Evaluation of the efficacy of rat renal ischemia-reperfusion injury after itaconic acid and its isomers treatment by contrast-enhanced ultrasound(CEUS)

doi:10.21203/rs.3.rs-4178858/v1

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Evaluation of the efficacy of rat renal ischemia-reperfusion injury after itaconic acid and its isomers treatment by contrast-enhanced ultrasound(CEUS)

https://doi.org/10.21203/rs.3.rs-4178858/v1

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Itaconic acid and its isomers citraconic acid and mesaconic acid are a recently discovered class of metabolites with anti-inflammatory and antioxidant effects. This study will investigate its role in ischemia-reperfusion-induced acute kidney injury, and use contrast-enhanced ultrasound to evaluate kidney function, in order to provide a new diagnostic method and treatment strategy for renal acute kidney injury. In this study, a rat model of renal ischemia-reperfusion was established, and itaconic acid, citraconic acid and mesaconic acid were given as preoperative intervention. After the operation, the rat kidneys were examined by contrast-enhanced ultrasound, biochemical analysis and pathological staining. The results showed that the intervention of itaconic acid, citraconic acid and mesaconic acid could effectively reduce renal ischemia-reperfusion injury through anti-inflammatory and antioxidant effects,and inhibiting cell pyroptosis. These findings suggest that itaconic acid, citraconic acid, and mesaconic acid may be effective strategies for the treatment of renal ischemia-reperfusion through Inflammation-related pyroptosis pathway.

Health sciences/Diseases

Health sciences/Nephrology

itaconic acid

citraconic acid

mesaconic acid

renal ischemia-reperfusion injury

contrast-enhanced ultrasound

Acute kidney injury (AKI) is a common disease (8–16% of hospital admissions) and a serious disease (a four-fold increase in hospital mortality), whose global incidence is growing. The high cost of treatment in the later stages of the disease and the risk of chronic kidney disease have made it a growing global health problem[1]. The main pathologic feature of AKI is a decreased glomerular filtration rate, resulting in a short period of reduced kidney function, which is characterized by tubular and vascular damage, as well as uncontrolled inflammatory responses[2]. However, the current treatment methods for AKI are still not satisfactory in clinic. AKI is related to surgery, drug therapy and decreased renal perfusion pressure, in which renal ischemia-reperfusion injury (IRI) is a major cause of acute renal injury and secondary renal function loss[3]. Because of the rapid development of AKI, early diagnosis of AKI is of great significance to reduce the mortality of AKI. Current diagnosis of acute kidney injury relies on measurements of serum creatinine (Cr) levels and urine output. However, the serum Cr level is affected by many factors such as diet and drug therapy, and the specificity and sensitivity of measuring urinary volume are not enough to be the diagnostic marker of AKI. These results may lead to an underestimation of the extent of damage to kidney function. Given the serious consequences of AKI, more and more researchers are focusing on early identification of patients with AKI in an attempt to improve their outcomes[4, 5]. At present, ultrasound technology has been increasingly applied to the diagnosis of various diseases[6], and it is a common means for clinical application to screen or diagnose diseases, especially in urology and other systems.

Contrast-enhanced ultrasound (CEUS) is a quantitative imaging technique. After injecting contrast agents into the body, high-echo microbubbles are used to enhance the contrast between the target position and surrounding tissue, which can more accurately reflect blood perfusion and increase the sensitivity and specificity of blood flow measurement[7, 8]. At present, the main component of clinically used ultrasound contrast agents is sulfur hexafluoride (SF6) microbubbles, which are not affected by glomerular filtration rate and tubule absorption, have no metabolic burden on the kidney, and do not increase the risk of kidney injury[9, 10]. Therefore, compared with enhanced computed tomography or enhanced magnetic resonance imaging, CEUS has great advantages in the diagnosis of kidney disease, and can safely assess renal perfusion and microcirculation in critically ill patients[11, 12]. In addition, the time-intensity curve (TIC) and quantitative parameters(i.e. peak intensity (Peak), time to peak (Tp), area under the curve (AUC) and mean transition time (MTT)) were obtained by quantifying the change of time-concentration of contrast agents in the region of interest (ROI).These are sensitive indicators of renal blood perfusion, which may help to assess renal tissue damage in the early stages of AKI[13–15]. It has been proved that CEUS examination combined with TIC curve analysis has high application value in evaluating renal blood perfusion on IRI[16, 17].

