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J Am Soc Nephrol 12:2652-2663, 2001
© 2001 American Society of Nephrology

Overexpression of Brain Natriuretic Peptide in Mice Ameliorates Immune-Mediated Renal Injury

Takayoshi Suganami, Masashi Mukoyama, Akira Sugawara, Kiyoshi Mori, Tetsuya Nagae, Masato Kasahara, Kensei Yahata, Hisashi Makino, Yuriko Fujinaga, Yoshihiro Ogawa, Issei Tanaka and Kazuwa Nakao

Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Correspondence to Dr. Masashi Mukoyama, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 81-75-751-4285; Fax: 81-75-771-9452; E-mail: muko{at}kuhp.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. One of major causes of end-stage renal disease is glomerulonephritis, the treatment of which remains difficult clinically. It has already been shown that transgenic mice that overexpress brain natriuretic peptide (BNP), with a potent vasorelaxing and natriuretic property, have ameliorated glomerular injury after subtotal nephrectomy. However, the role of natriuretic peptides in immune-mediated renal injury still remains unknown. Therefore, the effects of chronic excess of BNP on anti-glomerular basement membrane nephritis induced in BNP-transgenic mice (BNP-Tg) were investigated and the mechanisms how natriuretic peptides act on mesangial cells in vitro were explored. After induction of nephritis, severe albuminuria (~21-fold above baseline), tissue damage, including mesangial expansion and cell proliferation, and functional deterioration developed in nontransgenic littermates. In contrast, BNP-Tg exhibited much milder albuminuria (approximately fourfold above baseline), observed only at the initial phase, and with markedly ameliorated histologic and functional changes. Up-regulation of transforming growth factor-ß (TGF-ß) and monocyte chemoattractant protein-1 (MCP-1), as well as increased phosphorylation of extracellular signal-regulated kinase (ERK), were also significantly inhibited in the kidney of BNP-Tg. In cultured mesangial cells, natriuretic peptides counteracted the effects of angiotensin II with regard to ERK phosphorylation and fibrotic action. Because angiotensin II has been shown to play a pivotal role in the progression of nephritis through induction of TGF-ß and MCP-1 that may be ERK-dependent, the protective effects of BNP are likely to be exerted, at least partly, by antagonizing the renin-angiotensin system locally. The present study opens a possibility of a novel therapeutic potential of natriuretic peptides for treating immune-mediated renal injury.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
One of the major causes of end-stage renal disease is chronic glomerulonephritis (GN) (1). The mechanisms responsible for the progressive loss of renal function in this syndrome are largely unknown, and the management of GN with a worsening course remains difficult clinically. Among the models of experimental GN, anti-glomerular basement membrane (anti-GBM) GN has been most extensively studied, serving as a model for human Goodpasture syndrome (2). Especially, an accelerated form of anti-GBM GN is preferred to examine the long-term effects of interventions, because it leads to progressive renal injury that involves mesangial expansion and mesangial cell proliferation along with heavy proteinuria (2). Anti-GBM GN occurs in two distinct phases: a heterologous phase that occurs within minutes to hours and involves complement activation with neutrophil-mediated glomerular capillary damage and a delayed autologous phase resulting from host response that involves monocyte/macrophages and coagulation system activation (2). The course of GN is affected by multiple proinflammatory molecules (2), especially in the former phase, which is mostly dependent on Fc receptors (3), although there is a certain redundancy in multiple interacting mediation systems.

A variety of attempts of interventions have so far been reported to try to inhibit the progression of GN. Of them, several recent reports have shown that the renin-angiotensin system (RAS) is activated in various GN models, including anti-GBM GN (4), and that the blockade of angiotensin II (Ang II) generation or receptor signaling in GN results in amelioration of renal injury (4–6). A number of other humoral factors have been implicated in the progression of GN (2). Among them, monocyte chemoattractant protein-1 (MCP-1), a key molecule of macrophage chemotaxis and activation, is up-regulated in various GN models, and the extent of its up-regulation is associated well with the severity of proteinuria and tissue damage (4,7,8). Transforming growth factor-ß (TGF-ß) is also up-regulated in this GN model (4–6,9), potentially directing fibrogenesis in the kidney. Ang II has been shown to induce TGF-ß (10,11) and MCP-1 gene expression (12) in certain cultured cells, at least partly through the activation of extracellular signal-regulated kinase (ERK) (11,12). The inhibition of the RAS in vivo leads to attenuated up-regulation of MCP-1 and TGF-ß (4–6). Thus, MCP-1 and TGF-ß are likely important mediators of the progressive renal injury in GN, acting downstream of the activated RAS.

The natriuretic peptide family consisting of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) possesses potent diuretic, natriuretic, and vasorelaxing properties, thereby regulating cardiovascular homeostasis and renal function (13–17). ANP and BNP are secreted predominantly by the cardiac atrium and ventricle, respectively, upon cardiac overload (13,14,16). ANP and BNP share their receptor, a particulate guanylyl cyclase-coupled receptor, GC-A, and exert almost identical actions (13,17,18). Besides, they are thought to function, in general, to antagonize the RAS both systemically and locally (13). Administration of ANP has been shown to exert beneficial effects in experimental and clinical acute renal failure (19,20), but the long-term effects of natriuretic peptides on the kidney function are still poorly understood.

