The acute effect of angiotensin-converting enzyme inhibition (ACEi) on proximal convoluted tubule (PCT) function is well documented. However, the effect of chronic treatment is less known. The aim of this work was to evaluate the effect of chronic ACEi on PCT acidification (JHCO3−). Rats received enalapril (10 mg·kg−1·day−1, added to the drinking water) during 3 mo. Micropuncture experiments were performed to measure the effect of chronic ACEi on JHCO3−. Nitric oxide (NO·) synthesis in kidney cortex homogenates was assessed by quantifying the conversion of [14C]-l-arginine to [14C]-l-citrulline. Western blot analysis was performed to determine the abundances of V-H+ATPase and NHE3 isoform of the Na+/H+ exchanger in proximal brush-border membrane vesicles (BBMV). Enalapril treatment induced a ∼50% increase in JHCO3−. Luminal perfusion with ethyl-isopropyl amiloride (EIPA) 10−4M or bafilomycin 10−6M decreased JHCO3− by ∼60% and ∼30%, respectively, in both control and enalapril-treated rats. The effect of EIPA and bafilomycin on absolute JHCO3− was larger in enalapril-treated than in control rats. Acute inhibition of NO· synthesis with NG-nitro-l-arginine methyl esther abolished the enalapril-induced increase in JHCO3−. Cortex homogenates from enalapril-treated rats displayed a 46% increase in nitric oxide synthase (NOS) activity compared with those from untreated animals. Enalapril treatment did not affect the abundances of NHE3 and V-H+ATPase in BBMV. Our results suggest that PCT acidification is increased during chronic ACEi probably due to an increase in NO· synthesis, which would stimulate Na+/H+ exchange and electrogenic proton transport.
- Na+/H+ exchange
- micropuncture experiments
- NO· synthase activity
blockade of the renin-angiotensin system (RAS) with angiotensin-converting enzyme inhibitors (ACEi) is one of the most important tools for the treatment of chronic renal failure, diabetic nephropathy, and a number of cardiovascular disorders, including hypertension.
The physiological responses observed upon ACE inhibition are the result of combined effects of ACEi acting through two major pathways. On the one hand, there is inhibition of ANG II production, which causes hemodynamic effects, as well as the suppression of the ANG II-dependent generation of growth-promoting cytokines, free oxygen radicals, and fibrosis mediators in tissues (37). On the other hand, there is an increase in the levels of bioactive peptides that, under normal conditions, would be substrates for ACE-mediated proteolysis (11, 12). Indeed, ACE inhibition has been reported to increase the plasmatic concentration of angiotensin 1-7, an ANG I derivative which promotes bradykinin (BK) synthesis and consequently, nitric oxide (NO·) -dependent vasodilatation (5, 35). Moreover, the renoprotective action of ACEi following 5/6 reduction of renal mass depends on BK synthesis since BK inhibition with HOE140 abolishes the effect (26). Furthermore, the cardioprotective effects of ACEi are significantly decreased in knockout mice lacking the endothelial NO· synthase isoform (23).
The acute effects of ACEi on renal function have been extensively studied. Certainly, acute ACE inhibition has been reported to decrease proximal tubular Na+ reabsorption (22). However, the actions of chronic pharmacological ACE inhibition are less known. The development of knockout mice selectively lacking ACE expression has contributed toward distinguishing the roles of systemically and locally produced ANG II (14, 19). Despite the great significance of this model, the chronic pharmacological inhibition is still a valuable tool because it allows evaluating the system in conditions that resemble those present in humans.
Approximately 70% of Na+ reabsorption in the kidneys takes place at the proximal convoluted tubule (PCT), and 70% of it depends on bicarbonate (HCO3−) reabsorption, that is, H+ secretion (20). PCT acidification is a highly regulated process that involves the activity of different systems. A large fraction of HCO3− reabsorption is mediated by apical membrane electroneutral Na+/H+ exchange, mostly through the NHE3 isoform (4, 42, 45). The remaining fraction (∼30%) occurs through apical H+ electrogenic extrusion mediated by the V-H+-ATPase (45).
ANG II, NO·, and BK, all of them affected by ACEi, are actively involved in the regulation of PCT function, including H+ secretion (1, 29, 32, 36, 44). The aim of the present work was to evaluate PCT acidification after chronic ACE inhibition.
