A novel immunosuppressant FTY720 ameliorates proteinuria and alterations of intrarenal adrenomedullin in rats with autoimmune glomerulonephritis

Regulatory Peptides 127 (2005) 233 – 238 www.elsevier.com/locate/regpep

A novel immunosuppressant FTY720 ameliorates proteinuria and alterations of intrarenal adrenomedullin in rats with autoimmune glomerulonephritis Yi Shia, Fumiki Yoshiharab, Hajime Nakahamab,*, Naotsugu Ichimarua, Koji Yazawaa, Masaharu Sadac, Rieko Gotoc, Yuhei Kawanob, Toshiki Moriyamad, Shiro Takaharaa, Akihiko Okuyamaa, Kenji Kangawac b

a Department of Urology, Osaka University School of Medicine, Japan Division of Hypertension and Nephrology, National Cardiovascular Center, Fujishirodai 5-7-1, Suita 565-8565, Osaka, Japan c Research Institute, National Cardiovascular Center, Japan d School of Health and Sport Sciences, Osaka University, Japan

Received 15 June 2004; accepted 10 December 2004

Abstract FTY720 has been originally developed as a new immunosuppressive agent, which prolongs graft survival in organ transplantation. Adrenomedullin (AM) participates in the regulation of sodium homeostasis and has renoprotective effects. The possible involvement of renal AM in the pathophysiology of glomerulonephritis (GN) and the effect of FTY720 has been evaluated in rats. HgCl2 (1 mg/kg body weight) was inoculated subcutaneously 3 times/week for a total of 2 weeks. FTY720 (3 or 10 mg/kg) was inoculated subcutaneously daily. The proteinuria, urinary N-acetyl-beta-d-glucosaminidase (NAG) excretion and serum total cholesterol levels were increased and serum albumin level was reduced in rats with HgCl2-induced GN compared with controls. FTY720 reduced proteinuria (3 mg/kg: 25%; 10 mg/kg: 41%), urinary NAG excretion ( 11%; 52%) and total cholesterol level ( 21%; 55%) in a dose-dependent manner. Renal AM level and its mRNA expression were increased in rats with GN compared with controls (Peptide Cortex: +69%; Medulla: +82%; mRNA Cortex: +25%). Interestingly, FTY720 additionally increased these levels (Peptide Cortex: +38%; Medulla: +39%; mRNA Cortex: +20%). Renal AM levels correlated with urinary NAG excretion and creatinine clearance. These results suggest that FTY720 suppresses the renal damage in rats with GN and renal AM may participate in the pathophysiology of GN and the renoprotective effects of FTY720. D 2005 Elsevier B.V. All rights reserved. Keywords: Adrenomedullin; FTY720; Brown Norway rat; Mercuric chloride; Autoimmune glomerulonephritis

1. Introduction FTY720 is a synthetic compound that is chemically modified from Isaria sinclairii [1]. It is originally developed as a new immunosuppressive agent that remarkably prolongs graft survival in organ transplantation models without causing severe adverse reaction [2]. Induction of apoptosis in resting and activated lymphocytes is considered to be a

* Corresponding author. Tel.: +81 6 6833 5012; fax: +81 6 6872 7486. E-mail address: [email protected] (H. Nakahama). 0167-0115/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.12.013

mechanism of action [3]. We expected this compound might provide antiproteinuric potential, as do other immunosuppressants. We have shown that FK 506 (tacrolimus) is effective against the autoimmune glomerulonephritis (GN) in Brown Norway rats induced by mercuric chloride (HgCl2) administration [4,5]. In the present study, using the same animal model of autoimmune glomerulonephritis, we investigated the effect of FTY720 thereon. Adrenomedullin (AM) and its gene expression have been reported to be observed in the glomerulus, distal tubules, and medullary collecting duct cells in the kidney [6]. Immunohistochemical analysis of the human kidney

