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Am J Physiol Renal Physiol 286: F516-F525, 2004. First published October 28, 2003; doi:10.1152/ajprenal.00294.2003
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Overexpression of angiotensin type 2 receptor ameliorates glomerular injury in a mouse remnant kidney model

Naoko Hashimoto,1 Yohei Maeshima,1 Minoru Satoh,1 Masahiro Odawara,1 Hitoshi Sugiyama,1 Naoki Kashihara,2 Hiroaki Matsubara,3 Yasushi Yamasaki,1 and Hirofumi Makino1

1Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558; 2Division of Nephrology, Department of Internal Medicine, Kawasaki Medical School, Kurashiki 701-01; and 3Department of Medicine II, Kansai Medical School, Osaka 570-8506, Japan

Submitted 21 August 2003 ; accepted in final form 20 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Angiotensin II mediates the progression of renal disease through the type 1 receptor (AT1R). Recent studies have suggested that type 2 receptor (AT2R)-mediated signaling inhibits cell proliferation by counteracting the actions of AT1R. The aim of the present study was to determine the effect of AT2R overexpression on glomerular injury induced by 5/6 nephrectomy (5/6Nx). AT2R transgenic mice (AT2-Tg), overexpressing AT2R under the control of {alpha}-smooth muscle actin ({alpha}-SMA) promoter, and control wild-type mice (Wild) were subjected to 5/6Nx. In AT2-Tg mice, the glomerular expression of AT2R was upregulated after 5/6Nx. Urinary albumin excretion at 12 wk after 5/6Nx was decreased by 33.7% in AT2-Tg compared with Wild mice. Glomerular size in AT2-Tg mice was significantly smaller than in Wild mice after 5/6Nx (93.1 ± 3.0 vs. 103.3 ± 1.8 µm; P < 0.05). Immunohistochemistry revealed significant decreases in glomerular expression of platelet-derived growth factor-BB chain (PDGF-BB) and transforming growth factor-{beta}1 (TGF-{beta}1) in AT2-Tg with 5/6Nx compared with Wild mice. Urinary excretion of nitric oxide metabolites was increased 2.5-fold in AT2-Tg compared with Wild mice. EMSA showed that activation of early growth response gene-1, which induces the transcription of PDGF-BB and TGF-{beta}1, was decreased in AT2-Tg mice. These changes in AT2-Tg mice at 12 wk after 5/6Nx were blocked by the AT2R antagonist PD-123319. Taken together, our findings suggest that AT2R-mediated signaling may protect from glomerular injuries induced by 5/6Nx and that overexpression of AT2R may serve as a potential therapeutic strategy for glomerular disorders.

angiotensin II receptor; angiotensin II; transgenic mouse; nitric oxide

THE RENIN-ANGIOTENSIN SYSTEM (RAS) plays important roles in the cardiovascular system (13). All components of the RAS are also present in the kidneys and constitute the functional renal RAS (47). Angiotensin II (ANG II) has two major receptor isoforms, AT1R and AT2R (33). AT1Rs are expressed in the afferent and efferent arterioles, glomeruli, and proximal tubules in normal animals and humans (45). Studies with AT1R- and AT2R-selective antagonists have revealed that the known biological actions of ANG II are exclusively mediated via AT1R (33). AT1Rs regulate vasoconstriction and sodium as well as water reabsorption, and they also promote cellular hypertrophy, proliferation, and extracellular matrix (ECM) deposition in the kidney. Experimental (26) and clinical (40) studies using an AT1R antagonist (AT1RA) indicated the involvement of AT1R in the progression of renal disorders. The analysis of AT1a receptor knockout mice (15, 44) demonstrated the renoprotective effect of AT1R blockade. AT1RA prevents binding of ANG II to AT1R and increases levels of plasma renin and ANG II (12). Increased ANG II may potentially induce signaling through AT2R. This alternate signaling via AT2R could be an additional mechanism of renoprotection by AT1RA.

Recent studies have suggested that AT2R-mediated signaling induces inhibition of cell growth or apoptosis by counteracting the AT1R signal (49). The basal systolic blood pressure of AT2R-null mice is mildly elevated compared with wild-type (Wild) mice (48). Renal interstitial fibrosis was accelerated in AT2R-null mice during unilateral ureteral obstruction (27), suggesting its important role in the remodeling of renal interstitium. The AT2R is abundantly and widely expressed in fetal tissues (35), but it diminishes rapidly after birth. In the adult human renal cortex, expression of mRNA for AT2R was localized to interlobular arteries but not in glomeruli (34). However, the AT2R is thought to be expressed in glomeruli in response to RAS activation, such as during sodium depletion (39). The mechanism of AT2R signaling has not yet been fully elucidated.

