Regulatory Peptides 154 (2009) 44–53
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Natriuretic peptide/natriuretic peptide receptor-A (NPR-A) system has inhibitory effects in renal fibrosis in mice Toshio Nishikimi a,⁎, Chikako Inaba-Iemura a, Kimihiko Ishimura a, Kazuyoshi Tadokoro a, Shogo Koshikawa a, Keiko Ishikawa a, Kazumi Akimoto b, Yoshiyuki Hattori c, Kikuo Kasai c, Naoto Minamino d, Nobuyo Maeda e, Hiroaki Matsuoka a a
Department of Hypertension and Cardiorenal Medicine, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan Department of Laboratory Medicine, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan c Department of Endocrine and Metabolism, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan d Research Institute National Cardiovascular Center, Fujishirodai 5, Osaka 565-8565, Japan e Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7525, United States b
a r t i c l e
i n f o
Article history: Received 28 July 2008 Received in revised form 11 December 2008 Accepted 3 February 2009 Available online 15 February 2009 Keywords: Atrial natriuretic peptide Renal fibrosis cGMP Renin–angiotensin NF-kappaB
a b s t r a c t Object: This study was designed to examine whether natriuretic peptide/natriuretic peptide receptor-A (NPR-A) system attenuates renal fibrosis in a unilateral ureteral obstruction (UUO) model and also examined the mechanism involved. Methods: Three groups were studied: untreated UUO in wild-type mice; untreated UUO in NPR-A KO mice; and ANP treated (0.05 µg/kg/min) UUO in wild-type mice. We measured histological and immunohistochemical findings (α-SMA and F4/80), tissue cGMP levels, various mRNA expression levels by real-time PCR analysis, and transcription factor levels (AP-1 and NF-kappaB) in renal tissue. Results: Compared with wild-type UUO mice, NPRA-KO UUO mice had abnormal morphological findings (fibrous area: +26%, α-SMA expression: +30%) with lower tissue cGMP levels and increases in the mRNA expression levels of TGF-β, collagen I, collagen III, PAI-1, renin and angiotensinogen, whereas there were no differences in F4/80 positive cells or the mRNA expression levels of ICAM-1, osteopontin, or MCP-1 between the two groups. In contrast, ANP pre-treatment significantly improved morphological changes with increase of tissue cGMP levels and reduction in the mRNA expression level of TGF-β, collagen I, collagen III, PAI-1, ICAM-1, osteopontin, MCP-1, renin, and angiotensinogen. NPRA-KO UUO mice had higher AP-1 levels than wild-type UUO mice and ANP pre-treatment reduced AP-1 and NF-kappaB activity. Conclusion: The endogenous natriuretic peptide/NPR-A system may inhibit renal fibrosis partly via inhibition of the angiotensin/AP-1/TGF-β/collagen pathway and exogenous ANP pre-treatment may inhibit it partly via both the angiotensin/AP-1/TGF-β/collagen and NF-kappaB/inflammatory pathways. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Many neurohumoral factors, both vasodilatory factors and vasoconstrictory factors, play an important role in maintaining homeostasis of the cardiovascular system and renal function. Of these vasodilatory factors, a family of natriuretic peptides (NPs), e.g. atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), is involved [1]. ANP and BNP are produced and secreted by the heart and exert a variety of biological actions such as diuresis, natriuresis, vasorelaxation, and inhibition of renin and aldosterone secretion by binding natriuretic peptide receptor-A (NPR-A) [2].
⁎ Corresponding author. Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan. Tel.: +81 282 87 2149; fax: +81 282 86 1596. E-mail address:
[email protected] (T. Nishikimi). 0167-0115/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2009.02.006
Adding to these physiological actions, recent studies demonstrated that ANP and BNP regulate the growth of multiple cell types. In cultured vascular smooth muscle cells, NPs inhibit proliferation and migration induced by growth factor or angiotensin II, suggesting that NPs act as antiatherosclerotic factors [3]. In fact, doubly deficient NPR-A (−/−) and apolipoprotein E (ApoE) (−/−) mice showed greater atherosclerotic lesion size and more advanced plaque morphology compared with NPR-A (+/+) and ApoE (−/−) mice [4]. In cultured neonatal myocytes and fibroblasts, NPs also inhibit the growth of cardiac myocytes and fibroblasts [5,6]. A study using an NPR-A antagonist, HS-142-1, showed that endogenous ANP and BNP have antihypertrophic and antifibrotic actions [7]. Indeed, mice lacking NPR-A or BNP develop ventricular hypertrophy and fibrosis, respectively, independent of their blood pressure [8,9]. Taken together, these results suggest that ANP and BNP are autocrine and/ or paracrine antigrowth factors.
