Lycium barbarum polysaccharides attenuate rat anti-Thy-1 glomerulonephritis through mediating pyruvate dehydrogenase

Lycium barbarum polysaccharides attenuate rat anti-Thy-1 glomerulonephritis through mediating pyruvate dehydrogenase

Biomedicine & Pharmacotherapy 116 (2019) 109020 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevi...

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Biomedicine & Pharmacotherapy 116 (2019) 109020

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage:

Lycium barbarum polysaccharides attenuate rat anti-Thy-1 glomerulonephritis through mediating pyruvate dehydrogenase ⁎⁎

Ting Lua,1, Wen-e Zhaob,1, Fang Zhangc,1, Xiaohong Qid, Ye Yanga,e, , Chunyan Gua,

T ⁎


School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, 210023, Nanjing, China Department of Analysis and Testing Center, Nanjing Medical University, 210029, Nanjing, China c College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China d Department of Pathophysiology, Nanjing Medical University, Nanjing, 211166, China e School of Holistic Integrative Medicine, Nanjing University of Chinese Medicine, 210023, Nanjing, China b



Keywords: Lycium barbarum polysaccharides Anti-Thy 1 nephritis Glomerulonephritis Pyruvate metabolism

Glomerulonephritis is the major cause of chronic kidney disease characterized by mesangial cell proliferation and extracellular matrix deposition. The aim of this study was to investigate the effects of Lycium barbarum polysaccharides (LBPs) on anti-Thy 1 nephritis rats and explore the protective mechanism of LBPs. After the model of glomerulonephritis created by injecting anti-thymocyte serum (ATS), rats were treated with enalapril or LBPs for 8 weeks. The therapeutic effect was evaluated by detection of renal-related biochemical parameters, histological observation and markers of renal fibrosis. Moreover, RNA-seq analysis and experiments in vitro were employed to explore the signaling pathway involved in LBPs treatment. The results found that LBPs treatment significantly suppressed ATS-caused increment at levels of blood urea nitrogen, creatinine, proteinuria, PAI-1 protein expression, glomerular mesangial cell proliferation and extracellular matrix hyperplasia, along with reduction of creatinine clearance. RNA sequencing showed pyruvate metabolism acting as a potential signaling pathway, which was evidenced by the inhibitory effect on up-regulation of pyruvate dehydrogenase and PAI-1 levels via treatment with LBPs in vitro. LBPs are the promising agents for the management of glomerulonephritis through pyruvate metabolism signaling pathway.

1. Introduction Chronic kidney disease (CKD) is a public health problem that affects people from all over the world [1]. Complications of CKD include cardiovascular disease [2], hyperlipidemia [3], metabolic acidosis [4] and anemia [5], which contribute to the progression of CKD [6,7].The chronic glomerulonephritis (CG) is one of the leading causes of CKD, with high morbidity and mortality [8]. In clinical practice, CG manifests as proteinuria and initial mesangial cell proliferation, follows by different degrees of renal impairment, and eventually develops into

chronic renal failure [9–11]. In addition, CG is secondary to renal fibrogenesis with feature of abnormal deposition of extracellular matrix (ECM) [12]. As reported, podocyte injury occurs in glomerular diseases of different pathological types, accompanied by increased mesangial hyperplasia and mesangialstroma dilatation, which aggravates glomerulonephritis [13]. Mesangial proliferative glomerulonephritis (MsPGN) is a group of glomerular diseases characterized by diffuse mesangial cell proliferation and various degrees of mesangial matrix proliferation [14]. Currently, numerous western medicines have been proved to slow

