Diabetes
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published online January 31, 2007
Diabetes 56:968-974, 2007
DOI: 10.2337/db06-1136
© 2007 by the American Diabetes Association
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
db06-1136v1
56/4/968    most recent
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sebeková, K.
Right arrow Articles by Amann, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sebeková, K.
Right arrow Articles by Amann, K.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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

Katarína Sebeková1, Timo Eifert2, André Klassen3, August Heidland3, and Kerstin Amann2

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

Address correspondence and reprint requests to Katarína Sebeková MD, DSc, Slovak Medical University, Department of Clinical and Experimental Pharmacotherapy, Limbová 12, 833 03 Bratislava, Slovakia. E-mail: katarina.sebekova{at}szu.sk

Abbreviations: AOPP, advanced oxidation protein product; apoE, apolipoprotein E; AST, aspartate-aminotransferase; AT II, angiotensin II; GN, glomerulonephritis; GPX, glutathione peroxidase; GSI, glomerulosclerosis index; HE, hematoxylin/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-ß1, transforming growth factor ß1; TP, thromboxane-prostanoid endoperoxides; TxA2, thromboxane A2; TxB2, thromboxane B2; UNX, uninephrectomy; WBC, white blood cell


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thromboxane A2 (TxA2) is assumed to contribute to the development of diabetes 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–1 · day–1, was administered to UNX-OZR by gavage over 8 weeks (n = 8 each group). UNX lean rats (n = 12) and OZR rats that received placebo (OZR-PLAC, n = 8) served as controls. As compared with 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; NS). 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%, NS). 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 defense.

Thromboxane A2 (TxA2) induces platelet activation and is a powerful vasoconstrictor. It decreases renal blood flow, lowers single-nephron glomerular filtration rate, and potentiates the tubuloglomerular feedback (1,2). In vitro, TxA2 contracts isolated glomeruli and mesangial cells (3) and enhances proliferation of glomerular cells and the synthesis of extracellular matrix. It has chemoattractive properties, stimulates formation of adhesion molecules, and exerts immunological actions (4). Major cellular sources of TxA2 are platelets and—in disease—leukocytes, macrophages, vascular smooth muscle endothelial cells, mesangial cells, and immune cells (5). In vivo TxA2 is rapidly metabolized to the stable, inactive derivative 2,3-dinor-thromboxane B2 (TxB2).

The biological effects of TxA2 are mediated by a G protein–coupled thromboxane prostanoid receptor (TxA2-PGH2) that is activated by phospholipase C and an immediate rise of intracellular calcium (6). TxA2-PGH2 receptors (TPr) are expressed in blood vessels and renal microvessels, glomeruli, mesangial cells, thick ascending limbs, and collecting ducts (7). The receptor is activated not only by TxA2 but also by PGH2, 8-isoprostanes, hydroxyeicosatatraenoic acid (HETE), and products of oxygen formation (8–10). TPr play a decisive role in the hormonal effects of angiotensin II (AT II), endothelin, and arginine vasopressin. After deletion/antagonism of the TPr in mice, the AT II–induced intrarenal vasoconstriction and the enhanced oxygen free radical formation are abolished (10). Other factors activating TPr are interleukin-1 and -2, immune attack complexes, terminal complement components (C5b-C9), and endotoxins (4).

Thromboxane and other prostanoids have been implicated in the proinflammatory and oxidative events in cardiovascular diseases, as well as in experimental and human renal diseases. TxA2 formation was evaluated by TxB2 content in urine, isolated glomeruli, and tubulo-interstitial fluid, as well as by immune histochemistry. Enhanced levels have been observed in diabetic nephropathy, remnant kidney, immune-mediated renal diseases (anti–glomular basement membrane nephritis, active lupus nephritis, and renal allograft rejection), endotoxin-induced acute renal failure, and cyclosporine toxicity (4,5,11,12). Inhibition of thromboxane synthesis or blockade of the TPr ameliorated the functional/structural alterations in early stages of the aforementioned diseases (13–18). However, treatment with thromboxane synthase inhibitors may be followed by a rise of PGH2 in platelets, favoring a pro-aggregatory activity (19).

