uptake. Potassium flux in suspensions of freshly isolated rat proximal tubules was measured using an ion-selective electrode. Exposure of the tubules to hypoxia for only 2.5 min resulted in a rise in the passive leak rate of K+ but no decrease in active K+ uptake. The passive leak of K+ was associated with a 40% decrease in cell ATP content. The passive K+ efflux was inhibited by 5 mM Ba2+ (95%) and by 15 mM tetraethylammonium (85%) suggesting that K+ channels were the primary route of K+ movement. The effects of K+ channel blockade on the development of hypoxic injury were also examined. Tetraethylammonium and glibenclamide, an inhibitor of ATP-sensitive K+ channels, reduced hypoxic injury as assessed by the release of lactate dehydrogenase or measurement of DNA damage. These results suggest that activation of K+ channels is an early response to hypoxia and contributes to hypoxic renal injury.     

Effect of in vitro metabolic acidosis on luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport in rabbit kidney proximal tubules.

The aim of this study was to evaluate the role of the kidney in mediating the signals involved in adaptive changes in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis. Proximal tubular suspensions were prepared from rabbit kidney cortex and incubated in acidic (A) or control (C) media (pH 6.9 vs 7.4, 5% CO2) for 2 h. Brush border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from the tubular suspensions and studied for the activity of Na+/H+ exchange and Na+:HCO3- cotransport. Influx of 1 mM 22Na at 10 s (pH6 7.5, pH(i) 6.0) into BBM vesicles was 68% higher in group A compared to group C. The increment in Na+ influx in the group A was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.1 mM in C vs 9.2 in A and Vmax of 31 nmol/mg protein per min in group C vs 57 in A. Influx of 1 mM 22Na at 10 s (pH0 7.5, pH(i) 6.0, 20% CO2, 80% N2) into BLM vesicles was 83% higher in the group A compared to C. The HCO3-dependent increment in 22Na uptake in group A was 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid sensitive, suggesting that Na+:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 11.4 mM in C vs 13.6 in A and Vmax of 35 nmol/mg protein per min in C vs 64 in A. The presence of cyclohexamide during incubation in A medium had no effect on the increments in 22Na uptake in group A. We conclude that the adaptive increase in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis is acute and mediated via direct signal(s) at the level of renal tubule.     

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

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

Effects of inhibitors and substitutes for chloride in lumen on p-aminohippurate transport by isolated perfused rabbit renal proximal tubules

The transport step for p-aminohippurate (PAH) from cell to lumen across the luminal membrane of rabbit proximal tubules has not been adequately defined. To examine this process more closely, we determined the effects of possible transport inhibitors and substitutes for chloride on PAH secretion in isolated perfused S2 segments of rabbit proximal tubules. The addition of 4-acetamido-4'-isothiocyano-2,2' disulfonic stilbene (10(-4) M) to the perfusate irreversibly inhibited PAH secretion, whereas the addition of probenecid (10(-4) M) to the perfusate reversibly inhibited PAH secretion. PAH secretion was unaffected by thiocyanate replacement of chloride in the luminal perfusate, reversibly inhibited by 15 to 20% by methyl sulfate replacement, and irreversibly inhibited by isethionate replacement. Because the luminal membrane is at least as permeable to thiocyanate as to chloride, less permeable to methyl sulfate, and much less permeable to isethionate, these data suggest that the PAH transport step from cells to lumen does not require chloride in the lumen but does require a highly permeant anion. During inhibition of PAH transport from cells to lumen, PAH uptake across the basolateral membrane was also reduced, suggesting some type of feedback inhibition. The data are compatible with PAH transport across the luminal membrane by an anion exchanger, a potential-driven uniporter, both carriers, or a carrier that can function in both modes.     

Carbon dioxide causes exocytosis of vesicles containing H+ pumps in isolated perfused proximal and collecting tubules.

