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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal

Renoprotective Effect of TP0472993, a Novel and Selective 20-Hydroxyeicosatetraenoic Acid Synthesis Inhibitor, in Mouse Models of Renal Fibrosis

Takashi Hirata, Hiroki Ohara, Naoki Kojima, Hiroko Koretsune, Yoshitaka Hasegawa, Shoko Inatani and Teisuke Takahashi
Journal of Pharmacology and Experimental Therapeutics July 2023, 386 (1) 56-69; DOI: https://doi.org/10.1124/jpet.122.001521

Abstract

Kidney fibrosis is considered the essential pathophysiological process for the progression of chronic kidney disease (CKD) toward renal failure. 20-Hydroxyeicosatetraenoic acid (20-HETE) has crucial roles in modulating the vascular response in the kidney and the progression of albuminuria. However, the roles of 20-HETE in kidney fibrosis are largely unexplored. In the current research, we hypothesized that if 20-HETE has important roles in the progression of kidney fibrosis, 20-HETE synthesis inhibitors might be effective against kidney fibrosis. To verify our hypothesis, this study investigated the effect of a novel and selective 20-HETE synthesis inhibitor, TP0472993, on the development of kidney fibrosis after folic acid– and obstructive-induced nephropathy in mice. Chronic treatment with TP0472993 at doses of 0.3 and 3 mg/kg twice a day attenuated the degree of kidney fibrosis in the folic acid nephropathy and the unilateral ureteral obstruction (UUO) mice, as demonstrated by reductions in Masson’s trichrome staining and the renal collagen content. In addition, TP0472993 reduced renal inflammation, as demonstrated by markedly reducing interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) levels in the renal tissue. Chronic treatment with TP0472993 also reduced the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) in the kidney of UUO mice. Our observations indicate that inhibition of 20-HETE production with TP0472993 suppresses the kidney fibrosis progression via a reduction in the ERK1/2 and STAT3 signaling pathway, suggesting that 20-HETE synthesis inhibitors might be a novel treatment option against CKD.

SIGNIFICANCE STATEMENT In this study, we demonstrate that the pharmacological blockade of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis using TP0472993 suppresses the progression of kidney fibrosis after folic acid– and obstructive-induced nephropathy in mice, indicating that 20-HETE might have key roles in the pathogenesis of kidney fibrosis. TP0472993 has the potential to be a novel therapeutic approach against chronic kidney disease.

Introduction

Chronic kidney disease (CKD) has affected over 800 million individuals worldwide and has become a global public health concern (Kovesdy, 2022). CKD has shown to be an independent risk factor not only for progression to renal failure but also for cardiovascular events (Tonelli et al., 2006; Jankowski et al., 2021). To date, the renin-angiotensin system blockers are used as a standard care for CKD patients, but the effectiveness of these medicines for preventing the progression of CKD is moderate (Mann et al., 2008). Recently, it was shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors exert renoprotective effect in diabetic and nondiabetic CKD patients (Dharia et al., 2023). However, a residual risk of progression to end-stage renal disease remains due to incomplete suppression of kidney fibrosis by SGLT2 inhibitor (Kojima et al., 2013). Thus, there is a need to explore novel and effective therapeutic targets that arrest the progression of CKD.

Kidney fibrosis is widely considered an essential pathophysiological process for the development of CKD toward renal failure regardless of the initial etiology (Hodgkins and Schnaper, 2012). Kidney fibrosis is featured by exaggerated extracellular matrix accumulation and chronic inflammation in glomerular and tubulointerstitial compartments. This abnormal extracellular matrix deposition and unresolved fibrosis eventually results in the decline of glomerular filtration rate (GFR) and the loss of renal function. In fact, clinical evidence demonstrates that the extent of kidney fibrosis is closely associated with the loss of kidney function (Nath, 1992; Gewin, 2018). Thus, the inhibition of kidney fibrosis could be a promising therapeutic approach to arrest or delay the development of CKD.

Recent studies indicate that lipid mediators such as eicosanoids are involved in not only renal physiology but also the pathophysiology of kidney disease (Hao and Breyer, 2007). These lipid mediators are produced by specific enzymes and exert their biologic activities by binding to specific G protein–coupled receptors (GPRs). Accordingly, these enzymes or receptors have received much attention as an attractive therapeutic target for kidney diseases. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a major bioactive arachidonate metabolite in the kidney. Cytochrome P450 (P450) ω-hydroxylases, specifically CYP4F2 and CYP4A11, are the major isoforms that contribute to the renal 20-HETE production in human (Roman, 2002). It was reported that 20-HETE has crucial roles in renal physiology, including the modulating tubular function and vascular tone (Roman, 2002). In contrast, several reports suggested that upregulating the renal 20-HETE production is involved in the renal pathophysiology. In this regard, Rong et al. (2018) described that CYP4A expression in the kidney and urinary excretion of 20-HETE were elevated in rats with angiotensin II–induced hypertension along with the amount of urinary protein excretion. A study in streptozotocin-treated diabetic rats indicated that administration of an inhibitor of 20-HETE synthesis reduced renal hypertrophy and albuminuria (Eid et al., 2013). However, the role of 20-HETE in kidney fibrosis is largely unknown.

