Mesenchymal Stem Cells Attenuate Diabetic Nephropathy by Suppressing the ERK-ferroptosis-ROS axis

Mesenchymal Stem Cells Attenuate Diabetic Nephropathy by Suppressing the ERK-ferroptosis-ROS 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 Research Article Mesenchymal Stem Cells Attenuate Diabetic Nephropathy by Suppressing the ERK-ferroptosis-ROS axis Shuaijing Ma, Jing Li, Haiyan Wang, Yiming Wang, Shuang Peng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8603066/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Diabetic nephropathy (DN) is a major cause of end-stage renal disease with limited therapeutic options. As ferroptosis is a key mechanism of renal tubular injury in DN, this study investigates whether mesenchymal stem cells (MSCs) transplantation alleviates DN by inhibiting this form of cell death, although its precise mechanisms remain incompletely understood. Methods To investigate the therapeutic efficacy and mechanisms of human umbilical cord-derived MSCs (UMSCs) in diabetic nephropathy, we established a rat model of type 2 DN (T2DN) using a high-fat diet and streptozotocin. Oxidative stress was assessed via measurements of DNA, protein, and lipid oxidation. To elucidate the underlying mechanisms, RNA sequencing (RNA-seq) was performed to investigate the renal protective effects of UMSCs. Results UMSCs treatment significantly improved renal function and alleviated tubular injury in DN rats, concomitant with reduced mitochondrial dysfunction, iron overload, reactive oxygen species (ROS) accumulation, and ferroptosis. In vitro, UMSCs suppressed high glucose-induced mitochondrial dysfunction, oxidative stress and ferroptosis in renal tubular cells. RNA-seq and experimental findings identified the MAPK/ERK pathway as essential for this protection, confirmed by pharmacological activation/inhibition of p-ERK/ERK. Conclusions Targeting the ERK-ferroptosis-ROS axis in renal tubular epithelial cells represents a novel therapeutic strategy for DN. This strategy is supported by our finding that MSCs confer protection specifically by disrupting the p-ERK/ERK-GPX4/ACSL4 axis, thereby preventing glutathione depletion and lipid ROS accumulation. Mesenchymal stem cells ferroptosis Oxidative stress Renal tubular injury MAPK/ERK pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes mellitus and is the leading cause of chronic kidney disease and end-stage renal disease globally [ 1 – 3 ]. Traditional perspectives considered DN a "glomerulocentric" disease. However, recent research has revealed that tubular injury may precede glomerulopathy and play a critical role in the early stages of DN [ 4 , 5 ]. The pathogenesis of DN encompasses multifactorial mechanisms, including lipid metabolism dysregulation, hemodynamic abnormalities, inflammation, oxidative stress, cellular damage, and ferroptosis [ 6 ]. Ferroptosis induces renal tubular epithelial cell death, triggering pathological cascades through damage-associated molecular pattern release - activating innate immunity, disrupting tubular reabsorption causing proteinuria, and promoting renal fibrosis via epithelial-mesenchymal transition [ 5 , 7 ]. Proximal tubular reabsorption critically demands iron for ATP production [ 8 ], and dysregulated iron metabolism secondary to diabetic renal injury heightens oxidative stress and inflammatory responses, thereby potentiating renal damage [ 9 – 11 ]. The iron metabolism-oxidative stress vicious cycle constitutes the core mechanism underlying ferroptosis in renal tubular epithelial cells during DN. The above reports suggested the importance of ferroptosis in the pathological progression of diabetic nephropathy. Mesenchymal stem cells (MSCs) have garnered increasing attention as a novel regenerative therapy for DN. The therapeutic efficacy of MSCs transplantation for DN has been established in numerous preclinical studies and demonstrates promising outcomes in early-phase clinical trials [ 12 – 16 ]. However, the clinical translation of these therapies remains challenging, primarily due to the heterogeneous therapeutic responses resulting from unresolved mechanistic uncertainties. MSC-based therapies ameliorate diabetic nephropathy primarily via paracrine and immunomodulatory mechanisms, including anti-inflammatory, antioxidant, antifibrotic, and cellular protective effects, alongside promotion of angiogenesis, mitochondrial transfer, and tissues repair [ 16 , 17 ]. Nevertheless, the precise regulatory mechanisms underlying these therapeutic effects remain incompletely elucidated. Based on the established role of ferroptosis in driving renal tubular cell loss and dysfunction in diabetic nephropathy, we hypothesized that the beneficial effects of MSC therapy resulted from the modulation of tubular ferroptosis pathways. Recent studies report that MSCs and their derived exosomes mitigate acute multi-organ injury through ferroptosis regulation [ 18 – 22 ]. However, how MSCs treat chronic metabolic diseases such as DN by modulating ferroptosis is still unclear. To address this gap, we investigated whether MSCs ameliorate diabetic kidney injury by targeting the iron metabolism-ROS-ferroptosis positive feedback loop in renal tubular epithelial cells. We assessed dose-dependent therapeutic efficacy and explored ferroptosis-related molecular regulatory mechanisms. Materials and Methods Culture and Conditioned Medium Collection from UMSCs The primary human umbilical cord-derived MSCs (UMSCs) were provided by Cell Energy Life Sciences group CO. LTD (Initial ethical approval by Ethics Committee of Liaocheng People’s Hospital, Approval No. 2021105, and the donors had signed informed consent.). UMSCs were identified as described previously [ 23 ] (Supplementary Fig. S1 ). UMSCs of passage 4 or 5 were used in our experiments. Cells were cultured in a humidified incubator with 5% CO 2 at 37°C and passaged with trypsin/EDTA after reaching the confluence. Once the UMSCs reached 70–80% confluency, the medium was replaced with fresh full medium and harvested after 24h. Subsequently, UMSCs-CM were centrifuged at 3000 rpm for 20 min with 0.22µm filtration to remove detached MSCs and cell debris. Animal model and treatment protocols Sprague-Dawley (SD) rats were provided by Spefo (Beijing) Biotechnology Co., Ltd., and all animal procedures followed guidelines approved by the Chinese PLA General Hospital Ethics Committee (Approval No. 2022-x18-39). After acclimation, male SD rats (5–6 weeks old, 150–180 g) were fed a high-fat diet for 6 weeks to induce insulin resistance, followed by a 12–18 h fast and a single intraperitoneal injection of streptozotocin (STZ, 40 mg/kg). Blood glucose was monitored daily for three days. Rats with sustained glucose ≥ 16.7 mmol/L were considered type 2 diabetes (Supplementary Fig. S2 a). Type 2 diabetic nephropathy (T2DN) was confirmed when the 24-hour urinary protein (PRO) excretion exceeded 30 mg (Supplementary Fig. S2 b). On day 0, 35 rats meeting both criteria (GLU ≥ 16.7 mmol/L and 24-h PRO > 30 mg) were selected as established T2DN models (Supplementary Fig. S3 ). Subsequently, thirty of these T2DN rats were randomly selected and divided into 3 groups (Supplementary Fig. S3 , n = 10 per group): the DN model group (DN), which received no cell therapy; the low-dose umbilical cord mesenchymal stem cell group (UMSCs-LD), administered 2×10⁶ cells per rat; and the high-dose group (UMSCs-HD), administered 5×10⁶ cells per rat. Cell suspensions were delivered via intravenous injection once every two weeks, for a total of three injections. Meanwhile, both the DN model group and a normal control group of SD rats (Ctrl, n = 9) received equal volumes of PBS via the same route and schedule. The timelines for model induction and treatment administration are presented in Fig. 1 a. Animal anaesthesia and euthanasia All rats were euthanized at the designated experimental endpoints. Euthanasia was performed by inducing deep anesthesia via inhalation of 3% isoflurane (in 100% oxygen) in an induction chamber, followed by maintenance at this concentration until the cessation of breathing and cardiac function was verified. All procedures were performed in strict accordance with the animal welfare guidelines approved by the Chinese PLA General Hospital Ethics Committee. Anesthesia was not utilized at any other stage of the study. Biochemical test The 24-h urine samples were collected from rats at 4-week intervals up to week 20, and analyzed for urea nitrogen (BUN), 24-h urinary protein (PRO), and creatinine (Cr) levels in the supernatant according to the manufacturer's instructions (XR220PLUS, XinRui, China). Concurrently, blood samples were collected at 4-week intervals up to week 20 and analyzed for blood glucose, insulin, triglycerides (TG), and total cholesterol (CHOL) using Fully automatic biochemical analyzer (XR220PLUS, XinRui, China) according to the manufacturer's instructions. Pathological staining Renal tissues were fixed in 4% paraformaldehyde and conventionally paraffin embedded, with 5 µm pathology sections then being created. These were then sequentially immersed in 100%, 95%, 70% and 30% ethanol for 2 min each for hydration, and then immersed in water for 2 min. HE staining was used to observe the basic structure of the glomerulus, PAS staining was used to observe changes in glomerular thylakoids and basement membranes, and Masson's staining was used to observe the degree of renal mesenchymal Masson's staining was used to observe the degree of interstitial fibrosis. All stained sections were reviewed by a pathologist blinded to the experimental conditions. Three random fields of view were selected from each slide, and quantitative analysis was performed using ImageJ software for objective assessment. Immunohistochemistry (IHC) Kidney tissue sections were deparaffinized, rehydrated through graded ethanol and washed with PBS. Antigen retrieval was performed using 10 mM sodium citrate buffer for 20 min. Slides were fixed with 5% BSA for 1 hour at room temperature and then washed with PBS. For ACSL4 (22401-1-AP, Proteintech), GPX4 (67763-1-Ig, Proteintech), SLC7A11 (26864-1-AP, Proteintech), FTH1 (DF6278, Affinity), TFRC (AF8136, Beyotime, China), or p-ERK (4370S, Cell Signaling Technology, USA) primary antibodies were added to the slides and incubated overnight. Secondary antibodies were subsequently added and incubated at room temperature. DBA was then added for a 2-minute reaction, and the sections were reprobed with haematoxylin. The staining intensity was semiquantitatively analyzed using ImageJ software by evaluating at least three random 20× fields per section, which were selected by an independent pathologist (n = 5). Cell culture and intervention The human renal tubular epithelial cell line HK-2 was obtained from China Center for Type Culture Collection (CCTCC No. GDC0152, Wuhan, China) and cultured in MEM (C11095500BT, Gibco, USA) for serial subcultivation. Cells cultured in normal glucose (5 mM) for 72 h were designated the control group (NG). Cells cultured in high-glucose (30 mM) medium for 72 h were designated the high-glucose group (HG). Cells cultured in high-glucose (30 mM) medium plus UMSCs-CM for 72 h were designated the therapy group (HG + CM). Glucose (5 mM) plus mannitol (24.5 mM) was used as an osmotic control (Man). Additionally, to inhibit p-ERK activity, cells with HG medium were treated with 10nM GSK2606414 (HY-18072, MCE) for 72 h. To determine whether UMSCs trigger p-ERK/ERK pathway to inhibit ferroptosis, HG-cells were treated with UMSCs-CM and 10 µM MK-28 (HY-137207, MCE) for 72 h. ELISA detection of 8-OHdG The levels of 8-OHdG in kidney tissues and HK-2 cells were determined by ELISA kits (MM-0331H1, Meimian, Jiangsu, China) following the manufacturer’s instructions. Malondialdehyde (MDA) assay According to the MDA Assay Kit (S0131S, Beyotime), MDA levels in kidney tissues or HK-2 cells were measured after lysis and incubation using a microplate reader. Glutathione (GSH) assay Total GSH content in tissues or HK-2 cells were assessed using the Reduced Glutathione Content Assay Kit (BC1175, Solarbio) following the manufacturer’s instructions. Protein Carbonyl Content (PCO) Test The levels of PCO in kidney tissues and HK-2 cells were determined by Protein Carbonyl Content Assay Kit (BC1275, Solarbio) following the manufacturer’s instructions. Prussian Blue Staining (Enhance With DAB) Kidney tissues sections were deparaffinized, rehydrated with gradient ethanol, washed with PBS, stained with Prussian blue and subsequently reacted with the addition of DBA for 2 min, sections were re-stained with hematoxylin, sealed and observed under the microscope. For objective quantification, a blinded pathologist randomly selected three non-overlapping fields of view from each stained slide. Three fields of view were randomly selected from each stained slide by a blinded pathologist and quantified using ImageJ (n = 5). Iron array Total