Background: High level of serum fibroblast growth factor 23 (FGF23) is implicated in the development and progression of diabetic nephropathy (DN), making it a crucial factor in the pathogenesis of DN. FGF23 is also tightly correlated with inflammation in the progression of DN. The aim of this study was to explore whether the C-terminal C-tail of FGF23 (FGF23 ), an antagonist that can block the FGF23 signaling pathway by competing with intact FGF23, could exhibit a therapeutic effect on DN. C-tail Results: Biochemical data and histological examination showed that FGF23 administration ameliorated the functional and morphological abnormalities of db/db mice with DN without changing the levels of circulating FGF23 and phosphate. Evaluation of morphology and fibrosis by Masson’s trichrome staining and IHC staining of C-tail fibronectin, PCR, and western blot analysis showed that FGF23 prevents diabetes-induced fibrosis in db/db C-tail mice. Importantly, FGF23 decreased the levels of inflammatory cytokines in serum and renal tissues. C-tail Conclusion: FGF23 may improve diabetic nephropathy by decreasing inflammation and fibrosis in db/db mice, suggesting that blocking of FGF23 action remains an important therapeutic target for the prevention or attenuation of the progression of DN. Keywords: FGF23, Diabetic nephropathy, Inflammation, Fibrosis Background with the further progression of DN [5–7]. Abundant Diabetic nephropathy (DN) is one of the major causes clinical and experimental evidence indicate a positive of end-stage renal disease in diabetic patients in association of circulating FGF23’slevelsandthestage developed countries, mainly due to the increasing of DN [8–10]. Consistent with these findings, recent prevalence of type 2 diabetes [1, 2]. DN is character- research reported that the plasma FGF23 concentra- ized by proteinuria, glomerular hypertrophy, thickened tion is a risk factor of diabetic nephropathy (DN) basement membrane, podocytopenia, increased extra- triggering a series of pathological changes in kidney, cellular matrix protein deposition, and fibrosis . and a valuable and sensitive biomarker in several The mechanism involved in diabetes-induced renal acute and chronic disorders [11–14]. In the past diseaseiscomplex.Emergingevidencesuggeststhat decades, a significant body of evidence has demonstrated inflammatory mechanisms play an important role in that gradual elevation of FGF23 over time can result in theetiologyof DN. Notably, with relevance to our inflammation in patients with diabetes-induced nephropa- research, studies of the pathophysiology of DN found thy [15, 16]. Therefore, to explore strategies that block that the level of fibroblast growth factor 23 (FGF23) FGF23 action is an important therapeutic target in in plasma was significantly upregulated in DN, and preventing or attenuating the progression of DN and the the circulating level of FGF23 continued to increase occurrence of relative complications. Fibroblast growth factors (FGFs) are a family of * Correspondence: firstname.lastname@example.org; email@example.com polypeptides with diverse biological functions related to Xiaomin Zhang, Kaiwen Guo and Feng Xia contributed equally to this work. cellular development, differentiation, migration, and re- Department of Biopharmacy, School of Pharmacy, Wenzhou Medical pair . FGF23 belongs to the endocrine subfamily of University, Chashan Town, Wenzhou 325035, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. BMC Biotechnology (2018) 18:33 Page 2 of 9 FGFs that also includes FGF19 and FGF21. A multitude Methods of studies have demonstrated that FGF23 is a secretory Material C-tail molecule that is mainly produced by osteoblastic cells FGF23 was produced by the Key Laboratory of and was originally shown to function as a central regula- Biotechnology and Pharmaceutical Engineering of tor of phosphate (Pi) and vitamin D metabolism . Zhejiang Province, Wenzhou Medical University. FGF23 activity is regulated by proteolytic cleavage at the Antibodies against CD68, IL-6, fibronectin, and colla- 176 179 RXXR motif, located at the boundary between the gen IV were produced by Abcam (Cambridge, UK), and FGF core homology domain and the 72-residue-long antibodies against MCP-1 and