Abstract

   Acute kidney injury (AKI) is a significant global health issue, which
   is often caused by cisplatin therapy and characterized by mitochondrial
   dysfunction. Restoring mitochondrial homeostasis in tubular cells could
   exert therapeutic effects. Here, we investigated Slc25a21, a
   mitochondrial carrier, as a potential target for AKI intervention.
   Renal Slc25a21 expression is negatively associated with kidney function
   in both AKI patients and cisplatin-induced murine models. Sustaining
   renal expression of Slc25a21 slowed down AKI progression by reducing
   cellular apoptosis, necroptosis, and the inflammatory response, likely
   through its regulation of 2-oxoadipate conversion. Slc25a21 is highly
   expressed in proximal tubular epithelial cells, and its down-regulation
   contributes to compromised mitochondrial biogenesis and integrity, as
   well as impaired oxidative phosphorylation. Mechanistically, reduced
   Slc25a21 in AKI disrupts mitochondrial 2-oxoadipate transport,
   affecting related metabolites influx and the tricarboxylic acid cycle.
   These findings demonstrate a previously unappreciated metabolic
   function of Slc25a21 in tubular cells, and suggest that targeting
   mitochondrial metabolic homeostasis by sustaining Slc25a21 expression
   could be a potential novel therapeutic strategy for AKI.

   Subject terms: Pathogenesis, Acute kidney injury

Introduction

   Acute kidney injury (AKI) is defined as a rapid deterioration of renal
   function, manifested by an increase in serum creatinine level, with or
   without a decline in urine output [[42]1, [43]2]. It is estimated that
   over 13.3 million people worldwide suffer from AKI annually, which
   leads to approximately 1.7 million deaths [[44]3]. Notably, survivors
   of AKI are known to face a heightened risk of developing chronic kidney
   disease (CKD), with some cases progressing to end-stage renal disease
   (ESRD) [[45]4]. Cisplatin, a cytotoxic chemotherapeutic drug, is one of
   the most common causes of AKI [[46]5]. About 50–70% of patients
   receiving cisplatin chemotherapy may develop AKI [[47]6]. Among renal
   cells, proximal tubular epithelial cells are particularly susceptible
   to damage from cisplatin, and they typically show distinct pathological
   changes such as tubular dilatation, brush border loss, and cell
   necrosis [[48]7]. Despite various mechanisms for cisplatin-induced AKI
   have been proposed [[49]5, [50]7–[51]9], the precise underlying
   processes remain unclear and, most critically, effective interventions
   to prevent or reverse AKI remain very limited.

   Recent research has identified mitochondrial dysfunction as a primary
   factor in the development of AKI [[52]10, [53]11]. The kidney is the
   second most metabolically active organ in human body with an abundance
   of mitochondria, where ATP is predominantly synthesized via
   mitochondrial oxidative phosphorylation (OXPHOS) [[54]12].
   Specifically, glucose and lipid metabolism are instrumental in the
   generation of acetyl-CoA, a crucial substrate that fuels the
   tricarboxylic acid (TCA) cycle, thereby facilitating ATP synthesis
   [[55]2, [56]13]. In AKI, mitochondrial damage disrupts the normal
   mitochondrial OXPHOS, leading to a significant alteration in the
   kidney’s metabolic state [[57]14–[58]16]. This reprogramming of energy
   metabolism results in decreased cellular ATP levels and a concurrent
   increase in reactive oxidative species (ROS). These metabolic
   perturbations trigger tubular cell apoptosis and necroptosis as well as
   inflammatory response, which exacerbate the pathogenesis of AKI
   [[59]17, [60]18].

   Given these metabolic disruptions in AKI, several therapeutic
   strategies targeting renal metabolism, which focus on restoring energy
   balance and mitochondrial homeostasis, have been proposed in AKI. For
   instance, the administration of glutamine has been shown to boost the
   TCA cycle, thereby partially restoring energy production and
   attenuating the kidney damage typically observed in AKI [[61]19,
   [62]20]. Additionally, given its capacity to counteract the effects of
   acetyl-CoA depletion, sodium acetate supplementation has been also
   considered as a promising therapeutic avenue for ameliorating kidney
   injury [[63]21, [64]22].

   Mitochondrial 2-oxodicarboxylate carrier (ODC), also called SLC25A21,
   is essential for the translocation of 2-oxoadipate and 2-oxoglutarate
   across the mitochondrial inner membrane through a counter exchange
   mechanism [[65]23]. 2-oxoadipate, also known as 2-oxoadipic acid,
   entering the mitochondria is converted into acetyl-CoA, facilitating
   ATP production. Our previous study showed that overexpression of
   SLC25A21 in a yeast mitochondrial dysfunction model, substantially
   enhanced mitochondrial respiration, associated with elevated levels of
   complex IV and TCA-mediated ATP production [[66]24]. In contrast,
   SLC25A21 deficiency contributes to the accumulation of 2-oxoadipate,
   leading to mitochondrial dysfunction and neurotoxicity in spinal
   muscular atrophy–like disease [[67]25].

