Tea polypeptide ameliorates diabetic nephropathy through RAGE and

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Bioactive Constituents, Metabolites, and Functions

Tea polypeptide ameliorates diabetic nephropathy through RAGE and NF-#B signaling pathway in type 2 diabetes mice Xuming Deng, Lingli Sun, Xingfei Lai, Limin Xiang, Qiuhua Li, Wenji Zhang, lingzhi zhang, and shili sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04819 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 25, 2018

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Journal of Agricultural and Food Chemistry

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Tea polypeptide ameliorates diabetic nephropathy through

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RAGE and NF-κB signaling pathway in type 2 diabetes

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mice

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Xuming Deng1, 2#, Lingli Sun1,#, Xingfei Lai1, Limin Xiang1, Qiuhua Li1, Wenji Zhang1, ,

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Lingzhi Zhang2,*, and Shili Sun1,*

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1 Tea

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Key Laboratory of Tea Plant Resources Innovation & Utilization, Dafeng Road NO.6, Tianhe

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District, Guangzhou 510640, China

Research Institute, Guangdong Academy of Agricultural Sciences/ Guangdong Provincial

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2

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Guangzhou 510641, China

Department of Tea Science, College of Horticulture, South China Agricultural University,

12 13 14

# These

authors contributed equally to this work

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*

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[email protected] (Lingzhi Zhang)

Corresponding author: [email protected] (Shili Sun), Tel.: +86-20-8516-1045;

ACS Paragon Plus Environment


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Abstract: Diabetic nephropathy (DN) is a major complication of type 2 diabetes (T2D), which

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is a key determinant of mortality in the diabetic patients. Developing new therapeutic drugs

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which can not only control T2D but also prevent the development of DN is of great

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significance. We studied the therapeutic potential of Cuiyu tea polypeptides (TP), natural

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bioactive peptides isolated from a type of green tea, against DN and its underlying molecular

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mechanisms. TP (1000 mg/kg bw/day, p.o.) administration for five weeks significantly reduced

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the fasting blood glucose by 52.04

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(HFD/STZ)-induced (30 mg/kg bw) diabetic mice. Compared to model group, the serum

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insulin level of the TP group was decreased by 25.54 ± 6.06%, at the same time, the

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HOMA-IR, HOMA-IS and lipid levels showed different degrees of recovery (p < 0.05).

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Moreover, in TP group mice the total urinary protein, creatinine and urine nitrogen, all which

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can reflect the damage degree of the glomerular filtration function in a certain extent, were

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dramatically declined by 34.51 ± 2.65%, 42.24 ± 15.24% and 80.30 ± 6.01% compared to

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model

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PKCζ/JNK/NF-κB/TNF-α/iNOS and AGEs/RAGE/TGF-β1 pathways, upregulated the

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expression of podocin in the glomeruli, and decreased the release of pro-inflammatory

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cytokines. These results strongly indicate the therapeutic potential of TP against DN.

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Keywords: tea polypeptide; type 2 diabetes; diabetic nephropathy; AGEs; NF-κB

group

respectively.

±

9.23% in the high fat diet/streptozocin

Mechanistically,

TP


stimulated

the

polyol

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INTRODUCTION

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Type 2 diabetes (T2D) is thought to be a chronic metabolic disorder that currently affects

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numerous of individuals worldwide, with its prevalence increasing at an alarming rate. In

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addition, one-third of the individuals with T2D are undiagnosed, making it a global public

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health concern1. Poorly controlled diabetes is associated with various complications including

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nephropathy, atherosclerosis, retinopathy, and neuropathy2, 3, of which diabetic nephropathy

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(DN) has probably the most detrimental consequences resulting in dialysis and heightened

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cardiovascular risks4, 5. About 30% of the diabetic patients suffer from DN complications and

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eventually undergoing kidney dialysis or transplantation6. The conspicuous pathological

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changes were seen in DN, such as persistent albuminuria, altered creatinine clearance, tubule

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interstitial fibrosis and glomerular sclerosis7.

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The precise mechanisms by which dyslipidemia generates renal injury are yet to be elucidated.

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However, studies indicate the involvement of multiple pathways and mechanisms in the

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pathogenesis DN, which is also a major cause of diabetic mortality. Current evidence suggests

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that chronic inflammation may be an important mediator of the occurrence and progression of

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DN8. In DN model, persistent hyperglycemia activates the polyol pathway and accumulates

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advanced glycation end products (AGEs), leading to the activation of multiple signaling

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pathways like nuclear fraction kappa-beta (NF-κB), protein kinase C (PKC), and poly

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ADP-ribose polymerase (PARP) signals9. The activation of NF-κB can promotes the secretion

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of various pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 1β

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(IL-1β), interleukin (IL-6) etc., leading to the deterioration of DN10. Besides, the progression of

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DN is also influenced by factors like transforming growth factor (TGF-β1), angiotensin II and

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c-Jun N-terminal kinases (JNK), which can stimulate glomerular and tubular fibrogenesis11, 12.

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Furthermore, it has been reported that glucose-induced reactive oxygen species (ROS) induces ACS Paragon Plus Environment


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podocyte apoptosis and subsequent depletion which is an early trigger that leads to DN in

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mouse type 1 and type 2 diabetic models13.

