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.     

Sodium-dependent net urea transport in rat initial inner medullary collecting ducts.

We reported that feeding rats 8% protein for 3 wk induces net urea transport and morphologic changes in initial inner medullary collecting ducts (IMCDs) which are not present in rats fed 18% protein. In this study, we measured net urea transport in microperfused initial IMCDs from rats fed 8% protein for > or = 3 wk and tested the effect of inhibiting Na+/K(+)-ATPase activity and found that adding 1 mM ouabain to the bath reversibly inhibited net urea transport from 14 +/- 3 to 6 +/- 2 pmol/mm per min (P < 0.01), and that replacing potassium (with sodium) in the bath reversibly inhibited net urea transport from 18 +/- 3 to 5 +/- 0 pmol/mm per min (P < 0.01). Replacing perfusate sodium with N-methyl-D-glucamine reversibly inhibited net urea transport from 12 +/- 2 to 0 +/- 1 pmol/mm per min (P < 0.01), whereas replacing bath sodium had no significant effect on net urea transport. Adding 10 nM vasopressin to the bath exerted no significant effect on net urea transport. Finally, we measured Na+/K(+)-ATPase activity in initial and terminal IMCDs from rats fed 18% or 8% protein and found no significant difference in either subsegment. Thus, net urea transport in initial IMCDs from rats fed 8% protein for > or = 3 wk requires sodium in the lumen, is reduced by inhibiting Na+/K(+)-ATPase, and is unchanged by vasopressin or phloretin. These results suggest that net urea transport may occur via a novel, secondary active, sodium-urea cotransporter.     

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.     

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.     

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.     

Control of sodium and potassium transport in the cortical collecting duct of the rat. Effects of bradykinin, vasopressin, and deoxycorticosterone.

Several factors interact to maintain precise control of electrolyte transport in the mammalian cortical collecting duct. We have studied the effects of deoxycorticosterone, arginine vasopressin, and bradykinin on net transepithelial sodium and potassium transport in isolated, perfused rat cortical collecting ducts. Chronic administration of deoxycorticosterone to rats increased both sodium absorption and potassium secretion above very low basal levels. Consequently, deoxycorticosterone-treated rats were used for all remaining studies. Arginine vasopressin (10(-10) M in the bath) caused a sustained fourfold increase in net sodium absorption and a sustained threefold increase in net potassium secretion. Bradykinin (10(-9) M in the bath) caused a reversible 40-50% inhibition of net sodium absorption without affecting net potassium transport or the transepithelial potential difference. In the perfusate, up to 10(-6) M bradykinin had no effect. We conclude: As in rabbits, chronic deoxycorticosterone administration to rats increases sodium absorption and potassium secretion in cortical collecting ducts perfused in vitro. Arginine vasopressin causes a reversible increase in net potassium secretion and net sodium absorption. Bradykinin in the peritubular bathing solution reversibly inhibits net sodium absorption, possibly by affecting an electroneutral sodium transport pathway.     

Sodium, phosphate, glucose, bicarbonate, and alanine interactions in the isolated proximal convoluted tubule of the rabbit kidney.

Interactions among the transport systems involved with sodium, bicarbonate, glucose, phosphate, and alanine absorption in isolated segments of the rabbit proximal convoluted tubule were examined with radioisotopic techniques to measure glucose, phosphate, and fluid absorption rates. The composition of the perfusate and bath varied from normal, physiological fluids to fluids deficient in a single solute. The deletion of glucose from the perfusate increased the lumen-to-bath flux of phosphate from 5.51 +/- 1.15 to 8.32 +/- 1.34 pmol/mm-min (P less than 0.01). Similar changes occurred when glucose transport was inhibited by phlorizin 10 micron in the perfusate, The deletion of alanine from the perfusate increased the lumen-to-bath flux of phosphate from 6.55 +/- 1.08 to 9.00 +/- 1.30 pmol/mm-min (P less than 0.01) but did not affect glucose transport significantly, 80.1 +/- 10.1 vs. 72.5 +/- 5.4 pmol/mm-min. Replacement of intraluminal sodium with choline, elimination of potassium from the bath, and removal of bicarbonate from the lumen and bath each reduced glucose, phosphate, and fluid absorption. These data indicate that the proximal absorptive processes for glucose and for phosphate include elements that are dependent upon some function of sodium transport. Additionally, the effects on phosphate transport of deleting glucose or alanine occur independent of any changes in net sodium transport and are opposite the effects of deleting bicarbonate. These differences may relate to the observations that the transport of glucose and alanine is electrogenic while that of bicarbonate is not. Regardless of possible mechanisms, the data demonstrate that important changes in the absorption rates of different solutes handled significantly by the proximal convoluted tubule may occur in response to changes in specific components of proximal sodium transport.     