S18886, a polysubstituted tetrahydronaphthalene derivative, is a new and highly selective TPr antagonist with a long duration of action (20,21). It exerts potent antiplatelet and antivasoconstrictory effects via the TPr and antagonizes the actions of TxA2, as well as of other arachidonic acid products (prostaglandin PGH2, PGF2{alpha}, HETE acids, and isoprostanes) (9). Recently, it has been shown that S18886 inhibited the development of atherosclerosis in two rabbit models (22,23) and in apolipoprotein E–deficient (apoE–/–) mice, with or without streptozotocin (STZ)-induced diabetes (24,25), and acted renoprotectively in the rat STZ model of diabetes (26). Because type 2 diabetes is the main cause of end-stage renal failure in humans, we were interested in the effects of S18886 in a rat model of type 2 diabetes. The obese Zucker rat (OZR) model shares many characteristics of type 2 diabetes in humans, such as obesity, insulin resistance, moderate hyperglycemia, hyperlipidemia, and hypertension (27), and is also used for studies in diabetic nephropathy. To accelerate the renal injury, we performed a uninephrectomy (UNX), as has been previously suggested (28,29). Besides the functional and structural effects of S18886 on progressive renal injury in UNX-OZR, we focused our interest on parameters of oxidative stress and antioxidant defense.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The investigation was conducted according to the guidelines for studies using laboratory animals. The study protocol was approved by the Institutional Ethics Committee for Experimental Animals (Bratislava, Slovakia).

Rats.
Twenty-four infant male fa/fa OZR and 12 age- and sex-matched lean controls (LZR) were obtained from Charles River, Sulzfeld, Germany. They were housed four to six rats per cage, under conditions of controlled humidity, room temperature, and a 12-h light/dark cycle. The rats had free access to drinking water and a standard rat food (SP1; Top Dovo, Czech Republic). At the age of 4 weeks, all rats were subjected to UNX of the left kidney under intraperitoneal anesthesia (ketamin 75 mg/kg and xylazin 10 mg/kg; Spofa, Hlohovec, Slovakia).

Experimental protocol.
The study was designed as an interventional trial on primary prevention. On the day of surgery, the OZR were randomized into three groups (n = 8 each) and treatment was initiated. Two verum groups were administered 10 or 30 mg · kg–1 · day–1 of S18886 dissolved daily in tap water (OZR-S18886-10 and OZR-S18886-30, respectively). The third OZR (OZR-PLAC) and LZR control group received placebo (tap water). Oral gavage was performed once per day, 5 days a week, over 8 weeks. The gavage volume was adjusted to the body weight (recorded weekly).

Systolic blood pressure (SBP, tail-cuff plethysmography under light ether anesthesia) was monitored in 2-week intervals. Before sacrifice, the rats were placed in metabolic cages for stool-free 24-h urine collection for determination of proteinuria (pyrogallol red method) and transforming growth factor ß1 (TGF-ß1ELISA; Immmunotech, Marseille, France). Upon sacrifice, urine was collected from the bladder for 2,3-dinor-thromboxane B2 (2,3-dinor-TXB2) determination (RIA; Institute of Isotopes, Budapest, Hungary), reflecting TXA2 released from both platelets and intrarenal synthesis (30).

Blood samples for biochemistry (Vitros 250 analyzer; J&J, Rochester, NY) and hematology (Sysmex K-20; TOA Medical Electronics, Kobe, Japan) were collected under anesthesia from the abdominal aorta into Li-heparin and K2EDTA tubes, respectively. If not analyzed immediately, plasma, whole blood, and separated erythrocytes were stored at –70°C. Plasma levels of glucose, total cholesterol, triglycerides, creatinine, urea, albumin, total protein, aspartate-aminotransferase (AST) activity, and the urinary concentrations of urea and creatinine were analyzed. Creatinine and urea clearance were calculated. Glutathione peroxidase (GPX, whole blood) and superoxide dismutase activity (SOD, erythrocytes) were assessed by commercial kits (Randox, Crumlin, U.K.), and advanced oxidation protein products (AOPPs) were assessed according to the method of Witko-Sarsat et al. (31).