The pathological mechanism of renal IRI includes many factors, such as oxidative stress, inflammation, apoptosis and autophagy, etc., which lead to vascular dysfunction, immune system activation and renal tubular epithelial cell injury[18]. During ischemia, mitochondrial synthesis of Adenosine 5'-triphosphate (ATP) is impaired, leading to cell death; after ischemic tissue reperfusion, mitochondria produce a large number of reactive oxygen species (ROS) to directly oxidize cells and stimulate the release of pro-inflammatory mediators, leading to tissue injury[19]. Therefore, reducing ROS accumulation can effectively alleviate renal IRI[20–22]. Due to the multifactorial pathophysiological mechanism of renal IRI, there is no effective pharmacological intervention to prevent or reverse renal IRI[1]. Current studies have proposed dialysis treatment [23], alternative therapy[24], stem cell therapy[25–27], etc., but these treatments all have certain limitations. Based on this, more and more research is targeting the drug treatment of AKI[28–30].

Itaconic acid is a recently discovered endogenous metabolite, which is mainly involved in the regulation of cellular metabolism, and plays an important role in cellular immunity and inflammatory metabolism[31, 32]. It has antibacterial, anti-inflammatory and antioxidant properties[33–35]. For one thing, itaconic acid can reduce ROS production by inhibiting the mitochondrial tricarboxylic acid (TCA) cycle and succinate dehydrogenase (SDH) activity[36]. For another, itaconic acid is also released into the cytoplasm, where it can modify Kelch-like ECH-associated protein 1 (Keap1), activate nuclear factor erythroid 2-related factor 2 (Nrf2), and promote the expression of anti-inflammatory and antioxidant genes. In addition, itaconic acid can also induce the release of cytochrome c by inhibiting the activity of cytochrome c oxidase (Complex IV) in the respiratory chain, which stimulates the opening of mitochondrial permeability transition pore, thereby reducing the accumulation of intracellular Ca²⁺[37]. Itaconic acid's inhibition of nucleotide-binding oligomerization domain-like receptor protein-3 (NLRP3) inflammasome is also considered to be an important part of its anti-inflammatory mechanism[31]. It has been shown that the itaconic acid derivative 4-octyl itaconate (4-OI) can improve early renal fibrosis by inhibiting inflammation and reducing oxidative stress[38, 39]. However, the role of itaconic acid in the acute stage of renal injury has not been studied. Based on this, we established a renal IRI-induced AKI model to explore the therapeutic effect of exogenous itaconic acid on AKI.

Citraconic acid and mesaconic acid are isomers of itaconic acid, and they have similar biological properties[40], but there is still a lack of pre-clinical studies. In this study, we simultaneously investigated whether these two isomers have the same therapeutic effect as itaconic acid in renal IRI. In addition to biochemical and pathological examinations, CEUS was used to assess renal function from the imaging perspective, and quantitative parameters obtained by CEUS were used to assess the degree of kidney injury, providing a new and non-invasive examination technique for clinical assessment of AKI.

2.1. Experimental method

2.1.1Experimental animals and grouping

Our study was approved by the Ethics Committee of the Affiliated Hospital of Southwest Medical University(NO:20231127). A total of 61 healthy adult male Sprague-Dawley rats (250g ± 50g, 8–10 weeks of age) were provided by the Animal Experimental Center of Southwest Medical University (license number: SCXK(Sichuan)2023-0017).In accordance with The ARRIVE guidelines 2.0, the experimental rats were individually housed in single cages within the animal care facilities of the Imaging Center Laboratory at the Affiliated Hospital of Southwest Medical University. These facilities are equipped with air conditioning, light control, and other necessary equipment.The rats were randomly divided into five groups: (1) In the sham group (sham group), n = 10, each rat was injected with 2ml/kg/d of normal saline for three days before surgery. (2) In the renal ischemia-reperfusion group (IRI group), n = 12, each rat was intraperitoneally injected with 2ml/kg/ day of normal saline for 3 days before surgery. (3) Renal IRI + itaconic acid intervention group (Itaconic acid group), n = 13, each rat was given itaconic acid (C11530755, Macklin, Shanghai) 50mg/kg/day by intraperitoneal injection for 3 days before surgery. (4) Renal IRI + citraconic acid intervention group (Citraconic acid group), n = 13, each rat was given 50mg/kg/ day by intraperitoneal injection (C39535F63, ACMEC, Shanghai) for 3 days before surgery. (5) renal IRI + mesacanic acid intervention group (Mesacanic acid group), n = 13, each rat was given 50mg/kg/ day by intraperitoneal injection (C15385206, Macklin, Shanghai) for 3 days before surgery.