Recently, we have generated BNP-transgenic mice (BNP-Tg) that overexpress the mouse BNP gene in the liver and show a >100-fold increase in plasma BNP levels as well as elevated plasma and urinary guanosine 3',5'-cyclic monophosphate (cGMP) levels (21). Moreover, BNP-Tg revealed low BP and reduced heart weight (21), which indicates the usefulness of these mice to examine the long-term effects of natriuretic peptides in vivo. Using this mouse model, we have recently demonstrated that glomerular hypertrophy and mesangial expansion were markedly inhibited after subtotal nephrectomy (22), a widely used experimental model of renal failure with reduced nephron number involving hemodynamic abnormalities (23). However, the role of natriuretic peptides in immune-mediated renal injury, a major cause of clinical renal failure (1), still remains unknown. In this study, we induced an accelerated form of anti-GBM GN in BNP-Tg to examine the effects of chronic excess of BNP on immune-mediated renal injury. We also studied whether natriuretic peptides have direct actions on Ang II-treated mesangial cells (MC) in culture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All animal experiments were conducted in accordance with our institutional guidelines for animal research. Generation of BNP-Tg (line 55), harboring 20 copies of the transgene that are under the control of the human serum amyloid P component promoter, has been reported elsewhere (21,22). This promoter was active only in the liver after birth (21). In this study, BNP-Tg and their littermates, C57BL/6J nontransgenic mice (non-Tg), were used at 9 to 11 wk of age at the beginning of the study. Mice were fed on standard chow (CE-2 containing 0.5% NaCl; Japan Clea, Tokyo, Japan) and given water ad libitum. We maintained these animals under alternating 12-h cycles of light and dark.

Induction of Anti-GBM GN
The preparation of anti-GBM antiserum in rabbits was performed as reported elsewhere (4). In brief, glomeruli were isolated by differential sieving from the ddY mouse renal cortex and disrupted by sonication. The GBM was collected by centrifugation, emulsified with complete Freund’s adjuvant (CFA; Difco, Detroit, MI) and immunized in rabbits. An accelerated form of anti-GBM GN was induced in BNP-Tg (n = 28) and non-Tg (n = 33). Mice were immunized by an intraperitoneal injection of 0.5 mg per 20 g body weight of normal rabbit IgG (ICN, Aurora, OH) emulsified with CFA. Five days later, 0.3 ml per animal of anti-GBM antiserum (nephrotoxic serum [NTS]) or isovolume of control normal rabbit serum was injected from the femoral vein. Thereafter, mice were killed at days 1, 28, and 84. For a hydralazine administration group, nephritic non-Tg (n = 6) were given drinking water that contained 60 mg/L of hydralazine hydrochloride (Sigma, St. Louis, MO) from 1 wk before the induction of GN and killed at day 28.

Histology and Morphometric Analysis
For light microscopy, sagittal kidney sections were fixed by immersion in Carnoy’s solution followed by 4% buffered formaldehyde and embedded in paraffin. One-micron-thick sections were stained with periodic acid-Schiff. The glomerular cell number and the mesangial area were measured quantitatively with a computer-aided manipulator (KS400, Carl Zeiss Vision, Munich, Germany) by counting the nuclei and analyzing the periodic acid-Schiff-positive area within the glomerular tuft (22). More than 30 consecutive glomerular sections, randomly selected in each mouse by scanning from the outer cortex, were examined by two investigators without knowledge of the origin of the slides, and the mean values were calculated.

Immunohistochemistry
For immunohistochemical study of MCP-1, the kidney sections embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan) were snap frozen in acetone-dry ice, and 5-µm-thick cryostat sections were fixed in acetone. The sections were washed with phosphate-buffered saline, and treated with 0.9% H2O2 in methanol for 30 min to quench endogenous peroxidase activity. The specimens were incubated overnight at 4°C with goat anti-rat MCP-1 antibody, which cross-reacts with mouse MCP-1 (Santa Cruz Biotechnology, Santa Cruz, CA). For macrophage staining, 5-µm paraffin-embedded sections were immunohistochemically analyzed by use of rat monoclonal anti-mouse mac-2 antibody (Cedarlane, Hornby, Ontario, Canada). After incubation with biotin-conjugated second antibody, the specimens were processed by use of the avidin-biotin-peroxidase complex kit (Vector, Burlingame, CA) and developed with 3,3'-diaminobenzidine tetrahydrochloride (Kanto Chemical, Tokyo, Japan). More than 20 consecutive glomerular sections in each mouse were examined, and the mean number of macrophages in the glomerulus was calculated.

For immunofluorescence study of rabbit and mouse IgG and mouse complement C3, 1-µm-thick cryostat sections were fixed in acetone. The sections were washed with phosphate-buffered saline and incubated overnight at 4°C with FITC-labeled goat anti-rabbit IgG (Zymed, San Francisco, CA), anti-mouse IgG (Southern Biotechnology, Birmingham, AL), and anti-mouse C3 antibodies (ICN).

