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RANK-mediated NF-κB and MAPK signaling pathways
Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK signaling pathways Xiao-Wen Chen a, 1, Wen-Ting Liu a, 1, Yu-Xian Wang b, 1, Wen-Jing Chen a, Hong-Yu Li a, Yi-Hua Chen a, Xiao-Yan Du a, Fen-Fen Peng a, Wei-Dong Zhou a, Zhao-Zhong Xu c, Hai-Bo Long a,⁎ a b c
Department of Nephrology, ZhuJiang Hospital, Southern Medical University, Guangzhou, 510280, China Department of Gerontology, ZhuJiang Hospital, Southern Medical University, Guangzhou, 510280, China Department of Emergency, ZhuJiang Hospital, Southern Medical University, Guangzhou, 510280, China
a r t i c l e
i n f o
Article history: Received 4 January 2016 Received in revised form 7 March 2016 Accepted 14 March 2016 Available online xxxx Keywords: LJ Receptor activator for NF-κB ligand Receptor activator for NF-κB NF-κB Mitogen-activated protein kinase
a b s t r a c t Aims: The aim of this research was to investigate the effects of cyclopropanyldehydrocostunolide (also named LJ), a derivative of sesquiterpene lactones (SLs), on high glucose (HG)-induced podocyte injury and the associated molecular mechanisms. Methods: Differentiated mouse podocytes were incubated in different treatments. The migration and albumin filtration of podocytes were examined by Transwell filters. The protein and mRNA levels of MCP-1 were measured using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (q-PCR). Protein expression and phosphorylation were detected by western blot, and the nuclear translocation of NF-κB was performed with a confocal microscope. The gene expression of the receptor activator for NF-κB (RANK) was silenced by small interfering RNA (siRNA). Results: Our results showed that HG enhanced migration, albumin filtration and MCP-1 expression in podocytes. At the molecular level, HG promoted the phosphorylation of NF-κB/p65, IKKβ, IκBα, mitogen-activated protein kinase (MAPK) and the nuclear translocation of p65. LJ reversed the effects of HG in a dose-dependent manner. Furthermore, our data provided the first demonstration that the receptor activator for NF-κB ligand (RANKL) and its cognate receptor RANK were overexpressed in HG-induced podocytes and were downregulated by LJ. RANK siRNA also attenuated HG-induced podocyte injury and markedly inhibited the activation of NF-κB and MAPK signaling pathways. Conclusions: LJ attenuates HG-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK signaling pathways. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Diabetic nephropathy (DN), characterized by progressive albuminuria and a gradually declining glomerular filtration rate, has a dismal prognosis. It is one of the most common and severe microvascular complications of diabetes resulting from the leading risk of end-stage renal disease (Ritz, Rychlik, Locatelli, & Halimi, 1999). Despite being given multiple therapies for hyperglycemia and high blood pressure, numerous patients still suffer progressive and serious renal injury (Stolar, 2010). Thus, combating DN has become a major focus of research (Collins et al., 2014; Liu, 2013; Xu et al., 2013). Conflict of Interest: The authors declare no conflict of interest. ⁎ Corresponding author. E-mail address: [email protected] (H-B. Long). 1 These authors contributed equally to this study.
Podocytes are vitally important to the integrity of the glomerular filtration barrier. It has been reported that they can precede and predict the occurrence of proteinuria (Anil, Welsh, Saleem, & Menon, 2014) and may be an early pathological manifestation of DN (Meyer, Bennett, & Nelson, 1999). When podocytes suffer from injurious stimuli such as HG, TGF-β1 and adriamycin, they become more motile and leave the glomerular basement membrane, leading to the onset of proteinuria (Kang et al., 2010). Hence, finding a drug that can protect podocyte from injury is essential for the development of targeted, effective DN treatment. SLs, originally isolated from medicinal herbs of the Asteraceae family, have been examined for their anti-inflammatory, immunomodulatory, and anticancer effects (Viennois et al., 2014; Zhang et al., 2012). LJ is a derivative of SLs, i.e., cyclopropanyldehydrocostunolide. Our previous studies have shown that SL derivatives, including LJ (also named compound 1), can block the advanced oxidation protein
http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013 1056-8727/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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product (AOPP)-induced activation of the NF-κB pathway and the expression of MCP-1 in rat mesangial cells or podocytes (Wang et al., 2013; Zhao et al., 2015). However, little is known about whether LJ can attenuate migration and albumin filtration in HG-induced podocytes and what the underlying molecular mechanism is. RANKL, a type II membrane protein, belongs to the TNF superfamily and contains a C-terminal receptor-binding domain and a transmembrane region. RANK, a type I membrane protein that shares high homology with CD40, is one of the receptors for RANKL. Recently, Liu et al. (Liu et al., 2012) reported that RANK and RANKL are receptor-ligand complexes for the pathogenesis of podocyte injury. A genome-wide association study also revealed a polymorphism in the podocyte receptor, RANK, that leads to a decline in renal function in coronary patients (Leiherer et al., 2014). These results suggest that RANK is a promising therapeutic target in glomerular diseases. Moreover, emerging evidence shows that RANKL and RANK play a vital role in the migration of cancer cells and osteoblasts (Armstrong et al., 2008; Hsu et al., 2010; Jones et al., 2006). It has been demonstrated that parthenolide, which is a natural SL, can inhibit RANKL-mediated osteoclast differentiation and suppress IL-6/sIL6R-induced RANKL expression in fibroblast-like synoviocytes (Hashizume, Hayakawa, & Mihara, 2008; Kim, Cheon, Yoon, Lee, & Oh, 2014). In addition, NF-κB and MAPK pathways contribute to the migration of cancer cells and osteoblasts (Golden, Saria, & Hansen, 2015; Schramek et al., 2010; Song et al., 2014; Wang, Sun, Zhang, Wang, & Li, 2015). We can therefore speculate that LJ may attenuate the migration and albumin filtration of podocytes by regulating RANKL/RANK-mediated NF-κB and MAPK signaling pathways. In the present study, we investigated the effects of LJ and RANK on the migration and albumin filtration in HG-induced podocytes and examined the underlying molecular mechanisms involved in this process.
and transferred onto PVDF membranes (Millipore, Bedford, MA). The membrane was blocked for 1 h with Tris-buffered saline (TBS; 20 mM Tris–HCl, 140 Mm NaCl, PH7.6) containing 5% non-fat dry milk, washed with TBS containing 0.1% Tween-20, and incubated overnight at 4 °C with primary antibodies. The primary antibodies for RANKL rabbit mAb, RANK rabbit mAb and nephrin rabbit mAb were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,USA). IKKβ rabbit mAb, phospho-IKKβ rabbit mAb, p65 rabbit mAb, phospho-p65 rabbit mAb, phospho-IκBα rabbit mAb, p38 rabbit mAb, phospho-p38 mouse mAb, ERK rabbit mAb, phospho-ERK rabbit mAb, JNK rabbit mAb, and phospho-JNK rabbit mAb were purchased from Cell Signaling Technology (Cell Signaling Technologies, Danvers, MA). β-actin mouse mAb and HRP-conjugated secondary antibodies were purchased from EarthOx (EarthOx, LLC, San Francisco, CA,USA). The intensities of the protein bands were quantified by Quantity One 4.6.2 analysis software (Quantity One, Bio-Rad Laboratories, Inc., USA), which was provided with the Kodak 2000MM System (Eastman Kodak company, Rochester, NY, USA).
2. Materials and methods
Podocytes (5 × 10 4/well) were plated in a 12-well plate under various experimental conditions. After 24 h, the supernatants were assayed using a solid-phase quantitative sandwich ELISA kit for MCP-1, as described previously (Zhao et al., 2015).
