Mol Cell Biochem DOI 10.1007/s11010-015-2569-x

The wnt/b-catenin signaling pathway participates in rhein ameliorating kidney injury in DN mice Suyan Duan1 • Yingyi Wu2 • Chuanyan Zhao1 • Mingyu Chen1 • Yanggang Yuan1 Changying Xing1 • Bo Zhang1

•

Received: 4 May 2015 / Accepted: 3 September 2015 Ó Springer Science+Business Media New York 2015

Abstract The present study aimed to investigate the relationship between wnt/b-catenin signaling pathway and kidney impairment in diabetic nephropathy (DN) mice as well as the renoprotective effect of rhein (RH). Mice were randomly divided into four groups (n = 6): db/db mice treated with RH (DN ? RH), db/db mice (DN), db/m mice treated with RH (NC ? RH) and db/m mice (NC). RHtreated groups were administered orally at a daily dose 120 mg/kg. Mice were sacrificed after 12 weeks of treatments. In our study, increased albuminuria, together with weight gain and hyperglycemia was observed in the beginning of the study and continued to increase throughout the length of the study (12 weeks). Histopathologic changes were observed in the DN group. Expectedly, mice receiving the treatment with RH were protected from this injury. Meanwhile, the expression of nephrin, a podocytespecific marker, was significantly reduced while wnt1, p-GSK-3b/tGSK-3b, p-b-catenin/tb-catenin were higher in the DN group mice when analyzed by immunofluorescence and Western blotting. RH reversed these above changes. wnt/b-catenin signaling pathway participates in RH ameliorating kidney injury in DN mice. The manipulation of

& Bo Zhang [email protected] Suyan Duan [email protected] 1

Department of Nephrology, The First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital), Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China

2

Department of Rheumatology, The Wuxi 4th People’s Hospital (The Affiliated Hospital of Jiangnan University), 200 Huihe Road, Wuxi 214062, Jiangsu, China

RH might act as a promising therapeutic intervention for DN. Keywords Rhein  db/db mice  Diabetic nephropathy  wnt/b-catenin signaling pathway Abbreviations DN Diabetic nephropathy NC Normal control ESRD End-stage renal disease

Introduction Diabetes mellitus accounts for approximately one-third of all new cases of end-stage renal disease (ESRD) worldwide, and is the most common cause of ESRD in the United States where approximately 45 % of new cases of ESRD are secondary to diabetes mellitus. There is increasing evidence that the hemodynamic and metabolic pathways, inflammatory mechanism, cytokines, oxidative stress, genetic predisposition, and autophagy are functionally linked in the development of diabetic kidney disease [1, 2]. However, the mechanisms responsible for the development and progression of diabetic kidney disease remain further research. Many studies proved that the activation of wnt/b-catenin signaling may result in proteinuria and kidney injury [3–5]. However, to date, little is known about whether the wnt/b-catenin signaling pathway is associated with rhein (RH) preventing kidney injury in DN mice. In the present study, we investigated this hypothesis that wnt/b-catenin signaling pathway may play an important role in RH ameliorating kidney injury in DN

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mice and aimed at providing a reliable experiment basis for clinical practice.

Materials and methods Antibodies and reagents RH is a gift from Dr. Zhihong Liu (Jinling Hospital, Nanjing, China) and reversed-phase high-performance liquid chromatography assay showed that the purity of RH was over 92 %. Anti-nephrin, anti-wnt1, anti-pGSK-3b (Ser9), anti-GSK-3b, anti-b-catenin, and anti-PPAR-c were obtained from Abcam (Cambridge, MA) and anti-pbcatenin (Ser675) were from cell signaling (Beverly, MA).

Histopathological analysis and electron microscope Paraffin-embedded mouse kidney Sects. (3 lm thickness) were prepared by a routine procedure. Kidney sections were stained with periodic acid-Schiff (PAS) by light microscopy in a blinded manner. Electron microscope of kidney samples was carried out by routine procedures. Mouse kidneys were perfusion-fixed with 2.5 % glutaraldehyde in PBS and postfixed in aqueous 1 % osmium tetroxide. Specimens were dehydrated through an ethanol series, infiltrated in a 1:1 mixture of propylene oxidePolybed 812 epoxy resin (Polysciences, Warrington, PA), and then embedded. Ultrathin sections were stained with 2 % uranyl acetate followed by 1 % lead citrate. Sections were observed and photographed using a JEOL JEM 1010 transmission electron microscope (JEOL, Peabody, MA).