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.     

Metabolic acidosis stimulates H+ secretion in the rabbit outer medullary collecting duct (inner stripe) of the kidney.

The outer medullary collecting duct (OMCD) absorbs HCO3- at high rates, but it is not clear if it responds to metabolic acidosis to increase H+ secretion. We measured net HCO3- transport in isolated perfused OMCDs taken from deep in the inner stripes of kidneys from control and acidotic (NH4Cl-fed for 3 d) rabbits. We used specific inhibitors to characterize the mechanisms of HCO3- transport: 10 microM Sch 28080 or luminal K+ removal to inhibit P-type H+,K+-ATPase activity, and 5-10 nM bafilomycin A1 or 1-10 nM concanamycin A to inhibit H+-ATPase activity. The results were comparable using either of each pair of inhibitors, and allowed us to show in control rabbits that 65% of net HCO3- absorption depended on H+-ATPase (H flux), and 35% depended on H+,K+-ATPase (H,K flux). Tubules from acidotic rabbits showed higher rates of HCO3- absorption (16.8+/-0.3 vs. 12.8+/-0.2 pmol/min per mm, P < 0.01). There was no difference in the H,K flux (5.9+/-0.2 vs. 5.8+/-0.2 pmol/min per mm), whereas there was a 61% higher H flux in segments from acidotic rabbits (11.3+/-0.2 vs. 7.0+/-0.2 pmol/min per mm, P < 0.01). Transport was then measured in other OMCDs before and after incubation for 1 h at pH 6.8, followed by 2 h at pH 7.4 (in vitro metabolic acidosis). Acid incubation in vitro stimulated HCO3- absorption (12.3+/-0.3 to 16.2+/-0.3 pmol/min per mm, P < 0.01), while incubation at pH 7.4 for 3 h did not change basal rate (11.8+/-0.4 to 11.7+/-0.4 pmol/min per mm). After acid incubation the H,K flux did not change, (4.7+/-0.4 to 4.6+/-0.4 pmol/min per mm), however, there was a 60% increase in H flux (6.6+/-0.3 to 10.8+/-0.3 pmol/min per mm, P < 0.01). In OMCDs from acidotic animals, and in OMCDs incubated in acid in vitro, there was a higher basal rate and a further increase in HCO3- absorption (16.7+/-0.4 to 21.3+/-0.3 pmol/min per mm, P < 0.01) because of increased H flux (11.5+/-0.3 to 15.7+/-0.2 pmol/min per mm, P < 0.01) without any change in H,K flux (5.4+/-0.3 to 5.6+/-0.3 pmol/min per mm). These data indicate that HCO3- absorption (H+ secretion) in OMCD is stimulated by metabolic acidosis in vivo and in vitro by an increase in H+-ATPase-sensitive HCO3- absorption. The mechanism of adaptation may involve increased synthesis and exocytosis to the apical membrane of proton pumps. This adaptation helps maintain homeostasis during metabolic acidosis.     

Calcium transport in the thick ascending limb of Henle. Heterogeneity of function in the medullary and cortical segments.

