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Thromboxane A2 receptor antagonist (ONO‐8809) attenuates renal disorders caused by salt overload in stroke‐prone spontaneously hypertensive rats

Abstract Epidemiological and clinical studies have demonstrated that excessive salt intake causes severe hypertension and exacerbates organ derangement, such as in chronic kidney disease (CKD). In this study, we focused on evaluating the histological and gene expression effects in the kidneys of stroke‐prone spontaneously hypertensive rats (SHRSP) with a high salt intake and the thromboxane A2/ prostaglandin H2 receptor (TPR) blocker ONO‐8809. Six‐week‐old SHRSPs were divided into three groups and were fed normal chow containing 0.4% NaCl, 2.0%NaCl or 2.0%NaCl + ONO‐8809 (0.6 mg/kg p.o. daily). Histological analyses with immunohistochemistry and a gene expression assay with a DNA kidney microarray were performed after 8 weeks. The following changes were observed in SHRSPs with the high salt intake. Glomerular sclerotic changes were remarkably observed in the juxtamedullary cortex areas. The ED1, monocyte chemoattractant protein‐1 (MCP‐1), nitrotyrosine and hypoxia inducible factor 1α (HIF‐1α) staining areas were increased in the glomeruli and interstitial portion of the kidneys. The genes Tbxa2r (that encodes TPR), Prcp and Car7 were significantly underexpressed in the kidneys. The plasma 8‐isoprostane level was significantly elevated and was attenuated with the ONO‐8809 treatment. Thromboxane A2 (TXA2) and oxidative stress exaggerated renal dysfunction in the salt‐loaded SHRSPs, and ONO‐8809 as a TPR blocker suppressed these changes. Therefore, ONO‐8809 is a candidate drug to prevent CKD in hypertensive patients when CKD is associated with a high salt intake.

field of hypertension, and also as renal disorder models with salt sensitivity. [8][9][10][11] Dietary salt overloading could itself increase the generation of reactive oxygen species (ROS) in the kidneys and blood vessels over the course of hypertension development, 12,13 and ROS are related to inflammation processes in vascular impairment and renal disorders. 14,15 In these processes, thromboxane A 2 (TXA 2 ) plays a crucial role in the progression of chronic renal disability over the course of inflammation-implicated ROS in SHRSP 16 or SHR, 17 and the expression of TXA 2 and/or the prostaglandin endoperoxide (PGH 2 ) receptor (TPR) are shown to increase because of salt loading in the rat cortical kidney. 18,19 Thromboxane A 2 , or its stable analogue U-46,619, reduces renal blood flow (RBF) and the glomerular filtration rate (GFR), and potentiates tubuloglomerular feedback (TGF), 19,20 platelet aggregation 21 and monocyte chemoattractant protein-1 (MCP-1) production via the induction of nuclear factor (NF) κB and activating protein 1 (AP-1) binding activity, 22 vascular smooth muscle contraction, 23 and the production of extracellular matrix proteins. 24 The TXA 2 synthase inhibitor Dazmegrel increases prostaglandin E2 (PGE 2 ) synthesis, 20 which preserves kidney function. The TXA 2 synthetase antagonist OKY-046 leads to less chronic renal failure and glomerular sclerosis in immunologically created Dahl-salt sensitive rats given a high salt intake. 25 These findings indicate that the inflammation processes via ROS and TXA 2 are strongly associated with chronic renal-disorder changes caused by high salt loading. 26 However, one TPR antagonist, SQ-29548, has not shown any significant effect on PGE 2 production in the glomerulonephritis model rat. 27 Based on these findings, our study focused on the detailed histological analysis, oxidative stress and gene expression characteristics associated with kidney function using SHRSP with a high salt intake to evaluate the pharmacological effects of another TPR blocker, ONO-8809 and TXA 2 involvement during renal disorders.

| Blood pressure
The systolic blood pressures of SHRSPs at 6 weeks of age (the start of this experiment) and at 14 weeks of age (the end of the experiment) were not significantly different among groups, as shown in Table 1. The values at 6 weeks of age were 142 ± 6, 140 ± 4 and 144 ± 4 mm Hg in the low salt control (LSC), high salt (HS) and high salt + ONO-8809 (HS+ONO) groups, respectively. Body weights were significantly lower in HS group than those in LSC and HS+ONO groups, and the heart-body weight ratios were not significantly different among the groups at 14 weeks of age.

| Findings of the renal functional data
Both the urinary volume and protein excretion in the HS group were significantly higher than those in the LSC and HS+ONO groups. The water intakes in the HS group was higher than those in the LSC group and tended be a higher level than those in the HS+ONO groups at 14 weeks of age. The plasma creatinine levels in the HS group were higher, and creatinine clearance values were lower in the HS group than those of the LSC and HS+ONO groups, as shown in Table 1.

