Calcium transport in the rabbit superficial proximal convoluted tubule.

Calcium transport was studied in isolated S2 segments of rabbit superficial proximal convoluted tubules. 45Ca was added to the perfusate for measurement of lumen-to-bath flux (JlbCa), to the bath for bath-to-lumen flux (JblCa), and to both perfusate and bath for net flux (JnetCa). In these studies, the perfusate consisted of an equilibrium solution that was designed to minimize water flux or electrochemical potential differences (PD). Under these conditions, JlbCa (9.1 +/- 1.0 peq/mm X min) was not different from JblCa (7.3 +/- 1.3 peq/mm X min), and JnetCa was not different from zero, which suggests that calcium transport in the superficial proximal convoluted tubule is due primarily to passive transport. The efflux coefficient was 9.5 +/- 1.2 X 10(-5) cm/s, which was not significantly different from the influx coefficient, 7.0 +/- 1.3 X 10(-5) cm/s. When the PD was made positive or negative with use of different perfusates, net calcium absorption or secretion was demonstrated, respectively, which supports a major role for passive transport. These results indicate that in the superficial proximal convoluted tubule of the rabbit, passive driving forces are the major determinants of calcium transport.     

Calcium transport in the pars recta and thin descending limb of Henle of the rabbit, perfused in vitro.

Unidirectional calcium flux (JCa) in the superficial pars recta and thin descending limb of Henle (DLH) was examined by the isolated tubule microperfusion technic using 45Ca as the isotopic tracer. In the pars recta sequential measurements of lumen-to-bath flux (JlbCa) and bath-to-lumen flux (JblCa) revealed: JlbCa 22.4 +/- 4.18, JblCa 7.97 +/- 1.95, and calculated net efflux of calcium (JnetCa 13.0 +/- 1.74 peq min-1 mm-u. To measure JnetCa directly, 45Ca of identical specific activity was used to bathe and perfuse the tubule. These studies revealed: JlbCa 14.1 +/- 1.33, JnetCa 11.2 +/- 1.15, and calculated JblCa 2.91 +/- 0.49 peq min-1 mm-1. The addition of ouabain (10 microM) resulted in a rise in potential difference and a fall in water absorption, but not a statistically significant change in JnetCa. Tubules studies at 25 degrees C bath temperature, showed no significant JnetCa, and upon heating the bath to 37 degrees C, showed JnetCa of 3.75--5.00 peg min-1 mm-1. Unidirectional and net efflux studies in six DLH showed no significant transport of calcium. These studies demonstrate substantial active absorption of calcium by the superficial pars recta, which is not inhibitable by ouabain but is inhibited by lowering bath temperature to 25 degrees C. No significant calcium transport was found in the DLH using identical technics.     

Calcium and phosphate transport in isolated segments of rabbit Henle''s loop.

Calcium and phosphate transport was examined in rabbit thin descending, thin ascending, and thick ascending limbs of Henle by in vitro perfusion of isolated tubular segments. Permeability coefficients for these segments with 45Ca and 32PO4 were determined for both lumen-to-bath and bath-to-lumen directions. Both the thin descending and thin ascending limbs were found to be relatively impermeable to both 45Ca and 32PO4. In neither segment were we able to show evidence for net transport of calcium or phosphate. In contrast, the thick ascending limb of Henle showed a decrease in calcium lumen-to-bath concentration from 0.97 +/- 0.02 to 0.88 +/- 0.02 when perfused at 4.8 nl min-1. 45Ca lumen-to-bath and bath-to-lumen fluxes were 19.96 +/- 1.05 and 9.89 +/- 0.02 peq-min-1-cm-1, respectively, and the potential difference was +3.8 +/- 0.3 mV (lumen positive). The observed calcium flux ratio was significantly higher than that predicted by Ussing's equation. When ouabain was added to the bath the potential difference fell to +1.1 +/- 0.3 mV, whereas the calcium efflux was only slightly diminished (29.5 +/- 5.3-23.7 +/- 5.1 peq-cm-1-min-1). Ouabain had no effect on the influx of Ca across the thick ascending limb of Henle. There was no net transport of phosphate across the thick ascending limb. Phosphate permeability was exceedingly low bidirectionally across the thick ascending limb. Our findings indicate: (a) all segments of Henle's loop are relatively impermeable to calcium and phosphate; (b) net transport of phosphate seems to be absent in Henle's loop; (c) net calcium reabsorption, which cannot be explained by passive mechanisms, occurs in the thick ascending limb.     

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.     

