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 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.     

Chronic hyperkalemia impairs ammonium transport and accumulation in the inner medulla of the rat.

Previously we demonstrated in rats that chronic hyperkalemia had no effect on ammonium secretion by the proximal tubule in vivo but that high K+ concentrations inhibited ammonium absorption by the medullary thick ascending limb in vitro. These observations suggested that chronic hyperkalemia may reduce urinary ammonium excretion through effects on medullary transport events. To examine directly the effects of chronic hyperkalemia on medullary ammonium accumulation and collecting duct ammonium secretion, micropuncture experiments were performed in the inner medulla of Munich-Wistar rats pair fed a control or high-K+ diet for 7-13 d. In situ pH and total ammonia concentrations were measured to calculate NH3 concentrations for base and tip collecting duct and vasa recta. Chronic K+ loading was associated with significant systemic metabolic acidosis and a 40% decrease in urinary ammonium excretion. In control rats, 15% of excreted ammonium was secreted between base and tip collecting duct sites. In contrast, no net transport of ammonium was detected along the collecting duct in high-K+ rats. The decrease in collecting duct ammonium secretion in hyperkalemia was associated with a decrease in the NH3 concentration difference between vasa recta and collecting duct. The fall in the NH3 concentration difference across the collecting duct in high-K+ rats was due entirely to a decrease in [NH3] in the medullary interstitial fluid, with no change in [NH3] in the collecting duct. These results indicate that impaired accumulation of ammonium in the medullary interstitium, secondary to inhibition of ammonium absorption in the medullary thick ascending limb, may play an important role in reducing collecting duct ammonium secretion and urinary ammonium excretion during chronic hyperkalemia.     

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.     

Magnesium transport in the cortical thick ascending limb of Henle''s loop of the rabbit.

Isolated cortical thick ascending limbs of Henle's loop were perfused in order to directly evaluate magnesium transport in this segment. Transepithelial potential difference was altered by varying the NaCl concentration in perfusate and bath and adding 50 microM furosemide to the perfusate. Perfusion under standard conditions with isotonic solutions resulted in a mean transepithelial potential difference of +8.8 +/- 0.7 mV and net magnesium absorption at a rate of 0.32 +/- 0.06 pmol/mm per min. Perfusion with a hypotonic solution significantly increased potential difference and the net absorptive rate of magnesium, calcium, and potassium. Conversely, reversal of the polarity of the potential difference with low NaCl bath and luminal furosemide produced net secretion of magnesium, calcium, and potassium. Parathyroid hormone in a bath concentration of 1.0 U/ml increased magnesium absorption from 0.32 +/- 0.06 to 0.63 +/- 0.06 pmol/mm per min (P less than 0.001) and calcium from 0.52 +/- 0.08 to 0.97 +/- 0.08 pmol/mm per min (P less than 0.001). Dibutyryl cyclic AMP produced similar effects on both calcium and magnesium absorption. Increasing bath calcium concentration twofold significantly inhibited net calcium absorption from 0.79 +/- 0.27 to 0.16 +/- 0.02 pmol/mm per min but magnesium transport was unaffected. Increasing bath magnesium concentration twofold significantly inhibited net magnesium absorption from 0.56 +/- 0.14 to -0.09 +/- 0.13 pmol/mm per min but had no effect upon net calcium transport. Net absorption of magnesium was significantly increased with increased concentration in the perfusate but calcium transport was unchanged. Similarly, increasing perfusate calcium concentration produced an increase in net calcium transport but did not alter magnesium transport. These data indicate that this segment of the loop of Henle is an important site for magnesium transport. Transport is influenced by luminal and bath concentration and is stimulated by parathyroid hormone and cyclic AMP. The data do not provide support for the concept of an interactive process between calcium and magnesium, and suggest that the positive transepithelial voltage is an important driving force for net reabsorption of magnesium, as well as calcium and potassium in this segment.     

Cellular heterogeneity of ammonium ion transport across the basolateral membrane of the hamster medullary thick ascending limb of Henle's loop.