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Therefore, efficiently prevent both the initiation as well as progression of DN is utmost

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important issue in designing therapeutic strategies for diabetic patients. Previous studies have

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shown that anti-inflammatory agents have ameliorative effects on glomerular injury in diabetic

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rats, such as mycophenolate mofetil, retinoic acid etc.14. However, these conventional drugs

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often have numerous side-effects. Hence, it is imperatives for a novel therapeutic strategy

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targeting the specific molecular pathways of DN. Bioactive peptides obtained from medicinal

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plants and dietary sources have recently gained wide attention for their potential in treating and

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preventing various diseases. Peptides derived from the proteins dietary sources exhibit a wide

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range of physiological and biological effects15, including the anti-diabetic effects of certain

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polypeptides16-19. To the best of our knowledge, no study so far has reported anti-DN effects of

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bioactive peptides derived from medicinal plants and dietary sources.

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We isolated novel bioactive tea polypeptides (TPs) from the proteins of tea slag, and studied

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their therapeutic effects on DN in type 2 diabetic ICR mouse model induced by high fat

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diet/streptozotocin (HFD/STZ). We hypothesized that TPs protect the renal functions in

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HFD/STZ T2D mice by inhibiting receptor for advanced glycation end-product (RAGE),

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TGF-β1, podocin, protein kinase C zeta (PKCζ), c-Jun N-terminal kinases (JNK) and NF-κB

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pro-inflammatory signaling pathways.

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MATERIAL AND METHODS

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Chemicals

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Cuiyu green tea was obtained from Tea Research Institute, Guangdong Academy of

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Agricultural Sciences in China. Streptozotocin (STZ) was procured from Sigma-Aldrich ACS Paragon Plus Environment

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(Sigma-Aldrich, USA). Commercial kits/reagents for biochemical assays were bought from

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Jiancheng Inst. Biotechnology (Nanjing, China). All other chemicals and solvents were of

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analytical grade.

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Extraction of tea protein (TPR) and tea polypeptides (TP)

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Tea protein (TPR) was obtained using the alkali method as previously described. Briefly, 1 g of

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the Cuiyu green tea residue was crushed and extracted (liquid to solid ratio 20:1, v/v) with an

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aqueous solution containing 2.5% pectinase at 45 oC (pH 3.4) for 3 h. The crude extract was

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centrifuged at 1237 × g for 10 min and the supernatant was removed. The precipitate was

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dissolved in 0.1 M NaOH (liquid to solid ratio 50:1, v/v) and further extracted at 90 oC for 100

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min20. The mixture was cooled at room temperature and centrifuged (2200 × g, 10 min) to

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remove the debris, and the proteins in the supernatant were precipitated by dropwise addition

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of HCl for 0.5 h to a final pH of 2.5. The supernatant was removed by centrifugation, and the

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precipitate was collected in dialysis bag (MWCO = 500 Da), dialyzed with deionized water for

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24 h, and then centrifuged again to remove the supernatant. The protein precipitate was

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re-dissolved in 75% acetone (solid-liquid ratio 1:6), shaken in an oscillator for 10 min

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centrifuged and the steps were repeated once more. After a final wash with pure water, the

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supernatant containing the acetone was removed, and the resulting TPR (300 mg, yield 30%)

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was stored till analysis.

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To obtain the TP, 1 g of the TPR preparation was digested with 50 ml acidic protease solution

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(0.96 mg/ml, at pH 4) at 40 oC for 2 h, and centrifuged at 3155 × g for 15 min. The supernatant

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was concentrated in a rotary evaporator, and freeze-dried into the TP (420 mg, yield 20%).

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The Hitachi L-8900 Amino Acid Analyzer was used to determine the amino acid composition

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in TPR and TP. The peptides in the TP preparation were characterized by LC-MS (liquid



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chromatography-mass spectrometry), and the data was processed using Mascot 2.3 (Matrix

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Science) mass spectrometry software.

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Determination of amino acid composition of TP and TPR

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TP and TPR samples were hydrolyzed with 0.5 ml of 6 N HCl for 24 h at 112 °C, filtered

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through a 0.45 µm membrane filter prior to analysis. 10 µl of treated samples were derivatized

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6-aminoquinolyl-N-hydroxysuccinimidyl carbamate Waters AccQ·Fluor Reagent Kit. The

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amino acids of samples were separated and detected using reversed phase high performance

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liquid chromatography (Agilent Tech., USA) equipped with a Pico Tag column (3.9 × 300

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mm, 5 µm, Waters). The amount of amino acids was calculated, based on the sample amino

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acid peak area in comparison with that of standard. All analyses were repeated three times.

