Renal Effects of S18886 (Terutroban), a TP Receptor Antagonist, in an Experimental Model of Type 2 Diabetes

Diabetes In Press, published online January 31, 2007

Renal effects of S18886 (terutroban), a TP receptor antagonist, in an experimental model of type 2 diabetes

Received on 15 August 2006 and accepted in revised form 14 January 2007.

Katarína Šebeková1, Timo Eifert2, André Klassen3, August Heidland3, Kerstin Amann2

1Slovak Medical University, Department of Clinical and Experimental Pharmacotherapy, Bratislava, Slovakia; 2Department of Pathology, University of Erlangen-Nürnberg; and 3Department of Internal Medicine, University of Würzburg, Germany

Running head: TP antagonist improves diabetic nephropathy

Address for correspondence: Katarína Šebeková MD., DSc. Slovak Medical University Department of Clinical and Experimental Pharmacotherapy Limbová 12 833 03 Bratislava Slovakia Email: [email protected]

Word count (abstract): 200 Word count (main text): 3798

ABSTRACT

Thromboxane A2 (TxA2) is assumed to contribute to the development of diabetic complications, including nephropathy. We investigated whether the selective thromboxane- prostanoid endoperoxide receptor antagonist, S18886, ameliorates renal damage in uninephrectomized (UNX) obese Zucker rats (OZR). S18886, at doses of 10 (S18886-10) and 30 (S18886-30) mg/kg/d, was administered to UNX- OZR by gavage over 8 weeks (n=8 each group). UNX lean rats (n=12) and OZR, receiving placebo OZR-PLAC (n=8), served as controls. As compared to the OZR-PLAC, S18886 had no significant effect on the elevated blood pressure and the enhanced creatinine clearance, while augmented proteinuria was partially prevented (-12% and -37%, low and high dose respectively, n.s.). The increased excretion of transforming growth factor ß1 (TGF-ß1) and of the thromboxane metabolite 2,3-dinor thromboxane B2 (TxB2) was lowered (p<0.05). S18886 prevented both the enhanced mesangiolysis (p<0.01) in the OZR-PLAC as well as enlargement and degeneration of podocytes. In the blood S18886-30 augmented the antioxidant enzymes (p<0.01) and lessened the increase of plasma advanced oxidation protein products (-25%, n.s.). Body weight, hyperglycemia and dyslipidemia remained uninfluenced under both doses of treatment. S18886 has renoprotective properties in the model of UNX-OZR. It prevents mesangiolysis, reduces urinary TGF-ß1 and 2,3-dinor-TxB2 excretion, and enhances the antioxidative defence.

Key words: selective thromboxane-prostanoid endoperoxide (TP) receptor antagonist – diabetic nephropathy – proteinuria – mesangiolysis – glomerulosclerosis Abbreviations: AOPPs: advanced oxidation protein products, apoE: apolipoprotein E, AST: aspartate- aminotransferase, AT II: angiotensin II, GN: glomerulonephritis, GPX: glutathione peroxidase, GSI: glomerulosclerosis index, HE: hematoxilin/eosin, HETE: 12- hydroxyeicosatatraenoic acid, HMG-CoA: 3-hydroxymethyl-glutaryl-coenzyme A, LZR: lean Zucker rat, MGI: mesangiolysis index, OZR: obese Zucker rat, PAS: periodic Schiff’s acid, PLAC: placebo, PG: prostaglandin, SOD: superoxide dismutase, STZ: streptozotocin, TGF- β1: transforming growth factor β1, TP: thromboxane-prostanoid endoperoxides, TxA2: thromboxane A2, TxB2: thromboxane B2, UNX: uninephrectomy, WBC: white blood cells

Thromboxane A2 (TxA2) induces platelet evaluated by TxB2 content in urine, activation and is a powerful isolated glomeruli, tubulo-interstitial fluid vasoconstrictor. It decreases renal blood and by immune histochemistry. Enhanced flow, lowers single nephron glomerular levels have been observed in diabetic filtration rate and potentiates the nephropathy, remnant kidney, immune tubuloglomerular feedback (1,2). In vitro, mediated renal diseases (anti GBM TxA2 contracts isolated glomeruli and nephritis, active lupus nephritis and renal mesangial cells (3), enhances proliferation allograft rejection), endotoxin-induced of glomerular cells and the synthesis of acute renal failure and cyclosporine extracellular matrix. It has chemoattractive toxicity (4, 5, 11, 12). Inhibition of properties, stimulates formation of thromboxane synthesis or blockade of the adhesion molecules and exerts TPr ameliorated the functional/structural immunological actions (4). Major cellular alterations in early stages of the sources of TxA2 are platelets and – in aforementioned diseases (13-18). disease – leukocytes, macrophages, However, treatment with thromboxane vascular smooth muscle endothelial,, synthase inhibitors may be followed by a mesangial and immune cells (5). In vivo rise of PGH2 in platelets, favouring a pro- TxA2 is rapidly metabolised to the stable, aggregatory activity (19). inactive derivative 2,3-dinor-thromboxane S18886, a polysubstituted B2 (TxB2). tetrahydronaphthalene derivative, is a new The biological effects of TxA2 are and highly selective TPr antagonist with a mediated by a G protein-coupled long duration of action (20, 21). It exerts thromboxane prostanoid receptor (TxA2- potent anti-platelet and anti- PGH2) which is activated by phospholipase vasoconstrictory effects via the TPr and C and an immediate rise of intracellular antagonises the actions of TxA2, as well as calcium (6). TxA2-PGH2 receptors (TPr) of other arachidonic acid products are expressed in blood vessels and renal (prostaglandin PGH2, PGF2α, HETE acids microvessels, glomeruli, mesangial cells, and isoprostanes) (9). Recently it has been thick ascending limbs and collecting ducts shown that S18886 inhibited the (7). The receptor is activated not only by development of atherosclerosis in two TxA2 but also by PGH2, 8-isoprostanes, rabbit models (22, 23) and in hydroxyeicosatatraenoic acid (HETE) and apolipoprotein E-deficient (apoE-/-) mice, products of oxygen formation (8-10). TPr with or without streptozotocin (STZ)- play a decisive role in the hormonal effects induced diabetes (24, 25), and acted of angiotensin II (AT II), endothelin and renoprotectively in the rat STZ model of arginine vasopressin. After diabetes (26). Since type 2 diabetes is the deletion/antagonism of the TPr in mice, the main cause of end-stage renal failure in AT II-induced intrarenal vasoconstriction humans, we were interested in the effects and the enhanced oxygen free radical of S18886 in a rat model of type 2 formation are abolished (10). Other factors diabetes. The obese Zucker rat (OZR) activating TPr are interleukin-1 and -2, model shares many characteristics of type immune attack complexes, terminal 2 diabetes in humans, such as obesity, complement components (C5b-C9) and insulin resistance, moderate endotoxins (4). hyperglycemia, hyperlipidemia and Thromboxane and other prostanoids have hypertension (27) and is also used for been implicated in the proinflammatory studies in diabetic nephropahy. To and oxidative events in cardiovascular accelerate the renal injury, we performed a diseases, as well as in experimental and uninephrectomy (UNX), as has been human renal diseases. TxA2 formation, was previously suggested (28, 29). Besides the 4