iron levels in tissues were assessed using a tissue iron assay kit (BC4355, Solarbio, China) following the manufacturer’s instructions. Western blotting Western blotting was performed as previously described [ 24 ]. Proteins were extracted from renal tissues or cultured cells using RIPA lysis buffer (P0013B, Beyotime, China) with protease and phosphatase inhibitors, homogenized on ice, and centrifuged at 13,500 × g for 20 min at 4°C. Protein concentration was measured by BCA assay, and equal amounts (10 µg) were separated on SDS-PAGE and transferred to PVDF membranes. The membranes were then incubated overnight at 4°C with primary antibodies against PGC1α (ab317540, Abcam, USA), TFAM (22586-1-AP, Proteintech), ACSL4, GPX4, SLC7A11, FTH1, TFRC, p-ERK, ERK (4695S, Cell Signaling Technology), p-P38 (28796-1-AP, Proteintech), P38 (14064-1-AP, Proteintech), and β-actin (66009-1-Ig, Proteintech). After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Signals were detected by ECL and quantified using ImageJ. Target protein levels were normalized to β-actin. ATP level Following the procedure of ATP Assay Kit (S0026, Beyotime), the ATP level of kidney tissues and HK-2 cells was detected by chemiluminescence. Mitochondrial Membrane Potential (MMP) assay HK-2 cells were harvested and incubated with the JC-1 probe following the manufacturer’s protocol provided with the mitochondrial membrane potential assay kit (M8650, Solarbio). Subsequently, mitochondrial membrane potential was assessed via flow cytometry. MtDNA Copy Number Genomic DNA was isolated from kidney tissues or total cells pursuant to the instructions of the DNA extraction kit (DP304, TanGen, China). Subsequently, cycle threshold (Ct) values were acquired via quantitative real-time PCR (qPCR) performed with a standard system and procedure. Primer sequences are available in the Supplementary Methods. ROS level HK-2 cells were loaded with DCFH probe (S0033S, Beyotime) in situ according to the instructions and then incubated at 37°C for 15 min for staining, and photographed by laser confocal microscopy under microscopic observation or assessed via flow cytometry. RNA-seq and analysis Total RNA was isolated from rat kidney tissues using Trizol reagent (Invitrogen, USA) followed by RNeasy Mini Kit (Qiagen, USA) purification. rRNA-depleted RNA libraries were prepared with the NEBNext Directional RNA Library Prep Kit. First-strand cDNA was synthesized with random hexamers and M-MuLV reverse transcriptase, followed by second-strand synthesis using DNA Polymerase I and RNase H. Libraries were purified (AMPure XP beads), quality-checked (Agilent Bioanalyzer 2100), and clustered (cBot System, Illumina NovaSeq reagents). Paired-end sequencing was performed on an Illumina NovaSeq platform (Cnkingbio, China). Gene expression was quantified as FPKM. Differentially expressed genes were identified using DESeq2 (v1.30.0) with FDR correction (Benjamini-Hochberg method). Statistical analysis All results were analyzed using GraphPad Prism software (version 10.1.2) and expressed as means ± standard deviation (Mean ± SD). Statistical analysis was performed as indicated in the figure legends. For two group comparison, Student's t-test was performed. For multiple-group comparison, one-way ANOVA analysis was performed. Results UMSCs Mitigate Renal Pathological Manifestations in DN Rat Models To investigate the therapeutic potential of UMSCs in DN, we established a T2DN rat model (GLU ≥ 16.7 mmol/L and 24-h PRO > 30 mg) through a combination of high-fat diet for 6 weeks and intraperitoneal STZ administration. UMSCs were administered via triple tail vein injections (0, 2, and 4 week) at two dosage regimens: 2×10 6 cells/injection (Low-dose group, UMSCs-LD, n = 10) and 5×10 6 cells/injection (High-dose group, UMSCs-HD, n = 10) (Fig. 1 a). Biochemical results detected every four weeks during treatment are presented in Supplementary Fig. S4. At the 20-week endpoint, the DN cohort (n = 10) exhibited marked elevations in fasting blood glucose, glycated hemoglobin (HbA1c), 24h-urinary protein excretion (PRO), urine creatinine (UCr), and urea nitrogen (BUN), all of which were substantially attenuated by UMSCs therapy (Fig. 1 b, d–g). Although serum insulin levels were significantly reduced in the DN group, they returned to normal after UMSCs administration (Fig. 1 c), indicating that UMSCs ameliorate renal function in diabetic nephropathy. Moreover, the significant reduction in serum lipids (triglycerides and cholesterol) in DN rats following UMSC administration (Fig. 1 h, i) suggests that lipid-lowering is a novel aspect of their pleiotropic effects, beyond their established roles in improving renal function. Histopathological analysis revealed characteristic renal pathology in DN rats, including glomerular hypertrophy, tubular dilatation, and lipid vacuolization in proximal tubular cells (Fig. 1 j, k). PAS staining demonstrated tubular basement membrane thickening (Fig. 1 j, l), while Masson trichrome staining confirmed progressive interstitial fibrosis involving glomerulotubular junctions (Fig. 1 j, m). Remarkably, UMSCs administration ameliorated these structural anomalies, restoring glomerular morphology and reducing collagen deposition by 40–60% across histological metrics. To evaluate the long-term engraftment of the administered MSCs, we analyzed kidney tissues from DN rats at 20 weeks by immunofluorescence with an anti-human nuclear antigen antibody. No human cells were detected at this time point (Supplementary Fig. S5), indicating that the MSCs did not establish a persistent population in the renal niche under these conditions. Consequently, the sustained therapeutic benefits observed are unlikely to be mediated by direct cellular engraftment, suggesting the involvement of alternative mechanisms. These collective findings demonstrate the successful establishment of a pathologically relevant T2DN model, which recapitulates hallmark metabolic derangements (Fig. 1 b-i) and progressive nephropathy (Fig. 1 j-m). The data further establish the potent nephroprotective efficacy of UMSCs therapy. This intervention significantly mitigated characteristic functional impairments, including proteinuria and azotemia. Concurrently, it ameliorated critical histopathological manifestations, such as glomerular hypertrophy and tubulointerstitial fibrosis. In conclusion, the study validates a robust T2DN model and conclusively shows that UMSCs confer renoprotection by simultaneously alleviating both functional and structural deteriorations in the diabetic kidney. UMSCs Attenuate Ferroptosis-related Mitochondrial Dysfunction in DN Renal Tissues Ferroptosis is characterized by complex and dynamic mitochondrial damage. To investigate the role of mitochondrial dysfunction in the pathogenesis of DN, we first assessed mitochondrial function in renal tissues of diabetic nephropathy DN rats under high-glucose conditions. Results demonstrated significantly reduced ATP production in mitochondria isolated from DN rats, accompanied by decreased mitochondrial DNA (mtDNA) copy number (Fig. 2 a, b). UMSCs treatment significantly restored mitochondrial function. Western blot analysis of mitochondrial biogenesis markers PGC1α and TFAM revealed suppressed expression of both proteins in DN rat kidneys. This suppression was reversed following UMSCs treatment (Fig. 2 c–e), indicating that impaired mitochondrial biogenesis contributes critically to mitochondrial dysfunction during high glucose-induced renal damage, and that UMSCs may confer protection by enhancing mitochondrial biogenesis and functional recovery. Mitochondria constitute the primary cellular source of ROS [ 25 ]. Exposure to metabolic stressors (e.g., hyperglycemia and inflammation) induces mitochondrial damage, triggering excessive ROS generation [ 26 ] that promotes lipid peroxidation and accelerates ferroptosis. To investigate the role of oxidative stress in DN, we assessed renal oxidative stress markers in a rat model of DN. Compared with controls, DN rats exhibited significantly elevated levels of malondialdehyde (MDA), a terminal product of lipid peroxidation, along with depleted glutathione (GSH). Treatment with UMSCs markedly attenuated MDA accumulation and restored GSH levels (Fig. 2 f, g). Protein Carbonyl Content (PCO) analysis demonstrated increased PCO content in DN renal tissues, which was reversed by UMSC intervention, indicating amelioration of high glucose-induced protein oxidation (Fig. 2 h). Similarly, ELISA detected elevated 8-OHdG in DN kidneys, with UMSCs treatment significantly reducing this DNA oxidation biomarker (Fig. 2 i). Western blot analysis showed upregulated expression of ACSL4, a key enzyme in pro-ferroptosis lipid metabolism, in DN renal tissues. This elevation was attenuated following UMSCs administration (Fig. 2 j, k). Collectively, these results demonstrate a state of profound oxidative stress accompanied by enhanced lipid peroxidation in DN kidneys, strongly supporting the involvement of ferroptosis in the pathogenesis of DN. UMSCs Attenuate Ferroptosis in DN Rat Renal Tissues To investigate the potential mitigating effects of UMSCs on ferroptosis in DN, we initially examined renal iron deposition, a hallmark of ferroptosis. Prussian blue staining demonstrated pronounced iron accumulation within the renal tissues of DN rats, which was markedly attenuated upon UMSCs treatment (Fig. 3 a). Subsequent quantitative analysis of renal iron content provided corroborative evidence (Fig. 3 b), confirming a state of renal iron overload and disrupted iron homeostasis in the diabetic kidney. The iron deposition was notably prominent within tubular epithelial cells. To further elucidate the involvement of ferroptosis and the regulatory role of UMSCs, we evaluated the expression of key proteins governing ferroptosis pathways. IHC analysis revealed that renal tissues from DN rats exhibited substantially suppressed expression of the central ferroptosis inhibitors GPX4, SLC7A11, and FTH1, concomitant with a pronounced upregulation of the pro-ferroptosis iron transporter TFRC. These alterations were most evident within tubular compartments, underscoring the susceptibility of this segment to ferroptosis in DN. Systemic administration of UMSCs effectively restored the expression profiles of these markers toward normal levels (Fig. 3 c). Consistent with these findings, western blot analysis of renal protein confirmed the downregulation of GPX4, SLC7A11, and FTH1 protein expression and the upregulation of TFRC in DN kidneys. Importantly, UMSCs intervention significantly reversed these aberrant expression patterns (Fig. 3 d). These results confirm that ferroptosis is a key pathogenic mechanism in diabetic nephropathy, and umbilical cord-derived mesenchymal stem cells can effectively alleviate renal injury by regulating this pathway. UMSCs-CM Mitigates High Glucose-Induced Ferroptosis in HK-2 Cells Tubular injury constitutes a critical determinant of DN progression [ 10 , 11 , 27 ]. To investigate the underlying mechanisms, we evaluated the effects of high glucose on ferroptosis in human renal tubular epithelial cells HK-2 in vitro. After HK-2 cells were exposed to high glucose (30 mM; HG) for 72 hours, we observed ferroptosis-associated mitochondrial dysfunction characterized by diminished mitochondrial membrane potential (MMP, ΔΨm, Fig. 4 a and Supplementary Fig. S6a), reduced ATP production, and decreased mtDNA copy number. UMSCs-conditioned medium (UMSCs-CM) treatment (HG + CM) effectively restored membrane potential, enhanced ATP synthesis, and increased mtDNA copy number (Fig. 4 a-c), indicating rescue of glucose-stressed mitochondrial function. Western blot analysis revealed suppressed expression of mitochondrial biogenesis regulators PGC1α and TFAM in HG-exposed HK-2 cells. UMSCs-CM treatment significantly upregulated both proteins (Fig. 4 d-f and Supplementary Fig. S6b), suggesting that the therapeutic benefit of UMSCs-CM is likely mediated through the enhancement of mitochondrial biogenesis and the attenuation of high glucose-induced mitochondrial damage. Based on the established role of ferroptosis and mitochondrial dysfunction in high glucose-induced tubular injury, we further investigated the oxidative stress profile in HK-2 cells. Exposure of HK-2 cells to high glucose for 72 hours resulted in a profound redox imbalance, manifested as a significant elevation in the lipid peroxidation product MDA and a sharp decrease in the antioxidant GSH. Treatment with UMSCs-CM effectively reversed this HG-induced imbalance by attenuated MDA accumulation and normalized GSH levels (Fig. 4 g, h). Furthermore, PCO content, indicative of irreversible protein oxidation, was markedly elevated in HG-treated cells. UMSCs-CM intervention significantly attenuated this protein carbonylation (Fig. 4 i). Consistent with DNA oxidative damage, ELISA analysis of culture supernatants revealed elevated 