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 bath and luminal potassium concentration on ammonia production and secretion by mouse proximal tubules perfused in vitro.

To determine the effects of acute changes in K+ concentration in vitro on ammonia production and secretion by the proximal tubule, we studied mouse S2 segments perfused with and bathed in Krebs-Ringer bicarbonate buffers containing various K+ concentrations. All bath solutions contained L-glutamine as the ammoniagenic substrate. High bath and luminal K+ concentrations (8 mM), but not high luminal K+ concentration alone, inhibited total ammonia production rates by 26%, while low bath and luminal K+ concentrations (2 mM), but not low luminal K+ concentration alone, stimulated total ammonia production rates by 33%. The stimulation of ammonia production by low bath K+ concentration was not observed when L-glutamine was added to the luminal perfusion solution. On the other hand, high luminal K+ concentration stimulated, while low luminal K+ concentration inhibited, net luminal secretion of total ammonia in a way that was: (a) independent of total ammonia production rates, (b) independent of Na(+)-H+ exchange activity, and (c) not due to changes in transepithelial fluxes of total ammonia. These results suggest that luminal potassium concentration has a direct effect on cell-to-lumen transport of ammonia.     

Modulation of phosphate absorption by calcium in the rabbit proximal convoluted tubule.

Proximal convoluted (S2) and straight (S3) renal tubule segments were studied to determine the effect of Ca on lumen-to-bath phosphate flux (JlbPO4). Increasing bath and perfusate Ca from 1.8 to 3.6 mM enhanced JlbPO4 from 3.3 +/- 0.7 to 6.6 +/- 0.6 pmol/mm per min in S2 segments (P less than 0.001) but had no effect in S3 segments. Decreasing bath and perfusate Ca from 1.8 to 0.2 mM reduced JlbPO4 from 3.7 +/- 0.6 to 2.2 +/- 0.6 in S2 segments. These effects were unrelated to changes in fluid absorption and transepithelial potential difference. Increasing cytosolic Ca with a Ca ionophore, inhibiting the Ca-calmodulin complex with trifluoperazine, or applying the Ca channel blocker nifedipine had no effect on JlBPO4 in S2 segments. Increasing only bath Ca from 1.8 to 3.6 mM did not significantly affect JlbPO4. However, increasing only perfusate Ca enhanced JlbPO4 from 3.4 +/- 0.7 to 6.1 +/- 0.7 pmol/mm per min (P less than 0.005). Inhibition of hydrogen ion secretion, by using a low bicarbonate, low pH perfusate, both depressed base-line JlbPO4 and abolished the stimulatory effect of raising perfusate Ca. Net phosphate efflux (JnetPO4) also increased after ambient calcium levels were raised, ruling out a significant increase in PO4 backflux. When net sodium transport was abolished by reducing the bath temperature to 24 degrees C, JnetPO4 at normal ambient calcium was reduced and increasing ambient calcium failed to increase it, ruling out a simple physicochemical reaction wherein phosphate precipitates out of solution with calcium. The present studies provide direct evidence for a stimulatory effect of Ca on sodium-dependent PO4 absorption in the proximal convoluted tubule, exerted at the luminal membrane. It is postulated that Ca modulates the affinity of the PO4 transporter for the anion.     

Effects of potassium on ammonia transport by medullary thick ascending limb of the rat

Renal ammonium excretion is increased by potassium depletion and reduced by potassium loading. To determine whether changes in potassium concentration would alter ammonia transport in the medullary thick ascending limb (MAL), tubules from rats were perfused in vitro and effects of changes in K concentration within the physiological range (4-24 mM) were evaluated. Increasing K concentration from 4 to 24 mM in perfusate and bath inhibited total ammonia absorption by 50% and reduced the steady-state transepithelial NH+4 concentration gradient. The inhibition of total ammonia absorption was reversible and occurred when K replaced either Na or N-methyl-D-glucamine. Increasing K concentration in the luminal perfusate alone gave similar inhibition of total ammonia absorption. At 1-2 nl/min per mm perfusion rate, increasing K concentration in perfusion and bathing solutions had no significant effect on transepithelial voltage. With either 4 or 24 mM K in perfusate and bath, an increase in luminal perfusion rate markedly increased total ammonia absorption. Thus, both potassium concentration and luminal flow rate are important factors capable of regulating total ammonia transport by the MAL. Changes in systemic potassium balance may influence renal ammonium excretion by affecting NH+4 absorption in the MAL and altering the transfer of ammonia from loops of Henle to medullary collecting ducts.     

