Department of Pharmacology, Faculty of Medicine, University of Tübingen, D-72074 Tübingen, Germany
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ABSTRACT |
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Renal function was studied in mice of different ages. In metabolic cage experiments, the renal electrolyte excretion was similar in young (n = 8; 5- to 7-wk-old) and adult (n = 6; 20- to 22-wk-old) CD-1 (ICR) BR mice, whereas spontaneous drinking volume and urinary flow rate were significantly higher in the adult compared with the young mice. Subsequently, the renal functional reserve was investigated by amino acid (AA) infusion (10%) in anesthetized young (n = 8) and adult (n = 6) mice. Because the body weight of adult mice was significantly higher than that of young animals, one group of adult mice (n = 8) received 12.5% AA to ensure that the dose of AA related to body weight was similar in both groups. Young animals constantly infused with Ringer solution served as time controls (n = 8). Glomerular filtration rate (GFR) at baseline was similar in each group. Because of AA, GFR significantly increased in young mice but not in both groups of adult animals, whereas in time controls GFR remained constant. Urinary flow rate and sodium excretion were elevated by AA in young and adult mice. We conclude that in CD-1 mice the first signs of age-related changes in kidney function concern alterations in renal hemodynamics, whereas renal tubular function appears to be preserved.
kidney; amino acids; glomerular filtration rate; physiological aging
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INTRODUCTION |
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IT IS WELL KNOWN THAT INCREASING age is accompanied, even if no specific kidney disease is present, by changes in renal structure and function. Morphological studies in laboratory rats indicate that age-dependent glomerular injury is characterized by increased mesangial deposits, mesangial proliferation, thickening of the glomerular basement membrane, and, ultimately, glomerular sclerosis (7). These morphological changes result in profound functional alterations, including changes in renal hemodynamics and tubular function. Previous reports demonstrate an age-related decrease in baseline levels of both glomerular filtration rate (GFR) and renal plasma flow (1). A more sensitive indicator for the functional integrity of the kidney is the ability to increase GFR in response to intravenous amino acid (AA) infusion or a high-protein-containing meal. These procedures assess the so-called renal functional reserve or residual vasodilatory capacity of the kidney. In an earlier study in Sprague-Dawley rats, age dependency of renal function was described; in chronically catheterized conscious animals, the renal response to an intravenous glycine infusion was markedly diminished in the 22- to 24-month-old rats, indicating a loss of functional reserve in the aging kidney (2).
Advances in gene-targeted manipulations of the murine genome have provided new experimental approaches to the investigation of the structural, functional, and pathophysiological significance of different genes and their products. Indeed, numerous gene knockout and transgenic mouse models have been developed. Several of these models are employed in experiments on kidney function (5, 12, 13). In light of the age-dependent renal changes observed in rats, we found it of interest whether such alterations also occur in mice. In the present study, the functional changes in the aging kidney were evaluated in conscious and anesthetized CD-1 mice. Kidney function at baseline and renal response to an acutely administered intravenous AA load were investigated in young and adult mice.
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METHODS |
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Animals. Male CD-1 (ICR) BR mice weighing 25-40 g (Charles River, Sulzfeld, Germany) were used in the experiments. The animals were housed in a temperature- and humidity-controlled environment with a 12:12-h light-dark cycle. Mice were allowed free access to standard rodent chow (no. 1324, Altromin, Lage, Germany) and tap water. Experiments were performed in two groups of mice of different ages: young (YNG; 5-7 wk old) and adult (ADT; 20-22 wk old).
Metabolic cage experiments. Conscious mice were placed in metabolic cages (Techniplast, Ehret, Freiburg, Germany) from 7:30 AM to 3:30 PM with free access to tap water, but food was withheld. Water intake as well as urinary excretion of water, sodium, and potassium were monitored in both groups of young and adult mice.
