Nuciferine Alleviates Renal Injury by Inhibiting Inflammatory

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Nuciferine Alleviates Renal Injury by Inhibiting Inflammatory Responses in Fructose-Fed Rats Ming-Xing Wang, Xiao-Juan Zhao, Tian-Yu Chen, Yang-Liu Liu, Rui-Qing Jiao, Jian-Hua Zhang, Chun-Hua Ma, Jia-Hui Liu, Ying Pan,* and Ling-Dong Kong* State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210023, People’s Republic of China S Supporting Information *

ABSTRACT: Nuciferine is a major active component from the lotus leaf. This study examines the effects of nuciferine on fructose-induced renal injury and explores its possible mechanism. Rats consumed drinking water or 10% fructose for 12 weeks. Fructose-fed rats were orally treated with water or 7, 14, or 28 mg/kg of nuciferine for the last 6 weeks. HK-2 cells were exposed to 5 mM fructose alone or in combination with nuciferine (2.5−40 μM) for 24 h. Nuciferine significantly attenuated fructoseinduced hyperuricemia, dyslipidemia, and systemic inflammation in rats. More importantly, it alleviated renal pathological injury with proteinuria at 20 and 40 mg/kg (2.58 ± 0.97 and 2.48 ± 1.04 mg/mg·creatinine, P < 0.05) compared with fructose-vehicle group (4.10 ± 1.18 mg/mg·creatinine). Furthermore, nuciferine reduced TLR4, MyD88, PI3K, ILK, p-AKT, p-P65, and NLRP3 inflammasome protein levels (P < 0.05 for all) in the renal cortex of fructose-fed rats (14 and 28 mg/kg) and fructose-exposed HK-2 cells (5−40 μM), which is consistent with its reduction of inflammatory cytokines IL-1β, IL-6, TNF-α, and MCP-1 (P < 0.05 for all) in vivo and in vitro. These findings suggest that nuciferine alleviated fructose-induced inflammation by inhibiting TLR4/PI3K/NF-κB signaling and NLRP3 inflammasome activation in rat renal cortex and HK-2 cells, which may contribute to the improvement of renal injury. KEYWORDS: nuciferine, fructose-fed rats, fructose-exposed HK-2 cells, renal inflammation, renal injury

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INTRODUCTION Excess fructose consumption is considered as a potential cause of obesity, insulin resistance, dyslipidemia, and hyperuricemia,1 which may increase the risk for kidney injury.2 Indeed, toll-like receptor 4 (TLR4) signaling cascade contributes to renal dysfunction, inflammation, and injury.3 TLR4 deficiency decreases renal interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemotactic protein-1 (MCP-1) levels and alleviates kidney inflammation and podocytopathy in diabetic nephropathy of mice.4 TLR4 activation initiates inflammatory response by producing nuclear factor-κB (NFκB)-dependent inflammatory factors in which its adapter protein myeloid differentiation factor 88 (MyD88) is involved in renal ischemia-reperfusion injury.5 Phosphatidylinositol 3-kinase (PI3K) is necessary for NF-κB activation in lipopolysaccharideinduced TLR4 signaling in human microvascular endothelial cells.6 Integrin-linked kinase (ILK) as a candidate downstream effector in patients with proteinuria7 and interacts with its downstream regulator of protein kinase B (AKT) in a PI3Kdependent manner.8 This interaction increases the phosphorylation of IκB kinase α/β (IKKα/β), an inhibitor of kappa Bα (IκBα) and subsequent NF-κB.9 Additionally, NOD-like receptor family, pyrindomain containing 3 (NLRP3) inflammasome, composed of NLRP3, apoptosis-associated speck-like protein (ASC), and Caspase-1, drives the maturation of interleukin-1beta (IL-1β).10 IL-1β secretion is stimulated by polyhydroxylated metallofullerenols in macrophages through TLRs/MyD88/NF-κB pathway and NLRP3 inflammasome activation.11 Therefore, the suppression of TLR4/PI3K/NF-κB © 2016 American Chemical Society

signaling and NLRP3 inflammasome activation may alleviate fructose-induced renal inflammation and injury. Lotus (Nelumbo nucifera Gaertn.) has been used not only as a vegetable and food garnish but also as a traditional medicine in Asia. Lotus leaf products are widely advertised as weight control dietary supplement.12,13 Its active constituent nuciferine (Figure 1), an aromatic ring-containing alkaloid, is reported to reduce serum IL-6 and TNF-α levels and prevent hepatic steatosis in high fat diet-fed hamsters.14 Nuciferine also decreases aortic IL1β and MCP-1 levels in a mouse model of atherosclerosis.15 Our previous study showed that nuciferine alleviated renal inflam-

Figure 1. Chemical structure of nuciferine. Received: Revised: Accepted: Published: 7899

July 7, 2016 October 8, 2016 October 10, 2016 October 10, 2016 DOI: 10.1021/acs.jafc.6b03031 J. Agric. Food Chem. 2016, 64, 7899−7910

Article

Journal of Agricultural and Food Chemistry mation in hyperuricemic mice.16 However, the effects of nuciferine on fructose-induced renal inflammation and injury, as well as its possible mechanism, are still unknown. Therefore, this study was designed to investigate the effects of nuciferine on fructose-induced renal inflammation and injury in rats. The possible mechanism by which nuciferine might produce its nephroprotective effect was also investigated by detecting TLR4/PI3K/NF-κB signaling and NLRP3 inflammasome activation in the renal cortex of fructose-fed rats and fructoseexposed human renal proximal tubule epithelial cell line HK-2 cells.

