1First Department of Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-3192; and 2Medical Center, Shizuoka University, Shizuoka 422-8529, Japan
Submitted 11 February 2003 ; accepted in final form 7 October 2003
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ABSTRACT |
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dietary salt intake; angiotensin; transforming growth factor-
Previous studies indicated that ANG II expression is involved in the progression of kidney diseases (5, 10). Furthermore, angiotensin-converting enzyme inhibitors and ANG II receptor blockers exhibit renoprotective effects and delay the progression to end-stage renal disease in human and animal kidney diseases (6, 20, 31). In the kidney, ANG II causes contraction of efferent rather than afferent arterioles and increases intraglomerular pressure (8). Glomerular hypertension has been shown to lead to progressive renal injury through several mechanisms (33). For example, high glomerular pressure enlarges the radius of the pores in the glomerular basement membrane by a mechanism that is mediated, at least in part, by ANG II (19) and impairs the size-selective barrier of the membrane so that protein content in the glomerular filtrate increases, which in turn increases the endocytosis of proteins by tubular cells, resulting in a nephritogenic effect (32). Glomerular hypertension also induces stretch or shear stress in glomerular cells and increases the expression of transforming growth factor- (TGF-
), a major growth factor that underlies development of renal scarring resulting from pathological accumulation of extracellular matrix in chronic progressive kidney diseases (2). TGF-
exerts its biological effects through binding to specific cell surface receptor proteins composed of two transmembrane serine/threonine kinases, designated type I (T
RI) and type II (T
RII) TGF-
receptors (T
R). T
RII binds the ligand first, after which T
RI is recruited to form a heteromeric complex. ANG II-mediated expression of TGF-
in mesangial cells (12) and proximal tubular cells (43), and that of T
RII in proximal tubular cells (41), has also been reported. These data strongly suggest the pathogenic role of the RAS in the progression of renal sclerosis mediated by upregulation of TGF-
and T
R expression. However, it is also known that deoxycorticosterone acetate-salt-hypertensive rats exhibit high TGF-
expression in the kidney despite decreased renin activity (14).
Hypertension associated with high-salt intake can accelerate renal injury in patients with chronic progressive glomerulonephritis. However, a low-salt diet is not always necessary in patients with glomerulonephritis without overhydration or hypertension, and inappropriate restriction of salt intake should be avoided in patients with chronic renal failure and impaired urinary concentration ability because of the risk of dehydration. On the other hand, salt restriction is well known to enhance the circulatory RAS (13). At this stage, little is known about the effect of low- or high-salt intake per se, without changes in blood pressure, on the progression of glomerulonephritis. In the present study, we examined the effects of lowand high-salt intake on circulatory and intrarenal RAS, progression of glomerular lesions, and glomerular expression of TGF-1, T
RI, and T
RII in antithymocyte serum (ATS) nephritis. Our results showed that dietary salt intake modulated the progression of ATS nephritis by altering glomerular TGF-
, T
RI, and T
RII expression, which was due, at least in part, to alteration of glomerular AT1R and AT2R expression.
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MATERIALS AND METHODS |
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ATS was raised in a nonpregnant adult female England Suffolk sheep by subcutaneous immunization with 1 x 108 Wistar rat thymocytes in complete Freund's adjuvant, followed by four injections of 1 x 106 thymocytes in complete Freund's adjuvant at 4-wk intervals. Sera obtained 1 wk after the last injection were heat inactivated (56°C for 30 min) and absorbed three times with Wistar rat erythrocytes.