2.1.2 Establishment of animal model

Before surgery, the experimental rats were fasted for 12 hours but weren’t forbidden to drink. They were narcotized by intraperitoneally injecting with Xylazine (11.25mg/kg) and Zoletil® 50 (45mg/kg). The skin and muscle layers were cut along the right waist and the right kidney was ligatured and excised. The left kidney pedicle was freed and the kidney pedicle was clipped with a non-invasive artery clamp. After the kidney color changed, the incision was inserted into the abdominal cavity, and the incision was kept warm and moisturized for 45 minutes to establish the ischemia model. After 45 minutes, the arterial clip was released, and the color change of the kidney was observed. After confirming that the kidney was reperfused, the incision was closed layer by layer, and the reperfusion model was successfully established. After the operation, the rats were kept clean and fed freely. The left kidney of the rats in the sham group was only free of the kidney pedicle, and no arterial clamp was used to clamp the kidney pedicle, and other operations were the same as those in other groups.

2.1.3 CEUS

All the rats were examined by ultrasound 24 hours after the operation, and the channels for injecting ultrasound contrast agent and normal saline were established in advance. Sonovue contrast agent is configured as an SF6 microbubble suspension with 5ml of normal saline, which should be oscillated repeatedly before use. Siemens ACUSONS3000 ultrasonic diagnostic instrument (Siemens GMBH) and 9L4 frequency conversion linear array probe were used and the relevant parameters were set: frequency 4.0MHz, depth 3.5cm, Mechanical index 0.07. The contrast agent was injected rapidly at a dose of 0.4ml/kg through the tail vein tube, and then 0.2ml of normal saline was injected into the tube. The left kidney was observed continuously. The renal cortical region with an area of 0.05mm2 was delineated as ROI, and the analysis software autotracking contrast quantification (ACQ) was used for quantitative analysis. The relevant parameters (Peak, TP, AUC, MTT) were obtained and the corresponding TIC curves were drawn. All parameters were measured three times by the same operator.

2.1.4 Serum biochemical analysis

After CEUS, 3ml of blood was taken from the heart and the upper serum was taken after centrifugation. Serum Cr, Urea and Cystatin C (Cys-C) levels were detected respectively (Mindray BS-22000M biochemical detection pipeline).

2.1.5 Hematoxylin-eosin (HE) staining

After blood collection, the rats were euthanized with Sodium pentobarbital(120 mg/kg, intraperitoneal), and the right kidney tissues of the rats were quickly taken, half of which were fixed with tissue fixation solution and stored in the environment at 4℃, and half of which were frozen in liquid nitrogen and stored in the refrigerator at -80℃ for follow-up examination. The specimens were fixed with paraffin embedding, and HE staining was performed after the section. Pathological morphological changes of rat kidney sections were observed under the microscope.

2.1.6 Enzyme-linked immunosorbent assay (ELISA)

The concentrations of Interleukin (IL) -1β, IL-18, NLRP3, Keap1, Nrf2 and SDH were detected by ELISA. The kidney tissue of rats was accurately weighed, and the residual blood was removed. Phosphate buffer saline was added according to the mass volume ratio of 1:9, and then tissue homogenization was performed using a tissue grinder. The homogenate was centrifuged at 5000 g for 10 min, and the supernatant was taken to be measured. The actual concentration of the sample was measured and calculated according to the kit instructions.

2.1.7 Dihydroethidium (DHE) staining

The frozen sections of the rat kidney were restored to room temperature and then labeled and stained according to the instructions of the DHE detection kit (S0063, Beyotime Biotechnology, Shanghai). Scanning and browsing software was used for image acquisition of sections. Each section was observed at low power first, and then 100-fold and 200-fold microscopic images were collected respectively. A total of 3 fields of view were collected. The proportion of positive area per image was calculated using the Halo 101-WL-HALO-1 data analysis system (Indica Labs, USA).

2.1.8 Immunofluorescence staining of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)

ASC is an important component protein of NLRP3 inflammasome. In order to further verify whether itaconic acid and its isomer citraconic acid and mesaconic acid can affect the activation of the inflammasome, immunofluorescence staining of ASC was performed on frozen sections of rat kidney according to the instructions of the kit. After staining, scanning and browsing software was used to collect images of the sections. Each section was observed at low magnification before all tissues were observed, and then 100-fold and 200-fold microscopic images were collected respectively. A total of 3 fields of view were collected. The Halo data analysis system was used to calculate the proportion of positive area in each image.