8-OHdG levels in the HG group, which was substantially reduced upon UMSCs-CM treatment (Fig. 4 j). Underlying these specific macromolecular damages, flow cytometry analysis demonstrated significant intracellular ROS accumulation in HG-exposed cells, an effect that was effectively mitigated by UMSCs-CM administration (Fig. 4 k and Supplementary Fig. S7b). Collectively, these results demonstrate that UMSCs-CM attenuates high glucose-induced oxidative stress in renal tubular cells by reducing lipid peroxidation, protein carbonylation, DNA oxidation, and overall ROS burden. Western blot analysis revealed dysregulated ferroptosis markers in HG-exposed HK-2 cells, with significantly elevated protein expression of ACSL4 and TFRC and suppressed expression of GPX4, SLC7A11, and FTH1(Fig. 4 l–q and Supplementary Fig. S7c). These changes are consistent with the characteristic molecular profile of ferroptosis induction. Treatment with UMSCs-CM effectively reversed these expression changes (Fig. 4 l–q and Supplementary Fig. S7c), indicating attenuation of HG-induced ferroptosis in renal tubular epithelial cells. UMSCs Exert Therapeutic Effects via Suppression of the MAPK/ERK Pathway in DN renal tissues To elucidate molecular mechanisms underlying ferroptosis inhibition and renal protection of UMSCs, we conducted bulk RNA sequencing of rat kidney tissues. GSEA and GO pathway enrichment analyses revealed that genes associated with ferroptosis, iron ion homeostasis, and lipid metabolism were significantly upregulated in DN renal tissues (Fig. 5 a, b). Subsequent administration of UMSCs resulted in a significant downregulation of these pathogenic transcriptional profiles (Fig. 5 a, b), a finding that aligns coherently with our prior phenotypic and functional observations. Further analysis of the transcriptomic data indicated a pronounced activation of the MAPK/ERK signaling pathway in DN kidneys, as evidenced by an increased p-ERK/ERK ratio, which was substantially mitigated following UMSCs treatment (Fig. 5 b). As a key MAPK family member, ERK responds to growth factors, cytokines, and stress stimuli, regulating proliferation, differentiation, survival, and metabolism [ 28 ]. Based on these findings, we hypothesized that the therapeutic effect of UMSCs is mediated by inhibiting p-ERK/ERK activation. Western blot analysis of kidney lysates confirmed a significant increase in the phosphorylation levels of both ERK (p-ERK/ERK) and p38 (p-P38/P38) in the DN group compared to controls (Fig. 5 c-e). Importantly, intervention with UMSCs effectively reversed these changes, normalizing the phosphorylation status of these key MAPK nodes. This suppressive effect of UMSCs on ERK activation was further corroborated by IHC, which revealed intensified p-ERK signals in DN renal sections that were markedly reduced upon UMSCs administration (Fig. 5 f). To validate the translational relevance of these findings, we established an in vitro model using HK-2 cells cultured in high glucose. Consistent with the in vivo data, high glucose induced the phosphorylation of ERK and P38, which was attenuated by UMSCs-CM (Fig. 5 g–i ). These results demonstrated that UMSCs modulated the MAPK/ERK signaling under diabetic conditions. Combined transcriptomic, proteomic, and histological evidence indicated that UMSCs suppressed aberrant MAPK/ERK activation in diabetic kidneys. Given the established role of ERK signaling in ferroptosis and renal injury, our findings suggest that UMSCs mitigate ferroptosis and delay DN progression largely through the inhibition of the MAPK/ERK pathway. UMSCs Attenuate Ferroptosis-ROS loop via p-ERK/ERK Pathway Suppression in HK-2 cells To pharmacologically validate the role of p-ERK/ERK activation in high glucose-induced renal tubular ferroptosis, we utilized the selective p-ERK inhibitor GSK2606414 at a concentration of 10 µM. As anticipated, treatment with GSK2606414 markedly attenuated HG-stimulated ROS overproduction in HK-2 cells (Fig. 6 b, c). Concurrently, this inhibition of p-ERK signaling significantly suppressed the expression of the pro-ferroptosis protein ACSL4, while upregulating the key ferroptosis inhibitor GPX4 (Fig. 6 a–e). These data collectively confirm that pharmacological blockade of p-ERK effectively mitigates high glucose-induced ferroptosis in renal tubular cells. To further test pathway specificity in UMSCs-mediated protection, the p-ERK activator MK-28 (10 nM) was applied to UMSCs-CM-treated cells. MK-28 treatment effectively induced ERK phosphorylation (Fig. 6 f) and partially reversed the renoprotective effects of UMSCs-CM. Specifically, MK-28 co-treatment abolished the UMSCs-CM-mediated reductions in oxidative stress markers, including MDA, PCO, 8-OHdG, and intracellular ROS levels (Fig. 6 g–k). Furthermore, it diminished the restoration of GSH levels, indicating a compromised antioxidant capacity. In parallel, the activation of p-ERK attenuated the ferroptosis protection conferred by UMSCs-CM, as evidenced by a decrease in GPX4 expression and a concomitant increase in ACSL4 levels (Fig. 6 l–n). Using both inhibitor and activator approaches, we established that UMSC-mediated protection against HG-induced tubular ferroptosis is achieved by suppressing the p-ERK/ERK pathway, which reduces downstream oxidative stress (Fig. 7 ). Discussion In this study, we demonstrated the therapeutic efficacy of UMSCs in DN rats and elucidated the underlying molecular mechanisms. Under hyperglycemic conditions, pathological activation of the p-ERK/ERK signaling pathway in renal tubular epithelial cells drives ferroptosis through suppression of GPX4 and excessive ROS production, which results in oxidative damage to DNA, proteins, and lipids. UMSCs mitigate renal injury by inhibiting hyperglycemia-induced ERK phosphorylation, which in turn suppresses the ferroptosis-ROS feedback loop and attenuates oxidative stress (Fig. 7 ). Consistent with previous findings [ 13 – 15 ], UMSCs demonstrated therapeutic efficacy in high-fat diet fed/STZ-induced T2DN rats. Both the low-dose (2 × 10⁶ cells) and high-dose (5 × 10⁶ cells) groups exhibited significant renoprotective effects, as evidenced by reduced blood glucose, elevated insulin levels, and improved renal function. However, no dose-dependent relationship was observed. This absence of dose-dependence aligns with studies showing UMSC-mediated renoprotection in T1DN mice [ 29 ] and a phase 1 dose-escalation trial of allogeneic UMSCs for systemic lupus erythematosus (2×10⁶ to 4×10⁶ cells/kg) [ 30 ]. However, some animal models, such as chronic spinal cord injury in rats using lower doses (4×10⁵ to 10⁶ cells), have reported dose-dependent MSC effects [ 31 ]. The lack of dose-dependence observed here may be attributable to the relatively high UMSC doses administered, potentially exceeding the threshold for such effects in this model. Pathological assessment confirmed that our T2DN rats exhibited early-stage DN, lacking hallmark features of advanced disease such as Kimmelstiel-Wilson nodules and glomerulosclerosis. This is concordant with recent evidence indicating that tubular injury precedes and may be more critical than glomerular damage in DN progression [ 4 , 32 ]. Notably, we identified a significant lipid-lowering effect of UMSCs in our T2DN rat model. These T2DN rats displayed both hyperglycemia and dyslipidemia, yet UMSCs administration effectively reduced serum levels of triglycerides and total cholesterol, and concurrently decreased blood glucose. This finding suggests UMSCs mitigate diabetic kidney injury through multifaceted mechanisms, including potential direct modulation of lipid metabolism, and highlights their possible utility in clinical lipid management strategies. Our results demonstrate that DN rats exhibit renal tubular epithelial cell iron overload, mitochondrial dysfunction, and excessive oxidation affecting DNA, proteins, and lipids, culminating in ferroptosis. UMSCs ameliorated these abnormalities—including iron overload, mitochondrial dysfunction, elevated ROS, and ferroptosis—indicating their multi-target therapeutic potential. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation [ 33 ], is critically dependent on mitochondrial damage, which serves as both an initiating event and an amplifier of the process [ 34 , 35 ]. In chronic hyperglycemia, mitochondria undergo progressive damage [ 36 ], promoting excessive ROS production and heightened oxidative stress in renal tubular epithelial cells. Mitochondrial iron accumulation, dysfunctional respiratory chain-driven ROS generation, PUFA-rich membrane structures (notably phosphatidylserine), and inactivation of the key antioxidant enzyme GPX4 collectively establish a microenvironment permissive to mitochondrial ferroptosis [ 34 , 37 , 38 ]. ROS-induced lipid peroxidation, particularly of phosphatidylserine, leads to mitochondrial structural damage and functional collapse, thereby amplifying ferroptosis throughout the cell [ 34 , 37 ]. UMSCs exert their therapeutic effect by targeting multiple nodes within this pathogenic cascade, interrupting the 'mitochondria-iron overload-ROS-ferroptosis' axis to mitigate mitochondrial damage and suppress excessive ROS production. Our study identifies a critical and novel role for the MAPK/ERK pathway in mediating ferroptosis within renal tissues of DN rats treated with UMSCs. Current understanding indicates ferroptosis is regulated via key signaling axes (e.g., System Xc⁻-GSH-GPX4, ACSL4/LOX) modulated by pathways including AMPK, Akt/mTOR, MAPK, and p53/NRF2/ATF4 [ 9 , 34 , 38 – 40 ]. While the MAPK pathway typically regulates ferroptosis through oxidative stress-induced JNK/p38 activation—promoting ACSL4 expression or Lysyl Oxidase activity [ 9 , 34 , 38 – 40 ]—ERK has been shown to promote ferroptosis in some cancers by inhibiting System Xc⁻ [ 41 ]. In this study, RNA-seq analysis revealed that UMSCs suppress ferroptosis by inhibiting MAPK/ERK pathway. Pharmacological intervention in HG-injured HK-2 cells using the ERK phosphorylation inhibitor GSK2606414 or the ERK activator MK-28 demonstrated that ERK inhibition effectively attenuated HG-induced ROS overproduction and ferroptosis. Conversely, ERK activation diminished the therapeutic efficacy of UMSCs against HG-induced oxidative stress and ferroptosis. These findings indicate UMSCs mitigate ferroptosis in renal tubular epithelial cells by downregulating ERK and P38 phosphorylation within the MAPK pathway. By downregulating this pathway, the antioxidant system is activated, which in turn suppresses excessive ROS generation, limits the accumulation of MDA and 8-OHdG, and elevates levels of GSH and GPX4.Consequently, UMSCs inhibit HG-induced ferroptosis, ultimately alleviating renal injury. These data underscore the therapeutic potential of ERK inhibition in DN. Although ERK inhibitors are extensively investigated in oncology, clinical translation is hindered by compensatory feedback mechanisms, pathway reactivation, target-related toxicity, selectivity issues, and drug resistance, confining them primarily to preclinical and clinical trial phases [ 42 ]. Notably, ERK inhibition can trigger robust negative feedback, activating upstream nodes (e.g., RAF) or parallel pathways [ 42 ], limiting monotherapy efficacy and necessitating combination strategies. Our results suggest exploring combination therapies integrating ERK inhibitors with UMSCs, potentially offering novel avenues for cell therapy development and ERK-targeted drug design in DN. In addition, our results demonstrate that no human cells were detected in kidney tissues at 20 weeks after MSC transplantation (Supplementary Fig. S5). The absence of long-term engraftment suggests that the sustained therapeutic effect of UMSCs is not dependent on their direct differentiation or persistent presence within the kidney. Instead, these findings support the concept that UMSCs exert beneficial effects through transient yet potent secretory activities, thereby modifying the host cellular microenvironment and signaling landscape [ 43 ]. This mechanism is increasingly recognized in stem cell therapy, wherein exosomes, cytokines, and growth factors mediate the cross-talk between MSCs and injured tissues [ 43 ]. Several study limitations should be acknowledged. First, the specific molecular mediators released by UMSCs that suppress ERK activation have yet to be elucidated. Future analyses of the UMSCs secretome under hyperglycemic conditions may provide valuable insights. Second, although this study emphasizes the ERK–ferroptosis pathway, other signaling pathways such as TGF-β/Smad and NF-κB are also implicated in DN [ 44 ] and may be influenced by UMSCs [ 13 ], requiring further exploration. Conclusions In conclusion, this study elucidates the molecular mechanisms underlying UMSC efficacy, moving beyond descriptive accounts. Specifically, we demonstrate that UMSCs protect renal parenchymal cells by inhibiting MAPK/ERK