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«A sustained-release prostacyclin analog ONO-1301 ameliorates tubulointerstitial alterations in a mouse obstructive nephropathy model Tatsuyo Nasu1, ...»

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Articles in PresS. Am J Physiol Renal Physiol (March 14, 2012). doi:10.1152/ajprenal.00538.2011

Am J Phys-Renal Physiol ONO-1301 and unilateral ureteral obstruction

Nasu T, et al.

MS # F-00538-2011-R1

A sustained-release prostacyclin analog ONO-1301 ameliorates

tubulointerstitial alterations in a mouse obstructive nephropathy model

Tatsuyo Nasu1, Masaru Kinomura1, Katsuyuki Tanabe1, Hiroko Yamasaki1, Su Le Htay1, Daisuke Saito1, Norikazu Hinamoto1, Hiroyuki Watatani1, Haruyo Ujike1, Yoshinori Suzuki2, Takeshi Sugaya3, Hitoshi Sugiyama4, Yoshiki Sakai5, Kunio Matsumoto2, Yohei Maeshima1 and Hirofumi Makino1 10 Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan, 2Division of 12 Tumor Dynamics and Regulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan, 3CMIC Co., Ltd., Tokyo, Japan, 4Center for Chronic Kidney Disease 14 and Peritoneal Dialysis, Okayama University Graduate School of Medicine, Dentistry, 15 and Pharmaceutical Sciences, Okayama, Japan, Ono Pharmaceutical Co., Ltd., 16 Research Headquarters, Osaka, Japan.

21 Running title: ONO-1301 and unilateral ureteral obstruction (UUO) 24 Address for Correspondence:

25 Dr. Yohei Maeshima 26 Professor of Medicine, 27 Department of Chronic Kidney Disease and Cardiovascular disease, 28 Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 29 2-5-1 Shikata-cho, Okayama, 30 700-8558, JAPAN 31 Tel. No.: +81-86-235-7235 32 Fax No.: +81-86-222-5214 33 E-mail: ymaeshim@md.okayama-u.ac.jp Copyright © 2012 by the American Physiological Society.

Am J Phys-Renal Physiol ONO-1301 and unilateral ureteral obstruction Nasu T, et al.

MS # F-00538-2011-R1 Abstract 38 Tubulointerstitial injuries are crucial histological alterations that predict the 39 deterioration of renal function in chronic kidney disease. ONO-1301, a novel sustainedrelease prostacyclin analog, accompanied by thromboxane synthase activity, exerts 41 therapeutic effects on experimental pulmonary hypertension, lung fibrosis, 42 cardiomyopathy, and myocardial ischemia, partly associated with the induction of 43 hepatocyte growth factor (HGF). In the present study, we examined the therapeutic 44 efficacies of ONO-1301 on tubulointerstitial alterations induced by unilateral ureteral 45 obstruction (UUO). After inducing UUO in C57/BL6J mice, a single injection of

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47 suppressed interstitial fibrosis, accumulation of type I and III collagen, increase in the number of interstitial fibroblast-specific protein-1 (FSP-1)+ cells, and interstitial infiltration of monocytes/macrophages (F4/80+) in the obstructed kidneys (OBK; Day 7).

50 Treatment with SR-ONO significantly suppressed the increase of the renal levels of 51 pro-fibrotic factor TGF-β and phosphorylation of Smad2/3, and elevated the renal levels 52 of HGF in the OBK. In cultured mouse proximal tubular epithelial cells (mProx24), 53 ONO-1301 significantly ameliorated the expression of FSP-1 and α-smooth muscle 54 actin as well as phosphorylation of Smad3 and increased the expression of ZO-1 and

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59 together, our findings suggest the potential therapeutic efficacies of ONO-1301 in 60 suppressing tubulointerstitial alterations partly mediated via inducing HGF, an antifibrotic factor counteracting TGF-β.

62 Keywords: unilateral ureteral obstruction, prostacyclin, TGF-β1, HGF, FSP-1

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Introduction 65 Tubulointerstitial alterations are involved in the progression of chronic kidney 66 disease (CKD). The histological features are characterized by interstitial infiltration of 67 mononuclear cells, accumulation of myofibroblasts, proliferation of interstitial fibroblasts 68 and accumulation of extracellular matrix (ECM) proteins, ultimately leading to interstitial 69 fibrosis(3). In many forms of glomerular disorders, deterioration of renal function 70 correlates better with the degree of tubulointerstitial injury rather than that of glomerular 71 alterations(40). Therefore, the development of novel therapeutic strategies to inhibit 72 the progression of tubulointerstitial alterations of CKD patients is required.