Several lines of evidence suggested that the profibrotic signaling pathways, such as extracellular signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3), have crucial roles in the pathophysiological mechanisms underlying kidney fibrosis (Pat et al., 2003; Nagai et al., 2005; Bienaimé et al., 2016). Indeed, pharmacologic blockade of these signaling pathways has shown to attenuate the progression of kidney fibrosis in murine models of adriamycin and obstructive-induced nephropathy (Pang et al., 2010; Das et al., 2019). Recently, GPR75 has been reported as the receptor of 20-HETE, which is involved in the stimulation of the mitogen-activated protein kinase (MAPK)/ERK1/2 pathway in the vasculature (Garcia et al., 2017; Murtaza et al., 2022). Interestingly, it was reported that 20-HETE activates the ERK1/2 pathway in kidney epithelial cells, which is mainly involved in the interstitial fibrosis (Akbulut et al., 2009). Thus, we hypothesized that 20-HETE might contribute to the progression of kidney fibrosis, perhaps in a manner related to the stimulation of profibrotic signaling pathways, and that 20-HETE synthesis inhibitors could be effective for suppressing the progression of kidney fibrosis. Recently, we generated a novel and orally active 20-HETE synthesis inhibitor, 1-[4-({[6-(1H-pyrazol-5-yl)pyridin-3-yl]oxy}methyl)piperidin-1-yl]ethan-1-one (TP0472993) (Kawamura et al., 2022). Here, we examined the pharmacological effects of TP0472993 on 20-HETE production both in vitro and in vivo as well as its potential effect on the development of kidney fibrosis after folic acid (FA)- and obstructive-induced nephropathy in mice.

Materials and Methods

Compound

1-[4-({[6-(1H-pyrazol-5-yl)yridine-3-yl]oxy}methyl)piperidin-1-yl]ethan-1-one (TP0472993) (Fig. 1) was synthesized at Taisho Pharmaceutical Co., Ltd.

Fig. 1.
Fig. 1.

Chemical structure of TP0472993.

Animals

Crl:CD [Sprague-Dawley (SD)] rats (The Jackson Laboratory Japan, Inc., Kanagawa, Japan) were maintained in each plastic cage (two or three rats per cage) with wood chips and were given wooden blocks as enrichment. BALB/cAJcl (Balb/c) and C57BL/6JJcl (C57BL/6J) mice (CLEA Japan, Inc., Tokyo, Japan) were maintained in each plastic cage (five or eight mice per cage) with wood chips and were given the CAREFEEAZ (Hamri Co., Ltd., Ibaraki, Japan) as enrichment. Animals were supplied a regular rodent chow (MF; Oriental Yeast, Tokyo, Japan) and water ad libitum and kept in a 12-hour light/dark schedule. The room humidity (50% ± 20%) and temperature (23°C ± 3°C) were kept throughout the study. Except for the study of FA nephropathy model, age-matched animals were randomly assigned to each treatment group when these were obtained from the vendors. We selected group size considered necessary to statistically judge the effectiveness of compound based on the results of pilot experiments. All animal experiments were authorized by the Institutional Animal Care and Use Committee at Taisho Pharmaceutical Co., Ltd., which is accredited by Japan Pharmaceutical Information Center (certification number 21-026), and were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments.

Enzymatic Assays

Recombinant human CYP4F2 and CYP4A11 enzymes and EasyCYP Control (control protein) were obtained from Cypex Limited (Dundee, UK). Arachidonic acid (10 μM for CYP4F2 and 20 μM for CYP4A11) was mixed with 10 nM CYP4F2 or CYP4A11 enzymes in 100 mM KPO4 buffer [pH7.4] containing 150 μg/ml EasyCYP Control with vehicle (dimethylsulfoxide) or various concentrations of TP0472993. Reactions were started with 20 mM NADPH. After incubating at 37°C for 90 minutes (CYP4F2) or 30 minutes (CYP4A11), 10% formic acid was used to terminate the reactions. After adding ethanol, a part of samples was collected and mixed with the internal standard (20-HETE-d6) (Cayman Chemical, Ann Arbor, MI). After centrifugation (4°C, 140 × g, 10 minutes), the supernatants were analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS) to determine the 20-HETE concentration. The LC-MS/MS analysis was conducted according to a previous report (Kawamura et al., 2022). The IC50 values were calculated using a nonlinear least squares method by SAS9.2 (SAS Institute, Tokyo, Japan).

Selectivity Assays

The binding and enzyme assays for 55 receptors, channels, transporters, and enzymes were performed by Eurofins (Le Bois l’Evêque, France). TP0472993 was tested in duplicate at 30 μM.

Effects of TP0472993 on Renal and Plasma 20-HETE Levels in Mice and Rats

20-HETE synthase activity has been shown to be higher in renal microsomes prepared from male mice than those from female mice because of the upregulation of Cyp4a12a expression, which is induced by androgen (Muller et al., 2007). Therefore, in these experiments, we selected male animals, considering the influence by the higher production of 20-HETE related with androgen. We randomly assigned male Balb/c mice (8 weeks of age) into treatment groups receiving 0.5% methyl cellulose (MC) or TP0472993 (0.3–3 mg/kg) (n = 5 per group and timepoint). The mice were administered 0.5% MC or TP0472993 (suspension in 0.5% MC) via oral gavage. At 2 and 8 hours after administration, blood samples (500 μl) were collected in EDTA tubes via the postcaval vein under anesthesia with isoflurane. Then, the mice were killed by exsanguination, and both kidneys were harvested for the measurement of 20-HETE. All specimens were kept frozen until the measurement of the 20-HETE and TP0472993 concentrations.

We also randomly assigned male SD rats (8 weeks of age) to treatment groups receiving 0.5% MC or TP0472993 (0.3–10 mg/kg) (n = 5 per group and timepoint). The rats were administered 0.5% MC or TP0472993 (suspension in 0.5% MC) via oral gavage. At 4, 8, 12, 16, 20, and 24 hours after administration, 5 ml of blood sample was taken in EDTA tube via the postcaval vein under anesthesia with isoflurane. Then, the rats were euthanized by exsanguination, and left kidney was harvested for the measurement of 20-HETE. All specimens were kept frozen until the measurement of the 20-HETE and TP0472993 concentrations.

Determination of Renal 20-HETE Concentration

The kidney samples were homogenized in four times their weight of ethanol and 100 μl of 10 mM triphenylphosphine. A part of the homogenates was mixed with the internal standard (20-HETE-d8). After centrifugation (4°C, 3250 × g, 10 minutes), the concentration of 20-HETE in the supernatant was determined by LC-MS/MS. The LC-MS/MS analysis was conducted according to past experiments (Kawamura et al., 2022). The levels of renal 20-HETE were shown as ng/g of kidney weight. The lower limit of quantification (LLOQ) was 0.1 ng/g for the mouse kidney analysis and 0.2 ng/g for the rat kidney analysis. 20-HETE-d8 was synthesized at Taisho Pharmaceutical Co., Ltd.

