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Assessment of renal pathophysiological processes and protective effect of quercetin on contrast-induced acute kidney injury in type 1 diabetic mice using diffusion tensor imaging
REDOX REPORT
Z. WU ET AL.
Wu Ziqian a
Hu Jingyi a
Li Yanfei b
Yao Xiang c
Ouyang Siyu d
Ren Ke a
a Department of Radiology, Xiang’an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen Radiological Control Center, Xiamen, People’s Republic of China
b Cell Therapy Research Center, Xiamen Humanity Hospital, Xiamen, People’s Republic of China
c Department of Neurosurgery, Zhongshan Hospital of Xiamen University, Xiamen, People’s Republic of China
d Department of Radiology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, People’s Republic of China
CONTACT Ke Ren renke815@sina.com Department of Radiology, Xiang’an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen Radiological Control Center, Xiamen 361102, Fujian, People’s Republic of China
Supplemental data for this article can be accessed online at https://doi.org/10.1080/13510002.2024.2398380.

16 9 2024
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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

Purpose: To investigate the renal pathophysiological processes and protective effect of quercetin on contrast-induced acute kidney injury (CI–AKI) in mice with type 1 diabetic mellitus(DM) using diffusion tensor imaging(DTI).

Methods: Mice with DM were divided into two groups. In the diabetic + contrast medium(DCA) group, the changes of the mice kidneys were monitored at 1, 24, 48, and 72 h after the injection of iodixanol(4gI/kg). The mice in the diabetic + contrast medium + quercetin(DCA + QE) group were orally given different concentrations of quercetin for seven days before injection of iodixanol. In vitro experiments, renal tubular epithelial (HK-2) cells exposed to high glucose conditions were treated with various quercetin concentrations before treatment with iodixanol(250 mgI/mL).

Results: DTI-derived mean diffusivity(MD) and fractional anisotropy(FA) values can be used to evaluate CI-AKI effectively. Quercetin significantly increased the expression of Sirt 1 and reduced oxidative stress by increasing Nrf 2/HO-1/SOD1. The antiapoptotic effect of quercetin on CI-AKI was revealed by decreasing proteins level and by reducing the number of apoptosis-positive cells. In addition, flow cytometry indicated quercetin-mediated inhibition of M1 macrophage polarization in the CI-AKI.

Conclusions: DTI will be an effective noninvasive tool in diagnosing CI-AKI. Quercetin attenuates CI-AKI on the basis of DM through anti-oxidative stress, apoptosis, and inflammation.

KEYWORDS

Contrast-induced acute kidney injury
diabetic mellitus
diffusion tensor imaging
quercetin
Sirt1
the National Natural Sciences Foundation of China 10.13039/501100001809 82071886 the Scientific Research Foundation for Advanced Talents, Xiang’an Hospital of Xiamen University 10.13039/501100004300 PM201809170011 The authors gratefully acknowledge the financial supports from the National Natural Sciences Foundation of China [grant number 82071886], and the Scientific Research Foundation for Advanced Talents, Xiang’an Hospital of Xiamen University [grant number PM201809170011].
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pmc1. Introduction

Contrast-induced acute kidney injury(CI-AKI) occurring in enhanced computed tomography and angiographic studies can result in accelerated progression of chronic kidney disease(CKD) and necessary dialysis, along with substantial costs [1, 2]. Renal vasoconstriction induced ischemia, hypoxia, and direct toxicity of contrast media to renal tubular cells are currently thought to be the mechanisms of CI-AKI [3]. It is important to recognize that multiple factors can contribute to CI-AKI, which can be divided into patient-related and contrast-related risk factors. CKD and diabetic nephropathy(DN) are the two main patient-related factors [4]. Besides, DM may also be a risk factor for CI-AKI and the incidence of CI-AKI in non-diabetic patients is about 0.6–2.3%, while it ranges from 5.7% to 29.4% in diabetics [5]. Nevertheless, there is a lack of comprehensive evidence demonstrating the efficacy of compounds in mitigating the nephrotoxic effects of iodinated contrast agents in diabetic individuals. Therefore, further research is warranted to investigate potential compounds for the prevention of CI-AKI.

Quercetin, also known as 3,5,7,3′,4′-pentahydroxyflavone, is a natural flavonoid widely distributed in herbal medicines. It has been proved that quercetin has a protective effect on kidney function by alleviating renal toxicity, apoptosis, fibrosis, and inflammation [6, 7]. It has been demonstrated that chronic supplementation with quercetin can reduce cadmium ion-induced adverse kidney effects in rats by attenuating oxidative stress and upregulating silent information regulator 1(Sirt 1) [8]. Sirt 1, a NAD2+ deacetylase enzyme, exerts its protective function by deacetylating a wide range of substrates and transcription factors [9]. The beneficial effects of Sirt1 on renal injuries correlate with the activation of the nuclear factor erythroid 2-related factor 2(Nrf 2) and antioxidant response element(ARE) antioxidative pathways, which leads to overexpression of antioxidative enzymes such as hemeoxygenase-1(HO-1) and superoxide dismutase 1(SOD1) [10, 11]. There is evidence that quercetin can enhance Sirt1 activity, promote autophagy, and reduce sepsis-induced acute kidney injury [12]. However, the mechanisms underlying how quercetin prevents CI-AKI in diabetic patients are poorly understood.