| Indices representing oxidative stress in the plasma
The plasma thiobarbituric acid reactive substances (TBARS) levels, as an index of oxidative stress, were not significantly different between the LSC and HS groups, but were significantly lower in the HS+ONO group than those of the HS group. The plasma 8-isoprostane levels, which are another index of oxidative stress in HS, were significantly higher than those of the LSC group (Table 2).

| Findings of histological changes in the kidney caused by high salt and/or ONO-8809 intake
The glomerular sclerotic results, such as lesions in which the basement membrane and mesangial cells were increased and collapsed, were frequently observed in the juxtamedullary cortex areas of HS kidneys.
These changes were improved by ONO-8809 treatment. Onion skin lesions and fibrinoid necrotic changes in the interlobular arteries and afferent arterioles were observed more often in the HS group than the LSC group. The collagen fibre contents in the cortex were identified by measuring the blue area stained by Masson trichrome staining in the interstitial portion. The interstitial fibrotic changes were significantly higher in the HS group than those observed in the LSC group ( Figure 1 and Table 2).

| Immunohistochemical changes in the kidney caused by high salt and/or ONO-8809 intake
The existence of MCP-1, an inflammatory chemokines, was more evident in the collecting tubules, thick ascending limbs of Henle's loop, distal tubules and macula densa of the HS group than in the LSC group, and ONO-8809 administration decreased it in the HS+ONO group. The existence of nitrotyrosine, an oxidative stress marker showing peroxynitrite, was almost as common as MCP-1 ( Figure 2 and Table 2). Hypoxia inducible factor-1α (HIF-1α) was observed more in the large collecting tubules, thick ascending limbs of Henle's loop, distal tubules and the macula densa in the HS kidneys compared with those of the LSC group and was significantly reduced in HS+ONO kidneys ( Figure 2C and Table 2). The ED1-positive staining (representing macrophage infiltration) was more evident in the glomerular and tubulointerstitial areas with glomerular sclerosis, arteriolar onion skin and fibrinoid necrosis in the HS group than in the LSC group. Administration of ONO-8809 to the salt-loaded SHRSPs decreased these changes, as shown in Figure 3 and Table 2.

| Findings of the DNA microarray experiments
Three substantially upregulated and three substantially strikingly downregulated genes in the comparisons of HS with LSC and HS+ONO with HS are summarized in Table 3. In the comparison between HS and LSC, the two upregulated genes were Kcnv and Speg, and the two genes downregulated genes were Prcp and Tbxa2r. When these were compared with the HS+ONO/HSC comparison, Kcnv1 and Speg were decreased 0.25-to 4.0-fold, and Agtr2 (gene of angiotensin II receptor type 2) was significantly decreased by 0.016-fold.
On the other hand, Prcp, Tbxa2r and Car7 (gene of carbonic anhydrase 7) were significantly upregulated compared with HS and LSC.