Abbreviations: LBPs, Lycium barbarum polysaccharides; ATS, anti-thymocyte serum; Ena, enalapril; PAI-1, plasminogen activator inhibitor-1; CKD, chronic kidney disease; CG, chronic glomerulonephritis; ECM, extracellular matrix; MsGPN, Mesangial proliferative glomerulonephritis; MMC, murine mesangial cells; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; NC, normal control group; PBS, phosphate-buffered saline; DC, disease control; BUN, blood urea nitrogen; PAS, periodic acid Schiff; LG, low-glucose; HG, high-glucose; DCA, dichloroacetate sodium; PVDF, poly (vinylidene fluoride); SDS, Sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ccr, creatinine clearance; DEGs, differentially expressed genes; BP, biological process; CC, cellular component; MF, molecular function; DAG, directed acyclic graph; PDC, pyruvate dehydrogenase complex; PDKs, PDC kinases; PDPs, PDC phosphatases ⁎ Corresponding author. ⁎⁎ Corresponding author at: School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, 210023, Nanjing, China. E-mail addresses: [email protected] (Y. Yang), [email protected] (C. Gu). 1 These authors have contributed equally to this work. Received 19 February 2019; Received in revised form 20 May 2019; Accepted 21 May 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (

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Table 1 Effects of LBPs on renal function. Renal Function Plasma BUN (mmol/L) Plasma creatinine (μmol/L) Ccr (ml/min)

NC 5.31 ± 0.16 29.5 ± 0.93 2.38 ± 0.93


Ena *

6.01 ± 0.74 35.83 ± 4.31 2.04 ± 0.94

8.53 ± 1.67 37.33 ± 2.07* 1.32 ± 0.57*

One-way analysis of variance (ANOVA) was carried out and significant changes are shown.


LBPs #

P < 0.05 vs NC;


6.14 ± 0.77# 33.17 ± 9.41 2.04 ± 0.54

P < 0.05 vs DC.

Fig. 1. The levels of 24-h proteinuria in rats with anti-Thy 1 glomerulonephritis for 8 weeks. One-way analysis of variance (ANOVA) was carried out and significant differences among groups were shown in detail with P values.

2. Materials and methods

the progression of chronic nephropathy, but toxic side effects largely limit their clinical applications [15]. Chinese herbal medicines have abundant resources, which are widely used to treat kidney diseases for their features of multi-component, multi-target, and less side effects [16]. Lycium barbarum L. is an old shrub herb distributed in China, of which the ripe-dried berries have been officially recorded as “Gouqizi”, also called “Goji, Gojiberry, or Wolfberry” in English [17]. Polysaccharides are major components of Lycium barbarum with a variety of bioactivity [18]. Neuroprotective role of Lycium barbarum polysaccharides (LBPs) against ischemic injury was attributed to their dual roles in activation of NR2A and inhibition of NR2B signaling pathways [19]. Recently, a new study showed that LBPs exerted the positive effect on macrophage function [20]. In addition, LBPs were reported to improve cadmium induced damage, hyperglycemia-enhanced ischemic brain damage, as well as humoral and cellular immunity [21–23]. Many Chinese medicines, like Rhubarb, have been reported to treat chronic nephritis [24]. Meanwhile, polysaccharide extract of Chinese medicine, such as Astragalus polysaccharides, has been attracted attention in protecting against glomerulonephritis [25]. LBPs have been illustrated to ameliorate renal injury and inflammatory reaction in rabbits with diabetic nephropathy [26]. However, there is very little research focused on LBPs in renal fibrosis. In this study, to determine whether LBPs could be promising agents for the treatment of MsPGN, we used chronic anti-Thy 1 nephritis rats as a model of human MsPGN and found the possible mechanisms of LBPs treatment in glomerulonephritis.

2.1. Reagents Lycium barbarum fruits from Ningxia were fully dried and pulverized into powder. The preparation method comprises the following steps [27]: a proper amount of Lycium barbarum powder was weighed, followed by adding 80% ethanol according to a volume ratio of 1:3, refluxing at 80 °C for 2 h (repeating for 2 times), filtering with gauze, and taking the residue. The residues were further refluxed with petroleum ether: acetone solvent (1:1, in vol) at a volume ratio of 1:10 to decolorize at 55 °C for 1.5 h (repeating for 2 times), then filtered with gauze, and took the filter residue. Next, the residues were extracted 2 times with ultrapure water at a volume ratio of 1:10, followed by refluxing for 2 h at 105 °C, and filtering with gauze to obtain a polysaccharide filtrate. The filtrate was concentrated at 65 °C to about 1/3 of original volume, then precipitated overnight with anhydrous ethanol (1:3, in vol), dehydrated with anhydrous ethanol and acetone, and finally dried in a 50 °C vacuum drying chamber to obtain crude polysaccharides. The crude polysaccharides were dissolved in appropriate amount of ultrapure water and were deproteinized by Sevage method [28]. The experimental methods were as follows: the polysaccharides solution was placed in a large volume funnel, added with chloroform in the volume of 1/5 polysaccharides solution and the n-butanol in the volume of 1/25 polysaccharides solution, rocked repeatedly for 10 min, then centrifuged at 4 °C, 3500 rpm for 15 min to collect the upper polysaccharides liquid. The above steps were repeated for 10–15 times 2