Calcium transport was studied in medullary and cortical segments of the thick ascending limb of Henle perfused in vitro. 45Ca was added to the perfusate for measuring lumen-to-bath flux (JlbCa), to the bath for measuring bath-to-lumen flux (JblCa), or to both perfusate and bath for measuring net flux (JnetCa). In the medullary segment JlbCa exceeding JblCa and the efflux:influx coefficient ratio was not different from the value predicted from the observed potential difference (PD). In the cortical segments, however, efflux:influx coefficient ratio was greater than the value predicted from the PD, suggesting that calcium transport in this segment may be active, while it is passive in the medullary segment. Furosemide, which reversibly decreases PD in both cortical and medullary segments, inhibited JlbCa only in the medullary segment. Parathyroid hormone (PTH), on the other hand, had no effect on JnetCa in the medullary segment, but it significantly augmented JnetCa in the cortical segment. These results indicate that calcium transport in the thick ascending limb is heterogeneous. In the medullary segment it is passive, inhibited by furosemide and not influenced by PTH. In the cortical segment, however, calcium transport appears to be active, not inhibited by furosemide and stimulated by PTH.     

Adaptation of rabbit cortical collecting duct HCO3- transport to metabolic acidosis in vitro.

Net HCO3- transport in the rabbit kidney cortical collecting duct (CCD) is mediated by simultaneous H+ secretion and HCO3- secretion, most likely occurring in a alpha- and beta-intercalated cells (ICs), respectively. The polarity of net HCO3- transport is shifted from secretion to absorption after metabolic acidosis or acid incubation of the CCD. We investigated this adaptation by measuring net HCO3- flux before and after incubating CCDs 1 h at pH 6.8 followed by 2 h at pH 7.4. Acid incubation always reversed HCO3- flux from net secretion to absorption, whereas incubation for 3 h at pH 7.4 did not. Inhibition of alpha-IC function (bath CL- removal or DIDS, luminal bafilomycin) stimulated net HCO3- secretion by approximately 2 pmol/min per mm before acid incubation, whereas after incubation these agents inhibited net HCO3- absorption by approximately 5 pmol/min per mm. Inhibition of beta-IC function (luminal Cl- removal) inhibited HCO3- secretion by approximately 9 pmol/min per mm before incubation, whereas after incubation HCO3- absorption by only 3 pmol/min per mm. After acid incubation, luminal SCH28080 inhibited HCO3- absorption by only 5-15% vs the circa 90% inhibitory effect of bafilomycin. In outer CCDs, which contain fewer alpha-ICs than midcortical segments, the reversal in polarity of HCO3- flux was blunted after acid incubation. We conclude that the CCD adapts to low pH in vitro by downregulation HCO3- secretion in beta-ICs via decreased apical CL-/base exchang activity and upregulating HCO3- absorption in alpha-ICs via increased apical H+ -ATPase and basolateral CL-/base exchange activities. Whether or not there is a reversal of IC polarity or recruitment of gamma-ICs in this adaptation remains to be established.     

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.     

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.     

Pathways for HCO3-reabsorption in mouse medullary collecting duct segments

To determine pathways of HCO3- reabsorption in the collecting duct of the mouse kidney, the outer medullary collecting duct (OMCD) and the terminal inner medullary collecting duct (IMCDt) were dissected and perfused at 1 to 2 nL/min, and total CO2 was measured by microfluorometry. In the OMCD, net HCO3- flux (JtCO2) was 12.2 +/- 0.7 pmol/min/mm tubule length and decreased to 6.9 +/- 0.6 pmol/min/mm tubule length (n = 5) with 10 mol/L Schering 28080 (SCH) in perfusate (P < .001) and to 7.7 +/- 0.6 pmol/min/mm tubule length (P < .004; n = 4) with 50 micromol/L diethylstilbestrol (DES), an inhibitor of H+-adenosine triphosphatase; together they reduced JtCO2 to 3.7 +/- 0.2 pmol/min/mm tubule length (P = .0002; n = 4). In IMCDt, JtCO2 was 10.9 +/- 1.1 pmol/min/mm tubule length, and it decreased to 4.3 +/- 0.9 pmol/min/mm tubule length (n = 4) with 10 micromol/L SCH in perfusate (P < .05) and to 7.0 +/- 1.1 pmol/min/mm tubule length (P < .05; n = 4) with 50 micromol/L DES; together they decreased JtCO2 to 2.3 +/- 0.3 pmol/min/mm tubule length (P < .002; n = 4). Ouabain (1 mmol/L), an inhibitor of colonic H-K-adenosine triphosphatase (cHKA), in perfusate had no effect on JtCO2 in either segment. Northern hybridization studies showed a high level of expression of gastric HKA (gHKA) in outer medulla and a low level in inner medulla; cHKA expression was undetectable. Thus, in normal mouse OMCD and IMCDt, HCO3- reabsorption is predominantly mediated by gHKA and H+-adenosine triphosphatase and not cHKA. A third isoform of HKA could be present in mouse IMCDt.     

Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule.

Bicarbonate transport was studied in vivo by separate microperfusion experiments of early and late distal tubules. Total CO2 was measured by microcalorimetry and fluid absorption by 3H-inulin. Significant bicarbonate absorption was observed in all experimental conditions. Bicarbonate transport was load-dependent upon increasing the luminal bicarbonate concentration from 15 to 50 mM in both early and late distal tubule segments and remained constant at higher concentrations at a maximum rate of 100-110 pmol/min per mm. At low lumen bicarbonate concentrations (15 mM), higher rates of bicarbonate absorption were observed in early (32.9 +/- 4.57 pmol/min per mm) as compared to late distal tubules (10.7 +/- 3.1 pmol/min per mm). Amiloride and ethyl-isopropylamiloride both inhibited early but not late distal tubule bicarbonate absorption whereas acetazolamide blocked bicarbonate transport in both tubule segments. Fluid absorption was significantly reduced in both tubule segments by amiloride but only in early distal tubules by ethyl-isopropylamiloride. Substitution of lumen chloride by gluconate increased bicarbonate absorption in late but not in early distal tubules. Bafilomycin A1, an inhibitor of H-ATPase, inhibited late and also early distal tubule bicarbonate absorption, the latter at higher concentration. After 8 d on a low K diet, bicarbonate absorption increased significantly in both early and late distal tubules. Schering compound 28080, a potent H-K ATPase inhibitor, completely blocked this increment of bicarbonate absorption in late but not in early distal tubule. The data suggest bicarbonate absorption via Na(+)-H+ exchange and H-ATPase in early, but only by amiloride-insensitive H+ secretion (H-ATPase) in late distal tubules. The study also provides evidence for activation of K(+)-H+ exchange in late distal tubules of K depleted rats. Indirect evidence implies a component of chloride-dependent bicarbonate secretion in late distal tubules and suggests that net bicarbonate transport at this site results from bidirectional bicarbonate movement.     

Active proton secretion and potassium absorption in the rabbit outer medullary collecting duct. Functional evidence for proton-potassium-activated adenosine triphosphatase.

We examined the hypothesis that proton-potassium-activated adenosine triphosphatase (H-K-ATPase) mediates K absorption and acidification in the inner stripe of the outer medullary collecting duct (OMCDi). Rabbits were fed a low-K diet (0.55% K) for 7-14 d because we have demonstrated previously that this low-K diet stimulates K-absorptive flux by the OMCDi. Proton secretion was measured as net total CO2 flux (JTCO2) by microcalorimetry. After basal collections, either vehicle or an inhibitor of gastric H-K-ATPase, omeprazole (0.1 mM), was added to the perfusate during the second period. Addition of vehicle to the perfusate changed neither the transepithelial voltage (VT, in millivolts) nor the JTCO2. In contrast, the addition of omeprazole (0.1 mM) to the perfusate abolished JTCO2 (from 14.5 +/- 5.6 to -0.1 +/- 3.1 pmol.mm-1.min-1) without significantly affecting VT. In additional experiments, in 16 tubules there was significant net K absorption (JK) of 5.0 +/- 1.0 pmol.mm-1.min-1 during the basal period, which exceeded the rate of K absorption that could be attributed to a paracellular voltage-mediated pathway (JKP = 1.0 +/- 0.4 pmol.mm-1.min-1, P less than 0.01). Administration of vehicle did not significantly affect either VT or JK. However, omeprazole abolished JK (from 5.1 +/- 1.0 to 0.1 +/- 2.5 pmol.mm-1.min-1) without affecting VT or JNa. The present results demonstrate that the OMCDi possesses an active, omeprazole-sensitive acidification and K-absorptive mechanism. These findings are consistent with the presence of H-K-ATPase activity in this nephron segment.     