Kidney, heart, and aorta were removed after retrograde perfusion fixation with glutaraldehyde via the abdominal aorta as previously described (32).

Tissue preparation.
The kidneys were weighed and dissected in a plane perpendicular to the interpolar axis, yielding slices of 1-mm width. Ten small pieces of one kidney were selected by area weighted sampling for embedding in Epon-Araldite. Semithin (1 µm) slices were prepared and stained with methylene blue and basic fuchsin. The remaining tissue slices were embedded in paraffin; 4-µm sections were prepared and stained with hematoxylin/eosin (HE) and periodic Schiff's acid (PAS). Histomorphologic evaluation was performed by a single observer (T.E.) in a blinded manner.

Morphologic investigations
Semiquantitative indexes of kidney damage, i.e., mesangiolysis, glomerulosclerosis, tubulointerstitial and vascular damage.
Mesangiolysis, i.e., dissolution of the mesangial matrix and necrosis or apoptosis of mesangial cells due to immunologic, hemodynamic, or metabolic injury, can be regarded as an initial step in glomerular damage (33). The mesangiolysis score as an index of mesangiolytic damage was determined in PAS-stained paraffin sections and graded in 100 systematically subsampled glomeruli per animal using the following scoring system: score 0, no changes of capillaries; score 1, capillary dilatation <25% of the capillary convolute; score 2, capillary dilatation >25% of the capillary convolute or capillary aneurysms <50% of the capillary convolute; score 3, capillary aneurysms comprising 50–75% of the capillary convolute; and score 4, capillary aneurysms comprising >75% of the capillary convolute (33).

Mesangial matrix expansion is an initial event in diabetic glomerulopathy. The degree of mesangial matrix expansion was determined on PAS-stained paraffin sections adapting the semiquantitative scoring system (34). Using light microscopy at a magnification of x400, the glomerulosclerosis score of each animal was derived as the mean of 100 glomeruli. Severity of mesangial matrix expansion was expressed on an arbitrary scale from 0 to 4 with an individual glomeruli score as follows: grade 0, normal glomerulus; grade 1, presence of mesangial expansion/thickening of the basement membrane; grade 2, mild-to-moderate segmental hyalinosis/sclerosis involving less than 50% of the glomerular tuft; grade 3, diffuse glomerular hyalinosis/sclerosis >50% of the tuft; and grade 4, diffuse glomerulosclerosis with total tuft obliteration and/or collapse.

Tubulointerstitial and vascular damage was assessed on PAS-stained paraffin sections at a magnification of x100 using a similar scoring system (0–4) as described in detail (35).

Glomerular geometry.
Briefly, glomerular geometry was analyzed as follows: volume density (VV) of glomeruli and area density of glomerular tuft (AAT) were measured by point counting according to PP = AA = VV at a magnification of 400x on HE sections (33,36,37). Total area of glomerular tuft (AT) was then determined as AT = AAT x ACortex. The number of glomeruli per volume (NV) and the volume density (VV) of glomeruli were obtained using the formula: NV = k/ß x NA1.5/VV0.5 with k = 1.1 and ß = 1.382. The total number of glomeruli was derived from the total volume of the renal cortex and the number of glomeruli per cortex volume: Nglom = NV x VCortex. The mean glomerular tuft volume was determined according to v = ß/k x AT1.5 with ß = 1.382 and k = 1.1 (38).