Determination of Plasma 20-HETE Concentration

The plasma samples (150 μl) were spiked with the internal standard (20-HETE-d8) and applied to Bond Elut C18 cartridges (Agilent, Santa Clara, CA). Then, the extract was subjected to LC-MS/MS to quantify 20-HETE. The LC-MS/MS analysis was conducted according to a previous report (Kawamura et al., 2022). The LLOQ was 0.03 ng/ml for plasma 20-HETE concentration.

Determination of Plasma TP0472993 Concentration

Plasma TP0472993 concentrations were determined by an API4000 triple quadrupole mass spectrometer (AB SCIEX, Framingham, MA) equipped with an LC-20AD high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan). Chromatographic separation was performed on a Shim-pack XR-ODS column (3.0-mm I.D. × 30 mm, 2.2 μm; Shimadzu) using 0.1% acetic acid aqueous solution and acetonitrile as the mobile phase for binary gradient elution. Each analytical batch of samples was run with calibration standard and quality control samples, which were prepared by blank plasma and appropriate amount of TP0472993.

FA Nephropathy Model

Male C57BL/6J mice (9 weeks of age) were administered intraperitoneally with 0.3 M NaHCO3 (vehicle) or FA (250 mg/kg) (Sigma-Aldrich, St. Louis, MO). After 14 days of FA administration, they were killed by exsanguination under anesthesia with isoflurane, and the kidneys were harvested for the analysis of the expression of CYP4A and GPR75. In an intervention study, blood sample (100 μl) was drawn from the mice via tail vein after 2 days of FA injection, and blood urea nitrogen levels were determined by the Urea Nitrogen Colorimetric Detection Kit (Arbor Assays, Ann Arbor, MI). Based on the blood urea nitrogen level and body weight (Souza et al., 2015), FA nephropathy mice were randomly assigned to the following groups: vehicle (0.5% MC), TP0472993 (0.3 and 3 mg/kg), and losartan (n = 8 per group). The mice were administered 0.5% MC or TP0472993 (suspension in 0.5% MC) via oral gavage twice a day for 12 days beginning 2 days after the FA injection to avoid the potential inhibition of acute kidney injury. Losartan (0.1 mg/ml) (Tokyo Chemical Industry, Tokyo, Japan) was administered via drinking water. Previous studies have shown to be effective against kidney fibrosis in mouse models of nephrotoxic nephritis and obstructive nephropathy at 0.1 mg/ml of losartan (Ougaard et al., 2018; Song et al., 2019). After 14 days of FA administration, all animals were killed by exsanguination under anesthesia with isoflurane. Subsequently, the kidneys were collected for histopathological and biochemical assessment.

Obstructive Nephropathy Model

Female C57BL/6J mice (9 weeks of age) were performed unilateral ureteral obstruction (UUO) surgery. Under isoflurane anesthesia, the left ureter was occluded by two-point ligations and cut between them. After 1 or 7 days of surgery, mice were euthanized by exsanguination under anesthesia with isoflurane. Then, the obstructed kidney was harvested for analysis of CYP4A and GPR75 protein expression. Age-matched female C57BL/6J mice served as normal controls. In the intervention study, we randomly assigned the mice to the following treatment groups: vehicle (0.5% MC), TP0472993 (0.3 and 3 mg/kg), and losartan (n = 8 per group). The mice were administered 0.5% MC or TP0472993 (suspension in 0.5% MC) via oral gavage twice a day starting on the day of operation and for 7 days after the surgery. Losartan (0.1 mg/ml) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was administered via drinking water (Ougaard et al., 2018; Song et al., 2019). Age-matched, vehicle-treated female C57BL /6J mice served as normal controls. After 7 days of the surgery, all animals were killed by exsanguination under anesthesia with isoflurane, and the obstructed kidney was harvested for histopathological and biochemical analysis.

Effects of TP0472993 on Renal Hemodynamics in SD Rats

Male SD rats (12 weeks of age) were anesthetized with intraperitoneal injection of 50 mg/kg of thiobutabarbital (Inactin; Sigma-Aldrich) and warmed by heating pad to ensure a body temperature at 37°C. To facilitate breathing, PE-240 tubing was placed in the trachea. Then, the femoral artery and vein were cannulated with PE-50 tubing to measure the mean arterial pressure (MAP) and heart rate (HR) and for intravenous infusion. Renal blood flow (RBF) was assessed by placing an ultrasonic flow probe (Transonic Systems, Ithaca, NY) on the renal artery. After a 30-minute equilibration, the MAP, RBF, and HR were monitored over a 15-minute baseline period. Subsequently, the rats received vehicle (10% sulfobutylether-β-cyclodextrin) or a bolus injection of TP0472993 (1.7 mg/kg) followed by continuous intravenous infusion (0.55 mg/kg per hour) to maintain a blood TP0472993 concentration similar to the peak level obtained after oral administration of 3 mg/kg of TP0472993. The MAP, RBF, and HR were then recorded every 5 minutes for 15 minutes after drug administration. After experiments, all animals were killed by exsanguination under anesthesia with Inactin.

