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Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Correspondence to Dr. Hiroyuki Sasamura, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: +81-3-3353-1211; Fax: +81-3-3359-2745; E-mail: sasamura{at}mc.med.keio.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The efficacy of these inhibitors of the renin-angiotensin (R-A) system in ameliorating the disease attests to the importance of the R-A system in the pathophysiologic mechanisms of both hypertension and renal injury in this model. Indeed, several studies have suggested that renin levels are inappropriately high, considering the degree of hypertension in this strain, suggesting a causative role for the overactivated R-A system in the pathogenesis of the abnormalities in these rats, in comparison with normotensive control Wistar-Kyoto (WKY) rats (4,5).
Recent studies using knockout mice demonstrated that the R-A system also plays an important role in kidney development. Targeted deletion of components of the R-A system (angiotensinogen, ACE, or AT1 receptors) resulted in the development of kidneys with multiple abnormalities, including vascular hypertrophy, mesangial expansion, and tubular atrophy (for review, see reference 6). Interestingly, the same changes could be produced by treating newborn rats from 0 to 3 wk with ACEI or AT1R-Ant, suggesting that these 3 wk represent a critical time window for the developmental effect of the R-A system on kidney maturation (6).
In this study, we examined the effects of treatment of these rats with angiotensin inhibitors at a later stage of infancy, i.e., from weaning (3 wk) to puberty (10 wk), on the development of renal lesions at 6 mo, well after discontinuation of the treatments. Our results suggest that, unlike treatment from 0 to 3 wk, treatment from 3 to 10 wk suppresses the development of renal lesions in this model. Moreover, we found that permanent changes in the R-A system may be involved in the striking beneficial effects of these treatments.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Assays
The systolic BP and heart rate of conscious animals were measured by
tail-cuff plethysmography, using a Natsume KN-210 manometer (Tokyo, Japan).
Twenty-four-h urine collection was performed in metabolism cages, and urinary
protein concentrations were determined using an autoanalyzer. Rats were killed
by decapitation, and trunk blood was collected in prechilled beakers
containing 0.1 mM phenylmethylsulfonyl fluoride, 3.5 mM
ethylenediaminetetraacetate, and 0.12 mM pepstatin. Samples were immediately
centrifuged (3000 x g for 15 min) at 4°C and stored at
-20°C. Plasma renin activity (PRA) was determined by RIA of AngI formed by
incubation of plasma for 1 h at 37°C
(2). Plasma AngII levels were
analyzed by RIA as described previously
(8).
Histologic Studies
Kidneys and thoracic aortae were fixed in 10% phosphate-buffered formalin
and then embedded in paraffin blocks. Histologic sections from the rat kidneys
were stained with periodic acid-Schiff stain, and sections from aortae were
stained with Azan (Mitsubishi Kagaku, Tokyo, Japan). Slides were examined by
light microscopy, and renal histopathologic changes were scored as described
previously (2). For assessment
of glomerular damage, the number of glomeruli exhibiting focal or global
ischemic or proliferative damage was enumerated and expressed as a percentage
of the total number of glomeruli examined. Blood vessels were graded 0 to 4
for arteriolar sclerosis, on the basis of the severity of hyalinosis and
thickening of the vascular wall. Tubulointerstitial changes, including
interstitial inflammation and tubular atrophy, were assessed and graded 0 to
3, as follows: grade 1, involvement of <20% of the cortical interstitium;
grade 2, involvement of 20 to 40% of the interstitium; grade 3, involvement of
>40% of the interstitium.
Reverse Transcription-PCR Analysis of Renal Gene Expression
Total RNA was purified from the kidneys of five animals in each group,
using the acid guanidinium-phenol-chloroform method, and was quantified by
spectrophotometric measurement of absorbance at 260 nm. Renin and AT1 and AT2
receptor subtype mRNA were analyzed by reverse transcription-PCR (RT-PCR), as
reported by us previously
(2,9).