Semithin sections were qualitatively inspected for glomerular cellular changes, i.e., podocyte enlargment and degeneration, and mesangial or endothelial cell hyperplasia. Glomerular capillarization and cellularity were quantitatively evaluated using stereological techniques on five semithin sections per animal. At least 30 glomeruli per animal were investigated using the point counting method and a 100-point eyepiece (Integrationsplatte II; Zeiss, Jena, Germany) at a magnification of 1,000x (oil immersion) as previously described (32). Cell density of mesangial cells, endothelial cells, and podocytes was calculated from cell density per volume (NcV) and cell volume density (VcV) according to this equation: NcV = k/ß x NcA1.5/VcV0.5 with ß = 1.4 and k = 1 (32). The number of cells per glomerulus (Nend) was determined from cell density (NcV) and mean glomerular volume (Vglom): Nend = NcV x Vglom. Mean cell volume (mVend) was calculated from cell volume density (VcV), mean glomerular volume (Vglom), and number of cells per glomerulus (Nend) according to the formula: mVend = VcV x Vglom/Nend. The glomerular tuft volume was calculated as fractional capillary tuft volume x mean glomerular volume. The total capillary volume per glomerulus was calculated as volume capillary density x mean glomerular tuft volume.

Statistics.
Statistical analysis was performed using the SPSS version 8 statistical program. Unpaired Wilcoxon's test was used to compare the means between the LZR and OZR-PLAC groups. ANOVA was used to compare the means between the OZR groups. If P < 0.05 was obtained, post hoc Dunnett's test (comparison of verum groups versus OZR-PLAC) was performed, as were correlation and regression analyses. P < 0.05 (two-sided) was accepted as significant. Results are given as means ± SE; P values of Wilcoxon or Dunnett's test are indicated.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body weight, heart weight, and blood pressure.
At initiation of the treatment, mean body weight did not differ between the groups (data not given). Throughout the treatment period the OZR gained more weight than the LZR (Table 1). Thus, at time of death, the body weight of the OZR-PLAC was higher when compared with the LZR controls (P < 0.05). S18886 did not significantly affect this parameter. At the end of the experiment, mean SBP in the OZR-PLAC group was higher in comparison with the LZR (P < 0.05). In the S18886 groups, mean SBP did not significantly differ from the OZR-PLAC. Mean heart weight did not differ between the groups (data not shown).


View this table:
[in this window]
[in a new window]

  		TABLE 1 Body and organ weight, blood pressure, and renal function of UNX-LZR and UNX-OZR rats after 8 weeks of treatment

 
Kidney weight and renal function.
Mean kidney weight of the OZR-PLAC was higher compared with the LZR (P < 0.05) (Table 1). The kidney weight of animals treated with S18886-30 was significantly lower in comparison to the OZR-PLAC group (P < 0.01). Creatinine clearance was significantly higher in the OZR-PLAC in comparison to the LZR (P < 0.01), whereas plasma creatinine concentration was reduced (P < 0.01). Treatment resulted in a trend to lower creatinine clearance (NS). In the OZR-PLAC, the plasma urea concentration and the urinary excretion of urea were increased in comparison to the LZR (P < 0.01). In the S18886-30–treated group, the plasma urea concentration was lower in comparison to OZR-PLAC (P < 0.01), while the enhanced urea excretion observed in the OZR-PLAC was not significantly influenced by S18886. The OZR-PLAC showed a high diuresis (rise by a factor of four in comparison to LZR, P < 0.01). Treatment with S18886-30 induced a mild decrease (–31%, P > 0.05). Proteinuria of the OZR-PLAC group was fourfold higher when compared with the LZR (P < 0.01). In both treated groups, a mild to moderate reduction was observed (–12 and –37%, both doses, respectively, NS). The urinary TGF-ß1 excretion in the OZR-PLAC was sevenfold higher than in the LZR (P < 0.01). Treatment with both doses of S18886 was associated with significantly lower values (P < 0.05). Urinary 2,3-dinor-TxB2 excretion in the OZR-PLAC was threefold higher than in the LZR (P < 0.01) and tended to be lower in the treatment groups but reached significance (P < 0.05) only under S18886-30. There was a direct relationship between proteinuria and urinary 2,3-dinor-TxB2 excretion (r = 0.56, P < 0.01).