The epithelia of the medullary thick ascending limb (MAL) consists of two cell types, high (HBC) and low basolateral conductance (LBC) cell, depending on the K+ conductance of the basolateral membrane. The NH4+ conductance distinct from the K+ conductance has been suggested to exist in the proximal tubule, MAL cell, and Xenopus oocyte. The present study was designed to examine whether there is a conductive NH4+ transport system distinct from K+ conductance in two different cell types of the hamster MAL perfused in vitro. The basolateral membrane voltage (VB) was measured by impaling cells with conventional microelectrodes. Addition of NH4+ to the bath depolarized VB in a dose-dependent manner in both cell types. The response was maintained in the absence of HCO3-. When the VB deflection elicited upon 50 mM KCl or NH4Cl in the bath (delta VBK+ or delta VBNH4+) were compared, delta VBNH4+ was almost the same as delta VBK+ in the HBC cell, whereas the former was greater than the latter in the LBC. In the HBC cell, 10 mM Ba2+ in the bath equally suppressed both delta VBK+ and delta VBNH4+, whereas in the LBC cell it suppressed delta VBK+ with a small effect on delta VBNH4+, indicating that NH4+ is transported via a pathway distinct from Ba(2+)-sensitive K+ conductance. The VB deflection by NH4+ was unaffected by addition of 0.1 mM ouabain or 10 microM 5-nitro-2-(3-phenylpropylamino)-benzoate (a Cl- channel blocker) to the bath, excluding the contribution of the Na+, K+ pump or Cl- channel. An abrupt reduction of Na+ in the bath from 200 to 20 mM did not cause any changes in VB, suggesting that a nonselective cation channel may not account for the NH4+ transport. Amiloride at 10 microM inhibited delta VBNH4+ with a higher efficacy in the LBC cell. We conclude that a rheogenic NH4+ transport system independent from the K+ conductance exists in the basolateral membrane of the LBC cell of the hamster MAL, and may play some roles in the regulation of NH4+ transport.     

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.     

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.     

Consequences of potassium recycling in the renal medulla. Effects of ion transport by the medullary thick ascending limb of Henle''s loop.

The consequences of K recycling and accumulation in the renal medulla were examined by measuring the effect of elevated K concentration on ion transport by the medullary thick ascending limb of Henle's loop. Perfused and bathed in vitro, thick limbs from both mouse and rabbit displayed a graded, reversible reduction of transepithelial voltage after increasing K concentration from 5 to 10, 15, or 25 mM. The effect was reproducible whether osmolality was 328 or 445 mosmol/kg H2O, and whether K replaced Na or choline. Net chloride absorption and transepithelial voltage were reduced by almost 90% when ambient K concentration was 25 mM. When either lumen or bath K was increased to 25 mM, net Na absorption was reduced. There was spontaneous net K absorption when perfusate and bath K concentration was 5 mM. Analysis of transepithelial K transfer after imposition of chemical gradients demonstrated rectification in the absorptive direction. Absorption of K by this segment provides a means to maintain high medullary interstitial concentration. Accumulation of K in the outer medulla, by reducing NaCl absorption, would increase volume flow through the loop of Henle and increase Na and water delivery to the distal nephron. K recycling thus might provide optimum conditions for K secretion by the distal nephron.     

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 osmolality on bicarbonate absorption by medullary thick ascending limb of the rat.

Previously we demonstrated that arginine vasopressin (AVP) directly inhibits bicarbonate absorption (JHCO3, pmol/min per mm) in the medullary thick ascending limb (MTAL) of the rat. To determine whether changes in osmolality also may affect bicarbonate absorption, MTAL were studied in vitro with 25 mM HCO3- solutions. Control osmolality was 290 mosmol/kg H2O. In the absence of AVP, increasing osmolality to 560 in perfusate and bath by addition of 150 mM NaCl reduced JHCO3 from 13.7 to 4.5. With 2 x 10(-10) M AVP in the bath, adding 150 mM NaCl to perfusate and bath reduced JHCO3 from 6.9 to 0.6, while adding NaCl to the bath alone reduced JHCO3 from 7.1 to 0.5. Adding 150 mM NaCl to perfusate and bath caused a similar inhibition of JHCO3 in MTAL perfused with furosemide to inhibit net NaCl absorption. In the presence of AVP, adding 600 mM urea to perfusate and bath inhibited JHCO3 by 55%; adding 300 or 600 mM mannitol to perfusate and bath inhibited JHCO3 by 75%. The effects on JHCO3 were reversible and dissociable from changes in transepithelial voltage. Conclusions: (1) osmolality is a factor capable of regulating renal tubule bicarbonate absorption; (2) hypertonicity produced with NaCl, urea, or mannitol markedly inhibits bicarbonate absorption in the MTAL; (3) this inhibition occurs independent of, and is additive to, inhibition by vasopressin. Hypertonicity may shift TAL HCO3- absorption from medulla to cortex, thereby limiting delivery of bicarbonate to the medullary interstitium during antidiuresis.     

