# Purple Sweet Potato Color Attenuates Kidney Damage by Blocking VEGFR2/ROS/NLRP3 Signaling in High-Fat Diet-Treated Mice

Authors: Gui-Hong Zheng; Qun Shan; Jing-Jing Mu; Yong-Jian Wang; Zi-Feng Zhang; Shao-Hua Fan; Bin Hu; Meng-Qiu Li; Jun Xie; Ping Chen; Dong-Mei Wu; Jun Lu; Yuan-Lin Zheng
Journal: Oxidative Medicine and Cellular Longevity
 (2019)

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## Abstract

Our preliminary data showed that VEGFR2 upregulation promoted renal ROS overproduction in high-fat diet- (HFD-) treated mice. Given that ROS-induced NLRP3 activation plays a central role in the pathogenesis of type 2 diabetic kidney injury, we evaluate whether VEGFR2 upregulation induces type 2 diabetic kidney injury via ROS-mediated NLRP3 activation and further explore the underlying mechanism. Our results showed that VEGFR2 knockdown decreased ROS overproduction, blocked NLRP3-dependent inflammation, and alleviated kidney damage in HFD-treated mice. Treatment withα-lipoic acid, a scavenger of ROS, lowered ROS overproduction and alleviated NLRP3-triggered kidney injury of HFD-treated mice. Collectively, the VEGFR2/ROS/NLRP3 signal is a critical therapeutic strategy for the kidney injury of HFD-treated mice. Purple sweet potato color (PSPC), a natural anthocyanin, can exert renal protection by inhibiting ROS in HFD-treated mice. Here, we provide a novel mechanism of PSPC against renal damage in HFD-treated mice by downregulating VEGFR2 expression.


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## Body

## 1. Introduction

In recent years, type 2 diabetes mellitus (T2DM) has become more prevalent worldwide mainly due to excess calorie intake and low physical activity [1, 2]. Diabetic nephropathy (DN) is one of the major complications of T2DM and its pathological mechanism is still not clear [3]. Several lines of evidence have showed that oxidative stress and inflammation are the main pathological mechanisms of DN [4–7]. Further evidence shows that ROS-induced NLRP3 activation plays a key role in the pathological damage of DN [8]. Vascular endothelial growth factor receptor (VEGFR), a tyrosine kinase receptor, has been reported to trigger ROS generation and inflammation in many kinds of pathological angiogenesis in cancer, atherosclerosis, and diabetic retinopathy [9, 10]. Our preliminary data also found that upregulation of VEGFR2 promoted excessive ROS production in the kidney of high-fat diet- (HFD-) treated mice, but the underlying mechanism of the pathophysiologic process has not been fully elucidated. As a specific and high-affinity receptor for proinflammatory VEGF, VEGFR2 is originally recognized for many biological properties in vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and blood vessel formation [11, 12]. Based on our previous finding that ROS-induced NLRP3 activation accelerated the kidney damage in HFD-treated mice, we hypothesize that VEGFR2 may be involved in the renal pathological injury via ROS-activated NLRP3 inflammatory signaling in HFD-treated mice. Next, we confirm this hypothesis and explore the underlying mechanism of VEGFR2 mediating the renal injury in the HFD-treated mice.PSPC, a natural compound of flavonoids obtained fromIpomoea batatas, possesses multiple biological functions for preventing various diseases, such as cancers, atherosclerosis, and nonalcoholic fatty liver disease [8, 13–16]. Our previous data demonstrated that PSPC could prevent the pathological damage of the brain, liver, and kidney via antioxidative stress, anti-inflammation, and antiapoptosis in various animal models [8, 13, 16]. Recently, Shan et al. have found that PSPC relieves kidney damage by inhibiting NLRP3-induced inflammation [8]. In addition, various flavonoids have been shown to inhibit VEGFR2 expression and its downstream signaling in the pathological angiogenesis of tumors and diabetic retinopathy [17, 18]. Given our preliminary evidence showing that VEGFR2 stimulated ROS overproduction, the current study aims at investigating whether PSPC alleviates kidney injury by downregulating VEGFR2-mediated oxidation stress and inflammation and further explaining the pathological mechanism underlying kidney damage in HFD-treated mice.


## 2. Materials and Methods

### 2.1. Animal Administration

All experimental protocols and euthanasia procedures were approved by the Institutional Animal Care and Use Committee of Jiangsu Normal University (permit number: 16-0050, 6 March 2016). Eighty 8-week-old male ICR mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Mice were acclimated to laboratory conditions for 1 week before experimentation. Animals were housed at 23 ± 1°C and 55 ± 5% humidity, with free access to food and water. After acclimatization for 1 week, the mice were divided into two groups of 40 animals each: the chow diet (CD, control) group and the HFD group. Both received separate treatments for 20 weeks. Mice in the control group ate the chow diet (10% of energy as fat; D12450; Research Diets Inc., New Brunswick, NJ, USA), while mice in the HFD group ate the HFD diet (60% of energy as fat; D12492; Research Diets Inc., New Brunswick, NJ, USA) [19].


### 2.2. Adeno-Associated Virus (AAV) Infection and VEGFR2 Short Hairpin RNA (shRNA) Knockdown

Adeno-associated viral (AAV2/9) vectors encoding short hairpin RNAs (shRNAs) were purchased from HannBio Technology Co. (Shanghai, China). AAV2/9 infection was performed as previously described [20]. In brief, vectors expressed shRNAs containing VEGFR2 (5′-GCGATGAACTCACCATCATGG-3′), negative control (5′-GGAAGTCGTGAGAAGTAGAAT-3′) target sequences under the control of the human U6 promoter. In addition, the vectors also expressed enhanced GFP (EGFP) as a reporter to allow for visualization of transduced kidney cells. Kidney-specific VEGFR2 knockdown experiments were performed 16 weeks after HFD administration. For a knockdown experiment, each mouse was injected 10 μL of each vector (1 × 1012) in phosphate-buffered saline (PBS) into the right kidney via microinjection as described (using a 2 μL Hamilton syringe, 0.25 mL/min, injecting at 5 points on the sagittal plane 1.5 mm deep into the kidney). A schematic representation of the AAV2/9 used in this study is shown in Figure 1(a). The mice were sacrificed for subsequent experiments three weeks after injection.Figure 1
Kidney-specific VEGFR2 knockdown inhibits renal oxidative stress of the HFD-treated mice. (a) Schematic representation of the AAV used in this study. (b) VEGFR2 mRNA were determined by real time qPCR. Values are expressed as the ratio of VEGFR2 to GAPDH.∗∗∗p<0.001vs. the HFD scramble group. (c) Representative immunoblots for VEGFR2 and β-actin. (d) Relative density analysis of VEGFR2 protein bands. Relative densities are expressed as the ratio of VEGFR2 to β-actin. ∗∗∗p<0.001vs. the HFD scramble group. (e) Kidney sections were stained with EGFP and CY3 (green) and VEGFR2 (red) to visualize AAV2/9-infective cells, respectively (200x, scale bar, 100 μm). (f) Analysis of the relative intensity of EGFP-positive and VEGFR2-positive cells in the kidney sections, ∗∗∗p<0.001vs. the HFD scramble group. (g) Representative immunoblots for NOX4 and β-actin. (h) Relative density analysis of NOX4 protein bands. Relative densities are expressed as the ratio of NOX4 to β-actin. ∗∗∗p<0.001vs. the CD scramble group, and ###p<0.001vs. the HFD scramble group. (i) ROS levels were assessed by 2′,7′-dichlorofluorescein diacetate (DCF-DA). All values are expressed as mean ± SEM ((b–i), n=5). ∗∗∗p<0.001vs. the CD scramble group, and ###p<0.001vs. the HFD scramble group.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)


### 2.3.α-Lipoic Acid Administration

Mice were fed with CD or HFD for 16 weeks (20 mice per group), then orally administrated 150 mg/kgα-lipoic acid daily for 4 weeks (α-lipoic acid was dissolved in the corn oil) as described in previous literature [21]. Additional HFD-treated and CD-treated mice (10 mice per group) received the same dose of the solvent, corn oil (Sigma-Aldrich, St. Louis, Missouri, USA). After 4 weeks of α-lipoic acid treatment, blood and kidneys were obtained from the sacrificed mice for further experiments [21].


### 2.4. PSPC Administration

PSPC was purchased from Qingdao Pengyuan Natural Pigment Research Institute (Qingdao, China). Its major components are six acetylated anthocyanins, all of which are flavonoids (purity > 90%). The detailed components of PSPC have been previously described [16]. Based on the pilot experiment (see Supplementary Figure 2), forty 8-week-old male ICR mice were randomly divided into four groups of 10 animals each: chow diet (CD, control) group, HFD group, HFD + PSPC group, and CD + PSPC group. They received the following treatments for 20 weeks: Mice in the HFD + PSPC and CD + PSPC groups were orally administered 700 mg/kg/d of PSPC in distilled water containing 0.1% Tween 80 daily for 20 weeks according to our previous and pilot results. An equal volume of distilled water containing 0.1% Tween 80 was administered to mice in the CD groups. The dosage of PSPC used in this study was according to data in our previous reports and preliminary experiments (see Supplementary Figure 2) [8]. At the end of the experiment, blood and kidneys were obtained from the sacrificed mice for further experiments.The experimental procedures are shown in Supplementary Figure3.