Blood chemistry.
All OZR were moderately hyperglycemic, had elevated cholesterol, and markedly augmented triglyceride levels (Table 2). These conditions were not affected by either dosage. Plasma total protein concentrations did not differ significantly between the groups. The concentrations of plasma electrolytes and minerals as well as the erythrocyte and platelet count were within the normal range and did not differ between the groups (data not shown). The number of white blood cells (WBCs) and neutrophils (data not shown) was higher in the OZR-PLAC than in the LZR (P < 0.01), and neither dosage influenced this data significantly.


View this table:
[in this window]
[in a new window]

  		TABLE 2 Plasma total protein, glucose and lipid concentration, and data characterizing oxidative status in UNX-LZR and UNX-OZR rats after 8 weeks of treatment

 
Oxidative status.
Plasma AOPP levels were threefold higher in the OZR-PLAC in comparison with the LZR (P < 0.01) (Table 2). S18886-30 resulted in a trend to lower levels of AOPPs (–25%, NS). The activities of GPX and SOD were comparable in the OZR-PLAC and LZR. After the 8-week treatment, GPX activity was significantly higher in both treated groups (P < 0.01), whereas that of SOD reached a significant rise only in the OZR-S18886-30 group (P < 0.01).

Kidney morphology.
The mesangiolysis score was significantly higher in the OZR-PLAC than in the LZR (P < 0.01) (Fig. 1 and 2, Table 3). Treatment with S18886 completely prevented this alteration so that the mesangiolysis scores in the OZR treatment groups were comparable to the LZR. The glomerulosclerosis index showed a trend to higher levels in the OZR-PLAC. The S18886 treatment groups were not different from the LZR. Evaluation of the tubulointerstitial damage score revealed no significant differences between the groups (Table 3). Vascular damage score was significantly higher in OZR-PLAC than in LZR and OZR-S18886-10 (Table 3).


Figure 1
View larger version (132K):
[in this window]
[in a new window]

  		FIG. 1. Representative changes in glomerular morphology. Paraffin sections. A: Glomerulus of a LZR. B, D, and F: Glomeruli from placebo-treated OZR showing irregular structures of the capillary tuft with capillary widening, capillary dilatation, and dissolution of the mesangium (mesangiolysis score 1–2). C and E: Glomeruli of TP receptor antagonist S18886-treated OZR with nearly normal capillary structure and mesangium (C, low-dose treatment; E, high-dose treatment).

 

Figure 2
View larger version (103K):
[in this window]
[in a new window]

  		FIG. 2. Changes in glomerular cellularity and capillarization. Semithin sections. A: Glomerulus of an untreated lean Zucker control rat. B, D, and F: Glomeruli from placebo-treated obese Zucker rats showing markedly enlarged podocytes with cystic degeneration, slightly irregular capillaries, and mild mesangial cell hyperplasia. C and E: Glomeruli of TP receptor antagonist S18886-treated OZR with nearly normal cellular and capillary structure (C, low-dose treatment; E, high-dose treatment). Original magnification x40.

 

View this table:
[in this window]
[in a new window]

  		TABLE 3 Parameters of glomerular geometry in UNX-LZR and UNX-OZR rats after 8 weeks of treatment

 
As indicated in Table 3, mean glomerular volume was significantly higher in the OZR-PLAC and in the OZR-S18886-10 than in LZR. Minimal and maximal glomerular diameter and the area of capillary tuft were significantly higher in the OZR-PLAC. While the minimal glomerular diameter increased significantly under S18886-10, the maximal glomerular diameter and the area of the capillary tuft were not affected significantly. In the OZR-S18886-30 group, no significant changes were revealed. Mean glomerular cell number was significantly lower in the OZR-S18886-30 than in the LZR. It was comparable in the other treatment groups.