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LC-MS/MS analysis of TP structure characterization

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The LC-MS/MS analysis of TP was conducted in a TripleTOF5600 system (AB SCIEX)

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combined with nanospray III ion source (AB SCIEX, USA). The hydrolysate fractions were

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resuspended in mobile phase A (98% (v/v) water, 2% (v/v) acetonitrile, and 0.1% (v/v) of

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formic acid) and 10 μL of sample was loaded on C18 nanoLC trap column(100 µm × 3 cm, C18,

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3µm, 150Å) and washed by mobile phase A at 4 μL/min for 10 min. After 10 min of

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preconcentration, the trap column was automatically switched in-line onto a ChromXP C18

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column (75 μm × 15 cm, C18, 3μm, 120 Å). Mobile phase A contained 98% (v/v) water, 2%

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(v/v) acetonitrile, and 0.1% (v/v) of formic acid. Mobile phase B contained 2% (v/v) water,

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98% (v/v) acetonitrile, and 0.1% (v/v) of formic acid. Peptides were eluted with a linear


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gradient from 5% to 35% of solvent B over 90 min at a flow rate of 0.3 μL/min and running

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temperature of 25 ºC. The following conditions were used to characterize the TP peptides:

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spray voltage 2.5 kV, air curtain pressure 30 PSI, and atomization pressure 5 PSI. The data was

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processed using Mascot 2.3 (Matrix Science) mass spectrometry software and homologous

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protein analysis was performed with the protein database2 20160526 (15,566 sequences;

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4,692,812 residues).（Figure S1）

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Induction of T2D in a mouse model and treatment protocol

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All animal experiments were performed in accordance with the laboratory animal care and use

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guidelines strictly, and best efforts were made to minimize the pain of experimental animals.

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The experiment protocols were approved by the Animal Care & Welfare Committee of Tea

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Research Institute, Guangdong Academy of Agricultural Sciences. Sixty 8-week-old male ICR

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mice were purchased from the Beijing Vital River Laboratory Animal Technology Co. Ltd. All

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animals were maintained in a specific pathogen-free laboratory under the standard temperature

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(23 ± 2 oC) and relative humidity (55 ± 5%) and a 12 h light/dark cycle, with ad libitum access

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to food and water throughout the experiment.

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T2D was induced by the streptozotocin (STZ) method as previously described21. After 1-week

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acclimatization, the mice were randomized into the control and diabetic groups. The mice in

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the normal control (NC) group were fed standard diet (protein 18%, fat 4%, carbohydrate 62%,

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fiber 5%, minerals 8%, w: w; Beijing Huafukang Bioscience Co. Ltd, 1022) and those in the

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diabetic group were given a high-fat diet (HFD; 16.46% protein, 45.65% fat, 37.89%

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carbohydrates, as percentage of total kcal; Beijing Huafukang Bioscience Co. Ltd, D12451) ad

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libitum till the end of the experiment. After five weeks of dietary manipulation, the HFD-fed



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mice were injected intraperitoneally with a single dose of STZ (30 mg/kg bw in citrate buffer

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pH 4.4) once a week for three weeks without fasting. The control group mice were given

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equivalent amounts of citrate buffer and normal saline. Five days after the last STZ injection,

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the plasma glucose concentration of the model mice were measured after 12 h fasting period,

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and a concentration of 11~18 mM confirmed T2D. No significant damage was seen in the

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pancreas of these mice (Figure S2.)

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For the treatment regimen, four groups of mice (n = 10 per group) were respectively given oral

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doses of the following daily for 5 weeks: 1) NC – normal saline, 2) diabetic model control (MC)

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– normal saline, 3) diabetic TP – 1000 mg/kg body weight TP, and 4) diabetic TPR – 1000

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mg/kg body weight TPR. No side effects were observed based on the general behavior and

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mental state of mice treated with TP and TPR. After the 5-week treatment, the serum alanine

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aminotransferase (ALT) levels of the TP and TPR group mice were 11.0~31.0 IU/L. In

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addition, these extracts had no toxic effects in normal healthy mice given the same doses as the

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experimental mice (Figure S3.). The body weights, and total food and water consumption were

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monitored weekly.

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Preparation of serum and tissue homogenate

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After the last TP/TPR dose, all mice were fasted overnight, and their urine samples were

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collected the next day and maintained under mineral oil to avoid evaporation. The mice were

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sacrificed by carbon dioxide asphyxiation, and blood samples were collected by cardiac

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puncture. After leaving the samples undisturbed at room temperature for 30 min, the serum

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was separated by centrifuging at 877 × g for 20 min and all serum samples were stored at -80

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°C for further use. The animals were perfused trans-cardially through the ascending aorta with

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saline to remove any residual blood clots, and then the left kidney and liver were rapidly

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removed, weighed and washed thoroughly with phosphate buffer saline (PBS pH 7.4). The ACS Paragon Plus Environment

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tissues were homogenized in ice-cold PBS, and stored in liquid nitrogen for further

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biochemical and molecular assays. The right kidney was also removed and fixed in 10%

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buffered formalin, and embedded in paraffin for histopathological examination.

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Serum biochemical analysis

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Blood was collected from the tail veins once a week during the 5-week treatment period after

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12 h fasting, and the levels of fasting blood glucose (FGB) were measured using commercial

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assay kits (Nanjing, China). In addition, total cholesterol (TC), triglyceride (TG), low-density

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lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were

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measured using an Automatic Analyzer (Icubio, China). Urine was also collected on a weekly

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basis 24 h after each treatment, and total urinary protein levels were measured using suitable

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kits. A homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity

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(HOMA-IS) was used to evaluate insulin resistance and sensitivity respectively, with the

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following formulae: HOMA-IR = fasting serum insulin (mU/ml) × fasting plasma glucose

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(mM)/22.5, and HOMA-IS = 1/[fasting plasma glucose (mM) × fasting serum insulin (mU/ml)]

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22.