Clearance experiments. The animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (100 µg/g body wt; Sigma, Deisenhofen, Germany) and placed on a temperature-controlled heating table to maintain the body temperatures at 37.5°C. Supplemental doses of anesthesia (5 µg pentobarbital sodium/g body wt iv) were administered if required. A tracheostomy was performed to facilitate breathing. The right jugular vein was catheterized with PE-10 tubing for fluid infusion. The left carotid artery was cannulated with PE-10 tubing for continuous measurement of arterial blood pressure and blood sampling. The bladder was cannulated for urine collection with PE-50 tubing via a suprapubic incision. Tritiated inulin (10 µCi/ml; NEN, Dreieich, Germany) in Ringer solution [(in mM/l) 111 NaCl, 30 NaHCO3, and 4.7 KCl] was intravenously infused at 3 µl/min for assessment of GFR. Mice were allowed to recover from surgical procedures for 60 min before the clearance measurements were started. Via a second intravenous catheter, Ringer solution was infused at 2.5 µl/min during both baseline periods. Thereafter, Ringer solution was changed to a mixed solution of AA. A concentration of 10% AA was infused in young (AA-YNG) and adult (AA-ADT) mice. In an additional group of adult mice (AA-ADT-C), the concentration of AA solution was 12.5% to ensure that a reduced dose of AA related to body weight in adult animals would not confound the observations. An AA solution was administered at the same infusion rate, and another three experimental clearance periods (EP I, II, and III) were performed. Total infusion volume was ~1% of body wt. The 10% AA solution contained (in g/l) 3.8 L-isoleucine, 6.6 L-leucine, 9.3 L-lysine, 2.8 L-methionine, 4.1 L-phenylalanine, 4.6 L-threonine, 1.2 L-tryptophan, 4.1 L-valine, 9.2 L-arginine, 4.4 L-histidine, 7.7 aminoacetic acid, 14.3 L-alanine, 9.2 L-proline, 0.7 L-cysteine, 9.9 L-glutamic acid, 4.6 L-ornithine-L-aspartate, 5.9 L-serine, and 0.5 L-tyrosine. In an additional group of young mice, Ringer solution was maintained throughout the entire experiment as a vehicle control (VHC-CON). All infusion solutions contained 2.25% BSA. Per-period urine was collected for 30 min, with one blood sample taken at midpoint (40 µl).
Analyses, calculations, and statistics. The [3H]inulin radioactivity was measured by liquid scintillation counting (2550 TR, Canberra-Packard, Frankfurt, Germany), and GFR was calculated with the standard equation. Sodium and potassium concentrations were determined by flame photometry (ELEX 6361, Eppendorf, Hamburg, Germany). In clearance experiments, renal excretory and hemodynamic values were calculated per gram of kidney wet weight. The values of the first two periods were averaged to form one baseline value. The statistical significance between different groups at baseline and between baseline and experimental periods EP I-III within groups was calculated by ANOVA with Bonferroni's posttest. Values are expressed as means ± SE. A P value of <0.05 was considered statistically significant.
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RESULTS |
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With regard to the somatometric data, body weight was
significantly higher in the adult mice compared with the young animals (AA-ADT, 37.4 ± 0.6 g; AA-ADT-C, 37.5 ± 0.6 g;
AA-YNG, 30.5 ± 0.4 g). The same observation was made for
kidney wet weight (AA-ADT, 0.60 ± 0.04 g; AA-ADT-C,
0.59 ± 0.03 g; AA-YNG, 0.42 ± 0.02 g). However,
the ratio of kidney wet weight to body weight was
significantly higher in both groups of adult CD-1 mice compared with
the young animals (Fig. 1).
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The data obtained in conscious animals in metabolic cages are given in
Table 1. A significantly higher urinary
flow rate was observed in adult mice compared with the young animals.
Urinary flow rate was positively correlated in good quantitative
accordance with drinking volume. In contrast, the urinary excretion of
sodium and potassium was not significantly different in both groups of mice. Osmolality of the urine was 1.4-fold higher in young mice; however, because of interindividual variability, this difference did
not reach the level of significance.