Measurement of Circulating Anti-Rabbit IgG Antibody
The circulating anti-rabbit IgG antibody level in nephritic mice was measured by enzyme-linked immunosorbent assay (4). Ninety-six-well plates coated with normal rabbit IgG (ICN) were incubated with 1:5000 diluted serum from nephritic mice for 1 h. After being washed extensively with Tris-buffered saline containing 0.05% Tween 20, the plates were incubated with horseradish peroxidase-conjugated secondary antibody (Betyl, Montgomery, TX) and developed with 3,3',5,5'-tetramethyl benzidine (Betyl). The absorbance at 450 nm was analyzed with Microplate Reader 550 (Bio-Rad, Richmond, CA). Untreated mouse serum as a negative control gave an OD value <0.04.

BP Measurement
The systolic, mean, and diastolic BP were measured every 2 wk by a programmable sphygmomanometer (BP-98A, Softron, Tokyo, Japan) by use of the tail-cuff method (22). At least six readings were taken for each measurement when mice were in the conscious condition.

Blood and Urinary Parameter Measurements
Blood samples were obtained under pentobarbital anesthesia, and blood urea nitrogen, serum creatinine, and serum albumin levels were measured (22). For urine measurements, each mouse was separately housed in a metabolic cage (Shinano Manufacturing, Tokyo, Japan), and daily urine samples were collected at days 0, 4, 7, 14, 28, 56, and 84. Urinary albumin excretion was assayed with a murine albumin enzyme-linked immunosorbent assay kit (Exocell, Philadelphia, PA) (22).

Mesangial Cell Culture
Male Wistar-Kyoto rats at 20 wk of age were obtained from Shionogi Research Laboratories (Osaka, Japan). Cultured MC were prepared from glomeruli isolated by differential sieving and used at passages 7 to 10 (24). Cells were maintained in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) that contained 10% fetal calf serum (Sanko Junyaku, Tokyo, Japan), L-glutamine (4 mM), and antibiotics (24).

Proliferation Studies
Proliferation assay in cultured MC was performed with 3H-thymidine (Amersham, Arlington Heights, IL) as reported elsewhere (24). In brief, the effect of natriuretic peptides (ANP, BNP, and CNP, all from Peptide Institute, Osaka, Japan) on cell proliferation was studied in the presence of 3 ng/ml of platelet-derived growth factor (PDGF)-BB (Becton Dickinson Labware, Bedford, MA). Each agent was added to the medium of cultured MC for 24 h that were kept quiescent in serum-free conditions with insulin, transferrin, and selenium for 48 h in 24-well plates (24,25). After incubation with 3H-thymidine for final 8 h, the tracer incorporated into the cells was measured in a liquid scintillation counter.

Measurement of ERK Phosphorylation
The whole kidneys were excised from BNP-Tg and non-Tg at day 28 after induction of nephritis. Cultured MC kept quiescent for 6 h in 6-cm dishes were preincubated with ANP, BNP, CNP (1 µM each), 8-bromo-cGMP (1 mM) (Sigma), or vehicle for 10 min and stimulated by 100 nM of Ang II (Peptide Institute) for 5 min at 37°C. The whole kidney tissue and MC were lysed on ice in solution that contained 1 M Tris-HCl (pH 7.5), 12 mM ß-glycerophosphate, 0.1 M ethyleneglycol-bis(ß-aminoethyl ether)-N,N'-tetraacetic acid, 1 mM pyrophosphate, 5 mM NaF, 10 mg/ml aprotinin, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. The lysates were centrifuged at 15,000 x g for 20 min at 4°C, and the supernatants were mixed with Laemmli’s sample buffer (26). Samples (30 µg protein/lane) were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred onto Immobilon polyvinylidine difluoride filter (Millipore, Bedford, MA). After the filters were incubated with anti-phospho-ERK antibody or anti-ERK1/2 antibody (New England Biolabs, Boston, MA) for 2 h at room temperature, immunoblots were developed with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Bio-Rad, Richmond, CA) and a chemiluminescence kit (ECL one plus, Amersham).

Northern Blot Analysis
Total RNA from the whole kidney or MC was extracted by use of the acid guanidinium-phenol-chloroform method. As for cultured MC, after pretreatment with various concentrations of natriuretic peptides for 1 h, cells were stimulated with Ang II for 24 h. Northern blot analysis was performed as described elsewhere (24). In brief, 40 µg of total RNA was electrophoresed on a 1.0% agarose gel and transferred to a nylon membrane filter (Biodyne, Pall BioSupport, Port Washington, NY). The cDNA fragments for mouse TGF-ß1 (nucleotides [nt] 1142 to 1546), rat TGF-ß1 (nt 1141 to 1549), rat fibronectin (nt 619 to 1082), and rat MCP-1 (nt 121 to 652), which were prepared by reverse-transcription-PCR that used mouse and rat kidney mRNA, were used as probes (10,27–29). The filter was hybridized with 32P-dCTP-labeled probes, and autoradiography was performed with BAS-2500 system (Fuji Photo Film, Tokyo, Japan). As an internal control, the filter was rehybridized with a human GAPDH cDNA probe (Clontech, Palo Alto, CA).