Protocol 1. Male Wistar rats weighing 80 g were purchased from SLC (Hamamatsu, Japan). They were randomly assigned into the following four groups: group A, in which 30 rats were fed normal rat chow containing 0.39% sodium chloride, given tap water as drinking water, and injected with ATS through the tail vein; group B, in which 35 rats were fed a low-salt diet containing 0.03% sodium chloride, given tap water as drinking water, injected with furosemide (2 mg/kg ip) 7 days before ATS injection, and then injected with ATS; group C, in which 35 rats were fed normal chow but given saltwater containing 1% sodium chloride as drinking water, injected with deoxycorticosterone acetate (10 mg/kg sc) 14 and 7 days before ATS injection, and then injected with ATS; and group D, in which 28 rats treated similarly to group C received candesartan cilexetil (1 mg/kg) orally every day after ATS injection.
Before and 3, 7, 14, and 28 days after ATS injection, the rats were placed in metabolic cages for a 24-h urine collection under a 12:12-h light-dark cycle, and urinary protein excretion was measured using the Bradford method. Blood pressure was measured by the tail-cuff method in animals that were conscious and restrained. Under ether anesthesia, blood samples were collected from the abdominal aorta, and the kidneys were perfused with ice-cold sterile 0.1 M PBS at pH 7.4, dissected out, and subjected to histopathological and immunohistochemical analysis and glomerular isolation. Glomeruli were isolated using a graded sieving technique under sterile conditions (18).
Protocol 2. To investigate the role of glomerular AT2R on the progression of ATS nephritis, we compared the levels of proteinuria, renal lesions, cellular fibronection deposition, and glomerular expression of TGF-1 and its receptors in the following two groups. In group E, seven ATS nephritic rats on a low-salt intake were treated similarly to group B. In group F, seven ATS nephritic rats on a low-salt intake treated similarly to group B were implanted with microosmotic pumps (Alzet, Cupertino, CA) containing PD-123319, an AT2R blocker, at a dose of 100 mg/ml, in the subcutaneous tissue of the back region after ATS injection. Seven days after ATS injection, the levels of urinary protein excretion were measured, and the kidneys were subjected to histopathological and immunohistochemical analysis and glomerular isolation as described above.
Glomerular TGF-1 Production
Glomeruli (1 x 104) were kept in RPMI-1640 supplemented with penicillin and streptomycin and incubated under a 95% air-5% CO2 humidified atmosphere at 37°C. After a 24-h incubation, the medium was collected, centrifuged at 12,000 rpm for 10 min, and the supernatant was treated with 1 N HCl to activate latent TGF-1 to active TGF-
1 detectable by the kit and allocated for the measurement of total TGF-
1 by ELISA using Quantikine (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Plasma Renin Activity
Plasma renin activity was measured by determining the generation rate of angiotensin I per milliliter plasma per hour using an RIA kit (SRL, Tokyo, Japan).
Histopathological and Immunohistochemical Analyses
Kidney tissues were fixed in 4% paraformaldehyde in PBS and embedded in paraffin. Tissue sections (3-µm thick) were stained with periodic acid-Schiff for histopathological evaluation. Immunoreactivities for ED-1 and renin proteins were determined using the standard biotin-streptavidin-peroxidase method as described previously (9). Mouse anti-rat ED-1 antibody was purchased from Serotec (Raleigh, NC). Rabbit anti-mouse renin antibody was kindly provided by Dr. A. Fukamizu (Univ. of Tsukuba, Ibaraki, Japan). The secondary antibodies were affinity-purified biotinylated donkey anti-rabbit (Cortex Biochemical, San Leandro, CA) and donkey anti-mouse IgG (Cortex Biochemical). Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 30 min. Sections incubated with nonimmune rabbit sera instead of primary antibodies were used as controls. Nuclei were counterstained lightly with hematoxylin. All sections were stained under identical conditions together with a control incubation.
To evaluate the levels of extracellular matrix deposition before and after induction of nephritis, immunoreactivity for cellular fibronectin was determined by the immunofluorescence method in frozen sections as described previously (50). Mouse monoclonal antibody to ED-A of cellular fibronection, 1ST-9, was kindly provided by Dr. L. Zardi (Instituto Nazionale per la Ricerca sul Cancro, Genoa, Italy), and FITC-conjugated donkey anti-mouse IgG was purchased from Chemicon (Temecula, CA).
