Lactate drives maladaptive metabolic reprogramming via MRS2 inischemia–reperfusion-induced acute kidney injury

Lactate drives maladaptive metabolic reprogramming via MRS2 inischemia–reperfusion-induced acute kidney injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Lactate drives maladaptive metabolic reprogramming via MRS2 inischemia–reperfusion-induced acute kidney injury zhixin Yan, Annan Chen, Fang Li, Jian Zhang, Qiwen Xie, Gaoxiang Han, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8846937/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background Ischemia–reperfusion–induced acute kidney injury (I/R-AKI) causes profound bioenergetic collapse in renal proximal tubular epithelial cells, triggering a sustained shift from mitochondrial oxidative metabolism to glycolysis. Although lactate accumulation is a hallmark of this metabolic state, whether lactate actively drives mitochondrial dysfunction and enforces persistent metabolic reprogramming remains unclear. Methods Human renal biopsy specimens, murine I/R-AKI, and hypoxia/reoxygenation–challenged proximal tubular epithelial cells were used to investigate the lactate–MRS2 axis in I/R-AKI. Lactate signaling was inhibited by sodium oxamate, while MRS2 was suppressed using CPACC or siRNA-based approaches, including lipid nanoparticle–mediated siMRS2 delivery. Mitochondrial function and oxidative metabolism were assessed by oxygen consumption rate, ATP production, mitochondrial membrane potential, and tricarboxylic acid cycle (TCA) gene expression. Results I/R-AKI induced a pronounced bioenergetic deficit in proximal tubules, marked by disrupted mitochondrial homeostasis, suppressed TCA cycle activity and enhanced aerobic glycolysis. Glycolysis-derived lactate accumulated during reperfusion, disrupting mitochondrial oxidative metabolism, whereas inhibition of lactate production with sodium oxamate attenuated tubular injury and restored mitochondrial metabolic function. Mechanistically, lactate activated the mitochondrial Mg²⁺ channel MRS2, causing mitochondrial Mg²⁺ overload and sustained reliance on inefficient glycolysis. Targeting MRS2, either pharmacologically or via lipid nanoparticle–mediated siRNA delivery, normalized mitochondrial Mg²⁺ homeostasis, improved mitochondrial function and reinstated oxidative metabolism following I/R-AKI. Conclusion This study identifies a previously unrecognized lactate–MRS2 signaling axis that drives maladaptive metabolic reprogramming in ischemic AKI. Targeting MRS2 to restore mitochondrial Mg²⁺ homeostasis reinstates oxidative metabolism, breaks maladaptive metabolic reprogramming, and promotes renal recovery. acute kidney injury metabolic reprogramming lactate MRS2 mitochondrial homeostasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Acute kidney injury (AKI) is a common and severe clinical syndrome associated with high morbidity, mortality, and progression to chronic kidney disease. Ischemia–reperfusion injury (IRI) is one of its major causes, especially in kidney transplantation, cardiac surgery, and sepsis[ 1 ]. Among tubular segments, proximal tubular epithelial cells (PTECs) are uniquely vulnerable due to their exceptionally high ATP demand and reliance on mitochondrial oxidative metabolism. Even brief ischemic insults precipitate rapid ATP depletion, ionic imbalance, and bioenergetic failure, ultimately triggering cell death [ 2 ]. Despite extensive efforts to target mitochondria integrity and improve energy production, effective strategies to restore cellular bioenergetics and preserve tubular viability remain limited[ 3 – 7 ]. To survive transient ischemic stress, PTECs undergo a profound metabolic reprogramming, shifting from tricarboxylic acid (TCA) cycle–driven mitochondrial oxidative metabolism toward anaerobic glycolysis [ 8 – 10 ]. While this switch initially supports cell survival under hypoxic conditions, the persistence of glycolytic metabolism during reperfusion becomes maladaptive. This Warburg-like metabolic state results in inefficient ATP generation and exacerbates mitochondrial dysfunction [ 8 , 11 – 13 ]. However, the upstream signals that sustain this pathological metabolic state remain poorly defined. Lactate, traditionally considered as the end-product of glycolysis, has recently emerged as a multifunctional metabolic signal capable of modulating mitochondrial function [ 14 , 15 ], cellular bioenergetics[ 16 ], and stress responses[ 17 – 19 ]. Elevated lactate levels are consistently observed in patients and experimental models of AKI, correlating with disease severity, impaired tubular recovery, and progression towards renal fibrosis [ 13 , 20 – 22 ]. However, whether lactate directly drives maladaptive metabolic reprogramming in injured PTECs remains unresolved, and clarifying its functional consequences may reveal key determinants of energy failure and repair after AKI. Mitochondrial RNA splicing 2 protein (MRS2) is the predominant magnesium channel in the mitochondrial inner membrane and constitutes the principal pathway for Mg²⁺ entry into the mitochondrial matrix [ 23 , 24 ]. Mg²⁺ is essential for ATP stability and the activity of numerous metabolic enzymes in TCA cycle [ 25 , 26 ], and disruption of MRS2-mediated Mg²⁺ transport reprograms cellular metabolism[ 27 ]. Notably, MRS2 may also act as a metabolic effector responsive to lactate, thereby influencing mitochondrial function [ 16 , 27 ]. Nevertheless, the role of MRS2 in the kidney, particularly under ischemic conditions, remains largely unexplored. It is unclear whether lactate-mediated MRS2 activation contributes to mitochondrial dysfunction and metabolic reprogramming following I/R-AKI. Based on these observations, we hypothesized that lactate accumulation during I/R-AKI activates MRS2, leading to mitochondrial Mg²⁺ overload, and suppression of TCA cycle–driven oxidative metabolism. In this study, we identify lactate as a key driver of maladaptive metabolic reprogramming in ischemic renal tubular cells through activation of MRS2. Furthermore, pharmacological inhibition of MRS2 or lipid nanoparticle–mediated delivery of siMRS2 restores metabolic homeostasis and attenuates tubular injury following I/R-AKI. Materials and methods Human kidney samples Renal biopsy specimens were collected from patients diagnosed with acute tubular injury during routine diagnostic evaluation at Zhongshan Hospital. The study protocol was approved by the Institutional Ethics Committee, and written informed consent was obtained from all participants prior to sample collection (Approval No.: B2025-316R). Animal models of ischemia/reperfusion-induced acute kidney injury (I/R-AKI) Male C57BL/6 mice (8–10 weeks old, 20–25 g) were used as previously described[ 28 ]. Briefly, mice were anesthetized via intraperitoneal injection of 4% phenobarbital (10 mL/kg body weight). Bilateral renal pedicles were exposed through a midline abdominal incision and clamped using atraumatic vascular clamps for 19 minutes to induce ischemia. Reperfusion was initiated by releasing the clamps, after which the incision was closed. Sham-operated mice underwent the same procedure without clamping. Body temperature was maintained at 36°C throughout surgery using a heating pad. Kidneys and blood samples were collected 24 hours after reperfusion for subsequent analyses. For the sodium oxamate treatment, sodium oxamate (300 mg/kg, MCE) was administered via intraperitoneal injection three days before ischemia surgery and then injected once after ischemia. For the chloropentaammine cobalt (III)chloride (CPACC) treatment, CPACC (20 mg/kg, MACKLIN) was administered via intraperitoneal injection every three days for three times before ischemia surgery. All procedures were conducted in strict accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (8th edition). Mice were group-housed under specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle, controlled temperature (22 ± 2°C) and humidity (50% ± 10%), and provided with free access to standard chow and water. Every effort was made to minimize animal suffering and reduce the number of animals used, including the application of appropriate anesthesia and analgesics during surgical procedures. Preparation of Lipid nanoparticles (LNPs)-encapsulated siRNA LNPs were prepared using an ethanol-dilution microfluidic method. Briefly, the lipid phase was composed of an ionizable lipid, the structural lipid cholesterol, the phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and the helper lipid 1,2-dimyristoyl-rac-glycerol-methoxypolyethylene glycol-2000 (DMG-PEG2000), mixed in ethanol at a molar ratio of 50:10:38.5:1.5. Scramble and siMRS2 were individually dissolved in 25 mM sodium acetate buffer (pH 5.0) prior to formulation. The ethanolic lipid solution and the aqueous RNA solution were combined at a 1:3 volume ratio in a microfluidic mixer to facilitate self-assembly of LNPs. The resulting suspensions were dialyzed against PBS at room temperature for > 2 hours to remove ethanol and achieve buffer exchange. In vivo administration of LNPs For in vivo renal delivery, LNP-encapsulated scramble RNA or siMrs2 RNA was administered via retrograde pyeloureteral injection. Briefly, mice were anesthetized, and each kidney received 10 µg RNA in 50 µL sterile PBS through the renal pelvis using a microsyringe under direct visualization. LNPs were delivered 48 hours prior to ischemia induction to ensure sufficient renal uptake and gene silencing. Control mice received an equal volume and RNA dose of scramble RNA–loaded LNPs. Cell culture and treatment Immortalized mouse proximal tubular epithelial cells (mTECs; BeNa Culture Collection, BNCC, China) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum under standard conditions (37°C, 5% CO₂). For hypoxia/reoxygenation (H/R) treatment, mTECs were seeded at 1 × 10⁵ cells per well in 6-well plates and cultured until ~ 70% confluence. Cells were then incubated in serum-free RPMI 1640 under hypoxia (1% O₂, 5% CO₂, 94% N₂) for 24 hours, followed by reoxygenation for 0, 4, 8 hours under normoxia (21% O₂, 5% CO₂) [ 11 ]. Based on the severity of cellular injury observed across conditions, a reoxygenation period of 4 hours was selected for subsequent experiments. For lactate stimulation, mTECs were exposed to 20 mM L-lactic acid (L812604, MACKLIN) for 12 h. To block lactate uptake, cells were pretreated with the monocarboxylate transporter inhibitor AR-C155858 (2 µM; HY-13248, MCE) 1 h before lactate exposure. To inhibit lactate synthesis, mTECs were pretreated with the LDHA inhibitor GSK2837808A (100 nM; HY-100681, MCE) 1 h before H/R. For gene silencing, MRS2-specific siRNA (ZORIN) was transfected using Lipofectamine 3000 (Invitrogen), with a nonsilencing siRNA as the negative control. For overexpression, an MRS2 expression plasmid (ZORIN) was introduced using the same reagent, and sequence integrity was verified by DNA sequencing; the ZV304 vector served as control. Transfection efficiency was confirmed by qPCR and Western blotting. siRNA and plasmid sequences are listed in Supplementary Table S1 . Evaluation of kidney function Serum creatinine (SCr) and blood urea nitrogen (BUN) were measured using QuantiChrom Creatinine (DICT-500) and Urea (DIAG-100) Assay Kits (BioAssay Systems, USA) according to the manufacturer’s instructions. Histopathological examination Kidney tissues were fixed, paraffin-embedded, and sectioned at 4 µm thickness. Sections were stained with periodic acid–Schiff (PAS) reagent to evaluate tubular injury. Tubular injury score was performed in a blinded manner based on the percentage of tubules showing epithelial necrosis, brush border loss, cast formation, or tubular dilation, using a 0–4 scale: 0, none; 1, 75% affected[ 29 ]. Immunohistochemistry Paraffin-embedded kidney sections (4 µm) were deparaffinized, rehydrated, and subjected to antigen retrieval. After blocking with 1% bovine serum albumin (BSA; Sigma, B2064), sections were incubated overnight at 4°C with primary antibodies against MRS2 (ANT-148; Alomone Labs, 1:400). After washing, slides were incubated with HRP-conjugated secondary antibodies (Gene Tech, GK500710, 1:300) for 30 min at 37°C. Peroxidase activity was visualized using a DAB detection kit (Gene Tech, GK500710), and images were captured under a light microscope. Immunofluorescence Immunofluorescence staining was performed as described previously [ 30 ]. After deparaffinization, antigen retrieval, and blocking with 1% BSA, kidney sections were incubated overnight at 4°C with primary antibodies against MRS2 (ANT-148; Alomone Labs,1:400), Lotus Tetragonolobus Lectin(LTL) (ZE1102;Vector, 1:800), Uromucoid(THP) (ab207170;Abcam,1:1000), Thiazide-Sensitive NaCl Cotransporter (NCC) (AB3553;EMD Millipore,1:1000), Aquaporin 2(AQP2) (AB3274;Merck,1:1000) and KIM1 (AF1817; R&D, 1:200). The next day, slides were incubated with Alexa Fluor 488–, 594–conjugated secondary antibodies (Thermo Fisher Scientific,1:200) or Alexa Fluor 647-conjugated Streptavidin (YEASEN,1:200) for 1 hour at room temperature. Nuclei were counterstained with DAPI. Fluorescence was visualized using an FV3000 confocal laser scanning microscope (Olympus, Japan). TUNEL staining Apoptotic cells were detected using a TUNEL assay kit (C1089; Beyotime, China) according to the manufacturer’s instructions. After staining, nuclei were counterstained with DAPI, and TUNEL⁺/DAPI⁺ cells were quantified as apoptotic. Images were captured with an FV3000 confocal microscope (Olympus, Japan). Transmission electron microscopy (TEM) Fresh kidney tissues were fixed overnight in 2% paraformaldehyde and 2.5% glutaraldehyde at 4°C, followed by dehydration, embedding in epoxy resin, and sectioning according to standard protocols. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a JEOL 1010 transmission electron microscope (JEOL, Japan). Western blotting Proteins were extracted from kidney tissues or cell lysates using RIPA buffer. Equal protein amounts (40 µg) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 hour, membranes were incubated overnight at 4°C with primary antibodies. HRP-conjugated secondary antibodies were applied for 1 hour at room temperature. Primary antibodies used in this study were: anti-MRS2 (ANT-148; Alomone Labs, 1:1000), anti-KIM1 (AF1817; R&D, 1:1000), anti-HK2 (ab209847;Abcam,1:1000), anti-PFKP(13389-1-AP;proteintech, 1:1000), anti-PKM2(15822-1-AP;proteintech, 1:1000) and anti-LDHA(A21893;ABclonal, 1:1000). Signals were visualized using a chemiluminescent substrate, and band intensities were normalized to β-actin. The intensity of bands was quantified using Image J software. RNA extraction and quantitative real-time PCR Total RNA was extracted using TRIzol reagent (Invitrogen). Reverse transcription was performed using Superscript II (TOYOBO, Japan) and oligo(dT) primers. qRT-PCR was conducted using SYBR Green Master Mix (TOYOBO) on an ABI 7500 system. Gene expression levels were calculated using the 2⁻ΔΔCt method, with Actb as the reference gene. Primer sequences are provided in Supplementary Table S2. Cell viability assay and Annexin V-FITC/PI apoptosis assay Cell viability was assessed using the CCK-8 assay (C0038; Beyotime, China). mTECs were seeded in 96-well plates (5 × 10³ cells/well), treated with lactic acid (5–20 mM) for 24 h, and incubated with CCK-8 reagent for 1–4 h. Absorbance at 450 nm was measured using a microplate reader (BioTek, USA). Apoptosis was analyzed using Annexin V-FITC/PI staining (KeyGEN Biotech, China) as previously described[ 31 ]. Briefly, mTECs were stained according to the manufacturer’s instructions and analyzed by flow cytometry (Attune NxT; Thermo Fisher Scientific, USA). Cells were classified as viable (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), or necrotic (Annexin V⁻/PI⁺). The apoptotic rate was defined as the sum of early and late apoptotic cells. Data were analyzed using FlowJo software (Tree Star, USA). ATP measurement ATP levels in kidney tissues and mTECs were determined using an ATP Fluorometric Assay Kit (S0026; Beyotime, China) according to the manufacturer’s protocol and normalized to total protein. Mitochondrial membrane potential (MMP) assay MMP was evaluated using JC-1 (C2006; Beyotime, China). After incubation with JC-1 for 20 min at 37°C, red (Ex/Em: 525/590 nm) and green (Ex/Em: 490/530 nm) fluorescence intensities were measured using a Varioskan™ LUX reader, and the red/green ratio was calculated. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurement OCR and ECAR were measured using a Seahorse XFe96 Analyzer (Agilent, USA) with the Mito Stress Test and Glycolysis Stress Test kits. Cells (2 × 10³/well) were exposed to H/R or 20 mM lactic acid, with control medium as baseline. Sequential injections included oligomycin (1.5 µM), FCCP (1.0 µM), and rotenone/antimycin A (0.5 µM each) for OCR, and glucose (10 mM), oligomycin (1 µM), and 2-DG (50 mM) for ECAR. Data were analyzed using Agilent Seahorse Wave 2.6.1 and GraphPad Prism 8. Lactate measurement Lactate concentrations in kidney tissues, mTECs, and culture medium were quantified using a colorimetric assay kit (A019-2-1; Nanjing Jiancheng Bioengineering Institute, China) and normalized to total protein. Spectrofluorimetric analysis of mitochondrial Mg²⁺ flux Mitochondrial Mg²⁺ dynamics were measured using a Varioskan™ LUX microplate reader as previously described[ 16 , 27 ]. Cells were washed with Ca²⁺- and Mg²⁺-free DPBS (pH 7.4) and pelleted by centrifugation at 503 × g for 5 minutes at 4°C. Approximately 4–5 × 10⁶ cells were resuspended in 1.5 mL intracellular medium (ICM; 120 mM KCl, 10 mM NaCl, 1 mM KH₂PO₄, 20 mM HEPES-Tris, pH 7) and permeabilized with 40 µg/mL digitonin. The medium was supplemented with succinate (5 mM), ATP, and Mag-Fluo-4 AM(5 µM; MX4544; MKBio) probes for Mg²⁺measurement (Ex/Em:494/516nm). For measurement of mitochondrial Mg²⁺ level, after a baseline recording, mTECs were challenged with FCCP (2 mM) to uncouple mitochondrion and changes in extramitochondrial ion concentrations served as indicators of mitochondrial Mg²⁺. For measurement of mitochondrial Mg²⁺ uptake, mTECs were challenged with either a single Mg²⁺ bolus (1 mM), followed by FCCP addition (2 mM) to uncouple mitochondria. Mitochondrial Mg²⁺uptake rates were calculated from the initial linear portion of the fluorescence traces. All measurements were conducted at 37°C with constant stirring. Statistical analysis Data are presented as mean ± standard deviation (SD). Statistical analyses and visualization were performed using GraphPad Prism 8 (GraphPad Software, USA). Comparisons between two groups were made using unpaired t-tests. One-way ANOVA with Tukey’s multiple comparisons test was used for multi-group analysis, and two-way ANOVA with Tukey’s test was applied when two independent variables were involved. P ≤ 0.05 was considered statistically significant. Results Proximal tubules showed bioenergetic deficit and metabolic reprogramming toward aerobic glycolysis in I/R-AKI model The I/R-AKI model was successfully established, as indicated by elevated serum creatinine (SCr) and blood urea nitrogen (BUN) at 24 h (Figure S1 a, b), accompanied by pronounced tubular injury on PAS staining, increased apoptosis on TUNEL staining (Figure S1 c), and marked upregulation of the injury marker KIM-1 (Figure S1 d). Following I/R, kidneys exhibited a pronounced loss of bioenergetic capacity, reflected by reduced ATP levels (Fig. 1 a) and severe mitochondrial structural injury, including swelling and cristae disruption in proximal tubules (Fig. 1 b). Consistently, expression of key tricarboxylic acid (TCA) cycle enzymes was markedly suppressed (Fig. 1 c), indicating impaired mitochondrial oxidative metabolism. In parallel, glycolytic pathways were strongly upregulated (Fig. 1 d), and renal lactate levels remained elevated during reperfusion despite oxygen restoration (Fig. 1 e), demonstrating a shift toward aerobic glycolysis. To model I/R-AKI in vitro, hypoxia/reoxygenation (H/R) was applied to cultured proximal tubular epithelial cells (mTECs) (Figure S1 e,f). H/R markedly reduced oxygen consumption rate (OCR) while increasing extracellular acidification rate (ECAR) (Figs. 1 f,g). Notably, glycolytic gene expression (Fig. 1 h) and intracellular lactate levels (Fig. 1 i) remained elevated during reoxygenation, demonstrating a sustained Warburg-like phenotype in proximal tubules independent of oxygen availability. Collectively, these findings demonstrate that I/R-AKI causes profound mitochondrial bioenergetic failure and enforces a persistent metabolic shift from mitochondrial oxidative metabolism toward aerobic glycolysis in proximal tubules. Excess lactate disrupts mitochondrial homeostasis and inhibits TCA cycle activity Given the accumulation of lactate during I/R-AKI and H/R-induced injury, and its known role in regulating mitochondrial homeostasis[ 14 , 15 , 21 , 22 ], we next examined whether lactate contributes to mitochondrial dysfunction in tubular epithelial cells. Exogenous L-lactic acid (LA) treatment reduced cell viability in a concentration-dependent manner (Figure S2a), with 20 mM selected for subsequent experiments. Pharmacologic inhibition of monocarboxylate transporters (MCT1/2) with AR-C155858 significantly attenuated LA-induced apoptosis (Figure S2b), indicating that intracellular lactate is the primary driver of cytotoxicity. As expected, LA induced mitochondrial damage in mTECs, characterized by increased mitochondrial swelling (Figure S2c) and loss of mitochondrial membrane potential (Figure S2d). This was accompanied by decreased intracellular ATP levels (Figure S1 e) and downregulation of key TCA cycle enzymes (Figure S2f), consistent with impaired mitochondrial oxidative capacity. These findings indicate that intracellular lactate acts not merely as a metabolic byproduct but as a signaling metabolite that disrupts mitochondrial homeostasis, suppresses oxidative metabolism, and may reinforce maladaptive glycolytic dependence during I/R-AKI. Blocking lactate production restores mitochondrial metabolism and alleviates renal dysfunction in I/R-AKI mice To determine whether reducing endogenous lactate accumulation alleviates renal injury, sodium oxamate (an LDH inhibitor) was administered in vivo (Fig. 2 a). Oxamate significantly reduced renal lactate levels (Fig. 2 b), improved kidney function as reflected by decreased SCr (Fig. 2 c) and BUN levels (Fig. 2 d), and attenuated tubular injury and apoptosis (Fig. 2 e). KIM-1 expression was also reduced (Fig. 2 f). Importantly, oxamate restored mitochondrial ultrastructure (Fig. 2 g), increased ATP content (Fig. 2 h), and upregulated TCA cycle–related genes (Fig. 2 i), indicating improved mitochondrial function and oxidative metabolism. Consistently, inhibiting lactate production with the LDHA inhibitor GSK2837808A mitigated H/R-induced apoptosis in vitro, as evidenced by reduced cleaved caspase-3 and caspase-9 levels (Figure S3a). Notably, exogenous lactate supplementation during reoxygenation—but not during hypoxia—exacerbated cellular injury (Figure S3b,c). Together, these findings indicate that lactate is particularly detrimental during the recovery phase and reducing lactate production alleviates tubular injury by restoring mitochondrial metabolism. Lactate disrupts mitochondrial homeostasis and energy metabolism through activation of the mitochondrial Mg²⁺ channel MRS2 We next investigated the mechanism by which lactate perturbs mitochondrial function. Previous studies have reported that lactate promotes intracellular Mg²⁺ mobilization and activates MRS2[ 16 ], the principal mitochondrial Mg²⁺ uptake channel, suggesting a potential link between lactate signaling and Mg²⁺-dependent mitochondrial regulation. In mTECs, exogenous lactate significantly increased mitochondrial Mg²⁺ concentration (Fig. 3 a) and uptake (Fig. 3 b), indicating MRS2 activation. Silencing MRS2 with siRNA (Figure S4a,b) effectively improved mitochondrial membrane potential following LA stimulation (Fig. 3 c,d), restored OCR (Fig. 3 e,f), increased ATP content (Fig. 3 g), and reinstated expression of TCA cycle genes (Fig. 3 h). Conversely, MRS2 overexpression (Figure S4c,d) abolished the protective effects of LDHA inhibition on H/R-induced apoptosis(Fig. 3 i,j) and mitochondrial membrane potential (Fig. 3 k). Collectively, these findings demonstrate that lactate disrupts mitochondrial homeostasis and suppresses oxidative metabolism through activation of the MRS2 Mg²⁺ channel, identifying MRS2 as a key downstream effector of lactate signaling in tubular epithelial cells. MRS2 inhibition protects renal function and mitochondrial metabolism in I/R-AKI To evaluate the functional relevance of MRS2 in vivo, its expression was assessed in human kidney biopsy specimens with varying severity of acute tubular injury (ATI) and compared with that in adjacent non-tumorous renal tissue. Immunohistochemistry revealed marked upregulation of MRS2 in injured and dilated tubules (Fig. 4 a). In the murine kidney under physiological conditions, MRS2 was predominantly localized to proximal tubules (LTL⁺), with lower expression in distal segments (NCC⁺) and minimal presence in Henle’s loop (THP⁺) or collecting ducts (AQP2⁺) (Figure S5a). After I/R, MRS2 expression rose substantially across multiple tubular segments, including proximal and distal