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Fig. 2. Transmission electron microscopy study on renal cotex in experimental animals. (a) The ultrastructure of glomerulus in NC group. (b) The ultrastructure of glomerulus in DC group. (c) The ultrastructure of glomerulus in Ena treatment group. (d) The ultrastructure of glomerulus in LBPs treatment group. Original magnification × 6800. (e) The scores of renal glomerular basement membrane thickness. (f) The scores of foot processes width. One-way analysis of variance (ANOVA) was carried out. *P < 0.05 vs NC; #P < 0.05 vs DC.

2.3. Cell culture

until there was no obvious precipitation in the lower layer after centrifugation, and then dried to obtain the purified Lycium barbarum polysaccharides. Enalapril was supplied by SZYY Group Pharmaceutical Limited (Jiangsu, China). Dichloroacetate sodium was supplied by Adamas-beta (Shanghai, China). Unless otherwise indicated, other materials and chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Murine mesangial cells (MMCs) were kindly donated by Nanjing Key Laboratory of Pediatrics. The cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM; BI, Israel) of low glucose containing 10% fetal bovine serum (FBS; BI, Israel), 100 U/mL Penicillin and 100 μg/mL streptomycin at 37 °C in a 5% CO2 incubator. The cells grew in a 100 mm dish and were subcultured when rising to 80%–90% fusions. After a few days of adaptive culture, different dishes with MMCs were treated with high glucose in the presence or absence of LBPs or dichloroacetate sodium (DCA) for 48 h, and then the cells were collected for western blotting analysis.

2.2. Animals Eight-week-old male Sprague Dawley rats (specific pathogen free) weighing 180 g to 200 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Rats were maintained under standard conditions at 12 h light-dark cycle and a temperature-controlled environment (24 ± 2 °C), with free access to water and food in the Animal Center of Nanjing University of Chinese Medicine. All animals were handled in accordance with government-published recommendations for the Care and Use of laboratory animals. The animal experiment was approved by the Institutional Ethics Review Boards of Nanjing University of Chinese Medicine (Ethics number ACU-14 (20151123)).

2.4. Experimental groups and design Twenty-eight male SD rats were randomly assigned to four groups of 7 rats each: (1) normal control (NC) group, injection of equal volumes of phosphate-buffered saline (PBS); (2) disease control (DC) group, injection of ATS (1.75 mg/kg body weight BW); (3) enalapril (Ena) group (positive group), injection of ATS (1.75 mg/kg body weight BW) with oral gavage Ena (20 mg/kg/day); (4) LBPs group, injection of ATS with oral gavage LBPs (80 mg/kg/day). The rat anti-Thy 1 nephritis model was established by tail vein injection of ATS for one week. Then rats were given Ena or LBPs once daily for 8 weeks. The ATS was produced by immunizing New Zealand Rabbit with rat’s thymocyte cell 3

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Fig. 3. Effects of LBPs on improving renal fibrosis in experimental animals. (a) Light microscopy of glomerulus stained with PAS staining in NC group. (b) Light microscopy of glomerulus stained with PAS staining in DC group. (c) Light microscopy of glomerulus stained with PAS staining in Ena group. (d) Light microscopy of glomerulus stained with PAS staining in LBPs group. Original magnification × 400. (e) The score of glomerular extracellular matrix. (f) The level of PAI-1 protein expression analyzed by Western blot. The relative expression level of PAI-1 protein was measured by using GAPDH densitometric intensity as control. One-way analysis of variance (ANOVA) was carried out. *P < 0.05 vs NC; # P < 0.05 vs DC.