Platelet-derived growth factor-BB chain (PDGF-BB) and transforming growth factor-{beta}1 (TGF-{beta}1) are known to play critical roles in promoting mesangial cell proliferation and ECM accumulation, respectively. Blockade of these growth factors prevents glomerular injury (2, 20). Early growth response gene-1 (Egr-1; an immediate-early gene) acts as a transcription factor activating the transcription of PDGF-AA, -BB, and TGF-{beta}1 (5, 42). Egr-1 expression was closely linked to mesangial cell proliferation, and specific inhibition of Egr-1 results in the suppression of mesangial cell proliferation (4, 42). Several studies suggested that nitric oxide (NO) has multiple biological functions related to kidney (42, 46). NO inhibits mesangial cell growth, and this effect is partly mediated via suppression of Egr-1 expression (5, 42). In the 5/6 nephrectomy (5/6Nx) model, renal dysfunction is associated with disturbed NO production (41).

On glomerular injury, mesangial cells acquire the phenotype of myofibroblasts, characterized by the expression of {alpha}-smooth muscle actin ({alpha}-SMA) (19). Elevation of mesangial {alpha}-SMA precedes the development of glomerulosclerosis in the rat 5/6Nx model, indicating that phenotypic alteration of mesangial cells is associated with progressive renal disorders (8).

In the present study, we used AT2R transgenic mice (AT2-Tg), overexpressing mouse AT2R under the control of the mouse {alpha}-SMA promoter (53), to elucidate the role of the AT2R-mediated signaling in the 5/6Nx model. Our results indicate the protective role of AT2R-mediated signaling against progressive glomerular injuries.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental protocol. Male AT2-Tg mice (C57BL/6 background), overexpressing mouse AT2R under the control of the mouse {alpha}-SMA promoter (53), and male C57BL/6 mice (Wild) were used at the age of 8-10 wk. Mice were divided into subgroups (n = 6/group), and no mice died during the experimental period. Food and water were supplied ad libitum. The experimental protocol was approved by the Animal Ethics Review Committee of Okayama University Medical School. Renal ablation was performed as described previously (55). PD-123319 (30 mg·kg-1·day-1), an AT2R-specific antagonist, was infused using an Alzet osmotic pump (model 2002; Alza, Palo Alto, CA) from 6-12 wk after 5/6Nx in the AT2-Tg group. Body weight (BW) was measured every 3 wk, and kidney weight was measured just after death. Arterial blood pressure was measured every 3 wk using a programmable sphygmomanometer (BP-98A; Softron, Tokyo, Japan) by the tail-cuff method as described previously (50). Mean blood pressure (MBP) was calculated as (systolic pressure + 2 x diastolic pressure)/3. Twenty-four-hour urine samples were collected in metabolic cages every 6 wk. Immediately before death, blood samples were drawn from the retroorbital sinus.

Blood and urine examination. Blood urea nitrogen (BUN) and urinary creatinine levels were measured by SRL (Okayama, Japan). Urinary albumin concentration was determined with a murine microalbuminuria ELISA kit (Albuwell M; Exocell, Philadelphia, PA) following the instructions provided by the manufacturer. Urinary concentrations of total nitrate and nitrite (NOx) were determined by using a Nitrate/Nitrite assay kit (Cayman Chemical, Ann Arbor, MI). Deproteinized urine samples premixed with carrier solution (0.0007% EDTA and 0.03% NH4Cl) were used. Absorbance was detected at 540 nm using a microplate reader (model 550; Bio-Rad, Hercules, CA).

Histopathological assessment. At 6 or 12 wk after 5/6Nx, kidneys were removed, fixed in 10% buffered formalin, and embedded in paraffin. Sections (4 µm thick) were stained with periodic acid-Schiff (PAS) and Azan for light microscopic examination. The diameter of glomeruli, spanning from the vascular pole to the opposite Bowman's capsule, was measured using an objective micrometer (Olympus Optical, Tokyo, Japan). Glomerular cell number was determined by counting the nuclei within the glomerular tuft. Glomerular sclerosis was graded as follows: 0, none; +1, sclerotic change in <25% of the glomerulus; +2, from 25 to 50%; +3, >50% (51). The mean score per glomerulus in each kidney was determined as the sclerosis index. In each kidney, >30 glomerular cross sections were examined by 2 investigators and averaged.

