SMTP-44D Exerts Renoprotective Effects against Diabetic Nephropathy via Its Anti-inflammatory and Antioxidant Action

Abstract

Diabetic nephropathy (DN) is among the most serious diabetes-related microvascular complications, a disease with risks leading to end-stage kidney disease (ESKD). However, only limited DN treatment options are currently available. DN development and progression involve different pathological mechanisms, including inflammation and oxidative stress. Stachybotrys microspora is a fungus producing the triphenyl phenol SMTP-44D, which exhibits anti-inflammatory and antioxidant properties in several disease models. In this study, we aimed to evaluate the effects of SMTP-44D in a DN mouse model, which was created by removing the right kidney of 6-week-old db/db mice. We administered SMTP-44D for 10 weeks between weeks 6 and 16 of age to observe blood glucose levels, renal function parameters, inflammatory factors, oxidative stress markers, and histopathological characteristics. SMTP-44D treatment did not reduce blood glucose level but significantly decreased serum creatinine and urinary albumin as renal function parameters, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, and nicotinamide adenine dinucleotide phosphate oxidase-1 as inflammation and oxidative stress in the kidney. In addition, histopathological assessment revealed its preventive effect against glomerulosclerosis and local regenerative tubule. Therefore, we discovered that SMTP-44D might protect renal function without affecting blood glucose level in DN possibly via suppression of inflammation and oxidative stress. In conclusion, SMTP-44D could be a potential DN treatment agent, even in patients with poor glycemic control.

INTRODUCTION

Global diabetes mellitus (DM) prevalence has estimably exceeded 1 in 10 adults in 2021 and is expected to keep rising rapidly in the future.¹⁾ DM causes micro- and macrovascular complications. Diabetic nephropathy (DN) is one of the most common and serious microvascular complications, occurring in 30–40% and 15–25% of type 2 and 1 diabetics, respectively, of whom 5–10% eventually develop end-stage kidney disease (ESKD).^2,3)

The pathological features of glomerular lesions in DN consist of glomerular mesangium expansion, basement membrane thickening, podocyte loss, nodular glomerulosclerosis, and endothelial cell destruction.^4,5) These are followed by global glomerulosclerosis in the late DN phases.^4,5) Concerning renal tubulointerstitial lesions, renal tubulointerstitial injury precedes glomerular lesions, and tubular hypertrophy appears in the early DN stages, eventually progressing to interstitial fibrosis with tubular atrophy.^4,6) DN development and progression involve different pathological mechanisms, the renin–angiotensin–aldosterone system, the activation of transforming growth factor-β1 and protein kinase C, and the formation of advanced glycation end product (AGE) and reactive oxygen species.⁷⁾ These pathways induce oxidative stress and inflammation through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), nuclear factor-κB (NF-κB), and other factors.⁷⁾ Furthermore, the formation and deposition of AGE, as well as the increased expression of chemokines and cell adhesion molecules, appear to lead to the accumulation and activation of macrophages in the diabetic kidney. This promotes the progression of renal injury and renal functional decline.^4,8–10)

Stachybotrys microspora is a fungal species that produces a novel class of triprenyl phenols called SMTPs.^11,12) Previously, we demonstrated that SMTPs inhibit soluble epoxide hydrolase (sEH), an enzyme hydrolyzing epoxy fatty acids such as the anti-inflammatory signaling molecules epoxyeicosatrienoic acids (EETs) to the inactive dihydroxyeicosatrienoic acids (DHETs), exerting an anti-inflammatory activity.^12–16) SMTP-44D, an SMTP family molecule, reportedly exhibits anti-inflammatory and antioxidant properties and efficacy against several animal disease models,^17–20) although its DN-related effect remains unclear. In this study, we investigated the renoprotective effects of SMTP-44D in DN mice, with the aim of providing a potential model for future DN treatment.

MATERIALS AND METHODS

Materials and Reagents

We purchased isoflurane from Viatris Inc. (Canonsburg, PA, U.S.A.), LabAssay TM Creatinine, acetaminophen, 10% formalin neutral buffer solution (pH 7.4), and metformin from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), and Enrofloxacin (Baytril®) from Bayer AG (Leverkusen, Germany). We purchased the protease inhibitor cocktail from Sigma-Aldrich Co., LLC (St. Louis, MO, U.S.A.), RIPA buffer from Cayman Chemical Company (Ann Arbor, MI, U.S.A.), and normal saline solution (NS) from Otsuka Pharmaceutical Co., Ltd. (Osaka, Japan). Finally, we bought the monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) enzyme-linked immunosorbent assay (ELISA) kit from R&D systems Inc. (Minneapolis, MN, U.S.A.), NADPH oxidase 1 (NOX-1) ELISA kit from MyBioSource, Inc. (San Diego, CA, U.S.A.), Albumin ELISA kit from Ethos Biosciences, Inc. (Newtown Square, PA, U.S.A.), and BCA assay kit from Thermo Fisher Scientific Inc. (Waltham, MA, U.S.A.). SMTP-44D was generously donated by TMS Co., Ltd. (Tokyo, Japan).