phosphorylation, thereby disrupting the ferroptosis-ROS feedback loop. The mechanistic insight not only validates UMSC-based therapy for DN but also highlights the ERK/ferroptosis axis as a promising target. Consequently, targeting this pathway, whether through cell therapy or paracrine mediators, thus holds substantial translational potential. Abbreviations DN Diabetic nephropathy MSCs Mesenchymal stem cells T2DN Type 2 diabetic nephropathy UMSCs Human umbilical cord-derived MSCs RNA-seq RNA sequencing ROS Reactive oxygen species GPX4 Glutathione peroxidase 4 ACSL4 Acyl-CoA synthetase long-chain family member 4 UMSCs-CM UMSCs-conditioned medium STZ Streptozotocin GLU Blood glucose PRO 24-hour urinary protein PBS Phosphate-buffered saline BUN Urea nitrogen PRO 24h-urinary protein excretion UCr Urine creatinine TG triglycerides CHOL Total cholesterol SLC7A11 Solute Carrier Family 7 Member 11 TFRC Transferrin Receptor 1 FTH1 Ferritin Heavy Chain 1 8-OHdG 8-Hydroxy-2'-deoxyguanosine MDA Malondialdehyde GSH Glutathione PCO Protein carbonyl MMP Mitochondrial membrane potential MtDNA Mitochondrial DNA PGC1α Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha TFAM Mitochondrial transcription factor A Declarations Contributions Q.H., X.B., and R.C.Z. conceived and supervised the project. S.P. provided the primary UMSCs and contributed to the experimental design. S.M. and J.L. performed the experiments. All authors contributed to data analysis and interpretation. S.M., J.L., and R.C.Z. drafted the original manuscript and prepared the figures. All authors reviewed and revised the manuscript. Ethics approval and consent to participate UMSCs used in this study were provided by Cell Energy Life Sciences group CO. LTD. Animal Ethics declaration The animal experiment has been approved by the Animal Experimental Ethics Review Committee of the Chinese PLA General Hospital Ethics Committee (Title of the approved project: Research on the Mechanisms of Kidney Injury Repair. Approval No. 2022-x18-39. Date of approval: March 6, 2022). Conflict of interests Author S.P. is an employee of Cell Energy Life Sciences group CO. LTD; the other authors declare no conflict of interests. Competing Interests Author S.P. is an employee of Cell Energy Life Sciences group CO. LTD; the other authors declare no conflict of interests. Funding This work was supported by State Key Laboratory of Common Mechanism Research of Major Diseases Platform. Author Contribution Q.H., X.B., and R.C.Z. conceived and supervised the project. S.P. provided the primary UMSCs and contributed to the experimental design. S.M. and J.L. performed the experiments. All authors contributed to data analysis and interpretation. S.M., J.L., and R.C.Z. drafted the original manuscript and prepared the figures. All authors reviewed and revised the manuscript. Acknowledgement The authors declare that they have not use AI-generated work in this manuscript. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References International Diabetes Federation. IDF Diabetes Atlas, 10th edn. 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Stockwell BR. Ferroptosis turns 10: Emerging mechanisms, physiological functions, and therapeutic applications. Cell. 2022;185(14):2401–21. Singh DK, Winocour P, Farrington K. Oxidative stress in early diabetic nephropathy: fueling the fire. Nat Reviews Endocrinol. 2011;7(3):176–84. Dixon SJ, Olzmann JA. The cell biology of ferroptosis. Nat Rev Mol Cell Biol. 2024;25(6):424–42. Conrad M. Ferroptosis: when metabolism meets cell death. Physiol Rev. 2024;105(2):651–706. Wu T, Ji M, Li T, Luo L. The molecular and metabolic landscape of ferroptosis in respiratory diseases: Pharmacological aspects. J Pharm Anal. 2025;15(1):101050. Wang X, Tan X, Zhang J, Wu J, Shi H. The emerging roles of MAPK-AMPK in ferroptosis regulatory network. Cell Communication Signal. 2023;21(1):200. Wang K, Zhang X, Fan Y, Zhou L, Duan Y, Li S, et al. Reactivation of MAPK-SOX2 pathway confers ferroptosis sensitivity in KRASG12C inhibitor resistant tumors. Redox Biol. 2024;78:103419. Bahar ME, Kim HJ, Kim DR. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8603066","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601064095,"identity":"9828d967-4da8-49a7-93bc-9697762fb82c","order_by":0,"name":"Shuaijing Ma","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Shuaijing","middleName":"","lastName":"Ma","suffix":""},{"id":601064097,"identity":"ba83926d-16f2-4fda-9d35-9fc3996eadff","order_by":1,"name":"Jing Li","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""},{"id":601064098,"identity":"e7961d9a-c96d-4240-868d-861dae8b4dbb","order_by":2,"name":"Haiyan Wang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Wang","suffix":""},{"id":601064100,"identity":"0625760f-5a41-486a-97bb-b5c74a46c879","order_by":3,"name":"Yiming Wang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yiming","middleName":"","lastName":"Wang","suffix":""},{"id":601064101,"identity":"4f1ac260-e541-4df6-89bb-8088915dbf21","order_by":4,"name":"Shuang Peng","email":"","orcid":"","institution":"Cell Energy Life Sciences group CO. 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Animal experimental design; b-i. Changes in blood glucose, insulin(INS), HbA1c, urinary protein(PRO), creatinine(UCr), urea nitrogen(BUN), triglycerides(TG), and total cholesterol(CHOL) levels in DN rats treated with or without UMSCs; j. Representative pathological staining of kidney sections (Top panel: HE staining, Middle panel: PAS staining, Bottom panel: Masson staining, Scale bar=50μm); k. Quantification analysis of glomerular area; l. Quantitative percentage of PAS-positive area m. Quantitative analysis of collagen volume fraction, n=9/10; \u003cem\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8603066/v1/a47df7d9d624e2f8188573ae.jpg"},{"id":104404260,"identity":"698dc2e8-6ebc-4626-a1c0-7b1c72c0822f","added_by":"auto","created_at":"2026-03-11 12:19:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":794766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUMSCs alleviate renal ferroptosis related mitochondrial dysfunction in DN rats \u003c/strong\u003ea. ATP levels in rat renal tissues, n=4; b. MtDNA copy number in rat renal tissues, n=4; c-e. Protein expression of PGC1α and TFAM detected by western blot and quantitative analysis in rat renal tissues, n=3; f-i. MDA, GSH, PCO, and 8-OHdG level in rat renal tissues, n=4; j-k. Protein expression of ACSL4 detected by Western blot in rat renal tissues and quantitative analysis, n=3; \u003cem\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8603066/v1/f09ade467b9cdb169a856503.jpg"},{"id":104106669,"identity":"45634370-593b-4b45-a6f9-7d9261dfecb5","added_by":"auto","created_at":"2026-03-06 22:09:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2284038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUMSCs inhibit ferroptosis in renal tissues of rats with DN \u003c/strong\u003ea. Rat renal tissues Prussian blue staining (DAB-enhanced method) and statistical analysis (Scale bar=100μm), n=5; b. Fe levels in rat renal tissues, n=4; c. IHC staining for GPX4, SLC7A11, FTH1, and TFRC in rat renal tissues and quantitative analysis of positive areas (Scale bar=100μm), n=5; d. Protein expression of ferroptosis markers GPX4, SLC7A11, FTH1, and TFRC in rat renal tissues and quantitative analysis of relative protein expression, n=3; \u003cem\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8603066/v1/26c1b5ff943ce1b55fa1560d.jpg"},{"id":104106672,"identity":"a4177ad4-d199-47fa-861a-cdc0fdc8cbb3","added_by":"auto","created_at":"2026-03-06 22:09:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1339709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUMSCs-CM reduces high glucose-induced ferroptosis in HK-2 cells \u003c/strong\u003ea. HK-2 cells mitochondrial membrane potential detected by JC-1 probe, n=3; b. Total ATP level in HK-2 cells, n=3; c. mtDNA copy number in HK-2 cells, n=3; d-f. Protein expression of PGC1α and TFAM in HK-2 cells and quantitative analysis, n=3; g-i. MDA, GSH, and PCO level in HK-2 cells, n=3; j. Supernatant 8-OHdG level in HK-2 cells, n=3; k. Flow assay for ROS level in HK-2 cells; m-q. Protein expression of ferroptosis markers ACSL4, GPX4, SLC7A11, FTH1, and TFRC in HK-2 cells and quantitative analysis, n=3; \u003cem\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/em\u003e \u003cstrong\u003eNG:\u003c/strong\u003e Normal glucose; \u003cstrong\u003eMan:\u003c/strong\u003e Mannitol, osmotic control; \u003cstrong\u003eHG:\u003c/strong\u003eHigh glucose; \u003cstrong\u003eHG+CM:\u003c/strong\u003e High glucose plus UMSCs-CM.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8603066/v1/9e6176f3c2fc5b4034b4bc26.jpg"},{"id":104106665,"identity":"3c5d702f-6d66-4aaa-898b-19b2641d0791","added_by":"auto","created_at":"2026-03-06 22:09:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1650524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUMSCs exert therapeutic effects by inhibiting p-ERK/ERK activation in renal tissues with DN \u003c/strong\u003ea. GSEA analysis; b. GO analysis; c-e. Western blot detection of p-ERK/ERK, p-P38/P38 expression in rat kidney tissues and quantitative analysis of ratio, n=3; f. IHC detection of p-ERK expression in rat kidney tissues and quantitative analysis (Scale bar=100μm), n=3; g-i. Western blot detection of p-ERK/ERK, p-P38/P38 expression and quantitative analysis of ratio in HK-2 cells, n=3; \u003cem\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/em\u003e \u003cstrong\u003eNG:\u003c/strong\u003e Normal glucose; \u003cstrong\u003eMan:\u003c/strong\u003e Mannitol, osmotic control; \u003cstrong\u003eHG:\u003c/strong\u003e High glucose; \u003cstrong\u003eHG+CM:\u003c/strong\u003e High glucose plus UMSCs-CM.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8603066/v1/774b7cf8d8b6a236a9f514b9.jpg"},{"id":104106671,"identity":"9d421806-ad75-4ff6-a983-cef560038b01","added_by":"auto","created_at":"2026-03-06 22:09:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1286620,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUMSCs inhibit ferroptosis-ROS loop by suppressing p-ERK/ERK activation in HK-2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Flow assay of ROS levels in HK-2 cells after application of p-ERK inhibitor GSK2606414 (10μM) in HG group, n=3; b-e. Western blots analysis showing the expression change of p-ERK/ERK, ACSL4, GPX4 in HK-2 cells treated with p-ERK inhibitor GSK2606414 (10μM) and quantitative analysis, n=3; f. Western blot detection of p-ERK/ERK expression in HK-2 cells after application of p-ERK activator MK-28 (10nM) in UMSCs-CM-treated group and quantitative analysis of p-ERK/ERK ratio, n=3; g. MDA level in HK-2 cells, n=3; h. GSH level in HK-2 cells, n=3 i. PCO level in HK-2 cells, n=3; j. 8-OHdG level in HK-2 cell supernatant, n=3; k. Flow assay for ROS level in HK-2 cells, n=3; l-n. Western blot detection of ACSL 4, GPX4 protein expression in HK-2 cells and quantitative analysis, n=3; \u003cem\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/em\u003e \u003cstrong\u003eNG:\u003c/strong\u003e Normal glucose; \u003cstrong\u003eMan:\u003c/strong\u003e Mannitol, osmotic control; \u003cstrong\u003eHG:\u003c/strong\u003eHigh glucose; \u003cstrong\u003eHG+ GSK2606414:\u003c/strong\u003e High glucose plus p-ERK inhibitor GSK2606414 (10μM); \u003cstrong\u003eHG+CM:\u003c/strong\u003e High glucose plus UMSCs-CM; \u003cstrong\u003eHG+CM+MK-28: \u003c/strong\u003eHigh glucose plus UMSCs-CM and p-ERK activator MK-28 (10nM).