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Ren Fail
Ren Fail
Renal Failure
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39291665
10.1080/0886022X.2024.2403653
2403653
Version of Record
Research Article
Laboratory Study
4-Octyl itaconate attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis
M. Shao et al.
Shao Muqing ab
Chen Jiayao bc
Zhang Fuwei bd
Su Qian ab
Lin Xiaoqian bc
Wang Weiwei bd
Chen Caiyu e
Ren Hongmei e
Zheng Shuo e
Hui Suocheng bc
Qin Si a
Ni Yinxing a
Zhong Jian a
Yang Jian bc
a Department of Endocrinology, The Third Affiliated Hospital of Chongqing Medical University, Chongqing, China
b Research Center for Metabolic and Cardiovascular Diseases, The Third Affiliated Hospital of Chongqing Medical University, Chongqing, China
c Department of Clinical Nutrition, The Third Affiliated Hospital of Chongqing Medical University, Chongqing, China
d Department of Cardiology, The Third Affiliated Hospital of Chongqing Medical University, Chongqing, China
e Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing, China
Muqing Shao and Jiayao Chen contributed equally to this work.

Supplemental data for this article can be accessed online at https://doi.org/10.1080/0886022X.2024.2403653.

CONTACT Jian Yang jianyang@hospital.cqmu.edu.cn Research Center for Metabolic and Cardiovascular Diseases, The Third Affiliated Hospital of Chongqing Medical University, Chongqing, China
Jian Zhong zhongjian@hospital.cqmu.edu.cn Department of Endocrinology, The Third Affiliated Hospital of Chongqing Medical University, Chongqing, China
18 9 2024
2024
18 9 2024
46 2 24036532 1 2024
11 7 2024
8 9 2024
KnowledgeWorks Global Ltd.17 9 2024
published online in a building issue17 9 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

Abstract

Objectives: The aim of this study was to investigate the mechanism of itaconate’s potential effect in diabetic kidney disease.

Methods: Renal immune responsive gene 1 (IRG1) levels were measured in db/db mice and streptozotocin (STZ) + high-fat diet (HFD)-induced diabetic mice. Irg1 knockout mice were generated. db/db mice were treated with 4-octyl itaconate (4-OI, 50 mg/kg), a derivative of itaconate, for 4 weeks. Renal function and morphological changes were investigated. Ultrastructural alterations were determined by transmission electron microscopy.

Results: Renal IRG1 levels were reduced in two diabetic models. STZ+HFD-treated Irg1 knockout mice exhibited aggravated renal tubular injury and worsened renal function. Treatment with 4-OI lowered urinary albumin-to-creatinine ratio and blood urea nitrogen levels, and restored renal histological changes in db/db mice. It improved mitochondrial damage, increased expressions of peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM) in the renal cortex of db/db mice. These were confirmed in vitro; 4-OI improved high glucose-induced abnormal mitochondrial morphology and TFAM expression in HK-2 cells, effects that were inhibited by PGC-1α silencing. Moreover, 4-OI reduced the number of apoptotic cells in the renal cortex of db/db mice. Further study showed that 4-OI increased renal Nrf2 expression and decreased oxidative stress levels in db/db mice. In HK-2 cells, 4-OI decreased high glucose-induced mitochondrial ROS production, which was reversed by Nrf2 silencing. Nrf2 depletion also inhibited 4-OI-mediated regulation of PGC-1α, TFAM, and mitochondrial apoptotic protein expressions.

Conclusions: 4-OI attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis.

Keywords

Diabetic kidney disease
immune responsive gene 1
mitochondrial biogenesis
Nrf2
4-octyl itaconate
renal tubular injury
Program of Chongqing Medical University for Youth Innovation in Future Medicine This work was supported in part by grants from the Program of Chongqing Medical University for Youth Innovation in Future Medicine (W0085), Project of Chongqing Medical Talent Studio (2022), Postdoctoral Research Project of Chongqing of China (2021XM3104), Research Project of Chongqing Education Commission (KJQN202200433) and Program of The Third Affiliated Hospital of Chongqing Medical University (KY22040, KY22045).
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pmc1. Introduction

Diabetes, a leading cause of morbidity, mortality, and rising healthcare spending, has become a serious global public health problem [1]. Current data reveal a worldwide surge in diabetes cases. In 2021, approximately 529 million individuals were diagnosed with diabetes, with type 2 diabetes accounting for 90% of these cases. It is estimated that this number will more than double to approximately 1.31 billion by 2050 [2]. From 2020 to 2030, the prevalence of diabetes among Chinese adults aged 20–79 is expected to increase from 8.2% to 9.7%, with its economic impact outpacing gross domestic product growth [3]. In the United States, diabetes ranked 3rd among 154 health conditions in healthcare costs, estimated at $111.2 billion, with most costs covered by public insurance [4]. The high prevalence of diabetes imposes a substantial disease and economic burden worldwide [5].

