Suoquan Yishen Formula Inhibits Podocyte Epithelial-Mesenchymal Transition in Diabetic Kidney Disease by Regulating miR-16-5p/FGF7 axis

Suoquan Yishen Formula Inhibits Podocyte Epithelial-Mesenchymal Transition in Diabetic Kidney Disease by Regulating miR-16-5p/FGF7 axis | 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 Suoquan Yishen Formula Inhibits Podocyte Epithelial-Mesenchymal Transition in Diabetic Kidney Disease by Regulating miR-16-5p/FGF7 axis Shiqi Chen, Zhu Wu, Jin Zhang, Yuxin Lin, Jiaqi Xie, Xiaoyu Peng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9304192/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 Podocyte epithelial-mesenchymal transition (EMT) drives diabetic kidney disease (DKD). Suoquan Yishen Formula (SQYSF) shows clinical efficacy, but its mechanism is unclear. Objective To investigate SQYSF protection against high glucose-induced podocyte EMT via miR-16-5p/FGF7 axis. Methods SQYSF was analyzed by LC-MS/MS. A podocyte EMT model was induced by high glucose and treated with SQYSF. Cell viability and ultrastructure were evaluated. Expression levels of EMT markers, miR-16-5p, and FGF7 were measured via RT-qPCR, Western blot, and immunofluorescence. Candidate targets were identified through bulk RNA-seq. miR-16-5p localization determined by FISH. The miR-16-5p/FGF7 interaction was confirmed by dual-luciferase assay, and functional validation was performed using miR-16-5p mimics/inhibitor and FGF7 overexpression. Results LC-MS/MS analysis identified 21 bioactive compounds. SQYSF dose-dependently attenuated high glucose-induced podocyte EMT, mitochondrial damage, and significantly upregulated miR-16-5p expression(P < 0.05). Inhibition of miR-16-5p reversed the protective effects of SQYSF(P < 0.05). Bulk RNA-seq analysis identified FGF7 as a key target. FISH confirmed the cytoplasmic localization of miR-16-5p.The miR-16-5p/FGF7 direct interaction was confirmed, and SQYSF inhibited high glucose-induced FGF7 upregulation(P < 0.05). Importantly, FGF7 overexpression abolished SQYSF’ s efficacy(P < 0.05). miR-16-5p mimics rescued the pro-EMT effects of FGF7(P < 0.05). Conclusion SQYSF alleviates podocyte EMT by upregulating miR-16-5p to repress FGF7, revealing a novel DKD therapeutic target. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Endocrinology Biological sciences/Molecular biology Health sciences/Nephrology Diabetic kidney disease Suoquan Yishen Formula miR-16-5p FGF7 Epithelial-mesenchymal transition Podocyte Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Diabetic kidney disease (DKD) is a chronic kidney condition caused by diabetes and represents one of the major microvascular complications of diabetes[ 1 ]. DKD is among the most common chronic complications of diabetes worldwide, including in China[ 2 ]. The prevalence of DKD in patients with type 2 diabetes in China is 21.8%[ 3 ]. It is the leading cause of end-stage renal disease (ESRD), which requires dialysis or kidney transplantation, and also a significant contributor to mortality among diabetic patients[ 4 ]. According to data from the International Diabetes Federation (IDF), the number of people with diabetes worldwide exceeded 537 million in 2021, and the prevalence of DKD has shown a gradual increasing trend. This has become a growing public health challenge [ 5 ]. Current pharmacological treatments for DKD, such as RAAS blockers and SGLT2 inhibitors, can slow the decline of kidney function [ 6 ]. However, their efficacy is limited by individual variability and adverse effects, and they cannot halt disease progression, falling far short of meeting clinical needs[ 7 ]. In the onset and progression of DKD, the progressive decline in renal function is closely associated with renal fibrosis, characterized by the abnormal accumulation of extracellular matrix (ECM) proteins in the glomerular basement membrane and tubulointerstitium, ultimately leading to irreversible loss of kidney function[ 8 ]. Epithelial-mesenchymal transition (EMT) is one of the central mechanisms driving renal fibrosis[ 9 ]. Sustained hyperglycemia can alter the phenotype of normal renal cells, inducing a transition toward a pro-fibrotic mesenchymal phenotype through the EMT process [ 10 , 11 ]. Podocytes, as essential components of the glomerular filtration barrier, play a pivotal role in the pathogenesis of DKD [ 12 ]. A high-glucose environment can induce EMT in podocytes [ 13 ], causing them to lose epithelial characteristics, such as decreased expression of markers including E-cadherin and ZO-1, and acquire a mesenchymal phenotype, characterized by increased expression of markers such as N-cadherin, fibronectin, and α-SMA [ 14 ]. Pathologically, this manifests as alterations in podocyte morphology, loss of polarity, disruption of intercellular junctions, and cytoskeletal rearrangement, which consequently increase their susceptibility to detachment from the glomerular basement membrane [ 15 ]. Such structural and functional impairment ultimately promotes basement membrane thickening and ECM accumulation, driving glomerulosclerosis and proteinuria development [ 16 ]. Studies have shown that podocyte phenotypic transition participates in the early stage of podocyte loss and correlates significantly with the degree of glomerulosclerosis, proteinuria, and reduction in glomerular filtration rate [ 17 ]. Therefore, inhibiting podocyte EMT is considered a promising potential strategy for protecting podocyte integrity and delaying the fibrotic progression of DKD. MicroRNAs (miRNAs) are a class of non-coding RNA molecules that play a key regulatory role at the post-transcriptional level. Recent research has increasingly focused on miRNAs, recognizing them as central to deciphering the complex pathological networks in kidney diseases. They are widely involved in the pathophysiology of various renal conditions, including acute kidney injury, chronic kidney disease, DKD, and transplant kidney injury. Differential miRNA expression profiles may reflect the underlying mechanistic features of distinct diseases[ 18 ]. Their clinical translational potential is significant. The stability and sensitivity of miRNAs in body fluids make them superior non-invasive biomarkers compared to traditional markers like urinary albumin, demonstrating considerable diagnostic value (Wang et al., 2024). This potential has begun to be realized in clinical practice [ 19 ]. At the mechanistic level, miRNAs act as hubs within signaling pathways, providing critical entry points for elucidating the molecular mechanisms of diseases such as DKD [ 20 ]. Furthermore, their high targetability establishes a theoretical foundation for developing precise nucleic acid-based therapies [ 21 ]. For instance, exosomes derived from human urine stem cells (hUSCs) can deliver miR-16-5p, alleviating podocyte injury under high-glucose conditions and thereby halting the progression of DKD [ 22 ]. These findings collectively establish miR-16-5p as a crucial regulatory factor and a potential therapeutic target in DKD. Mature miRNAs, processed sequentially by the Drosha and Dicer complexes, are incorporated into the RNA-induced silencing complex (RISC). By guiding RISC to complementary target mRNAs, they post-transcriptionally repress gene expression primarily through mRNA degradation or translational inhibition[ 23 ]. Traditional Chinese medicine (TCM) formulations have gained attention for their multi-targeted approach in managing complex diseases like DKD[ 24 ]. For instance, Jia Wei Qingxin Lotus Seed Drink was shown to attenuate EMT in DKD by suppressing the JMJD1C/SP1/ZEB1 signaling pathway[ 25 ]. Other studies have confirmed the role of TCM compounds in inhibiting tubular ferroptosis and reducing inflammatory injury[ 26 ]. These findings highlight the diverse mechanisms through which TCM can target DKD pathology. Among these interventions, Suoquan Yishen Formula (SQYSF), a patented traditional Chinese medicinal compound authorized for the treatment of DKD (Patent No: ZL201610423677.5), has been used in clinical practice for over a decade. SQYSF comprises seven medicinal herbs, namely Alpinia oxyphylla Miq.(Yizhiren in Chinese), Lindera aggregata(Wuyao), Poria cocos (Fuling), Atractylodes lancea (Cangzhu), Salviae Miltiorrhizae(Danshen), Paeonia lactiflora(Chishao), and Trichosanthes kirilowii(Tianhuafen). Preliminary clinical studies have confirmed that SQYSF can effectively reduce proteinuria and improve renal function in patients with DKD, demonstrating clear clinical application value[ 27 ]. SQYSF has demonstrated efficacy in reducing proteinuria and ameliorating renal function in DKD models, potentially through inhibiting renal senescence and enhancing autophagy[ 28 ]. Despite these promising findings, the precise molecular targets and pathways through which it attenuates podocyte EMT are poorly defined. This study aims to systematically identify key downstream target genes of miR-16-5p through combined miRNA‑mRNA analysis, and to explore whether the Chinese herbal compound SQYSF inhibits high glucose‑induced EMT in podocytes by regulating the miR‑16‑5p/FGF7, thereby delaying the progression of DKD. Graphical Abstract 2 Material and methods 2.1 Chemicals and reagents The mouse glomerular podocyte cell line MPC5 (CL0098, Fenghui Biotechnology, China) was used in this study. Key reagents and kits included fetal bovine serum (Gibco, 10099141C), penicillin/streptomycin (Yuanpei Biotech, S110JV), a CCK-8 assay kit (Beyotime Biotechnology, C0038), a Dual-Luciferase Reporter Assay Kit (GenePharma, G06011), GP-transfect-Mate transfection reagent (GenePharma, G04009), the iScript™ cDNA Synthesis Kit (BIO-RAD, 1708891), the Hairpin-it™ microRNA and U6 snRNA Normalization RT-PCR Quantitation Kit (GenePharma, E22001), an RNA FISH Kit (Cy3) (GenePharma, F40211), the HyperFluor 488 TSA Fluorescence System Kit (APExBIO, K4301), and Fluoroshield Mounting Medium with DAPI (Abcam, ab104139). Antibodies against N-cadherin (ab18203), E-cadherin (ab231303), fibronectin (ab268020), ZO-1 (ab276131), α-SMA (ab124964), KGF/FGF7 (ab131162), β-tubulin (ab21058), and β-actin (ab49900) were all obtained from Abcam. 2.2 Preparation of SQSYF All components of the SQYSF were sourced from Beijing Bencao Fangyuan Pharmaceutical Technology Co., Ltd., and authenticated per the Chinese Pharmacopoeia (2020 edition). To prepare the extract, 6000 g of herbs underwent water extraction and alcohol precipitation. The concentrate was then lyophilized, yielding 1500 g of powder. For in vitro studies, 40 g of this powder was dissolved in 60 mL of sterile saline to make a 0.4 g/mL stock solution, which was filter-sterilized (0.22 µm) and stored at 4°C. 2.3 LC-MS/MS analysis LC-MS/MS analysis was conducted to profile chemical composition of SQYSF. The lyophilized extract (0.5 g) was reconstituted in 10 mL methanol-water (60:40, v/v), centrifuged, and the supernatant was analyzed. Separation was performed on a Waters ACQUITY UPLC I-Class Plus system with a HSS T3 column (100 mm × 2.1 mm, 1.8 µm) at 45°C, using a gradient of 0.1% formic acid in water (A) and acetonitrile (B) at 0.35 mL/min: 0–2 min (5% B), 2–4 min (5–30% B), 4–8 min (30–50% B), 8–10 min (50–80% B), 10–14 min (80–100% B), 14–15 min (100% B), 15.1–16 min (5% B). Mass detection used a Q-Exactive Orbitrap spectrometer (Thermo Scientific) with H-ESI source in positive/negative modes. Key settings: spray voltage 3.8 kV(+)/3.2 kV(-); sheath gas 35 arb; auxiliary gas 8 arb; capillary temp 320°C; full MS resolution 60,000 (m/z 100–1500); dd-MS2 resolution 15,000 with stepped NCE (10, 20, 40 eV). 