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8603066/v1/89338891c991771766ae82ab.jpg"},{"id":104106678,"identity":"959e7a82-e190-4383-8165-47ab1d70771c","added_by":"auto","created_at":"2026-03-06 22:09:22","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":483110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular mechanism of UMSCs in the treatment of diabetic nephropathy\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8603066/v1/b01a6d162b8ee2cc2cec7a77.jpg"},{"id":104779426,"identity":"22afab93-a0a4-408a-a4e6-fc4c19569993","added_by":"auto","created_at":"2026-03-17 07:40:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11249063,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8603066/v1/42dbb6d6-f49c-43eb-bd1c-47058c042ec1.pdf"},{"id":104403256,"identity":"7cc6b282-510e-4a22-afc8-3d058b63a0ec","added_by":"auto","created_at":"2026-03-11 12:17:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6854226,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8603066/v1/97d3df99719ba39653612356.docx"},{"id":104403763,"identity":"6559220a-7d9a-4aa1-80e4-c8207ee3f8e4","added_by":"auto","created_at":"2026-03-11 12:19:00","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":248104,"visible":true,"origin":"","legend":"","description":"","filename":"ARRIVEChecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8603066/v1/73b1df1e869c876f0be3ce5a.pdf"}],"financialInterests":"Competing interest reported. Author S.P. is an employee of Cell Energy Life Sciences group CO. LTD; the other authors declare no conflict of interests.","formattedTitle":"Mesenchymal Stem Cells Attenuate Diabetic Nephropathy by Suppressing the ERK-ferroptosis-ROS axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes mellitus and is the leading cause of chronic kidney disease and end-stage renal disease globally [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Traditional perspectives considered DN a \"glomerulocentric\" disease. However, recent research has revealed that tubular injury may precede glomerulopathy and play a critical role in the early stages of DN [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The pathogenesis of DN encompasses multifactorial mechanisms, including lipid metabolism dysregulation, hemodynamic abnormalities, inflammation, oxidative stress, cellular damage, and ferroptosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Ferroptosis induces renal tubular epithelial cell death, triggering pathological cascades through damage-associated molecular pattern release - activating innate immunity, disrupting tubular reabsorption causing proteinuria, and promoting renal fibrosis via epithelial-mesenchymal transition [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Proximal tubular reabsorption critically demands iron for ATP production [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and dysregulated iron metabolism secondary to diabetic renal injury heightens oxidative stress and inflammatory responses, thereby potentiating renal damage [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The iron metabolism-oxidative stress vicious cycle constitutes the core mechanism underlying ferroptosis in renal tubular epithelial cells during DN. The above reports suggested the importance of ferroptosis in the pathological progression of diabetic nephropathy.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells (MSCs) have garnered increasing attention as a novel regenerative therapy for DN. The therapeutic efficacy of MSCs transplantation for DN has been established in numerous preclinical studies and demonstrates promising outcomes in early-phase clinical trials [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the clinical translation of these therapies remains challenging, primarily due to the heterogeneous therapeutic responses resulting from unresolved mechanistic uncertainties. MSC-based therapies ameliorate diabetic nephropathy primarily via paracrine and immunomodulatory mechanisms, including anti-inflammatory, antioxidant, antifibrotic, and cellular protective effects, alongside promotion of angiogenesis, mitochondrial transfer, and tissues repair [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Nevertheless, the precise regulatory mechanisms underlying these therapeutic effects remain incompletely elucidated.\u003c/p\u003e \u003cp\u003eBased on the established role of ferroptosis in driving renal tubular cell loss and dysfunction in diabetic nephropathy, we hypothesized that the beneficial effects of MSC therapy resulted from the modulation of tubular ferroptosis pathways. Recent studies report that MSCs and their derived exosomes mitigate acute multi-organ injury through ferroptosis regulation [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, how MSCs treat chronic metabolic diseases such as DN by modulating ferroptosis is still unclear. To address this gap, we investigated whether MSCs ameliorate diabetic kidney injury by targeting the iron metabolism-ROS-ferroptosis positive feedback loop in renal tubular epithelial cells. We assessed dose-dependent therapeutic efficacy and explored ferroptosis-related molecular regulatory mechanisms.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCulture and Conditioned Medium Collection from UMSCs\u003c/h2\u003e \u003cp\u003e The primary human umbilical cord-derived MSCs (UMSCs) were provided by Cell Energy Life Sciences group CO. LTD (Initial ethical approval by Ethics Committee of Liaocheng People\u0026rsquo;s Hospital, Approval No. 2021105, and the donors had signed informed consent.). UMSCs were identified as described previously [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). UMSCs of passage 4 or 5 were used in our experiments. Cells were cultured in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C and passaged with trypsin/EDTA after reaching the confluence.\u003c/p\u003e \u003cp\u003eOnce the UMSCs reached 70\u0026ndash;80% confluency, the medium was replaced with fresh full medium and harvested after 24h. Subsequently, UMSCs-CM were centrifuged at 3000 rpm for 20 min with 0.22\u0026micro;m filtration to remove detached MSCs and cell debris.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal model and treatment protocols\u003c/h3\u003e\n\u003cp\u003e Sprague-Dawley (SD) rats were provided by Spefo (Beijing) Biotechnology Co., Ltd., and all animal procedures followed guidelines approved by the Chinese PLA General Hospital Ethics Committee (Approval No. 2022-x18-39). After acclimation, male SD rats (5\u0026ndash;6 weeks old, 150\u0026ndash;180 g) were fed a high-fat diet for 6 weeks to induce insulin resistance, followed by a 12\u0026ndash;18 h fast and a single intraperitoneal injection of streptozotocin (STZ, 40 mg/kg). Blood glucose was monitored daily for three days. Rats with sustained glucose\u0026thinsp;\u0026ge;\u0026thinsp;16.7 mmol/L were considered type 2 diabetes (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea). Type 2 diabetic nephropathy (T2DN) was confirmed when the 24-hour urinary protein (PRO) excretion exceeded 30 mg (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb). On day 0, 35 rats meeting both criteria (GLU\u0026thinsp;\u0026ge;\u0026thinsp;16.7 mmol/L and 24-h PRO\u0026thinsp;\u0026gt;\u0026thinsp;30 mg) were selected as established T2DN models (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSubsequently, thirty of these T2DN rats were randomly selected and divided into 3 groups (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, n\u0026thinsp;=\u0026thinsp;10 per group): the DN model group (DN), which received no cell therapy; the low-dose umbilical cord mesenchymal stem cell group (UMSCs-LD), administered 2\u0026times;10⁶ cells per rat; and the high-dose group (UMSCs-HD), administered 5\u0026times;10⁶ cells per rat. Cell suspensions were delivered via intravenous injection once every two weeks, for a total of three injections. Meanwhile, both the DN model group and a normal control group of SD rats (Ctrl, n\u0026thinsp;=\u0026thinsp;9) received equal volumes of PBS via the same route and schedule. The timelines for model induction and treatment administration are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAnimal anaesthesia and euthanasia\u003c/h3\u003e\n\u003cp\u003eAll rats were euthanized at the designated experimental endpoints. Euthanasia was performed by inducing deep anesthesia via inhalation of 3% isoflurane (in 100% oxygen) in an induction chamber, followed by maintenance at this concentration until the cessation of breathing and cardiac function was verified. All procedures were performed in strict accordance with the animal welfare guidelines approved by the Chinese PLA General Hospital Ethics Committee. Anesthesia was not utilized at any other stage of the study.\u003c/p\u003e\n\u003ch3\u003eBiochemical test\u003c/h3\u003e\n\u003cp\u003e The 24-h urine samples were collected from rats at 4-week intervals up to week 20, and analyzed for urea nitrogen (BUN), 24-h urinary protein (PRO), and creatinine (Cr) levels in the supernatant according to the manufacturer's instructions (XR220PLUS, XinRui, China). Concurrently, blood samples were collected at 4-week intervals up to week 20 and analyzed for blood glucose, insulin, triglycerides (TG), and total cholesterol (CHOL) using Fully automatic biochemical analyzer (XR220PLUS, XinRui, China) according to the manufacturer's instructions.\u003c/p\u003e\n\u003ch3\u003ePathological staining\u003c/h3\u003e\n\u003cp\u003eRenal tissues were fixed in 4% paraformaldehyde and conventionally paraffin embedded, with 5 \u0026micro;m pathology sections then being created. These were then sequentially immersed in 100%, 95%, 70% and 30% ethanol for 2 min each for hydration, and then immersed in water for 2 min. HE staining was used to observe the basic structure of the glomerulus, PAS staining was used to observe changes in glomerular thylakoids and basement membranes, and Masson's staining was used to observe the degree of renal mesenchymal Masson's staining was used to observe the degree of interstitial fibrosis. All stained sections were reviewed by a pathologist blinded to the experimental conditions. Three random fields of view were selected from each slide, and quantitative analysis was performed using ImageJ software for objective assessment.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eKidney tissue sections were deparaffinized, rehydrated through graded ethanol and washed with PBS. Antigen retrieval was performed using 10 mM sodium citrate buffer for 20 min. Slides were fixed with 5% BSA for 1 hour at room temperature and then washed with PBS. For ACSL4 (22401-1-AP, Proteintech), GPX4 (67763-1-Ig, Proteintech), SLC7A11 (26864-1-AP, Proteintech), FTH1 (DF6278, Affinity), TFRC (AF8136, Beyotime, China), or p-ERK (4370S, Cell Signaling Technology, USA) primary antibodies were added to the slides and incubated overnight. Secondary antibodies were subsequently added and incubated at room temperature. DBA was then added for a 2-minute reaction, and the sections were reprobed with haematoxylin. The staining intensity was semiquantitatively analyzed using ImageJ software by evaluating at least three random 20\u0026times; fields per section, which were selected by an independent pathologist (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture and intervention\u003c/h3\u003e\n\u003cp\u003eThe human renal tubular epithelial cell line HK-2 was obtained from China Center for Type Culture Collection (CCTCC No. GDC0152, Wuhan, China) and cultured in MEM (C11095500BT, Gibco, USA) for serial subcultivation. Cells cultured in normal glucose (5 mM) for 72 h were designated the control group (NG). Cells cultured in high-glucose (30 mM) medium for 72 h were designated the high-glucose group (HG). Cells cultured in high-glucose (30 mM) medium plus UMSCs-CM for 72 h were designated the therapy group (HG\u0026thinsp;+\u0026thinsp;CM). Glucose (5 mM) plus mannitol (24.5 mM) was used as an osmotic control (Man).\u003c/p\u003e \u003cp\u003eAdditionally, to inhibit p-ERK activity, cells with HG medium were treated with 10nM GSK2606414 (HY-18072, MCE) for 72 h. To determine whether UMSCs trigger p-ERK/ERK pathway to inhibit ferroptosis, HG-cells were treated with UMSCs-CM and 10 \u0026micro;M MK-28 (HY-137207, MCE) for 72 h.\u003c/p\u003e\n\u003ch3\u003eELISA detection of 8-OHdG\u003c/h3\u003e\n\u003cp\u003eThe levels of 8-OHdG in kidney tissues and HK-2 cells were determined by ELISA kits (MM-0331H1, Meimian, Jiangsu, China) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMalondialdehyde (MDA) assay\u003c/h2\u003e \u003cp\u003eAccording to the MDA Assay Kit (S0131S, Beyotime), MDA levels in kidney tissues or HK-2 cells were measured after lysis and incubation using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGlutathione (GSH) assay\u003c/h2\u003e \u003cp\u003eTotal GSH content in tissues or HK-2 cells were assessed using the Reduced Glutathione Content Assay Kit (BC1175, Solarbio) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eProtein Carbonyl Content (PCO) Test\u003c/h2\u003e \u003cp\u003eThe levels of PCO in kidney tissues and HK-2 cells were determined by Protein Carbonyl Content Assay Kit (BC1275, Solarbio) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePrussian Blue Staining (Enhance With DAB)\u003c/h2\u003e \u003cp\u003eKidney tissues sections were deparaffinized, rehydrated with gradient ethanol, washed with PBS, stained with Prussian blue and subsequently reacted with the addition of DBA for 2 min, sections were re-stained with hematoxylin, sealed and observed under the microscope. For objective quantification, a blinded pathologist randomly selected three non-overlapping fields of view from each stained slide. Three fields of view were randomly selected from each stained slide by a blinded pathologist and quantified using ImageJ (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIron array\u003c/h2\u003e \u003cp\u003eTotal iron levels in tissues were assessed using a tissue iron assay kit (BC4355, Solarbio, China) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eWestern blotting was performed as previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Proteins were extracted from renal tissues or cultured cells using RIPA lysis buffer (P0013B, Beyotime, China) with protease and