| DISCUSS ION
High salt consumption is one of the most important factors in the World Health Organization has recommended <5 g salt per day.
Salt is a popular additive to improve the taste of food, so it is not easy for people to keep to the recommended salt limit for health reasons.
To solve this problem, pathophysiological analyses using salt-loaded SHRSPs with hypertension as a model for CKD under the administration of the TPR antagonist, ONO-8809, as a pharmacological tool, including a detailed measurement of gene expression, were undertaken. The TPR antagonist ONO-8809 attenuated the renal inflammation and arteriolar sclerosis of in the SHRSPs caused by high salt intake, without significantly altering the blood pressure. It may have been useful to set up an additional experiment to measure the circadian rhythm, given that it did not significantly change the blood pressure. According to previous studies, metoprolol, a β-blocker, was highly effective when administrated at night, 29 valsartan, an AT1blocker, was highly effective when administrated before sleep, 30 and Janssen et al 31 reported that the circadian rhythms of blood pressure and heart rate were under sympathetic control in SHR and were not influenced by non-sympatholytic vasodilators. Therefore, it seems the absence of a significant difference in the blood pressures of rats fed a high-salt diet with or without ONO-8809 administration in the daytime was not caused by major mistakes in our study.
Urinary protein excretion and plasma creatinine levels were significantly higher in the HS group than in the LSC group. Creatinine clearance values decreased with a HS intake and improved under HS+ONO. The urinary protein excretion levels of HS+ONO SHRSPs tended to decrease compared with those in the HS group, but difference was not significant (Table 1). These data indicated that the  Note: Values are mean ± standard error of the mean. * or **, and # or ## indicate significant differences at P < .05 or P < .01 compared with the LSC values, and at P < .05 or P < .01 compared with the HS values, respectively, determined with a Jonckheere-Terpstra test with Bonferroni correction (glomerular finding scores and arteriolar lesion scores), and a one-way ANOVA test followed by a Tukey-Kramer for the other parameters. The SHRSP rats grouped in the LSC, HS and HS+ONO groups were given a 0.4% NaCl mixed diet, 2% NaCl mixed diet, and 2% NaCl mixed diet with 0.6 mg/kg ONO-8809 p.o. per day, respectively, for 8 weeks from 6 weeks of age.
TA B L E 2 Oxidative stress, histology and immunohistology of SHRSPs after 8 weeks of treatment The plasma TBARS levels, which indicate oxidative stress, were not different between the LSC and HS groups, but those of the HS+ONO group were significantly lower than those of the HS group.
Plasma 8-isoprostane levels in HS rats were significantly higher than those of the LSC group ( Table 2). The presence of MCP-1, an inflammatory chemokines, estimated by immunostaining with anti-MCP1 antibody, was more extensively observed in HS rats than in LSC rats, and ONO-8809 administration decreased it in the HS+ONO group ( Figure 2 and Table 2). Nitrotyrosine was more common in HS tissue than in LSC, and was significantly reduced in the tissue of HS+ONO rats ( Figure 2 and Table 2). Hypoxia inducible factor-1α staining areas were more widespread in HS kidneys than in those of LSC rats and were significantly reduced in the group of HS+ONO, as shown in Figure 2C and Table 2. These findings indicate that oxidative stress and hypoxia occur around the interlobular and afferent arterioles in the kidneys of salt-loaded SHRSP with intimal necrosis, fibrinoid necrosis and onion skin lesions and that ONO-8809 improved these conditions.
The ED1-positive staining for macrophage infiltration was observed more often in the glomerular and tubulointerstitial areas with glomerular sclerosis, arteriolar onion skin, and fibrinoid necrosis in the HS group than in the LSC group. Administering ONO-8809 to salt-loaded SHRSPs reduced macrophage infiltration, with less glomerulosclerosis, arteriolar onion skin and fibrinoid necrosis observed ( Figure 3 and Table 2).
Regarding the measurement of messenger RNA (mRNA) expression using the DNA microarray experiment, two genes were strikingly upregulated in the comparison between the HS and LSC groups: Kcnv1 and Speg; Agtr2 was not significantly upregulated.
One gene was substantially downregulated in the comparison be-   35 revealed TXA 2 receptor expression in the renal cortex, especially in the glomeruli, arteries, arterioles and in the renal medulla.
Knowledge regarding the distribution of TXA 2 receptor in the kidney could contribute to its role both in normal kidney physiology and as a mediator of kidney dysfunction and injury. That is, TxA 2 causes marked renal vasoconstriction, 32 reduces renal blood flow and glomerular filtration rate and stimulates mesangial cell contraction to cause proteinuria. 33 Therefore, these effects were attenuated by the treatment of ONO-8809 as a TPR blocker.
Using high salt-loaded SHRSPs with or without a TPR antagonist, the following findings were clarified. High-salt intake during hypertensive states facilitates the occurrence of CKD, and TXA 2 and ROS are the main causative factors in the pathophysiological course of the disease. Therefore, drugs such as TPR blockers will be useful for HS intake-associated CKD. The conclusions obtained from this experiment are summarized in Figure 4.

sion. A HS intake increases ROS and mineral corticoid receptors via
nicotinamide adenine dinucleotide phosphate oxidase, lowers superoxide dismutase, 37 increases TXA 2 , 36 accumulates macrophages via macrophage-colony stimulating factor/c-fms 38 and increases MCP-1 39,40 and inflammatory cytokines such as interleukin-6 and tumour necrosis factorα. 41 As a result, two major factors in hypertension, TXA 2 and ROS 13,33 exaggerate renal dysfunction and inflammation, 8,42 and cause CKD. 2 The upregulation of Kcnv1 and Speg, the downregulation of Prcp and Tbxa2r under a HS load, and the downregulation of Agtr2 under ONO-8809 load explain the objective and logical biochemical phenomena that occurred in this experiment.
In conclusion, TXA 2 and oxidative stress with hypertension exaggerate renal dysfunction and inflammation, leading to CKD in saltloaded SHRSPs. We strongly advocate that TPR blockers such as ONO-8809 be considered as candidate drugs to prevent CKD for hypertensive patients, particularly when this is associated with a HS intake. The findings of this experiment were suggestive of a positive effect of ONO-8809, but further research is required to obtain definite results.