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He Zhi-Gan containing lotus leaf with great clinical curative effects on insulin resistance and metabolic disorders in patients.18,19 The raw lotus leaf contains approximately 0.1−1.1% nuciferine,20,21 thus the dosage of nuciferine was approximately 19−190 mg/day for an adult human, which was approximately 2.0−19.6 mg/kg/day for rats by body surface area calculation. Our preliminary study showed the restoration of potassium oxonate-induced hyperuricemia kidney inflammation in mice by nuciferine at 10−40 mg/kg/day.16 The similar safe dose range (equivalent) used in animal studies was also reported by others.22,23 Thus, the doses of 7, 14, and 28 mg/kg/day in rats were chosen in the present study. Cell Culture. HK-2 cells obtained from Cell Bank of Chinese Academy of Science (Shanghai, P. R. China) were cultured in RPMI 1640 medium containing 10% FBS in a humidified atmosphere of 5% CO2 at 37 °C. Exponentially growing HK-2 cells were seeded at 1.0 × 106 cells/well in 6-well plates or at 1.0 × 105 cells/well in 96-well plates. After incubating the cells in serum-free media overnight, these cells were cultured in RPMI 1640 medium (containing 10% fetal bovine serum) supplemented with or without 5 mM fructose in the presence or absence of nuciferine. Although the peak concentration (Cmax) of blood nuciferine in rats after oral treatment was only about several micromoles (μM) with half-life time (T1/2) about several hours,24 the results from the determination in rats and mice showed the intestinal tract absorption and rapid tissue distribution of nuciferine,22,25 and the concentration in the kidney (about 80 nmol/g tissue) was the highest.25 Furthermore, nuciferine was primarily considered to be metabolized by hepatic cytochrome P450 enzymes.26 Accordingly, nuciferine may be maintained at a relative concentration in action time in renal cell microenvironment. In addition, based on the results from the preliminary experiments by us and others16,27,28 and optimal selection by MTT assay (Figure S2), we applied 2.5, 5, 10, 20, and 40 μM nuciferine to treat HK-2 cells for 24 h. To further understand the pharmacologic mechanism of nuciferine, especially focusing on the potent anti-inflammatory effect, the “nuciferine-only” control groups (20 and 40 μM) were carried out in cell experiments. For inflammatory cytokines and inflammation-related gene and protein assays, the conditioned cell culture supernatants and lysates were collected, respectively. For uric acid assay, HK-2 cells in 96-well plates were exposed with 5 mM fructose or 4 mg/dL uric acid with or without 10 μM nuciferine or 500 μM probenecid for 24 h. Cell culture supernatants and cell lysates were collected to determine the extracellular and intracellular uric acid contents, respectively. Biochemical Analysis. Serum concentrations of uric acid, creatinine, BUN, TG, HDL-C, and LDL-C, as well as urinary concentrations of uric acid, creatinine, urea nitrogen, albumin, and fructose in rats, were measured according to instructions of commercially available kits mentioned above. FEUA was detected as previously described.26 Serum endotoxin levels in rats were measured by the limulus amebocyte lysate assay kit and calculated by a standard curve range of 10−100 EU/L. IL-1β, IL-6, and TNF-α concentrations in serum and renal cortex of rats, as well as culture supernatants of HK-2 cells, were determined according to the manufacturer’s instructions of ELISA kits by microplate reader (TECAN, Safire 2, Switzerland). Histology. Renal cortex sections were fixed in 10% neutral-buffered formalin for 1 day and then put into 70% ethanol and embedded in paraffin. Sections (4 μm) were stained with H&E and examined under a light microscope. Cortical tissues were cut into 1 mm3 sections, fixed with 2.5% glutaraldehyde, rinsed in 0.1 M cacodylate buffer, postfixed for 2 h with 1% OSO4, dehydrated in a graded series of ethanol, and embedded in Araldite, using propylene oxide as an intermedium. Ultrathin sections were contrasted with 1% uranyl acetate and a solution of saturated lead citrate double and examined with a JEM 1010 TEM (JEOL, Tokyo, Japan) at 80 kV. Western Blot Analysis. Proteins were isolated from renal cortex tissues of rats and HK-2 cells, quantified, and subjected to Western blot as previously described.25 The rabbit polyclonal anti-MyD88, anti-PI3K, anti-ILK, anti-AKT, anti-p-AKT, anti-IKKα/β, anti-p-IKKα/β, antiIκBα, anti-NF-κB P65, anti-p-NF-κB p-P65, anti-NLRP3, anti-ASC, anti-MCP-1, and mouse polyclonal anti-p-IκBα antibodies were