2.1.9 TdT-mediated dUTP nick end labeling (TUNEL)

The renal tissue of rats stained by TUNEL was observed under a fluorescence microscope to detect the breakdown of nuclear DNA during apoptosis and determine the percentage of apoptotic cells in the renal tissue. The fixed specimens were embedded and stained according to the TUNEL test kit (1168479590, Roche Group). The Pannoramic 250 digital slicing scanner was used for scanning. Image collection and apoptotic cell analysis were carried out. And then manual counting was used to calculate the percentage (%) of apoptotic cells in the picture.

2.2. Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics 27.0 software, and the measurement data were expressed as mean ± standard deviation (𝑥 ± s). The Shapiro-Wilk test was used to check the normality of the distribution before the analysis. One-way ANOVA and Kruskal-Wallis test in non-parametric tests were used to compare indicators among measurement data sets. p < 0.05 was considered statistically significant.

3.1 CEUS parameters assessed renal function changes

We compared the IRI group and Sham group to confirm the effect of renal IRI on renal function in rats, and compared itaconic acid, citraconic acid and mesaconic acid intervention group and IRI group to study whether these substances have protective effects on renal IRI. Figure 1A shows the experimental flow of itaconic acid and its isomers citraconic acid and mesaconic acid in the intervention of IRI-induced AKI. Figure 1B shows the renal color changes during the establishment of the IRI model when we confirmed renal ischemia and reperfusion.

Firstly, the parameters of renal CEUS were statistically analyzed in each group. The results showed that compared with the sham group, the Peak value of the IRI group was increased (p < 0.001), while the Peak value of the itaconic acid group, citraconic acid group and mesaconic acid group was significantly decreased compared with the IRI group (p < 0.001) (Fig. 1C). Compared with sham group, Tp value of IRI group was increased (p < 0.01). The Tp value of mesaconic acid group was decreased compared with IRI group (p < 0.05), but there was no statistical significance in Tp value of the itaconic acid group and citraconic acid group compared with IRI group (p > 0.05) (Fig. 1D). Compared with the sham group, AUC value in IRI group was significantly increased (p < 0.001), while AUC value in the itaconic acid group, citraconic acid group and mesaconic acid group was significantly decreased compared with IRI group (p < 0.001) (Fig. 1E). Compared with the sham group, MTT value in IRI group was prolonged (p < 0.001), and MTT value in the itaconic acid group (p < 0.001), citraconic acid group (p < 0.01) and mesaconic acid group (p < 0.01) was shortened compared with IRI group (Fig. 1F). TIC curves drawn showed that the signal strength of contrast agent in the sham group and drug intervention group rapidly decreased to the basic level after reaching the peak, while that of contrast agent signal strength in IRI group decreased slowly after reaching the peak (Fig. 1G).

3.2 Itaconic acid, citraconic acid and mesaconic acid can reduce renal function injury caused by IRI

In order to evaluate whether the intervention of itaconic acid, citraconic acid and mesaconic acid can effectively reduce renal impairment, biochemical analysis was performed on rat serum to detect the levels of Urea, Cr and Cys-C. Compared with the sham group, the serum Urea level of rats in the IRI group was increased (p < 0.05), while the serum Urea level of rats in itaconic acid group (p < 0.01), mesaconic acid group (p < 0.001) and citraconic acid group (p < 0.05) was decreased compared with that of IRI group (Fig. 2A). Compared with the sham group, Cr level in IRI group was increased (p < 0.05), while Cr level in itaconic acid group (p < 0.05), mesaconic acid group (p < 0.05) and citraconic acid group (p < 0.001) was decreased compared with IRI group (Fig. 2B). The serum Cys-C level of rats in IRI group was higher than that in sham group (p < 0.05), while the serum Cys-C level in itaconic acid group (p < 0.05), citraconic acid group (p < 0.001) and mesaconic acid group (p < 0.01) was lower than that in IRI group (Fig. 2C). In order to further understand the degree of kidney injury in rats, HE staining was performed on the kidney tissues of rats in each group. The results showed that the renal tissue capsule in the sham group was relatively complete, and no connective tissue hyperplasia and inflammatory exudation were observed; the boundary between cortex and medulla was clear, and the renal corpuscle was complete and clear. The renal capsule cavity was clearly visible, and the capillary basement membrane or mesangial hyperplasia was not observed in the vascular globules. Renal tubular epithelial cell vacuolar degeneration, tubular epithelial cell degeneration and necrosis, tubular epithelial cell shedding, brush edge shedding, and tubular formation were observed in the IRI group, and the renal tissue lesions in the itaconic acid group, citraconic acid group and mesaconic acid group were relatively mild (Fig. 2D). It was proved that renal function was obviously improved after drug intervention. However, TUNEL staining results showed that there were no significant differences in renal cell apoptosis among all groups (p > 0.05) (Fig. 2E, Fig. 2F). The results of biochemical analysis and pathological staining of rat kidney showed that itaconic acid, citraconic acid and mesaconic acid could effectively reduce renal function injury caused by renal IRI.