functional and structural effects of S18886 β1ELISA, Immmunotech, Marseille, on progressive renal injury in UNX-OZR, France). At sacrifice, urine was collected we focused our interest on parameters of from the bladder for 2,3-dinor- oxidative stress and antioxidant defence. thromboxane B2 (2,3-dinor-TXB2) determination (RIA, Institute of Isotopes, RESEARCH DESIGN AND METHODS Budapest, Hungary), reflecting TXA2 released from both platelets and intrarenal The investigation was conducted according synthesis (30). to the guidelines for studies using Blood samples for biochemistry (Vitros laboratory animals. The study protocol was 250 analyzer, J&J, Rochester, USA) and approved by the Institutional Ethics haematology (Sysmex K-20, TOA Medical Committee for Experimental Animals Electronics, Kobe, Japan) were collected (Bratislava, Slovakia). under anaesthesia from the abdominal Rats: Twenty-four infant male fa/fa OZR aorta into Li-heparin and K2EDTA tubes, and 12 age- and sex-matched lean controls respectively. If not analysed immediately, (LZR) were obtained from Charles River, plasma, whole blood and separated red Sulzfeld, Germany. They were housed 4-6 blood cells were stored at -70oC. Plasma rats/cage, under conditions of controlled levels of glucose, total cholesterol, humidity, room temperature, and a 12 hour triglycerides, creatinine, urea, albumin, light/dark cycle. The rats had free access to total protein, aspartate-aminotransferase drinking water and a standard rat food (AST) activity and the urinary (SP1, Top Dovo, Czech Republic). At the concentrations of urea and creatinine were age of 4 weeks all rats were subjected to analysed. Creatinine and urea clearance UNX of the left kidney under i.p. were calculated. Glutathione peroxidase anaesthesia (ketamin: 75 mg/kg, and (GPX, whole blood) and superoxide xylazin, 10 mg/kg, Spofa, Hlohovec, dismutase activity (SOD, red blood cells) Slovakia). were assessed by commercial kits Experimental protocol: The study was (Randox, Crumlin, Great Britain), designed as an interventional trial on advanced oxidation protein products primary prevention. On the day of surgery (AOPPs) according to Witko-Sarsat et al. the OZR were randomised into 3 groups (31). (n=8 each) and treatment was initiated. Kidney, heart and aorta were removed Two verum groups were administered 10 after retrograde perfusion fixation with or 30 mg/kg/d of S18886 dissolved daily in glutaraldehyde via the abdominal aorta as tap water (OZR-S18886-10 and OZR- previously described (32). S18886-30, respectively). The third OZR Tissue preparation: The kidneys were (OZR-PLAC) and LZR control group weighed and dissected in a plane received placebo (tap water). Oral gavage perpendicular to the interpolar axis, was performed once per day, 5 days a yielding slices of 1 mm width. Ten small week, over 8 weeks. The gavage volume pieces of one kidney were selected by area was adjusted to the body weight (recorded weighted sampling for embedding in weekly). Epon-Araldite. Semithin (1 µm) slices Systolic blood pressure (SBP, tail-cuff were prepared and stained with methylene plethysmography under light ether blue and basic fuchsin. The remaining anaesthesia) was monitored in two-week tissue slices were embedded in paraffin; 4 intervals. Before sacrifice, the rats were µm sections were prepared and stained placed in metabolic cages for stool-free 24 with hematoxilin/eosin (HE) and periodic hour urine collection for determination of Schiff’s acid (PAS). Histomorphological proteinuria (pyrogallol red method) and evaluation was performed by a single transforming growth factor β1 (TGF- observer (T. Eifert) in a blinded manner. 5