### 2.5. Quantitative Real Time Polymerase Chain Reaction (qPCR)

Total mRNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Single-stranded cDNAs were amplified using the following primers: VEGFR2 (sense 5′-TACACAATTCAGAGCGATGTGTGGT-3′, antisense 5′-CTGGTTCCTCCAATGGGATATCTTC-3′) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sense 5′-GTCTTCCTGGGCAAGCAGTA-3′, antisense 5′-CTGGACAGAAACCCCACTTC-3′) (Sangon Biotech, Shanghai, China). VEGFR2 mRNA levels were analyzed by qPCR using BeyoFast™ SYBR Green qPCR Mix (2x, High ROX, Beyotime, Nantong, China) in a StepOnePlus instrument (Applied Biosystems, Carlsbad, CA, USA). Amplification was performed using an initial denaturation step at 95°C for 3 min, 40 cycles of 95°C for 15 s and 60°C for 30 s, and a final incubation at 72°C for 30 s. The relative levels of VEGFR2 mRNA were normalized to GAPDH mRNA and were analyzed by the comparative cycle threshold (Ct) method.


### 2.6. Tissue Homogenates

At the end of experiment, mice were deeply anesthetized and sacrificed. Kidney was promptly dissected and homogenized in 1 : 5 (w/v) 50 mM (pH 7.4) ice-cold Tris-buffered saline (TBS) containing a protease inhibitor cocktail (Sigma-Aldrich, MO, USA) with 10 strokes at 1200 rpm in a Teflon-glass Potter-Elvehjem homogenizer (Kontes Glass Inc., Vineland, NJ, USA). Homogenates were centrifuged at 12,000 ×g for 10 min, and the supernatant was used for the ROS assay and western blot analysis.


### 2.7. Assay of ROS

ROS was determined as described previously, based on the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) to 2′,7′-dichlorofluorescein (DCF) (Jiancheng Institute of Biotechnology, Nanjing, China) [15, 16]. Briefly, each renal supernatant was diluted into a 1% tissue homogenate in 1 × PBS and the supernatant was obtained by centrifugation at 12,000 ×g and 4°C. Samples or hydrogen peroxide standard (50 μL) were mixed with 100 μL of 10 μM DCFH-DA in 96-well plates and incubated for 20 min at 37°C in the dark. Fluorescence was measured at 480 nm excitation/530 nm emission on a Synergy H4 Hybrid Multi-Mode Microplate Reader (Biotek, Winooski, Vermont, USA). ROS formation was quantified from a DCF-standard curve. Data are expressed as pmol of DCF formed/min/mg protein.


### 2.8. Biochemical Analysis

At the end of experiment, the blood was obtained by caudal venipuncture. Each sample was divided into 2 parts: one for measuring serum creatinine and another for determining blood urea nitrogen concentrations using a commercial creatinine and urea nitrogen determination kit (Jiancheng Institute of Biotechnology, Nanjing, China) according to the manufacturer’s protocols.


### 2.9. Isolation of Cytoplasmic and Nuclear Proteins

The isolation of cytoplasmic and nuclear proteins was performed using commercial nuclear protein-cytoplasmic protein extraction kits (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s protocols. Briefly, weighed fresh kidneys were added to hypotonic buffer (3 mL/g), homogenized with a ball mill instrument (Retsch GmbH, Haan, Germany), incubated on ice for 15 min, and centrifuged for 10 min at 850 ×g at 4°C. The supernatants and pellets contained the cytoplasmic and nuclear fractions, respectively. The nuclear pellets were resuspended in 50μL Complete Lysis Buffer and vortexed, then incubated for 30 min at 4°C on a rocking platform at 150 rpm. After vortexing, the supernatant was obtained by centrifugation for 10 min at 14,000 ×g.


### 2.10. Western Blot Analysis

Western blot was conducted as in previously published articles [8, 22]. Total proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Nonspecific antigen binding sites were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at 22 ± 2°C. Primary antibodies used included rabbit anti-VEGFR2 (ab39256, 1 : 5000, Abcam, Cambridge, USA), rabbit anti-NADPH oxidase 4 (NOX4, BM4135, 1 : 400, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-NLRP3 (BM4490, 1 : 400, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-tumor necrosis factor (TNF) α (PB0270, 1 : 1000, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-interleukin- (IL-) 6 (BA4339, 1 : 1000, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-inducible nitric oxide synthase (iNOS, BA0362, 1 : 400, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-pro-IL1β (BM0962, 1 : 400, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-cleaved IL1β (16806-1-AP, 1 : 4000, Proteintech, Wuhan, China), mouse anti-nuclear factor- (NF-) κB p65 (Bsm-33059M, 1 : 2000, Bioss, Beijing, China), anti-rabbit procaspase 1 (22915-1-AP, 1 : 1000, Proteintech, Wuhan, China), and anti-rabbit cleaved caspase 1 (22915-1-AP, 1 : 1000, Proteintech, Wuhan, China). Membranes were incubated with primary antibodies overnight at 4°C, rinsed with TBST three times for 10 min, and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (SA00001-1/2, 1 : 5000, Proteintech, Wuhan, China) for 1 h. After washing, proteins were visualized on an Amersham Imager 600 (GE Healthcare UK Limited, UK) using LumiGLO® Reagent and Peroxide (Cell Signaling Technology, Beverly, MA, USA). The optic density (OD) values of detected bands were measured with the Scion Image analysis software (Scion, Frederick, MD, USA) and were normalized to mouse anti-β-actin (66009-1, 1 : 5000, Proteintech, Wuhan, China) or mouse anti-histone H3 (Bsm-33042M, 1 : 5000, Bioss, Beijing, China) as internal controls (OD detected protein/OD internal control). The specificity of each of the primary antibodies was evaluated by preincubation in the presence or absence of the corresponding blocking peptides or by omission of the primary antibodies.


### 2.11. Collection of Kidney Slices

At the end of experiment, mice were euthanized and perfused with sterile PBS to remove their blood. Kidneys were immediately fixed in 4% phosphate-buffered paraformaldehyde, dehydrated in 30% sucrose (Amresco, Ohio, USA), and then sectioned with a freezing microtome (Leica CM3050S, Biosystems, Nussloch, Germany) and stored at −80°C (DW-86L578J, Haier BioMedical, Qingdao, China) for dihydroethidium (DHE) staining, hematoxylin and eosin (HE) staining, and immunofluorescence.


### 2.12. Immunofluorescence Staining

Immunofluorescence staining of renal sections were conducted as previously described [8]. First, antigens were retrieved by boiling in citric acid buffer (pH 6.0) for 15 min. Nonspecific antigens were blocked with 5% bovine serum albumin for 1 h at room temperature. Samples were incubated with rabbit anti-VEGFR2 antibody (ab2349, 1 : 500, Abcam, Cambridge, UK) at 4°C overnight, and then goat anti-rabbit DyLight 488 Antibody (1031-02, 1 : 20,000, Vector Laboratories, Burlingame, CA, USA) or anti-rabbit CY3 antibody (CY-1300-1, 1 : 2000, Vector Laboratories, Burlingame, CA, USA) was added for 1 h. Subsequently, DAPI was applied for 8 min. Stained sections were imaged at 200x on a fluorescence microscope (Leica DM4000B, Leica Microsystems, Wetzlar, Germany), and quantitative analysis was performed using Image-Pro Plus 6.0 (IPP; Media Cybernetics, Rockville, MD, USA) [21].


### 2.13. HE Staining

Mice were euthanized and perfused with sterile PBS to remove their blood. Kidney were immediately fixed in 4% phosphate-buffered paraformaldehyde, dehydrated in 30% sucrose (Amresco, Ohio, USA), and then sectioned with a freezing microtome (Leica CM3050S, Biosystems, Nussloch, Germany) and stored at −80°C (DW-86L578J, Haier Biomedical, Qingdao, China). To assess renal morphology, 10μm sections were stained with HE (Sigma-Aldrich, St. Louis, MO, USA). Detailed staining procedures have been previously described [8]. Pathological changes in kidney tissues were observed under a light microscope, and renal damage was evaluated in a double-blind fashion by two kidney pathologists [23].


