Fisetin alleviates unilateral ureteral obstruction-induced renal interstitial fibrosis by inhibiting Src activation to block macrophage-myofibroblast transition

Fisetin alleviates unilateral ureteral obstruction-induced renal interstitial fibrosis by inhibiting Src activation to block macrophage-myofibroblast transition | 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 Article Fisetin alleviates unilateral ureteral obstruction-induced renal interstitial fibrosis by inhibiting Src activation to block macrophage-myofibroblast transition Yonghong Jian, Rong Li, Yuhan Wan, Wei Li, Ke Su, Yifei Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7548347/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fisetin, as a natural dietary flavonoid, exhibits multiple biological activities such as anti-inflammatory, anti-oxidant, and anti-tumorigenic activities. Previous studies have indicated that fisetin has potential renal protective effects in many animal models of kidney disease. However, the effect of fisetin on unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis (RIF) remains largely unknown. In our present study, we found that fisetin attenuated UUO-induced kidney injury by decreasing fibrotic lesion and the accumulation of extracellular matrix (ECM). The results showed that fisetin could effectively block macrophage-to-myofibroblast transition (MMT) in the kidneys of UUO mice in vivo and in transforming growth factor-β1(TGFb1)-stimulated bone marrow-derived macrophages (BMDM) in vitro. Molecular docking was employed to explore the interactions between fisetin and Src (a key mediator in MMT). The results indicated that fisetin could form hydrogen bonds and hydrophobic interactions with Src, thus binding effectively in the active pocket of Src and exhibiting strong affinity. Further in vivo and in vitro investigation demonstrated that fisetin inhibited activity of Src and subsequently lowered the phosphorylation levels of epidermal growth factor receptor (EGFR) at Tyr845, ERK1/2 and Smad3. In conclusion, this study revealed the mechanism by which fisetin blocked the progression of MMT by inhibiting the activationof Src, thus alleviating UUO-induced RIF. Therefore, fisetin might be a potential therapeutic agent for preventing and alleviating renal fibrosis. Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Nephrology Health sciences/Urology fisetin renal interstitial fibrosis (RIF) macrophage-to-myofibroblast transition(MMT) Src Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Kidney disease has a major impact on global health, and it is not only a direct cause of global morbidity and mortality but also a major risk factor for cardiovascular disease. 1 Patients suffering from chronic kidney disease (CKD) are clinically characterized by gradual loss of kidney function, and patients at the end stage have to rely on dialysis or kidney transplantation. CKD is largely preventable and requires great attention so as to formulate related global health policy. Fisetin is a plant-derived flavonoid compounds, and it is widely found in fruits and vegetables such as apples, onions, and cucumbers with its highest content in strawberries. 2 Fisetin has multiple pharmacological effects mainly including anti-inflammatory, anti-apoptotic,anti-oxidant,anti-tumor, anti-angiogenic effects. 3,4 More importantly, no side effects have been reported even at relatively high doses. 5 Previous physicochemical analysis has revealed that the concentrations of fisetin and its metabolites are high in mouse kidney, suggesting that it has potential to prevent and treat kidney diseases. 6 Fisetin has been reported to have renal protective effects against kidney diseases in animal models such as lipopolysaccharide (LPS)-induced acute kidney injury (AKI), 7 diabetic nephropathy, 8 renal ischemic reperfusion injury, 9 and hyperuricemic nephropathy. 10 – 11 However, the effect of fisetin on unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis (RIF) in mice remains unclear. Renal interstitial fibrosis (RIF), as the most common pathological lesion at the end stage of renal disease, is mainly characterized by myofibroblast proliferation, accumulation of extracellular matrix (ECM), and renal scar formation. 12 Myofibroblast, as a subgroup of activated fibroblasts, expresses αlpha-smooth muscle actin (α-SMA), and it is the main cell type responsible for the synthesis and deposition of fibrillar collagen during active tissue fibrosis. 13 As a heterogeneous population, myofibroblasts can derive from multiple sources such as proliferation of local resident fibroblasts, epithelial-to-mesenchymal transition (EMT), endothelial-to-mesenchymal transition, and differentiation from bone marrow. 14 Macrophages play a central role in the development of renal fibrosis. Macrophages derived from bone marrow cells can differentiate into a-SMA + myofibroblasts in injured kidney during a process termed as macrophage-to-myofibroblast transition (MMT). MMT is an important mechanism in the development process from renal chronic inflammation to fibrosis which is regulated by c-Src tyrosine kinase (Src) activation. Specific inhibition of MMT could significantly ameliorated unilateral ureteral obstruction (UUO)-induced renal fibrosis. 15 Fisetin has been reported to inhibit macrophage-mediated acute inflammatory responses. 16 However, whether fisetin affects the progression of MMT in the chronic kidney injury remains largely unknown. In the present study, we explored the effects of fisetin on UUO-induced RIF in mice. At the same time, we also predicted the target of fisetin by molecular docking with the key mediator Src, and then explored the mechanism of fisetin in RIF.The aim of this study was to investigate the prophylactic and therapeutic potentials of fisetin for RIF. 2. Material and methods 2.1 Reagents Fisetin was purchased from Sigma (St. Louis, MO, USA and stored at -20 ◦ C). Recombinant mouse transforming growth factor-β1(TGFb1) was purchased from Biolegend (San Diego, CA,USA). The primary antibodies against α-SMA, Collagen-I, and CD68 used for immuno-staining were obtained from Abcam (Cambridge, MA,USA). For western blotting analysis, antibodies against p-Src, Src, p-EGFR, EGFR, p-ERK1/2, ERK1/2, p-Smad3 and Smad3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-GAPDH was purchased from Santa Cruz (Santa Cruz, CA, USA). 2.2 Animal experiments Male C57BL/6J mice (8–10 weeks old, weighting 25 − 27 g) were housed in specific pathogen-free conditions at the Center for Animal Experiments of Wuhan University. UUO model was established by tying off the left ureter with silk suture, while sham surgery mice underwent the same procedures without left ureter ligation. After treatment, all mice were anesthetized and euthanized (Pentobarbital sodium 150 mg/kg, intraperitoneal injection), the injured kidneys of mice were collected on day 7 after UUO for subsequent experiments. As for the fisetin group, fisetin was dissolved in 20% PEG400, further diluted with 0.9% normal saline, and administered by gavage to mice with left ureter ligation at a dose of 100 mg/kg/d for 7 days, [10] whereas sham surgery group received only normal saline. All the animal experiments were approved by the Animal Ethics Review Board of Wuhan University and performed in accordance with the guidelines of the National Health and Medical Research Council of China. We confirm that the design and reporting of this study strictly adhered to the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). 2.3 Histological staining and evaluation Kidney samples were fixed in 4% paraformaldehyde (pH7.4) and then embedded in paraffin. Hematoxylin-eosin (HE) or Masson’s trichrome stainings of tissue sections (4µm) were performed according to the manufacturer’s instruction. Renal tubules with the following histopathological changes were considered as damaged /injured : brush border loss, tubular dilation and disruption, cast formation and cell lysis. Tissue damage was assessed in a blind manner and scored as the percentage of damaged/injured renal tubules: 0, no damage; 1, 75%.The degree of interstitial fibrosis was scored semiquantitatively as follows: 0 (no fibrosis), 1, 75% of interstitium. 17 The positively stained areas of renal interstitial fibrosis were quantified by Image J (NIH, Bethesda, MD,USA). 