scoring system (0-4) as described in detail Morphological investigations (35). Semiquantitative indices of kidney damage, Glomerular geometry: Briefly, glomerular i.e. mesangiolysis, glomerulosclerosis, geometry was analysed as follows: volume tubulointerstitial and vascular damage density (VV) of glomeruli and area density Mesangiolysis, i.e. dissolution of the of glomerular tuft (AAT) were measured by mesangial matrix and necrosis or apoptosis point counting according to PP=AA=VV at a of mesangial cells due to immunological, magnification of 400x on HE sections (33, hemodynamic or metabolic injury, can be 36, 37). Total area of glomerular tuft (AT) regarded as an initial step in glomerular was then determined as AT = AAT x ACortex. damage (33). The mesangiolysis score as The number of glomeruli per volume (NV) an index of mesangiolytic damage was and the volume density (VV) of glomeruli determined in PAS-stained paraffin were obtained using the formula: NV = k/ß 1.5 0.5 sections and graded in 100 systematically x NA / VV with k=1.1 and ß= 1.382. subsampled glomeruli per animal using the The total number of glomeruli was derived following scoring system: score 0: no from the total volume of the renal cortex changes of capillaries, score 1: capillary and the number of glomeruli per cortex dilatation <25% of the capillary convolute, volume: Nglom = NV x VCortex. The mean score 2: capillary dilatation >25% of the glomerular tuft volume was determined 1.5 capillary convolute or capillary aneurysms according to v = ß/k x AT with ß = 1.382 <50% of the capillary convolute, score 3: and k = 1.1 (38). capillary aneurysms comprising 50-75% of Semithin sections were qualitatively the capillary convolute, score 4: capillary inspected for glomerular cellular changes, aneurysms comprising >75% of the i.e. podocyte enlargment and degeneration, capillary convolute (33). and mesangial or endothelial cell Mesangial matrix expansion is an initial hyperplasia. Glomerular capillarisation and event in diabetic glomerulopathy. The cellularity were quantitatively evaluated degree of mesangial matrix expansion was using stereological techniques on 5 determined on PAS-stained paraffin semithin sections per animal. At least 30 sections adapting the semiquantitative glomeruli per animal were investigated scoring system (34). Using light using the point counting method and a 100 microscopy at a magnification of x400 the point eyepiece (Integrationsplatte II; Zeiss glomerulosclerosis score of each animal Co., Jena, Germany) at a magnification of was derived as the mean of 100 glomeruli. 1000x (oil immersion) as previously Severity of mesangial matrix expansion described (32). Cell density of mesangial, was expressed on an arbitrary scale from 0 endothelial cells and podocytes was to 4 with an individual glomeruli score as calculated from cell density per volume follows: grade 0: normal glomerulus, grade (NcV) and cell volume density (VcV) 1: presence of mesangial according to this equation: NcV = k/ß x 1.5 0.5 expansion/thickening of the basement NcA /VcV with ß = 1.4 and k = 1 (32). membrane, grade 2: mild to moderate The number of cells per glomerulus (Nend) segmental hyalinosis/sclerosis involving was determined from cell density (NcV) less than 50% of the glomerular tuft, grade and mean glomerular volume (Vglom): Nend 3: diffuse glomerular hyalinosis/sclerosis = NcV x Vglom. Mean cell volume (mVend) >50% of the tuft, and grade 4: diffuse was calculated from cell volume density glomerulosclerosis with total tuft (VcV), mean glomerular volume (Vglom) obliteration and/or collapse. and number of cells per glomerulus (Nend) Tubulointerstitial and vascular damage was according to the formula: mVend = VcV x assessed on PAS-stained paraffin sections Vglom / Nend. The glomerular tuft volume at a magnification of x100 using a similar was calculated as: fractional capillary tuft 6 volume x mean glomerular volume. The In the OZR-PLAC the plasma urea total capillary volume per glomerulus was concentration and the urinary excretion of calculated as: volume capillary density x urea were increased in comparison to the mean glomerular tuft volume. LZR (p<0.01). In the S18886-30 treated group, the plasma urea concentration was Statistics: Statistical analysis was lower in comparison to OZR-PLAC performed using the SPSS version 8 (p<0.01), while the enhanced urea statistical programme. Unpaired Wilcoxon excretion observed in the OZR-PLAC was test was used to compare the means not significantly influenced by S18886. between the LZR and OZR-PLAC groups. The OZR-PLAC showed a high diuresis ANOVA was used to compare the means (rise by a factor of 4 in comparison to between the OZR groups. If p<0.05 was LZR, p<0.01). Treatment with S18886-30 obtained, post hoc Dunnett’s test induced a mild decrease (-31%, p>0.05). (comparison of verum groups versus OZR- Proteinuria of the OZR-PLAC group was PLAC) was performed, as were correlation 4-fold higher when compared with the and regression analyses. P<0.05 (two- LZR (p<0.01). In both treated groups, a sided) was accepted as significant. Results mild to moderate reduction was observed are given as mean ± SE, p values of (-12% and –37%, both doses respectively, Wilcoxon or Dunnett’s test are indicated. n.s.). The urinary TGF- β1 excretion in the RESULTS OZR-PLAC was 7-fold higher than in the LZR (p<0.01). Treatment with both doses of S18886 was associated with Body weight , heart weight and blood pressure (Table 1): At initiation of the significantly lower values (p<0.05). treatment, mean body weight did not differ Urinary 2,3-dinor-TxB2 excretion in the between the groups (data not given). OZR-PLAC was 3-fold higher than in the Throughout the treatment period the OZR LZR (p<0.01) and tended to be lower in gained more weight than the LZR. Thus, at the treatment groups but reached sacrifice the body weight of the OZR- significance (p<0.05) only under S18886- PLAC was higher when compared with the 30. There was a direct relationship LZR controls (p<0.05). S18886 did not between proteinuria and urinary 2,3-dinor- significantly affect this parameter. At the TxB2 excretion (r=0.56, p<0.01). end of the experiment, mean SBP in the Blood chemistry (Table 2): All OZR were OZR-PLAC group was higher in moderately hyperglycaemic, had elevated comparison with the LZR (p<0.05). In the cholesterol and markedly augmented S18886 groups, mean SBP did not triglyceride levels. These conditions were significantly differ from the OZR-PLAC. not affected by either dosage. Plasma total Mean heart weight did not differ between protein concentrations did not differ the groups (data not shown). significantly between the groups. The concentrations of plasma electrolytes and Kidney weight and renal function (Table 1): Mean kidney weight of the OZR-PLAC minerals as well as the erythrocyte and was higher compared to the LZR (p<0.05). platelet count were within the normal The kidney weight of animals treated with range and did not differ between the S18886-30 was significantly lower in groups (data not shown). The number of comparison to the OZR-PLAC group white blood cells (WBC) and neutrophils (p<0.01). Creatinine clearance was (data not shown) was higher in the OZR- significantly higher in the OZR-PLAC in PLAC than in the LZR (p<0.01), and comparison to the LZR (p<0.01), while neither dosage influenced this data plasma creatinine concentration was significantly. reduced (p<0.01). Treatment resulted in a Oxidative status (Table 2): Plasma AOPP trend to lower creatinine clearance (n.s.). levels were 3-fold higher in the OZR- 7