GAPDH were purchased C-tail C-terminal tail of FGF23 (FGF23 ). This unique from Cell Signaling Technology (CST, USA). Quanti- C-terminal sequence mediates high affinity binding to Quik™ Urea (BUN) Quick Test strips for blood urea ni- the FGFR/Klotho receptor complex [20, 21]. Previous trogen (BUN) in urine and kits for serum creatinine C-tail study demonstrated that the isolated FGF23 could were purchased from Bioassay Systems (CA, USA). The interfere with FGF23 signaling by competing with the ELISA kit for microalbumin in urine was from Abcam full-length ligand for binding to the binary FGFR-Klotho (Cambridge, UK). ELISA kits for pro-inflammatory cyto- C-tail complex, making FGF23 a promising therapeutic kines (IL-6, TNFα, MCP-1, and IP-10) were obtained by agent for the treatment of diseases in which the overex- Thermo Fisher Scientific (NY, USA). The kit for reverse pression and accumulation of FGF23 induced the further transcription and the SYBR mix was purchased from progression of disease . Invitrogen (CA, USA). All other chemicals and reagents In the present study, we investigated the hypothesis used in these experiments were of analytical grade. C-tail that the FGF23 could ameliorate the development of DN as a competing antagonist of intact FGF23 in Animal experiments mouse models of T2D (db/db). Our findings indicated C57BLKS/J-Leprdb/Leprdb (db/db) and C57BLKS/ C-tail that the use of the FGF23 significantly improved J-Leprdb/m (db/m) male mice were obtained from the renal dysfunction and morphologic damage by redu- Model Animal Research Center of Nanjing University cing fibrosis and renal inflammation, although no sig- (Nanjing, China) for this study. The db/db mice were nificant decrease was observed in the plasma level of randomly and equally divided into two groups: vehicle C-tail FGF23 and hyperglycemia was not corrected. To our group (n = 10) and FGF23 group (n = 10). The ve- C-tail knowledge, this is the first demonstration of the pro- hicle group and FGF23 group (n = 10) were treated C-tail C-tail tective effect of FGF23 on diabetic nephropathy. with PBS or FGF23 (0.5 mg/kg), respectively, by C-tail Fig. 1 FGF23 treatment improved renal hypertrophy and dysfunction in db/db mice. a Treatment protocol for db/db mice wherein db/db C-tail mice received vehicle or FGF23 (0.5 mg/kg body weight (BW), every other day) for 12 weeks; b Photograph of representative mice and C-tail C-tail kidneys of db/m, db/db, and FGF23 group. c Kidney weight-to-body weight (KW/BW) ratio in db/m, db/db, and FGF23 group; d Renal function was determined by measuring excretion of creatinine and e the level of BUN and f micro-albumin in urine; g The levels of FGF23 in serum; h the levels of phosphorous (Pi) in serum Zhang et al. BMC Biotechnology (2018) 18:33 Page 3 of 9 intraperitoneal injection every other day for 12 weeks, as embedded in paraffin, and then the paraffin blocks were shown in Fig. 1a. The db/m littermates (n = 10) were treated cut into 5-μm pieces and transferred to slides. After with PBS, and served as the sham group. All mice were deparaffinization and rehydration, the 5 μm-thick serial housed under controlled conditions with free access to food sections were stained with hematoxylin and eosin (HE) and water. Blood glucose levels were monitored by using for routine histopathological observations. Periodic the Precision G Blood Glucose Testing System (Abbott acid-Schiff (PAS) and Masson’s trichrome staining was Laboratories, Abbott Park, IL) and the body weight was re- performed to determine collagen deposition and fibrosis. corded. After 12 weeks of treatment, all mice were kept in All of these sections were analyzed to evaluate tubular individual metabolic cages to allow 24 h of urine collection and glomerular injury. and then the blood samples were collected. After that the animals were sacrificed, kidney tissues were harvested for Immunofluorescence staining and immunohistochemistry subsequent studies. BUN and microalbumin in urine, serum Immunofluorescence (IF) Staining and Immunohisto- creatinine and proflammatory cytokines (IL-6, TNF-α, chemistry (IHC) were performed on 5-μm MCP-1 and IP-10) were measured by ELISA Kit according paraffin-embedded kidney sections. After deparaffiniza- to