Male Wistar rats with free access to food and tap water were used 3 mo after weaning as a control.
Enalapril (10 mg·kg−1 of body weight·day−1) was added to the drinking water, from weaning until the day when the kidneys were removed or the micropuncture experiments were performed, 3 mo after the start of the treatment. The concentration of enalapril in the water was adjusted every 3 days, according to the volume of ingested water and the body weight.
Rats were weighted and placed in metabolic cages with free access to preweighted food containing 0.3% Na and tap water during 24 h. Systolic blood pressure was measured by plesthymography using a pneumatic pulse transducer (Narco Bio Systems, Houston, TX). Urine sodium concentration was measured with a Na+-sensitive electrode autoanalizer (Roche/Hitachi 917).
The rats were anesthetized with Inactin (100 mg/kg ip) and prepared for micropuncture experiments as previously described (2). Briefly, the right jugular vein was cannulated to administer anesthesia and fluids, a cannula was placed in the trachea, and the left kidney was exposed through a flank incision. The right femoral artery was also cannulated to monitor blood pressure using a TSD104 blood pressure transducer interfaced to a DA100 differential amplifier (Biopac Systems, Goleta, CA) and to collect blood samples for pH and CO2 determination. Proximal convoluted tubules were impaled with double-barreled micropipettes, one barrel containing Sudan black-colored castor oil and the other the luminal perfusion solution. The oil column injected in the tubule lumen was split by a droplet of perfusion solution and a single-barreled pH-sensitive microelectrode, positioned two to three loops downstream, was used to continuously measure the pH of the droplet. The luminal perfusate contained (in mM): 75 NaCl, 4 KCl, 1 CaCl2, 1 H2KPO4, 1.2 MgSO4, 25 NaHCO3, 10 glucose, and 90 raffinose. To prevent shrinkage of the droplet placed in the tubular lumen because of the constant Na+ reabsorption, we replaced part of the luminal Na+ with 90 mM raffinose, an osmotically active solute that cannot be reabsorbed. In this way, the total volume of the injected droplet does not change with time. The solution was bubbled with 5% CO2-95% O2. Final pH of the luminal solution was adjusted to 7.4 and the osmolality, measured with a vapor pressure osmometer (model 5100C, Wescor, Logan, UT), was 290 mOsm × kg−1.
Liquid membrane pH-sensitive microelectrodes were made as previously described (3) using H+ ionophore, Cocktail A purchased from Fluka (Ronkonkoma, NY). Microelectrodes were calibrated in HCO3−-Ringer buffer and those with slopes of 54–58 mV/pH unit were selected. The voltage difference between the pH microelectrode and a reference calomel electrode placed in contact with the skinned tip of the tail is proportional to the pH of the luminal fluid. Voltages were measured with a high-impedance electrometer (FD 223; World Precision Instruments, Sarasota, FL).
At the end of the experiments, a blood sample from the femoral artery and a urine sample from the bladder were taken. Blood and urine pH and pCO2 were measured with a blood gas analyzer (AADEE, Buenos Aires, Argentina).
To calculate acidification rates, the log of ([HCO3− t] − [HCO3−∞]), where [HCO3−∞] and [HCO3− t] are the concentration of HCO3− at steady state and at time t, respectively, is plotted against time, in seconds. This plot can be fitted to a straight line, meaning that [HCO3−] approaches exponentially to its steady-state value. The slope of this line is the acidification rate constant (κ) (13, 27). Net HCO3− flux (JHCO3−) is calculated according to where r is the tubule radius (0.015 mm).
The following experimental procedures were performed in both control and enalapril-treated rats: 1) For acute NO· synthesis inhibition, rats received, through the jugular vein, a bolus injection of NG-nitro-l-arginine methyl esther (l-NAME), 16 mg/kg of body weight; 2) for inhibition of the V-H+ATPase, bafilomycin (10−6 M) was added to the luminal perfusion solution; and 3) for Na+/H+ exchange inhibition, ethyl-isopropyl amiloride (EIPA) (10−4 M) was added to the luminal perfusion solution.