tubules as well as Henle’s loop, indicating broad activation of this mitochondrial Mg²⁺ channel in response to ischemic stress (Figure S5a). Immunoblotting further corroborated the increase in MRS2 protein levels in I/R-AKI kidneys (Fig. 4 b). To determine whether MRS2 contributes causally to injury progression, mice were treated with CPACC, a MRS2 inhibitor, during I/R-AKI (Figure S5b). CPACC reduced renal MRS2 expression (Fig. 4 e) and significantly improved kidney function, as indicated by lower SCr (Fig. 4 c) and BUN (Fig. 4 d), reduced tubular injury and apoptosis (Fig. 4 e), and decreased KIM-1 expression (Fig. 4 f). CPACC also preserved mitochondrial ultrastructure (Fig. 4 g), increased renal ATP content (Fig. 4 h), restored TCA cycle gene expression (Fig. 4 i), and alleviated renal lactate accumulation (Fig. 4 j). These findings show that pharmacologic inhibition of MRS2 mitigates mitochondrial injury and reverses the shift from mitochondrial oxidative metabolism toward aerobic glycolysis, thereby promoting energy restoration in I/R-AKI. MRS2 knockdown preserves mitochondrial integrity and alleviates H/R-induced tubular injury In vitro, H/R markedly increased MRS2 expression (Fig. 5 a, Figure S6a) and enhanced mitochondrial Mg²⁺ uptake (Fig. 5 b,c), both of which were abolished by MRS2 knockdown. Silencing MRS2 significantly reduced H/R-induced apoptosis, as reflected by fewer Annexin V⁺ cells (Fig. 5 d), decreased levels of cleaved caspase-3 and caspase-9 (Figure S5b), and reduced KIM-1 expression (Figure S6c). MRS2 knockdown also improved mitochondrial membrane potential (Fig. 5 e, Figure S6d) and increased oxygen consumption rate, particularly the maximal respiratory capacity (Fig. 5 f,g). In parallel, it restored intracellular ATP levels (Fig. 5 h), and normalized the expression of TCA cycle enzymes (Fig. 5 i). Moreover, silencing MRS2 reduced the expression of key glycolytic enzymes (Fig. 5 j) and lowered intracellular lactate levels (Fig. 5 k), indicating an attenuation of aerobic glycolysis. Collectively, these findings demonstrate that MRS2 contributes to H/R-induced mitochondrial dysfunction and metabolic reprogramming, and that its silencing preserves mitochondrial integrity and supports cellular energy homeostasis. Targeted delivery of siMrs2 via LNP ameliorates I/R-AKI Given that genetic or pharmacological suppression of MRS2 ameliorated renal injury following I/R, we next evaluated whether lipid nanoparticle–mediated delivery of siRNA targeting Mrs2 (siMrs2-LNP) could serve as a therapeutic strategy. TEM analysis revealed that siMrs2-LNPs exhibited a uniform spherical morphology (Figure S7a), with an average diameter of ~ 100 nm and a polydispersity index < 0.2 (Figure S7b), indicating good formulation homogeneity. To achieve efficient and localized renal delivery, siMrs2-LNPs were administered by pelvic injection (Fig. 6 a). Time-course analyses showed a rapid decline in renal Mrs2 mRNA levels by 12 h after siMrs2-LNP administration, reaching maximal knockdown at 48 h and returning toward baseline by day 7 (Figure S7c). Consistent with transcript dynamics, MRS2 protein abundance decreased markedly at 12 h, reached its lowest levels at 48–72 h, and gradually recovered by day 7 (Figure S7d). Based on these pharmacodynamic kinetics, a 48h pretreatment interval was selected for subsequent studies. To further determine the optimal dosing regimen, we performed a dose–response analysis using 5, 10, and 15 µg per kidney. Administration of 10 µg resulted in a significantly greater reduction in renal MRS2 protein levels compared with 5 µg, whereas no further suppression was observed with 15 µg, indicating a plateau effect (Figure S7e). Therefore, a dose of 10 µg per kidney was used in all subsequent experiments. Importantly, this dosing strategy was well tolerated. Serum creatinine and blood urea nitrogen levels remained unchanged (Figure S7f,g). Consistently, PAS staining revealed no tubular injury, and TUNEL assays showed no increase in apoptotic cells following renal pelvic delivery of 10 µg LNP-siRNA (Figure S7h), indicating that the selected dose did not induce overt renal dysfunction. In addition, the renal specificity of pelvic-delivered LNPs was confirmed, as no appreciable reduction in MRS2 expression was observed in extra-renal tissues such as the liver or intestine (Figure S7i). Notably, siMRS2-LNP effectively reduced MRS2 expression across multiple tubular segments under both basal conditions and after I/R-AKI, as shown by co-staining with renal tubule markers (Figure S8). Pretreatment with siMrs2-LNP significantly attenuated I/R-induced kidney dysfunction, as evidenced by reduced serum creatinine (Fig. 6 b) and BUN levels (Fig. 6 c), diminished tubular injury and apoptosis (Fig. 6 d), and decreased KIM-1 expression (Fig. 6 e). This was accompanied by preservation of mitochondrial ultrastructure (Fig. 6 f), improved ATP production (Fig. 6 g), upregulation of TCA cycle–associated genes (Fig. 6 h), and decreased renal lactate accumulation (Fig. 6 i). Collectively, these results indicate that pre-ischemic siMrs2-LNP delivery confers significant prophylactic protection against I/R-AKI by reducing renal injury, improving mitochondrial integrity, improving mitochondrial oxidative metabolism and inhibiting the persistent activation of aerobic glycolysis. Discussion I/R-AKI provokes profound bioenergetic collapse in renal tubular epithelial cells, driving a metabolic shift from mitochondrial oxidative phosphorylation to glycolysis. Although this transition initially serves as an adaptive survival mechanism under hypoxic stress, persistent glycolytic reliance during reperfusion becomes maladaptive, exacerbating tubular injury and impairing recovery. The molecular determinants that sustain this pathological metabolic state have remained poorly understood. In this study, we identify lactate accumulation as a central mediator of maladaptive metabolic reprogramming through activation of the mitochondrial Mg²⁺ channel MRS2, which couples metabolic stress to mitochondrial dysfunction and persistent bioenergetic impairment. Renal tubular epithelial cells, particularly proximal tubules, rely predominantly on mitochondrial oxidative metabolism to meet their high energy demands under physiological conditions. Consistent with prior studies [ 20 , 32 – 34 ], we observed a characteristic metabolic reprogramming after I/R injury, marked by suppression of TCA cycle activity and sustained activation of aerobic glycolysis despite restoration of oxygen and blood supply. While this shift initially supports cell survival under hypoxic stress[ 35 ], its persistence during reperfusion impairs mitochondrial recovery, limits ATP restoration, and promotes maladaptive repair. Tubules that fail to re-establish oxidative metabolism remain locked in a low-efficiency glycolytic state[ 8 ], predisposing them to sustained injury and incomplete repair [ 11 , 12 , 21 , 22 , 36 , 37 ]. Although the metabolic phenotype of I/R-AKI is well recognized, the signals that maintain this maladaptive state during reperfusion have remained unclear. Lactate, once regarded solely as a glycolytic byproduct, is increasingly appreciated as a signaling metabolite that modulates mitochondrial function and cellular stress responses[ 14 – 16 , 18 , 19 , 38 ]. Our findings demonstrate that lactate accumulation directly enforces persistent glycolytic dependence by disrupting mitochondrial homeostasis, suppressing TCA cycle enzyme expression, and impairing ATP recovery. Importantly, inhibition of endogenous lactate production interrupted this pathological feedback loop, restored mitochondrial oxidative metabolism, and alleviated tubular injury, establishing lactate as an active driver rather than a passive marker of metabolic maladaptation in I/R-AKI. To identify the molecular sensor coupling lactate accumulation to mitochondrial dysfunction, we focused on MRS2, the principal Mg²⁺ channel of the mitochondrial inner membrane [ 16 , 27 ]. MRS2 was consistently upregulated across human acute tubular injury (ATI) biopsies, murine I/R-AKI kidneys, and H/R-challenged tubular cells. Lactate stimulation increased mitochondrial Mg²⁺ influx and MRS2 expression, leading to Mg²⁺ overload and mitochondrial depolarization. Genetic silencing or pharmacologic inhibition of MRS2 prevented Mg²⁺ accumulation, restored mitochondrial function, and reduced lactate buildup, whereas MRS2 overexpression abolished the protective effects of lactate synthesis inhibition. Together, these findings define a lactate/MRS2 regulatory axis that perpetuates maladaptive metabolic reprogramming during ischemic kidney injury. Recently, LNP–based RNA delivery has emerged as a promising approach for transient and efficient in vivo gene modulation. Building on these advances, we further demonstrated the translational feasibility of targeting the lactate–MRS2 axis in vivo by LNP–mediated siMrs2 delivery. Renal pelvic administration of siMrs2-LNPs achieved efficient and kidney-specific silencing of MRS2. Time-course and dose–response analyses showed that MRS2 suppression peaked between 48 and 72 hours and gradually returned toward baseline, enabling precise alignment of the therapeutic window with the onset of ischemic injury. Importantly, this delivery route showed well biosafety, as LNP injection did not induce renal injury or apoptosis and did not alter kidney function in sham-treated animals. Pre-ischemic MRS2 knockdown restored mitochondrial metabolism. These improvements translated into marked reductions in tubular apoptosis, along with significant amelioration of serum creatinine and BUN levels following I/R injury. Collectively, these findings not only validate MRS2 as a causal driver of metabolic maladaptation but also demonstrate that its transient inhibition confers prophylactic metabolic protection. Several limitations should be acknowledged. First, while our study demonstrates the protective effects of MRS2 inhibition in murine I/R-AKI and cultured tubular cells, validation in human tissues remains limited. Future studies should incorporate larger biopsy cohorts and longitudinal follow-up to establish clinical relevance. Second, the specificity, long-term safety, and potential off-target effects of pharmacologic inhibitor CPACC require further investigation. The development of tubule-specific MRS2 conditional knockout models will be essential to verify these findings. Third, our investigation focused on ischemic AKI; whether MRS2 plays similar roles in cisplatin-induced, septic, or diabetic kidney injury remains to be determined. Moreover, given that maladaptive metabolism underlies the transition from AKI to CKD, future work should explore whether chronic MRS2 activation contributes to fibrotic remodeling and irreversible decline in renal function. In conclusion, this study identifies a previously unrecognized lactate–MRS2 signaling axis that drives maladaptive metabolic reprogramming in I/R-AKI. We demonstrate that lactate accumulation during reperfusion activates the mitochondrial Mg²⁺ channel MRS2, leading to mitochondrial Mg²⁺ overload and suppression of TCA cycle–dependent oxidative metabolism. This cascade enforces persistent aerobic glycolysis, precipitating energy failure and tubular injury. Importantly, pharmacological or genetic inhibition of MRS2, including renal pelvic delivery of siMRS2-loaded lipid nanoparticles, restores mitochondrial oxidative metabolism and preserves mitochondrial integrity. Together, these findings establish mitochondrial Mg²⁺ homeostasis as a critical metabolic checkpoint in ischemic AKI and highlight MRS2 as a promising metabolic target for therapeutic intervention aimed at preventing maladaptive reprogramming and facilitating renal recovery after ischemic injury. Declarations Acknowledgements None. Author contributions Conceptualization: NNS, XQD, YQS. Methodology: ZXY, ANC, FL, JZ, WRZ, AY, SZ, YF, YL, YD. Investigation: ZXY, ANC, FL, JZ, QWX, GXH, WZC, QYG, YY, JLW, SJ (with in vivo studies conducted by NNS, ZXY, ANC, FL, JZ, QWX, GXH; in vitro experiments by WRZ, AY, WZC, QYG, YY, JLW; and pathological evaluation by YQS, JLW, SJ). Formal Analysis: NNS, YL. Data Curation: NNS, YL. Writing – Review & Editing: All authors. Validation: All authors. Supervision: NNS, XQD, YQS. Project Administration: NNS, XQD, YQS. Funding Acquisition: NNS, XQD, YQS. ZXY, ANC, and FL are designated as co-first authors, with ZXY recognized for her major contributions to experimental design, execution, and data analysis, and ANC and FL for initiating the project and performing all long-term in vivo studies. Fundings This work was supported by grants from Shanghai Science and Technology Innovation Action Plan (25ZR1401056, 25ZR1402064), the National Natural Science Foundation of China (82470717, 82070710 and 82200792), National Key Research and Development Program of China (2024YFE0199600), Shanghai Key Laboratory of Kidney and Blood Purification, Shanghai Science and Technology Commission (20DZ2271600). Data availability All data will be made available by the corresponding author upon reasonable request. Declaration of Interests Ethics approval and consent to participate The study protocol involving human participants was approved by the Ethics Committee of Zhongshan Hospital, Fudan University (Approval No.: B2025-316R). Written informed consent was obtained from all participants prior to the study. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Zhongshan Hospital, Fudan University (Approval No.: 2024-109). This study was approved by the IACUC of Zhongshan Hospital, Fudan University (Approval No.: 2024-109) and performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals. All efforts were undertaken to minimize animal suffering. Consent for publication All authors have approved the final manuscript for publication. Competing interests The authors declare that they have no competing interests. Author details 1. Department of Nephrology, Zhongshan Hospital, Fudan University, Shanghai, China; 2. Shanghai Medical Center of Kidney; Shanghai Institute of Kidney and Dialysis, Shanghai, China; 3. Shanghai Key Laboratory of Kidney and Blood Purification, Shanghai, China; 4. Department of Nephrology, Zhongshan Hospital (Xiamen), Fudan University, Fujian, China References Ostermann M, Lumlertgul N et al (2025) Acute kidney injury. Lancet 405:241–256. https://doi.org/10.1016/S0140-6736(24)02385-7 Pickkers P, Darmon M et al (2021) Acute kidney injury in the critically ill: an updated review on pathophysiology and management. Intensive Care Med 47. https://doi.org/10.1007/s00134-021-06454-7 . :835 – 50 Dare AJ, Bolton EA et al (2015) Protection against renal ischemia-reperfusion injury in vivo by the mitochondria targeted antioxidant MitoQ. Redox Biol 5:163–168. https://doi.org/10.1016/j.redox.2015.04.008 Perry HM, Huang L et al (2018) Dynamin-Related Protein 1 Deficiency Promotes Recovery from AKI. 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Kidney Int 101:987–1002. https://doi.org/10.1016/j.kint.2022.01.029 Izzo LT, Wellen KE (2019) Histone lactylation links metabolism and gene regulation. Nature 574:492–493. https://doi.org/10.1038/d41586-019-03122-1 Supplementary Files supplementaryCMLS.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revision 24 Mar, 2026 Reviewers agreed at journal 23 Feb, 2026 Reviewers invited by journal 20 Feb, 2026 Editor assigned by journal 12 Feb, 2026 First submitted to journal 10 Feb, 2026 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8846937","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594340120,"identity":"47d51d68-a242-4a79-9421-1b6b3e78b677","order_by":0,"name":"zhixin 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University","correspondingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2026-02-11 04:21:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8846937/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8846937/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103313643,"identity":"c8cddafb-401a-45b6-bd06-6ec80f47adde","added_by":"auto","created_at":"2026-02-24 10:30:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4679137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eI/R-AKI induces an energetic deficit and a glycolytic shift.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Measurement of ATP content in sham and I/R kidneys. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus sham group (n = 3). (b) Representative transmission electron microscopy (TEM) images of proximal tubules in sham and I/R kidneys. (c) Quantitative real-time PCR analysis of mRNA levels in sham and I/R kidneys. \u003cem\u003eActb\u003c/em\u003e was used as the loading control. ***\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.001 versus sham group (n=6). (d) Representative Western blot and quantitative data of glycolytic pathway. *p \u0026lt; 0.05, **p \u0026lt; 0.01 (n = 6). (e) Measurement of renal lactate content in sham and I/R -treated kidneys. ***p \u0026lt; 0.001 (n = 6). (f-i) The mTECs were challenged with H/R. (f) The oxygen consumption rate (OCR) (n=3). (g) The extracellular acidification rate (ECAR) (n=3). (h) Representative Western blot of glycolytic pathway at different time of reoxygenation. (i) Measurement of intracellular lactate content. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 6). Data were shown as mean ± SD. Statistical analysis was performed by unpaired t test (a,c,d,e) and one-way ANOVA with Dunnett's multiple comparisons test (i).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8846937/v1/c9034dfeb6341b8d1ace05cd.png"},{"id":103506604,"identity":"4f70babb-e3a0-4f7d-8eb6-d5955d9f50ba","added_by":"auto","created_at":"2026-02-26 13:37:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4880403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of lactate production improves renal function and restores energy metabolism following I/R-AKI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Schematic of sodium oxamate–mediated inhibition of lactate production in vivo and I/R-AKI experimental design. (b) Measurement of renal lactate content in sham, I/R and I/R with sodium oxamate-treated kidneys. *p \u0026lt; 0.05, **p \u0026lt; 0.01 (n = 4). (c) Measurement of serum creatinine (SCr) levels. ***p \u0026lt; 0.001(n=6). (d) Measurement of blood urea nitrogen (BUN) levels. ***p \u0026lt; 0.001 (n=5-6). (e) Representative micrographs of Periodic Acid-Schiff (PAS) and Terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) staining, with quantitative analysis of tubular injury scores and TUNEL-positive cells. ***p \u0026lt; 0.001 (n = 6). (f) Representative Western blot and quantitative data of KIM-1. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 6). (g) Representative TEM images of proximal tubules in vehicle and sodium oxamate-treated I/R-AKI kidneys. (h) Measurement of renal ATP content in vehicle and sodium oxamate-treated I/R-AKI kidneys. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 5). (i) Quantitative real-time PCR analysis of mRNA levels in vehicle and sodium oxamate-treated kidneys. \u003cem\u003eActb\u003c/em\u003e was used as the loading control. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus sham group (n=4-6). \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01 versus I/R group (n=5-6). Data were shown as mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparisons test (b-e, f, h) and two-way ANOVA with Tukey's multiple comparisons test(i).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8846937/v1/8cba5c0e047d72056806475a.png"},{"id":103505744,"identity":"c577c415-a7c0-4b69-9040-e1a553eea07b","added_by":"auto","created_at":"2026-02-26 13:32:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1650701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactate disrupts energy metabolism via MRS2 activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a-j) The mTECs were treated with 20 mM lactic acid for 12 h. (a) Permeabilized mTECs were pulsed with mitochondrial uncoupler, FCCP(2μM), and change in bath [Mg\u003csup\u003e2+\u003c/sup\u003e] due to mitochondrial Mg\u003csup\u003e2+\u003c/sup\u003e release. Representative traces show bath [Mg\u003csup\u003e2+\u003c/sup\u003e] and quantitative analysis of mitochondrial [Mg\u003csup\u003e2+\u003c/sup\u003e] ([Mg\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003em\u003c/sub\u003e). *p \u0026lt; 0.05 versus vehicle group (n=3). (b) Permeabilized mTECs were pulsed with 1mM Mg\u003csup\u003e2+\u003c/sup\u003e followed by 2μM FCCP. Representative traces show bath [Mg\u003csup\u003e2+\u003c/sup\u003e]. 1/τ depicts mitochondrial uptake rate. (c) Quantitative analysis of JC-1 staining fluorescence intensity (red to green ratio) via microplate reader. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 6). (d) Representative image of JC-1 staining. (e) Representative image of OCR. (f) Quantitative analysis of basal respiration, ATP-linked respiration, maximal respiration and proton leak. **p \u0026lt; 0.01(n=3). (g) Measurement of ATP content. *p \u0026lt; 0.05, ***p \u0026lt; 0.001 (n = 3). (h) Quantitative real-time PCR analysis of mRNA levels. \u003cem\u003eActb\u003c/em\u003e was used as the loading control. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus vehicle group (n = 6). \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01 versus LA group (n=5-6). (i-k) The mTECs were transfected with Mrs2 plasmid for 24h and treated with 100nM GSK 1h before H/R treatment. (i,j) Flow cytometry analysis for Annexin V\u003csup\u003e+\u003c/sup\u003e cells%. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n=3). (k) Quantitative analysis of JC-1 staining fluorescence intensity (red to green ratio) via microplate reader. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 3). Data were shown as geometric mean(a,b) and mean ± SD. Statistical analysis was performed by unpaired t test(a), one-way ANOVA with Tukey's multiple comparisons test(c,e-i,l) and two-way ANOVA with Tukey's multiple comparisons test(j).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8846937/v1/a736ad1d816d191fb657e5c9.png"},{"id":103313646,"identity":"2d69d3b3-f2a1-46cd-9b82-fd08c6187e67","added_by":"auto","created_at":"2026-02-24 10:30:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7659063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological inhibition of MRS2 alleviates tubular injury and bioenergetic deficit during I/R-AKI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Immunohistochemistry staining of MRS2 in kidney biopsies from patients with mild or severe ATI, compared with adjacent non-tumorous (ANT) renal tissue as control. (b) Representative Western blot and quantitative data of MRS2. *p \u0026lt; 0.05 versus sham group (n = 6). (c) Measurement of SCr levels. ***p \u0026lt; 0.001 (n=6). (d) Measurement of BUN levels. ***p \u0026lt; 0.001 (n=6). (e) Representative immunohistochemistry staining of MRS2, PAS and TUNEL staining, with quantitative analysis of MRS2, tubular injury scores and TUNEL-positive cells. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 4-6). (f) Representative Western blot and quantitative data of KIM-1. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 6). (g) Representative TEM images of proximal tubules in vehicle and CPACC-treated I/R-AKI kidneys. (h) Measurement of renal ATP content. *p \u0026lt; 0.05, **p \u0026lt; 0.01 (n = 6). (i) Quantitative real-time PCR analysis of mRNA levels. \u003cem\u003eActb\u003c/em\u003e was used as the loading control.\u0026nbsp; **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus sham group (n = 6). \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01 versus I/R group (n=6). (j) Measurement of renal lactate content. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 6). Data were shown as mean ± SD. Statistical analysis was performed by unpaired t test (b), one-way ANOVA with Tukey's multiple comparisons test(c-f,h,j) and two-way ANOVA with Tukey's multiple comparisons test(i).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8846937/v1/05f18e1299515e047d6c57d6.png"},{"id":103505685,"identity":"1846b317-1747-4282-83e7-48cbde3a873d","added_by":"auto","created_at":"2026-02-26 13:32:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2393403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of MRS2 attenuates apoptosis and restores energy metabolism in H/R-induced injury.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative Western blot and quantitative data of MRS2. ***p \u0026lt; 0.001 versus CTL group (n = 6). β-actin was used as the loading control. (b-l) mTECs were transfected with siMrs2 in H/R-induced injury. (b) Representative traces show bath [Mg\u003csup\u003e2+\u003c/sup\u003e] and quantitative analysis of mitochondrial [Mg\u003csup\u003e2+\u003c/sup\u003e] ([Mg\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003em\u003c/sub\u003e). **p \u0026lt; 0.01, ***p \u0026lt; 0.001(n=3). (c) Permeabilized mTECs were pulsed with 1mM Mg\u003csup\u003e2+\u003c/sup\u003e followed by 2μM FCCP. Representative traces show bath [Mg\u003csup\u003e2+\u003c/sup\u003e]. 1/τ depicts mitochondrial uptake rate. (d) Flow cytometry analysis for Annexin V\u003csup\u003e+\u003c/sup\u003e cells%. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n=6). (e) Representative image of JC-1 staining. (f) Representative image of OCR. (g) Quantitative analysis of maximal respiration. **p \u0026lt; 0.01 (n = 6). (h) Measurement of ATP content. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n=3). (i) Quantitative real-time PCR analysis of mRNA levels. \u003cem\u003eActb\u003c/em\u003e was used as the loading control.\u003csup\u003e #\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 versus H/R group (n = 3). (j) Representative Western blot of glycolytic pathway. (k) Measurement of lactate content. *p \u0026lt; 0.05, (n = 3). Data were shown as geometric mean (b, c) or mean ± SD. Statistical analysis was performed by unpaired t test (a,g,k), one-way ANOVA with Tukey's multiple comparisons test (b,d,h) and two-way ANOVA with Tukey's multiple comparisons test(i).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8846937/v1/6f8150929b9cc51b5f9fcc02.png"},{"id":103313647,"identity":"2276e54e-ba77-4dbf-af45-ef7680bde91a","added_by":"auto","created_at":"2026-02-24 10:30:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5708770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLNP-mediated siMRS2 delivery preserves renal function and mitochondrial integrity following I/R-AKI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Schematic of LNP-mediated siMRS2 delivery in vivo and the experimental design of I/R-AKI. (b) Measurement of SCr levels. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n=6). (c) Measurement of BUN levels. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n=6). (d)Representative immunohistochemistry staining of MRS2, PAS and TUNEL staining, with quantitative analysis of MRS2, tubular injury scores and TUNEL-positive cells. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 6). (e)Representative Western blot and quantitative analysis. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n=6). (f) Representative TEM images of proximal tubules in scramble-LNP and siMrs2-LNP-treated I/R-AKI kidneys. (g) Measurement of renal ATP content. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n=3). (h) Quantitative real-time PCR analysis of mRNA levels. \u003cem\u003eActb\u003c/em\u003e was used as the loading control.\u003csup\u003e \u003c/sup\u003e***p \u0026lt; 0.001 versus sham group(n=6). \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 versus I/R group (n = 6). (i)Measurement of renal lactate content. **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n=3). Data were shown as mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparisons test (b-g, i) and two-way ANOVA with Tukey's multiple comparisons test(h).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8846937/v1/672f79a870c954820b65fd79.png"},{"id":103509770,"identity":"9b9f035b-616f-4cb9-8186-2cbc665ea4bc","added_by":"auto","created_at":"2026-02-26 14:01:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28787792,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8846937/v1/2f7af486-ed78-4f2f-a52b-e5fae7eb3c26.pdf"},{"id":103313650,"identity":"9a34c0db-f9ff-453d-a281-96662292ce8e","added_by":"auto","created_at":"2026-02-24 10:30:11","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16463340,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryCMLS.docx","url":"https://assets-eu.researchsquare.com/files/rs-8846937/v1/5d9d984150dc9e2708c42235.docx"}],"financialInterests":"","formattedTitle":"Lactate drives maladaptive metabolic reprogramming via MRS2 inischemia–reperfusion-induced acute kidney injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute kidney injury (AKI) is a common and severe clinical syndrome associated with high morbidity, mortality, and progression to chronic kidney disease. Ischemia\u0026ndash;reperfusion injury (IRI) is one of its major causes, especially in kidney transplantation, cardiac surgery, and sepsis[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among tubular segments, proximal tubular epithelial cells (PTECs) are uniquely vulnerable due to their exceptionally high ATP demand and reliance on mitochondrial oxidative metabolism. Even brief ischemic insults precipitate rapid ATP depletion, ionic imbalance, and bioenergetic failure, ultimately triggering cell death [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite extensive efforts to target mitochondria integrity and improve energy production, effective strategies to restore cellular bioenergetics and preserve tubular viability remain limited[\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo survive transient ischemic stress, PTECs undergo a profound metabolic reprogramming, shifting from tricarboxylic acid (TCA) cycle\u0026ndash;driven mitochondrial oxidative metabolism toward anaerobic glycolysis [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. While this switch initially supports cell survival under hypoxic conditions, the persistence of glycolytic metabolism during reperfusion becomes maladaptive. This Warburg-like metabolic state results in inefficient ATP generation and exacerbates mitochondrial dysfunction [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the upstream signals that sustain this pathological metabolic state remain poorly defined.\u003c/p\u003e \u003cp\u003eLactate, traditionally considered as the end-product of glycolysis, has recently emerged as a multifunctional metabolic signal capable of modulating mitochondrial function [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], cellular bioenergetics[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and stress responses[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Elevated lactate levels are consistently observed in patients and experimental models of AKI, correlating with disease severity, impaired tubular recovery, and progression towards renal fibrosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, whether lactate directly drives maladaptive metabolic reprogramming in injured PTECs remains unresolved, and clarifying its functional consequences may reveal key determinants of energy failure and repair after AKI.\u003c/p\u003e \u003cp\u003eMitochondrial RNA splicing 2 protein (MRS2) is the predominant magnesium channel in the mitochondrial inner membrane and constitutes the principal pathway for Mg\u0026sup2;⁺ entry into the mitochondrial matrix [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Mg\u0026sup2;⁺ is essential for ATP stability and the activity of numerous metabolic enzymes in TCA cycle [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and disruption of MRS2-mediated Mg\u0026sup2;⁺ transport reprograms cellular metabolism[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Notably, MRS2 may also act as a metabolic effector responsive to lactate, thereby influencing mitochondrial function [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Nevertheless, the role of MRS2 in the kidney, particularly under ischemic conditions, remains largely unexplored. It is unclear whether lactate-mediated MRS2 activation contributes to mitochondrial dysfunction and metabolic reprogramming following I/R-AKI.\u003c/p\u003e \u003cp\u003eBased on these observations, we hypothesized that lactate accumulation during I/R-AKI activates MRS2, leading to mitochondrial Mg\u0026sup2;⁺ overload, and suppression of TCA cycle\u0026ndash;driven oxidative metabolism. In this study, we identify lactate as a key driver of maladaptive metabolic reprogramming in ischemic renal tubular cells through activation of MRS2. Furthermore, pharmacological inhibition of MRS2 or lipid nanoparticle\u0026ndash;mediated delivery of siMRS2 restores metabolic homeostasis and attenuates tubular injury following I/R-AKI.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman kidney samples\u003c/h2\u003e \u003cp\u003eRenal biopsy specimens were collected from patients diagnosed with acute tubular injury during routine diagnostic evaluation at Zhongshan Hospital. The study protocol was approved by the Institutional Ethics Committee, and written informed consent was obtained from all participants prior to sample collection (Approval No.: B2025-316R).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal models of ischemia/reperfusion-induced acute kidney injury (I/R-AKI)\u003c/h3\u003e\n\u003cp\u003eMale C57BL/6 mice (8\u0026ndash;10 weeks old, 20\u0026ndash;25 g) were used as previously described[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Briefly, mice were anesthetized via intraperitoneal injection of 4% phenobarbital (10 mL/kg body weight). Bilateral renal pedicles were exposed through a midline abdominal incision and clamped using atraumatic vascular clamps for 19 minutes to induce ischemia. Reperfusion was initiated by releasing the clamps, after which the incision was closed. Sham-operated mice underwent the same procedure without clamping. Body temperature was maintained at 36\u0026deg;C throughout surgery using a heating pad. Kidneys and blood samples were collected 24 hours after reperfusion for subsequent analyses. For the sodium oxamate treatment, sodium oxamate (300 mg/kg, MCE) was administered via intraperitoneal injection three days before ischemia surgery and then injected once after ischemia. For the chloropentaammine cobalt (III)chloride (CPACC) treatment, CPACC (20 mg/kg, MACKLIN) was administered via intraperitoneal injection every three days for three times before ischemia surgery. All procedures were conducted in strict accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (8th edition). Mice were group-housed under specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle, controlled temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and humidity (50% \u0026plusmn; 10%), and provided with free access to standard chow and water. Every effort was made to minimize animal suffering and reduce the number of animals used, including the application of appropriate anesthesia and analgesics during surgical procedures.\u003c/p\u003e\n\u003ch3\u003ePreparation of Lipid nanoparticles (LNPs)-encapsulated siRNA\u003c/h3\u003e\n\u003cp\u003eLNPs were prepared using an ethanol-dilution microfluidic method. Briefly, the lipid phase was composed of an ionizable lipid, the structural lipid cholesterol, the phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and the helper lipid 1,2-dimyristoyl-rac-glycerol-methoxypolyethylene glycol-2000 (DMG-PEG2000), mixed in ethanol at a molar ratio of 50:10:38.5:1.5. Scramble and siMRS2 were individually dissolved in 25 mM sodium acetate buffer (pH 5.0) prior to formulation. The ethanolic lipid solution and the aqueous RNA solution were combined at a 1:3 volume ratio in a microfluidic mixer to facilitate self-assembly of LNPs. The resulting suspensions were dialyzed against PBS at room temperature for \u0026gt;\u0026thinsp;2 hours to remove ethanol and achieve buffer exchange.\u003c/p\u003e\n\u003ch3\u003eIn vivo administration of LNPs\u003c/h3\u003e\n\u003cp\u003eFor in vivo renal delivery, LNP-encapsulated scramble RNA or siMrs2 RNA was administered via retrograde pyeloureteral injection. Briefly, mice were anesthetized, and each kidney received 10 \u0026micro;g RNA in 50 \u0026micro;L sterile PBS through the renal pelvis using a microsyringe under direct visualization. LNPs were delivered 48 hours prior to ischemia induction to ensure sufficient renal uptake and gene silencing. Control mice received an equal volume and RNA dose of scramble RNA\u0026ndash;loaded LNPs.\u003c/p\u003e\n\u003ch3\u003eCell culture and treatment\u003c/h3\u003e\n\u003cp\u003eImmortalized mouse proximal tubular epithelial cells (mTECs; BeNa Culture Collection, BNCC, China) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum under standard conditions (37\u0026deg;C, 5% CO₂). For hypoxia/reoxygenation (H/R) treatment, mTECs were seeded at 1 \u0026times; 10⁵ cells per well in 6-well plates and cultured until ~\u0026thinsp;70% confluence. Cells were then incubated in serum-free RPMI 1640 under hypoxia (1% O₂, 5% CO₂, 94% N₂) for 24 hours, followed by reoxygenation for 0, 4, 8 hours under normoxia (21% O₂, 5% CO₂) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Based on the severity of cellular injury observed across conditions, a reoxygenation period of 4 hours was selected for subsequent experiments. For lactate stimulation, mTECs were exposed to 20 mM L-lactic acid (L812604, MACKLIN) for 12 h. To block lactate uptake, cells were pretreated with the monocarboxylate transporter inhibitor AR-C155858 (2 \u0026micro;M; HY-13248, MCE) 1 h before lactate exposure. To inhibit lactate synthesis, mTECs were pretreated with the LDHA inhibitor GSK2837808A (100 nM; HY-100681, MCE) 1 h before H/R.