PLAC in comparison with the LZR of the mesangium were seen (Fig. 2). (p<0.01). S18886-30 resulted in a trend to These changes were not observed in either lower levels of AOPPs (-25%, n.s.). The treatment group. Quantitative analysis of activities of GPX and SOD were glomerular capillaries and cells revealed comparable in the OZR-PLAC and LZR. no difference in mean capillary tuft area After the 8-week treatment, GPX activity and volume, capillary length density, i.e. was significantly higher in both treated total length of all capillaries per glomerular groups (p<0.01), while that of SOD volume, podocyte number and mean reached a significant rise only in the OZR- podocyte volume. Podocyte number was S18886-30 group (p<0.01). highest, however, in the OZR-S18886-30 Kidney morphology (Fig. 1-2, Table 3). group (101±16.9 vs. 75.9±11.1 in OZR- The mesangiolysis score (Tab. 3, Fig. 1, 2) PLAC) indicating preservation of podocyte was significantly higher in the OZR-PLAC structure. In parallel, podocyte volume was than in the LZR (p<0.01). Treatment with highest in the OZR-PLAC and lowest in S18886 completely prevented this the OZR-S18886-10 group. alteration so that the mesangiolysis scores Survival, tolerability and toxicity: in the OZR treatment groups were Treatment with S18886 was well-tolerated. comparable to the LZR. The One OZR of the S18886-10 group died of glomerulosclerosis index showed a trend to asphyxia during the blood pressure higher levels in the OZR-PLAC. The measurement at week 5, most probably due S18886 treatment groups were not to an ether-induced depression of the different from the LZR. Evaluation of the respiratory centre. In the 8-week treatment tubulointerstitial damage score revealed no period no signs of hemato- or significant differences between the groups hepatotoxicity were observed (data not (Tab. 3). Vascular damage score was shown). significantly higher in OZR-PLAC than in LZR and OZR-S18886-10 (Tab. 3). DISCUSSION As indicated in Table 3, mean glomerular volume was significantly higher in the OZR represents one of the most important OZR-PLAC and in the OZR-S18886-10 animal models of nephropathy in type 2 than in LZR. Minimal and maximal diabetes. Since renal complications glomerular diameter and the area of develop rather late (at approximately 40 capillary tuft were significantly higher in weeks), their natural course can be the OZR-PLAC. While the minimal accelerated by UNX (28, 29), as performed glomerular diameter increased in our study. Eight weeks after UNX in the significantly under S18886-10, the OZR-PLAC, renal pathology was maximal glomerular diameter and the area characterised by an increase of kidney of the capillary tuft were not affected weight, glomerular size and mesangiolysis significantly. In the OZR-S18886-30 index, as well as cystic degeneration of group, no significant changes were podocytes. Creatinine clearance and revealed. Mean glomerular cell number diuresis were enhanced, as was the urinary was significantly lower in the OZR- excretion of protein, TGF-β1 and 2,3- S18886-30 than in the LZR. It was dinor-TxB2. S18886 dose-dependently comparable in the other treatment groups. hindered the functional and morphological On semithin sections in the glomeruli of alterations. The prevention of the OZR-PLAC marked cystic mesangiolysis and podocyte degeneration degeneration of podocytes (glomerular represents the most striking, new epithelial cells), slightly irregular observation, associated with an improved capillaries with thickened glomerular antioxidant defence in the blood. basement membrane and mild expansion 8