Noninvasive, reproducible and highly promising, diffusion tensor imaging (DTI) is one of the functional magnetic resonance imaging(fMRI) techniques. In terms of renal function and renal microstructure, DTI is a useful tool. The DTI provides two common quantitative metrics, including mean diffusivity(MD) and fractional anisotropy(FA), which have been proven to have significant correlation with kidney injury [13–15]. In particular, DTI can identify pathology in diabetes and distinguish different stages of the disease [16]. DTI may be a useful tool to assess the renal pathology of CI-AKI, including renal inflammation and edema.

Hence, to investigate the impact of QE on renal injury in diabetic mice induced by iodinated contrast agents and elucidate the underlying protective mechanism, we postulated that QE may mitigate the nephrotoxic effects through activation of the Sirt1/Nrf2/HO-1 pathway. Our findings revealed that exposure to iodinated contrast agents led to microcirculation disturbances in renal tissue, resulting in impaired water diffusion and filtration. QE can indeed regulate renal dysfunction caused by oxidative stress and apoptosis, thereby protecting diabetic mice from CI-AKI. Additionally, our findings suggested that DTI parameters MD and FA may serve as early indicators of renal functional changes. This study aims to offer a foundation and theoretical framework for further investigation into the potential of QE as a preventive compound against CI-AKI in diabetic patients.

2. Materials and methods

2.1 Animals and experimental design

All animal experiments were reviewed and approved by the Animal Ethics Committee of Xiamen University(Profile No. XMULAC20190005). Male C57BL/6 mice were housed under specific pathogen-free conditions at the Xiamen University Laboratory Animal Center and were randomly assigned to experimental animal groups. All animals were acclimatized 7 days before starting each experiment. All the mice were fasted for 24 h. A diabetic state was induced in 7-week-old mice by an intraperitoneal injection of streptozotocin(STZ) (Macklin, S6089) in citrate buffer, pH4.5 (50 mg/kg body weight /day), for five consecutive days. One week after the last injection, the mice with glucose levels >16.7 mmol/L were considered diabetic [17]. The mice with high glucose status were maintained for at least 4 weeks before subsequent experimentation.

Sixty mice were randomly divided into two primary groups. After dehydration for 24 h, the CI-AKI model was induced by Iodixanol-320(4gI/kg) injection through tail vein [18]. The mice in one primary group were used for the diabetic + contrast medium group(DCA): five mice obtained DTI data at 24 h pre-injection and 1, 24, 48, and 72 h after the injection of the contrast agent, and five mice randomly chosen from the remaining 25 mice were measured for the biochemical indicators and sacrificed for histological studies at each time point. The data at 24 h pre-injection was used as the baseline. And the other 30 mice were used as diabetic + contrast medium + quercetin(HPLC >97%, MAYA – R, 816042)group(DCA + QE): the mice were treated with different quercetin concentrations(25, 50, 75 mg/kg/d, p.o.)once a day, for a total of 7 days before applying Iodixanol. Animals from this group underwent DTI 24 h after the injection of contrast agent. More specific information is given in the workflow schematic in Figure 1. Figure 1. Main procedure of experiment workflow. Workflow shows the group designations, and the time points for fMRI data acquisition, blood and kidney tissue collections. STZ, streptozocin; fMRI, functional Magnetic Resonance Imaging.

2.2 Kidney functional magnetic resonance imaging

All MRI experiments were performed on a horizontal bore 9.4 T scanner operating on a Brucker Avance platform(Bruker 9.4 T MicroMRI, BioSpec 94/20USR)over the coronal renal region in a prone position, head-in first. The imaging protocol included T2-weighted images and DTI. DTI acquisition parameters are shown in Table 1. As DTI-derived metrics, FA and MD provide added information by gauging diffusion direction and degree of direction of directed diffusion [13, 14]. Quantitative regional DTI parameters were performed, using manually defined region-of-interest(ROI) delineated by three experienced radiologists who remained blinded to each group assignment. The intragroup correlation coefficient(ICC) was used to analyze the consistency of the three measurements. ROIs were manually placed in the rental cortex(CO), outer medulla(OM), and inner medulla(IM) according to a previously described approach [19] (Supplemental Fig.1). Table 1. Scanning parameters for DTI sequence.

Parameters/sequences	DTI	
Number of slices, n	18	
Section thickness, mm	0.5	
Repetition time, ms	4500	
Echo time, ms	23.5	
Orientation	Coronal	
Matrix	128×128	
Field of view, mm2	40×40	
Bandwidth(hertz perpixel)	250000	
Interslice dist, mm	0.5	
b-values(s/mm2)	0,1000	
diffusion directions	30	
Breathing protocol	Free	

2.3 Histopathology and immunohistochemistry

Soon after fMRI images were obtained, the mice were sacrificed with an overdose of anesthetics for 10 min. After the kidneys were fixed in 4% paraformaldehyde, they were dehydrated with an increasing gradient of ethanol and dimethylbenzene. Then kidneys were embedded in paraffin and sectioned at 4 μm for haematoxylin and eosin(HE) staining. Histological assessment of renal damage was performed according to the intensity of tubular vacuolization and degeneration, casts forming after contrast agent injection [20]. Based on a previously described method [21], the injury was examined by the percentage of damaged fields: grade 0, no damage; grade 1, < 25%; grade 2, 25-49%; grade 3, 50-75%; grade 4, > 75%. Five nonoverlapping fields of each renal compartment were observed.