Immunohistochemistry was performed using frozen sections as described previously (30, 44). Samples were incubated overnight at 4°C with polyclonal anti-mouse-{alpha}-SMA (Sigma, St. Louis, MO), polyclonal rabbit anti-mouse AT2R, PDGF-BB, and TGF-{beta}1 antibodies (Santa Cruz Biotechnology). Normal rabbit IgG was used as a negative control. Immunoperoxidase staining was carried out utilizing the Vectastain ABC Elite reagent kit (Vector Labs, Burlingame, CA). Diaminobenzidine was used as a chromogen. All slides were counterstained with hematoxylin. Expression of PDGF-BB and TGF-{beta}1 was graded semiquantitatively according to the degree of positive staining in the mesangial area (0 = 0-5%, +1 = 5-25%, +2 = 25-50%, +3 = 50-75%, +4 = 75-100%) as described previously (9), and the mean score per section was calculated.

RNA extraction and quantitative real-time RT-PCR. Glomeruli from each mouse were isolated by the fractional sieving technique as described previously (29, 31). Total RNA was extracted from glomeruli using the RNeasy Midi Kit (Qiagen, Chatsworth, CA) and stored at -80°C until use. Total RNA was subjected to RT with poly-d(T) primers and RT (RTG T-Primed First-Strand kit; Amersham Pharmacia Biotech, Piscataway, NJ). Quantitative real-time RT-PCR was used to quantify the amounts of AT2R mRNA. cDNA was diluted 1:5 with autoclaved deionized water, and 5 µl of the diluted cDNA were added to the Lightcycler-Mastermix, 0.3 µM specific primer, 3 mM MgCl2, and 2 µl of Master SYBR Green (Roche Diagnostics, Mannheim, Germany). This reaction mixture was filled up to a final volume of 20 µl with water. PCR reactions were carried out in a real-time PCR cycler (Lightcycler; Roche Diagnostics). The program was optimized and performed finally as denaturation at 95°C for 10 min followed by 40 cycles of amplification (95°C for 10 s, 62°C for 10 s, 72°C for 10 s). The temperature ramp rate was 20°C/s. At the end of each extension step, the fluorescence of each sample was measured to allow the quantification of the PCR products. After completion of the PCR, the melting curve of the product was measured by a temperature gradient from 60 to 95°C at 0.2°C/s with continuous fluorescence monitoring to produce a melting profile of the primers. The amount of PCR product was normalized with a housekeeping gene (GAPDH) to determine the relative expression ratio for AT2R mRNA in relation to GAPDH mRNA. The following oligonucleotide primers specific for mouse AT2R and GAPDH were used: AT2R, 5'-CAGAATTACCCGTGACCAAG-3' (forward) and 5'-TAAACACACTGCGGAGCTT-3' (reverse); GAPDH, forward 5'-ATGGTGAAGGTCGGTGTG-3' and reverse 5'-ACCAGTGGATGCAGGGAT-3'. Four independent experiments were performed.

Preparation of nuclear extracts from glomeruli. Isolated glomeluri were suspended in PBS and homogenized. Nuclear extracts were prepared as described previously (29, 44) and stored at -80°C until use. Protein concentration was determined using a protein assay reagent (Bio-Rad).

EMSA. To detect the DNA binding activity, a gel shift assay was performed as described previously (29, 44). Nuclear extract (15 µg) was incubated with 1 ng of radiolabeled DNA containing two tandem Egr-1 binding sites (5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3') in 20 µl of binding buffer [10 mM Tris·HCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 1 mM MgCl2, 4% glycerol, 50 mg/ml poly dI-dC] for 30 min. Reaction mixtures were then separated on a 4% SDS-polyacrylamide gel followed by autoradiography. The relative intensity of autoradiograms was determined by scanning densitometry. Four independent experiments were performed. A competition assay was performed with 100-fold excess of unlabeled consensus sequences of Egr-1 or mutant Egr-1 (5'-GGATCCAGCTAGGGCGAGCTAGGGCGA-3').

