Alterations in cell cycle and MAPK pathway contribute to transition from SMF-associated acute kidney injury to fibrosis: Field direction matters

Alterations in cell cycle and MAPK pathway contribute to transition from SMF-associated acute kidney injury to fibrosis: Field direction matters | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Alterations in cell cycle and MAPK pathway contribute to transition from SMF-associated acute kidney injury to fibrosis: Field direction matters Seong Min Lee, Saram Lee, Seong Joon Park, Kyu Hong Kim, Sunhwa Lee, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6392601/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Various mechanisms, including inflammation, oxidative stress, and apoptosis, are involved in the transition from acute kidney injury to chronic kidney disease (AKI − to − CKD). In this study, we aimed to determine the pathway linking acute injury and fibrosis under static magnetic fields (SMFs). Human tubular epithelial cells (hTECs) were cultured on SMF platforms (119 mT; outward vs. inward direction) for 3 days, followed by treatment with adenine and p38 MAPK inhibitor to verify the role of MAP-kinase pathway. We orally administered 2 mg of adenine to mice daily for 14 days (adenine-induced tubular nephropathy; AITN). Phospho-p38 was significantly elevated in hTECs cultured under inward SMFs compared with that cultured under outward SMFs. Inhibition of p38 MAPK reduced G1/S arrest and oxidative stress, exerted anti-apoptotic effects, and downregulated the expression of fibrosis markers under inward SMFs. Deposition of F4/80-positive cells, IL-17R, p53, and p38 was significantly increased in AITN mice. p38 MAPK inhibition under inward SMFs led to a decrease in fibronectin expression in adenine-treated hTECs. This study revealed that SMF-related AKI − to − CKD transition progresses with the direction of SMFs affecting the severity of injury, whereas p38 MAPK inhibition attenuates SMF-induced kidney injury and prevents fibrosis. Biological sciences/Cell biology/Cell death/Apoptosis Health sciences/Nephrology/Kidney diseases/Renal fibrosis AKI − to − CKD cell cycle mitogen-activated protein pathway static magnetic field Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The increasing utilization of magnetic resonance imaging (MRI) for diagnosing diseases has triggered heightened concerns regarding the potential effects of magnetic fields on human health. MRI machines typically used in hospitals have magnetic field strengths ranging from 3 to 9.4 Tesla [ 1 ]. Recently, to achieve higher resolutions, MRI machines with even stronger magnetic field strengths have been developed. Milovanovich et al. reported that an upward-oriented static magnetic field (SMF) induced brain edema and increased spleen cellularity, whereas the downward-oritented SMF led to liver inflammation and a reduction in serum granulocyte levels [ 2 ]. However, in a cisplatin-induced nephrotoxicity model, moderate SMFs at hundreds of mT attenuated kidney injury by reducing oxidative stress and inflammation [ 3 ]. The effects of SMFs can be variable according to their particular characteristics including shapes, strength, and directions, and cell types. The exact mechanisms also remain inconclusive owing to the complicated effects of magnetic fields on biological systems. Acute kidney injury (AKI) affects 5–10% of hospitalized patients, leading to kidney tissue damage, elevated blood creatinine levels, and reduced urine output [ 4 , 5 ]. Over the past decade, increasing evidence has indicated that AKI is a significant contributor to the development of chronic kidney disease (CKD) [ 6 ]. The AKI − to − CKD transition involves complex mechanisms primarily driven by maladaptive repair processes, including cell cycle arrest, inflammation, and fibrosis [ 6 ]. The p38 mitogen-activated protein kinase (MAPK), a serine/threonine protein kinase, is involved in crucial intracellular signal transduction pathways and plays a significant role in the pathogenesis of fibrosis [ 7 ]. Upon cellular stress, p38 MAPK can translocate to the nucleus and subsequently induce the activation of transcription factors contributing to the production of proinflammatory mediators [ 8 ]. Previous studies have demonstrated that inhibiting p38 MAPK may protect kidneys from injury and attenuate disease progression in various models [ 8 – 10 ]. p38 MAPK activation was reportedly associated with kidney interstitial fibrosis in a UUO mouse model, and the anti-fibrotic effect of p38 MAPK inhibition has been confirmed [ 10 ]. To our knowledge, there have been no reports regarding the differential effect of SMF directions on kidney tissues and proper damage mechanisms. In this study, we established an SMF-associated AKI model and observed the effects of field directions. Furthermore, we evaluated the role of p38 MAPK in AKI − to − CKD progression in a mouse model. Results SMFs induce tubular epithelial cell injury, especially under the inward direction Simulations were conducted under two conditions by changing the polarity of the magnets: outward, where the cells receive magnetic force from the bottom up, and inward, where the cells receive magnetic force from the top down. In the outward condition, the maximum value of magnetic flux density was approximately 121.4 mT in the x-y plane 1 mm above the magnet (the plane where the cells were located). Within the range of the magnet, the maximum magnetic flux density of approximately 121.4 mT was observed at the tip of the magnet, whereas the minimum magnetic flux density of approximately 105.6 mT was observed at the center of the magnet (the difference of 13.0%). Thesimulation results for the inward condition were identical to those for the outward condition, with the sole difference being the reversal of the direction of the magnetic flux density vector. Vertically upward and downward SMFs formed an outward and inward direction of SMFs, respectively (Fig. 1 a, b). To verify the reliability of the magnetic field simulation results, the simulated magnetic flux density values at each location were compared with the actual magnetic field measurements. The magnetic field strengths measured at 15, 30, and 45 mm along the y-axis of the magnet were approximately 114.8, 110, and 112.1 mT, respectively, and the simulation results at those points were 112.0, 106.1, and 112.1 mT, respectively, with an average error of approximately 2.0% between the measured and simulated results, i.e., the simulation results were considered reliable. Using a magnetic field analyzer, the intensity of the SMFs was measured to be approximately 119 mT under both orientations. We initially investigated the effects of SMFs at both early and late stages by culturing with SMFs for either 3 or 7 days. By the seventh day, the vast majority of cells were dead. Although the expression of kidney injury marker (NGAL) did not change significantly after 3 days of exposure to SMFs, we noticed an elevation in fibrosis markers (collagen 1, fibronectin, and VEGFR) and increase in pp38 protein levels. There was a pronounced increase in pp38 under inward SMFs compared with that under outward SMFs, suggesting that the modulation of p38 MAPK pathway by inward SMFs contributes to fibrosis (Fig. 1 c, d). The immunofluorescence assay revealed that exposure to both outward and inward SMFs for 3 days led to decreased expression of intracellular junction marker (E-cadherin) and increased levels of HIF-1α. This suggests that SMFs induced AKI-to-CKD transition by regulating inflammation and oxidative stress through HIF-1α signaling. HIF-1α elevation implies oxygen deprivation in cells exposed to SMFs (Fig. 1 e). Afterward, the mRNA expression of CDK4 and cyclin D1, key regulators of the G1/S phase in the cell cycle, was analyzed. CDK4 expression decreased under both inward and outward SMFs compared with that under the control, with no significant difference observed between the two conditions. However, cyclin D1 expression was significantly lower under inward SMFs than under outward SMFs (Fig. 1 f). In line with the significant increase in pp38 expression and decreased levels of cyclin D1 under inward SMFs, these findings imply that exposure to inward SMFs induced tubular epithelial injury by modulating the p38 MAPK pathway and inducing G1/S phase arrest. In particular, the direction of SMFs exerts a significant effect on these changes. p38 MAPK inhibitor protected against tubular damage induced by inward SMFs To examine the therapeutic potential of p38 MAPK inhibition in tubular epithelial cells exposed to inward SMFs, we treated human primary tubular epithelial cells (hTECs) with iP38 (0.1 µM, 1 µM) while concomitantly exposing them to inward SMFs for 3 days. p38 MAPK inhibitor treatment considerably reduced the expression of OGG1, a marker of DNA damage and oxidative stress, compared with inward SMFs alone. Additionally, fibrosis markers such as periostin, α-SMA, and fibronectin markedly increased with inward SMF exposure but iP38 treatment attenuated this increase (Fig. 2 a, b). The mRNA expression of CDK4 was downregulated by inward SMFs but was recovered following iP38 treatment. The increased OGG1 mRNA expression under inward SMFs also decreased after iP38 treatment (Fig. 2 c). This indicates that p38 MAPK inhibition prevented cell cycle arrest in the G1/S phase induced by inward SMFs. Cells in the G1 phase significantly increased under inward SMFs compared with those in the control group (51.57 ± 0.68 vs 66.05 ± 0.97, *** P < 0.001; control vs inward SMFs). However, the increase was reduced with iP38 treatment of 0.1 µM and 1 µM, respectively (66.05 ± 0.97 vs 62.7 ± 1.02, * P < 0.05, 59.35 ± 2.64, * P < 0.05; inward SMFs vs inward SMFs + iP38 0.1 µM, inward SMFs + iP38 1 µM). Additionally, cells in the sub G1 phase, indicating an increase in apoptotic cells, increased by 2.85-fold following inward SMF exposure compared with those in the control group (1.3 ± 0.15 vs 3.72 ± 0.04, *** P < 0.001; control vs inward SMFs). iP38 treatment significantly decreased the proportion of cells in the sub G1 phase in a dose-dependent manner (3.72 ± 0.04 vs 3.52 ± 0.05, ** P < 0.01, 2.68 ± 0.05, *** P < 0.001; inward SMFs vs inward SMFs + iP38 0.1 µM, inward SMFs + iP38 1 µM). Correspondingly, the p38 MAPK inhibitor mitigated the rise in apoptotic cells caused by inward SMFs, with a particularly notable reduction in early apoptotic cells (Fig. 2 d). Overall, these results suggest that inhibiting p38 MAPK may reduce the tubular epithelial injury driven by oxidative stress and G1/S arrest induced by inward SMFs. Inhibition of p38 MAPK mitigated fibrosis in adenine-induced tubular nephropathy (AITN) model To mimic CKD progression, we established an AITN model by administering adenine orally to mice daily for two weeks (Fig. 3 a). Adenine treatment significantly elevated the serum levels of blood urea nitrogen (BUN) and creatinine in ATIN models to 138.34 ± 4.03 mg/dL (*** P < 0.001) and 1.23 ± 0.07 mg/dL (*** P < 0.001), respectively (Fig. 3 b). Kidney injury was observed in the renal cortex tubules of mice exposed to adenine, characterized by the enlargement of the basement membrane and loss of the brush border. Moreover, Masson's trichrome staining revealed the advancement of tubular atrophy and interstitial fibrosis. NGAL expression and F4/80 + macrophage infiltration were increased in the adenine-treated group compared with those in the sham group. The upregulation of pro-inflammatory markers such as ICAM-1, IL-17R, and the apoptosis marker p53 was also observed, indicating the involvement of inflammation and programmed cell death in the progression of renal injury and fibrosis following adenine administration (Fig. 3 c, d). The protein levels of pp38 and KIM-1, markers indicating the progression of AKI − to − CKD transition, increased after adenine treatment (Fig. 3 e). To identify the effect of p38 MAPK on the inhibition of fibrosis in the AITN model under inward SMFs, we treated hTECs with adenine and a p38 MAPK inhibitor. Inward SMFs significantly increased the expression of fibronectin. Under conditions without SMF exposure, the increased expression of fibronectin caused by adenine treatment was not mitigated by inhibiting p38 MAPK. However, under inward SMFs, there was a notable reduction when treated with the p38 MAPK inhibitor in a dose-dependent manner (Fig. 3 f). Discussion In this study, we aimed to identify the pathway linking acute renal tubule injury under SMFs to fibrosis. First, we found that exposure to SMFs triggers tubular epithelial injury, which leads to fibrosis by altering the p38 MAPK pathway and inducing G1/S phase arrest. In addition, the direction of SMFs plays a significant role in these