Ammonia production by individual segments of the rat nephron

Ammonia production was measured directly in 10 segments of the rat nephron to determine the relative importance of the segments as sites of renal ammonia production. Tubules were microdissected from normal rats and rats drinking 0.28 M NH4Cl or 0.28 M NaHCO3 for 3-8 d. The segments were incubated in vitro with and without 2 mM glutamine. Ammonia concentrations in the incubation fluid were measured by microfluorometry to determine ammonia production rates. All segments produced ammonia from glutamine. In normal rats, production with glutamine was highest (greater than 5 pmol/min per mm) in the proximal convoluted (S-1), proximal straight (S-3), and distal convoluted tubules, and lowest (less than or equal to 2) in cortical and medullary collecting ducts and thin descending limbs. Metabolic acidosis increased production by 60% in the S-1 segment of the proximal convoluted tubule and by 150% in the S-2 segment of the proximal straight tubule without significant effect in any other segment. Bicarbonate loading decreased production by S-1 but had no effect on S-2 or S-3. Thus, acid-base changes altered production only in specific segments of the proximal tubule. We infer that the bulk of ammonia production occurs in the proximal tubules and that production by collecting ducts can account for only a few percent of renal ammonia production and excretion in the rat.     

Effect of formate on volume reabsorption in the rabbit proximal tubule.

Studies on microvillus membrane from rabbit kidney cortex suggest that chloride absorption may occur by chloride/formate exchange with recycling of formic acid by nonionic diffusion. We tested whether this transport mechanism participates in active NaCl reabsorption in the rabbit proximal tubule. In proximal tubule S2 segments perfused with low HCO-3 solutions, the addition of formate (0.25-0.5 mM) to the lumen and the bath increased volume reabsorption (JV) by 60%; the transepithelial potential difference remained unchanged. The effect of formate on JV was completely reversible and was inhibited both by ouabain and by luminal 4,4'-diisothiocyanostilbene-2,2'-disulfonate. Formate (0.5 mM) failed to stimulate JV in early proximal convoluted tubules perfused with high HCO-3 solutions. As measured by miniature glass pH microelectrodes, this lack of formate effect on JV was related to a less extensive acidification of the tubule fluid when high HCO-3 solutions were used as perfusate. These data suggest that chloride/formate exchange with recycling of formic acid by nonionic diffusion represents a mechanism for active, electroneutral NaCl reabsorption in the proximal tubule.     

Effects of Anion-Transport Inhibitors on NaCl Reabsorption in the Rat Superficial Proximal Convoluted Tubule

The effects of anion-transport inhibitors on volume reabsorption, and total CO(2) concentrations were examined by in vivo microperfusion of superficial proximal convoluted tubules of rats. The luminal perfusion solution was a high-chloride, low-bicarbonate solution like that in the in vivo late proximal tubule. The anion-transport inhibitors were only added to the luminal perfusion solutions.In tubules perfused with the control high-chloride solution, the rate of volume reabsorption (J(v)) was 2.3+/-0.2 nl/mm.min (n = 18), and the collected total CO(2) concentration was 4.0+/-0.3 mM. Furosemide (3 mM) caused a marked reduction in volume reabsorption to 0.8+/-0.3 nl/mm.min (n = 20) and only a slight increase in the total CO(2) concentration of collected samples of perfusate (7.8+/-0.5 mM). 0.8 mM acetazolamide caused a more pronounced rise in the collected total CO(2) concentrations to 10.7+/-0.5 mM but only a slight fall in J(v) to 1.7+/-0.3 nl/mm.min (n = 19). Hence, we inferred that inhibition of carbonic anhydrase only partially accounted for the inhibition of J(v) by furosemide. 4-acetamido-4'-iso-thiocyanato-stilbene-2,2'-disulphonic acid (0.1 mM), a well-characterized inhibitor of erythrocyte anion exchange mechanisms, also reduced J(v) to 1.6+/-0.3 nl/mm.min (n = 15) without changing the total CO(2) concentrations of the collected perfusates (3.6+/-0.4 mM). The effect of 4-acetamido-4'-iso-thiocyanato-stilbene-2,2'-disulphonic acid on volume reabsorption could not be explained by carbonic anhydrase inhibition because there was no increase in the total CO(2) concentration of the collected fluids. Furosemide did not significantly inhibit the rate of tracer glucose efflux out of the tubules, which suggests that the effect of furosemide on volume reabsorption was not a result of some nonspecific depression of active sodium transport. These results are discussed with respect to the possible effects of anion-transport inhibitors on the paracellular shunt pathway, active sodium reabsorption, and neutral sodium chloride transport.     