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.     

Inhibition of bicarbonate absorption by peptide hormones and cyclic adenosine monophosphate in rat medullary thick ascending limb.

In vitro microperfusion experiments were performed to examine the effects of peptide hormones on bicarbonate and ammonium transport by the medullary thick ascending limb (MTAL) of the rat. Arginine vasopressin (AVP; 2.8 X 10(-10) M in the bath) reduced bicarbonate absorption by 50% (from 7.8 to 3.7 pmol/min per mm). AVP caused a similar reduction in bicarbonate absorption in tubules perfused with 10(-4) M furosemide to inhibit net NaCl absorption. Glucagon (2 X 10(-9) M in the bath) also reduced bicarbonate absorption (from 11.7 to 7.6 pmol/min per mm). The inhibition of bicarbonate absorption could be reproduced with either exogenous 8-bromo-cAMP or forskolin. With 8-bromo-cAMP (10(-3) M) in the bath, addition of vasopressin to the bath did not significantly affect bicarbonate absorption. PTH significantly inhibited bicarbonate absorption, but the extent of inhibition was less than that observed with either AVP or glucagon. Vasopressin had no effect on net ammonium absorption in MTAL perfused and bathed with 4 mM NH4Cl. These findings indicate that: (a) vasopressin, glucagon, and PTH directly inhibit bicarbonate absorption in the MTAL of the rat; (b) this inhibition occurs independent of effects on net NaCl absorption and appears to be mediated in part by cAMP; and (c) HCO3- and NH4+ absorption can be regulated independently in the MTAL.     

Ammonia and urea transport by the excluded human colon.

1. Ammonia and urea transport across the colonic mucosa was studied by a perfusion technique in four subjects with colonic exclusion for chronic hepatic encephalopathy. 2. Reduction of luminal pH inhibited net and unidirectional transport of ammonia from lumen to plasma, but net absorption from high luminal concentrations persisted at low pH. 3. Neither addition of urea to the perfusate nor intravenous infusion of urea produced a consistent increase in the colonic excretion of ammonia when ammonia-free solutions were perfused. 4. In one subject intravenous infusion of (15N)-ammonium chloride produced rapid labelling of colonic effluent ammonia and within 60 min the specific enrichments of ammonia in effluent and in arterial plasma were approximately equal. 5. During perfusion of nitrogen-free solutions, only small amounts of urea appeared in the effluent, suggesing limited permeability of the colonic mucosa to urea. 6. These results are discussed in relation to the equilibration of ammonia across the colonic mucosa by both ionic and non-ionic diffusion. The lack of evidence of 'juxtamucosal' (as opposed to luminal) ureolysis is in contrast to other observations on the intact colon. The possible reasons for and implications of this discrepancy are discussed.     

Characterization of acidification in the cortical and medullary collecting tubule of the rabbit