2.1. Cell culture and treatment Conditionally immortalized mouse podocytes between passages 12 and 20 (a kind gift from Prof. Peter Mundel, Mount Sinai School of Medicine, through Prof. Tanqi Lou, the Third First Affiliated Hospital of Sun Yat-Sen University) were cultured as previously described (Shankland, Pippin, Reiser, & Mundel, 2007). Briefly, at a permissive temperature of 33 °C, undifferentiated podocytes were grown in RPMI1640 containing 10% FBS (Life Technologies, Rochester, NY, USA), penicillin (100 U/mL), streptomycin (100 mg/mL), and 50 IU/mL recombinant murine IFN-γ (Pepro Tech, USA). To induce differentiation, podocytes were cultured at 37 °C without IFN-γ for 10–14 days. For experiments, the differentiated podocytes were incubated in the presence of 5 mmoles (mM) D-glucose (NG), 30 mM D-glucose (HG) or 5 mM D-glucose + 25 mM mannitol (MA) at 37 °C for 3, 6, 12, and 24 h (h). In some experiments, the podocytes were divided into different groups and then treated with LJ of different concentrations in HG condition for 24 h. LJ was provided by Accendatech Co. Ltd (Tianjin, China). In other experiments, subconfluent podocyte cultures were transfected with RANK siRNA (50 nmoles) or siRNA control (50 nmoles) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The sequences of RANK siRNA were as follows: si-RANK1: sense: 5′-CCU CCU UGG AAA GCU AGA ATT-3′, antisense: 5′-UUC UAG CUU UCC AAG GAG GTT-3′; si-RANK2: sense: 5′-GGA GAG GCA UUA UGA GCA UTT-3′, antisense: 5′- AUG CUC AUA AUG CCU CUC CTT-3′; and si-RANK3: sense: 5′- GCG CAG ACU UCA CUC CAU AUU-3′, antisense: 5′UAU GGA GUG AAG UCU GCG CUU-3′. 2.2. Western blot analysis All samples were handled as described previously (Zhao et al., 2015). Total proteins were separated on 10% SDS-polyacrylamide gels
2.3. Confocal microscopy analysis Differentiated podocytes were fixed with 10% paraformaldehyde and permeabilized with 0.2% Trition X-100 in PBS. Then, the cells were blocked with 5% BSA. They were then incubated with NF-κB p65 rabbit mAb (Cell Signaling Technologies, Danvers, MA) for 2 h at 37 °C, AlexaFluor 488 goat anti-rabbit (Invitrogen) for 1 h at 37 °C, and then 4 ˊ, 6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. The cells were visualized under a Nikon C2 confocal microscope (Nikon corporation, Tokyo, Japan). 2.4. Enzyme-linked immunosorbent assay
2.5. Quantitative real-time PCR analysis The RNA from the podocytes in all samples was extracted as described previously (Zhao et al., 2015). The primers for qPCR were as follows: MCP-1 forward: 5′-CCC AAT GAG TAG GCT GGA GA-3′, reverse: 5′-TCT GGA CCC ATT CCT TCT TG-3′and GAPDH forward: 5′-ATT GTC AGC AAT GCA TCC TG-3′, reverse: 5 ′-ATG GAC TGT GGT CAT GAG CC-3′. All data were normalized to their GAPDH content, and expression levels were analyzed by the 2-DDCt method. 2.6. Cell migration assay Podocytes (5 × 104/well), transfected with or without RANK-siRNA (50 nM)/control- siRNA (50 nM), were plated on the top side of 8.0 μm pore polycarbonate Transwell filters (Corning, Lowell, MA) coated with type I collagen (Corning, Lowell, MA). Serum-free medium was used in the both chambers. Afterward, HG or MA and various concentrations of LJ were added. The cells were incubated at 37 °C for 24 h. The nonmigratory cells in the top chambers were removed with cotton swabs, and the migrated cells were fixed in 4% paraformaldehyde for 10 min, stained with 0.1% crystal violet (sigma), and counted under a microscope (Ni-U, Nikon Corporation, Tokyo, Japan). 2.7. Transwell albumin filtration assay Podocytes (5 × 103/well) were plated on the top side of 3.0 μm pore polycarbonate Transwell filters (Corning, Lowell, MA) coated with type I collagen (Corning, Lowell, MA). The cells were then transfected with or without RANK-siRNA (50 nM)/control- siRNA (50 nM) for 24 h and then
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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3. Results
or MA or HG for 24 h and then examined migration by Transwell filters. The results showed that HG significantly increased the migratory ability of podocytes across the pores of the Transwell filters (Fig. 1A and B). We next examined whether HG affects the barrier function of the podocyte monolayer. To this end, we used an albumin flux assay that assesses albumin filtration across the monolayer of cultured differentiated podocytes. As shown in Fig. 1C, HG increased albumin flux in podocytes more than NG did. To eliminate the influence of osmotic pressure, we set the MA group and found that osmotic pressure did not influence the results. MCP-1 is characterized as attracting macrophages and promoting inflammation in DN (Chow, Ozols, Nikolic-Paterson, Atkins, & Tesch, 2004; Kanamori et al., 2007). However, it also plays a noninflammatory role in enhancing the migratory capacity of podocytes. Thus, we measured the MCP-1 protein level using ELISA and detected MCP-1 RNA by q-PCR in HG-induced podocytes. The results demonstrated that HG significantly induced the expression of MCP-1 in protein and mRNA levels (Fig. 1D and E).
3.1. HG enhances migration, albumin filtration and MCP-1 expression in podocytes
3.2. The effect of HG on nephrin and the NF-κB and MAPK signaling pathways in podocytes
Podocytes after HG stimulation may become motile with an increased migratory capacity. To test this hypothesis, we treated podocytes with NG
Nephrin is a marker of normal podocytes. Its reduction reflects podocyte injury. As shown in Fig. 2A, nephrin was significantly
treated with HG or MA and various concentrations of LJ for 24 h. The medium in each upper chamber was replaced with 150 μL RPMI 1640, and 1 ml RPMI 1640 with 40 mg/ml bovine serum albumin was added to each bottom chamber. After incubation at 37 °C for 6 h, an aliquot of medium from the upper chamber was collected, and the concentrations of albumin were measured using a BCA protein assay kit (Thermo Fisher Scientific, Shanghai, China). 2.8. Statistical analysis The results were presented as the mean ± S.D. Differences among multiple groups were analyzed by one-way ANOVA followed by a t-test to detect between-group differences. A two-sided P b 0.05 was considered statistically significant. All analyses were performed using SPSS Statistics 20.0 (SPSS, Chicago, IL, USA).
Fig. 1. HG enhances migration, albumin filtration and MCP-1 expression in podocytes. Podocytes were treated with NG or MA or HG for 24 h before undergoing different examinations. The results were as follows: (A) Representative fields of migratory podocytes. They were stained with 0.1% crystal violet on the membrane and observed with a microscope (scale bars: 50 μm). (B) The numbers of migratory cells were counted under a microscope in different groups. (C) Albumin filtration of podocytes in different groups. (D) The secretion of MCP-1 in supernatants of podocytes in different groups was measured by ELISA. (E) The mRNA expression of MCP-1 in lysates of podocytes in different groups. All experiments were performed three times. Bars B–E show the mean expression in arbitrary units (error bars S.D.). *P b 0.05 compared with NG, t-test.
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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decreased in HG-induced podocytes at 12 and 24 h (Fig. 2A and D: d1), indicating the injury of podocytes. Inversely, proteins in the NF-κB signaling pathway, including phospho-p65, phospho-IKKβ, and phospho-IκBα, were enhanced by HG in a time-dependent manner, whereas IKKβ and p65 did not significantly change in any group (Fig. 2B and D: d2–d4). Similarly, to clarify the effects of HG on MAPK activation in podocytes, we investigated the phosphorylation of individual MAPKs and found that HG induced a rapid and sustained activation of p38, ERK and JNK without affecting their total levels (Fig. 2C and D: d5–d7).
with 0 μM, 2.5 μM, 5 μM, and 10 μM of LJ in HG condition for 24 h. Fig. 3A shows the structure of LJ. As shown in Fig. 3B–D, LJ improved the migration and albumin filtration of differentiated podocytes in a dose-dependent manner. After 24 h of exposure to 10 μM LJ, cell migration and albumin filtration reached the best improvement. The increase in MCP-1 in protein and mRNA levels in HG-induced podocytes were also blocked by LJ in a dose-dependent manner (Fig. 3E and F).
3.3. LJ suppresses HG-induced migration, albumin filtration and MCP-1 expression in podocytes
From the above, it is clear that NF-κB and MAPK signaling pathways were activated in HG-induced podocytes. Thus, we went on to measure these proteins to explore the effect of LJ on them. As shown in Fig. 4, LJ inhibited phospho-p65, phospho-IKKβ, phospho-IκBα (Fig. 4B and D: d2–d4) and the phosphorylation of p38, ERK
To examine whether LJ attenuated HG-induced migration, albumin filtration and MCP-1 expression in podocytes, we treated podocytes
3.4. LJ blocks NF-κB and MAPK signaling pathway activation but upregulates nephrin in HG-induced podocytes
Fig. 2. The effect of HG on nephrin and the NF-κB and MAPK signaling pathways in podocytes. Podocytes were treated with MA or HG for 3, 6, 12, and 24 h and NG for 24 h. Then, the cell supernatants were obtained for western blot. (A) Representative immunoblot of nephrin and β-actin. (B) Representative immunoblot of p-p65, p65, p-IKKβ, IKKβ, p-IκBα and β-actin. (C) Representative immunoblot of p-p38, p38, p-ERK, ERK, p-JNK, JNK and β-actin. (D) The ratios between nephrin and β-actin (d1), p-p65 and p65 (d2), p-IKKβ and IKKβ (d3), p-IκBα and β-actin (d4), p-p38 and p38 (d5), p-ERK and ERK (d6), and p-JNK and JNK (d7) in podocytes were calculated and indicated (mean and S.D., N = 3, *P b 0.05 compared with NG, #P b 0.05 compared with HG stimulation, t-test).