Statistical Analysis
Data are expressed as the mean ± SEM. Statistical analysis was performed by use of ANOVA followed by Scheffe’s test. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histologic Analysis
To evaluate the long-term outcome of tissue damage in BNP-Tg and non-Tg caused by anti-GBM GN, we examined renal histologic changes 84 d after the administration of NTS (Figure 1). Compared with non-Tg injected with normal rabbit serum (control non-Tg) (Figure 1A), non-Tg treated with NTS (nephritic non-Tg) showed marked glomerular changes, including glomerular hypercellularity and mesangial expansion with occasional glomerular crescents and global sclerosis (Figure 1C). The hypercellularity was remarkable in the mesangial area, but some infiltrating cells were also observed in the tubulointerstitium of nephritic non-Tg. In contrast, nephritic BNP-Tg exhibited only mild and segmental glomerular changes (Figure 1D) that were almost indistinguishable from BNP-Tg with normal serum injection (control BNP-Tg) (Figure 1B). Quantitative analysis revealed that the increase in glomerular cell number was marked in nephritic non-Tg, whereas it was significantly inhibited in nephritic BNP-Tg (+42% versus +9%, P < 0.01, n = 6) without a significant difference from the level in control BNP-Tg (Figure 1E). Similarly, the increase in mesangial area by NTS observed in non-Tg was markedly inhibited in BNP-Tg (+93% versus +13%, P < 0.01, n = 6) (Figure 1F). In control BNP-Tg, the glomerular cell number and mesangial area were slightly larger than those in control non-Tg but not statistically significantly (Figure 1, E and F). These findings indicate that histologic changes caused by NTS were much ameliorated in BNP-Tg, compared with those in nephritic non-Tg.



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  		Figure 1. Histologic examination of anti-glomerular basement membrane (anti-GBM) nephritis in mice. Representative views on light microscopy at day 84 of the kidneys from (A) nontransgenic mice injected with normal control serum (non-Tg, control), (B) brain natriuretic peptide (BNP)-transgenic mice (BNP-Tg) with control serum (Tg, control), (C) non-Tg injected with nephrotoxic serum (non-Tg, NTS), and (D) BNP-Tg with NTS (Tg, NTS) are shown. The kidney sections were stained with periodic acid-Schiff. The glomerular cell number (E) and the mesangial area (F) within the glomerular tuft were significantly increased only in nephritic non-Tg. Mean ± SEM. *P < 0.01, n = 6. Magnification, x200.

 
Immunologic Responses
To compare the immunologic responses between non-Tg and BNP-Tg after the induction of anti-GBM GN, we examined IgG and C3 deposition in the glomeruli of both groups. Immunofluorescence staining for heterologous rabbit IgG (Figure 2A) was observed linearly along the glomerular capillary wall in both non-Tg and BNP-Tg at day 1 after the induction of GN. There was no significant difference in the staining intensity between the two groups. At day 28, linear deposition of autologous mouse IgG was detected in the nephritic glomeruli from both groups (Figure 2B). Moreover, linear C3 deposition along the capillary wall was also observed at similar intensities at day 1 (Figure 2C) and day 28 (not shown). In control BNP-Tg and non-Tg with normal rabbit serum injection, no staining for IgG or C3 was seen (not shown). Furthermore, there was no significant difference in the amount of autologous mouse anti-rabbit IgG antibody between nephritic non-Tg and nephritic BNP-Tg at day 28 (0.247 ± 0.022 versus 0.260 ± 0.019 OD, n = 5). These findings suggest that the chronic excess of BNP neither significantly affects the process of heterologous antibody/complement deposition nor autologous antibody production.



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  		Figure 2. Immunofluorescence study for IgG and C3 in the kidneys after the induction of anti-GBM nephritis in non-Tg and BNP-Tg. (A) Linear deposition of rabbit IgG along the glomerular capillary wall was detected at day 1 in both groups. (B) At day 28, linear deposition of mouse IgG was observed in the glomerulus similarly to that of rabbit IgG. (C) Linear staining for C3 along the capillary wall was also noted at day 1 in both groups. Magnification, x200.

 
Macrophage Infiltration and Renal MCP-1 Expression
To further analyze the inflammatory response in anti-GBM GN, we investigated glomerular macrophage infiltration, which is regarded as a key event in GN that leads to renal injuries and proteinuria (7,8). Immunohistochemical analysis revealed an increased number of glomerular infiltration of mac-2-positive macrophages in non-Tg after the induction of GN (Figure 3A, inset). Nephritic BNP-Tg also showed an increase in glomerular macrophage infiltration at day 28, but the extent was significantly milder than nephritic non-Tg (P < 0.01, n = 6) (Figure 3A). Moreover, the increased glomerular macrophage infiltration was still observed in nephritic non-Tg at day 84, whereas it was abolished in nephritic BNP-Tg. The number of macrophages in the glomerulus was similar in control BNP-Tg and non-Tg. Next, we examined the protein abundance and the gene expression of MCP-1 in the kidney. The immunohistochemical staining of MCP-1 in the glomeruli was markedly increased in nephritic non-Tg at day 28, which was primarily detected in the mesangial area (Figure 3B). In contrast, the staining was significantly weaker in nephritic BNP-Tg (Figure 3C). In control BNP-Tg and non-Tg, only faint staining was seen in the glomeruli (data not shown). Furthermore, Northern blot analysis revealed that the renal MCP-1 gene expression was significantly up-regulated only in nephritic non-Tg (3.2-fold of untreated control non-Tg, P < 0.01, n = 5) but not in BNP-Tg (Figure 4, A and B). These findings indicate that the glomerular macrophage infiltration paralleled well with the degree of MCP-1 expression and that the alleviation in nephritic BNP-Tg possibly resulted from the inhibited up-regulation of MCP-1 expression.