\u003c/p\u003e \u003cp\u003eFor gene silencing, MRS2-specific siRNA (ZORIN) was transfected using Lipofectamine 3000 (Invitrogen), with a nonsilencing siRNA as the negative control. For overexpression, an MRS2 expression plasmid (ZORIN) was introduced using the same reagent, and sequence integrity was verified by DNA sequencing; the ZV304 vector served as control. Transfection efficiency was confirmed by qPCR and Western blotting. siRNA and plasmid sequences are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of kidney function\u003c/h2\u003e \u003cp\u003eSerum creatinine (SCr) and blood urea nitrogen (BUN) were measured using QuantiChrom Creatinine (DICT-500) and Urea (DIAG-100) Assay Kits (BioAssay Systems, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathological examination\u003c/h3\u003e\n\u003cp\u003eKidney tissues were fixed, paraffin-embedded, and sectioned at 4 \u0026micro;m thickness. Sections were stained with periodic acid\u0026ndash;Schiff (PAS) reagent to evaluate tubular injury. Tubular injury score was performed in a blinded manner based on the percentage of tubules showing epithelial necrosis, brush border loss, cast formation, or tubular dilation, using a 0\u0026ndash;4 scale: 0, none; 1, \u0026lt;\u0026thinsp;25%; 2, 25\u0026ndash;50%; 3, 50\u0026ndash;75%; and 4, \u0026gt;\u0026thinsp;75% affected[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eParaffin-embedded kidney sections (4 \u0026micro;m) were deparaffinized, rehydrated, and subjected to antigen retrieval. After blocking with 1% bovine serum albumin (BSA; Sigma, B2064), sections were incubated overnight at 4\u0026deg;C with primary antibodies against MRS2 (ANT-148; Alomone Labs, 1:400). After washing, slides were incubated with HRP-conjugated secondary antibodies (Gene Tech, GK500710, 1:300) for 30 min at 37\u0026deg;C. Peroxidase activity was visualized using a DAB detection kit (Gene Tech, GK500710), and images were captured under a light microscope.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed as described previously [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. After deparaffinization, antigen retrieval, and blocking with 1% BSA, kidney sections were incubated overnight at 4\u0026deg;C with primary antibodies against MRS2 (ANT-148; Alomone Labs,1:400), Lotus Tetragonolobus Lectin(LTL) (ZE1102;Vector, 1:800), Uromucoid(THP) (ab207170;Abcam,1:1000), Thiazide-Sensitive NaCl Cotransporter (NCC) (AB3553;EMD Millipore,1:1000), Aquaporin 2(AQP2) (AB3274;Merck,1:1000) and KIM1 (AF1817; R\u0026amp;D, 1:200). The next day, slides were incubated with Alexa Fluor 488\u0026ndash;, 594\u0026ndash;conjugated secondary antibodies (Thermo Fisher Scientific,1:200) or Alexa Fluor 647-conjugated Streptavidin (YEASEN,1:200) for 1 hour at room temperature. Nuclei were counterstained with DAPI. Fluorescence was visualized using an FV3000 confocal laser scanning microscope (Olympus, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL staining\u003c/h2\u003e \u003cp\u003eApoptotic cells were detected using a TUNEL assay kit (C1089; Beyotime, China) according to the manufacturer\u0026rsquo;s instructions. After staining, nuclei were counterstained with DAPI, and TUNEL⁺/DAPI⁺ cells were quantified as apoptotic. Images were captured with an FV3000 confocal microscope (Olympus, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eFresh kidney tissues were fixed overnight in 2% paraformaldehyde and 2.5% glutaraldehyde at 4\u0026deg;C, followed by dehydration, embedding in epoxy resin, and sectioning according to standard protocols. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a JEOL 1010 transmission electron microscope (JEOL, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eProteins were extracted from kidney tissues or cell lysates using RIPA buffer. Equal protein amounts (40 \u0026micro;g) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 hour, membranes were incubated overnight at 4\u0026deg;C with primary antibodies. HRP-conjugated secondary antibodies were applied for 1 hour at room temperature. Primary antibodies used in this study were: anti-MRS2 (ANT-148; Alomone Labs, 1:1000), anti-KIM1 (AF1817; R\u0026amp;D, 1:1000), anti-HK2 (ab209847;Abcam,1:1000), anti-PFKP(13389-1-AP;proteintech, 1:1000), anti-PKM2(15822-1-AP;proteintech, 1:1000) and anti-LDHA(A21893;ABclonal, 1:1000). Signals were visualized using a chemiluminescent substrate, and band intensities were normalized to β-actin. The intensity of bands was quantified using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol reagent (Invitrogen). Reverse transcription was performed using Superscript II (TOYOBO, Japan) and oligo(dT) primers. qRT-PCR was conducted using SYBR Green Master Mix (TOYOBO) on an ABI 7500 system. Gene expression levels were calculated using the 2⁻ΔΔCt method, with \u003cem\u003eActb\u003c/em\u003e as the reference gene. Primer sequences are provided in Supplementary Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay and Annexin V-FITC/PI apoptosis assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using the CCK-8 assay (C0038; Beyotime, China). mTECs were seeded in 96-well plates (5 \u0026times; 10\u0026sup3; cells/well), treated with lactic acid (5\u0026ndash;20 mM) for 24 h, and incubated with CCK-8 reagent for 1\u0026ndash;4 h. Absorbance at 450 nm was measured using a microplate reader (BioTek, USA).\u003c/p\u003e \u003cp\u003eApoptosis was analyzed using Annexin V-FITC/PI staining (KeyGEN Biotech, China) as previously described[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Briefly, mTECs were stained according to the manufacturer\u0026rsquo;s instructions and analyzed by flow cytometry (Attune NxT; Thermo Fisher Scientific, USA). Cells were classified as viable (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), or necrotic (Annexin V⁻/PI⁺). The apoptotic rate was defined as the sum of early and late apoptotic cells. Data were analyzed using FlowJo software (Tree Star, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eATP measurement\u003c/h2\u003e \u003cp\u003eATP levels in kidney tissues and mTECs were determined using an ATP Fluorometric Assay Kit (S0026; Beyotime, China) according to the manufacturer\u0026rsquo;s protocol and normalized to total protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial membrane potential (MMP) assay\u003c/h2\u003e \u003cp\u003eMMP was evaluated using JC-1 (C2006; Beyotime, China). After incubation with JC-1 for 20 min at 37\u0026deg;C, red (Ex/Em: 525/590 nm) and green (Ex/Em: 490/530 nm) fluorescence intensities were measured using a Varioskan\u0026trade; LUX reader, and the red/green ratio was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurement\u003c/h2\u003e \u003cp\u003eOCR and ECAR were measured using a Seahorse XFe96 Analyzer (Agilent, USA) with the Mito Stress Test and Glycolysis Stress Test kits. Cells (2 \u0026times; 10\u0026sup3;/well) were exposed to H/R or 20 mM lactic acid, with control medium as baseline. Sequential injections included oligomycin (1.5 \u0026micro;M), FCCP (1.0 \u0026micro;M), and rotenone/antimycin A (0.5 \u0026micro;M each) for OCR, and glucose (10 mM), oligomycin (1 \u0026micro;M), and 2-DG (50 mM) for ECAR. Data were analyzed using Agilent Seahorse Wave 2.6.1 and GraphPad Prism 8.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLactate measurement\u003c/h2\u003e \u003cp\u003eLactate concentrations in kidney tissues, mTECs, and culture medium were quantified using a colorimetric assay kit (A019-2-1; Nanjing Jiancheng Bioengineering Institute, China) and normalized to total protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSpectrofluorimetric analysis of mitochondrial Mg\u0026sup2;⁺ flux\u003c/h2\u003e \u003cp\u003eMitochondrial Mg\u0026sup2;⁺ dynamics were measured using a Varioskan\u0026trade; LUX microplate reader as previously described[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Cells were washed with Ca\u0026sup2;⁺- and Mg\u0026sup2;⁺-free DPBS (pH 7.4) and pelleted by centrifugation at 503 \u0026times; g for 5 minutes at 4\u0026deg;C. Approximately 4\u0026ndash;5 \u0026times; 10⁶ cells were resuspended in 1.5 mL intracellular medium (ICM; 120 mM KCl, 10 mM NaCl, 1 mM KH₂PO₄, 20 mM HEPES-Tris, pH 7) and permeabilized with 40 \u0026micro;g/mL digitonin. The medium was supplemented with succinate (5 mM), ATP, and Mag-Fluo-4 AM(5 \u0026micro;M; MX4544; MKBio) probes for Mg\u0026sup2;⁺measurement (Ex/Em:494/516nm). For measurement of mitochondrial Mg\u0026sup2;⁺ level, after a baseline recording, mTECs were challenged with FCCP (2 mM) to uncouple mitochondrion and changes in extramitochondrial ion concentrations served as indicators of mitochondrial Mg\u0026sup2;⁺. For measurement of mitochondrial Mg\u0026sup2;⁺ uptake, mTECs were challenged with either a single Mg\u0026sup2;⁺ bolus (1 mM), followed by FCCP addition (2 mM) to uncouple mitochondria. Mitochondrial Mg\u0026sup2;⁺uptake rates were calculated from the initial linear portion of the fluorescence traces. All measurements were conducted at 37\u0026deg;C with constant stirring.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses and visualization were performed using GraphPad Prism 8 (GraphPad Software, USA). Comparisons between two groups were made using unpaired t-tests. One-way ANOVA with Tukey\u0026rsquo;s multiple comparisons test was used for multi-group analysis, and two-way ANOVA with Tukey\u0026rsquo;s test was applied when two independent variables were involved. P\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eProximal tubules showed bioenergetic deficit and metabolic reprogramming toward aerobic glycolysis in I/R-AKI model\u003c/h2\u003e \u003cp\u003eThe I/R-AKI model was successfully established, as indicated by elevated serum creatinine (SCr) and blood urea nitrogen (BUN) at 24 h (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea, b), accompanied by pronounced tubular injury on PAS staining, increased apoptosis on TUNEL staining (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec), and marked upregulation of the injury marker KIM-1 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed). Following I/R, kidneys exhibited a pronounced loss of bioenergetic capacity, reflected by reduced ATP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and severe mitochondrial structural injury, including swelling and cristae disruption in proximal tubules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Consistently, expression of key tricarboxylic acid (TCA) cycle enzymes was markedly suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), indicating impaired mitochondrial oxidative metabolism. In parallel, glycolytic pathways were strongly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), and renal lactate levels remained elevated during reperfusion despite oxygen restoration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), demonstrating a shift toward aerobic glycolysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo model I/R-AKI in vitro, hypoxia/reoxygenation (H/R) was applied to cultured proximal tubular epithelial cells (mTECs) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ee,f). H/R markedly reduced oxygen consumption rate (OCR) while increasing extracellular acidification rate (ECAR) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef,g). Notably, glycolytic gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh) and intracellular lactate levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei) remained elevated during reoxygenation, demonstrating a sustained Warburg-like phenotype in proximal tubules independent of oxygen availability.\u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate that I/R-AKI causes profound mitochondrial bioenergetic failure and enforces a persistent metabolic shift from mitochondrial oxidative metabolism toward aerobic glycolysis in proximal tubules.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eExcess lactate disrupts mitochondrial homeostasis and inhibits TCA cycle activity\u003c/h2\u003e \u003cp\u003eGiven the accumulation of lactate during I/R-AKI and H/R-induced injury, and its known role in regulating mitochondrial homeostasis[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], we next examined whether lactate contributes to mitochondrial dysfunction in tubular epithelial cells. Exogenous L-lactic acid (LA) treatment reduced cell viability in a concentration-dependent manner (Figure S2a), with 20 mM selected for subsequent experiments. Pharmacologic inhibition of monocarboxylate transporters (MCT1/2) with AR-C155858 significantly attenuated LA-induced apoptosis (Figure S2b), indicating that intracellular lactate is the primary driver of cytotoxicity. As expected, LA induced mitochondrial damage in mTECs, characterized by increased mitochondrial swelling (Figure S2c) and loss of mitochondrial membrane potential (Figure S2d). This was accompanied by decreased intracellular ATP levels (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ee) and downregulation of key TCA cycle enzymes (Figure S2f), consistent with impaired mitochondrial oxidative capacity.