Animals and experimental design Immunofluorescent staining All animal studies were approved by the Ethical Committee of Nanjing Medical University. Seven-week-old healthy male db/db and db/m mice were purchased from Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). Mice were maintained under standard room temperature and regular 12 h photoperiods and were allowed free access to food and water. After one week of acclimatization, the mice were randomly divided into four groups (n = 6): db/db mice treated with RH (DN ? RH), db/db mice (DN), db/m mice treated with RH (NC ? RH) and db/m mice normal control (NC). Among the 8-week-old mice, RH-treated groups were administered orally at a daily dose 120 mg/kg, suspended in 0.5 % carboxymethyl cellulose sodium. Other groups were given 0.5 % solution of sodium carboxymethyl cellulose. Mice were monitored every four weeks using a blood glucose monitoring system (Bayer) and one drop of tail blood. Body weights were assessed every 2 weeks, and randomized urine samples were collected in metabolic cages every 4 weeks. All animals were sacrificed after 12-week treatment. At the end of the experiment and prior to sacrifice, blood samples were collected for biochemical studies, and samples of renal cortex were collected for histological assessments and Western blotting.

Plasma and urine determinations Serum concentrations of total cholesterol, triglyceride, urea nitrogen, creatinine, albumin, and cystatin-C were determined using an automatic biochemistry analyzer (OLYMPUS AU5400, Japan). Urinary albumin concentrations were measured using an ELISA Kit (Uscn Life Science Inc, Wuhan, China) according to the manufacturer’s method.

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Kidney cryosections were fixed with cold methanol/acetone (1:1) for 10 min at -20 °C. Following three extensive washings with PBS, the cells were blocked with 3 % bovine serum albumin in PBS buffer for 60 min at room temperature and then incubated with the anti-nephrin, antiwnt1, or anti-p-b-catenin followed by staining with FITC secondary antibody. Cells were double stained with 40 ,6diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i epifluorescence microscope equipped with a digital camera (DS-Ri1, Nikon). The fluorescence intensity was analyzed by Leica confocal software. Podocyte culture Conditionally immortalized human podocytes were derived and cultured as described previously [6]. The cells were grown at the permissive temperature of 33 °C in the presence of ITS in RPMI 1640 medium supplemented with 10 % FBS, penicillin(100 U/mL), streptomycin (100 mg/ mL) (Gibco, USA) to promote cell propagation with a cobblestone phenotype (undifferentiated) and then moved to the nonpermissive culture temperature of 37 °C under 5 % CO2 and 95 % air for 10–20 days to allow them to differentiate. Differentiated podocytes were used for the proposed experiments. For a proportion of experiments, differentiated podocytes were divided into four groups: the normal glucose group (NG: 5.5 mM of glucose), the osmotic control group (NG ? M: 5.5 mM of glucose and 24.5 mM of mannitol (sigma, USA)), the HG group (HG: 30 mM of glucose), and the HG ? 25 lg/mL RH (sigma, USA) group. Those groups were cultured for 48 h.

Mol Cell Biochem

Western blotting analysis Renal tissues were collected and frozen at -80 °C for Western blot analysis. Briefly, the total proteins extracted from renal cortex were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Nonspecific antibody binding was blocked by a preincubation of the membranes in 19 TBS containing 5 % skim milk for 1 h at room temperature. The membrane was incubated overnight with primary antibodies against nephrin, wnt1, p-bcatenin, b-catenin, glycogen synthase kinase-3b (GSK-3b), p-GSK-3b, PPAR-c, and GAPDH at 4 °C, followed by incubation with HRP-conjugated secondary antibodies for 1 h. Densitometric analysis of the images was performed using the Quantity One, and data are mean ± S.D. of three independent experiments.