All procedures involving animals were designed to minimize pain and suffering and have been conducted in conformity with the principles stated in NIH's Guide for the Care and Use of Laboratory Animals. The Laboratory Animal Care and Use Committee of the Universidad Nacional de General San Martín reviewed and approved these protocols for animal use.
Evaluation of NO· Synthesis by Determination of Conversion of [14C]-l-Arginine to [14C]-l-Citrulline
Three months after weaning, rats belonging to the different experimental groups were anesthetized by enfluran inhalation, and an abdominal incision was made. The kidneys were perfused with ice-cold saline solution (NaCl 0.9%) and removed from the animal; the cortex was then separated from the medulla with cold scissors on a Petri dish in contact with ice. Cortex samples were immediately frozen in liquid nitrogen and stored at −80°C until required. Homogenates were obtained by sonicating the cortex in 2 ml of ice-cold extraction buffer per gram of tissue. Extraction buffer contained 20 mM HEPES, 1 mM EDTA, 10 μg/ml leupeptin, 2 μg/ml aprotinin, 10 μg/ml pepstatin A, 1 mM dithiothreitol, 2 mM PMSF, and 280 mM sucrose; pH 7.4. Homogenates were centrifuged at 12,000 g, 4°C for 5 min. The supernatants were aliquoted for 1) determination of protein concentration by Lowry et al. (24) and 2) evaluation of nitric oxide synthase (NOS) activity by determination of conversion of [14C]-l-arginine to [14C]-l-citrulline.
The reaction was carried out by incubation of 25 μl of the sample homogenate plus 75 μl of assay buffer at 37°C for 5 min. Assay buffer contained 20 mM HEPES, 30 nM calmodulin, 5 μM tetrahydrobiopterin, 2 mM Cl2Ca, 38.5 μM l-arginine, 67 mM l-valine, 1 mM NADPH, and 1.7 μM [14C]-l-arginine; pH 7.4. Reaction was stopped by the addition of 300 μl ice-cold stop buffer (20 mM HEPES and 2 mM EDTA; pH 5.5) and the tubes were kept at 0°C for 5 min. To separate the arginine substrate from citrulline product, we used the acidic-cation exchange resin Dowex 50W, 200–400 mesh, 8/100 cross-linked in its Na+ complexed form. The separation was carried out by adding 600 μl of the resin (50% in water) under vortex agitation. The tubes were kept on ice for 10 min and then centrifuged at 400 g, 4°C for 5 min. Finally, 500-μl aliquots of supernatant were mixed to 1 ml of scintillation cocktail, and [14C]-l-citrulline activity was counted in a liquid scintillation counter (Packard-Prias). NOS activity was expressed as a production of [14C]-l-citrulline per minute per milligram of protein.
Brush-Border Membrane Vesicle Preparation
Renal cortex brush-border membrane vesicles (BBMV) from control and enalapril-treated rats were isolated using a previously described technique (17). Kidneys were removed and washed with cold HEPES-sucrose-EDTA (HSE) buffer (in mM: 50 sucrose, 10 Tris, 10 HEPES, 0.5 EDTA, pH 7.5). Cortexes were separated and homogenized. After differential centrifugation, the pellet containing BBMV was dissolved in HSE buffer with protease inhibitors (aprotinine, 10 μg/ml; leupeptine, 10 μg/ml; pepstatin A, 10 μg/ml; phenylmethylsulfonyl fluoride, 2 mM; and dithiothreitol, 1 mM). Protein concentration was determined according to Lowry et al. (24). The purity of brush-border membrane fraction was assessed by measuring the activity of γ-glutamyl transferase (30), which increased 10-fold in the BBMV compared with the original homogenate. To rule out the possibility of contamination with basolateral membrane, we measured the activity of Na+/K+ ATPase (33). As expected, Na+/K+ ATPase activity was not detectable in BBMV.
Quantification of V-H+ATPase
BBMV corresponding to 100 μg protein were electrophoresed on a 10% SDS-PAGE under reducing conditions, according to Laemmli (21), and transferred to a nitrocellulose membrane. The transblots were incubated with the rabbit polyclonal antibody against the V-H+ATPase subunit E (FL-226) (catalog no. sc-20946, Santa Cruz Biotechnology, Santa Cruz, CA) and, subsequently, with an alkaline phosphatase-labeled secondary antibody (goat anti-rabbit IgG from Santa Cruz Biotechnology).