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However, there have been few studies regarding the role of ANP and BNP in kidney disease. An earlier study suggested that hyperfiltration by ANP contributes to glomerulosclerosis [10]. In contrast, recent studies have shown that the natriuretic peptide/NPR-A/ cyclic 3′,5′ guanosine monophosphate (cGMP) system inhibits proliferation in mesangial cells induced by angiotensin II or endothelin-1 [1]. The effect of the ANP/NPR-A system negatively regulates mitogen-activated protein kinases/extracellular signalregulated protein kinases activity and the proliferation of mesangial cells through a cGMP-dependent-protein kinase pathway [12]. Furthermore, renal impairment was attenuated in BNP transgenic mice compared with wild-type mice in models of renal ablation, glomerulonephritis, and diabetic nephropathy [13–15], suggesting that overexpression of BNP has renoprotective effects. However, the pathophysiological role of the endogenous natriuretic peptide/NPR-A system in renal disease is not fully understood. In addition, there have not been any studies that investigated the effects of long-term ANP infusion on renal interstitial fibrosis. Unilateral ureteral obstruction (UUO) is an excellent model of interstitial fibrosis because it is normotensive, non-proteinuric and non-hyperlipidemic [16]. Renal interstitial fibrosis is one of the common histopathological features of progressive renal disease [16]. In addition, recent studies revealed that increased monocyte/macrophage infiltration, renin–angiotensin system, growth factor, and NF-kappaB activity are involved in the progression of renal fibrosis in this model [17]. Therefore, using this model, we tested the hypothesis that the ANP/NPR-A system contributes to renal interstitial fibrosis. To examine this hypothesis, we used a genetic approach in mice. We produced a model of UUO in mice with wild-type (+/+) and homozygous null mutants (−/−) of the NPRA gene and evaluated the severity of renal interstitial fibrosis. To confirm the results from these genetically engineered mice, we also evaluated the effect of chronic infusion of ANP on the renal interstitial fibrosis. In addition, we attempted to elucidate the mechanism by measuring histological and immunohistochemical findings (α-smooth muscle actin (α-SMA) and F4/80), tissue cGMP levels, various mRNA expression levels, and transcription factor levels [17].
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(each n = 6–8) after the left ureteral ligation. Because some genes and transcription factors were reported to be activated in earlier days after UUO, and the other were reported to be activated in late phase [21,22]. Kidney tissue samples collected at day 4 after UUO were used for cGMP, real-time polymerase chain reaction (PCR) analyses and transcription factor measurement. Kidney tissue samples collected at day 11 after UUO were used for histology, immunohistochemistry, immunofluorescent, and real-time PCR analyses study. Sham group operated without ureteral ligation was also produced. 2.3. Renal morphology The obstructed kidney was excised and immersed in neutralized formalin for histological examination. The area of fibrotic lesions in the interstitium (fibrosis area) was determined on sections stained by Masson's trichrome method to stain the collagen fibers (stained blue), using a computer-aided manipulator program as described previously [23]. 2.4. Immunohistochemical analysis Immunohistochemical analysis using an antibody against F4/80, a monocyte/macrophage marker, was performed as reported previously [24]. Quantification of F4/80-positive area was determined using a computer-aided manipulator program as described previously [23]. 2.5. Immunofluorescence study Kidney sections were incubated with anti-α-SMA (1:100) and Ecadherin antibodies (1:100) overnight at room temperature. After washing, second antibody conjugated with Alexa-fluor 647 and Rhodamine was added for 4 h at room temperature. Nuclei were labeled with 4′,6-diamidino-2-phenylindol (DAPI). Images were acquired and red scale intensity was measured as described above [23]. 2.6. Plasma ANP concentration