In brief, 1 µg of total RNA was reverse-transcribed in a reaction mixture
containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM
dNTP, 1 U of RNase inhibitor, 2.5 µM (50 pmol) random hexamers, and 2.5 U
of Moloney murine leukemia virus reverse transcriptase, in a volume of 20
µ1. The reverse-transcribed product was amplified with renin, AT1 receptor,
AT2 receptor, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense and
antisense primers, in a reaction mixture containing 10 mM Tris-HCl (pH 8.3),
50 mM KCl, 2 mM MgCl2, 0.2 mM dNTP, 15 pmol of each primer, 5
µCi of [32P]dCTP, and 2.5 U of Taq polymerase, using a
Perkin-Elmer-Cetus thermal cycler for 25 cycles. Renin primers were
5'-TGCCACCTTGTTGTGTGAGG-3' and
5'-ACCCGATGCGATTGTTATGCCG-3', corresponding to the sense and
antisense sequences of bases 851 to 870 and 1203 to 1224, respectively, in the
rat renin sequence. AT1 receptor primers were
5'-GGAAACAGCTTGGTGGTG-3' and
5'-GCACAATCGCCATAATTATCC-3', corresponding to the sense sequence
of bases 133 to 150 and the antisense sequence of bases 719 to 739 in the rat
AT1a and AT1b receptor sequences, respectively. AT2 receptor primers were
5'-ATGAAGGACAACTTCAGTTTTGC-3' and
5'-CAAGGGGAACTACATAAGATG-3', corresponding to the sense sequence
of bases 1 to 23 and the antisense sequence of bases 478 to 499, respectively.
GAPDH primers were 5'-TCCCTCAAGATTGTCAGCAA-3' and
5'-AGATCCACAACGGATACATT-3', corresponding to the sense sequence of
bases 451 to 470 and the antisense sequence of bases 739 to 758, respectively.
To assess the relative levels of AT1a and AT1b receptor-amplified PCR
products, the PCR products were incubated for 90 min at 37°C in the
presence of EcoRI (10 U). Because the AT1a receptor (but not the AT1b
receptor) contains an internal EcoRI site, EcoRI digestion
under these conditions results in two fragments (428 and 178 bp in length) in
the case of AT1a receptor DNA and one fragment (606 bp in length) in the case
of AT1b receptor DNA (2).
Levels of transforming growth factor-ß (TGF-ß) subtype mRNA were
assessed using primers specific for TGF-ß1, TGF-ß2, and TGF-ß3
isoforms, as described in detail by Gao et al.
(10). Preliminary experiments
confirmed that these PCR were performed within the linear phase of the PCR
amplification. Reaction products were resolved by electrophoresis through 8%
polyacrylamide gels. Gels were dried using a gel dryer, and incorporated
radioactivity in each band was quantified using a laser image analyzer (model
BAS 2000; Fuji Film Co., Tokyo, Japan).
Materials
Delapril and candesartan cilexetil were generously provided by Takeda
Chemical Industries (Osaka, Japan). RT-PCR and electrophoresis reagents were
obtained from Perkin Elmer (Branchburg, NJ) and Bio-Rad (Hercules, CA). Other
chemicals were from Sigma Chemical Co. (St. Louis, MO).
Statistical Analyses
Results are expressed as mean ± SEM. Statistical comparisons were
made by ANOVA, followed by Scheffé's
F test for comparisons between groups. Values of P < 0.05
were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After euthanasia of the rats at 30 wk, the hearts and aortae were examined. The heart weight/body weight ratios for the ACEI- and AT1R-Ant-treated groups were significantly (P < 0.05) decreased, compared with the other groups. However, no significant differences in the aortic wall thickness (media/lumen ratio) between the treated and untreated groups could be detected (Table 1).