To evaluate the difference in induction of ATS nephritis among the groups on normal-, low-, and high-salt intake, an additional five rats in each group were killed 24 h after ATS injection, and intraglomerular deposition of ATS (sheep IgG) and endogenous complement (rat C3) was investigated by the immunofluorescence method. FITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rat C3 were purchased from Cappel (Aurora, OH).
The number of ED-1-positive cells per glomerulus was counted under light microscopy at x400 magnification. At least 30 glomeruli/sample were evaluated, and the mean values were determined. The levels of mesangial matrix deposition and the staining intensity of renin, cellular fibronectin, ATS (sheep IgG), and rat C3 were graded using the following 0-4 scale in coded sections observed at x400 magnification: 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. Thirty randomly selected glomeruli were evaluated per sample, and the mean values of the staining scores were calculated. Evaluation of tissue sections was performed by a single investigator blinded to the experimental background.
Western Blot Analysis
Isolated glomeruli were dissolved in RIPA buffer (0.5% deoxycholate, 0.5% Triton X-100, 25 mM Tris·HCl, pH 7.4, 0.1% SDS, and 150 mM NaCl) at 4°C containing 1 mM PMSF, 1 µM leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin. Samples were denatured by boiling for 10 min in LDS sample buffer (Novex, San Diego, CA) and separated on 4-12% polyacrylamide gradient gels. To analyze differences in the levels of glomerular TRI, T
RII, AT1R, and AT2R expression among groups A, B, C, and D, samples obtained before and 7 days after ATS injection were applied on the same gel and examined in the same run. After electrophoresis, the proteins were electroblotted onto nitrocellulose membranes. The membranes were subsequently washed three times for 10 min each in TBS-T (20 mM Tris·HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20), incubated in TBS-T containing 5% skim milk for 1 h at room temperature to block nonspecific binding, and subsequently incubated with the primary antibody (10 µg/ml) overnight at 4°C. The primary antibodies used were rabbit anti-human T
RI and T
RII (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human AT1R and AT2R (Santa Cruz Biotechnology), and mouse anti-
-actin (Sigma, St. Louis, MO) antibodies. After the membranes were washed three times for 10 min each to remove excess antibodies, they were incubated with horse-radish peroxidase-conjugated rabbit anti-mouse IgG antibody (OEM Concepts, Toms River, NJ) or donkey anti-rabbit IgG (Amersham, Arlington Heights, IL) antibody diluted appropriately in TBS containing 5% skim milk for 1 h at room temperature. Antibodies bound to membrane-immobilized proteins were visualized by enhanced chemiluminescence (Amersham). Quantification of the band intensity was performed using National Institutes of Health Image software. The results were expressed as the densitometric ratios of T
RI, T
RII, AT1R, or AT2R relative to
-actin.
Measurement of Glomerular ANG II
The glomerular levels of ANG II were measured using RIA. Isolated glomeruli (1 x 104) were homogenized with 1 ml of ice-cold saline containing 0.1 N HCl and 5% aprotinin and centrifuged at 12,000 g at 4°C for 30 min. The supernatant was collected, florisil (Sigma) was added, and then it was centrifuged at 1,000 g for 2 min. The supernatant was discarded, and the pellet was washed three times with distilled water, followed by acetone containing 0.5 N HCl and petroleum ether. The extract was dried under vacuum, reconstituted in Tris buffer (pH 8.5), and used for RIA. The incubation mixture consisted of samples or standard and rabbit anti-human ANG II antibody (SRL). The anti-ANG II antibody used in the present study has been shown to have very low cross-reactivity (0.037%) with ANG I (11). The incubation was carried out at 4°C for 24 h, followed by the late addition of 125I-labeled ANG II and further incubation at 4°C for 8 h. To separate the bound complexes from free ligands, goat anti-rabbit IgG antibody and polyethylene glycol were added. The samples were incubated at 4°C for 1 h and then centrifuged at 2,000 g for 20 min. The radioactivity in the precipitate was counted using a -spectrometer (ARC950, Aloka, Tokyo, Japan).
