INTRODUCTION

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HYPERTENSION IS A MAJOR risk factor for renal injury; however, the mechanism underlying the development and progression of renal damage in hypertensive patients and experimental animals remains to be elucidated. ANG II, the main molecular effector of the renin-angiotensin system (RAS) and one of the most important regulators of systemic blood pressure, controls vasoconstriction, the facilitation of central sympathetic outflow and peripheral neurotransmission, and the release of vasopressin and aldosterone and has direct volume-retaining effects (12). ANG II also exhibits growth-promoting effects, particularly on the renal cells (31). Two major types of ANG II receptors (type 1 and type 2) have been identified and cloned to date (11, 16). The hemodynamic and nonhemodynamic effects of ANG II, however, are mediated primarily by the ANG II type 1 receptor (AT₁) (5, 28). Because of the pivotal role of ANG II, pharmacological blockade of the RAS recently has become a mainstay in the treatment of renal and cardiovascular diseases. The effects of RAS-inhibiting agents, such as ANG I-converting enzyme inhibitors (ACEI) and AT₁ antagonists (AT_1a), on histological and molecular renal alterations have been studied in vivo in various experimental models of hypertension (21, 22). We have reported previously that enhanced expression of the platelet-derived growth factor (PDGF) B-chain mRNA in the glomeruli preceded the appearance of histological changes in spontaneously hypertensive rats (SHR) and that the glomerular PDGF B-chain mRNA was suppressed by the administration of the ACEI cilazapril (33). In addition, treatment with cilazapril or the AT_1a, L-158,809, attenuated the expression of PDGF B-chain and transforming-growth factor (TGF)-1 mRNA in the glomeruli of deoxycorticosterone acetate (DOCA) salt-treated hypertensive rats without reducing blood pressure (25). Thus the renoprotective effects of RAS inhibitors are mediated in part by unknown mechanisms and are independent of the antihypertensive effects of the agents. To investigate the role of RAS on the hypertension and renal injury in salt-sensitive hypertension, we have evaluated the effects of chronic RAS inhibition on glomerular injury in Dahl salt-sensitive rats using an ACE inhibitor and an AT₁-receptor antagonist.

	METHODS

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Materials. Dahl-Iwai salt-sensitive (DS) and salt-resistant (DR) rats (5 wk old) were purchased from Japan SLC (Shizuoka, Japan). All rats were housed in climate-controlled metabolic cages with a 12:12-h light-dark cycle. The animals received a low (0.3%)-NaCl or high (8%)-NaCl diet (MF, Oriental Yeast, Tokyo, Japan), with water provided ad libitum.

Oligonucleotides for RT-PCR were synthesized with a model 380B DNA synthesizer (Applied Biosystems, Foster City, CA). The location of the oligonucleotides of primer pairs were as follows: PDGF B-chain, 1079-1098 and 1601-1620 (6); TGF-1, 1679-1699 and 1994-2014 (9); and PCNA, 197-216 and 715-734 (27). Cilazapril, an ACEI, and TCV-116, an AT_1a, were donated by Eisai Pharmaceutical (Tokyo, Japan) and Takeda Chemical Industries (Osaka, Japan), respectively. These drugs were dissolved in a 2% gum arabic solution.

Experimental protocol. At 7 wk of age, the DS rats were divided randomly into two groups. One group received a diet containing 0.3% NaCl (DSL; n = 6), the other group received a diet containing 8% NaCl (DSH) for 6 wk. The DSH rats were further divided into three groups that were given oral cilazapril (10 mg · kg¹ · day¹ in 0.3 ml of saline; cilazapril + DSH; n = 6), TCV-116 (1 mg · kg¹ · day¹ in 0.3 ml of saline; TCV + DSH; n = 6), or 0.3 ml of saline (control DSH; n = 14) once a day using gavage during this experimental period. A control group of DR rats also was fed a 0.3% NaCl diet (DRL; n = 6). Systolic blood pressure (SBP) and heart rate (HR) were measured weekly, at 9 AM, in conscious, restrained, and warmed rats using tail-cuff plethysmography (UR-5,000, Ueda Seisakusyo, Tokyo, Japan). Once a week, urine was collected over a 24-h period and used for the measurement of urinary protein excretion, N-acetyl--glucosaminidase (NAG) activity, and aldosterone excretion. Urinary protein excretion and NAG activity were determined using a Bio-Rad protein assay kit (Bio-Rad, Richmond, CA) and NAG test pack (Shionogi Pharmaceutical, Osaka, Japan), respectively. Urinary aldosterone excretion was determined using a SPAC-S-Aldosterone RIA kit (Daiichi Radioisotope, Tokyo, Japan). The rats were killed by decapitation after the 6-wk treatment, and the kidneys were removed quickly for histological assessment and RNA extraction. Trunk blood samples were collected and stored at 30°C until they were assayed for electrolytes, blood creatinine, uric acid, and total protein by an autoanalyzer system. Plasma renin activity (PRA) was determined by RIA for ANG I (Dinabot Radioisotope, Tokyo, Japan).