Renal function dependently. Possibly, the TPr antagonist The increased creatinine clearance in the prevented the amplification of OZR-PLAC, as compared to the LZR, thromboxane generation in platelets. The reflects hyperfiltration, preceding the positive relationship between urinary 2,3- sclerotic changes in the glomeruli (38). dinor-TxB2 excretion and proteinuria Administration of S18886 was associated suggests involvement of the TxA2 with a trend to a decline of creatinine metabolic pathway in the pathogenesis of clearance, similar to the effects of proteinuria. However, administration of inhibitors of the renin angiotensin system, aspirin (which lowers thromboxane indicating a beneficial response. As a synthesis) did not protect the diabetic consequence of hyperphagia, the plasma apoE-/- mice (27) or rats with other renal concentration of urea and its urinary diseases from progression. This suggests excretion rate were increased in the OZR- that the enhanced thromboxane formation PLAC, compared to the LZR, as was the may be less significant than the other body weight. S18886-30 lowered plasma ligands to the TPr (26). urea concentration, associated with a decrease (n.s.) of its urinary excretion rate. Oxidative stress Proteinuria was increased 4-fold in the An increased production of reactive OZR-PLAC. Both doses of S18886 oxygen species is assumed to play a central partially lowered proteinuria. Besides the role in the development of diabetic potential impact of altered intraglomerular nephropathy (41). Correspondingly, an hemodynamics, due to the mild (but enhanced expression of the NAD(P)H insignificant) decline in SBP, an improved oxidase subunit p47phox and an podocyte function of by TPr antagonism accumulation of nitrotyrosine (a marker of could be involved. oxidative/nitrosative stress) and 12- Administration of S18886-30 resulted in a lipoxygenase in the renal tissue, as well as moderate decline of the polyuria, an enhanced urinary excretion of HETE implicating an improved urinary and isoprostane F2α have been observed concentration mechanism and/or a (26, 41, 42). In the diabetic apoE-/- mice decreased fluid intake. In fact, TPr in the administration of S18886 attenuated all the hypothalamus have been shown to mediate above mentioned markers of oxidative the dipsogenic response to angiotensin II stress. The causal role of (39), which may be lessened by S18886 oxidative/nitrosative stress in renal injury treatment. is supported by the protective effect of an Urinary excretion of TGF-ß1 was markedly overexpression of SOD against early increased in the OZR-PLAC. This rise was diabetic glomerular lesions in transgenic prevented by the TPr antagonist, mice (43). corresponding to the reduction of In our study in the OZR-PLAC, circulating intrarenal accumulation of this cytokine by AOPP levels were increased 3-fold as S18886 in diabetic apoE-/- mice (26). compared to the LZR, highlighting the role Similar to studies in STZ diabetic rats (15) of an enhanced oxygen radical formation and in the Otsuka Long-Evans Tokushima in this model. A similar rise was observed Fatty strain (18), the urinary 2,3-dinor- in human diabetic and non-diabetic renal TxB2 excretion was significantly enhanced diseases (30). Since AOPPs are partly in the OZR-PLAC, indicating augmented derived from the myeloperoxidase production of TxA2. This could be reaction, their high levels point to an favoured by both enhanced glucose levels activation of phagocytic cells. Indeed, in (40) and stimulation of the angiotensin II our study, a rise in WBC count was type 1 receptor (12). S18886 lowered the observed. In human diabetics, an elevated urinary 2,3-dinor-TxB2 excretion dose- WBC count, even within the normal range, 9 is associated with the risk of complications glomerular injury. To our knowledge, this (44). Interestingly, with S18886-30, the is the first observation that a TPr elevated plasma AOPP levels were lower antagonist prevents this early feature of (-25%, n.s.). The lack of significance is diabetic nephropathy, a possible forerunner most likely due to a high interindividual of the ensuing nodular glomerulosclerosis variability. The activities of the antioxidant (49). In addition, S18886 prevented enzymes SOD and GPX did not differ degeneration of podocytes and at least between the LZR and OZR-PLAC. partly also hypertrophy and loss of However, S18886 dose-dependently podocytes, i.e. podocytopenia. The latter increased both antioxidants in the blood. may explain at least in part the mild This finding corresponds to the antiproteinuric effect of S18886. However, nephroprotective action of the rise of the early signs of glomerulosclerosis (i.e. lowered renal SOD after S18886 in the an increase of mesangial matrix with diabetic apoE-/- mice (26) as well as an capillary obliteration) were not overexpression of SOD in the db/db mice significantly altered, possibly due to the (43). short duration of the experiment. Also the glomerular volume was not reduced by the Renal morphology TPr antagonist, since thromboxane Interpretation of changes in experimental mimetics lower glomerular volume (3). It diabetic nephropathy is always limited by is conceivable that in this response a the lack of an adequate animal model relaxation of contracted mesangial cells which develops human-like lesions (45). was involved. The OZR rat is a widely used animal In summary, our data show that TPr model for diabetic nephropathy (29) antagonism with S18886 retards the although the glomerular changes in this progression of nephropathy in UNX-OZR. animal model differ in certain aspects from This is reflected by improved renal biopsy findings in human diabetic histomorphology with a prevention of nephropathy. In our OZR-PLAC renal mesangiolysis and podocyte degeneration, histology was characterized by a trend to reduced proteinuria, and a lower enlargement of the glomeruli with a urinary excretion of TGF-β1 and 2,3-dinor- significant increase of mesangiolysis (33), TxB2. In blood, the activity of antioxidant which is associated with a loss of enzymes was increased, while the mesangial cells, capillary dilatation and circulating AOPP levels were lower. All finally, formation of capillary aneurysms these data suggest the involvement of (46), as typically seen in inflammatory activated TPr in the pathogenesis of the glomerular diseases (33). In diabetic nephropathy in OZR. They further imply nephropathy it is postulated that the potential benefits of long-term TPr nodular glomerulosclerosis develops out of antagonism with S18886 in the clinical the mesangiolytic damage, in particular setting of type 2 diabetes. from repair of capillary micro-aneurysms (46-49). Besides an augmented glomerular pressure (due to hyperglycemia and UNX), ACKNOWLEDGEMENT an enhanced formation of TxA2 could be involved in the glomerular injury, since in The authors would like to thank Dr. S. experimental settings mesangiolysis was Corda and Dr. L. Lerond, Servier, Paris, associated with a rise in TxB2 secretion for their stimulating and constructive from mesangial cells (50). In our study support in drafting the manuscript. The blockade of TPr with S18886 completely skilful technical assistance of M. Klewer, prevented mesangiolysis, underlining its M. Ramming and S. Söllner is gratefully potential pathogenetic role in this acknowledged. This study was supported 10 in part by the Institut de Recherches Z2). Preliminary data of this work were Internationales Servier, Courbevoie, presented as poster at the World Congress France and by the DFG (SFB 423, project of Nephrology, Berlin, 2003. 11