Statistical Analysis
Data are expressed as means ± SD. Differences among groups were tested by analysis of variance with the Scheffé's correction for independent variables. A P value <0.05 denoted the presence of a statistically significant difference.
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RESULTS |
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The mean body weight of rats with ATS nephritis on a low-salt intake (group B) was less than that of other groups at all time points (Table 1). There were no significant differences in mean arterial blood pressure levels among the groups (Table 1).
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Induction of ATS Nephritis
No significant differences in the levels of glomerular deposition of ATS (sheep IgG) and rat C3 24 h after ATS induction were noted among rats on a normal (group A)-, low (group B)-, and high (group C)-salt intake (Fig. 1). It is suggested that the induction of ATS nephritis did not significantly differ among the groups.
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Urinary Protein Excretion
In rats with ATS nephritis on a normal-salt intake (group A) and those on a low-salt intake (group B), urinary protein excretion increased after ATS injection, reached a peak level on day 3, and decreased thereafter to normal levels by day 28 (Fig. 2). However, urinary protein excretion levels were lower in group B than group A throughout the study period. In rats with ATS nephritis on a high-salt intake (group C), urinary protein excretion levels were significantly higher than those of group A on days 14 and 28. Administration of candesartan cilexetil (group D) tended to reduce the level of proteinuria in rats with ATS nephritis on a high-salt intake on days 14 and 28, albeit insignificantly, compared with group C.
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Histopathological Findings
There were no differences in mesangial cellularity and mesangial matrix deposition among all groups before ATS injection. In rats with ATS nephritis on a normal-salt intake (group A), mesangiolysis was noted 3 days after ATS injection, which was followed by mesangial cell proliferation and mesangial matrix accumulation, reaching a peak level at day 7 and returning to almost normal levels by day 28. The severity of these glomerular lesions was less in rats on a low-salt intake (group B; Fig. 3, A and B). In rats with ATS nephritis on a high-salt intake (group C), enlargement of glomeruli and worsening of glomerular lesions were evident. Compared with group C, administration of candesartan cilexetil (group D) resulted in amelioration of the glomerular lesions.
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Cellular fibronectin was not detected in the glomeruli of all groups before induction of nephritis (data not shown). In rats with ATS nephritis on a normal-salt intake (group A), glomerular deposition of cellular fibronectin was increased significantly 7 days after induction of nephritis. The levels of glomerular deposition of cellular fibronectin were less severe in rats on a low-salt intake (group B) and more in rats on a high-salt intake (group D) compared with group A (Fig. 3C). Compared with group C, administration of candesartan cilexetil (group D) significantly reduced the deposition of cellular fibronectin.
Glomerular Infiltration of ED-1-Positive Macrophages
ATS injection was associated with a subsequent increase in glomerular infiltration of ED-1-positive macrophages, which reached peak levels at day 7 in all groups (Fig. 4). Rats with ATS nephritis on a high-salt intake (group C) showed persistent infiltration of macrophages in glomeruli and a significantly higher number of infiltrating macrophages 14 days after ATS injection compared with group A. Administration of candesartan cilexetil significantly suppressed glomerular macrophage infiltration in rats with ATS nephritis on a high-salt intake (group D) at days 3, 7, and 14 compared with those in group C.
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Glomerular TGF-1 Production
Glomerular production of total TGF-1 increased after ATS injection, reaching peak levels at day 7, but returned to normal levels by day 28 in all groups (Fig. 5). The glomerular TGF-
1 level was significantly lower in rats with ATS nephritis on a low-salt intake (group B) than in those on a normal-salt intake (group A)on days 3, 7, and 14. No significant differences in the levels of glomerular TGF-
1 were noted between rats in group A and those on a high-salt intake (group C). Administration of candesartan cilexetil significantly decreased TGF-
1 production in rats with ATS nephritis on a high-salt intake (group D) at days 3 and 14 compared with those of group C.