2. Methods This study was approved by our institutional Animal Care Committee, and all of the procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Arterial blood samples from mice 4 days after UUO production were rapidly withdrawn as described previously [25]. ANP levels were determined by radioimmunoassay after extraction of plasma using a Sep-Pak C-18 cartridge as described previously [26]. 2.7. Renal tissue cGMP level
2.1. Experimental animals and design The NPR-A (−/−), and (+/+) mice used in this study were N8 or N9 generation of mice backcrossed to C57BL/6, derived from the original mutants as described previously [18]. Mice were divided into three groups: NPR-A (+/+) (n = 19), NPR-A (−/−) (n = 13), and NPR-A (+/+) with ANP infusion (n = 19). Rat synthetic ANP (0.05 µg/ kg/min) (Peptide Institute, Osaka, Japan) was infused using osmotic minipump as previously reported [19]. ANP infusion was started 2 days before unilateral obstruction. This dose (0.05 µg/kg/min) does not change blood pressure, but attenuated vascular and ventricular remodeling in rats [20]. ANP infusion was continued until mice were euthanized. BP was measured noninvasively in conscious and restrained mice as described previously at 11 days after the UUO operation (n =4–5). 2.2. Obstructive kidney disease model A UUO kidney disease model was induced in both WT and NPR-A NO mice (20–25 g body wt, 3–6 mo of age) by left ureteral ligation as described previously [16]. For investigation of the role of ANP/NPR-A system in the early and late time points of obstructive kidney disease, three groups mice were killed on day 4 (each n = 7–11) and day 11
Renal tissue cGMP levels were measured using radioimmunoassay kits (cyclic GMP assay kit, Yamasa Shoyu Co., Chiba, Japan) after extraction of cold 70% ethanol as described previously [26]. 2.8. Measurement of transcription factor level Renal tissue homogenates were extracted using NE-PER® nuclear and cytoplasmic extraction reagents (Pierce Perbio Science, Erembodegem, Belgium) according to the manufacturer's protocol. Protease inhibitors were added to the cytoplasmic and nuclear extraction reagents at the following final concentrations: 0.5 mg/ml benzamidine, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 0.75 mM phenylmethylsulfonyl fluoride to cytoplasmic extracts and 2 mM to nuclear extracts. Phosphatase inhibitors were also added: 1 or 0.5 mM sodium orthovanadate and 2 or 1 mM sodium fluoride to cytoplasmic and nuclear extracts, respectively. Then, the activated NF-kappaB (P50 and P65) and activator protein-1 (AP-1) activities were measured by Chemiluminescent NF-kappaB Assay kit (Marligen Biosciences, UK and Ireland.) and AP-1 Transcription Factor microplate assay kit (Marligen Biosciences), respectively. This assay kit allows the measurement of activated transcription factors from nuclear extracts. The assay measures
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Table 1 BW and kidney weight/body weight in three groups at four days and eleven days after UUO production.
Number at 4 days BW at 4 days Kid W/BW at 4 days (normal side) Kid W/BW at 4 days (UUO side) Number at 11 days BW at 11 days Kid W/BW at 11 days (normal side) Kid W/BW at 11 days (UUO side)
Wild-type
NPR-A KO
ANP therapy in wild-type
11 24.6 ± 2.4 7.3 ± 0.9 6.1 ± 1.2 8 24.7 ± 1.5 7.1 ± 0.6 6.1 ± 1.0
7 24.4 ± 5.0 7.8 ± 0.8 6.2 ± 0.6 6 23.1 ± 1.2 7.5 ± 0.9 6.5 ± 2.7
11 23.3 ± 3.1 6.9 ± 0.7 6.2 ± 0.7 8 23.5 ± 1.1 6.8 ± 0.2 5.9 ± 0.8
BW, body weight; Kid W, kidney weight; UUO: unilateral ureteral obstruction.
the binding of transcription factors to their cognate DNA binding sequences.
2.9. Quantification of mRNA expression The gene expression levels of renin, angiotensinogen, angiotensin type-1 receptor (AT1-R), collagen I, collagen III, transforming growth factor-beta (TGF-β), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), osteopontin, and NPR-C in the renal tissue were determined by real-time quantitative reverse-transcription polymerase chain reaction with the use of ABI 7700 and specific primers as reported previously [26].