2.4 Animals Male Sprague-Dawley rats (220 ± 20g), supplied by Chengdu Dashuo Experimental Animal Co., Ltd. (License Nos. SCXK 2020-0030, SYXK 2022 − 225), were acclimatized for one week under SPF conditions before drug-containing serum preparation. All animal experiments were approved by the Ethics Committee of Hainan Medical University (Approval No. HYLL-2022-110) and were conducted in accordance with relevant ethical guidelines, which included provisions for post-procedural care and humane euthanasia. 2.5 Preparation of SQYSF-containing serum Rats were stratified by body weight and randomly assigned to two groups (n=10per group). Group A (control) received 0.9% NaCI via oral gavage, while Group Bwas administered the herbal formula SQYSF at a dose of 8 g crude drug/kg/day(equivalent to 4 times the clinical dose). Both groups were dosed twice daily (08:00and 14:00) for three consecutive days, with the gavage volume adjusted to 10 mL/kgbased on real-time body weight. Two hours after the final administration, rats wereanesthetized with an intraperitoneal injection of sodium pentobarbital (0.3%, 15mL/kg). Whole blood was then collected from the abdominal aorta using vacuumtubes. Serum was separated and stored at -80' C as drug-containing(Group B) orblank control (Group A) serum for subsequent analysis. 2.6 Bulk RNA-seq Total RNA was isolated from renal cortical tissues of [db/m, db/db, and SQYSF-treated db/db mice (n = 3). The resulting RNA libraries were purified and sequenced on a HiSeq3000 sequencer (Illumina, San Diego, CA, USA). Cell samples were sent to Genechem Co., Ltd. (Shanghai, China) for RNA-Seq analysis. DESeq2 software (1.20.0) was used to analyze DEGs, and the P value was corrected using the Benjamini&Hochberg method. The differentially expressed RNAs between groups were identified with |log2 (fold change) | >1 and P value < 0.05. 2.7 Cell Culture and transfection MPC5 cells were maintained in DMEM with 10% FBS and 1% penicillin-streptomycin at 37°C under 5% CO₂. For experiments, cells were divided into five groups: normal control, high glucose model, and HG groups co-treated with 2%, 4%, or 8% SQYSF-containing serum. After 24 h, cells were harvested for analysis. For transfection, cells at 50–70% confluence were transfected using GP-transfect-Mate. Plasmid DNA (500 ng/µL) or RNA oligonucleotides (miR-16-5p mimic/inhibitor or corresponding controls at 10 pmol/µL) were mixed with the transfection reagent in serum-free medium, incubated for 20 min at room temperature, and then applied to the cells. Culture medium was refreshed 24 h post-transfection, and cells were collected at indicated time points for subsequent assays. 2.8 CCK-8 (Cell Counting Kit-8) Assay Cell viability was measured by CCK-8 assay (Beyotime). MPC5 cells were seeded at 4×10⁴ cells/mL (200 µL/well) for 48 h, treated with 1–10% SQYSF-containing serum for 22 h, and then incubated with CCK-8 for 2 h. Absorbance (450 nm/650 nm reference) was read on a microplate reader to calculate viability. 2.9 Transmission Electron Microscopy (TEM) TEM samples were prepared by standard chemical fixation (2.5% glutaraldehyde and 1% OsO₄), dehydrated through a graded acetone series, and embedded in Epon-812 resin. Ultrathin sections (60–90 nm) were stained with uranyl acetate and lead citrate. Ultrastructural analysis was performed using a transmission electron microscope. 2.10 RT-qPCR Total RNA was isolated using the Animal Total RNA Isolation Kit according to the manufacturer’s instructions and eluted in 150 µL RNase-free water. cDNA was synthesized by reverse transcription in a 20 µL reaction system. Gene-specific primers were designed based on the PubMed database and synthesized by Sangon Biotech (Shanghai). Quantitative PCR was performed under the following conditions: 95°C for 2 min; 40 cycles of 95°C for 6 s and 60°C for 18 s; followed by a melting curve analysis from 65°C to 95°C (increment of 0.5°C every 5 s). All reactions were run in triplicate. Relative gene expression was calculated using the 2 − ΔΔCt method with GAPDH and U6 as internal controls. Primer sequences are listed in Supplement Table 1. 2.11 Western Blot Analysis Total protein was extracted from MPC5 cells, and its concentration was determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking, membranes were incubated overnight at 4°C with primary antibodies against α-SMA, Fibronectin, N-cadherin, E-cadherin, ZO-1, FGF7, β-Actin, and β-Tubulin (all at 1:1000 dilution). Following washes, membranes were incubated with an HRP-conjugated secondary antibody (1:10000 in 5% BSA) for 1 h at room temperature. Protein bands were visualized using Clarity™ Western ECL Substrate, imaged with an automated chemiluminescence system, and quantified using ImageJ software. 2.12 Immunofluorescence (IF) Assay Cells on coverslips were processed for immunofluorescence. Following fixation, permeabilization (Triton X-100), and blocking (5% BSA), samples were incubated overnight with primary antibody, followed by secondary antibody and TSA™ amplification. Nuclei were stained with DAPI. Images were captured using a Zeiss Axio Imager Z2 microscope (200×; FITC/DAPI channels) and analyzed with Zen Blue 3.3 software. 2.13 Fluorescence In Situ Hybridization (FISH) For FISH detection of miR-16-5p, cells on coverslips were fixed (4% PFA, 15 min), permeabilized, and hybridized overnight at 37°C with a denatured fluorescent probe (5 µM). Post-hybridization washes were performed with pre-heated buffers. Nuclei were stained with DAPI, and images were acquired at 600×magnification using a fluorescence imaging system. 2.14 Dual-Luciferase Reporter Assay To validate whether miR-16-5p directly targets the 3’-UTR of FGF7, wild-type (WT) and mutant (MUT) FGF7 3’-UTR reporter plasmids were constructed. MPC5 cells were seeded in 6-well plates and co-transfected with miR-16-5p mimic or negative control (NC) and the corresponding reporter plasmid using transfection reagent. After 24 h, cells were lysed and luciferase activities were measured using a dual-luciferase reporter assay system. Firefly luciferase activity was normalized to Renilla luciferase activity. Each experiment was performed in triplicate and repeated independently three times. 2.15 Statistical Analysis All data analyses were performed using GraphPad Prism software (version 10.0.0). Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA), and comparisons between two groups were performed using the Student's t-test. A P-value of less than 0.05 was considered statistically significant. 3 Results 3.1 LC-MS/MS analysis of SQYSF LC-MS/MS analysis characterized the chemical composition of SQYSF (Fig. 1A, B). A total of 21 compounds absorbed into the bloodstream were detected, and their herbal origins were identified in Supplementary Table 2. [insert Fig. 1.] 3.2 SQYSF-containing serum attenuates high glucose-induced podocyte EMT Under high glucose conditions, the EMT of podocytes is a key pathological alteration leading to their functional impairment and loss[ 29 ]. To further investigate the mechanism of SQYSF in DKD, this study focuses on its intervention effect on high glucose-induced podocyte EMT. To evaluate the cytotoxicity of SQYSF-containing serum, we performed CCK-8 assays to assess podocyte viability after treatment with varying concentrations (1%-10%) for 24 hours. No significant difference in cell viability was observed across all tested concentrations compared to the control group, indicating that SQYSF-containing serum is non-toxic within this range(Fig. 2A). Based on these findings, we selected 2%, 4%, and 8% SQYSF as low, medium, and high doses for subsequent experiments to investigate its pharmacological effects and potential dose-response relationship. To establish a reliable model of high glucose-induced podocyte injury, we first determined the optimal glucose concentration for inducing EMT. Western blot analysis revealed that exposure to 35 mM glucose significantly upregulated mesenchymal markers (fibronectin, N-cadherin, α-SMA) and downregulated epithelial markers (E-cadherin, ZO-1) at protein levels (Figure S1 A-F), confirming successful induction of EMT. Therefore, 35 mM glucose was adopted as the standard condition for constructing the diabetic nephropathy-like cellular model. TEM was employed to examine ultrastructural changes in podocytes under different treatments. In the high glucose group, podocytes exhibited marked mitochondrial damage, including swelling, cristae disruption, and vacuolization (Fig. 2B). In contrast, mitochondria in the control group maintained normal morphology. Notably, treatment with SQYSF-containing serum significantly ameliorated these ultrastructural abnormalities, restoring mitochondrial integrity and architecture, suggesting a protective effect against high glucose-induced organelle dysfunction. To further characterize the anti-EMT effects of SQYSF, we analyzed the expression of key EMT-related proteins and mRNAs. Western blotting (Fig. 2C,D) and RT-qPCR (Fig. 2E) analyses demonstrated that 35 mM glucose dramatically increased fibronectin, N-cadherin, and α-SMA expression while decreasing E-cadherin and ZO-1 levels. Treatment with 2%, 4%, or 8% SQYSF reversed these alterations in a dose-dependent manner, epithelial markers were restored, and mesenchymal markers were suppressed. Among the three doses, 4% SQYSF exhibited the most potent inhibitory effect on EMT, with maximal reversal of marker expression. Interestingly, the protective efficacy followed an inverted U-shaped dose-response curve, where higher concentrations (8%) showed reduced effectiveness compared to 4%. This suggests that 4% SQYSF represents the optimal concentration for exerting maximal anti-EMT activity. Collectively, these results demonstrate that SQYSF-containing serum effectively protects podocytes from high glucose-induced EMT, preserves mitochondrial ultrastructure, and exerts its beneficial effects in a dose-dependent manner, with 4% being the most effective concentration for downstream mechanistic investigations. [insert Fig. 2.] 3.3 SQYSF attenuates podocyte EMT by upregulating miR-16-5p Building upon the confirmed protective effect of SQYSF against podocyte EMT, this study aims to elucidate its underlying molecular mechanisms. Given the critical role of miR-16-5p in diabetic kidney disease (DKD)[ 30 ], we specifically focused on investigating the regulation of this miR-16-5p by SQYSF. RT-qPCR analysis revealed that HG (35 mM) significantly suppressed miR-16-5p levels compared to normal glucose (NG) control. Notably, SQYSF-containing serum reversed this downregulation in a dose-dependent manner, with 4% concentration showing the most potent effect (Fig. 3A). To determine whether miR-16-5p upregulation is functionally required for SQYSF-mediated protection, rescue experiments were performed using a miR-16-5p inhibitor. Efficient knockdown of miR-16-5p was confirmed by (Figure S1 G), and Western blot analysis showed that miR-16-5p inhibition markedly attenuated the ability of SQYSF to restore epithelial markers (E-cadherin and ZO-1) and suppress mesenchymal markers (Fibronectin, N-cadherin, and α-SMA) (Figs. 3B–G). These findings demonstrate that the anti-EMT effect of SQYSF in podocytes is, at least in part, dependent on its upregulation of miR-16-5p. [insert Fig. 3.] 