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Glomerular Expression of TRI and T
RII
Compared with control rats, the levels of glomerular TRI expression increased in rats with ATS nephritis on a normal-salt intake (group A) at day 7, whereas no remarkable changes were noted in the levels of glomerular T
RII expression (Fig. 6). These results were identical to our previous report (25).
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Before ATS injection, slight decreases in glomerular TRI and T
RII expression were noted in rats on a low-salt intake and a slight increase in glomerular T
RI was observed in rats on a high-salt intake (Fig. 7). Seven days after ATS injection, when the glomerular lesions were at maximum levels, the expression of glomerular T
RI was less in rats with ATS nephritis on a low-salt intake (group B) than that in ATS nephritic rats on a normal-salt intake (group A) (Fig. 7). In contrast, glomerular expression of both T
RI and T
RII increased markedly in rats with ATS nephritis on a high-salt intake (group C) at day 7. Administration of candesartan cilexetil reduced glomerular expression of T
RII in rats with ATS nephritis on a high-salt intake at day 7 (group D).
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Glomerular Expression of AT1R and AT2R
Compared with control rats, the levels of glomerular AT1R expression increased markedly in rats with ATS nephritis on a normal-salt intake (group A) at day 7 (Fig. 6). An increase on glomerular AT2R expression also was noted in group A at day 7.
Before ATS injection, no remarkable changes in glomerular AT1R and AT2R expression were observed in rats on a high- or low-salt intake compared with that in rats on a normal-salt intake (Fig. 7). However, 7 days after ATS injection, overexpression of glomerular AT1R was noted in rats with ATS nephritis on a high-salt intake (group C) compared with that in normal-salt-intake ATS nephritic rats (group A), whereas no marked change in glomerular AT1R expression was observed in rats with ATS nephritis on a low-salt intake (group B) (Fig. 7). The increase in glomerular AT1R expression in ATS nephritic rats on a high-salt intake at day 7 was not significant with administration of candesartan cilexetil (group D). In contrast, the levels of glomerular AT2R expression increased markedly in rats with ATS nephritis on a low-salt intake (group B) and decreased in those on a high-salt intake (group C) at day 7 (Fig. 7). Administration of candesartan cilexetil increased the levels of glomerular AT2R in high-salt-intake ATS nephritic rats (group D).
Plasma Renin Activity, Renal Renin Expression, and Glomerular ANG II Levels
Plasma renin activity was significantly higher in group B and lower in group C than that in group A before and after induction of ATS nephritis (Table 2). No significant differences in the levels of plasma renin activity were noted between high-salt-intake ATS nephritic rats treated with candesartan cilexetil (group D) and those untreated (group C). Immunoreactive renin protein was found in glomerular arterioles constituting the juxtaglomerular apparatus. The staining intensity of intrarenal renin roughly paralleled the levels of plasma renin activity in all groups (Table 2). Glomerular ANG II levels also roughly paralleled the levels of plasma renin activity and intrarenal renin staining intensity; it was significantly higher in rats with ATS nephritis on a low-salt intake (group B) and lower in those on a high-salt intake (group C) than those on a normal-salt intake (group A) (Table 2). In all samples obtained from high-salt-intake ATS nephritic rats treated with candesartan cilexetil at day 7 (group D; n = 6), the glomerular ANG II levels were lower than the lower limit for detection by the method used in the present study.