2.10. Western blot analysis We measured phospho-Smad2/3 protein levels in the renal tissue as reported previously [27].
2.11. Statistical analysis Data are expressed as means ± SD. Statistical analysis was done by analysis of variance (ANOVA), using Fisher's post hoc test for multiple comparisons. Differences were considered statistically significant at P b 0.05. Statistical analysis was performed with the use of STATVIEW version 5 (Abacus Concepts, Berkeley, CA). 3. Results 3.1. Physiological profiles of the three groups Physiological profiles of the three groups at day four and day eleven are shown in Table 1. There were no significant differences in body weight or kidney weight/body weight among the three groups at four days or eleven days after UUO operation. NPR-A KO mice (127 ± 4 mm Hg) had significantly higher BP than wild-type mice (114 ± 6 mm Hg, P b 0.05) and ANP pre-treated mice (110 ± 6 mm Hg, P b 0.05). However, there were no differences in blood pressure between wildtype mice and ANP pre-treated mice. 3.2. Interstitial fibrosis of UUO kidneys in the three groups The representative appearances of Masson's trichrome-stained sections 11 days after ureteral obstruction are shown in Fig. 1. Interstitial fibrosis of obstructed kidney was prominent in untreated wild-type mice compared with a normal kidney of sham-operated wild-type mice (Fig. 1A and B). In obstructed kidney of NPR-A KO mice, interstitial fibrosis was more prominent compared with that in wild-type mice (Fig. 1C). ANP pre-treatment significantly ameliorated the interstitial fibrosis in obstructed kidney compared with that in wild-type mice (Fig. 1D). The tubular structure was also relatively well preserved in ANP treated mice. In measurement of the fibrous area in obstructed kidneys by
Fig. 1. The morphological appearance of the interstitium by Masson's trichrome staining in normal kidney in sham-operated mice (A) and in obstructed kidneys in wild-type (B), NPR-A KO (C), and ANP-treated mice (D) 11 days after production of UUO are shown. The areas of the fibrotic lesions of the interstitium (fibrosis area) are shown in panel E. Data are expressed as mean ± SD. Number of mice was 6−8 in each group. **P b 0.01 vs sham-operated, †P b 0.01 vs wild-type, ‡P b 0.05 vs. NPR-A KO.
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computed quantitative analysis, NPR-A KO had a greater fibrous area than wild-type mice, and ANP infusion significantly reduced the fibrous area (Fig. 1E). 3.3. Interstitial α-SMA expression in UUO kidneys in the three groups Induction of interstitial myofibroblasts was assessed by immunohistochemical detection of α-SMA. Positive staining for α-SMA was seen only in vascular smooth muscle cells, but not in the interstitial space in normal kidney in sham-operated wild-type mice (Fig. 2A). Intense immunostaining of α-SMA was observed in the peritubular interstitium in addition to the vascular smooth muscle cells of the arterioles in obstructed kidney in untreated wild-type mice 11 days after ureteral ligation (Fig. 2B). More intense immunostaining of α-SMA was observed in the interstitial space in the obstructed kidney in NPR-A KO mice (Fig. 2C). ANP pre-treatment reduced the appearance of α-SMA-positive myofibroblasts in the interstitium (Fig. 2D). The results of quantitative analysis of α-SMA immunostaining are shown in Fig. 2E. The α-SMA positive area in obstructed kidneys was significantly greater in NPR-A KO mice than in wild-type mice, and ANP pre-treatment significantly reduced the positive area compared with obstructed kidneys in wildtype mice (Fig. 2E). 3.4. Interstitial F4/80 expression in UUO kidneys in the three groups F4/80 positive cells in the interstitium are shown in Fig. 3. The number of F4/80 positive cells in the interstitium was obviously increased in untreated wild-type mice compared with normal kidneys of sham-operated wild-type mice (Fig. 3A and B). Whereas there was no difference in the number of F4/80 positive cells between wild-type mice and NPR-A KO mice (Fig. 3C). ANP pre-treatment significantly
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decreased the number of F4/80 positive cells compared with the other two groups (Fig. 3D). The results of quantitative analysis of F4/80 positive area are shown in Fig. 3E. The F4/80 positive area in obstructed kidneys was similar in NPR-A KO mice and wild-type mice, whereas ANP pre-treatment significantly reduced the F4/80 positive area compared with obstructed kidneys in the other two groups (Fig. 2E).