effects. Second, we confirmed the involvement of the p38 MAPK pathway by demonstrating the protective effect of its inhibition against SMF-induced tubular damage, as evidenced by reduced cell cycle arrest, oxidative stress, apoptosis, and decreased expression of fibrosis markers. Third, we identified the increased expression of pp38 and kidney injury markers in a mouse AITN model. Furthermore, we observed a remarkable reduction in fibronectin expression through p38 MAPK inhibition in an in-vitro model of adenine treatment under inward SMFs. Overall, the study highlights the detrimental effects of SMF exposure on tubular epithelial cells, implicating the p38 MAPK pathway and cell cycle arrest in renal fibrosis, and suggests that the direction of magnetic fields plays an important role in the overall changes. Kidney fibrosis is widely recognized as a common pathological consequence of CKD. When kidneys are injured, local pericytes and fibroblasts activate, secreting inflammatory mediators and synthesizing extracellular matrix components such as collagens and fibronectin [ 11 ]. Fibrosis develops with persistent accumulation of extracellular matrix proteins in cases of severe damage, accelerating the advancement of CKD through TGF-β signaling [ 12 ]. Tissue hypoxia is common in CKD, and hypoxic signaling primarily involves HIFs, whose stability is increased under low-oxygen conditions owing to reduced prolyl hydroxylase-mediated degradation [ 13 ]. HIF-1α, a transcription factor, promotes collagen accumulation and facilitates the epithelial-to-mesenchymal transition [ 14 , 15 ]. Overexpression of HIF-1α in tubular epithelial cells promotes interstitial fibrosis in 5/6 nephrectomy mice [ 16 ], whereas silencing gene expression of HIF-1α reduced TGF-β induced epithelial-to-mesenchymal transition and angiotensin II-induced profibrotic effects in kidney cells [ 17 ]. Our experiment resulted in the upregulated expression of fibrosis markers and HIF-1α as well as the decreased expression of E-cadherin under inward SMFs, which is in line with previous studies. HIF-1α contributes to inflammation, kidney damage, and fibrosis, which results in AKI − to − CKD transition [ 18 ]. Reduced levels of E-cadherin are also associated with renal fibrosis and CKD progression [ 19 , 20 ]. In this study, we utilized the SMF platform to induce tubule injury and fibrosis. SMFs can induce various cellular effects, including cell proliferation, cell viability, and the cell cycle [ 21 , 22 ]. Previous studies have reported that the cellular effects induced by SMFs vary according to the intensity and the direction of magnetic fields as well as cell types [ 23 , 24 ]. Tian et al. discovered that upward SMFs of 0.2–1T could effectively decrease the cell numbers of human tumor cell lines MCF7 and GIST-T1, whereas downward SMFs did not produce a notable impact. Interestingly, the leukemia cell numbers were reduced by both upward and downward SMFs. This study also revealed that effects of SMFs were dependent on the direction of SMFs. The intensity in both vertically upward and downward SMFs was not different at all, which was beyond expectations. Vertically upward orientation resulted in the outward direction of SMFs, whereas vertically downward orientation resulted in the inward direction of SMFs. Inward SMFs upregulated the expression of fibrosis markers, pp38, and HIF-1α, whereas it decreased the expression of E-cadherin. Further research in various orientations of SMFs is necessary to clarify the role of SMF direction on renal tubular cells. The precise mechanisms underlying the different effects of SMF directions remain unclear [ 3 , 25 ]. Yu et al. [ 3 ] showed that although both upward and downward SMFs reduce oxidative stress in the kidney, downward SMFs offer stronger protective effects by reducing kidney inflammation, apoptosis, and cisplatin accumulation via decreased Oct2 levels; however, the precise mechanisms behind these effects remain elusive. Our findings suggest that exposure to inward SMFs upregulates the p38 MAPK pathway and induces G1/S phase arrest, resulting in tubular epithelial injury and subsequent fibrosis. Phosphorylated p38 MAPK, an essential pro-inflammatory element, is recognized for upregulating cytokines such as IL-6 and TNFα [ 26 ]. In the p38 MAPK pathway, the second messenger, reactive oxygen species (ROS) modulates MAPK activation via a positive feedback mechanism. This process, involving ROS generation and p38 activation, further enhances p53-mediated apoptosis [ 27 ]. AKI can lead to DNA damage caused by oxidative stress from ROS [ 28 ]. Under oxidative stress, 8-oxo-G is generated in DNA and subsequently released during repair by the DNA glycosylase OGG1 [ 29 ]. The expression of the OGG1 gene is likely upregulated in response to oxidative stress, which aligns with our observations. Activation of p38 by cellular stress commonly leads to cell cycle arrest or apoptosis [ 30 ]. Several reports have been shown that p38 activation results in G1 arrest [ 31 , 32 ]. AKI-induced DNA damage promotes the production of p21, which halts the cell cycle from G1 to S phase by binding to CDK4 and inhibiting the Cyclin D1/CDK4 complex. This action not only arrests cell cycle progression but also triggers the p53-dependent apoptosis pathway [ 33 , 34 ]. Suppressing p38 MAPK activity has been found to moderately decelerate disease progression. Li et al. and An et al. showed an elevation in pp38 MAPK after disease induction in both in vitro and in vivo models, alongside a dose-dependent reduction in fibrosis markers with the addition of a p38 MAPK inhibitor [ 35 – 37 ]. Lee et al. revealed a significant increase in phosphorylated p38 MAPK activity in a UUO mouse model and showed that inhibiting p38 MAPK led to a decrease in the mRNA expression of fibrosis markers [ 10 ]. Based on these findings, we examined the protective effects of p38 MAPK inhibition in inward SMF-induced tubular damage. Blockade of p38 MAPK not only reduced cell cycle arrest and apoptosis but also decreased the expression of fibrosis markers. This supports prior findings that the aggravation in kidney injury and fibrosis is, particularly mediated by the p38 MAPK pathway. To confirm the role of the p38 MAPK pathway in acute renal tubular injury and fibrosis, we established an adenine-induced nephrotoxicity model. When metabolized to 2,8-dihydroxyadenine (2,8-DHA), adenine accumulates crystal deposits in the renal tubules, contributing to CKD [ 38 ]. Orally ingested adenine tends to accumulate more extensively than other purines and has been frequently used to induce gradual kidney damage [ 38 ]. Adenine is rapidly metabolized to DHA, leading to crystal formation in the proximal tubule, which closely resembles human CKD [ 39 ]. Adenine treatment resulted in increased levels of kidney function biomarkers and profibrogenic markers [ 40 , 41 ]. This condition is characterized by diminished renal function, tubular dilation, infiltration of macrophages, and fibrosis [ 42 , 43 ]. In line with these observations, we validated these findings with histological analysis, indicating a significant increase in expression of ICAM-1, F4/80, NGAL, IL-17R, and p53. Furthermore, we assessed the effect of p38 MAPK inhibition of fibrosis in an in vitro AITN model under inward SMF exposure. This study opens up several avenues for further investigation. First, the effects of dynamic magnetic fields on tubular epithelial cells remain unexplored and will be a focus of future research. Second, the impact of inward SMFs and the inhibition of p38 MAPK in the AITN mouse model has not yet been fully confirmed, providing another important direction for subsequent studies. Acknowledging the scope for further exploration, we anticipate addressing these aspects in upcoming research efforts. In summary, our study reveals that acute tubular cell injury under inward SMFs, associated with inflammation, oxidative stress, and apoptosis, progresses to chronic fibrosis through maladaptive repair mechanisms, including G1/S cell cycle arrest and activation of the MAP kinase pathway (Fig. 4 ). Additionally, inhibiting p38 MAPK effectively mitigated inward SMF-induced kidney injury, preventing the progression to fibrosis, which is a common final pathway in the development of CKD. These findings indicate that targeting the p38 MAPK pathway may offer a promising therapeutic approach to control the progression from inward SMF-associated AKI − to − CKD. Methods Establishment and measurement of strength and directions of SMFs Human proximal tubular cells were placed between two ferrite magnets (length × width × height: 60 × 30 × 10 mm) positioned in different orientations: vertically upward SMFs opposing gravity vs. vertically downward SMFs aligning with gravity. Cells were exposed to SMFs for 3–7 days in a cell incubator (Eppendorf, Hamburg, Germany) at 37°C and 5% CO 2 . We used a digital Gauss meter (cat. MG-3002, Lutron Electronics, Coopersburg, PA, USA) to measure the magnetic field strength (mT; Tesla) within the culture plate, considering various configurations [ 1 ]. SMF simulation A magnetic field simulation was conducted utilizing the ANSYS 2023 R2 magnetostatic (Ansys Inc., USA) program. The geometry was constructed with two magnets (60 × 30 × 10 mm 3 ) spaced 15 mm apart on the z-axis. In this simulation, the coercive force was set to 3.5 × 10 5 A/m and the residual induction was set to 450 mT. Within the Magnetostatic program, the N and S poles of the magnet were set for the upward and downward SMF conditions. As both the Petri dish and the cells located between the magnets were non-magnetic objects that did not affect the SMF, the space between the magnets was set to air with an isotropic relative permeability of 1. The mesh of the magnets was composed of 972 hexahedron meshes, whereas the exterior region, excluding the magnets (dimensions: 100 × 70 × 65 mm³), constituted 81,895 tetrahedron meshes. Cell culture hTECs and HK-2 cells (CRL-2190, American Type Culture Collection, Manassas, VA, USA) were used in this study for in-vitro experiments. HK-2 cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (cat. S1480, BioWest, Nuaillé, France) and 1% penicillin/streptomycin (cat. 15140-122, Gibco, Billings, MT, USA). Primary hTECs were harvested and cultured following the procedures outlined in our previous studies [ 10 , 35 , 44 , 45 ]. According to the protocol approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 2110-026-1260), hTECs were isolated from normal tissue specimens obtained from resected kidneys of patients with renal cell carcinoma. Informed consent was obtained from all patients. All procedures were conducted in accordance with the ethical standards of the institutional and national research committee, as well as the 1964 Declaration of Helsinki and its subsequent amendments or comparable ethical guidelines. Following the dissection of the cortex, the tissues were minced and digested in Hank’s balanced salt solution containing collagenase (1.5 mg/mL; cat. SCR103, Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 1 h. A p38 MAPK inhibitor (iP38) (0.1 µM and 1 µM; SB203580, cat. S8307, Sigma-Aldrich) was used to investigate the AKI − to − CKD mechanism under SMFs. In addition, hTECs were treated with adenine (2 mM; cat. A8626, Sigma-Aldrich) within inward SMFs for 3 days to assess the impact of SMFs on the AITN model. Western blot analysis Proteins were extracted from the hTECs and kidney tissue using RIPA buffer (cat. RC2002-050-00, Biosesang, Yongin, South Korea, 150 mM NaCl; 100 mM Na 3 VO 4 ; 50 mM Tris; HCL, pH 7.3; 0.1 mM EDTA 1% (vol/vol) sodium deoxycholate; 1% (vol/vol) Triton X-100; and 0.2% NaF) with protease inhibitor (GeneDEPOT, Katy, TX, USA). BCA assay was used to standardize the protein lysates to equal concentration using Pierce BCA Protein Assay Kits (cat. 23227, Thermo Fisher Scientific, Waltham, MA, USA). The protein samples were separated in glycine-SDS buffer and then transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with a solution containing 5% skim milk and 2% BSA for 1 h at 25°C and incubated with primary antibodies (Supplementary Table S1 ). Next, the membranes were incubated with anti-mouse IgG (cat. 7076S, Cell Signaling Technology, Danvers, MA, USA) or anti-rabbit IgG (cat. 7074S, Cell Signaling Technology) for 1 h at 25°C. Target proteins were identified with the ImageQuant Las 4000 mini system (GE HealthCare, Chicago, IL, USA), and subsequent analysis was performed using ImageJ (v. 1.52, Wayne Rasband, National Institutes of Health). Immunofluorescence assay hTECs were cultured on 4-well culture slides (Nunc Lab-Tek II Chamber Slide System, cat. 154526, Thermo Fisher Scientific) with total media for 1 day. After replacing