### 2.14. DHE Staining

DHE staining was used to detect the ROS production in the kidney sections. Simply, 10μm-thick frozen renal sections were removed from −80°C storage and dried at 37°C for 30 min in an electric thermostatic incubator (Incucell, Germany), and then incubated in 1 × PBS for 15 min. Next, sections were incubated with 10 mol/L fluorescent-labeled DHE (Molecular Probes, Invitrogen, Carlsbad, CA, USA) in a lucifugal-humidified chamber at 37°C for 30 min, then stained with 4′,6′-diamidino-2-phenylindole (DAPI, Abcam, Cambridge, CA, USA). Images were obtained at 200x on a fluorescence microscope (Leica DM4000B, Leica Microsystems, Wetzlar, Germany). The average DHE fluorescence intensity was measured using IPP. Results were expressed as the ratio of the DHE-positive area and the DAPI.


### 2.15. Statistical Analysis

All statistical analyses were performed using SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance followed by Tukey’s honest significant difference test. Data are expressed as the mean ± standard error of mean (SEM). Statistical significance was set atp<0.05.


## 2.1. Animal Administration

All experimental protocols and euthanasia procedures were approved by the Institutional Animal Care and Use Committee of Jiangsu Normal University (permit number: 16-0050, 6 March 2016). Eighty 8-week-old male ICR mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Mice were acclimated to laboratory conditions for 1 week before experimentation. Animals were housed at 23 ± 1°C and 55 ± 5% humidity, with free access to food and water. After acclimatization for 1 week, the mice were divided into two groups of 40 animals each: the chow diet (CD, control) group and the HFD group. Both received separate treatments for 20 weeks. Mice in the control group ate the chow diet (10% of energy as fat; D12450; Research Diets Inc., New Brunswick, NJ, USA), while mice in the HFD group ate the HFD diet (60% of energy as fat; D12492; Research Diets Inc., New Brunswick, NJ, USA) [19].


## 2.2. Adeno-Associated Virus (AAV) Infection and VEGFR2 Short Hairpin RNA (shRNA) Knockdown

Adeno-associated viral (AAV2/9) vectors encoding short hairpin RNAs (shRNAs) were purchased from HannBio Technology Co. (Shanghai, China). AAV2/9 infection was performed as previously described [20]. In brief, vectors expressed shRNAs containing VEGFR2 (5′-GCGATGAACTCACCATCATGG-3′), negative control (5′-GGAAGTCGTGAGAAGTAGAAT-3′) target sequences under the control of the human U6 promoter. In addition, the vectors also expressed enhanced GFP (EGFP) as a reporter to allow for visualization of transduced kidney cells. Kidney-specific VEGFR2 knockdown experiments were performed 16 weeks after HFD administration. For a knockdown experiment, each mouse was injected 10 μL of each vector (1 × 1012) in phosphate-buffered saline (PBS) into the right kidney via microinjection as described (using a 2 μL Hamilton syringe, 0.25 mL/min, injecting at 5 points on the sagittal plane 1.5 mm deep into the kidney). A schematic representation of the AAV2/9 used in this study is shown in Figure 1(a). The mice were sacrificed for subsequent experiments three weeks after injection.Figure 1
Kidney-specific VEGFR2 knockdown inhibits renal oxidative stress of the HFD-treated mice. (a) Schematic representation of the AAV used in this study. (b) VEGFR2 mRNA were determined by real time qPCR. Values are expressed as the ratio of VEGFR2 to GAPDH.∗∗∗p<0.001vs. the HFD scramble group. (c) Representative immunoblots for VEGFR2 and β-actin. (d) Relative density analysis of VEGFR2 protein bands. Relative densities are expressed as the ratio of VEGFR2 to β-actin. ∗∗∗p<0.001vs. the HFD scramble group. (e) Kidney sections were stained with EGFP and CY3 (green) and VEGFR2 (red) to visualize AAV2/9-infective cells, respectively (200x, scale bar, 100 μm). (f) Analysis of the relative intensity of EGFP-positive and VEGFR2-positive cells in the kidney sections, ∗∗∗p<0.001vs. the HFD scramble group. (g) Representative immunoblots for NOX4 and β-actin. (h) Relative density analysis of NOX4 protein bands. Relative densities are expressed as the ratio of NOX4 to β-actin. ∗∗∗p<0.001vs. the CD scramble group, and ###p<0.001vs. the HFD scramble group. (i) ROS levels were assessed by 2′,7′-dichlorofluorescein diacetate (DCF-DA). All values are expressed as mean ± SEM ((b–i), n=5). ∗∗∗p<0.001vs. the CD scramble group, and ###p<0.001vs. the HFD scramble group.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)


## 2.3.α-Lipoic Acid Administration

Mice were fed with CD or HFD for 16 weeks (20 mice per group), then orally administrated 150 mg/kgα-lipoic acid daily for 4 weeks (α-lipoic acid was dissolved in the corn oil) as described in previous literature [21]. Additional HFD-treated and CD-treated mice (10 mice per group) received the same dose of the solvent, corn oil (Sigma-Aldrich, St. Louis, Missouri, USA). After 4 weeks of α-lipoic acid treatment, blood and kidneys were obtained from the sacrificed mice for further experiments [21].


## 2.4. PSPC Administration

PSPC was purchased from Qingdao Pengyuan Natural Pigment Research Institute (Qingdao, China). Its major components are six acetylated anthocyanins, all of which are flavonoids (purity > 90%). The detailed components of PSPC have been previously described [16]. Based on the pilot experiment (see Supplementary Figure 2), forty 8-week-old male ICR mice were randomly divided into four groups of 10 animals each: chow diet (CD, control) group, HFD group, HFD + PSPC group, and CD + PSPC group. They received the following treatments for 20 weeks: Mice in the HFD + PSPC and CD + PSPC groups were orally administered 700 mg/kg/d of PSPC in distilled water containing 0.1% Tween 80 daily for 20 weeks according to our previous and pilot results. An equal volume of distilled water containing 0.1% Tween 80 was administered to mice in the CD groups. The dosage of PSPC used in this study was according to data in our previous reports and preliminary experiments (see Supplementary Figure 2) [8]. At the end of the experiment, blood and kidneys were obtained from the sacrificed mice for further experiments.The experimental procedures are shown in Supplementary Figure3.


## 2.5. Quantitative Real Time Polymerase Chain Reaction (qPCR)

Total mRNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Single-stranded cDNAs were amplified using the following primers: VEGFR2 (sense 5′-TACACAATTCAGAGCGATGTGTGGT-3′, antisense 5′-CTGGTTCCTCCAATGGGATATCTTC-3′) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sense 5′-GTCTTCCTGGGCAAGCAGTA-3′, antisense 5′-CTGGACAGAAACCCCACTTC-3′) (Sangon Biotech, Shanghai, China). VEGFR2 mRNA levels were analyzed by qPCR using BeyoFast™ SYBR Green qPCR Mix (2x, High ROX, Beyotime, Nantong, China) in a StepOnePlus instrument (Applied Biosystems, Carlsbad, CA, USA). Amplification was performed using an initial denaturation step at 95°C for 3 min, 40 cycles of 95°C for 15 s and 60°C for 30 s, and a final incubation at 72°C for 30 s. The relative levels of VEGFR2 mRNA were normalized to GAPDH mRNA and were analyzed by the comparative cycle threshold (Ct) method.


## 2.6. Tissue Homogenates

At the end of experiment, mice were deeply anesthetized and sacrificed. Kidney was promptly dissected and homogenized in 1 : 5 (w/v) 50 mM (pH 7.4) ice-cold Tris-buffered saline (TBS) containing a protease inhibitor cocktail (Sigma-Aldrich, MO, USA) with 10 strokes at 1200 rpm in a Teflon-glass Potter-Elvehjem homogenizer (Kontes Glass Inc., Vineland, NJ, USA). Homogenates were centrifuged at 12,000 ×g for 10 min, and the supernatant was used for the ROS assay and western blot analysis.


## 2.7. Assay of ROS

ROS was determined as described previously, based on the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) to 2′,7′-dichlorofluorescein (DCF) (Jiancheng Institute of Biotechnology, Nanjing, China) [15, 16]. Briefly, each renal supernatant was diluted into a 1% tissue homogenate in 1 × PBS and the supernatant was obtained by centrifugation at 12,000 ×g and 4°C. Samples or hydrogen peroxide standard (50 μL) were mixed with 100 μL of 10 μM DCFH-DA in 96-well plates and incubated for 20 min at 37°C in the dark. Fluorescence was measured at 480 nm excitation/530 nm emission on a Synergy H4 Hybrid Multi-Mode Microplate Reader (Biotek, Winooski, Vermont, USA). ROS formation was quantified from a DCF-standard curve. Data are expressed as pmol of DCF formed/min/mg protein.


## 2.8. Biochemical Analysis

At the end of experiment, the blood was obtained by caudal venipuncture. Each sample was divided into 2 parts: one for measuring serum creatinine and another for determining blood urea nitrogen concentrations using a commercial creatinine and urea nitrogen determination kit (Jiancheng Institute of Biotechnology, Nanjing, China) according to the manufacturer’s protocols.