2.4 Immunohistochemistry(IHC) staining Sections were first deparaffinized, hydrated, and antigen-recovered, followed by incubation with specific primary antibodies against α-SMA (Abcam, MA,USA)) and Collagen-1 (BA0325, Bosterbio, USA). Five visual fields (×200 magnification) from each group were randomly selected and the percentage of positively stained areas was calculated using Image J software (NIH, Bethesda, MD,USA). 2.5 Immunofluorescence (IF) staining Immunofluorescence staining was performed on paraffin-embedded kidney sections (4 µm) and cultured bone marrow-derived macrophages (BMDMs). BMDMs were cultured on coverslips, washed 3 times with pre-chilled phosphate buffer saline(PBS), fixed in 4% paraformaldehyde, and blocked with PBS containing 10% normal donkey serum. Subsequently, sections and cell samples were incubated with primary antibodies against α-SMA and CD68 (Abcam, MA,USA). The samples were then stained with DAPI and observed under a confocal laser microscopy (Olympus FV1200, Tokyo, Japan). 2.6 Molecular docking of fisetin with Src Molecular docking was performed using the Schrodinger package (2018). The crystal structure of Src was obtained from RCSB Protein Data Bank (PDB ID: 4K11). The structure of fisetin was characterized and optimized by ChemDraw software to obtain the dominant conformation with the lowest energy. The fisetin was docked to the Src using Autodock Vina 1.1.2 software. The docking score reflected the affinity of fisetin to Src protein. 2.7 Generation of MMT cells from BMDMs Bone marrow cells were isolated from C57BL/6J mice by flushing the femur and tibia with Dulbecco’s modified Eagle’s medium (DMEM) /F12 medium. Cells were collected from suspension on the next day, incubated in DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 10 ng/ml macrophage colony-stimulating factor (M-CSF) for 5 day, and further differentiated into BMDMs. Macrophage-to-myofibroblast transition was triggered by TGFb1 (5ng/ml) in DMEM/ F12 medium containing 1% FBS to obtain myofibroblasts on day 5. 2.8 Cell viability assay Methylcyclopentadieny (MTT) assay was used to determine the toxicity of fisetin on BMDMs. BMDMs were cultured in 96-well plate for 24 h and later incubated with fisetin at various concentrations (5,10, 20, and 40 µM). After 5-day incubation, 10µL of MTT solution (5 mg/mL) was added to the medium, and the BMDMs were further incubated at 37°C for 4 h. After medium removal, 150µL dimethyl sulfoxide (DMSO) was added to the each well of 96-well plate. The plate was then shaken in the dark until the crystal was completely dissolved, and the absorbance was measured at 540 nm with a microplate reader (Molecular Devices, CA,USA). 2.9 Western blot Western blot analysis was performed on kidney tissues or BMDM lysates. The protein expressions of lysed samples were determined by Western blot analysis with primary antibodies against a-SMA, collagen-I, p-Src, Src, p-EGFR, EGFR, p-ERK1/2, ERK1/2, p-Smad3, Smad3 and GAPDH. GADPH was used as an internal control. Image J software (NIH, Bethesda, MD, USA) was used to quantify the intensity of protein bands. 2.10 Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from kidney samples or BMDMs using Trizol reagents (Invitrogen). cDNA was synthesized with 1 µg total RNA. Afterwards, the qRT-PCR was performed using SYBR Green PCR Master Mix. The relative expressions of target genes were calculated using the 2 −ΔΔCT method, and normalized to that of gene GAPDH. The primer sequences used in this study were presented in Supplementary Table 1. 2.11 Statistical analysis All data were expressed as the mean ± SEM. The differences between groups were estimated using Mann–Whitney U-test, or one-way ANOVA followed by the Bonferroni post-test. P < 0.05 was considered as statistically significant. The statistical analyses were performed using GRAPHPAD PRISM version 7.0 (GraphPad Software, USA). 3. Results 3.1 Fisetin allievates RIF in UUO mice To determine the effect of fisetin on RIF, a mouse model of UUO was established. Renal pathological test was performed on day 7 after UUO. HE and Masson’s trichrome staining showed that UUO mice exhibited widespread renal injury and tubulointerstitial fibrosis, but fisetin administration significantly reduced tissue injury and interstitial fibrosis in the obstructed kidney (Fig. 1 A). Immunohistochemical (HIC) staining (Fig. 1 B), qPCR (Fig. 1 C), and western blotting analysis (Fig. 1 D) showed that fisetin administration significantly down-regulated the expression of α-SMA and collagen-I in the obstructed kidney on day 7 after UUO. The above results indicated that fisetin could attenuate ECM deposition, renal interstitial fibrosis, thus protecting kidneys from UUO injury. 3.2 Fisetin suppresses RIF by inhibiting MMT in UUO mice Bone marrow cell-derived macrophages, as a source of myofibroblasts, can directly cause RIF. Considering the important role of MMT in RIF, we investigated the effect of fisetin on MMT process by detecting CD68 + a-SMA + cells derived from MMT in obstructed kidney tissues of mice via immunofluorescence staining. The results showed that obstructed kidney tissues of UUO mice exhibited a large number of infiltrated macrophages and MMT-derived CD68 + a-SMA + cells, whereas those of UUO mice treated with fisetin displayed a significantly reduced macrophage infiltration and MMT cell percentage (Fig. 2 ). Taken together, the above results suggested that fisetin could exert protective effect on kidney tissues of UUO mice by effectively blocking MMT. 3.3 Fisetin inhibits TGFb1-induced MMT in vitro To further verify the action mechanisms of fisetin on MMT, we stimulated BMDMs with TGFb1 for 5 days in vitro . MTT assay was used to determine the toxicity of fisetin on BMDMs. As shown in Fig. 3 A, fisetin (40 µM) inhibited the cell viability on day 5 in the absence or presence of TGFb1. In contrast, fisetin at the concentrations below 20 µM exhibited no cytotoxicity on BMDMs. Immunofluorescence staining showed that after 5-day TGFb1 stimulation, fisetin (20µM) significantly down-regulated the expression of α-SMA in BMDMs (Fig. 3 B). In addition, the mRNA (Fig. 3 C) and protein (Fig. 3 D) expression of α-SMA and Collagen-I were significantly down-regulated by fisetin in TGFb1-stimulated BMDMs. These results jointly suggested that fisetin could inhibit TGFβ1-induced MMT in BMDMs. 3.4 Fisetin is successfully docked to Src Src serves as a key mediator in the process of MMT. To investigate the interaction between fisetin and Src, we performed molecular docking of fisetin (Fig. 4 A) to crystal structure of Src. The results revealed that fisetin could be embedded well to the active pocket of Src, thus stabilizing the inactive conformation of the Src with a binding energy of -9.0kcal/mol, which might be attributed to fisetin’s hydrogen bonds and hydrophobic interactions with the Src. Three-dimensional interaction analysis revealed that the hydrogen bond interactions occurred between fisetin and animo acids MET341, ALA403 and ASP404 of Src, and that hydrophobic interactions were observed between hydrophobic functional groups of fisetin and animo acids LEU273, VAL281, LYS295, VAL323, ILE336, and LEU393 of Src(Fig. 4 B). All these results indicated that fisetin could interact with Src, and it might be a potential inhibitor of Src. 3.5 Fisetin inhibits Src activation in obstructed kidney To elucidate the mechanism by which fisetin protected against RIF, we investigated the effect of fisetin on Src activation in the obstructed kidney. The results showed that the phosphorylation level of Scr was significantly increased in the obstructed kidney, but fisetin administration significantly reduced the phosphorylation level of Src (Fig. 5 A). Previous studies have shown that Src can induce sustained fibrotic epidermal growth factor receptor (EGFR) transactivation by direct phosphorylation at Tyr845, and Src-mediated transactivation of EGFR and ERK1/2 phosphorylation can modulate Smad3 activation induced by TGFb1. 18 , 19 Considering this, we further examined the effect of fisetin on the activities of Src-mediated profibrotic molecules EGFR ,ERK1/2 and Smad3 in the obstructed kidney. The results showed that the phosphorylation levels of EGFR at Tyr845,ERK1/2 and Smad3 were significantly increased in the obstructed kidney, but their phosphorylation levels were significantly decreased under fisetin treatment (Fig. 5 B,C,D). These data suggested that fisetin could inhibit Src activation, and that fisetin might protect against MMT-derived RIF by blocking EGFR/ERK1/2/Smad3 signaling pathways. 