As the prevalence of diabetes continues to rise, diabetic kidney disease (DKD) has become a major global concern, affecting 20%–40% of individuals with diabetes [6]. DKD is recognized as a significant microvascular complication of diabetes, playing a crucial role in the progression of chronic kidney disease and end-stage renal disease [7]. The major pathological characteristics of DKD include glomerulosclerosis, a leading contributor to albuminuria, and renal fibrosis, a critical cause of renal function loss [8]. In DKD, hyperglycemia induces both direct and indirect damage to the kidney’s intrinsic cells, particularly podocytes and tubular cells, resulting in structural and functional abnormalities. Recent studies have focused on the potential role of renal tubular injury in DKD, demonstrating that tubular injury significantly contributes to renal dysfunction or failure in DKD [9,10]. These findings suggest that renal tubular injury is a crucial component of DKD pathophysiology and may be a primary cause of DKD development. Therefore, identifying potential therapeutic strategies to mitigate tubular injury is essential to slow the progression of DKD [11].

Itaconate was first identified in 1836 as a product of citrate distillation. It emerges as a primary metabolite synthesized de novo in the tricarboxylic acid cycle (TCA) cycle within activated macrophages, triggered by various factors such as lipopolysaccharide, Toll-like receptor agonists, and both type I and type II interferons [12]. These stimuli increase the levels of the enzyme cis-aconitate decarboxylase 1 (ACOD1), encoded by the immune-responsive gene 1 (IRG1). ACOD1 then facilitates the conversion of cis-aconitate into itaconate within the TCA cycle [13]. Research has demonstrated that itaconate exhibits beneficial effects in certain diseases through its physiological functions, such as anti-inflammatory and antioxidative stress properties [14,15]. Recently, the significance of itaconate in renal diseases has drawn increased interest. Irg1 deletion exacerbates renal inflammation and ischemia-reperfusion injury, whereas administration of itaconate protects against acute kidney injury and systemic inflammation [16]. A derivative of itaconate, 4-octyl itaconate (4-OI), ameliorates renal fibrosis in unilateral ureteral occlusion and adenine-induced fibrosis animal models [17]. However, the precise impact of itaconate in DKD remains uncertain. Additionally, nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the expression of many key antioxidant proteins [18]. Studies have shown that the Nrf2 signaling pathway plays a vital role in itaconate-mediated physiological effects [19,20]. However, whether Nrf2 is involved in the itaconate-mediated effects in DKD remains unclear.

Therefore, we investigated the underlying mechanism of itaconate’s potential protective effect in DKD in this study. Our findings indicate that the deletion of the Irg1 gene exacerbates renal tubular damage, whereas the administration of 4-OI alleviates renal tubular damage in diabetic mice models. Further investigations revealed that the protective effect of 4-OI on renal tubular damage in diabetic mice can be attributed, at least in part, to the activation of Nrf2 and the promotion of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-mediated mitochondrial biogenesis. This study helps elucidate the potential role of itaconate in ameliorating the progression of DKD.

2. Materials and methods

2.1. Mouse models

Germline global deletion of Irg1 (Irg1−/−) in C57BL/6J mice was obtained from Cyagen Biosciences (Guangzhou, China) and housed under specific pathogen-free conditions. Additionally, C57BL/6J and db/db mice were sourced from Beijing HFK Bioscience Co. Ltd (Beijing, China). 4-OI was acquired from MedchemExpress (NJ, USA). Animals were maintained and treated at the Animal Center of Daping Hospital. All mice were kept under standard laboratory conditions, with a 12-h light/dark cycle (lights on at 7:00 am), a room temperature of 24 °C, and relative humidity of 40%–70%. All animal care and experimental procedures adhered to the National Institutes of Health guidelines for laboratory animal welfare, focusing on minimizing discomfort and limiting the use of animals. The experimental protocols were approved by the Animal Care and Use Committee of the Third Military Medical University.

After being fed a high-fat diet (HFD) (60% fat, HFK Bioscience, Beijing, China) for four weeks, a total of 12 mice, including wild-type (WT, n = 6) and Irg1−/− (n = 6) mice, received intraperitoneal injections of streptozotocin (STZ) (Bioss, MA, USA) at 50 mg/kg per day for five consecutive days. They were then maintained on the HFD for an additional 12 weeks [21]. In another series of animal experiments, a total of 12 db/db mice (12 weeks old) were subjected to intraperitoneal administration of 4-OI at a dosage of 50 mg/kg (n = 6) or corn oil (n = 6) for four weeks, with administration occurring once every two days. Additionally, six C57BL/6J mice were treated with corn oil as the control group [22].

2.2. Cell culture and treatment

Human renal proximal tubule cells (HK-2), obtained from the Department of Cardiology, Daping Hospital (Chongqing, China), were cultured at 37 °C in an atmosphere containing 95% O2 and 5% CO2. The culture medium used was Dulbecco’s Modified Eagle’s Medium/F12, sourced from Gibco Life Science (Grand Island, NY), enriched with 10% fetal bovine serum and 1% penicillin-streptomycin (Beyotime, Shanghai, China) [23]. HK-2 cells were incubated with normal glucose (control, 5 mM), high glucose (HG, 30 mM), or HG + 4-OI (100 µM) for 24 h. Cells were collected at 80% confluence to analyze mRNA and protein expression. All samples were stored at −80 °C until use.

