Genomic and nongenomic stimulatory effect of aldosterone on H+-ATPase in proximal S3 segments.

The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H(+)-ATPase and on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in isolated proximal S3 segments of rats during superfusion with an Na(+)-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH(4)Cl pulse, was 0.064 ± 0.003 pH units/min (n = 17/74) and was abolished with concanamycin. Aldosterone (10(-12), 10(-10), 10(-8), or 10(-6) M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca(2+)](i) was 103 ± 2 nM (n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca(2+)](i) and after 6-min preincubation there was a new increase in [Ca(2+)](i) that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca(2+)](i) but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca(2+) chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H(+)-ATPase and on [Ca(2+)](i). The results are compatible with stimulation of the H(+)-ATPase by increases in [Ca(2+)](i) (at 10(-12)-10(-6) M aldosterone) and inhibition of the H(+)-ATPase by decreases in [Ca(2+)](i) (at 10(-12) or 10(-6) M aldosterone plus RU 486).

Effect of D-alpha-tocopherol on tubular nephron acidification by rats with induced diabetes mellitus.

The objective of the present study was to determine if treatment of diabetic rats with D-alpha-tocopherol could prevent the changes in glomerular and tubular function commonly observed in this disease. Sixty male Wistar rats divided into four groups were studied: control (C), control treated with D-alpha-tocopherol (C + T), diabetic (D), and diabetic treated with D-alpha-tocopherol (D + T). Treatment with D-alpha-tocopherol (40 mg/kg every other day, ip) was started three days after diabetes induction with streptozotocin (60 mg/kg, ip). Renal function studies and microperfusion measurements were performed 30 days after diabetes induction and the kidneys were removed for morphometric analyses. Data are reported as means +/- SEM. Glomerular filtration rate increased in D rats but decreased in D + T rats (C: 6.43 +/- 0.21; D: 7.74 +/- 0.45; D + T: 3.86 +/- 0.18 ml min-1 kg-1). Alterations of tubular acidification observed in bicarbonate absorption flux (JHCO3) and in acidification half-time (t/2) in group D were reversed in group D + T (JHCO3, C: 2.30 +/- 0.10; D: 3.28 +/- 0.22; D + T: 1.87 +/- 0.08 nmol cm-2 s-1; t/2, C: 4.75 +/- 0.20; D: 3.52 +/- 0.15; D + T: 5.92 +/- 0.19 s). Glomerular area was significantly increased in D, while D + T rats exhibited values similar to C, suggesting that the vitamin prevented the hypertrophic effect of hyperglycemia (C: 8334.21 +/- 112.05; D: 10,217.55 +/- 100.66; D + T: 8478.21 +/- 119.81 microm(2)). These results suggest that D-alpha-tocopherol is able to protect rats, at least in part, from the harmful effects of diabetes on renal function.

Effect of D-glucose and L-glutamate on the kinetics of bicarbonate reabsorption in rat proximal tubules.

The effect of D-glucose or L-glutamate on the kinetics of bicarbonate reabsorption in the early (EPT) and middle proximal tubule (MPT) was studied in vivo in Munich-Wistar rats by microperfusion techniques. The presence of 20 mM D-glucose in the lumen increased acidification half-time (t/2) (from 2.54 +/- 0.09 s to 3.11 +/- 0.17 s in EPT and from 4.75 +/- 0.20 s to 6.04 +/- 0.49 s in MPT). Bicarbonate reabsorption (JHCO3-) decreased as a consequence of this change (from 3.80 +/- 0.17 to 2.46 +/- 0.20 nmol cm-2 s-1 in EPT and from 2.30 +/- 0.10 to 1.64 +/- 0.10 nmol cm-2 s-1 in MPT). In this situation the basolateral membrane potential difference (BLMPD) in the MPT decreased from -41.6 +/- 2.47 to -29.7 +/- 2.45 mV and returned to control values after perfusion with D-glucose. The addition of 20 mM L-glutamate to the luminal perfusion caused an opposite effect, i.e., a decrease in t/2 (1.54 +/- 0.21 s in EPT and 3.25 +/- 0.26 s in MPT) and a consequent increase in JHCO3- in both segments (5.09 +/- 0.58 nmol cm-2 s-1 in EPT and 3.92 +/- 0.30 nmol cm-2 s-1 in MPT). The BLMPD of MPT increased during L-glutamate perfusion (-39.0 +/- 2.48 mV in control and -52.0 +/- 2.72 mV with L-glutamate) and returned to control values after perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of glucose on the kinetics of bicarbonate reabsorption in the early and middle proximal tubule.