3.5. Plasma ANP levels, renal tissue cGMP levels, and mRNA expressions of Renal NPR-C and ANP Wild-type UUO mice had slightly but significantly higher plasma ANP levels than sham-operated mice at 4 days after operation (Fig. 4A). NPR-A KO UUO mice had higher plasma ANP levels than wild-type UUO and sham-operated mice at 4 days after operation (Fig. 4A), because of lacking ANP receptor. ANP infusion increased plasma ANP levels compared with the other three groups (Fig. 4A). Wild-type UUO mice had higher renal tissue cGMP levels than sham-operated mice at 4 days after operation (Fig. 4B). Renal tissue cGMP levels at 4 days after operation were significantly lower in NPR-A KO mice than wild-type mice (Fig. 4B), because of lacking ANP/NPR-A/cGMP cascade. ANP infusion increased renal tissue cGMP levels compared with the other three groups. NPR-C mRNA expression was observed in the kidney of shamoperated mice. Wild-type UUO mice had higher NPR-C mRNA levels than sham-operated mice (Fig. 4C). However, there were no differences in NPR-C mRNA levels among the three UUO groups. Renal ANP mRNA expression was observed in the kidney of sham-operated mice. Wildtype UUO mice had higher renal ANP mRNA expression than shamoperated mice (Fig. 4D). However, there were no differences in renal ANP mRNA expression levels among the three UUO groups.
Fig. 2. Laser confocal microscopic images of α-SMA and E-cadherin deposition in the renal interstitium in normal kidney in sham-operated mice (A) and in obstructed kidneys in wild-type (B), NPR-A KO (C), and ANP-treated mice (D) 11 days after production of UUO are shown. Renal interstitium was labeled with rhodamine phalloidin for α-SMA (red) and Alexa-fluor for E-cadherin (green). Nuclei were labeled with DAPI (blue). The α-SMA positive area is shown in (E). Data are expressed as mean ± SD. Number of mice was 6−8 in each group. **P b 0.01 vs sham-operated, †P b 0.01 vs wild-type, ‡P b 0.05 vs. NPR-A KO. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. F4/80 positive cells in the interstitium in sham-operated mice (A) and wild-type (B), NPR-A KO (C), and ANP-treated mice (D) 11 days after production of UUO are shown. The F4/80-positive area was quantified with a computer-aided image manipulator. Data are expressed as mean ± SD. Number of mice was 6−8 in each group. **P b 0.01 vs sham-operated, †P b 0.01 vs wild-type, ‡P b 0.05 vs. NPR-A KO.
Fig. 4. Plasma ANP levels (A) in sham-operated mice and wild-type, NPR-A KO, and ANP-treated mice 4 days after production of UUO are shown. Renal tissue cGMP levels (B) in normal kidney in sham-operated mice and in obstructed kidneys in wild-type, NPR-A KO, and ANP-treated mice 4 days after production of UUO are shown. The mRNA expression levels of NPRC (C) and renal ANP (D) normalized by GAPDH mRNA levels in normal kidney in sham-operated mice and in obstructed kidney in wild-type, NPR-A KO, and ANP-treated mice 11 days after production of UUO are shown. Number of mice was 5−11 in each group. *P b 0.05 vs. sham-operated, **P b 0.01 vs. sham-operated, †P b 0.01 vs wild-type, ‡P b 0.05 vs. NPR-A KO.
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3.6. Transcription factor levels As reported, transcription factor level is activated earlier days after UUO operation, we measured activated transcription factor levels at four days after UUO production. The DNA-binding activity of activated AP-1 was enhanced (about 1.5 fold) in obstructed kidneys in wild-type mice compared with normal kidneys in sham-operated wild-type mice (Fig. 5A). In obstructed kidneys, the DNA-binding activity of activated AP-1 was significantly increased in NPR-A KO mice compared with wild-type mice, and ANP pre-treatment significantly decreased it (Fig. 5A). The DNA-binding activities of activated NF-kappaB were also enhanced (about 3–6 fold) in obstructed kidneys in wild-type mice compared with normal kidneys in sham-operated wild-type mice (Fig. 5B and C). In obstructed kidneys, there were no differences in the DNA-binding activity of NF-kappaB between wild-type mice and NPR-A KO mice (Fig. 5B and C). Whereas, ANP pre-treatment