REFERENCES

1. Baylis C: Effects of administered thromboxane on the intact, normal rat kidney. Renal Physiol 10:110-121, 1987 2. Wilcox CS, Welch WJ: Thromboxane synthase and TP receptor mRNA in rat kidney and brain: effects of salt intake and ANG II. Am J Physiol Renal Physiol 284:F525- F531, 2003 3. Scharschmidt LA, Lianos E, Dunn MJ: Arachidonate metabolites and the control of glomerular function. Fed Proc 42:3058-3063, 1983 4. Remuzzi G, Fitzgerald GA, Patrono C: Thromboxane synthesis and action within the kidney. Kidney Int 41:1483-1493, 1992 5. Datta PK, Lianos EA: Role of eicosanoids in glomerular, tubular, and vascular injury. In: Massry and Glassock’s Textbook of Nephrology. 4th ed. Massry SG, Glassock RJ Eds. Philadelphia, Lippincott Williams & Wilkins, 2001, p. 611-618 6. Menè P, Dubyak G, Abboud H, Scarpa A, Dunn MJ: Phospholipase C activation by prostaglandins and thromboxane A2 in cultured mesangial cells. Am J Physiol 255:F1058-F1069, 1988 7. Takahashi N, Takeuchi K, Abe T, Sugawara A, Abe K: Immunolocalization of rat thromboxane receptor in the kidney. Endocrinology 137:5170-5173, 1996 8. Ishizuka T, Kawakami M, Hidaka T, Matsuki Y, Takamizawa M, Suzuki K, Kurita A, Nakamura H: Stimulation with thromboxane A2 (TXA2) receptor agonist enhances ICAM-1, VCAM-1 or ELAM-1 expression by human vascular endothelial cells. Clin Exp Immunol 112:464-470, 1998 9. Audoly L, Rocca B, Fabre JE, Koller BH, Thomas D, Loeb AL, Coffman TM, FitzGerald GA. Cardiovascular responses to the isoprostanes i-PF2α-III and i-PE2-III are mediated via the thromboxane A2 receptor in vivo. Circulation 101:2833-2840, 2000 10. Kawada N, Dennehy K, Solis G, Modlinger P, Hamel R, Kawada JT, Aslam S, Moriyama T, Imai E, Welch WJ, Wilcox CS: TP receptors regulate renal hemodynamics during angiotensin II slow pressor response. Am J Physiol Renal Physiol 287:F753-F759, 2004 11. Brown GP, Venuto RC: Thromboxane receptors in human kidney tissues. Prostaglandins Other Lipid Mediat 57:179-188, 1999 12. Awad AS, Webb RL, Carey RM, Siragy HM: Increased renal production of angiotensin II and thromboxane B2 in conscious diabetic rats. Am J Hypertens 18:544- 548, 2005 13. Niwa T, Maeda K, Shibata M, Yamada K: Clinical effects of selective thromboxane A2 synthase inhibitor in patients with nephrotic syndrome. Clin Nephrol 30:276-281, 1988 14. Craven PA, Melhem MF, De Rubertis FR: Thromboxane in the pathogenesis of glomerular injury in diabetes. Kidney Int 42:937-946, 1992 15. Ogletree M, Harris D, Schumacher W, Webb ML, Misra RN: Pharmacological profile of BMS 180,291: a potent, long-acting, orally active thromboxane A2/prostaglandin endoperoxide receptor antagonist. J Pharmacol Exp Ther 264:570-578, 1992 16. Matsuo Y, Takagawa I, Koshida H, Kawabata T, Nakamura M, Ida T, Zhou L, Marumo, F: Antiproteinuric effect of a thromboxane receptor antagonist, S-1452, on diabetic nephropathy and murine lupus nephritis. Pharmacology 50:1-8, 1995 17. Giustina A, Perini P, Desenzani P, Bossoni S, Ianniello P, Milani M, Davi G, Romanelli G: Long-term treatment with the dual antithromboxane agent picotamide 12

decreases microalbuminuria in normotensive type 2 diabetic patients. Diabetes 47:423-430, 1998 18. Okumura M, Imanishi M, Okamura M, Hosoi M, Okada N, Konishi Y, Morikawa T, Miura K, Nakatani T, Fujii S: Role for thromboxane A2 from glomerular thrombi in nephropathy with type 2 diabetic rats. Life Sci 72:2695-2705, 2003 19. Fitzgerald DJ, Fragetta J, Fitzgerald GA: Prostaglandin endoperoxides modulate the response to thromboxane synthase inhibition during coronary thrombosis. J Clin Invest 82:1708-1713, 1988 20. Simonet S, Descombes JJ, Vallez MO, Dubuffet T, Lavielle G, Verbeuren TJ: S18886, a new thromboxane (TP)-receptor antagonist is the active isomer of S18204 in all species, except in the guinea-pig. Adv Exp Med Biol 433:173-176, 1996 21. Cimetière B, Dubuffet T, Landras C, Descombes JJ, Simonet S, Verbeuren TJ, Lavielle G: New tetrahydronaphthalene derivatives as combined thromboxane receptor antagonists and thromboxane synthase inhibitors. Bioorg Med Chem Lett 8:1381-1386, 1998 22. Viles-Gonzalez J, Fuster V, Corti R, Valdiviezo C, Hutter R, Corda S, Anand SX, Badimon JJ: Atherosclerosis regression and TP receptor inhibition: effect of S18886 on plaque size and composition – a magnetic resonance imaging study. Eur Heart J 26:1557-1561, 2005 23. Worth N, Berry C, Thomas A, Campbell JH: S18886, a selective TP receptor antagonist, inhibits development of atherosclerosis in rabbits. Atherosclerosis 183:65- 73, 2005 24. Cayatte AJ, Du Y, Oliver-Krasinski J, Lavielle G, Verbeuren TJ, Cohen RA: The thromboxane receptor antagonist S18886 but not aspirin inhibits atherogenesis in apoE-deficient mice: evidence that eicosanoids other than thromboxane contribute to atherosclerosis. Arterioscler Thromb Vasc Biol 20:1724-1728, 2000 25. Zuccollo A, Shi C, Mastroianni R, Maitland-Toolan KA, Weisbrod RM, Zang M, Xu S, Jiang B, Oliver-Krasinski JM, Cayatte AJ, Corda S, Lavielle G, Verbeuren TJ, Cohen RA: The thromboxane A2 receptor antagonist, S18886, prevents enhanced atherogenesis caused by diabetes mellitus. Circulation 112:3001-3008, 2005 26. Xu S, Jiang B, Maitland KA, Bayat H, Gu J, Nadler JL, Corda S, Lavielle G,Verbeuren TJ, Zuccollo A, Cohen RA: The thromboxane receptor antagonist S18886 attenuates renal oxidant stress and proteinuria in diabetic apolipoprotein E- deficient mice. Diabetes 55:110-119, 2006 27. Kasiske BL, O’Donnell MP, Keane WF: The Zucker rat model of obesity, insulin resistance, hyperlipidemia and renal injury. Hypertension 19 (Suppl 1):I110- I115, 1992 28. Van Zwieten PA, Kam KL, Pijl AJ, Hendriks MG, Beenen OH, Pfaffendorf M: Hypertensive diabetic rats in pharmacological studies. Pharmacol Res 33:95-105, 1996 29. Coimbra TM, Janssen U, Gröne HJ, T, Kunter U, Schmidt H, Brabant G, Floege J: Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Kidney Int 57:167-182, 2000 30. Witko-Sarsat V, Friedlander M, Nguyen Khoa T, Capeillere-Blandin C, Nguyen AT, Canteloup S, Dayer JM, Jungers P, Drueke T, Descamps-Latscha B: Advanced oxidation protein products as novel mediators of inflammation and monocyte activation in chronic renal failure. J Immunol 161:2524-2532, 1998 31. Benigni A, Chiabrando C, Perico N, Fanelli R, Patrono C, FitzGerald GA, Remuzzi G: Renal metabolism and urinary excretion of thromboxane B2 in the rat. Am J Physiol 257:F77-F85, 1989 13