Histological examination. For observation by light microscopy, a portion of each kidney was fixed in 10% buffered paraformaldehyde, embedded in paraffin, sectioned into 4-µm slices, and stained with periodic acid-Schiff (PAS) reagent. The diameters (µm) of 200-300 randomly selected glomeruli in each experimental group were measured using an objective micrometer (OB-M, Olympus Optical, Tokyo, Japan). Histograms of glomerular cross sections were compared among the five groups. The severity of glomerular sclerosis in the five groups was assessed using a mesangial injury score (26). A minimum of 100 glomeruli in each specimen was examined, and lesion severity was graded from 0 to 4+, according to the percentage of glomerular involvement. Thus a 1+ lesion represented an involvement of 25% of the glomerulus, whereas a 4+ lesion indicated that 100% of the glomerulus was involved. An injury score was obtained by multiplying the degree of damage (0 to 4+) with the percentage of the glomeruli with that type of injury (i.e., increase in mesangial matrix material or glomerulosclerosis). The extent of the injury in each tissue specimen then was calculated by adding these scores. In addition, glomerular cellularity was determined by counting total nuclear cells in each glomerulus (at least 100 glomeruli in each specimen). For immunofluorescence analyses, a portion of each kidney was immersed in OCT compound (TISSUE-TEK, Miles Scientific, London, UK) for flash freezing at 20°C in an N-hexane-dry ice-acetone bath. Thereafter, 5-µm sections were cut using a cryostat and stained by indirect immunofluorescence. The sections first were stained with rabbit anti-rat type III collagen antibody (Chemicon International, Temecula, CA), rabbit anti-bovine type IV collagen antibody (LSL, Tokyo, Japan), and rabbit anti-bovine type VI collagen antibody (LSL) for 60 min at room temperature. The samples then were incubated with a FITC-labeled anti-rabbit IgG antibody for 30 min at room temperature and examined using a fluorescence microscope (Olympus, Tokyo, Japan).

Separation of glomeruli and extraction of total RNA. The renal cortex was dissected out and minced in ice-cold PBS. Glomeruli were isolated using the graded sieving technique. The isolated glomeruli were rinsed with ice-cold PBS and treated with 2 mg/ml collagenase (Wako Pure, Osaka, Japan) in RPMI-1640 medium for 30 min at 37°C. Total RNA was extracted by the acid-guanidium-phenol-chloroform method. The final RNA pellets were washed with 70% ethanol and resuspended in 100 µl diethylpyrocarbonate-treated water. The RNA was quantified by measuring the absorbance at 260 nm and stored at 20°C until the assay.

Quantification of RNA expression by competitive RT-PCR. First, mutant cDNAs for competitive PCR were generated using the PCR MIMIC Construction Kit (Clontech, Palo Alto, CA). The mutant fragments for G3PDH and TGF-1 messages were 556-bp fragments long, and the mutant fragments for the PDGF B-chain and PCNA messages were 276-bp fragments long. Subsequently, the fragments were reamplified with four sets of specific primers to determine their ability to act as competitors for the native mRNAs. The obtained products were as follows: G3PDH, 596 bp; TGF-1, 598 bp; PDGF B-chain, 316 bp; and PCNA, 316 bp. These fragments were purified using a CHROME SPIN Column (Clontech) and diluted to 100 amol/µl with 10 µg/µl ultrapure glycogen.