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Enzyme-linked immunosorbent assay (ELISA)

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Insulin and AGEs levels in the serum and kidney were measured using commercial ELISA kits

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(Insulin: Sigma-Aldrich, USA; AGEs: Abcam, Cambridge, UK) according to the

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manufacturer’s instructions. Briefly, take 100 mg kidney tissue in EP tube with 1 ml of PBS

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(PH, 7.4), homogenized using a homogenizer (OMNI International, USA), after centrifuging

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(11187 × g, 5 min), the supernatant was collected and assayed immediately.

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Western blotting



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The kidney tissues were homogenized on ice for 10 s using a polytron tissue homogenizer, and

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then lysed in 0.5 ml ice-cold lysis buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1%

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Triton X-100, 1% sodium deoxycholate, 0.1% SDS, proteinase inhibitor (Roche Applied

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Science, Germany) and phosphatase inhibitor (Sigma-Aldrich, USA)]. The tissue samples were

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cracked use an ultrasonic dismembrator (Fisher Scientific, USA) at the speed of 2 greed for 30

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pulses, and stored at -80 °C for further analysis. Lowry method was used to determine the

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protein concentration of samples, and 50 µg of each sample was boiled in sodium dodecyl

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sulfate (SDS) sample loading buffer for 5 min. The samples were loaded into 10%

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SDS–polyacrylamide gel, and after electrophoresis for 1.5 h, were transferred to PVDF

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membranes (Millipore, USA). Block the membranes with 5% skim milk or BSA in TBST

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(Tris-buffered saline containing 0.1% Tween 20) at room temperature for 2 h. And then the

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membranes were incubated overnight at 4 °C with rabbit polyclonal antibodies against JNK,

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p-JNK, PKCζ p-PKCζ, NF-κB, p-NF-κB, inducible nitric oxide synthases (iNOS), TNF-α (all

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from Cell Signaling Technology), rabbit polyclonal antibodies (1:500) against Podocin,

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RAGE, TGF-β1 (all from Boster, China), and mouse polyclonal anti-β-actin (1:2000;

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Sigma-Aldrich). After washing thrice with TBST (5 min), the membrane was incubated with

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anti-mouse or anti-rabbit immunoglobulin G antibody conjugated to horseradish peroxidase

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(1:4000; Cell Signaling Technology, USA) for 2 h at room temperature. The positive signals

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were visualized using chemiluminiscence (ECL) detection kit (Thermo Scientific, USA),

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imaged with Chemi Doc system (Bio-Rad, USA), and quantified by NIH IMAGEJ software

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(Bethesda, MD, USA). The protein expression levels of p-PKCζ, p-JNK and p-NF-κB were

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normalized to that of PKCζ, JNK and NF-κB, while iNOS, Podocin, RAGE, TGF-β1 and

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TNF-α levels were normalized to β-actin (internal control).

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Histology and immunohistochemistry (IHC) ACS Paragon Plus Environment

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Formalin fixed kidney tissues were paraffin embedded, cut into sections, and stained with

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hematoxylin and eosin (HE) using standard protocols. For IHC, the sections were

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de-paraffinized with xylene for 2 times (each time more than 5 min), quenched with 3%

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hydrogen peroxide for 15 min, washed twice with PBS (PH 7.4), and then blocked for 1 h with

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10% normal goat serum (Invitrogen, USA) at room temperature. The sections were then

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incubated overnight at 4 °C with the primary antibodies, and then with the IHC Detection

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Reagent (Boster) against rabbit IgG for 30 min at room temperature. Developed the color using

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3, 30- diaminobenzidine (DAB), and photographed the images under a BX60 microscope

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(Olympus, Japan) fitted with a camera.

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Statistical analysis

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The results are presented as mean ± SEM. The means were compared by one-way ANOVA

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and Tukey’s test using the GraphPad Prism 7.0 for Windows (GraphPad Software Inc., San

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Diego, CA, USA). The level of confidence required for significance was set at p ≤ 0.05. All

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experiments were performed at least triplicates.

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RESULTS

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The amino acid composition of TP

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As shown in Table 1, although the total content of hydrolyzed amino acids was higher in TPR

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compared to TP (38.0 g/100 g vs 14.8 g/100 g), its total content of free amino acids was lower

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than that of TP (0.16 g/100 g vs 3.89 g/100 g).

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LC-MS/MS analysis of TP structure characterization

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The peptides in the TP were identified according to the observed total ion chromatogram

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(Figure S1) and MS/MS spectroscopy data. Protein score of the TP matched to that of ACS Paragon Plus Environment


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Flavonoids-3'-hydroxylase, Chloroplast stroma ascorbate peroxidase, Arginine decarboxylase,

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RNA polymerase II accessory factor and Beta-glucosidase 5 GH3 family. Thirteen major

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peptide components were sequenced. These peptides range from 7 to 31 residues. Specific

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peptide information is shown in Table 2.

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Effect of TP on body weight, water consumption, FBG and serum insulin

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No significant difference in the pre-treatment blood glucose levels across the different groups.