\u003c/p\u003e \u003cp\u003eThese findings indicate that intracellular lactate acts not merely as a metabolic byproduct but as a signaling metabolite that disrupts mitochondrial homeostasis, suppresses oxidative metabolism, and may reinforce maladaptive glycolytic dependence during I/R-AKI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eBlocking lactate production restores mitochondrial metabolism and alleviates renal dysfunction in I/R-AKI mice\u003c/h2\u003e \u003cp\u003eTo determine whether reducing endogenous lactate accumulation alleviates renal injury, sodium oxamate (an LDH inhibitor) was administered in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Oxamate significantly reduced renal lactate levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), improved kidney function as reflected by decreased SCr (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) and BUN levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), and attenuated tubular injury and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). KIM-1 expression was also reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Importantly, oxamate restored mitochondrial ultrastructure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg), increased ATP content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh), and upregulated TCA cycle\u0026ndash;related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei), indicating improved mitochondrial function and oxidative metabolism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsistently, inhibiting lactate production with the LDHA inhibitor GSK2837808A mitigated H/R-induced apoptosis in vitro, as evidenced by reduced cleaved caspase-3 and caspase-9 levels (Figure S3a). Notably, exogenous lactate supplementation during reoxygenation\u0026mdash;but not during hypoxia\u0026mdash;exacerbated cellular injury (Figure S3b,c).\u003c/p\u003e \u003cp\u003eTogether, these findings indicate that lactate is particularly detrimental during the recovery phase and reducing lactate production alleviates tubular injury by restoring mitochondrial metabolism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eLactate disrupts mitochondrial homeostasis and energy metabolism through activation of the mitochondrial Mg\u0026sup2;⁺ channel MRS2\u003c/h2\u003e \u003cp\u003eWe next investigated the mechanism by which lactate perturbs mitochondrial function. Previous studies have reported that lactate promotes intracellular Mg\u0026sup2;⁺ mobilization and activates MRS2[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the principal mitochondrial Mg\u0026sup2;⁺ uptake channel, suggesting a potential link between lactate signaling and Mg\u0026sup2;⁺-dependent mitochondrial regulation. In mTECs, exogenous lactate significantly increased mitochondrial Mg\u0026sup2;⁺ concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), indicating MRS2 activation. Silencing MRS2 with siRNA (Figure S4a,b) effectively improved mitochondrial membrane potential following LA stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d), restored OCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee,f), increased ATP content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg), and reinstated expression of TCA cycle genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). Conversely, MRS2 overexpression (Figure S4c,d) abolished the protective effects of LDHA inhibition on H/R-induced apoptosis(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei,j) and mitochondrial membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate that lactate disrupts mitochondrial homeostasis and suppresses oxidative metabolism through activation of the MRS2 Mg\u0026sup2;⁺ channel, identifying MRS2 as a key downstream effector of lactate signaling in tubular epithelial cells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eMRS2 inhibition protects renal function and mitochondrial metabolism in I/R-AKI\u003c/h2\u003e \u003cp\u003eTo evaluate the functional relevance of MRS2 in vivo, its expression was assessed in human kidney biopsy specimens with varying severity of acute tubular injury (ATI) and compared with that in adjacent non-tumorous renal tissue. Immunohistochemistry revealed marked upregulation of MRS2 in injured and dilated tubules (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In the murine kidney under physiological conditions, MRS2 was predominantly localized to proximal tubules (LTL⁺), with lower expression in distal segments (NCC⁺) and minimal presence in Henle\u0026rsquo;s loop (THP⁺) or collecting ducts (AQP2⁺) (Figure S5a). After I/R, MRS2 expression rose substantially across multiple tubular segments, including proximal and distal tubules as well as Henle\u0026rsquo;s loop, indicating broad activation of this mitochondrial Mg\u0026sup2;⁺ channel in response to ischemic stress (Figure S5a). Immunoblotting further corroborated the increase in MRS2 protein levels in I/R-AKI kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether MRS2 contributes causally to injury progression, mice were treated with CPACC, a MRS2 inhibitor, during I/R-AKI (Figure S5b). CPACC reduced renal MRS2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee) and significantly improved kidney function, as indicated by lower SCr (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and BUN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), reduced tubular injury and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), and decreased KIM-1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). CPACC also preserved mitochondrial ultrastructure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), increased renal ATP content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), restored TCA cycle gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei), and alleviated renal lactate accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003eThese findings show that pharmacologic inhibition of MRS2 mitigates mitochondrial injury and reverses the shift from mitochondrial oxidative metabolism toward aerobic glycolysis, thereby promoting energy restoration in I/R-AKI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eMRS2 knockdown preserves mitochondrial integrity and alleviates H/R-induced tubular injury\u003c/h2\u003e \u003cp\u003eIn vitro, H/R markedly increased MRS2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, Figure S6a) and enhanced mitochondrial Mg\u0026sup2;⁺ uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb,c), both of which were abolished by MRS2 knockdown. Silencing MRS2 significantly reduced H/R-induced apoptosis, as reflected by fewer Annexin V⁺ cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), decreased levels of cleaved caspase-3 and caspase-9 (Figure S5b), and reduced KIM-1 expression (Figure S6c). MRS2 knockdown also improved mitochondrial membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, Figure S6d) and increased oxygen consumption rate, particularly the maximal respiratory capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef,g). In parallel, it restored intracellular ATP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), and normalized the expression of TCA cycle enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). Moreover, silencing MRS2 reduced the expression of key glycolytic enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej) and lowered intracellular lactate levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek), indicating an attenuation of aerobic glycolysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate that MRS2 contributes to H/R-induced mitochondrial dysfunction and metabolic reprogramming, and that its silencing preserves mitochondrial integrity and supports cellular energy homeostasis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTargeted delivery of siMrs2 via LNP ameliorates I/R-AKI\u003c/h3\u003e\n\u003cp\u003eGiven that genetic or pharmacological suppression of MRS2 ameliorated renal injury following I/R, we next evaluated whether lipid nanoparticle\u0026ndash;mediated delivery of siRNA targeting Mrs2 (siMrs2-LNP) could serve as a therapeutic strategy. TEM analysis revealed that siMrs2-LNPs exhibited a uniform spherical morphology (Figure S7a), with an average diameter of ~\u0026thinsp;100 nm and a polydispersity index\u0026thinsp;\u0026lt;\u0026thinsp;0.2 (Figure S7b), indicating good formulation homogeneity. To achieve efficient and localized renal delivery, siMrs2-LNPs were administered by pelvic injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Time-course analyses showed a rapid decline in renal Mrs2 mRNA levels by 12 h after siMrs2-LNP administration, reaching maximal knockdown at 48 h and returning toward baseline by day 7 (Figure S7c). Consistent with transcript dynamics, MRS2 protein abundance decreased markedly at 12 h, reached its lowest levels at 48\u0026ndash;72 h, and gradually recovered by day 7 (Figure S7d). Based on these pharmacodynamic kinetics, a 48h pretreatment interval was selected for subsequent studies. To further determine the optimal dosing regimen, we performed a dose\u0026ndash;response analysis using 5, 10, and 15 \u0026micro;g per kidney. Administration of 10 \u0026micro;g resulted in a significantly greater reduction in renal MRS2 protein levels compared with 5 \u0026micro;g, whereas no further suppression was observed with 15 \u0026micro;g, indicating a plateau effect (Figure S7e). Therefore, a dose of 10 \u0026micro;g per kidney was used in all subsequent experiments. Importantly, this dosing strategy was well tolerated. Serum creatinine and blood urea nitrogen levels remained unchanged (Figure S7f,g). Consistently, PAS staining revealed no tubular injury, and TUNEL assays showed no increase in apoptotic cells following renal pelvic delivery of 10 \u0026micro;g LNP-siRNA (Figure S7h), indicating that the selected dose did not induce overt renal dysfunction. In addition, the renal specificity of pelvic-delivered LNPs was confirmed, as no appreciable reduction in MRS2 expression was observed in extra-renal tissues such as the liver or intestine (Figure S7i). Notably, siMRS2-LNP effectively reduced MRS2 expression across multiple tubular segments under both basal conditions and after I/R-AKI, as shown by co-staining with renal tubule markers (Figure S8).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePretreatment with siMrs2-LNP significantly attenuated I/R-induced kidney dysfunction, as evidenced by reduced serum creatinine (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) and BUN levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), diminished tubular injury and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), and decreased KIM-1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). This was accompanied by preservation of mitochondrial ultrastructure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), improved ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg), upregulation of TCA cycle\u0026ndash;associated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh), and decreased renal lactate accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003eCollectively, these results indicate that pre-ischemic siMrs2-LNP delivery confers significant prophylactic protection against I/R-AKI by reducing renal injury, improving mitochondrial integrity, improving mitochondrial oxidative metabolism and inhibiting the persistent activation of aerobic glycolysis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eI/R-AKI provokes profound bioenergetic collapse in renal tubular epithelial cells, driving a metabolic shift from mitochondrial oxidative phosphorylation to glycolysis. Although this transition initially serves as an adaptive survival mechanism under hypoxic stress, persistent glycolytic reliance during reperfusion becomes maladaptive, exacerbating tubular injury and impairing recovery. The molecular determinants that sustain this pathological metabolic state have remained poorly understood. In this study, we identify lactate accumulation as a central mediator of maladaptive metabolic reprogramming through activation of the mitochondrial Mg\u0026sup2;⁺ channel MRS2, which couples metabolic stress to mitochondrial dysfunction and persistent bioenergetic impairment.