On semithin sections in the glomeruli of the OZR-PLAC marked cystic degeneration of podocytes (glomerular epithelial cells), slightly irregular capillaries with thickened glomerular basement membrane and mild expansion of the mesangium were seen (Fig. 2). These changes were not observed in either treatment group. Quantitative analysis of glomerular capillaries and cells revealed no difference in mean capillary tuft area and volume, capillary length density, i.e., total length of all capillaries per glomerular volume, podocyte number, and mean podocyte volume. Podocyte number was highest, however, in the OZR-S18886-30 group (101 ± 16.9 vs. 75.9 ± 11.1 in OZR-PLAC) indicating preservation of podocyte structure. In parallel, podocyte volume was highest in the OZR-PLAC and lowest in the OZR-S18886-10 group.

Survival, tolerability, and toxicity.
Treatment with S18886 was well-tolerated. One OZR of the S18886-10 group died of asphyxia during the blood pressure measurement at week 5, most probably due to an ether-induced depression of the respiratory center. In the 8-week treatment period, no signs of hemato- or hepatotoxicity were observed (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
OZR represents one of the most important animal models of nephropathy in type 2 diabetes. Since renal complications develop rather late (at ~40 weeks), their natural course can be accelerated by UNX (28,29), as performed in our study. Eight weeks after UNX in the OZR-PLAC, renal pathology was characterized by an increase of kidney weight, glomerular size, and mesangiolysis index, as well as cystic degeneration of podocytes. Creatinine clearance and diuresis were enhanced, as was the urinary excretion of protein, TGF-ß1, and 2,3-dinor-TxB2. S18886 dose-dependently hindered the functional and morphologic alterations. The prevention of mesangiolysis and podocyte degeneration represents the most striking new observation, associated with an improved antioxidant defense in the blood.

Renal function.
The increased creatinine clearance in the OZR-PLAC, as compared with the LZR, reflects hyperfiltration, preceding the sclerotic changes in the glomeruli (38). Administration of S18886 was associated with a trend to a decline of creatinine clearance, similar to the effects of inhibitors of the renin angiotensin system, indicating a beneficial response. As a consequence of hyperphagia, the plasma concentration of urea and its urinary excretion rate were increased in the OZR-PLAC, as compared with the LZR, as was the body weight. S18886-30 lowered plasma urea concentration and was associated with a decrease (NS) in its urinary excretion rate. Proteinuria was increased fourfold in the OZR-PLAC. Both doses of S18886 partially lowered proteinuria. Besides the potential impact of altered intraglomerular hemodynamics, due to the mild (but insignificant) decline in SBP, an improved podocyte function by TPr antagonism could be involved.

Administration of S18886-30 resulted in a moderate decline of the polyuria, implicating an improved urinary concentration mechanism and/or a decreased fluid intake. In fact, TPr in the hypothalamus have been shown to mediate the dipsogenic response to angiotensin II (39), which may be lessened by S18886 treatment.

Urinary excretion of TGF-ß1 was markedly increased in the OZR-PLAC. This rise was prevented by the TPr antagonist, corresponding to the reduction of intrarenal accumulation of this cytokine by S18886 in diabetic apoE–/– mice (26).

Similar to studies in STZ diabetic rats (15) and in the Otsuka Long-Evans Tokushima Fatty strain (18), the urinary 2,3-dinor-TxB2 excretion was significantly enhanced in the OZR-PLAC, indicating augmented production of TxA2. This could be favored by both enhanced glucose levels (40) and stimulation of the angiotensin II type 1 receptor (12). S18886 lowered the urinary 2,3-dinor-TxB2 excretion dose-dependently. Possibly, the TPr antagonist prevented the amplification of thromboxane generation in platelets. The positive relationship between urinary 2,3-dinor-TxB2 excretion and proteinuria suggests involvement of the TxA2 metabolic pathway in the pathogenesis of proteinuria. However, administration of aspirin (which lowers thromboxane synthesis) did not protect the diabetic apoE–/– mice (27) or rats with other renal diseases from progression. This suggests that the enhanced thromboxane formation may be less significant than the other ligands to the TPr (26).