73 Unilateral ureteral obstruction (UUO) is a well-established experimental model 74 of progressive renal tubulointerstitial injuries. Previous reports have shown the 75 involvement of various mechanisms in chronic renal tubulointerstitial injuries in a UUO 76 model including pro-fibrotic transforming growth factor (TGF)-β1 (20), and inflammatory 77 cytokines and chemokines such as IL-6 or monocyte chemoattractant protein (MCP)More recently, the involvement of epithelial-mesenchymal transition (EMT) in 79 the development of tubulointerstitial fibrosis has been demonstrated(13, 24, 64). In the 80 process of EMT, epithelial cells dedifferentiate, lose epithelial cell surface markers 81 accompanied by the acquisition of mesenchymal marker proteins, resulting in the 82 formation of fibroblasts(53). The possibility of EMT as a contributor to renal interstitial 83 fibrosis including UUO(4), as well as the involvement of various factors including TGFβ1 in the process of EMT and interstitial fibrosis has also been reported(53).

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91 biological effects of HGF to counteract TGF-β1 and EMT play important roles in 92 attenuating renal fibrosis(12, 62).

93 Prostacyclin (prostaglandin I2: PGI2), a metabolite of arachidonic acid,

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95 aggregation and the proliferation of vascular smooth muscle cells(32, 33). In contrast, 96 thromboxane (TX)A2 exerts the opposite effects to PGI2, contributing to vascular 97 lesions(33). Currently, there are two known receptors mediating the biological effects 98 of PGI2, the IP receptor on the cell surface and the nuclear peroxisome proliferatoractivated receptor δ (PPARδ). Prostacyclin-deficient mice by genetic disruption of 100 prostaglandin I2 synthetase developed renal fibrosis and arterial sclerosis(63), 101 suggesting its important role in renal development and maintenance of a normal 102 structure. Previous reports have demonstrated the renoprotective effects of beraprost 103 sodium, a prostacyclin derivative, in various experimental models of renal disorders 104 (19, 49, 58) including a rat partial UUO(55) model.

105 ONO-1301 is a synthetic nonprostanoid IP agonist possessing a potent 106 inhibitory activity against TXA2 synthase(6, 10, 34, 46). Although administration of 107 prostacyclin generally leads to elevated plasma TXB2 levels, administration of ONOshowed inhibitory effects on the increase of TXB2 in the rat pulmonary 109 hypertension model(15), suggesting unique characteristics distinct from known 110 prostacyclin derivatives. ONO-1301 specifically interacts with the IP receptor, but not 111 with other types of prostaglandin receptors such as EP, TP or FP receptors(18).

112 Prostacyclin and its analogues are not stable in vivo, since 15-hydroxy PG 113 dehydrogenase metabolizes their prostanoid structures. Unlike prostacyclin, ONOlacks the typical prostanoid structures, including a five-membered ring and allylic 115 alcohol, thus leading to the improved biological and chemical stability of this compound 116 in vivo.

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117 The therapeutic effects of ONO-1301 in experimental models of pulmonary 118 fibrosis(34), pulmonary hypertension(15), and ischemic heart disease(37) have been 119 reported. The beneficial effect of ONO-1301 in treating cardiac ischemia was partly 120 mediated via up-regulation of HGF and vascular endothelial growth factor (VEGF), via 121 a cAMP-dependent pathway (37). More recently, a novel sustained-release ONO-1301 122 (SR-ONO) has been developed. A single subcutaneous injection of SR-ONO resulted 123 in sustained elevation of its circulating levels for 3 weeks(41).

124 A previous report has demonstrated the antinephritic effects of ONO-1301 125 (twice daily) in a rat anti-GBM nephritis model(6). More recently, we reported the 126 therapeutic effects of SR-ONO in a rat model of type 1 diabetic nephropathy(60).

127 However, the therapeutic effects of ONO-1301 on tubulointerstitial injuries have not 128 been demonstrated to date.

129 In the present study, we hypothesized that ONO-1301 may ameliorate 130 tubulointerstitial injuries in a mouse UUO model through the anti-fibrotic effects 131 mediated partly via the induction of HGF, blockade of EMT and by down-regulation of 132 TGF-β1 signaling.

136 Pharmaceutical Co., Ltd. (Osaka, Japan) as previously described(6, 10, 34). A slowrelease form of ONO-1301 (SR-ONO) was generated by encapsulating ONO-1301 with 138 poly (D, L-lactic-co-glycolic acid); PLGA as described previously(14, 41). (Please see 139 Supplementary Materials and methods.) 140 Experimental protocol: The experimental protocol was approved by the 141 Animal Ethics Review Committee of Okayama University Graduate School of Medicine, 142 Dentistry, and Pharmaceutical Sciences. Studies were performed in male C57/BL6 143 mice. The animals were fed a standard pellet laboratory diet and were provided with 144 water ad libitum. With the mice under pentobarbital anesthesia, the left ureter was 145 exposed through a mid-abdominal incision and ligated with 3-0 silk at two locations, 146 approximately 1 cm below the renal hilum, as described previously (50, 54). Sham 147 operated mice had their ureters manipulated, but not ligated. Weight-matched (20-22g) 148 10-week old mice were divided into four subgroups: (1) undiseased, (2) vehicle (PLGA 149 polymer containing no active substance)-treated UUO, (3) SR-ONO (3 mg/kg body 150 weight)-treated UUO, (4) SR-ONO (10 mg/kg body weight)-treated UUO (N = 5 for 151 each subgroup). Mice received subcutaneous injection of SR-ONO or vehicle buffer on 152 the day of initiation of UUO. Mice were sacrificed on Day 7, and the bilateral kidneys 153 and the serum were obtained.

