Although aminopeptidase A (APA), which is abundant in the kidneys, is responsible for metabolizing angiotensin II (Ang II), its association with salt sensitivity remains uncertain. We aimed to clarify the involvement of APA in salt-induced hypertension and renal damage.
Male Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats were fed low-salt (0.3%) or high-salt diet (8%) from 6 weeks of age for 12 weeks. Tail-cuff–measured blood pressure (BP), renal APA activity, renal Ang II levels, histologic renal damage, and APA immunoreactivity were periodically examined.
Systolic BP progressively increased only in DS rats given the high-salt diet (DS-8% rats). The DR-8% rats had approximately 3-fold higher renal APA activity than the rats given the low-salt diet (DR-0.3% rats) during the maintenance on the high-salt diet. However, although DS-8% rats also had 2.5-fold higher renal APA activity than DS-0.3% rats at 10 weeks, continuing the high-salt diet afterward suppressed the activity in DS-8% rats below the levels observed in DS-0.3% rats. High-salt diet reduced renal Ang II levels by 30% in DR rats, whereas it showed a small and nonsignificant decrease in DS rats. The number of injured glomeruli was markedly elevated in DS-8% rats after 10 weeks. The APA immunostaining in DS-8% rats was enhanced in glomeruli displaying mild damage, diminished in the severely injured glomeruli, and absent in lesions with hyalinization.
High-salt diet in DS rats increased renal APA activity, although renal injury remained mild, but then reduced it along with the progression of glomerulosclerosis, suggesting that reduced APA activity may be involved in the deterioration of salt-induced hypertension and renal injury. Am J Hypertens 2005;18:544–548 © 2005 American Journal of Hypertension, Ltd.
The renin-angiotensin system (RAS) plays an important role in regulating blood pressure (BP) as well as various physiologic and pathophysiologic phenomena such as electrolytic balance and extracellular fluid volume.1,2 Concentrations of angiotensin II (Ang II), the most active substance in RAS, depend on the balance between production and degradation. However, until now, little attention has been paid to the degradation of Ang II when evaluating the regulation of Ang II actions. Aminopeptidase A (APA, EC 22.214.171.124) is responsible for the N-terminal cleavage of Ang II, a hydrolytic event that serves as a rate-limiting step in angiotensin degradation.3 The hypotensive effect of purified APA and the hypertensive effect of APA inhibitors in vivo strongly suggest a possible involvement of APA in BP regulation.4,5 In addition, our recent findings that APA-deficient mice develop hypertension and that renal APA is involved in the development of hypertension in spontaneously hypertensive rats (SHR) also support this possibility.6,7
Epidemiologically, dietary salt is correlated with the prevalence and progression of essential hypertension.8–10 To examine the molecular mechanisms of salt-induced hypertension, the genetic rat model introduced by Dahl et al11, namely, the Dahl salt-sensitive rat [DS rat]) is of great use. Previous efforts to elucidate the mechanisms of hypertension in DS rats have shown various plausible candidates including stimulation of sympathetic nerves,12 deficiency in nitric oxide,13,14 and increased plasma endothelin-1 levels.15,16 The RAS also seems to be involved in the development of hypertension in DS rats. The DS rats are reported to have a low activity of systemic RAS accompanied with inappropriately activated renal RAS,17–19 indicating a significant role of local RAS in salt-induced hypertension. Because APA, which is highly expressed within the kidneys,20 could suppress local RAS activity by metabolizing Ang II, it is plausible that high renal RAS activity may be attributed to low renal APA activity. In addition, the finding that DS rats develop renal damage characterized by glomerulosclerosis,2,21 which appears to have an influence on APA activity, also leads us to presume a possible association of APA with the development or deterioration of salt-induced hypertension as well as renal injury. However, APA activity in DS rats has not been examined in the kidneys or elsewhere.
In this study, to examine our hypothesis that high-salt diet may reduce renal APA activity in DS rats and to assess the relationship between APA expression and glomerulosclerosis, we evaluated changes in renal APA activity and the histologic structure of the glomerulus in the kidneys of DS and of Dahl salt-resistant (DR) rats, which served as controls.