Quantification of NHE3
The general procedure is similar to that described above for quantification of V-H+ATPase, with the following modifications: BBMV samples corresponding to 40 μg protein were electrophoresed on 8% SDS-PAGE. Transblots were incubated with the primary antibody (MAB against isoform NHE3 catalog no. MAB3138, Chemicon International, Temecula, CA) and, subsequently, with the secondary antibody [Antibody anti-mouse IgG (H+L) alkaline phosphatase conjugate, catalog no. S372B from Promega, Madison, WI].
In the blots used to quantify NHE3, as well as in those used to determine the abundance of V-H+ATPase, the bands were visualized with BCIP/NBT color development substrate (Promega, Madison, WI). In all cases, membranes containing the same samples were incubated with the primary antibody against β-actin (Ab5), catalog no. 612656 from BD Biosciences. Band intensities were determined by densitometric analysis using ScionImage program (http://www.scioncorp.com) and were normalized to β-actin band intensities on the same blot (10).
Data are reported as means ± SE. Statistical significance was assessed using Student's t-test for unpaired data or ANOVA with the Newman-Keuls' a posteriori test when more than two experimental groups were compared (JHCO3− flux data). Differences were considered statistically significant at P < 0.05.
Table 1 shows body weight, blood pressure, sodium intake, and urinary sodium excretion (UvNa+) in control and enalapril-treated rats. Urinary Na+ excretion was lower in enalapril-treated than in control rats. However, in both groups, Na+ balance remained in equilibrium until the time when the micropuncture experiment data were collected (Na+ balance was measured the day previous to the experiment). The smaller UvNa+ observed in enalapril-treated rats could be due to their decreased Na+ intake, mostly attributable to a decrease in food intake displayed by these rats, as can be observed by their diminished protein intake.
Effect of Chronic ACE Inhibition on Acid-Base Parameters and Proximal Tubule Acidification Kinetics
Acid-base status was evaluated in blood samples taken from animals of the different experimental groups. Chronic treatment with enalapril caused no changes in acid-base status (Table 2), but it induced a significant increase in proximal tubule acidification, raising the net bicarbonate reabsorption (JHCO3−) 51% over the control value (Table 3).
Effect of Chronic ACE Inhibition on Na+-Dependent/EIPA-Sensitive and Na+-Independent/Bafilomycin-Sensitive, Proximal Tubule Acidification Kinetics
EIPA 10−4 M, added to the luminal perfusion solution, decreased JHCO3− by ∼60% in both control and enalapril-treated rats. The remaining JHCO3− after EIPA treatment was larger in enalapril-treated than in control rats. Luminal perfusion with bafilomycin 10−6 M induced a ∼30% decrease in JHCO3−, of both enalapril and control rats. The remaining flux after bafilomycin was also larger in enalapril-treated than in control animals. The sum of the bafilomycin-sensitive plus the EIPA-sensitive fraction of HCO3− reabsorption were close to the total JHCO3−(without inhibitors) in both control and enalapril-treated rats (Fig. 1 and Table 3).
Effect of Systemic NO· Synthesis Blockade on Enalapril-Induced Increase in Proximal Tubule Acidification
We tested the hypothesis that NO· was involved in the effect of chronic ACE inhibition on proximal tubule acidification kinetics. Nitric oxide synthesis was inhibited by systemic bolus injection of l-NAME during the micropuncture experiment. The addition of l-NAME did not change arterial blood pressure in any of the experimental groups (data not shown). Inhibition of NO· synthesis did not affect PCT acidification in untreated animals, but it abolished the increase in JHCO3− induced by chronic enalapril treatment (Table 3).
Effect of Chronic ACE Inhibition on the Conversion of [14C]l-Arginine to [14C]l-Citrulline in Kidney Cortex Homogenate
NOS activity was evaluated by measuring the conversion of a radiolabeled substrate of NOS, [14C]-l-arginine, to the product of the NO· synthesis reaction, [14C]-l-citrulline. Chronic treatment with enalapril significantly increased NOS activity (Fig. 2).