Statistical analysis. Results are expressed as means ± SE. Statistical analyses were performed using Student's t-test for unpaired samples and ANOVA to test for changes within groups over time. A P value <0.05 considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of {alpha}-SMA and AT2R. Using quantitative real-time RT-PCR, we investigated the mRNA expression of AT2R in glomeruli (Fig. 1). Although AT2R mRNA was not detectable in Wild mice, it was expressed in AT2-Tg mice and was elevated at 6 and 12 wk after 5/6Nx. The distribution pattern of {alpha}-SMA in glomeruli was studied by immunohistochemistry (Fig. 2A). The expression of {alpha}-SMA was not observed in the glomerulus of either Wild or AT2-Tg control groups (Fig. 2, AA and AD) but was detected only in the intrarenal arteries. The expression of {alpha}-SMA was upregulated in both groups at 6 wk after 5/6Nx, mainly in the mesangial area (Fig. 2, AB and AE). In the AT2-Tg group, {alpha}-SMA expression was decreased at 12 wk after 5/6 Nx (Fig. 2AF). Expression of AT2R protein was absent in glomeruli of the Wild group, as detected by immunohistochemistry (Fig. 2, BA-BC). Weak AT2R expression was observed in AT2-Tg control mice, and it was markedly upregulated in AT2-Tg mice at 6 wk and mildly upregulated at 12 wk after 5/6Nx, mainly in the mesangium (Fig. 2, BE and BF), similar to {alpha}-SMA. Because AT2Rs were overexpressed under the control of {alpha}-SMA promoter in AT2-Tg mice, we speculate that the distribution pattern of AT2Rs in glomeruli was in parallel with that of {alpha}-SMA in AT2-Tg mice.



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  		Fig. 1. Expression of AT2 receptor (AT2R) mRNA detected by real-time RT-PCR. Total RNA was extracted from glomeruli and subjected to the examination using quantitative real-time RT-PCR as described in MATERIALS AND METHODS. The amount of AT2R mRNA relative to GAPDH mRNA is shown. Results were expressed relative to AT2R transgenic mice (AT2-Tg) 6 (6W) and 12 wk (12W) after 5/6 nephrectomy (5/6Nx), which were arbitrarily assigned a value of 100%. Wild, wild-type mice; Cont, control.

 


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  		Fig. 2. Immunohistochemistry of {alpha}-smooth muscle actin (SMA) in kidneys of Wild and AT2-Tg mice. A: representative photomicro-graphs of Wild control (A), 6 (B) or 12 wk (C) after 5/6Nx, and AT2-Tg control (D), 6 (E) or 12 wk (F) after 5/6Nx are shown (original magnification: x400). B: immunohistochemistry of AT2R in kidneys of Wild and AT2-Tg mice. Representative photomicrographs of Wild control (A), 6 (B) or 12 wk (C) after 5/6Nx, and AT2-Tg control (D), 6 (E) or 12 wk (F) after 5/6Nx are shown (original magnification: x400).

 

Changes in arterial blood pressure and BW. Arterial blood pressure in Wild and AT2-Tg mice increased within 3 wk of 5/6Nx (Table 1). PD-123319 treatment did not affect the MBP of AT2-Tg mice after 5/6Nx. The increase in BW of Wild mice was not observed at time points later than 6 wk after 5/6Nx (Table 2). In AT2-Tg mice, BW did increase at time points later than 6 wk after 5/6Nx. The increase in BW was not observed at 6 wk after 5/6Nx in AT2-Tg mice treated by PD-123319, similar to 5/6Nx in Wild mice. The kidney weight (KW)-to-BW ratio (KW/BW) in AT2-Tg mice after 5/6Nx was significantly lower than in Wild mice (Table 2). However, the KW/BW of AT2-Tg mice treated by PD-123319 was elevated in Wild mice, similar to that after 5/6Nx.


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  		Table 1. Mean blood pressure

 

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  		Table 2. Body weight and kidney weight

 

Changes in BUN and urinary albumin excretion. To evaluate the effect of AT2R overexpression on 5/6Nx-induced alteration of biological functions, we measured BUN and urinary albumin excretion (UAE; Fig. 3). The basal levels of BUN and UAE were not significantly different between Wild and AT2-Tg mice (UAE: 71.6 ± 5.8 vs. 73.6 ± 9.6 µg/mg, respectively). In both groups, significant increases in BUN and UAE were observed at 12 wk after 5/6Nx, but they were not elevated in AT2-Tg compared with Wild mice (UAE: 128.8 ± 15.9 vs. 194.2 ± 17.0 µg/mg, respectively). This inhibitory effect on UAE and BUN elevation in AT2-Tg mice after 5/6Nx was abolished by PD-123319 treatment (UAE: 224.8 ± 12.4 µg/mg).