Extracted glomerular RNA was reverse transcribed using a GeneAmp RNA PCR kit (Perkin Elmer Cetus, Norwalk, CT). RT was performed with 10 ng of RNA per reaction using random hexamer (2.5 µM), reverse transcriptase (2.5 U/µl), and deoxynucleotide triphosphate (dNTP; 1 mM) at the following conditions: 42°C for 55 min, 99°C for 5 min, and 4°C for 5 min. The resulting cDNA was resuspended in 50 µl deionized, autoclaved water for competitive PCR analysis. The linear portion of the relationship between the native cDNAs and competitive mutant cDNAs then was determined for G3PDH, TGF-1, PDGF B-chain, and PCNA. For a preliminary analysis, a fixed amount (0.5 ng) of cDNA derived from control DSH RNA was coamplified with tenfold serial dilutions (1-10⁶) of the competitive mutant cDNAs using the four primer sets and the preceding PCR kit. For fine-tuned competitive PCR, another reamplification was performed using twofold serial dilutions (2¹-2⁹) of one of the dilution step of the preliminary PCR as a starting point (G3PDH, 10⁴; TGF-1, 10⁵; PDGF B-chain, 10⁴; PCNA, 10²). The competitive PCR was performed using the preceding PCR kit and a thermal cycler (TP Cycler-100, Toyobo, Osaka, Japan) under the following conditions: G3PDH and PDGF B-chain, 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min; TGF-1, 40 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min; and PCNA, 37 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min. Aliquots of the PCR products of cDNA and competitive mutant cDNA were electrophoresed on 1.5% agarose gels, visualized by ethidium bromide staining, and photographed using an instant positive/negative film (337; Polaroid, Cambridge, MA). The negatives were analyzed by a scanning densitometer (Scanning Imager 300-SX, Molecular Dynamics, Sunnyvale, CA), and the relative integrated density of each band was calculated by taking the absorbance multiplied by the surface area. Finally, the ratios between the densitometric readings of the native cDNA- and mutant cDNA-PCR products were plotted using logarithmic scale on the y-axis against the logarithmic dilutions of the mutant cDNA on the x-axis. After establishment of the working ranges in which a linear relationship existed, cDNAs from all individual samples were subjected to competitive PCR analysis, using the TGF-1, PDGF B-chain, and PCNA primers. Control analyses were carried out using G3PDH primers.

Statistical analysis. All results were expressed as means ± SE and statistically analyzed by ANOVA. P values <0.05 were accepted as statistically significant.

	RESULTS

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Time courses of SBP, HR, and body weight. In the control DSH group, the SBP increased markedly with age and was significantly higher than in the DRL and DSL animals throughout the ages of 7-13 wk and 8-13 wk, respectively (Fig. 1). The cilazapril + DSH and TCV + DSH animals generally did not exhibit significantly reduced SBP compared with the control DSH animals, except for 11-wk-old TCV + DSH animals. Moreover, no significant difference in SBP existed between the cilazapril + DSH and TCV + DSH groups. The HR was significantly lower in the DRL animals compared with the control DSH animals. Neither cilazapril nor TCV-116 treatment significantly altered the HR during the experimental period (data not shown). The animals' body weights throughout the observation period were as follows: DRL > DSL > control DSH > TCV + DSH > cilazapril + DSH (data in 13-wk-old animals are shown in Table 2). During the experimental period, the mortality rate in each group was as follows: control DSH, 14.3%; DSL, DRL, cilazapril + DSH, and TCV + DSH, 0%.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effects of salt-sensitive hypertension and treatment with renin-angiotensin system inhibitors on systolic blood pressure (SBP). In Dahl salt-sensitive (S) rats receiving a high-salt diet (control DSH; n = 12) SBP increased with age and was significantly higher than in low-salt-fed Dahl salt-resistant rats (DRL; n = 6) and Dahl S rats (DSL; n = 6) between the ages of 7-13 and 8-13 wk, respectively. In DSH animals treated with cilazapril (Cilaza + DSH; n = 6) or TCV-116 (TCV + DSH; n = 6), SBP did not decrease significantly, except at 11 wk. No significant differences existed between Cilaza + DSH and TCV + DSH animals. Bars represent mean ± SE. * P < 0.05 vs. control DSH; # P < 0.05 vs. DRL.

Changes in urine volume, urinary protein, and NAG excretion. The urine volume was highly increased in the control DSH animals compared with the DSL and DRL animals (Fig. 2A). The increase in urine volume also occurred in cilazapril + DSH and TCV + DSH animals. Especially in the cilazapril + DSH animals, the urine volume tended to increase between the ages of 9 and 12 wk. Both urinary protein and NAG excretion was significantly increased in control DSH animals compared with DRL and DSL animals (Fig. 2, B and C). TCV-116 treatment significantly reduced the increase in urinary protein and NAG excretion in the DSH animals. Cilazapril treatment did not significantly decrease these two parameters compared with control DSH animals.