   In this study, we investigated the role of SLC25A21 as a novel target
   for regulating metabolic processes in renal tubular cells, and
   mitigating kidney dysfunction in AKI. We reported the reduced
   expression of Slc25a21 in both AKI patients and mice induced by
   cisplatin, and showed that down-regulation of Slc25a21 was associated
   with kidney dysfunction in AKI. Using both in vivo AKI murine models
   and in vitro cisplatin-exposed mouse proximal tubule cells (TKPTS)
   lines, combined with metabolomics analysis, we further demonstrated
   that maintaining renal Slc25a21 expression protects renal tubules from
   cisplatin-induced damage and alleviates cell apoptosis and necroptosis
   as well as inflammation, thereby preventing the progression of AKI.
   These studies highlight the role for Slc25a21 in facilitating
   mitochondrial 2-oxoadipate transport, regulating metabolites influx and
   enhancing energy metabolism in AKI. Our findings might pave the way to
   design novel therapeutic interventions in AKI, targeting mitochondrial
   homeostasis.

Methods

Patients

   Renal biopsy samples were acquired from four patients diagnosed with
   acute tubular necrosis at Children’s Hospital of Nanjing Medical
   University, China. The AKI samples include four pediatric patients,
   with a median age of approximately 4 years, and a male-to-female ratio
   of 1:3. These children presented with AKI characterized by oliguria or
   anuria and elevated serum creatinine, caused by drug toxicity, sepsis,
   or ischemia. The pediatric patient details are provided in Table
   [68]S1. Normal renal tissues, also obtained from para-carcinoma kidney
   tissues, were collected from five children (<14 years old; the
   male-to-female ratio is 3:2.) who underwent renal carcinoma resection.

Animals

   8-week-old male C57BL/6 J mice were purchased from GemPharmatech
   (Nanjing, China) and maintained under a 12-hour light/ 12-hour dark
   cycle with free access to food and water in the animal facility of
   Nanjing Medical University (Nanjing, China). Slc25a21^AAV9 mice were
   generated by kidney multipoint in situ injection of adeno-associated
   virus 9 (AAV9) expressing Slc25a21 gene (pAAV-CMV-Slc25a21-HA-WPRE,
   4 × 10^11 virus genome copies), while AAV9 containing scrambled
   sequence served as control (NC^AAV9, pAAV-CMV-MCS-WPRE) (OBiO,
   Shanghai, China). The surgery procedure was performed following the
   instruction of previously reported study [[69]26]. To deliver the
   vector, 32 G, 50 µl syringes (Conpuvon Company, China) were used for
   multi-point in situ injection into the renal cortex. Slc25a21^DNA mice
   were generated through high-pressure tail-vein injection of 70 μg
   Slc25a21 plasmids within 7 sec. The empty vector (pcDNA3.1) served as
   negative control (NC^DNA). Cisplatin-induced AKI model was obtained by
   intraperitoneal injection of 25 mg/kg cisplatin (Sigma-Aldrich) and the
   same volume of saline were injected into the controls [[70]27]. Mice
   were sacrificed after 3 days of injection.

Cell culture

   Mouse proximal tubule cells (TKPTS; Cat# CRL-3361; RRID: CVCL_UJ13)
   lines were procured from ATCC, cultured in DMEM/F-12 medium (Gibco,
   319-075-CL) supplemented with 7% Fetal Bovine Serum and 0.6% insulin.
   The cells were maintained at 37 °C with 5% CO2 and 95% air. The TKPTS
   were transfected with Slc25a21 plasmids or empty vector (pcDNA3.1
   served as negative control) using JetPRIME® transfection reagent
   (Polyplus, 101000046) following the manufacturer’s instructions and
   then treated with cisplatin (10 µM) for 24 h. Cells were regularly
   monitored for mycoplasma via qPCR under ISO 17025, ensuring
   mycoplasma-free conditions.