In the turtle bladder it has recently been shown that CO2 stimulates H+ secretion, at least in part, by causing fusion of vesicles enriched in H+ pumps with the luminal plasma membrane. To test for the presence of this mechanism in the kidney we perfused collecting ducts and proximal straight tubules on the stage of an inverted epifluorescence microscope with fluorescein isothiocyanate dextran (70,000 mol wt) in CO2-free medium. After washout we noted punctate fluorescence in endocytic vesicles in some collecting ducts and in all proximal straight tubule cells. More cells took up fluorescent dextran in outer medullary than in cortical collecting ducts. Using the pH dependence of the excitation spectrum of fluorescein we found the pH of the vesicles to be acid (approximately pH 6). Addition of proton ionophores increased vesicular pH by 0.6 +/- 0.1 U, suggesting that the acidity of the vesicles was caused by H+ pumps. CO2 added to the medium (25 mmHg, pH 7.6 at 37 degrees C) reduced fluorescence intensity by 24 +/- 5% in cortical collecting ducts, 27 +/- 5% in medullary collecting ducts, and 25 +/- 5% in proximal straight tubules. Since this effect was prevented by the prior addition of colchicine to the bath, we believe that CO2 caused a decrease in cytoplasmic fluorescence by stimulating exocytotic fusion of the vesicles and thereby secretion of fluorescent dextran. This exocytotic fusion also occurred when tubules that were loaded with fluorescent dextran at a pCO2 of 37 mmHg were exposed isohydrically to a pCO2 of 114 mmHg; the mean decrease was 53 +/- 4%. We conclude that some cells in the collecting ducts and all cells in the proximal straight tubule incorporate fluorescent dextran into the apical cytoplasmic vesicles and acidify them with H+ pumps. CO2 causes fusion of these vesicles with the luminal membrane, but whether CO2 stimulates H+ secretion by increasing the number of functioning H+ pumps remains to be determined.     

Effect of quercetin on hypoxic injury in freshly isolated rat proximal tubules

The bioflavonoid quercetin, which has antioxidant properties, protects renal tubular epithelial cells from oxidant-induced injury by inhibiting lipid peroxidation. We examined the effect of quercetin on hypoxia-induced injury in freshly isolated rat renal proximal tubules. Hypoxia induced rapid loss of cellular ATP, followed by functional and structural alterations measured as a decrease in tubular potassium content and sequentially by an increase in lactate dehydrogenase release. Furthermore, hypoxia increased lipid peroxidation, measured as thiobarbituric acid-reactive substances. Quercetin significantly inhibited hypoxia-induced functional and structural tubular injury in addition to lipid peroxidation but did not alter hypoxia-induced ATP depletion. These results demonstrate the potency of the bioflavonoid quercetin in protecting proximal tubules from hypoxic injury, which is independent of tubular energy metabolism and may be related to the inhibition of lipid peroxidation.     

A comparison of the relative effectiveness of three transplant preservation fluids upon the integrity and function of rabbit proximal convoluted tubules perfused in vitro

Rabbits were anaesthetized and kidneys removed directly with no flush (group NF), or alternatively kidneys were flushed (group F) with sodium phosphate buffered 140 mmol/l sucrose (PBsuc 140), Collins C2 (C2) or Euro-Collins (EC) transplant preservation solutions and stored at 4 degrees C for 0-4, 24 or 48 h. Mid-cortical proximal convoluted tubule (PCT) segments were dissected from all groups and set up for microperfusion in vitro. Observations were made of tubule morphology, fluid reabsorption rate (Jv), bath leak of the glomerular marker iothalamate and transmural potential difference (p.d.), both at 37 degrees C and at 15-20 degrees C, in order to compare the relative effectiveness of the solutions in the preservation of tubule integrity and immediate function. Tubules from kidneys flushed with EC and C2 contained luminal debris and frequently had high bath leaks of iothalamate, both indicative of poor preservation of tubule integrity. In contrast, tubules from NF kidneys and PBsuc 140-flushed kidneys were free of luminal debris with a lower incidence of high iothalamate leaks. Tubules from PBsuc 140-flushed and EC-flushed kidneys after 0-4 and 24 h storage had Jv that were similar to those of NF kidneys. Tubules from all groups of flushed kidneys after 48 h cold storage and all tubules from C2-flushed kidneys showed reduced Jv values. In all cases Jv was reduced to approximately zero when the temperature was lowered to 15-20 degrees C. Transmural p.d. was similar in all groups except for particularly low p.d. values observed in tubules from C2-flushed kidneys after 48 h storage. These observations suggest that PBsuc 140 is more effective in the preservation of tubule function during prolonged cold storage than the glucose-based Collins solutions. The marked difference in the effectiveness of C2 and EC contraindicates the inclusion of magnesium (present in C2) for preservation of kidneys, as judged by experiments on the rabbit.     