\u003c/p\u003e \u003cp\u003eRenal tubular epithelial cells, particularly proximal tubules, rely predominantly on mitochondrial oxidative metabolism to meet their high energy demands under physiological conditions. Consistent with prior studies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], we observed a characteristic metabolic reprogramming after I/R injury, marked by suppression of TCA cycle activity and sustained activation of aerobic glycolysis despite restoration of oxygen and blood supply. While this shift initially supports cell survival under hypoxic stress[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], its persistence during reperfusion impairs mitochondrial recovery, limits ATP restoration, and promotes maladaptive repair. Tubules that fail to re-establish oxidative metabolism remain locked in a low-efficiency glycolytic state[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], predisposing them to sustained injury and incomplete repair [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Although the metabolic phenotype of I/R-AKI is well recognized, the signals that maintain this maladaptive state during reperfusion have remained unclear.\u003c/p\u003e \u003cp\u003eLactate, once regarded solely as a glycolytic byproduct, is increasingly appreciated as a signaling metabolite that modulates mitochondrial function and cellular stress responses[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our findings demonstrate that lactate accumulation directly enforces persistent glycolytic dependence by disrupting mitochondrial homeostasis, suppressing TCA cycle enzyme expression, and impairing ATP recovery. Importantly, inhibition of endogenous lactate production interrupted this pathological feedback loop, restored mitochondrial oxidative metabolism, and alleviated tubular injury, establishing lactate as an active driver rather than a passive marker of metabolic maladaptation in I/R-AKI.\u003c/p\u003e \u003cp\u003eTo identify the molecular sensor coupling lactate accumulation to mitochondrial dysfunction, we focused on MRS2, the principal Mg\u0026sup2;⁺ channel of the mitochondrial inner membrane [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. MRS2 was consistently upregulated across human acute tubular injury (ATI) biopsies, murine I/R-AKI kidneys, and H/R-challenged tubular cells. Lactate stimulation increased mitochondrial Mg\u0026sup2;⁺ influx and MRS2 expression, leading to Mg\u0026sup2;⁺ overload and mitochondrial depolarization. Genetic silencing or pharmacologic inhibition of MRS2 prevented Mg\u0026sup2;⁺ accumulation, restored mitochondrial function, and reduced lactate buildup, whereas MRS2 overexpression abolished the protective effects of lactate synthesis inhibition. Together, these findings define a lactate/MRS2 regulatory axis that perpetuates maladaptive metabolic reprogramming during ischemic kidney injury.\u003c/p\u003e \u003cp\u003eRecently, LNP\u0026ndash;based RNA delivery has emerged as a promising approach for transient and efficient in vivo gene modulation. Building on these advances, we further demonstrated the translational feasibility of targeting the lactate\u0026ndash;MRS2 axis in vivo by LNP\u0026ndash;mediated siMrs2 delivery. Renal pelvic administration of siMrs2-LNPs achieved efficient and kidney-specific silencing of MRS2. Time-course and dose\u0026ndash;response analyses showed that MRS2 suppression peaked between 48 and 72 hours and gradually returned toward baseline, enabling precise alignment of the therapeutic window with the onset of ischemic injury. Importantly, this delivery route showed well biosafety, as LNP injection did not induce renal injury or apoptosis and did not alter kidney function in sham-treated animals. Pre-ischemic MRS2 knockdown restored mitochondrial metabolism. These improvements translated into marked reductions in tubular apoptosis, along with significant amelioration of serum creatinine and BUN levels following I/R injury. Collectively, these findings not only validate MRS2 as a causal driver of metabolic maladaptation but also demonstrate that its transient inhibition confers prophylactic metabolic protection.\u003c/p\u003e \u003cp\u003eSeveral limitations should be acknowledged. First, while our study demonstrates the protective effects of MRS2 inhibition in murine I/R-AKI and cultured tubular cells, validation in human tissues remains limited. Future studies should incorporate larger biopsy cohorts and longitudinal follow-up to establish clinical relevance. Second, the specificity, long-term safety, and potential off-target effects of pharmacologic inhibitor CPACC require further investigation. The development of tubule-specific MRS2 conditional knockout models will be essential to verify these findings. Third, our investigation focused on ischemic AKI; whether MRS2 plays similar roles in cisplatin-induced, septic, or diabetic kidney injury remains to be determined. Moreover, given that maladaptive metabolism underlies the transition from AKI to CKD, future work should explore whether chronic MRS2 activation contributes to fibrotic remodeling and irreversible decline in renal function.\u003c/p\u003e \u003cp\u003eIn conclusion, this study identifies a previously unrecognized lactate\u0026ndash;MRS2 signaling axis that drives maladaptive metabolic reprogramming in I/R-AKI. We demonstrate that lactate accumulation during reperfusion activates the mitochondrial Mg\u0026sup2;⁺ channel MRS2, leading to mitochondrial Mg\u0026sup2;⁺ overload and suppression of TCA cycle\u0026ndash;dependent oxidative metabolism. This cascade enforces persistent aerobic glycolysis, precipitating energy failure and tubular injury. Importantly, pharmacological or genetic inhibition of MRS2, including renal pelvic delivery of siMRS2-loaded lipid nanoparticles, restores mitochondrial oxidative metabolism and preserves mitochondrial integrity. Together, these findings establish mitochondrial Mg\u0026sup2;⁺ homeostasis as a critical metabolic checkpoint in ischemic AKI and highlight MRS2 as a promising metabolic target for therapeutic intervention aimed at preventing maladaptive reprogramming and facilitating renal recovery after ischemic injury.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: NNS, XQD, YQS. Methodology: ZXY, ANC, FL, JZ, WRZ, AY, SZ, YF, YL, YD. Investigation: ZXY, ANC, FL, JZ, QWX, GXH, WZC, QYG, YY, JLW, SJ (with in vivo studies conducted by NNS, ZXY, ANC, FL, JZ, QWX, GXH; in vitro experiments by WRZ, AY, WZC, QYG, YY, JLW; and pathological evaluation by YQS, JLW, SJ). Formal Analysis: NNS, YL. Data Curation: NNS, YL. Writing \u0026ndash; Review \u0026amp; Editing: All authors. Validation: All authors. Supervision: NNS, XQD, YQS. Project Administration: NNS, XQD, YQS. Funding Acquisition: NNS, XQD, YQS. ZXY, ANC, and FL are designated as co-first authors, with ZXY recognized for her major contributions to experimental design, execution, and data analysis, and ANC and FL for initiating the project and performing all long-term in vivo studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from Shanghai Science and Technology Innovation Action Plan (25ZR1401056, 25ZR1402064), the National Natural Science Foundation of China (82470717, 82070710 and 82200792), National Key Research and Development Program of China (2024YFE0199600), Shanghai Key Laboratory of Kidney and Blood Purification, Shanghai Science and Technology Commission (20DZ2271600).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data will be made available by the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol involving human participants was approved by the Ethics Committee of Zhongshan Hospital, Fudan University (Approval No.: B2025-316R). Written informed consent was obtained from all participants prior to the study. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Zhongshan Hospital, Fudan University (Approval No.: 2024-109). This study was approved by the IACUC of Zhongshan Hospital, Fudan University (Approval No.: 2024-109) and performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals. All efforts were undertaken to minimize animal suffering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have approved the final manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Department of Nephrology, Zhongshan Hospital, Fudan University, Shanghai, China;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. Shanghai Medical Center of Kidney; Shanghai Institute of Kidney and Dialysis, Shanghai, China;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. Shanghai Key Laboratory of Kidney and Blood Purification, Shanghai, China;\u003c/p\u003e\n\u003cp\u003e4. Department of Nephrology, Zhongshan Hospital (Xiamen), Fudan University, Fujian, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOstermann M, Lumlertgul N et al (2025) Acute kidney injury. 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Nature 574:492\u0026ndash;493. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/d41586-019-03122-1\u003c/span\u003e\u003cspan address=\"10.1038/d41586-019-03122-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"acute kidney injury, metabolic reprogramming, lactate, MRS2, mitochondrial homeostasis","lastPublishedDoi":"10.21203/rs.3.rs-8846937/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8846937/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIschemia\u0026ndash;reperfusion\u0026ndash;induced acute kidney injury (I/R-AKI) causes profound bioenergetic collapse in renal proximal tubular epithelial cells, triggering a sustained shift from mitochondrial oxidative metabolism to glycolysis. Although lactate accumulation is a hallmark of this metabolic state, whether lactate actively drives mitochondrial dysfunction and enforces persistent metabolic reprogramming remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHuman renal biopsy specimens, murine I/R-AKI, and hypoxia/reoxygenation\u0026ndash;challenged proximal tubular epithelial cells were used to investigate the lactate\u0026ndash;MRS2 axis in I/R-AKI. Lactate signaling was inhibited by sodium oxamate, while MRS2 was suppressed using CPACC or siRNA-based approaches, including lipid nanoparticle\u0026ndash;mediated siMRS2 delivery. Mitochondrial function and oxidative metabolism were assessed by oxygen consumption rate, ATP production, mitochondrial membrane potential, and tricarboxylic acid cycle (TCA) gene expression.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eI/R-AKI induced a pronounced bioenergetic deficit in proximal tubules, marked by disrupted mitochondrial homeostasis, suppressed TCA cycle activity and enhanced aerobic glycolysis. Glycolysis-derived lactate accumulated during reperfusion, disrupting mitochondrial oxidative metabolism, whereas inhibition of lactate production with sodium oxamate attenuated tubular injury and restored mitochondrial metabolic function. Mechanistically, lactate activated the mitochondrial Mg\u0026sup2;⁺ channel MRS2, causing mitochondrial Mg\u0026sup2;⁺ overload and sustained reliance on inefficient glycolysis. Targeting MRS2, either pharmacologically or via lipid nanoparticle\u0026ndash;mediated siRNA delivery, normalized mitochondrial Mg\u0026sup2;⁺ homeostasis, improved mitochondrial function and reinstated oxidative metabolism following I/R-AKI.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study identifies a previously unrecognized \u003cb\u003elactate\u0026ndash;MRS2 signaling\u003c/b\u003e axis that drives maladaptive metabolic reprogramming in ischemic AKI. Targeting MRS2 to restore mitochondrial Mg\u0026sup2;⁺ homeostasis reinstates oxidative metabolism, breaks maladaptive metabolic reprogramming, and promotes renal recovery.\u003c/p\u003e","manuscriptTitle":"Lactate drives maladaptive metabolic reprogramming via MRS2 inischemia–reperfusion-induced acute kidney injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 10:30:05","doi":"10.21203/rs.3.rs-8846937/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2026-03-24T05:38:54+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-02-23T16:11:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-20T09:21:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-12T08:44:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2026-02-10T23:20:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5809d670-e290-480b-a1fb-50137c7ba43c","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T02:47:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 10:30:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8846937","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8846937","identity":"rs-8846937","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}