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  		Fig. 3. Urinary albumin/creatinine excretion (A) and blood urea nitrogen (B). Values are means ± SE; n = 6/group. Open bars, Wild mice; shaded bars, AT2-Tg mice; filled bars, PD-123319-treated AT2-Tg after 5/6Nx. *P < 0.05 vs. AT2-Tg mice at 12 wk after 5/6Nx.#P < 0.01 vs. Wild control mice.$P < 0.01 vs. AT2-Tg control mice.

 

Histological and morphometric analysis. Histological examination of the kidneys revealed no difference between Wild and AT2-Tg control mice (Fig. 4, AA and AE). At 12 wk after 5/6Nx, kidney sections from Wild mice exhibited glomerular hypertrophy, cellular proliferation, and glomerular sclerosis (Fig. 4, AB and AF). In AT2-Tg mice at 12 wk after 5/6Nx, these histological changes were milder than those in Wild mice (Fig. 4, AC and AG). Glomerular damage in AT2-Tg at 12 wk after 5/6Nx was similar to that in the Wild group after PD-123319 treatment (Fig. 4, AD and AH). Next, glomerular diameter, total cell number, and sclerosis index were analyzed. In the Wild group, these parameters were elevated at 12 wk after 5/6Nx (Fig. 4B). These changes were significantly suppressed in Wild vs. AT2-Tg mice (cell no.: 37.7 ± 2.8 vs. 31.5 ± 0.7; sclerosis index: 2.0 ± 0.13 vs. 1.5 ± 0.11; glomerular diameter: 103.4 ± 1.8 vs. 93.2 ± 3.0 µm). PD-123319 treatment induced an increase in these parameters in AT2-Tg mice after 5/6Nx (cell no.: 37.4 ± 2.78; sclerosis index: 1.94 ± 0.02; glomerular diameter: 105.0 ± 0.76 µm, respectively). These results indicate that histological changes associated with glomerular hypertrophy after 5/6Nx were significantly diminished in AT2-Tg mice. No significant difference was observed in renal interstitial fibrosis between Wild and AT2-Tg mice at 12 wk after 5/6Nx (data not shown).



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  		Fig. 4. A: histological appearance of glomeruli in Wild and AT2-Tg mice. Wild control (A and E), 12 wk after 5/6Nx (B and F), AT2-Tg 12 wk after 5/6Nx (C and G), AT2-Tg 12 wk after 5/6Nx treated with PD-123319 (D and H). Periodic acid-Schiff (PAS; A-D) and Azan-Mallory staining (E-H) (original magnification: x400). B: quantification of glomerular diameter (A), total cell number (B), and sclerosis index (C) in Wild and AT2-Tg mice. These histological parameters were determined as described in MATERIALS AND METHODS. Values are means ± SE; n = 6/group. Open bars, Wild mice; shaded bars, AT2-Tg mice; filled bars, PD-123319-treated AT2-Tg mice.#P < 0.05 vs. Wild control.$P < 0.05 vs. AT2-Tg control. *P < 0.05 vs. AT2-Tg at 12 wk after 5/6Nx.

 

Immunohistochemical analysis of glomerular PDGF-BB chain and TGF-{beta}1 expression. To further evaluate the involvement of AT2R in phenotypic alterations in glomeruli, the expression levels of PDGF-BB and TGF-{beta}1 were examined by immunohistochemistry (Fig. 5A). These growth factors act as key molecules in progressive glomerulosclerosis and mesangial cell proliferation after renal ablation (8, 18, 21). At 12 wk after 5/6Nx, the glomerular expression of PDGF-BB and TGF-{beta}1 was significantly increased in the Wild group (Fig. 5, AB and AG), whereas this effect was significantly inhibited in AT2-Tg mice (Fig. 5, AC and AH). These inhibitory effects were abolished by PD-123319 in the AT2-Tg group (Fig. 5, AD and AI). In both Wild and AT2-Tg groups, the staining score of PDGF-BB and TGF-{beta}1 was significantly increased at 6 wk after 5/6Nx (Fig. 5B). However, it was significantly decreased in AT2-Tg at 12 wk after 5/6Nx compared with the Wild group (PDGF-BB chain: 3.16 ± 0.20 vs. 1.06 ± 0.28; TGF-{beta}1: 2.80 ± 0.26 vs. 1.52 ± 0.13). These inhibitory effects were reversed by PD-123319 treatment in AT2-Tg mice at 12 wk after 5/6Nx (PDGF-BB: 2.36 ± 0.33; TGF-{beta}1: 2.57 ± 0.53, respectively).