Paraffin-embedded kidney sections were also used for immunohistochemical studies. The kidney tissue sections were dewaxed, rehydrated, subjected to antigen retrieval, and individually incubated with antibodies: anti-kidney injury molecule-1 antibody (KIM-1, 1:100, 14971S; Cell Signaling, China). After an overnight incubation at 4°C, the samples were incubated with secondary antibodies for 1 h at 24°C, treated with diaminobenzidine, and counterstained with haematoxylin. The KIM-1-positive area was measured using ImageJ software(U.S. National Institute of Health).

2.4 Renal function biomarkers

All mice were sacrificed at specified time points after MRI examinations, blood from the eye socked(0.5–1.0 mL) standing at room temperature (20–25°C) for 30 min and centrifuged at 4,000 rpm for 10 min, collecting serum for analysis. The amounts of blood urea nitrogen(BUN), serum creatinine(Cr) were tested at Wuhan Servicebio Technology Co., Ltd. The detailed procedure is described in the instruction manual of Leidu Creatinin Assay Kit (S03076) and Nitrogen Assay Kit (S03036).

2.5 Flow cytometry

As described in the previous method [22], the target leukocytes were incubated with antibodies(CD11b, 1:200, 742640; CD45, 1:200,560510; F4/80, 1:200, 565411; CD86, 1:200, 551396; CD206, 1:200, 569273) for 20 min at 4°C in darkness and analyzed by flow cytometry. Leukocyte, CD45+; Total macrophages, CD45+ F4/80+ CD11b+; M1 macrophage, CD45+ F4/80+ CD11b + CD86+; M2 macrophage, CD45+ F4/80+ CD11b + CD206 + . All antibodies were purchased from BD Pharmingen. Results were analyzed using FlowJo software(FlowJo LLC).

2.6 In vitro experiments

The human proximal tubule epithelial cell line HK-2 was purchased from Stem Cell Bank(Chinese Academy of Sciences, Shanghai, China). The culture medium (DMEM, PM150210; Procell, China) containing 10% fetal bovine serum(FBS, C04001-100; Biological Industries, U.S.A.) and antibiotics(100IU/mL penicillin and 100μg/mL streptomycin) was used for HK-2 cells culture. Cells were cultured at 37°C in a humidified atmosphere condition containing 95% O2 and 5% CO2. Prior to treatment, HK-2 cells were cultured in serum-free medium for 12 h for starvation. The culture medium with a high level of glucose to induce a DM model. The quercetin(10μM, 20μM, 30μM) was added to culture medium for 4 h before composing to contrast media. The method of contrast-induced HK-2 cells (250mgI/mL) was completed as the previously described [23], and HK-2 cells were collected for subsequent experiment at 72 h after incubating with iodixanol.

2.7 CCK-8 cytotoxic assay

In order to evaluate the protective effect of quercetin, HK-2 cell viability was quantified by a Cell Counting Kit-8(CCK-8) assay(GK10001, GLPBIO, U.S.A.). Briefly, during the logarithmic growth phase, HK-2 cells were planted to a plate with 96 wells at 5×103 cells/well. After treatment, cells were incubated with 10μL CCK-8 test solution for 4 h at 37°C. In final, by using a microplate reader(Thermo Fisher Scientific, Waltham, MA, U.S.A.), the absorbance value at 450 nm was counted.

2.8 Scratch test

HK-2 cells were seeded to 6-well dishes, and 1×106 cells were inoculated in every well and cultured to 80%−90% fusion degree. After treatment, the bottom of 6-well plates was gently scratched with 200 μL spear head, a vertical line, and a horizontal line were drawn, then the cells were cleaned with PBS for three times and culture with drug-containing medium. The final concentration of QE in each experimental group was 10, 20, 30 μM, respectively. An inverted microscope was employed to take pictures and take again at the same position 24 h later. Use ImageJ software to measure the scratch width, mobility% = (0 h scratch width/0 h scratch width – 24 h scratch width)/0 h scratch width × 100%.

2.9 Real-time polymerase chain reaction

The cell injury was also measured by gene expression of KIM-1 assesses by quantitative real-time polymerase chain reaction. Human KIM-1 primers were purchased from Brogene Biotechnology: forward, 5′-CTGCAGGGAGCAATAAGGAG – 3′; reverse, 5′-TCCAAAGGCCATCTGAAGAC – 3′. β-Actin was used as an internal reference to quantify the expression of target gene.

2.10 TUNEL assay

Terminal deoxynucleotidyl transferase-medicated deoxyuridine triphosphate nick end labeling (TUNEL) assay was performed to detect cell apoptosis. TUNEL staining was detected by TdT enzyme according to the manufacturer’s instructions (B0013, Lablead, China), and TUNEL-positive cells were calculated per 0.06 mm2.

