Interaction of angiotensin II and atrial natriuretic peptide on pH(i) regulation in MDCK cells.

The effect of ANG II and atrial natriuretic peptide (ANP) on intracellular pH (pH(i)) and cytosolic free calcium concentration ([Ca(2+)](i)) was investigated in Madin-Darby canine kidney cells by using the fluorescent probes 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (AM) and fura 2-AM or fluo 4-AM. pH(i) recovery rate was examined in the first 2 min after the acidification of pH(i) with a NH(4)Cl pulse. In the control situation, the pH(i) recovery rate was 0.088 +/- 0.014 pH units/min (n = 14); in the absence of external Na(+), this value was decreased. ANG II (10(-12) or 10(-9) M) caused an increase in this value, but ANG II (10(-7) M) decreased it. ANP (10(-6) M) or dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA)-AM (50 microM) alone did not affect this value but impaired both stimulatory and inhibitory effects of ANG II. ANG II (10(-12), 10(-9), or 10(-7) M) increased [Ca(2+)](i) progressively from 99 +/- 10 (n = 20) to 234 +/- 7 mM (n = 10). ANP or dimethyl-BAPTA-AM decreases [Ca(2+)](i), and the subsequent addition of ANG II caused a recovery of [Ca(2+)](i) but without reaching ANG II values found in the absence of these agents. The results indicate a role for [Ca(2+)](i) in regulating the process of pH(i) recovery mediated by the Na(+)/H(+) exchanger, stimulated/impaired by ANG II, and not affected by ANP or ANG II plus ANP. This hormonal interaction may represent physiologically relevant regulation in conditions of volume alterations in the intact animal.

Luminal arginine vasopressin stimulates Na(+)-H+ exchange and H(+)-ATPase in cortical distal tubule via V1 receptor.

Bicarbonate reabsorption was evaluated by stationary microperfusion of in vivo early distal (ED) and late distal (LD) segments of rat kidney. Intratubular pH was recorded by double-barreled H ion-exchange resin/reference (1 M KCl) microelectrodes for the determination of HCO3- reabsorption. In the presence of luminal arginine vasopressin (AVP, 10(-9) M), a significant increase in HCO3- reabsorption was observed both in ED (from 0.931 +/- 0.061 to 2.12 +/- 0.171 nmol.cm-2.s-1] and LD segments [from 0.542 +/- 0.086 to 1.67 +/- 0.111 nmol.cm-2.s-1]. The addition of the V1-receptor antagonist [(d (CH2)5, Tyr (Et)2) arginine vasopressin] (10(-5) M) to luminal perfusion blocked luminal AVP mediated stimulation in ED and LD segments. 5-(N, N-hexamethylene) amiloride (10(-4) M) added to luminal perfusion inhibited luminal AVP-mediated stimulation in ED (by 63.7%) and LD (by 34.1%) segments. The addition of Bafilomycin A1 (2 x 10(-7) M) to the luminal perfusion did not affect luminal AVP-mediated stimulation in ED segments, but reduced it (by 31.7%) in LD segments. Our results indicate that luminal AVP acts to stimulate the Na(+)-H+ exchange in ED and LD segments via activation of V1 receptors, as well as the vacuolar H(+)-ATPase in LD segments.

PCO2 in renal cortex.

In a number of recent investigations a renal cortical PCO2 higher than that of systemic blood was reported. We have studied this problem with the use of micro-Severinghaus electrodes based on antimony, H+ liquid ion exchange, and glass pH electrodes with an inner buffer solution containing 0.5 mg/ml carbonic anhydrase (CA). Measurements in renal cortical structures (renal tubules, star vessels, capillaries, and glomeruli in Munich-Wistar rats) were compared with determinations in renal vein or artery performed with the same electrode in sequence. No significant differences in PCO2 were found between cortical structures and renal vein in control rats, in metabolic alkalosis, respiratory acidosis and alkalosis, and after CA inhibition. Nevertheless, absolute PCO2 levels, which followed the PCO2 of systemic blood, were markedly different in these groups. Measurements of pH and PCO2 at the same tubule site were compatible with HCO3- determinations in tubule fluid in vitro (made with use of the Henderson-Hasselbalch equation) in control rats. When proximal tubules were pump-perfused in vivo with a solution containing 30 mM NaHCO3, measured PCO2 approached that of the perfusing solution at high pump rates, and approached the free-flow value as rates were reduced to zero, indicating that the CO2 generated in the lumen equilibrated rapidly across the epithelium. Reducing renal blood flow by aortic clamping reduced renal cortical PCO2. In conclusion, in a large number of experimental conditions renal cortical PCO2 was never higher than that measured in systemic blood.

Filtered load of buffer and renal H-ion secretion: mechanism of proximal tubule load dependence.

