Bone-derived FGF23 promotes diabetic renal fibrosis through FGFR4-dependent suppression of PPARγ

Bone-derived FGF23 promotes diabetic renal fibrosis through FGFR4-dependent suppression of PPARγ | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bone-derived FGF23 promotes diabetic renal fibrosis through FGFR4-dependent suppression of PPARγ Huixin Hao, Zhe Li, Angel Xu, Gui Lu, Donghong Hu, Hairuo Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9339648/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Renal fibrosis is a major pathological feature of diabetic nephropathy (DN), but the upstream signals linking diabetes to progressive renal fibrosis are still not well defined. Fibroblast growth factor 23 (FGF23) is a bone-derived endocrine factor, and its role in diabetic renal fibrosis remains unclear. Methods We used a streptozotocin (STZ)-induced diabetic mouse model to evaluate the effects of FGFR4 inhibition with BLU9931 and PPARγ activation with rosiglitazone in vivo. To examine epithelial-fibroblast communication in vitro, conditioned medium from recombinant FGF23 (rFGF23)-treated HK-2 cells was transferred to NRK-49F fibroblasts. Histology, immunostaining, Western blotting, RT-qPCR, ELISA, transcriptomic analysis, and protein interaction analysis were used to assess fibrosis-related signaling changes. Results STZ-treated mice showed clear renal injury and interstitial fibrosis, along with increased FGF23 expression in bone, accumulation of FGF23 protein in the kidney, increased renal FGFR4 expression, and decreased PPARγ expression. Renal Fgf23 mRNA was not detectable, suggesting that renal FGF23 was mainly derived from the circulation rather than local synthesis. Both BLU9931 and rosiglitazone reduced TGF-β1 signaling and alleviated renal fibrosis in diabetic mice. In vitro, conditioned medium from rFGF23-treated HK-2 cells promoted fibroblast activation, and this effect was weakened by FGFR4 inhibition or PPARγ activation. Conclusions Bone-derived FGF23 may promote diabetic renal fibrosis through FGFR4-related suppression of PPARγ and subsequent activation of TGF-β1 signaling. These findings suggest that the bone-kidney axis is involved in diabetic renal fibrosis and that the FGFR4/PPARγ pathway may represent a potential therapeutic target. Diabetic nephropathy Renal fibrosis FGF23 FGFR4 PPARγ Figures Figure 3 Figure 4 Figure 5 Figure 7 Introduction Diabetic nephropathy (DN) remains a major cause of chronic kidney disease and end-stage renal disease worldwide [ 1 ]. Despite improvements in glycemic control, blood pressure management, and kidney-protective therapy, many patients still develop progressive tubulointerstitial fibrosis [ 2 – 7 ]. Because fibrosis is closely associated with declining renal function, the upstream factors driving this process in diabetes are still unclear. Fibroblast growth factor 23 (FGF23) is an osteocyte-derived hormone that regulates phosphate and vitamin D homeostasis [ 8 , 9 ]. Recent studies have linked elevated circulating FGF23 to adverse cardiovascular and renal outcomes, including in patients with diabetes [ 10 – 13 ]. When Klotho expression is reduced, FGF23 may signal through FGFR4 and activate pathways related to inflammation and fibrosis [ 11 , 14 ]. However, whether this endocrine signal directly contributes to renal fibrosis in DN remains unclear. Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor with anti-inflammatory and anti-fibrotic effects in the kidney [ 15 – 17 ]. Previous studies have shown that PPARγ activity is reduced in diabetic kidney disease and that restoration of PPARγ signaling can alleviate fibrotic injury [ 18 , 19 ]. However, the upstream signals responsible for PPARγ suppression in DN are not well defined. We therefore investigated whether FGF23-FGFR4 signaling might contribute to renal fibrosis by reducing PPARγ activity and enhancing TGF-β1-driven epithelial-fibroblast crosstalk. In this study, we examined whether bone-derived FGF23 promotes diabetic renal fibrosis through FGFR4-dependent suppression of PPARγ. Using an STZ-induced diabetic mouse model together with an in vitro conditioned-medium system, we analyzed the relationship among FGF23, FGFR4, PPARγ, and TGF-β1 signaling and evaluated the effects of FGFR4 inhibition and PPARγ activation on renal fibrotic responses. Materials and methods Animals and experimental design Male C57BL/6J mice (8 weeks old; Experimental Animal Center of Southern Medical University) were housed under specific pathogen-free conditions with a 12-hour light/dark cycle and free access to food and water. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 70 mg/kg/day; Sigma-Aldrich) for 5 consecutive days after fresh dissolution in 0.1 M citrate buffer (pH 4.5). Fasting blood glucose was measured weekly, and mice with values > 16.7 mmol/L for 2 consecutive weeks were considered diabetic. At week 5 after STZ induction, mice were randomized into six groups (n = 5/group): non-diabetic vehicle (ND-Veh), ND + BLU9931, ND + rosiglitazone, STZ-diabetic vehicle (STZ-Veh), STZ + BLU9931, and STZ + rosiglitazone. BLU9931 (30 mg/kg, twice daily by oral gavage; Selleck) and rosiglitazone (5 mg/kg/day by oral gavage; Selleck) were administered for 3 weeks. All animal procedures were approved by the ethics review board at Nanfang Hospital, Southern Medical University (Approval no. NFYY20190930). Cell culture and paracrine crosstalk assays HK-2 human proximal tubular epithelial cells (ATCC, CRL-2190) and NRK-49F rat renal fibroblasts (ATCC, CRL-1570) were cultured in DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO₂. To evaluate epithelial-fibroblast communication, HK-2 cells were treated with recombinant human FGF23 (rFGF23, 100 ng/mL; R&D Systems) for 24 h. Cells were then washed three times with PBS and once with serum-free medium, followed by a 2-h serum-free chase to minimize carryover of exogenous rFGF23. Fresh serum-free medium was added for an additional 24 h, and the conditioned medium (CM) was collected and passed through a 0.22-µm filter before transfer to NRK-49F cells for 24 h. Where indicated, BLU9931 (0.5 µM) or rosiglitazone (5 µM) was added 30 min before rFGF23 treatment. Assessment of renal function and systemic FGF23 At week 8, mice were euthanized with pentobarbital sodium anesthesia (50 mg/kg, intraperitoneally), and blood was collected by cardiac puncture. Serum creatinine was measured using a colorimetric assay (Abcam, ab65340), renal neutrophil gelatinase-associated lipocalin (NGAL) was measured by ELISA (Cusabio, CSB-E12796m), and circulating FGF23 was measured using a mouse-specific ELISA kit (Merck Millipore, EZMFGF23-43K). Histological and morphometric analysis Paraffin-embedded kidney sections (4 µm) were stained with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and Masson's trichrome according to standard protocols (Servicebio). Interstitial fibrosis was scored semi-quantitatively as 0 (none), 1 ( 50%) according to the extent of fibrotic involvement. For quantitative analysis, five non-overlapping fields at 40× magnification were selected randomly from each section, and the percentage of trichrome-positive area was measured with ImageJ by two investigators blinded to group assignment. Immunohistochemistry and Immunofluorescence For immunohistochemistry, deparaffinized sections underwent heat-mediated antigen retrieval in citrate buffer (pH 6.0) and blocking with 5% bovine serum albumin. Sections were incubated overnight at 4°C with antibodies against FGF23 (R&D, MAB26291), FGFR4 (CST, 8562), Klotho (R&D, AF1819), and TGF-β1 (Abcam, ab215715) (all 1:100), followed by HRP-conjugated secondary antibodies (1:200) and DAB detection. For immunofluorescence, cells or tissue sections were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 5% bovine serum albumin. Samples were incubated overnight with antibodies against PPARγ (CST, 2443), α-SMA (CST, 14968), Vimentin (CST, 5741), Collagen I (CST, 72026), and EpCAM (Abcam, ab223582) (all 1:100), followed by Alexa Fluor-conjugated