Overexpression of angiotensin type 2 receptor ameliorates glomerular injury in a - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:16 |只看该作者 |倒序浏览 |打印

作者：Naoko Hashimoto, Yohei Maeshima, Minoru Satoh, Masahiro Odawara, Hitoshi Sugiyama, Naoki Kashihara, Hiroaki Matsubara, Yasushi Yamasaki, and Hirofumi Makino作者单位：1 Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558; 2 Division of Nephrology, Department of Internal Medicine, Kawasaki Medical School, Kurashiki 701-01; and 3 Department of Medicine II, Kansai Medical School, Osaka 570-850 J

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      【摘要】& a5 c1 w- O0 U, d9 B& K- e8 O/ B
   Angiotensin II mediates the progression of renal disease through the type 1 receptor (AT 1 R). Recent studies have suggested that type 2 receptor (AT 2 R)-mediated signaling inhibits cell proliferation by counteracting the actions of AT 1 R. The aim of the present study was to determine the effect of AT 2 R overexpression on glomerular injury induced by nephrectomy ( Nx). AT 2 R transgenic mice (AT 2 -Tg), overexpressing AT 2 R under the control of -smooth muscle actin ( -SMA) promoter, and control wild-type mice (Wild) were subjected to Nx. In AT 2 -Tg mice, the glomerular expression of AT 2 R was upregulated after Nx. Urinary albumin excretion at 12 wk after Nx was decreased by 33.7% in AT 2 -Tg compared with Wild mice. Glomerular size in AT 2 -Tg mice was significantly smaller than in Wild mice after Nx (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- 1 (TGF- 1 ) in AT 2 -Tg with Nx compared with Wild mice. Urinary excretion of nitric oxide metabolites was increased 2.5-fold in AT 2 -Tg compared with Wild mice. EMSA showed that activation of early growth response gene-1, which induces the transcription of PDGF-BB and TGF- 1, was decreased in AT 2 -Tg mice. These changes in AT 2 -Tg mice at 12 wk after Nx were blocked by the AT 2 R antagonist PD-123319. Taken together, our findings suggest that AT 2 R-mediated signaling may protect from glomerular injuries induced by Nx and that overexpression of AT 2 R may serve as a potential therapeutic strategy for glomerular disorders.
      【关键词】 angiotensin II receptor angiotensin II transgenic mouse nitric oxide* P7 O" H* u2 X8 C
   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, AT 1 R and AT 2 R ( 33 ). AT 1 Rs are expressed in the afferent and efferent arterioles, glomeruli, and proximal tubules in normal animals and humans ( 45 ). Studies with AT 1 R- and AT 2 R-selective antagonists have revealed that the known biological actions of ANG II are exclusively mediated via AT 1 R ( 33 ). AT 1 Rs 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 AT 1 R antagonist (AT 1 RA) indicated the involvement of AT 1 R in the progression of renal disorders. The analysis of AT 1a receptor knockout mice ( 15, 44 ) demonstrated the renoprotective effect of AT 1 R blockade. AT 1 RA prevents binding of ANG II to AT 1 R and increases levels of plasma renin and ANG II ( 12 ). Increased ANG II may potentially induce signaling through AT 2 R. This alternate signaling via AT 2 R could be an additional mechanism of renoprotection by AT 1 RA.- l* x7 R$ M  I0 _4 }1 o6 w& Y6 K
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Recent studies have suggested that AT 2 R-mediated signaling induces inhibition of cell growth or apoptosis by counteracting the AT 1 R signal ( 49 ). The basal systolic blood pressure of AT 2 R-null mice is mildly elevated compared with wild-type (Wild) mice ( 48 ). Renal interstitial fibrosis was accelerated in AT 2 R-null mice during unilateral ureteral obstruction ( 27 ), suggesting its important role in the remodeling of renal interstitium. The AT 2 R 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 AT 2 R was localized to interlobular arteries but not in glomeruli ( 34 ). However, the AT 2 R is thought to be expressed in glomeruli in response to RAS activation, such as during sodium depletion ( 39 ). The mechanism of AT 2 R signaling has not yet been fully elucidated.3 }$ M8 ^. L" v% V) B
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Platelet-derived growth factor-BB chain (PDGF-BB) and transforming growth factor- 1 (TGF- 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- 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 nephrectomy ( Nx) model, renal dysfunction is associated with disturbed NO production ( 41 ).% p8 _' i1 E$ j2 z4 N