with new total media, cells were exposed to SMFs for 3 days. Cells on culture slides were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, and permeabilized with 0.01% Triton X. Blocking agent containing 5% normal goat serum and 2% bovine serum albumin was used and incubated for 1 h at 25°C. The slides were stained with antibodies overnight at 4°C (Supplementary Table S1 ), followed by incubation with Alexa 488-conjugated goat anti-mouse (cat. A-11001, Thermo Fisher Scientific) and Alexa 555-conjugated goat anti-rabbit (cat. A-21428, Thermo Fisher Scientific) secondary antibodies for 1 h at 25°C. 4′,6-Diamidino-2-phenylindole (cat. D1306, Invitrogen, Waltham, MA, USA) was used for nuclear staining. Cell cycle analysis Cells were fixed with ice-cold 70% ethanol for at least 1 h at -20°C and then washed with cell staining buffer (cat. 420201, BioLegend, San Diego, CA, USA). Cells were stained with APC-conjugated Ki-67 antibody (cat. 350514, BioLegend, 5 µL /1×10 6 cells in 100 µL) for 30 min at room temperature in the dark. To ensure selective staining of DNA, cells were treated with 100 µg/mL ribonuclease (cat. GE6228, Glentham Life Sciences, Corsham, UK). Propidium iodide (PI) solution (cat. 421301, BioLegend) was used to assess DNA content in cell cycle analysis using flow cytometry. The cell cycle was evaluated through flow cytometry using a BD FACSLyric (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software (v10.8.1., BD Biosciences). RNA isolation and real-time qPCR analysis Total RNA from hTECs was extracted using the TRIzol reagent (Thermo Fischer Scientific) according to the manufacturer’s instructions. cDNA was synthesized from the total RNA of hTECs and amplified via PCR with a C1000 thermal cycler (Bio-Rad, Hercules, CA, USA). Afterward, qPCR was performed on a 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) with the following thermal cycling conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95˚C for 15 s and 60°C for 1 min, 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. Relative gene expression levels were quantified using the comparative CT (ΔΔCT) method, with GAPDH serving as the normalization control. The forward and reverse primer sequences used in this study are listed in Supplementary Table S2. Cell apoptosis assay To assess cell apoptosis and necrosis, the Annexin V/propidium iodide FITC apoptosis kit (cat. 556547, BD Biosciences) was used for flow cytometry, following the manufacturer’s instructions. After SMF exposure, harvested cells were washed with PBS, resuspended in 1X binding buffer (100 µL), and stained with FITC-conjugated Annexin V (3.5 µL) and PI (3.5 µL, 50 mg/mL). Subsequently, the cells were incubated for 15 min at 25°C in the dark. Flow cytometry data were acquired using the BD FACSCanto (BD Biosciences) and analyzed with FlowJo software (v10.8.1., BD Biosciences). AITN model C57BL/6 mice (male, 8 weeks old, n = 5 per group) were obtained from KOATECH (South Korea) and adenine (2 mg/mouse, cat. A8626, Sigma-Aldrich) was orally administered daily for 2 weeks. After 2 weeks, the mice were anesthetized by intraperitoneal injection of Zoletil™ (30 mg/kg; Virbac, Carros, France) and xylazine (Rompun; 10 mg/kg; Bayer, Leverkusen, Germany), followed by sacrifice via abdominal aortic puncture for blood collection [ 46 ]. BUN (mg/dL) and creatinine (mg/dL) concentrations were measured to assess renal function using an autoanalyzer (HITACHI7180, Hitachi Chemical Industries, Tokyo, Japan). All animal studies were performed under the guidance of the Institutional Animal Care and Use Committee (IACUC: 24-0057-S1A1) of Seoul National University Hospital and conducted in accordance with the National Research Council’s Guidelines for the Care and Use of Laboratory Animals. All methods for animal experiments are reported according to the ARRIVE guidelines ( https://arriveguidelines.org ). Immunohistochemistry Kidney tissues were fixed in 10% buffered formalin and embedded in paraffin. Then, 4-µm- thick sections were cut from the paraffin blocks for dehydration and rehydration, involving a series of xylene treatments followed by decreasing concentrations of ethanol and water. For the antigen retrieval, the kidney sections were microwaved in sodium citrate buffer. To block endogenous peroxidase activity, a 3% hydrogen peroxide solution diluted in methyl alcohol was applied. The sections were incubated overnight at 4°C with primary antibodies (Supplementary Table S1 ). Subsequently, Dako Envision + System-HRP-labeled polymer anti-mouse (cat. K4001, Agilent DAKO, Santa Clara, CA, USA) and anti-rabbit (cat. K4003, Agilent DAKO) were applied and incubated for 1 h at 25°C. Periodic acid-Schiff staining was performed to evaluate tubular injury scores based on the percentage of the affected area, with cell nuclei counterstained using Mayer’s hematoxylin (Sigma-Aldrich). The degree of tubular injury was assessed as previously described. The injury scores were categorized as follows: 0 for no damage, 1 for 1–10% injured area, 2 for 11–25%, 3 for 26–75%, and 4 for more than 75% injured area [ 42 , 47 , 48 ]. A Leica inverted microscope (Leica Camera, Wetzlar, Germany) and the LAS-4000 software (Leica Camera) were used to analyze and quantify the affected area. Statistical analysis The data are presented as means with standard error of the mean. Each experiment was repeated at least three times independently. Statistical analysis was conducted using an unpaired two-tailed Student’s t-test in GraphPad Prism 10.0 (GraphPad Software Inc., San Diego, CA, USA). A p-value of less than 0.05 was considered significant (* P < 0.05, ** P < 0.01, *** P < 0.001). Declarations Ethics Approval Statement All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Seoul National University Hospital (IACUC approval number: 24-0057-S1A1). Human kidney biopsy samples were collected with approval from the Institutional Review Board of Seoul National University Hospital (IRB approval number: 2110-026-1260), and informed consent was obtained from all patients. Additional Information Competing Interests Statement The authors declare no competing interests. Funding This research was funded by the National Research Foundation of Korea grant (NRF-2023R1A2C2006651) Author Contribution SML, SRL, SHL, EJB, KDY, JWL, YSK, RHC, and SHY designed the experiments. SML, SRL, SJP, and KHK performed the research. All authors analyzed the data. SML and SRL wrote the paper. RHC and SHY designed and supervised the project. All authors reviewed the manuscript. Acknowledgement Illustrations in Figure 3a and Figure 4 were created with BioRender.com. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References References1. Zhang, L. et al. 1 T moderate intensity static magnetic field affects Akt/mTOR pathway and increases the antitumor efficacy of mTOR inhibitors in CNE-2Z cells. Sci. Bull. 60 , 2120–2128 (2015) Milovanovich, I. D. et al. Homogeneous static magnetic field of different orientation induces biological changes in subacutely exposed mice. Environ. Sci. Pollut. Res. Int. 23 , 1584–1597 (2016) Yu, X. et al. Static magnetic fields protect against cisplatin-induced kidney toxicity. Antioxidants (Basel) 12 , 73 (2022) Hsu, R. K., McCulloch, C. E., Dudley, R. A., Lo, L. J. & Hsu, C. Y. Temporal changes in incidence of dialysis-requiring AKI. J. Am. Soc. Nephrol. 24 , 37–42 (2013) Guo, R. et al. The road from AKI to CKD: molecular mechanisms and therapeutic targets of ferroptosis. Cell Death Dis. 14 , 426 (2023) Coca, S. G., Singanamala, S. & Parikh, C. R. Chronic kidney disease after acute kidney injury: a systematic review and meta-analysis. Kidney Int. 81 , 442–448 (2012) Sugiyama, N., Kohno, M. & Yokoyama, T. Inhibition of the p38 MAPK pathway ameliorates renal fibrosis in an NPHP2 mouse model. Nephrol. Dial. Transplant. 27 , 1351–1358 (2012) Tao, Y. et al. Nr4a1 promotes renal interstitial fibrosis by regulating the p38 MAPK phosphorylation. Mol. Med. 29 , 63 (2023) Wang, D. et al. Inhibition of p38 MAPK attenuates renal atrophy and fibrosis in a murine renal artery stenosis model. Am. J. Physiol. Renal Physiol. 304 , F938–F947 (2013) Lee, J. et al. p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients. PLOS One 14 , e0213981 (2019) Falke, L. L., Gholizadeh, S., Goldschmeding, R., Kok, R. J. & Nguyen, T. Q. Diverse origins of the myofibroblast—implications for kidney fibrosis. Nat. Rev. Nephrol. 11, 233–244 (2015) Reiss, A. B. et al. Fibrosis in chronic kidney disease: pathophysiology and therapeutic targets. J. Clin. Med. 13, 1881 (2024) Nakanishi, T. & Kuragano, T. Growing concerns about using hypoxia-inducible factor prolyl hydroxylase inhibitors for the treatment of renal anemia. Clin. Kidney J. 17, sfae051 (2024) Liu, K. H. et al. Hypoxia stimulates the epithelial-to-mesenchymal transition in lung cancer cells through accumulation of nuclear β-catenin. Anticancer Res. 38, 6299–6308 (2018) Hu, J. et al. Hypoxia inducible factor-1α mediates the profibrotic effect of albumin in renal tubular cells. Sci. Rep. 7, 15878 (2017) Kimura, K. et al. Stable expression of HIF-1alpha in tubular epithelial cells promotes interstitial fibrosis. Am. J. Physiol. Renal Physiol. 295, F1023–F1029 (2008) Wang, Z. et al. Silencing of hypoxia-inducible factor-1α gene attenuates chronic ischemic renal injury in two-kidney, one-clip rats. Am. J. Physiol. Renal Physiol. 306, F1236–F1242 (2014) Yu, L. et al. HIF-1α alleviates high-glucose-induced renal tubular cell injury by promoting Parkin/PINK1-mediated mitophagy. Front. Med. (Lausanne) 8, 803874 (2021) Shu, S. et al. Hypoxia and hypoxia-inducible factors in kidney injury and repair. Cells 8, 207 (2019) Conde, E. et al. HIF-1α induction during reperfusion avoids maladaptive repair after renal ischemia/reperfusion involving miR127-3p. Sci. Rep. 7, 41099 (2017) Wang, J. et al. Inhibition of viability, proliferation, cytokines secretion, surface antigen expression, and adipogenic and osteogenic differentiation of adipose-derived stem cells by seven-day exposure to 0.5 T static magnetic fields. Stem Cells Int. 2016, 7168175 (2016) Luo, Y. et al. Moderate intensity static magnetic fields affect mitotic spindles and increase the antitumor efficacy of 5-FU and Taxol. Bioelectrochemistry 109, 31–40 (2016) Sullivan, K., Balin, A. K. & Allen, R. G. Effects of static magnetic fields on the growth of various types of human cells. Bioelectromagnetics 32 , 140–147 (2011) Tian, X. et al. Magnetic field direction differentially impacts the growth of different cell types. Electromagn. Biol. Med. 37 , 114–125 (2018) Yang, X. et al. An upward 9.4 T static magnetic field inhibits DNA synthesis and increases ROS-P53 to suppress lung cancer growth. Transl. Oncol. 14 , 101103 (2021) Ganguly, P., Macleod, T., Wong, C., Harland, M. & McGonagle, D. Revisiting P38 mitogen-activated protein kinases (MAPK) in inflammatory arthritis: A narrative of the emergence of MAPK-activated protein kinase inhibitors (MK2i). Pharmaceuticals (Basel) 16 , 1286 (2023) Yue, J. & López, J. M. Understanding MAPK signaling pathways in apoptosis. Int. J. Mol. Sci. 21 , 2346 (2020) van der Slikke, E. C. et al. Sepsis is associated with mitochondrial DNA damage and a reduced mitochondrial mass in the kidney of patients with sepsis-AKI. Crit. Care 25 , 36 (2021) Chernikov, A. V., Gudkov, S. V., Usacheva, A. M. & Bruskov, V. I. Exogenous 8-oxo-7,8-dihydro-2′-deoxyguanosine: biomedical properties, mechanisms of action, and therapeutic potential. Biochemistry (Mosc) 82 , 1686–1701 (2017) Kim, J. Y. et al. Involvement of p38 mitogen-activated protein kinase in the cell growth inhibition by sodium arsenite. J. Cell. Physiol. 190 , 29–37 (2002) Whitaker, R. H. & Cook, J. G. Stress relief techniques: p38 MAPK determines the balance of cell cycle and apoptosis pathways. Biomolecules 11 , 1444 (2021) Faust, D. et al. Differential p38-dependent signalling in response to cellular stress and mitogenic stimulation in fibroblasts. Cell Commun. Signal. 10 , 6 (2012) Zamir-Nasta, T., Razi, M., Shapour, H. & Malekinejad, H. Roles of p21, p53, cyclin D1, CDK-4, estrogen receptor α in aflatoxin b1-induced cytotoxicity in testicular tissue of mice. Environ. Toxicol. 33 , 385–395 (2018) Amin, M., Razi, M., Sarrafzadeh-Rezaei, F., Shalizar Jalali, A. & Najafi, G. Berberine inhibits experimental varicocele-induced cell cycle arrest via regulating cyclin D1, cdk4 and p21 proteins expression in rat testicles. Andrologia 50 , e12984 (2018) An, J. N. et al. Periostin induces kidney fibrosis after acute kidney injury via the p38 MAPK pathway. Am. J. Physiol. Renal Physiol. 316 , F426–F437 (2019) Li, J. et al. Inhibition of p38 mitogen-activated protein kinase and transforming growth factor-beta1/Smad signaling pathways modulates the development of fibrosis in adriamycin-induced nephropathy. Am. J. Pathol. 