secondary antibodies (1:200). Nuclei were stained with DAPI. Fluorescence intensity was quantified with ImageJ in five random 40× fields per sample by an investigator blinded to treatment group. RT-qPCR and Western blotting Total RNA was extracted using TRIzol (Invitrogen, 15596018) and reverse-transcribed using PrimeScript RT Master Mix (Takara, RR036A). Quantitative PCR was performed with TB Green Premix Ex Taq II (Takara, RR820A) on a StepOnePlus Real-Time PCR System. Gene expression was normalized to GAPDH and calculated using the 2^-ΔΔCt method. Primer sequences are listed in Supplementary Table 1. For Western blotting, total protein was extracted in RIPA buffer containing protease and phosphatase inhibitors (Roche, 04693159001). Equal amounts of protein (50 µg) were separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% bovine serum albumin, and incubated overnight with primary antibodies against PPARγ (CST, 2443), TGF-β1 (Abcam, ab215715), Collagen I (CST, 72026), Vimentin (CST, 5741), α-SMA (CST, 14968), and GAPDH (Arigo, ARG10119). After incubation with HRP-conjugated secondary antibodies (1:5000), signals were detected with ECL reagent (Bio-Rad, 1705061) and quantified by densitometry using ImageJ. Statistical analysis Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 9.0. Two-group comparisons were made with unpaired two-tailed Student's t-tests, and comparisons among multiple groups were made with one-way ANOVA followed by Tukey's post hoc test. A two-sided P value < 0.05 was considered statistically significant. Results STZ-induced diabetes causes renal injury and fibrosis An STZ-induced diabetic mouse model was established to assess renal fibrotic changes (Supplementary Fig. 1A). STZ treatment resulted in sustained hyperglycemia (Fig. 1 A) and was associated with increased renal NGAL and serum creatinine levels, indicating renal injury (Fig. 1 B, C). GO analysis of the GSE218563 dataset showed enrichment of metabolic and profibrotic pathways (Supplementary Fig. 1B). Histological examination with PAS, HE, and Masson's trichrome staining demonstrated glomerular injury, tubular dilatation, and increased interstitial collagen deposition in diabetic kidneys (Fig. 1 D). These findings were accompanied by increased Acta2 , Col1a1 , and Vim mRNA expression (Supplementary Fig. 1C-E), as well as higher α-SMA, Collagen I, and Vimentin protein levels by immunofluorescence and Western blotting (Fig. 1 E-L). FGF23-FGFR4 signaling is activated in the bone-kidney axis We next evaluated whether the FGF23-FGFR4 pathway was altered in diabetic kidneys. RT-qPCR showed that renal Fgfr4 mRNA was increased, whereas renal Fgf23 transcripts were undetectable (Fig. 2 A, Supplementary Fig. 2A). Klotho expression was reduced (Supplementary Fig. 2B). In contrast, Western blotting, immunohistochemistry, and ELISA demonstrated increased FGF23 protein levels in the kidney and circulation (Fig. 2 B-E, Supplementary Fig. 2C-F). Single-cell RNA sequencing localized Fgfr4 predominantly to renal epithelial cells and again showed no detectable intrarenal Fgf23 expression (Fig. 2 F-H). Bone tissue from diabetic mice showed increased FGF23 expression (Fig. 2 I, J). Together, these findings support the possibility that renal FGF23 in diabetes reflects systemic input rather than local synthesis. FGFR4 inhibition attenuates renal fibrosis in vivo To test the role of FGFR4 in diabetic renal fibrosis, diabetic mice were treated with the selective FGFR4 inhibitor BLU9931 (Supplementary Fig. 3A). Molecular docking supported binding between FGF23 and FGFR4 (Fig. 3 A). BLU9931 reduced the Masson's trichrome-positive fibrotic area in diabetic kidneys (Fig. 3 B, C). Immunofluorescence showed that BLU9931 also reduced the diabetes-associated increases in α-SMA, Collagen I, and Vimentin (Fig. 3 D-H). Protein interaction analysis identified TGF-β1 as a central node linked to FGF23 and FGFR4 (Supplementary Fig. 3B). Consistent with this, Western blotting and immunohistochemistry showed that FGFR4 inhibition reduced TGF-β1 expression together with fibrotic marker expression (Fig. 3 I-L, Supplementary Fig. 3C, D). FGF23-stimulated tubular epithelial cells promote fibroblast activation through paracrine signaling We then used an HK-2/NRK-49F conditioned-medium system to examine epithelial-fibroblast communication (Fig. 4 A). Treatment of HK-2 cells with rFGF23 increased Fgfr4 expression and TGF-β1 secretion (Fig. 4 B, C). Conditioned medium from rFGF23-treated HK-2 cells induced α-SMA, Collagen I, and Vimentin expression in NRK-49F cells (Fig. 4 D-L), indicating fibroblast activation. These effects were substantially reduced when HK-2 cells were pretreated with BLU9931. Western blot analysis further showed that rFGF23-induced TGF-β1 production in HK-2 cells was FGFR4 dependent (Supplementary Fig. 3E, F). Rosiglitazone alleviates renal fibrosis by restoring PPARγ activity KEGG analysis of the GSE218563 dataset identified the PPAR signaling pathway as one of the most downregulated pathways in diabetic kidneys (Fig. 5 A). The rosiglitazone treatment protocol is shown in Supplementary Fig. 4A, and STRING analysis suggested a regulatory relationship between PPARG and TGFB1 (Supplementary Fig. 4B). In diabetic mice, rosiglitazone reduced interstitial fibrosis on Masson's trichrome staining (Fig. 5 B, C). Immunofluorescence showed that the diabetes-associated decrease in tubular PPARγ expression, together with the increases in α-SMA, Collagen I, and Vimentin, was reversed by rosiglitazone treatment (Fig. 5 D-I). Similar changes were confirmed by Western blotting and immunohistochemistry, which also showed reduced TGF-β1 expression after rosiglitazone treatment (Fig. 5 J-M, Supplementary Fig. 4C, D). Rosiglitazone blocks rFGF23-induced profibrotic signaling To further define the relationship between FGF23 and PPARγ, HK-2 cells were treated with rFGF23 in the presence or absence of rosiglitazone (Fig. 6 A). Rosiglitazone increased PPARG mRNA expression and reduced rFGF23-induced TGF-β1 secretion in HK-2 cells (Fig. 6 B, C). In the conditioned-medium model, rosiglitazone pretreatment of HK-2 cells restored PPARγ expression and attenuated the ability of rFGF23-conditioned medium to induce α-SMA, Collagen I, and Vimentin expression in NRK-49F cells (Fig. 6 D-L). Western blotting confirmed that rosiglitazone reduced downstream TGF-β1 production in rFGF23-treated HK-2 cells (Supplementary Fig. 4E, F). Proposed mechanism: FGF23/FGFR4/PPARγ/TGF-β1 signaling in diabetic renal fibrosis Taken together, our findings support a model in which increased bone-derived FGF23 in diabetes reaches the kidney through the circulation and acts on epithelial FGFR4. This is associated with reduced PPARγ expression and increased TGF-β1 production, which in turn promotes fibroblast activation and extracellular matrix deposition. Both FGFR4 inhibition and PPARγ activation interrupted this signaling pattern and attenuated fibrotic responses (Fig. 7 ). Discussion In this study, diabetic mice showed renal fibrosis together with increased bone and circulating FGF23, increased renal FGFR4 expression, and reduced PPARγ expression. FGFR4 inhibition and PPARγ activation both attenuated fibrotic changes in vivo. In addition, conditioned-medium experiments showed that FGF23-stimulated tubular epithelial cells promoted fibroblast activation through paracrine signaling. Together, these results support the involvement of the FGF23-FGFR4-PPARγ-TGF-β1 pathway in diabetic renal fibrosis. FGF23 is best known as a regulator of phosphate homeostasis [ 8 , 9 ], but growing evidence suggests that it may also contribute to tissue injury outside bone [ 10 , 12 , 20 , 21 ]. In the present study, renal Fgf23 mRNA was undetectable, whereas FGF23 protein was increased in the kidney and FGF23 expression in bone was elevated. This pattern suggests that renal FGF23 in diabetic mice is mainly derived from the circulation. At the same time, renal FGFR4 expression increased and Klotho expression decreased, supporting the possibility