View larger version (15K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of salt-sensitive hypertension and treatment with renin-angiotensin system inhibitors on urine volume, urinary protein, and N-acetyl--glucosaminidase (NAG) excretion. For a description of experimental groups, see Fig. 1. Control DSH animals exhibited an extremely elevated urine volume compared with DRL and DSL animals (A). In Cilaza + DSH and TCV + DSH animals, the urine volume varied but tended to increase in Cilaza + DSH animals between the ages of 9 and 12 wk. Urinary protein excretion in the control DSH group increased significantly compared with the DRL and DSL groups (B). Increase of urinary protein excretion was significantly lower in TCV + DSH animals than in control DSH animals. Urinary NAG excretion, which was significantly elevated in control DSH animals, also was suppressed in TCV + DSH animals (C). Bars represent mean ± SE. * P < 0.05 vs. control DSH; # P < 0.05 vs. Cilaza + DSH.

Evaluation of renal function and RAS. The effects of a high-salt diet and treatment with cilazapril or TCV-116 on numerous indicators of renal function were evaluated at the end of the experimental period (i.e., in 13-wk-old animals). The levels of serum sodium, potassium, chloride, total protein, and urea nitrogen in the trunk blood did not differ significantly among the groups (Table 1). The serum creatinine levels, however, were significantly elevated in control DSH animals compared with the DSL and DRL animals. Treatment with cilazapril or TCV-116 prevented this elevation. Serum uric acid levels were lower in the DSL group than in the control DSH group. The PRA level under TCV-116 treatment was increased significantly above those of control DSH animals, which was rather elevated compared with DSL or DRL despite the high-salt treatment. Finally, urinary aldosterone levels were analyzed in 7- and 12-wk-old animals (Table 1). DRL animals that had received a low-salt diet for 5 wk demonstrated markedly increased aldosterone levels. A high-salt diet, in contrast, reduced urinary aldosterone concentrations to undetectable levels, independent of the treatment with cilazapril or TCV-116.

Organ weights and body weight ratios. We also analyzed the body weights and organ weights of the five groups at the end of the experimental period. The kidney weights were significantly lower among DSL, DRL, and cilazapril + DSH animals compared with the control DSH animals (Table 2). The kidney weights of TCV + DSH animals, however, were significantly higher than those of the cilazapril + DSH animals. Heart weight was significantly lower in the DSL and DRL groups than in the control DSH group and was not affected by cilazapril or TCV-116. Kidney weight-to-body weight and heart weight-to-body weight ratios were significantly lower in DSL and DRL groups than in the control DSH group, and these ratios were not affected by either drug.

Histological findings. The glomerular structures in the five experimental groups were compared using light microscopy (Fig. 3). DSL and DRL animals showed normal glomeruli. Control DSH animals, in contrast, exhibited severely damaged glomeruli characterized by mesangial expansion, increases in the mesangial matrix accompanied by sclerotic changes, and cell proliferation. In animals treated with cilazapril or TCV-116, the extent of the glomerular injury was markedly decreased compared with the control DSH animals. These findings were confirmed by mesangial-injury scoring and analysis of glomerular cellularity. Both of these parameters were elevated in the control DSH animals compared with DSL and DRL animals (Table 3). Moreover, the glomerular cellularity was significantly lower in DRL than in DSL animals. Treatment with cilazapril or TCV-116 significantly improved both indicators of glomerular injury to the same extent. We also examined the glomerular sizes in the five groups. The glomerular size distribution indicated that the glomeruli in DRL animals tended to be smaller and those in TCV + DSH animals tended to be larger among the five experimental groups (Fig. 4). Finally, as a indicator of the extracellular matrix (ECM), glomerular collagen (type III, IV, VI) expression was assessed by immunofluorescence. Among the three types of collagens examined, type III collagen was strongly expressed in the mesangial region of control DSH animals, but not of DSL and DRL animals (Fig. 5). Treatment with TCV-116 markedly suppressed the expression of type III collagen. The expression of type IV and VI collagens did not differ significantly among the five groups.