3.3. Itaconic acid, citraconic acid and mesaconic acid reduce IRI by reducing the activation of NLRP3 and the release of inflammatory cytokines

4-OI has been shown to inhibit lactate dehydrogenase release, IL-18 release, ASC speck formation, and gasdermin D and IL-1β processing, all of which are indicators of NLRP3 activation[41]. To verify whether non-derived itaconic acid and its isomers have the same anti-inflammatory effects, we examined the levels of inflammatory cytokines IL-1β and IL-18 in rat kidneys. Compared with the sham operation group, IL-1β concentration in the kidney of the IRI group was significantly increased (p < 0.01). Compared with the IRI group, the renal IL-1β concentration in the itaconic acid group and mesaconic acid group was decreased (p < 0.05). There was no statistically significant difference in IL-1β concentration in the kidney of rats in the citraconic acid group (Fig. 3A). Compared with the sham group, IL-18 concentration in the kidney of the IRI group was increased (p < 0.01). IL-18 concentration in the kidney of rats in the itaconic acid group (p < 0.05), citraconic acid group (p < 0.05) and mesaconic acid group (p < 0.01) was lower than that in the IRI group (Fig. 3B).

We further tested the levels of the NLRP3 inflammasome in the rat kidneys using an ELISA kit. Compared with the sham group, NLRP3 concentration in the kidney of rats in the IRI group was significantly increased (P < 0.01). Compared with the IRI group, the concentration of NLRP3 in the kidney of rats in itaconic acid group (P < 0.05), citraconic acid group (P < 0.05) and mesaconic acid group (P < 0.01) was decreased (Fig. 3C).

In addition, the frozen sections of rat kidney tissue were stained with ASC spot immunofluorescence, and the positive expression of ASC spots was green. The results showed that compared with the sham group, the expression of ASC spot protein in the renal IRI group was significantly increased (P < 0.01). The expression of ASC spot protein in the itaconic acid group, citraconic acid group and mesaconic acid group was significantly reduced compared with that in the renal IRI group (P < 0.01) (Fig. 3D, Fig. 3E).

3.4 Itaconic acid, citraconic acid and mesaconic acid can reduce IRI-induced AKI by inhibiting oxidative stress

Next, we measured the concentration of SDH in the rat kidney tissue. Compared with the sham group, SDH concentration in the kidneys of rats in the IRI group was decreased (p < 0.01). SDH concentration in the itaconic acid group and mesaconic acid group was increased (p < 0.05) compared with the IRI group. There was no statistical significance in SDH concentration in the kidney of rats in the citraconic acid group (p > 0.05) (Fig. 4A). The SDH in renal tissue of rats decreased after IRI. The addition of itaconic acid and mesaconic acid could increase the SDH level of rats, but citraconic acid had little effect on the SDH level.

We used DHE probes to measure ROS levels in the rats' kidneys. The results showed that compared with the sham group, the proportion of red fluorescence area in the tissue of rats in the IRI group was significantly increased, with statistically significant significance (P < 0.01). Compared with the IRI group, the proportion of red fluorescence area in renal tissue of rats in itaconic acid, citraconic acid and mesaconic acid groups was significantly decreased (P < 0.01) (Fig. 4B, Fig. 4C). The results indicated that ROS concentration in renal tissues increased significantly after renal IRI, and ROS concentration in renal tissues of rats treated with itaconic acid, citraconic acid and mesaconic acid decreased significantly.