that non-canonical FGF23 signaling is involved in the diabetic kidney. Another important observation in this study was the association between FGFR4 signaling and reduced PPARγ activity. PPARγ has recognized anti-inflammatory and anti-fibrotic effects in the kidney [ 15 , 18 , 22 – 24 ]. In our models, reduced PPARγ expression was accompanied by activation of TGF-β1 signaling and fibroblast activation, whereas rosiglitazone restored PPARγ expression and attenuated these changes. These results suggest that suppression of PPARγ is an important part of the profibrotic response downstream of FGF23-FGFR4 signaling. Our conditioned medium experiments further suggest that tubular epithelial cells participate in the downstream effect of FGF23 on renal fibroblasts. After rFGF23 stimulation, HK-2 cells showed increased TGF-β1 production, and conditioned medium from these cells promoted the expression of α-SMA, Collagen I, and Vimentin in NRK-49F cells. This effect was reduced by BLU9931 or rosiglitazone. These findings support a role for epithelial-fibroblast communication in the fibrotic response associated with FGF23 signaling. This study has several limitations. First, the work relied mainly on pharmacologic approaches, and genetic models targeting Fgfr4 or Pparg in renal epithelial cells would provide stronger causal evidence. Second, the STZ model reflects type 1 diabetes and does not fully represent the metabolic setting of type 2 diabetic kidney disease. Third, although our data support an endocrine contribution of bone-derived FGF23, direct lineage-tracing or tissue-specific approaches would be needed to define its source more clearly. Finally, validation in human renal tissue or clinical samples would further strengthen the clinical relevance of these findings. Conclusions Our findings suggest that increased bone-derived FGF23 contributes to diabetic renal fibrosis through FGFR4-associated suppression of PPARγ and activation of TGF-β1 signaling. These results link endocrine signaling from bone to epithelial-fibroblast communication in the kidney and provide a possible mechanism for the role of FGF23 in fibrosis progression in DN. The FGFR4-PPARγ pathway may be a potential therapeutic target in diabetic renal disease. Abbreviations DN Diabetic nephropathy STZ Streptozotocin FGF23 Fibroblast growth factor 23 rFGF23 Recombinant human FGF23 FGFR4 Fibroblast growth factor receptor 4 PPARγ Peroxisome proliferator-activated receptor gamma TGF-β1 Transforming growth factor-β1 HK-2 Human kidney proximal tubular epithelial cells CM Conditioned medium NRK-49F Normal rat kidney fibroblast cells ELISA Enzyme-linked immunosorbent assay IHC Immunohistochemistry PAS Periodic acid-Schiff HE Hematoxylin and eosin RT-qPCR Real-time quantitative PCR EpCAM Epithelial cell adhesion molecule NGAL Neutrophil gelatinase-associated lipocalin GO Gene ontology KEGG Kyoto encyclopedia of genes and genomes STRING Search tool for the retrieval of interacting genes/proteins ECM Extracellular matrix Declarations Acknowledgements We thank Hairuo Lin and Angel Xu for their intellectual input and technical support during this study. We also thank the staff of the Core Facility Center and the Laboratory Animal Center at Southern Medical University for assistance with animal breeding and husbandry. Author contributions HH and HL conceived the study, performed the investigation, and drafted the manuscript. HL contributed to formal analysis and validation. AX revised the manuscript. HH, ZL, AX, GL, DH, and HL contributed to methodology and resources. HL supervised the study, curated data, and acquired funding. HH coordinated the project. All authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (No. 82272602). Additional institutional support was provided by Southern Medical University and Johns Hopkins University. Data availability The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All experiments were performed in accordance with our institutional guidelines for animal research, which conform to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication, 8th Edition, 2011). Approval for this study was granted by the ethics review board at Nanfang Hospital, Southern Medical University (Guangzhou, China). (Approval no. NFYY20190930). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Gupta S, Dominguez M, Golestaneh L. Diabetic Kidney Disease: An Update. Med Clin North Am. 2023; 107(4):689–705. Reiss AB, Jacob B, Zubair A, Srivastava A, Johnson M, De Leon J. Fibrosis in Chronic Kidney Disease: Pathophysiology and Therapeutic Targets. J Clin Med. 2024; 13(7). Kalantar-Zadeh K, Jafar TH, Nitsch D, Neuen BL, Perkovic V. Chronic kidney disease. Lancet. 2021; 398(10302):786–802. Furman BL. Streptozotocin-Induced Diabetic Models in Mice and Rats. Curr Protoc. 2021; 1(4):e78. Humphreys BD. Mechanisms of Renal Fibrosis. Annu Rev Physiol. 2018; 80:309–326. Machowska A, Carrero JJ, Lindholm B, Stenvinkel P. Therapeutics targeting persistent inflammation in chronic kidney disease. Transl Res. 2016; 167(1):204–213. Thomas MC, Brownlee M, Susztak K, Sharma K, Jandeleit-Dahm KA, Zoungas S, et al. Diabetic kidney disease. Nat Rev Dis Primers. 2015; 1:15018. Agoro R, White KE. Regulation of FGF23 production and phosphate metabolism by bone-kidney interactions. Nat Rev Nephrol. 2023; 19(3):185-193. Cipriani C, Minisola S, Colangelo L, V DEM, Ferrone F, Biamonte F, et al. FGF23 functions and disease. Minerva Endocrinol (Torino). 2022; 47(4):437-448. Simic P, Babitt JL. Regulation of FGF23: Beyond Bone. Curr Osteoporos Rep. 2021; 19(6):563-573. Hao H, Ma S, Zheng C, Wang Q, Lin H, Chen Z, et al. Excessive fibroblast growth factor 23 promotes renal fibrosis in mice with type 2 cardiorenal syndrome. Aging (Albany NY). 2021; 13(2):2982–3009. Smith ER, Holt SG, Hewitson TD. FGF23 activates injury-primed renal fibroblasts via FGFR4-dependent signalling and enhancement of TGF-beta autoinduction. Int J Biochem Cell Biol. 2017; 92:63–78. Faul C, Amaral AP, Oskouei B, Hu MC, Sloan A, Isakova T, et al. FGF23 induces left ventricular hypertrophy. J Clin Invest. 2011; 121(11):4393–4408. Gutierrez OM, Januzzi JL, Isakova T, Laliberte K, Smith K, Collerone G, et al. Fibroblast growth factor 23 and left ventricular hypertrophy in chronic kidney disease. Circulation. 2009; 119(19):2545–2552. Kokeny G, Calvier L, Hansmann G. PPARgamma and TGFbeta-Major Regulators of Metabolism, Inflammation, and Fibrosis in the Lungs and Kidneys. Int J Mol Sci. 2021; 22(19). Yang J, Zhou Y, Guan Y. PPARgamma as a therapeutic target in diabetic nephropathy and other renal diseases. Curr Opin Nephrol Hypertens. 2012; 21(1):97–105. Zhang Y, Guan Y. PPAR-gamma agonists and diabetic nephropathy. Curr Diab Rep. 2005; 5(6):470–475. Sharma V, Patial V. Peroxisome proliferator-activated receptor gamma and its natural agonists in the treatment of kidney diseases. Front Pharmacol. 2022; 13:991059. Ivanova EA, Myasoedova VA, Melnichenko AA, Orekhov AN. Peroxisome Proliferator-Activated Receptor (PPAR) Gamma Agonists as Therapeutic Agents for Cardiovascular Disorders: Focus on Atherosclerosis. Curr Pharm Des. 2017; 23(7):1119–1124. Sun T, Yu X. FGF23 Actions in CKD-MBD and other Organs During CKD. Curr Med Chem. 2023; 30(7):841-856. Andrukhova O, Slavic S, Smorodchenko A, Zeitz U, Shalhoub V, Lanske B, et al. FGF23 regulates renal sodium handling and blood pressure. EMBO Mol Med. 2014; 6(6):744–759. Zhang X, Li L, Xue R, Liang D, Wang Y, Yang W, et al. Artemether Attenuates High Glucose-Induced Inflammation and Fibrogenesis in Renal Tubular Epithelial Cells by Modulating the TGF-beta/Smad Pathway Via PPARgamma Activation. J Diabetes Res. 2025; 2025:5052561. Wei JY, Hu MY, Chen XQ, Lei FY, Wei JS, Chen J, et al. Rosiglitazone attenuates hypoxia-induced renal cell apoptosis by inhibiting NF-kappaB signaling pathway in a PPARgamma-dependent manner. Ren Fail. 2022; 44(1):2056–2065. Li L, Fu H, Liu Y. The fibrogenic niche in kidney fibrosis: components and mechanisms. Nat Rev Nephrol. 2022; 18(9):545–557. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9339648","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":639402859,"identity":"cc750031-156e-4eca-9964-a857af105ec6","order_by":0,"name":"Huixin Hao","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huixin","middleName":"","lastName":"Hao","suffix":""},{"id":639402860,"identity":"bcfcb49c-cc56-4b51-a269-043dbaed10d2","order_by":1,"name":"Zhe Li","email":"","orcid":"","institution":"Guangzhou Eighth People's Hospital, Affiliated to Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Li","suffix":""},{"id":639402862,"identity":"f4099c97-1f2f-4d22-bf7c-571cc18fccfa","order_by":2,"name":"Angel Xu","email":"","orcid":"","institution":"University of British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Angel","middleName":"","lastName":"Xu","suffix":""},{"id":639402864,"identity":"54903324-d805-4d61-b096-d0c4215c6ecd","order_by":3,"name":"Gui Lu","email":"","orcid":"","institution":"Xinxiang Central Hospital, Affiliated to Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Gui","middleName":"","lastName":"Lu","suffix":""},{"id":639402866,"identity":"f85dfd75-d0c7-4ea7-9225-12b6a87e6523","order_by":4,"name":"Donghong Hu","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Donghong","middleName":"","lastName":"Hu","suffix":""},{"id":639402868,"identity":"365f060e-e448-423f-b43b-185470c791ba","order_by":5,"name":"Hairuo Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACAwbGNiBlA+HxkKAljSQtDGxA6jAJWszZD7c9+FFx3l53RgLjg7dtDPLmhLRY9iS2G/acuZ247UYCs+HcNgbDnQ2EHHYgsU2Ct+12gtmNBDZp3jaGBIMDhLScf9gm+bftnD1QC/tv4rTcSGwDGn6AEegwNmYitTxsN5Y5k5y47czDZsk55yQMNxB2WPqzh28q7OzNjicf/PCmzEaeoC1IgLEBSEgQr34UjIJRMApGAW4AALeiQqJiOnBqAAAAAElFTkSuQmCC","orcid":"","institution":"Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Hairuo","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2026-04-07 05:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9339648/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9339648/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109317631,"identity":"58cddff6-c70e-434d-9429-311938ae5785","added_by":"auto","created_at":"2026-05-15 12:50:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1224869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of FGFR4 attenuated diabetic renal fibrosis in vivo.\u003c/strong\u003e (A) Molecular docking model showing the predicted interaction between FGF23 and FGFR4. (B, C) Representative Masson's trichrome staining and quantification of renal interstitial fibrosis (n = 5 per group). Scale bar: 50 μm. (D) Immunofluorescence staining for FGFR4, α-SMA, Collagen I, and Vimentin in kidney sections. Scale bars: 50 μm for FGFR4 and α-SMA; 100 μm for Collagen I and Vimentin. (E-H) Quantification of fluorescence intensity for the indicated markers (n = 5 per group). (I) Western blot analysis of TGF-β1, Collagen I, and Vimentin in kidney lysates. (J-L) Densitometric analysis normalized to GAPDH (n = 3 per group). Data are presented as mean ± SEM. \u003cem\u003e*p\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**p \u003c/em\u003e\u0026lt; 0.01, \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9339648/v1/a84062c19eb47211f713eaf6.png"},{"id":109405288,"identity":"d5c4b4ad-2ee4-48af-a521-96f7826d4a95","added_by":"auto","created_at":"2026-05-17 13:16:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1292607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConditioned medium from rFGF23-treated HK-2 cells promoted fibroblast activation.\u003c/strong\u003e (A) Schematic diagram of the HK-2/NRK-49F conditioned medium experiment. (B, C) \u003cem\u003eFgfr4\u003c/em\u003e mRNA expression and secreted TGF-β1 levels in HK-2 cells after rFGF23 stimulation with or without BLU9931 (n = 3-5 per group). (D) Immunofluorescence staining of FGFR4 in HK-2 cells and α-SMA, Collagen I, and Vimentin in NRK-49F cells. Scale bar: 100 μm. (E-H) Quantification of fluorescence intensity (n = 5 per group). (I-L) Western blot analysis and densitometric quantification of fibrotic markers in NRK-49F cells (n = 3 per group). Data are presented as mean ± SEM. \u003cem\u003e*p\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01, \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9339648/v1/959d5d701f81b612958733ce.png"},{"id":109317634,"identity":"d63940de-ff3a-423f-b996-b23f407c0a32","added_by":"auto","created_at":"2026-05-15 12:50:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1371105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRosiglitazone attenuates renal fibrosis and restores tubular PPARγ expression.\u003c/strong\u003e (A) KEGG pathway analysis showing downregulation of the PPAR signaling pathway in diabetic kidneys. (B, C) Representative Masson's trichrome staining and quantification of renal fibrosis (n = 5 per group). Scale bar: 50 μm. (D) Co-immunofluorescence staining for PPARγ and EpCAM in renal tubules. Scale bar: 50 μm. (E) Immunofluorescence staining for α-SMA, Collagen I, and Vimentin. Scale bars: 50 μm for α-SMA; 100 μm for Collagen I and Vimentin. (F-I) Quantification of EpCAM\u003csup\u003e+\u003c/sup\u003ePPARγ\u003csup\u003e+\u003c/sup\u003e cells and fluorescence intensity for fibrotic markers (n = 5 per group). (J-M) Western blot analysis and densitometric quantification of TGF-β1, Vimentin, and Collagen I (n = 3 per group). Data are presented as mean ± SEM. \u003cem\u003e*p\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**p\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9339648/v1/f9d0de51547de60806b26834.png"},{"id":109317636,"identity":"5d73e803-7319-40d0-bd05-b65d7ca9eb78","added_by":"auto","created_at":"2026-05-15 12:50:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":281402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed mechanism of bone-derived FGF23 in diabetic renal fibrosis.\u003c/strong\u003e Under diabetic conditions, increased bone-derived FGF23 enters the circulation and activates FGFR4 in tubular epithelial cells, leading to reduced PPARγ activity and increased TGF-β1 production. The resulting paracrine signaling promotes fibroblast activation and extracellular matrix deposition. FGFR4 inhibition with BLU9931 or PPARγ activation with rosiglitazone partly interrupts this pathway.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9339648/v1/44eda80d3b16df67b26551cd.png"},{"id":109405746,"identity":"e336f47f-914d-475c-9cbb-b0780a979ce5","added_by":"auto","created_at":"2026-05-17 13:20:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4363404,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9339648/v1/47bcdea5-6a98-4c1e-985c-769bdab2dd2d.pdf"},{"id":109317628,"identity":"114bd0d0-58e2-4aa9-a10d-5e23e0a2d10c","added_by":"auto","created_at":"2026-05-15 12:50:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":836485,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialBMCNephrology.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9339648/v1/d1f2a2a0f0f76947154d59a6.pdf"},{"id":109317629,"identity":"ccf96098-6b84-4b59-9a8a-c21a920dc362","added_by":"auto","created_at":"2026-05-15 12:50:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":859982,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2.Fulllengthoriginalunprocessedblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9339648/v1/e26a81983563eb83f6673758.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bone-derived FGF23 promotes diabetic renal fibrosis through FGFR4-dependent suppression of PPARγ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic nephropathy (DN) remains a major cause of chronic kidney disease and end-stage renal disease worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite improvements in glycemic control, blood pressure management, and kidney-protective therapy, many patients still develop progressive tubulointerstitial fibrosis [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Because fibrosis is closely associated with declining renal function, the upstream factors driving this process in diabetes are still unclear.