2.3. Analysis of urine and blood samples

Urine samples were collected over a 24-h period for analysis. The concentration of albumin in the urine was determined using an ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Urine creatinine was measured using a commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The urinary albumin to creatinine ratio (UACR) was calculated by dividing the concentration of urine albumin (mg/dL) by the concentration of urine creatinine (g/dL).

For blood sample analysis, the collected samples were centrifuged at 1000 rpm for 15 min. The extracted plasma was then used to measure blood urea nitrogen (BUN) levels using commercially available kits according to the manufacturer’s guidelines (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.4. Histological analysis

In this segment of the study, renal tissues were preserved using 4% paraformaldehyde (Servicebio, Wuhan, China) for 24 h at 4 °C. After fixation, 4 μm thick tissue slices were generated from paraffin-embedded samples and stained with hematoxylin and eosin (HE) (Solarbio, Beijing, China). Tubular injury was quantified using a specific scoring system ranging from 0 (indicating no injury) to 5 (representing more than 75% of tubules injured) [24]. Additionally, renal fibrosis was evaluated by staining the sections with Masson’s trichrome solution (Solarbio, Beijing, China). Interstitial collagen deposition was assessed and scored based on the percentage of staining observed, ranging from 0 (no staining) to 4 (75 to 100% staining) [24]. Kidney sections were also stained with periodic acid-Schiff (PAS) (Solarbio, Beijing, China) to observe tubular structural damage and mesangial matrix expansion.

2.5. Transmission electron microscopy

The study further examined ultrastructural alterations in renal tissues, specifically focusing on mitochondrial changes. Initially, these tissues were fixed using a specialized transmission electron microscopy (TEM) fixative (Servicebio, Wuhan, China) at 4 °C. The samples were then incubated with a solution containing 1% OsO4 in 0.1 M phosphate buffer (pH 7.4) at room temperature for 2 h. Ultrathin sections were stained with a 2% solution of uranyl acetate in alcohol for 8 min. The sections were observed using a TEM (Hitachi HT7800, Tokyo, Japan). Mitochondrial area, length, and width were measured using ImageJ. Mitochondrial length/width (a ratio between major axis and minor axis of an ellipse equivalent to object, measure of mitochondrial length) and average mitochondrial area (the average surface area of individual mitochondria within a cell) were assessed [25].

2.6. TUNEL assay

The experimental procedure involved the use of a one-step TUNEL apoptosis assay kit to conduct terminal deoxynucleotidyl TUNEL staining (Beyotime, Shanghai, China). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China). Images were observed under a fluorescence microscope (Eclipse Ti-U; Nikon Corporation, Tokyo, Japan) and analyzed using ImageJ software.

2.7. Measurement of oxidative stress

To assess oxidative stress levels, particularly reactive oxygen species (ROS) in kidney tissues, a specific fluorescent dye, dihydroethidium (5 μM; Beyotime, Shanghai, China), was utilized. For HK-2 cells, mitochondrial ROS production was quantified using the MitoSOX Red mitochondrial superoxide indicator (5 μM; Invitrogen, Grand Island, NY). After washing with PBS, photographs were observed using a fluorescence microscope (Eclipse Ti-U; Nikon Corporation, Tokyo, Japan).

2.8. Detection of mitochondrial fragmentation

The evaluation of mitochondrial morphology in HK-2 cells was conducted by incubating the cells with MitoTracker Red CMXRos solution (100 nM; Invitrogen, Grand Island, NY) for 15–25 min at 37 °C. After incubation, the cells were washed with phosphate buffered saline (PBS) and examined using a laser scanning confocal microscope (BX53, Olympus Corporation, Tokyo, Japan).

2.9. Western blot analysis

Protein expression in kidney tissues and HK-2 cells was analyzed using Western blotting. Tissues and cells were lysed in RIPA buffer (Beyotime, Shanghai, China). Denaturation was then carried out by adding loading buffer to the lysates. The proteins were separated on a 10%–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes (Amersham Life Science, Arlington, TX). The membranes were blocked with nonfat dry milk at room temperature for 1 h, followed by incubation with primary antibodies overnight at 4 °C. Afterward, membranes were incubated with infrared-labeled secondary antibodies for 1 h at room temperature. The antibodies used are detailed in Supplementary—S1. The Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) was used to detect bound complexes. Image analysis was performed using Odyssey Application Software to measure integrated intensities [26,27].

2.10. Real-time quantitative PCR

Real-time quantitative PCR was employed to assess gene expression. Total RNA was extracted from tissues and cells using TRIzol reagent (TaKaRa, Tokyo, Japan). Two micrograms of this RNA were used as a template to synthesize cDNA, which served for the amplification of target genes. The specific primers for gene expression analysis are listed in Supplementary Table S2. The PCR conditions were as follows: initial denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and elongation at 72 °C for 45 s. Gene expression levels were normalized to GAPDH expression.