(24 h x60 min) [35]. The kidney tissues of rats were cut into several sections, and the pieces of cortex were fixed in 10% neutral-buffered formalin (pH 7.4) for periodic acid Schiff (PAS) staining and in glutaraldehyde for electron microscopy, respectively. The remaining cortices were frozen in liquid nitrogen rapidly and then stored at -80 °C for protein and RNA extraction. The mesangial cell injury was induced by high glucose culture, which stimulated the proliferation of mesangial cells, release of inflammatory factor and collagen accumulation [36,37]. MMCs were divided into 6 dishes: (1) the low-glucose (LG) group cultured in the lowglucose medium (5.6 mM); (2) the high-glucose (HG) group cultured in the high-glucose medium (25 mM) [38]; (3) the LBPs or LBPs 1 group cultured with LBPs (50 μg/mL) in the high-glucose medium; (4) the LBPs 2 group cultured with LBPs (100 μg/mL) in the high-glucose medium; (5) DCA group cultured with DCA (5 mM) in the high-glucose medium; (6) LBPs + DCA group cultured with LBPs (50 μg/mL) and DCA (5 mM) in the high-glucose medium. The doses of LBPs [39] and DCA [40] in cell experiments selected on the basis of previous literature reports. After intervention for 48 h, the cell proteins were extracted for western blot analysis.

suspension [29], which binds to a Thy 1-like antigen on the surface of renal mesangial cells and leads to mesangial cell proliferation, accumulation of the mesangial matrix followed by proteinuria [30,31]. The doses of LBPs were selected on the basis of previous literature reports and partly our pilot studies [32,33]. Biochemical and histopathological approaches were taken to trace the progression of chronic nephritis for each group after the model established. On day 7 after injection of ATS, urine samples (24 h) were collected from rats housed in metabolic cages once a week for 8 weeks. The urine samples were collected and frozen at −20 °C, especially, 24 h urinary protein was detected by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). After 8 weeks of treatment, the rats were anesthetized and blood was immediately collected from aorta abdominalis. Blood urea nitrogen (BUN), creatinine levels and calculated creatinine clearance (Ccr) are important indicators for evaluating renal function [34]. The plasma BUN and creatinine levels were detected with standard laboratory procedures. Urine creatinine was measured by QuantiChrom™ creatinine assay kit (Bio Assay System, Hayward, CA, USA). Ccr was calculated using the formula (urine creatinine levels/plasma creatinine levels) x 24 urine volume (ML) / 4

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Fig. 4. Large scale gene expression profiles between DC and LBPs groups. (a) The volcano plot was used for enabling visualization of the relationship between fold change and statistical significance. The red point in the plot represented upregulated RNA (fold change > 2, P < 0.05) and the green point indicated downregulated RNA (fold change < 2, P < 0.05), while the blue point demonstrated RNA with no statistical differences (fold change < 2, P > 0.05). (b) The overlap represented 19 differentially expressed gene common to the three different groups in the Venn diagram. (c) GO classification of target genes. The 30 most significantly enriched GO terms for whole kidney. The first 10 bits of GO enrichment analysis were chosen as the main nodes of DAG, and the depth of color represented the degree of enrichment. (d) KEGG pathway classification of target genes. The Y-axis showed the name of pathway and the X-axis represented the Rich factor. The point size indicated the number of differentially expressed genes in one pathway, and the color of the point corresponded to the range of the Q value. The 20 most significant up-regulated pathways (LBPs VS DC).

field occupying each glomerulus, and grade from 0 to 4 (0, 0–4%; 1, 5–24%; 2, 25–49%; 3, 50–74%; 4, 75% to more) [31].