Oxidative stress.
An increased production of reactive oxygen species is assumed to play a central role in the development of diabetic nephropathy (41). Correspondingly, an enhanced expression of the NAD(P)H oxidase subunit p47phox and an accumulation of nitrotyrosine (a marker of oxidative/nitrosative stress) and 12-lipoxygenase in the renal tissue, as well as an enhanced urinary excretion of HETE and isoprostane F2{alpha}, have been observed (26,41,42). In the diabetic apoE–/– mice, administration of S18886 attenuated all of the above-mentioned markers of oxidative stress. The causal role of oxidative/nitrosative stress in renal injury is supported by the protective effect of an overexpression of SOD against early diabetic glomerular lesions in transgenic mice (43).

In our study in the OZR-PLAC, circulating AOPP levels were increased threefold as compared with the LZR, highlighting the role of an enhanced oxygen radical formation in this model. A similar rise was observed in human diabetic and nondiabetic renal diseases (30). Because AOPPs are partly derived from the myeloperoxidase reaction, their high levels point to an activation of phagocytic cells. Indeed, in our study, a rise in WBC count was observed. In humans with diabetes, an elevated WBC count, even within the normal range, is associated with the risk of complications (44). Interestingly, with S18886-30, the elevated plasma AOPP levels were lower (–25%, NS). The lack of significance is most likely due to a high interindividual variability. The activities of the antioxidant enzymes SOD and GPX did not differ between the LZR and OZR-PLAC. However, S18886 dose-dependently increased both antioxidants in the blood. This finding corresponds to the nephroprotective action of the rise of lowered renal SOD after S18886 in the diabetic apoE–/– mice (26) as well as an overexpression of SOD in the db/db mice (43).

Renal morphology.
Interpretation of changes in experimental diabetic nephropathy is always limited by the lack of an adequate animal model that develops human-like lesions (45). The OZR is a widely used animal model for diabetic nephropathy (29), although the glomerular changes in this animal model differ in certain aspects from biopsy findings in human diabetic nephropathy. In our OZR-PLAC, renal histology was characterized by enlargement of the glomeruli with a significant increase of mesangiolysis (33), which is associated with a loss of mesangial cells, capillary dilatation, and, finally, formation of capillary aneurysms (46), as typically seen in inflammatory glomerular diseases (33). In diabetic nephropathy, it is postulated that the nodular glomerulosclerosis develops out of the mesangiolytic damage, in particular from repair of capillary micro-aneurysms (46–49). Besides an augmented glomerular pressure (due to hyperglycemia and UNX), an enhanced formation of TxA2 could be involved in the glomerular injury, since in experimental settings mesangiolysis was associated with a rise in TxB2 secretion from mesangial cells (50). In our study, blockade of TPr with S18886 completely prevented mesangiolysis, underscoring its potential pathogenetic role in this glomerular injury. To our knowledge, this is the first observation that a TPr antagonist prevents this early feature of diabetic nephropathy, a possible forerunner of the ensuing nodular glomerulosclerosis (49). In addition, S18886 prevented degeneration of podocytes and at least partly also hypertrophy and loss of podocytes, i.e., podocytopenia. The latter may explain at least in part the mild antiproteinuric effect of S18886. However, the early signs of glomerulosclerosis (i.e., an increase of mesangial matrix with capillary obliteration) were not significantly altered, possibly due to the short duration of the experiment. Moreover, the glomerular volume was not reduced by the TPr antagonist, since thromboxane mimetics lower glomerular volume (3). It is conceivable that in this response a relaxation of contracted mesangial cells was involved.

In summary, our data show that TPr antagonism with S18886 retards the progression of nephropathy in UNX-OZR. This is reflected by improved renal histomorphology with a prevention of mesangiolysis and podocyte degeneration, a trend to reduced proteinuria, and a lower urinary excretion of TGF-ß1 and 2,3-dinor-TxB2. In blood, the activity of antioxidant enzymes was increased, while the circulating AOPP levels were lower. All these data suggest the involvement of activated TPr in the pathogenesis of the nephropathy in OZR. They further imply potential benefits of long-term TPr antagonism with S18886 in the clinical setting of type 2 diabetes.