On glomerular injury, mesangial cells acquire the phenotype of myofibroblasts, characterized by the expression of -smooth muscle actin ( -SMA) ( 19 ). Elevation of mesangial -SMA precedes the development of glomerulosclerosis in the rat Nx model, indicating that phenotypic alteration of mesangial cells is associated with progressive renal disorders ( 8 ).! }5 h$ @+ b( K, [! v8 k7 U

In the present study, we used AT 2 R transgenic mice (AT 2 -Tg), overexpressing mouse AT 2 R under the control of the mouse -SMA promoter ( 53 ), to elucidate the role of the AT 2 R-mediated signaling in the Nx model. Our results indicate the protective role of AT 2 R-mediated signaling against progressive glomerular injuries.9 j4 C8 o0 R# n0 k% |$ }: [/ t

MATERIALS AND METHODS* i0 |' @0 p- o7 y$ Y" O0 }

Experimental protocol. Male AT 2 -Tg mice (C57BL/6 background), overexpressing mouse AT 2 R under the control of the mouse -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 AT 2 R-specific antagonist, was infused using an Alzet osmotic pump (model 2002; Alza, Palo Alto, CA) from 6-12 wk after Nx in the AT 2 -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.
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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% NH 4 Cl) were used. Absorbance was detected at 540 nm using a microplate reader (model 550; Bio-Rad, Hercules, CA).7 Q6 }6 a0 ?# Q4 r2 M/ Z5 y

Histopathological assessment. At 6 or 12 wk after Nx, 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
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Immunohistochemistry was performed using frozen sections as described previously ( 30, 44 ). Samples were incubated overnight at 4°C with polyclonal anti-mouse- -SMA (Sigma, St. Louis, MO), polyclonal rabbit anti-mouse AT 2 R, PDGF-BB, and TGF- 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- 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.9 D% |! w. H2 W/ c* M6 g

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 AT 2 R 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 MgCl 2, 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 AT 2 R mRNA in relation to GAPDH mRNA. The following oligonucleotide primers specific for mouse AT 2 R and GAPDH were used: AT 2 R, 5'-CAGAATTACCCGTGACCAAG-3' (forward) and 5'-TAAACACACTGCGGAGCTT-3' (reverse); GAPDH, forward 5'-ATGGTGAAGGTCGGTGTG-3' and reverse 5'-ACCAGTGGATGCAGGGAT-3'. Four independent experiments were performed.; Y/ ?. {! r/ @8 A% e! T* x% ^
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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)." o9 a6 @# Q7 \; |8 h+ z; E2 z

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'-GGATCCA GCGGGGGCG A GCGGGGGCG A-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 MgCl 2, 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'-GGATCCA GCTAGGGCG A GCTAGGGCG A-3').( A; i' Q4 ^6 e+ ]# R" C0 }
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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

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Expression of -SMA and AT 2 R. Using quantitative real-time RT-PCR, we investigated the mRNA expression of AT 2 R in glomeruli ( Fig. 1 ). Although AT 2 R mRNA was not detectable in Wild mice, it was expressed in AT 2 -Tg mice and was elevated at 6 and 12 wk after Nx. The distribution pattern of -SMA in glomeruli was studied by immunohistochemistry ( Fig. 2 A ). The expression of -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 -SMA was upregulated in both groups at 6 wk after Nx, mainly in the mesangial area ( Fig. 2, AB and AE ). In the AT2-Tg group, -SMA expression was decreased at 12 wk after Nx ( Fig. 2 AF ). Expression of AT 2 R protein was absent in glomeruli of the Wild group, as detected by immunohistochemistry ( Fig. 2, BA-BC ). Weak AT 2 R expression was observed in AT 2 -Tg control mice, and it was markedly upregulated in AT 2 -Tg mice at 6 wk and mildly upregulated at 12 wk after Nx, mainly in the mesangium ( Fig. 2, BE and BF ), similar to -SMA. Because AT 2 Rs were overexpressed under the control of -SMA promoter in AT 2 -Tg mice, we speculate that the distribution pattern of AT 2 Rs in glomeruli was in parallel with that of -SMA in AT 2 -Tg mice.
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Fig. 1. Expression of AT 2 receptor (AT 2 R) 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 AT 2 R mRNA relative to GAPDH mRNA is shown. Results were expressed relative to AT 2 R transgenic mice (AT 2 -Tg) 6 (6W) and 12 wk (12W) after nephrectomy ( Nx), which were arbitrarily assigned a value of 100%. Wild, wild-type mice; Cont, control.
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Fig. 2. Immunohistochemistry of -smooth muscle actin (SMA) in kidneys of Wild and AT 2 -Tg mice. A : representative photomicro-graphs of Wild control ( A ), 6 ( B ) or 12 wk ( C ) after Nx, and AT 2 -Tg control ( D ), 6 ( E ) or 12 wk ( F ) after Nx are shown (original magnification: x 400). B : immunohistochemistry of AT 2 R in kidneys of Wild and AT 2 -Tg mice. Representative photomicrographs of Wild control ( A ), 6 ( B ) or 12 wk ( C ) after Nx, and AT 2 -Tg control ( D ), 6 ( E ) or 12 wk ( F ) after Nx are shown (original magnification: x 400).
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Changes in arterial blood pressure and BW. Arterial blood pressure in Wild and AT 2 -Tg mice increased within 3 wk of Nx ( Table 1 ). PD-123319 treatment did not affect the MBP of AT 2 -Tg mice after Nx. The increase in BW of Wild mice was not observed at time points later than 6 wk after Nx ( Table 2 ). In AT 2 -Tg mice, BW did increase at time points later than 6 wk after Nx. The increase in BW was not observed at 6 wk after Nx in AT 2 -Tg mice treated by PD-123319, similar to Nx in Wild mice. The kidney weight (KW)-to-BW ratio (KW/BW) in AT 2 -Tg mice after Nx was significantly lower than in Wild mice ( Table 2 ). However, the KW/BW of AT 2 -Tg mice treated by PD-123319 was elevated in Wild mice, similar to that after Nx.
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Table 1. Mean blood pressure

