2018 Volume 41 Issue 9 Pages 1430-1439
Tribulus terrestris L. (Zygophyllaceae) (TT) is usually used as a cardiotonic, diuretic, and aphrodisiac, as well as for herbal post-stroke rehabilitation in traditional Chinese medicine. However, little is known about the renoprotective effects of TT on obesity-related glomerulopathy (ORG). In this study, 340 monomeric compounds were identified from TT extracts obtained with ethyl acetate combined with 50% methanol. In vitro, IC50 of TT was 912.01 mg/L, and the appropriate concentration of TT against oxidized-low density lipoprotein (ox-LDL) induced human renal glomerular endothelial cells (HRGECs) was 4 mg/L. TT significantly increased the viability (63.2%) and migration (2.33-fold increase) of HRGECs. ORG model rats were induced by a chronic high-fat diet (45%) for 20 weeks and were then treated with TT extract (2.8 g/kg/d) for 8 weeks. Subsequently, the kidneys were removed and their differentially expressed protein profile was identified using two-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-TOF MS. Molecular categorization and functional analysis of bioinformatic annotation suggested that excessive energy metabolism, decreased response to stress and low immunity were the potential etiologies of ORG. After TT administration for 8 weeks, body weight, blood pressure, serum cystatin C and cholesterol were decreased. Additionally, TT significantly enhanced the resistance of rats to ORG, decreased energy consumption and the hemorrhagic tendency, and improved the response to acute phase reactants and immunity. In conclusion, TT may play a protective role against ORG in rats.
Considering the rapid increase in the incidence of obesity worldwide, obesity has become an independent risk factor for chronic kidney disease (CKD).1) The prevalence of obesity-related glomerulopathy (ORG) is increasing in parallel with the worldwide obesity epidemic.2) An increased glomerular filtration rate, renal plasma flow, filtration fraction and tubular sodium reabsorption are considered to be the main renal physiologic changes due to obesity.2) Adipokines and ectopic lipid accumulation in the kidney promote maladaptive responses of renal cells to the mechanical forces of hyperfiltration, leading to podocyte depletion, proteinuria, focal segmental glomerulosclerosis (FSGS) and interstitial fibrosis. Although some patients with morbid obesity do not demonstrate overt clinical renal manifestations, several glomerular lesions (an increased mesangial matrix, mesangial cell proliferation, podocyte hypertrophy, glomerulomegaly, and focal and segmental glomerulosclerosis) are known to occur.3) The pathologic features of ORG include glomerulomegaly and FSGS, particularly the perihilar variant, and the degree of foot process effacement in ORG is usually less than that in primary FSGS. However, the exact change in the molecular expression profile of ORG remains unclear.
Herbal medicines demonstrate good compatibility with the human body and have stood the test of time because of their safety, efficacy, cultural acceptability, and few side effects. Tribulus terrestris L. (Zygophyllaceae) (TT) is an herb with a wide distribution in subtropical regions. TT is usually used as a cardiotonic, diuretic, aphrodisiac, antioxidant, weight reducer, and herbal rehabilitation after stroke in traditional Chinese medicine.4) However, little attention has been paid to the renoprotective effects of TT in ORG. Here, we induced an injured glomerular endothelium model via oxidized-low density lipoprotein (ox-LDL) in vitro and an ORG rat model by feeding a chronic high-fat diet for 20 weeks in vivo, and further treatment with TT. We focused on the alteration of the pathophysiology and change in the protein expression profile in the kidneys of ORG rats using two-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-TOF MS, and explored the renoprotective effect of TT against ORG in rats as well as its underlying mechanism.
TT fruit was purchased from Tian Jiang Pharmaceutical Co., Ltd. (Jiangyin, Jiangsu, China) in October 2015 and identified by Prof. Feng Li in the Pharmacy College, Shandong University of Traditional Chinese Medicine. According to our pre-experiment, TT was air-dried, stir-fried until it had a slightly golden peel, ultra-fine pulverized, soaked in 70% ethanol for 30 min and then extracted with heating reflux method 3 times for 2.5 h every time. The extracts were then filtered and combined with the soluble part. Crude extracts were concentrated using rotary evaporator (Puredu, Shanghai, China), and then extracted and recovered with ethyl acetate combined with 50% methanol under negative pressure.