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Effects of Prepubescent Treatment with Angiotensin Inhibitors on
Proteinuria and Renal Histologic Changes at 30 Wk in SHRSP/Izm Rats
Urinary protein levels were examined at 14 and 30 wk, as shown in
Figure 2a. At 14 wk,
proteinuria had not yet developed in SHRSP/Izm rats (comparative values for
normotensive WKY/Izm rats were 5.4 ± 1.3 mg/100 g per d). In contrast,
marked proteinuria was evident at age 30 wk for the control rats. This
development of proteinuria was completely suppressed in the ACEI- and
AT1R-Ant-treated groups (to 6.5 ± 1.2 and 7.9 ± 2.9 mg/100 g per
d, respectively), whereas no such suppression was observed in the
hydralazine-treated group.
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The histologic findings for the different groups are presented in Figure 2b. As evident from the histologic scoring, suppression of the glomerular, vascular, and interstitial changes were observed in the ACEI- and AT1R-Ant-treated groups, compared with either control or hydralazine-treated rats. Representative histologic sections are shown in Figure 3. In the control SHRSP and hydralazine-treated rats, prominent thickening of small to medium-sized arteries were observed. These changes were dramatically reduced in the ACEI- and AT1R-Ant-treated groups.
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Effects of Prepubescent Treatment with Angiotensin Inhibitors on PRA
and Plasma AngII Levels at 30 Wk in SHRSP/Izm Rats
PRA values for the different groups were as follows: control, 13.1 ±
2.4 ng/ml per h; ACEI, 6.0 ± 1.3 ng/ml per h; AT1R-Ant, 6.3 ±
0.6 ng/ml per h; hydralazine, 15.7 ± 6.7 ng/ml per h. These data
revealed that PRA was decreased to <50% in the angiotensin
inhibitor-treated groups. To confirm these findings, the changes in plasma
AngII levels were also assayed and yielded consistent results, as demonstrated
in Figure 4.
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Effects of Prepubescent Treatment with Angiotensin Inhibitors on
Renal Gene Expression at 30 Wk in SHRSP/Izm Rats
Renal expression of renin mRNA was examined using our previously reported
RT-PCR method. As presented in Figures
5 and
6, renin/GAPDH mRNA ratios were
reduced to <50% in the angiotensin inhibitor-treated groups. In contrast,
no such changes in the mRNA for angiotensin receptor subtypes (AT1a, AT1b, and
AT2) were detected. TGF-ß1 mRNA levels were also significantly
(P < 0.05) reduced in the angiotensin inhibitor-treated groups,
whereas no significant changes in TGF-ß2 or TGF-ß3 mRNA levels were
observed (Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Other studies have shown that the use of ACEI or AT1R-Ant during a later period (up to 2 mo after birth) can attenuate the full development of hypertension in spontaneously hypertensive (SHR) rats (12,13,14,15). Wu and Berecek (13) reported that treatment of newborn rats with the ACEI captopril for 2 mo also attenuated central responses to AngI and AngII, suggesting alterations in the central R-A system. In other studies, the same group also noted decreases in plasma arginine vasopressin (AVP) levels (16) and alterations in endothelial function in these rats (17). However, the exact mechanisms for the changes in BP are still unclear.
In this study, we focused on the effects of angiotensin inhibitor treatment of prepubescent rats on the nephrosclerosis observed in SHRSP rats. These rats were derived from the parent SHR strain by selection of rats with a propensity to develop stroke and malignant nephrosclerosis while receiving a high-salt diet (18). With a normal-salt diet, these rats do not develop fulminant hypertension but develop nephrosclerosis with histologic changes very similar to the changes observed in human benign hypertensive nephrosclerosis, including characteristic sclerosis of small to medium-sized arteries in the kidney. We previously showed that the renal changes are not evident at 14 wk but are well developed after 22 wk (2).