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revealed adrenomedullin specific receptor immunoreactivity in the juxtaglomerular arteries, the glomerular capillaries and chief cells of the collecting duct [7]. These results suggest that adrenomedullin and its receptor system in the kidney may be involved in the regulation of renal hemodynamics, glomerular filtration and tubular Na+ homeostasis in the experimental model of renal disease. There is also evidence for a role for adrenomedullin in mesangial cell biology. Mesangial cells grown in primary culture synthesize adrenomedullin, which is stimulated by TNF alpha [8], IL-1 beta [9] and under hypoxic conditions [10]. Adrenomedullin increases cAMP levels in mesangial cells [11] leading to inhibit their proliferation [12], migration [13], reactive oxygen generation [9] and macrophage infiltration [9]. In addition, adrenomedullin has been reported to stimulate hyaluronic acid, an important extracellular matrix component, release from mesangial cells through p38 kinase and PI3-kinase pathways [14]. These data suggest that there may be a role for adrenomedullin not only in the pathophysiology of mesangial cell proliferation and matrix biology, but also in protecting the renal glomeruli from inflammatory reactions or immune injuries. However, the pathophysiological significance of renal AM and the possible involvement of renal AM in the effect of immunosuppressant in rats with glomerulonephritis have not been elucidated. In the present study, we evaluated the pathophysiological significance of renal AM in glomerulonephritis and whether a novel immunosuppressant FTY720 alters renal AM synthesis.

were treated with HgCl2 alone; group 3, rats were treated with HgCl2 and FTY720 (3 mg/kg body weight); group 4, rats were treated with HgCl2 and FTY720 (10 mg/kg body weight); and group 5, FTY720 (10 mg/kg body weight) alone. On day 13 of the experimental protocol, all rats were housed overnight in metabolic cages in order to collect urine. Serum and urine samples were obtained from all animals on day 14. Animals were sacrificed on day 14. Serum albumin, creatinine, total cholesterol, blood urea nitrogen, urinary protein, urinary N-acetyl-beta-d-glucosaminidase (NAG), urinary creatinine and urinary sodium were determined using an automatic analyzer (Hitachi 7170, Hitachi, Japan). 2.5. Radioimmunoassay for renal AM AM levels in the renal cortex and the renal medulla were measured using radioimmunoassay as described previously [15]. 2.6. Northern blot analysis An EcoRI/NaeI restriction fragment of rat AM cDNA corresponding to nucleotides 153 to 436 was used as the rat AM cDNA probe. Total RNA (20 Ag/lane) for AM mRNA evaluation was denatured, electrophoresed and transferred to nylon membrane. For hybridization with the cDNA probe, conditions for hybridization and washing have been previously described [15]. 2.7. Statistical analysis

2. Materials and methods 2.1. Animals Six-week-old male Brown Norway rats were obtained from Oriental Yeast Co. Ltd., Osaka, Japan. They weighed 150–160 g at the start of the experiments. They were fed standard food and tap water ad libitum.

Data are expressed as meansFSD. The one-way ANOVA test, followed by multiple comparisons test, was used for intergroup comparisons. Differences were considered significant at a level of pb0.05. Correlation coefficients were calculated using linear regression analysis.

3. Results 2.2. HgCl2 administration HgCl2 (1 mg/kg body weight) was dissolved in normal saline and inoculated subcutaneously 3 times/week for a total of 2 weeks.

3.1. Characteristics of HgCl2-induced autoimmune glomerulonephritis

FTY720 (donated by Novartis Pharmaceuticals Ltd, Basel, Switzerland) was suspended in normal saline and inoculated subcutaneously daily.

With subcutaneous inoculation of HgCl2, a progressive proteinuria developed from 7 days onwards. The proteinuria stabilized at around 600 mg/24 h. Concomitant with the increase in proteinuria, there was a severe drop in serum albumin levels, reaching around 2 g/dL at day 14. A concomitant rise in serum total cholesterol was observed, reaching above 200 mg/dL at day 14.