Fig. 1 The effects of rhein on body weight in each group of mice. Values are presented as the mean ± SD (n = 6 in each group). DN diabetic nephropathy, RH rhein, NC normal control; 0 week of X axis means we start the rhein treatment with the 8-week-old mice. 4 P \ 0.01 versus the NC group, *P \ 0.01 versus the DN group

Statistical analysis

Rhein reduces UACR in db/db mice

The statistical analyses were performed using the SPSS 20.0 statistical software package (SPSS). All results are expressed as the mean ± standard deviation (SD). Oneway analysis of variance (ANOVA) and Mann–Whitney U analysis were used to perform comparisons among the different groups, and P \ 0.05 was considered to be statistically significant. Correlation analysis was calculated using the Spearman rank-order correlation, and P \ 0.05 was considered to be statistically significant.

Microalbuminuria is considered the earliest clinical indicator in the early stage of DN. 8-week-old db/db diabetic mice had a gradually elevated urinary albumin-to-creatinine ratio (UACR), which indicated a aggravated diabetic renal pathological changes with aging in this study. As shown in Fig. 3, after treatment for 4 weeks, albuminuria was significantly decreased in the RH-treated diabetic db/ db mice compared with db/db control mice. During 12 weeks, albuminuria continued decreasing in the RHtreated db/db mice.

Results Rhein reduces fat weight in db/db mice At 8 weeks of age, db/db mice became more obese than db/m mice [(47.9 ± 1.34) vs. (19.3 ± 1.13)g, P \ 0.01]. After 4 weeks of treatment, RH reduced the body weight of db/db mice when compared to vehicle control [(52.3 ± 2.02) vs. (56.3 ± 1.92)g, P \ 0.01]. A weight-loss plateau occurs after 8 weeks of RH treatment with an obvious decline in obesity levels. No diarrhea in the animals occurred during the study. The data suggested that RH may decrease the fat mass in vivo (as shown in Fig. 1; Table 1). Rhein ameliorates blood glucose The db/db mice exhibited higher fasting blood glucose levels and appeared to trend ascendantly when compared to NC during the 12-week experiment (as shown in Fig. 2). The RH-treated group showed lower fasting glucose levels than vehicle control after four weeks of treatment, but it was still higher than NC. There were no significant differences between db/m mice with or without RH treated.

Other changes after rhein treatment in db/db mice As a model for diabetic dyslipidemia, the total cholesterol (TC) and triglyceride (TG) in db/db mice were higher than control mice blood with a statistically significance (P \ 0.01) (Table 2). TC and TG fell slightly after RH treatment with no statistical significance (P [ 0.05). And the RH-treat db/m mice showed no difference in TC and TG with the NC. Concentration of blood urea nitrogen (BUN) was higher than in normal mice with the statistically significant (P \ 0.01). Although BUN seemed lower than in vehicle control after RH treatment, the differences were not statistically significant (P [ 0.05). Other serum biochemical indice (albumin, serum creatinine, and cystatin-C) did not differ among the four groups (P [ 0.05). Rhein ameliorates renal histopathologic changes Figure 4 shows representative microphotographs of renal Periodic acid-Schiff (PAS) staining. Using light microscopy, we evaluated the control group had no significant pathological changes. When compared with db/m mice

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Mol Cell Biochem Table 1 The effects of rhein on body weight in each group of mice The 8th week ? 0 week

2 weeks

4 weeks

6 weeks

8 weeks

10 weeks

12 weeks

DN ? RH

48.0 ± 1.14

49.7 ± 2.20

53.3 ± 2.02*

52.0 ± 2.59*

53.0 ± 1.93*

53.4 ± 1.60*

53.8 ± 1.41*

DN

47.9 ± 1.344

51.4 ± 3.024

56.3 ± 1.924

56.6 ± 0.934

57.5 ± 0.884

57.8 ± 1.624

58.4 ± 2.554

NC ? RH

19.6 ± 0.67

25.3 ± 1.12

25.5 ± 0.75

25.4 ± 2.53

27.3 ± 1.38

28.0 ± 0.94

28.8 ± 0.89

NC

19.3 ± 1.13

26.9 ± 0.53

27.4 ± 0.41

29.7 ± 1.05

30.4 ± 0.69

30.6 ± 0.76

30.8 ± 0.94

Values are presented as the mean ± SD (n = 6 in each group) DN diabetic nephropathy, RH rhein, NC normal control; 0 week of X axis means we start the rhein treatment with the 8-week-old mice 4