3.6 Fisetin inhibits TGFb1-induced MMT in BMDMs by suppressing Src/EGFR/ERK1/2/Smad3 signaling pathways Src activity is essential for the activation of TGFβ1signaling pathway. To further investigate the effect of fisetin on Src activity in the process of MMT, we examined the activities of Src, EGFR, ERK1/2 and Smad3 in TGFb1-stimulated BMDMs on day 5, As shown in Fig. 6 A, TGFb1 stimulation induced the phosphorylation level of Scr, while the increased phosphorylation level of Src was inhibited by fisetin administration. We also examined the effect of fisetin on phosphorylation levels of EGFR at Tyr845,ERK1/2 and Smad3. As shown in Fig. 6 B,C,D, fisetin administration reduced their phosphorylation levels. These results further confirmed that fisetin might protect the obstructed kidney tissues against MMT-derived RIF potentially by inhibiting Src and subsequently down-regulating EGFR/ERK1/2/Smad3 signaling pathway. 4. Discussion The progression of RIF is complex and irreversible. Although the pathogenesis of RIF has been extensively studied, there is still a lack of effective prevention and treatment methods at present. 20 Therefore, it is urgent to develop safe and effective therapies to prevent against RIF, thereby improving the prognosis of CKD. In 2018, the World Health Organization introduced traditional Chinese medicine into its global medical plan. Various plant extracts inhibiting the renal fibrosis progression such as flavonoids have been reported and attracted wide attention. 21 Fisetin, as a natural dietary flavonoid, has been reported to have renoprotective effects in some animal models of kidney injury. Our current study indicated that fisetin could attenuate ECM deposition, renal interstitial fibrosis, thus protecting kidneys from UUO injury by inhibiting Src activation to block MMT. Macrophage infiltration is a common feature of active fibrotic lesions, and it is significantly correlated with the degree of RIF. 22 Renal macrophages include tissue-inherent macrophages and bone marrow-derived monocytes/macrophages. When the kidney is damaged, the tissue-inherent macrophages can not proliferate, and most of the recruited macrophages come from bone marrow. Macrophages are heterogeneous cells with various phenotypes under tissue damage conditions when the local microenvironment is affected by multiple factors such as pathogens, cellular damage, hypoxia, and tissue repair processes. 23 Pro-inflammatory macrophages (M1) mainly appear in the early stage of inflammation. With the alleviation of inflammation or injury, anti-inflammatory macrophages (M2) gradually play a dominant role and secrete a large number of anti-inflammatory factors such as IL-10 to exert anti-inflammatory function. In contrast, unresolved or sustained inflammation enhances the production and activation of profibrotic factors such as TGFβ1 in the damaged tissue. These persistently activated profibrotic factors induce the transition of macrophages from an M2 to a-SMA + myofibroblasts (a process termed as macrophage-to-myofibroblast transition, MMT), promote fibrosis and disease progression. 24 , 25 MMT is an important reason for the development of CKD into end stage renal disease(ESRD). Previous studies have revealed that MMT cells (CD68 + α-SMA + ) are significantly expressed in active areas of fibrotic lesions, and that a large proportion of myofibroblasts in the UUO model of renal fibrosis come from the bone marrow-derived macrophages. 26 , 27 Key factors,such as FABP4 and Pou4f1, involved in MMT can influence UUO-induced RIF. 28 , 29 In addition, resveratrol, a bioactive component of blueberries, also has been reported to attenuate RIF by inhibiting MMT. 30 In this study, our results revealed that fisetin could protect against UUO-induced RIF by regulating MMT.Our findings also confirmed the potential prophylactic and therapeutic capability of active phytoconstituent in RIF. MMT, as a newly discovered cell phenotype and functional transformation mechanism, mainly depends on the continuous activation of TGFb1/Smad3 signaling pathway. Some other signaling pathways such as EGFR and IL-4/IL-4R are also involved in MMT. 15 Src is a non-receptor tyrosine kinase, and it can be induced by a variety of cytokines and growth factors,including TGFβ1. Single-cell RNA sequencing analysis in previous study has indicated that Src is at the center of differentially expressed gene network in TGFβ1-induced MMT. 31 In fact, Src serves as a key mediator of MMT-related signaling pathways during RIF. Inhibition of Src could significantly suppress MMT-driven RIF in the UUO model. It has been reported that the main mechanism by which flavonoids exert their biological effects lies in the direct access to signaling kinases in the cytoplasm and the inhibition of the activities of signaling kinases driving biological processes. In a previous study, the relationship between the structure of flavonoids (quercetin, apigenin, and catechin) and their abilities to inhibit Src-family kinase activity were examined by using molecular docking. The results showed that the hydroxyl groups of flavonoids formed hydrogen bonds with the residues at the kinase catalytic site, thus inhibiting the activity of Src-family kinase. 32 Based on these findings, we speculated that fisetin, also known as 3,3',4',7-Tetrahydroxyflavone, might be able to form specific interaction with Src. To confirm our speculation, we performed molecular docking between fisetin and Src protein structures. The molecular docking results showed that fisetin exhibited strong affinity to Src, and that fisetin was embedded well into the active pocket to form hydrogen bond and hydrophobic interactions with Src.These results suggested that fisetin might be a potential inhibitor of Src, and that fisetin might regulate MMT by inhibiting Src activity. Therefore, we further examined the activation and expression of Src in the obstructed kidney and in TGFb1-stimulated bone marrow-derived macrophages. Both in vivo and in vitro study results showed that fisetin significantly inhibited activity of Src. Src has been reported to participate in the regulation and coupling of several signaling pathways related to MMT. In the classical TGFb1/Smad3 signaling pathways, Src can be activated by TGFb1. Meanwhile, TGFb1 triggered Smad3 binding to the 3ʹ untranslated region( UTR) of Src gene to increase its transcription. Src was also found to be involved in the activation of EGFR signaling pathway. TGFβ1 can induce sustained EGFR transactivation in a Src-mediated non-ligand-dependent manner. Src can induce sustained phosphorylation of EGFR at Tyr845, which is essential for EGFR to exert its cellular functions. 18 , 19 , 33 , 34 ERK1/2 pathway is the downstream of EGFR transactivation. Moreover, Src-mediated transactivation of EGFR and ERK1/2 phosphorylation involve late Smad3 activation induced by TGFb1.TGFβ1-initiated ECM accumulation is partially mediated by Src/EGFR/ERK 1/2 / Smad3 signaling pathway. Consistent with previous studies, our results showed that fisetin serving as Src inhibitor significantly inhibited the phosphorylation levels of EGFR at Tyr845, ERK1/2 and Smad3 in the obstructed kidney. Similar results were also observed in the TGFb1-induced MMT in BMDMs. Collectively, these findings demonstrated that fisetin could block MMT by inhibiting Src activation and subsequently down-regulating EGFR/ERK1/2/Smad3 signaling pathway. In conclusion, our findings revealed the mechanism by which fisetin could alleviate UUO-induced RIF by blocking MMT, and this mechanism might be related to the inhibition of Src activation and downstream EGFR/ERK1/2/Smad3 signaling pathway. Therefore, fisetin exhibit a great potential to prevent and treat RIF. It is worth noting that Src is the only target protein used to investigate the effect of feisetin in this study. In the future, molecular docking with more relevant target proteins can be used to further explore the potential mechanism by which fisetin alleviates renal fibrosis. Abbreviations UUO unilateral ureteral obstruction RIF renal interstitial fibrosis MMT macrophage-to-myofibroblast transition BMDM bone marrow-derived macrophages CKD chronic kidney disease ECM extracellular matrix EMT epithelial-to-mesenchymal transition α-SMA αlpha-smooth muscle actin TGFb1 transforming growth factor-β1 EGFR epidermal growth factor receptor LPS lipopolysaccharide AKI acute kidney injury M-CSF macrophage colony-stimulating factor HE Hematoxylin-eosin MTT Methylcyclopentadieny IHC Immunohistochemistry IF Immunofluorescence Declarations Conflicts of interest The authors declare no conflict of interest. Funding Declaration This work was supported by Natural Science Foundation of Hubei Province (No. 2021CFB360, 2023AFB145) and National Natural Science Foundation of China (No.2023AFB145). The funding body played no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Author Contribution Yonghong Jian, Yifei Yang and KeSu conceived and designed the research; Yifei Yang, Yonghong Jiang and Rong Li performed experiments; Yuhan Wan and Wei Li established UUO model of mice; Yifei Yang and Yonghong Jian analyzed data; Yonghong Jian completed the molecular docking analysis; Yifei Yang, Ke Su wrote the paper. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 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Wright, K.A. Watson, L.J. McGuffin, J.A. Lovegrove, J.M. Gibbins, GRID and docking analyses reveal a molecular basis for flavonoid inhibition of Src family kinase activity, J Nutr Biochem , 2015, 26 , 1156-1165. H.M. Chen, J.J. Dai, R. Zhu, et al., Parathyroid hormone-related protein induces fibronectin up-regulation in rat mesangial cells through reactive oxygen species/Src/EGFR signaling, Bioscience Rep, 2019, 39. K. Sato, Cellular functions regulated by phosphorylation of EGFR on Tyr845, Int J Mol Sci, 2013, 14, 10761-10790. Table Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.tif westernblot.docx GraphicAbstract.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":510871,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFisetin alleviates UUO-induced renal interstitial fibrosis (RIF) in mice.\u003c/strong\u003e(A) Representative photomicrographs of kidney tissues stained with hematoxylin and eosin (HE) and Masson’s trichrome. Scale bar = 50 μm. (B) Renal damage scores.\u003cbr\u003e\n(C) Interstitial fibrosis scores. (D) Representative immunohistochemical staining of α-SMA and collagen-I in kidney tissue sections. Scale bar = 50 μm.(E, F) Quantitative analysis of renal interstitial deposition of (E) α-SMA and (F) collagen-I.(G, H) mRNA expression levels of (G) α-SMA and (H) collagen-I in mouse kidney tissues, as determined by RT-qPCR.(I) Representative Western blot images showing protein expression levels of α-SMA and collagen-I in the kidneys.(J, K) Densitometric quantification of (J) α-SMA and (K) collagen-I protein expression normalized to GAPDH. Data were expressed as mean ± SEM (n = 6). *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01; ***,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001 compared with sham group. \u003csup\u003e#\u003c/sup\u003e, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; \u003csup\u003e##\u003c/sup\u003e, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and\u003csup\u003e ###\u003c/sup\u003e,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001 compared with UUO mice.ns means no significance.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7548347/v1/d363a9a4393531dfb6dcbd4a.png"},{"id":93595974,"identity":"c786ebed-228c-4663-879d-e449f7259857","added_by":"auto","created_at":"2025-10-15 13:44:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":270708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFisetin inhibits MMT in kidneys of UUO mice. \u003c/strong\u003e(A) Representative immunofluorescence micrographs showing CD68⁺α-SMA⁺cells (indicating macrophage-to-myofibroblast transition, MMT) in kidney tissues from each group. CD68 (green) labels macrophages; α-SMA (red) labels myofibroblasts. Yellow signal indicates co-localization (CD68⁺α-SMA⁺ MMT cells). Scale bar = 50 μm.(B) Quantitative analysis of CD68⁺ cells in renal tissues.(C) Quantitative analysis of the percentage of α-SMA⁺ cells among CD68⁺ cells (MMT proportion). Data are expressed as mean ± SEM (n = 6). *,\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 compared with sham group. \u003csup\u003e#\u003c/sup\u003e, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05;\u003csup\u003e ##\u003c/sup\u003e, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and \u003csup\u003e###\u003c/sup\u003e, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared with UUO mice.ns means no significance.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7548347/v1/bd788a84c75241b4cd11006e.png"},{"id":93593956,"identity":"d25eae45-45d7-4012-a488-04928de6810a","added_by":"auto","created_at":"2025-10-15 13:20:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":207421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFisetin inhibits MMT in TGFb1-stimulad BMDMs in vitro. \u003c/strong\u003e(A) Cytotoxicity of fisetin on BMDMs was assessed by MTT assay. (B)\u0026nbsp;Representative immunofluorescence images of α-SMA and collagen-I in TGF-β1-stimulated BMDMs at day 5. Scale bar = 50 μm. (C, D)\u0026nbsp;mRNA expression levels of\u0026nbsp;(C)\u0026nbsp;α-SMA and\u0026nbsp;(D)\u0026nbsp;collagen-I in TGF-β1-stimulated BMDMs, determined by RT-qPCR. (E) Representative Western blot images showing protein expression of α-SMA and collagen-I in TGF-β1-stimulated BMDMs. (F, G)\u0026nbsp;Protein expression levels of\u0026nbsp;(F)\u0026nbsp;α-SMA and\u0026nbsp;(G)\u0026nbsp;collagen-I were quantified by densitometry and normalized to GAPDH. Data are expressed as mean ± SEM from 3 independent experiments. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared with non-treated BMDMs.\u003csup\u003e #\u003c/sup\u003e, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; \u003csup\u003e##\u003c/sup\u003e, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e###\u003c/sup\u003e, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared with TGFb1-stimulad BMDMs. ns means no significance.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7548347/v1/8efe2fd3d027c4e28da874b4.png"},{"id":93594911,"identity":"98a3172d-fc0d-476d-a417-f24fe06e8027","added_by":"auto","created_at":"2025-10-15 13:28:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":146302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking of fisetin with Src.\u003c/strong\u003e (A) Structures of fisetin. (B) The three-dimensional diagram displays the interaction of fisetin with Src and bound amino acid residues.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7548347/v1/15eb0d4b58c5c2ebf02c5041.png"},{"id":93595096,"identity":"cab7a4eb-b3fd-478c-9f1e-5e4f6c09272d","added_by":"auto","created_at":"2025-10-15 13:36:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":212874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFisetin inhibits Src/EGFR/ERK1/2/Smad3 signaling pathway in the obstructed kidney.\u003c/strong\u003e (A) Representative Western blot images of kidney tissues from each group, probing for p-Src and Src. (B) Densitometric quantification of p-Src protein levels normalized to GAPDH and total Src. (C) Representative Western blot images probing for p-EGFR (Tyr845) and EGFR. (D) Densitometric quantification of p-EGFR (Tyr845) normalized to GAPDH and total EGFR. (E) Representative Western blot images probing for p-ERK1/2 and ERK1/2. (F) Densitometric quantification of p-ERK1/2 normalized to GAPDH and total ERK1/2. (G) Representative Western blot images probing for p-Smad3 and Smad3. (H) Densitometric quantification of p-Smad3 normalized to GAPDH and total Smad3. Data are expressed as mean ± SEM from 3 independent experiments. *, P \u0026lt; 0.05; **, P \u0026lt; 0.01; ***, P \u0026lt; 0.001 compared with sham group. #, P \u0026lt; 0.05; ##, P \u0026lt; 0.01; and ###, P \u0026lt; 0.001 compared with UUO mice.ns means no significance.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7548347/v1/7c4aef60ad3e6e626e6b3076.png"},{"id":93593962,"identity":"8f71f684-bfed-406a-a5e2-d4dc63942a2f","added_by":"auto","created_at":"2025-10-15 13:20:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":224757,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFisetin inhibits MMT by suppressing the Src/EGFR/ERK1/2/Smad3 signaling pathway\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A) Representative Western blot images of TGF-β1-stimulated BMDMs (day 5) probing for p-Src and Src. (B) Densitometric quantification of p-Src protein levels normalized to GAPDH and total Src. (C) Blots probing for p-EGFR (Tyr845) and EGFR. (D) Densitometric quantification of p-EGFR (Tyr845) normalized to GAPDH and total EGFR. (E) Blots probing for p-ERK1/2 and ERK1/2. (F) Densitometric quantification of p-ERK1/2 normalized to GAPDH and total ERK1/2. (G) Blots probing for p-Smad3 and Smad3. (H) Densitometric quantification of p-Smad3 normalized to GAPDH and total Smad3. Data are expressed as mean ± SEM from 3 independent experiments. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared with non-treated BMDMs. \u003csup\u003e#\u003c/sup\u003e, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; \u003csup\u003e##\u003c/sup\u003e, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; \u003csup\u003e###\u003c/sup\u003e, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 compared with TGFb1-stimulad BMDMs.ns means no significance.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7548347/v1/1c695d516750300ab1b9e640.png"},{"id":95802222,"identity":"23a6270a-a132-4312-839c-87dde0539cdc","added_by":"auto","created_at":"2025-11-13 08:27:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2531528,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7548347/v1/8fd38686-3edb-470d-9281-b6f4c4530e59.pdf"},{"id":93595098,"identity":"69cd3f33-cfb4-4ba8-8eab-79e0ab40a0bf","added_by":"auto","created_at":"2025-10-15 13:36:34","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8759148,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7548347/v1/53412243f215ad6e572398b8.tif"},{"id":93594926,"identity":"211741e6-a305-44c3-a5f4-0a490575b10b","added_by":"auto","created_at":"2025-10-15 13:28:35","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7788257,"visible":true,"origin":"","legend":"","description":"","filename":"westernblot.docx","url":"https://assets-eu.researchsquare.com/files/rs-7548347/v1/ac24ff47c187f1f8fb2728db.docx"},{"id":93593960,"identity":"2bf27646-8ba5-475f-bf9b-b3c3a3a6f065","added_by":"auto","created_at":"2025-10-15 13:20:34","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":100782,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7548347/v1/4dffb0c7ee5c900882cd8460.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fisetin alleviates unilateral ureteral obstruction-induced renal interstitial fibrosis by inhibiting Src activation to block macrophage-myofibroblast transition","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eKidney disease has a major impact on global health, and it is not only a direct cause of global morbidity and mortality but also a major risk factor for cardiovascular disease.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Patients suffering from chronic kidney disease (CKD) are clinically characterized by gradual loss of kidney function, and patients at the end stage have to rely on dialysis or kidney transplantation. CKD is largely preventable and requires great attention so as to formulate related global health policy.\u003c/p\u003e\u003cp\u003eFisetin is a plant-derived flavonoid compounds, and it is widely found in fruits and vegetables such as apples, onions, and cucumbers with its highest content in strawberries.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Fisetin has multiple pharmacological effects mainly including anti-inflammatory, anti-apoptotic,anti-oxidant,anti-tumor, anti-angiogenic effects. \u003csup\u003e3,4\u003c/sup\u003e More importantly, no side effects have been reported even at relatively high doses.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Previous physicochemical analysis has revealed that the concentrations of fisetin and its metabolites are high in mouse kidney, suggesting that it has potential to prevent and treat kidney diseases.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Fisetin has been reported to have renal protective effects against kidney diseases in animal models such as lipopolysaccharide (LPS)-induced acute kidney injury (AKI),\u003csup\u003e7\u003c/sup\u003e diabetic nephropathy,\u003csup\u003e8\u003c/sup\u003e renal ischemic reperfusion injury, \u003csup\u003e9\u003c/sup\u003eand hyperuricemic nephropathy.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e However, the effect of fisetin on unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis (RIF) in mice remains unclear.\u003c/p\u003e\u003cp\u003eRenal interstitial fibrosis (RIF), as the most common pathological lesion at the end stage of renal disease, is mainly characterized by myofibroblast proliferation, accumulation of extracellular matrix (ECM), and renal scar formation.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Myofibroblast, as a subgroup of activated fibroblasts, expresses αlpha-smooth muscle actin (α-SMA), and it is the main cell type responsible for the synthesis and deposition of fibrillar collagen during active tissue fibrosis.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e As a heterogeneous population, myofibroblasts can derive from multiple sources such as proliferation of local resident fibroblasts, epithelial-to-mesenchymal transition (EMT), endothelial-to-mesenchymal transition, and differentiation from bone marrow.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eMacrophages play a central role in the development of renal fibrosis. Macrophages derived from bone marrow cells can differentiate into a-SMA\u003csup\u003e+\u003c/sup\u003e myofibroblasts in injured kidney during a process termed as macrophage-to-myofibroblast transition (MMT). MMT is an important mechanism in the development process from renal chronic inflammation to fibrosis which is regulated by c-Src tyrosine kinase (Src) activation. Specific inhibition of MMT could significantly ameliorated unilateral ureteral obstruction (UUO)-induced renal fibrosis.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Fisetin has been reported to inhibit macrophage-mediated acute inflammatory responses.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e However, whether fisetin affects the progression of MMT in the chronic kidney injury remains largely unknown.\u003c/p\u003e\u003cp\u003eIn the present study, we explored the effects of fisetin on UUO-induced RIF in mice. At the same time, we also predicted the target of fisetin by molecular docking with the key mediator Src, and then explored the mechanism of fisetin in RIF.The aim of this study was to investigate the prophylactic and therapeutic potentials of fisetin for RIF.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Reagents\u003c/h2\u003e\u003cp\u003eFisetin was purchased from Sigma (St. Louis, MO, USA and stored at -20\u003csup\u003e◦\u003c/sup\u003eC). Recombinant mouse transforming growth factor-β1(TGFb1) was purchased from Biolegend (San Diego, CA,USA). The primary antibodies against α-SMA, Collagen-I, and CD68 used for immuno-staining were obtained from Abcam (Cambridge, MA,USA). For western blotting analysis, antibodies against p-Src, Src, p-EGFR, EGFR, p-ERK1/2, ERK1/2, p-Smad3 and Smad3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-GAPDH was purchased from Santa Cruz (Santa Cruz, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Animal experiments\u003c/h2\u003e\u003cp\u003eMale C57BL/6J mice (8\u0026ndash;10 weeks old, weighting 25\u0026thinsp;\u0026minus;\u0026thinsp;27 g) were housed in specific pathogen-free conditions at the Center for Animal Experiments of Wuhan University. UUO model was established by tying off the left ureter with silk suture, while sham surgery mice underwent the same procedures without left ureter ligation. After treatment, all mice were anesthetized and euthanized (Pentobarbital sodium 150 mg/kg, intraperitoneal injection), the injured kidneys of mice were collected on day 7 after UUO for subsequent experiments. As for the fisetin group, fisetin was dissolved in 20% PEG400, further diluted with 0.9% normal saline, and administered by gavage to mice with left ureter ligation at a dose of 100 mg/kg/d for 7 days,\u003csup\u003e[10]\u003c/sup\u003e whereas sham surgery group received only normal saline. All the animal experiments were approved by the Animal Ethics Review Board of Wuhan University and performed in accordance with the guidelines of the National Health and Medical Research Council of China. We confirm that the design and reporting of this study strictly adhered to the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Histological staining and evaluation\u003c/h2\u003e\u003cp\u003eKidney samples were fixed in 4% paraformaldehyde (pH7.4) and then embedded in paraffin. Hematoxylin-eosin (HE) or Masson\u0026rsquo;s trichrome stainings of tissue sections (4\u0026micro;m) were performed according to the manufacturer\u0026rsquo;s instruction. Renal tubules with the following histopathological changes were considered as damaged /injured : brush border loss, tubular dilation and disruption, cast formation and cell lysis. Tissue damage was assessed in a blind manner and scored as the percentage of damaged/injured renal tubules: 0, no damage; 1, \u0026lt;\u0026thinsp;25%; 2, 25\u0026ndash;50%; 3, 50\u0026ndash;75%; 4, \u0026gt;\u0026thinsp;75%.The degree of interstitial fibrosis was scored semiquantitatively as follows: 0 (no fibrosis), 1, \u0026lt;\u0026thinsp;25%; 2, 25\u0026ndash;50%; 3, 50\u0026ndash;75%; 4, \u0026gt;\u0026thinsp;75% of interstitium.