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Fig. 3. LJ suppresses HG-induced migration, albumin filtration and MCP-1 expression in podocytes. Podocytes were treated with different concentrations of LJ in the HG condition for 24 h. Then, different examinations were performed. The results were as follows: (A) The structure of LJ. (B) Representative fields of migratory podocytes (scale bars: 50 μm). (C) The numbers of migratory cells were counted under a microscope in different groups. (D) Albumin filtration of podocytes in different groups. (E) The secretion of MCP-1 in supernatants of podocytes in different groups were measured by ELISA. (F) The mRNA expression of MCP-1 in lysates of podocytes in different groups. All experiments were performed three times. Bars C–F show the mean expression in arbitrary units (error bars S.D.). #P b 0.05 compared with HG stimulation, t-test.
and JNK (Fig. 4C and D: d5–d7), whereas it upregulated nephrin (Fig. 4A and D: d1) in HG-induced podocytes in a dose-dependent manner. The total levels of p65, IKKβ, p38, ERK and JNK did not significantly change in any group. In addition to the NF-κB western blot assays, we performed the immunofluorescence staining of p65 in HG-induced podocytes with or without LJ. Our results showed that in the absence of HG, most p65 that was unphosphorylated and inactive was localized to the cytoplasm. Upon HG stimulation for 24 h, almost all p65 was localized to the nucleus, but the nuclear translocation of p65 did not occur with the co-treatment of 10 μM LJ and HG (Fig. 4E). 3.5. RANKL and RANK are upregulated in HG-induced podocytes but downregulated by LJ It has been reported that puromycin aminonucleoside (PAN) induces the expression of RANKL and RANK in podocytes (Liu et al.,
2012), but little is known about whether HG can also do so. To solve this problem, we investigated the protein level of RANKL and RANK in HG-induced podocytes. As shown in Fig. 5, HG significantly upregulated RANKL and RANK in a time-dependent manner (Fig. 5A and B). Moreover, we found that LJ downregulated RANKL and RANK in a dose-dependent manner when we treated podocytes with HG and LJ for 24 h (Fig. 5C and D). 3.6. RANK is important for the LJ-mediated inhibition of HG-induced migration, albumin filtration and MCP-1 expression in podocytes To shed light on the importance of RANKL and RANK in the protection of HG-induced podocytes by LJ, we knocked down RANK by siRNA. First, we transfected podocytes with siRNA control or three different siRNA sequences of RANK (si-RANK1, si-RANK2, si-RANK3), respectively, for 24 h and then treated these podocytes with HG for
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Fig. 4. LJ blocks NF-κB and MAPK signaling pathway activation but upregulates nephrin in HG-induced podocytes. Podocytes were treated with different concentrations of LJ in the HG condition for 24 h. Then, western blot and immunofluorescence were performed. The results were as follows: (A) Representative immunoblot of nephrin and β-actin. (B) Representative immunoblot of p-p65, p65, p-IKKβ, IKKβ, p-IκBα and β-actin. (C) Representative immunoblot of p-p38, p38, p-ERK, ERK, p-JNK, JNK and β-actin. (D) The ratios between nephrin and β-actin (d1), p-p65 and p65 (d2), p-IKKβ and IKKβ (d3), p-IκBα and β-actin (d4), p-p38 and p38 (d5), p-ERK and ERK (d6), and p-JNK and JNK (d7) in podocytes were calculated and indicated (mean and S.D., N = 3, *P b 0.05 compared with NG, #P b 0.05 compared with HG stimulation, t-test). (E) Representative fields of the localization of p65 in podocytes under a confocal microscope (scale bars: 50 μm).
24 h. After transient transfection, the cell supernatants of the podocytes were obtained for western blot to detect the most efficient sequence of si-RANK. The results showed that si-RANK3 was the most efficient sequence compared to siRNA control (Fig. 6A and B). Furthermore, we found that the endogenous knockdown of RANK attenuated the migration and albumin filtration of podocytes compared with the HG-stimulated cells transfected with siRNA control. Subsequently, we treated the cells transfected with si-RANK with 10 μM LJ and found that the inhibition of migration and albumin filtration was similar to that in the siRNA-RANK-treated group (Fig. 6C–E). Intriguingly, the protein and mRNA levels of MCP-1 were also inhibited by si-RANK (Fig. 6F and G).
3.7. RANK is important for the LJ-mediated inactivation of the NF-κB and MAPK pathways To further elucidate the molecular mechanisms by which LJ influenced podocytes, we silenced RANK in the podocytes and treated them with 10 μM LJ. Then, we examined the proteins via western blot. Fig. 7 shows that RANKL, RANK (Fig. 7A and D: d1–d2), phospho-p65, phosphor-IKKβ, phosphor-IκBα (Fig. 7B and D: d4–d6) and the phosphorylation of p38, ERK and JNK (Fig. 7C and D: d7–d9) were significantly attenuated but that nephrin (Fig. 7A and D: d3) increased slightly when RANK was silenced. The result of the immunofluorescence staining of p65 also showed that the nuclear translocation of
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Fig. 5. RANKL and RANK are upregulated in HG-induced podocytes but downregulated by LJ. Podocytes were treated with MA or HG for 3, 6, 12, 24 h and NG for 24 h or treated with different concentrations of LJ in the HG condition for 24 h. Then, the cell supernatants were obtained for western blot. (A, C) Representative immunoblot of RANKL, RANK and β-actin. (B, D) The ratios between RANKL and β-actin (b1, d1), RANK and β-actin (b2, d2) in podocytes were calculated and indicated (mean and S.D., N = 3, *P b 0.05 compared with NG, #P b 0.05 compared with HG stimulation, t-test).
p65 was blocked in the absence of RANK (Fig. 7E). Moreover, the effects of HG on the activation of NF-κB and MAPK pathways were also reversed by the co-administration of LJ and siRNA-RANK. 4. Discussion Podocytes play a vital role in DN. When exposed to HG, podocytes may increase their motility and lose their morphological structure, which undoubtedly impairs the integrity of the glomerular filtration barrier and increase paracellular albumin flux across podocyte monolayers, thereby causing proteinuria (Kang et al., 2010; Muller-Krebs et al., 2015). Inevitably, MCP-1, an important protein that is correlated with progression and renal outcomes in DN (Chow et al., 2004; Kanamori et al., 2007), will occur as a multifunctional cytokine simultaneously. On the one hand, MCP-1 attracts macrophage migration into the diabetic kidney and promote inflammation in DN (Chow et al., 2004). On the other hand, MCP-1 can interact with its receptor that is predominantly localized to podocytes, contributing to migration and the albumin filtration of podocytes (Lee et al., 2009). This process leads to vicious circle of increasing migration, strengthening proteinuria and enhancing MCP-1. In our study, we confirmed that HG induced this vicious circle, and we further demonstrated for the first time that LJ prevented the effects of HG in a dosedependent manner. How can LJ achieve success? We focused on the key point: podocyte migration. Studies have increasingly demonstrated that RANKL and RANK direct the migration of cancer cells and osteoblasts (Armstrong et al., 2008; Hsu et al., 2010; Jones et al., 2006). Moreover, Liu et al. reported that RANKL and RANK were overexpressed in PAN-induced podocytes (Liu et al., 2012). In their report, they found that the knockdown of RANK alone did not induce podocyte apoptosis but mildly increased the apoptosis of podocytes exposed to PAN, which had no statistical significance. However, the application of exogenous RANKL significantly reduced the apoptosis of podocytes transfected with or without RANK siRNA when exposed to PAN. Their
results showed that RANK played an insignificant role in the protection of podocytes, and RANKL reduced podocyte apoptosis independently of RANK. Thus, it is controversial whether RANKL acts on RANK to protect podocytes from apoptosis. In our opinion, RANKL may exert its anti-apoptotic effect on PAN-induced podocytes by binding the decoy receptor osteoprotegerin (OPG) (Blazquez-Medela, Lopez-Novoa, & Martinez-Salgado, 2011), which acts as a survival factor for endothelial cells (Malyankar et al., 2000), pancreatic β-cells (Schrader et al., 2007) and tubular cells (Candido, 2014; Lorz et al., 2008). Contrary to the findings of Liu et al., Stefan Kiechl et al. (Kiechl et al., 2013) found that RANKL was a significant and independent risk predictor of type 2 diabetes mellitus, and when they blocked RANK, the RANKL-mediated upregulation of NF-κB activation and proinflammatory genes was completely inhibited. Similar to this report, our data showed that RANKL and RANK were damage factors in podocytes exposed to HG. Our results provided the first demonstration, to our knowledge, that RANKL and RANK were upregulated in HG-induced podocytes and that silencing RANK significantly reduced migration, albumin filtration and MCP-1 expression in podocytes. Hence, we infer that RANK may be the key point on which LJ acts to protect podocytes from injury. As a downstream effector of RANKL-RANK, the NF-κB pathway is involved in the pathogenesis of diabetic mellitus (Kiechl et al., 2013; Oguiza et al., 2015). Normally, NF-κB is kept in an inactive state by the inhibitory subunit IκB in the cytosol. When external stimulation occurs, the IκB kinase (IKK) complex phosphorylates IκB molecules and promotes their degradation. This leads to the release of NF-κB and its subsequent translocation into the nucleus, where it promotes the transcription of gene-encoding inflammatory mediators, such as MCP-1 (Amann, Tinzmann, & Angelkort, 2003; Mezzano et al., 2004). Additionally, the MAPKs (JNK, ERK and p38) influence inflammatory gene expression in DN (Kang, Song, et al., 2010) and have been reported to be activated by RANKL and associated with cell migration (Tang, Zhang, Tang, Qi, & Jiang, 2011; Wang et al., 2015). Therefore, to explore the underlying molecular mechanisms that
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Fig. 6. RANK is important for the LJ-mediated inhibition of HG-induced migration, albumin filtration and MCP-1 expression in podocytes. (A) Representative immunoblot of RANK in lysates of podocytes after 24 h of transfection with siRNA control or three different siRNA sequences of RANK and subsequent 24 h treatment with HG. (B) The ratio between RANK and β-actin in podocytes was calculated and indicated (mean and S.D., N = 3, #P b 0.05 compared with si-CON, t-test). RANK in podocytes was silenced by siRNA for 48 h. Then, these podocytes were treated with HG or/and 10 μM LJ for 24 h. Different examinations were performed, and the results were as follows: (C) Representative fields of migratory podocytes (scale bars: 50 μm). (D) The numbers of migratory cells were counted under a microscope (mean and S.D., N = 3, #P b 0.05 compared with si-CON, t-test). (E) Albumin filtration of podocytes in different groups. (F) The secretion of MCP-1 in supernatants of podocytes. (G) The mRNA expression of MCP-1 in lysates of podocytes. All experiments were performed three times. Bars E–G show the mean expression in arbitrary units (error bars S.D.). *P b 0.05 compared with NG, #P b 0.05 compared with HG stimulation, t-test.