Table 2. Body weight and kidney weight! [6 `) s7 e, n# @6 }9 w' N6 E6 t. n

Changes in BUN and urinary albumin excretion. To evaluate the effect of AT 2 R overexpression on Nx-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 AT 2 -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 Nx, but they were not elevated in AT 2 -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 AT 2 -Tg mice after Nx was abolished by PD-123319 treatment (UAE: 224.8 ± 12.4 µg/mg).% ?: r% ^, ^" i" K$ X1 r
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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, AT 2 -Tg mice; filled bars, PD-123319-treated AT 2 -Tg after Nx. * P 2 _6 V( c/ }6 z2 F* }* `
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Histological and morphometric analysis. Histological examination of the kidneys revealed no difference between Wild and AT 2 -Tg control mice ( Fig. 4, AA and AE ). At 12 wk after Nx, kidney sections from Wild mice exhibited glomerular hypertrophy, cellular proliferation, and glomerular sclerosis ( Fig. 4, AB and AF ). In AT 2 -Tg mice at 12 wk after Nx, these histological changes were milder than those in Wild mice ( Fig. 4, AC and AG ). Glomerular damage in AT 2 -Tg at 12 wk after Nx 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 Nx ( Fig. 4 B ). These changes were significantly suppressed in Wild vs. AT 2 -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 AT 2 -Tg mice after Nx (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 Nx were significantly diminished in AT 2 -Tg mice. No significant difference was observed in renal interstitial fibrosis between Wild and AT 2 -Tg mice at 12 wk after Nx (data not shown).

Fig. 4. A : histological appearance of glomeruli in Wild and AT 2 -Tg mice. Wild control ( A and E ), 12 wk after Nx ( B and F ), AT 2 -Tg 12 wk after Nx ( C and G ), AT 2 -Tg 12 wk after Nx treated with PD-123319 ( D and H ). Periodic acid-Schiff (PAS; A - D ) and Azan-Mallory staining ( E - H ) (original magnification: x 400). B : quantification of glomerular diameter ( A ), total cell number ( B ), and sclerosis index ( C ) in Wild and AT 2 -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, AT 2 -Tg mice; filled bars, PD-123319-treated AT 2 -Tg mice. # P