MATERIALS AND METHODS

Chemicals. Nuciferine was obtained from Nanjing Spring & Autumn Biological Engineering (Nanjing, P. R. China) for animal experiment (>90.0%) or National Institutes for Food and Drug Control (Beijing, P. R. China) for cell experiment (>99.9%). Probenecid (>98%) was purchased from MedChem Express (Monmouth Junction, NJ, U.S.A.). Fructose was obtained from Jiakangyuan Technology (Beijing, P. R. China) for animal experiment and from Sigma-Aldrich (St Louis, MO, U.S.A.) for cell experiment. Trizol reagent was obtained from Invitrogen (Carlsbad, CA, U.S.A.). Assay kits of uric acid, creatinine, blood urea nitrogen (BUN), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), and hematoxylin-eosin (H&E) reagents were obtained from Jiancheng Biotech (Nanjing, P. R. China). Assay kit of intracellular uric acid was purchased from Invitrogen (Carlsbad, CA, U.S.A.). ELISA kits for IL-1β, IL-6, and TNF-α were purchased from ExCell Bio. (Shanghai, P. R. China). Urinary albumin ELISA kit was purchased from Exocell Inc. (Philadelphia, PA, U.S.A.). Fructose assay kit (EFRU-100) was purchased from Bioassay Systems (Hayward, CA, U.S.A.). Endotoxin assay kit (E-TOXATE) was purchased from Sigma-Aldrich (St Louis, MO, U.S.A.). Animals and Experimental Design. All experimental animal protocols were approved by the Institutional Animal Care Committee at the Nanjing University and the China Council on Animal Care at Nanjing University. Male Sprague-Dawley rats (200 ± 20 g) were purchased from the experimental Animal Center of Nanjing Medical University (SCXK2013-0005). The animals were housed individually and maintained on a 12 h light/dark cycle at a temperature of 22−24 °C with ad libitum access to drinking water and a standard diet (China Experimental Animal Food Standard, GB 14924.3-2001). The diet contained moisture (≤10%), crude protein (≥18%), crude fat (≥4%), crude fiber (≥5%), ash (≤8%), calcium (1.0−1.8%), and phosphorus (0.6−1.2%). After a 1 wk acclimatization period, 50 rats were randomly divided into two groups: control group (n = 10) drank normally (available ad libitum), and fructose-fed group (n = 40) were given 10% fructose in drinking water for 12 wks. After 6 wks, renal function was evaluated (data in Table S1), and animals with renal dysfunction were defined to model success. Then, these fructose-fed rats with renal dysfunction were further divided into four subgroups (n = 10/group), receiving water (fructose-vehicle group), or nuciferine 7, 14, and 28 mg/kg daily for the next 6 wks, respectively. Nuciferine was suspended in water and disrupted by sonication and then immediately administered to rats intragastrically. At end of the experiment, 24 h urine samples were collected as previously described.16 Then, rats were food deprived for 12 h and euthanized by sodium pentobarbital anesthesia. Blood samples were collected from celiac aorta just prior to sacrifice and centrifuged at 3000×g for 10 min at 4 °C to get serum samples stored at −80 °C until assays. Renal cortex tissues in rats were dissected quickly on ice and immediately fixed for H&E staining and transmission electron microscope (TEM) analysis or frozen and stored at −80 °C for gene and protein assays. The animal experimental setup is outlined schematically in Figure S1. The clinic dosages of raw lotus leaf for adults are 3−10 g/day according to the State Pharmacopoeia of People’s Republic of China,17 and 30 g/day according to He-Ye Jiang-Zhi decoction as well as Shen7900

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Journal of Agricultural and Food Chemistry Table 1. Effects of Nuciferine on Serum and Urine Parameters in Fructose-Fed Ratsa Parameters Serum uric acid, mg/dL creatinine, mg/dL BUN, mg/dL triglyceride, mg/dL LDL cholesterol, mg/dL HDL cholesterol, mg/dL interleukin-1β,ng/L interleukin-6, ng/L tumor necrosis factor-α, ng/L endotoxin, EU/L Urine albumin, mg/mg·creatinine fructose, nmol/mg·creatinine FEUA, % a

fructose-fed rats control

vehicle

nuciferine 7 mg/kg

nuciferine 14 mg/kg

nuciferine 28 mg/kg

1.97 ± 0.57 b 1.08 ± 0.12 b 11.4 ± 2.34 b 100 ± 8.09 b 17.9 ± 4.89 c 37.7 ± 11.9 ab 8.67 ± 2.89 b 26.5 ± 4.98 b 27.4 ± 4.04 b 83.0 ± 6.51 b

2.60 ± 0.38 a 1.39 ± 0.38 a 14.0 ± 1.63 a 132 ± 44.1 a 43.0 ± 10.2 a 27.2 ± 8.16 b 14.2 ± 3.03 a 32.9 ± 3.22 a 36.0 ± 4.80 a 98.1 ± 10.3 a

2.12 ± 0.58 ab 1.11 ± 0.13 b 10.0 ± 0.97 b 114 ± 24.5 ab 35.7 ± 4.97 ab 33.4 ± 10.7 ab 10.8 ± 3.39 ab 30.9 ± 4.44 ab 34.5 ± 8.16 ab 94.3 ± 7.91 a