P \ 0.01 versus the NC group, * P \ 0.01 versus the DN group

treatment markedly inhibited podocyte foot process effacement and decreased the glomerular basement thickness (Fig. 4b). Rhein increases expression of nephrin and decreases wnt1 and p-b-catenin expression in db/db mice

Fig. 2 The effects of rhein on blood glucose in each group of mice. Values are presented as the mean ± SD (n = 6 in each group). 4 P \ 0.01 versus the DN group, *P \ 0.01 versus the NC group

Nephrin, as important slit diaphragm proteins in podocytes, has been identified as playing a pivotal role in the maintenance of podocyte integrity. We quantified the expression levels of nephrin by immunofluorescence. As shown in Fig. 5a, the protein levels of nephrin were significantly reduced in the DN group mice. In addition, the expression of wnt1 and p-b-catenin, protein of wnt/b-catenin pathway, were observed. RH treatment markedly decreased wnt1 and p-b-catenin expression compared with DN control animals, as shown by immunofluorescence (P \ 0.05, Fig. 5b, c). These results indicate that RH protects against podocyte injury by increasing the expression levels of nephrin and it also decreases wnt1 and p-b-catenin expression in DN mice. Rhein regulates the expressions of wnt/b-catenin pathway, GSK3b, nephrin, and PPAR-c in DN mice

Fig. 3 The effects of rhein on urinary albumin-creatine ratio (UACR) in each group of mice. Values are presented as the mean ± SD (n = 6 in each group). 4P \ 0.05 versus the DN group, *P \ 0.05 versus the NC group

(Fig. 4a, PAS, NC), the marked glomerular enlargement, lobulation, and mesangial expansion with accumulation of extracellular matrix observed in db/db mice (Fig. 4a, PAS, DN) was clearly improved on RH treatment (Fig. 4a, PAS, DN ? RH). Ultramicro-morphological changes in the podocyte foot processes were observed using electron microscopy (Fig. 4a, EM). The picture showed the remarkable enhanced thickness of the glomerular basement membrane and podocyte effacement in the DN mice. RH

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Compared with the NC, we investigated that the wnt/bcatenin signaling pathway could be activated and the protein expression of nephrin and PPAR-c be attenuated in the renal cortex of db/db mice (P \ 0.05). The administration of RH decreased the expression of wnt/b-catenin and upregulated nephrin and PPAR-c compared with the untreated DN group (P \ 0.05), as demonstrated by Western blot analysis (Fig. 6). These results indicated that in the DN model of db/db mice, the podocyte marker nephrin and PPAR-c were reduced. The expression of p-GSK3b and p-b-catenin were increasing, while the total GSK3b and total b-catenin showed no difference. RH protected the podocytes and improved proteinuria, most likely through the suppression of the wnt/b-catenin signaling pathway.

Mol Cell Biochem Table 2 Influence of rhein on the general parameters in experimental mice TC (mmol/L)

TG (mmol/L)

ALB (g/L)

BUN (mmol/L)

CREA (lmol/L)

Cys-C (mg/L)

DN ? RH

4.43 ± 0.28

1.36 ± 0.27

32.43 ± 0.67

7.72 ± 1.03

12.02 ± 1.56

0.138 ± 0.02

DN

4.57 ± 0.34

1.88 ± 0.42

32.04 ± 2.06

9.31 ± 0.55

11.81 ± 1.76

0.098 ± 0.052

NC ? RH

2.80 ± 0.35

0.62 ± 0.08

28.95 ± 2.54

7.85 ± 2.08

9.29 ± 4.22

0.140 ± 0.076

10.57 ± 1.52

0.147 ± 0.069

NC

3.23 ± 0.83

a

b

0.76 ± 0.05

31.78 ± 0.27

7.77 ± 0.32

c

Values are presented as the mean ± SD (n = 6 in each group) TC total cholesterol, TG triglyceride, ALB albumin, BUN blood urea nitrogen, CREA serum creatinine, Cys-C cystatin-C a