Western Blot Analysis

Kidney tissues were lysed on ice using RIPA buffer (Thermo Fisher Scientific, Waltham, MA) containing protease and phosphatase inhibitors (Thermo Fisher Scientific). After centrifugation (4°C, 11,100 × g, 15 minutes), the supernatants (50 or 20 micrograms of protein) were electrophoresed in 5%–20% polyacrylamide gels. The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. After 1 hour of incubation at room temperature in blocking solution (Tris-buffered saline with 0.1% Tween 20 containing 5% skim milk or bovine serum albumin), the membranes were reacted with either an anti–cytochrome P450 4A/CYP4A11 antibody (Ab) (1:1,000, ab3573; Abcam, Cambridge, MA), an anti–GPR75 Ab (1:500, LS-C109745; LSBio, Seattle, WA), an anti–ERK1/2 Ab (1:1,000, 4695; Cell Signaling Technology, Danvers, MA), an anti–phosphorylated ERK1/2 Ab (1:1,000, 9101; Cell Signaling Technology), an anti–STAT3 Ab (1:2,000, 4904; Cell Signaling Technology), an anti–phosphorylated STAT3 Ab (1:2,000, ab76315; Abcam), or an anti–alpha tubulin Ab (1:10,000, ab176560; Abcam) overnight at 4°C. Then, the blots were reacted with a secondary Ab conjugated with horseradish peroxidase (1:10,000, ab205718; Abcam) for 30 minutes at room temperature. The blots were developed by an ECL Prime Western Blotting Detection Reagent (Cytiva, Marlborough, MA), and the band intensities were quantified by a FUSION imaging system (Vilber-Lourmat, Marne-la-Vallée, France).

Sircol Collagen Assay

Kidney tissues were lysed and centrifuged as described above. The supernatants were assessed by the Sircol Collagen Assay Kit (Biocolor, Antrim, UK) to determine the collagen concentration. The concentration of total protein was measured using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). The collagen content in each sample was expressed as milligrams of collagen per gram of total protein.

Enzyme-Linked Immunosorbent Assay

Kidney tissues were lysed and centrifuged as described above. The interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) levels in the supernatants were measured using DuoSet ELISA (enzyme-linked immunosorbent assay) (R&D Systems, Minneapolis, MN).

Histochemistry

The kidneys were fixed by immersion overnight at 4°C in 4% paraformaldehyde and sucrose in phosphate-buffered saline (PBS). The kidneys were then placed overnight at 4°C in PBS with 30% sucrose. Thereafter, the tissues were frozen in OCT embedding medium (Tissue-Tek, Torrance, CA). To determine the degree of renal fibrosis, five-micrometer frozen sections were prepared and subjected to Masson’s trichrome staining using a Trichrome Stain Kit (ScyTek Laboratories, Logan, UT). Ten randomly chosen nonoverlapping fields were collected by a BZ-X710 microscope (Keyence, Osaka, Japan), and fibrotic areas were quantified using the BZ-X Analyzer (Keyence).

Statistics

Unless otherwise described, data are presented as mean ± S.E.M. The variance homogeneity was tested using the F-test or Bartlett’s test. The Aspin-Welch t test or Student’s t test was used to determine the significance of differences between two groups. The Steel’s test or Dunnett’s test was performed to verify the significance of differences among multiple groups. The SAS9.2 was used for statistical evaluation. A value of P < 0.05 was defined as statistically significant.

Results

In Vitro Characterization of TP0472993

As previously reported, the IC50 values of TP0472993 against 20-HETE formation in human and rat renal microsomes were 29 and 347 nM, respectively (Kawamura et al., 2022) (Table 1). To further characterize the inhibitory profiles of TP0472993 for the primary 20-HETE-producing P450 isoforms in human, we additionally evaluated the IC50 values for CYP4F2 and CYP4A11. TP0472993 concentration dependently inhibited 20-HETE production by CYP4F2 and CYP4A11 (Fig. 2, A and B). The IC50 values of TP0472993 for CYP4F2 and CYP4A11 were 37 and 425 nM, respectively. In contrast, TP0472993 at a concentration of 30 μM had no apparent affinity for 55 representative receptors, enzymes, ion channels, and transporters (Table 2).

View this table:
TABLE 1

Effect of TP0472993 on 20-HETE formation by CYP4F2, CYP4A11, and human and rat renal microsomes

The values are expressed as the geometric means of the results from at least two assays.

Fig. 2.
Fig. 2.

Effects of TP0472993 on the metabolism of arachidonic acid by CYP4F2 (A) and CYP4A11 (B) enzymes responsible for the formation of 20-HETE in human kidneys. Results are expressed as a percentage of the control value. The values are expressed as the mean of two independent experiments performed in duplicate.

View this table:
TABLE 2

Selectivity of TP0472993 at 30 μM over 55 receptors, ion channels, transporters, and enzymes

The values are presented as the means from two experiments.

Effects of TP0472993 on Renal 20-HETE Levels in Balb/c Mice

To investigate the effects of TP0472993 on 20-HETE levels in the kidney of mice, Balb/c mice were subjected to a single oral administration of TP0472993. A representative chromatogram showing the 20-HETE standard and the typical renal 20-HETE level in Balb/c mice treated with the vehicle or TP0472993 is presented in Fig. 3. 20-HETE was quantifiable in the kidney of Balb/c mice, but the Balb/c mice had no quantifiable 20-HETE in their plasma (data not shown). The renal 20-HETE levels in Balb/c mice at 2 and 8 hours after administration are presented in Fig. 4A. The renal 20-HETE levels dose-dependently decreased at 2 and 8 hours after the administration of TP0472993, and a significant inhibitory effect was found at doses of more than 0.3 mg/kg. The relationship between the plasma TP0472993 concentrations and the percentage inhibition of renal 20-HETE formation for each individual animal are presented in Fig. 4B. These data revealed that TP0472993 consistently inhibited the renal 20-HETE formation in Balb/c mice in a drug concentration-dependent manner.

Fig. 3.
Fig. 3.

Comparison of the chromatograms of 20-HETE in kidney extracts from mice treated with the vehicle or TP0472993. Panels A and B present typical chromatograms for the internal standard (20-HETE-d8) and the 20-HETE standard, respectively. Panels C and D present typical chromatograms for 20-HETE in kidney extracts from Balb/c mice treated with the vehicle and TP0472993 (3 mg/kg), respectively.

Fig. 4.
Fig. 4.