Animals

We purchased 5-week-old male C57BL/6J and db/db mice (a type 2 diabetes model with a leptin receptor mutation) from CLEA Japan, Inc. (Tokyo, Japan). We acclimatized the animals to the laboratory conditions for 1 week prior to the start of the experiments, and housed them all in an air-conditioned animal facility maintained at 23 ± 2°C, with 50 ± 20% of relative humidity and a 12-h light/dark cycle (lights on between 8:00 a.m. and 8:00 p.m.), with ad libitum access to water and food, except for the experimental periods. All animal procedures were approved by the Committee on Animal Care and Welfare of Showa University (Permit Number: 324030) and conformed to the U.S. National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

DN Mouse Model Creation

We established the DN mouse model by removing the right kidney of 6-week-old male db/db mice under isoflurane anesthesia, as previously described.²¹⁾ We used male C57BL/6J mice as the control that underwent the same surgical procedure (n = 8). After surgery, we subcutaneously administered the animals acetaminophen (75 mg/kg) and enrofloxacin (5 mg/kg). We divided the db/db mice into four groups: DN, SMTP-44D (0.3, 3, or 30 mg/kg)-treated DN, and metformin (300 mg/kg)-treated DN groups (n = 6–8 for each group). We administered SMTP-44D intraperitoneally every 2 d or metformin orally and daily for a total of 10 weeks between the ages of 6 and 16 weeks. Mice in the control and DN groups received equivalent volumes of NS intraperitoneally. SMTP-44D reportedly affects diabetic neuropathy and diabetic retinopathy models dose-dependently, with significant effects observed at a dosage of 30 mg/kg administered at the same interval.^17,19) Therefore, in this study, we selected 30 mg/kg as the maximum dose. Finally, we set the metformin dosage (300 mg/kg) based on the previously described effective doses.^22,23)

Physiological Measurements and Sample Collection

We placed the animals in metabolic cages one day prior to urine collection for 24 h. Afterward, we weighed and sacrificed the mice and collected the left kidney and blood for subsequent assays. We measured their blood glucose levels using Medisafe Mini GR-102 (TERUMO, Tokyo, Japan). We centrifuged the blood and urine samples at 25°C and 800 × g for 15 min. We obtained the respective serum and urine supernatants and stored them at −20°C. Following blood and urine collection, we removed the left kidney and weighed the kidney after exfoliating its fa in NS. Finally, we cut the kidney samples into half, flash-freezing one-half in liquid nitrogen for homogenization (storing them at −80°C) and fixing the other in 10% formalin neutral buffer solution.

Renal Function Parameters

We evaluated serum creatinine (SCr) and urinary albumin (UAlb) as described previously.^16,24) We carried out all operations strictly according to the manufacturer’s instructions. We calculated UAlb using the following formula: UAlb (mg/h/kg) = (albumin concentration in urine [μg/mL] × urine volume [μL]) / (collection time [h] × body weight of the mouse [kg] × 10⁶).

ELISA

We extracted total protein from the kidney tissue by homogenizing it with RIPA buffer on ice, followed by centrifugation at 4°C and 1500 × g for 15 min. We collected the supernatant and measured the MCP-1, ICAM-1, and NOX-1 levels. We conducted all procedures in strict accordance with the manufacturer’s instructions. We normalized the level by total protein content of each sample, expressed as the levels per mg of total protein content.

Histopathological Assessment

We fixed the kidney tissues from each group in 10% neutral buffered formalin and subsequently stained them with Periodic Acid-Schiff (PAS) and Hematoxylin–Eosin (H&E). We imaged PAS- and H&E-stained sections at 100× and 50× magnifications, respectively. We assessed PAS staining as described previously²⁵⁾ by counting the number of scleroglomerulus (PAS-positive area of >50%) in the upper half of the kidney, where approximately 100 central, relatively large glomeruli were counted. The severity of glomerulosclerosis was scored as follows: 0, n < 5; 1, n = 6–25; and 2, n = 26–50, where ̔n̕ represents the number of scleroglomeruli counted in each kidney section. We evaluated H&E staining based on local regenerative tubule severity, as described previously.^26,27) The data are expressed as the presence (+) or absence (–) of regenerated renal tubules in the kidneys of each group.

Statistical Analysis

We represented the results as the mean ± standard error of the mean (S.E.M.). We evaluated the continuous variables such as body weight, blood glucose level, SCr, UAlb, MCP-1, ICAM-1, and NOX-1 using one-way ANOVA followed by Bonferroni correction. The ordinal variable glomerulosclerosis score and the binary variable local regenerative tubules severity were evaluated using Kruskal–Wallis test followed by Steel. We considered p-values of p < 0.05 as statistically significant.