In our study, treatment of prepubescent rats in the developmental stage of hypertension (from 3 to 10 wk) resulted in a decrease in the BP plateau reached after 14 wk, consistent with the results of studies using the parent SHR strain. The fact that this phenomenon was observed with both ACEI and AT1R-Ant demonstrated that the effect was attributable to inhibition of angiotensin actions at the AT1 receptor. Although ACEI also decrease the degradation of bradykinin and diminish stimulation of AT2 receptors (19), the mirroring of ACEI effects by AT1R-Ant makes it unlikely that these actions were involved in the observed effects. Moreover, this effect was not observed with the vasodilator hydralazine, demonstrating that the decrease in BP per se during the treatment period was not the main cause of this phenomenon. Of interest, we found that the heart weight/body weight ratios were lower in the angiotensin inhibitor-treated groups but aortic wall hypertrophy was not attenuated by treatment with angiotensin inhibitors. These results are similar to those observed using SHR rats (20).
We next examined proteinuria and renal histologic lesions in the different groups. In the angiotensin inhibitor-treated groups, the levels of proteinuria were similar to the levels observed for normotensive WKY/Izm rats of the same age. In other words, the development of proteinuria was completely suppressed by the interventions. Histologic examinations also revealed dramatic improvements in the renal lesions. In particular, marked renal arteriolar hypertrophy was observed in both the SHRSP control and hydralazine-treated groups but was virtually absent in the ACEI- and AT1R-Ant-treated groups. The development of nephrosclerosis in SHRSP rats is known to be accompanied by increases in TGF-ß expression (3). In concert with the improvements in the histologic changes, reductions in TGF-ß1 mRNA levels were observed in the kidneys of the angiotensin inhibitor-treated rats in this study.
Next, we examined potential mechanisms for the observed changes. As noted in the introduction, the inhability to suppress renin activity has been implicated in the pathogenesis of the changes observed in SHRSP rats (4). In these rats, PRA increases progressively and is always significantly higher than that in normotensive WKY rats (5). We therefore examined the R-A system in these rats and found that both PRA and plasma AngII levels were significantly reduced, compared with control SHRSP rats. Similar results were found at the mRNA level, suggesting that the excess renin production characteristic of this strain was attenuated in the treated rats.
It has been demonstrated that high levels of renin and hence of AngII are associated with cerebrovascular, renal, and cardiac lesions in SHRSP rats and that there is a correlation between PRA and the severity of cerebrovascular (4) and renal (4,21) damage. Therefore, it is possible that the decreases in PRA and AngII levels observed in our angiotensin inhibitor-treated rats played an important role in the observed beneficial effects of treatments with these agents.
It is presently unclear why treatment from 3 to 10 wk of age caused a sustained decrease in R-A activity in these rats. As mentioned above, the time window of 0 to 3 wk after birth has been demonstrated to correspond to the time window for the actions of the R-A system on the development of normal kidney morphologic features. We speculate that the time period from 3 to 10 wk may include the time window for the maturation of the adult R-A system. One possibility is that the R-A system has a positive feedback effect on its own development at this stage; therefore, blockade of the R-A system during this time period may result in permanent attenuation of the R-A system. Interestingly, a recent study by St. Lezin et al. (22), using congenic SHR strains, demonstrated that the susceptibility to hypertension-induced renal damage in SHR rats is genetically determined by a region on chromosome 1q. It would be interesting to determine whether genes affecting the activity and maturation of the R-A system are located in this region.