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significantly decreased the DNA-binding activity of NF-kappaB compared with wild-type mice (Fig. 5B and C). 3.7. Renal gene expression levels in the three groups As reported, since some genes are upregulated earlier after UUO operation, and the other genes are upregulated later, we measured mRNA levels at both four and eleven days after UUO operation. In obstructed kidneys in wild-type mice at four days after operation, the mRNA expression levels of renin, angiotensinogen, and the AT1-R/GAPDH ratio were increased (1.5–3.0 fold) compared with those in normal kidney in sham-operated wild-type mice (Fig. 6A, B and C). In obstructed kidneys, NPR-A KO mice had higher mRNA expression levels of these genes (2.2–3.5 fold) than wild-type mice (Fig. 6A, B and C). ANP pre-treatment significantly reduced these increased mRNA expression levels compared with wild-type mice and NPR-A KO mice (Fig. 6A, B and C). The mRNA expression levels of this gene at eleven days show similar trend; however, there was no difference in the mRNA expression levels of renin between wild type and NPR-A KO mice. The mRNA expression levels of TGF-β, collagen I, and collagen III/ GAPDH ratio at four days after operation were significantly increased in obstructed kidneys in wild-type mice (10–30 fold) compared with those in normal kidney in sham-operated mice (Fig. 6D, E and F). In obstructed kidneys, NPR-A KO mice had higher mRNA expression levels of these genes (18–75 fold) than wild-type mice (Fig. 6D, E and F). ANP pre-treatment significantly reduced these increased mRNA expression levels compared with wild-type mice (Fig. 6D, E, and F). The mRNA expression levels of this gene at eleven days show similar trend; however, there was no difference in the mRNA expression levels of TGF-β between wild type and NPR-A KO mice. The mRNA expression levels of MCP-1, TNF-α, and the osteopontin/GAPDH ratio at four days after operation were also significantly increased in obstructed kidneys in wild-type mice (20–100 fold) compared with those in normal kidney in sham-operated wild-type mice (Fig. 6G, H and I). There were no differences in these genes between wild-type mice and NPR-A KO mice (Fig. 6G, H, and I). Whereas ANP pre-treatment significantly decreased these expression levels of these genes compared with wild-type mice (Fig. 6G, H, and I). At eleven days the mRNA expression level of osteopontin appeared to decrease, whereas the mRNA expression level of TNF-α and MCP-1 appeared to increase. The difference among the groups showed the similar trend. 3.8. Phospho-Smad2/3 protein levels Western blot analysis revealed that the levels of Smad2/3 phosphorylation in renal tissues were increased in wild-type UUO mice compared with those in sham-operated mice. NPR-A KO mice had similar Smad2/3 phosphorylation levels compared with wild-type mice. ANP pre-treatment significantly decreased the level of Smad2/3 phosphorylation compared with sham-operated mice (Fig. 7A,B). 4. Discussion
Fig. 5. The transcription factor activity of AP-1 (A), NF-kappaB (p50) and NF-kappaB (p65) in normal kidney in sham-operated mice and in obstructed kidney in wild-type, NPR-A KO, and ANP-treated mice 4 days after production of UUO are shown. Data are expressed as mean ± SD. Number of mice was 7−11 in each group. *P b 0.05 vs. sham-operated, †P b 0.01 vs wild-type, ‡P b 0.05 vs. NPR-A KO.
In the present study, we demonstrated for the first time that NPR-A KO mice have more severe interstitial fibrosis in a UUO model than wild-type mice, and that chronic ANP administration attenuates this fibrosis. The increase in interstitial fibrosis in NPR-A KO mice is associated with decreased renal tissue cGMP levels and increased mRNA expression levels of angiotensinogen, renin, AT1-R, TGF-β, collagen I, and collagen III, and increased AP-1 levels; however, there were no differences in F4/80 positive cells, inflammation related gene expression, or NF-kappaB levels between wild-type mice and NPR-A KO mice. Whereas, ANP administration significantly increased plasma ANP and renal tissue cGMP levels and attenuated increased renin–