The extract was analyzed using an LC-electrospray ionization (ESI)-MS/MS system equipped with a UPLC (Shim-pack UFLC ShimADZU CBM20A system) and MS (Applied Biosystems 4500 QTRAP), and the analytical conditions were optimized. Briefly, for HPLC, the conditions were as follows: a WatersACQUITY UPLC HSS T3 C18 column, 2.1 mm×100 mm; solvent system: water (0.1% formic acid) and acetonitrile (0.1% formic acid); gradient program: (95 : 5, v/v) at 0 min, (5 : 95, v/v) at 11.0 min, (5 : 95, v/v) at 12.0 min, (95 : 5, v/v) at 12.1 min, (95 : 5, v/v) at 15.0 min; flow rate, 0.4 mL/min; temperature, 40°C; injection volume, 5 µL; MS, ESI; source temperature, 550°C; ionspray voltage, 5500.0 V; and curtain gas, 25 psi. Collision-activated dissociation was high. The data obtained were processed using Analyst 1.6.1 software. Structure analysis of the compounds referenced the MassBank, Knapsack, HMDB, MoTo DB and METLIN databases.
The extracts were filtered and concentrated under reduced pressure to a relative density of 1.25 (75°C) and then spray dried to obtain 20 g raw herbs/g granules.
Determination of the IC50 and Appropriate Concentration of TTThe IC50 of TT was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Human renal glomerular endothelial cells (HRGECs) were donated by Professor Ju Liu from Shandong Qianfoshan Hospital. HRGECs were cultured in a basal endothelial cell medium (EBM-2, ScienCell, U.S.A.) with the EGM-2-MV Bullet Kit and 5% fetal bovine serum (FBS). Third to eighth passage cells were used in the experiments. HRGECs (2000/200 µL per well) were plated in 96-well plates in serum-free Dulbecco’s modified Eagle’s medium (DMEM)/F12 (negative control), complete endothelial cell medium (ECM) (positive control), or TT (1000000, 10000, 1000, 100, 1, 0.1, 0.01, 0.001 and 0.0001 mg/L separately) for 24 h and treated with MTT for the last 4 h. The absorbance was measured at 562 nm with a reference wavelength of 630 nm.
To determine the appropriate concentration of TT, HRGECs (2000/200 µL per well) were pre-incubated for 60 min with TT (100, 20, 4, 1, and 0.4 mg/L separately) and then injured endothelium model was then established by applying 100 mg/L ox-LDL (Union-Biology Co., Ltd., Beijing, China) for 24 h. Cellular viability was determined using the MTT assay as previously described.
Cell Migration AssayThe cell migration assay was performed with a Transwell insert system (6.5 mm diameter inserts with 8.0 µm pores in a polycarbonate membrane situated in wells of 24-well polystyrene, tissue culture-treated plates, Corning, Corelle, NY, U.S.A.). The Transwell inserts were precoated with Matrigel (1 : 8) at 4°C overnight and hydrated with serum-free DMEM/F12 at 37°C in a 5% CO2 incubator for 30 min. The HRGEC suspension was added at 20000 cells per insert. Drugs were added to the Transwell inserts as usual, and ECM in the lower chamber. At the end of the observation period, the cells on the upper surface of the insert were removed. The migrated cells on the bottom side were fixed in absolute ethyl alcohol and stained with hematoxylin–eosin (HE). Cells were counted from four random fields under a microscope at 100× magnification. The cell migration experiments were repeated three times.
AnimalsThe study was approved by the Faculty of Medicine & Health Sciences Ethics Committee for Animal Research, Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Jinan, China). In the present study, 40 male Wistar rats (8-week-old) were purchased from Shandong Lukang Pharmaceutical Group Co., Ltd. (certificate: SCXK (Lu) 20130001). All rats were housed in an air-conditioned room with a 12 h light/dark cycle at a temperature of 21±1°C and humidity of 50±5% and had access to their diet and water ad libitum. After a 1-week adaptation to the environment, rats were randomly divided into the high-fat diet (HFD) group (n=32) and control group (n=8). Control group rats were fed a standard chow diet (Lukang, Jining, China, consisting of 11.5% energy as fat, 20.8% as protein, and 67.7% as carbohydrates), while HFD rats were fed a high-fat diet (Lukang, Jining, China, providing 45% of energy as fat, 17% as protein, and 38% as carbohydrates) for 20 weeks.
Body weight and blood pressure were monitored every week. Systolic and diastolic pressure were detected by the non-invasive rat tail method. Briefly, rats were placed in the ALC-heating thermostats (HTP, Shanghai Alcott Biotech Co., LTD., China) animal system, which was heated to dilate the rat tail artery, and were data measured using the ALC-NIBP (Shanghai Alcott Biotech Co., LTD., China), noninvasive blood pressure analysis system. The measurements were repeated 5 times in parallel and the average blood pressure value was then calculated.