2.11 Western Blot

The protein from kidney and HK-2 cells was separated in 7.5%, 10%, or 12.5% sodium dodecyl sulphate gel electrophoresis, and polyvinylidene membranes were incubated in primary antibodies at 4°C overnight. After washing, membranes were incubated with horseradish peroxidase-labeled conjugated secondary antibodies (ab6712, Abcam) for 1 h. The immunostained protein bands were visualized with an enhanced chemiluminescence system and analyzed using Image J software. The primary antibodies used in the study were as follows: Sirt1(ab189494, Abcam); Nrf 2(ab92946, Abcam); Bax(abs130057, absin); Bcl-2(NB100-56101, Novus Biologicals); HO-1(A19062, ABclonal); SOD1(WL01846, Wanleibio), cleaved caspase-3(WL02117, Wanleibio). β-Actin was used as an internal control.

2.12 Statistical Analysis

All statistical analyses were performed using Prism 8(GraphPad). Initially, we conducted normality tests on the data. For normally distributed data, we used parametric tests; for skewed data, we applied the Wilcoxon signed-rank test. Experiments were analyzed using a one-way repeated measures ANOVA based on time (baseline, 1, 24, 48, 72 h post-contrast injection) or condition (dose of QE). Post hoc tests were performed with Benjamini and Hochberg's correction for multiple comparisons and effect sizes were estimated by calculating Cohen’s d and eta squared. Spearman correlation analysis was used to study the linkage between fMRI and pathology. Only correlations whose statistical significance withstood multiple testing of FDR correction were considered. For all tests, the FDR-corrected statistical significance level was set at P < 0.05; and FDR was set at P = 0.05.

3. Results

3.1 DTI parameters MD and FA can dynamically reflect the changes of renal function

Prior to commencing the experiment, the weight and blood sugar levels of the two cohorts of mice were reevaluated (Supplemental Fig.2). The MD and FA values between the two groups of mice are presented in Supplemental Table 1 and their corresponding representative images are shown in Figure 2. Figure 2. Renal MD, FA maps in representative mice from the two groups and the changing trend of MD and FA values. The window and level setting for each map were kept the same. MD, mean diffusivity; FA, fractional anisotropy; Cont, control; CI-AKI, contrast-induced acute kidney injury; CI-AKI + LQE, contrast medium plus 25 mg/kg quercetin-treated group; CI-AKI + IQE, contrast medium plus 50 mg/kg quercetin-treated group; CI-AKI + HQE, contrast medium plus 75 mg/kg quercetin-treated group.

DTI facilitates measurement of not only the magnitude of Brownian water motion by means of MD but also the directionality that is defined as FA. The consistency of the measurement results of the two parameters of MD and FA is good, which were 0.84 and 0.81 respectively. Within the DCA group, MD and FA values were severely decreased at 24 h in renal CO (P = 0.041 and P = 0.040, Cohen's d = 1.098 and Cohen's d = 0.980), IM (P = 0.030 and P < 0.001, Cohen's d = 1.369 and Cohen's d = 1.633) when compared with baseline values. In the 24 h group, there was still a difference in renal OM FA values (P < 0.001, Cohen's d = 1.700) but not in MD values(P = 0.067). In addition to the FA value(P = 0.040, Cohen's d = 1.476) of renal IM, MD and FA increased toward baseline within 48 h. Thus, the difference was the greatest between the baseline group with 24 h group.

Within the DCA + QE group, experimental data revealed significant differences between CI-AKI + IQE, CI-AKI + HQE with CI-AKI for FA in OM (P = 0.008, P < 0.001, Cohen's d = 2.147 and Cohen's d = 1.767), IM (P = 0.020, P < 0.001, Cohen's d = 0.365 and Cohen's d = 0.284). MD values were significantly higher in the CI-AKI + IQE, CI-AKI + HQE groups compared with the CI-AKI group in CO (P = 0.039, P = 0.004, Cohen's d = 1.518 and Cohen's d = 1.237). No significant difference was found in other groups. In summary, quercetin relieved restriction of renal water molecule movement induced by exposure to contrast medium in DM. According to the DTI images, MD and FA values are highly effective MR biomarkers to evaluate renal function in CI-AKI with DM.

3.2 Quercetin can reduce acute kidney injury induced by iodoxanol

After administration of contrast medium, tubular dilatation was observed at 1 h. Kidney injury with intraepithelial vacuolar degeneration and interstitial inflammation started at 1 h, progressing in number and size over 24 h, followed by a gradual decline. At 24 h, the proximal convoluted tubular epithelial cells became swollen and part of the distal convoluted tubular lumens became narrow. After that, gradually declined.

Within 72 h after the injection of contrast medium, the exfoliation of renal tubular epithelial cells and disruption of tubular structure can be observed. The total severity scores were significantly increased at 24 h in CO(P < 0.001, Cohen's d = 6.197), OM(P < 0.001, Cohen's d = 8.003), IM(P = 0.030, Cohen's d = 2.982) when compared with baseline scores (Figure 3A and B). OM exhibited the most severe pathological changes among the ROIs [24], so we further evaluated the expression of Kim-1 in OM. Kim-1 was mainly expressed in the nucleus of tubular cells. After iodixanol injection, Kim-1 was transiently upregulated at 24 h and confined to a period of time (Figure 3C and D). Figure 3. (A-D) Histopathological changes of kidney (×400) and immunohistochemistry (IHC) (×200)of Kim-1 in the DCA group. (A) The black stars show cytoplasmic vacuoles. The black arrows indicate interstitial infiltration and tubular desquamation, necrosis, or atrophy. (B) Histological score. (C-D) Representative images and quantification of Kim-1 staining in kidney OM. (E-H) Histopathological changes of kidney (×400) and IHC (×200)of Kim-1 in the DCA + QE group. (E-F) Histological pathology of kidney and histological score. (G-H) Representative images and quantification of Kim-1 staining in kidney OM. Data were presented as mean± standard error. * P < 0.05.