## 2.9. Isolation of Cytoplasmic and Nuclear Proteins

The isolation of cytoplasmic and nuclear proteins was performed using commercial nuclear protein-cytoplasmic protein extraction kits (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s protocols. Briefly, weighed fresh kidneys were added to hypotonic buffer (3 mL/g), homogenized with a ball mill instrument (Retsch GmbH, Haan, Germany), incubated on ice for 15 min, and centrifuged for 10 min at 850 ×g at 4°C. The supernatants and pellets contained the cytoplasmic and nuclear fractions, respectively. The nuclear pellets were resuspended in 50μL Complete Lysis Buffer and vortexed, then incubated for 30 min at 4°C on a rocking platform at 150 rpm. After vortexing, the supernatant was obtained by centrifugation for 10 min at 14,000 ×g.


## 2.10. Western Blot Analysis

Western blot was conducted as in previously published articles [8, 22]. Total proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Nonspecific antigen binding sites were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at 22 ± 2°C. Primary antibodies used included rabbit anti-VEGFR2 (ab39256, 1 : 5000, Abcam, Cambridge, USA), rabbit anti-NADPH oxidase 4 (NOX4, BM4135, 1 : 400, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-NLRP3 (BM4490, 1 : 400, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-tumor necrosis factor (TNF) α (PB0270, 1 : 1000, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-interleukin- (IL-) 6 (BA4339, 1 : 1000, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-inducible nitric oxide synthase (iNOS, BA0362, 1 : 400, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-pro-IL1β (BM0962, 1 : 400, Boster Biological Technology Ltd., Wuhan, China), rabbit anti-cleaved IL1β (16806-1-AP, 1 : 4000, Proteintech, Wuhan, China), mouse anti-nuclear factor- (NF-) κB p65 (Bsm-33059M, 1 : 2000, Bioss, Beijing, China), anti-rabbit procaspase 1 (22915-1-AP, 1 : 1000, Proteintech, Wuhan, China), and anti-rabbit cleaved caspase 1 (22915-1-AP, 1 : 1000, Proteintech, Wuhan, China). Membranes were incubated with primary antibodies overnight at 4°C, rinsed with TBST three times for 10 min, and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (SA00001-1/2, 1 : 5000, Proteintech, Wuhan, China) for 1 h. After washing, proteins were visualized on an Amersham Imager 600 (GE Healthcare UK Limited, UK) using LumiGLO® Reagent and Peroxide (Cell Signaling Technology, Beverly, MA, USA). The optic density (OD) values of detected bands were measured with the Scion Image analysis software (Scion, Frederick, MD, USA) and were normalized to mouse anti-β-actin (66009-1, 1 : 5000, Proteintech, Wuhan, China) or mouse anti-histone H3 (Bsm-33042M, 1 : 5000, Bioss, Beijing, China) as internal controls (OD detected protein/OD internal control). The specificity of each of the primary antibodies was evaluated by preincubation in the presence or absence of the corresponding blocking peptides or by omission of the primary antibodies.


## 2.11. Collection of Kidney Slices

At the end of experiment, mice were euthanized and perfused with sterile PBS to remove their blood. Kidneys were immediately fixed in 4% phosphate-buffered paraformaldehyde, dehydrated in 30% sucrose (Amresco, Ohio, USA), and then sectioned with a freezing microtome (Leica CM3050S, Biosystems, Nussloch, Germany) and stored at −80°C (DW-86L578J, Haier BioMedical, Qingdao, China) for dihydroethidium (DHE) staining, hematoxylin and eosin (HE) staining, and immunofluorescence.


## 2.12. Immunofluorescence Staining

Immunofluorescence staining of renal sections were conducted as previously described [8]. First, antigens were retrieved by boiling in citric acid buffer (pH 6.0) for 15 min. Nonspecific antigens were blocked with 5% bovine serum albumin for 1 h at room temperature. Samples were incubated with rabbit anti-VEGFR2 antibody (ab2349, 1 : 500, Abcam, Cambridge, UK) at 4°C overnight, and then goat anti-rabbit DyLight 488 Antibody (1031-02, 1 : 20,000, Vector Laboratories, Burlingame, CA, USA) or anti-rabbit CY3 antibody (CY-1300-1, 1 : 2000, Vector Laboratories, Burlingame, CA, USA) was added for 1 h. Subsequently, DAPI was applied for 8 min. Stained sections were imaged at 200x on a fluorescence microscope (Leica DM4000B, Leica Microsystems, Wetzlar, Germany), and quantitative analysis was performed using Image-Pro Plus 6.0 (IPP; Media Cybernetics, Rockville, MD, USA) [21].


## 2.13. HE Staining

Mice were euthanized and perfused with sterile PBS to remove their blood. Kidney were immediately fixed in 4% phosphate-buffered paraformaldehyde, dehydrated in 30% sucrose (Amresco, Ohio, USA), and then sectioned with a freezing microtome (Leica CM3050S, Biosystems, Nussloch, Germany) and stored at −80°C (DW-86L578J, Haier Biomedical, Qingdao, China). To assess renal morphology, 10μm sections were stained with HE (Sigma-Aldrich, St. Louis, MO, USA). Detailed staining procedures have been previously described [8]. Pathological changes in kidney tissues were observed under a light microscope, and renal damage was evaluated in a double-blind fashion by two kidney pathologists [23].


## 2.14. DHE Staining

DHE staining was used to detect the ROS production in the kidney sections. Simply, 10μm-thick frozen renal sections were removed from −80°C storage and dried at 37°C for 30 min in an electric thermostatic incubator (Incucell, Germany), and then incubated in 1 × PBS for 15 min. Next, sections were incubated with 10 mol/L fluorescent-labeled DHE (Molecular Probes, Invitrogen, Carlsbad, CA, USA) in a lucifugal-humidified chamber at 37°C for 30 min, then stained with 4′,6′-diamidino-2-phenylindole (DAPI, Abcam, Cambridge, CA, USA). Images were obtained at 200x on a fluorescence microscope (Leica DM4000B, Leica Microsystems, Wetzlar, Germany). The average DHE fluorescence intensity was measured using IPP. Results were expressed as the ratio of the DHE-positive area and the DAPI.


## 2.15. Statistical Analysis

All statistical analyses were performed using SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance followed by Tukey’s honest significant difference test. Data are expressed as the mean ± standard error of mean (SEM). Statistical significance was set atp<0.05.


## 3. Results

### 3.1. Kidney-Specific VEGFR2 Knockdown Inhibits Renal Oxidative Stress of HFD-Treated Mice

The immunofluorescence density of EGFP expressed by AAV2/9 reached a peak after a three-week transfection in the kidney of HFD-treated mice (Supplementary Figure1(a)–1(c). So, an experiment of kidney-specific VEGFR2 knockdown was conducted after a three-week injection (Figures 1(b)–1(f)). Then, we evaluated the knockdown effect of VEGFR2 by qPCR, western blot, and immunofluorescence (Figures 1(b)–1(f)). In comparison with the HFD scramble group, VEGFR2 mRNA in the HFD VEGFR2 shRNA group was remarkably reduced to less than 70% (Figure 1(b)). Similar results were obtained from western blot and immunofluorescence analysis of VEGFR2 protein (vs. the HFD scramble group, Figures 1(c)–1(f)). However, no significantly different EGFP expression was observed between scramble and VEGFR2 shRNA in the kidney of HFD-treated mice (Figures 1(e) and 1(f)). These results indicated that the VEGFR2 gene was successfully knocked down in the kidney of mice (vs. the HFD scramble group, Figures 1(b)–1(f)). Further, NOX4 protein expression and ROS production were markedly suppressed by VEGFR2 knockdown in the kidney of HFD-treated mice (vs. the HFD scramble group, Figures 1(g)–1(i)). However, no remarkable differences of NOX4 protein expression and ROS levels were observed between CD VEGFR2 shRNA and scramble in CD-treated mice (Figures 1(g)–1(i)). These results demonstrated that ROS overproduction was induced by VEGFR2 upregulation in the kidney of HFD-treated mice.