The serum glucose level was measured with a OneTouch UltraEasy blood glucose meter (Johnson, Shanghai, China) and the level of serum cystatin C was determined by enzyme-linked immunosorbent assay (ELISA) (10135R, BioYun, Shanghai, China) every five weeks. After 20 weeks, rats with a body weight that was 25% more than (≥) the mean value of the control group and with abnormal levels of serum cystatin C were considered to fit the ORG model; screening showed that only 14 rats were ORG rats. ORG rats were randomly divided into the TT and ORG groups (both n=7). The phase out of the remaining rats was performed via an anesthetic overdose and sacrifice (sodium pentobarbital, 80 mg/kg, intraperitoneally (i.p.)).
Further, rats in the TT group were administered TT (2.8 g TT extracts/kg body weight/d) intragastrically for 8 weeks, and rats in the ORG and control groups were intragastrically given the same volume of saline for the same duration. The respective diets continued throughout the period of drug administration.
Tissue Harvest and Sample PreparationRats were sacrificed at the end of the 28th week. Blood was collected by venipuncture, and the kidneys were removed on ice as soon as possible after anesthesia with sodium pentobarbital (40 mg/kg, i.p.). The kidneys were removed immediately and divided into three parts: one part was fixed in neutral formaldehyde for HE staining and Masson staining, another part was fixed in 2.5% glutaraldehyde solution for ultrastructural observation with a transmission electron microscope (TEM) and the third was homogenized for 2-dimensional electrophoresis and Western blotting. Renal tissue was suspended in 0.5 mL of isoelectric focusing (IEF) buffer containing 40 mM Tris, 5 M urea, 2 M thiourea, 4% CHAPS, 10 mM 1,4- dithiothreitol (DTT), 1.0 mM ethylenediaminetetraacetic acid (EDTA), and 1% phenylmethylsulfonyl fluoride (PMSF) (Beyotime Bio, Shanghai, China). Tissue was torn into small pieces and lysed for approximately 30 s, followed by centrifugation at 12000 rpm at 4°C for 20 min. The protein content of the supernatant was determined using BCA protein assay kit (Beyotime, Nantong, China) to evaluate 100 µg of protein from each sample.
Two-Dimensional ElectrophoresisAccording to GE Healthcare’s two-dimensional electrophoresis experimental instructions, samples (100 µg protein) were separated by 2-dimensional electrophoresis (2-DE), employing an IPGphor multiple sample IEF device (GE Healthcare) in the first dimension, and a Criterion Dodeca cell (Bio-Rad) in the second dimension. Briefly, samples were loaded on to 24 cm dehydrated precast immobilized pH gradient (IPG) strips (GE Healthcare), and the strips were rehydrated overnight under 50 V. IEF was performed at 20°C with the following parameters: 300 V, 0.5 h; 700 V, 0.5 h; 1500 V, 1.5 h; graduate 9000 V, 3 h; 9000 V, 4 h; and 52000 V/h. The IPG strips were then incubated sequentially with sodium dodecyl sulfate (SDS) in 10 mL of equilibration buffer (6 M urea, 2% SDS, 50 mM Tris–HCl, pH 8.8, 30% glycerol) containing 1% dithiothreitol (for reduction) and 2% iodoacetamide (for alkylating conditions) for 15 min each at 22°C. Thereafter, electrophoresis was performed at 17 W for 4.5 h at 4°C using precast 8–16% polyacrylamide gels in Tris-glycine-SDS buffer (25 mM Tris–HCl, 192 mM glycine, 0.1% SDS, pH 8.3).5)
Image AnalysisThe gels were fixed in 10% acetic acid, 40% ethanol, and 0.068% sodium acetate overnight, placed in sensitizing solution (30% ethanol and 30 mM Na2S2O3) and stained with silver nitrate for 1 h using a procedure compatible with mass spectrometry.6) Silver-stained gels were dried between cellophane followed by scanning for comparative proteins and MS analysis using ImageMaster 2D platinum 5.0 (GE) software.
To select differentially abundant protein spots for mass spectrometry, normalized spot volumes were subjected to statistical analysis using in-built tools in the Totallab SameSpots software. The spot volumes were log 2 transformed and the spot-wise standard deviation, arithmetic mean, and coefficient of variation (CV) values of the standard abundance values were calculated for each spot. The protein spots, avoiding unclear and over-dense spots with a differential abundance greater than 1.5 (LOD>1.5), were submitted for mass spectrometry identification. 61 cases were included.
Mass Spectrometry AnalysisMALDI-TOF-TOF data were acquired using an Autoflex speed™ III 200 TOF-MS (Bruker Daltonik, Germany) equipped with a Smart Beam™ laser and a LIFT-TOF/TOF unit. Data acquisition and data processing were performed using the Flex Control 3.0 and Flex Analysis 3.0 software (Bruker Daltonik). All of the spectra were obtained using the default mode with a UV wavelength of 355 nm, acceleration voltage of 20 kV, repetition rate of 200 Hz and best detection resolution of 1500 Da. The scanning quality range was 700–3200 Da.