32. Schwarz U, Amann K, Orth SR, Simonaviciene A, Wessels S, Ritz E: Effect of 1,25 (OH) 2 vitamin D3 on glomerulosclerosis in subtotally nephrectomized rats. Kidney Int 53:1696-1705, 1998 33. Dimmler A, Haas CS, Cho S, Hattler M, Forster C, Peters H, Schocklmann HO, Amann K: Laser capture microdissection and real-time PCR for analysis of glomerular endothelin-1 gene expression in mesangiolysis of rat and anti-Thy 1.1 and murine Habu Snake Venom glomerulonephritis. Diagn Mol Pathol 12:108-117, 2003 34. El Nahas AM, Bassett AH, Cope GH, Le Carpentier JE: Role of growth hormone in the development of experimental renal scarring. Kidney Int 40:29-34, 1991 35. Veniant M, Heudes D, Clozel JP, Bruneval P, Menard J: Calcium blockade versus ACE inhibition in clipped and unclipped kidneys of 2K-1C rats. Kidney Int 46:421- 429, 1994 36. Weibel ER: Measuring through the microscope: development and evolution of stereological methods. J Microsc 155:393-403, 1989 37. Gross ML, Ritz E, Schoof A, Helmke B, Parkman A, Tulp O, Munter K, Amann K: Renal damage in the SHR/N-cp type 2 diabetes model: comparison of an angiotensin- converting enzyme inhibitor and endothelin receptor blocker. Lab Invest 83:1267- 1277, 2003 38. Hostetter TH, Troy JL, Brenner BM: Glomerular hemodynamics in experimental diabetes mellitus. Kidney Int 19:410-415, 1981 39. Kitiyakara C, Welch WJ, Verbalis JG, Wilcox CS: Role of thromboxane in the dipsogenic response to central angiotensin II. Am J Physiol Regul Integr Comp Physiol 282:R865-R869, 2002 40. De Rubertis FR, Craven PA: Eicosanoids in the pathogenesis of the functional and structural alterations of the kidney in diabetes. Am K Kidney Dis 22:727-735, 1993 41. Thuraisingham RC, Nott CA, Dodd SM, Yaqoob MM: Increased nitrotyrosine staining in kidneys from patients with diabetic nephropathy. Kidney Int 57:1968-1972, 2000 42. Onozato ML, Tojo A, Goto A, Fujita T, Wilcox CS: Oxidative stress and nitric oxide synthase in rat diabetic nephropathy: effects of ACEI and ARB. Kidney Int 61:186- 194, 2002 43. De Rubertis FR, Craven PA, Melhem MF, Salah EM: Attenuation of renal injury in db/db mice overexpressing superoxide dismutase: evidence for reduced superoxide- nitric oxide interaction. Diabetes 53:762-768, 2004 44. Tong PC, Lee KF, So WY, Ng MH, Chan WB, Lo MK, Chan NN, Chan JC: White blood cell count is associated with macro- and microvascular complications in Chinese patients with type 2 diabetes. Diabetes Care 27:216-222, 2004 45. Breyer MD, Bottinger E, Brosius FC, Coffman TM, Fogo A, Harris RC, Heilig CW, Sharma K. Diabetic nephropathy: of mice and men. Adv Chronic Kidney Dis 12:128- 145, 2005 46. Morita T, Yamamoto T, Churg J: Mesangiolysis: An update. Am J Kidney Dis 31:4559-4573, 1998 47. Morita T, Churg J. Mesangiolysis. Kidney Int 24:1-9, 1983 48. Saito Y, Kida H, Takeda S, Yoshimura M, Yokoyama H, Koshino Y, Hattori N. Mesangiolysis in diabetic glomeruli: its role in the formation of nodular lesions. Kidney Int 34:389-396, 1988 49. Stout LC, Kumar S, Whorton EB. Focal mesangiolysis and the pathogenesis of the Kimmelstiel-Wilson nodule. Hum Pathol 24:77-89, 1993 14

50. Radeke HH, Kuster S, Kaever V, Resch K: Effects of cyclosporin and FK-506 on glomerular mesangial cells. Evidence for direct inhibition of thromboxane synthase by low cyclosporin concentrations. Eur J Clin Pharmacol 44 (Suppl 1):S11-S16, 1993 15

Table 1. Body and organ weight, blood pressure and renal function of unilaterally nephrectomized (UNX) obese Zucker (OZR) and lean control (LZR) rats after 8 weeks treatment