3.4 SQYSF attenuates podocyte EMT by downregulating FGF7 To systematically investigate the molecular mechanisms underlying the therapeutic effects of SQYSF through miR-16-5p-mediated regulation, we first focused on identifying downstream target genes whose expression decreases following treatment, consistent with the upregulation of miR-16-5p. Based on this miRNA-mRNA regulatory principle[ 31 ], we performed transcriptome sequencing followed by integrative bioinformatic and experimental analyses. A hierarchical clustering heatmap of the top 20 most significantly differentially expressed genes revealed distinct global expression patterns across experimental groups (Fig. 4A). Genes differentially expressed among the control, model, and SQYSF-treated groups were clustered based on their expression profiles. Notably, FGF7 exhibited a marked upregulation in the model group compared to controls, which was progressively reversed by SQYSF intervention, a pattern consistent with its potential regulation by the upregulated miR-16-5p and supporting its selection as a candidate downstream target gene implicated in disease progression and drug response. To characterize the transcriptional changes during disease development, two volcano plot were generated (Figs. 4B,C). This analysis revealed a set of significantly upregulated genes, including FGF7, suggesting its activation under pathological conditions. Further evaluation of the regulatory effect of SQYSF was performed by comparing the treated group with the model group. The resulting volcano plot showed that multiple disease-associated genes were downregulated after treatment, with FGF7 being notably suppressed, supporting its responsiveness to pharmacological intervention. To validate these transcriptomic findings, we examined FGF7 expression at both protein and mRNA levels using Western blotting and qRT-PCR. Consistent with sequencing data, high glucose stimulation significantly induced FGF7 expression, whereas SQYSF treatment dose-dependently attenuated both FGF7 protein and mRNA levels(Figs. 4D,F). Collectively, these results demonstrate that FGF7, predicted as a potential target of miR-16-5p, is activated in the diabetic nephropathy model and effectively suppressed by SQYSF, and experimental validation supports a critical role for FGF7 in DKD. To further confirm the functional role of FGF7 in mediating the effects of SQYSF, we performed a rescue experiment by overexpressing FGF7 in high glucose-treated cells. Western blot analysis (Figs. 5A,B) revealed that 35 mM glucose significantly upregulated fibronectin, N-cadherin, and α-SMA expression, while decreasing E-cadherin and ZO-1 levels. Treatment with 4% SQYSF effectively reversed these changes, restoring epithelial markers and suppressing mesenchymal markers. However, when FGF7 was overexpressed via pcDNA3.1-FGF7 transfection(Figures S1 H-J), the protective effects of SQYSF were significantly attenuated. Specifically, fibronectin, N-cadherin, and α-SMA remained elevated, whereas E-cadherin and ZO-1 expression failed to recover, indicating that FGF7 overexpression counteracts anti-EMT actions of SQYSF. Immunofluorescence staining also corroborated these findings(Figs. 5C,D). In control cells, E-cadherin was localized at cell membranes, forming intact junctions. Under high glucose conditions, E-cadherin expression decreased, and membrane localization was disrupted. SQYSF treatment restored E-cadherin membrane distribution, but this effect was abolished upon FGF7 overexpression. Similarly, N-cadherin, which is typically associated with mesenchymal phenotype, was strongly induced by high glucose and reduced by SQYSF; however, its expression was maintained at high levels in the SQYSF + OE-FGF7 group. Collectively, these results demonstrate that FGF7 plays a critical role in the pathogenesis of high glucose-induced EMT and that its overexpression can rescue the beneficial effects of SQYSF, supporting the hypothesis that FGF7 is a key downstream target through which SQYSF exerts its protective functions. [insert Fig. 4.] [insert Fig. 5.] 3.5 miR-16-5p inhibits podocyte EMT by directly targeting FGF7 To investigate the subcellular localization and potential functional interaction between miR-16-5p and its predicted target FGF7, we first performed fluorescence in situ hybridization (FISH) using a specific probe for mouse miR-16-5p(Fig. 6A). miR-16-5p signals were predominantly localized in the cytoplasm, suggesting that its regulatory activity likely occurs within this compartment. This cytoplasmic enrichment supports the possibility of direct post-transcriptional regulation of FGF7 via miRNA-mediated mechanisms. Based on bioinformatic prediction from TargetScan, a putative binding site for miR-16-5p was identified in the 3’-UTR of FGF7 mRNA (Figure S1 K). To validate this interaction, we constructed luciferase reporter vectors containing either the wild-type (WT) or mutant (MUT) 3’-UTR of FGF7 and co-transfected them with miR-16-5p mimics or negative control (mimics NC) into podocytes(Fig. 6B). Firefly luciferase activity was normalized to Renilla luciferase (internal control), and values were further normalized to the mimics NC group. The results (Fig. 6C) showed that miR-16-5p mimics significantly reduced luciferase activity in the WT-FGF7 group, whereas no significant suppression was observed in the MUT-FGF7 group, indicating that the inhibitory effect is dependent on the specific seed sequence match. These findings confirm the direct and specific targeting of FGF7 by miR-16-5p. To confirm the functional relationship between miR-16-5p and FGF7, we modulated their expression and assessed EMT marker changes. Overexpression of FGF7 (via pcDNA3.1-FGF7) led to a significant increase in mesenchymal markers (fibronectin, N-cadherin, α-SMA) and a concomitant decrease in epithelial markers (E-cadherin, ZO-1) compared to the empty vector control. In contrast, transfection with miR-16-5p mimics reversed these changes, promoting an epithelial phenotype by upregulating E-cadherin and ZO-1 while downregulating fibronectin, N-cadherin, and α-SMA. Notably, when miR-16-5p mimics were co-transfected with pcDNA3.1-FGF7, the pro-EMT effects induced by FGF7 overexpression were significantly attenuated, epithelial markers were partially restored, and mesenchymal markers were suppressed, demonstrating a mutual antagonistic relationship between miR-16-5p and FGF7 in regulating podocyte EMT(Figs. 6D,I). Finally, to determine whether the suppressive effect of SQYSF on FGF7 is mediated through miR-16-5p, we performed a loss-of-function rescue experiment. MPC5 cells were pretreated with a miR-16-5p inhibitor or negative control (inhibitor NC) prior to administration of 4% SQYSF under high glucose conditions. SQYSF alone significantly downregulated FGF7 protein expression compared to the high glucose group, consistent with its protective role. However, this inhibitory effect was markedly reversed upon knockdown of miR-16-5p; FGF7 levels remained elevated despite SQYSF treatment(Figs. 6J,K). Collectively, our data elucidate a protective mechanism whereby SQYSF upregulates miR-16-5p, which in turn directly represses FGF7, thereby mitigating high glucose–induced podocyte EMT. [insert Fig. 6.] 4 Discussion DKD is a severe chronic complication of diabetes and represents a leading cause of chronic kidney disease (CKD). The pathogenesis of DKD is marked by a substantial increase in ECM accumulation, which leads to thickening of the glomerular and tubular basement membranes[ 32 ]. Furthermore, the EMT has been identified as playing a major role in the advancement of basement membrane thickening in DKD[ 33 ]. Currently, there are no effective therapeutic agents to combat this condition, and therapeutic options to halt this progression are limited[ 34 ]. Therefore, exploring novel therapeutic targets and intervention strategies is of great importance. In this context, our study elucidates a previously unrecognized mechanism by which the traditional Chinese medicine formula SQYSF exerts its renoprotective effects. We demonstrate that SQYSF attenuates high glucose-induced podocyte EMT primarily by upregulating miR-16-5p, which directly targets and suppresses FGF7, thereby identifying the miR-16-5p/FGF7 axis as a critical pathway mediating the therapeutic action of SQYSF. The most significant finding of this research is the identification and functional validation of the miR-16-5p/FGF7 axis. We first established that SQYSF-containing serum dose-dependently reversed high glucose-induced podocyte EMT, with concurrent restoration of mitochondrial ultrastructure, highlighting its cytoprotective potential. Subsequent investigations revealed that SQYSF significantly upregulated the expression of miR-16-5p, which was suppressed under high glucose conditions. Functional rescue experiments using a miR-16-5p inhibitor confirmed that the anti-EMT effect of SQYSF is largely dependent on its ability to elevate miR-16-5p levels, positioning miR-16-5p as a functional effector rather than merely a biomarker. Among the numerous miRNAs involved in the development and progression of DKD, miR-16-5p has attracted significant attention due to its well-documented renal protective functions as confirmed by multiple studies. miRNAs serve as central regulatory factors within gene expression networks. They modulate gene expression at the post-transcriptional level by binding to the 3' untranslated region (3'UTR) of target mRNAs, thereby influencing cellular biological behaviors[ 35 ]. A key innovation of this work lies in the discovery of FGF7 as a direct and functionally relevant target of miR-16-5p in podocytes under diabetic conditions. FGF7 is a functionally distinct member of the FGF family. Its classical biological characteristic is its specific secretion by mesenchymal cells and selective binding to the FGFR1b and FGFR2b receptor isoforms on the surface of epithelial cells, thereby mediating unidirectional “mesenchymal-to-epithelial” cellular communication[ 36 ]. This property endows FGF7 with a pivotal role in embryonic development, particularly in orchestrating the morphogenesis of organs such as the kidneys and liver[ 37 ]. During renal development, FGF7 modulates ureteric bud signaling to support the survival and aggregation of renal progenitor cells, thereby facilitating nephron formation[ 38 ]. Current research has largely concentrated on its roles in cancer drug resistance, cell proliferation, glucose metabolism, and inflammatory responses[ 39 , 40 ]. While the role of FGF7 in developmental biology is well-established, and its potential value as a biomarker or therapeutic target for kidney regeneration has been suggested[ 41 ], its function in the pathogenesis of DKD remains largely unexplored, and the specific role of FGF7 in the pathological progression of podocyte EMT remains an uncharted area. To address this knowledge gap, our study, for the first time, identifies FGF7 as a critical research target in DKD-associated podocyte EMT. We aim to systematically investigate its potential role and underlying molecular mechanisms in high glucose-induced podocyte EMT. Our findings not only reveal the potential of FGF7 as a novel target in DKD, but also extend the paradigm of miRNA-mediated post-transcriptional regulation to the mechanistic study of