169 , 1527–1540 (2006) Stambe, C. et al. Blockade of p38alpha MAPK ameliorates acute inflammatory renal injury in rat anti-GBM glomerulonephritis. J. Am. Soc. Nephrol. 14 , 338–351 (2003) Choi, J. et al. In vivo longitudinal 920 nm two-photon intravital kidney imaging of a dynamic 2,8-dha crystal formation and tubular deterioration in the adenine-induced chronic kidney disease mouse model. Biomed. Opt. Express 14 , 1647–1658 (2023) Khan, M. A. et al. Adenine overload induces ferroptosis in human primary proximal tubular epithelial cells. Cell Death Dis. 13 , 104 (2022) Awad, A. M., Saleh, M. A., Abu-Elsaad, N. M. & Ibrahim, T. M. Erlotinib can halt adenine induced nephrotoxicity in mice through modulating ERK1/2, STAT3, p53 and apoptotic pathways. Sci. Rep. 10 , 11524 (2020) Jia, T. et al. A novel model of adenine-induced tubulointerstitial nephropathy in mice. BMC Nephrol. 14 , 116 (2013) Yoo, K. D. et al. Role of the CCL20/CCR6 axis in tubular epithelial cell injury: kidney-specific translational insights from acute kidney injury to chronic kidney disease. FASEB J. 38 , e23407 (2024) Park, J. Y. et al. Blockade of STAT3 signaling alleviates the progression of acute kidney injury to chronic kidney disease through antiapoptosis. Am. J. Physiol. Renal Physiol. 322 , F553–F572 (2022) [published correction appears in Am. J. Physiol. Renal Physiol. 323, F700 (2022)] Lee, J. et al. Antibiotic-induced intestinal microbiota depletion can attenuate the acute kidney injury to chronic kidney disease transition via NADPH oxidase 2 and trimethylamine-N-oxide inhibition. Kidney Int. 105 , 1239–1253 (2024) Park, S. et al. Elevated bilirubin levels are associated with a better renal prognosis and ameliorate kidney fibrosis. PLOS One 12 , e0172434 (2017) An, J. N. et al . cMet agonistic antibody attenuates apoptosis in ischaemia-reperfusion-induced kidney injury. J. Cell. Mol. Med. 24 , 5640-5651 (2020) Jang, H. R. et al. Early exposure to germs modifies kidney damage and inflammation after experimental ischemia-reperfusion injury. Am. J. Physiol. Renal Physiol. 297 , F1457–F1465 (2009) Yoo, K. D. et al. Chemokine receptor 5 blockade modulates macrophage trafficking in renal ischaemic-reperfusion injury. J. Cell. Mol. Med. 24 , 5515–5527 (2020) Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 10 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 08 May, 2025 Reviews received at journal 07 May, 2025 Reviewers agreed at journal 02 May, 2025 Reviews received at journal 24 Apr, 2025 Reviewers agreed at journal 23 Apr, 2025 Reviewers invited by journal 22 Apr, 2025 Editor assigned by journal 22 Apr, 2025 Editor invited by journal 11 Apr, 2025 Submission checks completed at journal 10 Apr, 2025 First submitted to journal 10 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6392601","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":447494177,"identity":"f39716ee-6463-4f22-9408-216077ba6810","order_by":0,"name":"Seong Min Lee","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Seong","middleName":"Min","lastName":"Lee","suffix":""},{"id":447494178,"identity":"1f85ba3c-c51b-44e0-9d3d-7375c88b6ed5","order_by":1,"name":"Saram Lee","email":"","orcid":"","institution":"Seoul National University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Saram","middleName":"","lastName":"Lee","suffix":""},{"id":447494179,"identity":"9103683e-5ef7-4f94-aec2-c141223159fa","order_by":2,"name":"Seong Joon Park","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Seong","middleName":"Joon","lastName":"Park","suffix":""},{"id":447494180,"identity":"140b5a36-7aef-447a-9b63-22b302e223ed","order_by":3,"name":"Kyu Hong Kim","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Kyu","middleName":"Hong","lastName":"Kim","suffix":""},{"id":447494181,"identity":"c9b85a2b-e7fa-409f-bac6-52074363b169","order_by":4,"name":"Sunhwa Lee","email":"","orcid":"","institution":"Kangwon National University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sunhwa","middleName":"","lastName":"Lee","suffix":""},{"id":447494182,"identity":"a94e46c4-22a4-411a-9194-c950d8af8b3f","order_by":5,"name":"Eunjin Bae","email":"","orcid":"","institution":"Gyeongsang National University Changwon Hospital","correspondingAuthor":false,"prefix":"","firstName":"Eunjin","middleName":"","lastName":"Bae","suffix":""},{"id":447494183,"identity":"fc67428e-346c-4e7f-8033-1d73a4c8460e","order_by":6,"name":"Kyung Don Yoo","email":"","orcid":"","institution":"University of Ulsan College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kyung","middleName":"Don","lastName":"Yoo","suffix":""},{"id":447494184,"identity":"702fc7ef-8f7f-4337-b6a9-9362c84d830f","order_by":7,"name":"Jae Wook Lee","email":"","orcid":"","institution":"National Cancer Center of Korea","correspondingAuthor":false,"prefix":"","firstName":"Jae","middleName":"Wook","lastName":"Lee","suffix":""},{"id":447494186,"identity":"223e747f-4b3a-4a6c-b1c4-69998d383481","order_by":8,"name":"Daehan Kim","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Daehan","middleName":"","lastName":"Kim","suffix":""},{"id":447494187,"identity":"17059f6e-4649-48bf-81dc-709dfaeeb90d","order_by":9,"name":"Joong Yull Park","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Joong","middleName":"Yull","lastName":"Park","suffix":""},{"id":447494188,"identity":"25fb35e8-0a7f-43de-8d11-d07213b62a0d","order_by":10,"name":"Yon Su Kim","email":"","orcid":"","institution":"Seoul National University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yon","middleName":"Su","lastName":"Kim","suffix":""},{"id":447494189,"identity":"f176d19f-1563-449d-853c-cd07284a2814","order_by":11,"name":"Ran-Hui Cha","email":"","orcid":"","institution":"Seoul National University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ran-Hui","middleName":"","lastName":"Cha","suffix":""},{"id":447494190,"identity":"bce43a60-1a13-42e9-9d0c-18efec511c1a","order_by":12,"name":"Seung Hee Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYFACHvbPfyrY5GBcA2K0sDHwnOEzJlELb5tcYgPRWgzOnz32QLLNLH3+7B4Dhh81DMbmDYS03MhLNzA4l5a74c4ZA8aeYwxmMgcIauExkEgoO5a7QSLHgIG3gcFGgrDDzhhIHGD7ny4/I8eA8S9RWg7kmEk2tLElMNzIMWAG2mJGUIvkjRxjY4YzbIYbbqQVHJY5JmFMUAvf+TOGjxkq2OTlZyRvfPimxsZwBiEtCgeQOEA2QTsYGOQbCKsZBaNgFIyCkQ4AzWU8OAYTzmQAAAAASUVORK5CYII=","orcid":"","institution":"Seoul National University Medical Research Center","correspondingAuthor":true,"prefix":"","firstName":"Seung","middleName":"Hee","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-04-07 09:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6392601/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6392601/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-09077-w","type":"published","date":"2025-07-10T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81374366,"identity":"ab88c217-82f8-459b-beaf-f74794433ee2","added_by":"auto","created_at":"2025-04-25 11:11:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5258111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInward SMF exposure induced tubular epithelial injury by modulating the MAP-kinase pathway and triggering G1/S phase arrest.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b) Schematic design of experiments and magnetic field distribution. hTECs were exposed to outward SMFs (a) or inward SMFs (b) for 3 or 7 days. (c, d) Western blot representative images (c) and quantification (d) of collagen 1 (Col 1), fibronectin (FN), NGAL, pp38, and VEGFR. (e) Reduced expression of E-cadherin and elevated expression of HIF-1α were observed via immunofluorescence staining under exposure to SMFs for 3 days, particularly in the inward direction. Scale bars, 100 mm (×100). (f) Relative mRNA expression levels of CDK4 and cyclin D1 (n = 10 in each group). The experiments were independently replicated at least three times, with the data presented as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FIgure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6392601/v1/afef9b7881ac200820ac4877.jpg"},{"id":81374776,"identity":"ddcd67fe-0625-43df-a5ff-7d86e421f9ab","added_by":"auto","created_at":"2025-04-25 11:19:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5075744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of p38 protected from inward SMF-induced tubular damage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b) Western blot representative images (a) and quantification (b) for OGG1, periostin, alpha smooth muscle actin (α-SMA), and fibronectin (FN). (c) mRNA levels of CDK4 and OGG1 were analyzed using real-time qPCR (n = 4 in each group). (d) Representative images of cell cycle analysis by PI and Ki-67 staining (n = 3–6 in each group). (e) Proportions of HK-2 cells in each phase of the cell cycle quantified as percentages. (f) Increased ratios of apoptotic cells were observed with inward SMF exposure, which decreased following treatment with a p38 MAPK inhibitor. Early apoptosis, late apoptosis, necrosis, and combined cell proportions were assessed under inward SMF exposure and p38 inhibition. The experiments were independently replicated at least three times, and the data are presented as mean ± SEM. Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FIgure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6392601/v1/d066956f72138fa1cba93f46.jpg"},{"id":81374369,"identity":"fd4b4f82-647b-4421-b279-5c916c53f9c1","added_by":"auto","created_at":"2025-04-25 11:11:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9212446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ep38 MAPK inhibitor attenuated fibrosis in AITN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Schematic diagram of adenine administration in AITN. (b) Kidney function was assessed by measuring BUN and blood creatinine levels (n = 5 in each group). (c, d) PAS staining, along with IHC analysis, revealed upregulation of ICAM-1, F4/80, NGAL, IL-17R, and p53 following adenine administration. Interstitial fibrosis was determined by MT staining in the adenine group (n = 5 in each group). Scale bars, 250 mm (×40), 100 mm (×100). (e) Increased expression of pp38 and KIM-1 in kidney tissue in the AITN group with western blot analysis. (f) Increased expression of fibronectin under inward SMF condition was significantly decreased with p38 MAPK inhibitor treatment in a dose-dependent manner. The experiments were independently replicated at least three times, with the data presented as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FIgure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6392601/v1/f4f868c2825b9584d6938953.jpg"},{"id":81374363,"identity":"6d1b5def-60c8-43f7-a9d7-ba2f8a99c526","added_by":"auto","created_at":"2025-04-25 11:11:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1623223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of cell cycle alterations and MAP-kinase pathway involvement in the transition from SMF-induced acute renal tubular injury to fibrosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSMF-driven damage exacerbated ROS-induced apoptosis in renal tubule injury, ultimately leading to fibrosis.\u003c/p\u003e","description":"","filename":"FIgure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6392601/v1/8f2ce062746b375953246637.jpg"},{"id":86699423,"identity":"957442e3-d718-4d7c-81ac-110176b11195","added_by":"auto","created_at":"2025-07-14 16:09:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22189357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6392601/v1/e68165d8-55da-4445-b0e1-ac83986b6769.pdf"},{"id":81375604,"identity":"a2d936ac-6258-4d52-8b3b-9fae5800a0a2","added_by":"auto","created_at":"2025-04-25 11:27:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":20400,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6392601/v1/2b376fd890f7281a24998551.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Alterations in cell cycle and MAPK pathway contribute to transition from SMF-associated acute kidney injury to fibrosis: Field direction matters","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe increasing utilization of magnetic resonance imaging (MRI) for diagnosing diseases has triggered heightened concerns regarding the potential effects of magnetic fields on human health. MRI machines typically used in hospitals have magnetic field strengths ranging from 3 to 9.4 Tesla [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recently, to achieve higher resolutions, MRI machines with even stronger magnetic field strengths have been developed. Milovanovich et al. reported that an upward-oriented static magnetic field (SMF) induced brain edema and increased spleen cellularity, whereas the downward-oritented SMF led to liver inflammation and a reduction in serum granulocyte levels [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, in a cisplatin-induced nephrotoxicity model, moderate SMFs at hundreds of mT attenuated kidney injury by reducing oxidative stress and inflammation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The effects of SMFs can be variable according to their particular characteristics including shapes, strength, and directions, and cell types. The exact mechanisms also remain inconclusive owing to the complicated effects of magnetic fields on biological systems.