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  		Fig. 5. A: immunohistochemistry of platelet-derived growth factor-BB chain (PDGF-BB) and transforming growth factor-{beta}1 (TGF-{beta}1). PDGF-BB staining in Wild control (A), 12 wk after 5/6Nx (B), AT2-Tg 12 wk after 5/6Nx (C), AT2-Tg 12 wk after 5/6Nx treated with PD-123319 (D), and negative control (E) of Wild mice 12 wk after 5/6Nx. TGF-{beta}1 staining in Wild control (F), 12 wk after 5/6Nx (G), AT2-Tg 12 wk after 5/6 Nx (H), AT2-Tg 12 wk after 5/6Nx treated with PD-123319 (I), and negative control (J) of Wild mice at 12 wk after 5/6Nx is shown (original magnification: x400). B: semiquantitative analysis of glomerular expression of PDGF-BB (A) and TGF-{beta}1 (B). The staining score was calculated as described in MATERIALS AND METHODS. Values are means ± SE; n = 6/group. Open bars, Wild mice; shaded bars, AT2-Tg mice; filled bars, PD-123319-treated AT2-Tg mice.#P < 0.05 vs. Wild control mice.$P < 0.05 vs. AT2-Tg control mice. *P < 0.01 vs. AT2-Tg mice at 12 wk after 5/6Nx.

 

Urinary NOx excretion. Urinary excretion of NOx was significantly increased in AT2-Tg compared with Wild mice at week 0 [Fig. 6; 0.47 ± 0.10 (Wild) vs. 1.16 ± 0.14 µmol/ml (AT2-Tg)]. At 12 wk after 5/6Nx in the Wild group, NOx excretion showed a significant reduction compared with the Wild control (0.33 ± 0.04 vs. 0.47 ± 0.10 µmol/mg). At 6 wk after 5/6Nx in AT2-Tg mice, the amount of urinary NOx was significantly increased compared with AT2-Tg control, but at 12 wk after 5/6Nx it returned to the AT2-Tg control level (AT2-Tg at 6 wk after 5/6Nx: 1.95 ± 0.23 µmol/mg vs. AT2-Tg at 12 wk after 5/6Nx; 1.21 ± 0.13 µmol/mg vs. AT2-Tg at week 0, 1.16 ± 0.14 µmol/mg). PD-123319 treatment decreased urinary NOx to the level of the Wild group at 12 wk after 5/6Nx (AT2-Tg at 12 wk after 5/6Nx treated by PD-123319: 0.45 ± 0.04 µmol/mg vs. Wild mice at 12 wk after 5/6Nx, 0.33 ± 0.04 µmol/mg).



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  		Fig. 6. Urinary total nitrates and nitrites (NOx)/creatinine excretion (24 h). Values are means ± SE; n = 6/group. {circ}, Wild control; {square}, AT2-Tg control; {bullet}, Wild after 5/6Nx; {blacksquare}, AT2-Tg after 5/6Nx; {blacktriangleup}, PD-123319-treated AT2-Tg after 5/6Nx. *P < 0.05 vs. Wild control. **P < 0.05 vs. Wild after 5/6Nx.#P < 0.05 vs. AT2-Tg control.$P < 0.05 vs. AT2-Tg after 5/6Nx.

 

EMSA analysis of Egr-1 DNA binding activity. DNA binding activity of the Egr-1 was assessed by EMSA (Fig. 7A, left). DNA binding activity of Egr-1was increased in the Wild group after 5/6Nx (lanes 2 and 3), but it was significantly attenuated in AT2-Tg (lanes 5 and 6), especially at 12 wk after 5/6Nx. This attenuation was reversed by PD-123319 treatment (lane 7). Administration of PBS in the AT2-Tg group did not alter Egr-1 activity (lane 8). To demonstrate the specificity of Egr-1 binding, a competition assay was performed (Fig. 7A, right). The addition of 100-fold excess of unlabeled Egr-1 consensus probe abolished the binding of Egr-1 in the Wild group at 12 wk after 5/6Nx, whereas the unlabeled mutant Egr-1 probe had no effect (lanes 2 and 3). Scanning densitometry was performed to quantitate Egr-1 activation (Fig. 7B).