View larger version (122K): [in this window] [in a new window]   Fig. 3.   Effects of salt-sensitive hypertension and treatment with renin-angiotensin system inhibitors on glomerular structure. For a description of experimental groups, see Fig. 1. Light microscopy of glomeruli in each group demonstrated that control DSH animals (A) exhibited severely damaged glomeruli characterized by mesangial expansion, increased mesangial matrix and sclerotic changes, and cell proliferation. DSL (B) and DRL (C) animals showed almost intact glomeruli. In Cilaza + DSH (D) and TCV + DSH (E) animals, glomerular injury was much less severe than in control DSH animals.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effects of salt-sensitive hypertension and treatment with renin-angiotensin system inhibitors on glomerular diameter. For a description of experimental groups, see Fig. 1. Frequency distribution of glomerular size demonstrated the tendency that DRL animals had smaller and TCV + DSH animals had larger glomeruli among the experimental groups.

View larger version (145K): [in this window] [in a new window]   Fig. 5.   Analysis of glomerular collagen expression by immunofluorescence. For a description of experimental groups, see Fig. 1. Type III collagen (top row) was strongly expressed in the mesangium of control DSH animals (A) but not in the glomeruli of DSL (B) and DRL animals (C). In comparison with the cilazapril (D) treatment, TCV-116 (E) markedly suppressed the expression of type III collagen in DSH. Expression of type IV (middle) and VI (bottom) collagens did not differ significantly among the 5 groups.

Quantification of glomerular expression of PDGF B-chain, TGF-1, and PCNA mRNAs. To quantify the glomerular expression of PDGF B-chain, TGF-1, and PCNA by competitive RT-PCR, we first determined the linear range of the ratios of coamplified mutant cDNAs and native cDNAs reverse transcribed from glomerular RNA as described in METHODS (Fig. 6A). For the quantification of all native cDNA samples, we chose the following logarithmic dilutions of the mutant cDNAs: 2⁷ (7.8 × 10³ amol/µl) for G3PDH, 2⁵ (3.125 × 10⁴ amol/µl) for PDGF B-chain, 2⁵ (3.125 × 10⁵ amol/µl) for TGF-1, and 2⁶ (1.56 × 10⁴ amol/µl) for PCNA. For the competitive PCR reactions, 2 µl of these dilutions were added to 2 µl of each native cDNA (0.25 ng/µl). The resulting PCR products were quantified by densitometric scanning as described in the METHODS (Fig. 6B). The results demonstrated that control DSH animals exhibited significantly enhanced levels of glomerular PDGF B-chain and PCNA mRNAs than did DSL and DRL animals. TCV-116 treatment significantly reduced the levels of glomerular PCNA and TGF-1 compared with the control DSH group. The PDGF B-chain level reduced by TCV-116 treatment but not significantly. Cilazapril treatment also reduced the levels of glomerular PDGF B-chain, TGF-1, and PCNA compared with control DSH animals, but these differences were not statistically significant. G3PDH levels did not differ significantly among the groups.

View larger version (41K): [in this window] [in a new window]   View larger version (43K): [in this window] [in a new window]   Fig. 6.   Quantification of glomerular mRNA expression by competitive RT-PCR. For a description of experimental groups, see Fig. 1. A: linear range in the ratios of coamplified PCR products was determined using native cDNA that was obtained by reverse transcription of glomerular RNA from a control DSH animal and competitive mutant cDNAs for G3PDH, platelet-derived growth factor (PDGF) B-chain, transforming growth factor (TGF)-1, and proliferating cell nuclear antigen (PCNA) at serial logarithmic dilutions of 21-29. B: for quantification of native cDNA samples, competitive PCR was performed by mixing a constant amount (2 µl) of native cDNA (0.25 ng/µl) with the following dilutions of mutant cDNAs: 27 (7.8 × 103 amol/µl) for G3PDH, 25 (3.125 × 104 amol/µl) for PDGF B-chain, 25 (3.125 × 105 amol/µl) for TGF-1, and 26 (1.56 × 104 amol/µl) for PCNA. Densitometric quantification of the PCR products demonstrated that control DSH animals exhibited significantly higher levels of glomerular PDGF B-chain and PCNA than did DSL and DRL animals. TCV-116 treatment significantly reduced the levels of glomerular PCNA and TGF-1 compared with the control DSH animals. G3PDH levels did not differ significantly among the groups. Bars represent means ± SE. Representative blots are shown above each graph, lane 1-5, control DSH, DSL, DRL, Cilaza + DSH, and TCV + DSH. * P < 0.05 vs. control DSH; ** P < 0.01 vs. control DSH. MM, molecular wt marker.