Renal bicarbonate reabsorption in the rat. III. Distal tubule perfusion study of load dependence and bicarbonate permeability.

Using continuous microperfusion techniques, we studied the load dependence of bicarbonate reabsorption along cortical distal tubules of the rat kidney and their bicarbonate permeability. Net bicarbonate transport was evaluated from changes in tracer inulin concentrations and total CO2 measurements by microcalorimetry. Bicarbonate permeability was estimated from the flux of total CO2 along known electrochemical gradients into bicarbonate-and chloride-free perfusion solution containing 10(-4) M acetazolamide. Transepithelial potential differences were measured with conventional glass microelectrodes. Significant net bicarbonate reabsorption occurred at luminal bicarbonate levels from 5 to 25 mM, and at perfusion rates from 5 to 30 nl/min. Bicarbonate reabsorption increased in a load-dependent manner, both during increments in luminal bicarbonate concentration or perfusion rate, reaching saturation at a load of 250 pmol/min with a maximal reabsorption rate of approximately 75 pmol/min.mm. Rate of bicarbonate reabsorption was flow dependent at luminal concentrations of 10 but not at 25 mM. During chronic metabolic alkalosis, maximal rates of reabsorption were significantly reduced to 33 pmol/min.mm. The bicarbonate permeability was 2.32 +/- 0.13 x 10(-5) cm/s in control rats, and 2.65 +/- 0.26 x 10(-5) cm/s in volume-expanded rats. Our data indicate that at physiological bicarbonate concentrations in the distal tubule passive bicarbonate fluxes account for only 16-21% of net fluxes. At high luminal bicarbonate concentrations, passive bicarbonate reabsorption contributes moderately to net reabsorption of this anion.     

Endogenous production of angiotensin II modulates rat proximal tubule transport.

There is evidence that angiotensin II is synthesized by the proximal tubule and secreted into the tubular lumen. This study examined the functional significance of endogenously produced angiotensin II on proximal tubule transport in male Sprague-Dawley rats. Addition of 10(-11), 10(-8), and 10(-6) M angiotensin II to the lumen of proximal convoluted tubules perfused in vivo had no effect on the rate of fluid reabsorption. The absence of an effect of exogenous luminal angiotensin II could be due to its endogenous production and luminal secretion. Luminal 10(-8) M Dup 753 (an angiotensin II receptor antagonist) resulted in a 35% decrease in proximal tubule fluid reabsorption when compared to control (Jv = 1.64 +/- 0.12 nl/mm.min vs. 2.55 +/- 0.32 nl/mm.min, P < 0.05). Similarly, luminal 10(-4) M enalaprilat, an angiotensin converting enzyme inhibitor, decreased fluid reabsorption by 40% (Jv = 1.53 +/- 0.23 nl/mm.min vs. 2.55 +/- 0.32 nl/mm.min, P < 0.05). When 10(-11) or 10(-8) M exogenous angiotensin II was added to enalaprilat (10(-4) M) in the luminal perfusate, fluid reabsorption returned to its baseline rate (Jv = 2.78 +/- 0.35 nl/mm.min). Thus, addition of exogenous angiotensin II stimulates proximal tubule transport when endogenous production is inhibited. These experiments show that endogenously produced angiotensin II modulates fluid transport in the proximal tubule independent of systemic angiotensin II.     

Bicarbonate secretion in vivo by rat distal tubules during alkalosis induced by dietary chloride restriction and alkali loading.