regulate cell migration, albumin filtration and MCP-1 expression mediated by LJ, we measured the protein levels of RANKL, RANK, NF-κB and MAPK signaling pathways. Our results indicated that LJ dose-dependently inhibits RANKL, RANK, phospho-IKKβ, phosphorIκBα, phospho-p65, phospho-p38, phospho-ERK and phospho-JNK in HG-induced podocytes. Furthermore, we found that silencing RANK significantly blocked the activation of NF-κB and MAPK pathways and when we treated podocytes with si-RANK and LJ in HG condition, the inhibition was similar to that in the siRNA-RANK-treated group. It is possible that RANK is a key point for LJ-mediated inhibition of NF-κB and MAPK pathways. However, the exact regulatory mechanism by which LJ acts on RANK requires further exploration. Nephrin is a marker of normal podocytes, controlling the slit diaphragm structure that is the critical factor of the size permselec-
tivity of the glomerular filtration barrier (Kestila et al., 1998; Tryggvason & Wartiovaara, 2001). A reduction in nephrin protein and mRNA levels has been reported in DN, which reflects the injury of podocytes (Doublier et al., 2003; Langham et al., 2002). In our experiment, nephrin was decreased in HG-induced podocytes in a time-dependent manner. Meanwhile, LJ dose-dependently increased nephrin. However, when we silenced RANK in HG-induced podocytes, nephrin was only slightly upregulated. Taken together, our results indicate that nephrin participates in the safeguarding of HG-induced podocytes by LJ. In summary, we demonstrated for the first time that RANKL and RANK were overexpressed in HG-induced podocytes and that LJ attenuated HG-induced migration, albumin filtration and MCP-1 expression by blocking RANKL, RANK and the downstream NF-κB
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
X-W. Chen et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
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Fig. 7. RANK is important for the LJ-mediated inactivation of the NF-κB and MAPK pathways. RANK in podocytes was silenced by siRNA. Then, these podocytes were treated with HG or/and 10 μM LJ for 24 h. Western blot and immunofluorescence were performed, and the results were as follows: (A) Representative immunoblot of RANKL, RANK, nephrin and β-actin in lysates of podocytes. (B) Representative immunoblot of p-p65, p65, p-IKKβ, IKKβ, p-IκBα and β-actin in lysates of podocytes. (C) Representative immunoblot of p-p38, p38, p-ERK, ERK, p-JNK, JNK and β-actin in lysates of podocytes. (D) The ratios between RANKL and β-actin (d1), RANK and β-actin (d2), nephrin and β-actin (d3), p-p65 and p65 (d4), p-IKKβ and IKKβ (d5), p-IκBα and β-actin (d6), p-p38 and p38 (d7), p-ERK and ERK (d8), and p-JNK and JNK (d9) in podocytes were calculated and indicated (mean and S.D., N = 3, *P b 0.05 compared with control, #P b 0.05 compared with HG stimulation, t-test). (E) Representative fields of the localization of p65 in podocytes under confocal microscope (scale bars: 50 μm).
and MAPK signaling pathways. These findings suggest the protective role of LJ in HG-induced podocytes, which encourage us to continue to investigate the beneficial effects of LJ in protecting against DN.
ZZX performed the graph preparation and statistical analysis. HBL, XWC conceived of the study and helped draft the manuscript. All authors read and approved the final manuscript.
Author contributions
Acknowledgements
XWC, WTL and YXW conducted the molecular and cellular studies. XWC and WTL drafted the manuscript. WJC, HYL, YHC, XYD, FFP, WDZ,
This work was supported by the National Natural Science Foundation of China (NSFC; no. 81072848 to Hai-Bo Long), the
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Science and Technology Planning Project of Guangdong Province, China (no. 2013B021800149, 2014A020210011 and 2015A020211012 to Hai-Bo Long), the Natural Science Foundation of Guangdong Province, China (no. 2014A030310065 to Fen-Fen Peng), and the Science and Technology Planning Project of Guangzhou, China (no. 201510010137 to Hai-Bo Long). We thank Accendatech Co. Ltd for providing us LJ and thank Prof. Tanqi Lou for providing us podocytes. References Amann, B., Tinzmann, R., & Angelkort, B. (2003). ACE inhibitors improve diabetic nephropathy through suppression of renal MCP-1. Diabetes Care, 26, 2421–2425. Anil, K. P., Welsh, G. I., Saleem, M. A., & Menon, R. K. (2014). Molecular and cellular events mediating glomerular podocyte dysfunction and depletion in diabetes mellitus. Frontiers in Endocrinology, 5, 151. Armstrong, A. P., Miller, R. E., Jones, J. C., Zhang, J., Keller, E. T., & Dougall, W. C. (2008). RANKL acts directly on RANK-expressing prostate tumor cells and mediates migration and expression of tumor metastasis genes. Prostate, 68, 92–104. Blazquez-Medela, A. M., Lopez-Novoa, J. M., & Martinez-Salgado, C. (2011). Osteoprotegerin and diabetes-associated pathologies. Current Molecular Medicine, 11, 401–416. Candido, R. (2014). The osteoprotegerin/tumor necrosis factor related apoptosisinducing ligand axis in the kidney. Current Opinion in Nephrology and Hypertension, 23, 69–74. Chow, F., Ozols, E., Nikolic-Paterson, D. J., Atkins, R. C., & Tesch, G. H. (2004). Macrophages in mouse type 2 diabetic nephropathy: Correlation with diabetic state and progressive renal injury. Kidney International, 65, 116–128. Collins, A. J., Foley, R. N., Chavers, B., Gilbertson, D., Herzog, C., Ishani, A., ... Agodoa, L. (2014). US Renal Data System 2013 Annual Data Report. American Journal of Kidney Diseases, 63(1 Suppl.), A7. Doublier, S., Salvidio, G., Lupia, E., Ruotsalainen, V., Verzola, D., Deferrari, G., & Camussi, G. (2003). Nephrin expression is reduced in human diabetic nephropathy: Evidence for a distinct role for glycated albumin and angiotensin II. Diabetes, 52, 1023–1030. Golden, D., Saria, E. A., & Hansen, M. F. (2015). Regulation of osteoblast migration involving receptor activator of nuclear factor (NF)-Kappa B (RANK) signaling. Journal of Cellular Physiology, 230(12), 2951–2960. Hashizume, M., Hayakawa, N., & Mihara, M. (2008). IL-6 trans-signalling directly induces RANKL on fibroblast-like synovial cells and is involved in RANKL induction by TNF-alpha and IL-17. Rheumatology (Oxford), 47, 1635–1640. Hsu, C. J., Lin, T. Y., Kuo, C. C., Tsai, C. H., Lin, M. Z., Hsu, H. C., ... Tang, C. H. (2010). Involvement of integrin up-regulation in RANKL/RANK pathway of chondrosarcomas migration. Journal of Cellular Biochemistry, 111, 138–147. Jones, D. H., Nakashima, T., Sanchez, O. H., Kozieradzki, I., Komarova, S. V., Sarosi, I., ... Penninger, J. M. (2006). Regulation of cancer cell migration and bone metastasis by RANKL. Nature, 440, 692–696. Kanamori, H., Matsubara, T., Mima, A., Sumi, E., Nagai, K., Takahashi, T., ... Arai, H (2007). Inhibition of MCP-1/CCR2 pathway ameliorates the development of diabetic nephropathy. Biochemical and Biophysical Research Communications, 360, 772–777. Kang, Y. S., Li, Y., Dai, C., Kiss, L. P., Wu, C., & Liu, Y. (2010). Inhibition of integrin-linked kinase blocks podocyte epithelial-mesenchymal transition and ameliorates proteinuria. Kidney International, 78, 363–373. Kang, Y. S., Song, H. K., Lee, M. H., Ko, G. J., Han, J. Y., Han, S. Y., ... Cha, D. R. (2010). Visfatin is upregulated in type-2 diabetic rats and targets renal cells. Kidney International, 78, 170–181. Kestilä, M., Lenkkeri, U., Männikkö, M., Lamerdin, J., McCready, P., Putaala, H., ... Tryggvason, K. (1998). Positionally cloned gene for a novel glomerular protein– nephrin–is mutated in congenital nephrotic syndrome. Molecular Cell, 1, 575–582. Kiechl, S., Wittmann, J., Giaccari, A., Knoflach, M., Willeit, P., Bozec, A., ... Schett, G. (2013). Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents development of diabetes mellitus. Nature Medicine, 19, 358–363. Kim, J. Y., Cheon, Y. H., Yoon, K. H., Lee, M. S., & Oh, J. (2014). Parthenolide inhibits osteoclast differentiation and bone resorbing activity by down-regulation of NFATc1 induction and c-Fos stability, during RANKL-mediated osteoclastogenesis. BMB Reports, 47, 451–456. Langham, R. G., Kelly, D. J., Cox, A. J., Thomson, N. M., Holthofer, H., Zaoui, P., ... Gilbert, R. E. (2002). Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: Effects of angiotensin converting enzyme inhibition. Diabetologia, 45, 1572–1576. Lee, E. Y., Chung, C. H., Khoury, C. C., Yeo, T. K., Pyagay, P. E., Wang, A., & Chen, S. (2009). The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-beta,