\u003c/p\u003e \u003cp\u003eAcute kidney injury (AKI) affects 5\u0026ndash;10% of hospitalized patients, leading to kidney tissue damage, elevated blood creatinine levels, and reduced urine output [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Over the past decade, increasing evidence has indicated that AKI is a significant contributor to the development of chronic kidney disease (CKD) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The AKI\u0026thinsp;\u0026minus;\u0026thinsp;to \u0026minus;\u0026thinsp;CKD transition involves complex mechanisms primarily driven by maladaptive repair processes, including cell cycle arrest, inflammation, and fibrosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe p38 mitogen-activated protein kinase (MAPK), a serine/threonine protein kinase, is involved in crucial intracellular signal transduction pathways and plays a significant role in the pathogenesis of fibrosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Upon cellular stress, p38 MAPK can translocate to the nucleus and subsequently induce the activation of transcription factors contributing to the production of proinflammatory mediators [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previous studies have demonstrated that inhibiting p38 MAPK may protect kidneys from injury and attenuate disease progression in various models [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. p38 MAPK activation was reportedly associated with kidney interstitial fibrosis in a UUO mouse model, and the anti-fibrotic effect of p38 MAPK inhibition has been confirmed [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo our knowledge, there have been no reports regarding the differential effect of SMF directions on kidney tissues and proper damage mechanisms. In this study, we established an SMF-associated AKI model and observed the effects of field directions. Furthermore, we evaluated the role of p38 MAPK in AKI\u0026thinsp;\u0026minus;\u0026thinsp;to \u0026minus;\u0026thinsp;CKD progression in a mouse model.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSMFs induce tubular epithelial cell injury, especially under the inward direction\u003c/h2\u003e \u003cp\u003eSimulations were conducted under two conditions by changing the polarity of the magnets: outward, where the cells receive magnetic force from the bottom up, and inward, where the cells receive magnetic force from the top down. In the outward condition, the maximum value of magnetic flux density was approximately 121.4 mT in the x-y plane 1 mm above the magnet (the plane where the cells were located). Within the range of the magnet, the maximum magnetic flux density of approximately 121.4 mT was observed at the tip of the magnet, whereas the minimum magnetic flux density of approximately 105.6 mT was observed at the center of the magnet (the difference of 13.0%). Thesimulation results for the inward condition were identical to those for the outward condition, with the sole difference being the reversal of the direction of the magnetic flux density vector. Vertically upward and downward SMFs formed an outward and inward direction of SMFs, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). To verify the reliability of the magnetic field simulation results, the simulated magnetic flux density values at each location were compared with the actual magnetic field measurements. The magnetic field strengths measured at 15, 30, and 45 mm along the y-axis of the magnet were approximately 114.8, 110, and 112.1 mT, respectively, and the simulation results at those points were 112.0, 106.1, and 112.1 mT, respectively, with an average error of approximately 2.0% between the measured and simulated results, i.e., the simulation results were considered reliable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing a magnetic field analyzer, the intensity of the SMFs was measured to be approximately 119 mT under both orientations. We initially investigated the effects of SMFs at both early and late stages by culturing with SMFs for either 3 or 7 days. By the seventh day, the vast majority of cells were dead. Although the expression of kidney injury marker (NGAL) did not change significantly after 3 days of exposure to SMFs, we noticed an elevation in fibrosis markers (collagen 1, fibronectin, and VEGFR) and increase in pp38 protein levels. There was a pronounced increase in pp38 under inward SMFs compared with that under outward SMFs, suggesting that the modulation of p38 MAPK pathway by inward SMFs contributes to fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). The immunofluorescence assay revealed that exposure to both outward and inward SMFs for 3 days led to decreased expression of intracellular junction marker (E-cadherin) and increased levels of HIF-1α. This suggests that SMFs induced AKI-to-CKD transition by regulating inflammation and oxidative stress through HIF-1α signaling. HIF-1α elevation implies oxygen deprivation in cells exposed to SMFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Afterward, the mRNA expression of CDK4 and cyclin D1, key regulators of the G1/S phase in the cell cycle, was analyzed. CDK4 expression decreased under both inward and outward SMFs compared with that under the control, with no significant difference observed between the two conditions. However, cyclin D1 expression was significantly lower under inward SMFs than under outward SMFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). In line with the significant increase in pp38 expression and decreased levels of cyclin D1 under inward SMFs, these findings imply that exposure to inward SMFs induced tubular epithelial injury by modulating the p38 MAPK pathway and inducing G1/S phase arrest. In particular, the direction of SMFs exerts a significant effect on these changes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ep38 MAPK inhibitor protected against tubular damage induced by inward SMFs\u003c/h3\u003e\n\u003cp\u003eTo examine the therapeutic potential of p38 MAPK inhibition in tubular epithelial cells exposed to inward SMFs, we treated human primary tubular epithelial cells (hTECs) with iP38 (0.1 \u0026micro;M, 1 \u0026micro;M) while concomitantly exposing them to inward SMFs for 3 days. p38 MAPK inhibitor treatment considerably reduced the expression of OGG1, a marker of DNA damage and oxidative stress, compared with inward SMFs alone. Additionally, fibrosis markers such as periostin, α-SMA, and fibronectin markedly increased with inward SMF exposure but iP38 treatment attenuated this increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). The mRNA expression of CDK4 was downregulated by inward SMFs but was recovered following iP38 treatment. The increased OGG1 mRNA expression under inward SMFs also decreased after iP38 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). This indicates that p38 MAPK inhibition prevented cell cycle arrest in the G1/S phase induced by inward SMFs. Cells in the G1 phase significantly increased under inward SMFs compared with those in the control group (51.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 vs 66.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; control vs inward SMFs). However, the increase was reduced with iP38 treatment of 0.1 \u0026micro;M and 1 \u0026micro;M, respectively (66.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97 vs 62.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 59.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; inward SMFs vs inward SMFs\u0026thinsp;+\u0026thinsp;iP38 0.1 \u0026micro;M, inward SMFs\u0026thinsp;+\u0026thinsp;iP38 1 \u0026micro;M). Additionally, cells in the sub G1 phase, indicating an increase in apoptotic cells, increased by 2.85-fold following inward SMF exposure compared with those in the control group (1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 vs 3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; control vs inward SMFs). iP38 treatment significantly decreased the proportion of cells in the sub G1 phase in a dose-dependent manner (3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 vs 3.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, 2.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; inward SMFs vs inward SMFs\u0026thinsp;+\u0026thinsp;iP38 0.1 \u0026micro;M, inward SMFs\u0026thinsp;+\u0026thinsp;iP38 1 \u0026micro;M). Correspondingly, the p38 MAPK inhibitor mitigated the rise in apoptotic cells caused by inward SMFs, with a particularly notable reduction in early apoptotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Overall, these results suggest that inhibiting p38 MAPK may reduce the tubular epithelial injury driven by oxidative stress and G1/S arrest induced by inward SMFs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eInhibition of p38 MAPK mitigated fibrosis in adenine-induced tubular nephropathy (AITN) model\u003c/h3\u003e\n\u003cp\u003eTo mimic CKD progression, we established an AITN model by administering adenine orally to mice daily for two weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Adenine treatment significantly elevated the serum levels of blood urea nitrogen (BUN) and creatinine in ATIN models to 138.34\u0026thinsp;\u0026plusmn;\u0026thinsp;4.03 mg/dL (***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mg/dL (***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Kidney injury was observed in the renal cortex tubules of mice exposed to adenine, characterized by the enlargement of the basement membrane and loss of the brush border. Moreover, Masson's trichrome staining revealed the advancement of tubular atrophy and interstitial fibrosis. NGAL expression and F4/80\u003csup\u003e+\u003c/sup\u003e macrophage infiltration were increased in the adenine-treated group compared with those in the sham group. The upregulation of pro-inflammatory markers such as ICAM-1, IL-17R, and the apoptosis marker p53 was also observed, indicating the involvement of inflammation and programmed cell death in the progression of renal injury and fibrosis following adenine administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). The protein levels of pp38 and KIM-1, markers indicating the progression of AKI\u0026thinsp;\u0026minus;\u0026thinsp;to \u0026minus;\u0026thinsp;CKD transition, increased after adenine treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). To identify the effect of p38 MAPK on the inhibition of fibrosis in the AITN model under inward SMFs, we treated hTECs with adenine and a p38 MAPK inhibitor. Inward SMFs significantly increased the expression of fibronectin. Under conditions without SMF exposure, the increased expression of fibronectin caused by adenine treatment was not mitigated by inhibiting p38 MAPK. However, under inward SMFs, there was a notable reduction when treated with the p38 MAPK inhibitor in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we aimed to identify the pathway linking acute renal tubule injury under SMFs to fibrosis. First, we found that exposure to SMFs triggers tubular epithelial injury, which leads to fibrosis by altering the p38 MAPK pathway and inducing G1/S phase arrest. In addition, the direction of SMFs plays a significant role in these effects. Second, we confirmed the involvement of the p38 MAPK pathway by demonstrating the protective effect of its inhibition against SMF-induced tubular damage, as evidenced by reduced cell cycle arrest, oxidative stress, apoptosis, and decreased expression of fibrosis markers. Third, we identified the increased expression of pp38 and kidney injury markers in a mouse AITN model. Furthermore, we observed a remarkable reduction in fibronectin expression through p38 MAPK inhibition in an in-vitro model of adenine treatment under inward SMFs. Overall, the study highlights the detrimental effects of SMF exposure on tubular epithelial cells, implicating the p38 MAPK pathway and cell cycle arrest in renal fibrosis, and suggests that the direction of magnetic fields plays an important role in the overall changes.