3.5 Itaconic acid, citraconic acid and mesaconic acid reduce IRI induced AKI by Keap1-Nrf2 axis

In order to further study the effect of itaconic acid and its isomers citraconic acid and mesaconic acid on the renal IRI signaling pathway, we detected Nrf2 and Keap1 concentrations in rat kidneys. Compared with the sham operation group, the Nrf2 concentration in the IRI group was decreased (p < 0.01), while the Nrf2 concentration in the drug intervention group was significantly increased (p < 0.05) compared with the IRI group (Fig. 5A). Compared with the sham group, the Keap1 concentration in the kidneys of rats in the IRI group was increased (p < 0.01), while the Keap1 concentration in the itaconic acid group (p < 0.01), mesaconic acid group (p < 0.01) and citraconic acid group (p < 0.05) was decreased compared with the IRI group (Fig. 5B). These results indicate that itaconic acid, citraconic acid and mesaconic acid may reduce tissue damage by activating Keap1-Nrf2 pathway.

AKI is a syndrome with multiple etiologies that is characterized by rapid deterioration of kidney function occurring over a period of hours to days[4]. It is also a common complication of many critical diseases, with an incidence of about 13–73%[11]. Renal injury is affected by various pathological factors, and renal IRI is generally believed to be the basic pathogenesis of AKI. Ischemia-reperfusion is the temporary loss of blood flow and tissue perfusion followed by its restoration. Because blood flow stops, cells lack enough oxygen to synthesize ATP[42]. Renal IRI is closely related to ROS production, intracellular calcium overload, inflammation and apoptosis[43–45].

A key component of inflammation is the activation and recruitment of freely circulating white blood cells in the blood pool, resulting in impaired renal microcirculation and slowed blood flow in the renal cortex[46]. Ischemia and hypoxia in renal tissue lead to massive ROS release, and inflammatory cells release proinflammatory factors, recruiting large numbers of leukocytes. Microbubbles are engulfed by activated leukocytes, and the engulfed ones are still acoustically active, thus allowing the detection of microbubbles on ultrasound imaging[47]. The microbubbles exist in the inflammation area for a long time, resulting in slower clearance, and the Tp value increases, MTT prolongation, Paek value and AUC value increase, which are reflected in the TIC curve as a slow rise, and then a slow decline after reaching the peak.

The renal tissue inflammation in itaconic acid, citraconic acid and mesaconic acid groups was significantly alleviated, and the parameters showed a decrease in Tp value, a decrease in MTT value, and a decrease in Paek value and AUC value compared with the renal IRI group, which was reflected in the TIC curve showing a rapid rise in the curve and a rapid decline after reaching the peak value, basically consistent with the previous study results of Sun Xiaoying et al.[16]. CEUS can reflect the renal function status through the changes of Tp, peak, AUC, MTT value and TIC curve at the early stage of the kidney, and the changes of the indicators are consistent with the pathological results, which indicates that we may timely detect the risk of renal IRI through early CEUS examination. At the same time, when CEUS was measured after drug treatment, all the indicators showed remission compared with that in the untreated group, indicating that CEUS can be used as an effective means to evaluate the efficacy of renal IRI in partial drug treatment. Therefore, CEUS can be used as a new non-invasive test to evaluate renal function[48].

In our experimental results, we detected the concentration of SDH in the kidney of rats, and the results showed that the concentration of SDH in the kidney of rats decreased after IRI, itaconic acid and mesaconic acid inhibited the activity of SDH, and the concentration of SDH in the kidney of rats increased compensatively, while citraconic acid almost did not increase the concentration of SDH. Itaconic acid is thought to limit inflammation by inhibiting SDH [49, 50]. The similar structure of itaconic acid and succinic acid can directly competitively inhibit the enzymatic activity of SDH[33, 51]. Previous studies have shown that itaconate can act as a mitochondrial regulator to control redox metabolism and improve cerebral IRI in mice. The main mechanism is to reduce tissue oxidative stress damage by inhibiting the activity of SDH and reducing ROS levels during reperfusion in vivo[52]. Itaconic acid and its isomers inhibit SDH activity to limit ROS production and reduce the release of inflammatory factors at the injury site. In vitro studies have shown that itaconate has a strong inhibitory effect on SDH, while citraconate has no inhibitory effect on SDH, but it does reveal that mesaconate has a moderate inhibitory effect on SDH[40]. However, in the study of He, W. et al., it was believed that mesaconate had no inhibitory effect on SDH[53]. In our results, citraconic acid did not inhibit SDH, but still mitigated kidney damage, and we speculate that citraconic acid may inhibit oxidative stress through other pathways.