The pathology was significantly less severe in mice given 50 mg/kg quercetin during the same period. Scored renal injuries at CO and OM were milder in CI-AKI + IQE (vs. CI-AKI) group (P = 0.009, P < 0.001, Cohen's d = 3.378 and Cohen's d = 3.708). In particular, the number of intraepithelial vacuolar degeneration was significantly reduced (Figure 3E and F). Expression of Kim-1 intensified after iodixanol injection was attenuated by quercetin. Mice in the CI-AKI + IQE group demonstrated a significantly lower staining of Kim-1, compared with the CI-AKI group (P < 0.001, Cohen's d = 5.173). However, it did not recover to the level when no contrast medium was injected (P = 0.024) (Figure 3G and H).

3.3 Correlation Between Functional Magnetic Resonance Imaging and Pathology

MD vs Histology and Kidney injury molecule-1 A negative correlation was found between MD and the histological score (CO, r = −0.64, P < 0.001; OM, r = −0.59, P < 0.001; IM, r = −0.68, P < 0.001) (Figure 4A–C). In addition, Kim-1 exhibited moderate negative correlation with MD (r = −0.62, P < 0.001) (Figure 4G). More severe kidney damage is associated with lower MD values. Figure 4. Correlation of the MD and FA values with histological score and Kim-1 expression.

FA vs Histology and Kidney injury molecule-1 FA in renal CO, OM, and IM negatively correlated with the corresponding histological score (r = −0.73, P < 0.001; r = −0.61, P < 0.001; r = −0.63, P < 0.001, respectively) (Figure 4D–F). The correlation between FA values and Kim-1 expression was also moderate (r = −0.61, P < 0.001) (Figure 4H).

3.4 Quercetin improved the contrast induced renal function changes

Renal function was evaluated according to the BUN and serum Cr. The concentrations of BUN and serum Cr were significantly increased 24, 48 h after iodixanol injection in the DCA group. (P = 0.002, P < 0.001; P < 0.001, P < 0.001). And the differences in serum Cr were still present at 72 h(P < 0.001) (Supplemental Fig.3 A-B, Table 2).

In the DCA + QE group, serum Cr and BUN levels were lower in the CI-AKI + IQE compared to the CI-AKI group (P = 0.007; P = 0.017, respectively), whereas there was no significant difference between CI-AKI and CI-AKI + LQE groups (P = 0.947; P = 0.0989, respectively) (Supplemental Fig.3 C-D, Table 3). Thus, appropriate doses quercetin ameliorated acute renal dysfunction after exposure to contrast medium in DM.

3.5 Quercetin attenuated M1 polarization of macrophages in kidney of DM mice with CI-AKI

To investigate the effects of quercetin on inflammatory macrophage in DM with contrast medium, the expression of macrophage markers was detected by flow cytometry. M1 macrophages promote the inflammation in kidney of AKI, whereas M2 macrophages inhibit inflammation. CD45 is a leukocyte common antigen marker. Then, total macrophages were identified according to the dual presence of CD11b and F4/80 in live cells [25, 26]. Therefore, we used flow cytometry to detect the changes of macrophages polarity in kidney of CI-AKI after quercetin administration in DM. Samples from mice injected with contrast medium were used for analysis 24 h after injection. Although CD206 (Marker of M2 macrophage) positive macrophages were not increased following quercetin treatment in CI-AKI kidney, we were surprised to find that CD86 (Marker of M1 macrophage) positive macrophages were decreased. Studies had shown that in the early stages of acute renal injury, a predominance of M1 macrophages is observed, promoting tubular necrosis and inflammation, but in later stages a higher content of M2 macrophages is observed to resolve inflammation and participate in tissue remodeling [27]. Therefore, in the early stages of the CI-AKI in DM, quercetin may exert anti-inflammatory effects by reducing the proportion of M1 macrophages (Figure 5). Figure 5. Flow cytometry analysis. Leukocyte, CD45+; Total macrophages, CD45+ F4/80+ CD11b+; M1 macrophage, CD45+ F4/80+ CD11b + CD 86+; M2 macrophage, CD45+ F4/80+ CD11b + CD206+. Gate setting: CD45+→F4/80+, CD11b+→CD86+ or CD206+. (A) Normal mice. M1 macrophage, Q3: CD45+ F4/80+ CD11b + CD 86+, accounting for 20.5%. (B)Diabetes mice, M1 macrophage accounting for 17.5%. (C)Diabetes mice with contrast medium, M1 macrophage, accounting for 33.8%. (D)Diabetes mice contrast medium plus 25 mg/kg quercetin, M1 macrophage accounting for 31.0%. (E)Diabetes mice contrast medium plus 50 mg/kg quercetin, M1 macrophage accounting for 16.2%. (F)Diabetes mice contrast medium plus 75 mg/kg quercetin, M1 macrophage accounting for 18.4%.