traditional Chinese medicine formulations. Furthermore, integrated transcriptomic sequencing and bioinformatic analysis corroborate the pivotal position of the miR-16-5p/FGF7 axis within the regulatory network of DKD. Through an integrative approach combining transcriptomic analysis and bioinformatics prediction, we identified FGF7 as a potential target of miR-16-5p. This interaction was conclusively verified by dual-luciferase reporter assay, confirming specific binding of miR-16-5p to the 3'-UTR of FGF7 mRNA. The functional significance of this mechanism was cemented by a series of rescue experiments. Collectively, these findings delineate an axis whereby SQYSF inhibits EMT mechanistically through the upregulation of miR-16-5p and the subsequent downregulation of its target, FGF7. The proposed mechanism is schematically illustrated in Fig. 7, depicting the pathway from SQYSF intervention to the inhibition of podocyte EMT via the miR-16-5p/FGF7 axis. We acknowledge several limitations of the present study. Firstly, the findings are primarily derived from in vitro experiments using MPC5 podocyte cells. Although the use of drug-containing serum enhances translational relevance by reflecting processed herbal components, future validation in in vivo diabetic animal models is essential. Secondly, while we have established the centrality of the miR-16-5p/FGF7 axis, traditional Chinese medicine formulas like SQYSF are characterized by multi-component, multi-target, and multi-pathway actions. It is plausible that SQYSF exerts its overall protective effect through additional parallel or synergistic mechanisms independent of this mechanism, as suggested by the partial restoration of epithelial phenotype even upon miR-16-5p inhibition. This study elucidates the cellular mechanism of the holistic formula. The next step is to bridge the chemical and biological findings by identifying which of the LC-MS/MS-characterized constituents are primarily responsible for modulating the miR-16-5p/FGF7 axis. Several limitations of this work should be acknowledged. First, although the use of SQYSF-containing serum in cultured podocytes partially recapitulates the metabolic fate of the herbal formula in vivo, the present findings are primarily derived from in vitro experiments. Whether SQYSF exerts renoprotective effects through the miR-16-5p/FGF7 axis in vivo requires further validation in diabetic animal models such as db/db mice or STZ-induced diabetic rats. Second, the bulk RNA-seq data were generated from renal cortical tissues of db/db mice, which comprise heterogeneous cell populations including glomerular and tubular compartments. While the functional role of FGF7 was specifically validated in podocytes, its cell-type-specific expression pattern and regulation by SQYSF in the diabetic kidney remain to be clarified using approaches such as immunofluorescence co-localization or single-cell sequencing. Third, although our rescue experiments demonstrate that upregulation of miR-16-5p is a critical mediator of SQYSF action, the partial restoration of epithelial markers upon miR-16-5p inhibition suggests the existence of additional, miR-16-5p-independent mechanisms. As a multi-herb formulation, SQYSF is inherently capable of modulating multiple signaling pathways, and these parallel mechanisms warrant further investigation. Fourth, an inverted U-shaped dose–response was observed, with 4% SQYSF-containing serum exhibiting greater efficacy than 8%. Whether this phenomenon reflects receptor saturation, feedback inhibition, or mild cytotoxicity remains unresolved and merits further exploration. Fifth, although FGF7 is conventionally regarded as an epithelial-protective growth factor, our study reveals a pro-EMT role under diabetic conditions. The context-dependent functional switch of FGF7 and its upstream regulatory networks in DKD pathogenesis remain poorly understood and require systematic investigation. Despite these limitations, this study provides the first evidence linking the miR-16-5p/FGF7 axis to podocyte EMT and identifies this axis as a novel mechanistic target of SQYSF, thereby laying a foundation for subsequent in vivo validation and phytochemical interrogation. 5 Conclusion This study provides evidence that SQYSF mitigates high glucose-induced podocyte EMT by activating the miR-16-5p/FGF7 axis. These findings not only decipher a key molecular mechanism underlying the renal protective effect of SQYSF but also contribute to the broader field by identifying a novel miRNA-target pair involved in DKD pathogenesis. Future studies focusing on the active compounds of SQYSF and their in vivo validation will be crucial for further development and clinical translation. [insert Fig. 7] Declarations CRediT authorship contribution statement Shi-qi Chen : Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing–original draft, Writing–review and editing. Zhu Wu : Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing–original draft, Writing–review and editing. Jing Zhang : Data curation, Supervision, Writing–review and editing. Yu-Xin Lin : Software, Supervision, Writing–review and editing. Jia-Qi Xie : Investigation, Methodology, Writing–review and editing. Xiao-yu Peng : Investigation, Writing–review and editing. De-hui Yin : Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Writing–review and editing. Ye Zhu : Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing–review and editing. Funding Financial support for this this article was provided by National Natural Science Foundation of China (82274464, 82360917);Natural Science Foundation of Hainan Province (823MS147). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments Everyone who contributed significantly to this study has been listed. Data availability Data will be made available on request References Feng, X. S. et al. CKD Prevalence Among Patients With and Without Type 2 Diabetes: Regional Differences in the United States. Kidney Med. 4 (1), 100385 (2022). Zhang, L. et al. Trends in Chronic Kidney Disease in China. N. Engl. 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M., Huang, C. & Pollock, C. A. MicroRNA as novel biomarkers and therapeutic targets in diabetic kidney disease: An update. FASEB bioAdvances . 1 (6), 375–388 (2019). Damase, T. R. et al. The Limitless Future of RNA Therapeutics. Front. Bioeng. Biotechnol. 9 , 628137 (2021). Duan, Y. R. et al. Exosomal microRNA-16-5p from human urine-derived stem cells ameliorates diabetic nephropathy through protection of podocyte. J. Cell. Mol. Med. 25 (23), 10798–10813 (2021). Cai, X., Hagedorn, C. H. & Cullen, B. R. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs, RNA (2004). (New York, N.Y.) 10 (12) 1957–1966 . Shen, S. et al. Advances in Traditional Chinese Medicine research in diabetic kidney disease treatment. Pharm. Biol. 62 (1), 222–232 (2024). Xie, J. et al. Jia Wei Qingxin Lotus Seed Drink ameliorates epithelial mesenchymal transition injury in diabetic kidney disease via inhibition of JMJD1C/SP1/ZEB1 signaling pathway. Phytomedicine: Int. J. phytotherapy phytopharmacology . 135 , 156142 (2024). Chen, D. Q. et al. Tangshen Formula alleviates inflammatory injury against aged diabetic kidney disease through modulating gut microbiota composition and related amino acid metabolism. Exp. Gerontol. 188 , 112393 (2024). Cai, P. et al. Suoquan pill for the treatment of diabetic nephropathy: A protocol for systematic review and meta-analysis. Medicine 100 (17), e25613 (2021). Yan, Z. et al. SuoquanYishen formula improves renal cellular senescence by inhibiting YTHDF1-Rubicon axis to promote autophagy in diabetic kidney disease. Front. Pharmacol. 16 , 1543277 (2025). Zha, D. Q., Gao, P. & Wu, X. Y. Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes. World J. diabetes . 16 (10), 109782 (2025). Assmann, T. S. et al. Circulating miRNAs in diabetic kidney disease: case-control study and in silico analyses. Acta Diabetol. 56 (1), 55–65 (2019). Rai, B., Srivastava, J. & Saxena, P. The Functional Role of microRNAs and mRNAs in Diabetic Kidney Disease: A Review. Curr. Diabetes. Rev. 20 (6), e201023222412 (2024). Adeva-Andany, M. M. & Carneiro-Freire, N. Biochemical composition of the glomerular extracellular matrix in patients with diabetic kidney disease. World J. diabetes . 13 (7), 498–520 (2022). Li, Y. et al. PTEN-induced partial epithelial-mesenchymal transition drives diabetic kidney disease. J. Clin. Investig. 129 (3), 1129–1151 (2019). Tang, G. et al. Clinical efficacies, underlying mechanisms and molecular targets of Chinese medicines for diabetic nephropathy treatment and management. Acta Pharm. Sinica B . 11 (9), 2749–2767 (2021). Kim, H., Lee, Y. Y. & Kim, V. N. The biogenesis and regulation of animal microRNAs, Nature reviews. Mol. cell. biology . 26 (4), 276–296 (2025). Del Corral, R. D. & Morales, A. V. The Multiple Roles of FGF Signaling in the Developing Spinal Cord. Front. cell. Dev. biology . 5 , 58 (2017). Zinkle, A. & Mohammadi, M. Structural Biology of the FGF7 Subfamily. Front. Genet. 10 , 102 (2019). Maddaluno, L., Urwyler, C. & Werner, S. Fibroblast growth factors: key players in regeneration and tissue repair. Dev. (Cambridge England) . 144 (22), 4047–4060 (2017). Meng, H. et al. FGF7 enhances the expression of ACE2 in human islet organoids aggravating SARS-CoV-2 infection. Signal. Transduct. Target. therapy . 9 (1), 104 (2024). Feng, S. et al. Cancer-associated fibroblast-secreted FGF7 as an ovarian cancer progression promoter. J. translational Med. 22 (1), 280 (2024). Trivedi, N. & Kumar, D. Fibroblast growth factor and kidney disease: Updates for emerging novel therapeutics. J. Cell. Physiol. 236 (12), 7909–7925 (2021). Additional Declarations No competing interests reported. 