\u003c/p\u003e \u003cp\u003eFibroblast growth factor 23 (FGF23) is an osteocyte-derived hormone that regulates phosphate and vitamin D homeostasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recent studies have linked elevated circulating FGF23 to adverse cardiovascular and renal outcomes, including in patients with diabetes [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. When Klotho expression is reduced, FGF23 may signal through FGFR4 and activate pathways related to inflammation and fibrosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, whether this endocrine signal directly contributes to renal fibrosis in DN remains unclear.\u003c/p\u003e \u003cp\u003ePeroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor with anti-inflammatory and anti-fibrotic effects in the kidney [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Previous studies have shown that PPARγ activity is reduced in diabetic kidney disease and that restoration of PPARγ signaling can alleviate fibrotic injury [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the upstream signals responsible for PPARγ suppression in DN are not well defined. We therefore investigated whether FGF23-FGFR4 signaling might contribute to renal fibrosis by reducing PPARγ activity and enhancing TGF-β1-driven epithelial-fibroblast crosstalk.\u003c/p\u003e \u003cp\u003eIn this study, we examined whether bone-derived FGF23 promotes diabetic renal fibrosis through FGFR4-dependent suppression of PPARγ. Using an STZ-induced diabetic mouse model together with an in vitro conditioned-medium system, we analyzed the relationship among FGF23, FGFR4, PPARγ, and TGF-β1 signaling and evaluated the effects of FGFR4 inhibition and PPARγ activation on renal fibrotic responses.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and experimental design\u003c/h2\u003e \u003cp\u003eMale C57BL/6J mice (8 weeks old; Experimental Animal Center of Southern Medical University) were housed under specific pathogen-free conditions with a 12-hour light/dark cycle and free access to food and water. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 70 mg/kg/day; Sigma-Aldrich) for 5 consecutive days after fresh dissolution in 0.1 M citrate buffer (pH 4.5). Fasting blood glucose was measured weekly, and mice with values\u0026thinsp;\u0026gt;\u0026thinsp;16.7 mmol/L for 2 consecutive weeks were considered diabetic.\u003c/p\u003e \u003cp\u003eAt week 5 after STZ induction, mice were randomized into six groups (n\u0026thinsp;=\u0026thinsp;5/group): non-diabetic vehicle (ND-Veh), ND\u0026thinsp;+\u0026thinsp;BLU9931, ND\u0026thinsp;+\u0026thinsp;rosiglitazone, STZ-diabetic vehicle (STZ-Veh), STZ\u0026thinsp;+\u0026thinsp;BLU9931, and STZ\u0026thinsp;+\u0026thinsp;rosiglitazone. BLU9931 (30 mg/kg, twice daily by oral gavage; Selleck) and rosiglitazone (5 mg/kg/day by oral gavage; Selleck) were administered for 3 weeks. All animal procedures were approved by the ethics review board at Nanfang Hospital, Southern Medical University (Approval no. NFYY20190930).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture and paracrine crosstalk assays\u003c/h3\u003e\n\u003cp\u003eHK-2 human proximal tubular epithelial cells (ATCC, CRL-2190) and NRK-49F rat renal fibroblasts (ATCC, CRL-1570) were cultured in DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37\u0026deg;C in 5% CO₂. To evaluate epithelial-fibroblast communication, HK-2 cells were treated with recombinant human FGF23 (rFGF23, 100 ng/mL; R\u0026amp;D Systems) for 24 h. Cells were then washed three times with PBS and once with serum-free medium, followed by a 2-h serum-free chase to minimize carryover of exogenous rFGF23. Fresh serum-free medium was added for an additional 24 h, and the conditioned medium (CM) was collected and passed through a 0.22-\u0026micro;m filter before transfer to NRK-49F cells for 24 h. Where indicated, BLU9931 (0.5 \u0026micro;M) or rosiglitazone (5 \u0026micro;M) was added 30 min before rFGF23 treatment.\u003c/p\u003e\n\u003ch3\u003eAssessment of renal function and systemic FGF23\u003c/h3\u003e\n\u003cp\u003eAt week 8, mice were euthanized with pentobarbital sodium anesthesia (50 mg/kg, intraperitoneally), and blood was collected by cardiac puncture. Serum creatinine was measured using a colorimetric assay (Abcam, ab65340), renal neutrophil gelatinase-associated lipocalin (NGAL) was measured by ELISA (Cusabio, CSB-E12796m), and circulating FGF23 was measured using a mouse-specific ELISA kit (Merck Millipore, EZMFGF23-43K).\u003c/p\u003e\n\u003ch3\u003eHistological and morphometric analysis\u003c/h3\u003e\n\u003cp\u003eParaffin-embedded kidney sections (4 \u0026micro;m) were stained with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and Masson's trichrome according to standard protocols (Servicebio). Interstitial fibrosis was scored semi-quantitatively as 0 (none), 1 (\u0026lt;\u0026thinsp;25%), 2 (25%-50%), or 3 (\u0026gt;\u0026thinsp;50%) according to the extent of fibrotic involvement. For quantitative analysis, five non-overlapping fields at 40\u0026times; magnification were selected randomly from each section, and the percentage of trichrome-positive area was measured with ImageJ by two investigators blinded to group assignment.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry and Immunofluorescence\u003c/h3\u003e\n\u003cp\u003eFor immunohistochemistry, deparaffinized sections underwent heat-mediated antigen retrieval in citrate buffer (pH 6.0) and blocking with 5% bovine serum albumin. Sections were incubated overnight at 4\u0026deg;C with antibodies against FGF23 (R\u0026amp;D, MAB26291), FGFR4 (CST, 8562), Klotho (R\u0026amp;D, AF1819), and TGF-β1 (Abcam, ab215715) (all 1:100), followed by HRP-conjugated secondary antibodies (1:200) and DAB detection.\u003c/p\u003e \u003cp\u003eFor immunofluorescence, cells or tissue sections were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 5% bovine serum albumin. Samples were incubated overnight with antibodies against PPARγ (CST, 2443), α-SMA (CST, 14968), Vimentin (CST, 5741), Collagen I (CST, 72026), and EpCAM (Abcam, ab223582) (all 1:100), followed by Alexa Fluor-conjugated secondary antibodies (1:200). Nuclei were stained with DAPI. Fluorescence intensity was quantified with ImageJ in five random 40\u0026times; fields per sample by an investigator blinded to treatment group.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRT-qPCR and Western blotting\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol (Invitrogen, 15596018) and reverse-transcribed using PrimeScript RT Master Mix (Takara, RR036A). Quantitative PCR was performed with TB Green Premix Ex Taq II (Takara, RR820A) on a StepOnePlus Real-Time PCR System. Gene expression was normalized to GAPDH and calculated using the 2^-ΔΔCt method. Primer sequences are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eFor Western blotting, total protein was extracted in RIPA buffer containing protease and phosphatase inhibitors (Roche, 04693159001). Equal amounts of protein (50 \u0026micro;g) were separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% bovine serum albumin, and incubated overnight with primary antibodies against PPARγ (CST, 2443), TGF-β1 (Abcam, ab215715), Collagen I (CST, 72026), Vimentin (CST, 5741), α-SMA (CST, 14968), and GAPDH (Arigo, ARG10119). After incubation with HRP-conjugated secondary antibodies (1:5000), signals were detected with ECL reagent (Bio-Rad, 1705061) and quantified by densitometry using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analyses were performed using GraphPad Prism 9.0. Two-group comparisons were made with unpaired two-tailed Student's t-tests, and comparisons among multiple groups were made with one-way ANOVA followed by Tukey's post hoc test. A two-sided P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSTZ-induced diabetes causes renal injury and fibrosis\u003c/h2\u003e \u003cp\u003eAn STZ-induced diabetic mouse model was established to assess renal fibrotic changes (Supplementary Fig.