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Following HFD/STZ diabetes induction, the mice developed significant hyperglycemia with

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steady increase in the levels of blood glucose (~10 mM) compared to the normal group. The

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mice in all treatment groups had similar level of FBG, significantly higher than that of NC

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group (Fig.1A; p < 0.01). While treatment with TP or TPR did not significantly affect FBG

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levels during the first week after administration, the latter began to decline after three weeks of

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treatment. After 5 weeks, FBG levels decreased by 52.04 ± 9.23% (from 16.3 mM to 8.46

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mM) in the TP group, compared to that of the MC group (~15.61 mM). Although TPR also

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alleviated the hyperglycemic symptoms, the FBG of the TP group was lower by 2.75 mM

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compared to the TPR group (Fig.1A). The serum insulin level of the TP group was reduced by

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25.54 ± 6.06% compared to the MC group (6.36 ± 0.03 vs 8.59 ± 0.49mU/L, Fig. 1B), while

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TPR did not have a significant effect (8.36 ± 0.34, Fig. 1B). Furthermore, the body weight of

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the T2D mice was significantly lower than that of normal mice (Fig. 1C), and TP and TPR

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treatment significantly reduced water intake but had no effect on food consumption in the

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diabetic mice (Fig. 1D).

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Effects of TP on insulin tolerance and lipid levels in serum

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To further evaluate the ameliorative effects of TP on the diabetic phenotype, we analyzed the

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HOMA-IR, HOMA-IS and lipid levels in mice serum (Fig. 2). Compared to the MC group, the ACS Paragon Plus Environment

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HOMA-IR values were significantly lower in all treatment groups (p < 0.05; Fig. 2A).

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Furthermore, a significant increase in HOMA-IS values was observed in the TP and TPR

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groups relative to the MC group (p < 0.01; Fig. 2B). These results indicated that TP can

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ameliorate insulin resistance in diabetic mice. The effects of TP and TPR on different serum

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biochemical parameters are shown in Fig. 2C-F. The TC and TG levels were considerably

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higher in the MC group compared to the controls (p < 0.05), and were both significantly

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downregulated by TP and TPR treatment (p < 0.05 for both), with serum TG levels decreasing

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by 75.0%~86.1% (Fig. 2C, D). The T2D mice exhibited significant escalation in the

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LDL-cholesterol (p < 0.01) levels with simultaneous depletion of HDL-cholesterol (p < 0.01)

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compared to the normal mice, and while HDL was increased by 14.3%~17.6% to almost

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normal levels following TP and TPR treatment (Fig. 2E), LDL was reduced by 44.8% by TP

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(Fig. 2F) in the diabetic mice.

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Renal dysfunction of T2D mice was markedly ameliorated by TP

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The diabetic mice exhibited a significant increase in renal mass and volume, and TP treatment

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significantly restored both kidney appearance and kidney mass to body mass ratio to near

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normal status (Fig. 3A, B). In addition, a 3-5 fold increase was observed in total urinary protein

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excretion in the MC mice compared to the control mice, which were restored to baseline levels

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by TP and TPR (p < 0.01; Fig. 3C) with greater effect of TP. Furthermore, diabetes-induced

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histopathological damage of the kidneys was alleviated by TP and TPR (Fig. 3D). Since the

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glomerular endothelial cells are vital for the functional maintenance of glomerular filtration,

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we also analyzed the number of cells in the glomeruli (Fig. 3E). The MC mice showed

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significantly fewer glomerular cells compared to the control mice (26.2 ± 2.18 vs 52.00 ± 3.59

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cells per glomerulus, p < 0.01), which was reversed by both TP (39.00 ± 1.14 cells per



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glomerulus) and TPR (35.20 ± 1.36 cells per glomerulus; p < 0.05 for both vs MC group, Fig.

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3E). In addition, all treated groups had significantly lower glomerular hypertrophy (3038.05 ±

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129.75 μm2 for TP; 3194.54 ± 100.47 μm2 for TPR; 4330.83 ± 208.33 μm2 for MC; 2478.21 ±

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116.68 μm2 for NC; p < 0.05, Fig. 3F). Creatinine, urine nitrogen and the ratio of the two can

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reflect the damage degree of the glomerular filtration function in a certain extent. Significant

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decreases in creatinine and urine nitrogen in serum were observed in TP or TPR group

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compared to MC group (Fig. 4).

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Podocytes express various cytoskeletal proteins e.g. the slit diaphragm (SD)-localized podocin

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which maintain the SD and foot processes integrity, and any disruption in these proteins result

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in podocyte injury, foot process effacement and albuminuria. As shown in Fig. 5, podocin

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levels were significantly decreased in diabetic mice, while TP and TPR treatment significantly

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increased the same (p < 0.05).

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TP attenuated renal AGEs accumulation and re-established normal expression of RAGE

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Interaction between AGEs and their receptors (RAGE) play important roles in the initiation

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and progression of diabetes mellitus and diabetic complications23,

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AGEs in diabetic patients may be an important mediator of the detrimental effect of

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hyperglycemia. In addition, glomerular IgG deposition is also seen in diabetes due to the

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increased expression levels of circulating antibody targeted modified proteins. Therefore, the

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protective effects of TP on DN could be due to the reversal of AGEs accumulation. Higher

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levels of AGEs were seen in the kidneys of MC mice (4.64 ± 0.27-fold increase compared to

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NC group, p < 0.01), which was attenuated not only by TPR (1.63 ± 0.06-fold increase

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compared to NC group; p < 0.05 vs MC group), but also by TP (1.44 ± 0.03-fold increase

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compared to NC group; p < 0.05 vs MC group) (Fig. 6). Given that AGEs exert inflammatory


24.