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angiotensin/TGF-β/collagen related gene activity, increased AP-1 levels, increased F4/80 positive cells, increased inflammation related gene expression, and increased NF-kappaB levels. These results suggest that the endogenous ANP/NPR-A/cGMP system has antifibrotic effects partly via inhibition of renin–angiotensin system in the kidney. Furthermore, exogenous ANP had an anti-fibrotic effect partly via inhibition of the renin–angiotensin system and inflammatory cascade. This is the first report of a potent in vivo anti-fibrotic action of ANP in the kidney. Recent studies revealed that the ANP/NPR-A system has inhibitory effects in cardiac hypertrophy and fibrosis. However, there have been few studies on the effect of the ANP/NPR-A system on renal fibrosis and the pathophysiology of renal disease. Earlier studies showed that renal responses to ANP are largely dependent on ANP-induced alterations in renal hemodynamics [28]. At the glomerulus, ANP-induced afferent arteriolar dilatation and efferent arteriolar constriction produce a rise in the glomerular capillary hydraulic pressure and thus leading to glomerular hyperfiltration [28]. This ANP-induced hyperfiltration has been suggested
to promote glomerulosclerosis [10]. Indeed, chronic administration of ANP worsened the glomerulosclerosis [29] and long-term administration of ANP antagonist, HS-142-1, improved nephropathy in a rat diabetic model [30]. However, recent studies demonstrated that natriuretic peptide inhibits cell growth of mesangial cells, vascular smooth muscle cells and fibroblasts [2,11,12], suggesting that natriuretic peptide may protect against the progression of renal disease. In fact, overexpression of BNP in mice attenuated renal injury in a model of renal ablation, glomerulonephritis, and diabetic nephropathy [13–15]. Since BNP level in this transgenic mice model is extremely higher than that in wild-type mice and the possibility that some compensation in these genetic mice might affect the renal effect cannot be excluded. Therefore, whether the endogenous ANP/NPR-A system has renoprotective effect or whether administration of ANP attenuates renal fibrosis remains unknown. Multiple factors have been identified as possible mechanisms by which chronic urethral obstruction leads to interstitial fibrosis [16,17]. The activation of the renin–angiotensin system by enhanced expression of the renin–angiotensin gene is a key step [17,31], as demonstrated by the
Fig. 6. The mRNA expression levels of renin (A), ATI-R (B), angiotensinogen (C), TGF-β (D), collagen I (E), collagen III (F), MCP-1 (G), osteopontin (H), and TNF-α (I) normalized by GAPDH mRNA levels in normal kidney in sham-operated mice and in obstructed kidney in wild-type, NPR-A KO, and ANP-treated mice 4 and 11 days after production of UUO are shown. Data are expressed as mean ± SD. Number of mice was 6−11 in each group. *P b 0.05 vs. wild-type mice, **P b 0.01 vs. wild-type mice, †P b 0.05 vs. NPR-A KO mice.
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Fig. 6 (continued).
effects of the interruption of this system resulting in profound structural and functional amelioration [32,33]. Of note, the activation of the renin– angiotensin system is thought to occur locally in the kidney, with its circulating counterpart un-changed, or rather suppressed, as a result of systemic hypervolemia [31]. After this activation, the stimulation of the downstream cascade involving multiple growth factors such as TGF-β/Smad2/3 has been proposed to play a pivotal role in disease progression [30]. The mechanisms that initiate the cascade of growth factor activation are not well understood but may be dependent on the
Fig. 7. The phospho-Smad2/3 levels in normal kidney in sham-operated mice and in obstructed kidney in wild-type, NPR-A KO, and ANP-treated mice 11 days after production of UUO are shown. Data are expressed as mean ± SD. Number of mice was 3 in each group. *P b 0.05 vs. sham-operated, **P b 0.01 vs. sham-operated, †P b 0.01 vs wild-type, ‡P b 0.05 vs. NPR-A KO.