### 3.2. Kidney-Specific VEGFR2 Knockdown Alleviates NRLP3-Dependent Inflammation in the Kidney of the HFD-Treated Mice

Next, to verify whether VEGFR2 upregulation induced NRLP3-dependent inflammatory response, and ultimately resulting in kidney injury in the HFD-treated mice, we investigated the NRLP3 activation, NF-κB p65 nuclear translocation, and its inflammatory cascade protein expression. Western blot analysis showed that kidney-specific VEGFR2 knockdown notably inhibited NRLP3 activation and remarkably decreased the ratio of cleaved to procaspase and cleaved to pro-IL1β induced by HFD adminstration in the mice (vs. the HFD scramble group, Figures 2(a) and 2(b)). Additionally, further data showed that kidney-specific VEGFR2 knockdown markedly reduced the nuclear NF-κB p65, TNFα, IL6, and iNOS expression (vs. the HFD scramble group, Figures 2(c)–2(f)). However, no significant difference was observed in NRLP3 activation, NF-κB p65 nuclear translocation, TNFα, IL6, and iNOS expression, and the ratio of cleaved to procaspase 1 and cleaved to pro-IL1β between the HFD VEGFR2 shRNA and CD scramble group. These results indicated that kidney-specific VEGFR2 knockdown alleviated NRLP3-dependent inflammation in the kidney of the HFD-treated mice.Figure 2
Kidney-specific VEGFR2 knockdown alleviates NRLP3-dependent inflammation in the kidney of the HFD-treated mice. (a) Representative immunoblots for NLRP3, procaspase 1, cleaved caspase 1, pro-IL1β, cleaved IL1β, and β-actin. (b) Relative density analysis of the NLRP3, cleaved caspase 1, and cleaved IL1β protein bands. Relative densities are expressed as the ratio of NRLP3 to β-actin, cleaved caspase 1 to procaspase 1 (expressed as cleaved/procaspase 1), and cleaved IL1β to pro-IL1β (expressed as cleaved/pro-IL1β). (c) Representative immunoblots for cytoplasmic NF-κB p65 (C-NF-κB p65), nuclear NF-κB p65 (N-NF-κB p65), β-actin, and histone H3 (H3). (d) Relative density analysis of the C-NF-κB p65 and N-NF-κB p65 protein bands. Relative densities are expressed as the ratio of C-NF-κB p65 to β-actin and N-NF-κB p65 to H3. (e) Representative immunoblots for TNFα, IL6, iNOS, and β-actin. (f) Relative density analysis of the TNFα, IL6, and iNOS protein bands. Relative densities are expressed as the ratio of TNFα, IL6, and iNOS to β-actin. All values are expressed as mean ± SEM ((a–f), n=5). ∗∗p<0.01 and ∗∗∗p<0.001vs. the CD scramble group, and ##p<0.01 and ###p<0.001vs. the HFD scramble group.
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### 3.3. Kidney-Specific VEGFR2 Knockdown Alleviates Kidney Injury of HFD-Treated Mice

Then, to explore whether VEGFR2 upregulation accelerated kidney injury in the HFD-treated mice, we assessed kidney function by HE staining and determination of serum creatinine and blood urea nitrogen concentrations (Figures3(a)–3(f)). HE staining showed that VEGFR2 knockdown obviously improved loosened kidney structure, glomerulus hypertrophy, swelled tubules, severe inflammatory cell accumulation, and thickened basement-membranes induced by HFD (vs. the HFD scramble group, Figures 3(a)–3(e)). Moreover, the enhancements of the glomerulus and Bowman’s capsule diameter, tubule diameter, glomerulus and Bowman’s capsule area, tubulointerstitial lesion score, and serum creatinine and blood urea nitrogen levels induced by HFD were remarkably depressed by VEGFR2 knockdown (vs. the HFD scramble group, Figures 3(a)–3(f)). In addition, no obvious changes were displayed in the aforementioned parameters between the CD VEGFR2 shRNA and CD scramble group (Figures 3(a)–3(f)). These results suggested that kidney VEGFR2 upregulation significantly deteriorated the kidney injury in the HFD-treated mice.Figure 3
Kidney-specific VEGFR2 knockdown alleviates the kidney injury of HFD-treated mice. (a) Kidney sections were stained by hematoxylin-eosin; the black arrow represents the accumulation of inflammatory cells (200x, scale bar, 100μm). (b) Glomerulus and Bowman’s capsule diameter; the red arrow represents blood glomerulus, and the green arrow represents Bowman’s capsule. (c) Tubule diameter; the blue arrow represents proximal tubules, and the yellow arrow represents the distal tubules. (d) Glomerulus and Bowman’s capsule area. (e) Tubulointerstitial lesion score. (F) Serum creatinine and blood urea nitrogen determination. All values are expressed as mean ± SEM ((a–e), n=5). ∗∗∗p<0.001vs. the CD scramble group, and ##p<0.01 and ###p<0.001vs. the HFD scramble group.
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### 3.4.α-Lipoic Acid Decreases ROS-Triggered NRLP3-Dependent Inflammation in the Kidney of HFD-Treated Mice

To explore whether ROS triggered NRLP3-dependent inflammation stimulated by VEGFR2 upregulation in the kidney of HFD-treated mice, we determined the effect ofα-lipoic acid on ROS production, NRLP3 activation, and nuclear NF-κB and its downstream inflammatory protein expression. DHE staining showed that α-lipoic acid significantly inhibited ROS production in the kidney of HFD-treated mice (vs. HFD + α-lipoic acid, Figures 4(a) and 4(b)). Further western blot analysis showed that α-lipoic acid markedly depressed the NRLP3 activation and reduced the ratio of cleaved to procaspase 1 and cleaved to pro-IL1β in the kidney of HFD-treated mice (vs. HFD + α-lipoic acid, Figures 4(c) and 4(d)). In addition, α-lipoic acid significantly blocked NF-κB p65 nuclear translocation and its downstream TNFα, IL6, and iNOS expression in the kidney of HFD-treated mice (vs. HFD + α-lipoic acid, Figures 4(e)–4(h)). However, no significant variations of ROS levels and the abovementioned protein expression were observed between the CD + α-lipoic acid and CD + vehicle group. Thus, ROS was confirmed to be a mediator of VEGFR2-stimulated NRLP3 activation and its dependent inflammation in the kidney of HFD-treated mice.Figure 4
α-Lipoic acid decreases ROS-triggered inflammation in the kidney of HFD-treated mice. (a) ROS levels were assessed by dihydroethidium (DHE) staining. (b) DHE fluorescence intensity was measured by the IPP, and results are expressed by the ratio of the integrated optical density of the DHE-positive area to the DAPI. (c) Representative immunoblots for NLRP3, procaspase 1, cleaved caspase 1, pro-IL1β, cleaved IL1β, and β-actin. (d) Relative density analysis of NLRP3, cleaved caspase 1, and cleaved IL1β protein bands. Relative densities are expressed as the ratio of NRLP3 to β-actin, cleaved caspase 1 to procaspase 1 (expressed as cleaved/procaspase 1), and cleaved IL1β to Pro-IL 1β (expressed as cleaved/pro-IL1β). (e) Representative immunoblots for cytoplasmic NF-κB p65 (C-NF-κB p65), nuclear NF-κB p65 (N-NF-κB p65), β-actin, and histone H3 (H3). (f) Relative density analysis of the C-NF-κB p65 and N-NF-κB p65 protein bands. Relative densities are expressed as the ratio of C-NF-κB p65 to β-actin and N-NF-κB p65 to H3. (g) Representative immunoblots for TNFα, IL6, iNOS, and β-actin. (h) Relative density analysis of TNFα, IL6, and iNOS protein bands. Relative densities are expressed as the ratio of TNFα, IL6, and iNOS to β-actin. All values are expressed as mean ± SEM ((a–h), n=5). ∗∗∗p<0.001vs. the CD vehicle group, and ###p<0.001vs. the HFD vehicle group.
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### 3.5.α-Lipoic Acid Alleviates NRLP3-Dependent Kidney Injury in HFD-Treated Mice

Additionally, we explored whetherα-lipoic acid alleviates NRLP3-dependent kidney injury in the HFD-treated mice. We determined the kidney function by HE staining and serum creatinine and blood urea nitrogen determination (Figures 5(a)–5(f)). HE staining showed that α-lipoic acid significantly improved loosened kidney structure, glomerular hypertrophy, swelled tubules, abundant inflammatory cell accumulation, thicker basement-membranes, and tubulointerstitial lesion score of HFD-treated mice (vs. the HFD + vehicle group, Figures 5(a)–5(e)). Moreover, further analysis showed that α-lipoic acid significantly lowered the glomerulus and Bowman’s capsule diameter, tubule diameter, the glomerulus and Bowman’s capsule area, tubulointerstitial lesion score, and serum creatinine and blood urea nitrogen levels of HFD-treated mice (vs. the HFD + vehicle group, Figures 5(a)–5(f)). However, no noticeable changes were disclosed in the abovementioned indicators between the CD + α-lipoic acid and CD + vehicle group (Figures 5(a)–5(f)). These results suggested that α-lipoic acid alleviates NRLP3-dependent kidney injury in HFD-treated mice.Figure 5
α-Lipoic acid alleviates NRLP3-dependent kidney injury in HFD-treated mice. (a) Kidney sections were stained by hematoxylin-eosin; the black arrow represents the accumulation of inflammatory cells (200x, scale bar, 100 μm). (b) Glomerulus and Bowman’s capsule diameter; the red arrow represents blood glomerulus, and the green arrow represents Bowman’s capsule. (c) Tubule diameter; the blue arrow represents proximal tubules, and the yellow arrow represents the distal tubules. (d) Glomerulus and Bowman’s capsule area. (e) Tubulointerstitial lesion score. (f) Serum creatinine and blood urea nitrogen determination. All values are expressed as mean ± SEM ((a–e), n=5). ∗∗∗p<0.001vs. the CD vehicle group, and ###p<0.001vs. the HFD vehicle group.
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### 3.6. PSPC Administration Inhibits ROS-Triggered NRLP3 Inflammation in HFD-Treated Mice