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  		Fig. 7. A: EMSA of early growth response gene-1 (Egr-1). Left: lane 1, nuclear extracts from Wild control mice; lane 2, Wild mice at 6 wk after 5/6Nx; lane 3, Wild mice at 12 wk after 5/6Nx; lane 4, AT2-Tg control mice; lane 5, AT2-Tg mice at 6 wk after 5/6Nx; lane 6, AT2-Tg mice at 12 wk after 5/6Nx; lane 7, AT2-Tg mice at 12 wk after 5/6Nx treated by PD-123319; lane 8, AT2-Tg mice at 12 wk after 5/6Nx treated with PBS. Right: competition experiments of Egr-1 binding. Lane 1, nuclear extracts from Wild mice at 12 wk after 5/6Nx; lane 2, pretreated with 100x excess cold Egr-1; lane 3, pretreated with 100x excess cold mutant Egr-1. Arrow, position of Egr-1-DNA complex. Representative results of 3 independent experiments are shown. B: scanning densitometry of Egr-1 activation in Wild and AT2-Tg mice. Open bars, Wild mice; shaded bars, AT2-Tg mice; filled bars, PD-123319-treated AT2-Tg mice.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we used transgenic mice overexpressing AT2R under the control of the {alpha}-SMA promoter (53). These mice were more resistant to the functional and morphological alterations induced by 5/6Nx than Wild mice. Although the level of AT2R mRNA in AT2-Tg at 12 wk after 5/6Nx was similar to 6 wk after 5/6Nx, the protein level of AT2-Tg at 12 wk was weaker than at 6 wk after 5/6Nx. A possible explanation could be altered stability of AT2-Tg mRNA, altered efficiency in translation, or diminished cell surface distribution of AT2R. The BUN level and UAE were decreased at 12 wk after 5/6Nx in AT2-Tg mice. The parameters of glomerular impairment after 5/6Nx, such as hypertrophy, hypercellularity, and sclerosis, were also improved in AT2-Tg mice. Previous reports demonstrated different outcomes after subtotal renal mass ablation in mice (10, 11, 24), and a study utilizing C57BL/6 mice failed to show a significant difference in renal function. In that study, glomerular hypertrophy and glomerulosclerosis were mildly increased after subtotal renal mass ablation (24). We speculate that the different outcome can be attributed to the difference in age and gender of the mice used, the method used for renal mass ablation after uninephrectomy, and the interval between uninephrectomy and the ablation of two poles of the remaining kidney. Recent study using PD-123319 and/or AT1RA (valsartan) demonstrated the protective effect of PD-123319 in a rat subtotal nephrectomy model (3). In that study, expression of AT1R mRNA or protein was decreased in nephrectomized rats, and that of AT2R mRNA was elevated in nephrectomized rats compared with control rats (3). Interestingly, the AT2R protein level was not altered after subtotal nephrectomy (3), and these results show similarities with our present findings of a discrepancy between AT2R mRNA and protein level after subtotal nephrectomy. AT2R blockade resulted in a reduced increase in proteinuria induced by subtotal nephrectomy, but to a lesser degree compared with valsartan treatment (3). Because AT2R is "overexpressed" under the control of {alpha}-SMA promoter in AT2-Tg mice, the number of AT2Rs expressed on activated mesangial cells is markedly higher than the physiological level (as evidenced by the comparison with Wild 5/6Nx mice) and thus leads to reduced glomerular injuries compared with Wild control mice in the present study. Interestingly, our results showed deterioration of glomerular injuries by treatment with PD-123319, further supporting the potential beneficial effect of AT2R overexpression in the setting of subtotal nephrectomy. Reduction of systemic blood pressure is known to be beneficial for the kidney (26). Because there was no difference in initial MBP after 5/6Nx between Wild and AT2-Tg mice, we speculate that the renoprotective effect is mediated by AT2R-mediated signaling and not directly by the hemodynamic effect.

Alterations in TGF-{beta}1 and PDGF-BB expression, two major growth factors involved in progressive renal disorders (2, 8, 21), were studied. The glomerular expression of PDGF-BB and TGF-{beta}1 in 5/6Nx mice was significantly suppressed in AT2-Tg mice, consistent with the postulated role of PDGF-BB and TGF-{beta}1 as mediators of glomerular hypertrophy and injury. A previous study demonstrated that administration of the angiontensin-converting enzyme inhibitor ramipril and the AT1R blocker valsartan blunted the increase in TGF-{beta}1 mRNA and attenuated glomerulosclerosis in a rat subtotal nephrectomy model (54). The inhibitory effect of valsartan on TGF-{beta}1 expression induced by subtotal nephrectomy (54) may be attributed, at least in part, to enhanced AT2R signaling in addition to AT1R blockade, considering our findings obtained by AT2-Tg mice.