Ouabain and lithium decrease acidification in open-circuited bladders by eliminating the electrical gradient favoring acidification. The effect of ouabain and lithium on acidification in cortical and medullary collecting tubules derived from starved New Zealand white rabbits was studied by using the techniques of isolated nephron microperfusion and microcalorimetric determination of total CO2 flux. Bath and perfusion solutions were symmetric throughout all studies, and solutions contained 25 meq of bicarbonate and were bubbled with 93.3% O2/6.7% CO2 gas mixtures. In cortical collecting tubules, ouabain (10(-8) M) addition to bath resulted in a decrease in both potential difference (PD), from -16.4 to -2.2 mV (P less than 0.001), and total CO2 flux (JTCO2), from +6.0 to 1.5 pmol/mm per min (P less than 0.005). In medullary collecting tubules neither PD nor JTCO2 changed with the addition of ouabain in either 10(-8) or 10(-4) M concentration. Replacement of 40 mM NaCl with 40 mM LiCl in both perfusate and bath in cortical collecting tubules resulted in decreases in both PD, from -11.6 to 0.4 mV (P less than 0.005), and JTCO2, from +10.8 to +4.2 pmol/mm per min (P less than 0.025). This substitution had no effect on medullary collecting tubules. When control flux rates were plotted against animal bladder urine pH, both medullary and cortical tubules showed good inverse correlation between these variables, with higher values of flux rate for the medullary tubules. The data support a role for transepithelial PD in acidification in the cortical collecting tubule and also suggest that both cortical and medullary segments of the collecting tubule participate when urinary acidification is increased during starvation in the rabbit.     

Effect of dimethylaminoethanol, an inhibitor of betaine production, on the disposition of choline in the rat kidney

The choline metabolite betaine has been shown to be an important organic osmoregulatory solute in the kidney. The isolated perfused rat kidney and kidney slice incubations were used to investigate the effect of 2-dimethylaminoethanol (DMAE), a choline oxidase inhibitor, on the renal excretion and metabolism of choline. In the isolated perfused kidney, [14C]choline, at an initial perfusate concentration of 300 microM, was effectively removed from the perfusate over 25 min, with nearly all the 14C in the perfusate accounted for by betaine during the remainder of the 90-min perfusion. DMAE at concentrations of 3.0 or 5.0 mM significantly decreased the rate of removal of [14C]choline from the perfusate and the rate of addition of [14C]betaine to the perfusate, yet [14C]betaine remained the only metabolite of choline in perfusate and urine. In kidney tissue slice experiments, conversion of [14C]choline to [14C]betaine was found in cortical, outer medullary and inner medullary regions of rat kidney. DMAE at 5.0 mM significantly inhibited [14C]betaine production in each of the three regions studied. These data show that DMAE is an effective inhibitor of betaine production by the kidney and, as such, may be an important agent for the study of osmoregulation by the kidney.     

Response of the renal K+-conserving mechanism to kaliuretic stimuli: evidence for a direct kaliuretic effect by furosemide

Isolated perfused kidneys from rats given a K+-free diet for 3 days demonstrate striking renal K+ conservation by a mechanism that is independent of perfusate and renal tissue K+, aldosterone, urine pH, urine flow, and sodium and anion excretion. To examine the effect of several kaliuretic stimuli on this intrinsic renal K+-conserving mechanism, kidneys from K+-depleted rats perfused with 3.5 mmol/L K+ were subjected to an experimental maneuver after 45 minutes of perfusion under normal conditions. Fractional potassium excretion (FEK) remained stable in time controls during a subsequent 45 minutes of perfusion. Acidification of the perfusate to pH 6.96 by addition of HCI had no significant effect on FEK. Alkalinization to pH 7.7 by either addition of NaHCO3 or a reduction in PCO2 resulted in significant kaliuresis (FEK 26% vs. 15%), which appeared to be a direct result of systemic alkalinization. Addition of Na2SO4 to the perfusate also produced significant kaliuresis (FEK 29%), which could not be dissociated from the magnitude of the natriuresis. Although both alkalosis and sulfate increased K+ excretion by depleted kidneys, FEK was much lower than with kidneys from normal rats perfused at pH 7.4 (FEK 51%). Thus the intrinsic renal K+-conserving mechanism dramatically diminishes the kidneys' kaliuretic potency and probably accounts for the blunted kaliuretic effect of these stimuli during K+ depletion in vivo. An increase in sodium excretion and urine flow rate achieved by lowering the perfusate albumin concentration, increasing the perfusate pressure, or adding the diuretics hydrochlorothiazide and furosemide resulted in significant kaliuresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bicarbonate secretion and chloride absorption by rabbit cortical collecting ducts. Role of chloride/bicarbonate exchange.