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Effect of AT2R Blocker on Rats with ATS Nephritis on a Low-Salt Intake
Because the decrease in urinary protein excretion and amelioration of renal lesions were associated with increased glomerular AT2R expression in ATS nephritic rats on a low-salt intake, we investigated the effect of PD-123319, an AT2R blocker, on the progression of ATS nephritis under a low-salt intake. Administration of PD-123319 did not affect blood pressure levels and changes in body weight during the course of ATS nephritis in rats on a low-salt intake (Table 3). Significant increases in mesangial cell proliferation and matrix accumulation, urinary protein excretion (group E, 31.2 mg/day, vs. group F, 63.2 mg/day, P < 0.05), deposition of cellular fibronectin, and production of glomerular TGF-1 were noted in low-salt-intake ATS nephritic rats treated with PD-123319 (group F) compared with those untreated (group E) 7 days after ATS injection (Fig. 8). No remarkable differences in glomerular T
RI expression were noted between groups E and F. However, glomerular expression of T
RII increased significantly in low-salt-intake ATS nephritic rats treated with PD-123319 (group F) at day 7 (Fig. 9). The data for immunohistochemical staining of T
RI and T
RII also showed similar results; no remarkable changes in T
RI and marked increases in T
RII in glomeruli of PD-123319-treated, low-salt-intake nephritic rats on day 7 (data not shown). These results suggest that increased glomerular AT2R expression in low-salt-intake nephritic rats played some protective role in the progression of ATS nephritis.
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DISCUSSION |
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Induction of ATS nephritis was not significantly different among rats on a normal-, low-, and high-salt intake 24 h after ATS injection. However, exacerbation of nephritis with increased glomerular expression of AT1R, TRI, and T
RII, and decreased glomerular expression of AT2R were noted in ATS nephritic rats on a high-salt intake. Glomerular TGF-
1 expression also increased in these rats, although it was not more than the level seen in nephritic rats on a normal-salt intake. A high-salt intake has been shown to increase AT1R expression in glomeruli, afferent arterioles, mesenteric arteries, and aorta (35, 46). In this context, Nickenig et al. (25) reported that sodium chloride upregulated AT1R expression directly in vascular endothelial cells. In addition, ANG II-mediated induction of TGF-
and T
R expression has been shown in mesangial cells (12) and proximal tubular cells (41, 43). Considering the fact that glomerular ANG II levels were not increased in rats with ATS nephritis on a high-salt intake, the increased glomerular AT1R expression seems to explain the increased activity of glomerular RAS, resulting in exacerbation of ATS nephritis with increased glomerular TGF-
and T
R expression during a high-salt intake. Amelioration of nephritis with decreased glomerular TGF-
and T
RII expression in high-salt-intake nephritic rats treated with candesartan cilexetil, an AT1R blocker, further supports this concept. The marked decrease of glomerular ANG II may be also involved in the amelioration of nephritis resulted from treatment with candesartan cilexetil. Previous studies showed that administration of an AT1R blocker decreased renal ANG II levels in ANG II-infused rats (48). ANG II-mediated expression of angiotensinogen was also demonstrated in cardiomyocytes (39). The decrease of glomerular AT2R expression noted in nephritic rats on a high-salt intake might also play some additive role in the aggravation of nephritis, because most signals mediated by AT2R are antagonistic to those mediated by AT1R (22).
In rats on a low-salt intake, progression of ATS nephritis was mitigated and glomerular expression levels of TGF- and T
RI were decreased markedly, although no remarkable difference in glomerular T
RII expression was noted. A low-salt intake has been shown to increase the expression of AT1R in proximal tubular cells (4) and adrenal cortical cells (37), resulting in increases of sodium reabsorption and aldosterone secretion, respectively, due to increased responsiveness to ANG II in these target cells. It was also reported that a low-salt intake decreases expression of AT1R in afferent arterioles, mesenteric arteries, and aorta (4, 35), resulting in decreases of ANG II-mediated vascular contraction (35). On the other hand, the effect of a low-salt intake on glomerular AT1R expression has been controversial. Amiri and Garcia (1) reported that glomerular AT1R expression did not change after a low-salt intake. However, Cheng et al. (4) reported a decrease in the expression during a low-salt intake. In the present study, no significant change was noted in the expression of glomerular AT1R in ATS nephritic rats on a low-salt intake compared with those on a normal-salt intake, when progression of ATS nephritis and glomerular expression of TGF-
and T
RI were mitigated in low-salt-intake nephritic rats. The glomerular ANG II levels paralleled renin expression and were high in rats with ATS nephritis on a low-salt intake. Considered together, these results suggest that the ameliorative effect of a low-salt intake was not attributable to the reduced glomerular ANG II activity mediated by AT1R.