Effect of prostaglandin E2 on chloride transport across the rabbit thick ascending limb of Henle. Selective inhibitions of the medullary portion.

Prostaglandins are present in large quantities in the kidney and have been shown to directly affect transepithelial transport. The present studies were designed to examine whether prostaglandin E2 could affect chloride transport across the thick ascending limb of Henle. Isolated segments of the cortical and medullary thick ascending limb of Henle were perfused in vitro and the transepithelial voltage and net chloride flux were measured. Exposure of the medullary thick ascending limb to 2 microM prostaglandin E2 resulted in a fall in net chloride transport of 40--50% with a concomitant fall in voltage. In contrast, net chloride transport in the cortical thick ascending limb was not affected by prostaglandin E2. Under similar conditions, the medullary thick ascending limb possessed twice the capacity to transport chloride than did the cortical thick ascending limb. The results suggest that endogenous renal prostaglandins may play a modulating role in the addition of salt to the renal medullary interstitium and may, under some circumstances, by chloruretic.     

Effects of vasopressin antagonist on vasopressin binding, adenylate cyclase activation, and water flux.

We studied the effect of an arginine vasopressin (AVP) analogue, (1-[beta-mercapto-beta, beta-cyclopentamethylenepropionic acid],2-O-ethyltyrosine, 4-valine)AVP(d[CH2]5Tyr[Et]VAVP), on the stimulation of adenylate cyclase by various hormones in the isolated nephron segments and 3H-AVP binding to renal papillary membranes from the rat. The net water flux across the renal cortical collecting tubules of the rabbit was also examined. We found that d(CH2)5Tyr(Et)VAVP significantly inhibited adenylate cyclase activation by AVP in cortical, medullary, and papillary collecting tubules and in the medullary thick ascending limb. In contrast, the AVP analogue did not alter the stimulation of adenylate cyclase by parathyroid hormone in the cortical thick ascending limb, by glucagon in the medullary thick ascending limb, and by calcitonin in cortical collecting tubules. In addition, d(CH2)5Tyr(Et)VAVP blocked [3H]AVP binding to renal papillary membranes. The enhanced net water transport induced by AVP in isolated, perfused rabbit cortical collecting tubules also was completely blocked by this AVP analogue. These results indicate that d(CH2)5Tyr(Et)VAVP specifically antagonizes the cellular action of AVP on the medullary thick ascending limb and on the cortical, medullary, and papillary collecting tubules. Evidence is also presented for competitive antagonism as the cellular mechanism of action.     

Adenylate cyclase responsiveness to hormones in various portions of the human nephron.

The action sites for parathyroid hormone (PTH), salmon calcitonin (SCT), and arginine-vasopressin (AVP) were investigated along the human nephron by measuring adenylate cyclase activity, using a single tubule in vitro microassay. Well-localized segments of tubule were isolated by microdissection from five human kidneys unsuitable for transplantation. PTH (10 IU/ml) increased adenylate cyclase activity in the convoluted and the straight proximal tubule, in the medullary and cortical portions of the thick ascending limb, and in the early portion of the distal convoluted tubule (corresponding stimulated:basal activity ratios were 64, 19, 10, 18, and 22, respectively). SCT (10 ng/ml) increased adenylate cyclase activity in the medullary and cortical portions of the thick ascending limb, in the early portion of the distal convoluted tubule, and, to a lesser extent, in the cortical and the medullay collecting tubule (activity ratios were 7, 14, 15, 3, and 3, respectively). AVP (1 microM) stimulated adenylate cyclase activity in the terminal nephron segments only, i.e., the late portion of the distal convoluted tubule, the cortical and medullary portions of the collecting tubule (activity ratios 81, 51, and 97, respectively). As measured in one experiment, nearly one-half maximal responses were obtained with 0.1 IU/ml PTH or 0.3 ng/ml SCT in thick ascending limbs and with 1 nM AVP in collecting tubules, suggesting that enzyme sensitivity to hormones as well preserved under the conditions used in this study.     