P \ 0.01,b P \ 0.01,c P \ 0.01, versus the DN group

Fig. 4 The influence of rhein on renal histopathological changes in each group of mice. a PAS stainings of the indicated tissue (9400); morphological changes in the podocyte foot process were examined using electron microscopy (EM, 920,000). b Thickness of the glomerular basement membrane (GBM) of the four groups. Values are presented as the mean ± SD (n = 5 in each group). *P \ 0.05 versus the DN group

Rhein regulates the expressions of wnt/b-catenin pathway, GSK3b and PPAR-cin cultured podocytes Effect of RH on HG-induced podocyte injury was detected by Western blot. Cells incubated with NG, NG ? M, HG, and HG combined with RH (25 lg/mL) for 48 h. Dosage of RH used in our experiments was selected according to previous studies [7]. As shown in Fig. 7, the levels of nephrin and PPARc protein expression were significantly increased with RH treatment as compared in HG-induced podocyte damage. The protein in the wnt/b-catenin signaling pathway, including wnt1, b-catenin, were significantly down-regulated by RH treatment (Fig. 7). Furthermore, RH treatment efficiently suppressed p-GSK3b and p-b-catenin, while the total GSK3b and total b-catenin showed no difference.

Discussion To investigate the effect of RH, db/db mice were used. The spontaneous mutant strain C57BLKS/J db/db mice have a db gene mutation, a splicing mutation caused by a point mutation in the downstream intron of the leptin receptor gene, and so they are unresponsive to leptin. Leptin is a peptide hormone secreted by adipocytes, and it is involved in eating behavior and energy homeostasis [8]. In this study, db/db mice showed diabetic characteristics, such as excessive body weight gain and hyperglycemia, compared with db/m mice. In 1980s, rhubarb, a traditional Chinese medicinal herb, was first used to treat the patients who acquired DN by a Japanese scholar. Hence, ample studies find the major renoprotective components of rhubarb are RH, emodin, and chrysophanol, especially RH. RH (4,5-

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Fig. 5 The influence of rhein on the expression of nephrin, wnt1 and p-b-catenin. a–c Immunofluorescence staining of nephrin, wnt1 and p-b-catenin in the four groups (9400). d Quantitative immunofluorescence analysis of nephrin, wnt1, and p-b-catenin expression in the

four groups. Values are presented as the mean ± SD (n = 5). 4 P \ 0.05 versus the DN group, *P \ 0.05 versus the NC group

dihydroxyl-2-carboxylic-9,10-dihydrodiketoanthracene, a free anthraquinone compound isolated from rhubarb, has extensive pharmacological actions, including the antibacterial, antiviral, anti-inflammatory, antiproliferative, and antifibrosis properties [9]. In our study, increased albuminuria, together with weight gain and hyperglycemia, was observed as early as the beginning of the study and continued to increase throughout the length of the study (12 weeks). The marked glomerular enlargement, lobulation, and glomerular mesangial matrix expansion were also observed in db/db mice as well as the enhanced thickness

of the glomerular basement membrane and podocyte effacement. The protein levels of nephrin were significantly reduced while wnt1 and p-b-catenin enhanced by immunofluorescence in the DN group mice. Expectedly, mice receiving the treatment with RH were protected from these changes. Twelve-week RH treatment markedly lost weight, attenuated hyperglycemia, hyperlipidemia, and albuminuria in the db/db mice. In addition, RH ameliorated renal histopathologic changes, inhibited podocyte foot process effacement and decreased the glomerular basement thickness. These results indicate RH indeed protects