RESULTS

SMTP-44D Did Not Affect Body Weight and Blood Glucose Levels

To obtain basic information on the SMTP-44D effects, we assessed the body weight and blood glucose levels and observed that they were significantly higher in the DN group compared with the control (Fig. 1). We observed no significant changes in body weight in either the SMTP-44D (30 mg/kg)-treated or the metformin-treated DN groups compared with the DN group, while the blood glucose levels significantly decreased by 29.9% in the metformin-treated DN group, with no observed changes in the SMTP-44D (30 mg/kg)-treated DN group.

Fig. 1. SMTP-44D Did Not Affect Body Weight and Blood Glucose Levels

Body weight and blood glucose levels of each group are shown. All data represent the mean ± S.E.M. (n = 8 mice/group), and one-way ANOVA with Bonferroni was used for comparisons. S.E.M.: standard error of the mean.

SMTP-44D Exerted Renoprotective Effects

To evaluate the renoprotective effects of SMTP-44D, we assessed the SCr and UAlb levels, which were both significantly higher at 131 and 211% in the DN group compared with the control (Fig. 2). In contrast, the levels of SCr and UAlb were significantly lower in both the SMTP-44D (30 mg/kg)-treated and metformin-treated DN groups compared with the DN group, with reductions of 37.4 and 50.7% for SMTP-44D (30 mg/kg), and 22.2 and 90.8% for metformin, respectively. However, renal function was not significantly improved in the SMTP-44D (0.3 mg/kg)-treated and SMTP-44D (3 mg/kg)-treated DN groups.

Fig. 2. SMTP-44D Had Renoprotective Effects

The levels of SCr and UAlb of each group are shown. All data represent the mean ± S.E.M. (n = 6–8 mice/group), and one-way ANOVA with Bonferroni was used for comparisons. S.E.M.: standard error of the mean.

SMTP-44D Displayed Anti-inflammatory and Antioxidant Effects

To evaluate how SMTP-44D affects DN-associated inflammation and oxidative stress, we assessed the MCP-1, ICAM-1, and NOX-1 levels (Fig. 3). The expression levels of these proteins were significantly increased by 178, 81.4, and 15.0%, respectively, in the DN group compared with the control, and were significantly decreased both in the SMTP-44D (30 mg/kg)-treated DN group (34.5, 25.3, and 27.1%, respectively) and the metformin-treated DN groups (23.0, 31.7, and 13.4%, respectively) compared with the DN group.

Fig. 3. SMTP-44D Exerted Anti-inflammatory and Antioxidant Effects

MCP-1, ICAM-1, and NOX-1 protein level are shown. All data represent the mean ± S.E.M. (n = 7–8 mice/group), and one-way ANOVA with Bonferroni was used for comparisons. S.E.M.: standard error of the mean.

SMTP-44D Reduced Glomerulosclerosis

To evaluate how SMTP-44D impacts the glomeruli, we assessed the degree of glomerulosclerosis by imaging sclerotic glomerular presence within the glomerular structures using PAS staining (Fig. 4A). The degree of glomerulosclerosis was significantly higher in the DN group compared with the control, while we observed no significant changes in the metformin-treated DN group compared with the DN group (Fig. 4B). By contrast, the SMTP-44D (30 mg/kg)-treated DN group displayed a significant reduction in glomerulosclerosis by 64.3% compared with the DN group.

Fig. 4. SMTP-44D Reduced Glomerulosclerosis

(A) Representative images of PAS staining of kidney sections. Scale bar, 100 μm. The arrow indicates sclerotic glomeruli. (B) Kidney section glomerulosclerosis scores in all groups. PAS staining was scored by the number of scleroglomeruli (with PAS positive area of >50%) in upper half of the kidney, where approximately 100 central (relatively large) glomeruli were counted. Score 0,n < 5; 1, n = 6–25; and 2, n = 26–50. All data represent the mean ± S.E.M. (n = 8 mice/group), and Kruskal–Wallis test with Steel was used for comparisons. PAS: Periodic Acid-Schiff; S.E.M.: standard error of the mean.

SMTP-44D Prevented Tubular Injury

To evaluate how SMTP-44D affects tubular injury, we assessed local regenerative tubule severity via H&E staining (Fig. 5A). Local regenerative tubule severity was significantly higher in the DN group than in the control (Fig. 5B: 75% of tubules positive for regeneration). However, while we observed no significant improvement in the metformin-treated DN group (50%), severity was significantly reduced in the SMTP-44D (30 mg/kg)-treated DN group (12.5%) compared with the DN group.

Fig. 5. SMTP-44D Prevented Tubular Injury

(A) Representative H&E staining images of kidney sections Scale bar, 200 μm. The arrows indicate local regenerative tubules. (B) Local regenerative tubule severity of kidney sections in all groups. H&E staining was used to evaluate the presence (+) or absence (–) of regenerated renal tubules in the kidneys of each group (n = 8 mice/group), and Kruskal–Wallis test with Steel was used for comparisons. H&E: Hematoxylin–Eosin.