the manufacture’s instructions. All experiments were con- tion and rehydration, the slides were placed in citric acid ducted in accordance with the National Institutes of Health buffer and microwaved for 3 min at 900 W, 150 W for guidelines and with approval of the Wenzhou Medical 8 min, followed by cooling for 30–60 min at room University Institutional Animal Care and Use Committee. temperature. Next, all kidney slides were treated with 3% H O for 30 min and then blocked with 5% BSA for 2 2 Histopathological examination 60 min at 37 °C. The slides were then incubated over- The harvested kidney tissue samples were fixed over- night with primary antibodies at 4 °C in a wet box, using night in 4% paraformaldehyde. Then, the tissues were an HRP-conjugated goat anti-rabbit or mouse secondary C-tail Fig. 2 FGF23 treatment strikingly improved renal glomerular lesions in db/db Mice. a H & E staining of renal tissues; b PAS staining (indicating tubular injury and mesangial expansion; c-d Immunochemistry for NPHS2 and WT1, two biomarkers of podocytes; e Electron microscope images to observe the morphology of podocytes. Original magnification ×10000; f Mesangial matrix index statistically obtained from Panel B; g-h Semi-quantitative analysis for the NPHS2 and WT1-positive area; i-j Mean GBM thickness and podocyte effacement were semi-quantitatively analyzed based on Panel e Zhang et al. BMC Biotechnology (2018) 18:33 Page 4 of 9 antibody. For Immunofluorescence Staining, we used a severe diabetes-induced renal changes that are charac- secondary antibody conjugated to FITC and the nuclei terized by increased renal/glomerular volume, renal were stained using 40,6-diamidino-2-phenylindole hypotrophy, a high level of creatinine in plasma, and the (DAPI). Sections in the slides were examined using an abnormal excretion of microalbumin and BUN in urine inverted Leica DM inverted microscope and SPOT cam- [26–28]. Therefore, db/db mice were ideal to evaluate C-tail era system. the therapeutic effect of FGF23 on diabetic nephrop- C-tail athy. We tried 0.1 and 0.5 and 2.5 mg/kg FGF23 in Transmission electron microscopy the db/db mice and investigated the effects of different C-tail The morphological changes of renal tissue were ob- doses of FGF23 on the renal-function using three served by transmission electron microscopy (TEM) as biomarkers (serum creatinine, blood urea nitrogen done previously . Briefly, the kidney tissues were (BUN), and albumin). The results showed that 0.5 mg/ C-tail fixed, dehydrated, and sealed with an acetone/resin mix- kg FGF23 showed the best therapeutic effects on ture in an oven at 60 °C for 24 h. Then, the kidney tis- renal-function in DN mice (Additional file 1 Figure S1). sues were cut into semi-thin sections (1 μm) and stained After 12 weeks of treatment with 0.5 mg/kg C-tail with toluidine blue. Later, the samples were cut into FGF23 , plasma glucose levels, body weight, renal ultra-thin sections, stained with lead nitrate and uranyl morphology, and the kidney weight/body weight (KW/ acetate, and then examined with a transmission electron BW) ratio were analyzed. The results showed that C-tail microscope H7500 (Hitachi, Tokyo, Japan). FGF23 treatment did not change body weight or glucose levels (data not shown), however, morphological Real-time PCR changes of renal hypertrophy in db/db mice were signifi- C-tail Total RNA samples were isolated from renal tissue using cantly improved in the FGF23 group (Fig. 1b, c). an RNA extraction kit (Beijing Tiangen Biotech, China). The renal functions were evaluated by determining the Real-time RT-PCR was performed using a 2-step levels of three biomarkers of renal injuries (serum cre- M-MLV Platinum SYBR Green qPCR SuperMix-UDG atinine, blood urea nitrogen (BUN), and albumin). The kit (Invitrogen). All kits were used strictly according to levels of serum creatinine, blood urea nitrogen (BUN), the manufacturer’s instructions. Amplification of glycer- and albumin in db/db mice were much higher than the aldehyde 3-phosphate dehydrogenase (GAPDH) was per- levels in db/m mice, which indicated a severe decline of formed as an endogenous control to normalize the renal functions. However, the increases in these bio- C-tail amount of total RNA in each