phosphatase inhibitors, homogenized on ice, and centrifuged at 13,500 \u0026times; g for 20 min at 4\u0026deg;C. Protein concentration was measured by BCA assay, and equal amounts (10 \u0026micro;g) were separated on SDS-PAGE and transferred to PVDF membranes. The membranes were then incubated overnight at 4\u0026deg;C with primary antibodies against PGC1α (ab317540, Abcam, USA), TFAM (22586-1-AP, Proteintech), ACSL4, GPX4, SLC7A11, FTH1, TFRC, p-ERK, ERK (4695S, Cell Signaling Technology), p-P38 (28796-1-AP, Proteintech), P38 (14064-1-AP, Proteintech), and β-actin (66009-1-Ig, Proteintech). After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Signals were detected by ECL and quantified using ImageJ. Target protein levels were normalized to β-actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eATP level\u003c/h2\u003e \u003cp\u003eFollowing the procedure of ATP Assay Kit (S0026, Beyotime), the ATP level of kidney tissues and HK-2 cells was detected by chemiluminescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Membrane Potential (MMP) assay\u003c/h2\u003e \u003cp\u003eHK-2 cells were harvested and incubated with the JC-1 probe following the manufacturer\u0026rsquo;s protocol provided with the mitochondrial membrane potential assay kit (M8650, Solarbio). Subsequently, mitochondrial membrane potential was assessed via flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMtDNA Copy Number\u003c/h2\u003e \u003cp\u003eGenomic DNA was isolated from kidney tissues or total cells pursuant to the instructions of the DNA extraction kit (DP304, TanGen, China). Subsequently, cycle threshold (Ct) values were acquired via quantitative real-time PCR (qPCR) performed with a standard system and procedure. Primer sequences are available in the Supplementary Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eROS level\u003c/h2\u003e \u003cp\u003eHK-2 cells were loaded with DCFH probe (S0033S, Beyotime) in situ according to the instructions and then incubated at 37\u0026deg;C for 15 min for staining, and photographed by laser confocal microscopy under microscopic observation or assessed via flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq and analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from rat kidney tissues using Trizol reagent (Invitrogen, USA) followed by RNeasy Mini Kit (Qiagen, USA) purification. rRNA-depleted RNA libraries were prepared with the NEBNext Directional RNA Library Prep Kit. First-strand cDNA was synthesized with random hexamers and M-MuLV reverse transcriptase, followed by second-strand synthesis using DNA Polymerase I and RNase H. Libraries were purified (AMPure XP beads), quality-checked (Agilent Bioanalyzer 2100), and clustered (cBot System, Illumina NovaSeq reagents). Paired-end sequencing was performed on an Illumina NovaSeq platform (Cnkingbio, China). Gene expression was quantified as FPKM. Differentially expressed genes were identified using DESeq2 (v1.30.0) with FDR correction (Benjamini-Hochberg method).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results were analyzed using GraphPad Prism software (version 10.1.2) and expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). Statistical analysis was performed as indicated in the figure legends. For two group comparison, Student's t-test was performed. For multiple-group comparison, one-way ANOVA analysis was performed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eUMSCs Mitigate Renal Pathological Manifestations in DN Rat Models\u003c/h2\u003e \u003cp\u003eTo investigate the therapeutic potential of UMSCs in DN, we established a T2DN rat model (GLU\u0026thinsp;\u0026ge;\u0026thinsp;16.7 mmol/L and 24-h PRO\u0026thinsp;\u0026gt;\u0026thinsp;30 mg) through a combination of high-fat diet for 6 weeks and intraperitoneal STZ administration. UMSCs were administered via triple tail vein injections (0, 2, and 4 week) at two dosage regimens: 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/injection (Low-dose group, UMSCs-LD, n\u0026thinsp;=\u0026thinsp;10) and 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/injection (High-dose group, UMSCs-HD, n\u0026thinsp;=\u0026thinsp;10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Biochemical results detected every four weeks during treatment are presented in Supplementary Fig. S4. At the 20-week endpoint, the DN cohort (n\u0026thinsp;=\u0026thinsp;10) exhibited marked elevations in fasting blood glucose, glycated hemoglobin (HbA1c), 24h-urinary protein excretion (PRO), urine creatinine (UCr), and urea nitrogen (BUN), all of which were substantially attenuated by UMSCs therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, d\u0026ndash;g). Although serum insulin levels were significantly reduced in the DN group, they returned to normal after UMSCs administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), indicating that UMSCs ameliorate renal function in diabetic nephropathy. Moreover, the significant reduction in serum lipids (triglycerides and cholesterol) in DN rats following UMSC administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh, i) suggests that lipid-lowering is a novel aspect of their pleiotropic effects, beyond their established roles in improving renal function.\u003c/p\u003e \u003cp\u003eHistopathological analysis revealed characteristic renal pathology in DN rats, including glomerular hypertrophy, tubular dilatation, and lipid vacuolization in proximal tubular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, k). PAS staining demonstrated tubular basement membrane thickening (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, l), while Masson trichrome staining confirmed progressive interstitial fibrosis involving glomerulotubular junctions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, m). Remarkably, UMSCs administration ameliorated these structural anomalies, restoring glomerular morphology and reducing collagen deposition by 40\u0026ndash;60% across histological metrics. To evaluate the long-term engraftment of the administered MSCs, we analyzed kidney tissues from DN rats at 20 weeks by immunofluorescence with an anti-human nuclear antigen antibody. No human cells were detected at this time point (Supplementary Fig. S5), indicating that the MSCs did not establish a persistent population in the renal niche under these conditions. Consequently, the sustained therapeutic benefits observed are unlikely to be mediated by direct cellular engraftment, suggesting the involvement of alternative mechanisms.\u003c/p\u003e \u003cp\u003eThese collective findings demonstrate the successful establishment of a pathologically relevant T2DN model, which recapitulates hallmark metabolic derangements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-i) and progressive nephropathy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej-m). The data further establish the potent nephroprotective efficacy of UMSCs therapy. This intervention significantly mitigated characteristic functional impairments, including proteinuria and azotemia. Concurrently, it ameliorated critical histopathological manifestations, such as glomerular hypertrophy and tubulointerstitial fibrosis. In conclusion, the study validates a robust T2DN model and conclusively shows that UMSCs confer renoprotection by simultaneously alleviating both functional and structural deteriorations in the diabetic kidney.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eUMSCs Attenuate Ferroptosis-related Mitochondrial Dysfunction in DN Renal Tissues\u003c/h2\u003e \u003cp\u003eFerroptosis is characterized by complex and dynamic mitochondrial damage. To investigate the role of mitochondrial dysfunction in the pathogenesis of DN, we first assessed mitochondrial function in renal tissues of diabetic nephropathy DN rats under high-glucose conditions. Results demonstrated significantly reduced ATP production in mitochondria isolated from DN rats, accompanied by decreased mitochondrial DNA (mtDNA) copy number (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). UMSCs treatment significantly restored mitochondrial function. Western blot analysis of mitochondrial biogenesis markers PGC1α and TFAM revealed suppressed expression of both proteins in DN rat kidneys. This suppression was reversed following UMSCs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u0026ndash;e), indicating that impaired mitochondrial biogenesis contributes critically to mitochondrial dysfunction during high glucose-induced renal damage, and that UMSCs may confer protection by enhancing mitochondrial biogenesis and functional recovery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMitochondria constitute the primary cellular source of ROS [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Exposure to metabolic stressors (e.g., hyperglycemia and inflammation) induces mitochondrial damage, triggering excessive ROS generation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] that promotes lipid peroxidation and accelerates ferroptosis. To investigate the role of oxidative stress in DN, we assessed renal oxidative stress markers in a rat model of DN. Compared with controls, DN rats exhibited significantly elevated levels of malondialdehyde (MDA), a terminal product of lipid peroxidation, along with depleted glutathione (GSH). Treatment with UMSCs markedly attenuated MDA accumulation and restored GSH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g). Protein Carbonyl Content (PCO) analysis demonstrated increased PCO content in DN renal tissues, which was reversed by UMSC intervention, indicating amelioration of high glucose-induced protein oxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Similarly, ELISA detected elevated 8-OHdG in DN kidneys, with UMSCs treatment significantly reducing this DNA oxidation biomarker (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Western blot analysis showed upregulated expression of ACSL4, a key enzyme in pro-ferroptosis lipid metabolism, in DN renal tissues. This elevation was attenuated following UMSCs administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej, k). Collectively, these results demonstrate a state of profound oxidative stress accompanied by enhanced lipid peroxidation in DN kidneys, strongly supporting the involvement of ferroptosis in the pathogenesis of DN.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eUMSCs Attenuate Ferroptosis in DN Rat Renal Tissues\u003c/h2\u003e \u003cp\u003eTo investigate the potential mitigating effects of UMSCs on ferroptosis in DN, we initially examined renal iron deposition, a hallmark of ferroptosis. Prussian blue staining demonstrated pronounced iron accumulation within the renal tissues of DN rats, which was markedly attenuated upon UMSCs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Subsequent quantitative analysis of renal iron content provided corroborative evidence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), confirming a state of renal iron overload and disrupted iron homeostasis in the diabetic kidney. The iron deposition was notably prominent within tubular epithelial cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the involvement of ferroptosis and the regulatory role of UMSCs, we evaluated the expression of key proteins governing ferroptosis pathways. IHC analysis revealed that renal tissues from DN rats exhibited substantially suppressed expression of the central ferroptosis inhibitors GPX4, SLC7A11, and FTH1, concomitant with a pronounced upregulation of the pro-ferroptosis iron transporter TFRC. These alterations were most evident within tubular compartments, underscoring the susceptibility of this segment to ferroptosis in DN. Systemic administration of UMSCs effectively restored the expression profiles of these markers toward normal levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Consistent with these findings, western blot analysis of renal protein confirmed the downregulation of GPX4, SLC7A11, and FTH1 protein expression and the upregulation of TFRC in DN kidneys. Importantly, UMSCs intervention significantly reversed these aberrant expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). These results confirm that ferroptosis is a key pathogenic mechanism in diabetic nephropathy, and umbilical cord-derived mesenchymal stem cells can effectively alleviate renal injury by regulating this pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eUMSCs-CM Mitigates High Glucose-Induced Ferroptosis in HK-2 Cells\u003c/h2\u003e \u003cp\u003eTubular injury constitutes a critical determinant of DN progression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To investigate the underlying mechanisms, we evaluated the effects of high glucose on ferroptosis in human renal tubular epithelial cells HK-2 in vitro. After HK-2 cells were exposed to high glucose (30 mM; HG) for 72 hours, we observed ferroptosis-associated mitochondrial dysfunction characterized by diminished