Cell viability assay

   TKPTS cells were pre-seeded in a 96-well plate and transfected with
   Slc25a21 plasmids or empty vector (pcDNA3.1 as a negative control)
   after cisplatin (10 µM) treatment for 24 h. Cell viability was assessed
   using Cell Counting Kit-8 (CCK-8) (ApexBio, K1018) in accordance with
   the manufacturer’s instructions. Following the respective treatments,
   10 μl of the CCK-8 solution was added to each well of the plates (care
   should be taken to avoid introducing bubbles into the wells, as they
   can interfere with absorbance) and incubated for 2 h at 37 °C. The
   absorbance was measured at 450 nm using a microplate reader. Cell
   viability was calculated using the following formula:

   Cell viability (%) = [(As - Ab) / (Ac - Ab)] x 100%, where As is the
   absorbance of the tested vectors (absorbance of the well containing
   cells, culture medium, CCK-8, and tested vectors), Ab is the absorbance
   of the blank (absorbance of the well containing culture medium and
   CCK-8), and Ac is the absorbance of the control (absorbance of the well
   containing cells, culture medium, and CCK-8).

Western blotting analysis

   Total proteins from renal tissue (25–30 mg) or cultured cells (10^6)
   were extracted using RIPA lysis buffer (Beyotime, P0013K) containing
   the protease inhibitor cocktail (Roche, 04693132001). The lysate
   concentrations were determined using the BCA protein assay kit
   (Thermo-Fisher, 23227) and separated by sodium dodecyl
   sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following gel
   electrophoresis, the proteins were transferred onto polyvinylidene
   fluoride (PVDF) membranes, which were then blocked with 5% non-fat milk
   powder for 1 h at room temperature (RT). Subsequently, the membranes
   were incubated with primary antibodies overnight at 4 °C. The primary
   antibodies used in this study included: SLC25A21 antibody (Thermo
   Fisher Scientific, PA5-101672; RRID: AB_2851106); NGAL antibody (Abcam,
   ab63929; RRID: AB_1140965); Mouse KIM-1 Antibody (R&D Systems, AF1817;
   RRID: AB_2116446); BAX (Cell Signaling Technology, 2772; RRID:
   AB_10695870); Cleaved Caspase3 (Cell Signaling Technology, 9664; RRID:
   AB_2070042); P53 (Cell Signaling Technology, 9282; RRID: AB_331476);
   β-Actin Rabbit mAb (High Dilution) (ABclonal, AC026; RRID: AB_2768234);
   α-Tubulin Mouse mAb (ABclonal, AC012; RRID: AB_2768341); Mouse anti
   HA-tag mAb (ABclonal, AE008; RRID: AB_2770404); NDUFB8 Rabbit mAb
   (ABclonal, A19732; RRID: AB_2938519); SDHB Rabbit mAb (ABclonal, A1809;
   RRID: AB_2861883); UQCRC2 Rabbit mAb (ABclonal, A4366; RRID:
   AB_2863250); COXIV Rabbit pAb (ABclonal, A6564; RRID: AB_2767158);
   ATP5A1 Rabbit mAb (ABclonal, A11217; RRID: AB_2861524); Anti-mtTFA
   antibody - Mitochondrial Marker (Abcam, ab47517; RRID: AB_945799);
   PGC1α Rabbit mAb (ABclonal, A20995; RRID: AB_3096101); ACAA2 Rabbit mAb
   (ABclonal, A0664; RRID: AB_3096102); DHTKD1 Rabbit pAb (ABclonal,
   A8369; RRID: AB_2769161); Citrate synthetase Rabbit mAb (ABclonal,
   A23371; RRID: AB_3096103); OGDH Rabbit mAb (ABclonal, [71]A22163; RRID:
   AB_3096104); SUCLG2 Rabbit pAb (ABclonal, A19872; RRID: AB_2862784);
   ACO2 Rabbit mAb (ABclonal, A4524; RRID: AB_2863288); MT-ND1 Rabbit mAb
   (ABclonal, A9743; RRID: AB_3096106); ATP6 Rabbit pAb (ABclonal, A8193;
   RRID: AB_2768510); MT-CYB Rabbit pAb (ABclonal, A17966; RRID:
   AB_2861768); COX1 Rabbit mAb (ABclonal, A23123; RRID: AB_3096107);
   Membrane Integrity WB Antibody Cocktail (Abcam, ab110414; RRID:
   AB_2687585). PIPK1/RIP Rabbit pAb (ABclonal, A7414; RRID: AB_ 2767944),
   PIPK3 Rabbit pAb (ABclonal, A25050; RRID: AB_3662682), MLKL Rabbit mAb
   (ABclonal, A26436; RRID: AB_3662683). The PVDF membranes were then
   incubated with HRP-tagged secondary antibodies (HRP-conjugated Mouse
   Anti-Rabbit IgG (H + L), AS079; RRID: AB_2864059; HRP-conjugated Goat
   Anti-Mouse IgG Light Chain, AS062; RRID: AB_2864056) for 1 h at RT. The
   bands were visualized using an enhanced chemiluminescence kit (Tanon,
   180-501) on a Tanon 5200 Chemiluminescent Imaging System (Tanon,
   China). Band quantification was performed using Image J software (RRID:
   SCR_003070) and normalized to loading controls.

