| Background and ObjectivesDiabetic mellitus (DM) is the third chronic non-communicable diseases after cardiovascular disease and tumor in the developed countries. In the recent years, the prevalence of DM is increasing rapidly in our contry, from 5.5% in 2000 to 11.6% in 2010, which has become a public health problem that greatly harm people’s health in our country and increase our national health burden1. Diabetic nephropathy (DN) is one of the most common and severe chronic complications of DM as well as the single most important cause of end-stage renal disease (ESRD) throughout the world2,3. Approximately 30% of patients with type 1 DM develop DN, and approximately 25 to 30% of patients with type 2 DM will develop overt DN4’5. DN is characterized by progressive albuminuria and a gradual decline in glomerular filtration rate6,7. Diverse pathogenic mechanisms contribute to DN, including advanced glycation end products8, activation of protein kinase C9, overexpression of different growth factors10 and release of inflammatory mediators11,12. However, the exact mechanism is still not fully understood nowadays, leading to the limitation of treatment of DN. Even if we adopt a full range of comprehensive therapy such as lowering blood sugar, making blood pressure and blood fat under control, improving the way of life and using angiotensin Ⅱ receptor blockers or angiotensin converting enzyme inhibitors, we still cannot stop the DN patients eventually progress to ESRD13. Thus, It is imperative to deepen the study of pathogenesis of DN and find effective methods to the therapy of DN.The glomerular filtration barrier is composed of glomerular endothelial, glomerular basement membrane (GBM) and podocytes. Glomerular podocytes is a terminally differentiated cell, which is attached to the outside of the GBM and play an vital role in maintaining the glomerular filtration barrier structure and function. In the previous studies, podocyte injury has been observed in the kidneys of diabetic patients and animal models, can precede and predict the occurrence of proteinuria and may be a predictor of early DN When podocytes suffer from injurious stimuli such as high glucose (HG),transforming growth factor (TGF)-β1 and adriamycin, they will occur damage performances such as changing of normal structure, becoming more motile, merging or disappearance and releasing of inflammatory factors, leading to the onset of proteinuria. Hence, clarifying the molecularnism of podocyte injury and finding a drug that can protect podocyte from injury are very essential for the development of targeted, effective DN treatment.Receptor activator of Nuclear factor-κB (RANK) and receptor activator of Nuclear factor-κB ligand (RANKL), which belong to the TNF superfamily, have been extensively studied in osteoclasts and immunoragulation cells. Lots of researches indicated that binding of RANK initiates a signaling cascade that activates NF-κB and mitogen-activated protein kinases (MAPK), which induce osteoclastogenesis and lead to osteoclast-related diseases such as osteoporosis. Recent studies have shown that RANKL and RANK may also involved in the occurrence and development of chronic kidney disease. Liu et al.found that RANKL and RANK were overexpressed in focal segmental glomerular sclerosis (FSGS), IgA nephropathy (IgAN) and membranous nephropathy (MN), and RANK was specifically expressed in podocytes. Moreover, their further animal experiments showed that RANKL and RANK were overexpressed in the kidneys of 5/6 nephrectomy model of glomerular sclerosis and puro amino nucleoside-induced rat model23’24. After that, a genome-wide association study also revealed a polymorphism in the podocyte receptor RANK that leads to a decline in renal function in coronary patients25. It suggests that RANKL/RANK may play an important role in the pathophysiology of DN. However, little is known about the potential role of RANKL and RANK in the development of DN. Recently, Kiechl et al.26found that, as the chief up-streaming molecules of NF-kB, RANKL and RANK play a pivotal role in the pathophysiology of DM. Our previous results showed that NF-κB and p38MAPK pathways were activated in advanced oxidation protein products (AOPP)-induced podocytes and subsequently inflammatory factor monocyte chemoattractant protein (MCP)-1 was also