Spectral Analysis and Protein IdentificationPost-acquisition two step calibration was automatically performed in FlexAnalysis using a standard peptide calibration mixture (Bruker Daltonics) for external calibration, and then, an additional post-acquisition internal calibration was performed to obtain better mass accuracies. Ubiquitously presented auto-digested tryptic mass values visible in all spectra were employed as the internal calibration. The background masses (matrix, metal adducts, tryptic peptides from contaminating α-keratins) were automatically subtracted from the selected list and not carried on for the further analysis. Additionally, irrelevant picks derived from putative contamination were removed by pairwise comparison of all of the analyzed spectra.7) Dalton flexAnalysis (Bruker) was used to filter the baseline peaks and identify the signal peaks. For protein identification, peptide masses were transferred to the BioTools 3.2 interface (Bruker Daltonik) to search in NCBI databases using an in-house MASCOT search engine (version 2.3.0.2, Matrix Science Ltd.). The following conditions were queried: 1) Peptide mass range: 800–4000 Da; 2) Error between the apparent isoelectric point (PI) and relative molecular weight values: unlimited; 3) Primary MS quality error: 50 ppm; 4) Secondary MS quality error: 0.5 Da; 5) Enzyme fragment incomplete site (missing enzyme site): 1; 6) Species: rattus; 7) Charge: +1; 8) Isotope peaks: monoisotopic; 9) Global modification: Carbamidomethyl; and 10) Variable modification: Oxidation. Protein identification was confirmed according to the sequence information obtained by MS/MS analysis in the “LIFT” mode. When the protein score approached a threshold of 51, LIFT analysis was performed as an additional average to determine the protein.8) The integrated analysis of compound identification of TT and proteomic data with Cytoscape 3.2.1 software.
Western Blotting AnalysisFor validation of the MALDI-TOF-TOF MS results, PYC, C1QBP and KNT1 were selected for use in perform a Western blot assay. The remaining renal lysates were subjected to SDS polyacrylamide gel electrophoresis (PAGE) using 12% polyacrylamide resolving gels, and precisely 40 µg of protein was loaded onto the gels. After electrophoresis, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, which were then blocked with 5% nonfat dry milk in phosphate buffered saline (PBS)-0.05% Tween-20 (PBS-T) for 1 h at room temperature and incubated at 4°C with gentle shaking overnight with primary antibodies (PYC, 16588-1-AP, 1 : 1000; C1QBP, 24474-1-AP, 1 : 1200; KNT1, 11926-1-AP, 1 : 2000, Proteintech, Wuhan, China). After being washed with PBS-T, the blots were incubated with horseradish peroxidase conjugated to goat anti-rabbit immunoglobulin G (IgG) (1 : 20000) for 1 h. They were then incubated with 0.4 mL of Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany) and exposed for 60 s using Fluor Chem Q system.
Statistical AnalysisStatistical analyses were performed by using SPSS 21.0. All data are presented as the means±standard deviation (S.D.). Data were analyzed by one-way ANOVA, followed by Student’s t-tests. Significant differences were accepted when the p-value was less than 0.05.
An HPLC chromatogram of the TT extracts obtained with ethyl acetate is shown in Fig. 1A. A total of 340 monomeric compounds were identified, including amino acids, carbohydrates, terpenoids, alkaloids, phenylpropanoid, flavonoids, organic acids, indole and its derivatives, phytohormones, fatty acids, and 52 unknown compounds (Figs. 1B and C, Supplementary Table S1). Among them, the main components related to renoprotection in ORG were apparently flavonoids (Isorhamnetin O-rutinoside, Tricetin O-hexoside, Selgin O-hexoside, Hyperin, Luteolin 5-O-hexoside, Isoquercitrin, O-methyl quercetin O-hexoside, hesperetin 5-O-glucoside), choline (acetylcholine and carbacholine) and vitamins (nicotinic acid and D-pantothenic acid).
* p<0.05, vs. Ang II-induced HRGECs; ** p<0.01, vs. ox-LDL-induced HRGECs. (Color figure can be accessed in the online version.)
According to the results of the MTT assay, IC50 of TT was 912.01 mg/L (Fig. 1D) and an appropriate concentration of TT against ox-LDL induced HRGECs was 4 mg/L (Fig. 1E) in vitro. After treatment with ox-LDL for 24 h, the cell viability of HRGECs was decreased by 39.2% (p<0.05). Following TT treatment, the cell viability of HRGECs was increased (63.2% increase after 4 mg/L TT treatment) (p<0.05) (Fig. 1E). Treating HRGECs with ox-LDL for 24 h significantly suppressed their migration (the migration was decreased by 61.3%; p<0.05), which we speculated to be the possible reason for the increased renal permeability in ORG. By contrast, the number of migrated cells was obviously increased after TT treatment (2.33-fold increase after 4 mg/L TT treatment) (p<0.05), which might have contributed to the recovery from ORG (Fig. 2). We do not believe that TT promotes HRGECs migration becuase the migration of HRGECs was weak and TT did not improve the migration of HRGECs in the absence of ox-LDL in our cellular pre-experiments. However, TT released the inhibition of HRGECs migration induced by ox-LDL.