Materials and methods
Studies were commenced using 4-week-old male DS and DR rats maintained by Japan SLC Inc. (Shizuoka, Japan). Rats were housed in our animal facility on a 12-h light, 12-h dark cycle and given free access to food and water. A total of 42 rats of each strain were initially fed a low-salt diet (CE-2, CLEA Japan, Inc., Tokyo, Japan) containing 0.3% NaCl until the age of 6 weeks and then randomly divided into seven groups (n = 6 per group). Throughout the experiment, DS and DR rats of groups 2 to 4 were kept on a low-salt diet (0.3% NaCl); these rats were designated DS-0.3% and DR-0.3% rats, respectively. Those in groups 5 to 7 were fed a high-salt diet containing 8% NaCl (MF; Oriental Yeast, Tokyo, Japan) from the age of 6 weeks until the end of the experiment; these rats were designated as DS-8% and DR-8% rats. Rats were killed for biochemical and histochemical analyses at 6 weeks (group 1), 10 weeks (groups 2 and 5), 14 weeks (groups 3 and 6), and 18 weeks of age (groups 4 and 7). All experiments were approved by the local Animal Care Committee of Nagoya University Graduate School and were performed in accordance with the Guidelines for Animal Experimentation of Nagoya University.
Measurement of BP
Systolic BP (SBP) of conscious animals was measured with a pneumatic tail-cuff device (Softron BP98A, Tokyo, Japan) according to the method previously reported.6,7 The metal chamber was pre-warmed and maintained at approximately 37°C and the procedure was carried out once per week in all animals. The values obtained from five consecutive measurements were averaged and recorded as the BP for a given rat at each time point.
Rats were killed by decapitation, and the right kidney was rapidly removed and dissected on an ice-cold plate into cortex and medulla. One-half of each tissue sample was used for measuring Ang II; the samples were immediately homogenized on ice in an acid ethanol solution (80% vol/vol /0.1 N HCl) containing a mixture of peptidase inhibitors (0.1 mmol/L phenylmethylsulfonylfluoride, 25 mmol/L EDTA, 0.44 mmol/L o-phenanthroline, 1 mmol/L 4-chloromercuribenzonic acid, 0.12 mmol/L pepstatin A) and a renin inhibitor.20 Homogenates were centrifuged at 4°C for 10 min at 2000 g and the supernatants were stored at −80°C until assay.
The other half of each tissue sample was immediately frozen in liquid nitrogen and stored at −80°C until analysis for APA activity. The left kidney was used for histologic and immunohistochemical studies as described below.
Measurement of renal Ang II levels
Concentrations of Ang II in the kidney homogenates were measured by radioimmunoassay (SRL, Inc., Tokyo, Japan) and the values were corrected by protein concentrations assessed using the bicinchoninic acid (BCA) method.
Measurement of renal APA activity
Samples stored at −80°C were homogenized at 4°C in 0.25% buffered sucrose and centrifuged at 3000 g for 10 min to remove cellular debris. The supernatant was then centrifuged at 105,000 g for 20 min to collect crude membrane fractions. Each pellet was well dissolved in 50 mmol/L Tris-HCl (pH 7.5) with 0.2% Triton-X 100. Protein concentrations were measured using the BCA method. The APA activity in crude membrane preparations was determined as previously described.6,7 Briefly, L-glutamyl-p-nitroanilide was added to the samples as a substrate for APA, with incubation at 37°C for 30 min, and the optical density of the supernatant at 405 nm was then measured with a spectrophotometer (Shimadzu Co., Kyoto, Japan). The APA activity was expressed as picomoles of released p-nitroaniline per minute, designated for a unit and corrected with division by protein concentrations. All samples were measured in duplicate, including blank controls.
After rats were killed, the left kidneys were immediately fixed in 10% phosphate-buffered formalin. Fixed samples were embedded in paraffin, sectioned at 3 μm thickness, and stained with hematoxylin and eosin for histologic examination. Renal histopathologic changes were scored as previously described.21 For evaluating glomerular damage, >50 glomeruli per kidney were examined, and the percentage displaying damage was used as an index. Two investigators who were blinded to the group to which each rat belonged evaluated all tissue samples.
Renal tissue blocks embedded in paraffin were sectioned at 4-μm thickness and immunostained using rabbit anti-APA polyclonal antibody by means of the streptavidin-biotin-peroxidase method.22 After chromogenic development, the slides were then counterstained with Meyer's hematoxylin. Sections not exposed to primary antibody were always included as controls.