increased27. Thus, we speculated that RANKL and RANK are also overexpressed in podocytes of DN and promote podocyte injury by mediating NF-κB and MAPK pathways.Cyclopropanyldehydrocostunolide (also named LJ), a sesquiterpene lactone derivative that is derived from our national unique traditional Chinese medicine Aplotaxis auriculata, is synthesized independently by our team with features of wide source, low price, easy to be synthetic and valid ingredient stability. In pharmacodynamics, our previous results showed that LJ inhibited inflammation in AOPP-induced podocytes and meanwhile it blocked IKK/NF-κB pathway27. It suggested that ①L may protect injured podocytes; ②LJ may has the similar drug function mechanism with some sesquiterpene lactone derivative such as parthenolide. However, is the protective role of LJ limited in regulating IKK/NF-κB pathway? Recently, Kim et al.29reported that parthenolide downregulated RANK in the RANKL-induced osteoclasts. Then dose LJ protect podocytes by regulating RANKL/RANK and the other pathways? It need to study further. Hence, we studied the protective role of LJ in HG-induced podocytes and the possible underlying molecular mechanism in this subject.In sum, we conducted db/db mice and conditionally immortalized mouse podocyte as research objects to study the role of RANKL and RANK in podocyte injury of type 2 DN in vitro and vivo, which clarified the molecular mechanism how RANKL/RANK mediated NF-κB and MAPK pathways to promote podocyte injury and then contribute to the development of DN. Furthermore, we intervened HG-induced podocytes with LJ to explore the protective role of LJ in HG-induced podocytes and how it regulate the RANKL/RANK-mediated NF-κB and MAPK pathways. Our results not only help to deepen the understanding of the mechanism of podocyte injury in type 2 DN but also provide a possible drug to treat DN and provide experimental foundation and theoretical basis for identifying a clinical candidate to the treatment of DN.Contents and methods1. RANKL and RANK promote podocyte injury by mediating NF-κB and MAPK pathways1.1 Animal level1.1.1 Groups and treatments:db/db mice (7 weeks age) in a C57BL/KsJ (BKS.Cg-Dock7m+/+Leprdb/J) background and their age-matched, normal, wild-type db/m littermates were obtained from the Animal Model Research Center of Nanjing University. Mice were housed in the Laboratory Animal Center of Sun Yat-Sen University. After 1 week of acclimatization, random plasma glucose in excess of 16.7mmol/L is required to the success model of DM in db/db mice. During feeding, we should dynamically detect weight, diet, drink, fasting blood glucose (FBS) and urinary albumin to creatinine ratio (ACR) to confirm the success of DN model. At the age of 20 weeks, db/db mice were sacrificed for this experiment by devoting their blood, urine and kidneys with 1% pentobarbital.1.1.2 Detection indices:Blood samples were used for detecting FBS, fasting insulin (FINS), serum creatinine (Scr), blood urea nitrogen (BUN), triglyceride (TG) and total cholesterol(TC). Urine samples were used for examining the protein of MCP-1 by ELISA. Kidneys were used for detecting the mRNA of MCP-1 by q-PCR, stained with Periodic acid-Schiff (PAS) for mesangial analyses and transmission electron microscopy (TEM) analyses. RANKL, RANK and nephrin were detected by immunohistochemistry. RANKL, RANK, p-IκBα, IκBα, p-NF-κB/p-p65, NF-κB/p65, p-p38 and p38 were examined by Western blot.1.2 Cellular level1.2.1 Podocyte culture:Conditionally immortalized mouse podocytes between passages 12 and 20 (a kind gift from Prof. Peter Mundel were cultured as previously described.) Briefly, at a permissive temperature of 33 ℃, undifferentiated podocytes were grown in RPMI1640 containing 10%FBS penicillin (100 IU/mL), streptomycin (100 mg/mL), and 50 IU/mL recombinant murine IFN-y. To induce differentiation, podocytes were cultured at 37℃ without IFN-y for 10-14 days.1.2.2 Cytomorphology observation:Podocytes at 33℃ with IFN-y for 4 days and at 37℃ without IFN-y for 10-14 days were observed and photographed under inverted microscope.1.2.3 The effect of HG on podocyte injury1.2.3.1 Groups and treatments:The differentiated podocytes at 37℃ were incubated in the presence of 5 mM D-glucose (NG),30 mM D-glucose (HG) or 5 mM D-glucose+25 mM mannitol (MA) for 24h after synchronization for 