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miR-16-5p/FGF7 axis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eDiabetic kidney disease (DKD) is a chronic kidney condition caused by diabetes and represents one of the major microvascular complications of diabetes[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. DKD is among the most common chronic complications of diabetes worldwide, including in China[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The prevalence of DKD in patients with type 2 diabetes in China is 21.8%[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It is the leading cause of end-stage renal disease (ESRD), which requires dialysis or kidney transplantation, and also a significant contributor to mortality among diabetic patients[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. According to data from the International Diabetes Federation (IDF), the number of people with diabetes worldwide exceeded 537\u0026nbsp;million in 2021, and the prevalence of DKD has shown a gradual increasing trend. This has become a growing public health challenge [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Current pharmacological treatments for DKD, such as RAAS blockers and SGLT2 inhibitors, can slow the decline of kidney function [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, their efficacy is limited by individual variability and adverse effects, and they cannot halt disease progression, falling far short of meeting clinical needs[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the onset and progression of DKD, the progressive decline in renal function is closely associated with renal fibrosis, characterized by the abnormal accumulation of extracellular matrix (ECM) proteins in the glomerular basement membrane and tubulointerstitium, ultimately leading to irreversible loss of kidney function[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Epithelial-mesenchymal transition (EMT) is one of the central mechanisms driving renal fibrosis[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Sustained hyperglycemia can alter the phenotype of normal renal cells, inducing a transition toward a pro-fibrotic mesenchymal phenotype through the EMT process [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Podocytes, as essential components of the glomerular filtration barrier, play a pivotal role in the pathogenesis of DKD [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A high-glucose environment can induce EMT in podocytes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], causing them to lose epithelial characteristics, such as decreased expression of markers including E-cadherin and ZO-1, and acquire a mesenchymal phenotype, characterized by increased expression of markers such as N-cadherin, fibronectin, and α-SMA [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Pathologically, this manifests as alterations in podocyte morphology, loss of polarity, disruption of intercellular junctions, and cytoskeletal rearrangement, which consequently increase their susceptibility to detachment from the glomerular basement membrane [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Such structural and functional impairment ultimately promotes basement membrane thickening and ECM accumulation, driving glomerulosclerosis and proteinuria development [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Studies have shown that podocyte phenotypic transition participates in the early stage of podocyte loss and correlates significantly with the degree of glomerulosclerosis, proteinuria, and reduction in glomerular filtration rate [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, inhibiting podocyte EMT is considered a promising potential strategy for protecting podocyte integrity and delaying the fibrotic progression of DKD.\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) are a class of non-coding RNA molecules that play a key regulatory role at the post-transcriptional level. Recent research has increasingly focused on miRNAs, recognizing them as central to deciphering the complex pathological networks in kidney diseases. They are widely involved in the pathophysiology of various renal conditions, including acute kidney injury, chronic kidney disease, DKD, and transplant kidney injury. Differential miRNA expression profiles may reflect the underlying mechanistic features of distinct diseases[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Their clinical translational potential is significant. The stability and sensitivity of miRNAs in body fluids make them superior non-invasive biomarkers compared to traditional markers like urinary albumin, demonstrating considerable diagnostic value (Wang et al., 2024). This potential has begun to be realized in clinical practice [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. At the mechanistic level, miRNAs act as hubs within signaling pathways, providing critical entry points for elucidating the molecular mechanisms of diseases such as DKD [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, their high targetability establishes a theoretical foundation for developing precise nucleic acid-based therapies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For instance, exosomes derived from human urine stem cells (hUSCs) can deliver miR-16-5p, alleviating podocyte injury under high-glucose conditions and thereby halting the progression of DKD [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These findings collectively establish miR-16-5p as a crucial regulatory factor and a potential therapeutic target in DKD. Mature miRNAs, processed sequentially by the Drosha and Dicer complexes, are incorporated into the RNA-induced silencing complex (RISC). By guiding RISC to complementary target mRNAs, they post-transcriptionally repress gene expression primarily through mRNA degradation or translational inhibition[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTraditional Chinese medicine (TCM) formulations have gained attention for their multi-targeted approach in managing complex diseases like DKD[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For instance, Jia Wei Qingxin Lotus Seed Drink was shown to attenuate EMT in DKD by suppressing the JMJD1C/SP1/ZEB1 signaling pathway[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Other studies have confirmed the role of TCM compounds in inhibiting tubular ferroptosis and reducing inflammatory injury[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These findings highlight the diverse mechanisms through which TCM can target DKD pathology. Among these interventions, Suoquan Yishen Formula (SQYSF), a patented traditional Chinese medicinal compound authorized for the treatment of DKD (Patent No: ZL201610423677.5), has been used in clinical practice for over a decade. SQYSF comprises seven medicinal herbs, namely Alpinia oxyphylla Miq.(Yizhiren in Chinese), Lindera aggregata(Wuyao), Poria cocos (Fuling), Atractylodes lancea (Cangzhu), Salviae Miltiorrhizae(Danshen), Paeonia lactiflora(Chishao), and Trichosanthes kirilowii(Tianhuafen). Preliminary clinical studies have confirmed that SQYSF can effectively reduce proteinuria and improve renal function in patients with DKD, demonstrating clear clinical application value[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. SQYSF has demonstrated efficacy in reducing proteinuria and ameliorating renal function in DKD models, potentially through inhibiting renal senescence and enhancing autophagy[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Despite these promising findings, the precise molecular targets and pathways through which it attenuates podocyte EMT are poorly defined.\u003c/p\u003e \u003cp\u003eThis study aims to systematically identify key downstream target genes of miR-16-5p through combined miRNA‑mRNA analysis, and to explore whether the Chinese herbal compound SQYSF inhibits high glucose‑induced EMT in podocytes by regulating the miR‑16‑5p/FGF7, thereby delaying the progression of DKD.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGraphical Abstract\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2 Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals and reagents\u003c/h2\u003e \u003cp\u003eThe mouse glomerular podocyte cell line MPC5 (CL0098, Fenghui Biotechnology, China) was used in this study. Key reagents and kits included fetal bovine serum (Gibco, 10099141C), penicillin/streptomycin (Yuanpei Biotech, S110JV), a CCK-8 assay kit (Beyotime Biotechnology, C0038), a Dual-Luciferase Reporter Assay Kit (GenePharma, G06011), GP-transfect-Mate transfection reagent (GenePharma, G04009), the iScript\u0026trade; cDNA Synthesis Kit (BIO-RAD, 1708891), the Hairpin-it\u0026trade; microRNA and U6 snRNA Normalization RT-PCR Quantitation Kit (GenePharma, E22001), an RNA FISH Kit (Cy3) (GenePharma, F40211), the HyperFluor 488 TSA Fluorescence System Kit (APExBIO, K4301), and Fluoroshield Mounting Medium with DAPI (Abcam, ab104139). Antibodies against N-cadherin (ab18203), E-cadherin (ab231303), fibronectin (ab268020), ZO-1 (ab276131), α-SMA (ab124964), KGF/FGF7 (ab131162), β-tubulin (ab21058), and β-actin (ab49900) were all obtained from Abcam.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of SQSYF\u003c/h2\u003e \u003cp\u003eAll components of the SQYSF were sourced from Beijing Bencao Fangyuan Pharmaceutical Technology Co., Ltd., and authenticated per the Chinese Pharmacopoeia (2020 edition). To prepare the extract, 6000 g of herbs underwent water extraction and alcohol precipitation. The concentrate was then lyophilized, yielding 1500 g of powder. For in vitro studies, 40 g of this powder was dissolved in 60 mL of sterile saline to make a 0.4 g/mL stock solution, which was filter-sterilized (0.22 \u0026micro;m) and stored at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 LC-MS/MS analysis\u003c/h2\u003e \u003cp\u003eLC-MS/MS analysis was conducted to profile chemical composition of SQYSF. The lyophilized extract (0.5 g) was reconstituted in 10 mL methanol-water (60:40, v/v), centrifuged, and the supernatant was analyzed. Separation was performed on a Waters ACQUITY UPLC I-Class Plus system with a HSS T3 column (100 mm \u0026times; 2.1 mm, 1.8 \u0026micro;m) at 45\u0026deg;C, using a gradient of 0.1% formic acid in water (A) and acetonitrile (B) at 0.35 mL/min: 0\u0026ndash;2 min (5% B), 2\u0026ndash;4 min (5\u0026ndash;30% B), 4\u0026ndash;8 min (30\u0026ndash;50% B), 8\u0026ndash;10 min (50\u0026ndash;80% B), 10\u0026ndash;14 min (80\u0026ndash;100% B), 14\u0026ndash;15 min (100% B), 15.1\u0026ndash;16 min (5% B). Mass detection used a Q-Exactive Orbitrap spectrometer (Thermo Scientific) with H-ESI source in positive/negative modes. Key settings: spray voltage 3.8 kV(+)/3.2 kV(-); sheath gas 35 arb; auxiliary gas 8 arb; capillary temp 320\u0026deg;C; full MS resolution 60,000 (m/z 100\u0026ndash;1500); dd-MS2 resolution 15,000 with stepped NCE (10, 20, 40 eV).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Animals\u003c/h2\u003e \u003cp\u003eMale Sprague-Dawley rats (220\u0026thinsp;\u0026plusmn;\u0026thinsp;20g), supplied by Chengdu Dashuo Experimental Animal Co., Ltd. (License Nos. SCXK 2020-0030, SYXK 2022\u0026thinsp;\u0026minus;\u0026thinsp;225), were acclimatized for one week under SPF conditions before drug-containing serum preparation. All animal experiments were approved by the Ethics Committee of Hainan Medical University (Approval No. HYLL-2022-110) and were conducted in accordance with relevant ethical guidelines, which included provisions for post-procedural care and humane euthanasia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Preparation of SQYSF-containing serum\u003c/h2\u003e \u003cp\u003eRats were stratified by body weight and randomly assigned to two groups (n=10per group). Group A (control) received 0.9% NaCI via oral gavage, while Group Bwas administered the herbal formula SQYSF at a dose of 8 g crude drug/kg/day(equivalent to 4 times the clinical dose). Both groups were dosed twice daily (08:00and 14:00) for three consecutive days, with the gavage volume adjusted to 10 mL/kgbased on real-time body weight. Two hours after the final administration, rats wereanesthetized with an intraperitoneal injection of sodium pentobarbital (0.3%, 15mL/kg). Whole blood was then collected from the abdominal aorta using vacuumtubes. Serum was separated and stored at -80' C as drug-containing(Group B) orblank control (Group A) serum for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.6 Bulk RNA-seq\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from renal cortical tissues of [db/m, db/db, and SQYSF-treated db/db mice (n\u0026thinsp;=\u0026thinsp;3). The resulting RNA libraries were purified and sequenced on a HiSeq3000 sequencer (Illumina, San Diego, CA, USA). Cell samples were sent to Genechem Co., Ltd. (Shanghai, China) for RNA-Seq analysis. DESeq2 software (1.20.0) was used to analyze DEGs, and the P value was corrected using the Benjamini\u0026amp;Hochberg method. The differentially expressed RNAs between groups were