Effect of Chronic ACE Inhibition on the Abundances of NHE3 and V-H+ATPase in Kidney Cortex BBMV
The 31-kDa band, corresponding to the V-H+ATPase, was detected with a monoclonal antibody in BBMV from both control and enalapril-treated rats. As shown in Fig. 3, the normalized band intensities from both groups are not significantly different, indicating that chronic ACE inhibition did not affect the levels of expression of V-H+ATPase. The abundance of the NHE3 isoform of Na+/H+ exchanger was also determined. The amount of NHE3 was the same in enalapril-treated compared with control animals (Fig. 4). The quantification of the expression was performed using vesicles obtained from kidneys of three rats from each group.
The aim of this work was to study the effect of chronic ACE inhibition on proximal tubule HCO3− reabsorption. Since chronic ACE inhibition is the treatment of choice to deal with several renal and cardiovascular disorders in humans, it is relevant to evaluate its effects on a key mechanism for acid-base balance and sodium and volume reabsorption.
In this study, we found that chronic treatment with enalapril increased HCO3− reabsorption in PCT of young adult rats. This effect was abolished by intravenous bolus delivery of the NO· synthesis inhibitor l-NAME, which decreased the JHCO3− of enalapril-treated rats to levels similar to those observed in control rats exposed to l-NAME. The HCO3− flux, in control animals, was not statistically changed after l-NAME. In contrast, l-NAME induced a significant fall in JHCO3− in enalapril-treated animals, which suggests that the effect of chronic ACE inhibition involves the activation of the NO· pathway. The interaction between renin-angiotensin system and the path of NO· has been extensively studied (8, 23, 34, 38). We have previously shown that acute perfusion of the peritubular capillaries with an antagonist of NO· synthesis caused a decrease in H+-ion flux (7, 9). Moreover, it has been demonstrated that NO· stimulates absolute and fractional reabsorption in PCT (8, 15). Thus, in the present work, the lack of effect of l-NAME on PCT acidification observed in the control rats was unexpected. A possible explanation to this conflicting result could be that the effective concentration of l-NAME at the peritubular capillaries is lower when the antagonist is administered systemically than when it is added to the peritubular perfusate. In enalapril-treated animals, where there was an increase of NOS activity, the effect of the small dose of l-NAME used could be more apparent than in the control rats. Still, it is noteworthy that l-NAME was very close to reach a statistically significant effect (inhibition) on PCT acidification of untreated animals (P = 0.05). On the other hand, NOS activity, evaluated as the formation of [14C]-l-citrulline from [14C]-l-arginine, was increased, supporting the hypothesis that NO· plays a role in the effect of chronic enalapril treatment.
Both components of PCT acidification, the EIPA-sensitive Na+/H+ exchange and the bafilomycin-sensitive V-H+ATPase, were affected to the same extent by chronic treatment with enalapril. The absolute HCO3− flux reduction after EIPA was larger in enalapril-treated than in control rats. However, the relative decreases in JHCO3− with bafilomycin and EIPA were ∼30% and ∼70% in untreated and enalapril-treated animals, respectively. This suggests that chronic ACE inhibition affected both transport systems in a similar way. It has been reported that NHE3 expression in kidney is positively regulated by NO· (39). Moreover, inhibition of NO· synthesis with the antagonist LNMMA, induces a decrease in the absolute proximal reabsorption that can be counteracted with l-arginine, the natural substrate of NOS (8). We looked over changes in NHE3 and V-H+ATPase expressions as possibly responsible for the increase in HCO3− reabsorption. The abundances of NHE3 and V-H+ATPase in BBMV were the same in both groups of animals. Thus, the effect of chronic ACE inhibition seems to be independent of the expression of these transporters and should be the result of an increased activity of the Na+/H+ exchanger and the V-H+ATPase.