1. The kinetics of bicarbonate reabsorption in the early (EPT) and middle proximal tubule (MPT) was studied in Munich Wistar rats by means of microperfusion techniques. 2. The early proximal tubule (EPT) had a higher capacity of reabsorbing bicarbonate than the middle segment. The calculated bicarbonate flow (JHCO3) was 4.47 nmol s-1 cm-2 in the EPT and 2.30 nmol s-1 cm-2 in the MPT, and the half time of injected bicarbonate (t/2) was less in the EPT (2.24 s) than in the MPT (5.20 s). 3. The presence of 20 mM glucose in the lumen led to a reduction in the velocity of tubular acidification in the EPT (3.11 s) and caused a decrease in JHCO3 in both segments (2.46 nmol s-1 cm-2 in the EPT and 1.64 nmol s-1 cm-2 in the MPT). 4. These alterations may be the result either of lumen-to-cell sodium gradient dissipation or electrical changes induced by sodium-glucose cotransport that may depolarize the basolateral membrane leading to intracellular alkalinization. This effect may then impair the Na+/H+ exchanger, thus decreasing bicarbonate reabsorption.

Impairment of the renal tubular acidification kinetics by lithium.

We studied the kinetics of HCO3- reabsorption in the middle proximal (MPT) and distal convoluted tubules (DCT) by measuring continuously intratubular pH with Sb-microelectrodes in stopped-flow microperfusion (HCO3-, 30 mM Ringer) experiments. Male Wistar rats (240-280 g) were injected ip with LiCl (4 mEq kg-1 day-1) for 4 days (Li) and were compared to controls (C). Steady-state pH increased in MPT from 6.64 +/- 0.02 (57) to 6.89 +/- 0.02 (45), mean +/- SEM (number of measurements) on tissue from 13 rats in each group, and from 6.87 +/- 0.05 (30) to 7.08 +/- 0.01 (63) in DCT. HCO3- reabsorption decreased from 1.32 +/- 0.08 (57) to 0.96 +/- 0.04 (45) nmol cm-2 s-1 in MPT and from 0.85 +/- 0.07 (30) to 0.45 +/- 0.04 (63) in DCT. These data indicate that lithium affected the acidification mechanism in MPT and DCT, probably through an impairment of the Na(+)-H+ antiport in both tubular segments.

Proximal tubular acidification in metabolic alkalosis.

Metabolic alkalosis was induced in rats by acute bicarbonate loading in the presence and absence of extracellular volume (ECV) expansion. Proximal tubular acidification was studied by stopped-flow microperfusion and the determination of luminal pH by antimony microelectrodes. Stationary pH and bicarbonate were markedly increased in proximal tubules of alkalotic rats, and acidification half-times were increased, leading to a fall in net bicarbonate reabsorption (JHCO-3) to 36% of control values. Net H+ ion secretion (JH+) into phosphate buffer fell to 56% of controls, whereas alkalinization t/2 and H+ fluxes, measured during perfusion with acid phosphate, were unaltered. ECV expansion reduced proximal JHCO-3 36% in control rats, without affecting JH+, and caused a fall in alkalinization t/2, compatible with increased bicarbonate back-flux into the lumen. However, it did not affect JHCO3 and JH+ in alkalotic rats. Carbonic anhydrase inhibition did not significantly reduce acidification in alkalotic rats. An analysis of the components of bicarbonate reabsorption showed that alkalosis reduced catalyzed H+ -ion secretion to 7% of the control values. It is concluded that proximal H+ ion secretion is significantly reduced in metabolic alkalosis leading to lower JHCO-3. No evidence for modification of H+/HCO-3 apparent permeabilities was obtained. The effects of alkalosis were not significantly altered by ECV expansion or by carbonic anhydrase inhibition.