Sodium chloride, urea, and water transport in the thin ascending limb of Henle. Generation of osmotic gradients by passive diffusion of solutes

Studies were designed to examine whether the thin ascending limb of Henle (tALH) decreases its luminal solute concentration by an active or a passive transport process. In all experiments isolated segments of rabbit tALH were perfused in vitro. When tubules were perfused with solutions identical to the bath, active transport of NaCl was excluded by the following: (a) osmolality of the collected fluid remained unchanged and the same as the bath. (b) net water reabsorption could not be demonstrated, and (c) transtubular potential difference was zero. Isotopic permeability coefficients (x 10(-5) cm s-1) were calculated from the disappearance rate of the respective isotope added to the perfusate. These values indicate that tALH is moderately permeable to [14C]urea (6.97 +/- 1.95) while having a higher permeability to 22Na (25.5 +/- 1.8) and [not readable: see text]Cl (117 +/- 9.1) than any other segment similarly studied. The influx (bath-to-lumen) isotopic permeabilities were not statistically different from the above efflux permeabilities. Osmotic water permeability was immeasurably small. When tALH were perfused with a 600 mosmol/liter solution predominantly of NaCl against a 600 mosmol/liter bath in which 50% of osmolality was NaCl and 50% urea (to simulate in vivo papillary interstitium), the collected fluid osmolality was decreased significantly below that of the bath (300 mosmol/liter/mm of tubule). The decrease in osmolality was due to greater efflux of NaCl as compared to influx of urea. We conclude that active transport of salt by the tALH was not detected by the experimental protocol of the current studies, and that the unique membrane characteristics of tALH allows for generation of osmotic gradients (lumen less concentrated than adjacent surroundings) on purely passive mechanisms when perfused with isosmolal salt solutions in a bath with appropriate salt and urea concentrations. These findings are consistent with the passive counter-current model previously proposed from this laboratory.     

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.     

Vanadate stimulates the N-ethylmaleimide-sensitive adenosine triphosphatase in rat nephron

Vanadate has been used in many cellular systems to elucidate mechanisms of enzyme action. Vanadate inhibits Na-K adenosine triphosphatase (ATPase) activity in many tissues. In isolated collecting tubule it inhibits sodium transport and vasopressin-stimulated water flux, the latter presumably distal to cyclic AMP formation. Depending upon the tissue studied, vanadate also stimulates a variety of cellular reactions including adenylate cyclase, glucose oxidation and glycogen synthesis. We studied the effect of varying concentrations of vanadate on N-ethylmaleimide (NEM)-sensitive ATPase activity in microdissected segments of rat nephron. In proximal convoluted tubule and in cortical, medullary and papillary collecting ducts vanadate had no effect on enzyme activity. In medullary and cortical thick ascending limbs, however, vanadate significantly stimulated NEM-sensitive ATPase activity (medullary thick ascending limb, 241 +/- 14 pmol/mm/hr vs. 531 +/- 74 pmol/mm/hr; control vs. (1 mM) vanadate, respectively; n = 14, P less than 0.01). The stimulatory effect of vanadate on NEM-sensitive ATPase activity was present at 5 microM vanadate, a concentration that inhibited Na-K ATPase activity approximately 80%. Metabolic acidosis also stimulated enzyme activity in the thick ascending limb, and the effect of vanadate was not additive. Metabolic alkalosis had no effect on NEM-sensitive ATPase in the thick ascending limb, but the stimulatory effect of vanadate was still seen. These data document that the NEM-sensitive ATPase in thick ascending limb is different from that found in other nonmammalian proton secretory epithelia which are vanadate inhibitable. The results with vanadate plus metabolic acidosis suggest that both are acting via the same mechanism.     

Renal action of a novel uricosuric diuretic, S-8666. II. Effects on Cl- and urate transport in isolated perfused rabbit renal tubules

We have studied the effect of the two enantiomeric forms of the diuretic agent, S-8666 [6,7-dichloro-5-(N,N-dimethylsulfamoyl)-2,3-dihydro-2-benzofuran carboxylic acid], on the Cl- transport across the cortical thick ascending limb of Henle's loop (CAL) and on urate transport across the proximal tubule, using the in vitro microperfusion technique of individual tubular segments isolated from the rabbit kidney. S-8666 in the lumen reduced significantly the lumen-positive voltage in CAL. The suppression of lumen-positive voltage was instantaneous, and the effects were reversible when the drug was eliminated from the perfusate. These effects were observed with the (S-)-enantiomer of S-8666 but not with the (R+)-enantiomer. S-8666 did not affect the lumen-positive voltage when it was added to the bathing fluid. The lumen to bath 36Cl flux in CAL also was reduced by addition of S-8666 to the perfusate. S-8666 in the lumen inhibited the lumen-to-bath [14C]urate flux in both proximal convoluted tubule and proximal straight tubule. It also reduced the bath-to-lumen urate flux when it was added to the bath. Enantioselectivity was not found for these inhibitory effects on urate transport of S-8666. We conclude: 1) the (S-)-enantiomer of S-8666, but not the (R+)-enantiomer, has a direct effect on the Cl- transport in the CAL, acting from the luminal side and 2) both enantiomers of S-8666 inhibit urate to transport in proximal tubules.     