Acute ACE inhibition has been associated with a decrease in Na+ and fluid reabsorption in PCT (22). We found that Na+ excretion is smaller in enalapril-treated than in control rats. This correlated to the decrease in Na+ intake observed in enalapril-treated rats. This difference could result from the small food intake displayed by enalapril-treated rats, as reflected by their reduced protein intake and body weight. Although PCT acidification does not represent the totality of volume reabsorption in PCT, it drives the largest fraction of it and thus can be used as a good approximation to the Na+ and water reabsorption at this segment of the nephron (6). It has been reported that genetically modified mice (ACE2/2), lacking renal brush-border-associated ACE, display normal PCT reabsorption or even an increased fractional reabsorption (14, 19). These results, as well as our data, suggest that the effects of acute ACE inhibition would be different from those caused by chronic ACE inhibition. We do not have an explanation for the increase in JHCO3− observed upon chronic ACE inhibition and the lack of effect of ACE deficiency on volume reabsorption of ACE2/2 mice. It is possible that the diminished volume load observed in ACE2/2 mice, as a consequence of the fall in the single-nephron glomerular filtration rate, induces a huge increase in fractional reabsorption, which is not reflected in the absolute reabsorption. On the other hand, we found that our experimental pharmacological model induced an increase in NOS activity and presumably increased NO· production, to which we ascribe the effect of increased HCO3− reabsorption.
Low concentrations of ANG II were reported to increase HCO3− reabsorption through activation of AT1A receptors (16, 48). Multiple lines of evidence have suggested that there are alternative pathways for ANG II generation in the heart, large arteries, and the kidney that do not require ACE (28). This ACE-independent alternative formation of ANG II occurs in rat arteries at very high concentrations of ANG I (18). ANG I levels increase upon ACE inhibition; thus, it is possible that the ANG II produced through alternative pathways reaches a final concentration that stimulate HCO3− reabsorption (16). Acute ACE inhibition could not elicit the same effect.
No changes were detected in acid-base status of the enalapril-treated rats, indicating that the effect of ACE inhibition on proximal acidification does not affect the overall acid-base balance. Thus compensatory regulation of acid secretion must occur in other segments of the nephron after chronic ACEi treatment. ANG II has been reported to increase the abundances of NHE3 and the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1) at medullary thick ascending limb, as well as the expression of the B1 subunit of the H+ATPase at the collecting duct (41). In addition, NO· and cGMP inhibit H+-ATPase activity in cortical collecting ducts (39). Moreover, Ortiz et al. (31) demonstrated that NO· inhibits HCO3− reabsorption in thick ascending limb and outer medullary collecting duct (43). Thus, the increase in NO· synthesis that we found in enalapril-treated rats could induce a decrease in H+ secretion at these distal segments, which would compensate the increase in HCO3− reabsorption at the proximal segments.
Chronic ACE inhibition might have effects not necessarily related to ANG II concentration. Indeed, ACE is involved in the degradation of BK; thus, the use of ACE inhibitors such as enalapril could increase BK concentration (46) promoting NO· release. On the other hand, ACE inhibition induces an increase in plasmatic concentration of angiotensin 1-7, an ANG I derivative that promotes BK and NO·-dependent vasodilatation (5, 35).
Perspectives and Significance
The main point that should be emphasized is the fact that chronic and acute ACE inhibitions elicit different responses in PCT acidification. It is possible that the initial effects of ACE inhibition are the decreases in HCO3−, Na+, and volume reabsorption at both proximal and distal segments of the nephron. Indeed, acute ACE inhibition induces a marked increase in diuresis and natriuresis (25, 47). The initial volume depletion could induce a small negative sodium balance, which should signal an increase in PCT fractional reabsorption and, like the aldosterone escape phenomenon (40), could trigger an increase in proximal Na+/H+ exchange and electrogenic H+ transport. The increased activity of these transporters would depend on NO· synthesis. The increase in proximal HCO3− reabsorption must necessarily turn on mechanisms of regulation at the distal nephron to adjust acid-base equilibrium and overall water balance. If all of the segments of the nephron were affected by ACE inhibition in the same way (i.e., HCO3− and Na+ loss), then we would expect chronic ACEi treatment to cause metabolic acidosis and volume contraction. However, these side effects have not been reported in chronically ACEi-treated human patients. It would be interesting to determine whether ACE knockout animals display changes in the NO· pathway.
This work was supported by Agencia Nacional de Promoción Científica y Tecnológica Grant PICT 05-08305, Universidad Nacional de General San Martín Grant SB06/045, and Consejo Nacional de Investigaciones Científicas y Técnicas Grant PIP 0851.
The authors would like to thank Dr. N. Basso, Dr. N. L.Yeyati, and Dr. A. A. Altamirano for critical reading of this manuscript.
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