\u0026nbsp;1A). STZ treatment resulted in sustained hyperglycemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and was associated with increased renal NGAL and serum creatinine levels, indicating renal injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). GO analysis of the GSE218563 dataset showed enrichment of metabolic and profibrotic pathways (Supplementary Fig.\u0026nbsp;1B). Histological examination with PAS, HE, and Masson's trichrome staining demonstrated glomerular injury, tubular dilatation, and increased interstitial collagen deposition in diabetic kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These findings were accompanied by increased \u003cem\u003eActa2\u003c/em\u003e, \u003cem\u003eCol1a1\u003c/em\u003e, and \u003cem\u003eVim\u003c/em\u003e mRNA expression (Supplementary Fig.\u0026nbsp;1C-E), as well as higher α-SMA, Collagen I, and Vimentin protein levels by immunofluorescence and Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFGF23-FGFR4 signaling is activated in the bone-kidney axis\u003c/h2\u003e \u003cp\u003eWe next evaluated whether the FGF23-FGFR4 pathway was altered in diabetic kidneys. RT-qPCR showed that renal \u003cem\u003eFgfr4\u003c/em\u003e mRNA was increased, whereas renal \u003cem\u003eFgf23\u003c/em\u003e transcripts were undetectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;2A). Klotho expression was reduced (Supplementary Fig.\u0026nbsp;2B). In contrast, Western blotting, immunohistochemistry, and ELISA demonstrated increased FGF23 protein levels in the kidney and circulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E, Supplementary Fig.\u0026nbsp;2C-F). Single-cell RNA sequencing localized Fgfr4 predominantly to renal epithelial cells and again showed no detectable intrarenal Fgf23 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H). Bone tissue from diabetic mice showed increased FGF23 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J). Together, these findings support the possibility that renal FGF23 in diabetes reflects systemic input rather than local synthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFGFR4 inhibition attenuates renal fibrosis in vivo\u003c/h2\u003e \u003cp\u003eTo test the role of FGFR4 in diabetic renal fibrosis, diabetic mice were treated with the selective FGFR4 inhibitor BLU9931 (Supplementary Fig.\u0026nbsp;3A). Molecular docking supported binding between FGF23 and FGFR4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). BLU9931 reduced the Masson's trichrome-positive fibrotic area in diabetic kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Immunofluorescence showed that BLU9931 also reduced the diabetes-associated increases in α-SMA, Collagen I, and Vimentin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-H). Protein interaction analysis identified TGF-β1 as a central node linked to FGF23 and FGFR4 (Supplementary Fig.\u0026nbsp;3B). Consistent with this, Western blotting and immunohistochemistry showed that FGFR4 inhibition reduced TGF-β1 expression together with fibrotic marker expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-L, Supplementary Fig.\u0026nbsp;3C, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFGF23-stimulated tubular epithelial cells promote fibroblast activation through paracrine signaling\u003c/h2\u003e \u003cp\u003eWe then used an HK-2/NRK-49F conditioned-medium system to examine epithelial-fibroblast communication (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Treatment of HK-2 cells with rFGF23 increased \u003cem\u003eFgfr4\u003c/em\u003e expression and TGF-β1 secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Conditioned medium from rFGF23-treated HK-2 cells induced α-SMA, Collagen I, and Vimentin expression in NRK-49F cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-L), indicating fibroblast activation. These effects were substantially reduced when HK-2 cells were pretreated with BLU9931. Western blot analysis further showed that rFGF23-induced TGF-β1 production in HK-2 cells was FGFR4 dependent (Supplementary Fig.\u0026nbsp;3E, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRosiglitazone alleviates renal fibrosis by restoring PPARγ activity\u003c/h2\u003e \u003cp\u003eKEGG analysis of the GSE218563 dataset identified the PPAR signaling pathway as one of the most downregulated pathways in diabetic kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The rosiglitazone treatment protocol is shown in Supplementary Fig.\u0026nbsp;4A, and STRING analysis suggested a regulatory relationship between PPARG and TGFB1 (Supplementary Fig.\u0026nbsp;4B). In diabetic mice, rosiglitazone reduced interstitial fibrosis on Masson's trichrome staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). Immunofluorescence showed that the diabetes-associated decrease in tubular PPARγ expression, together with the increases in α-SMA, Collagen I, and Vimentin, was reversed by rosiglitazone treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-I). Similar changes were confirmed by Western blotting and immunohistochemistry, which also showed reduced TGF-β1 expression after rosiglitazone treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ-M, Supplementary Fig.\u0026nbsp;4C, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRosiglitazone blocks rFGF23-induced profibrotic signaling\u003c/h2\u003e \u003cp\u003eTo further define the relationship between FGF23 and PPARγ, HK-2 cells were treated with rFGF23 in the presence or absence of rosiglitazone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Rosiglitazone increased PPARG mRNA expression and reduced rFGF23-induced TGF-β1 secretion in HK-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). In the conditioned-medium model, rosiglitazone pretreatment of HK-2 cells restored PPARγ expression and attenuated the ability of rFGF23-conditioned medium to induce α-SMA, Collagen I, and Vimentin expression in NRK-49F cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-L). Western blotting confirmed that rosiglitazone reduced downstream TGF-β1 production in rFGF23-treated HK-2 cells (Supplementary Fig.\u0026nbsp;4E, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eProposed mechanism: FGF23/FGFR4/PPARγ/TGF-β1 signaling in diabetic renal fibrosis\u003c/h2\u003e \u003cp\u003eTaken together, our findings support a model in which increased bone-derived FGF23 in diabetes reaches the kidney through the circulation and acts on epithelial FGFR4. This is associated with reduced PPARγ expression and increased TGF-β1 production, which in turn promotes fibroblast activation and extracellular matrix deposition. Both FGFR4 inhibition and PPARγ activation interrupted this signaling pattern and attenuated fibrotic responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, diabetic mice showed renal fibrosis together with increased bone and circulating FGF23, increased renal FGFR4 expression, and reduced PPARγ expression. FGFR4 inhibition and PPARγ activation both attenuated fibrotic changes in vivo. In addition, conditioned-medium experiments showed that FGF23-stimulated tubular epithelial cells promoted fibroblast activation through paracrine signaling. Together, these results support the involvement of the FGF23-FGFR4-PPARγ-TGF-β1 pathway in diabetic renal fibrosis.\u003c/p\u003e \u003cp\u003eFGF23 is best known as a regulator of phosphate homeostasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], but growing evidence suggests that it may also contribute to tissue injury outside bone [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the present study, renal \u003cem\u003eFgf23\u003c/em\u003e mRNA was undetectable, whereas FGF23 protein was increased in the kidney and FGF23 expression in bone was elevated. This pattern suggests that renal FGF23 in diabetic mice is mainly derived from the circulation. At the same time, renal FGFR4 expression increased and Klotho expression decreased, supporting the possibility that non-canonical FGF23 signaling is involved in the diabetic kidney.