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  		Figure 3. Immunohistochemical analyses for infiltrating macrophages and monocyte chemoattractant protein-1 (MCP-1) in the glomeruli of anti-GBM nephritis. (A) The kidney sections at days 28 and 84 were stained with anti-mouse mac-2 antibody. A marked increase in macrophage infiltration was observed in nephritic non-Tg, whereas it was significantly attenuated in nephritic BNP-Tg both at days 28 and 84. Mean ± SEM. *P < 0.05, **P < 0.01, n = 6. (Inset) An arrow denotes a macrophage. (B) Immunohistochemical examination for MCP-1 revealed increased staining in the glomeruli of nephritic non-Tg at day 28. (C) MCP-1 staining was significantly weaker in the glomeruli of nephritic BNP-Tg. Magnification, x200.

 


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  		Figure 4. Renal gene expression of MCP-1 and transforming growth factor ß1 (TGF-ß1) in anti-GBM nephritis. (A) Representative Northern blots of MCP-1 and TGF-ß1 at day 28 after induction of nephritis are shown. Forty micrograms of total RNA from the whole kidney were separated on an agarose gel and probed for MCP-1 and TGF-ß1. Quantitative analyses of Northern blots for MCP-1 (B) and TGF-ß1 (C) revealed significant up-regulation of these messages in nephritic non-Tg but not in BNP-Tg. Mean ± SEM. *P < 0.01, n =5.

 
Renal TGF-ß1 Expression
Renal fibrosis characterized by glomerulosclerosis and tubulointerstitial fibrosis is a final common pathway to the end-stage renal disease, regardless of the initiating insult. TGF-ß has been shown to play a crucial role in renal fibrogenesis (30). To assess the extent toward fibrosis in anti-GBM GN among the groups, we examined the renal TGF-ß1 gene expression at day 28 (Figure 4, A and C). The TGF-ß1 expression was significantly up-regulated in nephritic non-Tg, compared with that in control non-Tg (2.2-fold, P < 0.01, n = 5), whereas it was not significantly increased in BNP-Tg. These results agree well with the modest degree of mesangial expansion and tubulointerstitial damage in nephritic BNP-Tg (Figure 1). These findings strongly suggest that reduced gene expression of TGF-ß as well as MCP-1 in BNP-Tg plays an important role in the amelioration of tissue damage induced by NTS.

ERK Phosphorylation in the Kidney of Anti-GBM GN
Activation of ERK plays an important role in the induction of TGF-ß1 and MCP-1 expression (11,12), and sustained activation of glomerular ERK has been observed in anti-GBM GN (31). By Western blotting technique, the level of phosphorylated ERK in nephritic non-Tg significantly increased, compared with that in control non-Tg (threefold, P < 0.01, n = 5) (Figure 5). Nephritic BNP-Tg also showed an increase in ERK phosphorylation, but the extent was significantly milder than that in nephritic non-Tg (P < 0.05, n = 5). In control BNP-Tg, the phosphorylated ERK level was slightly lower than that in control non-Tg but not statistically significantly. These findings indicate that ERK phosphorylation is attenuated in the kidney of nephritic BNP-Tg and suggest that the inhibited up-regulation of MCP-1 and TGF-ß in the kidney of nephritic BNP-Tg may be due to reduced ERK activation.



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  		Figure 5. Extracellular signal-regulated kinase (ERK) phosphorylation in the kidney of anti-GBM nephritis. (A) Representative Western blots of phosphorylated ERK and total ERK at day 28 after induction of nephritis. (B) The quantified relative phospho-ERK/total ERK levels calculated with densitometer. Equal amounts of protein (30 µg/lane) from the whole kidney were subjected to Western blot analysis by use of anti-phospho-ERK and anti-ERK1/2 antibodies. Mean ± SEM. *P < 0.05, **P < 0.01, n =5.

 
Blood and Urinary Parameters
To evaluate the functional alterations in anti-GBM GN, we examined urinary albumin excretion and serum albumin and blood urea nitrogen levels, together with creatinine clearance (Figure 6). At basal states, there was no significant difference in these parameters between control non-Tg and BNP-Tg. After induction of GN, urinary albumin excretion in nephritic non-Tg significantly increased by day 4, compared with that in control non-Tg, peaked at day 14 (~21-fold above baseline), and persisted until day 84 (Figure 6A). In nephritic BNP-Tg, on the other hand, a modest increase in urinary albumin excretion (up to approximately fourfold above baseline) was observed only in the initial phase (days 4 to 7), and thereafter proteinuria was markedly suppressed throughout the course of GN (Figure 6A). At day 84, nephritic non-Tg revealed significantly lower serum albumin and higher blood urea nitrogen levels than control non-Tg and nephritic BNP-Tg, along with significantly reduced creatinine clearance (Figure 6, B through D). Thus, the functional impairment became manifest in nephritic non-Tg, whereas the renal function was well preserved in nephritic BNP-Tg.