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Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK signaling pathways Xiao-Wen Chen a, 1, Wen-Ting Liu a, 1, Yu-Xian Wang b, 1, Wen-Jing Chen a, Hong-Yu Li a, Yi-Hua Chen a, Xiao-Yan Du a, Fen-Fen Peng a, Wei-Dong Zhou a, Zhao-Zhong Xu c, Hai-Bo Long a,⁎ a b c
Department of Nephrology, ZhuJiang Hospital, Southern Medical University, Guangzhou, 510280, China Department of Gerontology, ZhuJiang Hospital, Southern Medical University, Guangzhou, 510280, China Department of Emergency, ZhuJiang Hospital, Southern Medical University, Guangzhou, 510280, China
a r t i c l e
i n f o
Article history: Received 4 January 2016 Received in revised form 7 March 2016 Accepted 14 March 2016 Available online xxxx Keywords: LJ Receptor activator for NF-κB ligand Receptor activator for NF-κB NF-κB Mitogen-activated protein kinase
a b s t r a c t Aims: The aim of this research was to investigate the effects of cyclopropanyldehydrocostunolide (also named LJ), a derivative of sesquiterpene lactones (SLs), on high glucose (HG)-induced podocyte injury and the associated molecular mechanisms. Methods: Differentiated mouse podocytes were incubated in different treatments. The migration and albumin filtration of podocytes were examined by Transwell filters. The protein and mRNA levels of MCP-1 were measured using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (q-PCR). Protein expression and phosphorylation were detected by western blot, and the nuclear translocation of NF-κB was performed with a confocal microscope. The gene expression of the receptor activator for NF-κB (RANK) was silenced by small interfering RNA (siRNA). Results: Our results showed that HG enhanced migration, albumin filtration and MCP-1 expression in podocytes. At the molecular level, HG promoted the phosphorylation of NF-κB/p65, IKKβ, IκBα, mitogen-activated protein kinase (MAPK) and the nuclear translocation of p65. LJ reversed the effects of HG in a dose-dependent manner. Furthermore, our data provided the first demonstration that the receptor activator for NF-κB ligand (RANKL) and its cognate receptor RANK were overexpressed in HG-induced podocytes and were downregulated by LJ. RANK siRNA also attenuated HG-induced podocyte injury and markedly inhibited the activation of NF-κB and MAPK signaling pathways. Conclusions: LJ attenuates HG-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK signaling pathways. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Diabetic nephropathy (DN), characterized by progressive albuminuria and a gradually declining glomerular filtration rate, has a dismal prognosis. It is one of the most common and severe microvascular complications of diabetes resulting from the leading risk of end-stage renal disease (Ritz, Rychlik, Locatelli, & Halimi, 1999). Despite being given multiple therapies for hyperglycemia and high blood pressure, numerous patients still suffer progressive and serious renal injury (Stolar, 2010). Thus, combating DN has become a major focus of research (Collins et al., 2014; Liu, 2013; Xu et al., 2013). Conflict of Interest: The authors declare no conflict of interest. ⁎ Corresponding author. E-mail address: [email protected] (H-B. Long). 1 These authors contributed equally to this study.
Podocytes are vitally important to the integrity of the glomerular filtration barrier. It has been reported that they can precede and predict the occurrence of proteinuria (Anil, Welsh, Saleem, & Menon, 2014) and may be an early pathological manifestation of DN (Meyer, Bennett, & Nelson, 1999). When podocytes suffer from injurious stimuli such as HG, TGF-β1 and adriamycin, they become more motile and leave the glomerular basement membrane, leading to the onset of proteinuria (Kang et al., 2010). Hence, finding a drug that can protect podocyte from injury is essential for the development of targeted, effective DN treatment. SLs, originally isolated from medicinal herbs of the Asteraceae family, have been examined for their anti-inflammatory, immunomodulatory, and anticancer effects (Viennois et al., 2014; Zhang et al., 2012). LJ is a derivative of SLs, i.e., cyclopropanyldehydrocostunolide. Our previous studies have shown that SL derivatives, including LJ (also named compound 1), can block the advanced oxidation protein
http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013 1056-8727/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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X-W. Chen et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
product (AOPP)-induced activation of the NF-κB pathway and the expression of MCP-1 in rat mesangial cells or podocytes (Wang et al., 2013; Zhao et al., 2015). However, little is known about whether LJ can attenuate migration and albumin filtration in HG-induced podocytes and what the underlying molecular mechanism is. RANKL, a type II membrane protein, belongs to the TNF superfamily and contains a C-terminal receptor-binding domain and a transmembrane region. RANK, a type I membrane protein that shares high homology with CD40, is one of the receptors for RANKL. Recently, Liu et al. (Liu et al., 2012) reported that RANK and RANKL are receptor-ligand complexes for the pathogenesis of podocyte injury. A genome-wide association study also revealed a polymorphism in the podocyte receptor, RANK, that leads to a decline in renal function in coronary patients (Leiherer et al., 2014). These results suggest that RANK is a promising therapeutic target in glomerular diseases. Moreover, emerging evidence shows that RANKL and RANK play a vital role in the migration of cancer cells and osteoblasts (Armstrong et al., 2008; Hsu et al., 2010; Jones et al., 2006). It has been demonstrated that parthenolide, which is a natural SL, can inhibit RANKL-mediated osteoclast differentiation and suppress IL-6/sIL6R-induced RANKL expression in fibroblast-like synoviocytes (Hashizume, Hayakawa, & Mihara, 2008; Kim, Cheon, Yoon, Lee, & Oh, 2014). In addition, NF-κB and MAPK pathways contribute to the migration of cancer cells and osteoblasts (Golden, Saria, & Hansen, 2015; Schramek et al., 2010; Song et al., 2014; Wang, Sun, Zhang, Wang, & Li, 2015). We can therefore speculate that LJ may attenuate the migration and albumin filtration of podocytes by regulating RANKL/RANK-mediated NF-κB and MAPK signaling pathways. In the present study, we investigated the effects of LJ and RANK on the migration and albumin filtration in HG-induced podocytes and examined the underlying molecular mechanisms involved in this process.
and transferred onto PVDF membranes (Millipore, Bedford, MA). The membrane was blocked for 1 h with Tris-buffered saline (TBS; 20 mM Tris–HCl, 140 Mm NaCl, PH7.6) containing 5% non-fat dry milk, washed with TBS containing 0.1% Tween-20, and incubated overnight at 4 °C with primary antibodies. The primary antibodies for RANKL rabbit mAb, RANK rabbit mAb and nephrin rabbit mAb were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,USA). IKKβ rabbit mAb, phospho-IKKβ rabbit mAb, p65 rabbit mAb, phospho-p65 rabbit mAb, phospho-IκBα rabbit mAb, p38 rabbit mAb, phospho-p38 mouse mAb, ERK rabbit mAb, phospho-ERK rabbit mAb, JNK rabbit mAb, and phospho-JNK rabbit mAb were purchased from Cell Signaling Technology (Cell Signaling Technologies, Danvers, MA). β-actin mouse mAb and HRP-conjugated secondary antibodies were purchased from EarthOx (EarthOx, LLC, San Francisco, CA,USA). The intensities of the protein bands were quantified by Quantity One 4.6.2 analysis software (Quantity One, Bio-Rad Laboratories, Inc., USA), which was provided with the Kodak 2000MM System (Eastman Kodak company, Rochester, NY, USA).
2. Materials and methods
Podocytes (5 × 10 4/well) were plated in a 12-well plate under various experimental conditions. After 24 h, the supernatants were assayed using a solid-phase quantitative sandwich ELISA kit for MCP-1, as described previously (Zhao et al., 2015).