	DISCUSSION

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Dahl rats are considered a useful model of human salt-sensitive hypertension, because high dietary sodium levels exaggerate the development of hypertension in strains that are genetically predisposed to hypertension (7). Although the pathogenesis of salt-sensitive hypertension in these animals remains to be elucidated, genetic factors (7), as well as nitric oxide (14), have been implicated in determining the individual's sensitivity to salt ingestion. Several lines of evidence implicate the kidney as a primary determinant of arterial blood pressure in Dahl rats (8). The mechanism underlying hypertensive renal damage, including nephrosclerosis, however, is unknown, as are the factors that contribute to the progression of hypertension-related nephro- and glomerulosclerosis. We therefore investigated the potential therapeutic effects of RAS inhibition by administration with either ACEI (cilazapril) or AT_1a (TCV-116) to examine the role of RAS in glomerular damage observed in high-salt-treated DS rats. Some of our experimental results, the histological analyses and analysis of ECM expression patterns by immunofluorescence, demonstrated that salt-sensitive hypertension was complicated with significant glomerular injury. In contrast, the inhibition of the RAS by both cilazapril and TCV-116 significantly attenuated these histological changes using a light microscope. The renoprotective effects of these agents were independent of their antihypertensive actions. These findings suggest that glomerular injury in salt-sensitive hypertension is attributable to the activation of RAS.

In vitro analyses found that, among the components of the RAS, ANG II directly stimulates the growth of renal mesangial cells via AT₁ receptors (5). In in vivo models of renal disease caused by excessive ANG II expression, mesangial matrix expansion rather than cell proliferation usually occurs, indicating that the increased protein synthesis is a dominant response of mesangial cells to ANG II in vivo (26). Moreover, in vivo transfection of the genes for renin and angiotensinogen into the glomeruli has been shown to induce mesangial matrix expansion (2). These observations suggest that the increased protein synthesis by mesangial cells in response to ANG II may play a key role in the development of glomerulosclerosis. This fibrogenic effect of ANG II likely is mediated by TGF- (20). Transfection of TGF-1 into the kidney induces glomerulosclerosis characterized by the accumulation of ECM proteins, supporting the hypothesis that an increase in TGF-1 expression also is responsible for glomerulosclerosis in vivo (17). Renal TGF-1 gene expression reportedly is enhanced in several renal disease models, including glomerulonephritis (4) and obstructive nephropathy (19), as well as in some models of hypertensive nephropathy, including DOCA salt hypertensive rats (21) and spontaneously hypertensive stroke-prone (SHRSP) rats in the malignant phase (22). However, the role of TGF-1 in salt-sensitive hypertension has not yet been determined. In this study, we detected the enhanced levels of TGF-1 and type III collagen in the glomeruli of rats with salt-sensitive hypertension. Conversely, RAS inhibition by TCV-116 significantly reduced the glomerular TGF-1 level accompanied by the suppression of type III collagen without antihypertensive effect. The TCV-116 treatment also significantly suppressed the mRNA level of glomerular PCNA. PCNA is a nuclear protein that is expressed from late G1 through the M phase of the cell cycle (27). PCNA expression previously has been shown by immunostaining to be elevated in ANG II-infused Sprague-Dawley rats (18). These findings indicate that RAS may contribute to glomerular cell proliferation in salt-sensitive hypertension in vivo. This hypothesis is consistent with the change of glomerular cellularity due to RAS blockade using TCV-116. We further investigated the glomerular mRNA levels of the PDGF B-chain, a growth factor that accelerates mesangial cell proliferation (1). The PDGF B-chain mRNA levels were increased significantly in animals with salt-sensitive hypertension. In contrast to the change of PCNA level, however, RAS inhibition did not significantly alter the glomerular PDGF B-chain level. These findings suggest that cell proliferation in high-salt-treated DS rats may be associated with factors other than the PDGF B-chain, at least in this chronic hypertensive phase.