1.87 ± 0.14 b 1.05 ± 0.14 b 9.63 ± 2.18 b 98.6 ± 8.62 b 32.5 ± 6.14 b 38.0 ± 2.12 ab 9.62 ± 4.03 b 28.3 ± 2.54 ab 32.3 ± 7.92 ab 91.8 ± 8.26 ab

1.87 ± 0.29 b 0.93 ± 0.34 b 10.2 ± 3.06 b 93.7 ± 8.94 b 32.7 ± 3.54 b 41.7 ± 6.17 a 8.89 ± 4.02 b 27.9 ± 4.36 b 27.1 ± 6.16 b 88.8 ± 7.01 ab

2.19 ± 1.09 c 7.72 ± 2.79 c 21.6 ± 3.68 a

4.10 ± 1.18 a 15.6 ± 6.24 b 15.8 ± 2.21 b

3.61 ± 1.19 ab 17.1 ± 4.58 b 20.1 ± 4.28 ab

2.58 ± 0.97 bc 16.6 ± 9.92 ab 22.0 ± 3.07 a

2.48 ± 1.04 bc 25.7 ± 9.35 a 23.8 ± 4.41 a

Values are means ± SD, n = 6−8. Values in the same row with different superscript letters differ significantly, P < 0.05 (Duncan’s test).

purchased from Cell Signaling Technology, whereas anti-GLUT5 (glucose transporter 5) from Abcam, antiketohexokinase (KHK) from Sigma-Aldrich, anti-TLR4, anti-Caspase-1, and anti-GAPDH from Santa Cruz were also purchased. Protein loading was assessed by immunoblotting with the use of rabbit anti-GAPDH. All target protein signals were visualized by the enhanced chemiluminescence and quantified via densitometry using ImageJ (version 1.42q). RNA Isolation and qPCR. Total RNA in cells was isolated using Trizol reagent according to the manufacturer’s instructions. RNA concentration was detected with absorbance measurements at 260 nm. RNA purity and integrity were confirmed by formaldehyde-agarose gel electrophoresis followed by visualization with ethidium bromide. The cDNA was synthesized by the HiScript II Q RT SuperMix for qPCR kit (Vazyme, P. R. China), according to the manufacturer’s protocol. qPCR was performed using iTaq Universal SYBR Green Super Mix (Bio-Rad, U.S.A.) on a CFX96 real-time PCR detection system (Bio-Rad). Primers (Table S2) were synthesized by GenePharma (Shanghai, P. R. China). The amount of target genes was normalized with GAPDH mRNA to determine the relative expression ratio. Statistical Analysis. All data were expressed as means ± SD. Oneway analysis of variance (ANOVA) followed by Duncan’s multiple-rage test (SPSS 19.0) was performed to analyze the differences among groups. In addition, unpaired Student’s t-test was performed to analyze the differences between the data of two groups in Table S1. Two-way ANOVA followed by LSD test was performed in cell experiments to confirm the specific effect and molecular mechanism underlying the anti-inflammatory activity of nuciferine. P < 0.05 was considered statistically significant.

Nuciferine Alleviates Renal Dysfunction, Inflammation, and Injury in Fructose-Fed Rats. Nuciferine (14 and 28 mg/kg) significantly decreased serum creatinine and BUN levels but increased FEUA (P < 0.05 for all) in fructose-fed rats compared with the fructose-vehicle group (Table 1). Of note, tubular edema and inflammatory cell infiltration of renal interstitium were observed (Figure 2A) with podocyte foot process fusion and effacement (Figure 2B) in the fructose-vehicle group compared with the control group. Consistently, urinary albumin levels (P < 0.05) were increased, exhibiting podocyte injury in this animal model. Nuciferine (14 and 28 mg/kg) robustly alleviated kidney inflammatory damage, severe podocyte foot process fusion and effacement (Figure 2A,B), and proteinuria (P < 0.05) (Table 1) in fructose-fed rats. Nuciferine (28 mg/kg) significantly decreased renal cortex IL1β, IL-6, TNF-α/ and MCP-1 levels (P < 0.05 for all) in fructosefed rats (Figure 2C−F). In addition, serum endotoxin concentrations (P < 0.05) in the fructose-vehicle group were remarkably increased compared with the control group (Table 1). Nuciferine at 28 mg/kg had a tendency of reduction in serum endotoxin concentrations (P = 0.07) in fructose-fed rats (Table 1). Nuciferine Suppresses TLR4/PI3K/NF-κB Signaling and NLRP3 Inflammasome Activation in Renal Cortex of Fructose-Fed Rats. Compared with the control group, renal cortex TLR4, MyD88, PI3K, ILK, and p-AKT protein levels (P < 0.05 for all) were significantly up-regulated in the fructosevehicle group (Figure 3). Meanwhile, renal cortex IKKα, IKKβ, IκBα, and P65 phosphorylation levels (P < 0.05 for all) were also increased in this animal model (Figure 4). Renal cortex NLRP3 inflammasome activation characterized by the elevation of NLRP3, ASC, and Caspase-1 protein levels (Figure 5) were observed in fructose-vehicle group compared with control group. More importantly, 14 and 28 mg/kg nuciferine significantly down-regulated renal cortex protein levels of TLR4, MyD88, PI3K, ILK, and p-AKT (P < 0.05 for all) (Figure 3) in fructosefed rats compared with the fructose-vehicle group. Simultaneously, 14 and 28 mg/kg nuciferine reduced IKKα, IKKβ, IκBα, and P65 phosphorylation levels (Figure 4) and suppressed NLRP3 inflammasome activation (P < 0.05 for all) (Figure 5) in renal cortex of fructose-fed rats.