Additionally, the ratio of mitochondrial DNA to genomic DNA (mtDNA/gDNA) was determined using qPCR. This involved the amplification of a mitochondrial gene (ND1 in humans or mice) and a nuclear gene (18S rRNA in humans or mice). Primer sequences for these genes are provided in Supplementary Table S2.

2.11. Specific siRNA transfection

HK-2 cells were transfected with siRNA targeting human Nrf2 or PGC-1α (Ribobio, Guangzhou, China) using Lipofectamine 2000 Reagent (Invitrogen Life Technologies, Carlsbad, CA). Non-silencing scrambled siRNA served as a negative control. After 48 h, cells were collected to assess the efficiency of siRNA-mediated gene silencing.

2.12. Statistical analysis

Statistical analysis was carried out with data presented as mean ± standard error of the mean. Repeated-measures ANOVA or a two-tailed paired t-test was utilized for comparisons within groups, depending on the number of groups. One-way factorial ANOVA with the Holm-Sidak test, or a t-test, was applied for comparisons between groups. A p value less than 0.05 was considered statistically significant.

3. Results

3.1. Decreased IRG1 expression in diabetic animal models

To evaluate the potential role of itaconate in DKD, we initially investigated the renal mRNA and protein levels of IRG1 in two diabetic mice models, namely db/db mice and high-fat diet-fed mice with streptozotocin-induced diabetes. Quantitative RT-PCR and immunoblotting revealed notably reduced IRG1 mRNA and protein levels in the renal cortex of db/db mice compared to controls (Figure 1(A,B)). Similar reductions were observed in high-fat diet plus streptozotocin (HFD + STZ) diabetic mice (Figure 1(C,D)). These findings suggest a possible involvement of IRG1 in the progression of DKD.

Figure 1. Renal IRG1 expression in db/db mice and HFD + STZ-induced diabetic mice. The mRNA (A) and protein (B) expression of IRG1 in the renal cortex of db/db mice. IRG1 levels were normalized to GAPDH (*p < 0.05 vs. control, n = 6/group). The mRNA (C) and protein (D) expression levels of IRG1 in the renal cortex of HFD + STZ -induced diabetic mice. IRG1 expression was normalized to GAPDH (*p < 0.05 vs. control, n = 6/group).

3.2. Irg1 deletion aggravated renal tubular injury and worsened renal functions in diabetic mice

To assess the role of IRG1 in DKD, mice with a germline global deletion of Irg1 (Irg1−/−) were developed and subsequently subjected to a HFD and STZ to induce DKD (Figure 2(A,B)). When compared to wild-type (WT) counterparts, HFD + STZ-treated Irg1−/− mice exhibited no significant alterations in blood glucose levels or body weight (Figure 2(C,D)). We then examined the impact of Irg1 deletion on renal function in these diabetic mice. Notably, HFD + STZ treatment significantly elevated the kidney/body weight ratio in Irg1−/−mice beyond that observed in WT mice (Figure 2(E)). Additionally, both serum blood urea nitrogen (BUN) levels and urinary albumin-to-creatinine ratio (UACR) were markedly higher in diabetic Irg1−/−mice compared to diabetic WT mice (Figure 2(F,G)). Histological analysis revealed significant morphological changes in the kidneys of diabetic Irg1−/− mice, such as glomerular enlargement, renal tubule dilation, mesangial matrix deposition, and tubulointerstitial fibrosis (Figure 2(H)). Moreover, levels of neutrophil gelatinase-associated lipocalin (NGAL), a biomarker of renal tubular damage, were found to be increased in the renal cortex of diabetic Irg1−/− mice compared to diabetic WT mice (Figure 2(I)). These findings indicate that deletion of Irg1 exacerbates renal tubular damage and impairs renal function in diabetic mice.

Figure 2. Renal tubular injury in diabetic Irg1−/−mice. (A) Diagram of the administration of a HFD and STZ in WT and Irg1−/− mice. After being fed an HFD for four weeks, WT and Irg1−/− mice were intraperitoneally injected with STZ at 50 mg/kg each day for five consecutive days, followed by maintenance on an HFD for an additional 12 weeks. (B) Identification of Irg1−/− mice. IRG1 protein expression was determined by immunoblotting in the renal cortex of WT and Irg1−/− mice (*p < 0.05 vs. WT (DKD), n = 6/group). Fasting glucose levels (C) and body weight (D) in diabetic Irg1−/− mice and their WT littermates (n = 6/group). Kidney weight to body weight ratio (KW/BW) (E), serum blood urea nitrogen (BUN) (F) and urine albumin-to-creatinine ratio (UACR) (G) in diabetic WT and Irg1−/− mice(*p < 0.05 vs.WT (DKD), n = 6/group). (H) Representative images of HE (upper) and Masson staining (lower) and quantitative analysis of the kidneys of diabetic Irg1−/− and WT mice. Tubular epithelial cells exhibit swelling and vacuolar changes, with weakened eosinophilia, appearing pale (black arrows). Some tubular lumens are dilated, and cuboidal epithelial cells transform into flattened cells (green arrows). The interstitial area shows mild infiltration of inflammatory cells (yellow arrows). Renal tubulointerstitial shows fibrous deposition (blue arrows). Renal tubular injury and interstitial collagen deposition were quantified (scale bar = 50 μm, magnification 400×) (*p < 0.05 vs. WT (DKD), n = 6/group). (I) The protein expression of neutrophil gelatinase-associated lipocalin (NGAL) in the renal cortex of diabetic WT and Irg1−/− mice (*p < 0.05 vs. WT (DKD), n = 4/group).