DISCUSSION

In this study, we demonstrated that SMTP-44D protects mice from DN by inhibiting inflammation and oxidative stress without affecting blood glucose level. Specifically, blood glucose levels did not significantly decrease in the SMTP-44D group compared with the DN group (Fig. 1), consistent with the previous observation that SMTP-44D does not affect blood glucose level in the mouse model of diabetic neuropathy.¹⁹⁾ SMTP-44D treatment significantly reduced the level of the renal failure parameters SCr and UAlb at a dose of 30 mg/kg (Fig. 2). Conversely, we observed no beneficial effects at lower doses of SMTP-44D (0.3 and 3 mg/kg), where SCr levels even slightly increased. Although we have no evidence to explain this result, the fact that kidney function is impaired in sEH-deficient mice compared with wildtype animals after renal ischemia-reperfusion injury²⁸⁾ suggests an implication. After ischemia-reperfusion injury in sEH-deficient mice, renal level of 20-hydroxyeicosatetraenoic acid, a Cyp4a12a-dependent metabolite that aggravates inflammation, increases along with the increase in EET, a Cyp2c/Cyp2j-dependent anti-inflammatory mediator.²⁸⁾ Thus, further investigation is required to understand the dose-dependent profile of the SMTP-44D action, particularly in regard to such mediators involved in the control of inflammation.

SMTP-44D at a dose of 30 mg/kg decreased MCP-1, ICAM-1, and NOX-1 protein levels in the kidney (Fig. 3). In addition, histopathological analysis demonstrated that SMTP-44D prevented glomerulosclerosis and tubular injury (Figs. 4, 5). Thus, SMTP-44D demonstrated therapeutic effects on DN, exhibiting anti-inflammatory and antioxidant activities in the kidneys of DN mice. However, the detailed mechanisms underlying its renoprotective effects remain unclear. SMTP-44D reportedly inhibits sEH.¹²⁾ Previous studies suggested that sEH genetic deficiency or its pharmacological inhibition (e.g., using the sEH inhibitor trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid or podocyte-specific sEH disruption) might offer potential benefits in improving renal function in DN.^29,30) In addition, SMTP-44D reportedly possesses antioxidant properties, which are beneficial for various diseases.^18–20)

EETs are among the metabolic targets of sEH, and SMTP-44D inhibits sEH, thereby increasing the ratio of EETs to their corresponding DHETs.^12,20) The mechanism of EET to exert anti-inflammatory effects can involve inhibition of NF-κB activation and activation of peroxisome proliferator-activated receptor γ (PPARγ) by acting as its ligand.^12,31,32) The decrease in the levels of the NF-κB-driven proteins MCP-1, ICAM-1, and NOX-1 in the SMTP-44D-treated group (Fig. 3) is consistent with this idea.

In addition to the C-terminal epoxide hydrolase activity (sEH-H), sEH has an N-terminal lipid phosphatase activity (sEH-P). Unlike 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA), which selectively inhibits sEH-H, SMTP-44D targets both sEH-H and sEH-P.¹²⁾ However, the precise function of sEH-P has yet to be fully elucidated.³³⁾ A previous study suggested that the inhibition of sEH phosphatase activity could maintain endothelial nitric oxide synthase activation, leading to increased nitric oxide production.³⁴⁾ Since nitric oxide promotes vasodilation,⁵⁾ inhibition of sEH-P may be protective to DN development. In addition, lysophosphatidic acids (LPAs, excellent sEH-P substrates) stabilize nuclear factor erythroid 2-related factor 2 (Nrf2) through LPA receptor activation and directly activate PPARγ as PPARγ ligands.^33,35–37) Both Nrf2 and PPARγ are nuclear transcription factors that induce the transcription of various antioxidant genes.³⁸⁾ These pathways might be involved in the antioxidant effects of SMTP-44D. Furthermore, upregulation of PPARγ expression like that observed for the sEH inhibitor AUDA³⁹⁾ may be involved in the SMTP-44D action.

AGE accumulation in the kidneys has been implicated in the progression of kidney diseases including DN, chronic kidney disease (CKD), and ESKD.^40–42) The formation and accumulation of AGEs are accelerated under diabetic conditions, and these products persist in tissues for prolonged periods in patients with diabetes.^43–46) AGEs exert their effects by binding to the receptor for advanced glycation end products (RAGE), consisting of the AGE/RAGE signaling pathway, which is implicated in oxidative stress and inflammation particularly via NOX-1 and NF-κB activation.^47,48) Previous studies demonstrated that SMTP-44D attenuates the interaction between AGEs and RAGE,⁴⁹⁾ which might be protective to the development of DN.