renin–angiotensin system [31,34]. In the present study, production of UUO actually increased renal interstitial fibrosis, and associated with increased mRNA expression level of renin, angiotensinogen, AT1-R, TGF-β, collagen I, and collagen III, and increased phosphorylated Smad2/3 levels suggesting an important role of the renin/angiotensin/TGF-β/Smad2/3 axis in this model. Interestingly, the UUO model in NPR-A KO mice had more severe renal interstitial fibrosis compared with wild-type mice and increased fibrosis is associated with slightly but significantly increased mRNA expression levels of renin, angiotensinogen, and AT1-R. In addition, long-term ANP infusion significantly attenuated renal interstitial fibrosis and gene expression levels of renin, angiotensinogen, and AT1-R. Previous studies demonstrated that natriuretic peptides antagonize not only systemic but also local actions of the renin–angiotensin system in a cGMPdependent manner [1,2]. For example, ANP inhibited the angiotensin IIinduced proliferation of mesangial cells, -hypertrophic response of vascular smooth muscle cells, and -collagen synthesis in fibroblasts [1,3,5,6]. Indeed, in this study renal tissue cGMP levels were significantly lower in NPR-A KO mice compared with wild-type mice and that longterm ANP infusion significantly increased tissue cGMP levels. However, the difference of renal cGMP levels in obstructed kidney between NPR-A knockout (KO) mice and wild type mice is modest and apparently UUO operation increased renal tissue cGMP levels even in NPR-A KO mice. The exact mechanism of increase of renal cGMP levels in UUO model in NPR-A KO mice remains unknown at present; however, other system such as inducible nitric oxide may be involved in this increase. Further studies are necessary to elucidate the exact mechanism and role of increase of cGMP beside the NPR-A/cGMP system. In the present study, we observed that there was interstitial monocyte/macrophage infiltration, elevated NF-kappaB activity, and upregulation of proinflammatory genes after UUO production in wild-type mice. In this UUO model, monocyte/macrophage infiltration plays an important role in the process of interstitial fibrosis [21,22]. A recent study revealed that the mRNA levels of cytokine genes such as TNF-α were significantly higher in NPR-A KO-mice hearts as compared with age-matched wild-type mice [35]. In
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parallel, NF-kappaB binding activity was greater in the nuclear extract of NPR-A KO-mice hearts as compared with wild-type-mice hearts. Whereas cardiac tissue cGMP levels were drastically reduced in NPR-A KO-mice hearts as compared with the age-matched wildtype mice [35]. These findings suggest that ablation of NPR-A/cGMP signaling activates inflammatory cytokines, probably via NF-kappaB mediated signaling pathways. In the present study, there were no differences in the mRNA expression level of proinflammatory cytokine genes, NF-kappaB activation, or monocyte/macrophage infiltration in the obstructed kidneys between wild-type mice and NPR-A KO mice. The discrepancy between the two studies may be explained by the degree of the differences in tissue cGMP level. Cardiac tissue ANP levels in the heart were substantially higher than that in the kidney. In parallel, cardiac tissue cGMP levels were markedly higher than those in the kidney. Therefore, the difference in tissue cGMP levels between NPR-A KO mice and wild-type mice is greater in the heart than in the kidney. Thus, the small differences in renal tissue cGMP levels might not be sufficient to affect NF-kappaB transcription factor and/or proinflammatory cytokine gene expression levels. In addition, recent studies have shown that ANP inhibits cytokine mRNA expression via inhibition of NF-kappaB activation in microglial cells, endothelial cells, and macrophages in vitro [36–38]. ANP pre-treatment also inhibits NF-kappaB activation via inhibition of phosphorylation and degradation of the IkappaB-α protein in rats induced by LPS and in rats induced by sodium overload [39,40]. Taken together, ANP pre-treatment, if it is sufficient to elevate cGMP levels, may attenuate the inflammatory cascade partly via inhibition of transcription factor activation such as NF-kappaB. In the present study, NPR-A KO mice after UUO had higher blood pressure levels than wild-type UUO mice. Whereas there was no difference in blood pressure level between wild-type mice and ANP pre-treated mice, which are consistent with a previous report showing no effect of ANP infusion rate (0.05 µg/kg/min) on blood pressure [20]. Fern et al. [41] demonstrated that the mRNA expression levels of angiotensin closely correlated with the renal interstitial fibrotic response in UUO nephropathy, an effect independent of systemic blood pressure, suggesting the importance of renin/ angiotensin cascade in fibrosis of UUO model. Thus, we consider the low possibility that the blood pressure level affected the present results even if it was present. In the present study renal ANP mRNA expression was observed in the kidney of sham-operated mice. UUO operation increased the renal ANP mRNA expression in mice. However, there were no differences in renal ANP mRNA expression levels among the three UUO groups. Previous studies have shown that renal ANP mRNA is regulated independently from the heart and renal produced ANP may have renoprotective effect [42,43]. Therefore, severer fibrotic change of NPRA KO mice may be due to both circulating and renal ANP deficiency. In summary, the data presented here shows that the endogenous ANP/NPR-A/cGMP systems and exogenous ANP administration possess anti-fibrotic effect in a UUO model. This effect seems to be related to inhibition of the renin–angiotensin/TGF-β/collagen and NF-kappaB/proinflammatory cascades. As a clinical implication, the present result may have an important possibility to promote new strategies for human kidney disease. The strong neutral endopeptidase inhibitor or orally active BNP or ANP compound may be used for renal disease in the clinical setting in the future. Acknowledgments This work was supported in part by Scientific Research Grants-in-Aid 14570692, 18590787 and 20590837 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan, by the Research Grant for
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