Furosemide directly stimulates prostaglandin E2 production in the thick ascending limb of Henle's loop

Studies were conducted to investigate direct effects of loop diuretics on prostaglandin E2 (PGE2) production using microdissected nephron segments. At first, the effect of indomethacin on the diuretic response to furosemide was re-evaluated in anesthetized rats. Indomethacin significantly attenuated the diuretic, natriuretic and chloruretic effects of furosemide without significantly affecting inulin and p-aminohippurate clearance or filtration fraction. But, in nondiuretic states, indomethacin had no significant effects on these parameters. Furosemide, ethacrynic acid and bumetanide significantly increased PGE2 production in cortical and medullary thick ascending limbs of Henle's loop (P less than .001), but not PGE2 production in the cortical and outer medullary collecting tubules. The effect of furosemide on PGE2 production in CTAL was dose-dependent, and higher concentrations of of furosemide than 10(-6) M significantly increased PGE2 production. On the other hand, chlorothiazide showed no PGE2 productive stimulation in these four nephron segments. This study demonstrates that the enhanced PGE2 production in the thick ascending limb of Henle's loop by furosemide and other loop diuretics is one possible mechanism of these drugs.     

Insulin binding sites in various segments of the rabbit nephron.

Insulin binds specifically to basolateral renal cortical membranes and modifies tubular electrolyte transport, but the target sites of this hormone in the nephron have not been identified. Using a microassay that permits measurement of hormone binding in discrete tubule segments we have determined the binding sites of 125I-insulin along the rabbit nephron. Assays were performed under conditions that minimize insulin degradation, and specific binding was measured as the difference between 125I-insulin bound in the presence or absence of excess (10(-5) M) unlabeled hormone. Insulin monoiodinated in position A14 was used in all assays. Specific insulin binding (attomol . cm-1 +/- SE) was highest in the distal convoluted tubule (180.5 +/- 15.0) and medullary thick ascending limb of Henle's loop (132.9 +/- 14.6), followed by the proximal convoluted and straight tubule. When expressed per milligram protein, insulin binding capacity was highest along the entire thick ascending limb (medullary and cortical portions) and the distal convoluted tubule, i.e., the "diluting segment" (congruent to 10(-13) mol . mg protein-1), and was lower (congruent to 4 X 10(-14) mol . mg protein-1), and remarkably similar, in all other nephron segments. Binding specificity was verified in competition studies with unlabeled insulin, insulin analogues (proinsulin and desoctapeptide insulin), and unrelated hormones (glucagon, 1-34 parathyroid hormone, prolactin, follicle-stimulating hormone). In addition, serum containing antiinsulin receptor antibody from two patients with type B insulin resistance syndrome markedly inhibited insulin binding to isolated tubules. Whether calculated per unit tubule length or protein content, insulin binding is highest in the thick ascending limb and the distal convoluted tubule, the same nephron sites where a regulatory role in sodium transport has been postulated for this hormone.     

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.     

Sodium Chloride and Water Transport in the Medullary Thick Ascending Limb of Henle. EVIDENCE FOR ACTIVE CHLORIDE TRANSPORT

Transport of NaCl and water was examined in the rabbit medullary thick ascending limb of Henle (ALH) by perfusing isolated segments of these nephrons in vitro. Osmotic water permeability was evaluated by perfusing tubules against imposed osmotic gradients. In these experiments the net transport of fluid remained at zero when segments of thick ALH were perfused with isotonic ultrafiltrate in a bath of rabbit serum in which the serum osmolality was increased by the addition of either 239±8 mosmol/liter of raffinose or 232±17 mosmol of NaCl indicating that the thick ascending limb of Henle is impermeant to osmotic flow of water. When these tubules were perfused at slow rates with isosmolal ultrafiltrate of same rabbit serum as used for the bath, the effluent osmolality was consistently lowered to concentrations less than the perfusate and the bath. That this decrease in collected fluid osmolality represented salt transport was demonstrated in a separate set of experiments in which it was shown that the sodium and chloride concentrations decreased to 0.79±0.02 and 0.77±0.02 respectively when compared with the perfusion fluid concentrations. In each instance the simultaneously determined transtubular potential difference (PD) revealed the lumen to be positive with the magnitude dependent on the perfusion rate. At flow rates above 2 nl·min^-1, the mean transtubular PD was stable and equal to 6.70±0.34 mv. At stop-flow conditions this PD became more positive. Ouabain and cooling reversibly decreased the magnitude of this PD. The transtubular PD remained positive, 3.3±0.2 mV, when complete substitution of Na by choline was carried out in both the perfusion fluid and the bathing media. These results are interpreted to indicate that the active transport process is primarily an electrogenic chloride mechanism. The isotopic permeability coefficient for Na was 6.27±0.38 × 10^-5 cm·s^-1 indicating that the thick ALH is approximately as permeable to Na as the proximal convoluted tubule. The chloride permeability coefficient for the thick ALH was 1.06±0.12 × 10^-5 cm·s^-1 which is significantly less than the chloride permeability of the proximal tubule.     