3.6 Quercetin significantly activated Nrf 2-related antioxidant pathways in the kidney of diabetic mice and attenuated apoptosis induced by contrast agents

To investigate the molecular mechanisms associated with CI-AKI and protective effects of quercetin in DM mice with CI-AKI. The expression of Sirt1/Nrf 2/HO-1/SOD1 signaling proteins and apoptosis proteins were analyzed (Figure 6). Figure 6. Immunoblot analysis and quantification of Sirt 1, Nrf 2, HO-1, SOD1, Bax, cleaved caspase-3, Bcl-2. Relative densitometry analysis of the ratios of proteins to β−actin was expressed as mean±standard error. *P < 0.05, *P < 0.01, ***P < 0.001.

Immunoblot analysis showed that CI-AKI activates Sirt1 signaling pathway (Figure 6B–E), and the kidney of 24 h or 48 h group mice had a higher level of proapoptotic protein, namely, cleaved caspase-3 and Bax, and lower expression of anti-apoptotic protein Bcl-2 than that of baseline mice (Figure 6F–H).

To elucidate the therapeutic of quercetin in CI-AKI, we further examined the activation of Sirt1 signaling after quercetin administration. Western blot showed that quercetin significantly increased the protein level of Sirt1 and the activity of Nrf 2/HO-1/SOD 1 in the kidney of DM model with CI-AKI, indicating that quercetin may reduce oxidative stress by activating Sirt1 signaling pathway (Figure 6J–M). Furthermore, the protein level of Bcl-2 was significantly increased, and those of Bax, cleaved caspase-3 were significantly lower in quercetin-treated groups compared with the CI-AKI group (Figure 6N–P). This finding in vivo elucidated that quercetin protected kidney through reducing apoptosis after iodixanol injection in DM mice.

3.7 Quercetin attenuated the damage of HK-2 cells stimulated by contrast agent

The HK-2 cells under high glucose were pretreated with quercetin for 4 h before treatment with iodixanol. Cell morphology was observed making use of the phase-contrast microphase, and we found that HK-2 cells in the Cont group expressed a normal adherent growth, showing full stating. Notably, the morphology of HK-2 cells became longer and looser when stimulated with contrast medium, however, it was sight improvement after 20μM quercetin administration(P < 0.001) (Figure 7A,B). The scratch ratio in the cells treated with contrast medium was decreased to 23% (P < 0.001), compared with cells in the Cont group. The scratch ratio was increased in the treatment with 20 and 30 μM quercetin (P < 0.001, P = 0.038, respectively) (Figure 7D,E). We detected the expression of Kim-1 after quercetin administration by real-time PCR. The results showed that quercetin significantly decreased the mRNA expression of Kim-1 in HK-2 cells stimulated by contrast medium under high glucose, suggesting the excellent anti-injury effect of quercetin (Figure 7C). In addition, to further verify that Sirt1 signaling was involved in renoprotection against CI-AKI under high glucose conditions, western blot was applied to HK-2 cells. The results demonstrated that pretreatment with quercetin upregulated the expression of Sirt1, Nrf 2, HO-1, SOD1 in the iodixanol-treated HK-2 cells. These signaling play an important role in inhibiting oxidative stress. However, we found that the protective effect of quercetin was reversed when the concentration of quercetin reached 30 μM. The apoptosis in the HK-2 cells was examined using immunoblotting (cleaved Cas 3, Bax, Bcl-2) and TUNEL staining. Pretreatment with quercetin mitigated the iodixanol-induced enhanced levels of cleaved Cas 3, Bax, and decreased levels of Bcl-2, the number of TUNEL-positive cells. (Figure 7F–H) (Supplemental Fig.4). Figure 7. Effects of quercetin on cell toxicity induced by iodixanol under high glucose conditions. (A-B) Cell morphology (×100) and quantitative analysis of the percentage of viable cells in different groups. (C)The relative mRNA expression level of Kim-1 in various groups were examined by qRT-PCR. (D-E)Images of representative scratch test from each group at time 0 h,72 h (×100) and statistical analysis of the scratch test. (F)Effects of quercetin on the expression of Sirt 1, Nrf 2, HO-1, SOD1, and apoptosis proteins in vitro. (G-H)Apoptosis was also evaluated by TUNEL staining (×200). *P < 0.05 vs. Model, ***P < 0.001 vs. Model. Cont, control; Model, contrast-induced acute kidney injury model; M + QE 10μM, model + 10 μM quercetin; M + QE 20 μM, model + 20 μM quercetin; M + QE 30 μM, model + 30 μM quercetin.

4. Discussion

In patients undergoing imaging studies or interventional cardiac procedures using iodinated contrast media, CI-AKI remains a concern. There are currently no effective drugs that can protect against CI-AKI. For high-risk patients with CI-AKI, finding new molecular target(s) is thus crucial to the development of therapies.