To examine in vivo the separate effects on distal tubule JtCO2, of dietary chloride restriction, bicarbonate loading, and changes in luminal chloride concentration, we microperfused distal tubules at a physiologic flow rate (8 nl/min) with solutions containing either 45 or 0 mM chloride (after gluconate substitution). Rats were fed a diet containing zero, minimal, or normal amounts of chloride, while drinking either water or a solution of 0.15 M sodium bicarbonate. Neither extracellular fluid volume contraction nor negative chloride balance ensued. Analysis of covariance with repeated measures demonstrated that dietary chloride, drinking sodium bicarbonate, and perfusion with either 45 mM or zero chloride, each have separate and significant modulating effects on distal tubule bicarbonate secretion. During mild alkalemia, there is modest bicarbonate secretion which is significantly different from zero (-9.9 +/- 3.2 pmol.min-1.mm-1, P less than 0.01), and which is suppressed after perfusion with zero chloride. In contrast, during more pronounced metabolic alkalosis after supplemental bicarbonate drinking, the bicarbonate secretory flux is brisk (-26 +/- 3 pmol.min-1.mm-1) and significantly different from zero and persists (-11 +/- 3 pmol.min-1.mm-1) even during perfusion with zero luminal chloride. Accordingly, in this two-day model of alkalosis induced by dietary chloride restriction, there is regulatory secretion of bicarbonate by distal tubules in vivo which is modulated by luminal chloride concentration.     

Anion dependence of rabbit medullary collecting duct acidification.

Rabbit medullary collecting duct (MCD) acidification has been demonstrated to occur by means of a sodium-independent, aldosterone-stimulated mechanism. We have examined the anionic dependence of this process by means of the isolated perfused tubule technique. Total replacement of perfusate chloride with gluconate enhanced tubular bicarbonate reabsorption (JHCO3), from a basal rate of 10.7 +/- 1.0 pmol X mm-1 X min-1 to a rate of 15.01 +/- 1.0 pmol X mm-1 X min-1. Removal of bath chloride, with and without removal of perfusate chloride completely abolished acidification. Bath, but not luminal 4-acetamido-4' isothiocyano-2,2'-disulfonic stilbene provoked a marked decrease in JHCO3 from 10.1 +/- 1.2 pmol X mm-1 X min-1 to 2.3 +/- 0.3 pmol X mm-1 X min-1. Measurement of chloride reabsorptive rate (JCl) revealed colinearity between JHCO3 (9.18 +/- 0.9 pmol X mm-1 X min-1) and JCl (9.75 +/- 1.18 pmol X mm-1 X min-1). We propose a model of mammalian distal nephron acidification in which (a) cellular base exit is effected by means of a basolateral membrane Cl-base exchanger and (b) net electroneutrality of electrogenic proton secretion is maintained by the parallel movement of an anionic species, functionally chloride.     

Different mechanisms of action of acetazolamide and parathyroid hormone on proximal tubular absorption of fluid and 5,5-dimethyl-2,4-oxazolidinedione

The effects of acetazolamide (Diamox), parathyroid hormone (PTH) and dibutyrylcyclic-adenosine 3':5'-monophosphate (db-cAMP) on fluid and 5,5-dimethyl-2,4-oxazolidinedione (DMO) absorption in the rat proximal convoluted tubule were studied by using microperfusion methods. The rate of tubular absorption of DMO was used to estimate the rate of hydrogen ion secretion. When the tubular and the peritubular capillaries were perfused simultaneously with bicarbonate-free Ringer's solution containing DMO, the rate of DMO absorption (JDMO) was 140 +/- 15.7 pmol/min . mm, a value comparable to the rate of absorption of bicarbonate and glycodiazine, and net fluid absorption (JV) was 2.20 +/- 0.19 nl/min . mm. Administration of PTH (10(-6) M) to the capillary perfusate caused a decrease of JDMO by 23% and a decrease of JV by 28%. Similar results were observed when db-cAMP (10(-4) M) was administered to the luminal perfusate. Addition of acetazolamide (10(-4) M) to the luminal perfusate caused a decrease of JDMO by 66% and a decrease of JV by 45%. The effect of either PTH or db-cAMP was additive to the maximal effect of acetazolamide. However, the effect of PTH was not additive to the effect of db-cAMP. Thus, the results suggest that PTH and acetazolamide have different mechanisms of action on fluid and DMO absorption by proximal tubule and that cAMP mediates the effect of PTH.     