2.4. Experimental design

3.2. Effects of FTY720 on serum and urine chemistry

Forty animals were randomly divided into 5 groups of 8 animals: group 1, rats received vehicle only; group 2, rats

All animals were alive at the termination of the experiment. Effects of FTY720 (10 mg/kg body weight)

2.3. FTY720 administration

Y. Shi et al. / Regulatory Peptides 127 (2005) 233–238

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Table 1 Effects of HgCl2 and FTY720 on serum and urine chemistry Group HgCl2 FTY720

1 – –

2 + –

3 + 3 mg/kg

4 + 10 mg/kg

5 – 10 mg/kg

Serum Alb, g/dL BUN, mg/dL Tcho, mg/dL CRN, mg/dL

4.0F0.2 24.4F4.1 60.4F4.8 0.35F0.02

1.9F0.2a 34.0F13.2 242.3F30.7a 0.33F0.10

1.6F0.2a 40.8F10.4a 190.9F42.5a,b 0.29F0.05

1.7F0.3a 28.7F10.4 109.3F40.1a,b,c 0.27F0.10

2.6F0.1a,b,c,d 21.0F2.0c 48.6F4.6b,c,d 0.29F0.02

Urine P, mg/dL Ccr, mL/min NAG, U/day Na+, mEq/day

11.8F3.0 1.4F0.2 0.2F0.1 1.4F0.4

672.6F124.7a 1.1F0.5 1.3F0.6a 2.0F0.4

504.4F82.5a 0.9F0.3 1.2F0.5a 1.2F0.6b

395.7F217.6a,b 1.2F0.4 0.6F0.3b 1.7F0.5

4.4F1.2b,c,d 1.4F0.1 0.2F0.1b,c 1.5F0.2

Data are expressed as meansFSD. The One-way ANOVA test, followed by multiple comparisons test, was used for intergroup comparisons. Alb, albumin; BUN, blood urea nitrogen; Tcho, total cholesterol; CRN, creatinine; P, Protein; Ccr, creatinine clearance; NAG, N-acetyl-beta-d-glucosaminidase. a pb0.05 vs. group 1. b pb0.05 vs. group 2. c pb0.05 vs. group 3. d pb0.05 vs. group 4.

shown in Table 1. Treatment with FTY720 (10 mg/ body weight) resulted in a significant reduction in urinary protein excretion, total cholesterol levels and urinary excretion of NAG. The effects of FTY720 these parameters were dose dependent. Despite the

reduction in proteinuria, serum albumin levels were reduced in the treated rats (groups 3 and 4) compared with controls (group 1). The reduced albumin levels were also noted in rats treated only with FTY720 (group 5).

p<0.0001

p<0.0001 p<0.01 p<0.01 p<0.001 Cortical total AM conc. (fmol/mg)

Medullary total AM conc. (fmol/mg)

p<0.05 p<0.01 p<0.0001

1.2 0.8 0.4 0 p<0.001

1.6 1.2 0.8 0.4 0

p<0.05

p<0.0001

p<0.05

p<0.01

p<0.01 Cortical mature AM conc. (fmol/mg)

Medullary mature AM conc. (fmol/mg)

are kg the the on

1.2

0.8

0.4

0

HgCl2

-

+

+

-

FTY720

0

0

10

10

1.0 0.8 0.6 0.4 0.2 0

HgCl2

-

+

+

-

FTY720

0

0

10

10

mg/kg

mg/kg

mg/kg mg/kg

Fig. 1. Renal cortical and medullary adrenomedullin (AM) levels.

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HgCl2

-

+

FTY720

0

0

+

-

10

10

mg/kg

mg/kg

AM GAPDH p<0.05

AM/ GAPDH (%)

p<0.05 p<0.05 150 100 50

0 HgCl2 FTY720 n

0

+ 0

4

4

+ 10

10

mg/kg

mg/kg

4

4

Urinary NAG excretion (U/day)

Fig. 2. Representative Northern blots showing adrenomedullin (AM) mRNA expressions in the renal cortex in rats.