\u003c/p\u003e \u003cp\u003eKidney fibrosis is widely recognized as a common pathological consequence of CKD. When kidneys are injured, local pericytes and fibroblasts activate, secreting inflammatory mediators and synthesizing extracellular matrix components such as collagens and fibronectin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Fibrosis develops with persistent accumulation of extracellular matrix proteins in cases of severe damage, accelerating the advancement of CKD through TGF-β signaling [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Tissue hypoxia is common in CKD, and hypoxic signaling primarily involves HIFs, whose stability is increased under low-oxygen conditions owing to reduced prolyl hydroxylase-mediated degradation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. HIF-1α, a transcription factor, promotes collagen accumulation and facilitates the epithelial-to-mesenchymal transition [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Overexpression of HIF-1α in tubular epithelial cells promotes interstitial fibrosis in 5/6 nephrectomy mice [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], whereas silencing gene expression of HIF-1α reduced TGF-β induced epithelial-to-mesenchymal transition and angiotensin II-induced profibrotic effects in kidney cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our experiment resulted in the upregulated expression of fibrosis markers and HIF-1α as well as the decreased expression of E-cadherin under inward SMFs, which is in line with previous studies. HIF-1α contributes to inflammation, kidney damage, and fibrosis, which results in AKI\u0026thinsp;\u0026minus;\u0026thinsp;to \u0026minus;\u0026thinsp;CKD transition [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Reduced levels of E-cadherin are also associated with renal fibrosis and CKD progression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we utilized the SMF platform to induce tubule injury and fibrosis. SMFs can induce various cellular effects, including cell proliferation, cell viability, and the cell cycle [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Previous studies have reported that the cellular effects induced by SMFs vary according to the intensity and the direction of magnetic fields as well as cell types [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Tian et al. discovered that upward SMFs of 0.2\u0026ndash;1T could effectively decrease the cell numbers of human tumor cell lines MCF7 and GIST-T1, whereas downward SMFs did not produce a notable impact. Interestingly, the leukemia cell numbers were reduced by both upward and downward SMFs. This study also revealed that effects of SMFs were dependent on the direction of SMFs. The intensity in both vertically upward and downward SMFs was not different at all, which was beyond expectations. Vertically upward orientation resulted in the outward direction of SMFs, whereas vertically downward orientation resulted in the inward direction of SMFs. Inward SMFs upregulated the expression of fibrosis markers, pp38, and HIF-1α, whereas it decreased the expression of E-cadherin. Further research in various orientations of SMFs is necessary to clarify the role of SMF direction on renal tubular cells.\u003c/p\u003e \u003cp\u003eThe precise mechanisms underlying the different effects of SMF directions remain unclear [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Yu et al. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] showed that although both upward and downward SMFs reduce oxidative stress in the kidney, downward SMFs offer stronger protective effects by reducing kidney inflammation, apoptosis, and cisplatin accumulation via decreased Oct2 levels; however, the precise mechanisms behind these effects remain elusive. Our findings suggest that exposure to inward SMFs upregulates the p38 MAPK pathway and induces G1/S phase arrest, resulting in tubular epithelial injury and subsequent fibrosis. Phosphorylated p38 MAPK, an essential pro-inflammatory element, is recognized for upregulating cytokines such as IL-6 and TNFα [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the p38 MAPK pathway, the second messenger, reactive oxygen species (ROS) modulates MAPK activation via a positive feedback mechanism. This process, involving ROS generation and p38 activation, further enhances p53-mediated apoptosis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAKI can lead to DNA damage caused by oxidative stress from ROS [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Under oxidative stress, 8-oxo-G is generated in DNA and subsequently released during repair by the DNA glycosylase OGG1 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The expression of the OGG1 gene is likely upregulated in response to oxidative stress, which aligns with our observations. Activation of p38 by cellular stress commonly leads to cell cycle arrest or apoptosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Several reports have been shown that p38 activation results in G1 arrest [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. AKI-induced DNA damage promotes the production of p21, which halts the cell cycle from G1 to S phase by binding to CDK4 and inhibiting the Cyclin D1/CDK4 complex. This action not only arrests cell cycle progression but also triggers the p53-dependent apoptosis pathway [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSuppressing p38 MAPK activity has been found to moderately decelerate disease progression. Li et al. and An et al. showed an elevation in pp38 MAPK after disease induction in both in vitro and in vivo models, alongside a dose-dependent reduction in fibrosis markers with the addition of a p38 MAPK inhibitor [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Lee et al. revealed a significant increase in phosphorylated p38 MAPK activity in a UUO mouse model and showed that inhibiting p38 MAPK led to a decrease in the mRNA expression of fibrosis markers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Based on these findings, we examined the protective effects of p38 MAPK inhibition in inward SMF-induced tubular damage. Blockade of p38 MAPK not only reduced cell cycle arrest and apoptosis but also decreased the expression of fibrosis markers. This supports prior findings that the aggravation in kidney injury and fibrosis is, particularly mediated by the p38 MAPK pathway.\u003c/p\u003e \u003cp\u003eTo confirm the role of the p38 MAPK pathway in acute renal tubular injury and fibrosis, we established an adenine-induced nephrotoxicity model. When metabolized to 2,8-dihydroxyadenine (2,8-DHA), adenine accumulates crystal deposits in the renal tubules, contributing to CKD [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Orally ingested adenine tends to accumulate more extensively than other purines and has been frequently used to induce gradual kidney damage [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Adenine is rapidly metabolized to DHA, leading to crystal formation in the proximal tubule, which closely resembles human CKD [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Adenine treatment resulted in increased levels of kidney function biomarkers and profibrogenic markers [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This condition is characterized by diminished renal function, tubular dilation, infiltration of macrophages, and fibrosis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In line with these observations, we validated these findings with histological analysis, indicating a significant increase in expression of ICAM-1, F4/80, NGAL, IL-17R, and p53. Furthermore, we assessed the effect of p38 MAPK inhibition of fibrosis in an in vitro AITN model under inward SMF exposure.\u003c/p\u003e \u003cp\u003eThis study opens up several avenues for further investigation. First, the effects of dynamic magnetic fields on tubular epithelial cells remain unexplored and will be a focus of future research. Second, the impact of inward SMFs and the inhibition of p38 MAPK in the AITN mouse model has not yet been fully confirmed, providing another important direction for subsequent studies. Acknowledging the scope for further exploration, we anticipate addressing these aspects in upcoming research efforts.\u003c/p\u003e \u003cp\u003eIn summary, our study reveals that acute tubular cell injury under inward SMFs, associated with inflammation, oxidative stress, and apoptosis, progresses to chronic fibrosis through maladaptive repair mechanisms, including G1/S cell cycle arrest and activation of the MAP kinase pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Additionally, inhibiting p38 MAPK effectively mitigated inward SMF-induced kidney injury, preventing the progression to fibrosis, which is a common final pathway in the development of CKD. These findings indicate that targeting the p38 MAPK pathway may offer a promising therapeutic approach to control the progression from inward SMF-associated AKI\u0026thinsp;\u0026minus;\u0026thinsp;to \u0026minus;\u0026thinsp;CKD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment and measurement of strength and directions of SMFs\u003c/h2\u003e \u003cp\u003eHuman proximal tubular cells were placed between two ferrite magnets (length \u0026times; width \u0026times; height: 60 \u0026times; 30 \u0026times; 10 mm) positioned in different orientations: vertically upward SMFs opposing gravity vs. vertically downward SMFs aligning with gravity. Cells were exposed to SMFs for 3\u0026ndash;7 days in a cell incubator (Eppendorf, Hamburg, Germany) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. We used a digital Gauss meter (cat. MG-3002, Lutron Electronics, Coopersburg, PA, USA) to measure the magnetic field strength (mT; Tesla) within the culture plate, considering various configurations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSMF simulation\u003c/h3\u003e\n\u003cp\u003eA magnetic field simulation was conducted utilizing the ANSYS 2023 R2 magnetostatic (Ansys Inc., USA) program. The geometry was constructed with two magnets (60 \u0026times; 30 \u0026times; 10 mm\u003csup\u003e3\u003c/sup\u003e) spaced 15 mm apart on the z-axis. In this simulation, the coercive force was set to 3.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e A/m and the residual induction was set to 450 mT. Within the Magnetostatic program, the N and S poles of the magnet were set for the upward and downward SMF conditions. As both the Petri dish and the cells located between the magnets were non-magnetic objects that did not affect the SMF, the space between the magnets was set to air with an isotropic relative permeability of 1. The mesh of the magnets was composed of 972 hexahedron meshes, whereas the exterior region, excluding the magnets (dimensions: 100 \u0026times; 70 \u0026times; 65 mm\u0026sup3;), constituted 81,895 tetrahedron meshes.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003ehTECs and HK-2 cells (CRL-2190, American Type Culture Collection, Manassas, VA, USA) were used in this study for in-vitro experiments. HK-2 cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (cat. S1480, BioWest, Nuaill\u0026eacute;, France) and 1% penicillin/streptomycin (cat. 15140-122, Gibco, Billings, MT, USA). Primary hTECs were harvested and cultured following the procedures outlined in our previous studies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. According to the protocol approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 2110-026-1260), hTECs were isolated from normal tissue specimens obtained from resected kidneys of patients with renal cell carcinoma. Informed consent was obtained from all patients. All procedures were conducted in accordance with the ethical standards of the institutional and national research committee, as well as the 1964 Declaration of Helsinki and its subsequent amendments or comparable ethical guidelines. Following the dissection of the cortex, the tissues were minced and digested in Hank\u0026rsquo;s balanced salt solution containing collagenase (1.5 mg/mL; cat. SCR103, Sigma-Aldrich, St. Louis, MO, USA) at 37\u0026deg;C for 1 h. A p38 MAPK inhibitor (iP38) (0.1 \u0026micro;M and 1 \u0026micro;M; SB203580, cat. S8307, Sigma-Aldrich) was used to investigate the AKI\u0026thinsp;\u0026minus;\u0026thinsp;to \u0026minus;\u0026thinsp;CKD mechanism under SMFs. In addition, hTECs were treated with adenine (2 mM; cat. A8626, Sigma-Aldrich) within inward SMFs for 3 days to assess the impact of SMFs on the AITN model.