3.3. 4-OI attenuated renal tubular injury and improved renal function in db/db mice

To ascertain if exogenous itaconate could mitigate DKD, we utilized 4-OI, a synthetic itaconate derivative known for its cell membrane permeability (Figure 3(A)) [28–30]. Unlike the control group, db/db mice aged 16 weeks exhibited elevated blood glucose and body weight, along with increased kidney/body weight ratios, BUN levels and UACR. A four-week treatment with 4-OI resulted in reductions in the heightened BUN levels and UACR. However, this intervention did not significantly affect blood glucose levels or body weight in the db/db mice (Figure 3(B–F)).

Figure 3. Effect of 4-OI on renal tubular injury in db/db mice. (A) Diagram of the administration of 4-OI in db/db mice. Twelve-week-old db/db mice were intraperitoneally administered 4-OI at a concentration of 50 mg/kg every two days for four weeks. Fasting glucose levels (B) and body weight (C) in db/db mice treated with 4-OI for four weeks (*p < 0.05 vs. control, n = 6/group). Kidney weight to body weight ratio (KW/BW) (D), serum blood urea nitrogen (BUN) (E), and urine albumin to creatinine ratio (UACR) (F) in db/db mice treated with 4-OI for four weeks (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 6/group). (G) Protein expression of neutrophil gelatinase-associated lipocalin (NGAL) in the renal cortex of db/db mice treated with 4-OI for four weeks (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 4/group). (H) Representative images from three groups stained with hematoxylin and eosin (H&E), Masson’s trichrome staining, and periodic acid-Schiff (PAS) staining. Renal tubular injury and interstitial collagen deposition were quantified. Tubular epithelial cells exhibit swelling and vacuolar changes, with weakened eosinophilia, appearing pale (black arrows). Some tubular lumens are dilated, and cuboidal epithelial cells transform into flattened cells (green arrows). The interstitial area shows mild infiltration of inflammatory cells (yellow arrows). Renal tubulointerstitial shows fibrous deposition (blue arrows) (scale bar = 50 μm, magnification 400×) (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 6/group).

Additionally, treatment with 4-OI normalized the protein expression of NGAL in the renal cortex of db/db mice (Figure 3(G)). Hematoxylin and eosin (HE) and Masson’s trichrome staining revealed that db/db mice, compared to control mice, had increased renal tubule dilatation, mesangial matrix deposition, and interstitial collagen accumulation, all of which were ameliorated by 4-OI treatment (Figure 3(H)). Furthermore, PAS staining highlighted structural changes in renal tubules of db/db mice, including varying extents of tubular dilatation and brush border fusion; these alterations were mitigated following 4-OI treatment (Figure 3(H)). These results suggest that 4-OI administration effectively reduces renal tubular damage and enhances renal function in db/db mice.

3.4. 4-OI promoted mitochondrial biogenesis in renal tubular cells of db/db mice

Research indicates that mitochondrial dysfunction plays a crucial role in the development of DKD [31]. Itaconate is synthesized within mitochondria [32]. Consequently, we explored whether 4-OI exerts its renoprotective effects in db/db mice by modulating mitochondrial dynamics. Ultrastructural analyses using transmission electron microscopy (TEM) revealed that, compared to control mice, db/db mice exhibited significant mitochondrial damage in tubular cells, characterized by cristae disorder or disappearance, mitochondrial swelling, and deformation. These abnormalities were ameliorated following treatment with 4-OI (Figure 4(A)).