reaction and the relative markers were significantly attenuated by FGF23 ad- expression of target mRNA was calculated according to ministration, suggesting that the treatment with C-tail the − 2ΔΔCt method. FGF23 could improve the renal functions of DN (Fig. 1d, f). Western blot analysis Clinical studies observed markedly elevated levels of Proteins were extracted from mice renal tissue using intact FGF23 and phosphorous (Pi) in serum in diabetic RIPA buffer supplemented with protease and phosphat- patients with nephropathy [29, 30], suggesting these two ase inhibitors. The amount of total protein was detected factors can serve as biomarkers to predict the progres- using a BCA protein assay kit, and homogenates with sion of diabetic nephropathy [30, 31]. In this study, we equal amount of proteins were subjected to also examined the levels of intact FGF23 and phosphate SDS-polyacrylamide gel electrophoresis and then trans- in serum, and found that compared with db/db mice, ferred to polyvinylidene difluoride (PVDF) membranes. the plasma levels of FGF23 and phosphate in the C-tail The blots were blocked with 10% milk in PBS for 1 h at FGF23 group were slightly decreased, but not sig- room temperature, and then incubated with the primary nificantly different, suggesting the observed therapeutic antibodies for 12 h at 4 °C, washed and treated with effect was independent of the levels of circulating FGF23 PBST and HRP-conjugated secondary antibodies. Finally, and phosphorous (Pi) (Fig. 1g, h). chemiluminescence (ECL) was used to detect the bands C-tail when exposed to X-ray film. FGF23 improved renal glomerular lesions in db/db mice Results and Discussion Previous reports showed that the deposition of collagen C-tail FGF23 administration improved renal functions in in glomerulars has a central role in renal injury and may db/db mice contribute to the development of diabetic kidney disease. The db/db mouse, a genetic model of type 2 diabetes, Thus, hematoxylin and eosin (HE) and periodic acid– has been characterized as a model for obesity, sustained Schiff staining (PAS) were performed to examine the C-tail hyperglycemia, and hyperinsulinemia [23–25]. Recently, effects of FGF23 on the histologic changes and col- this animal model has been reported to present with lagen deposition in kidneys in db/db mice. Consistent Zhang et al. BMC Biotechnology (2018) 18:33 Page 5 of 9 C-tail with improved renal function with treatment with (Fig. 2g-j). These data suggested that FGF23 provided C-tail FGF23 , the data demonstrated that compared with therapeutic effect in slowing the progression of diabetic db/db mice, marked glomerular sclerosis, mesangial ex- nephropathy via the protection of podocytes. pansion, and collagen deposition in kidney sections were C-tail C-tail prevented by FGF23 treatment (Fig. 2a, b and Fig. 2f). FGF23 prevents diabetes-induced fibrosis in db/db Podocytes are associated with increased susceptibility to mice kidney injury after exposure to sustained hyperglycemia, DN is characterized by a pronounced collagen depos- and the loss or apoptosis of podocytes has been implicated ition and glomerular sclerosis, which facilitated the de- in the occurrence of proteinuria [32–34]. Therefore, we velopment of renal fibrotic lesions . Thus, the C-tail next investigated the protective effect of FGF23 in progression of the fibrosis plays a fundamental role in podocyte damage. We conducted immunochemistry diabetic nephropathy, and the abrogation or improve- (IHC) of NPHS2 and WT1, bio-markers of podocytes, to ment of fibrosis may alleviate DN. In the present study, C-tail measure the loss of podocytes (Fig. 2c, d). We also per- we investigated the effect of FGF23 administration formed transmission electron microscopy (TEM) to ob- on fibrosis. Renal glomerular fibrosis was examined by serve the infusion and effacement of podocytes, as well as Masson’s trichrome staining and IHC staining of fibro- the thickening of the basement membrane (Fig. 2e). nectin was used to evaluate morphology and fibrosis. Semi-quantitative analysis of IHC staining and TEM re- The results indicated that compared with db/db mice, C-tail sults revealed significant depletion of podocytes and thick- mice that received FGF23 treatment