Effects of TP0472993 on renal 20-HETE levels in Balb/c mice. (A) Renal 20-HETE levels at 2 and 8 hours after a single oral administration of TP0472993 at different doses. The mean values ± S.E.M. from five mice per group are presented. *P < 0.05 vs. the corresponding value measured in the vehicle control (Steel’s test). (B) PK/PD analysis of TP0472993. The PD effects were determined using the percentage inhibition of renal 20-HETE in TP0472993-treated animals compared with corresponding vehicle-treated animals. Data below the limit of quantification were calculated as LLOQ (0.1 ng/g) in this analysis. The PD effects were plotted against the corresponding plasma TP0472993 concentrations.

Effects of TP0472993 on Kidney Fibrosis in FA Nephropathy Mice

A comparison of the renal expressions of CYP4A and GPR75 proteins in normal and FA nephropathy mice is presented in Fig. 5, A and B. In comparison with normal mice, the renal protein levels of CYP4A, which is the 20-HETE-producing enzyme, increased by 5.5-fold in the FA nephropathy mice after 14 days of FA injection. The renal protein levels of GPR75, the receptor to which 20-HETE binds, also increased by 3.5-fold in these animals. The effect of TP0472993 treatment on the development of kidney fibrosis in FA nephropathy mice is presented in Fig. 5, C–E. Tubulointerstitial fibrosis was seen in vehicle-treated FA nephropathy mice (Fig. 5C). The percentage of Masson’s trichrome-positive area was 4.7-fold higher in the FA nephropathy mice received vehicle than in normal mice. Chronic treatment of the mice with 3 mg/kg of TP0472993 significantly reduced renal fibrosis by 66% in the percentage of the areas stained with Masson’s trichrome in comparison with the levels measured in the mice received vehicle (Fig. 5D). The renal collagen content was markedly greater in the FA nephropathy mice that received vehicle in comparison with the normal mice. Chronic treatment of the mice with 0.3 and 3 mg/kg of TP0472993 markedly reduced renal collagen content (Fig. 5E). However, chronic administration of losartan did not show significant effects on the percentage of Masson’s trichrome-positive area and the renal collagen content (Fig. 5, D and E). No significant differences were found in plasma creatine concentrations between normal and vehicle-treated FA nephropathy mice at 14 days after FA injection (data not shown).

Fig. 5.
Fig. 5.

Effects of TP0472993 on renal fibrosis in FA nephropathy mice. (A) Western blots showing the expression of CYP4A and GPR75 in kidneys collected from normal and FA nephropathy mice at 14 days after vehicle (0.3 M NaHCO3) or FA injection. (B) The levels of protein were normalized according to the α-tubulin level and expressed as a ratio of that in normal mice. The mean values ± S.E.M. from three mice per group are presented. *P < 0.05 vs. the corresponding value measured in normal mice (Student’s t test). (C) Representative images of kidney tissue stained with Masson’s trichrome in normal control and FA nephropathy mice treated with the vehicle (0.5% MC) or TP0472993 (3 mg/kg) twice a day by oral gavage or losartan (0.1 mg/ml) through drinking water for 12 days. Scale bars represent 100 μm. (D) A quantitative analysis of the blue-stained area was performed on 10 random nonoverlapping fields showing the kidney tissue. The mean values ± S.E.M. from seven to eight mice per group are presented. ###P < 0.001 vs. normal control (Aspin-Welch’s t test). **P < 0.01 vs. vehicle control (Steel’s test). (E) The collagen content in kidney homogenates was quantified using the Sircol collagen assay. The mean values ± S.E.M. from eight mice per group are presented. ###P < 0.001 vs. normal control (Student’s t test). ***P < 0.001, *P < 0.05 vs. vehicle control (Dunnett’s test).

Effects of TP0472993 on Renal Inflammatory Cytokine Levels in FA Nephropathy Mice

The effects of TP0472993 on the renal cytokine levels in the FA nephropathy mice are presented in Fig. 6. In comparison with normal mice, the IL-1β and TNF-α levels in the kidney were markedly elevated in vehicle-treated FA nephropathy mice. Chronic treatment of the mice with 3 mg/kg of TP0472993 significantly reduced the renal IL-1β and TNF-α levels. In contrast, chronic treatment with losartan did not show significant effect on IL-1β and TNF-α levels.

Fig. 6.
Fig. 6.

Effects of TP0472993 on renal IL-1β (A) and TNF-α (B) levels in FA nephropathy mice. The mean values ± S.E.M. from eight mice per group are presented. ###P < 0.001 vs. the corresponding value measured in normal control (Student’s t test). *P < 0.05 vs. the corresponding value measured in vehicle control (Dunnett’s test).

Effects of TP0472993 on Kidney Fibrosis in UUO Mice

A comparison of CYP4A and GPR75 protein expression in the kidneys of normal and UUO mice is presented in Fig. 7, A and B. In comparison with normal mice, the renal CYP4A protein level had increased significantly in UUO mice after 7 days of the surgery. Similarly, the renal GPR75 protein level was remarkably higher in UUO mice than those seen in normal mice. The effect of TP0472993 on the development of kidney fibrosis in UUO mice is presented in Fig. 7, C–E. The kidneys of vehicle-treated UUO mice showed severe renal interstitial fibrosis (Fig. 7C). The percentage of areas stained with Masson’s trichrome was 6.4-fold greater in the UUO mice that received vehicle than in normal mice at 7 days after surgery. Chronic treatment of the mice with 3 mg/kg of TP0472993 significantly reduced kidney fibrosis by 61% in comparison with the UUO mice that received vehicle (Fig. 7D). The renal collagen content was markedly greater in the UUO mice that received vehicle in comparison with the normal mice. Chronic treatment of the mice with 3 mg/kg of TP0472993 markedly reduced the renal collagen content by 43%, in comparison with the values found in the mice receiving the vehicle (Fig. 7E). Similarly, losartan also significantly reduced the renal collagen content by 27%, in comparison with the values measured in the UUO mice that received vehicle.

Fig. 7.
Fig. 7.