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Mol Cell Biochem Fig. 6 The influence of rhein on the expression of nephrin, GSK3b, wnt/b-catenin pathway, and PPAR-c in DN mice. Expressions of wnt/b-catenin pathway, nephrin, and PPAR-c in the renal cortex of the mice groups, as detected by Western blot analysis (a). Nephrin (b), wnt1 (c), b-catenin and GSK3b (d), p-b-catenin and p-GSK3b (e) and PPAR-c (f). Values are presented as the mean ± SD (n = 3). 4P \ 0.05 versus the DN ? Rh group, *P \ 0.05 versus the DN group

podocytes to attenuate albuminuria in diabetic nephropathy (DN). In early stages of DN, podocyte number is markedly reduced, which in turn leads to injury to the integrity of the glomerular filtration barrier. Podocyte injury has been identified as a key role in processes of DN, which is characterized by decreased expression of slit diaphragmassociated proteins, nephrin and podocin and increased albumin filtration [10–12]. Nephrin expression is a marker of normal podocyte and the loss of podocytes correlating closely with disease progression [13]. Activation of the canonical wnt signaling pathway plays an important role in

mediating podocyte dysfunction. Canonical wnt signaling upregulates expression of direct T-cell factor/lymphoid enhancer factor-1 (TCF/LEF-1) target genes that modulate cell proliferation, differentiation, survival, and apoptosis. wnt ligands, such as wnt1, bind to a co-receptor complex consisting of frizzled (Fz) receptors and LRP6, causing phosphorylation of LRP6 and recruitment of a degradation complex consisting of casein kinase 1 (CK-1), GSK-3b and adenopolyposis coli protein (APC). In the absence of wnt ligand, this kinase complex phosphorylates b-catenin, leading to degradation of b-catenin in the cytoplasm. Phosphorylation and degradation of b-catenin are

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Mol Cell Biochem Fig. 7 The influence of rhein on the expression of nephrin, GSK3b, wnt/b-catenin pathway and PPAR-c in cultured podocytes. Expressions of wnt/ b-catenin pathway, nephrin and PPAR-c in the mouse podocyte groups, as detected by Western blot analysis (a). Podocytes were cultured in medium of normal glucose, normal glucose with D-mannitol, high glucose and high glucose with 25 lg/ mL rhein for 48 h. Nephrin (b), wnt1 (c), p-b-catenin (d), p-GSK3b (e) and PPAR-c (f). Values are presented as the mean ± SD (n = 3). *P \ 0.05 versus the NG group

prevented when wnt ligands activate the pathway, and the stabilized b-catenin translocates to the nucleus and dimerizes with the TCF-Groucho complex, activating transcription of corresponding direct target genes [14]. Research has shown that increased wnt/b-catenin activities contribute to proteinuria and glomerulosclerosis development [3, 15]. In our study, we observed the expression of nephrin, wnt1, b-catenin, GSK-3b, PPAR-c in both kidney cortex of db/db mice and cultured podocytes, with the upregulated expression of wnt1, p-b-catenin, p-GSK-3b and down-regulated expression of nephrin and PPAR-c. In kidney cortex of db/db mice and podocytes, the expression of wnt1, p-GSK-3b/tGSK-3b, and p-b-catenin/tb-catenin is higher than NC. These results indicate that the phosphorylation of key proteins in wnt/b-catenin protects kidney from injury. RH may contribute to reversing wnt/b-catenin

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signaling pathway during the amelioration of podocyte injury in DN following RH treatment. Our results, that reduction in nephrin expression of DN mice and HG-induced podocytes indicating podocytes injury, while RH attenuated HG-induced wnt1 and p-b-catenin signaling pathway to improve nephrin expression, supported the theory. GSK-3b is known to modulate cell survival and apoptosis through multiple intracellular signaling pathways [16]. GSK-3b also plays an important role in canonical wnt signaling pathway. GSK-3b exists in a multimeric complex with APC, axin and b-catenin, where GSK-3b phosphorylates the N terminal Ser/Thr of b-catenin leading to its degradation mediated by ubiquitin/proteasomes. Activation of wnt signaling leads to the phosphorylation of GSK3b at Ser-9, thereby inhibiting its activity. GSK-3b inactivation