Metformin is the most widely prescribed treatment for diabetes worldwide, primarily exerting its effects by activating AMPK to improve insulin resistance and lower blood glucose.^50,51) This mechanism is known to secondarily confer anti-inflammatory and antioxidant benefits. In fact, we observed reductions in blood glucose levels and renal levels of MCP-1, ICAM-1, and NOX-1 in metformin-treated animals. Nevertheless, unlike SMTP-44D, which improved the histological glomerulosclerosis score without reducing blood glucose, metformin’s impact on histological improvement was only marginal and not statistically significant (Fig. 4). One of the possible explanations for this difference is that the partial reduction in blood glucose levels in metformin-treated animals (Fig. 1) is insufficient to prevent tissue-level damages (Figs. 4, 5). A direct intervention on the renal inflammatory/oxidative stress pathways, as demonstrated by SMTP-44D, may be a more powerful way for preventing early tissue damage than the indirect glycemic control. Therefore, blood glucose-lowering effects alone are insufficient to prevent the onset of DN, and interventions targeting the causes of its progression—such as inflammation and oxidative stress, which were shown to be effective in this study, as well as diurnal blood glucose fluctuations,⁵²⁾ hyperinsulinemia,⁵³⁾ and metabolic memory⁴⁶⁾—may contribute to its suppression.

Although we elucidated that SMTP-44D exerts renoprotective effects via anti-inflammation and antioxidant pathways, several uncertainties remain. First, the DN mouse model used in this study is an artificially accelerated model designed to rapidly induce renal pathology, primarily through heightened inflammation and oxidative stress. Consequently, it may not fully recapitulate the complex, chronic, and multifactorial pathophysiology of human DN, which develops over many years. Second, this study exclusively used male mice. Given the well-documented sex dimorphism in the progression of DN, where factors such as sex hormones, inflammation, and oxidative stress pathways exhibit sex-specific differences, the lack of female subjects limit the generalizability of our findings. This design choice, while aiming to reduce experimental variability in this initial investigation, may overlook essential sex-specific pathophysiological mechanisms that could influence the efficacy of SMTP-44D.⁵⁴⁾ Third, the experimental period was limited to 10 weeks (6–16 weeks of age). At the end of the study, the mice displayed early- to mid-stage DN, as evidenced by elevated serum creatinine and albuminuria, along with initial histopathological changes (Figs. 2, 4, 5). However, this model had not progressed to the advanced stages characterized by severe uremic complications and extensive renal fibrosis. Therefore, the observed effects of SMTP-44D should be strictly interpreted as evidence of a preventive or early-interventional strategy in DN treatment, rather than a therapeutic effect on late-stage disease. Fourth, while the present study demonstrated that SMTP-44D suppresses inflammation (MCP-1, ICAM-1) and oxidative stress (NOX-1) in the diabetic kidney (Fig. 3), the definitive mechanism underlying its renoprotective effect was not fully elucidated. Although we discussed the potential roles of sEH inhibition and AGE-RAGE blocking effect of SMTP-44D based on the previous literature, these pathways were not directly evaluated (e.g., by measuring EET/DHET ratios or NF-κB signaling). Therefore, our conclusion that the effect is possibly mediated by the suppression of inflammation and oxidative stress should be interpreted as a suggested pathway based on the expression profiles of key mediators. In addition, although this study demonstrated that SMTP-44D exerts anti-inflammatory and antioxidant effects in whole kidney tissue, the precise mechanism and location of these effects are not fully defined. Specifically, the lack of data on the extent of inflammatory/immune cell infiltration (e.g., macrophage density using F4/80 or T-cell markers) and the localized expression of pro-inflammatory cytokines (e.g., immunohistochemical detection of IL-6 and TNF-α) limits our ability to conclude that the magnitude of the observed reductions in MCP-1, ICAM-1, and NOX-1 is pharmacologically sufficient to mediate the protection of renal function and histological changes. Fifth, the assessment of tubular protection relied primarily on the semi-quantitative evaluation of local regenerative tubule severity via H&E staining (Fig. 5). While the reduction in the presence of these regenerative structures suggests a preventive effect against progressive tubular injury and atrophy,^26,27) corresponding positive data from quantitative urinary biomarkers such as KIM-1 and NGAL are lacking. A direct correlation between the observed histological protection and established molecular markers of tubular stress could not be demonstrated, which represents a limitation in the comprehensiveness of tubular injury assessment. Moreover, limitations remain regarding the pharmacokinetic (PK) and pharmacodynamic (PD) aspects. Previous studies have shown that they competitively inhibit the C-terminal domain of sEH and non-competitively inhibit the N-terminal domain.¹²⁾ However, the pharmacological rationale for the effective 30 mg/kg dose is currently limited, as comprehensive PK and PD data remain insufficient. The dose was chosen based on demonstrated efficacy in other diabetic complication models.^17,19) Further investigation is warranted to precisely determine the optimal dosing regimen in relation to achieving therapeutic concentration at the renal target site. Therefore, given the numerous limitations of this study, further research is required to establish the efficacy of SMTP-44D in DN, including the evaluation of additional DN models and elucidation of the primary mechanisms of action of SMTP-44D in the kidney.

In concluion, our results show that SMTP-44D could attenuate DN progression by the anti-inflammatory and antioxidant effects in the kidney. In this study, we suggest that SMTP-44D might exhibit renoprotective effects that are not related to blood glucose reduction, providing a novel strategy for the treatment of DN.