3

3 R=0.54 p<0.01

2

2

1

1

0

0 0

Creatinine clearance (mL/min)

R=0.49 p<0.01

0

0.2 0.4 0.6 0.8 1.0 1.2 Medullary mature AM conc. (fmol/mg)

2.0

2.0

1.6

1.6

1.2

1.2

0.8

0.8 R=-0.61 p<0.001

0.4

R=-0.40 p<0.05

0.4

0

0.2 0.4 0.6 0.8 1.0 1.2 Cortical mature AM conc. (fmol/mg)

0 0

0.2 0.4 0.6 0.8 1.0 1.2 Medullary mature AM conc. (fmol/mg)

0

0.2 0.4 0.6 0.8 1.0 Cortical mature AM conc. (fmol/mg)

1.2

Fig. 3. Correlations between renal tissue adrenomedullin (AM) levels in the renal cortex and medulla and urinary N-acetyl-beta-d-glucosaminidase (NAG) excretion and creatinine clearance (CCr).

Y. Shi et al. / Regulatory Peptides 127 (2005) 233–238

3.3. Effects of FTY720 (10 mg/kg body weight) on renal tissue adrenomedullin (AM) levels HgCl2 and FTY720 independently increased renal tissue AM levels significantly. Coadministration of HgCl2 and FTY720 resulted in a synergistic increase of plasma and renal tissue AM levels (Fig. 1). AM mRNA expressions in the renal cortex was also higher in the rats of groups 2, 4 and 5, the highest being in group 4 (Fig. 2). Significant correlations were found between renal cortical and medullary mature AM levels and urinary NAG excretion and creatinine clearance (Fig. 3). Although similar positive correlations existed between renal AM levels and urinary sodium excretion, they did not reach a statistical significance (data not shown).

4. Discussion Mercuric chloride (HgCl2) induces a lymphoproliferative disorder and autoimmune glomerulonephritis in Brown Norway rats [16]. In the kidney, early deposition of antiglomerular basement membrane antibodies occurs and is associated with heavy proteinuria. Subsequently, a superimposed immune complex type glomerulonephritis occurs. It has often been proposed as having many of the features of membranous nephropathy [17]. Immunosuppressive drug therapy can be expected to be beneficial in the treatment of autoimmune glomerulonephritis. We have reported that FK 506, a potent immunosuppressive agent with a similar mode of action to cyclosporine A, is effective both in the prevention and the treatment of mercuric chloride (HgCl2) induced autoimmune glomerulonephritis in Brown Norway rats [4,5]. FTY720 is an analogue of a fungal metabolite, Myriocin (ISP-1), which is obtained from culture filtrates of an ascomycete, I. sinclairii [1]. Although ISP-1 produces severe digestive disorders in experimental animals, it shows potent immunosuppressive activity in vitro. Therefore, ISP1 was chemically modified to be less toxic. FTY720 is one of the compounds generated by modification of ISP-1. The chemical structure of FTY720 is 2-amino-2-[2-(4-octylphenyl)ethyl]-1.3-propanediol hydrochloride; C19H33NO2 HCl and its molecular weight is 343.94. FTY720 has a completely different chemical structure from other immunosuppressants such as cyclosporine A or FK 506 [2]. The immunosuppressive mechanism of FTY720 is supposed to be due to induction of apoptosis of the lymphocytes, because FTY720 induced chromatin condensation formation of apoptotic bodies and DNA fragmentation in lymphocytes [3]. It has been reported that FTY720 exhibits beneficial effects on various kinds of organ transplantations in the rat system [2]. We have previously reported that FTY720 exerts anticarcinogenic effects against prostate cancer cells possibly involving modulation of mitogenic signaling, cell-cycle regulators, induction of G1 arrest and apoptotic cell death [18].