In our study, the major findings were as follows: (1) DTI-derived MD and FA are effective MR biomarkers to evaluate early changes of the renal function in CI-AKI with DM. (2) Quercetin protects against iodixanol-induced AKI in the kidney (in vivo) and HK-2 cells (in vitro) under high glucose, and (3) quercetin effectively attenuates oxidative stress, apoptosis, and inflammation caused by iodixanol via activation of Sirt1. In the present study, we investigated the effects of quercetin treatment on CI-AKI model. As a result of iodixanol administration, renal function was decreased and renal tubular injury was observed. Quercetin administration before iodixanol offered renoprotective effects for the kidney and HK-2 cells, as confirmed by improved renal function lessened histopathological injury, reduced the proportion of M1 macrophages, and reduced oxidative stress in kidney tissues and HK-2 cells.

Our choice of 9.4 T MRI was based on the fact that a higher magnetic field intensity can enhance MRI image quality and acquisition ability. In addition, small internal lesions and cancer can be detected earlier with the increased MRI imaging resolution. In DTI measurements, the b-value quantifies water molecule diffusion in various directions. Higher b-values, like 1000 s/mm², enhance MD measurement accuracy by minimizing isotropic scattering and better distinguishing tissue anisotropy [28]. DTI demonstrated diffusion properties with higher cortical than medullary MD values and inversely significantly higher FA values in the IM than in the OM and CO. The findings were in accordance with previous researches and renal anatomy [16, 29]. Due to radial orientation of the renal tubules and small blood vessels in the medulla, as well as the much lower blood perfusion in the medulla than cortex, lower medullary MD may occur. Additionally, the radial arrangement of tubules in the pyramids of renal medulla may contribute to the higher FA of water molecule diffusion than that in the cortex where glomeruli limit the direction of water molecule diffusion [30]. According to the results of the present study, renal function and histopathological changes were reflected in the MD and FA values early. Figure 4 shows the negative correlations between MD, FA, and renal pathology scores. MD and FA values were minimized at 24 h after iodixanol injection in the DCA group, and then gradually returned to baseline. This may be due to the following reasons: (1) As a result of the direct toxicity of the contrast agent, the renal tubular epithelial cells swell and the amount of vacuoles increases, altering the amount of water within the cell, which limits the diffusion of renal water and decreases the MD [31]. (2) The following explanations are why FA might be reduced. First of all, this result from glomerular lesions disrupts the normal structure and function of glomerular filtration membranes, resulting in macromolecular proteins and red blood cells being filtered into tubules. Secondly, there is a possibility that cellular debris and macromolecules might congest the tubules, impairing directed diffusion. Finally, damage to the renal parenchyma can cause reductions in renal blood volume, renal blood flow, and medulla microcirculation [32]. In previous studies, 99% of the injected iodine contrast medium was removed through the urine within 24 h. The pathological results and FA of the medulla, however, showed that the damage caused by the contrast agent persisted for 72 h after it was administered. In our previous studies, our team has exploited the isoosmolality of iodixanol and documented the impact of contrast agent viscosity on animal renal function. It was observed that an increase in contrast agent viscosity corresponded to a heightened severity of renal damage [33]. It is likely to be that DM is an additional activator of CI-AKI, increasing the viscosity of blood and other body fluids. Furthermore, the presence of DM causes the kidney's oxygen consumption to remain high and increase the sensitivity to diffusion and perfusion [22]. Within the DCA + QE group, with increasing quercetin dose, MD and FA values began to slowly recover. Probably because of its antioxidant properties, quercetin reduces inflammation in the kidneys. In addition, quercetin reduces edema by increasing blood volume and urine output [7]. As such, it is possible to detect changes in renal with CI-AKI early in combination with DTI.

DTI parameters can be skewed by low signal-to-noise ratios, image artifacts, and magnetic field gradient imperfections, particularly diffusion gradient inhomogeneity, which can overestimate anisotropic parameters like FA [34]. The BSD-DTI method, which directly measures spatially correlated b-matrix elements, is suggested as a final correction step for these errors in DTI data [35]. Despite the lack of benchmarks for mice kidney DTI indicators, we must standardize the measurement process, minimize spatial errors, and ensure values are as accurate as possible to properly reflect kidney changes and assess QE's protective effect on CI-AKI.

In DM models particularly, the abnormal DTI finding may reflect various pathological changes that occur after contrast medium administration. Injection of iodixanol resulted in kidney injury with vacuolar degeneration and interstitial inflammation as seen in HE staining. In addition, mice showed abnormal kidney functions with elevated serum BUN and creatinine levels, which was mitigated by quercetin administration. Kidney injury markers Kim-1, which had minimal expression in the baseline group, were remarkably upregulated in the CI-AKI group. It causes CI-AKI to occur more frequently, while high glucose states trap the antioxidative system in the body, which causes inflammation and worsened endothelial function [36]. Quercetin effectively suppressed the upregulation of the molecular in injured kidneys. Previous studies utilized the MRI-Magritek Mobile Universal Surface Explore with a stable gradient of 24 T/m for diffusion MRI to characterize living cells, analyzing the time-dependent diffusion coefficient to determine self-diffusion coefficients, sizes, and mole fractions of extracellular water, cytoplasm, and cellular structures [37]. This may help us explore in more depth. Taken together, these results confirm that quercetin exhibited certain renoprotective effect in our CI-AKI model.