The accumulation of

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and oxidative effects in DN via RAGE, we also analyzed the renal expression of RAGE.

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Compared to the NC group, both AGEs and RAGE were significantly upregulated in the

312

diabetic kidney, and decreased following TP treatment (Fig. 6).

313

TP improved renal fibrosis through reducing TGF-β1 production

314

The pro-inflammatory cytokine TGF-β1 is regulated by AGEs, and plays an important role in

315

kidney diseases, including DN25. Therefore, we further examined the possible involvement of

316

the TGF-β1 signaling pathway in the ameliorative effects of TP on DN pathologies (Fig. 7).

317

Renal expression of TGF-β1 significantly decreased in the diabetic mice treated with TP (0.59

318

± 0.07 vs 2.01 ± 0.05 fold increase compared to NC group) as well as TPR (0.70 ± 0.01

319

nmol/mg of protein) (p < 0.05; Fig. 7B, C). In addition, a strong in situ expression of TGF-β1

320

was seen in the kidney biopsies of diabetic mice, which was almost undetectable in the

321

drug-treated mice (Fig. 7A, D).

322

Effects of TP on the NF-κB signaling pathway in kidneys

323

Studies show the central role of inflammatory pathways in the development of various diabetic

324

complications. NF-κB pathway is activated in DN model26, and transcriptionally activates

325

genes encoding inflammatory and proliferative proteins involved in DN pathogenesis27. A

326

significant up-regulation in NF-κB phosphorylation was observed in the kidney homogenates

327

of T2D mice when compared to that of NC mice (p < 0.01; Fig. 8). Although both TP and TPR

328

treatment down-regulated (p < 0.01) p-NF-κB levels, the effect of TP was more evident. The

329

expression levels of renal p-PKCζ and p-JNK, which are known to activate NF-κB, were

330

significantly higher in the diabetic mice compared to NC mice (p < 0.01; Fig. 8). TP and TPR

331

treatment significantly restored p-PKCζ and p-JNK to baseline levels (p < 0.05), with TP

332

showing a greater effect than TPR on p-JNK levels (Fig. 8C). Furthermore, in MC group mice, ACS Paragon Plus Environment


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a significant escalation in the levels of TNF-α and iNOS (p < 0.01), which were restored by TP

334

and TPR treatments (Fig. 9).

335

DISCUSSION

336

Diabetic nephropathy (DN), as a serious complication of type 2 diabetes (T2D), is one of the

337

leading causes of death in diabetic patients28. Current therapy for diabetes focuses on the

338

intensive control of blood glucose, but neglects the renal complications associated with

339

hyperglycemia. We found that T2D mice modeled by a 6-week long HFD/STZ induction

340

exhibited hyperglycemia, insulin resistance, renal structural and function abnormalities and

341

increased renal inflammation, all of which are the critical features of DN. These results are in

342

agreement with Danda et al (2005), who successfully developed a model of DN in HFD/STZ

343

induced T2D rats, with hyperglycemia, insulin resistance and renal dysfunction and structural

344

remodeling, which simulated the human disease29. TP treatment however significantly

345

improved the levels of FBG, HOMA-IR, HOMA-IS and serum lipids in the T2D mice, and

346

significantly restored the serum and urine parameters to baseline values.

347

Hyperglycemia-induced formation of AGEs is an important source of oxidative free radicals9.

348

AGEs stimulate several cellular responses such as inflammation, matrix production and

349

fibrogenesis, by binding to their specific receptors (RAGE) expressed on various cells, like the

350

glomerular mesangial cells30. Moreover, the immune complex deposition in the glomerular

351

have been considered associated with an increased level of circulating antibodies against

352

AGEs, and may play a pivotal role in albuminuria and tissue injury31, 32. TP inhibited AGE

353

accumulation, downregulated RAGE expression, and decreased glomerular IgG deposition.

354

Therefore, the inhibition of AGEs formation by TP may largely contribute to the amelioration


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355

of DN symptoms. Furthermore, in DN models, gene deletion or pharmacologic blockade of

356

RAGE can effectively prevent early renal dysfunction and glomerular structure changes33.

357

The AGEs-RAGE system upregulates TGF-β1, a potent pro-fibrotic cytokine involved in the

358

progression of diabetic renal tubule interstitial fibrosis, in glomerular mesangial cells34. The

359

expression level of TGF-β1 is increased in diabetic patients35, 36, as well as in experimental

360

models of diabetic kidney disease37-39. It stimulates the production of several extracellular

361

matrix (ECM) proteins that accumulate in the podocytes of the diabetic kidney, such as type IV

362

collagen, fibronectin and laminin, and further increases TGF-β production by the podocytes

363

and mesangial cells40-42. In the present study, the expression levels of TGF-β1 increased

364

significantly in the renal tissue of MC mice, while treatment with TP significantly reduced its

365

levels.

366

Podocytes are glomerular cells occurring specifically in the Bowman's capsule that wraps

367

around capillaries of the glomerulus, and are attached to the lateral side of the glomerular

368

basement membrane (GBM). The cells are inter-connected by the slit diaphragm (SD) of

369

podocytes to form a barrier against protein loss43,

370

nephrin, cluster of differentiation 2-associated protein (CD2AP) and other related proteins, and

371

proteinuria is initiated with podocin downregulation, followed by that of nephrin and CD2AP.