Figure 4. Effect of 4-OI on mitochondrial biogenesis in renal tubular cells of db/db mice. (A) Mitochondrial morphology in renal tubular cells of db/db mice treated with 4-OI for four weeks observed via transmission electron microscopy (TEM). The average area of mitochondria and maximum length-width ratio were measured using ImageJ software. Red arrows indicate mitochondria with fragmented cristae, and yellow arrows indicate mitochondria with indistinct crista structures (scale bar = 2 μm, magnification 10,000×) (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 5/group). (B) mRNA expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in the renal cortex of db/db mice treated with 4-OI for four weeks (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 6/group). (C, D) Protein expression of PGC-1α and mitochondrial transcription factor A (TFAM) in the renal cortex of db/db mice treated with 4-OI for four weeks (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 4/group). (E) Mitochondrial DNA to genomic DNA (mtDNA/gDNA) ratio in the renal cortex of db/db mice treated with 4-OI for four weeks (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 6/group). (F) Protein expression of PGC-1α in HK-2 cells treated with PGC-1α siRNA for 48 h (*p < 0.05 vs. control). (G) After transfection with PGC-1α siRNA for 48 h, HK-2 cells were incubated with high glucose (HG, 30 mM) and HG + 4-OI (100 µM) for 24 h. Mitochondria were stained with MitoTracker Red CMXRos. (Scale bar = 10 μm, magnification 1000×). (H) After transfection with PGC-1α siRNA for 48 h, HK-2 cells were incubated with high glucose (HG, 30 mM) and HG + 4-OI (100 µM) for 24 h. TFAM protein expression was detected by immunoblotting (*p < 0.05 vs. control, #p < 0.05 vs. HG, &p < 0.05 vs. HG + 4-OI, %p< 0.05 vs. HG+ PGC-1α siRNA, n = 4/group). (I) Renal cell apoptosis determined by TUNEL staining in the renal cortex of db/db mice treated with 4-OI for four weeks (scale bar = 20 μm, magnification 200×) (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 5/group). (J,K) Protein expressions of Cytc and BCL-2-associated X protein (Bax) in the renal cortex of db/db mice treated with 4-OI for four weeks (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 4/group).

Numerous studies have established that the regulation of mitochondrial biogenesis plays a critical role in the development and potential prevention of DKD [33]. Our research showed that db/db mice demonstrated increased mRNA and protein levels of PGC-1α, a key marker of mitochondrial biogenesis, in the renal cortex compared to control mice. This effect was partially reversed by treatment with 4-OI (Figure 4(B,C)). Additionally, 4-OI treatment was observed to restore the expression of mitochondrial transcription factor A (TFAM) and normalize the mitochondrial DNA (mtDNA) to genomic DNA (gDNA) ratio in the renal cortex of db/db mice (Figure 4(D,E)).

To confirm the impact of 4-OI on mitochondrial biogenesis, we knocked down the expression of PGC-1α in vitro (Figure 4(F)). MitoTracker Red staining illustrated that, compared to control cells with filamentous mitochondria, high glucose (HG)-incubated HK-2 cells exhibited mitochondria with shortened rod shapes, suggesting increased mitochondrial fragmentation. This condition was ameliorated by 4-OI treatment. Additionally, cells treated with HG and PGC-1α siRNA displayed more pronounced mitochondrial structural damage; however, the effect of 4-OI was reversed by treatment with PGC-1α siRNA. Furthermore, 4-OI also elevated the reduced TFAM protein levels induced by HG, an effect that was reversed by PGC-1α silencing (Figure 4(G,H)).

Mitochondria are critical regulators of cellular apoptosis [34], and apoptosis of renal tubule cells is a significant pathological feature of DKD. To further assess the effects of 4-OI on mitochondrial function, we conducted an investigation into cell apoptosis. This study demonstrated a statistically significant increase in the number of apoptotic cells within the renal cortex of db/db mice compared to controls. Importantly, this increase was attenuated by treatment with 4-OI (Figure 4(I)). Moreover, the levels of mitochondrial apoptotic proteins, specifically cytochrome C (Cytc) and BCL-2-associated X protein (Bax), were elevated in db/db mice but were reduced following treatment with 4-OI (Figure 4(J,K)). These findings suggest that the renoprotective effect of 4-OI in DKD may be mediated, at least in part, through the modulation of mitochondrial biogenesis.

3.5. 4-OI attenuated renal tubular injury in DKD by activating Nrf2

Nrf2 has been shown to play a vital role in itaconate-mediated effects [35]. This led us to hypothesize that the protective effect of 4-OI on DKD may be mediated through the regulation of Nrf2. The findings of this study indicate a decrease in Nrf2 protein expression and an increase in oxidative stress levels in the renal cortex of db/db mice compared to control animals. However, these effects were reversed following the administration of 4-OI (Figure 5(A,B)). Additionally, the expressions of renal superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2), which are primarily associated with intracellular metabolic response and oxidative stress [36], were restored in mice treated with 4-OI (Figure 5(C,D)).