exhibited ening basement membrane in db/db mice. Interestingly, improved diabetes-induced fibrosis, suggesting that the C-tail C-tail administration of FGF23 improved podocyte damages effect of FGF23 on DN may be anti-fibrotic (Fig. 3a). C-tail Fig. 3 FGF23 administration improved diabetes-induced fibrosis in db/db mice. a Masson’s trichrome staining and IHC staining of fibronectin were performed to assess renal fibrosis, arrows indicate the extracellular matrix; b-g The relative mRNA levels of pro-fibrotic genes, including fibronectin, collagen IV, αSMA, Smad3, TGF-β1 and CTGF, as analyzed by RT-PCR Zhang et al. BMC Biotechnology (2018) 18:33 Page 6 of 9 C-tail Fig. 4 The effects of FGF23 administration on the levels of profibrotic proteins in kidneys. a-b Western blot analysis of fibronectin, COL IV, αSMA, Smad3, and TGF-β1; c-g Semi-quantitative western blot analysis of the data in panel A & B, GAPDH expression was used for the normalization of protein loading C-tail Fig. 5 FGF23 decreased the levels of inflammatory cytokines in serum in db/db mice. ELISA analysis of IL-6 a, TNFα b, IP-10 c, MCP-1 d C-tail protein levels in serum collected from db/m, db/db, and FGF23 group Zhang et al. BMC Biotechnology (2018) 18:33 Page 7 of 9 To investigate this further, we conducted PCR analysis expression of these diabetes-induced fibrosis-related pro- to measure the expression of genes related to fibrosis. teins (Fig. 4c-g). Compared with db/m mice, the levels of profibrotic In summary, the evaluation of fibrosis in renal tissue genes, including fibronectin, collagen IV, α-smooth and the expression of profibrotic genes and proteins C-tail muscle actin (αSMA), Smad3, transforming growth confirmed that FGF23 showed renoprotective effect factor-beta 1 (TGF-β1), and connective tissue growth by preventing the progression of fibrosis. factor (CTGF) in kidneys were upregulated in db/db C-tail mice. However, the up-regulation of these genes were FGF23 decreased the levels of inflammatory cytokines C-tail significantly down-regulated by FGF23 administra- in serum tion (Fig. 3b-g). Inflammation is implicated in the development and pro- C-tail To further confirm that FGF23 is effective against gression of diabetic nephropathy, and is believed to be a type 2 diabetes-induced renal fibrosis, western blot crucial factor in the pathogenesis of DN and associated analysis was performed to determine the effect of with adverse outcomes in many clinical settings [36–38]. C-tail FGF23 administration on the levels of profibrotic The alleviation or inhibition of the occurrence or devel- proteins in kidneys (Fig. 4a, b). Consistent with the opment of inflammation is a potential target for the above analysis of profibrotic gene expression, treatment of diabetic nephropathy. Thus, we investigated semi-quantitative western blot analysis of fibronectin, the plasma levels of pro-inflammatory cytokines COL IV, αSMA, Smad3 and TGF-β1 showed that com- interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), pared with the db/m mice, the levels of these monocyte chemotactic peptide-1 (MCP-1), and fibrosis-related proteins were markedly increased in db/db interferon-inducible protein-10 (IP-10), secreted from ei- mice, suggesting that db/db mice exhibited more severe ther inflammatory cells or renal resident cells. The levels C-tail collagen deposition and FGF23 could suppress the of plasma pro-inflammatory cytokines in db/db mice C-tail Fig. 6 Inflammation in renal tissue induced by diabetes was partly prevented by FGF23 in db/db mice. a IF analysis of renal IL-6 expression (original magnification 200×) and IHC analysis of renal CD68 expression (original magnification 400×); b-f Relative renal levels C-tail of TNFα, MCP-1, PAI-1, CD68, and IL-6 mRNAs in db/m, db/db, and FGF23 group. Representative western blots for TNFα,MCP-1,and C-tail PAI-1 g and CD68, IL-6, and NFkB h pJNK/JNK i, in lysates of renal tissues from db/m, db/db, and FGF23 group and semi-quantitation for corresponding proteins j-o Zhang et al. BMC Biotechnology (2018) 18:33 Page 8 of 9 were much higher than those of db/m mice. However, Additional file the observed amplification of these pro-inflammatory Additional file 1: Figure S1. The effects of different doses of