INTRODUCTION

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AS PART OF THE NORMAL AGING PROCESS, the kidney develops a progressive deterioration of several structures and functions (7, 9). Glomerular filtration rate, renal blood flow, and concentrating ability decrease with age (8, 21, 26, 29). Aging also affects tubular function, although the mechanisms affected are less well defined (20). Under normal conditions, Na⁺/H⁺ exchange (NHE) accounts for ~65% of the proximal tubular acidification (18). The control of the exchanger is very complex and depends on many factors including, among others, the renin-angiotensin system (RAS) (30), endothelium-derived relaxing factor (EDRF) (2, 25, 32), and parathyroid hormone (6). There is downregulation of the renal RAS with age, affecting renin mRNA and angiotensin-converting enzyme (17). In addition, renal hemodynamics of senescent rats seem to be more dependent on the EDRF than in younger animals (13). Kinsella and Sacktor (19) found a decrease of NHE activity in brush-border membrane vesicles (BBMV) from the renal cortex of kidneys from senile animals. On the other hand, Ikuma et al. (15) detected a decrease in the activity of the NHE in jejunal villus cells from senescent rats.

In the present work using in vivo and in vitro techniques, we studied the kinetics of proximal tubular acidification of aged rats. We performed micropuncture experiments with simultaneous luminal and peritubular perfusion, thus avoiding the effect of extratubular factors, and we evaluated the kinetics of the NHE of BBMV from the same population of rats. Our results suggest that proximal tubular acidification capacity in the aging rats is impaired.

	METHODS

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Groups of Rats

Blood Acid-Base Status

In Vitro Experiments

BBMV. BBMV from the renal cortex were isolated using a technique previously described (14). The vesicle pellet obtained after differential centrifugation was dissolved in HEPES-sucrose-EDTA (HSE) buffer (50 mM sucrose, 10 mM Tris, 10 mM HEPES, 0.5 mM EDTA, pH 7.5). Protein concentration was determined according to Lowry et al. (23). The purity of the brush border membrane fraction was assessed measuring the activity of Na⁺-K⁺-ATPase (27) and of glutamyl transferase in the vesicle pellet. Na⁺-K⁺-ATPase activity was not detectable, but the activity of glutamyl transferase increased 10-fold compared with the original homogenate. Vesicles were prepared freshly from four animals for each experiment.

NHE kinetics. Transport was measured fluorometrically according to Igarreta et al. (14). Vesicles dissolved in HSE were loaded up to a final concentration of 150 mM Na-gluconate at least 90 min before kinetic studies. Briefly, 20 µl of the vesicle preparation were diluted into 2 ml of external buffer (50 mM sucrose, 10 mM HEPES, 10 mM Tris, 150 mM N-methyl-D-glucamine gluconate, and 6 µM acridine orange) at pH 7.5. The addition of the vesicles to the external medium promotes a Na⁺ efflux with H⁺ exchange. This causes a reduction of the external fluorescence, until it reaches a minimum steady-state level. The BBMV were exposed to Na gluconate in concentrations from 1.5 to 100 mM added to the external buffer, and the recovery of the external fluorescence was recorded. The initial rate of fluorescence recovery for each Na-gluconate concentration was used to measure the maximal velocity (V_max) and Michaelis constant (K_m) of the NHE (14). Polymethacrylate cuvettes were used for the determination of external fluorescence, with constant stirring at 25°C under a flow of dry air in the measuring chamber.

Western blot. BBMV corresponding to 35 µg protein were resuspended in sample buffer. Samples were heated at 100°C for 2 min and were then spotted in a discontinuous gel, with 7% polyacrylamide and 0.1% SDS. After its development, proteins were transferred to a nitrocellulose membrane and blocked in PBS-0.02% Tween 20 (PBST) with 5% nonfat milk during 1 h at room temperature. The blot was incubated overnight at 4°C with the first antibody (MAB against isoform NHE3, catalog number MAB3138, Chemicon International) diluted 1:500 in PBST. After five washes in PBST, the membrane was incubated with the second antibody (Biotinylated Anti-Mouse IgG, Sigma-Aldrich) diluted 1:500 for 1 h at room temperature. After four washes with PBST and an additional one with PBS, the membrane was incubated for 1 h with extravidin peroxidase (Sigma-Aldrich) diluted 1:500 in PBS, and after five washes with PBS, the proteins were visualized using peroxidase substrate (Sigma FAST DAB Tablet Set, Sigma-Aldrich). Membranes were scanned, and the band intensities were quantified using the MD Image Quant Software (version 3.3).

In Vivo Experiments

Micropuncture technique. Rats were anesthesized with pentobarbital sodium (50 mg/kg body wt ip), placed on a thermostatically controlled heated table, and prepared by standard micropuncture techniques (3, 24). The kinetics of acidification in proximal convoluted tubule (PCT) were studied by simultaneous luminal and peritubular perfusion with continuous measurement of intratubular pH as previously described (4). Briefly, the PCT was perfused by means of a double-barrelled micropipette, one barrel filled with Sudan-Black-colored castor oil and the other with the perfusion solution (in mM: 75 NaCl, 5 KCl, 1 CaCl₂, 20 HNaPO, 1.25 MgSO, 10 glucose, 90 raffinose, pH 7.4, osmolality adjusted to 290 mosmol/kgH₂O water with raffinose). Peritubular capillaries were simultaneously perfused with single micropipettes filled with (in mM): 105 NaCl, 5 KCl, 1 CaCl₂, 20 HNaPO, 1.25 MgSO, and 10 glucose, pH 7.4. A fluid droplet of phosphate-Ringer buffer was injected between oil columns in the tubule lumen, and luminal pH changes were measured with an H⁺-sensitive resin microelectrode (5) (Fluka, Cocktail A). The difference between [H₂NaPO₄] at steady state (ss) and [H₂NaPO₄] at time t diminishes exponentially with an acidification rate constant (k) (11). Net H⁺ secretion (J_H⁺) was calculated as: J_H⁺= ([H₂NaPO₄]_t=ss  [H₂NaPO₄]_t=0) × k × (r/2), where r is the lumen radius of the tubule (15 µm in control and 19 µm in the aging rats), and [H₂NaPO₄]_t=0 and [H₂NaPO₄]_t=ss are the concentration of the injected phosphate at time 0 and at steady state, respectively. Microelectrodes were calibrated in phosphate-Ringer buffer pH 7 and 8, at the beginning and at the end of every group of acidificacation curves. The slope of microelectrodes was 56 ± 2 mV/pH unit.