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e The positively stained areas of renal interstitial fibrosis were quantified by Image J (NIH, Bethesda, MD,USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Immunohistochemistry(IHC) staining\u003c/h2\u003e\u003cp\u003eSections were first deparaffinized, hydrated, and antigen-recovered, followed by incubation with specific primary antibodies against α-SMA (Abcam, MA,USA)) and Collagen-1 (BA0325, Bosterbio, USA). Five visual fields (\u0026times;200 magnification) from each group were randomly selected and the percentage of positively stained areas was calculated using Image J software (NIH, Bethesda, MD,USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Immunofluorescence (IF) staining\u003c/h2\u003e\u003cp\u003eImmunofluorescence staining was performed on paraffin-embedded kidney sections (4 \u0026micro;m) and cultured bone marrow-derived macrophages (BMDMs). BMDMs were cultured on coverslips, washed 3 times with pre-chilled phosphate buffer saline(PBS), fixed in 4% paraformaldehyde, and blocked with PBS containing 10% normal donkey serum. Subsequently, sections and cell samples were incubated with primary antibodies against α-SMA and CD68 (Abcam, MA,USA). The samples were then stained with DAPI and observed under a confocal laser microscopy (Olympus FV1200, Tokyo, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Molecular docking of fisetin with Src\u003c/h2\u003e\u003cp\u003eMolecular docking was performed using the Schrodinger package (2018). The crystal structure of Src was obtained from RCSB Protein Data Bank (PDB ID: 4K11). The structure of fisetin was characterized and optimized by ChemDraw software to obtain the dominant conformation with the lowest energy. The fisetin was docked to the Src using Autodock Vina 1.1.2 software. The docking score reflected the affinity of fisetin to Src protein.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Generation of MMT cells from BMDMs\u003c/h2\u003e\u003cp\u003eBone marrow cells were isolated from C57BL/6J mice by flushing the femur and tibia with Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) /F12 medium. Cells were collected from suspension on the next day, incubated in DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 10 ng/ml macrophage colony-stimulating factor (M-CSF) for 5 day, and further differentiated into BMDMs. Macrophage-to-myofibroblast transition was triggered by TGFb1 (5ng/ml) in DMEM/ F12 medium containing 1% FBS to obtain myofibroblasts on day 5.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Cell viability assay\u003c/h2\u003e\u003cp\u003eMethylcyclopentadieny (MTT) assay was used to determine the toxicity of fisetin on BMDMs. BMDMs were cultured in 96-well plate for 24 h and later incubated with fisetin at various concentrations (5,10, 20, and 40 \u0026micro;M). After 5-day incubation, 10\u0026micro;L of MTT solution (5 mg/mL) was added to the medium, and the BMDMs were further incubated at 37\u0026deg;C for 4 h. After medium removal, 150\u0026micro;L dimethyl sulfoxide (DMSO) was added to the each well of 96-well plate. The plate was then shaken in the dark until the crystal was completely dissolved, and the absorbance was measured at 540 nm with a microplate reader (Molecular Devices, CA,USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Western blot\u003c/h2\u003e\u003cp\u003eWestern blot analysis was performed on kidney tissues or BMDM lysates. The protein expressions of lysed samples were determined by Western blot analysis with primary antibodies against a-SMA, collagen-I, p-Src, Src, p-EGFR, EGFR, p-ERK1/2, ERK1/2, p-Smad3, Smad3 and GAPDH. GADPH was used as an internal control. Image J software (NIH, Bethesda, MD, USA) was used to quantify the intensity of protein bands.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from kidney samples or BMDMs using Trizol reagents (Invitrogen). cDNA was synthesized with 1 \u0026micro;g total RNA. Afterwards, the qRT-PCR was performed using SYBR Green PCR Master Mix. The relative expressions of target genes were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003emethod, and normalized to that of gene GAPDH. The primer sequences used in this study were presented in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The differences between groups were estimated using Mann\u0026ndash;Whitney U-test, or one-way ANOVA followed by the Bonferroni post-test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant. The statistical analyses were performed using GRAPHPAD PRISM version 7.0 (GraphPad Software, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Fisetin allievates RIF in UUO mice\u003c/h2\u003e\u003cp\u003eTo determine the effect of fisetin on RIF, a mouse model of UUO was established. Renal pathological test was performed on day 7 after UUO. HE and Masson\u0026rsquo;s trichrome staining showed that UUO mice exhibited widespread renal injury and tubulointerstitial fibrosis, but fisetin administration significantly reduced tissue injury and interstitial fibrosis in the obstructed kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Immunohistochemical (HIC) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and western blotting analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) showed that fisetin administration significantly down-regulated the expression of α-SMA and collagen-I in the obstructed kidney on day 7 after UUO. The above results indicated that fisetin could attenuate ECM deposition, renal interstitial fibrosis, thus protecting kidneys from UUO injury.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Fisetin suppresses RIF by inhibiting MMT in UUO mice\u003c/h2\u003e\u003cp\u003eBone marrow cell-derived macrophages, as a source of myofibroblasts, can directly cause RIF. Considering the important role of MMT in RIF, we investigated the effect of fisetin on MMT process by detecting CD68\u003csup\u003e+\u003c/sup\u003ea-SMA\u003csup\u003e+\u003c/sup\u003e cells derived from MMT in obstructed kidney tissues of mice via immunofluorescence staining. The results showed that obstructed kidney tissues of UUO mice exhibited a large number of infiltrated macrophages and MMT-derived CD68\u003csup\u003e+\u003c/sup\u003ea-SMA\u003csup\u003e+\u003c/sup\u003e cells, whereas those of UUO mice treated with fisetin displayed a significantly reduced macrophage infiltration and MMT cell percentage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Taken together, the above results suggested that fisetin could exert protective effect on kidney tissues of UUO mice by effectively blocking MMT.