2.5. Histological analysis Electron microscopy is a common method for the morphological diagnosis of glomerular diseases. Renal cortices were pre-fixed with 2.5% glutaraldehyde, dehydrated in an ethanol series and embedded in an epoxy resin. Then the tissues were polymerizated and made into ultrathin sections for electron microscopy [41]. The sections were visualized and imaged using a transmission electron microscope (Tecnai G2 Spirit Bio TWIN, Hillsboro, Oregon, USA). Morphological changes of renal cortices were observed by electron microscopy, including thickness of glomerular basement membrane, numbers of mesangial cells, structure of foot process, degree of foot process fusion, etc. Renal cortices fixed with 10% paraformaldehyde was dehydrated, embedded in paraffin, cut into 4-μm-thick sections, and stained with periodic acid-Schiff (PAS) for morphological analysis [42]. The intensity of glomerular damage was determined in 20 glomeruli per sample in a blinded manner. The images of glomeruli were captured using an AxioVertA 1 digital camera, and the area of PAS-positive glomeruli was digitally analyzed using a computer-assisted color image analysis system (Image J). The extent of glomerular matrix expansion was scored semi-quantitatively according to area of positive staining

2.6. Western blot analysis To extract protein, renal cortex tissues were homogenized in RIPA lysis buffer (Fcmacs, Jiangsu, China) with protease inhibitor cocktail (Yeasen, Shanghai, China). Protein concentration was determined by using BCA Protein Assay kit (Thermo Fisher, New York, USA). 40 μg of total protein were separated by 12% SDS-PAGE and transferred to PVDF membrane. After blocking with 5% non-fat milk at room temperature for 1 h, blots were probed with primary antibody for rabbit polyclonal anti-plasminogen activator inhibitor-1 (PAI-1) antibody (Abcam, Cambridge, UK), rabbit polyclonal anti-pyruvate dehydrogenase antibody (Cell Signaling Technology, Massachusetts, USA), rabbit monoclonal anti-GAPDH antibody (Cell Signaling, USA) overnight at 4 °C, respectively. Blotting of GAPDH was used as internal control. Subsequently, the membranes were incubated in secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature followed by reaction with chemiluminescence reagent. Proteins were visualized with the High-sig ECL Western Blotting Substrate (Tannon, 5

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Fig. 5. Western blotting indicating that pyruvate metabolism was inhibited by LBPs in vivo and in vitro. (a) Expression of pyruvate dehydrogenase in renal cortex. The relative expression level of pyruvate dehydrogenase protein was measured by using GAPDH densitometric intensity as control. (b–d) The expressions of pyruvate dehydrogenase and PAI-1 in MMCs. The relative expression level of PAI-1 or pyruvate dehydrogenase protein were measured by using GAPDH densitometric intensity as control. LG: 5.6 mM; HG: 25 mM; LBPs 1: 50 μg/mL; LBPs 2: 100 μg/mL; DCA: 5 mM. One-way analysis of variance (ANOVA) was carried out. *P < 0.05 vs NC/LG; #P < 0.05 vs DC/HG; □P < 0.05 vs LBPs.

3. Results

Shanghai, China). The immunostaining band was quantified using Image J software. The relative expression level of PAI-1 or pyruvate dehydrogenase protein were measured by using GAPDH densitometric intensity as control for each sample. This ratio was set at unity for NC or LG samples and other lanes on the same gel are expressed as multiple of this value. All blots were running at least three times.

3.1. LBPs improve the renal function of rats with anti-Thy 1 nephritis and decrease degree of renal fibrosis 3.1.1. Renal function parameters Exposure to ATS led to a significant elevation of plasma BUN and creatinine levels in DC group, as well as remarkable reductive Ccr levels (P < 0.05 vs NC group), which indicated the successful establishment of glomerular injury model. The BUN levels were significantly decreased in LBPs treatment group compared to DC group (P < 0.05). Meanwhile, the levels of Ccr after LBPs treatment were elevated, but not reached statistical significance (Table 1). The DC rats showed a progressive ascend of 24 -h proteinuria during the following 8 weeks compared with NC group. The amount of 24 -h proteinuria was extremely reduced in rats with LBPs treatment relative to the DC rats (P < 0.05) (Fig. 1).