Data are means ± SD. Statistical analysis for comparison of data among groups was conducted with a one-way or two-way analysis of variance with a Scheffé post hoc test. A value of P < .05 was considered statistically significant.
Figure 1 illustrates age-related changes in SBP in DS and DR rats taking low- or high-salt diet. In all animals, at the age of 6 weeks SBP was approximately 130 mm Hg. High-salt diet induced systolic hypertension to levels of SBP >150 mm Hg in DS rats at 10 weeks and progressively increased SBP while these animals were on high-salt diets, whereas DS-0.3%, DR-0.3%, and DR-8% rats remained normotensive throughout the experiments. The SBP in DS-8% rats increased by 38.1 ± 6.8, 79 ± 8.8, and 113.6 ± 9.6 mm Hg at 10, 14, and 18 weeks of age, respectively.
Age-related changes in blood pressure in Dahl salt-sensitive (DS) and salt-resistant (DR) rats. Systolic blood pressure was measured weekly in DS and DR rats from 5 to 18 weeks of age. High-salt (8%) loading was started at the age of 6 weeks. *P < .05 v values in DS rats fed low-salt diets (DS-0.3% rats); †P < .05 v DR rats fed high-salt diets (DR-8% rats).
We then evaluated the influence of high-salt diet on renal APA activity in DS and DR rats (Fig. 2). Before taking each diet, renal APA activity of DS rats was nearly identical with that of DR rats. In contrast to the unaltered APA activity in DR-0.3% rats, APA activity in DR-8% rats was increased by approximately 3-fold at 10 weeks and remained so over the 8-week period. The DS rats at 10 weeks showed a trend similar to that in DR rats, indicating that APA activity was 2.5-fold greater in DS-8% rats than DS-0.3% rats. However, in contrast to the small increase in renal APA activity in DS-0.3% rats, further high-salt diet to DS-8% rats then decreased the activity after 10 weeks, which fell below the levels in DS-0.3% rats at 18 weeks.
Age-related changes in renal aminopeptidase A (APA) activity. The APA activity was measured in Dahl salt-sensitive (DS) rats fed 8%-salt (closed circles) and 0.3%-salt (open circles) as well as Dahl salt-resistant (DR) rats fed 8%-salt diet (closed squares) and 0.3%-salt (open squares). Data are mean ± SD values (n = 6 at each data point). *P < .05 v values in DS-0.3% or DR-0.3% rats; †P < .05 v values in DR-0.3% or DR-8% rats. Abbreviations as in Fig. 1.
We then measured renal Ang II levels in DS and DR rats (Fig. 3). At basal levels before taking high-salt diet, DR rats had higher renal Ang II levels than DS rats (321 ± 29 v 200 ± 20 fmol/g). In DR rats, high-salt intake suppressed renal Ang II levels significantly at 10 weeks (305 ± 15 v 212 ± 16 fmol/g), which could be also observed at 14 and 18 weeks. In DS rats, however, renal Ang II levels did not appear to decrease during maintenance of the high-salt diet.
Age-related changes in renal angiotensin II (Ang II) levels. Levels of Ang II were measured in Dahl salt-sensitive (DS) rats fed 8%-salt (closed circles) and 0.3%-salt (open circles) as well as Dahl salt-resistant (DR) rats fed 8%-salt (closed squares) and 0.3%-salt (open squares). Data are mean ± SD values (n = 6 at each data point). *P < .05 v values in DS-0.3% or DR-0.3% rats; †P < .05 v values in DR-0.3% or DR-8% rats. Abbreviations as in Fig. 1.
To investigate the influence of the high-salt diet on the kidneys, we assessed the histologic changes in kidney glomeruli. The DS-8% rats showed glomerulosclerosis with mesangial matrix increment at 10 weeks, which further advanced while these animals continued the high-salt diet, accompanied by expansion of glomerular size (Fig. 4A). Severe tubulointerstitial damage with tubular dilation and arteriosclerosis with thrombi were also noted in 18-week-old DS-8% rats (data not shown). The DS-0.3% and DR rats showed no histopathologic alterations in the kidneys at any time (data not shown). Figure 4C shows the percentage of glomeruli displaying fibrosis, hypercellularity, and hyalinization as a score of glomerular injury. In contrast to a few damaged glomeruli in DS-0.3% rats throughout the examination period, DS-8% rats showed marked increase in the population of injured glomeruli after 10 weeks. Fibrosis and hypercellularity could be observed in approximately 75% of all glomeruli in DS-8% rats at 10 weeks, which did not increase further until 18 weeks. Hyalinization increased from 25% at 10 weeks to 89% at 18 weeks in DS-8% rats.