An important issue concerns the cause-and-effect relationship between nephrosclerosis and BP. The fact that hypertension precedes the development of nephrosclerosis in this model (2) suggests that hypertension is the cause of the renal damage, not vice versa. The question of whether the reduction in BP per se in the ACEI- and AT1R-Ant-treated rats contributed to the amelioration of the renal changes thus arises. Interestingly, Nakamura et al. (3) administered hydralazine to SHRSP rats from 12 to 24 wk of age and reduced the BP to approximately 160 mmHg, which is similar to the values for our ACEI- and AT1R-Ant-treated groups. Those authors found that BP reduction was effective in reducing both proteinuria and histologic changes in their rats, which suggests that the lower BP in our treated groups played a significant role in the suppression of nephrosclerosis. However, the same group found that treatment with AT1R-Ant from 12 to 24 wk had a greater effect in abolishing the nephrosclerotic changes, suggesting that the combination of BP reduction and R-A system inhibition had the greatest nephroprotective effect. These findings are compatible with the assumption that BP reduction played a significant role in the attenuation of renal changes in our rats, with further protection being afforded by suppression of the R-A system.
Another interesting question is why the BP increased to reach values comparable to those for control untreated animals after the cessation of hydralazine therapy. Both Christensen et al. (23) and King et al. (24) reported a washout period for hydralazine of approximately 2 to 3 wk in rats, after which the hypotensive effect disappears. This probably explains why the effects of hydralazine were only temporary in this study. A related question is why the ACEI and AT1R-Ant had longer effects on BP reduction. One possibility is related to the "vascular amplifier" hypothesis. This hypothesis relates the establishment of hypertension to a positive feedback loop involving vascular hypertrophy and increases in BP, and it is the subject of ongoing debate (25). Interestingly, Adams et al. (15) reported that treatment of SHR rats either from 4 to 9 wk or from 4 to 14 wk resulted in long-term attenuation of SHR rat hindquarter resistance properties to values similar to those for normotensive WKY rats. Those authors proposed that the prevention of peripheral vascular changes in the young rats by the angiotensin inhibitors during this susceptible period could inhibit the development of the vascular amplifier mechanism, resulting in the observed long-term reductions in BP. However, Morton et al. (14) reported that treatment of SHR rats with either captopril or losartan from 3 to 7 wk of age resulted in decreased BP in the absence of changes in the mesenteric resistance artery media/lumen ratio. Those authors concluded that the persistent hypotensive effect was not related to the vascular structural changes. Therefore, the precise role of the vascular amplifier mechanism in the observed changes remains unclear. As mentioned above, Berecek and co-workers (13,16,17) have postulated that alterations in the central R-A system, decreases in plasma AVP levels, and alterations in endothelial function may be involved in the persistent hypotensive effect. These possibilities are not mutually exclusive, and we speculate that changes in the central and peripheral R-A systems, changes in the levels of other hormones such as AVP, and vascular functional and structural changes could all contribute to the sustained antihypertensive and nephroprotective effects of angiotensin inhibitors administered during the critical period in rat maturity.
One important clinical implication of this study is the possibility that patients with genetic susceptibility to renal disease might be treated for their innate susceptibility by intervention at an appropriate stage, before the signs of the disease become apparent. The timing of this intervention may be quite early, i.e., during the preadolescent stage. Of interest, studies by Raizada and co-workers (26,27) showed that a single injection of a retroviral vector containing antisense sequences for ACE or the AT1 receptor could cause permanent blockade of the R-A system. Moreover, the antisense sequence is integrated into the genome and is then transmitted to offspring, resulting in blockade of the R-A system in the F1 and F2 generations (26). Although these methods are effective in providing permanent cardiovascular protection in both the parents and the offspring (27) and offer possibilities for a total cure for hypertension and its sequelae, the effects may be too drastic and long-lasting for use in human subjects. The administration of orally active compounds for a limited time may be a more acceptable option. Obviously, it is not presently possible to extrapolate our results to other types of renal disease. However, the results do provide us with reasons to think that, in the future, genetic susceptibility to renal disease may be treated with appropriate interventions at an earlier stage of development. Because several renal diseases, including the most common autosomal dominant disease (polycystic kidney disease), have strong genetic components, we speculate that earlier intervention, perhaps with angiotensin inhibitors, may shift the therapeutic paradigm for renal disease away from the management of established disease and toward more effective disease prevention.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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