A–G: HE staining of migrated HRGECs. HRGECs were preincubated for 60 min with TT 100 mg/L TT (A), 20 mg/L TT (B), 4 mg/L TT (C), 1 mg/L TT (D) and 0.4 mg/L TT (E) separately and then induced by applying 100 mg/L ox-LDL (H) for 24 h; untreated HRGECs were used as the normal control (G). H: Quantity of migrated cells. * p<0.05, vs. Ang II-induced HRGECs; ** p<0.01, vs. ox-LDL-induced HRGECs. (Color figure can be accessed in the online version.)
The serum glucose and triglyceride levels, and routine urine test values remained unchanged; however, the body weight, body weight gain, blood pressure, serum cystatin C, peripheral renal white adipose tissue weight, and cholesterol of ORG rats were higher than those of control rats (p<0.05). After TT administration for 8 weeks, body weight, body weight gain, peripheral renal white adipose tissue weight blood pressure, serum cystatin C, and cholesterol were decreased (p<0.05). The general status of rats is shown in Table 1.
| TT | ORG | Control | |
|---|---|---|---|
| Final body weight, g | 838.26±18.10# | 1015.97±167.53* | 679.47±21.24 |
| Body-weight gain, g/d | 2.88±0.40# | 3.36±0.38* | 2.03±0.19 |
| Systolic/diastolic pressure, mmHg | 154.60±13.12/117.43±12.74# | 171.63±10.59/132.25±10.21* | 135.13±12.64/100.86±9.62 |
| Serum cystatin C, mg/L | 0.16±0.02# | 0.23±0.04* | 0.11±0.02 |
| Peripheral renal white adipose tissue weight, g | 2.75±0.31# | 3.81±0.32* | 0.67±0.08 |
| Glucose, mmol/L | 4.25±0.54 | 4.27±0.31 | 4.16±0.38 |
| Triglyceride, mmol/L | 0.80±0.16 | 0.85±0.22 | 0.76±0.11 |
| Cholesterol, mmol/L | 1.68±0.32# | 2.13±0.29* | 1.45±0.21 |
Data are mean±S.D. * p<0.05, vs. control group; # p<0.05, vs. ORG group.
HE staining, Masson staining and TEM observation showed the renal morphological change (Fig. 3). HE staining showed glomerular hypertrophy, a widened mesangial region and partial focal and segmental glomerulosclerosis in ORG rats, and Masson staining showed darker collagen deposition in the interstitium. After 8 weeks of TT administration, the renal morphology improved and collagen deposition decreased.
Arrows show the glomerular hypertrophy, widened mesangial region and collagen deposition in the interstitium. Scale bar=50 µm. (Color figure can be accessed in the online version.)
The mechanism underlying the association between overnutrition and renal lesions remains unclear. Identification of the molecular changes and biomarkers in obesity nephropathy is very demanding, and as a consequence, identification of novel therapeutic targets and determining options for its management are of vital importance. Therefore, in the present study, 2-DE PAGE/silver staining profiling was used to screen for differentially expressed proteins that were associated with ORG.
Approximately 2000–2292 protein spots were separated in the 2-DE gels/silver staining profiling (Figs. 4A–D). Among them, the abundances of 140 proteins were increased and those of 101 proteins were decreased between ORG rats and control rats that receiveda standard chow diet; 137 proteins were decreased and 118 proteins increased after the TT treatment for 8 weeks (Supplementary Table S2). Among these proteins, 61 proteins were further identified using MALDI-TOF-TOF MS, and 35 proteins were studied after repetitive points were excluded (Table 2). After molecular categorization and primary functional analysis, the possible corresponding pathways are shown in Fig. 4E. The relationship between the compound identification of TT and proteomic data was shown in Fig. 4F. Three proteins (PYC, C1QBP and KNT1) were selected to validate the results of MALDI-TOF-TOF mass spectrometry by Western blotting (Fig. 5).