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RESULTS Nuciferine Relieves Hyperuricemia, Dyslipidemia, and Systemic Inflammation in Fructose-Fed Rats. Fructose feeding significantly increased serum uric acid, TG, LDL-C, IL1β, IL-6, and TNF-α levels (P < 0.05 for all), and decreased serum HDL-C levels (P < 0.05) compared with the control group (Table 1). Nuciferine (14 and 28 mg/kg) significantly attenuated fructose-induced change of serum uric acid and lipid concentrations (P < 0.05 for all) in rats compared with the fructose-vehicle group (Table 1). Only 28 mg/kg nuciferine decreased serum IL-1β, IL-6, and TNF-α levels (P < 0.05 for all) in fructose-fed rats (Table 1). However, there were no significant differences in body weight (P = 0.68) and food intake (P = 0.89) between these animal groups (Table S3). 7901

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Figure 2. Effects of nuciferine on pathological injury and inflammatory cytokine levels in renal cortex of fructose-fed rats. (A) H&E staining (magnification, ×200), Scale bar = 50 μm. (B) Podocyte representative transmission electron microscope photomicrographs (magnification, ×25 000), Scale bar =1 μm. Renal cortex levels of IL-1β (C), IL-6 (D), and TNF-α (E) were determined by ELISA kits, respectively. (F) Representative Western blot bands and relative MCP-1 protein levels. Values are means ± SD, n = 6−8. Labeled means without a common letter are significantly different, P < 0.05 (Duncan’s test). 7902

DOI: 10.1021/acs.jafc.6b03031 J. Agric. Food Chem. 2016, 64, 7899−7910

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Figure 3. Effects of nuciferine on protein levels of TLR4, MyD88, PI3K, ILK, and p-AKT in the renal cortex of fructose-fed rats. (A) Representative Western blot bands of TLR4, MyD88, PI3K, ILK, p-AKT, and AKT in the renal cortex of rats. Protein relative levels of TLR4 (B), MyD88 (C), PI3K (D), and ILK (E) were normalized to GAPDH. (F) Graph showed changes in the ratio of p-AKT/AKT. Values are means ± SD, n = 6−8. Labeled means without a common letter are significantly different, P < 0.05 (Duncan’s test).

Nuciferine Inhibits TLR4/PI3K/NF-κB Signaling and NLRP3 Inflammasome Activation in Fructose-Exposed HK-2 Cells. To further confirm these in vivo results, HK-2 cells were incubated with 5 mM fructose alone or in combination with nuciferine (2.5, 5, 10, 20, and 40 μM) for 24 h. Culture supernatant IL-1β, IL-6, and TNF-α levels, as well as cell lysate MCP-1 levels (P < 0.05 for all) were increased in fructoseexposed HK-2 cells compared with the control group (Figure 6). Consistently, mRNA and protein levels of cell lysate TLR4 were up-regulated in this cell model. MyD88, PI3K, ILK, and p-AKT protein levels were also increased in fructose-exposed HK-2 cells (Figure 7) (P < 0.05 for all). Furthermore, p-IKKα, p-IKKβ, and p-P65 (Figure 8), as well as NLRP3, ASC, and Caspase-1 (P < 0.05 for all) (Figure 9) were also increased in lysates of fructoseexposed HK-2 cells compared with the control group. Nuciferine dose-dependently inhibited intracellular TLR4/PI3K/NF-κB signaling and NLRP3 inflammasome activation, which is consistent with the reduction of IL-1β, IL-6, TNF-α, and MCP-1 levels (P < 0.05 for all) in fructose-exposed HK-2 cells (Figures 7−9). In addition, the results from two-way ANOVA also supported the inhibitory effect of nuciferine on P65 protein phosphorylation (F(2, 30) = 25.59; P < 0.001) and NLRP3 protein levels (F(2, 30) = 16.06; P < 0.001) in HK-2 cells exposed to fructose or not (Figure 10), although this inhibition had more potential in fructose-exposed HK-2 cells (nuciferine ×

fructose interaction p-P65 F(2, 30) = 7.435; P < 0.01 and NLRP3 F(2, 30) = 4.056; P < 0.05). Nuciferine Reduces Fructose Load in Renal Cortex of Fructose-Fed Rats But Fails To Change Intracellular Uric Acid Levels in Fructose-Exposed HK-2 Cells. GLUT5 is a specific and main transporter for renal fructose reabsorption in rat renal tubular cell,29 and its expression is regulated by fructose.30 KHK mediates the first step in fructose metabolism by rapidly phosphorylating fructose without rate-limiting control, commonly causing intracellular ATP depletion and uric acid production.31 As reported previously, the KHK-dependent metabolism and uric acid of fructose could induce proinflammatory mediators in HK-2 cells.16,32 Therefore, the effect of nuciferine on fructose load, transportion, and metabolism was investigated in renal cells. High levels of urine fructose were detected in fructose-vehicle rats compared with the control group. Nuciferine (28 mg/kg) enhanced urinary fructose excretion in fructose-fed rats (Table 1). Furthermore, renal cortex GLUT5 protein levels were increased in fructose-fed rats, which were significantly attenuated by 28 mg/kg nuciferine (Figure S3A,B). Consistently, mRNA levels of cell lysate GLUT5 were up-regulated in 5 mM fructoseexposed HK-2 cells compared with the control group (Figure S3D). Nuciferine (5, 10, and 20 μM) remarkably restored fructose-induced change of GLUT5 mRNA levels in HK-2 cells (Figure S3D). These data indicated that the enhancement of 7903