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.     

Human complement protein D catabolism by the rat kidney.

Factor D (D) is an essential component of the alternative complement pathway. To determine whether D is catabolized by the kidney and, if so, at what site, we studied the renal handling of human D by in vivo nephron microperfusion and in vitro perfusion of rat kidneys. Human D was purified and labeled with 125I. Individual nephrons were perfused in vivo at varying rates with perfusate that contained 125I-D and [14C]inulin. When nephrons were perfused from proximal sites with perfusate 125I-D in a concentration of 3.0 micrograms/ml, urinary recovery of 125I-D increased (P less than 0.05) from 57.7 +/- 5.0 to 74.4 +/- 2.5% as tubule fluid flow rate was increased from 10 to 40 nl/min; recovery of 125I-D was less than (P less than 0.001) [14C]inulin recovery at all perfusion rates. At 20 nl/min, an increase in perfusate 125I-D concentration from 1.5 to 3.0 micrograms/ml was associated with an increase (P less than 0.001) in urinary 125I-D recovery (42.1 +/- 4.0 vs. 65.8 +/- 2.6%). Similarly, the addition of unlabeled D, 30 micrograms/ml, to 125I-D, 3.0 micrograms/ml, increased urinary 125I-D recovery (95.3 +/- 2.1%) at 20 nl/min. When nephrons were perfused from early distal segments at 10 nl/min, 125I-D recovery (91.2 +/- 4.3%) did not differ from [14C]inulin recovery (95.8 +/- 1.3%). In the isolated perfused filtering kidney, the concentration of intact 125I-D in the perfusate declined 60.3 +/- 14.6% over 1 h. 83.4 +/- 6.3% of the decrement in 125I-D was catabolized by the kidney; the remainder was excreted in the urine as intact D. When glomerular filtration was prevented by increasing perfusate albumin concentration to 16 g/dl, perfusate intact (125I-D) remained unchanged over 1 h. These data show that human D is catabolized by the kidney via glomerular filtration and reabsorption by the proximal nephron. Reabsorption of D appears to be a saturable process.     

Transport of ammonia in the rabbit cortical collecting tubule.

Nonionic diffusion and diffusion equilibrium of ammonia have been generally accepted as the mechanism of urinary ammonium excretion. However, these characteristics have not been examined directly in vitro. In the present studies, nonionic diffusion and diffusion equilibrium of ammonia were examined in rabbit cortical collecting tubules perfused in vitro. Collected fluid ammonium and pH were measured in tubules exposed to chemical gradients of NH3/NH+4. In tubules perfused with an acid perfusate free of ammonia and bathed with solutions containing NH4Cl, collected fluid ammonia failed to equilibrate across the epithelium except at slow flow rates. The estimated apparent permeability coefficient to NH3 was approximately 5 X 10(-3) cm/s. Predominant nonionic diffusion of NH3, rather than transport of NH+4, was indicated by alkalinization of luminal fluid in tubules exposed to peritubular NH4Cl and by the relative influence of peritubular NH+4 and NH3 on ammonia entry. In tubules perfused with an acid solution containing NH4Cl, little loss of ammonium was detectable, indicating a low permeability to NH+4. In contrast to the restricted diffusion of NH3 in cortical collecting tubules, proximal convoluted tubules exhibited a much higher apparent permeability to NH3. In conclusion, nonionic diffusion of NH3 accounted for most ammonium transport in the proximal convoluted tubule and in the cortical collecting tubule. However, there was relatively restricted diffusion in the collecting tubules; this may account for the failure of whole kidney ammonium excretion to obey quantitatively the predictions of nonionic diffusion and diffusion equilibrium of ammonia.     

Basolateral membrane Na+/H+ antiport, Na+/base cotransport, and Na+-independent Cl-/base exchange in the rabbit S3 proximal tubule.