2.7. Transcriptiomic RNA-sequencing The RNA-seq and sample analysis were performed by Xinyinzi Biotech (Shanghai, China). Briefly, mRNA was extracted from the total RNA after removing the rRNA. This project is Rattus_norvegicus ref RNAseq data sequencing and analysis technology service. After constructing RNA sequencing library, Hi Seq X-Ten was utilized for PE150 sequencing. Then, data were analyzed to find differentially expressed genes, following strict data quality control.

3.1.2. Electron microscopy The glomerular structures, including the podocytes, were well maintained in the control samples (Fig. 2a). In contrast, besides diffuse thickening of the glomerular basement membrane, DC group showed extensive fusion and effacement of the foot processes (Fig. 2b). Ena group and LBPs group revealed irregular thickening of the glomerular basement membranes, and fusion and effacement of podocytes in some areas compared to NC group, which indicated that the damage was

2.8. Statistical Results are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to evaluate the data between different groups. In detail, post-hoc analysis of multiple groups with LSD was applied to look for significant changes in the data. For all analyses, the SPSS 20.0 software was employed and P value < 0.05 was considered significant. 6

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(P < 0.05) (Fig. 5c). Further to validate LBPs acting on nephritis through pyruvate dehydrogenase, DCA, as an agonist of pyruvate dehydrogenase by inhibiting pyruvate dehydrogenase kinase, was conducted to stimulate the activation of pyruvate dehydrogenase [45]. We found that LBPs treatment could significantly reduce PAI-1 expression by 42%, which significantly increased approximate 40% by DCA (P < 0.05) (Fig. 5d). Collectively, these results demonstrated that pyruvate dehydrogenase played an important role in the nephritis model, by which LBPs treatment could restore the function of kidney and alleviate renal fibrosis.

partially reversed (Fig. 2c & d). Fig. 2e reflected a graphical representation of renal glomerular basement membrane thickness for each group. Fig. 2f displayed a graphical representation of foot processes width for each group. A significant (P < 0.05) decrease was seen in rats treated with either Ena or LBPs, compared to DC animals (Fig. 2e & f). 3.1.3. PAS staining Representative glomeruli stained with PAS were shown in Fig. 3. Normal tubular structures, mesangial cells and capsular space were observed in the samples of NC group. In contrast with NC group, the glomeruli from the DC rats showed marked accumulation of ECM expressed as PAS-positive material compared to normal glomeruli (Fig. 3a & b). Ena or LBPs treatment produced impressive decreases in glomerular matrix accumulation (Fig. 3c & d). Fig. 3e demonstrated the graphical representation of the matrix score for each group. A significant (P < 0.05) decrease of 48.3% and 47.1% in PAS staining was observedin rats treated with Ena and LBPs respectively, compared with DC (Fig. 3e).