DOI: 10.1021/acs.jafc.6b03031 J. Agric. Food Chem. 2016, 64, 7899−7910

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Figure 4. Effects of nuciferine on protein phosphorylation levels of IKKα, IKKβ, IκBα and P65 in renal cortex of fructose-fed rats. (A) Representative Western blot bands of p-IKKα, IKKα, p-IKKβ and IKKβ. (B, C) Graphs showed changes in the ratios of p-IKKα/IKKα and p-IKKβ/IKKβ. (D) Representative Western blot bands of p-IκBα, IκBα, p-P65 and P65. (E, F) Graphs showed changes in the ratios of p- IκBα/IκBα and p-P65/P65. Values are means ± SD, n = 6−8. Labeled means without a common letter are significantly different, P < 0.05 (Duncan’s test).

Figure 5. Effects of nuciferine on protein levels of NLRP3, ASC and Caspase-1 in renal cortex of fructose-fed rats. (A) Representative Western blot bands of NLRP3, ASC, and Caspase-1 in the renal cortex of rats. Protein relative levels of NLRP3 (B), ASC (C), and Caspase-1 (D) were normalized to GAPDH. Values are means ± SD, n = 6−8. Labeled means without a common letter are significantly different, P < 0.05 (Duncan’s test).

renal fructose excretion by nuciferine may result from its suppression of GLUT5. On the other hand, renal cortex KHK protein levels were also up-regulated in fructose-fed rats, which were remarkably restored by 28 mg/kg nuciferine (Figure S3C). Both isoforms of KHK mRNA were detected in lysates of HK-2 cells, and KHK-C

isoform was more predominant than KHK-A. In line with in vivo results, cell lysate KHK mRNA levels were up-regulated in 5 mM fructose-exposed HK-2 cells compared with the control group (Figure S3E,F). Nuciferine (5, 10, and 20 μM) remarkably downregulated KHK mRNA levels in this cell model (Figure S3E,F). 7904

DOI: 10.1021/acs.jafc.6b03031 J. Agric. Food Chem. 2016, 64, 7899−7910

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Figure 6. Effects of nuciferine on IL-1β, IL-6, TNF-α, and MCP-1 levels in fructose-exposed HK-2 cells. HK-2 cells were exposed to 5 mM fructose alone or in combination with 2.5, 5, 10, 20, and 40 μM nuciferine for 24 h. Culture supernatant levels of IL-1β (A), IL-6 (B), and TNF-α (C) were measured by ELISA kits, respectively. (D) Representative Western blot bands and relative cell lysate MCP-1 protein levels. Values are means ± SD, n = 3. Labeled means without a common letter are significantly different, P < 0.05 (Duncan’s test).

Proteinuria in patients with kidney injury is common in a positive correlation with urinary MCP-1.35 MCP-1 promotes monocyte and macrophage activation to increase IL-6 and TNFα expression.36 MCP-1 hyperexpression in tubular cells is observed to be correlated mainly with the magnitude of proteinuria and interstitial cell infiltration in patients with type 2 diabetes and overt nephropathy.37 MCP-1 knockout mice show the improvement of proteinuria.38 In fact, fructose induces MCP1 production in human kidney proximal tubular cells32 and increases renal IL-6 and TNF-α levels in rats with kidney damage.39 Nuciferine is reported to decrease IL-6 and TNF-α levels in oleic acid-exposed HepG2 cells and high-fat diet-fed hamsters14,28 or aortic IL-1β and MCP-1 levels in the mouse model of atherosclerosis.15 In the present study, nuciferine also decreased IL-1β, IL-6, TNF-α, and MCP-1 levels in the renal cortex of fructose-fed rats and fructose-exposed HK-2 cells. TLR4 signaling is suggested to be a mediator of kidney injury.40 TLR4 activation causes PI3K-MyD88 complex formation.41 ILK and AKT interaction modulates canonical NF-κB pathway in angiotensin II-induced renal inflammation.16 TLR4 inhibitor effectively blocks soluble the uric acid-induced increase of TLR4, NLRP3, Caspase-1, and IL-1β expression in human primary renal proximal tubule epithelial cells.42 In our previous study, nuciferine suppressed renal TLR4 signaling in the mouse model of hyperuricemia.16 In the present study, nuciferine was found to down-regulate protein levels of TLR4, as well as MyD88, PI3K, ILK, p-AKT, and p-P65 in the renal cortex of fructose-fed rats and fructose-exposed HK-2 cells. Furthermore, the inhibition of TLR4 expression at transcriptional levels was observed in fructose-exposed HK-2 cells treated with nuciferine, indicating the regulatory effect of nuciferine on TLR4 inflammatory signaling. Of note, TLR4-mediated inflammation signaling can positively activate its transcription. Further study is needed to investigate whether nuciferine has a direct transcriptional inhibition of TLR4. Moreover, nuciferine significantly