2.1. Cell culture and treatment Conditionally immortalized mouse podocytes between passages 12 and 20 (a kind gift from Prof. Peter Mundel, Mount Sinai School of Medicine, through Prof. Tanqi Lou, the Third First Affiliated Hospital of Sun Yat-Sen University) were cultured as previously described (Shankland, Pippin, Reiser, & Mundel, 2007). Briefly, at a permissive temperature of 33 °C, undifferentiated podocytes were grown in RPMI1640 containing 10% FBS (Life Technologies, Rochester, NY, USA), penicillin (100 U/mL), streptomycin (100 mg/mL), and 50 IU/mL recombinant murine IFN-γ (Pepro Tech, USA). To induce differentiation, podocytes were cultured at 37 °C without IFN-γ for 10–14 days. For experiments, the differentiated podocytes were incubated in the presence of 5 mmoles (mM) D-glucose (NG), 30 mM D-glucose (HG) or 5 mM D-glucose + 25 mM mannitol (MA) at 37 °C for 3, 6, 12, and 24 h (h). In some experiments, the podocytes were divided into different groups and then treated with LJ of different concentrations in HG condition for 24 h. LJ was provided by Accendatech Co. Ltd (Tianjin, China). In other experiments, subconfluent podocyte cultures were transfected with RANK siRNA (50 nmoles) or siRNA control (50 nmoles) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The sequences of RANK siRNA were as follows: si-RANK1: sense: 5′-CCU CCU UGG AAA GCU AGA ATT-3′, antisense: 5′-UUC UAG CUU UCC AAG GAG GTT-3′; si-RANK2: sense: 5′-GGA GAG GCA UUA UGA GCA UTT-3′, antisense: 5′- AUG CUC AUA AUG CCU CUC CTT-3′; and si-RANK3: sense: 5′- GCG CAG ACU UCA CUC CAU AUU-3′, antisense: 5′UAU GGA GUG AAG UCU GCG CUU-3′. 2.2. Western blot analysis All samples were handled as described previously (Zhao et al., 2015). Total proteins were separated on 10% SDS-polyacrylamide gels
2.3. Confocal microscopy analysis Differentiated podocytes were fixed with 10% paraformaldehyde and permeabilized with 0.2% Trition X-100 in PBS. Then, the cells were blocked with 5% BSA. They were then incubated with NF-κB p65 rabbit mAb (Cell Signaling Technologies, Danvers, MA) for 2 h at 37 °C, AlexaFluor 488 goat anti-rabbit (Invitrogen) for 1 h at 37 °C, and then 4 ˊ, 6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. The cells were visualized under a Nikon C2 confocal microscope (Nikon corporation, Tokyo, Japan). 2.4. Enzyme-linked immunosorbent assay
2.5. Quantitative real-time PCR analysis The RNA from the podocytes in all samples was extracted as described previously (Zhao et al., 2015). The primers for qPCR were as follows: MCP-1 forward: 5′-CCC AAT GAG TAG GCT GGA GA-3′, reverse: 5′-TCT GGA CCC ATT CCT TCT TG-3′and GAPDH forward: 5′-ATT GTC AGC AAT GCA TCC TG-3′, reverse: 5 ′-ATG GAC TGT GGT CAT GAG CC-3′. All data were normalized to their GAPDH content, and expression levels were analyzed by the 2-DDCt method. 2.6. Cell migration assay Podocytes (5 × 104/well), transfected with or without RANK-siRNA (50 nM)/control- siRNA (50 nM), were plated on the top side of 8.0 μm pore polycarbonate Transwell filters (Corning, Lowell, MA) coated with type I collagen (Corning, Lowell, MA). Serum-free medium was used in the both chambers. Afterward, HG or MA and various concentrations of LJ were added. The cells were incubated at 37 °C for 24 h. The nonmigratory cells in the top chambers were removed with cotton swabs, and the migrated cells were fixed in 4% paraformaldehyde for 10 min, stained with 0.1% crystal violet (sigma), and counted under a microscope (Ni-U, Nikon Corporation, Tokyo, Japan). 2.7. Transwell albumin filtration assay Podocytes (5 × 103/well) were plated on the top side of 3.0 μm pore polycarbonate Transwell filters (Corning, Lowell, MA) coated with type I collagen (Corning, Lowell, MA). The cells were then transfected with or without RANK-siRNA (50 nM)/control- siRNA (50 nM) for 24 h and then
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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3. Results
or MA or HG for 24 h and then examined migration by Transwell filters. The results showed that HG significantly increased the migratory ability of podocytes across the pores of the Transwell filters (Fig. 1A and B). We next examined whether HG affects the barrier function of the podocyte monolayer. To this end, we used an albumin flux assay that assesses albumin filtration across the monolayer of cultured differentiated podocytes. As shown in Fig. 1C, HG increased albumin flux in podocytes more than NG did. To eliminate the influence of osmotic pressure, we set the MA group and found that osmotic pressure did not influence the results. MCP-1 is characterized as attracting macrophages and promoting inflammation in DN (Chow, Ozols, Nikolic-Paterson, Atkins, & Tesch, 2004; Kanamori et al., 2007). However, it also plays a noninflammatory role in enhancing the migratory capacity of podocytes. Thus, we measured the MCP-1 protein level using ELISA and detected MCP-1 RNA by q-PCR in HG-induced podocytes. The results demonstrated that HG significantly induced the expression of MCP-1 in protein and mRNA levels (Fig. 1D and E).
3.1. HG enhances migration, albumin filtration and MCP-1 expression in podocytes
3.2. The effect of HG on nephrin and the NF-κB and MAPK signaling pathways in podocytes
Podocytes after HG stimulation may become motile with an increased migratory capacity. To test this hypothesis, we treated podocytes with NG
Nephrin is a marker of normal podocytes. Its reduction reflects podocyte injury. As shown in Fig. 2A, nephrin was significantly
treated with HG or MA and various concentrations of LJ for 24 h. The medium in each upper chamber was replaced with 150 μL RPMI 1640, and 1 ml RPMI 1640 with 40 mg/ml bovine serum albumin was added to each bottom chamber. After incubation at 37 °C for 6 h, an aliquot of medium from the upper chamber was collected, and the concentrations of albumin were measured using a BCA protein assay kit (Thermo Fisher Scientific, Shanghai, China). 2.8. Statistical analysis The results were presented as the mean ± S.D. Differences among multiple groups were analyzed by one-way ANOVA followed by a t-test to detect between-group differences. A two-sided P b 0.05 was considered statistically significant. All analyses were performed using SPSS Statistics 20.0 (SPSS, Chicago, IL, USA).
Fig. 1. HG enhances migration, albumin filtration and MCP-1 expression in podocytes. Podocytes were treated with NG or MA or HG for 24 h before undergoing different examinations. The results were as follows: (A) Representative fields of migratory podocytes. They were stained with 0.1% crystal violet on the membrane and observed with a microscope (scale bars: 50 μm). (B) The numbers of migratory cells were counted under a microscope in different groups. (C) Albumin filtration of podocytes in different groups. (D) The secretion of MCP-1 in supernatants of podocytes in different groups was measured by ELISA. (E) The mRNA expression of MCP-1 in lysates of podocytes in different groups. All experiments were performed three times. Bars B–E show the mean expression in arbitrary units (error bars S.D.). *P b 0.05 compared with NG, t-test.
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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decreased in HG-induced podocytes at 12 and 24 h (Fig. 2A and D: d1), indicating the injury of podocytes. Inversely, proteins in the NF-κB signaling pathway, including phospho-p65, phospho-IKKβ, and phospho-IκBα, were enhanced by HG in a time-dependent manner, whereas IKKβ and p65 did not significantly change in any group (Fig. 2B and D: d2–d4). Similarly, to clarify the effects of HG on MAPK activation in podocytes, we investigated the phosphorylation of individual MAPKs and found that HG induced a rapid and sustained activation of p38, ERK and JNK without affecting their total levels (Fig. 2C and D: d5–d7).
with 0 μM, 2.5 μM, 5 μM, and 10 μM of LJ in HG condition for 24 h. Fig. 3A shows the structure of LJ. As shown in Fig. 3B–D, LJ improved the migration and albumin filtration of differentiated podocytes in a dose-dependent manner. After 24 h of exposure to 10 μM LJ, cell migration and albumin filtration reached the best improvement. The increase in MCP-1 in protein and mRNA levels in HG-induced podocytes were also blocked by LJ in a dose-dependent manner (Fig. 3E and F).
3.3. LJ suppresses HG-induced migration, albumin filtration and MCP-1 expression in podocytes
From the above, it is clear that NF-κB and MAPK signaling pathways were activated in HG-induced podocytes. Thus, we went on to measure these proteins to explore the effect of LJ on them. As shown in Fig. 4, LJ inhibited phospho-p65, phospho-IKKβ, phospho-IκBα (Fig. 4B and D: d2–d4) and the phosphorylation of p38, ERK
To examine whether LJ attenuated HG-induced migration, albumin filtration and MCP-1 expression in podocytes, we treated podocytes
3.4. LJ blocks NF-κB and MAPK signaling pathway activation but upregulates nephrin in HG-induced podocytes
Fig. 2. The effect of HG on nephrin and the NF-κB and MAPK signaling pathways in podocytes. Podocytes were treated with MA or HG for 3, 6, 12, and 24 h and NG for 24 h. Then, the cell supernatants were obtained for western blot. (A) Representative immunoblot of nephrin and β-actin. (B) Representative immunoblot of p-p65, p65, p-IKKβ, IKKβ, p-IκBα and β-actin. (C) Representative immunoblot of p-p38, p38, p-ERK, ERK, p-JNK, JNK and β-actin. (D) The ratios between nephrin and β-actin (d1), p-p65 and p65 (d2), p-IKKβ and IKKβ (d3), p-IκBα and β-actin (d4), p-p38 and p38 (d5), p-ERK and ERK (d6), and p-JNK and JNK (d7) in podocytes were calculated and indicated (mean and S.D., N = 3, *P b 0.05 compared with NG, #P b 0.05 compared with HG stimulation, t-test).