High-salt treatment usually suppresses the renin secretion from the juxtaglomerular apparatus; however, it is noted that the suppression of PRA caused by high-salt intake is blunted in DS rats compared with DR rats in the literature (10). Von Lutterotti et al. reported that PRA gradually increases after 4 wk of high salt loading in DS rats, presumably secondary to the glomerulosclerosis and renal arterial injury observed in salt-loaded DS rats with hypertension (29). In our experiment, the period of blood sampling for PRA was 6 wk after the initiation of high-salt treatment. If the sampling for renin were right after the high-salt treatment, the suppression of PRA might be apparent. The chronic high-salt-treated DS rats in our study showed markedly elevated blood pressure and histopathologically severe arterial and renal damages. When the data are taken into consideration, it is presumable that the RAS is one of the major hypertensinogenic factors related to the kidney of DS rats in the chronic phase. Furthermore, PRA was significantly elevated in the TCV-116 group more than the cilazapril group. This finding was also agreeable with the results of the previous literature, which had demonstrated the other hypertension model, with DOCA salt hypertension (21) or SHRSP rats (22), indicating that the AT₁ receptor plays a role in the negative feedback regulation of renal renin secretion and that AT_1a is a more potent inhibitor of this regulatory mechanism than ACEI, although neither drug reduced the SBP. The urinary aldosterone excretion of DR rats was increased after 5 wk on a low-salt diet, whereas it was suppressed completely in DS rats receiving a high-salt diet; however, the differences between the RAS-inhibitory ways by ACEI and AT_1a were not found as difference of urinary aldosterone level. The suppression of aldosterone secretion in high-salt-treated DS rats was probably associated with not only the renin-ANG II-aldosterone cascade but also adrenocortical suppression due to the extremely hypertensive state during the chronic experimental period. Additionally, the blockade of adrenocortical AT_1B receptors, which stimulate aldosterone secretion, is also involved in the suppression of aldosterone under the TCV-116 treatment (30).

Although the degree of the histological improvement of the glomerular injury between ACEI and AT_1a is not significantly different, the glomeruli in the TCV + DSH or cilazapril + DSH group have been damaged compared with those in the low-salt-treated groups. During our experimental period, the effect of AT_1a, including significant suppression of glomerular TGF-1 and ECM levels, did not seem to take advantage of the histologically renoprotective effect in this salt-sensitive hypertension model; however, in the AT_1a group, the reduction of proteinuria and the attenuation of urinary NAG excretion, which also indicates the protection of the interstitial injury, will strongly prevent the further progression of glomerulosclerosis. It is expected that a long study or a high-doses study, whether or not accompanied by antihypertensive action, will make differences in not only the glomerular TGF-1 and ECM levels but also in the histological changes between the two RAS-inhibitory groups. O'Donnell et al. (24) reported that long-term (22 wk) enalapril treatment (50-200 mg/l drinking water), with reduction of blood pressure, did not affect albuminuria, glomerulosclerosis, and glomerular hemodynamics, suggesting that ACEI may not affect the course of renal disease in a setting of high-salt intake of DS rats (24), whereas there have been no reports of a long-term experiment using AT_1a in high salt-treated DS rats. The difference of renoprotective action between ACEI and AT_1a may be associated with the blocking manner of ANG II action. AT_1a is able to inhibit the action of ANG II produced via any ANG II synthetic system other than ACE (3). Moreover, the relatively enhanced AT₂ effect by chronic AT₁ blockade (15, 23, 32) may contribute to the renoprotection in the AT_1a group. In the ACEI group, the enhanced bradykinin induced by the blockade of kininase II may play a small role in the reduction of proteinuria in high salt-treated DS rats.

The glomerular diameters tended to be increased in DS rats receiving a high-salt diet compared with the low salt-treated DR rats. Interestingly, TCV-116 treatment but not cilazapril treatment tended to further increase the glomerular diameter despite improving the associated glomerulosclerosis and proliferative changes. It is possible that this tendency to increase glomerular size in the TCV-116-treated group is associated with the relaxation of glomeruli induced by the withdrawal from the mesangial contractile actions of ANG II via AT_1a receptors (5); however, in this study, the precise mechanism inducing this tendency was not clarified.

In conclusion, we observed that the RAS blockade significantly attenuated the glomerular injury in high- salt-treated DS rats independently of the antihypertensive effect. In particular, the reduction of the levels of glomerular TGF-1, PCNA and ECM, and proteinuria was prominent on the TCV-116 treatment. This study indicates that the glomerular-originated TGF-1 affected this glomerular injury and that the chronic treatment by TCV-116 may be more beneficial for renoprotection in DS hypertensive rats.

Perspectives