12h.1.2.3.2 Detection indices:Cell migration and albumin influx assay were detected by Trans well migration assay. The mRNA and protein of MCP-1 were examined by q-PCR and Western blot. Nephrin was detected by Western blot.1.2.4 The expression of RANKL and RANK in HG-induced podocytes1.2.4.1 Groups and treatments:To induce differentiation, podocytes were cultured at 37℃ without IFN-y for 14 days and then synchronized for 12h. Groups were divided into 9 groups:NG group, MA groups (5 mM D-glucose and 25 mM mannitol stimulated for 3,6,12,24h) and HG groups (30 mM D-glucose stimulated for 3,6, 12,24h).1.2.4.2 Detection indices:RANKL and RANK were examined by Western blot. 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).1.2.5 The effect of RANK silence in HG-induced podocyte injury1.2.5.1 Groups and treatments:The differentiated podocytes at 37℃ for 14 days were synchronized for 12h. Then, podocytes were treated with HG and transfected with si- RANK1, si-RANK2, si-RANK3 or si-CON for 24h to select the most efficient sequence. The selected sequence was used for the next experiment and the groups were HG+si-CON and HG+si-RANK.1.2.5.2 Detection indices:The protein level of RANK was detected by Western blot. Cell migration and albumin influx assay were detected by Transwell migration assay. The mRNA and protein of MCP-1 and nephrin were also examined.1.2.6 The effect of NF-κB and MAPK pathway in HG-induced podocyte1.2.6.1 Groups and treatments:The groups did the same to 1.2.4.1. MA and HG intervene podocytes for 3,6,12,24h respectively.1.2.6.2 Detection indices:p-p65, p65, p-IKKβ, IKKβ, p-IκBa, p-p38, p38, p-ERK, ERK, p-JNK and JNK were detected by Western blot. The intensities of the protein bands were quantified by Quantity One 4.6.2 analysis software which was provided with the Kodak 2000MM System.1.2.7 The effect of RANK silence in NF-kB and MAPK pathway1.2.7.1 Groups and treatments:The differentiated podocytes at 37℃ for 14 days were treated with HG and transfected with si-RANK or si-CON for 24h.1.2.7.2 Detection indices:RANKL, RANK, nephrin, NF-κB and MAPK pathways associated proteins were detected by Western blot. Nuclear translocation of NF-κB /p65 was examined by immunofluorescence under Nikon C2 confocal microscope (Nikon corporation, Tokyo, Japan).2. LJ ameliorates HG-induced podocyte injury and regulates RANKL/RANK-mediated NF-κB and MAPK pathways2.1 Dose-toxicity curve of LJ to podocytesLJ intervenes podocytes in a multiple growth of 8 concentrations at 0,1.25μM,2.5μM,5μM to 80μM. Then cell survival rate of podocytes was examined by MTT assay with microplate reader.2.2 The effect of LJ in HG-induced podocyte injury2.2.1 Groups and treatments:The differentiated podocytes were grouped into 5 groups such as NG group, HG group, HG+2.5μM group, HG+5μM group and HG+10μM group and treated with their relative interventions such as HG or/and LJ.2.2.2 Detection indices:The indices did the same to chapter one 2(3). Cell migration, albumin influx assay, the expression of MCP-1 and nephrin were examined.2.3 The effect of LJ in RANKL/RANK-mediated NF-κB and MAPK pathways2.3.1 Groups and treatments:The groups did the same to 2.2.1. Podocytes were treated with HG or/and LJ for 24h.2.3.2 Detection indices:The indices did the same to 1.2.7.2. RANKL, RANK, nephrin, NF-kB and MAPK pathways associated proteins and nuclear translocation of NF-κB /p65 were detected.3. StatisticsThe results are 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< 0.05 was considered significant. All analyses were performed using IBM SPSS Statistics 20.0 (SPSS, Chicago, IL, USA).Results1. RANKL and RANK promote podocyte injury by mediating NF-κB and MAPK pathways1.1 Animal level1.1.1 Biochemical indicator, inflammation and histopathology in miceAt the end of the experiment, FBG, FINS, Scr, BUN, TC, TG, ACR, and body weight (BW) levels were significantly increased in the db/db mice compared with the db/m mice. With age, ACR was increased dramatically in db/db mice but remained stable in db/m mice. The db/db mice showed hyaline degeneration in the glomerular afferent arteries and mesangial expansion with major accumulation of mesangial matrix in the glomerulus. The mesangial expansion score was significantly higher in db/db mice than in db/m mice. Using electron microscopy, we noted that in db/db mice, foot process