Representative histologic sections (A) and aminopeptidase A immunostaining (B) of the glomeruli in Dahl salt-sensitive (DS) rats. Kidneys were obtained from DS rats before starting high-salt diet at the age of 6 weeks (a) and those fed high-salt diet (DS-8% rats) at 10 weeks (b), 14 weeks (c), and 18 weeks (d). Bars indicate 50 μm. (C) Percent ratio of damaged glomeruli with fibrosis (open bars), hypercellularity (closed bars), and hyalinization (hatched bars) in DS rats. Data are mean ± SD values (n = 6 at each data point). *P < .05 v values in DS-0.3% rats; #P < .05 v values in DS-8% rats at 10 weeks.
To gain insight into the mechanisms regulating renal APA activity in DS rats and to examine the relationship between the glomerular injury and APA expression, we then performed immunohistochemical studies of APA in the kidneys. The APA immunostaining in DS-8% rats was enhanced in glomeruli displaying mild damage with fibrosis or hypercellularity, especially in glomerular and visceral epithelium, at 10 weeks (Fig. 4B, a and b); in contrast, it was diminished in the severely injured glomeruli with advanced fibrosis or hypercellularity and was undetectable in lesions with hyalinization at 14 and 18 weeks (Fig. 4B, c and d). Immunoreactivity of APA also was found in the proximal tubules and vascular wall in DS-8% rats, which showed no apparent changes at any time points (data not shown). The APA immunostaining pattern in the glomeruli or elsewhere in DS-0.3% rats did not change throughout the experiment (data not shown).
The Dahl salt-sensitive rat, a model of human salt-sensitive hypertension, is known to have activated local RAS in the kidneys.17–19 In the present study, we aimed to examine whether low Ang II metabolizing activity is associated with the activated local RAS in DS rats. To test this hypothesis, we focused our attention on renal APA, an enzyme responsible for Ang II metabolism. Because APA is highly expressed in the glomeruli,20 which show sclerosal lesion in DS rats consuming a high-salt diet, we also evaluated the relation between renal APA expression and renal damage.
We first demonstrated that SBP was increased in DS-8% rats at 10 weeks and over the period of high-salt intake but was unaltered in DS-0.3% and DR rats, which was similar to previous reports.11,19 In contrast to our initial hypothesis that high-salt diet may reduce renal APA activity in hypertensive DS rats, high-salt diet initially increased renal APA activity in DS-8% rats at 10 weeks and similarly in normotensive DR-8% rats. However, further administration of high-salt diet caused diverse effects on DS and DR rats; after 10 weeks, high-salt diet decreased APA activity in DS rats, whereas it retained high APA activity in DR rats. The fact that high dietary salt increased renal APA activity in DR rats (which means enhanced metabolism of Ang II) may contribute to the mechanism of protecting DR rats from hypertension in adaptation to high salt consumption. The observed decrease in renal APA activity in DS-8% rats after 10 weeks, which was consistent with our hypothesis, seemed to suggest that failure to upregulate renal APA activity in response to dietary salt could partially account for the pathogenesis of salt-induced hypertension. In addition, the result that even low-salt diet showed an upward trend of renal APA activity in DS rats might also indicate a dysfunctional regulation of APA responding to dietary salt in DS rats.