| Animals and experimental design
Six-week-old male SHRSPs were purchased from Kindai University by Ono Pharmaceutical Co. Ltd. It was administrated at a dose of 0.6 mg/kg dissolved in 0.9% saline and was given once per day using a gastric tube to the HS+ONO group rats. The dose was determined using dose-dependency data obtained from a pilot study, although a dose of 0.3 mg/kg/day was used in some studies. 16,43 All rats were weighed, and the systolic arterial blood pressure was

| Content measurements in urine and blood samples
The protein, creatinine (Wako Chemicals), sodium and potassium

| Histological studies of sclerotic kidney changes
The kidneys, once excised from the body, were decapsulated, and longitudinally cut by fine surgical scissors for the histological and immunohistochemical studies. The tissue was fixed with 10% formalin neutral buffer solution (Wako Chemicals), and embedded in paraffin wax, sliced 2-3 μm thick, and then mounted on glass slides.
Each specimen was stained with periodic acid for the Schiff (PAS) and Masson trichrome staining methods. Thirty superficial and juxtamedullary glomeruli in each kidney specimen sample were observed with a light microscope (ECLIPSE E800; Nikon) at 400× magnification.
Glomerular findings using the PAS staining specimens were graded from 0 to +4 using a semiquantitative score base on the following criteria: 0, without any histological change; +4, sclerotic change by more than 75% of the glomerulus. The glomerular damage index is an average of grades assigned to 60 glomeruli. 44 The fibrotic results of the arteriolar lesions in the kidney were graded from 0 to +4 using the PAS staining specimens. The grades were based on both the severity of the vascular wall thickness and the extent of fibrinoid necrosis in afferent arterioles, interlobular arterioles and small arteries, as follows: grade 0, normal findings in the vessel; +4, severe sclerotic changes with fibrinoid necrosis. The vascular lesion score was determined as an average of the grades assigned to 30 fields. 44 To evaluate the interstitial fibrosis, 20 fields for each section were assessed on Masson trichrome-stained sections using a microscope at 200× magnification. Interstitial blue stained areas excluding glomeruli, Bowman's capsules and large vessels were counted, and the percentage of the total area in the specimen was calculated using a computer-assisted morphometric analysing method with a digital microscope controller (VB-7000; Keyence) and software for image analysis software (Image J software version 1.43q; National Institutes of Health; http://rsb.info. nih.gov/ij/). To determine the mean number of filtrating macrophages/monocytes (ED1-positive cells) in the renal cortex, 20 fields in the tubulointerstitium or 20 glomeruli for each section were counted using the above-mentioned microscope at 400× magnification. In the evaluation of immunoperoxidase staining for MCP-1, nitrotyrosine and HIF-1α, each tubulointerstitial grid field of the renal cortex was counted, and the staining percentage of the total area was calculated using a computer-assisted morphometric analysing method with a digital microscope controller and software for image analysis software (Image J software version 1.43q).

| DNA microarray analysis of the kidney cortex
Small pieces taken from the cortical kidney of three rats per group were homogenized at a pitch speed of 22 strokes/second for 2 minutes (twice) in a 2-mL plastic tube with 5-mm diameter glass beads using a Qiagen Tissue Lyser (Retsch). The total RNA was extracted with a RNeasy Mini kit (QIAGEN) according to the manufacturer's protocol. The RNA quality was checked with RNA Nano Chips (Agilent Technologies) using an Agilent 2100 Bioanalyzer, and the RNA was then used in the microarray experiments.
To examine the gene expression profiles in rat kidneys, we syn- (Biometric Research Branch). 46 We used the Benjamini-Hochberg procedure to control the false discovery rate (FDR) <0.05, and a significance level (P < .01) for each probe was set using a oneway ANOVA and Tukey-Kramer multiple-comparison test. Genes obtained from rat groups were compared between HS/LSC and (HS+ONO)/HS and are shown as values of fold changes.

| Statistical analysis
All results except for the glomerular finding scores and arteriolar lesion scores are expressed as the mean ± standard error of the mean (SEM). To determine the significant differences among treatment groups for the glomerular scores and arteriolar lesion scores, we performed Jonckheere-Terpstra tests with Bonferroni correction.
Comparisons among the means of multiple groups were analysed by a one-way ANOVA and Tukey-Kramer multiple comparison test. In all tests, the differences were considered statistically significant at the value of P < .05.

| DATA AVA I L A B I LIT Y S TATE M E NT
The data that support the findings of this study are available from the corresponding author, H.H., on reasonable request, and openly available in each repository at reference number .

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.