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Fig. 3. LJ suppresses HG-induced migration, albumin filtration and MCP-1 expression in podocytes. Podocytes were treated with different concentrations of LJ in the HG condition for 24 h. Then, different examinations were performed. The results were as follows: (A) The structure of LJ. (B) Representative fields of migratory podocytes (scale bars: 50 μm). (C) The numbers of migratory cells were counted under a microscope in different groups. (D) Albumin filtration of podocytes in different groups. (E) The secretion of MCP-1 in supernatants of podocytes in different groups were measured by ELISA. (F) The mRNA expression of MCP-1 in lysates of podocytes in different groups. All experiments were performed three times. Bars C–F show the mean expression in arbitrary units (error bars S.D.). #P b 0.05 compared with HG stimulation, t-test.
and JNK (Fig. 4C and D: d5–d7), whereas it upregulated nephrin (Fig. 4A and D: d1) in HG-induced podocytes in a dose-dependent manner. The total levels of p65, IKKβ, p38, ERK and JNK did not significantly change in any group. In addition to the NF-κB western blot assays, we performed the immunofluorescence staining of p65 in HG-induced podocytes with or without LJ. Our results showed that in the absence of HG, most p65 that was unphosphorylated and inactive was localized to the cytoplasm. Upon HG stimulation for 24 h, almost all p65 was localized to the nucleus, but the nuclear translocation of p65 did not occur with the co-treatment of 10 μM LJ and HG (Fig. 4E). 3.5. RANKL and RANK are upregulated in HG-induced podocytes but downregulated by LJ It has been reported that puromycin aminonucleoside (PAN) induces the expression of RANKL and RANK in podocytes (Liu et al.,
2012), but little is known about whether HG can also do so. To solve this problem, we investigated the protein level of RANKL and RANK in HG-induced podocytes. As shown in Fig. 5, HG significantly upregulated RANKL and RANK in a time-dependent manner (Fig. 5A and B). Moreover, we found that LJ downregulated RANKL and RANK in a dose-dependent manner when we treated podocytes with HG and LJ for 24 h (Fig. 5C and D). 3.6. RANK is important for the LJ-mediated inhibition of HG-induced migration, albumin filtration and MCP-1 expression in podocytes To shed light on the importance of RANKL and RANK in the protection of HG-induced podocytes by LJ, we knocked down RANK by siRNA. First, we transfected podocytes with siRNA control or three different siRNA sequences of RANK (si-RANK1, si-RANK2, si-RANK3), respectively, for 24 h and then treated these podocytes with HG for
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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X-W. Chen et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
Fig. 4. LJ blocks NF-κB and MAPK signaling pathway activation but upregulates nephrin in HG-induced podocytes. Podocytes were treated with different concentrations of LJ in the HG condition for 24 h. Then, western blot and immunofluorescence were performed. The results were as follows: (A) Representative immunoblot of nephrin and β-actin. (B) Representative immunoblot of p-p65, p65, p-IKKβ, IKKβ, p-IκBα and β-actin. (C) Representative immunoblot of p-p38, p38, p-ERK, ERK, p-JNK, JNK and β-actin. (D) The ratios between nephrin and β-actin (d1), p-p65 and p65 (d2), p-IKKβ and IKKβ (d3), p-IκBα and β-actin (d4), p-p38 and p38 (d5), p-ERK and ERK (d6), and p-JNK and JNK (d7) in podocytes were calculated and indicated (mean and S.D., N = 3, *P b 0.05 compared with NG, #P b 0.05 compared with HG stimulation, t-test). (E) Representative fields of the localization of p65 in podocytes under a confocal microscope (scale bars: 50 μm).
24 h. After transient transfection, the cell supernatants of the podocytes were obtained for western blot to detect the most efficient sequence of si-RANK. The results showed that si-RANK3 was the most efficient sequence compared to siRNA control (Fig. 6A and B). Furthermore, we found that the endogenous knockdown of RANK attenuated the migration and albumin filtration of podocytes compared with the HG-stimulated cells transfected with siRNA control. Subsequently, we treated the cells transfected with si-RANK with 10 μM LJ and found that the inhibition of migration and albumin filtration was similar to that in the siRNA-RANK-treated group (Fig. 6C–E). Intriguingly, the protein and mRNA levels of MCP-1 were also inhibited by si-RANK (Fig. 6F and G).
3.7. RANK is important for the LJ-mediated inactivation of the NF-κB and MAPK pathways To further elucidate the molecular mechanisms by which LJ influenced podocytes, we silenced RANK in the podocytes and treated them with 10 μM LJ. Then, we examined the proteins via western blot. Fig. 7 shows that RANKL, RANK (Fig. 7A and D: d1–d2), phospho-p65, phosphor-IKKβ, phosphor-IκBα (Fig. 7B and D: d4–d6) and the phosphorylation of p38, ERK and JNK (Fig. 7C and D: d7–d9) were significantly attenuated but that nephrin (Fig. 7A and D: d3) increased slightly when RANK was silenced. The result of the immunofluorescence staining of p65 also showed that the nuclear translocation of
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Fig. 5. RANKL and RANK are upregulated in HG-induced podocytes but downregulated by LJ. Podocytes were treated with MA or HG for 3, 6, 12, 24 h and NG for 24 h or treated with different concentrations of LJ in the HG condition for 24 h. Then, the cell supernatants were obtained for western blot. (A, C) Representative immunoblot of RANKL, RANK and β-actin. (B, D) The ratios between RANKL and β-actin (b1, d1), RANK and β-actin (b2, d2) in podocytes were calculated and indicated (mean and S.D., N = 3, *P b 0.05 compared with NG, #P b 0.05 compared with HG stimulation, t-test).
p65 was blocked in the absence of RANK (Fig. 7E). Moreover, the effects of HG on the activation of NF-κB and MAPK pathways were also reversed by the co-administration of LJ and siRNA-RANK. 4. Discussion Podocytes play a vital role in DN. When exposed to HG, podocytes may increase their motility and lose their morphological structure, which undoubtedly impairs the integrity of the glomerular filtration barrier and increase paracellular albumin flux across podocyte monolayers, thereby causing proteinuria (Kang et al., 2010; Muller-Krebs et al., 2015). Inevitably, MCP-1, an important protein that is correlated with progression and renal outcomes in DN (Chow et al., 2004; Kanamori et al., 2007), will occur as a multifunctional cytokine simultaneously. On the one hand, MCP-1 attracts macrophage migration into the diabetic kidney and promote inflammation in DN (Chow et al., 2004). On the other hand, MCP-1 can interact with its receptor that is predominantly localized to podocytes, contributing to migration and the albumin filtration of podocytes (Lee et al., 2009). This process leads to vicious circle of increasing migration, strengthening proteinuria and enhancing MCP-1. In our study, we confirmed that HG induced this vicious circle, and we further demonstrated for the first time that LJ prevented the effects of HG in a dosedependent manner. How can LJ achieve success? We focused on the key point: podocyte migration. Studies have increasingly demonstrated that RANKL and RANK direct the migration of cancer cells and osteoblasts (Armstrong et al., 2008; Hsu et al., 2010; Jones et al., 2006). Moreover, Liu et al. reported that RANKL and RANK were overexpressed in PAN-induced podocytes (Liu et al., 2012). In their report, they found that the knockdown of RANK alone did not induce podocyte apoptosis but mildly increased the apoptosis of podocytes exposed to PAN, which had no statistical significance. However, the application of exogenous RANKL significantly reduced the apoptosis of podocytes transfected with or without RANK siRNA when exposed to PAN. Their
results showed that RANK played an insignificant role in the protection of podocytes, and RANKL reduced podocyte apoptosis independently of RANK. Thus, it is controversial whether RANKL acts on RANK to protect podocytes from apoptosis. In our opinion, RANKL may exert its anti-apoptotic effect on PAN-induced podocytes by binding the decoy receptor osteoprotegerin (OPG) (Blazquez-Medela, Lopez-Novoa, & Martinez-Salgado, 2011), which acts as a survival factor for endothelial cells (Malyankar et al., 2000), pancreatic β-cells (Schrader et al., 2007) and tubular cells (Candido, 2014; Lorz et al., 2008). Contrary to the findings of Liu et al., Stefan Kiechl et al. (Kiechl et al., 2013) found that RANKL was a significant and independent risk predictor of type 2 diabetes mellitus, and when they blocked RANK, the RANKL-mediated upregulation of NF-κB activation and proinflammatory genes was completely inhibited. Similar to this report, our data showed that RANKL and RANK were damage factors in podocytes exposed to HG. Our results provided the first demonstration, to our knowledge, that RANKL and RANK were upregulated in HG-induced podocytes and that silencing RANK significantly reduced migration, albumin filtration and MCP-1 expression in podocytes. Hence, we infer that RANK may be the key point on which LJ acts to protect podocytes from injury. As a downstream effector of RANKL-RANK, the NF-κB pathway is involved in the pathogenesis of diabetic mellitus (Kiechl et al., 2013; Oguiza et al., 2015). Normally, NF-κB is kept in an inactive state by the inhibitory subunit IκB in the cytosol. When external stimulation occurs, the IκB kinase (IKK) complex phosphorylates IκB molecules and promotes their degradation. This leads to the release of NF-κB and its subsequent translocation into the nucleus, where it promotes the transcription of gene-encoding inflammatory mediators, such as MCP-1 (Amann, Tinzmann, & Angelkort, 2003; Mezzano et al., 2004). Additionally, the MAPKs (JNK, ERK and p38) influence inflammatory gene expression in DN (Kang, Song, et al., 2010) and have been reported to be activated by RANKL and associated with cell migration (Tang, Zhang, Tang, Qi, & Jiang, 2011; Wang et al., 2015). Therefore, to explore the underlying molecular mechanisms that
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Fig. 6. RANK is important for the LJ-mediated inhibition of HG-induced migration, albumin filtration and MCP-1 expression in podocytes. (A) Representative immunoblot of RANK in lysates of podocytes after 24 h of transfection with siRNA control or three different siRNA sequences of RANK and subsequent 24 h treatment with HG. (B) The ratio between RANK and β-actin in podocytes was calculated and indicated (mean and S.D., N = 3, #P b 0.05 compared with si-CON, t-test). RANK in podocytes was silenced by siRNA for 48 h. Then, these podocytes were treated with HG or/and 10 μM LJ for 24 h. Different examinations were performed, and the results were as follows: (C) Representative fields of migratory podocytes (scale bars: 50 μm). (D) The numbers of migratory cells were counted under a microscope (mean and S.D., N = 3, #P b 0.05 compared with si-CON, t-test). (E) Albumin filtration of podocytes in different groups. (F) The secretion of MCP-1 in supernatants of podocytes. (G) The mRNA expression of MCP-1 in lysates of podocytes. All experiments were performed three times. Bars E–G show the mean expression in arbitrary units (error bars S.D.). *P b 0.05 compared with NG, #P b 0.05 compared with HG stimulation, t-test.