Immunohistochemical analysis of glomerular PDGF-BB chain and TGF- 1 expression. To further evaluate the involvement of AT 2 R in phenotypic alterations in glomeruli, the expression levels of PDGF-BB and TGF- 1 were examined by immunohistochemistry ( Fig. 5 A ). These growth factors act as key molecules in progressive glomerulosclerosis and mesangial cell proliferation after renal ablation ( 8, 18, 21 ). At 12 wk after Nx, the glomerular expression of PDGF-BB and TGF- 1 was significantly increased in the Wild group ( Fig. 5, AB and AG ), whereas this effect was significantly inhibited in AT 2 -Tg mice ( Fig. 5, AC and AH ). These inhibitory effects were abolished by PD-123319 in the AT 2 -Tg group ( Fig. 5, AD and AI ). In both Wild and AT 2 -Tg groups, the staining score of PDGF-BB and TGF - 1 was significantly increased at 6 wk after Nx ( Fig. 5 B ). However, it was significantly decreased in AT 2 -Tg at 12 wk after Nx compared with the Wild group (PDGF-BB chain: 3.16 ± 0.20 vs. 1.06 ± 0.28; TGF - 1 : 2.80 ± 0.26 vs. 1.52 ± 0.13). These inhibitory effects were reversed by PD-123319 treatment in AT 2 -Tg mice at 12 wk after Nx (PDGF-BB: 2.36 ± 0.33; TGF - 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- 1 (TGF- 1 ). PDGF-BB staining in Wild control ( A ), 12 wk after Nx ( B ), AT 2 -Tg 12 wk after Nx ( C ), AT 2 -Tg 12 wk after Nx treated with PD-123319 ( D ), and negative control ( E ) of Wild mice 12 wk after Nx. TGF- 1 staining in Wild control ( F ), 12 wk after Nx ( G ), AT 2 -Tg 12 wk after Nx ( H ), AT 2 -Tg 12 wk after Nx treated with PD-123319 ( I ), and negative control ( J ) of Wild mice at 12 wk after Nx is shown (original magnification: x 400). B : semiquantitative analysis of glomerular expression of PDGF-BB ( A ) and TGF- 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, AT 2 -Tg mice; filled bars, PD-123319-treated AT 2 -Tg mice. # P
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Urinary NOx excretion. Urinary excretion of NOx was significantly increased in AT 2 -Tg compared with Wild mice at week 0 [ Fig. 6; 0.47 ± 0.10 (Wild) vs. 1.16 ± 0.14 µmol/ml (AT 2 -Tg)]. At 12 wk after Nx 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 Nx in AT 2 -Tg mice, the amount of urinary NOx was significantly increased compared with AT 2 -Tg control, but at 12 wk after Nx it returned to the AT 2 -Tg control level (AT 2 -Tg at 6 wk after Nx: 1.95 ± 0.23 µmol/mg vs. AT 2 -Tg at 12 wk after Nx; 1.21 ± 0.13 µmol/mg vs. AT 2 -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 Nx (AT 2 -Tg at 12 wk after Nx treated by PD-123319: 0.45 ± 0.04 µmol/mg vs. Wild mice at 12 wk after Nx, 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., Wild control;, AT 2 -Tg control;, Wild after Nx;, AT 2 -Tg after Nx;, PD-123319-treated AT 2 -Tg after Nx. * P

EMSA analysis of Egr-1 DNA binding activity. DNA binding activity of the Egr-1 was assessed by EMSA ( Fig. 7 A, left ). DNA binding activity of Egr-1was increased in the Wild group after Nx ( lanes 2 and 3 ), but it was significantly attenuated in AT 2 -Tg ( lanes 5 and 6 ), especially at 12 wk after Nx. This attenuation was reversed by PD-123319 treatment ( lane 7 ). Administration of PBS in the AT 2 -Tg group did not alter Egr-1 activity ( lane 8 ). To demonstrate the specificity of Egr-1 binding, a competition assay was performed ( Fig. 7 A, 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 Nx, whereas the unlabeled mutant Egr-1 probe had no effect ( lanes 2 and 3 ). Scanning densitometry was performed to quantitate Egr-1 activation ( Fig. 7 B ).0 x! K! q. Z7 W