Figure 5. Nrf2 mediates the effects of 4-OI-improved mitochondrial biogenesis. (A) Protein expression of Nrf2 in the renal cortex of db/db mice treated with 4-OI for four weeks (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 6/group). (B) Fluorescence microscopy images and quantification of renal reactive oxygen species (ROS) production, determined by DHE staining, in the renal cortex of db/db mice treated with 4-OI for four weeks. (Scale bar = 20 μm, magnification 200×) (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 5/group). (C,D) Protein expression of superoxide dismutase 1 (SOD1) and SOD2 in the renal cortex of db/db mice treated with 4-OI for four weeks (*p < 0.05 vs. control, #p < 0.05 vs. db/db, n = 6/group). (E) Representative images of HK-2 cells after MitoSOX staining. HK-2 cells were transfected with Nrf2 siRNA for 48 h and then incubated with high glucose (HG, 30 mM) and 4-OI (100 µM) for 24 h (scale bar = 20 μm, magnification 400×). (F) Protein expression of Nrf2 in HK-2 cells treated with Nrf2 siRNA for 48 h (*p < 0.05 vs. control). (G,H) HK-2 cells were transfected with Nrf2 siRNA for 48 h and then incubated with high glucose (HG, 30 mM) and 4-OI (100 µM) for 24 h. PGC-1α and TFAM protein expressions were detected by immunoblotting (*p < 0.05 vs. control, #p < 0.05 vs. HG, &p < 0.05 vs. HG + 4-OI, n = 4/group). (I) HK-2 cells were transfected with Nrf2 siRNA for 48 h and then incubated with high glucose (HG, 30 mM) and 4-OI (100 µM) for 24 h. The mitochondrial DNA to genomic DNA (mtDNA/gDNA) ratio was measured (*p < 0.05 vs. control, #p < 0.05 vs. HG, &p < 0.05 vs. HG + 4-OI, n = 6/group). (J,K) HK-2 cells were transfected with Nrf2 siRNA for 48 h and then incubated with high glucose (HG, 30 mM) and 4-OI (100 µM) for 24 h. Protein expression levels of Cytc and BCL-2-associated X protein (Bax) were detected by immunoblotting (*p < 0.05 vs. control, #p < 0.05 vs. HG, &p < 0.05 vs. HG + 4-OI, n = 6/group).

The role of Nrf2 in the 4-OI-mediated enhancement of mitochondrial biogenesis was further verified through in vitro studies. In HK-2 cells, 4-OI treatment reduced high glucose (HG)-induced mitochondrial reactive oxygen species (ROS) production, as shown by MitoSOX staining, while Nrf2 silencing inhibited this effect. Additionally, the HG+ PGC-1α siRNA group exhibited more severe ROS production (Figure 5(E)). Furthermore, Nrf2 depletion reversed the 4-OI-mediated increases in PGC-1α and TFAM expression and the mtDNA/gDNA ratio in HG-incubated HK-2 cells (Figure 5(F–I)). Moreover, the decreased expression of Cytc and Bax in HG-incubated HK-2 cells mediated by 4-OI was also restored by Nrf2 silencing (Figure 5(J,K)). These findings suggest that 4-OI activates Nrf2 and promotes PGC-1α-mediated mitochondrial biogenesis, leading to reduced oxidative stress and apoptosis in injured tubule cells in DKD.

4. Discussion

Itaconate, a naturally occurring unsaturated dicarboxylic acid derived from the TCA cycle via IRG1 catalysis of cis-aconitate [37], has garnered increasing attention due to its various beneficial physiological functions, including anti-inflammatory and antibacterial/antiviral properties [37,38]. Interestingly, several studies have reported that itaconate exhibits protective effects against certain kidney diseases, such as promoting survival in mice subjected to lethal renal ischemia, protecting against renal ischemia-reperfusion injury and systemic inflammation, and ameliorating renal fibrosis [16,17]. Itaconate also mitigates renal damage induced by other diseases, including lupus and sepsis, and mediates protective effects in the kidney induced by other factors, such as sodium-glucose co-transporter 2 (SGLT2) inhibitors [39–41]. Conversely, Irg1 deficiency has been shown to promote renal damage, including renal inflammation and ischemia-reperfusion injury [16]. However, the specific role of itaconate in DKD remains to be fully elucidated. Our current study reveals a decrease in both the mRNA and protein levels of IRG1 in the renal cortex of two diabetic mice models, with Irg1 deletion exacerbating renal tubular injury and impairing renal function in diabetic mice. Notably, 4-OI, a cell-permeable itaconate derivative, exerts a protective effect against renal tubular injury in db/db mice. It is crucial to note that this effect is not attributable to alterations in blood glucose levels or body weight, as 4-OI treatment did not modify these parameters in diabetic mice. It should be noted that the measurement of IRG1 levels is relevant as it highlights the endogenous pathway for itaconate production, while the use of 4-OI demonstrates the protective effects that can be achieved through this pathway, thereby connecting IRG1 function with the therapeutic potential of 4-OI.

In recent years, in-depth investigations into the intricate pathophysiological mechanisms contributing to renal tubular injury in DKD have unveiled new insights and potential treatment approaches [42]. The kidney, comparable to the heart in metabolic demand, requires a high rate of metabolism [43], primarily due to the extensive energy needs of the renal tubules, which depend heavily on mitochondrial activity [44]. Numerous investigations have demonstrated the critical role that mitochondrial dysfunction plays in the etiology of diabetic renal tubular injury [45,46]. In particular, mitochondrial biogenesis, an essential aspect of mitochondrial quality control, is increasingly recognized for its significant impact on DKD progression [47]. ‘Mitochondrial biogenesis’ refers to the cellular process involved in the generation and operation of new mitochondria, facilitating adaptation to increased energy demands under abnormal conditions [48]. PGC-1α, a well-known transcriptional coactivator, orchestrates mitochondrial biogenesis and is a pivotal regulator of energy homeostasis [49,50]. PGC-1α is abundant in organs such as the kidney, which has a high specific metabolic rate [51,52]. The development of DKD is linked to mitochondrial biogenesis dysfunction in renal tubular cells [53]. Conversely, exogenous plant compounds such as resveratrol and lycopene exert protective effects, including ameliorating podocyte damage in diabetic mice and attenuating D-galactose-induced renal injury, by improving mitochondrial biogenesis [54,55]. Endogenous factors such as sirtuin 3 also protect against renal damage by enhancing mitochondrial biogenesis [56]. Our study revealed that treatment with 4-OI improved mitochondrial structure, increased the expression of PGC-1α and TFAM, and enhanced the mtDNA/gDNA ratio in the renal cortex of db/db mice. Furthermore, in vitro investigations provided additional evidence that suppression of PGC-1α reversed the beneficial effects of 4-OI on mitochondrial biogenesis in high glucose-treated HK-2 cells. These results indicate that 4-OI protects against renal tubular injury in DKD by improving mitochondrial biogenesis.