Measurement of glomerular filtration rate using inulin prepared from Vernonia herbacea, a Brazilian native species.

Vernonia herbacea (Vell.) Rusby (Asteraceae) is a perennial herb native to the cerrado vegetation of tropical areas in Brazil, which accumulates inulin in the underground reserve organs. The aim of this paper was to determine whether the inulin extracted from V. herbacea could replace commercial inulin for the measurement of glomerular filtration rate (GFR). Underground organs of vegetative plants were collected from a preserved area of the Brazilian cerrado. The inulin fraction utilized was obtained by ethanol precipitation after discarding the high molecular mass fructans in the freeze-thawing precipitate. GFR was determined in male Wistar rats anesthetized with inactin (100 mg/kg), which received intravenously commercial inulin obtained from Dahlia sp (Sigma) or Vernonia herbacea inulin (30 mg/100 g) as a priming dose and 0.05 mg min-1 100 g-1 as a sustaining dose in isotonic saline at the rate of 0/055 ml/min. Clearance was determined during 3 periods, with urine collected from the bladder and blood from the carotid artery. There was no significant difference in the GFR measured by clearance in inulin from both sources even when the plasma concentration of inulin from V. herbacea was doubled. The mean arterial pressure did not vary after the application of both inulins, indicating that they do not produce systemic side effects. The filtered load and the excreted amount of inulin from V. herbacea were equal, showing that the substance is not influenced by tubular function. These results demonstrate that the inulin from V. herbacea can substitute for imported inulin for the determination of GFR and in experiments of kidney microperfusion as a marker of tubular water reabsorption.

Interactions of ANP and ANG II in tubular nephron acidification.

In order to examine the effects and the interaction of angiotensin II (ANG II, 1 pM) and atrial natriuretic peptide (ANP, 1 microM) on the kinetics of bicarbonate reabsorption in the rat middle proximal tubule, we performed in vivo experiments using a stopped-flow microperfusion technique with the determination of lumen pH by Sb microelectrodes. These studies confirmed that ANG II added to the luminal or peritubular capillary perfusion fluid stimulates proximal bicarbonate reabsorption and showed that ANP alone does not affect this process, but impairs the stimulation caused by ANG II. We also studied the effects and the interaction of these hormones in cortical distal nephron acidification. Bicarbonate reabsorption was evaluated by the acidification kinetic technique in early (ED) and late (LD) distal tubules in rats during in vivo stopped-flow microperfusion experiments. The intratubular pH was measured with a double-barreled microelectrode with H(+)-sensitive resin. The results indicate that ANG II acted by stimulating Na+/H+ exchange in ED (81%) and LD (54%) segments via activation of AT1 receptors, as well as vacuolar H(+)-ATPase in LD segments (33%). ANP did not affect bicarbonate reabsorption in either segment and, as opposed to what was seen in the proximal tubule, did not impair the stimulation caused by ANG II. To investigate the mechanism of action of these hormones in more detail, we studied cell pH dependence on ANG II and ANP in MDCK cells using the fluorescent probe BCECF. We showed that the velocity of cell pH recovery was almost abolished in the absence of Na+, indicating that it is dependent on Na+/H+ exchange. ANP (1 microM) alone had no effect on this recovery but reversed both the acceleration of H+ extrusion at low ANG II levels (1 pM and 1 nM), and inhibition of H+ extrusion at higher ANG II levels (100 nM). To obtain more information on the mechanism of interaction of these hormones, we also studied their effects on the regulation of intracellular free calcium concentration, [Ca2+]i, monitored with the fluorescent probe Fura-2 in MDCK cells in suspension. The data indicate that the addition of increasing concentrations of ANG II (1 pM to 1 microM) to the cell suspension led to a progressive increase in [Ca2+]i to 2-3 times the basal level. In contrast, the addition of ANP (1 microM) to the cell suspension led to a very rapid 60% decrease in [Ca2+]i and reduced the increase elicited by ANG II, thus modulating the effect of ANG II on [Ca2+]i. These results may indicate a role of [Ca2+]i in the regulation of the H+ extrusion process mediated by Na+/H+ exchange and stimulated/impaired by ANG II. The data are compatible with stimulation of Na+/H+ exchange by increases of [Ca2+]i in the lower range, and inhibition at high [Ca2+]i levels.