These results suggested the possible protection by nuciferine through controlling fructose metabolism in renal cells. In the present study, decreased FEUA was observed in fructosefed rats with hyperurcemia (Table 1), which were attenuated by 14 and 28 mg/kg nuciferine (P < 0.05 for all), possibly showing the uricosuric effect of nuciferine in this animal model. However, upon exposure to 5 mM fructose, intracellular uric acid contents of HK-2 cells were significantly increased compared with the control group (Figure S4). However, 10 μM nuciferine failed to affect intracellular uric acid levels in 5 mM fructose-exposed HK2 cells (Figure S4), indicating that renal cell protection of nuciferine against fructose induction may have uric acidindependent mechanism.

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DISCUSSION Fructose has been a part of the human diet. The estimated mean consumption of fructose in the U.S. is ∼54.7 g/day, while higher (∼72.8 g/day) in adolescents.33 Despite the limited clinical evidence,34 there are more in vivo and in vitro experimental data supporting that excess fructose consumption causes renal dysfunction, inflammation, and injury.1 Lotus leaf has a long history from its food and medicinal uses. Its active constituent nuciferine possesses anti-inflammation, antihyperuricemia, antidyslipidemia, and antihyperinsulinemia.14−16,27 Nuciferine is absorbed through the intestinal tract and distributed into tissues rapidly with high concentrations in the kidney.25 It also shows its renal cell protection through anti-inflammation in vivo and in vitro.16,28 In the present study, nuciferine relieved fructoseevoked hyperuricemia, hyperlipidemia, and inflammation in rats, which is consistent with its alleviation of kidney pathology, podocyte injury, and proteinuria. These observations demonstrated that nuciferine had the nephroprotective effect in fructose-fed rats. Thus, the supplementation with nuciferine may be useful for the alleviation of fructose-driven podocyte injury and proteinuria. 7905

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Figure 7. Effects of nuciferine on TLR4 mRNA and protein levels, MyD88, PI3K, ILK, and p-AKT protein levels in fructose-exposed HK-2 cells. HK-2 cells were exposed to 5 mM fructose alone or in combination with 2.5, 5, 10, 20, and 40 μM nuciferine for 24 h. (A) mRNA levels of cell lysate TLR4. (B) Representative Western blot bands of cell lysate TLR4, MyD88, PI3K, ILK, p-AKT, and AKT. Protein relative levels of TLR4 (C), MyD88 (D), PI3K (E), and ILK (F) were normalized to GAPDH. (G) Graph shows changes in the ratio of p-AKT/AKT. Values are means ± SD, n = 3. Labeled means without a common letter are significantly different, P < 0.05 (Duncan’s test).

fed rats characterized by the elevation of FEUA. However, nuciferine did not change intracellular uric acid levels in fructoseexposed HK-2 cells. The similar results were also detected in fructose-exposed HK-2 cells treated with 500 μM probenecid, a clinical uricosuric agent (data not shown). Furthermore, intracellular uric acid contents were significantly increased in HK-2 cells after extracellular supplement of 4 mg/mL uric acid in culture media, simulating hyperuricemia state (data not shown). Nuciferine and probenecid significantly reduced intracellular uric acid contents in this condition (data not shown). These different results from the in vivo and in vitro experiments may result from the lack of specific membranous structures in culture HK-2 cells compared with proximal tubule cells in vivo. In fact, multiple transporters are selectively distributed in apical or basal membrane of proximal tubule cells to cooperatively regulate uric acid secretion and reabsorption.44 The different way of nuciferine to affect uric acid neo-produced by fructose

suppressed renal cortex NLRP3 inflammasome activation in fructose-fed rats. The similar effects of nuciferine were also observed in fructose-exposed HK-2 cells. Therefore, the blockade of renal cortex TLR4/PI3K/NF-κB signaling and NLRP3 inflammasome activation by nuciferine may alleviate fructose-induced renal inflammation and injury in rats. Renal fructose reabsorption is regulated by GLUT5 in rat renal tubular cell.29 As a key metabolite of KHK-dependent fructose metabolism, uric acid is hypothesized to have a causal role in fructose-induced disease.43 In the present study, fructose caused high urine fructose levels in rats with up-regulation of GLUT5 and KHK expression levels in fructose-fed rats and fructoseexposed HK-2 cells. Nuciferine reduced fructose-induced increase of GLUT5 and KHK expression levels in renal cortex of rats or HK-2 cells, resulting in promotion of renal fructose excretion and control of renal cell fructose metabolism. It is noted that nuciferine exhibited the uricosuric effect in fructose7906

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Figure 8. Effects of nuciferine on protein phosphorylation levels of IKKα, IKKβ, and P65 in fructose-exposed HK-2 cells. HK-2 cells were exposed to 5 mM fructose alone or in combination with 2.5, 5, 10, 20, and 40 μM nuciferine for 24 h. (A) Representative Western blot bands of cell lysate p-IKKα, IKKα, p-IKKβ, IKKβ, p-P65, and P65. (B−D) Graphs show changes in the ratios of p-IKKα/IKKα, p-IKKβ/IKKβ, and p-P65/P65. Values are means ± SD, n = 3. Labeled means without a common letter are significantly different, P < 0.05 (Duncan’s test).