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In the present study, FTY720 has been shown to ameliorate HgCl2-induced autoimmune glomerulonephritis in a dose-dependent manner. The degree of suppression of proteinuria was smaller compared to that exerted by FK 506, which virtually provided complete prevention of autoimmune glomerulonephritis and consequent proteinuria [4]. Different mechanisms and degree of immunosuppression between these two drugs may account for the difference in their antiproteinuric effects. FTY720 per se induced body weight loss, reduced serum albumin and serum total cholesterol (Table 1). Taken all together, FTY720 might be considered as a drug of choice for glomerulonephritis when other more potent immunosuppressants are not effective or when they should be withdrawn because of serious side effects. AM has been reported to have a possible role in human glomerulonephritis. In a study with patients with IgA nephropathy [19], the plasma AM level was higher in patients with IgA nephropathy than in healthy volunteers. The plasma AM concentration was positively correlated with serum creatinine and blood urea nitrogen and with fractional excretions of sodium and potassium. When the disease activity was scored based on the histological findings of renal biopsy specimen, the urinary AM level was lower in the high-activity group as compared with the low-activity one, which suggests that urinary AM may reflect the activity in IgA nephropathy. It has also been shown that plasma AM was positively correlated and urinary AM was negatively correlated with the degree of proteinuria in patients with a variety of types of glomerulonephritis [20]. Kinoshita et al. measured plasma and urine levels of AM in patients with chronic glomerulonephritis including IgA nephropathy, minor glomerulopathy, focal glomerular sclerosis, membranous nephropathy and lupus nephritis, one-third of whom showed nephrotic syndrome. Contrary to the previous reports, the plasma AM level in the patients with chronic glomerulonephritis did not differ from that in control subjects, whereas the urinary AM concentration was significantly less for the patients with chronic glomerulonephritis. The plasma AM concentration was negatively correlated with plasma renin activity and aldosterone concentration. The urinary concentrations of AM showed a significant correlation with the urinary excretion of sodium [21]. The findings suggested that AM might have a role in the regulation of urinary sodium excretion as paracrine or autocrine factors, in addition to the endocrine actions of plasma AM in patients with chronic glomerulonephritis. Chronic infusion of AM have been reported to reduce proteinuria, glomerular injury score and renal TGF-beta mRNA expression in Dahl saltsensitive rats, suggesting that its infusion has renoprotective effects in this type of hypertension model [22]. Our present findings suggest that renal AM is increased in compensation for sodium retention tendency in nephrotic syndrome and for renal damage in glomerulonephritis. The mechanisms by which FTY720 reduces proteinuria remain to be elucidated. Reduced immune response might be the main mechanism.

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Interestingly, FTY720 additionally increased the renal levels of AM peptide and mRNA expression in concurrence with the significant reduction of proteinuria, urinary NAG excretion and serum total cholesterol level in rats with HgCl2-induced autoimmune glomerulonephritis. We have very recently reported that the renal AM level was increased in rats with HgCl2-induced autoimmune glomerulonephritis and that mycophenolate mofetil almost completely suppressed the increase of renal AM level in concurrence with the suppression of renal damage [23]. These results suggest that the reduction of tissue AM level due to a drug administration may reflect the effect of its administration on the tissue damage. In contrast, our results that FTY720 additionally increased renal AM levels in the rats with autoimmune glomerulonephritis, although it suppressed renal damage, suggest that the alteration of renal AM levels under FTY720 administration might not reflect its effect on HgCl2-induced autoimmune glomerulonephritis. Taken together with our other result that FTY720 induces renal cortical AM transcription and increases renal AM levels in control rats, there may be a possibility that FTY720 increases renal AM production directly and the increased AM may participate in the reduction of proteinuria by FTY720 administration in the rats with HgCl2induced autoimmune glomerulonephritis. However, we previously reported that AM secretion from cultured rat cardiomyocytes was increased with the exposure to oxidative stress induced by hydrogen peroxide in association with the increase of lactate dehyrogenase leakage due to a cell reaction for the severe damage of cell structure [24]. Thus, we could not deny the possibility that the increased renal AM level induced by FTY720 administration in the present study might be due to some kind of adverse effects of FTY720, although there was no apparent renal damage in rats treated with FTY720 alone in our protocol. Further study is necessary to reveal the mechanism and significance of increased renal AM under the administration of FTY720 in glomerulonephritis.

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