There is a significant role for Sirt1 in the pathogenesis and development of CI-AKI. In addition to deacetylating histones, Sirt1 regulates the activity of multiple transcription factors and proteins, which involves mitochondrial biogenesis, redox homeostasis, and inflammation [38]. There have been extensive research looking into the role of Sirt1 in acute and chronic kidney diseases due to its high expression in renal tissues [39, 40]. The research indicated that activating the Nrf 2 antioxidative pathway leads to increased HO-1 and SOD1 expression, which is associated with Sirt1's protective effects on diabetic renal injuries [10]. A crucial adaptive system for maintaining cellular redox homeostasis and enhancing oxidative stress resistance is Nrf 2/HO-1 regulation [41]. There is still much to learn about the specific mechanisms behind CI-AKI, but previous studies have shown that oxidative stress plays a key role in its development [42]. According to our western blot analysis, the CI-AKI with DM deactivated Nrf 2 and Sirt1 while quercetin induced their expression in this study. Moreover, our results were validated in vitro and in vivo. As noted above, quercetin reversed the depletion of Nrf 2/HO-1 by activating Sirt1, suggesting the Sirt1-Nrf 2-HO-1 pathway may be involved in the oxidative stress process in CI-AKI with DM.

As a result of inflammatory infiltration in the kidney, cellular apoptosis occurs, leading to the loss of renal epithelial cells that characterize acute kidney disease [43]. A growing body of evidence indicates that the activation caspase-3 and Bax and inhibition of Bcl-2 are responsible for apoptosis of renal tubular cells. And the effect of quercetin on these factors also has been explored previously [44]. In addition to TUNEL staining, we evaluated the effect of quercetin on cleaved caspase-3, Bcl-2, and Bax levels. There was a significant reduction in TUNEL-positive cells following quercetin treatment. It also suppressed the cleavage caspase-3, Bax and preserved Bcl-2 in the kidneys of CI-AKI. Therefore, quercetin inhibited CI-AKI with DM pathogenesis by inhibiting tubular epithelial cell apoptosis. Previous studies used magnetic resonance to detect cell signals in suspension and determine the effective diffusion coefficient, aiding in distinguishing between cell necrosis and apoptosis [45]. As part of the renal injury process, macrophages play an important role as inflammatory cells. The biological effects of macrophages in kidney injury are determined by their polarization into pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. As well as inflammatory injury, polarized macrophages are involved in renal tissue repair [46]. As a result of these findings, we hypothesized quercetin's protective effect may be due to its control over macrophage polarization. Flow cytometry verified our conjecture, the administration of quercetin reduced kidney injury by inhibiting M1 macrophage polarization. In unilateral ureteral obstruction animal models, quercetin has been shown to possess anti-inflammatory effects by inhibiting the M1 macrophage responses [47]. In conclusion, these results suggest quercetin's anti-inflammatory effects may be related to its inhibition of macrophage polarization in M1. However, we did not observe the activation or inhibition of quercetin on M2 macrophages. Macrophages display different phenotypes depending on the time after injury, with M1 dominance initially(1–3 days post-injury), an M2 tilt during repair, and phenotype switching following adoptive transfer [48]. The mice in the DCA + QE group were selected 24 h after contrast medium injection, so no changes in M2 were observed, and further research is needed to understand the impact.

It is nevertheless necessary to discuss potential limitations of the present study. (1) As a result of using a DM procedure, the CI-AKI model became more complex. (2) Free-breathing DTI scanning was performed due to poor time efficiency of respiratory-triggered methods. We compared the mean values of ROIs drawn manually in different parts of the kidney and at different locations within the same kidney, which makes our analysis prone to error. Spatial systematic errors caused by uneven gradients overestimate changes in renal DTI parameters. (3) Due to renal heterogeneity or various dysfunctions, the use of a single biomarker may not be sufficient to determine CI-AKI. In subsequent experiments, potential markers such as urinary KIM-1 and Neutrophil gelatinase-associated lipocalin(NGAL) can be added and combined with MD and FA conducts correlation analysis to make the research more credible. (4) The effect of quercetin can be more fully confirmed using Sirt1 knockout mice or Sirt1 inhibitors. Furthermore, since quercetin possesses many pharmacological benefits, it is worth investigating whether quercetin protects against CI-AKI through any other mechanism. In conclusion, for both CI-AKI pathogenesis and quercetin's potential as a therapeutic, further molecular mechanisms studies are needed.

Our study is the first to show that 9.4 T DTI can noninvasively assess early renal function changes and QE's protective effects in the CI-AKI model. FA and MD differences emerged within 24 h post-iodine contrast injection, aligning with renal tissue pathology and effectively indicating CI-AKI severity. In addition, the pretreatment with quercetin can attenuate contrast-induced oxidative stress, apoptosis, and inflammation associated with CI-AKI in DM. Quercetin may exert its protective effects by activating Sirt1/Nrf2/HO-1 signaling pathways, at least in part. In the future, we plan to use the BSD-DTI method to correct potential systematic errors from uneven gradients and employ gene knockout technology to investigate QE's protective mechanism, providing more comprehensive preclinical evidence.

Supplementary Material

Supplementary file.docx

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Echical approval

All applicable international, national guidelines for the care and use of animals were followed.

Author contribution statement

Ziqian Wu conceived the idea and wrote. Jingyi Hu contributed to the DTI data analyzing. Yanfei Li and Xiang Yao contributed to diabetes model making. Siyu Ouyang searched the literatures. Ke Ren guided the design of this study and revised the manuscript.

Disclosure statement

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