Vacuolar H+-ATPase is a large multi-subunit protein that mediates ATP-driven vectorial H+ transport across the membranes. It is widely distributed and present in virtually all eukaryotic cells in intracellular membranes or in the plasma membrane of specialized cells. In subcellular organelles, ATPase is responsible for the acidification of the vesicular interior, which requires an intraorganellar acidic pH to maintain optimal enzyme activity. Control of vacuolar H+-ATPase depends on the potential difference across the membrane in which the proton ATPase is inserted. Since the transport performed by H+-ATPase is electrogenic, translocation of H+-ions across the membranes by the pump creates a lumen-positive voltage in the absence of a neutralizing current, generating an electrochemical potential gradient that limits the activity of H+-ATPase. In many intracellular organelles and cell plasma membranes, this potential difference established by the ATPase gradient is normally dissipated by a parallel and passive Cl- movement, which provides an electric shunt compensating for the positive charge transferred by the pump. The underlying mechanisms for the differences in the requirement for chloride by different tissues have not yet been adequately identified, and there is still some controversy as to the molecular identity of the associated Cl--conducting proteins. Several candidates have been identified: the ClC family members, which may or may not mediate nCl-/H+ exchange, and the cystic fibrosis transmembrane conductance regulator. In this review, we discuss some tissues where the association between H+-ATPase and chloride channels has been demonstrated and plays a relevant physiologic role.

The interaction between H+ extrusion via H+-ATPase and Cl- conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH4Cl acid pulse was 0.136 ± 0.008 pH units/min (dpHi/dt) in Na+ Ringer and 0.032 ± 0.003 in the absence of Na+ (0 Na+). With 0 Na+ plus the Cl- channel inhibitor NPPB (10 µM), recovery was reduced to 0.014 ± 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl- channels, increased dpHi/dt in 0 Na+ to 0.061 ± 0.009 and also in the presence of 46 nM concanamycin and 50 µM Schering 28080. Since it is thought that the Cl- dependence of H+-ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 µM valinomycin at high (100 mM) K+ was tested in our cells. In Na+ Ringer, dpHi/dt was increased, but no effect was detected in 0 Na+ Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl- channels on dpHi/dt was not due to an adverse electrical gradient. The effect of 100 µM ATP was studied in 0 Na+ Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 µM Bapta. We have shown that H+-ATPase present in MDCK C11 cells depends on Cl- ions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H+ transport.

In the present study we determined the effect of chronic diet supplementation with n-3 PUFA on renal function of healthy and cachectic subjects by providing fish oil (1 g/kg body weight) to female rats throughout pregnancy and lactation and then to their offspring post-weaning and examined its effect on renal function parameters during their adulthood. The animals were divided into four groups of 5-10 rats in each group: control, control supplemented with fish oil (P), cachectic Walker 256 tumor-bearing (W), and W supplemented with fish oil (WP). Food intake was significantly lower in the W group compared to control (12.66 ± 4.24 vs 25.30 ± 1.07 g/day). Treatment with fish oil significantly reversed this reduction (22.70 ± 2.94 g/day). Tumor growth rate was markedly reduced in the P group (16.41 ± 2.09 for WP vs 24.06 ± 2.64 g for W). WP group showed a significant increase in mean glomerular filtration rate compared to P and control (1.520 ± 0.214 ml min-1 kg body weight-1; P < 0.05). Tumor-bearing groups had low urine osmolality compared to control rats. The fractional sodium excretion decreased in the W group compared to control (0.43 ± 0.16 vs 2.99 ± 0.87%; P < 0.05), and partially recovered in the WP group (0.90 ± 0.20%). In summary, the chronic supplementation with fish oil used in this study increased the amount of fat in the diet by only 0.1%, but caused remarkable changes in tumor growth rate and cachexia, also showing a renoprotective function.

The present paper reviews work from our laboratories evaluating the importance of adrenal cortical hormones in acidification by proximal and cortical distal tubules. Proximal acidification was determined by stationary microperfusion, and measurement of bicarbonate reabsorption using luminal pH determination was performed with H+-ion-sensitive microelectrodes. Rats were adrenalectomized (ADX) 48 h before the experiments, and corticosteroids (aldosterone (A), corticosterone (B), and 18-OH corticosterone (18-OH-B)) were injected intramuscularly 100 and 40 min before the experiments. In ADX rats stationary pH increased significantly to 7.03 as compared to sham-operated rats (6.78). Bicarbonate reabsorption decreased from 2.65 &plusmn; 0.18 in sham-operated rats to 0.50 &plusmn; 0.07 nmol cm-2 s-1 after ADX. The administration of the three hormones stimulated proximal tubule acidification, reaching, however, only 47.2% of the sham values in aldosterone-treated rats. Distal nephron acidification was studied by measuring urine minus blood pCO2 differences (U-B pCO2) in bicarbonate-loaded rats treated as above. This pCO2 difference is used as a measure of the distal nephron ability to secrete H+ ions into an alkaline urine. U-B pCO2 decreased significantly from 39.9 &plusmn; 1.26 to 11.9 &plusmn; 1.99 mmHg in ADX rats. When corticosteroids were given to ADX rats before the experiment, U-B pCO2 increased significantly, but reached control levels only when aldosterone (two 3-&micro;g doses per rat) plus corticosterone (220 &micro;g) were given together. In order to control for the effect of aldosterone on distal transepithelial potential difference one group of rats was treated with amiloride, which blocks distal sodium channels. Amiloride-treated rats still showed a significant reduction in U-B pCO2 after ADX. Only corticosterone and 18-OH-B but not aldosterone increased U-B pCO2 back to the levels of sham-operated rats. These results show that corticosteroids stimulate renal tubule acidification both in proximal and distal nephrons and provide some clues about the mechanism of action of these steroids