Itaconic acid can also reduce the activation of NLRP3 inflammasome by modifying a specific cysteine on NLRP3[41]. This conclusion was also confirmed by ELISA test of NLRP3, and citraconic acid and mesaconic acid also showed inhibitory effects on NLRP3. The NLRP3 inflammasome is composed of a variety of protein complexes, including the protein ASC. They assemble into inflammatory bodies in response to inflammatory stimulation, leading to tissue damage[54]. The experimental results showed that the exogenous addition of itaconic acid, citraconic acid and mesaconic acid all reduced the expression of ASC patches, inhibited the activation of NLRP3 inflammasome, reduced the release of IL-18, and all of them except citraconic acid reduced the expression of pro-inflammatory factor IL-1β, thus alleviating the acute kidney injury caused by renal IRI. NLRP3 inflammasome is a polymeric complex composed of cytoplasmic sensor, caspase activation, ASC spot-like protein and pro caspase-1. Inflammasome is stimulated by DAMPs and triggers inflammation by activating caspase-1. Caspase-1 cuts the N-terminal sequence of GSDMD, causing it to bind to the membrane to produce membrane pores, resulting in cell pyroptosis. Activated caspase-1 also promotes the release of inflammatory cytokines IL-1β and IL-18 [52, 55–57], leading to interstitial immune cell infiltration and renal tubule injury[58]. It can be observed from the experimental results of this study that after renal IRI in rats, the expression of ASC spot protein is significantly increased, the activation of NLRP3 inflammation is increased, and the release of inflammatory factors IL-1β and IL-18 is increased. After treatment with itaconic acid and its isomers, the activation of NLRP3 inflammasome and the release of inflammatory factors IL-1β and IL-18 were reduced in the intervention group. It can be seen that itaconic acid and its isomers can not only alleviate kidney injury by inhibiting oxidative stress, but also relieve renal IRI by inhibiting inflammation-related pyroptosis.

In our experimental study, the concentration of Keap1 in the kidney tissues of rats in itaconic acid, citraconic acid and mesaconic acid groups was significantly lower than that in the IRI group, while the concentration of Nrf2 in the kidney tissues of rats in itaconic acid, citraconic acid and mesaconic acid groups was significantly higher, which proved that not only itaconic acid could inhibit inflammation by activating Nrf2, but also citraconic acid and mesaconic acid, as isomers, can exert anti-inflammatory effects through the Keap1-Nrf2 pathway. Studies have shown that reduced Nrf2 levels are found in many kidney diseases with high levels of ROS[59]. This is consistent with our experimental results that Nrf2 can counteract ROS-mediated tissue damage[60]. Nrf2 is a major transcription factor in the inflammatory response and in controlling the antioxidant response that is necessary to maintain cellular redox homeostasis, and since oxidative stress is also a key driver of various kidney diseases, Nrf2 has been reported to prevent kidney disease by down-regulating the production of ROS[61]. Under normal conditions, Nrf2, as a combination with Keap1, promotes its ubiquitination and proteasome degradation. Itaconic acid directly modifies proteins through the alkylation of cysteine residues, leading to the conformational change of the NRF2-KEap1 complex, inhibiting its degradation of Nrf2, and increasing the expression of Nrf2, which has antioxidant and anti-inflammatory capabilities[49]. Studies have shown that citraconic acid is the strongest electrophilic among the three isomers, resulting in the strongest activation of Nrf2[40]. In our experimental results, we did not compare the differences in the activation of Nrf2 by the three isomers, which may be related to the differences in membrane permeability and cell absorption of the three substances, and the specific reasons for the differences need to be further studied.

In addition to its appeal mechanism, itaconic acid induces the activation of activating transcription factor 3, which participates in the body's anti-inflammatory effects[49, 62, 63]. Besides, itaconic acid selectively inhibits dioxygenase to inhibit inflammation[64]. Itaconic acid also plays an important anti-inflammatory role as a glycolysis inhibitor[65](Fig. 6). These pathways could not be verified in our study, but it cannot be ruled out that they also play a key role in reducing renal IRI. At present, itaconic acid has shown good therapeutic effects in many animal disease models such as sepsis and pulmonary fibrosis[66–69]. Various itaconic acid derivatives such as 4-OI and dimethyl itaconic acid exhibit similar biological properties[70, 71]. Fumaric acid, which has a similar structure to itaconic acid, is already used clinically to treat multiple sclerosis and psoriasis. In our study, the isomers of itaconic acid and itaconic acid were used as experimental drugs to intervene in renal IRI, and it was found that they were also feasible in the treatment of acute kidney injury, which proved that itaconic acid and its various derivatives and isomers have great research value and application prospects in the field of disease treatment.