When the filtered load of buffers like bicarbonate or phosphate is increased by elevating GFR or buffer concentration in plasma, the overall renal reabsorption of bicarbonate or the formation of titratable acidity are markedly increased. The same happens when buffer concentration or flow rate are varied during proximal microperfusion. We have recently studied the mechanisms of this functional dependence. We have observed that the rate of bicarbonate reabsorption is always proportional to luminal buffer concentration when a stationary fluid column is injected into the proximal lumen. H-ion secretion is also proportional to luminal levels of non-bicarbonate buffers. Using a pH-stat technique adapted to renal tubules, we have shown that H-ion secretion is dependent on proximal pH independently of the used buffer species. A kinetic analysis of these data shows a non-linear relationship between luminal H+ and H+ secretion, compatible with carrier mediated transport.

Filtered load of buffer and renal H-ion secretion: mechanism of proximal tubule load dependence.

When the filtered load of buffers like bicarbonate or phosphate is increased by elevating GFR or buffer concentration in plasma, the overall renal reabsorption of bicarbonate or the formation of titratable acidity are markedly increased. The same happens when buffer concentration or flow rate are varied during proximal microperfusion. We have recently studied the mechanisms of this functional dependence. We have observed that the rate of bicarbonate reabsorption is always proportional to luminal buffer concentration when a stationary fluid column is injected into the proximal lumen. H-ion secretion is also proportional to luminal levels of non-bicarbonate buffers. Using a pH-stat technique adapted to renal tubules, we have shown that H-ion secretion is dependent on proximal pH independently of the used buffer species. A kinetic analysis of these data shows a non-linear relationship between luminal H+ and H+ secretion, compatible with carrier mediated transport.

Verapamil effect on renal function of normotensive and hypertensive rats.

The effect of verapamil, a Ca++ antagonist drug, on renal function and proximal fluid reabsorption in normal and hypertensive (GII) rats was studied. During intravenous infusion of verapamil, mean arterial pressure (MAP) fell significantly in both groups, 23% more in hypertensive than in normotensive rats. Glomerular filtration rate (GFR) was significantly higher in hypertensive rats and also increased significantly in this group during verapamil infusion. Effective renal plasma flow (ERPF) was similar in both groups and did not change significantly during verapamil infusion. The increase in urine flow, Na+ and Ca++ excretion was higher in hypertensive than in normotensive rats during verapamil infusion. When 10(-5) M verapamil was added to the luminal perfusate of proximal tubules, fluid reabsorption was reduced to 64% in normotensive and to 42% in hypertensive rats. When added to capillary perfusate, fluid reabsorption was almost completely but reversibly inhibited (92% in normotensive and 83% in hypertensive rats). Our findings indicate a direct effect of verapamil on renal Na+ and possibly also on Ca++ reabsorption, suggesting involvement of the Na+-Ca++ countertransport system. The greater effect of verapamil on Na+ excretion in hypertensive rats was not due to increased action on proximal Na+ reabsorption.

Proximal tubular HCO3-, H+ and fluid transport during maleate-induced acidification defect.

The mechanism of tubular acidification was studied in proximal tubular acidification defect induced in rats by acute parenteral infusion of maleate (200 mg/kg), which causes diuresis and bicarbonaturia. Proximal tubular bicarbonate reabsorption and H+ ion secretion were determined by stopped-flow microperfusion and measurement of luminal pH by Sb microelectrodes. Stationary pH increased in proximal tubule from 6.78 to 7.25 and bicarbonate reabsorption decreased from 1.32 to 0.51 nmol/cm2 X s. In these segments, mean cell PD fell from -66.6 to -20.2 mV, while Jv as estimated by the Gertz technique fell to 15% of controls. A similar impairment of acidification was observed during luminal and capillary perfusion with phosphate Ringer's. Since H+-ion efflux from the lumen was not significantly increased and both acidification and alkalinization half-times (t/2) were increased, no evidence for an increase in passive permeability for H+/HCO3- was obtained. The increased t/2 found during luminal perfusion with acid phosphate indicates, according to an electrical analog model, a reduction in pump series conductance. These results show that maleate affects both proximal Na+ and H+ transport; this effect may be ascribed to impairment of sodium-dependent transport systems in the brush-border membrane.

Role of luminal buffers in renal tubular acidification.

The acidification of kinetics of artificial solutions containing buffers of different permeancy were studied in rat proximal tubules by means of stationary microperfusion techniques. Luminal pH changes were measured by antimony microelectrodes and used to calculate net rates of acidification and the approach to steady-state pH levels. For most buffer species, tracer efflux out of the lumen was compared with changes in buffer concentration as derived from calculations based on the Henderson Hasselbalch equation. Steady-state luminal pH was similar for most buffer systems studied. However, secretory hydrogen ion fluxes into the lumen were significantly higher for permeant than for less permeant buffers. The most likely explanation is that permeant buffers behave as "open" systems maintaining constant low diffusible acid levels in the lumen, whereas impermeant buffers behave as "closed" systems in which non-ionized acid levels are maintained at higher levels. A behavior consistent with this thesis was directly demonstrated for glycodiazine and, to a lesser degree, for DMO. In contrast, phosphate and creatinine behave like buffers in a "closed" system. Characteristics of proximal tubular acidification, of buffer reabsorption, and the effect thereupon of carbonic anhydrase inhibitors are satisfactorily explained by an essential role of (1) hydrogen ion secretion, (2) pK differences, and (3) different permeance of the non-ionized buffer species. However, specific transport mechanisms may, in addition, also contribute to differences in transepithelial buffer movement.