effacement occurred in most of the podocytes, and the thickness of the GBM significantly increased compared with the db/m mice. The urinary concentrations of MCP-1 and the MCP-1 mRNA level of kidneys were significantly increased in db/db mice compared with db/m mice.1.1.2 RANKL and RANK were overexpressed in the kidneys of db/db miceCompared with db/m mice, db/db mice had a significantly increased level of immunohistochemical staining for RANKL and RANK. We found that RANKL and RANK were mainly expressed in the glomeruli, along the glomerular capillary loop in db/db mice. Moreover, the protein levels of RANKL and RANK were also significantly higher in the kidneys of db/db mice.1.1.3 NF-κB and MAPK pathway were activated in the kidneys of db/db miceCompared with db/m mice, the protein level of p-IκBa, p-NF-κB/p-p65 and p-p38 were significantly increased but the degradation of IκBa was significantly decreased. The total level of NF-icB/p65 and p38 were not changed.1.2 Cellular level1.2.1 The effect of HG on podocyte injuryCompared with NG group, HG significantly increased the migratory ability of podocytes across the pores of the Transwell filters and enhanced albumin flux in podocytes. Moreover, the protein and mRNA level of MCP-1 were significantly increased. However, nephrin was significantly reduced.1.2.2 The expression of RANKL and RANK in HG-induced podocytesCompared with NG group, HG significantly increased RANKL and RANK in a time-dependent manner. But the MA groups were not changed.1.2.3 The effect of RANK silence in HG-induced podocyte injuryCompared with si-CON group, si-RANK significantly reduced the cell migration, albumin flux and expression of MCP-1 but slightly upregulated nephrin in HG-induced podocytes.1.2.4 The effect of NF-κB and MAPK pathway in HG-induced podocyteCompared with NG groups, proteins in the NF-κB signaling pathway, including p-p65, p-IKKβ, and p-IκBa, were enhanced by HG in a time-dependent manner, whereas IKKβ and p65 did not significantly change in any group. Similarly, HG induced a rapid and sustained activation of p38, ERK and JNK without affecting their total levels.1.2.5 The effect of RANK silence in NF-κB and MAPK pathwayCompared with si-CON group, RANKL, RANK, p-p65, p-IKKβ, p-IκBa and the phosphorylation of p38, ERK and JNK were significantly attenuated when RANK was silenced. The result of the immunofluorescence staining of p65 also showed that the nuclear translocation of p65 was blocked in the absence of RANK.2. LJ ameliorates HG-induced podocyte injury and regulates RANKL/RANK-mediated NF-κB and MAPK pathways2.1 Dose-toxicity curve of LJ to podocytesThe dose-toxicity curve of LJ to podocytes shaped into "S". The plateau period duration is between 0 to lOμM. With raised concentrations, cell viability decreased greatly. Based on calculation and analysis, IC50 of LJ was 18.898μM and IC10 of LJ was 10.232μM.2.2 The effect of LJ in HG-induced podocyte injuryLJ 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.2.3 The effect of LJ in RANKL/RANK-mediated NF-kB and MAPK pathwaysCompared with HG groups, LJ inhibited the protein expression of RANKL, RANK, p-p65, p-IKKβ, p-IκBα, p-p38, p-ERK and p-JNK. Whereas the total level of p65, IKKP, p38, ERK and JNK were not changed remarkably. The nuclear translocation of p65 was blocked by 10μM LJ.Conclusions1. In vivo study, RANKL and RANK are overexpressed and their downstream NF-κB and MAPK pathways are activated in kidneys of DN model. In vitro study, HG enhances migration, increases albumin filtration, upregulates MCP-1 expression and downregulate nephrin in podocytes via RANKL/RANK-mediated NF-κB and MAPK pathways, suggesting that the activation of NF-κB and MAPK pathways that mediated by RANKL/RANK may be a new mechanism of podocyte injury in DN.2. In vitro study, LJ inhibits RANKL/RANK-mediated NF-κB and MAPK pathways and reversed the effects of HG in podocyte injury such as attenuating migration, decreasing albumin filtration, downregulating MCP-1 expression and upregulate nephrin in podocytes, which indicate that LJ may protect HG-induced podocyte by suppressing RANKL/RANK-mediated NF-κB and MAPK pathways. This will provide experimental foundation and theoretical basis for continuing to study the mechanism how LJ protect podocytes in DN. |
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