Furthermore, to estimate whether PSPC administration alleviates ROS-triggered NRLP3 inflammation by inhibiting VEGFR2 upregulation in HFD-treated mice, we determined the VEGFR2, NOX4, NRLP3 expression, and ROS levels. Immunofluorescence analysis showed that PSPC significantly reduced the VEGFR2-positive staining area in the kidney of HFD-treated mice (vs. CD group, Figures 6(a) and 6(b)). Further results showed that PSPC administration markedly inhibited NOX4 expression and ROS production in the kidney of HFD-treated mice (vs. the CD group, Figures 6(c)–6(e)). Moreover, western blot analysis showed that PSPC notably reduced NLRP3 expression and the ratio of cleaved to procaspase 1 and cleaved to pro-IL1β in the HFD-treated mice (vs. the CD group, Figures 6(f) and 6(g)). However, no marked difference of these indicators was observed between the CD + PSPC group and CD group. These results indicated that PSPC administration inhibits ROS-triggered NRLP3 activation by inhibiting VEGFR2 upregulation in HFD-treated mice.Figure 6
PSPC administration inhibits ROS-triggered NRLP3 inflammation in HFD-treated mice. (a) Kidney sections were stained with FITC to visualize VEGFR2-positive cell staining (green) (200x, scale bar, 100μm). (b) Analysis of the relative intensity of VEGFR2-positive cells in the kidney sections. (c) Representative immunoblot for NOX4 and β-actin. (d) Relative density analysis of the NOX4 protein bands. Relative densities are expressed as the ratio of NOX4 to β-actin. (e) The ROS levels were assessed by 2′,7′-dichlorofluorescein diacetate (DCF-DA) in the kidney of HFD-treated mice. (f) Representative immunoblots for NLRP3, procaspase 1, cleaved caspase 1, pro-IL1β, cleaved IL1β, and β-actin. (g) The relative density analysis of the NLRP3, cleaved caspase 1, and cleaved IL1β protein bands. The relative densities are expressed as the ratio of NRLP3 to β-actin, cleaved caspase 1 to procaspase 1 (expressed as cleaved/procaspase 1), and cleaved IL1β to pro-IL1β (expressed as cleaved/pro-IL1β). All values are expressed as mean ± SEM ((a–g), n=5). ∗∗∗p<0.001vs. the CD group, and ###p<0.001vs. the HFD group. (h) Kidney sections were stained by hematoxylin-eosin; the black arrow represents the accumulation of inflammatory cells in the kidney (200x, scale bar, 100 μm). (i) Glomerulus and Bowman’s capsule diameter were determined by Image-Pro Plus (IPP); the red arrow represents blood glomerulus, and the green arrow represents Bowman’s capsule. (j) Tubule diameter was measured by IPP; the blue arrow represents proximal tubules, and the yellow arrow represents the distal tubules. (k) Glomerulus and Bowman’s capsule area were determined by IPP. (l) Serum creatinine and blood urea nitrogen determination. (m) Schematic diagram of PSPC on VEGFR2/ROS/NRLP3 signaling in the kidney of HFD-treated mice. All values are expressed as mean ± SEM ((a–e), n=5). ∗∗∗p<0.001vs. the CD group, and ###p<0.001vs. the HFD group.
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(m)Finally, to explore whether PSPC administration relieved NRLP3-dependent kidney injury in HFD-treated mice, we estimated the kidney function by HE staining and determination of serum creatinine and blood urea nitrogen concentrations. HE staining showed that PSPC administration significantly improved loosened kidney structure, glomerulus hypertrophy, swelled tubules, severe inflammatory cells, and the thickened basement-membranes induced by HFD (vs. the CD group, Figures 6(h)–6(l)). Moreover, the increment of glomerulus diameter, Bowman’s capsule diameter, tubule diameter, glomerulus and Bowman’ capsule area, and serum creatinine and blood urea nitrogen induced by HFD were markedly inhibited by PSPC administration in the HFD + PSPC group (vs. the CD group, Figures 6(h)–6(l)). However, no significant difference in these biomarkers was found between the CD + PSPC and CD group. These results indicated that PSPC administration remarkably alleviated kidney injury by inhibiting VEGFR2 expression in the HFD-treated mice.


## 3.1. Kidney-Specific VEGFR2 Knockdown Inhibits Renal Oxidative Stress of HFD-Treated Mice

The immunofluorescence density of EGFP expressed by AAV2/9 reached a peak after a three-week transfection in the kidney of HFD-treated mice (Supplementary Figure1(a)–1(c). So, an experiment of kidney-specific VEGFR2 knockdown was conducted after a three-week injection (Figures 1(b)–1(f)). Then, we evaluated the knockdown effect of VEGFR2 by qPCR, western blot, and immunofluorescence (Figures 1(b)–1(f)). In comparison with the HFD scramble group, VEGFR2 mRNA in the HFD VEGFR2 shRNA group was remarkably reduced to less than 70% (Figure 1(b)). Similar results were obtained from western blot and immunofluorescence analysis of VEGFR2 protein (vs. the HFD scramble group, Figures 1(c)–1(f)). However, no significantly different EGFP expression was observed between scramble and VEGFR2 shRNA in the kidney of HFD-treated mice (Figures 1(e) and 1(f)). These results indicated that the VEGFR2 gene was successfully knocked down in the kidney of mice (vs. the HFD scramble group, Figures 1(b)–1(f)). Further, NOX4 protein expression and ROS production were markedly suppressed by VEGFR2 knockdown in the kidney of HFD-treated mice (vs. the HFD scramble group, Figures 1(g)–1(i)). However, no remarkable differences of NOX4 protein expression and ROS levels were observed between CD VEGFR2 shRNA and scramble in CD-treated mice (Figures 1(g)–1(i)). These results demonstrated that ROS overproduction was induced by VEGFR2 upregulation in the kidney of HFD-treated mice.


## 3.2. Kidney-Specific VEGFR2 Knockdown Alleviates NRLP3-Dependent Inflammation in the Kidney of the HFD-Treated Mice

Next, to verify whether VEGFR2 upregulation induced NRLP3-dependent inflammatory response, and ultimately resulting in kidney injury in the HFD-treated mice, we investigated the NRLP3 activation, NF-κB p65 nuclear translocation, and its inflammatory cascade protein expression. Western blot analysis showed that kidney-specific VEGFR2 knockdown notably inhibited NRLP3 activation and remarkably decreased the ratio of cleaved to procaspase and cleaved to pro-IL1β induced by HFD adminstration in the mice (vs. the HFD scramble group, Figures 2(a) and 2(b)). Additionally, further data showed that kidney-specific VEGFR2 knockdown markedly reduced the nuclear NF-κB p65, TNFα, IL6, and iNOS expression (vs. the HFD scramble group, Figures 2(c)–2(f)). However, no significant difference was observed in NRLP3 activation, NF-κB p65 nuclear translocation, TNFα, IL6, and iNOS expression, and the ratio of cleaved to procaspase 1 and cleaved to pro-IL1β between the HFD VEGFR2 shRNA and CD scramble group. These results indicated that kidney-specific VEGFR2 knockdown alleviated NRLP3-dependent inflammation in the kidney of the HFD-treated mice.Figure 2
Kidney-specific VEGFR2 knockdown alleviates NRLP3-dependent inflammation in the kidney of the HFD-treated mice. (a) Representative immunoblots for NLRP3, procaspase 1, cleaved caspase 1, pro-IL1β, cleaved IL1β, and β-actin. (b) Relative density analysis of the NLRP3, cleaved caspase 1, and cleaved IL1β protein bands. Relative densities are expressed as the ratio of NRLP3 to β-actin, cleaved caspase 1 to procaspase 1 (expressed as cleaved/procaspase 1), and cleaved IL1β to pro-IL1β (expressed as cleaved/pro-IL1β). (c) Representative immunoblots for cytoplasmic NF-κB p65 (C-NF-κB p65), nuclear NF-κB p65 (N-NF-κB p65), β-actin, and histone H3 (H3). (d) Relative density analysis of the C-NF-κB p65 and N-NF-κB p65 protein bands. Relative densities are expressed as the ratio of C-NF-κB p65 to β-actin and N-NF-κB p65 to H3. (e) Representative immunoblots for TNFα, IL6, iNOS, and β-actin. (f) Relative density analysis of the TNFα, IL6, and iNOS protein bands. Relative densities are expressed as the ratio of TNFα, IL6, and iNOS to β-actin. All values are expressed as mean ± SEM ((a–f), n=5). ∗∗p<0.01 and ∗∗∗p<0.001vs. the CD scramble group, and ##p<0.01 and ###p<0.001vs. the HFD scramble group.
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## 3.3. Kidney-Specific VEGFR2 Knockdown Alleviates Kidney Injury of HFD-Treated Mice