Egr-1 is a member of the family of immediate-early genes (42). It is rapidly and transiently induced by a variety of mitogens (52). Egr-1 binds to DNA through three zinc-finger domains (6) and transactivates downstream genes such as PDGF-AA, PDGF-BB (22), and TGF-{beta}1 (28). In cultured mesangial cells, antisense oligonucleotides directed against Egr-1 mRNA have been reported to block mitogen-induced Egr-1 expression and inhibit proliferation (16). Egr-1 induction was also observed in parallel with mesangial cell proliferation in vivo in anti-Thy1.1 nephritis (43), uninephrectomy (36), and ischemia-reperfusion (38) models. In the present study, Egr-1 DNA binding activity in glomeruli was induced after 5/6Nx in Wild mice. In contrast, the increase in Egr-1 DNA binding induced by 5/6Nx in AT2-Tg mice was less than its increase in Wild mice. Inhibition of Egr-1 was abolished by PD-123319 treatment, suggesting that AT2R-mediated signaling may inactivate Egr-1 activity.

Renal NO synthase (NOS) activity has been reported to decrease in a rat 5/6Nx model (16). In this model, stimulation of NO production resulted in normalization of creatinine clearance, suggesting that diminished NO production may be partially responsible for renal impairment (1). Furthermore, neuronal (n)NOS expression was downregulated in the cortex after 5/6Nx, and this reduction was blocked by an AT1RA (41). A recent report demonstrated that stimulation of renal AT2R activated nNOS (46). We showed that urinary excretion of NOx was significantly increased in AT2-Tg mice at 6 wk after 5/6Nx. Activation of nNOS through AT2R signaling may be the mechanism of increased NO production. NO is involved in vasodilatation, cell growth, apoptosis, and inflammation (14). NO inhibits growth of mesangial cells by cGMP-dependent as well as, more importantly, by cGMP-independent pathways, including interference with Egr-1 (25). The cGMP-independent pathway is mediated through nitrosylation of protein thiol groups involved in complexing of Zn2+/Cd2+ with subsequent formation of disulfide bonds (25). It is therefore conceivable that NO destroys the Cys2/His2-type zinc-fingers of the Egr-1 protein and prevents DNA binding. We speculate that Egr-1 binding activity is potentially inhibited through the AT2R-NO pathway. In addition, NO possesses various biological functions, including the regulation of hemodynamics, tubuloglomerular feedback, renin release, and sodium as well as water excretion (23). It is possible that the renoprotective effect in AT2-Tg mice after 5/6Nx is mediated by the effect of NO on renal hemodynamics.

AT2R-mediated signaling also activates protein tyrosine phosphatase, resulting in the inactivation of ERK, which is activated by AT1R and growth factors (37). Serine/threonine phosphatase 2A activation and consequent ERK inactivation through AT2R have also been reported (17). Previous reports demonstrated that ERK activity in the hearts of AT2-Tg mice was decreased (32), suggesting the role of AT2R in inhibiting ERK activation. ANG II activates Egr-1 through the activation of ERK via AT1R (7). The downregulation of Egr-1 activity observed in AT2-Tg mice after 5/6Nx might be potentially mediated via ERK inactivation induced by AT2R signaling.

In conclusion, our findings demonstrated that the glomerular hypercellularity and glomerulosclerosis induced by renal mass ablation were significantly diminished in AT2-Tg mice. This renoprotective effect may be attributed to the inhibition of Egr-1 activity and increased NO production induced via enhanced AT2R signaling. These results strongly imply that AT2R signaling may influence the pathogenesis and remodeling in renal diseases and that a further understanding of this pathway may contribute to the development of novel therapeutic approaches for renal diseases.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Part of this study was presented at the 34th annual meeting of the American Society of Nephrology and has been published in abstract form (J Am Soc Nephrol 12: 591A, 2001).

This study was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. Y. Maeshima is a recipient of the 2002 Research Award from the KANAE Foundation for Life and Socio-Medical Science, the 2002 Young Investigator Award from the Japan Society of Cardiovascular Endocrinology and Metabolism, and the 2003 Research Award from the Kobayashi Magobei Memorial Foundation for Medical Science.


   ACKNOWLEDGMENTS
 
We thank A. Morinaga and Y. Ishimaru for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Maeshima, Dept. of Medicine and Clinical Science, Okayama Univ. Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan (E-mail: ymaeshim{at}md.okayama-u.ac.jp).

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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