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Fisetin inhibits TGFb1-induced MMT \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo further verify the action mechanisms of fisetin on MMT, we stimulated BMDMs with TGFb1 for 5 days \u003cem\u003ein vitro\u003c/em\u003e. MTT assay was used to determine the toxicity of fisetin on BMDMs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, fisetin (40 \u0026micro;M) inhibited the cell viability on day 5 in the absence or presence of TGFb1. In contrast, fisetin at the concentrations below 20 \u0026micro;M exhibited no cytotoxicity on BMDMs. Immunofluorescence staining showed that after 5-day TGFb1 stimulation, fisetin (20\u0026micro;M) significantly down-regulated the expression of α-SMA in BMDMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In addition, the mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) expression of α-SMA and Collagen-I were significantly down-regulated by fisetin in TGFb1-stimulated BMDMs. These results jointly suggested that fisetin could inhibit TGFβ1-induced MMT in BMDMs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Fisetin is successfully docked to Src\u003c/h2\u003e\u003cp\u003eSrc serves as a key mediator in the process of MMT. To investigate the interaction between fisetin and Src, we performed molecular docking of fisetin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) to crystal structure of Src. The results revealed that fisetin could be embedded well to the active pocket of Src, thus stabilizing the inactive conformation of the Src with a binding energy of -9.0kcal/mol, which might be attributed to fisetin\u0026rsquo;s hydrogen bonds and hydrophobic interactions with the Src. Three-dimensional interaction analysis revealed that the hydrogen bond interactions occurred between fisetin and animo acids MET341, ALA403 and ASP404 of Src, and that hydrophobic interactions were observed between hydrophobic functional groups of fisetin and animo acids LEU273, VAL281, LYS295, VAL323, ILE336, and LEU393 of Src(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). All these results indicated that fisetin could interact with Src, and it might be a potential inhibitor of Src.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Fisetin inhibits Src activation in obstructed kidney\u003c/h2\u003e\u003cp\u003eTo elucidate the mechanism by which fisetin protected against RIF, we investigated the effect of fisetin on Src activation in the obstructed kidney. The results showed that the phosphorylation level of Scr was significantly increased in the obstructed kidney, but fisetin administration significantly reduced the phosphorylation level of Src (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Previous studies have shown that Src can induce sustained fibrotic epidermal growth factor receptor (EGFR) transactivation by direct phosphorylation at Tyr845, and Src-mediated transactivation of EGFR and ERK1/2 phosphorylation can modulate Smad3 activation induced by TGFb1.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Considering this, we further examined the effect of fisetin on the activities of Src-mediated profibrotic molecules EGFR ,ERK1/2 and Smad3 in the obstructed kidney. The results showed that the phosphorylation levels of EGFR at Tyr845,ERK1/2 and Smad3 were significantly increased in the obstructed kidney, but their phosphorylation levels were significantly decreased under fisetin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,C,D). These data suggested that fisetin could inhibit Src activation, and that fisetin might protect against MMT-derived RIF by blocking EGFR/ERK1/2/Smad3 signaling pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Fisetin inhibits TGFb1-induced MMT in BMDMs by suppressing Src/EGFR/ERK1/2/Smad3 signaling pathways\u003c/h2\u003e\u003cp\u003eSrc activity is essential for the activation of TGFβ1signaling pathway. To further investigate the effect of fisetin on Src activity in the process of MMT, we examined the activities of Src, EGFR, ERK1/2 and Smad3 in TGFb1-stimulated BMDMs on day 5, As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, TGFb1 stimulation induced the phosphorylation level of Scr, while the increased phosphorylation level of Src was inhibited by fisetin administration. We also examined the effect of fisetin on phosphorylation levels of EGFR at Tyr845,ERK1/2 and Smad3. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,C,D, fisetin administration reduced their phosphorylation levels. These results further confirmed that fisetin might protect the obstructed kidney tissues against MMT-derived RIF potentially by inhibiting Src and subsequently down-regulating EGFR/ERK1/2/Smad3 signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe progression of RIF is complex and irreversible. Although the pathogenesis of RIF has been extensively studied, there is still a lack of effective prevention and treatment methods at present.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Therefore, it is urgent to develop safe and effective therapies to prevent against RIF, thereby improving the prognosis of CKD. In 2018, the World Health Organization introduced traditional Chinese medicine into its global medical plan. Various plant extracts inhibiting the renal fibrosis progression such as flavonoids have been reported and attracted wide attention.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Fisetin, as a natural dietary flavonoid, has been reported to have renoprotective effects in some animal models of kidney injury. Our current study indicated that fisetin could attenuate ECM deposition, renal interstitial fibrosis, thus protecting kidneys from UUO injury by inhibiting Src activation to block MMT.\u003c/p\u003e\u003cp\u003eMacrophage infiltration is a common feature of active fibrotic lesions, and it is significantly correlated with the degree of RIF.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Renal macrophages include tissue-inherent macrophages and bone marrow-derived monocytes/macrophages. When the kidney is damaged, the tissue-inherent macrophages can not proliferate, and most of the recruited macrophages come from bone marrow. Macrophages are heterogeneous cells with various phenotypes under tissue damage conditions when the local microenvironment is affected by multiple factors such as pathogens, cellular damage, hypoxia, and tissue repair processes. \u003csup\u003e23\u003c/sup\u003e Pro-inflammatory macrophages (M1) mainly appear in the early stage of inflammation. With the alleviation of inflammation or injury, anti-inflammatory macrophages (M2) gradually play a dominant role and secrete a large number of anti-inflammatory factors such as IL-10 to exert anti-inflammatory function. In contrast, unresolved or sustained inflammation enhances the production and activation of profibrotic factors such as TGFβ1 in the damaged tissue. These persistently activated profibrotic factors induce the transition of macrophages from an M2 to a-SMA\u003csup\u003e+\u003c/sup\u003e myofibroblasts (a process termed as macrophage-to-myofibroblast transition, MMT), promote fibrosis and disease progression.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e MMT is an important reason for the development of CKD into end stage renal disease(ESRD). Previous studies have revealed that MMT cells (CD68\u003csup\u003e+\u003c/sup\u003eα-SMA\u003csup\u003e+\u003c/sup\u003e) are significantly expressed in active areas of fibrotic lesions, and that a large proportion of myofibroblasts in the UUO model of renal fibrosis come from the bone marrow-derived macrophages.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Key factors,such as FABP4 and Pou4f1, involved in MMT can influence UUO-induced RIF.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e In addition, resveratrol, a bioactive component of blueberries, also has been reported to attenuate RIF by inhibiting MMT.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e In this study, our results revealed that fisetin could protect against UUO-induced RIF by regulating MMT.Our findings also confirmed the potential prophylactic and therapeutic capability of active phytoconstituent in RIF.\u003c/p\u003e\u003cp\u003eMMT, as a newly discovered cell phenotype and functional transformation mechanism, mainly depends on the continuous activation of TGFb1/Smad3 signaling pathway. Some other signaling pathways such as EGFR and IL-4/IL-4R are also involved in MMT.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Src is a non-receptor tyrosine kinase, and it can be induced by a variety of cytokines and growth factors,including TGFβ1. Single-cell RNA sequencing analysis in previous study has indicated that Src is at the center of differentially expressed gene network in TGFβ1-induced MMT.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e In fact, Src serves as a key mediator of MMT-related signaling pathways during RIF. Inhibition of Src could significantly suppress MMT-driven RIF in the UUO model.\u003c/p\u003e\u003cp\u003eIt has been reported that the main mechanism by which flavonoids exert their biological effects lies in the direct access to signaling kinases in the cytoplasm and the inhibition of the activities of signaling kinases driving biological processes. In a previous study, the relationship between the structure of flavonoids (quercetin, apigenin, and catechin) and their abilities to inhibit Src-family kinase activity were examined by using molecular docking. The results showed that the hydroxyl groups of flavonoids formed hydrogen bonds with the residues at the kinase catalytic site, thus inhibiting the activity of Src-family kinase.