4. Discussion CG is a kidney disorder caused by various pathogenic factors, characterized by edema, hematuria, proteinuria, and hypertension. An important pathological feature of glomerulonephritis is the abnormal proliferation of mesangial cells which ultimately leads to irreversible glomerulosclerosis and even progression to renal failure [46]. MsPGN has been regarded as the most primary glomerulonephritis [47]. Since a rat experimental model for chronically progressive MsPGN was established by Cheng et al [48], some common pathological phenomena have been observed in experimental glomerulonephritis including massive proteinuria, increased expression of collagen, mesangial cell proliferation and mesangial matrix expansion. Anti-Thy 1 nephritis model is recognized as the best animal model to mimic the pathological characteristics of human MsPGN, which produces extensive mesangial cell proliferation and ECM accumulation [49]. In this study, anti-Thy 1 antibody administration have induced typical scenario of mesangial proliferative glomerulonephritis as reflected by the following characteristics: remarkable enhanced levels of proteinuria, serum creatinine and BUN, pronounced depletion of Ccr, along with the overt alteration of histomorphology like mesangial matrix hyperplasia, foot process fusion and ECM dilatation of the glomeruli. In addition, the expression of PAI-1 associated with renal fibrosis was significantly increased in disease animals. LBPs, the most important active component in Lycium barbarum, has been proved to possess the functions of promoting immunity, antiaging, anti-tumor, scavenging free radical, etc. [17,50]. Recently, some experiments in vivo indicatedthat LBPs are the compounds with relevant renal-protective properties [26]. In this study, we investigated the role of LBPs in inhibiting mesangial proliferation and figured out the potential mechanism. LBPs treatment manifested beneficial effects on maintaining renal function, for it produced the marked reduction in urinary protein excretion, serum creatinine and BUN levels, as well as significantly increased Ccr compared to DC group. Meanwhile, pathological changes in impaired kidney after modeling were sharply extenuated in LBPs group. In addition, PAI-1 expression was remarkably decreased in the rats with LBPs treatment. According to the results of RNA-sequencing, we speculated that pyruvate metabolism was the potential signaling pathway in CG, which could be activated by LBPs treatment. The RNA-seq analyses showed pronounced alterations in 293 up-regulated and 204 down-regulated mRNA between LBPs and DC groups. The most significantly up-regulation pathway is pyruvate metabolism. Based on the collected data, we conducted cell experiments for further verification. Consistent with the results in vivo, the results of cell experiments strongly implied that pyruvate dehydrogenase was the target of LBPs, which partially proved by application of DCA. Pyruvate is an intermediate product of glucose metabolism, which is considered to be the most “influential” compound of all energy metabolites and plays an important role in the metabolism of the three nutrients by converting sugar, fat, and amino acids through the circulation of acetyl CoA and tricarboxylic acid [51]. The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible oxidation of pyruvate to acetyl CoA, of which the reaction is primarily regulated by PDC kinases

3.1.4. Western blot analysis Massive studies have confirmed a strong association between elevated levels of PAI-1 and degrees of glomerular sclerosis and renal fibrosis [43]. Excessive PAI-1 protects ECM proteins like Type I collagen from proteolytic degradation and contributes to excessive accumulation of collagen in the kidney [44]. By performing western blotting, we found the expression of PAI-1 significantly increased by 51.3% in DC group compared to NC group (P < 0.05), and LBPs treatment inhibited PAI-1 expression by 19.5% but not reaching statistical significance (Fig. 3f). Taken together, these data indicated that LBPs have apparent effects on improving renal metabolism, renal histopathology and renal fibrosis in experimental anti-thy1-induced CG. 3.2. The potential signaling pathway of LBPs is explored through RNA-seq 3.2.1. RNA-seq analysis In order to gain further insights into the potential signaling pathway involved in LBPs treatment for the nephritis, differentially expressed genes (DEGs) among the groups were obtained and analyzed. As a result, we identified 293 up-regulated and 204 down-regulated mRNA varied remarkably between LBPs and DC groups (Fig. 4a). The Venn diagram showed that 19 overlaps were obtained in three sample types (Fig. 4b). Representative GO (Gene ontology) Biological Process terms selected from the most enriched charts was displayed in Fig. 4c. We identified that the three most significantly enriched GO terms for biological process (BP), cellular component (CC) and molecular function (MF) were related to metabolic process, organelle and catalytic activity, respectively. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification of target genes contained the top 20 most significantly enriched pathways, and the most enriched pathway was focusing on pyruvate metabolism (Fig. 4d). 3.2.2. Western blot analysis On the basis of gene expression profile, we tested the signaling pathway of pyruvate metabolism by western blotting. Consistent with the results of gene expression profile, pyruvate dehydrogenase was readily detected by western blot in kidney cortex tissues of rats with anti-Thy1 nephritis. The expression levels of pyruvate dehydrogenase were significantly decreased by 38.2% in rats treated with LBPs compared to DC group (P < 0.05) (Fig. 5a). To further confirm LBPs improving chronic nephritis by activating pyruvate metabolism signaling pathway, we performed cell experiments in vitro. LBPs significantly blocked the up-regulation of pyruvate dehydrogenase expression by 47.4% induced by high glucose (P < 0.05) (Fig. 5b). We also examined the expression of PAI-1, which was significantly reversed by LBPs 7

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(PDKs) and phosphatases (PDPs) [52]. DCA, as an inhibitor of PDK [53], has been used extensively to promote PDC activity in the laboratory and clinically. Aberrant pyruvate metabolism is of great importance in cancer, neurodegeneration, diabetes, heart failure and other diseases [54–57]. Recent experimental studies have found that both ischemic and nephrotoxic renal injury could cause significant and sustained depletion of pyruvate, and pyruvate therapy is capable of reducing renal injury, which may be due to the decrease of inflammation and preserving the enzymatic machinery essential for cell viability [58,59]. Pyruvate dehydrogenase kinase 4 exerts an inhibitory effect on cisplatin-induced acute renal injury, suggesting that it may be a therapeutic target for alleviating acute renal injury [60].