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eProteins were extracted from the hTECs and kidney tissue using RIPA buffer (cat. RC2002-050-00, Biosesang, Yongin, South Korea, 150 mM NaCl; 100 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e; 50 mM Tris; HCL, pH 7.3; 0.1 mM EDTA 1% (vol/vol) sodium deoxycholate; 1% (vol/vol) Triton X-100; and 0.2% NaF) with protease inhibitor (GeneDEPOT, Katy, TX, USA). BCA assay was used to standardize the protein lysates to equal concentration using Pierce BCA Protein Assay Kits (cat. 23227, Thermo Fisher Scientific, Waltham, MA, USA). The protein samples were separated in glycine-SDS buffer and then transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with a solution containing 5% skim milk and 2% BSA for 1 h at 25\u0026deg;C and incubated with primary antibodies (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Next, the membranes were incubated with anti-mouse IgG (cat. 7076S, Cell Signaling Technology, Danvers, MA, USA) or anti-rabbit IgG (cat. 7074S, Cell Signaling Technology) for 1 h at 25\u0026deg;C. Target proteins were identified with the ImageQuant Las 4000 mini system (GE HealthCare, Chicago, IL, USA), and subsequent analysis was performed using ImageJ (v. 1.52, Wayne Rasband, National Institutes of Health).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence assay\u003c/h2\u003e \u003cp\u003ehTECs were cultured on 4-well culture slides (Nunc Lab-Tek II Chamber Slide System, cat. 154526, Thermo Fisher Scientific) with total media for 1 day. After replacing with new total media, cells were exposed to SMFs for 3 days. Cells on culture slides were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, and permeabilized with 0.01% Triton X. Blocking agent containing 5% normal goat serum and 2% bovine serum albumin was used and incubated for 1 h at 25\u0026deg;C. The slides were stained with antibodies overnight at 4\u0026deg;C (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), followed by incubation with Alexa 488-conjugated goat anti-mouse (cat. A-11001, Thermo Fisher Scientific) and Alexa 555-conjugated goat anti-rabbit (cat. A-21428, Thermo Fisher Scientific) secondary antibodies for 1 h at 25\u0026deg;C. 4\u0026prime;,6-Diamidino-2-phenylindole (cat. D1306, Invitrogen, Waltham, MA, USA) was used for nuclear staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eCells were fixed with ice-cold 70% ethanol for at least 1 h at -20\u0026deg;C and then washed with cell staining buffer (cat. 420201, BioLegend, San Diego, CA, USA). Cells were stained with APC-conjugated Ki-67 antibody (cat. 350514, BioLegend, 5 \u0026micro;L /1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells in 100 \u0026micro;L) for 30 min at room temperature in the dark. To ensure selective staining of DNA, cells were treated with 100 \u0026micro;g/mL ribonuclease (cat. GE6228, Glentham Life Sciences, Corsham, UK). Propidium iodide (PI) solution (cat. 421301, BioLegend) was used to assess DNA content in cell cycle analysis using flow cytometry. The cell cycle was evaluated through flow cytometry using a BD FACSLyric (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software (v10.8.1., BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and real-time qPCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA from hTECs was extracted using the TRIzol reagent (Thermo Fischer Scientific) according to the manufacturer\u0026rsquo;s instructions. cDNA was synthesized from the total RNA of hTECs and amplified via PCR with a C1000 thermal cycler (Bio-Rad, Hercules, CA, USA). Afterward, qPCR was performed on a 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) with the following thermal cycling conditions: 50\u0026deg;C for 2 min, 95\u0026deg;C for 10 min, 40 cycles of 95˚C for 15 s and 60\u0026deg;C for 1 min, 95\u0026deg;C for 15 s, 60\u0026deg;C for 1 min, 95\u0026deg;C for 30 s, and 60\u0026deg;C for 15 s. Relative gene expression levels were quantified using the comparative CT (ΔΔCT) method, with GAPDH serving as the normalization control. The forward and reverse primer sequences used in this study are listed in Supplementary Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell apoptosis assay\u003c/h2\u003e \u003cp\u003eTo assess cell apoptosis and necrosis, the Annexin V/propidium iodide FITC apoptosis kit (cat. 556547, BD Biosciences) was used for flow cytometry, following the manufacturer\u0026rsquo;s instructions. After SMF exposure, harvested cells were washed with PBS, resuspended in 1X binding buffer (100 \u0026micro;L), and stained with FITC-conjugated Annexin V (3.5 \u0026micro;L) and PI (3.5 \u0026micro;L, 50 mg/mL). Subsequently, the cells were incubated for 15 min at 25\u0026deg;C in the dark. Flow cytometry data were acquired using the BD FACSCanto (BD Biosciences) and analyzed with FlowJo software (v10.8.1., BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAITN model\u003c/h2\u003e \u003cp\u003eC57BL/6 mice (male, 8 weeks old, n\u0026thinsp;=\u0026thinsp;5 per group) were obtained from KOATECH (South Korea) and adenine (2 mg/mouse, cat. A8626, Sigma-Aldrich) was orally administered daily for 2 weeks. After 2 weeks, the mice were anesthetized by intraperitoneal injection of Zoletil\u0026trade; (30 mg/kg; Virbac, Carros, France) and xylazine (Rompun; 10 mg/kg; Bayer, Leverkusen, Germany), followed by sacrifice via abdominal aortic puncture for blood collection [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. BUN (mg/dL) and creatinine (mg/dL) concentrations were measured to assess renal function using an autoanalyzer (HITACHI7180, Hitachi Chemical Industries, Tokyo, Japan). All animal studies were performed under the guidance of the Institutional Animal Care and Use Committee (IACUC: 24-0057-S1A1) of Seoul National University Hospital and conducted in accordance with the National Research Council\u0026rsquo;s Guidelines for the Care and Use of Laboratory Animals. All methods for animal experiments are reported according to the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eKidney tissues were fixed in 10% buffered formalin and embedded in paraffin. Then, 4-\u0026micro;m- thick sections were cut from the paraffin blocks for dehydration and rehydration, involving a series of xylene treatments followed by decreasing concentrations of ethanol and water. For the antigen retrieval, the kidney sections were microwaved in sodium citrate buffer. To block endogenous peroxidase activity, a 3% hydrogen peroxide solution diluted in methyl alcohol was applied. The sections were incubated overnight at 4\u0026deg;C with primary antibodies (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Subsequently, Dako Envision\u0026thinsp;+\u0026thinsp;System-HRP-labeled polymer anti-mouse (cat. K4001, Agilent DAKO, Santa Clara, CA, USA) and anti-rabbit (cat. K4003, Agilent DAKO) were applied and incubated for 1 h at 25\u0026deg;C. Periodic acid-Schiff staining was performed to evaluate tubular injury scores based on the percentage of the affected area, with cell nuclei counterstained using Mayer\u0026rsquo;s hematoxylin (Sigma-Aldrich). The degree of tubular injury was assessed as previously described. The injury scores were categorized as follows: 0 for no damage, 1 for 1\u0026ndash;10% injured area, 2 for 11\u0026ndash;25%, 3 for 26\u0026ndash;75%, and 4 for more than 75% injured area [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. A Leica inverted microscope (Leica Camera, Wetzlar, Germany) and the LAS-4000 software (Leica Camera) were used to analyze and quantify the affected area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are presented as means with standard error of the mean. Each experiment was repeated at least three times independently. Statistical analysis was conducted using an unpaired two-tailed Student\u0026rsquo;s t-test in GraphPad Prism 10.0 (GraphPad Software Inc., San Diego, CA, USA). A p-value of less than 0.05 was considered significant (*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEthics Approval Statement\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Seoul National University Hospital (IACUC approval number: 24-0057-S1A1). Human kidney biopsy samples were collected with approval from the Institutional Review Board of Seoul National University Hospital (IRB approval number: 2110-026-1260), and informed consent was obtained from all patients.\u003c/p\u003e \u003c/div\u003e\u003ch2\u003e \u003cb\u003eAdditional Information\u003c/b\u003e \u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by the National Research Foundation of Korea grant (NRF-2023R1A2C2006651)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSML, SRL, SHL, EJB, KDY, JWL, YSK, RHC, and SHY designed the experiments. SML, SRL, SJP, and KHK performed the research. All authors analyzed the data. SML and SRL wrote the paper. RHC and SHY designed and supervised the project. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eIllustrations in Figure 3a and Figure 4 were created with BioRender.com.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eReferences1. Zhang, L. \u003cem\u003eet al.\u003c/em\u003e 1 T moderate intensity static magnetic field affects Akt/mTOR pathway and increases the antitumor efficacy of mTOR inhibitors in CNE-2Z cells. \u003cem\u003eSci. Bull.\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 2120\u0026ndash;2128 (2015)\u003c/li\u003e\n\u003cli\u003eMilovanovich, I. D. \u003cem\u003eet al.\u003c/em\u003e Homogeneous static magnetic field of different orientation induces biological changes in subacutely exposed mice. \u003cem\u003eEnviron. Sci. Pollut. Res. Int.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1584\u0026ndash;1597 (2016)\u003c/li\u003e\n\u003cli\u003eYu, X. \u003cem\u003eet al.\u003c/em\u003e Static magnetic fields protect against cisplatin-induced kidney toxicity. \u003cem\u003eAntioxidants (Basel)\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 73 (2022)\u003c/li\u003e\n\u003cli\u003eHsu, R. K., McCulloch, C. E., Dudley, R. A., Lo, L. J. \u0026amp; Hsu, C. Y. Temporal changes in incidence of dialysis-requiring AKI. \u003cem\u003eJ. Am. Soc. Nephrol.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 37\u0026ndash;42 (2013)\u003c/li\u003e\n\u003cli\u003eGuo, R. \u003cem\u003eet al.\u003c/em\u003e The road from AKI to CKD: molecular mechanisms and therapeutic targets of ferroptosis. \u003cem\u003eCell Death Dis.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 426 (2023)\u003c/li\u003e\n\u003cli\u003eCoca, S. G., Singanamala, S. \u0026amp; Parikh, C. R. Chronic kidney disease after acute kidney injury: a systematic review and meta-analysis. \u003cem\u003eKidney Int.\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 442\u0026ndash;448 (2012)\u003c/li\u003e\n\u003cli\u003eSugiyama, N., Kohno, M. \u0026amp; Yokoyama, T. Inhibition of the p38 MAPK pathway ameliorates renal fibrosis in an NPHP2 mouse model. \u003cem\u003eNephrol. Dial. Transplant.\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1351\u0026ndash;1358 (2012)\u003c/li\u003e\n\u003cli\u003eTao, Y. \u003cem\u003eet al.\u003c/em\u003e Nr4a1 promotes renal interstitial fibrosis by regulating the p38 MAPK phosphorylation. \u003cem\u003eMol. Med.\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 63 (2023)\u003c/li\u003e\n\u003cli\u003eWang, D. \u003cem\u003eet al.\u003c/em\u003e Inhibition of p38 MAPK attenuates renal atrophy and fibrosis in a murine renal artery stenosis model. \u003cem\u003eAm. J. Physiol. Renal Physiol.\u003c/em\u003e \u003cstrong\u003e304\u003c/strong\u003e, F938\u0026ndash;F947 (2013)\u003c/li\u003e\n\u003cli\u003eLee, J. \u003cem\u003eet al.\u003c/em\u003e p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients. \u003cem\u003ePLOS One\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, e0213981 (2019)\u003c/li\u003e\n\u003cli\u003eFalke, L. L., Gholizadeh, S., Goldschmeding, R., Kok, R. J. \u0026amp; Nguyen, T. Q. Diverse origins of the myofibroblast\u0026mdash;implications for kidney fibrosis. Nat. Rev. Nephrol. 11, 233\u0026ndash;244 (2015)\u003c/li\u003e\n\u003cli\u003eReiss, A. B. et al. Fibrosis in chronic kidney disease: pathophysiology and therapeutic targets. J. Clin. Med. 13, 1881 (2024)\u003c/li\u003e\n\u003cli\u003eNakanishi, T. \u0026amp; Kuragano, T. Growing concerns about using hypoxia-inducible factor prolyl hydroxylase inhibitors for the treatment of renal anemia. Clin. Kidney J. 17, sfae051 (2024)\u003c/li\u003e\n\u003cli\u003eLiu, K. H. et al. Hypoxia stimulates the epithelial-to-mesenchymal transition in lung cancer cells through accumulation of nuclear \u0026beta;-catenin. Anticancer Res. 38, 6299\u0026ndash;6308 (2018)\u003c/li\u003e\n\u003cli\u003eHu, J. et al. Hypoxia inducible factor-1\u0026alpha; mediates the profibrotic effect of albumin in renal tubular cells. Sci. Rep. 7, 15878 (2017)\u003c/li\u003e\n\u003cli\u003eKimura, K. et al. Stable expression of HIF-1alpha in tubular epithelial cells promotes interstitial fibrosis. Am. J. Physiol. Renal Physiol. 295, F1023\u0026ndash;F1029 (2008)\u003c/li\u003e\n\u003cli\u003eWang, Z. et al. Silencing of hypoxia-inducible factor-1\u0026alpha; gene attenuates chronic ischemic renal injury in two-kidney, one-clip rats. Am. J. Physiol. Renal Physiol. 