Effects of TP0472993 on renal fibrosis in UUO mice. (A) Western blots showing the expression of CYP4A and GPR75 in kidneys collected from normal and UUO mice at the indicated number of days after ligation. (B) The levels of protein were normalized using α-tubulin and expressed as a ratio of the values in normal mice. The mean values ± S.E.M. from three mice per group are presented. ***P < 0.001, *P < 0.05 vs. the corresponding value measured in normal mice (Dunnett’s test). (C) Representative images of the kidney tissue stained with Masson’s trichrome in normal control and UUO mice treated with the vehicle (0.5% MC) or TP0472993 (3 mg/kg) twice a day by oral gavage or losartan (0.1 mg/ml) through drinking water for 7 days. Scale bars represent 100 μm. (D) A quantitative analysis of the blue-stained area was performed using 10 random nonoverlapping fields showing the kidney tissue. The mean values ± S.E.M. from seven to eight mice per group are presented. ###P < 0.001 vs. normal control (Aspin-Welch’s t test). **P < 0.01, *P < 0.05 vs. vehicle control (Steel’s test). (E) The total collagen content in kidney homogenates was quantified using the Sircol collagen assay. The mean values ± S.E.M. from eight mice per group are presented. ###P < 0.001 vs. normal control (Aspin-Welch’s t test). ***P < 0.001 vs. vehicle control (Dunnett’s test).

Effects of TP0472993 on Renal Inflammatory Cytokine Levels in UUO Mice

The effects of TP0472993 on cytokine levels in the kidney of UUO mice are presented in Fig. 8. Renal IL-1β and TNF-α levels were higher in the UUO mice that received vehicle than in normal mice. Chronic treatment of the mice with 3 mg/kg of TP0472993 markedly reduced the renal IL-1β and TNF-α levels. In comparison with UUO mice receiving the vehicle, chronic treatment with losartan reduced these cytokines levels.

Fig. 8.
Fig. 8.

Effects of TP0472993 on renal IL-1β (A) and TNF-α (B) levels in UUO mice. The mean values ± S.E.M. from seven to eight mice per group are presented. ###P < 0.001, #P < 0.05 vs. the corresponding value measured in the normal control (Aspin-Welch’s t test). *P < 0.05 (Steel’s test), †P < 0.05 (Dunnett’s test) vs. the corresponding value measured in the vehicle control.

Effects of TP0472993 on ERK1/2 and STAT3 Phosphorylation in UUO Mice

The effects of TP0472993 on ERK1/2 and STAT3 phosphorylation in UUO mice are presented in Fig. 9. In comparison with normal mice, the expression of phosphorylated ERK1/2 was elevated in the kidney of UUO mice that received vehicle (Fig. 9, A and B). Chronic treatment of the mice with TP0472993 markedly suppressed ERK1/2 phosphorylation. Interestingly, the total ERK1/2 expression was elevated in the kidneys of UUO mice receiving the vehicle as well compared with that in normal mice, but chronic treatment with TP0472993 did not affect its expression. The renal expression of phosphorylated STAT3 was barely detected in normal mice (Fig. 9C), although it was increased in vehicle-treated UUO mice (Fig. 9, C and D). Chronic administration of TP0472993 suppressed STAT3 phosphorylation in UUO mice.

Fig. 9.
Fig. 9.

Effects of TP0472993 on ERK1/2 and STAT3 phosphorylation in UUO mice. (A) Representative western blots show the expression of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2) in different groups, with α-tubulin used as a loading control. (B) Relative levels of ERK1/2 phosphorylation are expressed as a ratio of p-ERK1/2 to t-ERK1/2. The mean values ± S.E.M. from eight mice per group are presented. ###P < 0.001 vs. normal control (Student’s t test). **P < 0.01 vs. vehicle control (Student’s t test). (C) Representative western blots show the expression of phosphorylated STAT3 (p-STAT3) and total STAT3 (t-STAT3) in different groups, with α-tubulin used as a loading control. (D) Relative levels of STAT3 phosphorylation are expressed as the ratio of p-STAT3 to t-STAT3. The mean values ± S.E.M. from eight mice per group are presented. ###P < 0.001 vs. normal control (Aspin-Welch’s t test). *P < 0.05 vs. vehicle control (Student’s t test).

Effects of TP0472993 on Renal and Plasma 20-HETE Levels in SD Rats

The effect of TP0472993 on 20-HETE levels in the kidney of SD rats is presented in Fig. 10A. Treatment with TP0472993 significantly decreased the renal 20-HETE levels at 4 hours after administration, and this inhibitory effect lasted up to 24 hours after administration at doses greater than 3 mg/kg. The plasma 20-HETE levels from the same cohort of rats are presented in Fig. 10C. Treatment with TP0472993 (0.3 to 10 mg/kg) decreased the plasma 20-HETE levels in a dose-response fashion. The temporal patterns of 20-HETE reduction were comparable between the kidney and plasma, and a sustained reduction was observed for over 24 hours after administration at 10 mg/kg of dose. The relationships between the plasma TP0472993 concentrations and the percentage inhibition of 20-HETE in the kidney and plasma for each individual animal are presented in Fig. 10, B and D. These data showed that TP0472993 consistently reduced the renal and plasma 20-HETE levels in a drug concentration-dependent fashion.

Fig. 10.
Fig. 10.

Effects of TP0472993 on renal and plasma 20-HETE levels in SD rats. (A) Renal 20-HETE levels at 4, 8, 12, 16, 20, and 24 hours after a single oral administration of TP0472993 at different doses. The mean values ± S.E.M. from five rats per group are presented. *P < 0.05 vs. the corresponding value measured in the vehicle control (Steel’s test). (B) PK/PD analysis for the effect on renal 20-HETE. The percentage of inhibition of renal 20-HETE was plotted against the corresponding plasma TP0472993 concentration. Data below the limit of quantification were calculated as LLOQ (0.2 ng/g) in this analysis. (C) Plasma 20-HETE levels at 4, 8, 12, 16, 20, and 24 hours after a single oral administration of TP0472993 at different doses. The mean values ± S.E.M. from five rats per group are presented. *P < 0.05 vs. the corresponding value measured in the vehicle control (Steel’s test). (D) PK/PD analysis for the effect on plasma 20-HETE. The percentage of inhibition of plasma 20-HETE was plotted against the corresponding plasma TP0472993 concentration.