372

Podocin plays an important role in stabilizing nephrin and CD2AP in the SD. TP

373

administration reduced albuminuria, attenuated pathological renal damage and restored

374

glomerular podocin levels in the T2D mice. These findings indicate that TP relieves renal

375

dysfunction via its protective effects on the glomerular podocytes.

376

Recent studies focusing on the cellular and molecular etiologies of DN have gauged

377

inflammation as its key patho-physiological mechanism26. Hyperglycemic conditions are

378

known to increase macrophage infiltration and activation, which further aggravate

44.

The SD mainly consists of podocin,



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379

hyperglycemia and contribute to the development of renal injury and sclerosis45. NF- κ B, a

380

ubiquitous transcription factor, is a major regulator of the inflammatory response and is

381

activated by many DN-specific stimuli. Furthermore, growing evidence indicates that NF-κB is

382

central to many interrelated pathways involved in the structural and functional changes

383

observed in DN, such as activation of the renin–angiotensin system, AGE accumulation, and

384

NADPH-dependent oxidative stress10, 46. Schmid et al correlated the upregulation of NF-κB

385

with the inflammatory response in DN patients47, and activation of NF- κ B has also been

386

observed in the renal cortical tissue of animals with STZ-induced diabetes48. Consistent with

387

these findings, we found a significant increase in the nuclear translocation of NF-κB p65 in the

388

T2D mice, which was effectively inhibited by TP.

389

The activated NF- κ B, i.e. free NF- κ B p65 subunit, transcriptionally activates downstream

390

effector genes, such as those encoding TGF-β1, TNF-α, iNOS, pro-inflammatory cytokines and

391

chemokines49. PKC, JNK50, and PKCζ51 activate NF-κB in different cell types. JNK, which

392

regulates environmental stress and inflammatory cytokines52, triggers insulin resistance by

393

phosphorylating the c-Jun protein. This causes insulin deficiency and ultimately an increase in

394

glucose levels, thereby contributing to the progression of DN53,

395

upregulation of TNF-α and iNOS, as well as the activation of PKCζ and JNK in the diabetic

396

kidneys, indicating a possible post-transcriptional mechanism of suppressing inflammation and

397

fibrosis.

398

At the same time, the tea polypeptide extraction yield is relatively low, and the extraction

399

process needs to be optimized to improve the yield of tea polypeptide. TP is a kind of

400

polypeptide mixture containing peptides with different molecular weights, which is the reason

401

for the high dose (1000 mg/kg BW, p.o.) used in this study. Compared with the monomer

402

compounds, the mixture experiment materials need higher dose55, 56. The substance and exact ACS Paragon Plus Environment

54.

TP inhibited the

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structure of TP involved in the above functions remains to be elucidated. At present, there have

404

been no specific drugs that really target diabetic nephropathy. Current treatments have been

405

mainly focused on managing blood glucose and insulin levels57. Thus, we are not set positive

406

control group in this study. Therefore, a profound study and discussion on these problems

407

remain important topics in the further of our research.

408

Taken together, TP attenuated pathological renal damage via hypoglycemic, insulin-sensitizing

409

and anti-inflammatory in the DN mouse model of HFD/STZ-induced T2D. The hypoglycemic

410

and

411

AGEs/RAGE/TGF-β1 signaling pathway, and the upregulation of podocin in the glomeruli. TP

412

specifically suppressed the NF- κ B pathway-mediated renal dysfunction in these mice,

413

indicating that the NF- κB pathway is a potential therapeutic target in DN. Although there is

414

unable to compare the efficacy of TP with clinical drugs, tea polypeptide, as a natural and

415

nontoxic polypeptide, is still a promising candidate for the treatment diabetic nephropathy.

416

Acknowledgments: This work was supported by the National Natural Science Foundation of

417

China (NO. 81803236, 31800295). Guangdong Science and Technology program (NO.

418

2017A070702004, 2016B090918118, 2017A020224015), Natural Science Foundation of

419

Guangdong Province (NO. 2017A030310504), the Guangdong Provincial Agriculture

420

Department program (NO. 2017LM2151), Science and Technology Board of Qingyuan (NO.

421

2016A005), the President Foundation of Guangdong Academy of Agricultural Sciences (NO.

422

201534 and 201720) and the Open Fund of Guangdong Provincial Key Laboratory of Tea

423

Plant Resources Innovation & Utilization (NO. 201702).

insulin-sensitizing

effects

were

likely

mediated


by

stimulation

of

the


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Author Contributions: Lingzhi Zhang, Lingli Sun, Xingfei Lai and Shili Sun conceived and

425

designed the experiments. Xuming Deng performed the experiments. Xuming Deng and Limin

426

Xiang analyzed the data. Xingfei Lai, Qiuhua Li and Wenji Zhang contributed reagents and

427

participated in animal surgery. Xuming Deng wrote the paper, and Lingli Sun and Shili Sun

428

critically revised the manuscript. All authors approved the final version of the manuscript.

429

Conflicts of Interest: The authors declare no conflict of interest.