Hypothalamic stimulation and electrolyte excretion: a micropuncture study.

The anterior part of the lateral hypothalamus was stimulated by injection of carbachol through a stereotaxically implanted cannula in the rat. Re-collection micropuncture experiments showed that this procedure, which leads to diuresis and natriuresis with only transient changes in glomerular filtration rate and renal plasma flow, reduced the TF/P inulin ratio along proximal and distal tubules without significant alteration of single nephron glomerular filtration rate in most experimental groups. Fractional proximal sodium reabsorption was significantly reduced from 0.54 +/- 0.02 to 0.34 +/- 0.05. Treatment with DOCA, vasopressin, and oxytocin caused natriuresis, but additional hypothalamic stimulation (HS) led to further reduction in TF/P inulin ratio and proximal fractional sodium reabsorption from 0.42 +/- 0.03 to 0.33 +/- 0.03. Fluid transport across proximal and distal epithelium was also studied by the split-droplet method. It was markedly reduced in both segments after HS. During hormone treatment only distal segments showed reduction of fluid transport by HS. These experiments indicate that HS caused inhibition of fluid transport in the proximal and distal tubule. This effect was only partly due to the liberation of neurohypophysial hormones, since during their administration an additional effect of HS was still observed.

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.

Kinetics of luminal acidification in cortical tubules of the rat kidney.

1. Some kinetic aspects of renal tubular acidification were studied in proximal and distal tubules of the rat kidney by combining stationary microperfusion methods and continuous measurements of luminal pH changes of phosphate or bicarbonate buffers by means of antimony electrodes. The analysis included the measurement of steady-state pH, steady-state buffer concentrations and acidification half-times. From these data, net rates of tubular bicarbonate reabsorption and of H ion secretion were obtained since it was shown that the rate of phosphate acidification provides a realistic estimate of H ion secretion. 2. Experiments were performed in control rats, in animals undergoing metabolic acidosis or alkalosis and in control and acidotic rats receiving the carbonic anydrase inhibitor Diamox. 3. In all experiments, the rates of tubular bicarbonate reabsorption and of phosphate acidification (H ion secretion) were proportional to luminal buffer levels. The changes of luminal acid concentrations followed first-order kinetics. 4. Steady-state transepithelial pH differences were reduced in metabolic alkalosis and after diamox but augmented during metabolic acidosis. 5. Acidification half-times were prolonged in metabolic acidosis and after Diamox but remained similar to control levels in metabolic alkalosis. 6. From the observation that both bicarbonate reabsorption and phosphate acidification are similarly affected by these experimental manoeuvres, it is concluded that H ion secretion plays a key role in both transport processes.

Kinetics of potassium transport across single distal tubules of rat kidney.

1. The transport of potassium across the distal tubular epithelium was studied in vivo in rats on a normal potassium intake and in rats in which distal tubular potassium secretion was either stimulated by potassium loading or the I.V. administration of a 5% sodium bicarbonate solution or in which potassium secretion was suppressed by dietary deprivation of potassium or sodium.2. (42)K was used to measure unidirectional fluxes across the luminal and peritubular cell membranes and to assess the magnitude of cellular potassium partaking in the transport process. This was accomplished by the simultaneous perfusion of the peritubular capillary network with (42)K-Ringer and of the distal tubular lumen with initially tracer-free solution. From the steady-state flux and the time course of tracer washout into the lumen after discontinuing the peritubular perfusion, unidirectional fluxes, rate coefficients of ion transfer and cellular transport pools could be measured.3. Transepithelial movement of potassium involves mixing with a variable cellular potassium transport pool. The latter is significantly elevated in conditions of enhanced distal tubular potassium secretion; cellular potassium labelling is reduced in conditions in which potassium secretion has been suppressed by potassium deprivation.4. Evidence is presented that changes in the peritubular transport pattern are primarily responsible for modifications of potassium translocation. Thus, stimulation of potassium secretion is associated with increased peritubular potassium uptake; a reduced potassium uptake across the peritubular cell membrane accounts for the fall in potassium secretion in potassium-depleted animals. Whereas passive entry of potassium across the peritubular membrane is augmented in potassium-loaded animals, the induction of metabolic alkalosis by the administration of 5% sodium bicarbonate stimulates active potassium uptake across the peritubular cell membrane. Sodium deprivation stimulates active reabsorptive transfer of potassium from the tubular lumen.