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.     

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.     

Effect of vasopressin on electrical potential difference and chloride transport in mouse medullary thick ascending limb of Henle''s loop.

Medullary thick ascending limbs of Henle's loop of the Swiss-Webster mouse were perfused in vitro with an isotonic perfusate and a Ringer's bathing medium. In five studies, addition of a supramaximal concentration of synthetic arginine vasopressin (AVP) to the bathing medium resulted in an increase in electrical potential difference (PD) from 5.0 +/- 1.5 mV, lumen positive, to 10.7 +/- 1.4 mV (P < 0.001). When AVP was removed, the PD returned to 2.6 +/- 0.9 mV (P < 0.001), then increased again to 6.9 +/- 1.7 mV (P < 0.01) when AVP was added a second time. A significant, but submaximal, increase in PD of 2.3 +/- 0.6 MV (P < 0.05) was observed in five medullary thick ascending limbs when AVP was added to the bathing medium at a concentration of 10 microunits/ml. This increase was approximately one-third of the response observed at a concentration of 100 microunits/ml in the same tubule. No further increment in PD was observed in five medullary thick ascending limbs when the AVP concentration was increased from 100 to 1,000 microunits/ml. In seven thick ascendcing limbs, the effect of AVP on PD was reproduced by the addition of 8-[p-chlorophenylthio]-cyclic 3',5'-adenosine monophosphate to the bathing medium at a final concentration of 0.1 mM. AVP increased unidirectional chloride flux from lumen to bath from 29.3 +/- 3.2 to 69.8 +/- 6.2 peq/cm per s (P < 0.001) in spite of an increase in the lumen positive PD from 1.6 +/- 0.5 mV to 7.0 +/- 0.6 mV (P < 0.001). Unidirectional chloride flux from bath to lumen was not affected by AVP. In another series of experiments, net chloride flux increased from 15.6 +/- 3.0 to 41.7 +/- 5.3 peq/cm per s (P < 0.05) after addition of AVP. The effect of AVP on hydraulic water permeability (Lp) was examined by adding raffinose to the bathing medium in both the presence and the absence of AVP. The calculated Lp of 16 +/- 2 nm/s per atm in the absence of AVP, although very low, was significantly different from zero (P < 0.01). However, the Lp did not increase significantly when AVP was added to the bathing medium. These results suggest that AVP has a second site of action in the kidney to increase chloride transport by the medullary thick ascending limb in addition to its well-known effect on the water permeability of the collecting tubule. The former effect would contribute to urinary concentrating ability by increasing the axial osmotic gradient in the renal medulla.     

Angiotensin II binding sites in individual segments of the rat nephron.

The sites of action of angiotensin II along the nephron are not well defined and both proximal and distal effects are suggested. Using a microassay that permits measurement of hormone binding in discrete tubule segments, we determined the binding sites of 125I-angiotensin II along the nephron of Sprague-Dawley rats. Specific binding in proximal convoluted tubule (PCT) (at 25 degrees C, pH 7.4) was linearly related to tubule length and saturable, with an apparent maximal binding capacity of approximately 300 amol X cm-1. Binding specificity was verified in competition experiments that revealed significant (P less than 0.001) and comparable competition for radioligand binding by angiotensin II and angiotensin precursor, metabolite, and analogues, whereas unrelated peptides of similar size (bradykinin, ACTH [1-10]) were without effect. The profile of specific angiotensin II binding along the nephron was: PCT, 216 +/- 13; pars recta, 86 +/- 14; medullary thick ascending limb of Henle's loop, 46 +/- 8; cortical thick ascending limb of Henle's loop, 77 +/- 8; distal convoluted tubule, 49 +/- 10; cortical collecting tubule, 15 +/- 1; medullary collecting tubule, 32 +/- 7 amol X cm-1. These results indicate the presence of specific angiotensin II binding sites in all tubule segments studied, but binding capacity was highest in the proximal convoluted tubule, in agreement with transport studies that localize the effects of the hormone in this segment.     

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.