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5. Conclusion In conclusion, our data demonstrate that LBPs could significantly decrease proteinuria and renal fibrosis in anti-Thy 1 nephritis model. Importantly, this study provides the evidence that LBPs play a key role in protecting kidneys by activating pyruvate metabolic signaling pathway. LBPs supplementation may be a therapeutic strategy for human MsPGN. Author contributions Chunyan Gu and Ye Yang designed the research. Ting Lu wrote the manuscript. Ting Lu, Wen-e Zhao, Fang Zhang and Xiaohong Qi conducted the experiments and the data analysis. Chunyan Gu edited the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare no competing interests. Acknowledgments This work was supported by National Natural Science Foundation of China81670200, 81600177, 81770220 (to YY & CG); Natural Science Foundation of Jiangsu ProvinceBK20161041 (to CG); The 2016 outstanding youth fund of Jiangsu ProvinceBK20160048 (to YY); Innovation Team of Six Talent Peaks Project in Jiangsu ProvinceTDSWYY-015 (to CG); The Priority Academic Program Development of Jiangsu Higher Education Institutions for Chinese Medicine; National Natural Science Foundation of China81773837 (to FZ). References [1] M.C. Starr, S.R. Hingorani, Prematurity and future kidney health, Curr. Opin. Pediatr. 30 (2) (2018) 228–235. [2] L. Di Lullo, A. House, A. Gorini, A. Santoboni, D. Russo, C. Ronco, Chronic kidney disease and cardiovascular complications, Heart Fail. Rev. 20 (3) (2014) 259–272. [3] P.G. Gupta, Alfred Tau Liang Man, Ryan Eyn Kidd Fenwick, Eva K. Tham, YihChung Sabanayagam, Charumathi Wong, Tien Yin Cheng, Ching-Yu Lamoureux, L. Ecosse, Risk of incident cardiovascular disease and cardiovascular risk factors in first and second-generation Indians: the Singapore Indian eye study, Sci. Rep. 8 (1) (2018). [4] C. Chazot, G. Jean, D. Joly, Complications métaboliques de l’insuffisance rénale chronique, Néphrol. Thérap. 13 (6) (2017) 6S30–6S36. [5] W. McClellan, S.L. Aronoff, W.K. Bolton, S. Hood, D.L. Lorber, K.L. Tang, T.F. Tse, B. Wasserman, M. Leiserowitz, The prevalence of anemia in patients with chronic kidney disease, Curr. Med. Res. Opin. 20 (9) (2004) 1501–1510. [6] A. Kanso, R. Thomas, J.R. Sedor, Chronic kidney disease and its complications, Prim. Care Clin. Off. Pract. 35 (2) (2008) 329–344. [7] E. Moţa, Daniela CanăRuiu, Natalia Istrate, Renal Anemia - risk factor for chronic kidney disease, Curr. Health Sci. J. 39 (4) (2013) 214–217. [8] M.-W. Welker, N. Weiler, W.O. Bechstein, E. Herrmann, C. Betz, M. Schöffauer, S. Zeuzem, C. Sarrazin, K. Amann, O. Jung, Key role of renal biopsy in management of progressive chronic kidney disease in liver graft recipients, J. Nephrol. (2018). [9] S. Klahr, G. Schreiner, I. Ichikawa, The progression of renal disease, J. Urol. 141 (3) (1989) 680–681. [10] J.X. Wan, Y. Chen, D.W. Jiang, Clinical efficacy and safety of sequential treatment with alprostadil and beraprost sodium for chronic renal failure induced by chronic


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