FGF23C-tail factors induced by diabetes was remarkably abrogated by on the renal-functions in db/db mice. A: blood urea nitrogen (BUN); B: C-tail the FGF23 treatment, suggesting that FGF23 may serum creatinine (CREA); C: microalbumin (mALB). (DOCX 144 kb) protect against diabetic nephropathy by inhibiting inflammation (Fig. 5a-d). Abbreviations BUN: Blood urea nitrogen; CTGF: Connective tissue growth factor; FGF23: Fibroblast growth factor 23; FGFR: Fibroblast growth factor receptor; C-tail FGF23 decreased the levels of inflammatory cytokines GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; HE: Hematoxylin and eosin; IF: Immunofluorescence; IHC: Immunohistochemistry; IL-6: Interleukin- in renal tissues C-tail 6; IP10: Interferon-inducible protein-10; MCP-1: Monocyte chemotactic In order to investigate the effect of FGF23 peptide-1; NF-kB: Nuclearfactor-kappaB; PAI-1: Plasminogen activator administration on inflammation in the renal tissues of inhibitor-1; PAS: Periodic acid-Shiff; TEM: Transmission electron microscopy; TNFα: Tumor necrosis factor α; TNF-β1: Tumor necrosis factor-beta1; db/db mice, we next determined the levels of two αSMA: A-smooth muscle actin typical inflammatory markers, IL-6 and CD68, in the kidneys. As shown in Fig. 6a,the renalexpression Acknowledgments levels of IL-6 and CD68 were increased in db/db The authors would like to thank Dr. Xiaokun Li for critically reading the C-tail manuscript and for making thoughtful suggestions. mice and were greatly suppressed by FGF23 treatment. Consistent with the immuno-staining of Funding kidney sections, the mRNA levels of pro-inflammatory This work was supported, in part, by grants from Natural Science Foundation of Zhejiang LY15H300004 (to J.N.); Natural Science Foundation of Zhejiang genes, including TNF-a, MCP-1, plasminogen activa- Province LQ15H310005 (to L.S.) Science and Technology Project of Wenzhou tor inhibitor-1 (PA1–1),CD68and IL-6,wereupregu- (Y20140724, Y20140734, Y20160163, Y20170165). lated in db/db mice. However, the upregulation of C-tail Authors’ contributions these genes was downregulated by FGF23 treat- XZ performed the animal experiment, analyzed the data, and prepared a ment (Fig. 6b-f). Consistent with the changes of these draft of the manuscript, KG carried HE staining, PAS, immunohistochemistry pro-inflammatory genes, western blot confirmed that and immunofluorescence, FX performed analysis of gene expression by real- time quantitative PCR, XZ carried out analysis of protein expression by west- the levels of pro-inflammatory proteins induced by C-tail ern blot, MJ carried out the expression and purification of FGF23 , ZH and diabetes in db/db mice were markedly reduced by JN designed the project and analyzed the data, with JN finalizing the paper. C-tail FGF23 treatment (Fig. 6g, h). Moreover, we All authors read and approved the final manuscript. determined that the expression of NF-kB, a key regu- Ethics approval lator of inflammation that controls the expression of Human subjects were not involved in this study. This study involved the use hundreds of pro-inflammatory genes, and the phos- of animals. The experimental protocol was pre-approved by the IACUC (Insti- phorylation of JNK, another master regulator of in- tutional Animal Care and Use Committee) at the Wenzhou Medical University. flammation and an important upstream regulator of transcription factors in a variety of cell types [39–41]were Competing interests C-tail increased in db/db mice, and were inhibited by FGF23 The authors declare that they have no competing interests. treatment (Fig. 6h, i). The above data were consistent with previous observa- Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in tion of increased expression of cytokines in DN and published maps and institutional affiliations. marked activation of the NFκB pathway in kidneys from human DN and mouse disease models. Overall, Received: 13 September 2017 Accepted: 18 May 2018 C-tail FGF23 exerts direct or indirect effects on inflam- mation to protect kidneys from diabetes-induced References nephropathy. 1. 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BMC Biotechnology – Springer Journals
Published: May 30, 2018
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