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In the clearance experiments, urinary flow rate at baseline did not
significantly vary in the different groups. Baseline GFR was slightly
lower in adult (AA-ADT) compared with young mice, even though the
difference did not reach the level of significance (Fig.
2). The most pronounced difference
between young and adult animals was observed during infusion of AA;
although infusion of AA significantly increased GFR in young mice by
almost 1.6-fold, no AA-induced hyperfiltration at all occurred in the
adult animals, indicating an age-related reduction in renal reserve
capacity. The lack of GFR increase due to AA infusion was observed in
adult mice (AA-ADT) infused with 10% AA and in adult mice (AA-ADT-C) in which the dose of AA (12.5%) was corrected for higher body weight.
As expected, there was no significant change in GFR in the VHC-CON
group on constant Ringer infusion (Fig. 2). In contrast to GFR, urinary
flow rate increased to the same extent in response to AA in both young
and adult mice (Table 2). However, the
differences only partially reach the level of significance because of
interindividual variability. In the VHC-CON group, no significant
variation in urinary flow rate was observed. In each group, mean
arterial blood pressure slightly but significantly increased by ~10%
after the baseline period (Table 2). Concomitantly, heart rate was
slightly elevated in all groups with time (Table 2). At baseline,
absolute as well as fractional urinary sodium excretion was similar in each group (Table 2). The AA infusion induced an increase in absolute
renal sodium excretion in both young and adult mice. However, the
effect was more pronounced in young compared with adult animals (Table
2). In contrast, fractional sodium excretion showed a quantitatively
similar tendency to increase because of AA in both age groups (Table
2). As expected, both absolute and fractional urinary sodium excretion
remained constant throughout the entire experiment in the VHC-CON group
continuously infused with Ringer solution (Table 2).
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DISCUSSION |
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The present experiments were undertaken to compare renal function in young and adult laboratory mice. In the first set of experiments, renal excretory function was observed in metabolic cage experiments. Because animals were studied without surgical intervention and in a conscious and normovolemic state, the observations were assumed to be representative of the physiological condition. In the second set of experiments, anesthesia was employed to additionally assess GFR. The time control experiments showed constant values of GFR and of electrolyte and volume excretion, indicating stable experimental conditions. Mean arterial blood pressure and heart rate tended to increase in the course of the experiments. However, the observations of kidney function were not confounded by changes in systemic hemodynamics, because these alterations occurred in each of the four groups, including the time control group in which, in addition, no effects related to changes in systemic hemodynamics, such as pressure diuresis, were observed.
Urinary electrolyte excretion was similarly irrespective of age, whereas urinary flow rate was significantly higher and urine osmolality was lower in the adult mice. This might indicate that the renal concentrating ability is decreased in the aging kidney. Such an idea is supported by earlier studies in humans and rats (3, 10, 14). One of these studies showed that in humans the maximal urinary osmolality was inversely related to age by some 20% when subjects in their 40s and 50s were compared with subjects between 60 and 79 yr old (14). In that study, the age-related concentrating defect was not correlated with changes in GFR, which was suggested by several investigators. Also, other mechanisms were postulated to account for the concentrating defect of the aged kidney, including an increase in medullary blood flow resulting, by means of washout phenomena of medullary tonicity, in a decline in the efficacy of the countercurrent system (14) or defects in electrolyte transport processes. In the present study, we observed lower, although not significantly lower, GFR in adult mice as measured in the clearance experiments at baseline. With regard to the observations during infusion of AA, the regulation of tubular transport did not appear to be changed in adult mice compared with the young animals, because the increase in fractional sodium excretion was almost identical. However, because of interindividual variability, the differences between baseline and AA infusion did not reach the level of significance in these groups. Taken together, the data of the present study suggest that changes in renal hemodynamics contribute to the age-related decrease in concentrating ability rather than impairment of tubular transport processes. However, it has to be taken into account that in the present study the age of the adult mice was not as advanced as it was in earlier studies. The factors responsible for the age-related changes in renal sodium handling reported by other authors remain incompletely understood because all major determinants of renal sodium handling, i.e., renal hemodynamics, the renin-angiotensin-aldosterone system, and atrial natriuretic peptide, were demonstrated to vary significantly with age (4).