430

Supplementary Materials: The following are available online at www.mdpi.com/link, Figure

431

S1: The total ion chromatogram of TP. Figure S2: IHC staining of pancreas in all experimental

432

groups. Figure S3: No toxic effect of TP and TPR in normal healthy mice.

433 434

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Figure legends

619

Figure 1. Effect of TP treatment on fasting blood glucose levels (A), serum insulin levels (B),

620

body weight (C), water consumption (D) and food consumption (E) that were measured once a

621

week after mice were fasted for 12 h before blood collection. Serum insulin levels were

622

measured by ELISA. Each value represents the mean ± SEM (n =10). Values with different

623

letters (a−c) differ from each other significantly (P < 0.05).

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Figure 2. Effect of TP treatments on insulin resistance, insulin sensitivity and serum lipid

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levels. (A) HOMA-IR (Homeostasis Model Assessment of Insulin Resistance) = fasting serum

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insulin (mIU/ml) × fasting plasma glucose (mmol/L)/22.5. (B) HOMA-IS (Homeostasis Model

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Assessment of Insulin Sensitivity) = 1 / [fasting plasma glucose (mmol/L) × fasting serum

628

insulin (mU/ml) ]. Serum levels of TC (C), TG (D), HDL-C (E), LDL-C (F) after 12 h fasting

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at the end of the 5-week treatment. Each value represents the mean ± SEM (n = 10); Values

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with different letters (a−c) differ from each other significantly (P < 0.05).



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Figure 3. TP treatment ameliorates renal dysfunction in diabetic mice. (A) Representative

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images of the kidneys in all groups. (B) Kidney/body weight (%) was measured after 5-week

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treatment. (C) Quantification of total urinary protein excretion (μg/ml) 24 h after each

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treatment once a week. (D) Representative H&E staining images of the glomerulus (a - NC, b -

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MC, c - TP, and d - TPR groups; scale bar 20 μm). (E, F) Comparison of the glomerular cell

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number and glomerular area (μm2) across groups (average of 20 glomeruli per mouse; scale bar

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20 μm). The data are presented as means ± SEM (n = 10); Values with different letters (a−c)

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differ from each other significantly (P < 0.05).

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Figure 4. The effect of TP on urine nitrogen and creatinine in serum. (A) Urine nitrogen in

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serum; (B) Creatinine in serum; (C) Ratio of urine nitrogen and creatinine. The data are

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presented as means ± SEM (n = 8); Values with different letters (a−c) differ from each other

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significantly (P < 0.05).

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Figure 5. TP ameliorated the structural abnormalities of DN. (A) Representative pictures of

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IHC of Podocin in kidney sections (a - NC, b - MC, c - TP, and d - TPR groups; scale bar 20

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μm). (B) Western blotting images showing renal Podocin levels. Quantification of Podocin

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expression in Western blots (C) and IHC (D). Each value represents the mean ± SEM (n = 8; 4

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independent experiments); Values with different letters (a−c) differ from each other

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Figure 6. TP prevented diabetes-induced accumulation of AGEs and led to decreased RAGE

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levels in the glomeruli. (A) Representative pictures of IHC staining of RAGE in kidney

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sections (a - NC, b - MC, c - TP, and d - TPR groups; scale bar 40 μm). (B) Western blotting

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images of renal AGE and RAGE levels. (C) Fold increase in renal AGE levels relative to that

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in NC mice by ELISA. Quantification of RAGE levels in Western blotting (D) and IHC (E). ACS Paragon Plus Environment

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The data are presented as means ± SEM (n = 9; 3 independent experiments); Values with

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different letters (a−c) differ from each other significantly (P < 0.05).

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Figure 7. TP decreased TGF-β1 content in the kidney. (A) Representative pictures of IHC

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staining of TGF-β1 in kidney sections (a - NC, b - MC, c - TP, and d - TPR groups; scale bar

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40 μm). (B) Western blotting images of renal TGF-β1 levels. Quantification of TGF-β1 in

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Western blotting (C) and IHC (D). The data are presented as means ± SEM (n = 9; 3

660

independent experiments); Values with different letters (a−c) differ from each other

661


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Figure 8. Effect of TP treatment on the NF-κB signaling pathway. (A) Western blotting

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images of p- PKCζ, PKCζ, p-JNK, JNK, p-NF-κB and NF-κB expression. (B) Relative

664

expression of p-PKCζ, p-JNK and p-NF-κB to PKCζ, JNK and NF-κB levels respectively. The

665

data are presented as means ± SEM (n = 9; 3 independent experiments); Values with different

666

letters (a−c) differ from each other significantly (P < 0.05).

667

Figure 9. Effect of TP treatment on inflammatory factor expression levels. (A) Western

668

blotting images of iNOS and TNF-α. (B) Relative expression of iNOS and TNF-α to β-actin.

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The data are presented as means ± SEM (n = 8; 4 independent experiments); Values with

670

different letters (a−c) differ from each other significantly (P < 0.05).

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Table 1.

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The amino acid composition of tea polypeptides and tea protein Amino acid

Content of hydrolyzed amino acid

Content of free amino acid（

（g/100g）

g/100g）

name TP

TPR

TP

TPR

Aspartic acid

1.82±0.05

4.14±0.02

0.32±0.02

Tea polypeptide ameliorates diabetic nephropathy through RAGE and

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