Several recent studies have demonstrated the important role of AT2R in the repair process of various organs with tissue injuries. Increased expression of AT2R has been reported in postischemic kidney, heart, and brain lesions (17, 26, 47). Accelerated fibrosis and collagen deposition develop in the renal interstitium of AT2R null mutant mice during ureteral obstruction (21). Upregulation of glomerular AT2R was also demonstrated in rats on a low-salt intake (30). In the present study, the increase of glomerular AT2R expression was noted in ATS nephritic rats on a low-salt intake. AT2R blockade by PD-123319 resulted in aggravation of nephritis and increased glomerular expression of TGF-1 and T
RII in these rats. These data suggest that the increased glomerular AT2R expression induced by a low-salt intake played a role in amelioration of ATS nephritis through, at least in part, downregulation of glomerular TGF-
1 and T
RII expression. Su et al. (38) reported that overexpression of AT2R altered T
RII expression in vascular smooth muscle cells of spontaneous hypertensive rats and these changes might be attributable to the imbalance between AT1R and AT2R abundance.
Our results also showed that candesartan cilexetil significantly reduced glomerular macrophage infiltration in ATS nephritis. ANG II has been shown to induce the expression of monocyte chemoattractant protein-1 in mesangial cells (36) and on activation-regulated normal T cell expressed and secreted (RANTES) in glomerular endothelial cells (42). TGF- also has a chemoattractant activity for neutrophils, T cells, monocytes, and fibroblasts (34). Hilgers et al. (8) reported that valsartan reduced the expression of monocyte chemoattractant protein-1 and interstitial infiltration of macrophages in the two-kidney one-clip model. Because macrophages have AT1R (29), it is reasonable to conclude that candesartan cilexetil inhibited the infiltration of these cells by reducing the expression of chemoattractants and might also have suppressed the activity of these proteins through AT1R inhibition. Westerhuis et al. (40) reported that depletion of macrophages significantly reduced glomerular matrix expansion in rats with anti-Thy-1 nephritis, a nephritis model induced by an antibody that injures rat mesangial cells similar to the ATS nephritis model used in the present study. Recently, however, using mice with bone marrow lacking AT1R gene expression, Nishida et al. (27) demonstrated that AT1R on macrophages that infiltrated the interstitium in kidneys with unilateral ureteral obstruction acts to preserve renal parenchymal architecture and suppress the development of interstitial fibrosis. These data suggest the species- or disease-specific role of infiltrating macrophages in the progression of kidney diseases.
In conclusion, we have demonstrated in the present study that a high-salt intake aggravated progression of ATS nephritis and was associated with increased glomerular expression of TGF-, T
RI, T
RII, and AT1R and decreased glomerular expression of AT2R and that treatment with candesartan cilexetil reduced proteinuria, ameliorated glomerular lesions, reduced glomerular expression of TGF-
1 and T
RII, and increased glomerular expression of AT2R. A low-salt intake ameliorated progression of ATS nephritis and was associated with decreased glomerular expression of TGF-
and T
RI and increased glomerular expression of AT2R. Blocking of AT2R by PD-123319 aggravated the glomerular lesions and increased glomerular TGF-
1 and T
RII expression. Our results suggest that increased glomerular AT1R and decreased glomerular AT2R expression are involved in the aggravation of ATS nephritis under a high-salt intake and that upregulation of glomerular AT2R may participate in the amelioration of the nephritis under a low-salt intake.
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GRANTS |
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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