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  		Figure 6. Urinary and blood parameters after induction of anti-GBM nephritis. (A) Changes in urinary albumin excretion during the course of nephritis. The open and closed circles represent control BNP-Tg and non-Tg, respectively. The open and closed triangles represent nephritic BNP-Tg and non-Tg, respectively, indicating marked amelioration in BNP-Tg throughout the course. Mean ± SEM. *P < 0.05, **P < 0.01 versus each baseline level; {dagger}P < 0.01 versus nephritic non-Tg at each time point. n = 6. Comparison in the levels of (B) serum albumin, (C) blood urea nitrogen (BUN), and (D) creatinine clearance at day 84 revealed significant deterioration of these parameters only in nephritic non-Tg but not in BNP-Tg. Mean ± SEM. *P < 0.05, **P < 0.01, n =6.

 
BNP-Tg tended to exhibit an increased urine volume compared with non-Tg at the baseline level (1.34 ± 0.08 versus 1.17 ± 0.07 ml/d), although the difference did not achieve a statistical significance. After the induction of GN, the urine volume increased significantly in both groups, and the change was rather pronounced in non-Tg (1.53 ± 0.15 and 1.94 ± 0.11 ml/d in BNP-Tg and non-Tg, respectively, at day 28). Therefore, it seems unlikely that the preserved renal function in BNP-Tg resulted simply from the diuretic effect of BNP during the course of nephritis.

Effects of Hydralazine Administration
Analyses so far have suggested that chronic excess of BNP prevents the progression of renal injury in anti-GBM GN. To explore whether it was due to systemic hypotension observed in BNP-Tg, we examined the effects of systemic BP reduction with hydralazine administration in nephritic non-Tg. Baseline BP in BNP-Tg was significantly lower than that in non-Tg, as described elsewhere (systolic, 86.2 ± 1.2 versus 101.7 ± 0.7 mm Hg, P < 0.01) (21,22). There were no significant BP changes after the induction of GN throughout the study period in the two groups. In the group of hydralazine administration, despite effective reduction in systemic BP to the level comparable to that of BNP-Tg (Figure 7A), this treatment failed to inhibit the increase in albuminuria and the decrease in creatinine clearance induced by NTS (Figure 7, B and C). At the same time, there was no significant improvement in renal histology (data not shown). These results indicate that systemic hypotension in BNP-Tg may not play a critical role in the preventive effects in anti-GBM GN. Rather, this observation has raised a possibility that the effects of BNP were mediated by direct actions on renal cells and infiltrating cells or by indirect actions through the altered regulation of other humoral factors or local hemodynamics.



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  		Figure 7. Effects of hydralazine administration on (A) BP, (B) urinary albumin excretion, and (C) creatinine clearance in anti-GBM nephritis. (A) Administration of hydralazine in nephritic non-Tg resulted in effective reduction in BP throughout the course, to a level comparable to that of BNP-Tg. Chronic hydralazine treatment in nephritic non-Tg failed to inhibit increase in urinary albumin excretion (B) and decrease in creatinine clearance (C). Hyd, hydralazine-treated group. Mean ± SEM. {dagger}P < 0.01 versus hydralazine-treated nephritic non-Tg at each time point. *P < 0.05, **P < 0.01, n = 6.

 
Effects of Natriuretic Peptides in Cultured MC
We next addressed direct actions exerted by natriuretic peptides using cultured MC. First, we investigated the effects of natriuretic peptides on the gene expression of TGF-ß1 and its downstream molecule, fibronectin, after stimulation by Ang II. Pretreatment with ANP dose-dependently inhibited Ang II-dependent TGF-ß1 mRNA induction (Figure 8A). Natriuretic peptides as well as a membrane-permeable analog of cGMP, which is their intracellular signaling molecule, effectively abolished the up-regulation of TGF-ß1 gene expression (Figure 8B). Essentially equal results were obtained on fibronectin gene expression (Figure 8B).



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  		Figure 8. Anti-fibrotic and anti-proliferative effects of natriuretic peptides in cultured rat mesangial cells (MC). (A) Northern blot analysis for TGF-ß1 mRNA expression. Atrial natriuretic peptide (ANP) dose-dependently inhibited angiotensin (Ang) II-induced TGF-ß1 gene expression. (B) Inhibitory effects of natriuretic peptides and cGMP on Ang II-induced TGF-ß1 and fibronectin gene expression. (C) Inhibitory effects of natriuretic peptides on MC proliferation. Cells were stimulated with platelet-derived growth factor (PDGF)-BB for 24 h with or without pretreatment by natriuretic peptides. The count of 3H-thymidine incorporated into cells was increased 3.7-fold by PDGF stimulation (630 ± 16 to 2312 ± 177 cpm/104 cells). Natriuretic peptides dose-dependently inhibited PDGF-induced DNA synthesis in MC. (D) Inhibitory effects of natriuretic peptides on ERK phosphorylation. Natriuretic peptides and cGMP significantly inhibited Ang II- or PDGF-induced phosphorylation of ERK. Bas, basal; A, ANP; B, BNP; C, CNP; G, 8-bromo-cGMP. Mean ± SEM. *P < 0.01 versus basal, #P < 0.01 versus Ang II or PDGF alone, n =4.

 
Next, we examined whether BNP has an inhibitory effect on MC proliferation as has been reported elsewhere for ANP (25). BNP and ANP had a similar dose-dependent inhibitory effect on DNA synthesis induced by PDGF (Figure 8C), a growth factor that is postulated to play an important role in MC proliferation in various glomerular diseases (32). CNP also exerted a potent inhibitory effect on MC proliferation in culture.