The basolateral membrane Na+ and Cl(-)-dependent acid-base transport processes were studied in the isolated perfused rabbit S3 proximal straight tubule. Intracellular pH (pHi) was measured with 2'7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and a microfluorometer coupled to the tubule perfusion apparatus. Reduction of basolateral HCO3- from 25 to 5 mM caused pHi to decrease at a rate of 0.81 pH/min. Approximately 50% of this rate was Na+-dependent, 30% Cl(-)-dependent and 20% Na+ and Cl(-)-independent. Two basolateral Na+-dependent acid base transport pathways were detected: (a) an amiloride-sensitive Na+/H+ antiporter and (b) a stilbene-sensitive Na+/base cotransporter. No evidence was found for a Na+-dependent Cl-/base exchanger. The Cl(-)-dependent component of basolateral base efflux was mediated by a stilbene-sensitive Na+-independent Cl-/base exchange pathway. The results suggest that the acid base transport pathways of the basolateral membrane of the S3 proximal tubule differ from more proximal nephron segments.     

Inhibition of Renal Metabolism. Relative effects of arsenate on sodium, phosphate, and glucose transport by the rabbit proximal tubule.

These studies examine the inhibitory effects of arsenate on the transport of sodium, phosphate, glucose, and para-aminohippurate (PAH) as well as oxidative metabolism by proximal convoluted tubules from the rabbit kidney. Transport rates were measured with radioisotopes in isolated and perfused segments. Metabolic activity was monitored through oxygen-consumption rates and HADH fluorescence in parallel studies in suspensions of cortical tubules. The addition of 1mM arsenate to the perfusate reduced fluid absorption rates from 1.24 +/- 0.17 to 0.66 +/- 0.19 nl/nm.min (P < 0.01) and lumen-to-bath phosphate transport from 9.93 +/- 3.47 to 4.25 +/- 1.08 pmol/mm.min (P < 0.01). Similar concentrations of arsenate reduced glucose transport only slightly from 66.1 +/- 6.0 to 56.8 +/-4 4.6 pmol/mm.min (P < 0.05) and had no effect of PAH secretion. Removing phosphate from the perfusate did not affect the net transport of sodium or glucose. In suspensions of tubules, arsenate increased oxygen consumption rates by 20.5 +/- 2.9% and decreased NADH fluorescence by 10.8 +/- 1.5%. These effects on metabolism were concentration dependent and magnified in the presence of ouabain. The data indicate that arsenate's main effect is to uncouple oxidative phosphorylation, and that graded uncoupling of oxidative metabolism causes graded reductions in the net transport of both sodium and phosphate. Glucose transport is inhibited only slightly and PAH secretion is not affected. Thus, partial as opposed to complete inhibition of metabolism reveals that different relationships exist between net sodium transport and the transport of phosphate, glucose, and PAH by the proximal renal tubule.     

Luminal chloride modulates rat distal tubule bidirectional bicarbonate flux in vivo.

The effects of replacing luminal chloride with gluconate on distal tubule bicarbonate transport were studied in vivo in normally fed rats, overnight-fasted rats, and rats made mildly alkalotic by administration of desoxycorticosterone acetate (DOCA). In paired microperfusions of the same tubule with 0 or 55 mM Cl at 25 nl/min, net secretion of bicarbonate by distal tubules of fed rats was inhibited by chloride replacement. Zero chloride perfusion in DOCA rats also resulted in an inhibition of net bicarbonate secretion at 25 nl/min. In contrast, replacement of 45 mM chloride also perfused at 25 nl/min in fasted rats caused an increase in net bicarbonate reabsorption. To further characterize the effects of changes in luminal chloride, experiments were undertaken in fasted rats with 0, 45, and 100 mM chloride-containing solutions perfused at 8 and 25 nl/min. Perfusion with zero Cl resulted in net bicarbonate reabsorption at 8 nl/min that increased markedly with high flow, whereas bicarbonate reabsorption did not change significantly during perfusion at high flow with a 45-mM Cl perfusate. In marked contrast, perfusion with a 100-mM Cl solution resulted in only minimal bicarbonate reabsorption at 8 nl/min with significant secretion observed at high flow. Thus, chloride-free perfusates inhibit bicarbonate secretion and enhance bicarbonate reabsorption, while high chloride perfusates elicit net bicarbonate secretion in usually reabsorbing distal tubules.