Effects of TP0472993 on MAP, RBF, and HR in SD Rats

To assess the effects of TP0472993 on renal hemodynamic parameters at a therapeutic dose, TP0472993 was administered intravenously as a 1.7 mg/kg bolus, followed by a constant infusion at 0.55 mg/kg/h, in SD rats. In preliminary experiments, this dose of TP0472993 was found to be sufficient to maintain a plasma exposure at the same level as the peak concentration (Cmax) achieved from a 3 mg/kg oral dosing in SD rats (data not shown). As shown in Fig. 11, the administration of TP0472993 did not affect the MAP, RBF, or HR in SD rats.

Fig. 11.
Fig. 11.

Effects of TP0472993 on MAP, RBF, and HR in SD rats. (A) Schematic of the experimental protocol. MAP (B), RBF (D), and HR (F) were measured every 5 minutes for 15 minutes before and after the intravenous infusion of the vehicle (10% sulfobutylether-β-cyclodextrin) or TP0472993. The percent changes from baseline in MAP (C), RBF (E), and HR (G) after intravenous infusion of the vehicle or TP0472993 are shown. The mean values ± S.E.M. from four to five rats per group are presented.

Discussion

In the current research, we evaluated the pharmacological effect of TP0472993 against 20-HETE production in vitro and in vivo as well as its potential effect on the development of kidney fibrosis after FA- and obstructive-induced nephropathy in mice. TP0472993 has been shown to inhibit the production of 20-HETE in kidney microsomes from human and rat, and the IC50 values were 29 and 347 nM, respectively (Kawamura et al., 2022). We confirmed that TP0472993 inhibited 20-HETE production by CYP4F2 and CYP4A11, and the IC50 values were 37 and 425 nM, respectively. In contrast, TP0472993 did not affect the activities of P450 isoforms responsible for human hepatic drug metabolism at a concentration of up to 10 μM (Kawamura et al., 2022). Furthermore, we also confirmed that TP0472993 at a concentration of 30 μM did not exert an apparent inhibitory effect on a wide range of receptors, enzymes, ion channels, and transporters. These findings indicate that TP0472993 is a highly selective inhibitor of 20-HETE–producing P450 enzymes.

So far, some potent and highly selective inhibitors of 20-HETE synthesis have been developed such as HET0016 and TS-011 (Miyata et al., 2001, 2005). However, these compounds have a limitation of the use for patients with chronic disease such as CKD due to not being orally available. Our results indicated that oral administration of TP0472993 suppressed renal 20-HETE production in mice and rats in a dose-response fashion and completely reduced the renal 20-HETE levels at a dose of 3 mg/kg for up to 8 and 24 hours after administration in mice and rats, respectively. These results suggest that TP0472993 has more attractive properties than previous 20-HETE synthesis inhibitors in view of clinical applications such as oral availability leading to the better adherence for CKD patients. Furthermore, the blockade of 20-HETE formation in the kidneys of mice and rats was closely related to the plasma exposure of TP0472993, suggesting that TP0472993 has shown an excellent pharmacokinetic-pharmacodynamic (PK/PD) correlation. We also found that the levels of renal 20-HETE in SD rats changed during the 24-hour experimental period, with peak levels detected at 20 hours after dosing. Although the reasons for the transient 20-HETE elevation remain to be elucidated, one likely explanation for these findings is feeding-associated changes in the expression of CYP4A (Hardwick, 2008). Of note, TP0472993 inhibited renal 20-HETE formation to the same degree regardless of the basal 20-HETE level. This finding indicates that the inhibitory effects of TP0472993 on renal 20-HETE formation may not be affected by the CYP4A activity and/or expression.

The renal CYP4A protein expression increased along with the progression of kidney fibrosis in mouse models of FA and obstructive nephropathy, suggesting that the 20-HETE production via CYP4A is elevated in fibrotic kidneys. Interestingly, we also found that the renal expressions of CYP4A and GPR75 increased on day 1 and persistently increased up to day 7 postsurgery in UUO mice. Altogether, these findings indicate that the 20-HETE–GPR75 pathway may be involved in the initiation and development of kidney fibrosis. In support of our hypothesis, the current experiment revealed that the blockade of 20-HETE formation using TP0472993 significantly reduced renal fibrosis, as demonstrated by reductions in Masson’s trichrome-positive area and renal collagen content in FA nephropathy and UUO mice. Our findings indicate a possible involvement of 20-HETE in the progression of kidney fibrosis in both animal models. These results further imply that 20-HETE synthesis inhibitors have potential to delay the progression of kidney fibrosis in CKD patients. On the other hand, we found that plasma creatinine levels in FA nephropathy mice were similar to those in normal mice at 14 days after FA injection, indicating that the degree of renal fibrosis in this model was modest and not sufficient to impair renal function. Thus, further studies in CKD models with impairment of kidney function are necessary to determine whether TP0472993 improves renal function.

Considerable data indicate that chronic inflammation is tightly involved in the pathogenesis of kidney fibrosis in CKD (Lv et al., 2018). Our results indicate that renal IL-1β and TNF-α expression was elevated in FA nephropathy and UUO mice. Chronic treatment with TP0472993 significantly attenuated renal IL-1β and TNF-α expression in both animal models. These observations indicate that the antifibrotic effect of TP0472993 might, at least in part, be mediated by the inhibition of kidney inflammation. Profibrotic signaling pathways, including ERK1/2 and STAT3, have shown to play key roles in the pathophysiological processes involved in kidney inflammation and fibrosis (Tao et al., 2019; Park et al., 2022). Thus, we further studied the effect of TP0472993 on these profibrotic signaling pathways in UUO mice. In our study, phosphorylated ERK1/2 and STAT3 expression in the kidney was markedly increased in UUO mice along with the development of kidney fibrosis. Chronic treatment with TP0472993 significantly inhibited ERK1/2 and STAT3 phosphorylation in kidney tissue. These findings are in agreement with the observation that the chronic inhibition of 20-HETE synthesis decreases renal ERK1/2 phosphorylation in cystic rats (Park et al., 2009). Several investigators have reported that 20-HETE induces inflammation in endothelial cells through GPR75 (Garcia et al., 2017; Rocic and Schwartzman, 2018). Moreover, 20-HETE induces ERK1/2 and STAT3 activation through a GPR75-dependent mechanism (Gkouveris et al., 2014; Garcia et al., 2017; Murtaza et al., 2022). In this regard, we found that renal GPR75 expression elevated in UUO mice during the development of kidney fibrosis. Although additional studies are necessary to address this issue, blockade of 20-HETE production with TP0472993 appears to suppress the development of kidney fibrosis by attenuating kidney inflammation mediated via the ERK1/2 and STAT3 pathways through GPR75.