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 Nx; lane 3, Wild mice at 12 wk after Nx; lane 4, AT 2 -Tg control mice; lane 5, AT 2 -Tg mice at 6 wk after Nx; lane 6, AT 2 -Tg mice at 12 wk after Nx; lane 7, AT 2 -Tg mice at 12 wk after Nx treated by PD-123319; lane 8, AT 2 -Tg mice at 12 wk after Nx treated with PBS. Right : competition experiments of Egr-1 binding. Lane 1, nuclear extracts from Wild mice at 12 wk after Nx; lane 2, pretreated with 100 x excess cold Egr-1; lane 3, pretreated with 100 x 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 AT 2 -Tg mice. Open bars, Wild mice; shaded bars, AT 2 -Tg mice; filled bars, PD-123319-treated AT 2 -Tg mice.+ b; M4 \" V; V, @( [; b
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DISCUSSION
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In the present study, we used transgenic mice overexpressing AT 2 R under the control of the -SMA promoter ( 53 ). These mice were more resistant to the functional and morphological alterations induced by Nx than Wild mice. Although the level of AT 2 R mRNA in AT 2 -Tg at 12 wk after Nx was similar to 6 wk after Nx, the protein level of AT 2 -Tg at 12 wk was weaker than at 6 wk after Nx. A possible explanation could be altered stability of AT 2 -Tg mRNA, altered efficiency in translation, or diminished cell surface distribution of AT 2 R. The BUN level and UAE were decreased at 12 wk after Nx in AT 2 -Tg mice. The parameters of glomerular impairment after Nx, such as hypertrophy, hypercellularity, and sclerosis, were also improved in AT 2 -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 AT 1 RA (valsartan) demonstrated the protective effect of PD-123319 in a rat subtotal nephrectomy model ( 3 ). In that study, expression of AT 1 R mRNA or protein was decreased in nephrectomized rats, and that of AT 2 R mRNA was elevated in nephrectomized rats compared with control rats ( 3 ). Interestingly, the AT 2 R protein level was not altered after subtotal nephrectomy ( 3 ), and these results show similarities with our present findings of a discrepancy between AT 2 R mRNA and protein level after subtotal nephrectomy. AT 2 R blockade resulted in a reduced increase in proteinuria induced by subtotal nephrectomy, but to a lesser degree compared with valsartan treatment ( 3 ). Because AT 2 R is "overexpressed" under the control of -SMA promoter in AT 2 -Tg mice, the number of AT 2 Rs expressed on activated mesangial cells is markedly higher than the physiological level (as evidenced by the comparison with Wild Nx 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 AT 2 R 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 Nx between Wild and AT 2 -Tg mice, we speculate that the renoprotective effect is mediated by AT 2 R-mediated signaling and not directly by the hemodynamic effect.$ P- i# V$ x, t
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Alterations in TGF- 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- 1 in Nx mice was significantly suppressed in AT 2 -Tg mice, consistent with the postulated role of PDGF-BB and TGF- 1 as mediators of glomerular hypertrophy and injury. A previous study demonstrated that administration of the angiontensin-converting enzyme inhibitor ramipril and the AT 1 R blocker valsartan blunted the increase in TGF- 1 mRNA and attenuated glomerulosclerosis in a rat subtotal nephrectomy model ( 54 ). The inhibitory effect of valsartan on TGF- 1 expression induced by subtotal nephrectomy ( 54 ) may be attributed, at least in part, to enhanced AT 2 R signaling in addition to AT 1 R blockade, considering our findings obtained by AT 2 -Tg mice.7 M% N9 ^1 N: v3 S, P+ F

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- 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 Nx in Wild mice. In contrast, the increase in Egr-1 DNA binding induced by Nx in AT 2 -Tg mice was less than its increase in Wild mice. Inhibition of Egr-1 was abolished by PD-123319 treatment, suggesting that AT 2 R-mediated signaling may inactivate Egr-1 activity.
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Renal NO synthase (NOS) activity has been reported to decrease in a rat Nx 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 Nx, and this reduction was blocked by an AT 1 RA ( 41 ). A recent report demonstrated that stimulation of renal AT 2 R activated nNOS ( 46 ). We showed that urinary excretion of NOx was significantly increased in AT 2 -Tg mice at 6 wk after Nx. Activation of nNOS through AT 2 R 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 Zn 2  /Cd 2  with subsequent formation of disulfide bonds ( 25 ). It is therefore conceivable that NO destroys the Cys 2 /His 2 -type zinc-fingers of the Egr-1 protein and prevents DNA binding. We speculate that Egr-1 binding activity is potentially inhibited through the AT 2 R-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 AT 2 -Tg mice after Nx is mediated by the effect of NO on renal hemodynamics.

AT 2 R-mediated signaling also activates protein tyrosine phosphatase, resulting in the inactivation of ERK, which is activated by AT 1 R and growth factors ( 37 ). Serine/threonine phosphatase 2A activation and consequent ERK inactivation through AT 2 R have also been reported ( 17 ). Previous reports demonstrated that ERK activity in the hearts of AT 2 -Tg mice was decreased ( 32 ), suggesting the role of AT 2 R in inhibiting ERK activation. ANG II activates Egr-1 through the activation of ERK via AT 1 R ( 7 ). The downregulation of Egr-1 activity observed in AT 2 -Tg mice after Nx might be potentially mediated via ERK inactivation induced by AT 2 R signaling.! @; U$ s" T9 b& Y

In conclusion, our findings demonstrated that the glomerular hypercellularity and glomerulosclerosis induced by renal mass ablation were significantly diminished in AT 2 -Tg mice. This renoprotective effect may be attributed to the inhibition of Egr-1 activity and increased NO production induced via enhanced AT 2 R signaling. These results strongly imply that AT 2 R 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.% y& }; I" Z5 H. t
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GRANTS
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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).
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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.. M9 l% e2 Q# |
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ACKNOWLEDGMENTS5 f" l0 |$ U$ M

We thank A. Morinaga and Y. Ishimaru for technical assistance.6 N6 C# p+ _' ]' ~. S+ ?8 ^0 {
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