identified with |log2 (fold change) | \u0026gt;1 and P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell Culture and transfection\u003c/h2\u003e \u003cp\u003eMPC5 cells were maintained in DMEM with 10% FBS and 1% penicillin-streptomycin at 37\u0026deg;C under 5% CO₂. For experiments, cells were divided into five groups: normal control, high glucose model, and HG groups co-treated with 2%, 4%, or 8% SQYSF-containing serum. After 24 h, cells were harvested for analysis. For transfection, cells at 50\u0026ndash;70% confluence were transfected using GP-transfect-Mate. Plasmid DNA (500 ng/\u0026micro;L) or RNA oligonucleotides (miR-16-5p mimic/inhibitor or corresponding controls at 10 pmol/\u0026micro;L) were mixed with the transfection reagent in serum-free medium, incubated for 20 min at room temperature, and then applied to the cells. Culture medium was refreshed 24 h post-transfection, and cells were collected at indicated time points for subsequent assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 CCK-8 (Cell Counting Kit-8) Assay\u003c/h2\u003e \u003cp\u003eCell viability was measured by CCK-8 assay (Beyotime). MPC5 cells were seeded at 4\u0026times;10⁴ cells/mL (200 \u0026micro;L/well) for 48 h, treated with 1\u0026ndash;10% SQYSF-containing serum for 22 h, and then incubated with CCK-8 for 2 h. Absorbance (450 nm/650 nm reference) was read on a microplate reader to calculate viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Transmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eTEM samples were prepared by standard chemical fixation (2.5% glutaraldehyde and 1% OsO₄), dehydrated through a graded acetone series, and embedded in Epon-812 resin. Ultrathin sections (60\u0026ndash;90 nm) were stained with uranyl acetate and lead citrate. Ultrastructural analysis was performed using a transmission electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 RT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated using the Animal Total RNA Isolation Kit according to the manufacturer\u0026rsquo;s instructions and eluted in 150 \u0026micro;L RNase-free water. cDNA was synthesized by reverse transcription in a 20 \u0026micro;L reaction system. Gene-specific primers were designed based on the PubMed database and synthesized by Sangon Biotech (Shanghai). Quantitative PCR was performed under the following conditions: 95\u0026deg;C for 2 min; 40 cycles of 95\u0026deg;C for 6 s and 60\u0026deg;C for 18 s; followed by a melting curve analysis from 65\u0026deg;C to 95\u0026deg;C (increment of 0.5\u0026deg;C every 5 s). All reactions were run in triplicate. Relative gene expression was calculated using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method with GAPDH and U6 as internal controls. Primer sequences are listed in Supplement Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Western Blot Analysis\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from MPC5 cells, and its concentration was determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking, membranes were incubated overnight at 4\u0026deg;C with primary antibodies against α-SMA, Fibronectin, N-cadherin, E-cadherin, ZO-1, FGF7, β-Actin, and β-Tubulin (all at 1:1000 dilution). Following washes, membranes were incubated with an HRP-conjugated secondary antibody (1:10000 in 5% BSA) for 1 h at room temperature. Protein bands were visualized using Clarity\u0026trade; Western ECL Substrate, imaged with an automated chemiluminescence system, and quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Immunofluorescence (IF) Assay\u003c/h2\u003e \u003cp\u003eCells on coverslips were processed for immunofluorescence. Following fixation, permeabilization (Triton X-100), and blocking (5% BSA), samples were incubated overnight with primary antibody, followed by secondary antibody and TSA\u0026trade; amplification. Nuclei were stained with DAPI. Images were captured using a Zeiss Axio Imager Z2 microscope (200\u0026times;; FITC/DAPI channels) and analyzed with Zen Blue 3.3 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Fluorescence In Situ Hybridization (FISH)\u003c/h2\u003e \u003cp\u003eFor FISH detection of miR-16-5p, cells on coverslips were fixed (4% PFA, 15 min), permeabilized, and hybridized overnight at 37\u0026deg;C with a denatured fluorescent probe (5 \u0026micro;M). Post-hybridization washes were performed with pre-heated buffers. Nuclei were stained with DAPI, and images were acquired at 600\u0026times;magnification using a fluorescence imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Dual-Luciferase Reporter Assay\u003c/h2\u003e \u003cp\u003eTo validate whether miR-16-5p directly targets the 3\u0026rsquo;-UTR of FGF7, wild-type (WT) and mutant (MUT) FGF7 3\u0026rsquo;-UTR reporter plasmids were constructed. MPC5 cells were seeded in 6-well plates and co-transfected with miR-16-5p mimic or negative control (NC) and the corresponding reporter plasmid using transfection reagent. After 24 h, cells were lysed and luciferase activities were measured using a dual-luciferase reporter assay system. Firefly luciferase activity was normalized to Renilla luciferase activity. Each experiment was performed in triplicate and repeated independently three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll data analyses were performed using GraphPad Prism software (version 10.0.0). Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA), and comparisons between two groups were performed using the Student's t-test. A P-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 LC-MS/MS analysis of SQYSF\u003c/h2\u003e \u003cp\u003eLC-MS/MS analysis characterized the chemical composition of SQYSF (Fig.\u0026nbsp;1A, B). A total of 21 compounds absorbed into the bloodstream were detected, and their herbal origins were identified in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e[insert Fig.\u0026nbsp;1.]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 SQYSF-containing serum attenuates high glucose-induced podocyte EMT\u003c/h2\u003e \u003cp\u003eUnder high glucose conditions, the EMT of podocytes is a key pathological alteration leading to their functional impairment and loss[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To further investigate the mechanism of SQYSF in DKD, this study focuses on its intervention effect on high glucose-induced podocyte EMT. To evaluate the cytotoxicity of SQYSF-containing serum, we performed CCK-8 assays to assess podocyte viability after treatment with varying concentrations (1%-10%) for 24 hours. No significant difference in cell viability was observed across all tested concentrations compared to the control group, indicating that SQYSF-containing serum is non-toxic within this range(Fig.\u0026nbsp;2A). Based on these findings, we selected 2%, 4%, and 8% SQYSF as low, medium, and high doses for subsequent experiments to investigate its pharmacological effects and potential dose-response relationship. To establish a reliable model of high glucose-induced podocyte injury, we first determined the optimal glucose concentration for inducing EMT. Western blot analysis revealed that exposure to 35 mM glucose significantly upregulated mesenchymal markers (fibronectin, N-cadherin, α-SMA) and downregulated epithelial markers (E-cadherin, ZO-1) at protein levels (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-F), confirming successful induction of EMT. Therefore, 35 mM glucose was adopted as the standard condition for constructing the diabetic nephropathy-like cellular model. TEM was employed to examine ultrastructural changes in podocytes under different treatments. In the high glucose group, podocytes exhibited marked mitochondrial damage, including swelling, cristae disruption, and vacuolization (Fig.\u0026nbsp;2B). In contrast, mitochondria in the control group maintained normal morphology. Notably, treatment with SQYSF-containing serum significantly ameliorated these ultrastructural abnormalities, restoring mitochondrial integrity and architecture, suggesting a protective effect against high glucose-induced organelle dysfunction. To further characterize the anti-EMT effects of SQYSF, we analyzed the expression of key EMT-related proteins and mRNAs. Western blotting (Fig.\u0026nbsp;2C,D) and RT-qPCR (Fig.\u0026nbsp;2E) analyses demonstrated that 35 mM glucose dramatically increased fibronectin, N-cadherin, and α-SMA expression while decreasing E-cadherin and ZO-1 levels. Treatment with 2%, 4%, or 8% SQYSF reversed these alterations in a dose-dependent manner, epithelial markers were restored, and mesenchymal markers were suppressed. Among the three doses, 4% SQYSF exhibited the most potent inhibitory effect on EMT, with maximal reversal of marker expression. Interestingly, the protective efficacy followed an inverted U-shaped dose-response curve, where higher concentrations (8%) showed reduced effectiveness compared to 4%. This suggests that 4% SQYSF represents the optimal concentration for exerting maximal anti-EMT activity. Collectively, these results demonstrate that SQYSF-containing serum effectively protects podocytes from high glucose-induced EMT, preserves mitochondrial ultrastructure, and exerts its beneficial effects in a dose-dependent manner, with 4% being the most effective concentration for downstream mechanistic investigations.\u003c/p\u003e \u003cp\u003e[insert Fig.\u0026nbsp;2.]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 SQYSF attenuates podocyte EMT by upregulating miR-16-5p\u003c/h2\u003e \u003cp\u003eBuilding upon the confirmed protective effect of SQYSF against podocyte EMT, this study aims to elucidate its underlying molecular mechanisms. Given the critical role of miR-16-5p in diabetic kidney disease (DKD)[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], we specifically focused on investigating the regulation of this miR-16-5p by SQYSF. RT-qPCR analysis revealed that HG (35 mM) significantly suppressed miR-16-5p levels compared to normal glucose (NG) control. Notably, SQYSF-containing serum reversed this downregulation in a dose-dependent manner, with 4% concentration showing the most potent effect (Fig.\u0026nbsp;3A). To determine whether miR-16-5p upregulation is functionally required for SQYSF-mediated protection, rescue experiments were performed using a miR-16-5p inhibitor. Efficient knockdown of miR-16-5p was confirmed by (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG), and Western blot analysis showed that miR-16-5p inhibition markedly attenuated the ability of SQYSF to restore epithelial markers (E-cadherin and ZO-1) and suppress mesenchymal markers (Fibronectin, N-cadherin, and α-SMA) (Figs.\u0026nbsp;3B\u0026ndash;G). These findings demonstrate that the anti-EMT effect of SQYSF in podocytes is, at least in part, dependent on its upregulation of miR-16-5p.\u003c/p\u003e \u003cp\u003e[insert Fig.\u0026nbsp;3.]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 SQYSF attenuates podocyte EMT by downregulating FGF7\u003c/h2\u003e \u003cp\u003eTo systematically investigate the molecular mechanisms underlying the therapeutic effects of SQYSF through miR-16-5p-mediated regulation, we first focused on identifying downstream target genes whose expression decreases following treatment, consistent with the upregulation of miR-16-5p. Based on this miRNA-mRNA regulatory principle[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], we performed transcriptome sequencing followed by integrative bioinformatic and experimental analyses. A hierarchical clustering heatmap of the top 20 most significantly differentially expressed genes revealed distinct global expression patterns across experimental groups (Fig.