Acknowledgments

This study was supported by the Sasakawa Scientific Research Grant from The Japan Science Society (T.A.), by the Japan Society for the Promotion of Science KAKENHI, Grant Number: 22K08318 and by the Takeda Science Foundation (K.S.). The authors express their gratitude to TMS Co., Ltd. (Tokyo, Japan) for kindly providing SMTP-44D.

DECLARATION

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1)  Sun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, Stein C, Basit A, Chan JCN, Mbanya JC, Pavkov ME, Ramachandaran A, Wild SH, James S, Herman WH, Zhang P, Bommer C, Kuo S, Boyko EJ, Magliano DJ. IDF diabetes atlas: global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045. Diabetes Res. Clin. Pract., 183, 109119 (2022).
• 2)  Kundu A, Gali S, Sharma S, Park JH, Kyung SY, Kacew S, Kim IS, Lee KY, Kim HS. Tenovin-1 ameliorates renal fibrosis in high-fat-diet-induced diabetic nephropathy via antioxidant and anti-inflammatory pathways. Antioxidants, 11, 1812 (2022).
• 3)  Li HD, You YK, Shao BY, Wu WF, Wang YF, Guo JB, Meng XM, Chen H. Roles and crosstalks of macrophages in diabetic nephropathy. Front. Immunol., 13, 1015142 (2022).
• 4)  Chen J, Liu Q, He J, Li Y. Immune responses in diabetic nephropathy: pathogenic mechanisms and therapeutic target. Front. Immunol., 13, 958790 (2022).
• 5)  Cheng H, Harris RC. Renal endothelial dysfunction in diabetic nephropathy. Cardiovasc. Hematol. Disord. Drug Targets, 14, 22–33 (2014).
• 6)  Zhang K, Fu Z, Zhang Y, Chen X, Cai G, Hong Q. The role of cellular crosstalk in the progression of diabetic nephropathy. Front. Endocrinol. (Lausanne), 14, 1173933 (2023).
• 7)  Samsu N. Diabetic nephropathy: challenges in pathogenesis, diagnosis, and treatment. BioMed Res. Int., 2021, 1497449 (2021).
• 8)  Calle P, Hotter G. Macrophage Phenotype and Fibrosis in Diabetic Nephropathy. Int. J. Mol. Sci., 21, 2806 (2020).
• 9)  Chang TT, Chen JW. The role of chemokines and chemokine receptors in diabetic nephropathy. Int. J. Mol. Sci., 21, 3172 (2020).
• 10)  Tesch GH. Role of macrophages in complications of type 2 diabetes. Clin. Exp. Pharmacol. Physiol., 34, 1016–1019 (2007).
• 11)  Hasumi K, Yamamichi S, Harada T. Small-molecule modulators of zymogen activation in the fibrinolytic and coagulation systems. FEBS J., 277, 3675–3687 (2010).
• 12)  Matsumoto N, Suzuki E, Ishikawa M, Shirafuji T, Hasumi K. Soluble epoxide hydrolase as an anti-inflammatory target of the thrombolytic stroke drug SMTP-7. J. Biol. Chem., 289, 35826–35838 (2014).
• 13)  Matsumoto N, Suzuki E, Tsujihara K, Nishimura Y, Hasumi K. Structure-activity relationships of the plasminogen modulator SMTP with respect to the inhibition of soluble epoxide hydrolase. J. Antibiot., 68, 685–690 (2015).
• 14)  Shibata K, Hashimoto T, Nobe K, Hasumi K, Honda K. A novel finding of a low-molecular-weight compound, SMTP-7, having thrombolytic and anti-inflammatory effects in cerebral infarction of mice. Naunyn Schmiedebergs Arch. Pharmacol., 382, 245–253 (2010).
• 15)  Shibata K, Hashimoto T, Hasumi K, Honda K, Nobe K. Evaluation of the effects of a new series of SMTPs in the acetic acid-induced embolic cerebral infarct mouse model. Eur. J. Pharmacol., 818, 221–227 (2018).
• 16)  Shibata K, Hashimoto T, Hasumi K, Nobe K. Potent efficacy of Stachybotrys microspora triprenyl phenol-7, a small molecule having anti-inflammatory and antioxidant activities, in a mouse model of acute kidney injury. Eur. J. Pharmacol., 910, 174496 (2021).
• 17)  Ishibashi M, Shibata K, Terasaki M, Saito Y, Yamagishi S, Hasumi K, Nobe K. SMTP-44D alleviates diabetic retinopathy by suppressing inflammation and oxidative stress in in vivo and in vitro models. J. Pharmacol. Sci., 157, 57–64 (2025).