Then, to explore whether VEGFR2 upregulation accelerated kidney injury in the HFD-treated mice, we assessed kidney function by HE staining and determination of serum creatinine and blood urea nitrogen concentrations (Figures3(a)–3(f)). HE staining showed that VEGFR2 knockdown obviously improved loosened kidney structure, glomerulus hypertrophy, swelled tubules, severe inflammatory cell accumulation, and thickened basement-membranes induced by HFD (vs. the HFD scramble group, Figures 3(a)–3(e)). Moreover, the enhancements of the glomerulus and Bowman’s capsule diameter, tubule diameter, glomerulus and Bowman’s capsule area, tubulointerstitial lesion score, and serum creatinine and blood urea nitrogen levels induced by HFD were remarkably depressed by VEGFR2 knockdown (vs. the HFD scramble group, Figures 3(a)–3(f)). In addition, no obvious changes were displayed in the aforementioned parameters between the CD VEGFR2 shRNA and CD scramble group (Figures 3(a)–3(f)). These results suggested that kidney VEGFR2 upregulation significantly deteriorated the kidney injury in the HFD-treated mice.Figure 3
Kidney-specific VEGFR2 knockdown alleviates the kidney injury of HFD-treated mice. (a) Kidney sections were stained by hematoxylin-eosin; the black arrow represents the accumulation of inflammatory cells (200x, scale bar, 100μm). (b) Glomerulus and Bowman’s capsule diameter; the red arrow represents blood glomerulus, and the green arrow represents Bowman’s capsule. (c) Tubule diameter; the blue arrow represents proximal tubules, and the yellow arrow represents the distal tubules. (d) Glomerulus and Bowman’s capsule area. (e) Tubulointerstitial lesion score. (F) Serum creatinine and blood urea nitrogen determination. All values are expressed as mean ± SEM ((a–e), n=5). ∗∗∗p<0.001vs. the CD scramble group, and ##p<0.01 and ###p<0.001vs. the HFD scramble group.
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## 3.4.α-Lipoic Acid Decreases ROS-Triggered NRLP3-Dependent Inflammation in the Kidney of HFD-Treated Mice

To explore whether ROS triggered NRLP3-dependent inflammation stimulated by VEGFR2 upregulation in the kidney of HFD-treated mice, we determined the effect ofα-lipoic acid on ROS production, NRLP3 activation, and nuclear NF-κB and its downstream inflammatory protein expression. DHE staining showed that α-lipoic acid significantly inhibited ROS production in the kidney of HFD-treated mice (vs. HFD + α-lipoic acid, Figures 4(a) and 4(b)). Further western blot analysis showed that α-lipoic acid markedly depressed the NRLP3 activation and reduced the ratio of cleaved to procaspase 1 and cleaved to pro-IL1β in the kidney of HFD-treated mice (vs. HFD + α-lipoic acid, Figures 4(c) and 4(d)). In addition, α-lipoic acid significantly blocked NF-κB p65 nuclear translocation and its downstream TNFα, IL6, and iNOS expression in the kidney of HFD-treated mice (vs. HFD + α-lipoic acid, Figures 4(e)–4(h)). However, no significant variations of ROS levels and the abovementioned protein expression were observed between the CD + α-lipoic acid and CD + vehicle group. Thus, ROS was confirmed to be a mediator of VEGFR2-stimulated NRLP3 activation and its dependent inflammation in the kidney of HFD-treated mice.Figure 4
α-Lipoic acid decreases ROS-triggered inflammation in the kidney of HFD-treated mice. (a) ROS levels were assessed by dihydroethidium (DHE) staining. (b) DHE fluorescence intensity was measured by the IPP, and results are expressed by the ratio of the integrated optical density of the DHE-positive area to the DAPI. (c) Representative immunoblots for NLRP3, procaspase 1, cleaved caspase 1, pro-IL1β, cleaved IL1β, and β-actin. (d) Relative density analysis of NLRP3, cleaved caspase 1, and cleaved IL1β protein bands. Relative densities are expressed as the ratio of NRLP3 to β-actin, cleaved caspase 1 to procaspase 1 (expressed as cleaved/procaspase 1), and cleaved IL1β to Pro-IL 1β (expressed as cleaved/pro-IL1β). (e) Representative immunoblots for cytoplasmic NF-κB p65 (C-NF-κB p65), nuclear NF-κB p65 (N-NF-κB p65), β-actin, and histone H3 (H3). (f) Relative density analysis of the C-NF-κB p65 and N-NF-κB p65 protein bands. Relative densities are expressed as the ratio of C-NF-κB p65 to β-actin and N-NF-κB p65 to H3. (g) Representative immunoblots for TNFα, IL6, iNOS, and β-actin. (h) Relative density analysis of TNFα, IL6, and iNOS protein bands. Relative densities are expressed as the ratio of TNFα, IL6, and iNOS to β-actin. All values are expressed as mean ± SEM ((a–h), n=5). ∗∗∗p<0.001vs. the CD vehicle group, and ###p<0.001vs. the HFD vehicle group.
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## 3.5.α-Lipoic Acid Alleviates NRLP3-Dependent Kidney Injury in HFD-Treated Mice

Additionally, we explored whetherα-lipoic acid alleviates NRLP3-dependent kidney injury in the HFD-treated mice. We determined the kidney function by HE staining and serum creatinine and blood urea nitrogen determination (Figures 5(a)–5(f)). HE staining showed that α-lipoic acid significantly improved loosened kidney structure, glomerular hypertrophy, swelled tubules, abundant inflammatory cell accumulation, thicker basement-membranes, and tubulointerstitial lesion score of HFD-treated mice (vs. the HFD + vehicle group, Figures 5(a)–5(e)). Moreover, further analysis showed that α-lipoic acid significantly lowered the glomerulus and Bowman’s capsule diameter, tubule diameter, the glomerulus and Bowman’s capsule area, tubulointerstitial lesion score, and serum creatinine and blood urea nitrogen levels of HFD-treated mice (vs. the HFD + vehicle group, Figures 5(a)–5(f)). However, no noticeable changes were disclosed in the abovementioned indicators between the CD + α-lipoic acid and CD + vehicle group (Figures 5(a)–5(f)). These results suggested that α-lipoic acid alleviates NRLP3-dependent kidney injury in HFD-treated mice.Figure 5
α-Lipoic acid alleviates NRLP3-dependent kidney injury in HFD-treated mice. (a) Kidney sections were stained by hematoxylin-eosin; the black arrow represents the accumulation of inflammatory cells (200x, scale bar, 100 μm). (b) Glomerulus and Bowman’s capsule diameter; the red arrow represents blood glomerulus, and the green arrow represents Bowman’s capsule. (c) Tubule diameter; the blue arrow represents proximal tubules, and the yellow arrow represents the distal tubules. (d) Glomerulus and Bowman’s capsule area. (e) Tubulointerstitial lesion score. (f) Serum creatinine and blood urea nitrogen determination. All values are expressed as mean ± SEM ((a–e), n=5). ∗∗∗p<0.001vs. the CD vehicle group, and ###p<0.001vs. the HFD vehicle group.
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## 3.6. PSPC Administration Inhibits ROS-Triggered NRLP3 Inflammation in HFD-Treated Mice