The innovation of this study: First, fumaric acid, which is similar in structure to itaconic acid, has been applied in clinical treatment, and itaconic acid and its derivatives also have similar biological properties to fumaric acid. As a popular drug in preclinical studies on pharmacological intervention and pathological models, itaconic acid has demonstrated its great application value in a variety of pathological models. It has a promising clinical translational prospect as a therapeutic strategy to reduce IRI. Second, this study also studied the intervention effect of citraconic acid and mesaconic acid, isomers of itaconic acid, on IRI. Citraconic acid and mesaconic acid have anti-inflammatory and antioxidant effects similar to itaconic acid, but at present, there are few data on the application of these two substances in experimental studies. This study is the first time to apply these two substances in the model of treating AKI. The experimental data of citraconic acid and mesaconic acid in the treatment of these diseases are relatively lacking, and provide more possible new strategies for the treatment of IRI induced AKI. Third, CEUS technology was used in this study to monitor blood perfusion of renal IRI in real time, and quantitative TIC parameters (Peak, Tp, AUC, MTT) of CEUS technology were compared with biochemical indicators and pathological results reflecting renal function status. We found that CEUS can reflect the AKI condition of the kidney after IRI in an early, accurate and effective manner. The combination of CEUS results with AKI biomarkers and pathological results can provide a new means for the early diagnosis and prognosis assessment of ischemia-reperfusion induced AKI.

Limitations of this experiment: First, due to the small sample size, TUNEL staining in each group was significantly different, resulting in no statistical significance. Second, only a one-time point of 24 hours was selected for detection in this study, and multiple time points could be selected for dynamic detection of rat kidney changes in subsequent experiments. Third, this study only studied the similar anti-inflammatory and antioxidant mechanisms of itaconic acid, citraconic acid and mesaconic acid, and did not quantitatively compare whether these three substances had different protective effects on renal IRI.

Itaconic acid and its isomers citraconic acid and mesaconic acid have similar inhibitory effects on inflammation-related pyroptosis, improve the antioxidant capacity of kidney cells, and may alleviate renal injury caused by IRI through the Keap1-Nrf2 pathway, providing a new strategy for the treatment of AKI. CEUS can effectively evaluate the renal cortical blood perfusion of renal IRI, and CEUS can quantitatively evaluate the changes of renal function after drug intervention, providing an effective examination method for clinical diagnosis, treatment and prognosis assessment of this disease.

Author Contribution

Bin Tang and Zhijian Luo wrote the main manuscript text. Rong Zhang and Dongmei Zhang and Mingxing L i prepared figures. All authors reviewed the manuscript.Dai Yan conceptualized this manuscript,such as the experimental design and steps , and revised the original manuscript.

Data Availability

The data availability statement ：The data that support the findings of this study are available from the corresponding author upon reasonable request.all authors have agreed with share data;Data is provided within the manuscript information files.

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No competing interests reported.

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Evaluation of the efficacy of rat renal ischemia-reperfusion injury after itaconic acid and its isomers treatment by contrast-enhanced ultrasound(CEUS)

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Experimental method

2.1.1Experimental animals and grouping

2.1.2 Establishment of animal model

2.1.3 CEUS

2.1.4 Serum biochemical analysis

2.1.5 Hematoxylin-eosin (HE) staining

2.1.6 Enzyme-linked immunosorbent assay (ELISA)

2.1.7 Dihydroethidium (DHE) staining

2.1.8 Immunofluorescence staining of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)

2.1.9 TdT-mediated dUTP nick end labeling (TUNEL)

2.2. Statistical analysis

3. Results

3.1 CEUS parameters assessed renal function changes

3.2 Itaconic acid, citraconic acid and mesaconic acid can reduce renal function injury caused by IRI

3.4 Itaconic acid, citraconic acid and mesaconic acid can reduce IRI-induced AKI by inhibiting oxidative stress

3.5 Itaconic acid, citraconic acid and mesaconic acid reduce IRI induced AKI by Keap1-Nrf2 axis

4. Discussion

5. Conclusion

Declarations

Author Contribution

Data Availability

References

Additional Declarations

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