• 18)  Shi X, Ohta Y, Shang J, Morihara R, Nakano Y, Fukui Y, Liu X, Feng T, Huang Y, Sato K, Takemoto M, Hishikawa N, Yamashita T, Suzuki E, Hasumi K, Abe K. Neuroprotective effects of SMTP-44D in mice stroke model in relation to neurovascular unit and trophic coupling. J. Neurosci. Res., 96, 1887–1899 (2018).
• 19)  Shinouchi R, Shibata K, Hashimoto T, Jono S, Hasumi K, Nobe K. SMTP-44D improves diabetic neuropathy symptoms in mice through its antioxidant and anti-inflammatory activities. Pharmacol. Res. Perspect., 8, e00648 (2020).
• 20)  Shinouchi R, Shibata K, Jono S, Hasumi K, Nobe K. SMTP-44D exerts antioxidant and anti-inflammatory effects through its soluble epoxide hydrolase inhibitory action in immortalized mouse schwann cells upon high glucose treatment. Int. J. Mol. Sci., 23, 5187 (2022).
• 21)  Tesch GH, Lim AKH. Recent insights into diabetic renal injury from the db/db mouse model of type 2 diabetic nephropathy. Am. J. Physiol. Renal Physiol., 300, F301–F310 (2011).
• 22)  Huang MQ, Zhou CJ, Zhang YP, Zhang XQ, Xu W, Lin J, Wang PJ. Salvianolic acid B ameliorates hyperglycemia and dyslipidemia in db/db mice through the AMPK pathway. Cell. Physiol. Biochem., 40, 933–943 (2016).
• 23)  Neschen S, Scheerer M, Seelig A, Huypens P, Schultheiss J, Wu M, Wurst W, Rathkolb B, Suhre K, Wolf E, Beckers J, Hrabé de Angelis M. Metformin supports the antidiabetic effect of a sodium glucose cotransporter 2 inhibitor by suppressing endogenous glucose production in diabetic mice. Diabetes, 64, 284–290 (2015).
• 24)  Hashimoto T, Shibata K, Honda K, Nobe K. Acetic acid treatment causes renal inflammation and chronic kidney disease in mice. J. Pharmacol. Sci., 146, 160–168 (2021).
• 25)  Tervaert TW, Mooyaart AL, Amann K, Cohen AH, Cook HT, Drachenberg CB, Ferrario F, Fogo AB, Haas M, de Heer E, Joh K, Noel LH, Radhakrishnan J, Seshan SV, Bajema IM, Bruijn JA. Pathologic classification of diabetic nephropathy. J. Am. Soc. Nephrol., 21, 556–563 (2010).
• 26)  Fujigaki Y. Different modes of renal proximal tubule regeneration in health and disease. World J. Nephrol., 1, 92–99 (2012).
• 27)  Yoshida M, Honma S. Regeneration of injured renal tubules. J. Pharmacol. Sci., 124, 117–122 (2014).
• 28)  Zhu Y, Blum M, Hoff U, Wesser T, Fechner M, Westphal C, Gürgen D, Catar RA, Philippe A, Wu K, Bubalo G, Rothe M, Weldon SM, Dragun D, Schunck WH. Renal ischemia/reperfusion injury in soluble epoxide hydrolase-deficient mice. PLOS ONE, 11, e0145645 (2016).
• 29)  Bettaieb A, Koike S, Hsu MF, Ito Y, Chahed S, Bachaalany S, Gruzdev A, Calvo-Rubio M, Lee KSS, Inceoglu B, Imig JD, Villalba JM, Zeldin DC, Hammock BD, Haj FG. Soluble epoxide hydrolase in podocytes is a significant contributor to renal function under hyperglycemia. Biochim. Biophys. Acta, Gen. Subj., 1861 (11 Pt. A), 2758–2765 (2017).
• 30)  Jiang XS, Xiang XY, Chen XM, He JL, Liu T, Gan H, Du XG. Inhibition of soluble epoxide hydrolase attenuates renal tubular mitochondrial dysfunction and ER stress by restoring autophagic flux in diabetic nephropathy. Cell Death Dis., 11, 385 (2020).
• 31)  Liu Y, Zhang Y, Schmelzer K, Lee TS, Fang X, Zhu Y, Spector AA, Gill S, Morisseau C, Hammock BD, Shyy JYJ. The antiinflammatory effect of laminar flow: the role of PPARγ, epoxyeicosatrienoic acids, and soluble epoxide hydrolase. Proc. Natl. Acad. Sci. U.S.A., 102, 16747–16752 (2005).
• 32)  Wang X, Li L, Wang H, Xiao F, Ning Q. Epoxyeicosatrienoic acids alleviate methionine-choline-deficient diet-induced non-alcoholic steatohepatitis in mice. Scand. J. Immunol., 90, e12791 (2019).
• 33)  Leuillier M, Duflot T, Ménoret S, et al. CRISPR/Cas9-mediated inactivation of the phosphatase activity of soluble epoxide hydrolase prevents obesity and cardiac ischemic injury. J. Adv. Res., 43, 163–174 (2023).
• 34)  Hou HH, Liao YJ, Hsiao SH, Shyue SK, Lee TS. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase. Sci. Rep., 5, 13524 (2015).