We also assessed renal Ang II levels in DS and DR rats taking high- or low-salt diet. High-salt diet decreased renal Ang II levels in DR rats, whereas in DS-8% rats no decrease in renal Ang II was observed; this was difficult to integrate, at least in part, with the APA activity results. In contrast to the reasonable observation that renal Ang II levels in DR rats on high-salt diet were reduced concomitantly with an increase in APA activity, Ang II levels in DS-8% rats at 10 weeks did not decrease despite the increased APA activity. Although speculative, as demonstrated by the finding that high-salt diet increases intrarenal angiotensinogen levels in DS rats,19 it is possible that local RAS may produce excessive Ang II in the strain, overcoming the increase in APA activity. Furthermore, although renal Ang II levels might not always reflect the systemic BP, the relation between those and the BP profile also was perplexing. The Ang II levels in DS-8% rats showed no changes despite the progression of hypertension. Likewise, although DR-0.3% rats had higher Ang II levels than DS-8% rats and DR-8% rats, BP in DR-0.3% rats was normotensive and equivalent to that in DR-8% rats. One possible explanation for this discrepancy is that Ang II receptors or the signal pathways may be specifically regulated by dietary salt in each strain. Otherwise, Ang II levels may be differentially regulated within the intrarenal compartment, which have diverse effects on systematic BP, because we measured Ang II levels in the homogenates extracted from whole kidneys. In addition, we could not deny the possibility that factors other than RAS play more critical roles in regulating BP.
Our present data confirmed the previous report showing the increased glomerulosclerosis with advancing hypertension in DS rats2,21,23 and provided novel evidence that APA expression would be related to the severity of glomerulosclerosis. Kidneys in DS-8% rats showed glomerulosclerosis with mesangial matrix increment including mild fibrosis or hypercellularity at 10 weeks, which had enhanced APA immunoreactivity. Maintaining high-salt diet after 10 weeks further increased the number of injured glomeruli not only with fibrosis and hypercellularity but also with hyalinization. The APA staining decreased in the severely injured glomeruli and finally disappeared in lesions with hyalinization. The DS-0.3% rats showed no histopathologic renal injury and no apparent changes in APA immunostaining. The time-related change in the intensity of APA immunoreactivity in the kidneys was well consistent with that in renal APA activity in DS-8% rats. High-salt diet would enhance APA expression, while causing little or no damage to the kidneys. However, once glomerular injury became worse, high-salt diet could no longer maintain high APA expression. Our study is not able to determine whether the decrease in APA activity only reflects the severity of glomerulosclerosis or functions in the development of glomerulosclerosis through failure to metabolize Ang II. The inhibition of the RAS activity has been reported to attenuate glomerular injury independently of the antihypertensive effects in DS rats,21 suggesting that local Ang II plays a key role in the pathogenesis of glomerulosclerosis. In addition to raising glomerular capillary pressure, Ang II develops glomerulosclerosis through various mechanisms including enhanced cellular proliferation and overexpression of chemotactic factors and cell adhesion molecules.2,24–26 We therefore anticipated that reduced APA activity in DS-8% rats may be associated with deterioration of glomerulosclerosis and hypertension by enhancing these Ang II actions. In any event, the findings suggest that the sclerotic glomeruli progressively lose the ability to express APA. These results may also indicate the damaged Ang II signaling pathway in DS rats. Without salt loading, DS rats manifested a slowly progressive rise in APA accompanied by trivial degrees of glomerular hyalinization. This is what we might expect if these animals are adapting to a need to downregulate an overactive RAS. Yet, their Ang II levels were low throughout, at inelastic levels that could not be further lowered by high salt. It is as if Ang II receptors were locked in an “on” position, unresponsive to actual low Ang II levels that were maximally inactivated by rising APA. With salt loading, the effects might easily spill over to calcium channels, thereby setting off a fibrosis response. If this condition also prevails in the peripheral arterial tree, then it could explain the inexorable rise in BP in DS rats. Hence, the key defect in DS rats might be a constant inappropriate second messenger signaling by faulty Ang II receptors.
During the preparation of the present article, interesting results concerning the effects of high-salt diet on aminopeptidase N (APN) in DS rats were reported.27 The substance APN, an enzyme which further degrades Ang III converted from Ang II by APA, was abundantly expressed in the proximal tubules. High-salt diet for 10 days increased renal APN activity in Sprague-Dawley rats, whereas there was no change in DS rats, suggesting that increased APN activity may contribute to salt adaptation. Although the time points investigated were different between that study and ours, degrading activity of Ang II and its metabolite Ang III would be associated with salt sensitivity.
In conclusion, we have demonstrated that high-salt diet increased renal APA expression when renal injury was mild, but then decreased it along with the progression of glomerulosclerosis in DS rats only. Although further studies are required such as to examine whether APA reduction is causative or resultant of renal injury, reduced APA activity may be involved in the deterioration of salt-induced hypertension and renal injury.