154 For blockade of endogenous HGF, neutralizing rabbit anti-rat/mouse HGF 155 antibody (30) was used. Rat recombinant HGF was purified from the culture medium of 156 Chinese hamster ovary cells transfected with plasmid for expression of rat HGF(57).

157 Polyclonal antibody against rat HGF was raised in rabbits by immunizing with rat 158 recombinant HGF(42). Immunoglobulin G (IgGs) was purified using protein-A 159 Sepharose (Sigma Aldrich, Inc., Missouri, USA). Anti-rat HGF IgG at 3 μg/ml almost

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160 completely neutralizes the biological activity of rat and mouse HGF at 10 ng/ml. The 161 dose and interval for administration of anti-rat HGF IgG in mice were determined by 162 another experiment to examine the inhibitory effects of anti-rat/mouse HGF IgG on liver 163 regeneration in mice (data not shown). Male C57BL/6 mice (5 weeks old) receiving 164 UUO were subcutaneously injected with SR-ONO (10mg/kg BW) or vehicle buffer once 165 on the day of initiation of UUO. The SR-ONO-treated UUO mice were randomly divided 166 into two groups and received intraperitoneal injections of either rabbit anti-rat HGF IgG 167 (n = 5) or normal rabbit IgG (n = 5) every other day (250 μg/mouse). Mice were 168 sacrificed on Day 7 and the bilateral kidneys and the serum were obtained. No mice 169 died and no signs of apparent exhaustion were observed during the experimental 170 period.

171 Blood examination. Blood urea nitrogen (BUN) and serum creatinine levels 172 were measured by SRL, Inc. (Okayama, Japan). Serum creatinine levels were 173 measured by the enzymatic colorimetric method as described (5). Serum ONO-1301 174 level was measured by liquid chromatography tandem mass spectrometry assay (41).

175 Histological analysis. At 7 days after the induction of UUO, kidneys were 176 removed, fixed in 10% buffered formalin and embedded in paraffin. Sections (4 μm 177 thick) were stained with Masson’s trichrome for light microscopic observation. Bluestained interstitial fibrotic areas were assessed by computer-image analysis using 179 Lumina Vision software (Mitani, Fukui, Japan). Fifteen consecutive fields were 180 randomly selected in the renal cortex and evaluated at x400 magnification. Tubular 181 cells, tubular lumen, glomeruli or blood vessels were not included. This fraction 182 represented the relative interstitial volume. Results were expressed as a percentage of 183 the measured area, which represented the interstitial space and was determined as the 184 relative volume (Vv) of the interstitium(50). Histological evaluation was performed in a 185 blinded fashion by two investigators and averaged.

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186 Immunohistochemistry. Immunohistochemistry was performed using 4 μm 187 frozen sections as described previously(8, 26, 39, 56). Briefly, frozen kidney sections 188 were incubated with polyclonal rabbit anti-human FSP-1 (S100A4) antibody (1:100 189 dilution, A5114; DAKO Cytomation Inc., CA) and rat anti-mouse F4/80 antibody (1:200 190 dilution, A3-1; Serotec, Oxford, UK), and exposed to secondary antibody, Immun-Star 191 goat anti-rabbit (GAR)-HRP Conjugate (1:200 dilution, 170-5046; Bio-rad, Hercules, 192 CA), HRP-labeled goat anti-rat IgG (Millipore, Billerica, MA). Diaminobenzidine was

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194 interstitial monocyte/macrophage infiltration was determined by counting the number of F4/80+ cells in the renal cortex. The number of F4/80+ cells was determined in 15 196 randomly selected non-overlapping fields (x400) in each section of the individual mouse renal cortex. The average number of F4/80+ cells from 5 separate animals was

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199 tubulointerstitium was determined (x400). For immunohistochemistry of mouse IP 200 receptors, formalin (10%)-fixed, paraffin-embedded sections (4-μm) were used. After 201 deparaffinization, sections were incubated with rabbit polyclonal anti-IP receptor 202 antibody (1:100 dilution; Cayman Chemical, Ann Arbor, MI) followed by incubation with 203 biotinylated-secondary antibody, and immunoperoxidase staining was carried out.

204 Diaminobenzidine was used as a chromogen. All slides were counterstained with 205 hematoxylin. (Please see Supplementary Materials and methods.) 206 Immunofluorescence. Immunofluorescent staining was performed as 207 described previously(9, 39, 56) to assess interstitial accumulation of type I and III 208 collagen. (Please see Supplementary Materials and methods.)

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