Atrial natriuretic peptide modulates the effect of angiotensin II on the concentration of free calcium in the cytosol of Mandin-Darby canine kidney cells.

The effect of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular free calcium concentration [Ca2+]i was investigated in Mandin-Darby canine kidney (MDCK) cells in culture. Changes in [Ca2+]i were monitored fluorometrically with the Ca(2+)-sensitive probe fura-2/AM at 37 degrees C using a Perkin-Elmer LS-5 spectrofluorimeter (excitation 340/380 nm, slit 3 nm; emission 520 nm, slit 10 nm). MDCK cells exhibited a mean baseline [Ca2+]i of 98 +/- 10 nM. The addition of increasing concentrations of ANG II (1 pM to 1 microM) to the cell suspension led to a progressive increase in [Ca2+]i to 2-3 times basal levels. In contrast, addition of 1 microM ANP to the cell suspension led to a very rapid 60% decrease in [Ca2+]i. The addition of 1 pM to 1 microM ANG II immediately after 1 microM ANP caused an increase in [Ca2+]i which never exceeded the basal level in the absence of ANP. The data indicate that ANG II increases cell [Ca2+]i, as expected, and provide the new observation that ANP reduces [Ca2+]i in these cells. Furthermore, ANP reduces the increase in [Ca2+]i elicited by ANG II, thus modulating the effect of ANG II on [Ca2+]i.

Action of ANP on the nongenomic dose-dependent biphasic effect of aldosterone on NHE1 in proximal S3 segment.

The rapid (2 min) nongenomic effects of aldosterone (ALDO) and/or spironolactone (MR antagonist), RU 486 (GR antagonist), atrial natriuretic peptide (ANP) and dimethyl-BAPTA (BAPTA) on the intracellular pH recovery rate (pHirr) via NHE1 (basolateral Na⁺/H⁺ exchanger isoform), after the acid load induced by NH4Cl, and on the cytosolic free calcium concentration ([Ca²âº](i)) were investigated in the proximal S3 segment isolated from rats, by the probes BCECF-AM and FLUO-4-AM, respectively. The basal pHi was 7.15±0.008 and the basal pHirr was 0.195±0.012 pH units/min (number of tubules/number of tubular areas=16/96). Our results confirmed the rapid biphasic effect of ALDO on NHE1: ALDO (10⁻¹² M) increases the pHirr to approximately 59% of control value, and ALDO (10⁻6 M) decreases it to approximately 49%. Spironolactone did not change these effects, but RU 486 inhibited the stimulatory effect and maintained the inhibitory effect. ANP (10⁻6 M) or BAPTA (5×10⁻5 M) alone had no significant effect on NHE1 but prevented both effects of ALDO on this exchanger. The basal [Ca²âº](i) was 104±3 nM (15), and ALDO (10⁻¹² or 10⁻6 M) increased the basal [Ca²âº](i) to approximately 50% or 124%, respectively. RU 486, ANP and BAPTA decreased the [Ca²âº](i) and inhibited the stimulatory effect of both doses of ALDO. The results suggest the involvement of GR on the nongenomic effects of ALDO and indicate a pHirr-regulating role for [Ca²âº](i) that is mediated by NHE1, stimulated/impaired by ALDO, and affected by ANP or BAPTA with ALDO. The observed nongenomic hormonal interaction in the S3 segment may represent a rapid and physiologically relevant regulatory mechanism in the intact animal under conditions of volume alterations.

Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na+/H+ exchanger in proximal S3 segment: role of cytosolic calcium.

The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na+/H+ exchanger and on the [Ca2+]i were investigated in isolated rat S3 segment. Aldosterone [10(-12), 10(-10), or 10(-8) M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10(-6) M with 1-h, 15- or 2-min pi) decreased it (these effects were prevented by HOE694 but not by S3226). After 1 min of aldosterone pi, there was a transient and dose-dependent increase of the [Ca2+]i and after 6-min pi there was a new increase of [Ca2+]i that persisted after 1 h. Spironolactone, actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min pi) but inhibited the effects of aldosterone (1-h pi) on pHirr and on [Ca2+]i. RU 486 prevented the stimulatory effect of aldosterone (10(-12) M, 15- or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10(-6) M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca2+]i to an inhibitory effect. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on [Ca2+]i and on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca2+]i in the lower range (at 10(-12) M aldosterone) and inhibition by increases at high levels (at 10(-6) M aldosterone) or decreases in [Ca2+]i (at 10(-6) M aldosterone plus RU 486).

Signaling pathways in the biphasic effect of ANG II on Na+/H+ exchanger in T84 cells.

The effect of ANG II on pH(i), [Ca(2+)](i) and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH(i) via the Na(+)/H(+) exchanger was examined in the first 2 min following the acidification of pH(i) with a NH(4)Cl pulse. In the control situation, the pH(i) recovery rate was 0.118 +/- 0.001 (n = 52) pH units/min and ANG II (10(-12) M or 10(-9) M) increased this value (by 106% or 32%, respectively) but ANG II (10(-7) M) decreased it to 47%. The control [Ca(2+)](i) was 99 +/- 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH(i) recovery and [Ca(2+)](i). To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na(+)/H(+) exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10(-12) - 10(-7) M ANG II) and by increases of [Ca(2+)](i) in the lower range (at 10(-12) M ANG II) and 2) inhibition of the exchanger at high [Ca(2+)](i) levels (at 10(-9) - 10(-7) M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.

Effect of arginine vasopressin and ANP on intracellular pH and cytosolic free [Ca2+] regulation in MDCK cells.

BACKGROUND: The effects of arginine vasopressin (AVP) on intracellular pH (pHi) are not clearly defined, and may vary with cell membrane surface and the hormonal doses being studied. Since cytosolic free calcium concentration ([Ca2+]i) has an important effect on cellular H+ extrusion and it was shown that AVP increases [Ca2+]i while atrial natriuretic peptide (ANP) reduces it, there may be some interaction between AVP and ANP during the regulation of pHi. METHODS: The effects of AVP and/or ANP on pHi and [Ca2+]i were investigated in Madin-Darby canine kidney (MDCK) cells by the fluorescent probes BCECF-AM and Fluo 4-AM, respectively. The pHi recovery rate was examined in the first two minutes following the acidification of pHi with a NH4Cl pulse. RESULTS: AVP (10(-12) or 10(-9) mol/L) stimulated the rate of the Na+-dependent pHi recovery, but AVP (10(-6) mol/L) impaired it. At the apical membrane surface, specific V1 or V2 receptor antagonists did not alter the effects of AVP. At the basolateral membrane surface, the V1 antagonist returned both the stimulatory and inhibitory effects of AVP to control levels, and the V2 antagonist converted the inhibitory effect of AVP to a stimulatory effect. ANP (10(-6) mol/L) or dimethyl-BAPTA-AM (50 micromol/L) impaired both the stimulatory and inhibitory effects of AVP. AVP increased [Ca2+]i in a dose-dependent manner. ANP or dimethyl-BAPTA-AM decreased [Ca2+]i, and the subsequent addition of AVP caused only a partial recovery of [Ca2+]i. CONCLUSIONS: The results are compatible with stimulation of the Na+/H+ exchanger by increases of [Ca2+]i in the lower range (at 10(-12) or 10(-9) mol/L AVP, via basolateral V1 receptors) and inhibition at high [Ca2+]i levels (at 10(-6) mol/L AVP, via basolateral V1 and V2 receptors). ANP, by impairing the path causing the increase in [Ca2+]i, blocks both the stimulatory and inhibitory effects of AVP on Na+-dependent pHi recovery.