UNX-LZR UNX-OZR PLAC S18886-10 S18886-30 (n=12) (n=8) (n=7) (n=8) * Body wt t8 (g) 338±5 448±13 427±12 429±17 Body wt gain (g) 167±4 282±17† 267±15 274±15 Kidney wt (g) 2.15±0.07 2.43±0.09* 2.36±0.12 2.01±0.07§ SBP (mm Hg) 91.7±1.7 116.9±5.0* 107.9±2.9 107.1±2.4 P-Crea (µmol/l) 52.5±1.0 44.6±1.4† 41.2±1.1 44.7±1.3 † ClCrea (ml/min) 0.73±0.06 1.14±0.05 1.03±0.06 0.99±0.08 P-Urea (mmol/l) 8.0±0.4 13.4±0.5† 11.0±1.3 9.0±0.4§ U-Urea (mmol/24 h) 4.8±0.4 15.2±1.2† 12.1±0.7 11.8±1.4 Proteinuria (mg/24 h) 6.0±0.4 25.7±1.8† 22.6±7.7 16.2±4.0 Diuresis (ml/24h) 9.0±1.1 41.9±3.2† 36.7±6.3 29.2±2.8 † # # U-TGF-β1 (nmol/24h) 100±20 694±207 195±60 183±45 † # U-TXB2/crea (ng/µmol) 0.55±0.06 1.46±0.27 0.82±0.05 0.71±0.04

PLAC: placebo; wt: weight; t8: 8 weeks after treatment; SBP: systolic blood pressure; P-Crea: plasma creatinine; Clcrea: creatinine clearance; P-Urea: plasma urea, U-Urea: urinary excretion of urea; U-TGF-β1: urinary excretion of transforming growth factor β1; U-TxB2: urinary excretion of thromboxane B2; *: p<0.05 vs. LZR; †: p<0.01 vs. LZR (Wilcoxon test); #: p<0.05 vs. OZR-PLAC; §: p<0.01 vs. OZR-PLAC (Dunnett’s test). Table 2. Plasma total protein, glucose and lipid concentration, and data characterizing oxidative status in unilaterally nephrectomized (UNX) obese Zucker (OZR) and lean control (LZR) rats after 8 weeks treatment

UNX-LZR UNX-OZR PLAC S18886-10 S18886-30 (n=12) (n=8) (n=7) (n=8) Glucose (mmol/l) 15.7±1.7 25.2±1.3a* 23.3±1.3 26.8±1.5 Cholesterol (mmol/l) 2.30±0.12 3.36±0.12* 3.72±0.16 3.58±0.31 Triglycerides (mmol/l) 0.98±0.09 3.98±0.41* 3.92±0.69 3.50±0.59 Total protein (g/l) 62.1±1.0 59.1±1.2 62.4±0.8 62.6±1.6 WBC (x.103.µl-1) 4.61±0.43 6.01±0.35* 6.27±1.04 6.28±1.91 AOPPs (µmol/l) 57±5 171±23* 179±37 127±14 SOD (U/g Hb) 2299±91 2419±88 2757±157 2843±122† GPX (U/g Hb) 1145±135 1008±42 1486±84† 1568±119†

PLAC: placebo; WBC: white blood cell count; AOPPs: advanced oxidation protein products in plasma; SOD: superoxide dismutase activity; GPX: glutathione peroxidase activity; *: p<0.01 vs. LZR (Wilcoxon test); †: p<0.01 vs. OZR-PLAC (Dunnett’s test). 16

Table. 3: Parameters of glomerular geometry in unilaterally nephrectomized (UNX) obese Zucker (OZR) and lean control (LZR) rats after 8 weeks treatment

UNX-LZR UNX-OZR PLAC S18886-10 S18886-30 (n=12) (n=8) (n=7) (n=8) Mesangiolysis score 0.30±0.028 0.46±0.045† 0.31±0.035# 0.29±0.017§

Glomerulosclerosis score 0.25±0.028 0.32±0.044 0.25±0.025 0.22±0.026

Tubulointerstitial damage score 0.74±0.17 0.85±0.36 0.58±0.19 0.58±0.17

Vascular damage score 0.05±0.03 0.23±0.14* 0.03±0.03# 0.16±0.23

Glomerular cell number 61.0±1.96 56.8±2.27 59.8±2.99 55.0±1.11*

Maximal glomerular diameter 134±1.7 149±2.1† 157±3.9†,# 152±3.0 † (µm) Minimal glomerular diameter 108±1.1 121±0.8† 129±2.5# 124±3.1† (µm) Mean glomerular tuft area 10335±227 12152±235† 12784±431 11692±410+ (µm²) Mean glomerular tuft volume 1321±43 1682±49† 1818±94 1590±83≠ (µm³)

*: p<0.05 vs. LZR; †: p<0.01 vs. LZR (Wilcoxon test); #: p<0.05 vs. OZR-PLAC; §: p<0.01 vs. OZR-PLAC; +: p<0.05 vs. OZR-S18886-10; ≠: p<0.01 vs. OZR-S18886-10(Dunnett’s test) 17

FIGURE LEGENDS

Fig. 1: Representative changes in glomerular morphology.

Paraffin sections. A: Glomerulus of a lean Zucker rat; B, D, F: Glomeruli from placebo treated obese Zucker rats showing irregular structures of the capillary tuft with capillary widening, capillary dilatation and dissolution of the mesangium (mesangiolysis score 1-2), C, E: Glomeruli of TP receptor antagonist S18886 treated obese Zucker rats with nearly normal capillary structure and mesangium (C: low dose, and E: high dose treatment).

Fig. 2: Changes in glomerular cellularity and capillarisation

Semithin sections. A: Glomerulus of an untreated lean Zucker control rat; B, D, F: Glomeruli from placebo treated obese Zucker rats showing markedly enlarged podocytes with cystic degeneration, slightly irregular capillaries and mild mesangial cell hyperplasia; C, E: Glomeruli of TP receptor antagonist S18886 treated obese Zucker rats with nearly normal cellular and capillary structure (C: low dose, and E: high dose treatment). Original magnification 1:40.