Effect of steviol on para-aminohippurate transport by isolated perfused rabbit renal proximal tubule

An inhibitory effect of steviol, metabolite of the natural sweetener stevioside, on transepithelial transport of p-aminohippurate (J(PAH)) was observed in isolated S(2) segments of rabbit renal proximal tubules using in vitro microperfusion. Addition of steviol (0.01--0.25 mM) to the bathing medium significantly depressed J(PAH) (approximately 50--90%). This inhibitory effect was dose-dependent and was maximum at a concentration of 0.05 mM. To further examine this effect, a steviol concentration (0.01 mM) that produced approximately 50% inhibition of J(PAH), was chosen. Addition of 0.01 mM steviol to the bathing medium significantly depressed J(PAH) by about 50 to 60%. Steviol at the same concentration (0.01 mM), when present in the tubule lumen, had no significant effect on J(PAH). Addition of 0.01 mM steviol to lumen and bath simultaneously, produced a slightly greater inhibitory effect compared with addition to bath alone (60 versus 70%). A higher concentration of steviol, 0.05 mM (which maximally inhibited J(PAH) when on the basolateral side), was required on the luminal side than on the basolateral side before an inhibitory effect was observed. To further examine the mechanism by which steviol inhibited J(PAH), its effect on Na(+)-K(+) ATPase activity and ATP content was determined. Steviol at concentrations of 0.01 and 0.05 mM had no effect on Na(+)-K(+) ATPase activity or cell ATP content. Kinetic analyses indicated that steviol can competitively inhibit PAH transport at the basolateral membrane. The present study clearly showed that steviol can have a direct inhibitory effect on renal tubular transport by competitive binding with organic anion transporter.     

The effect of external potassium concentration on leucocyte cation transport in vitro.

1. Sodium and potassium transport rates in human leucocytes were measured in vitro at different external potassium concentrations. 2. At nominally zero external potassium concentrations, the ouabain-sensitive sodium efflux was reduced to less than 20% of its maximum value. There was evidence that under these conditions a ouabain-sensitive sodium-sodium exchange occurs. 3. Both total and ouabain-insensitive potassium influx increased with increasing external potassium concentration. The ouabain-sensitive potassium influx showed saturation. 4. Ouabain-insensitive potassium efflux was also stimulated by increasing the external potassium concentration, suggesting significant potassium-potassium exchange at physiological external potassium concentrations.     

Bicarbonate reabsorption and electrophysiology of proximal tubules in uninephrectomized rats.

1. The kinetics of acidification of luminal fluid in hypertrophied proximal tubules after unilateral nephrectomy was studied by stationary microperfusion and continuous measurement of luminal pH with antimony microelectrodes. 2. Trans-epithelial and basolateral membrane electrical potential differences were measured in order to detect modifications in electrogenic transport mechanisms under these conditions. 3. The values of stationary pH and HCO3- concentration were significantly lower, the mean acidification half-time was not different and net reabsorptive HCO3- fluxes in proximal tubules were significantly increased in uninephrectomized rats. According to an electrical analogue model, these results suggest (a) a reduction in the internal series resistance of the H+ pump, caused perhaps by an increased density of pump sites, and (b) an increase in the protonmotive force of the pump. 4. The trans-epithelial electrical potential difference measured in free flow conditions was significantly more lumen-positive in uninephrectomized rats. The trans-epithelial electrical potential difference measured during intraluminal perfusion with Ringer solution containing 30 mmol/l HCO3- was significantly more negative in all groups studied. In uninephrectomized rats treated with acetazolamide, the trans-epithelial electrical potential difference was more lumen-negative than that in untreated uninephrectomized rats. These results are compatible with a steeper transepithelial Cl- gradient as well as with electrogenic, active H+ secretion. 5. There was no significant difference in the basolateral electrical potential difference between control and uninephrectomized rats. 6. In conclusion, our data show an increase in the transport rates of HCO3- in the proximal tubule of uninephrectomized rats, which may be due to an increase in the density of transporters in the brush-border membrane, and an increased ability of the transport mechanism to create H+ gradients.     

Bicarbonate transport by rabbit cortical collecting tubules. Effect of acid and alkali loads in vivo on transport in vitro.

Rabbit cortical collecting tubules were perfused in vitro to investigate the control of bicarbonate transport. Bicarbonate was measured by microcalorimetry as total CO2. The perfusate and bath were identical solutions containing 25 mM bicarbonate at pH 7.4. The mean pH of the urine in the bladders of untreated rabbits at the time they were killed was 7.4. Their individual tubules, studied in vitro, either absorbed or secreted bicarbonate, and, combining the results, there was on the average no significant net transport. When the rabbits were treated with NH4Cl the day before the experiment, their urine was acidic and their tubules studied in vitro absorbed bicarbonate (i.e., there was net lumen-to-bath transport). In contrast, when the rabbits were treated with NaHCO3, their urine was significantly more alkaline, and their tubules studied in vitro generally secreted bicarbonate (i.e., net bath-to-lumen transport). Thus, the direction of bicarbonate transport by cortical collecting tubules studied under standard conditions in vitro correlated with the urine pH and was determined by the preceding treatment of the animals in vivo with acidifying or alkalinizing salts. These results demonstrate a previously unrecognized mechanism which contributes to the control of urinary bicarbonate excretion.