ERK phosphorylation is regarded as a key event in the induction of TGF-ß1 and MCP-1 expression (11,12) as well as cell proliferation (26,31). To address the intracellular mechanisms by which natriuretic peptides inhibit TGF-ß1 expression and MC proliferation, we examined the effect of natriuretic peptides on the ERK phosphorylation induced by Ang II or PDGF (Figure 8D). Preincubation with natriuretic peptides or cGMP completely inhibited Ang II-induced ERK phosphorylation. Similar significant inhibition was observed on PDGF-induced ERK phosphorylation. These results indicate that natriuretic peptides inhibit not only MC proliferation and extracellular matrix induction but also their upstream event, the phosphorylation of ERK, probably through the elevation of intracellular cGMP levels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated the effects of chronic excess of BNP on tissue damage and functional deterioration in an accelerated form of anti-GBM GN. In non-Tg, mesangial proliferation and expansion, glomerular macrophage infiltration, glomerulosclerosis, and tubulointerstitial injury accompanied by heavy proteinuria and decreased creatinine clearance were observed after induction of GN, which agrees well with studies elsewhere (2–9). In a striking contrast, these changes were all markedly ameliorated in nephritic BNP-Tg. Such amelioration was associated with significantly reduced up-regulation of MCP-1 and TGF-ß, both of which have been postulated to play a critical role in the progression of various kinds of experimental and clinical nephropathies by inducing macrophage infiltration and extracellular matrix accumulation (4–9,30). Neutralization of MCP-1 in anti-GBM GN has been demonstrated to cause a dramatic decrease in crescent formation and in interstitial fibrosis (8,33). The functional role of TGF-ß in the pathogenesis of this particular GN model has yet to be documented, but marked prevention by blocking of this cytokine in other GN models (34) provides a potential role in postinflammatory fibrosis of the kidney in general (30). Therefore, it seems likely that the protective effect of BNP in this model resulted from the inhibition of renal MCP-1 and TGF-ß induction.

Several lines of evidence have shown that Ang II is causative of progressive renal injury in experimental GN by inducing MCP-1 and TGF-ß, as demonstrated by alleviation associated with the inhibition of Ang II generation as well as the pharmacologic blockade or genetic disruption of the Ang II type 1 receptor (4–6). Therefore, to explain how BNP-Tg resulted in the amelioration of GN, we investigated whether natriuretic peptides counteract the effects of Ang II, using cultured MC. As shown in Figure 8, natriuretic peptides inhibited TGF-ß gene up-regulation and its upstream event, ERK phosphorylation, induced by Ang II. These findings suggest that BNP may have exerted a protective effect on tissue damage in anti-GBM GN by at least partly antagonizing the actions of Ang II at the cellular level locally.

Sustained activation of glomerular ERK has already been demonstrated in anti-GBM GN in rats, providing a possible mechanism of the long-term proliferative response to immune injury in this disease model (31). Previous reports have shown that ANP inhibits growth factor-induced ERK phosphorylation in cultured MC (26), through the up-regulation of an ERK phosphatase, MKP-1, in a cGMP-dependent manner (35). The present study revealed that the phosphorylation of ERK is attenuated in the kidney of nephritic BNP-Tg (Figure 5) and that BNP also has an inhibitory effect on ERK phosphorylation in cultured MC (Figure 8). Recently, nitric oxide has also been shown to exert an inhibitory action on mesangial ERK activation in a cGMP-dependent manner (36). Moreover, the lack of endothelial nitric oxide synthase in mice has been demonstrated to result in aggravation of anti-GBM GN (37). In the present study, natriuretic peptides inhibited Ang II-induced up-regulation of TGF-ß and fibronectin gene expression. Because TGF-ß requires ERK phosphorylation for its own induction (11) and in turn activates ERK in certain cell lines (38), natriuretic peptides may inhibit this autoinduction loop of TGF-ß. In addition, the activation of the ERK pathway appears to be important for Ang II-induced MCP-1 expression as well (12). The finding in the present study is the first demonstration of natriuretic peptides as anti-fibrotic agents in renal cells, and the results are consistent with our recent report that cardiac fibrosis in response to ventricular pressure overload is accelerated in mice that lack BNP (39). Furthermore, evidence so far obtained in vitro using MC, vascular smooth muscle cells, cardiocytes, and fibroblasts has given a notion that natriuretic peptides generally counteract the actions of Ang II (13,40). The present study may provide a further support in vivo for this hypothesis.

We have recently reported that glomerular hypertrophy and mesangial expansion were significantly inhibited in BNP-Tg after subtotal nephrectomy (22), suggesting a potential beneficial effect of natriuretic peptides on chronic renal insufficiency with the reduced number of functional nephrons. Accumulating evidence has shown that the Ang II/TGF-ß cascade is a critical mediator in renal pathogenesis associated with hemodynamic abnormalities such as the renal ablation model (41). Although molecular mechanisms for the amelioration exerted by BNP in this model have not been defined in detail, it is conceivable that the inhibition of this cascade would have worked as well to prevent the progression of glomerular injury (22).