regulate cell migration, albumin filtration and MCP-1 expression mediated by LJ, we measured the protein levels of RANKL, RANK, NF-κB and MAPK signaling pathways. Our results indicated that LJ dose-dependently inhibits RANKL, RANK, phospho-IKKβ, phosphorIκBα, phospho-p65, phospho-p38, phospho-ERK and phospho-JNK in HG-induced podocytes. Furthermore, we found that silencing RANK significantly blocked the activation of NF-κB and MAPK pathways and when we treated podocytes with si-RANK and LJ in HG condition, the inhibition was similar to that in the siRNA-RANK-treated group. It is possible that RANK is a key point for LJ-mediated inhibition of NF-κB and MAPK pathways. However, the exact regulatory mechanism by which LJ acts on RANK requires further exploration. Nephrin is a marker of normal podocytes, controlling the slit diaphragm structure that is the critical factor of the size permselec-
tivity of the glomerular filtration barrier (Kestila et al., 1998; Tryggvason & Wartiovaara, 2001). A reduction in nephrin protein and mRNA levels has been reported in DN, which reflects the injury of podocytes (Doublier et al., 2003; Langham et al., 2002). In our experiment, nephrin was decreased in HG-induced podocytes in a time-dependent manner. Meanwhile, LJ dose-dependently increased nephrin. However, when we silenced RANK in HG-induced podocytes, nephrin was only slightly upregulated. Taken together, our results indicate that nephrin participates in the safeguarding of HG-induced podocytes by LJ. In summary, we demonstrated for the first time that RANKL and RANK were overexpressed in HG-induced podocytes and that LJ attenuated HG-induced migration, albumin filtration and MCP-1 expression by blocking RANKL, RANK and the downstream NF-κB
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Fig. 7. RANK is important for the LJ-mediated inactivation of the NF-κB and MAPK pathways. RANK in podocytes was silenced by siRNA. Then, these podocytes were treated with HG or/and 10 μM LJ for 24 h. Western blot and immunofluorescence were performed, and the results were as follows: (A) Representative immunoblot of RANKL, RANK, nephrin and β-actin in lysates of podocytes. (B) Representative immunoblot of p-p65, p65, p-IKKβ, IKKβ, p-IκBα and β-actin in lysates of podocytes. (C) Representative immunoblot of p-p38, p38, p-ERK, ERK, p-JNK, JNK and β-actin in lysates of podocytes. (D) The ratios between RANKL and β-actin (d1), RANK and β-actin (d2), nephrin and β-actin (d3), p-p65 and p65 (d4), p-IKKβ and IKKβ (d5), p-IκBα and β-actin (d6), p-p38 and p38 (d7), p-ERK and ERK (d8), and p-JNK and JNK (d9) in podocytes were calculated and indicated (mean and S.D., N = 3, *P b 0.05 compared with control, #P b 0.05 compared with HG stimulation, t-test). (E) Representative fields of the localization of p65 in podocytes under confocal microscope (scale bars: 50 μm).
and MAPK signaling pathways. These findings suggest the protective role of LJ in HG-induced podocytes, which encourage us to continue to investigate the beneficial effects of LJ in protecting against DN.
ZZX performed the graph preparation and statistical analysis. HBL, XWC conceived of the study and helped draft the manuscript. All authors read and approved the final manuscript.
Author contributions
Acknowledgements
XWC, WTL and YXW conducted the molecular and cellular studies. XWC and WTL drafted the manuscript. WJC, HYL, YHC, XYD, FFP, WDZ,
This work was supported by the National Natural Science Foundation of China (NSFC; no. 81072848 to Hai-Bo Long), the
Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013
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Science and Technology Planning Project of Guangdong Province, China (no. 2013B021800149, 2014A020210011 and 2015A020211012 to Hai-Bo Long), the Natural Science Foundation of Guangdong Province, China (no. 2014A030310065 to Fen-Fen Peng), and the Science and Technology Planning Project of Guangzhou, China (no. 201510010137 to Hai-Bo Long). We thank Accendatech Co. Ltd for providing us LJ and thank Prof. Tanqi Lou for providing us podocytes. References Amann, B., Tinzmann, R., & Angelkort, B. (2003). ACE inhibitors improve diabetic nephropathy through suppression of renal MCP-1. Diabetes Care, 26, 2421–2425. Anil, K. P., Welsh, G. I., Saleem, M. A., & Menon, R. K. (2014). Molecular and cellular events mediating glomerular podocyte dysfunction and depletion in diabetes mellitus. Frontiers in Endocrinology, 5, 151. Armstrong, A. P., Miller, R. E., Jones, J. C., Zhang, J., Keller, E. T., & Dougall, W. C. (2008). RANKL acts directly on RANK-expressing prostate tumor cells and mediates migration and expression of tumor metastasis genes. Prostate, 68, 92–104. Blazquez-Medela, A. M., Lopez-Novoa, J. M., & Martinez-Salgado, C. (2011). Osteoprotegerin and diabetes-associated pathologies. Current Molecular Medicine, 11, 401–416. Candido, R. (2014). The osteoprotegerin/tumor necrosis factor related apoptosisinducing ligand axis in the kidney. Current Opinion in Nephrology and Hypertension, 23, 69–74. Chow, F., Ozols, E., Nikolic-Paterson, D. J., Atkins, R. C., & Tesch, G. H. (2004). Macrophages in mouse type 2 diabetic nephropathy: Correlation with diabetic state and progressive renal injury. Kidney International, 65, 116–128. Collins, A. J., Foley, R. N., Chavers, B., Gilbertson, D., Herzog, C., Ishani, A., ... Agodoa, L. (2014). US Renal Data System 2013 Annual Data Report. American Journal of Kidney Diseases, 63(1 Suppl.), A7. Doublier, S., Salvidio, G., Lupia, E., Ruotsalainen, V., Verzola, D., Deferrari, G., & Camussi, G. (2003). Nephrin expression is reduced in human diabetic nephropathy: Evidence for a distinct role for glycated albumin and angiotensin II. Diabetes, 52, 1023–1030. Golden, D., Saria, E. A., & Hansen, M. F. (2015). Regulation of osteoblast migration involving receptor activator of nuclear factor (NF)-Kappa B (RANK) signaling. Journal of Cellular Physiology, 230(12), 2951–2960. Hashizume, M., Hayakawa, N., & Mihara, M. (2008). IL-6 trans-signalling directly induces RANKL on fibroblast-like synovial cells and is involved in RANKL induction by TNF-alpha and IL-17. Rheumatology (Oxford), 47, 1635–1640. Hsu, C. J., Lin, T. Y., Kuo, C. C., Tsai, C. H., Lin, M. Z., Hsu, H. C., ... Tang, C. H. (2010). Involvement of integrin up-regulation in RANKL/RANK pathway of chondrosarcomas migration. Journal of Cellular Biochemistry, 111, 138–147. Jones, D. H., Nakashima, T., Sanchez, O. H., Kozieradzki, I., Komarova, S. V., Sarosi, I., ... Penninger, J. M. (2006). Regulation of cancer cell migration and bone metastasis by RANKL. Nature, 440, 692–696. Kanamori, H., Matsubara, T., Mima, A., Sumi, E., Nagai, K., Takahashi, T., ... Arai, H (2007). Inhibition of MCP-1/CCR2 pathway ameliorates the development of diabetic nephropathy. Biochemical and Biophysical Research Communications, 360, 772–777. Kang, Y. S., Li, Y., Dai, C., Kiss, L. P., Wu, C., & Liu, Y. (2010). Inhibition of integrin-linked kinase blocks podocyte epithelial-mesenchymal transition and ameliorates proteinuria. Kidney International, 78, 363–373. Kang, Y. S., Song, H. K., Lee, M. H., Ko, G. J., Han, J. Y., Han, S. Y., ... Cha, D. R. (2010). Visfatin is upregulated in type-2 diabetic rats and targets renal cells. Kidney International, 78, 170–181. Kestilä, M., Lenkkeri, U., Männikkö, M., Lamerdin, J., McCready, P., Putaala, H., ... Tryggvason, K. (1998). Positionally cloned gene for a novel glomerular protein– nephrin–is mutated in congenital nephrotic syndrome. Molecular Cell, 1, 575–582. Kiechl, S., Wittmann, J., Giaccari, A., Knoflach, M., Willeit, P., Bozec, A., ... Schett, G. (2013). Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents development of diabetes mellitus. Nature Medicine, 19, 358–363. Kim, J. Y., Cheon, Y. H., Yoon, K. H., Lee, M. S., & Oh, J. (2014). Parthenolide inhibits osteoclast differentiation and bone resorbing activity by down-regulation of NFATc1 induction and c-Fos stability, during RANKL-mediated osteoclastogenesis. BMB Reports, 47, 451–456. Langham, R. G., Kelly, D. J., Cox, A. J., Thomson, N. M., Holthofer, H., Zaoui, P., ... Gilbert, R. E. (2002). Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: Effects of angiotensin converting enzyme inhibition. Diabetologia, 45, 1572–1576. Lee, E. Y., Chung, C. H., Khoury, C. C., Yeo, T. K., Pyagay, P. E., Wang, A., & Chen, S. (2009). The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-beta,
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Please cite this article as: Chen, X-W., et al., Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-κB and MAPK si..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.03.013