Cortical collecting ducts (CCD) from rabbits treated with deoxycorticosterone (DOC) actively secrete bicarbonate at high rates. To investigate the mechanism of bicarbonate secretion, we measured bicarbonate and chloride transport in CCD from rabbits treated with DOC for 9-24 d. Removal of chloride (replaced with gluconate) from both perfusate and bath inhibited bicarbonate secretion without changing transepithelial voltage. Removal of chloride only from the bath increased bicarbonate secretion, while removal of chloride only from the perfusate inhibited secretion. In contrast to the effect of removing chloride, removal of sodium from both the perfusate and bath (replacement with N-methyl-D-glucamine) did not change the rate of bicarbonate secretion. The rate of bicarbonate secretion equaled the rate of chloride absorption in tubules bathed with 0.1 mM ouabain to inhibit any cation-dependent chloride transport. Under these conditions, chloride absorption occurred against an electrochemical gradient. Removal of bicarbonate from both the perfusate and bath inhibited chloride absorption. Removal of bicarbonate only from the bath inhibited chloride absorption, while removal of bicarbonate from the lumen stimulated chloride absorption. We conclude that CCD from DOC-treated rabbits actively secrete bicarbonate and actively absorb chloride by an electroneutral mechanism involving 1:1 chloride/bicarbonate exchange. The process is independent of sodium.     

KCl co-transport across the basolateral membrane of rabbit renal proximal straight tubules.

Mammalian renal proximal tubules reabsorb large amounts of chloride. Mechanisms of the transcellular chloride transport are poorly understood. To determine whether KCl co-transport exists in the basolateral membrane of mammalian renal proximal tubule, isolated rabbit proximal straight tubules (S2 segment) were perfused in vitro, and intracellular activities of potassium and chloride (aKi, aCli) were measured by double-barreled ion-selective microelectrodes. aCli did not change when basolateral membrane voltage was altered by application of a direct current through perfusion pipette. aCli changes in response to bath chloride elimination were not affected by current application as well, indicating that the basolateral chloride transport is electroneutral. An increase in potassium concentration of the bath fluid from 5 to 20 mM reversibly increased aCli by 10 mM. This response of aCli to a change in the bath potassium concentration was also observed when luminal chloride was removed, or ambient sodium was totally removed. aKi significantly decreased by 5 mM when chloride was removed from the bath. These data demonstrate the existence of an electroneutral Na+-independent KCl co-transport in the basolateral membrane of the rabbit proximal tubule. Calculated electrochemical driving force was favorable for the movement of KCl from the cell to the peritubular fluid.     

An in vivo microperfusion study of distal tubule bicarbonate reabsorption in normal and ammonium chloride rats.

For many years it has been thought that distal nephron hydrogen ion secretion can be importantly modulated by factors such as sodium delivery, sodium avidity, and potassium stores. Free flow micropuncture studies have also indicated that the rate of bicarbonate delivery may also alter the rate of bicarbonate reabsorption. The present studies were undertaken to examine possible luminal influences on total CO2 reabsorption in microperfused distal tubules in the rat in vivo. Tubules from normal and acidotic rats were perfused with five solutions in a manner that induced changes in bicarbonate load, sodium and potassium fluxes (JNa, JK), and luminal sulfate concentration. in each collected perfusate, simultaneous analyses were undertaken to determine water reabsorption, Na, and K concentrations using graphite furnace atomic absorption spectroscopy and total CO2 by microcalorimetry. Using factorial analysis of covariance to account for confounding effects on total CO2 flux (JtCO2) such as water reabsorption, distal tubules of acidotic rats reabsorbed CO2 in the range of 50-112 pmol X min-1 X mm-1 X These JtCO2 values were not significantly correlated with HCO3 load, JNa, or JK despite changes in the latter from net reabsorption to net secretion. Distal tubules of rats with normal acid-base status had JtCO2 values which were neither significantly different from zero nor correlated with changes in JK and JNa. Further, doubling the load from 250-500 pmol/min (by doubling the perfusion rate of 25-mM HCO3 solutions) did not stimulate JtCO2 in these normal animals. Accordingly, these acute in vivo microperfusion studies indicate for the first time that neither load nor potassium or sodium fluxes are important modulators of distal tubule bicarbonate reabsorption.