Statistics

	RESULTS

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Acid-Base Parameters

NHE Kinetics

Western Blot

View larger version (19K): [in this window] [in a new window]   Fig. 1.   A: densitometric determination of the bands with Image Quant is expressed as arbitrary densitometry units (ADU). B: Western immunoblot of brush-border membrane vesicles with an antibody against the isoform Na+/H+ exchange 3 of the Na+-H+ exchanger. Vesicles were obtained from the kidney cortex of young (3 mo) and aging (22 mo) rats. This shows a representative immunoblot of 2 independent experiments with similar findings.

Micropuncture Experiments

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View larger version (7K): [in this window] [in a new window]   Fig. 2.   Proximal proton flux (JH+) in young (3 mo) (n = 15) and aging (22 mo) (n = 21) rats during simultaneous luminal and peritubular microperfusion with phosphate-Ringer solution. Values are means ± SE. *P < 0.05 vs. young rats (Student's t-test).

	DISCUSSION

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The present work shows that aging impairs proximal tubular acidification. This impairment is probably due to a decrease in the activity and abundance of brush border NHE. However, this defect did not affect blood acid-base status because blood pH, PCO₂, and [HCO]_p were normal. Our results agree with those of Prasad et al. (28), who did not detect blood pH differences between 6-mo and 24-mo-old rats. Frassetto et al. (10) showed that acid-base status of the blood changes in adult humans. From young adulthood to old age, men and women develop a low grade metabolic acidosis attributed by the authors to the age-related renal insufficiency (10). We have evaluated the acid-base status of a small group of six young and six senile anesthetized rats, whereas Frassetto et al. (10) performed a more extensive study designed to detect small changes in acid-base composition of 64 individuals, ranging from 17 to 74 yr old. Thus the lack of evidence of alterations of the acid-base status in aging rats could be due to the experimental design. Another factor that could contribute to the difference observed between aging humans and rats is the levels of insulin-like growth factor 1 (IGF-1) and growth hormones. IGF-1, a growth factor that stimulates apical NHE in proximal tubules (16), declines in aging humans but does not decline in aging rats (12, 31). The absence of acid-base defects in basal conditions does not exclude a defect in HCO handling by the kidney of the aging rats, which would be manifest after an acid load (28).

In agreement with Kinsella and Sacktor (19), we found that both the amount of the NHE3 isoform measured by densitometry after isolation by Western blot and the kinetics of the NHE in BBMV were conspicuously diminished in 22-mo-old rats.

A direct approach to evaluate the capacity of acidification of the proximal tubule is by using micropuncture techniques. These results are independent of glomerular filtration rate or any other hemodynamic or systemic variable, because luminal and peritubular perfusion is simultaneously performed. We found that aging rats had a significant decrease in the capacity of proximal tubular acidification, mostly due to a reduction of the acidification rate constant. Thus aging rats would have an intrinsic defect in the proximal tubular acidification.

We found a larger reduction of the NHE activity in vitro than in PCT acidification parameters measured in vivo. In the proximal tubule, NHE accounts for ~65% of apical membrane proton secretion, and ~35% is mediated by an H⁺-ATPase (18). The difference between in vitro and in vivo observations suggests that an alternative mechanism(s) of proximal tubule acidification would take over the fall of the NHE activity. A possible mechanism involved could be an increase in the activity of H⁺-ATPase compensating, in part, by the impairment in the NHE activity. On the other hand, because several paracrine and autocrine systems regulate the activity of the NHE in the proximal tubule (2, 6, 25, 30, 32), it is possible that part of the difference between in vivo and in vitro observations results from the participation of regulatory mechanisms present in the intact whole cell. This could be in accordance with results obtained by Ikuma et al. (15) studying the effect of aging on intracellular pH (pH_i) regulation in jejunal villus cells. They found that after inducing cytoplasmic acidification, the relationship between pH_i and external Na⁺ concentration showed a V_max of alkalinization that was only 20% lower in senescent than in young rats. It is important to point out that the NHE3 isoform, responsible for NHE in the apical membrane of PCT, is also present in the brush border of small intestine epithelial cells (33). Moreover, Lorenz et al. (22) found a 38% decrease in proximal fluid reabsorption in homozygous NHE3/ knocknout mice compared with the wild type. These results indicate that an important fraction of Na and volume reabsorption in the PCT is independent of the NHE at least under conditions where the exchange is absent or poorly expressed. Our results suggest that despite the large reduction in the NHE expression and activity, as determined by in vitro experiments and Western blot, bicarbonate claim in PCT of aging rats would not be reduced to the same magnitude.

In conclusion, aging rats showed an impaired PCT acidification capacity, probably as a result of a decrease in the activity and abundance of the NHE. However, the quantitative difference between in vivo and in vitro results could indicate the presence of a compensatory regulatory mechanism(s) acting in the proximal cell in vivo or an increase in the activity of other acidifying mechanisms present in the PCT, as in the H⁺-ATPase.

Perspectives