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Based on these findings, we speculated that fisetin, also known as 3,3',4',7-Tetrahydroxyflavone, might be able to form specific interaction with Src. To confirm our speculation, we performed molecular docking between fisetin and Src protein structures. The molecular docking results showed that fisetin exhibited strong affinity to Src, and that fisetin was embedded well into the active pocket to form hydrogen bond and hydrophobic interactions with Src.These results suggested that fisetin might be a potential inhibitor of Src, and that fisetin might regulate MMT by inhibiting Src activity. Therefore, we further examined the activation and expression of Src in the obstructed kidney and in TGFb1-stimulated bone marrow-derived macrophages. Both \u003cem\u003ein vivo and in vitro\u003c/em\u003e study results showed that fisetin significantly inhibited activity of Src.\u003c/p\u003e\u003cp\u003eSrc has been reported to participate in the regulation and coupling of several signaling pathways related to MMT. In the classical TGFb1/Smad3 signaling pathways, Src can be activated by TGFb1. Meanwhile, TGFb1 triggered Smad3 binding to the 3ʹ untranslated region( UTR) of Src gene to increase its transcription. Src was also found to be involved in the activation of EGFR signaling pathway. TGFβ1 can induce sustained EGFR transactivation in a Src-mediated non-ligand-dependent manner. Src can induce sustained phosphorylation of EGFR at Tyr845, which is essential for EGFR to exert its cellular functions.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e ERK1/2 pathway is the downstream of EGFR transactivation. Moreover, Src-mediated transactivation of EGFR and ERK1/2 phosphorylation involve late Smad3 activation induced by TGFb1.TGFβ1-initiated ECM accumulation is partially mediated by Src/EGFR/ERK\u003c/p\u003e\u003cp\u003e1/2\u003cb\u003e/\u003c/b\u003eSmad3 signaling pathway. Consistent with previous studies, our results showed that fisetin serving as Src inhibitor significantly inhibited the phosphorylation levels of EGFR at Tyr845, ERK1/2 and Smad3 in the obstructed kidney. Similar results were also observed in the TGFb1-induced MMT in BMDMs. Collectively, these findings demonstrated that fisetin could block MMT by inhibiting Src activation and subsequently down-regulating EGFR/ERK1/2/Smad3 signaling pathway.\u003c/p\u003e\u003cp\u003eIn conclusion, our findings revealed the mechanism by which fisetin could alleviate UUO-induced RIF by blocking MMT, and this mechanism might be related to the inhibition of Src activation and downstream EGFR/ERK1/2/Smad3 signaling pathway. Therefore, fisetin exhibit a great potential to prevent and treat RIF. It is worth noting that Src is the only target protein used to investigate the effect of feisetin in this study. In the future, molecular docking with more relevant target proteins can be used to further explore the potential mechanism by which fisetin alleviates renal fibrosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eUUO unilateral ureteral obstruction\u003c/p\u003e\n\u003cp\u003eRIF renal interstitial fibrosis\u003c/p\u003e\n\u003cp\u003eMMT macrophage-to-myofibroblast transition\u003c/p\u003e\n\u003cp\u003eBMDM bone marrow-derived macrophages\u003c/p\u003e\n\u003cp\u003eCKD chronic kidney disease \u003c/p\u003e\n\u003cp\u003eECM extracellular matrix\u003c/p\u003e\n\u003cp\u003eEMT epithelial-to-mesenchymal transition\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-SMA \u0026alpha;lpha-smooth muscle actin\u003c/p\u003e\n\u003cp\u003eTGFb1 transforming growth factor-\u0026beta;1\u003c/p\u003e\n\u003cp\u003eEGFR epidermal growth factor receptor \u003c/p\u003e\n\u003cp\u003eLPS lipopolysaccharide \u003c/p\u003e\n\u003cp\u003eAKI acute kidney injury\u003c/p\u003e\n\u003cp\u003eM-CSF macrophage colony-stimulating factor \u003c/p\u003e\n\u003cp\u003eHE Hematoxylin-eosin \u003c/p\u003e\n\u003cp\u003eMTT Methylcyclopentadieny\u003c/p\u003e\n\u003cp\u003eIHC Immunohistochemistry\u003c/p\u003e\n\u003cp\u003eIF Immunofluorescence \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eThis work was supported by Natural Science Foundation of Hubei Province (No. 2021CFB360, 2023AFB145) and National Natural Science Foundation of China (No.2023AFB145). The funding body played no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eYonghong Jian, Yifei Yang and KeSu conceived and designed the research; Yifei Yang, Yonghong Jiang and Rong Li performed experiments; Yuhan Wan and Wei Li established UUO model of mice; Yifei Yang and Yonghong Jian analyzed data; Yonghong Jian completed the molecular docking analysis; Yifei Yang, Ke Su wrote the paper.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGlobal, regional, and national burden of chronic kidney disease, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017, \u003cem\u003eLancet\u003c/em\u003e, 2020, \u003cstrong\u003e395\u003c/strong\u003e, 709-733.\u003c/li\u003e\n\u003cli\u003eM.J. 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Sato, Cellular functions regulated by phosphorylation of EGFR on Tyr845, Int J Mol Sci, 2013, 14, 10761-10790.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"fisetin, renal interstitial fibrosis (RIF), macrophage-to-myofibroblast transition(MMT), Src","lastPublishedDoi":"10.21203/rs.3.rs-7548347/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7548347/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFisetin, as a natural dietary flavonoid, exhibits multiple biological activities such as anti-inflammatory, anti-oxidant, and anti-tumorigenic activities. Previous studies have indicated that fisetin has potential renal protective effects in many animal models of kidney disease. However, the effect of fisetin on unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis (RIF) remains largely unknown. In our present study, we found that fisetin attenuated UUO-induced kidney injury by \u0026nbsp;decreasing fibrotic lesion and the accumulation of extracellular matrix (ECM). The results showed that fisetin could effectively block macrophage-to-myofibroblast transition (MMT) in the kidneys of UUO mice\u003cem\u003e in vivo\u003c/em\u003e and in transforming growth factor-β1(TGFb1)-stimulated bone marrow-derived macrophages (BMDM) \u003cem\u003ein vitro. \u003c/em\u003eMolecular docking was employed to explore the interactions between fisetin and Src (a key mediator in MMT). The results indicated that fisetin could form hydrogen bonds and hydrophobic interactions with Src, thus binding effectively in the active pocket of Src and exhibiting strong affinity. Further \u003cem\u003ein vivo and in vitro \u003c/em\u003einvestigation demonstrated that fisetin inhibited activity of Src and subsequently lowered the phosphorylation levels of epidermal growth factor receptor (EGFR) at Tyr845, ERK1/2 and Smad3. In conclusion, this study revealed the mechanism by which fisetin\u003cem\u003e \u003c/em\u003eblocked the progression of MMT by inhibiting the activationof Src, thus alleviating UUO-induced RIF. Therefore, fisetin might be a potential therapeutic agent for preventing and alleviating renal fibrosis.\u003c/p\u003e","manuscriptTitle":"Fisetin alleviates unilateral ureteral obstruction-induced renal interstitial fibrosis by inhibiting Src activation to block macrophage-myofibroblast transition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 13:20:29","doi":"10.21203/rs.3.rs-7548347/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"31699bfa-0414-443f-9a54-290bfdf75db0","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56145171,"name":"Health sciences/Diseases"},{"id":56145172,"name":"Biological sciences/Drug discovery"},{"id":56145173,"name":"Health sciences/Nephrology"},{"id":56145174,"name":"Health sciences/Urology"}],"tags":[],"updatedAt":"2025-11-13T01:38:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 13:20:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7548347","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7548347","identity":"rs-7548347","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}