We also compared the renoprotective effects of TP0472993 with those observed in mice treated with losartan, which is a conventional therapy for CKD. In agreement with past observations (Nishida et al., 2012; Gu et al., 2020), losartan prevented the progression of kidney fibrosis in UUO mice. However, it only slightly and nonsignificantly reduced renal fibrosis in FA nephropathy mice. Reasons behind the failure of losartan to attenuate renal injury in FA nephropathy mice remain to be determined. Previous investigators have reported that plasma renin activity fell to low levels after the injection of FA in rats (Helmchen et al., 1972). Therefore, we suspect that losartan might have a small or limited renoprotective effect in FA nephropathy mice because of a low activity of the renin angiotensin system after FA injection. Conversely, losartan reportedly slowed the progression of kidney diseases in various CKD models by altering renal hemodynamics (Mihailovic-Stanojevic et al., 2009; García Trejo et al., 2017). 20-HETE has a strong vasoconstrictive action that contributes to RBF autoregulation and renal vascular response (Roman, 2002), suggesting that 20-HETE synthesis inhibitors have a potential risk to alter renal hemodynamics. We also considered the possibility that the renoprotective effect of TP0472993 acts through the alteration of renal hemodynamics. However, in this study, the acute infusion of TP0472993 did not affect the MAP, RBF, or HR within the pharmacological dosing range in SD rats. Altogether, the current observations indicate that the chronic inhibition of 20-HETE synthesis with TP0472993 suppresses kidney fibrosis by attenuating kidney inflammation and profibrotic signaling pathways without altering renal hemodynamics.

In clinical trials, PD biomarkers are important for validating target engagement, demonstrating pharmacological activity, and identifying appropriate dosages. The present results suggest that TP0472993 decreases plasma 20-HETE levels in SD rats according to a dose-response relationship that is similar to that observed for renal 20-HETE. Thus, the PD effects of TP0472993 on 20-HETE levels were nearly equivalent between kidney and plasma, suggesting the potential of plasma 20-HETE as a PD biomarker for the inhibition of renal 20-HETE in future clinical trials. Identifying patients in whom 20-HETE synthesis inhibitors are effective is also important for the development of drugs targeting a heterogeneous disease such as CKD. Recent clinical reports indicate that the level of serum 20-HETE is elevated in CKD patients and is independently related to CKD progression (Afshinnia et al., 2020). In addition, serum 20-HETE concentrations are reportedly correlated with a decline in estimated glomerular filtration rate (eGFR) in autosomal dominant polycystic kidney disease patients (Klawitter et al., 2014). These observations suggest that plasma 20-HETE level can be used as a potential marker for stratifying CKD patients according to the likelihood of a better response to treatment with 20-HETE synthesis inhibitors.

In conclusion, this study suggests that the chronic treatment with a selective inhibitor of 20-HETE synthesis, TP0472993, suppresses the progression of kidney fibrosis after FA- and obstructive-induced nephropathy in mice. These findings indicate that 20-HETE has crucial roles in the progression of kidney fibrosis and that 20-HETE synthesis inhibitor TP0472993 is an attractive therapeutic option for CKD patients.

Acknowledgments

The authors thank Emi Gunji and Takuya Takeda for technical support and Hiroaki Tanaka and Koichi Kumasaka for providing us with TP0472993.

Data Availability

The authors declare that all of the data supporting the findings of this research are contained within the manuscript.

Authorship Contributions

Participated in research design: Hirata, Kojima, Takahashi.

Conducted experiments: Hirata, Ohara, Kojima, Koretsune, Hasegawa.

Performed data analysis: Hirata, Ohara, Hasegawa.

Wrote or contributed to the writing of the manuscript: Hirata, Kojima, Inatani, Takahashi.

Footnotes

    • Received November 21, 2022.
    • Accepted April 17, 2023.

  • This research received no external funding.

  • No author has an actual or perceived conflict of interest with the contents of this manuscript.

  • dx.doi.org/10.1124/jpet.122.001521.

Abbreviations

CKD
chronic kidney disease
ERK1/2
extracellular signal-regulated kinase 1/2
FA
folic acid
GPR
G protein–coupled receptor
20-HETE
20-hydroxyeicosatetraenoic acid
HR
heart rate
IL-1β
interleukin-1β
LC-MS/MS
liquid chromatography–tandem mass spectrometry
LLOQ
lower limit of quantification
MAP
mean arterial pressure
MC
methyl cellulose
P450
cytochrome P450
PD
pharmacodynamic
PK
pharmacokinetic
RBF
renal blood flow
SD
Sprague-Dawley
STAT3
signal transducer and activator of transcription 3
TNF-α
tumor necrosis factor alpha
UUO
unilateral ureteral obstruction

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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal

20-HETE Synthesis Inhibitor Suppresses Renal Fibrosis

Takashi Hirata, Hiroki Ohara, Naoki Kojima, Hiroko Koretsune, Yoshitaka Hasegawa, Shoko Inatani and Teisuke Takahashi
Journal of Pharmacology and Experimental Therapeutics July 1, 2023, 386 (1) 56-69; DOI: https://doi.org/10.1124/jpet.122.001521
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