A and B: differences in expression between the kidney of the ORG group and control group; C and D: differences in expression between the kidneys of the TT group and ORG group; E: the possible corresponding pathways; F: the relationship between the compound identification of TT and proteomic data. (Color figure can be accessed in the online version.)
| Name of protein | Abbrev. | Spot No. | Fold change (ORG/Control) | Spot No. | Fold change (TT/ORG) | MASCOT score | MW | PI | Function |
|---|---|---|---|---|---|---|---|---|---|
| Actin | ACTB | K21, K52, K60 | 1.977+, 4.217+, 1000000+ | — | — | 491, 254, 148 | 42052 | 5.29 | Cytoskeleton |
| Alpha-aminoadipic semialdehyde dehydrogenase | AL7A1 | — | — | I09 | 4.296− | 245 | 25313 | 7.72 | Cellular osmolyte and methyl donor |
| Alpha-enolase | ENOA | K29, K43 | 3.580+, 1000000+ | — | — | 291, 377 | 47440 | 6.16 | Energy generation (Glycolysis) |
| Alcohol dehydrogenase [NADP+] | AK1A1 | K74 | 3.241+ | — | — | 418 | 36711 | 6.84 | Catalyzes reduction of aldehydes to alcohols and activates procarcinogens |
| ATP synthase subunit beta | ATPB | K51, K82 | 3.532+, 3.492+ | I53, I87 | 3.262−, 1000000− | 773, 237 | 56318 | 5.19 | Energy generation |
| ATP synthase subunit gamma | ATPG | K64 | 1.753+ | — | — | 232 | 30229 | 8.87 | Energy generation |
| Cathepsin B | CATB | K103 | 1.677+ | — | — | 464 | 38358 | 5.36 | Intracellular degradation and turnover of proteins |
| Delta-1-pyrroline-5-carboxylate dehydrogenase | AL4A1 | K61 | 3.921+ | I65 | 4.008− | 348 | 62286 | 7.14 | Energy generation |
| Fructose-1,6-bisphosphatase 1 | F16P1 | K35 | 3.430+ | — | — | 453 | 40040 | 5.54 | Energy generation (Glycogen synthesis) |
| Fructose-bisphosphate aldolase B | ALDOB | K71 | 6.635+ | — | — | 297 | 40049 | 8.66 | Energy generation |
| Glutamate dehydrogenase 1 | DHE3 | K08 | 2.454+ | I15 | 5.500− | 341 | 61719 | 8.05 | Amino acid metabolism |
| Glyceraldehyde-3-phosphate dehydrogenase | G3P | K46 | 4.210+ | — | — | 312 | 36090 | 8.14 | Energy generation |
| Heat shock protein HSP 90 | HS90B | K14 | 4.673+ | — | — | 261 | 83571 | 4.97 | Molecular chaperone |
| NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10 | NDUAA10 | K39 | 2.429+ | — | — | 501 | 40753 | 7.64 | Energy generation |
| Pyruvate carboxylase | PYC | K01, K03 | 2.246+, 3.282+ | I01, I05 | 2.137−, 1.964− | 450, 395 | 130436 | 6.34 | Energy generation (Gluconeogenesis) |
| Phosphoenolpyruvate carboxykinase [GTP] | PCKGC | K02 | 1.853+ | J22 | 1.588+ | 245 | 70112 | 6.09 | Energy generation |
| Peroxiredoxin-1 | PRDX1 | K112 | 3.157+ | — | — | 497 | 22323 | 8.27 | Redox regulation |
| Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 2 | PYRD2 | — | — | I40 | 2.189− | 304 | 63410 | 8.42 | Redox regulation |
| Spectrin alpha chain | SPTA2 | K12 | 1.686+ | I20 | 1.719− | 454 | 285261 | 5.20 | Cytoskeleton |
| Alpha-1-antiproteinase | A1AT | L26, L32 | 1000000−, 6.982− | J41 | 1000000+ | 518 | 46278 | 5.70 | Acute phase reactants and inflammation |
| Alpha-1-macroglobulin | A1M | L57, L58 | 1000000−, 5.344− | J25, J77 | 1000000+, 8.605+ | 142, 336, 255 | 168388 | 6.46 | Protease inhibitor |
| Chloride intracellular channel protein 4 | CLIC4 | L74 | 2.227− | — | — | 329 | 28843 | 5.92 | Angiogenesis |
| Complement component 1 Q subcomponent-binding protein | C1QBP | L70 | 2.507− | J89 | 2.559+ | 247 | 31320 | 4.77 | Immunity |
| C-Reactive protein | CRP | L76 | 4.582− | — | — | 137 | 25737 | 4.89 | Immunity |
| Endoplasmin | ENPL | — | — | J05 | 3.112+ | 397 | 92998 | 4.72 | Molecular chaperone |
| Fibrinogen gamma chain | FIBG | L37 | 1000000− | J52 | 1000000+ | 470 | 51228 | 5.62 | Hemostasis |
| Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 | GBB1 | — | — | J93 | 1000000+ | 299 | 38151 | 5.60 | Transmembrane signaling |
| Hemopexin | HEMO | L06 | 3.312− | J11 | 3.802+ | 83 | 52060 | 7.58 | Acute phase reactants |
| Major urinary protein | MUP | L94, L97, L98, L99 | 5.418−, 4.809−, 2.503−, 1.908− | J117 | 2.517+ | 262, 269, 269 | 21009 | 5.85 | Bind and release pheromones |
| Peroxiredoxin-2 | PRDX2 | L88 | 1000000− | — | — | 308 | 21941 | 5.34 | Redox regulation |
| Serine protease inhibitor A3L | SPA3L | L14 | 1000000− | — | — | 450 | 46419 | 5.48 | Protease inhibitor |
| Serine protease inhibitor A3N | SPA3N | L24 | 1000000− | — | — | 524 | 46793 | 5.33 | Protease inhibitor |
| Sulfotransferase 1C2A | S1C2A | L92 | 7.002− | — | — | 229 | 35178 | 7.00 | Sulfate metabolism and detoxication |
| Tubulin alpha-1B chain | TBA1B | L22 | 4.698− | J33 | 4.805+ | 364 | 50804 | 4.94 | Cytoskeleton |
| T-Kininogen 1 | KNT1 | — | — | J20 | 1000000+ | 315 | 48828 | 6.08 | Induction of hypotension, natriuresis and diuresis |
The differentially expressed protein profile in kidneys of ORG rats was identified using two-dimension electrophoresis coupled with MALDI-TOF-TOF MS. This table shows the detailed description of all the differentially expressed proteins along with their full name, abbreviated name, accession number, Ratio of HFD/Control & HFD/TT, MASCOT score, mass, pI values and function.
* p<0.05, vs. control group; # p<0.05, vs. control group.
Some proteins were identified in multiple spots, which might reflect posttranslational protein modification processing. Protein spots K51, K82, I53, I87 and K64 were simultaneously identified as ATP synthase; spots L94, L97, L98, L99 and J117 were identified as a major urinary protein; spots K01, K03, I01 and I05 were identified as a pyruvate carboxylase (PYC); spots L57, L58, J25 and J77 were identified as α-1-macroglobulin; spots L26, L32 and J41 were identified as α-1-antiproteinase; and spots K21, K52 and K60 were simultaneously identified as actin. Among them, the proteins involved in energy metabolism (including the citrate cycle, pyruvate metabolism and oxidative phosphorylation) were the most abundant, such as ATP synthase (K51, K82, I53, I87 and K64), α-enolase (K29 and K43), δ-1-pyrroline-5-carboxylate dehydrogenase (K61 and I65), fructose-1,6-bisphosphatase 1 (K35), fructose-bisphosphate aldolase B (K71), glyceraldehyde-3-phosphate dehydrogenase (K46), reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (K39), PYC (K01, K03, I01 and I05) and phosphoenolpyruvate carboxykinase [GTP] (K02 and J22). The next most abundant were proteins related to acute phase reactants and immunity, such as α-1-antiproteinase (L26, L32 and J41), complement component 1 Q subcomponent-binding protein (L70 and J89), C-reactive protein (L76) and hemopexin (L06 and J11); the cytoskeleton, such as actin (K21, K52 and K60), spectrin (K12 and I20) and tubulin (L22 and J33); redox regulation, such as peroxiredoxin-1 (K112), pyridine nucleotide-disulfide oxidoreductase domain (I40), peroxiredoxin-2 (L88) and alcohol dehydrogenase [NADP+] (K74); and protease inhibitor, such as serine protease inhibitors (L14 and L24) and α-1-macroglobulin (L57, L58, J25 and J77). Other proteins included molecular chaperones (heat shock protein HSP 90 and endoplasmin) and those involved with signaling and pheromone transmission (major urinary protein, guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit β-1 and T-kininogen 1), amino acid metabolism and degradation (glutamate dehydrogenase 1 and Cathepsin B) and hemostasis (fibrinogen γ chain).