\u003c/p\u003e \u003cp\u003eAnother important observation in this study was the association between FGFR4 signaling and reduced PPARγ activity. PPARγ has recognized anti-inflammatory and anti-fibrotic effects in the kidney [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In our models, reduced PPARγ expression was accompanied by activation of TGF-β1 signaling and fibroblast activation, whereas rosiglitazone restored PPARγ expression and attenuated these changes. These results suggest that suppression of PPARγ is an important part of the profibrotic response downstream of FGF23-FGFR4 signaling.\u003c/p\u003e \u003cp\u003eOur conditioned medium experiments further suggest that tubular epithelial cells participate in the downstream effect of FGF23 on renal fibroblasts. After rFGF23 stimulation, HK-2 cells showed increased TGF-β1 production, and conditioned medium from these cells promoted the expression of α-SMA, Collagen I, and Vimentin in NRK-49F cells. This effect was reduced by BLU9931 or rosiglitazone. These findings support a role for epithelial-fibroblast communication in the fibrotic response associated with FGF23 signaling.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, the work relied mainly on pharmacologic approaches, and genetic models targeting \u003cem\u003eFgfr4\u003c/em\u003e or \u003cem\u003ePparg\u003c/em\u003e in renal epithelial cells would provide stronger causal evidence. Second, the STZ model reflects type 1 diabetes and does not fully represent the metabolic setting of type 2 diabetic kidney disease. Third, although our data support an endocrine contribution of bone-derived FGF23, direct lineage-tracing or tissue-specific approaches would be needed to define its source more clearly. Finally, validation in human renal tissue or clinical samples would further strengthen the clinical relevance of these findings.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings suggest that increased bone-derived FGF23 contributes to diabetic renal fibrosis through FGFR4-associated suppression of PPARγ and activation of TGF-β1 signaling. These results link endocrine signaling from bone to epithelial-fibroblast communication in the kidney and provide a possible mechanism for the role of FGF23 in fibrosis progression in DN. The FGFR4-PPARγ pathway may be a potential therapeutic target in diabetic renal disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDN \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Diabetic nephropathy\u003c/p\u003e\n\u003cp\u003eSTZ \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Streptozotocin\u003c/p\u003e\n\u003cp\u003eFGF23 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Fibroblast growth factor 23\u003c/p\u003e\n\u003cp\u003erFGF23 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Recombinant human FGF23\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFGFR4 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fibroblast growth factor receptor 4\u003c/p\u003e\n\u003cp\u003ePPAR\u0026gamma; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Peroxisome proliferator-activated receptor gamma\u003c/p\u003e\n\u003cp\u003eTGF-\u0026beta;1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Transforming growth factor-\u0026beta;1\u003c/p\u003e\n\u003cp\u003eHK-2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Human kidney proximal tubular epithelial cells\u003c/p\u003e\n\u003cp\u003eCM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Conditioned medium\u003c/p\u003e\n\u003cp\u003eNRK-49F \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Normal rat kidney fibroblast cells\u003c/p\u003e\n\u003cp\u003eELISA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Enzyme-linked immunosorbent assay\u003c/p\u003e\n\u003cp\u003eIHC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Immunohistochemistry\u003c/p\u003e\n\u003cp\u003ePAS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Periodic acid-Schiff\u003c/p\u003e\n\u003cp\u003eHE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003eRT-qPCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Real-time quantitative PCR\u003c/p\u003e\n\u003cp\u003eEpCAM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Epithelial cell adhesion molecule\u003c/p\u003e\n\u003cp\u003eNGAL \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Neutrophil gelatinase-associated lipocalin\u003c/p\u003e\n\u003cp\u003eGO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Gene ontology\u003c/p\u003e\n\u003cp\u003eKEGG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Kyoto encyclopedia of genes and genomes\u003c/p\u003e\n\u003cp\u003eSTRING \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Search tool for the retrieval of interacting genes/proteins\u003c/p\u003e\n\u003cp\u003eECM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Extracellular matrix\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Hairuo Lin and Angel Xu for their intellectual input and technical support during this study. We also thank the staff of the Core Facility Center and the Laboratory Animal Center at Southern Medical University for assistance with animal breeding and husbandry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHH and HL conceived the study, performed the investigation, and drafted the manuscript. HL contributed to formal analysis and validation. AX revised the manuscript. HH, ZL, AX, GL, DH, and HL contributed to methodology and resources. HL supervised the study, curated data, and acquired funding. HH coordinated the project. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 82272602). Additional institutional support was provided by Southern Medical University and Johns Hopkins University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in accordance with our institutional guidelines for animal research, which conform to the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e (National Institutes of Health Publication, 8th Edition, 2011). Approval for this study was granted by the ethics review board at Nanfang Hospital, Southern Medical University (Guangzhou, China). (Approval no. NFYY20190930).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGupta S, Dominguez M, Golestaneh L. Diabetic Kidney Disease: An Update. \u003cem\u003eMed Clin North Am. \u003c/em\u003e2023; 107(4):689\u0026ndash;705.\u003c/li\u003e\n\u003cli\u003eReiss AB, Jacob B, Zubair A, Srivastava A, Johnson M, De Leon J. Fibrosis in Chronic Kidney Disease: Pathophysiology and Therapeutic Targets. \u003cem\u003eJ Clin Med. \u003c/em\u003e2024; 13(7).\u003c/li\u003e\n\u003cli\u003eKalantar-Zadeh K, Jafar TH, Nitsch D, Neuen BL, Perkovic V. Chronic kidney disease. \u003cem\u003eLancet. \u003c/em\u003e2021; 398(10302):786\u0026ndash;802.\u003c/li\u003e\n\u003cli\u003eFurman BL. Streptozotocin-Induced Diabetic Models in Mice and Rats. \u003cem\u003eCurr Protoc. \u003c/em\u003e2021; 1(4):e78.\u003c/li\u003e\n\u003cli\u003eHumphreys BD. Mechanisms of Renal Fibrosis. \u003cem\u003eAnnu Rev Physiol. \u003c/em\u003e2018; 80:309\u0026ndash;326.\u003c/li\u003e\n\u003cli\u003eMachowska A, Carrero JJ, Lindholm B, Stenvinkel P. Therapeutics targeting persistent inflammation in chronic kidney disease. \u003cem\u003eTransl Res. \u003c/em\u003e2016; 167(1):204\u0026ndash;213.\u003c/li\u003e\n\u003cli\u003eThomas MC, Brownlee M, Susztak K, Sharma K, Jandeleit-Dahm KA, Zoungas S, et al. Diabetic kidney disease. \u003cem\u003eNat Rev Dis Primers. \u003c/em\u003e2015; 1:15018.\u003c/li\u003e\n\u003cli\u003eAgoro R, White KE. Regulation of FGF23 production and phosphate metabolism by bone-kidney interactions. \u003cem\u003eNat Rev Nephrol. \u003c/em\u003e2023; 19(3):185-193.\u003c/li\u003e\n\u003cli\u003eCipriani C, Minisola S, Colangelo L, V DEM, Ferrone F, Biamonte F, et al. FGF23 functions and disease. \u003cem\u003eMinerva Endocrinol (Torino). \u003c/em\u003e2022; 47(4):437-448.\u003c/li\u003e\n\u003cli\u003eSimic P, Babitt JL. Regulation of FGF23: Beyond Bone. \u003cem\u003eCurr Osteoporos Rep. \u003c/em\u003e2021; 19(6):563-573.