mitochondrial membrane potential (MMP, ΔΨm, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Supplementary Fig. S6a), reduced ATP production, and decreased mtDNA copy number. UMSCs-conditioned medium (UMSCs-CM) treatment (HG\u0026thinsp;+\u0026thinsp;CM) effectively restored membrane potential, enhanced ATP synthesis, and increased mtDNA copy number (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c), indicating rescue of glucose-stressed mitochondrial function. Western blot analysis revealed suppressed expression of mitochondrial biogenesis regulators PGC1α and TFAM in HG-exposed HK-2 cells. UMSCs-CM treatment significantly upregulated both proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f and Supplementary Fig. S6b), suggesting that the therapeutic benefit of UMSCs-CM is likely mediated through the enhancement of mitochondrial biogenesis and the attenuation of high glucose-induced mitochondrial damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the established role of ferroptosis and mitochondrial dysfunction in high glucose-induced tubular injury, we further investigated the oxidative stress profile in HK-2 cells. Exposure of HK-2 cells to high glucose for 72 hours resulted in a profound redox imbalance, manifested as a significant elevation in the lipid peroxidation product MDA and a sharp decrease in the antioxidant GSH. Treatment with UMSCs-CM effectively reversed this HG-induced imbalance by attenuated MDA accumulation and normalized GSH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, h). Furthermore, PCO content, indicative of irreversible protein oxidation, was markedly elevated in HG-treated cells. UMSCs-CM intervention significantly attenuated this protein carbonylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Consistent with DNA oxidative damage, ELISA analysis of culture supernatants revealed elevated 8-OHdG levels in the HG group, which was substantially reduced upon UMSCs-CM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). Underlying these specific macromolecular damages, flow cytometry analysis demonstrated significant intracellular ROS accumulation in HG-exposed cells, an effect that was effectively mitigated by UMSCs-CM administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek and Supplementary Fig. S7b). Collectively, these results demonstrate that UMSCs-CM attenuates high glucose-induced oxidative stress in renal tubular cells by reducing lipid peroxidation, protein carbonylation, DNA oxidation, and overall ROS burden.\u003c/p\u003e \u003cp\u003eWestern blot analysis revealed dysregulated ferroptosis markers in HG-exposed HK-2 cells, with significantly elevated protein expression of ACSL4 and TFRC and suppressed expression of GPX4, SLC7A11, and FTH1(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el\u0026ndash;q and Supplementary Fig. S7c). These changes are consistent with the characteristic molecular profile of ferroptosis induction. Treatment with UMSCs-CM effectively reversed these expression changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el\u0026ndash;q and Supplementary Fig. S7c), indicating attenuation of HG-induced ferroptosis in renal tubular epithelial cells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eUMSCs Exert Therapeutic Effects via Suppression of the MAPK/ERK Pathway in DN renal tissues\u003c/h2\u003e \u003cp\u003eTo elucidate molecular mechanisms underlying ferroptosis inhibition and renal protection of UMSCs, we conducted bulk RNA sequencing of rat kidney tissues. GSEA and GO pathway enrichment analyses revealed that genes associated with ferroptosis, iron ion homeostasis, and lipid metabolism were significantly upregulated in DN renal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). Subsequent administration of UMSCs resulted in a significant downregulation of these pathogenic transcriptional profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b), a finding that aligns coherently with our prior phenotypic and functional observations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analysis of the transcriptomic data indicated a pronounced activation of the MAPK/ERK signaling pathway in DN kidneys, as evidenced by an increased p-ERK/ERK ratio, which was substantially mitigated following UMSCs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). As a key MAPK family member, ERK responds to growth factors, cytokines, and stress stimuli, regulating proliferation, differentiation, survival, and metabolism [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Based on these findings, we hypothesized that the therapeutic effect of UMSCs is mediated by inhibiting p-ERK/ERK activation. Western blot analysis of kidney lysates confirmed a significant increase in the phosphorylation levels of both ERK (p-ERK/ERK) and p38 (p-P38/P38) in the DN group compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e). Importantly, intervention with UMSCs effectively reversed these changes, normalizing the phosphorylation status of these key MAPK nodes. This suppressive effect of UMSCs on ERK activation was further corroborated by IHC, which revealed intensified p-ERK signals in DN renal sections that were markedly reduced upon UMSCs administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eTo validate the translational relevance of these findings, we established an in vitro model using HK-2 cells cultured in high glucose. Consistent with the in vivo data, high glucose induced the phosphorylation of ERK and P38, which was attenuated by UMSCs-CM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg\u0026ndash;i ). These results demonstrated that UMSCs modulated the MAPK/ERK signaling under diabetic conditions. Combined transcriptomic, proteomic, and histological evidence indicated that UMSCs suppressed aberrant MAPK/ERK activation in diabetic kidneys. Given the established role of ERK signaling in ferroptosis and renal injury, our findings suggest that UMSCs mitigate ferroptosis and delay DN progression largely through the inhibition of the MAPK/ERK pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eUMSCs Attenuate Ferroptosis-ROS loop via p-ERK/ERK Pathway Suppression in HK-2 cells\u003c/h2\u003e \u003cp\u003eTo pharmacologically validate the role of p-ERK/ERK activation in high glucose-induced renal tubular ferroptosis, we utilized the selective p-ERK inhibitor GSK2606414 at a concentration of 10 \u0026micro;M. As anticipated, treatment with GSK2606414 markedly attenuated HG-stimulated ROS overproduction in HK-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c). Concurrently, this inhibition of p-ERK signaling significantly suppressed the expression of the pro-ferroptosis protein ACSL4, while upregulating the key ferroptosis inhibitor GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u0026ndash;e). These data collectively confirm that pharmacological blockade of p-ERK effectively mitigates high glucose-induced ferroptosis in renal tubular cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further test pathway specificity in UMSCs-mediated protection, the p-ERK activator MK-28 (10 nM) was applied to UMSCs-CM-treated cells. MK-28 treatment effectively induced ERK phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef) and partially reversed the renoprotective effects of UMSCs-CM. Specifically, MK-28 co-treatment abolished the UMSCs-CM-mediated reductions in oxidative stress markers, including MDA, PCO, 8-OHdG, and intracellular ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg\u0026ndash;k). Furthermore, it diminished the restoration of GSH levels, indicating a compromised antioxidant capacity. In parallel, the activation of p-ERK attenuated the ferroptosis protection conferred by UMSCs-CM, as evidenced by a decrease in GPX4 expression and a concomitant increase in ACSL4 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003el\u0026ndash;n). Using both inhibitor and activator approaches, we established that UMSC-mediated protection against HG-induced tubular ferroptosis is achieved by suppressing the p-ERK/ERK pathway, which reduces downstream oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated the therapeutic efficacy of UMSCs in DN rats and elucidated the underlying molecular mechanisms. Under hyperglycemic conditions, pathological activation of the p-ERK/ERK signaling pathway in renal tubular epithelial cells drives ferroptosis through suppression of GPX4 and excessive ROS production, which results in oxidative damage to DNA, proteins, and lipids. UMSCs mitigate renal injury by inhibiting hyperglycemia-induced ERK phosphorylation, which in turn suppresses the ferroptosis-ROS feedback loop and attenuates oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsistent with previous findings [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], UMSCs demonstrated therapeutic efficacy in high-fat diet fed/STZ-induced T2DN rats. Both the low-dose (2 \u0026times; 10⁶ cells) and high-dose (5 \u0026times; 10⁶ cells) groups exhibited significant renoprotective effects, as evidenced by reduced blood glucose, elevated insulin levels, and improved renal function. However, no dose-dependent relationship was observed. This absence of dose-dependence aligns with studies showing UMSC-mediated renoprotection in T1DN mice [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and a phase 1 dose-escalation trial of allogeneic UMSCs for systemic lupus erythematosus (2\u0026times;10⁶ to 4\u0026times;10⁶ cells/kg) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, some animal models, such as chronic spinal cord injury in rats using lower doses (4\u0026times;10⁵ to 10⁶ cells), have reported dose-dependent MSC effects [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The lack of dose-dependence observed here may be attributable to the relatively high UMSC doses administered, potentially exceeding the threshold for such effects in this model. Pathological assessment confirmed that our T2DN rats exhibited early-stage DN, lacking hallmark features of advanced disease such as Kimmelstiel-Wilson nodules and glomerulosclerosis. This is concordant with recent evidence indicating that tubular injury precedes and may be more critical than glomerular damage in DN progression [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Notably, we identified a significant lipid-lowering effect of UMSCs in our T2DN rat model. These T2DN rats displayed both hyperglycemia and dyslipidemia, yet UMSCs administration effectively reduced serum levels of triglycerides and total cholesterol, and concurrently decreased blood glucose. This finding suggests UMSCs mitigate diabetic kidney injury through multifaceted mechanisms, including potential direct modulation of lipid metabolism, and highlights their possible utility in clinical lipid management strategies.\u003c/p\u003e \u003cp\u003eOur results demonstrate that DN rats exhibit renal tubular epithelial cell iron overload, mitochondrial dysfunction, and excessive oxidation affecting DNA, proteins, and lipids, culminating in ferroptosis. UMSCs ameliorated these abnormalities\u0026mdash;including iron overload, mitochondrial dysfunction, elevated ROS, and ferroptosis\u0026mdash;indicating their multi-target therapeutic potential. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], is critically dependent on mitochondrial damage, which serves as both an initiating event and an amplifier of the process [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In chronic hyperglycemia, mitochondria undergo progressive damage [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], promoting excessive ROS production and heightened oxidative stress in renal tubular epithelial cells. Mitochondrial iron accumulation, dysfunctional respiratory chain-driven ROS generation, PUFA-rich membrane structures (notably phosphatidylserine), and inactivation of the key antioxidant enzyme GPX4 collectively establish a microenvironment permissive to mitochondrial ferroptosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. ROS-induced lipid peroxidation, particularly of phosphatidylserine, leads to mitochondrial structural damage and functional collapse, thereby amplifying ferroptosis throughout the cell [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. UMSCs exert their therapeutic effect by targeting multiple nodes within this pathogenic cascade, interrupting the 'mitochondria-iron overload-ROS-ferroptosis' axis to mitigate mitochondrial damage and suppress excessive ROS production.