The main focus of the present study was the renal functional reserve in mice; the significant increase in GFR in young mice caused by infusion of 10% AA solution was almost completely absent in the adult animals. Because the AA solution was not factored for body weight, it may be argued that dose differences, which were some 20% larger compared with those in the young animals, might be responsible for the lack of response in the adult animals. Therefore, we addressed this potential factor by infusing an additional group of adult mice with a 12.5% AA solution. Also, in these mice, the observation that no GFR increase due to AA infusion occurred supports the hypothesis that the renal functional reserve is substantially diminished in adult mice.
The result of an age-dependent, AA-induced increase in GFR is in accordance with previous studies in rats (2) but is in contrast to a trial in humans (6). In the latter, renal hemodynamics were compared before and after an AA infusion in healthy young and elderly subjects. The study demonstrated that the median of the rise in inulin clearance was not significantly different in these subjects. The authors concluded that renal functional reserve is also demonstrable in older age, at least in humans. Thus age-dependent loss of kidney function appears to develop more rapidly and more severely in rodents than in humans. Renal failure is a significant cause of death in some rat strains (15). The present study demonstrates that the age-dependent decrease in kidney function in rats holds true also for mice. Remarkably, the physiological increase in GFR due to AA infusion in younger mice was clearly more pronounced than the increase reported in rats (2, 11). In addition, the age-related decline in renal reserve capacity in mice appears to be more progressive, because the age differences in the present study were not that large. Notwithstanding, strain differences in the aging process of the kidney have to be considered (8). These experiments demonstrated that aging patterns can differ greatly in animals of different genotypes. Taken together, the data from the present experiments suggest caution in the interpretation of renal functional results in mice. Potentially age-dependent kidney damage should be taken into consideration.
The present assessment of renal functional reserve was conducted in animals under general anesthesia. It may be argued that observations of renal hemodynamics under these conditions do not truly reflect renal vascular responsivity because age-dependent changes may also occur in the responsiveness of the autonomic nervous system. However, the excretory function in time controls was stable throughout the entire experiment, and the increase in GFR during AA infusion was clearly demonstrated. Thus the state of consciousness should not have confounded the present results. To overcome the possible influence of anesthesia, experiments using either an acute AA infusion in chronically instrumented animals or the feeding of a high-protein meal to conscious mice should be performed to investigate the renal functional reserve.
Interestingly, the kidney weight-to-body weight ratio was significantly higher in adult CD-1 mice than in young animals. This might reflect an attempt to compensate for the loss of function with advancing age. This observation has been also found previously in two different mice strains (C57BL/6 and CBA/HT6J) and corresponds with renal hypertrophy (8). That observation and the data of the present study seem to be in contrast to the known fact that aging is associated with a loss of renal mass (9). Because in the present study the age of the adult mice was less advanced, beginning kidney damage might not be so pronounced as to induce reduction of renal mass.
We conclude that in CD-1 mice, the first signs of age-related changes in kidney function concern alterations in renal hemodynamics, whereas renal tubular function does not decline with age. These observations should be considered when experiments in renal function in mice are designed.
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ACKNOWLEDGEMENTS |
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This study was supported by the Deutsche Forschungsgemeinschaft (Mu 1297/1-2) and the Federal Ministry of Education, Science, Research and Technology (01EC9405).
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. Luippold, Faculty of Medicine, Dept. of Pharmacology, Univ. of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany (E-mail:gerd.luippold{at}uni-tuebingen.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 11, 2001;10.1152/ajprenal.00134.2001
Received 1 May 2001; accepted in final form 10 December 2001.
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