Mol Cell Biochem

resulting in dephosphorylation and stabilization of b-catenin protein in the cytosol [17]. The increased levels of bcatenin lead to its nuclear translocation, and its interaction with transcription factors LEF/TCF (lymphoid enhancer factor/T-cell factor) activates the expression of target genes [18]. In our DN models, we observed that the expression level of nephrin, a podocyte marker, was significantly decreased and that the expressions of phosphorylated GSK3b and b-catenin were increased compared with NC, while the total GSK3b and b-catenin showed no difference. RH treatment abrogated the protein overexpression and phosphorylation of wnt/b-catenin signaling induced by db/ db mice. These results are likely contributing to the protection of podocyte and against albuminuria by RH, which are consistent with previous clinic results of mice. An assessment from bench to bedside showed that apart from improving glycemic control in type 2 DM, PPAR-c agonists, as insulin sensitizers, have beneficial effects on other components of the metabolic syndrome which contribute toward a renoprotective effect [19]. Welsh et al. [20] showed a direct effect of local insulin-receptor signaling on podocyte function in vitro and in vivo. These indicate RH which can upregulate PPAR-c as an insulin sensitizer to ameliorate podocyte injury in DN mice. Besides, our study demonstrated that the up-regulation of wnt/b-catenin signaling was involved in diabetes, accompanied by the mitigation of the protein overexpression and phosphorylation of wnt/b-catenin signaling after RH treatment. It is reasonable to assume that apart from acting as a PPAR-c agonist, the influence of RH treatment in decreasing the wnt/b-catenin phosphorylation contribute, at least in part, to the protection of DN. Above all, these data underscore the RH may repair HGinduced podocyte injury through the GSK3b-wnt/b-Catenin/PPAR-c pathway and thus exert its therapeutic effect in DN contribute to reversing wnt/b-catenin signaling pathway during the amelioration of podocyte injury in DN mice following RH treatment. First, we assume that DN podocyte injury is mediated by the serine/threonine protein kinase GSK-3b phosphorylation-wnt/b-catenin signaling pathway. Phosphorylation of GSK-3b leads to inhibition of GSK-3b thus resulting in the stimulation of the wnt pathway [21, 22]. In addition, the rationale for this process is supported by the fact that GSK-3b participates in podocytes’ EMT in the HG environment [23]. Down-regulating GSK-3b expression decreased b-catenin and Snail expression and reversed HG-induced podocytes EMT. Our data observed increased expression of wnt1 and p-b-catenin in HG-induced podocytes and DN mice, an effect that was attenuated with downregulation of p-GSK3b expression by RH. Second, PPAR-c, one of downstream target genes of wnt/b-catenin signaling pathway, is a promising target in the therapy of DN [24, 25]. In our study, PPAR-c

protein expressions are significantly decreased in podocytes exposed to HG which also induced reduction in nephrin expression indicating podocytes injury. We consider that RH serves as a GSK3b antagonist to activate GSK3b phosphorylation, leading to accumulated downstream wnt 1a and b-catenin decreased and the target PPAR-c increased in HG destroyed podocytes. Moreover, the effect of RH is similar to PPAR-c agonists which had been thought to be a promising candidate for strengthening the therapy of DN. However, our findings are only based on in vivo and partly in vitro data, and further in vitro studies are required in the future. Acknowledgments We thank Dr. Zhihong Liu in Jinling Hospital for providing rhein in this study, and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. This work was supported by grants from the National Natural Science Foundation of China (81170660/H0509,81370815/H0509, to Changying Xing; 81100512/H0510, to Bo Zhang; 81300573, to Yuanyang Gang), the Ph.D. Programs Foundation of the Ministry of Education of China (2011BAI10B06, 2011BAI10B08, to Changying Xing), the Natural Science Foundation of Jiangsu (LJ201125, BK20151588, BL2012032, BL2014080, to Changying Xing; BK20131030, to Yuanyang Gang). Authors Contribution Suyan Duan designed the study and drafted the paper. Bo Zhang was involved in revising the paper. Suyan Duan, Yingyi Wu, Chuanyan Zhao and Yuming Chen carried out the full experiment. Yanggang Yuan and Changying Xing were responsible for study oversight, data collection, analysis. All authors have read the final paper and approved the submission. Compliance with ethical standards Conflicts of Interest

No conflicts of interest.

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