\u003c/p\u003e \u003cp\u003eOur study identifies a critical and novel role for the MAPK/ERK pathway in mediating ferroptosis within renal tissues of DN rats treated with UMSCs. Current understanding indicates ferroptosis is regulated via key signaling axes (e.g., System Xc⁻-GSH-GPX4, ACSL4/LOX) modulated by pathways including AMPK, Akt/mTOR, MAPK, and p53/NRF2/ATF4 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. While the MAPK pathway typically regulates ferroptosis through oxidative stress-induced JNK/p38 activation\u0026mdash;promoting ACSL4 expression or Lysyl Oxidase activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u0026mdash;ERK has been shown to promote ferroptosis in some cancers by inhibiting System Xc⁻ [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In this study, RNA-seq analysis revealed that UMSCs suppress ferroptosis by inhibiting MAPK/ERK pathway. Pharmacological intervention in HG-injured HK-2 cells using the ERK phosphorylation inhibitor GSK2606414 or the ERK activator MK-28 demonstrated that ERK inhibition effectively attenuated HG-induced ROS overproduction and ferroptosis. Conversely, ERK activation diminished the therapeutic efficacy of UMSCs against HG-induced oxidative stress and ferroptosis. These findings indicate UMSCs mitigate ferroptosis in renal tubular epithelial cells by downregulating ERK and P38 phosphorylation within the MAPK pathway. By downregulating this pathway, the antioxidant system is activated, which in turn suppresses excessive ROS generation, limits the accumulation of MDA and 8-OHdG, and elevates levels of GSH and GPX4.Consequently, UMSCs inhibit HG-induced ferroptosis, ultimately alleviating renal injury. These data underscore the therapeutic potential of ERK inhibition in DN. Although ERK inhibitors are extensively investigated in oncology, clinical translation is hindered by compensatory feedback mechanisms, pathway reactivation, target-related toxicity, selectivity issues, and drug resistance, confining them primarily to preclinical and clinical trial phases [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Notably, ERK inhibition can trigger robust negative feedback, activating upstream nodes (e.g., RAF) or parallel pathways [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], limiting monotherapy efficacy and necessitating combination strategies. Our results suggest exploring combination therapies integrating ERK inhibitors with UMSCs, potentially offering novel avenues for cell therapy development and ERK-targeted drug design in DN.\u003c/p\u003e \u003cp\u003eIn addition, our results demonstrate that no human cells were detected in kidney tissues at 20 weeks after MSC transplantation (Supplementary Fig. S5). The absence of long-term engraftment suggests that the sustained therapeutic effect of UMSCs is not dependent on their direct differentiation or persistent presence within the kidney. Instead, these findings support the concept that UMSCs exert beneficial effects through transient yet potent secretory activities, thereby modifying the host cellular microenvironment and signaling landscape [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. This mechanism is increasingly recognized in stem cell therapy, wherein exosomes, cytokines, and growth factors mediate the cross-talk between MSCs and injured tissues [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Several study limitations should be acknowledged. First, the specific molecular mediators released by UMSCs that suppress ERK activation have yet to be elucidated. Future analyses of the UMSCs secretome under hyperglycemic conditions may provide valuable insights. Second, although this study emphasizes the ERK\u0026ndash;ferroptosis pathway, other signaling pathways such as TGF-β/Smad and NF-κB are also implicated in DN [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and may be influenced by UMSCs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], requiring further exploration.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study elucidates the molecular mechanisms underlying UMSC efficacy, moving beyond descriptive accounts. Specifically, we demonstrate that UMSCs protect renal parenchymal cells by inhibiting MAPK/ERK phosphorylation, thereby disrupting the ferroptosis-ROS feedback loop. The mechanistic insight not only validates UMSC-based therapy for DN but also highlights the ERK/ferroptosis axis as a promising target. Consequently, targeting this pathway, whether through cell therapy or paracrine mediators, thus holds substantial translational potential.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDiabetic nephropathy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMesenchymal stem cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eT2DN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eType 2 diabetic nephropathy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUMSCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman umbilical cord-derived MSCs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRNA-seq\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRNA sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPX4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutathione peroxidase 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACSL4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcyl-CoA synthetase long-chain family member 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUMSCs-CM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUMSCs-conditioned medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTZ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStreptozotocin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGLU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood glucose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePRO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e24-hour urinary protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBUN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUrea nitrogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePRO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e24h-urinary protein excretion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUCr\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUrine creatinine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etriglycerides\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCHOL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal cholesterol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSLC7A11\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSolute Carrier Family 7 Member 11\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFRC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransferrin Receptor 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFTH1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFerritin Heavy Chain 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e8-OHdG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e8-Hydroxy-2'-deoxyguanosine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutathione\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein carbonyl\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial membrane potential\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMtDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGC1α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFAM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial transcription factor A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eContributions\u003c/h2\u003e \u003cp\u003eQ.H., X.B., and R.C.Z. conceived and supervised the project. S.P. provided the primary UMSCs and contributed to the experimental design. S.M. and J.L. performed the experiments. All authors contributed to data analysis and interpretation. S.M., J.L., and R.C.Z. drafted the original manuscript and prepared the figures. All authors reviewed and revised the manuscript.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eUMSCs used in this study were provided by Cell Energy Life Sciences group CO. LTD.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAnimal Ethics declaration\u003c/h2\u003e \u003cp\u003e The animal experiment has been approved by the Animal Experimental Ethics Review Committee of the Chinese PLA General Hospital Ethics Committee (Title of the approved project: Research on the Mechanisms of Kidney Injury Repair. Approval No. 2022-x18-39. Date of approval: March 6, 2022).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of interests\u003c/h2\u003e \u003cp\u003eAuthor S.P. is an employee of Cell Energy Life Sciences group CO. LTD; the other authors declare no conflict of interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eAuthor S.P. is an employee of Cell Energy Life Sciences group CO. LTD; the other authors declare no conflict of interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by State Key Laboratory of Common Mechanism Research of Major Diseases Platform.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQ.H., X.B., and R.C.Z. conceived and supervised the project. S.P. provided the primary UMSCs and contributed to the experimental design. S.M. and J.L. performed the experiments. All authors contributed to data analysis and interpretation. S.M., J.L., and R.C.Z. drafted the original manuscript and prepared the figures. All authors reviewed and revised the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors declare that they have not use AI-generated work in this manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eInternational Diabetes Federation. IDF Diabetes Atlas, 10th edn. 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Cell Communication Signal. 2023;21(1):200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K, Zhang X, Fan Y, Zhou L, Duan Y, Li S, et al. Reactivation of MAPK-SOX2 pathway confers ferroptosis sensitivity in KRASG12C inhibitor resistant tumors. Redox Biol. 2024;78:103419.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBahar ME, Kim HJ, Kim DR. Targeting the RAS/RAF/MAPK pathway for cancer therapy: from mechanism to clinical studies. Signal Transduct Target Therapy. 2023;8(1):455.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia-Aponte OF, Kahlenberg S, Kouroupis D, Egger D, Kasper C. Effects of Hydrogels on Mesenchymal Stem/Stromal Cells Paracrine Activity and Extracellular Vesicles Production. J Extracell Vesicles. 2025;14(3):e70057.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnsari Z, Chaurasia A, Neha, Sharma N, Bachheti RK, Gupta PC. Exploring inflammatory and fibrotic mechanisms driving diabetic nephropathy progression. Cytokine Growth Factor Rev. 2025;84:120\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal stem cells, ferroptosis, Oxidative stress, Renal tubular injury, MAPK/ERK pathway","lastPublishedDoi":"10.21203/rs.3.rs-8603066/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8603066/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDiabetic nephropathy (DN) is a major cause of end-stage renal disease with limited therapeutic options. As ferroptosis is a key mechanism of renal tubular injury in DN, this study investigates whether mesenchymal stem cells (MSCs) transplantation alleviates DN by inhibiting this form of cell death, although its precise mechanisms remain incompletely understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo investigate the therapeutic efficacy and mechanisms of human umbilical cord-derived MSCs (UMSCs) in diabetic nephropathy, we established a rat model of type 2 DN (T2DN) using a high-fat diet and streptozotocin. Oxidative stress was assessed via measurements of DNA, protein, and lipid oxidation. To elucidate the underlying mechanisms, RNA sequencing (RNA-seq) was performed to investigate the renal protective effects of UMSCs.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUMSCs treatment significantly improved renal function and alleviated tubular injury in DN rats, concomitant with reduced mitochondrial dysfunction, iron overload, reactive oxygen species (ROS) accumulation, and ferroptosis. In vitro, UMSCs suppressed high glucose-induced mitochondrial dysfunction, oxidative stress and ferroptosis in renal tubular cells. RNA-seq and experimental findings identified the MAPK/ERK pathway as essential for this protection, confirmed by pharmacological activation/inhibition of p-ERK/ERK.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTargeting the ERK-ferroptosis-ROS axis in renal tubular epithelial cells represents a novel therapeutic strategy for DN. This strategy is supported by our finding that MSCs confer protection specifically by disrupting the p-ERK/ERK-GPX4/ACSL4 axis, thereby preventing glutathione depletion and lipid ROS accumulation.\u003c/p\u003e","manuscriptTitle":"Mesenchymal Stem Cells Attenuate Diabetic Nephropathy by Suppressing the ERK-ferroptosis-ROS axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 22:09:13","doi":"10.21203/rs.3.rs-8603066/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"965cace9-9fe5-4e5e-b4cf-a66747a8ec7a","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-06T22:09:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 22:09:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8603066","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8603066","identity":"rs-8603066","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}