Long-term high fat diets induced increased renal energy metabolism and cytoskeletal hypertrophy. The increased energy metabolism, on the one hand, accelerated the generation of ATP and met the renal tissue needs. On the other hand, a large increase in energy metabolism might lead to hypoxia due to excessive oxygen consumption, accumulation of metabolic wastes and mitochondrial damage. Several studies reported increased acetylation of PYC and enhanced PYC activity in the liver of high fat diet-induced mice, which are considered to be contributing factors in the development of diabetes.9–11) Similar results were observed for the PYC level in the kidney in the present study. The activity of PYC represents the level of renal gluconeogenesis, which is usually at a low level and enhanced at the time of starvation. In this study, excessive intake of fat increased free fatty acids in the serum and enhanced gluconeogenesis in the kidney. The enhancement of renal gluconeogenesis may promote the secretion of ammonia in renal tubules and osmotic diuresis. Furthermore, recent studies suggest that PYC is involved in tumorigenesis.12) Our results suggested that the PYC level was effectively decreased in the kidney after the TT extract treatment, which was helpful to maintain renal function and reduce the risk of developing diabetes and cancer in rats. The increased metabolic demands in ORG rats led to renal cytoskeletal hyperplasia and glomerular hyperfiltration,13) with compensatory hypertrophy of the kidney and glomerulus.14) Cytoskeletal hypertrophy led to decreased migration, podocyte lesions and injurd glomerular filtration barrier. Excessive proliferation of actin often changes the ability of podocyte foot processes to respond to dynamic changes in the pressure and shape of the capillary walls. After more podocyte loss and glomerulosclerosis, podocyte hypertrophy occurred with glomerular capillary hypertension ultimately leading to progressive glomerulosclerosis.15) Changes in NDUAA10 and AK1A1 expression have also been identified in some proteomic studies performed on hypertensive rats,16–20) revealing the association between hypertension and the development of ORG.
Meanwhile, overnutrition led to decreased expression of proteins related to acute phase reactants, immunity, hemostasis, transmembrane signaling and pheromone transmission, suggesting a decreased response to stress, low immunity and hemorrhagic tendency. Five spots were simultaneously identified as major urinary proteins (Mup) in the present study, also known as α2u-globulins, which have often been viewed as the cause of proteinuria and are known to be pheromone transporters. Mup positively influences the production of testosterone, growth hormone and thyroxine.21) Insufficient Mup implies a weakened capability for stress reactions and reduction of male sexual capacity, and Mup has been shown to be associated with the regulation of energy expenditure in mice. It was reported that genetically induced obese, diabetic mice produce thirty times less Mup RNA than their lean siblings,22) which was consistent with our study. C1QBP is considered to be an important regulator of lipid homeostasis that regulates both aerobic and anaerobic energy metabolism.23) Simultaneously, C1QBP regulates monocyte/macrophage chemotaxis and aggregation activities via activation of protein kinase C (PKC) and mediates inflammation and immune responses.24) Our data suggested a decreased C1QBP level in the kidney induced by the chronic high fat diet. The C1QBP level was effectively increased in the kidney after TT extract administration, which we believe, was beneficial for maintaining lipid homeostasis and improving the immune response of rats.
Notably, the alcohol dehydrogenase [NADP+] and cathepsin B levels were increased. Alcohol dehydrogenase [NADP+] catalyzes the reduction of aldehydes to alcohols and activates procarcinogens. Cathepsin B is a key enzymatic protein in lysosomal permeabilization and proapoptotic protein for mediating mitochondrial dysfunction25) and has been proposed to be a potentially effective biomarker for a variety of cancers.26) The increase of alcohol dehydrogenase [NADP+] and cathepsin B indicated that the possibility of tumorigenesis was increased. These pathological changes might be the etiology of nephropathy.
After TT extract administration for 8 weeks, body weight, blood pressure and serum cholesterol were decreased and some of the proteins involved in energy metabolism were decreased. Simultaneously, the expression levels of proteins related to acute phase reactants, immunity, hemostasis, transmembrane signaling and pheromone transmission caused by overnutrition were increased. The increase of Mup indicated elevation of androgen levels. T-kininogen 1 (KNT1) is a well-recognized vasodilator substance that belongs to the kallikrein-kinin system. KNT1 participates in induction of hypotension, natriuresis and diuresis. Although the chronic high fat diet did not induce a lowered level of KNT1, notably, KNT1 was greatly increased (1000000-fold change compared to ORG rats) after 8 weeks of TT administration in this study. Since TT demonstrated a powerful efficacy on ORG, we hypothesized that KNT1 might be one of the targets of TT.
In this study, 340 monomeric compounds were identified from TT. In vitro, the IC50 of TT was 912.01 mg/L and an appropriate concentration of TT against ox-LDL was 4 mg/L. TT significantly increased the viability and migration of HRGECs.
Molecular categorization and functional analysis suggested that excessive energy metabolism, decreased response to stress and lower immunity might be the potential etiologies of ORG. Conclusively, ORG rats demonstrated tumorigenesis and a hemorrhagic tendency. The health condition of ORG rats improved after TT treatment for 8 weeks. Reducing hypertension, decreasing energy consumption and the hemorrhagic tendency, and improving acute phase reactants and immunity might be the targets of TT.
This work was funded by National Natural Science Foundation of China No. 81573916 and No. 81673807; and Shandong Province ‘Taishan Scholar’ Construction Project Funds No. 2018-35.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