306, F1236\u0026ndash;F1242 (2014)\u003c/li\u003e\n\u003cli\u003eYu, L. et al. HIF-1\u0026alpha; alleviates high-glucose-induced renal tubular cell injury by promoting Parkin/PINK1-mediated mitophagy. Front. Med. (Lausanne) 8, 803874 (2021)\u003c/li\u003e\n\u003cli\u003eShu, S. et al. Hypoxia and hypoxia-inducible factors in kidney injury and repair. Cells 8, 207 (2019)\u003c/li\u003e\n\u003cli\u003eConde, E. et al. HIF-1\u0026alpha; induction during reperfusion avoids maladaptive repair after renal ischemia/reperfusion involving miR127-3p. Sci. Rep. 7, 41099 (2017)\u003c/li\u003e\n\u003cli\u003eWang, J. et al. Inhibition of viability, proliferation, cytokines secretion, surface antigen expression, and adipogenic and osteogenic differentiation of adipose-derived stem cells by seven-day exposure to 0.5 T static magnetic fields. Stem Cells Int. 2016, 7168175 (2016)\u003c/li\u003e\n\u003cli\u003eLuo, Y. et al. Moderate intensity static magnetic fields affect mitotic spindles and increase the antitumor efficacy of 5-FU and Taxol. Bioelectrochemistry 109, 31\u0026ndash;40 (2016)\u003c/li\u003e\n\u003cli\u003eSullivan, K., Balin, A. K. \u0026amp; Allen, R. G. Effects of static magnetic fields on the growth of various types of human cells. \u003cem\u003eBioelectromagnetics\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 140\u0026ndash;147 (2011)\u003c/li\u003e\n\u003cli\u003eTian, X. \u003cem\u003eet al.\u003c/em\u003e Magnetic field direction differentially impacts the growth of different cell types. \u003cem\u003eElectromagn. Biol. Med.\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 114\u0026ndash;125 (2018)\u003c/li\u003e\n\u003cli\u003eYang, X. \u003cem\u003eet al.\u003c/em\u003e An upward 9.4 T static magnetic field inhibits DNA synthesis and increases ROS-P53 to suppress lung cancer growth. \u003cem\u003eTransl. Oncol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 101103 (2021)\u003c/li\u003e\n\u003cli\u003eGanguly, P., Macleod, T., Wong, C., Harland, M. \u0026amp; McGonagle, D. Revisiting P38 mitogen-activated protein kinases (MAPK) in inflammatory arthritis: A narrative of the emergence of MAPK-activated protein kinase inhibitors (MK2i). \u003cem\u003ePharmaceuticals (Basel)\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1286 (2023)\u003c/li\u003e\n\u003cli\u003eYue, J. \u0026amp; L\u0026oacute;pez, J. M. Understanding MAPK signaling pathways in apoptosis. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 2346 (2020)\u003c/li\u003e\n\u003cli\u003evan der Slikke, E. C. \u003cem\u003eet al.\u003c/em\u003e Sepsis is associated with mitochondrial DNA damage and a reduced mitochondrial mass in the kidney of patients with sepsis-AKI. \u003cem\u003eCrit. Care\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 36 (2021)\u003c/li\u003e\n\u003cli\u003eChernikov, A. V., Gudkov, S. V., Usacheva, A. M. \u0026amp; Bruskov, V. I. Exogenous 8-oxo-7,8-dihydro-2\u0026prime;-deoxyguanosine: biomedical properties, mechanisms of action, and therapeutic potential. \u003cem\u003eBiochemistry (Mosc)\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 1686\u0026ndash;1701 (2017)\u003c/li\u003e\n\u003cli\u003eKim, J. Y. \u003cem\u003eet al.\u003c/em\u003e Involvement of p38 mitogen-activated protein kinase in the cell growth inhibition by sodium arsenite. \u003cem\u003eJ. Cell. Physiol.\u003c/em\u003e \u003cstrong\u003e190\u003c/strong\u003e, 29\u0026ndash;37 (2002)\u003c/li\u003e\n\u003cli\u003eWhitaker, R. H. \u0026amp; Cook, J. G. Stress relief techniques: p38 MAPK determines the balance of cell cycle and apoptosis pathways. \u003cem\u003eBiomolecules\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1444 (2021)\u003c/li\u003e\n\u003cli\u003eFaust, D. \u003cem\u003eet al.\u003c/em\u003e Differential p38-dependent signalling in response to cellular stress and mitogenic stimulation in fibroblasts. \u003cem\u003eCell Commun. Signal.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 6 (2012)\u003c/li\u003e\n\u003cli\u003eZamir-Nasta, T., Razi, M., Shapour, H. \u0026amp; Malekinejad, H. Roles of p21, p53, cyclin D1, CDK-4, estrogen receptor \u0026alpha; in aflatoxin b1-induced cytotoxicity in testicular tissue of mice. \u003cem\u003eEnviron. Toxicol.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 385\u0026ndash;395 (2018)\u003c/li\u003e\n\u003cli\u003eAmin, M., Razi, M., Sarrafzadeh-Rezaei, F., Shalizar Jalali, A. \u0026amp; Najafi, G. Berberine inhibits experimental varicocele-induced cell cycle arrest via regulating cyclin D1, cdk4 and p21 proteins expression in rat testicles. \u003cem\u003eAndrologia\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, e12984 (2018)\u003c/li\u003e\n\u003cli\u003eAn, J. N. \u003cem\u003eet al.\u003c/em\u003e Periostin induces kidney fibrosis after acute kidney injury via the p38 MAPK pathway. \u003cem\u003eAm. J. Physiol. Renal Physiol.\u003c/em\u003e \u003cstrong\u003e316\u003c/strong\u003e, F426\u0026ndash;F437 (2019)\u003c/li\u003e\n\u003cli\u003eLi, J. \u003cem\u003eet al.\u003c/em\u003e Inhibition of p38 mitogen-activated protein kinase and transforming growth factor-beta1/Smad signaling pathways modulates the development of fibrosis in adriamycin-induced nephropathy. \u003cem\u003eAm. J. Pathol.\u003c/em\u003e \u003cstrong\u003e169\u003c/strong\u003e, 1527\u0026ndash;1540 (2006)\u003c/li\u003e\n\u003cli\u003eStambe, C. \u003cem\u003eet al.\u003c/em\u003e Blockade of p38alpha MAPK ameliorates acute inflammatory renal injury in rat anti-GBM glomerulonephritis. \u003cem\u003eJ. Am. Soc. Nephrol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 338\u0026ndash;351 (2003)\u003c/li\u003e\n\u003cli\u003eChoi, J. \u003cem\u003eet al.\u003c/em\u003e In vivo longitudinal 920 nm two-photon intravital kidney imaging of a dynamic 2,8-dha crystal formation and tubular deterioration in the adenine-induced chronic kidney disease mouse model. \u003cem\u003eBiomed. Opt. Express\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1647\u0026ndash;1658 (2023)\u003c/li\u003e\n\u003cli\u003eKhan, M. A. \u003cem\u003eet al.\u003c/em\u003e Adenine overload induces ferroptosis in human primary proximal tubular epithelial cells. \u003cem\u003eCell Death Dis.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 104 (2022)\u003c/li\u003e\n\u003cli\u003eAwad, A. M., Saleh, M. A., Abu-Elsaad, N. M. \u0026amp; Ibrahim, T. M. Erlotinib can halt adenine induced nephrotoxicity in mice through modulating ERK1/2, STAT3, p53 and apoptotic pathways. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 11524 (2020)\u003c/li\u003e\n\u003cli\u003eJia, T. \u003cem\u003eet al.\u003c/em\u003e A novel model of adenine-induced tubulointerstitial nephropathy in mice. \u003cem\u003eBMC Nephrol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 116 (2013)\u003c/li\u003e\n\u003cli\u003eYoo, K. D. \u003cem\u003eet al.\u003c/em\u003e Role of the CCL20/CCR6 axis in tubular epithelial cell injury: kidney-specific translational insights from acute kidney injury to chronic kidney disease. \u003cem\u003eFASEB J.\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, e23407 (2024)\u003c/li\u003e\n\u003cli\u003ePark, J. Y. \u003cem\u003eet al.\u003c/em\u003e Blockade of STAT3 signaling alleviates the progression of acute kidney injury to chronic kidney disease through antiapoptosis. \u003cem\u003eAm. J. Physiol. Renal Physiol.\u003c/em\u003e \u003cstrong\u003e322\u003c/strong\u003e, F553\u0026ndash;F572 (2022) [published correction appears in \u003cem\u003eAm. J. Physiol. Renal Physiol.\u003c/em\u003e \u003cstrong\u003e323,\u003c/strong\u003e F700 (2022)]\u003c/li\u003e\n\u003cli\u003eLee, J. \u003cem\u003eet al.\u003c/em\u003e Antibiotic-induced intestinal microbiota depletion can attenuate the acute kidney injury to chronic kidney disease transition via NADPH oxidase 2 and trimethylamine-N-oxide inhibition. \u003cem\u003eKidney Int.\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 1239\u0026ndash;1253 (2024)\u003c/li\u003e\n\u003cli\u003ePark, S. \u003cem\u003eet al.\u003c/em\u003e Elevated bilirubin levels are associated with a better renal prognosis and ameliorate kidney fibrosis. \u003cem\u003ePLOS One\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e0172434 (2017)\u003c/li\u003e\n\u003cli\u003eAn, J. N. \u003cem\u003eet al\u003c/em\u003e. cMet agonistic antibody attenuates apoptosis in ischaemia-reperfusion-induced kidney injury. \u003cem\u003eJ. Cell. Mol. Med.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 5640-5651 (2020)\u003c/li\u003e\n\u003cli\u003eJang, H. R. \u003cem\u003eet al.\u003c/em\u003e Early exposure to germs modifies kidney damage and inflammation after experimental ischemia-reperfusion injury. \u003cem\u003eAm. J. Physiol. Renal Physiol.\u003c/em\u003e \u003cstrong\u003e297\u003c/strong\u003e, F1457\u0026ndash;F1465 (2009)\u003c/li\u003e\n\u003cli\u003eYoo, K. D. \u003cem\u003eet al.\u003c/em\u003e Chemokine receptor 5 blockade modulates macrophage trafficking in renal ischaemic-reperfusion injury. \u003cem\u003eJ. Cell. Mol. Med.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 5515\u0026ndash;5527 (2020)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"AKI − to − CKD, cell cycle, mitogen-activated protein pathway, static magnetic field","lastPublishedDoi":"10.21203/rs.3.rs-6392601/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6392601/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVarious mechanisms, including inflammation, oxidative stress, and apoptosis, are involved in the transition from acute kidney injury to chronic kidney disease (AKI\u0026thinsp;\u0026minus;\u0026thinsp;to \u0026minus;\u0026thinsp;CKD). In this study, we aimed to determine the pathway linking acute injury and fibrosis under static magnetic fields (SMFs). Human tubular epithelial cells (hTECs) were cultured on SMF platforms (119 mT; outward vs. inward direction) for 3 days, followed by treatment with adenine and p38 MAPK inhibitor to verify the role of MAP-kinase pathway. We orally administered 2 mg of adenine to mice daily for 14 days (adenine-induced tubular nephropathy; AITN). Phospho-p38 was significantly elevated in hTECs cultured under inward SMFs compared with that cultured under outward SMFs. Inhibition of p38 MAPK reduced G1/S arrest and oxidative stress, exerted anti-apoptotic effects, and downregulated the expression of fibrosis markers under inward SMFs. Deposition of F4/80-positive cells, IL-17R, p53, and p38 was significantly increased in AITN mice. p38 MAPK inhibition under inward SMFs led to a decrease in fibronectin expression in adenine-treated hTECs. This study revealed that SMF-related AKI\u0026thinsp;\u0026minus;\u0026thinsp;to \u0026minus;\u0026thinsp;CKD transition progresses with the direction of SMFs affecting the severity of injury, whereas p38 MAPK inhibition attenuates SMF-induced kidney injury and prevents fibrosis.\u003c/p\u003e","manuscriptTitle":"Alterations in cell cycle and MAPK pathway contribute to transition from SMF-associated acute kidney injury to fibrosis: Field direction matters","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 11:11:29","doi":"10.21203/rs.3.rs-6392601/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-08T08:05:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-07T09:42:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12081844060930538959223547556698643481","date":"2025-05-03T00:55:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-24T13:06:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82417206501625949202888887437029315982","date":"2025-04-23T23:26:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-23T02:43:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-23T02:39:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-11T09:18:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-11T01:43:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-11T01:42:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7715178-41f7-476f-a8a9-6d3c27253ca2","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47631577,"name":"Biological sciences/Cell biology/Cell death/Apoptosis"},{"id":47631578,"name":"Health sciences/Nephrology/Kidney diseases/Renal fibrosis"}],"tags":[],"updatedAt":"2025-07-14T16:03:29+00:00","versionOfRecord":{"articleIdentity":"rs-6392601","link":"https://doi.org/10.1038/s41598-025-09077-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-10 15:57:45","publishedOnDateReadable":"July 10th, 2025"},"versionCreatedAt":"2025-04-25 11:11:29","video":"","vorDoi":"10.1038/s41598-025-09077-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-09077-w","workflowStages":[]},"version":"v1","identity":"rs-6392601","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6392601","identity":"rs-6392601","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}