Peritubular pH and PCO'2 in renal tubular acidification.

The influence of peritubular capillary pH and PCO'2 on renal tubular acidification was studied in rats by luminal and peritubular perfusion techniques. Luminal stopped-flow microperfusions were carried out with bicarbonate or alkaline phosphate solutions and luminal pH continuously measured by antimony micorelectrodes. Peritubular calpillary microperfusions were carried out with mammalian Ringer solution kept at different pH and PCO'2. The acidification process was assessed in terms of 1)maximal pH differences, 2)rates of pH change, and 3)rates of bicarbinate reabsorption or H'+ ion secretion. During peritubular perfusions at physiological pH and PCO'2 tubular acidifying capacity was maintained at near-normal levels. Perfusingcapillaries at high pH and low PCO'2, especially with bicarbonate Ringer, acidification was markedly depressed; it was moderately reduced at a peritubular pH of 5.6. At a capillary pH of 7.4, acidification was similiar at low and physiological PCO'2and enhanced at elevated PCO'2. Systemic respiratory acidosis enhanced acidification in the proximal tubule, but reduced it in distal segments.

Sodium in renal tubular acidification kinetics.

Renal proximal tubules and their peritubular capillaries were perfused with mammalian Ringer solutions containing different sodium concentrations. In stop-flow microperfusion experiments, the pH was measured by means of antimony microelectrodes, permitting calculation of rates of H ion secretion and bicarbonate reabsorption. These rates, as well as transepithelial pH and bicarbonate gradients, were significantly reduced at ambient concentrations of 20 and 4 meq/liter Na+. However, even at the lowest sodium concentrations (4 meq/liter), H ion secretion was still 74%, and bicarbonate reabsorption of 64% of control rates. In similar conditions, sodium reabsorption as measured by the split-droplet technique fell to practically zero. Ouabain, 10(-3) M, in capillaries reduced bicarbonate reabsorption by 31%, and 3 X 10(-4) M furosemide in lumen and capillaries reduced acidification by 29%. At pH 8--9 in capillaries, sodium transport was normal while acidification was markedly reduced. These data show that low sodium levels impair renal tubular acidification, but they do not support a rigid coupling of these transport processes.

Renal acidification defect induced by lithium in control and acidotic rats.

1. The aim of this work was to contribute to a better understanding of the mechanism by which Li+ administration impairs renal tubular acidification. 2. The kinetics of tubular acidification in the mid-proximal and distal convoluted tubules were studied by measuring intratubular pH using Sb microelectrodes during stopped-flow microperfusion with Krebs-Ringer bicarbonate (30 mmol/l) buffer. Four groups of male Wistar rats were utilized in this study: control, control plus lithium (C + Li+; one intraperitoneal injection of LiCl of 4 mmol l-1 day-1 kg-1 for 4 days), acidotic and acidotic plus lithium (A + Li+). 3. In C + Li+ rats, the half-time of acidification was significantly higher than in control rats (P less than 0.01), in both the mid-proximal tubule (11.3 +/- 0.34 vs 6.73 +/- 0.22 s) and the distal convoluted tubule (17.5 +/- 0.31 vs 11.5 +/- 1.02 s), and net HCO3- reabsorption was lower in both the mid-proximal tubule and the distal convoluted tubule. The effects of Li+ on tubular acidification kinetics were similar in acidotic rats. 4. A net Na+ flux, as measured by the Gertz split-droplet method, was significantly decreased in the mid-proximal tubule (P less than 0.01) in C + Li+ rats compared with control rats (2.14 +/- 0.17 vs 4.07 +/- 0.39 nmol s-1 cm-2). 5. The transepithelial potential difference in the distal convoluted tubule was significantly lower (P less than 0.01) in C + Li+ rats than in control rats (-7.50 +/- 1.50 vs -20.5 +/- 1.12 mV).(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of the renal handling of H+ in diabetic rats.