• 35)  McIntyre TM, Pontsler AV, Silva AR, St. Hilaire A, Xu Y, Hinshaw JC, Zimmerman GA, Hama K, Aoki J, Arai H, Prestwich GD. Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPARγ agonist. Proc. Natl. Acad. Sci. U.S.A., 100, 131–136 (2003).
• 36)  Morisseau C, Schebb NH, Dong H, Ulu A, Aronov PA, Hammock BD. Role of soluble epoxide hydrolase phosphatase activity in the metabolism of lysophosphatidic acids. Biochem. Biophys. Res. Commun., 419, 796–800 (2012).
• 37)  Venkatraman G, Benesch MGK, Tang X, Dewald J, McMullen TPW, Brindley DN. Lysophosphatidate signaling stabilizes Nrf2 and increases the expression of genes involved in drug resistance and oxidative stress responses: implications for cancer treatment. FASEB J., 29, 772–785 (2015).
• 38)  Lee C. Collaborative power of Nrf2 and PPARγ activators against metabolic and drug-induced oxidative injury. Oxid. Med. Cell. Longev., 2017, 1378175 (2017).
• 39)  Dai N, Yang C, Fan Q, Wang M, Liu X, Zhao H, Zhao C. The anti-inflammatory effect of soluble epoxide hydrolase inhibitor and 14, 15-EET in Kawasaki Disease through PPARγ/STAT1 signaling pathway. Front Pediatr., 8, 451 (2020).
• 40)  Fukami K, Taguchi K, Yamagishi S, Okuda S. Receptor for advanced glycation endproducts and progressive kidney disease. Curr. Opin. Nephrol. Hypertens., 24, 54–60 (2015).
• 41)  Koyama H, Nishizawa Y. AGEs/RAGE in CKD: irreversible metabolic memory road toward CVD? Eur. J. Clin. Invest., 40, 623–635 (2010).
• 42)  Steenbeke M, Speeckaert R, Desmedt S, Glorieux G, Delanghe JR, Speeckaert MM. The role of advanced glycation end products and its soluble receptor in kidney diseases. Int. J. Mol. Sci., 23, 3439 (2022).
• 43)  Busch M, Franke S, Rüster C, Wolf G. Advanced glycation end-products and the kidney. Eur. J. Clin. Invest., 40, 742–755 (2010).
• 44)  Singh R, Barden A, Mori T, Beilin L. Advanced glycation end-products: a review. Diabetologia, 44, 129–146 (2001).
• 45)  Nowotny K, Jung T, Höhn A, Weber D, Grune T. Advanced glycation end products and oxidative stress in type 2 diabetes mellitus. Biomolecules, 5, 194–222 (2015).
• 46)  Yamazaki T, Mimura I, Tanaka T, Nangaku M. Treatment of diabetic kidney disease: current and future. Diabetes Metab. J., 45, 11–26 (2021).
• 47)  Kay AM, Simpson CL, Stewart JA Jr. The role of AGE/RAGE signaling in diabetes-mediated vascular calcification. J. Diabetes Res., 2016, 6809703 (2016).
• 48)  Lee J, Yun JS, Ko SH. Advanced glycation end products and their effect on vascular complications in type 2 diabetes mellitus. Nutrients, 14, 3086 (2022).
• 49)  Terasaki M, Shibata K, Mori Y, Saito T, Matsui T, Ohara M, Fukui T, Hasumi K, Higashimoto Y, Nobe K, Yamagishi S. SMTP-44D inhibits atherosclerotic plaque formation in apolipoprotein-E null mice partly by suppressing the AGEs-RAGE axis. Int. J. Mol. Sci., 24, 6505 (2023).
• 50)  LaMoia TE, Shulman GI. Cellular and Molecular Mechanisms of Metformin Action. Endocr. Rev., 42, 77–96 (2021).
• 51)  Medkova D, Hollerova A, Blahova J, Marsalek P, Mares J, Hodkovicova N, Doubkova V, Hesova R, Tichy F, Faldyna M, Tastan Y, Kotoucek J, Svobodova Z, Lakdawala P. Medicine designed to combat diseases of affluence affects the early development of fish. How do plastic microparticles contribute? Sci. Total Environ., 904, 166378 (2023).
• 52)  Zhang Y, Yang S, Cui X, Yang J, Zheng M, Jia J, Han F, Yang X, Wang J, Guo Z, Chang B, Chang B. Hyperinsulinemia can cause kidney disease in the IGT stage of OLETF rats via the INS/IRS-1/PI3-K/Akt signaling pathway. J. Diabetes Res., 2019, 4709715 (2019).
• 53)  Fu X, Zhang J, Huang X, Mo Z, Sang Z, Duan W, Huang W. Curcumin antagonizes glucose fluctuation-induced renal injury by inhibiting aerobic glycolysis via the miR-489/LDHA pathway. Mediators Inflamm., 2021, 6104529 (2021).
• 54)  Loeffler I, Ziller N. Sex-related aspects in diabetic kidney disease—an update. J. Clin. Med., 12, 2834 (2023).