\u0026nbsp;4A). Genes differentially expressed among the control, model, and SQYSF-treated groups were clustered based on their expression profiles. Notably, FGF7 exhibited a marked upregulation in the model group compared to controls, which was progressively reversed by SQYSF intervention, a pattern consistent with its potential regulation by the upregulated miR-16-5p and supporting its selection as a candidate downstream target gene implicated in disease progression and drug response. To characterize the transcriptional changes during disease development, two volcano plot were generated (Figs.\u0026nbsp;4B,C). This analysis revealed a set of significantly upregulated genes, including FGF7, suggesting its activation under pathological conditions. Further evaluation of the regulatory effect of SQYSF was performed by comparing the treated group with the model group. The resulting volcano plot showed that multiple disease-associated genes were downregulated after treatment, with FGF7 being notably suppressed, supporting its responsiveness to pharmacological intervention. To validate these transcriptomic findings, we examined FGF7 expression at both protein and mRNA levels using Western blotting and qRT-PCR. Consistent with sequencing data, high glucose stimulation significantly induced FGF7 expression, whereas SQYSF treatment dose-dependently attenuated both FGF7 protein and mRNA levels(Figs.\u0026nbsp;4D,F). Collectively, these results demonstrate that FGF7, predicted as a potential target of miR-16-5p, is activated in the diabetic nephropathy model and effectively suppressed by SQYSF, and experimental validation supports a critical role for FGF7 in DKD.\u003c/p\u003e \u003cp\u003eTo further confirm the functional role of FGF7 in mediating the effects of SQYSF, we performed a rescue experiment by overexpressing FGF7 in high glucose-treated cells. Western blot analysis (Figs.\u0026nbsp;5A,B) revealed that 35 mM glucose significantly upregulated fibronectin, N-cadherin, and α-SMA expression, while decreasing E-cadherin and ZO-1 levels. Treatment with 4% SQYSF effectively reversed these changes, restoring epithelial markers and suppressing mesenchymal markers. However, when FGF7 was overexpressed via pcDNA3.1-FGF7 transfection(Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eH-J), the protective effects of SQYSF were significantly attenuated. Specifically, fibronectin, N-cadherin, and α-SMA remained elevated, whereas E-cadherin and ZO-1 expression failed to recover, indicating that FGF7 overexpression counteracts anti-EMT actions of SQYSF. Immunofluorescence staining also corroborated these findings(Figs.\u0026nbsp;5C,D). In control cells, E-cadherin was localized at cell membranes, forming intact junctions. Under high glucose conditions, E-cadherin expression decreased, and membrane localization was disrupted. SQYSF treatment restored E-cadherin membrane distribution, but this effect was abolished upon FGF7 overexpression. Similarly, N-cadherin, which is typically associated with mesenchymal phenotype, was strongly induced by high glucose and reduced by SQYSF; however, its expression was maintained at high levels in the SQYSF\u0026thinsp;+\u0026thinsp;OE-FGF7 group. Collectively, these results demonstrate that FGF7 plays a critical role in the pathogenesis of high glucose-induced EMT and that its overexpression can rescue the beneficial effects of SQYSF, supporting the hypothesis that FGF7 is a key downstream target through which SQYSF exerts its protective functions.\u003c/p\u003e \u003cp\u003e[insert Fig.\u0026nbsp;4.]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e[insert Fig.\u0026nbsp;5.]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 miR-16-5p inhibits podocyte EMT by directly targeting FGF7\u003c/h2\u003e \u003cp\u003eTo investigate the subcellular localization and potential functional interaction between miR-16-5p and its predicted target FGF7, we first performed fluorescence in situ hybridization (FISH) using a specific probe for mouse miR-16-5p(Fig.\u0026nbsp;6A). miR-16-5p signals were predominantly localized in the cytoplasm, suggesting that its regulatory activity likely occurs within this compartment. This cytoplasmic enrichment supports the possibility of direct post-transcriptional regulation of FGF7 via miRNA-mediated mechanisms. Based on bioinformatic prediction from TargetScan, a putative binding site for miR-16-5p was identified in the 3\u0026rsquo;-UTR of FGF7 mRNA (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eK). To validate this interaction, we constructed luciferase reporter vectors containing either the wild-type (WT) or mutant (MUT) 3\u0026rsquo;-UTR of FGF7 and co-transfected them with miR-16-5p mimics or negative control (mimics NC) into podocytes(Fig.\u0026nbsp;6B). Firefly luciferase activity was normalized to Renilla luciferase (internal control), and values were further normalized to the mimics NC group. The results (Fig.\u0026nbsp;6C) showed that miR-16-5p mimics significantly reduced luciferase activity in the WT-FGF7 group, whereas no significant suppression was observed in the MUT-FGF7 group, indicating that the inhibitory effect is dependent on the specific seed sequence match. These findings confirm the direct and specific targeting of FGF7 by miR-16-5p. To confirm the functional relationship between miR-16-5p and FGF7, we modulated their expression and assessed EMT marker changes. Overexpression of FGF7 (via pcDNA3.1-FGF7) led to a significant increase in mesenchymal markers (fibronectin, N-cadherin, α-SMA) and a concomitant decrease in epithelial markers (E-cadherin, ZO-1) compared to the empty vector control. In contrast, transfection with miR-16-5p mimics reversed these changes, promoting an epithelial phenotype by upregulating E-cadherin and ZO-1 while downregulating fibronectin, N-cadherin, and α-SMA. Notably, when miR-16-5p mimics were co-transfected with pcDNA3.1-FGF7, the pro-EMT effects induced by FGF7 overexpression were significantly attenuated, epithelial markers were partially restored, and mesenchymal markers were suppressed, demonstrating a mutual antagonistic relationship between miR-16-5p and FGF7 in regulating podocyte EMT(Figs.\u0026nbsp;6D,I). Finally, to determine whether the suppressive effect of SQYSF on FGF7 is mediated through miR-16-5p, we performed a loss-of-function rescue experiment. MPC5 cells were pretreated with a miR-16-5p inhibitor or negative control (inhibitor NC) prior to administration of 4% SQYSF under high glucose conditions. SQYSF alone significantly downregulated FGF7 protein expression compared to the high glucose group, consistent with its protective role. However, this inhibitory effect was markedly reversed upon knockdown of miR-16-5p; FGF7 levels remained elevated despite SQYSF treatment(Figs.\u0026nbsp;6J,K). Collectively, our data elucidate a protective mechanism whereby SQYSF upregulates miR-16-5p, which in turn directly represses FGF7, thereby mitigating high glucose\u0026ndash;induced podocyte EMT.\u003c/p\u003e \u003cp\u003e[insert Fig.\u0026nbsp;6.]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eDKD is a severe chronic complication of diabetes and represents a leading cause of chronic kidney disease (CKD). The pathogenesis of DKD is marked by a substantial increase in ECM accumulation, which leads to thickening of the glomerular and tubular basement membranes[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Furthermore, the EMT has been identified as playing a major role in the advancement of basement membrane thickening in DKD[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Currently, there are no effective therapeutic agents to combat this condition, and therapeutic options to halt this progression are limited[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, exploring novel therapeutic targets and intervention strategies is of great importance. In this context, our study elucidates a previously unrecognized mechanism by which the traditional Chinese medicine formula SQYSF exerts its renoprotective effects. We demonstrate that SQYSF attenuates high glucose-induced podocyte EMT primarily by upregulating miR-16-5p, which directly targets and suppresses FGF7, thereby identifying the miR-16-5p/FGF7 axis as a critical pathway mediating the therapeutic action of SQYSF.\u003c/p\u003e \u003cp\u003eThe most significant finding of this research is the identification and functional validation of the miR-16-5p/FGF7 axis. We first established that SQYSF-containing serum dose-dependently reversed high glucose-induced podocyte EMT, with concurrent restoration of mitochondrial ultrastructure, highlighting its cytoprotective potential. Subsequent investigations revealed that SQYSF significantly upregulated the expression of miR-16-5p, which was suppressed under high glucose conditions. Functional rescue experiments using a miR-16-5p inhibitor confirmed that the anti-EMT effect of SQYSF is largely dependent on its ability to elevate miR-16-5p levels, positioning miR-16-5p as a functional effector rather than merely a biomarker. Among the numerous miRNAs involved in the development and progression of DKD, miR-16-5p has attracted significant attention due to its well-documented renal protective functions as confirmed by multiple studies. miRNAs serve as central regulatory factors within gene expression networks. They modulate gene expression at the post-transcriptional level by binding to the 3' untranslated region (3'UTR) of target mRNAs, thereby influencing cellular biological behaviors[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA key innovation of this work lies in the discovery of FGF7 as a direct and functionally relevant target of miR-16-5p in podocytes under diabetic conditions. FGF7 is a functionally distinct member of the FGF family. Its classical biological characteristic is its specific secretion by mesenchymal cells and selective binding to the FGFR1b and FGFR2b receptor isoforms on the surface of epithelial cells, thereby mediating unidirectional \u0026ldquo;mesenchymal-to-epithelial\u0026rdquo; cellular communication[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This property endows FGF7 with a pivotal role in embryonic development, particularly in orchestrating the morphogenesis of organs such as the kidneys and liver[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. During renal development, FGF7 modulates ureteric bud signaling to support the survival and aggregation of renal progenitor cells, thereby facilitating nephron formation[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Current research has largely concentrated on its roles in cancer drug resistance, cell proliferation, glucose metabolism, and inflammatory responses[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. While the role of FGF7 in developmental biology is well-established, and its potential value as a biomarker or therapeutic target for kidney regeneration has been suggested[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], its function in the pathogenesis of DKD remains largely unexplored, and the specific role of FGF7 in the pathological progression of podocyte EMT remains an uncharted area. To address this knowledge gap, our study, for the first time, identifies FGF7 as a critical research target in DKD-associated podocyte EMT. We aim to systematically investigate its potential role and underlying molecular mechanisms in high glucose-induced podocyte EMT. Our findings not only reveal the potential of FGF7 as a novel target in DKD, but also extend the paradigm of miRNA-mediated post-transcriptional regulation to the mechanistic study of traditional Chinese medicine formulations. Furthermore, integrated transcriptomic sequencing and bioinformatic analysis corroborate the pivotal position of the miR-16-5p/FGF7 axis within the regulatory network of DKD. Through an integrative approach combining transcriptomic analysis and bioinformatics prediction, we identified FGF7 as a potential target of miR-16-5p. This interaction was conclusively verified by dual-luciferase reporter assay, confirming specific binding of miR-16-5p to the 3'-UTR of FGF7 mRNA. The functional significance of this mechanism was cemented by a series of rescue experiments. Collectively, these findings delineate an axis whereby SQYSF inhibits EMT mechanistically through the upregulation of miR-16-5p and the subsequent downregulation of its target, FGF7. The proposed mechanism is schematically illustrated in Fig.