Characteristics of salt and water transport in superficial and juxtamedullary straight segments of proximal tubules.

The purpose of the present studies was to characterize the nature of salt and water transport out of the superficial (SF) and juxtamedullary (JM) straight segments of rabbit proximal tubules as examined by in vitro microperfusion techniques. When the perfusate consisted of a solution simulating ultrafiltrate of plasma, there were no differences between SF and JM straight tubules in either net reabsorption of fluid (SF=0.47 nl/mm per min; JM=0.56 nl/mm per min) or in transtubular potential difference (PD) (SF=-2.1 mV; JM=-1.8 mV). Removal of glucose and alanine from the perfusate had no effect on the magnitude of the PD in either straight segment. Ouabain decreased both the net reabsorptive rates and the PD. Isosmolal replacement of NaCL by Na-cyclamate (a presumed impermeant anion) in the perfusate and the bath caused an increase in luminal negativity in both segments wheras similar substitution of NaCL by choline-CL (nontransported cation) changed the PD TO NEAR ZERO. These studies, therefore, suggest that sodium is transported out of the proximal straight tubules by an active noncoupled process that generates a PD (electrogenic process). When the perfusate consisted of a solution with a high chloride concentration (resulting from greater HCO3 than CI reabsorption in the proximal convoluted tubule), different PDs in SF and JM tubules were generated: SF=+1.6 plus or minus 0.2 mV; JM=-1.3 plus or minus 0.3 mV. This difference in PD was attributed to relative differences in Na and CI permeabilities in these two segments. Electrophysiological and isotopic estimates of the chloride to sodium permeability revealed that the SF tubule is about twice as permeant to chloride than to sodium whereas the JM tubules are approximately twice as permeant to sodium than to chloride. It is concluded that the mechanism of active sodium transport in the straight segment of proximal tubule differs from that of the convoluted segment and that both the SF and JM straight segments differ from each other with respect os sodium and chloride permeability.     

Role of adenosine triphosphate (ATP) and NaK ATPase in the inhibition of proximal tubule transport with intracellular cystine loading.

Cellular cystine loading with cystine dimethyl ester inhibits volume absorption, transepithelial potential difference, glucose transport, and bicarbonate transport in proximal convoluted tubules perfused in vitro. This study examined the roles of ATP and NaK ATPase in this in vitro model of the Fanconi syndrome of cystinosis. Intracellular ATP was measured using the luciferin-luciferase assay. Intracellular ATP was reduced by 60% in proximal convoluted tubules incubated with 0.5 mM cystine dimethyl ester for 15 min at 37 degrees C (P less than 0.001). Incubation of cystine loaded tubules with 1 mM exogenous ATP increased intracellular ATP to levels not significantly different than that of controls. On the other hand, Vmax NaK ATPase activity was unchanged even though the incubation times and the concentration of cystine dimethyl ester were doubled to 30 min and 1 mM, respectively. In proximal convoluted tubules perfused in vitro, 0.5 mM cystine dimethyl ester resulted in an 89% inhibition in volume absorption (0.81 +/- 0.14 to 0.09 +/- 0.09 nl/mm.min), while there was only a 45% inhibition in volume absorption (P less than 0.01) due to cellular cystine loading in the presence of 1 mM lumen and bath ATP (0.94 +/- 0.05 to 0.52 +/- 0.11 nl/mm.min). These data demonstrate that proximal tubule cellular cystine loading decreases cellular ATP concentration, but does not directly inhibit NaK ATPase activity. The inhibition in transport and decrease in intracellular ATP due to cellular cystine loading was ameliorated by exogenous ATP. These data are consistent with cellular ATP depletion playing a major role in the inhibition of proximal tubule transport due to intracellular cystine loading.     

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.     

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.