\u003c/li\u003e\n\u003cli\u003eHao H, Ma S, Zheng C, Wang Q, Lin H, Chen Z, et al. Excessive fibroblast growth factor 23 promotes renal fibrosis in mice with type 2 cardiorenal syndrome. \u003cem\u003eAging (Albany NY). \u003c/em\u003e2021; 13(2):2982\u0026ndash;3009.\u003c/li\u003e\n\u003cli\u003eSmith ER, Holt SG, Hewitson TD. FGF23 activates injury-primed renal fibroblasts via FGFR4-dependent signalling and enhancement of TGF-beta autoinduction. \u003cem\u003eInt J Biochem Cell Biol. \u003c/em\u003e2017; 92:63\u0026ndash;78.\u003c/li\u003e\n\u003cli\u003eFaul C, Amaral AP, Oskouei B, Hu MC, Sloan A, Isakova T, et al. FGF23 induces left ventricular hypertrophy. \u003cem\u003eJ Clin Invest. \u003c/em\u003e2011; 121(11):4393\u0026ndash;4408.\u003c/li\u003e\n\u003cli\u003eGutierrez OM, Januzzi JL, Isakova T, Laliberte K, Smith K, Collerone G, et al. Fibroblast growth factor 23 and left ventricular hypertrophy in chronic kidney disease. \u003cem\u003eCirculation. \u003c/em\u003e2009; 119(19):2545\u0026ndash;2552.\u003c/li\u003e\n\u003cli\u003eKokeny G, Calvier L, Hansmann G. PPARgamma and TGFbeta-Major Regulators of Metabolism, Inflammation, and Fibrosis in the Lungs and Kidneys. \u003cem\u003eInt J Mol Sci. \u003c/em\u003e2021; 22(19).\u003c/li\u003e\n\u003cli\u003eYang J, Zhou Y, Guan Y. PPARgamma as a therapeutic target in diabetic nephropathy and other renal diseases. \u003cem\u003eCurr Opin Nephrol Hypertens. \u003c/em\u003e2012; 21(1):97\u0026ndash;105.\u003c/li\u003e\n\u003cli\u003eZhang Y, Guan Y. PPAR-gamma agonists and diabetic nephropathy. \u003cem\u003eCurr Diab Rep. \u003c/em\u003e2005; 5(6):470\u0026ndash;475.\u003c/li\u003e\n\u003cli\u003eSharma V, Patial V. Peroxisome proliferator-activated receptor gamma and its natural agonists in the treatment of kidney diseases. \u003cem\u003eFront Pharmacol. \u003c/em\u003e2022; 13:991059.\u003c/li\u003e\n\u003cli\u003eIvanova EA, Myasoedova VA, Melnichenko AA, Orekhov AN. Peroxisome Proliferator-Activated Receptor (PPAR) Gamma Agonists as Therapeutic Agents for Cardiovascular Disorders: Focus on Atherosclerosis. \u003cem\u003eCurr Pharm Des. \u003c/em\u003e2017; 23(7):1119\u0026ndash;1124.\u003c/li\u003e\n\u003cli\u003eSun T, Yu X. FGF23 Actions in CKD-MBD and other Organs During CKD. \u003cem\u003eCurr Med Chem. \u003c/em\u003e2023; 30(7):841-856.\u003c/li\u003e\n\u003cli\u003eAndrukhova O, Slavic S, Smorodchenko A, Zeitz U, Shalhoub V, Lanske B, et al. FGF23 regulates renal sodium handling and blood pressure. \u003cem\u003eEMBO Mol Med. \u003c/em\u003e2014; 6(6):744\u0026ndash;759.\u003c/li\u003e\n\u003cli\u003eZhang X, Li L, Xue R, Liang D, Wang Y, Yang W, et al. Artemether Attenuates High Glucose-Induced Inflammation and Fibrogenesis in Renal Tubular Epithelial Cells by Modulating the TGF-beta/Smad Pathway Via PPARgamma Activation. \u003cem\u003eJ Diabetes Res. \u003c/em\u003e2025; 2025:5052561.\u003c/li\u003e\n\u003cli\u003eWei JY, Hu MY, Chen XQ, Lei FY, Wei JS, Chen J, et al. Rosiglitazone attenuates hypoxia-induced renal cell apoptosis by inhibiting NF-kappaB signaling pathway in a PPARgamma-dependent manner. \u003cem\u003eRen Fail. \u003c/em\u003e2022; 44(1):2056\u0026ndash;2065.\u003c/li\u003e\n\u003cli\u003eLi L, Fu H, Liu Y. The fibrogenic niche in kidney fibrosis: components and mechanisms. \u003cem\u003eNat Rev Nephrol. \u003c/em\u003e2022; 18(9):545\u0026ndash;557.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-nephrology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnep","sideBox":"Learn more about [BMC Nephrology](http://bmcnephrol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bnep/default.aspx","title":"BMC Nephrology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Diabetic nephropathy, Renal fibrosis, FGF23, FGFR4, PPARγ","lastPublishedDoi":"10.21203/rs.3.rs-9339648/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9339648/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRenal fibrosis is a major pathological feature of diabetic nephropathy (DN), but the upstream signals linking diabetes to progressive renal fibrosis are still not well defined. Fibroblast growth factor 23 (FGF23) is a bone-derived endocrine factor, and its role in diabetic renal fibrosis remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe used a streptozotocin (STZ)-induced diabetic mouse model to evaluate the effects of FGFR4 inhibition with BLU9931 and PPARγ activation with rosiglitazone in vivo. To examine epithelial-fibroblast communication in vitro, conditioned medium from recombinant FGF23 (rFGF23)-treated HK-2 cells was transferred to NRK-49F fibroblasts. Histology, immunostaining, Western blotting, RT-qPCR, ELISA, transcriptomic analysis, and protein interaction analysis were used to assess fibrosis-related signaling changes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSTZ-treated mice showed clear renal injury and interstitial fibrosis, along with increased FGF23 expression in bone, accumulation of FGF23 protein in the kidney, increased renal FGFR4 expression, and decreased PPARγ expression. Renal \u003cem\u003eFgf23\u003c/em\u003e mRNA was not detectable, suggesting that renal FGF23 was mainly derived from the circulation rather than local synthesis. Both BLU9931 and rosiglitazone reduced TGF-β1 signaling and alleviated renal fibrosis in diabetic mice. In vitro, conditioned medium from rFGF23-treated HK-2 cells promoted fibroblast activation, and this effect was weakened by FGFR4 inhibition or PPARγ activation.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eBone-derived FGF23 may promote diabetic renal fibrosis through FGFR4-related suppression of PPARγ and subsequent activation of TGF-β1 signaling. These findings suggest that the bone-kidney axis is involved in diabetic renal fibrosis and that the FGFR4/PPARγ pathway may represent a potential therapeutic target.\u003c/p\u003e","manuscriptTitle":"Bone-derived FGF23 promotes diabetic renal fibrosis through FGFR4-dependent suppression of PPARγ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-15 12:50:35","doi":"10.21203/rs.3.rs-9339648/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-10T15:24:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"134101812898755488936186828145901934843","date":"2026-05-08T06:44:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207271861688915187871841101608590114626","date":"2026-05-07T11:22:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235412431433897250831123733483997585151","date":"2026-05-06T14:51:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-06T11:33:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-26T11:06:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-21T14:34:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-20T16:10:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Nephrology","date":"2026-04-20T15:07:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-nephrology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnep","sideBox":"Learn more about [BMC Nephrology](http://bmcnephrol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bnep/default.aspx","title":"BMC Nephrology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"383f4cd5-051b-4eff-aad7-28e9a5c5796c","owner":[],"postedDate":"May 15th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-10T15:24:57+00:00","index":78,"fulltext":""},{"type":"reviewerAgreed","content":"134101812898755488936186828145901934843","date":"2026-05-08T06:44:04+00:00","index":75,"fulltext":""},{"type":"reviewerAgreed","content":"207271861688915187871841101608590114626","date":"2026-05-07T11:22:07+00:00","index":74,"fulltext":""},{"type":"reviewerAgreed","content":"235412431433897250831123733483997585151","date":"2026-05-06T14:51:41+00:00","index":69,"fulltext":""},{"type":"reviewersInvited","content":"40","date":"2026-05-06T11:33:02+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T12:50:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-15 12:50:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9339648","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9339648","identity":"rs-9339648","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}