Renal acid excretion and proximal and distal nephron acidification were evaluated 20 days after induction of diabetes, in rats, by intraperitoneal injection of streptozotocin (45 mg/kg). Titratable acidity in urine was measured by microtitration and ammonium excretion (NH4+) by spectrophotometry. Proximal tubular acidification was evaluated by the kinetics of reabsorption of perfused HCO3-. Distal nephron acidification was evaluated by measuring urine - blood pCO2 differences under alkaline overload. The net acid excretion (titratable acidity + NH4+ - HCO3-) was higher (p < 0.001) in diabetic rats (9.82+/-0.65 micromol/min/kg, n = 26) than in the control group (6.34+/-0.14, n = 24). Proximal HCO3- reabsorption was also higher (p < 0.001) in diabetic rats (8.38+/-0.11 nmol/cm2/s, n = 12) than in the control group (2.30+/-0.10, n = 22); however, evaluation of distal nephron H+ secretion by urine - blood pCO2 methodology was similar in both groups. We concluded that in rats with induced diabetes mellitus there is an increased rate of proximal HCO3- reabsorption, possibly effected by a higher density of Na+/H+ antiporter in the luminal membrane of the proximal tubule and by an increased proton-motive force of the H+ secretory mechanism. The higher rates of H+ secretion generate lower stationary proximal luminal pH and probably maintain the blood pH within the physiological range.

Effect of luminal angiotensin II and ANP on early and late cortical distal tubule HCO3- reabsorption.

Bicarbonate reabsorption was evaluated by stationary microperfusion "in vivo" early distal (ED) and late distal (LD) segments of at kidney. Intratubular pH was recorded by double-barreled of H+ exchange resin/reference (1 M KCl) microelectrodes for the determination of HCO3- reabsorption. In the presence of angiotensin II (ANG II) (10(-12) M), a significant increase in HCO3- reabsorption was observed both in ED (from 0.930 +/- 0.060 to 2.64 +/- 0.210 nmol.cm-2.s-1 in luminally perfused tubules and from 0.850 +/- 0.040 to 2.03 +/- 0.210 nmol.cm-2.s-1 during capillary perfusion) and LD segments from 0.310 +/- 0.130 to 2.16 +/- 0.151 nmol.cm-2.s-1 during luminal perfusion and from 0.530 +/- 0.031 to 2.16 +/- 0.211 nmol.cm-2.s-1 with capillary perfusion). The addition of the AT1-receptor antagonist losartan (10(-6) M) to luminal perfusion blocked luminal ANG II-mediated stimulation in ED and LD segments. 5-(N,N-hexamethylene)amiloride (10(-4) M) added to luminal perfusion inhibited luminal ANG II-mediated stimulation in ED (by 81%) and LD (by 54%) segments. The addition of bafilomycin A1 (2 x 10(-7) M) to luminal perfusion does not affect luminal ANG II-mediated stimulation in ED segments but reduces it in LD segments (by 33%). During the addition of atrial natriuretic peptide (ANP) (10(-6) M) or ANG II plus ANP in both segments, no significant differences in HCO3- reabsorption were observed. Our results indicate that luminal ANG II acts to stimulate Na+/H+ exchange in ED and LD segments via activation of AT1 receptors, as well as the vacuolar H(+)-adenosinetriphosphatase in LD segments. ANP does not affect HCO3- reabsorption in either ED or LD segments and does not impair the stimulation caused by ANG II.