Apoptosis of renal tubule cells is a significant manifestation of DKD, with mitochondria playing a crucial role in tubular injury [33,57]. Mitochondrial abnormalities in tubular cells in DKD are often accompanied by increased apoptosis [58,59]. Bax, a pro-apoptotic member of the Bcl-2 protein family, is vital in regulating programmed cell death [60]. Studies have shown that Bax permeabilizes the outer mitochondrial membrane and drives cells toward apoptosis by facilitating the release of pro-apoptotic factors such as Cytc and activating caspase-3 [61]. Our results showed that db/db mice had an increased number of apoptotic cells and elevated expressions of Cytc and Bax in the renal cortex, which were reduced by treatment with 4-OI. Furthermore, the 4-OI-mediated decrease in Cytc and Bax expression in high glucose-incubated HK-2 cells was reversed by Nrf2 silencing. These findings suggest that the protective effect of 4-OI on renal tubular injury in DKD may be, at least in part, due to its ability to reduce tubular cell apoptosis.

Multiple studies have emphasized the cytoprotective function of Nrf2, particularly in regulating redox homeostasis [62]. The Nrf2 pathway has been implicated in various itaconate-mediated biological activities, including antioxidant and anti-inflammatory effects [63,64]. Dimethyl itaconate or 4-OI, via the Nrf2 pathway, protects against renal ischemia-reperfusion injury and lipopolysaccharide-induced acute kidney injury [16,65]. Furthermore, Nrf2 plays a crucial role in mitochondrial quality control and the regulation of key mitochondrial functions, such as reactive oxygen species (ROS) production and energy synthesis [66]. Both PGC-1α and TFAM are essential for mitochondrial biogenesis [67,68]. Studies have shown that Nrf2 activation increases the expressions of PGC-1α and TFAM, thereby promoting mitochondrial biogenesis [69–71]. In our current study, we found that 4-OI treatment improved Nrf2 protein expression and reduced renal oxidative stress levels in the renal cortex of db/db mice. In vitro studies further demonstrated that Nrf2 depletion reversed the 4-OI-mediated increases in PGC-1α and TFAM levels in high glucose-incubated HK-2 cells. These findings suggest that Nrf2 may regulate 4-OI-improved mitochondrial biogenesis by increasing PGC-1α and TFAM expression.

5. Conclusion

In conclusion, our study demonstrates that 4-OI, a derivative of itaconate, attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis (Figure 6). These findings enhance our understanding of the potential role of itaconate in managing DKD and provide novel insights into translational medicine. Specifically, they suggest a new therapeutic approach for managing DKD, highlighting the importance of mitochondrial biogenesis and the role of Nrf2 in mitigating renal tubular injury.

Figure 6. Schematic representation of the improvement of 4-OI on renal tubular injury in diabetic kidney disease. Treatment with 4-OI activates Nrf2 and promotes PGC-1α-mediated mitochondrial biogenesis, leading to reduced oxidative stress and apoptosis in injured tubule cells, subsequently attenuating renal tubular injury in db/db mice. 4-OI: 4-octyl itaconate; DKD: diabetic kidney disease; Nrf2: nuclear factor erythroid 2-related factor 2; PGC-1α: peroxisome-proliferator-activated receptor γ coactivator-1α; ROS: reactive oxygen species.

Ethics approval and consent to participate

The Laboratory Animal Welfare and Ethics Committee of the Third Military University approved with the research design of this animal experiment (AMUWE20223395), which follows the National Guidelines for laboratory Animals.

Supplementary Material

4_Supplementary Material_0711.docx

Figure 2.tif

Figure 6.tif

Figure 3.tif

Figure 5.tif

Figure 4.tif

5_Figure 1_0711.tif

Author contributions

J.Y., J.Z. and F.W.Z. conceived and designed the experiments. M.Q.S, J.Y.C, F.W.Z., Q.S., X.Q.L., W.W.W., C.Y.C., H.M.R., S.Z., S.Q. and S.C.H. performed the experiments and analyzed the data. M.Q.S wrote the manuscript. J.Y., F.W.Z., Y.X.N and J.Z. revised the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.
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