\u0026nbsp;7, depicting the pathway from SQYSF intervention to the inhibition of podocyte EMT via the miR-16-5p/FGF7 axis.\u003c/p\u003e \u003cp\u003eWe acknowledge several limitations of the present study. Firstly, the findings are primarily derived from in vitro experiments using MPC5 podocyte cells. Although the use of drug-containing serum enhances translational relevance by reflecting processed herbal components, future validation in in vivo diabetic animal models is essential. Secondly, while we have established the centrality of the miR-16-5p/FGF7 axis, traditional Chinese medicine formulas like SQYSF are characterized by multi-component, multi-target, and multi-pathway actions. It is plausible that SQYSF exerts its overall protective effect through additional parallel or synergistic mechanisms independent of this mechanism, as suggested by the partial restoration of epithelial phenotype even upon miR-16-5p inhibition. This study elucidates the cellular mechanism of the holistic formula. The next step is to bridge the chemical and biological findings by identifying which of the LC-MS/MS-characterized constituents are primarily responsible for modulating the miR-16-5p/FGF7 axis.\u003c/p\u003e \u003cp\u003eSeveral limitations of this work should be acknowledged. First, although the use of SQYSF-containing serum in cultured podocytes partially recapitulates the metabolic fate of the herbal formula in vivo, the present findings are primarily derived from in vitro experiments. Whether SQYSF exerts renoprotective effects through the miR-16-5p/FGF7 axis in vivo requires further validation in diabetic animal models such as db/db mice or STZ-induced diabetic rats. Second, the bulk RNA-seq data were generated from renal cortical tissues of db/db mice, which comprise heterogeneous cell populations including glomerular and tubular compartments. While the functional role of FGF7 was specifically validated in podocytes, its cell-type-specific expression pattern and regulation by SQYSF in the diabetic kidney remain to be clarified using approaches such as immunofluorescence co-localization or single-cell sequencing. Third, although our rescue experiments demonstrate that upregulation of miR-16-5p is a critical mediator of SQYSF action, the partial restoration of epithelial markers upon miR-16-5p inhibition suggests the existence of additional, miR-16-5p-independent mechanisms. As a multi-herb formulation, SQYSF is inherently capable of modulating multiple signaling pathways, and these parallel mechanisms warrant further investigation. Fourth, an inverted U-shaped dose\u0026ndash;response was observed, with 4% SQYSF-containing serum exhibiting greater efficacy than 8%. Whether this phenomenon reflects receptor saturation, feedback inhibition, or mild cytotoxicity remains unresolved and merits further exploration. Fifth, although FGF7 is conventionally regarded as an epithelial-protective growth factor, our study reveals a pro-EMT role under diabetic conditions. The context-dependent functional switch of FGF7 and its upstream regulatory networks in DKD pathogenesis remain poorly understood and require systematic investigation. Despite these limitations, this study provides the first evidence linking the miR-16-5p/FGF7 axis to podocyte EMT and identifies this axis as a novel mechanistic target of SQYSF, thereby laying a foundation for subsequent in vivo validation and phytochemical interrogation.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis study provides evidence that SQYSF mitigates high glucose-induced podocyte EMT by activating the miR-16-5p/FGF7 axis. These findings not only decipher a key molecular mechanism underlying the renal protective effect of SQYSF but also contribute to the broader field by identifying a novel miRNA-target pair involved in DKD pathogenesis. Future studies focusing on the active compounds of SQYSF and their in vivo validation will be crucial for further development and clinical translation.\u003c/p\u003e \u003cp\u003e[insert Fig.\u0026nbsp;7]\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShi-qi Chen\u003c/strong\u003e: Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing\u0026ndash;original draft, Writing\u0026ndash;review and editing.\u003cstrong\u003e Zhu Wu\u003c/strong\u003e: Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing\u0026ndash;original draft, Writing\u0026ndash;review and editing. \u003cstrong\u003eJing Zhang\u003c/strong\u003e: Data curation, Supervision, Writing\u0026ndash;review and editing. \u003cstrong\u003eYu-Xin Lin\u003c/strong\u003e: Software, Supervision, Writing\u0026ndash;review and editing. \u003cstrong\u003eJia-Qi Xie\u003c/strong\u003e: Investigation, Methodology, Writing\u0026ndash;review and editing. \u003cstrong\u003eXiao-yu Peng\u003c/strong\u003e: Investigation, Writing\u0026ndash;review and editing.\u003cstrong\u003e De-hui Yin\u003c/strong\u003e: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Writing\u0026ndash;review and editing. \u003cstrong\u003eYe Zhu\u003c/strong\u003e: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing\u0026ndash;review and editing.\u003c/p\u003e\n\u003cp id=\"_Toc27679\"\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinancial support for this this article was provided by National Natural Science Foundation of China (82274464, 82360917);Natural Science Foundation of Hainan Province (823MS147).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEveryone who contributed significantly to this study has been listed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFeng, X. 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Physiol.\u003c/em\u003e \u003cb\u003e236\u003c/b\u003e (12), 7909\u0026ndash;7925 (2021).\u003c/span\u003e\u003c/li\u003e\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Diabetic kidney disease, Suoquan Yishen Formula, miR-16-5p, FGF7, Epithelial-mesenchymal transition, Podocyte","lastPublishedDoi":"10.21203/rs.3.rs-9304192/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9304192/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePodocyte epithelial-mesenchymal transition (EMT) drives diabetic kidney disease (DKD). Suoquan Yishen Formula (SQYSF) shows clinical efficacy, but its mechanism is unclear.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo investigate SQYSF protection against high glucose-induced podocyte EMT via miR-16-5p/FGF7 axis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSQYSF was analyzed by LC-MS/MS. A podocyte EMT model was induced by high glucose and treated with SQYSF. Cell viability and ultrastructure were evaluated. Expression levels of EMT markers, miR-16-5p, and FGF7 were measured via RT-qPCR, Western blot, and immunofluorescence. Candidate targets were identified through bulk RNA-seq.\u0026nbsp;miR-16-5p localization determined by FISH. The miR-16-5p/FGF7 interaction was confirmed by dual-luciferase assay, and functional validation was performed using miR-16-5p mimics/inhibitor and FGF7 overexpression.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eLC-MS/MS analysis identified 21 bioactive compounds. SQYSF dose-dependently attenuated high glucose-induced podocyte EMT, mitochondrial damage, and significantly upregulated miR-16-5p expression(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Inhibition of miR-16-5p reversed the protective effects of SQYSF(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Bulk RNA-seq analysis identified FGF7 as a key target. FISH confirmed the cytoplasmic localization of miR-16-5p.The miR-16-5p/FGF7 direct interaction was confirmed, and SQYSF inhibited high glucose-induced FGF7 upregulation(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Importantly, FGF7 overexpression abolished SQYSF\u0026rsquo; s efficacy(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). miR-16-5p mimics rescued the pro-EMT effects of FGF7(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eSQYSF alleviates podocyte EMT by upregulating miR-16-5p to repress FGF7, revealing a novel DKD therapeutic target.\u003c/p\u003e","manuscriptTitle":"Suoquan Yishen Formula Inhibits Podocyte Epithelial-Mesenchymal Transition in Diabetic Kidney Disease by Regulating miR-16-5p/FGF7 axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 11:59:17","doi":"10.21203/rs.3.rs-9304192/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-12T22:42:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T10:13:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78958750624232553007887820096033423731","date":"2026-05-01T08:28:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262112958560131302882565912826198052701","date":"2026-04-30T09:12:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-29T06:13:40+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-09T07:34:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-03T05:19:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-03T05:18:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-02T13:54:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b7a9cdd-a954-469e-b415-0e81aa5fcad6","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-12T22:42:06+00:00","index":68,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T10:13:52+00:00","index":67,"fulltext":""},{"type":"reviewerAgreed","content":"78958750624232553007887820096033423731","date":"2026-05-01T08:28:19+00:00","index":64,"fulltext":""},{"type":"reviewerAgreed","content":"262112958560131302882565912826198052701","date":"2026-04-30T09:12:09+00:00","index":63,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67456651,"name":"Biological sciences/Cell biology"},{"id":67456654,"name":"Health sciences/Diseases"},{"id":67456655,"name":"Health sciences/Endocrinology"},{"id":67456656,"name":"Biological sciences/Molecular biology"},{"id":67456657,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2026-05-07T11:59:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 11:59:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9304192","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9304192","identity":"rs-9304192","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}