Figure 9. Effects of nuciferine on NLRP3, ASC, and Caspase-1 protein levels in fructose-exposed HK-2 cells. HK-2 cells were exposed to 5 mM fructose alone or in combination with 2.5, 5, 10, 20, and 40 μM nuciferine for 24 h. (A) Representative Western blot bands of cell lysate NLRP3, ASC, and Caspase-1. Protein relative levels of NLRP3 (B), ASC (C), and Caspase-1 (D) were normalized to GAPDH. Values are means ± SD, n = 3. Labeled means without a common letter are significantly different, P < 0.05 (Duncan’s test).

present study observed high serum levels of endotoxin in fructose-fed rats. The data indicated that fructose may induce the permeation of bacterial endotoxin to the kidney, possibly activating inflammatory signaling to injure kidney. Nuciferine tended to reduce serum endotoxin levels in fructose-fed rats, which may be due to its passive absorption in the small intestine.47 In fact, the direct inhibitory effects of nuciferine on TLR4/PI3K/NF-κB signaling, NLRP3 inflammasome activation, and pro-inflammatory cytokine production and/or secretion were observed in fructose-exposed HK-2 cells. Therefore, the effects of nuciferine might be mediated partly

metabolism or supplemented extracellularly in HK-2 cells could not evaluate its uricosuric ability of nucifeine in vivo but could suggest its possible protection against ex-renal uric acid. Thus, the anti-inflammatory effect of nuciferine without lowing intracellular uric acid suggested that nuciferine may have uric acid-independent mechanism involving its renal cell protection against fructose. High-fructose diet can induce intestinal bacterial overgrowth and increase intestinal permeability, producing a marked increase in plasma endotoxin levels.45 Actually, endotoxin seems to activate TLR4 signaling and NLRP3 inflammasome.46 The 7907

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Figure 10. Effect of nuciferine on p-P65 and NLRP3 protein levels in HK-2 cells. HK-2 cells were exposed to 5 mM fructose or not and in combination with 20 and 40 μM nuciferine or not for 24 h. (A) Representative Western blot bands of cell lysates p-P65 and NLRP3. The ratio of p-P65/P65 (B) and protein relative levels of NLRP3 (C) were normalized to GAPDH. Values are means ± SD, n = 6. #P < 0.05, ###P < 0.001 as compared with the vehicle control group; ***P < 0.001 as compared with the fructose vehicle group, respectively (two-way ANOVA followed by LSD test).

Funding

by reducing endotoxins to suppress inflammatory signaling in fructose-induced renal inflammation and injury in rats. Lotus leaf is widely used in recipes and health food and is officially listed in the Chinese Pharmacopoeia.17 Currently, a variety of health products containing lotus leaf are available. Lotus leaf dietary weight loss supplements are becoming increasingly popular. Considering the remarkable biological activity and safety, nuciferine is a promising drug candidate and food additive. Further studies are needed to understand its precise mechanisms and to characterize the exact role of nuciferine in the prevention and treatment of renal injury associated with high-fructose induction in female and male subjects.

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This work was supported by the National Basic Research Program of China 973 Program No.2012CB517600 (No. 2012CB517602), National Natural Science Foundation of China (NSFC No. 81373788, J1103512), and Changjiang Scholars and Innovative Research Team in University (IRT_14R271020), P. R. China. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AKT, protein kinase B; ASC, apoptosis-associated speck-like protein; BUN, blood urea nitrogen; FEUA, fraction excretion of uric acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT5, glucose transporter 5; H&E, hematoxylin-eosin; HDLC, high density lipoprotein cholesterol; IL-1β, interleukin-1beta; IL-6, interleukin-6; ILK, integrin-linked kinase; KHK, ketohexokinase; LDL-C, low density lipoprotein cholesterol; MCP-1, monocyte chemotactic protein-1; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor kappa B; NLRP3, NOD-like receptor family, pyrindomain containing 3; PI3K, phosphatidylinositol 3-kinase; TEM, transmission electron micrograph; TG, triglyceride; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor-α

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b03031. Table S1, body weight, kidney function parameters in rats fed with 10% fructose for 6 wk; Table S2, sequence of primers used for qPCR analysis; Table S3, effects of nuciferine on body weight and 24 h food intake in fructose-fed rats. Figure S1, schematic outline of the animal experiments; Figure S2, effects of nuciferine on cell viability in HK-2 cells; Figure S3, effects of nuciferine on protein and mRNA levels of GLUT5 and KHK in renal cortex of fructose-fed rats and fructose-exposed HK-2 cells; Figure S4, effects of nuciferine on intracellular uric acid contents in fructose-exposed HK-2 cells (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Telephone: +86-25-89681373. Fax: +86-25-83594691. *E-mail: [email protected]. Telephone: +86-25-83594691. Fax: +86-25-83594691. 7908

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