Concerning the beneficial effects of natriuretic peptides on immune-mediated renal injury, we have to consider the influence of natriuretic peptides on the immune system. Indeed, there is no doubt that inflammatory cells as well as complement activation play a critical role in the acute phase of anti-GBM GN (2). It should be also considered that the impact in the acute phase of the disease contributes to the outcome in the chronic phase. In this study, however, we could not detect apparent differences in renal histology in the acute phase of anti-GBM GN between non-Tg and BNP-Tg. In addition, we observed similar deposition of anti-GBM IgG and C3 at day 1 (Figure 2) as well as a similar course of proteinuria for the first 4 d (Figure 6), when Fc receptors have been shown to play a more important role than Ang II (3). Nevertheless, the effects of BNP in the acute phase may still have affected the changes in the later phase, because its receptors are expressed in thymocytes and macrophages (42,43). It has been reported that ANP inhibits not only cell proliferation of thymocytes (42) but also lipopolysaccharide-stimulated tumor necrosis factor-{alpha} production in macrophages via cGMP (43). Of note, this cytokine has been shown to play a key role in the recruitment of inflammatory cells and the subsequent development of crescents and proteinuria in a murine model of anti-GBM GN (44). Therefore, it is possible that the protective effects of BNP observed in the present study might be caused partly through its actions on macrophages and thymocytes. Further studies are needed to explore the effects of BNP on the immune system during the course of anti-GBM GN.

Whether the sustained reduction of systemic BP in BNP-Tg contributed to the observed effects is another issue to be addressed. To answer this question, we used chronic hydralazine administration. As shown in Figure 7, hydralazine treatment resulted in effective BP reduction but failed to inhibit functional and histologic worsening in nephritic non-Tg, the findings consistent with a report elsewhere (4). These results suggest that the systemic hypotension may not be critically contributing to the effects exerted by BNP.

The natriuretic peptide system consists of three endogenous ligands and two biologically active receptors: GC-A, which is activated mainly by ANP and BNP; and GC-B, which is rather specific to CNP (13,17,18). We already reported that BNP-Tg with higher copy numbers of the transgene showed marked skeletal overgrowth (45), in which BNP likely activates the physiologic CNP/GC-B pathway in the bone to stimulate endochondral ossification (46). The BNP-Tg line 55 used in the present study revealed mild skeletal phenotypes (45). It is important to clarify, therefore, whether the beneficial effects of BNP observed in this study are GC-A-dependent or GC-B-dependent. In the kidney, BNP and ANP are equally potent for activation of GC-A, which is distributed widely in the mesangium, capillary, and tubules, whereas CNP selectively activates GC-B, whose expression is detected mainly in the tubular system (40). In the present study, CNP had potent antiproliferative and antifibrotic effects in vitro on cultured MC (Figure 8). This may be explained by up-regulation of GC-B for certain cell lines in culture conditions (18). Recently, it has been reported that short-term infusion of CNP in rats ameliorates glomerular changes in anti-Thy1 GN (47). The antagonistic effect of CNP on the proliferative action of PDGF has also been reported in fibroblasts through the regulation of receptor phosphorylation (48). Although the distribution of GC-A and GC-B in the glomeruli and tubulointerstitium in GN remains unclear, GC-B might play important roles. Analyses using the mice crossed between BNP-Tg and GC-A-null mice (46) would give answers to this question.

The blockade of the RAS with angiotensin-converting enzyme inhibitors or Ang II AT1 receptor antagonists results in alleviation of renal injury in various GN models (4–6). Although such therapeutic approaches are clinically useful in treating various renal diseases such as chronic GN and diabetic nephropathy, the use of these agents is sometimes hampered by several adverse conditions, including progression of renal insufficiency (40). From this study, it is likely that natriuretic peptides not only act against the RAS but also inhibit several other cascades of growth factor activation. Moreover, natriuretic peptides will provide additional beneficial effects such as potent diuresis and possibly anti-inflammation. However, whether natriuretic peptides or agonists of this system prove beneficial clinically for treating immune-mediated renal diseases should await further investigation.

In summary, we demonstrate that chronic excess of BNP ameliorates the histologic and functional alterations caused by NTS. The results also suggest that the renoprotective effects of BNP in anti-GBM GN are not due to systemic BP reduction and might be applicable in other types of immune-mediated renal injuries. Because we have not examined whether BNP has renoprotective effects on the progression of GN even after GN is established, further study is needed to clarify the therapeutic usefulness of BNP. Nevertheless, our findings can open up the possibility of a novel therapeutic strategy for chronic GN, as well as an innovative application of natriuretic peptides to disorders other than congestive heart failure (49,50), acute renal failure (20), and hypertension.


   Acknowledgments
 
We gratefully acknowledge Dr. T. Sugaya and Dr. Y. Hisada (Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., Osaka, Japan) for valuable technical advice; J. Nakamura, A. Wada, and Y. Oki for technical assistance; and S. Doi and A. Sonoda for secretarial assistance. This work was supported in part by research grants from the Japanese Ministry of Education, Science, Sports and Culture, the Japanese Ministry of Health and Welfare, "Research for the Future" (RFTF) of Japan Society for the Promotion of Science, Smoking Research Foundation, Research Foundation for Community Medicine "Research Meeting on Hypertension and Arteriosclerosis," the Tanabe Medical Frontier Conference, and the Salt Science Research Foundation.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication March 5, 2001. Accepted for publication June 23, 2001.




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