   ACKNOWLEDGMENTS
 
This study was supported in part by the Institut de Recherches Internationales Servier, Courbevoie, France, and by the DFG (Deutsche Forschungsgemeinschaft) (SFB 423, project Z2).

Preliminary data of this work were presented in poster form at the World Congress of Nephrology, Berlin, 2003.

The authors would like to thank Dr. S. Corda and Dr. L. Lerond, Servier, Paris, for their stimulating and constructive support in drafting the manuscript. The skillful technical assistance of M. Klewer, M. Ramming, and S. Söllner is gratefully acknowledged.


   FOOTNOTES
 
Published ahead of print at http://diabetes.diabetesjournals.org on 31 January 2007. DOI: 10.2337/db06-1136.

A.H. received financial support from Servier, France, to conduct this experimental investigation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication August 15, 2006 and accepted in revised form January 14, 2007


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Baylis C: Effects of administered thromboxane on the intact, normal rat kidney. Renal Physiol 10:110–121, 1987[Medline]
  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[Abstract/Free Full Text]
  3. Scharschmidt LA, Lianos E, Dunn MJ: Arachidonate metabolites and the control of glomerular function. Fed Proc 42:3058–3063, 1983[Medline]
  4. Remuzzi G, Fitzgerald GA, Patrono C: Thromboxane synthesis and action within the kidney. Kidney Int 41:1483–1493, 1992[Medline]
  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[Abstract]
  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[Medline]
  9. Audoly L, Rocca B, Fabre JE, Koller BH, Thomas D, Loeb AL, Coffman TM, FitzGerald GA: Cardiovascular responses to the isoprostanes i-PF2-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[Abstract/Free Full Text]
  11. Brown GP, Venuto RC: Thromboxane receptors in human kidney tissues. Prostaglandins Other Lipid Mediat 57:179–188, 1999[Medline]
  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[Medline]
  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[Medline]
  14. Craven PA, Melhem MF, De Rubertis FR: Thromboxane in the pathogenesis of glomerular injury in diabetes. Kidney Int 42:937–946, 1992[Medline]
  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[Medline]
  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 decreases microalbuminuria in normotensive type 2 diabetic patients. Diabetes 47:423–430, 1998[Abstract]
  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[Medline]
  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[Medline]
  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[Medline]
  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[Abstract/Free Full Text]
  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[Medline]
  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[Abstract/Free Full Text]
  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[Abstract/Free Full Text]
  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[Medline]
  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[Medline]
  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[Abstract/Free Full Text]
  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
  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[Medline]
  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[Medline]
  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[Medline]
  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[Medline]
  36. Weibel ER: Measuring through the microscope: development and evolution of stereological methods. J Microsc 155:393–403, 1989[Medline]
  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[Medline]
  38. Hostetter TH, Troy JL, Brenner BM: Glomerular hemodynamics in experimental diabetes mellitus. Kidney Int 19:410–415, 1981[Medline]
  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[Abstract/Free Full Text]
  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[Medline]
  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[Medline]
  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[Abstract/Free Full Text]
  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[Abstract/Free Full Text]
  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[Medline]
  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[Medline]
  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[Medline]
  49. Stout LC, Kumar S, Whorton EB: Focal mesangiolysis and the pathogenesis of the Kimmelstiel-Wilson nodule. Hum Pathol 24:77–89, 1993[Medline]
  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

Add to CiteULike CiteULike   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Nephrol Dial TransplantHome page
P. Boor, K. Sebekova, T. Ostendorf, and J. Floege
Treatment targets in renal fibrosis
Nephrol. Dial. Transplant., September 21, 2007; (2007) gfm393v1.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
db06-1136v1
56/4/968    most recent
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sebeková, K.
Right arrow Articles by Amann, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sebeková, K.
Right arrow Articles by Amann, K.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Diabetes Diabetes Care Clinical Diabetes Diabetes Spectrum