Furthermore, to estimate whether PSPC administration alleviates ROS-triggered NRLP3 inflammation by inhibiting VEGFR2 upregulation in HFD-treated mice, we determined the VEGFR2, NOX4, NRLP3 expression, and ROS levels. Immunofluorescence analysis showed that PSPC significantly reduced the VEGFR2-positive staining area in the kidney of HFD-treated mice (vs. CD group, Figures 6(a) and 6(b)). Further results showed that PSPC administration markedly inhibited NOX4 expression and ROS production in the kidney of HFD-treated mice (vs. the CD group, Figures 6(c)–6(e)). Moreover, western blot analysis showed that PSPC notably reduced NLRP3 expression and the ratio of cleaved to procaspase 1 and cleaved to pro-IL1β in the HFD-treated mice (vs. the CD group, Figures 6(f) and 6(g)). However, no marked difference of these indicators was observed between the CD + PSPC group and CD group. These results indicated that PSPC administration inhibits ROS-triggered NRLP3 activation by inhibiting VEGFR2 upregulation in HFD-treated mice.Figure 6
PSPC administration inhibits ROS-triggered NRLP3 inflammation in HFD-treated mice. (a) Kidney sections were stained with FITC to visualize VEGFR2-positive cell staining (green) (200x, scale bar, 100μm). (b) Analysis of the relative intensity of VEGFR2-positive cells in the kidney sections. (c) Representative immunoblot for NOX4 and β-actin. (d) Relative density analysis of the NOX4 protein bands. Relative densities are expressed as the ratio of NOX4 to β-actin. (e) The ROS levels were assessed by 2′,7′-dichlorofluorescein diacetate (DCF-DA) in the kidney of HFD-treated mice. (f) Representative immunoblots for NLRP3, procaspase 1, cleaved caspase 1, pro-IL1β, cleaved IL1β, and β-actin. (g) The relative density analysis of the NLRP3, cleaved caspase 1, and cleaved IL1β protein bands. The relative densities are expressed as the ratio of NRLP3 to β-actin, cleaved caspase 1 to procaspase 1 (expressed as cleaved/procaspase 1), and cleaved IL1β to pro-IL1β (expressed as cleaved/pro-IL1β). All values are expressed as mean ± SEM ((a–g), n=5). ∗∗∗p<0.001vs. the CD group, and ###p<0.001vs. the HFD group. (h) Kidney sections were stained by hematoxylin-eosin; the black arrow represents the accumulation of inflammatory cells in the kidney (200x, scale bar, 100 μm). (i) Glomerulus and Bowman’s capsule diameter were determined by Image-Pro Plus (IPP); the red arrow represents blood glomerulus, and the green arrow represents Bowman’s capsule. (j) Tubule diameter was measured by IPP; the blue arrow represents proximal tubules, and the yellow arrow represents the distal tubules. (k) Glomerulus and Bowman’s capsule area were determined by IPP. (l) Serum creatinine and blood urea nitrogen determination. (m) Schematic diagram of PSPC on VEGFR2/ROS/NRLP3 signaling in the kidney of HFD-treated mice. All values are expressed as mean ± SEM ((a–e), n=5). ∗∗∗p<0.001vs. the CD group, and ###p<0.001vs. the HFD group.
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(m)Finally, to explore whether PSPC administration relieved NRLP3-dependent kidney injury in HFD-treated mice, we estimated the kidney function by HE staining and determination of serum creatinine and blood urea nitrogen concentrations. HE staining showed that PSPC administration significantly improved loosened kidney structure, glomerulus hypertrophy, swelled tubules, severe inflammatory cells, and the thickened basement-membranes induced by HFD (vs. the CD group, Figures 6(h)–6(l)). Moreover, the increment of glomerulus diameter, Bowman’s capsule diameter, tubule diameter, glomerulus and Bowman’ capsule area, and serum creatinine and blood urea nitrogen induced by HFD were markedly inhibited by PSPC administration in the HFD + PSPC group (vs. the CD group, Figures 6(h)–6(l)). However, no significant difference in these biomarkers was found between the CD + PSPC and CD group. These results indicated that PSPC administration remarkably alleviated kidney injury by inhibiting VEGFR2 expression in the HFD-treated mice.


## 4. Discussion

Oxidative stress and inflammation induced by HFD have been reported to cause renal damage in the T2DM [24, 25], but the potential regulatory mechanism is still obscure. The present findings show that VEGFR2 upregulation in the kidney of HFD-treated mice promotes ROS overproduction, consequently induces NLRP3-dependent inflammatory responses, and ultimately leads to kidney damage.Initially, experimental observation found that VEGFR2 upregulation increased the NOX4 expression and ROS levels in the kidney of HFD-treated mice, which agree with previous evidence that VEGF induced ROS overproduction in the pathological angiogenesis [9]. NOX4 is a member of the NADPH oxidase family, and a main source for generating ROS in multiple physiological and pathological processes, such as cellular signaling, cell proliferation, neurodegeneration, and diabetes mellitus [26, 27]. Many studies have shown that NOX4 is activated by various growth factors and cytokines, and it aggravates renal injury in the DN, which is similar to the findings in our present study [27–31]. Additionally, NOX4 is an important mediator for VEGF-mediated feedback reduction of ROS overproduction in pathological angiogenesis and inflammations [9, 10, 32]. Given that ROS is a major trigger for NRLP3-dependent signaling, further results showed that NF-κB p65 nuclear translocation and the expression of NRLP3, IL1β, TNFα, IL6, and iNOS protein were markedly enhanced by VEGFR2 upregulation in the kidney of the HFD-treated mice compared with the CD-treated mice. The NRLP3 inflammasome is known as NLRP3 or cryopyrin, and it belongs to the leucine-rich-containing family, pyrin domain-containing-3 [33]. It could sense a metabolic dangerous stressor and initiates the recruitment of mature caspase 1, which activates IL1β release. As previous studies reported that excessive ROS is an effective activator for NRLP3 activation in many inflammatory diseases, such as in cancers and autoimmune diseases [32–35], this study obtained highly consistent results. In addition, NRLP3 has been reported as an initiator for inflammation in obesity-related disorders such as diabetic neurodegenerative disease, diabetic nonalcoholic fatty liver, and DN [33], and chronic inflammation is the key link for the etiopathogenesis of obesity and T2DM [34]. Based on the aforementioned literature, further studies demonstrated that VEGFR2 upregulation aggravated kidney injury by increasing the glomerulus, Bowman’s capsule, and tubule diameter, as well as serum creatinine and blood urea nitrogen levels. Thus, a comprehensive assessment of serum creatinine levels, blood urea nitrogen levels, and pathological changes could provide a reliable reference for evaluating kidney injury in chronic and acute kidney diseases [35]. These results indicated that VEGFR2 induces kidney injury at least partly via ROS-mediated NLRP3 activation.Additionally, to further verify whether VEGFR2 upregulation stimulated NLRP3 activation via excessive ROS in the kidney of HFD-treated mice, the effect ofα-lipoic acid, a stronger ROS scavenger, on the kidney was determined after 4 weeks of administration at a dose of 150 mg/kg per day in the HFD-treated mice. Results showed that α-lipoic acid attenuated the kidney injury by reducing ROS levels, NLRP3 activation, NF-κB p65 nuclear translocation, and expression of its downstream inflammatory cytokines, such as IL1β, TNFα, IL6, and iNOS. Similar to these findings, previous studies also indicated that α-lipoic acid strongly reversed oxidative stress by directly or indirectly reacting to ROS in various disease models related to oxidative stress such as neurodegeneration, diabetes mellitus, and ischemic injury [19], and that the ROS scavenger effectively blocked NRLP3 activation, NF-κB nuclear translocation, and downstream inflammatory cytokine expression [36, 37]. As an important inflammatory transduction factor, NF-κB activation promotes proinflammatory gene translocation in numerous renal diseases such as systemic lupus erythematosus, ischemic nephropathy, aged nephropathy, and DN [38, 39], which is consistent with our present results. Moreover, the ROS scavenger has been shown to significantly decrease serum creatinine and blood urea nitrogen levels and to markedly relieve the glomerular hypertrophy and aggregation of inflammatory cells [40]. Therefore, scavenging of ROS by α-lipoic acid reduced the kidney injury by not only blocking NLRP3 activation, but also inhibiting NF-κB nuclear translocation and downstream inflammatory cytokine expression in the kidney of HFD-treated mice.PSPC, a natural anthocyanin extracted from purple sweet potato, has been demonstrated to possess many biofunctions, such as antioxidative stress, antiapoptosis, and anti-inflammation, against cognitive disorders, nonalcoholic fatty liver disease, atherosclerosis, and DN [8, 13–16]. Based on our previous study of the protective effect of PSPC on kidney injury by reducing excessive ROS-mediated NLRP3 activation in HFD-treated mice [8], the present study was aimed at investigating the protective action of PSPC on kidney injury by targeting VEGFR2 via ROS-mediated NLRP3 activation inflammatory signaling. Our main observation disclosed that PSPC alleviated the kidney injury by downregulating the VEGFR2 expression. We also observed the inhibition of ROS-mediated NLRP3 activation, as well as the decrease of NF-κB p65 nuclear translocation and its downstream inflammatory cascades.Although our results clearly demonstrated potential molecular mechanisms underlying the HFD-induced and PSPC-alleviated kidney injury in HFD-treated mice, there are some limitations. Studies in food chemistry showed that PSPC is a multicomponent mixture of anthocyanins [41]. We were not able to determine which anthocyanins protected against kidney injury induced by HFD and in what ways and with what molecular mechanisms. Indeed, the pharmacological functions of detailed anthocyanins of PSPC depressing kidney injury induced by HFD and the underlying molecular mechanisms are still obscure. Therefore, the separation of every anthocyanin in PSPC and the elucidation of the pharmacological mechanisms underlying the protective effects of every anthocyanin of PSPC on kidney injury induced by high-fat diet will be regarded as the focus of further research. Moreover, the relationships between VEGFR2/ROS/NRLP3 upregulation and injuries of renal intrinsic cells, such as endothelial cells, mesangial cells, podocytes, and renal tubular epithelial cells, remain unclear. Therefore, further experiments will explore these molecular mechanisms.In summary, our study provides compelling evidence that the kidney VEGFR2 knockdown alleviated kidney injury by inhibiting inflammation in HFD-treated mice. Furthermore, PSPC relieved renal damage at least partly via the VEGFR2/ROS/NRLP3 signaling axis, which suggested that VEGFR2 may be a potential target for PSPC preventing or treating kidney injury induced by high-fat diet (Figure6(m)).



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*Source: 5189819-2019-01-21.xml*