Quantitative real-time polymerase chain reaction (qRT-PCR)

   Total RNA from kidney tissues (25–30 mg) and cultured cells (10^6) was
   extracted using Trizol reagent (TaKaRA Biotechnology, 9109) following
   the manufacturer’s instructions. Subsequently, 1 μg of total RNA was
   reverse transcribed to cDNA using HiScript II Q RT SuperMix for qRT-PCR
   (Vazyme Biotechnology, R222-01) according to the manufacturer’s
   protocol. The resulting cDNA was then subjected to qRT-PCR
   amplification using SYBR Green master mix (Vazyme, q111-02/03) on a
   LightCycler®96 system (Roche, Germany). The temperature conditions were
   executed according to the manufacturer’s instructions for the SYBR
   Green master mix. Relative mRNA expression levels were calculated using
   the 2^-∆∆Ct method and normalized to β-Actin expression. Primer
   sequences used in the study were obtained from the Primer Bank and are
   listed in Table [72]S2.

Mitochondrial DNA copy number

   Total DNA was extracted from Kidney tissue using DNeasy Tissue Kit
   (QIAGEN Sciences, 69506) following the manufacturer’s instructions. The
   primer designed by Primer 5 software was listed in Table [73]S2.
   qRT-PCR was performed for the determination of mtDNA copy number.
   Relative mtDNA copy number was calculated using the 2^-∆∆Ct method and
   normalized by Rsp18.

Oxygen Consumption Rate (OCR) measurement

   The measurement of oxygen consumption rate was conducted using the
   Seahorse XF-96 Extracellular Flux Analyzer (Seahorse Bioscience,
   Copenhagen, Denmark). Briefly, TKPTS cells (10^4) were initially were
   pre-seeded in a Seahorse assay 96-well plate and transfected with
   Slc25a21 plasmids or empty vector (pcDNA3.1 as a negative control).
   After 6 h, cisplatin (10 µM) was added to the cell culture medium for
   24 h. On the day of the experiment, the cell medium was replaced by
   Seahorse assay medium, and cells were incubated at 37 °C in a
   CO[2]-free incubator for 1 h prior to assessing the basal OCR. Half an
   hour before the experiment, the sensor was placed into the XF-96
   instrument, and calibration was initiated. After calibration, the
   calibration fluid plate was replaced with the cell plate, and
   measurements began. Basal oxygen consumption was recorded for 20 min,
   and the inhibitors were subsequently introduced as follows [[74]1]:
   Oligomycin (1 µM), which blocks the proton channel of the Fo portion of
   ATP synthase, thereby inhibiting ATP synthesis, was employed to assess
   the coupling of ATP synthesis with the respiratory rate [[75]2].
   Carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP, 0.5 µM), a
   compound that dissipates the proton gradient across the mitochondrial
   membrane, was used to determine the maximal respiration rate [[76]3].
   Rotenone (ROT, 0.5 µM) and antimycin A (0.5 µM), inhibitors of Complex
   I and Complex III, respectively, were employed to measure the
   non-mitochondrial oxygen consumption rate. All these compounds were
   automatically injected into each well by the XF-96 instrument. The
   determined cellular bioenergetics parameters were the following: basal
   and maximal respiration.

Reactive oxygen species (ROS) measurement

   The assessment of ROS production was determined using MitoSOX^TM Red
   mitochondrial superoxide indicator for live-cell imaging (Invitrogen,
   [77]M36008) according to the manufacturer’s instructions. Briefly,
   TKPTS cells were pre-seeded in a 12-well plate and transfected with
   Slc25a21 plasmids or empty vector (pcDNA3.1 as a negative control)
   after cisplatin (10 µM) treatment for 24 h. 5 µM MitoSOX^TM reagent was
   applied to cover the cells, followed by incubation for 10 min at 37 °C,
   protected from light. The cells were then washed three times with warm
   buffer, and ROS levels were determined by flow cytometry at 510/580 nm.

ELISA assay of IL-18 and TNFα

   The concentrations of IL-18 and TNFα in the serum of mice measured by
   Mouse IL-18 ELISA Kit (ABclonal, RK00104) and Mouse TNFα ELISA Kit
   (ExCell Bio, EM008-96) in accordance with the manufacturer’s
   instructions. Briefly, prepared all necessary reagents, working
   standards, and samples. Washed each well for 40 s, then repeated. Added
   100 μl of standard/sample for each well. Utilized the provided adhesive
   sealer to cover the wells and incubated the plate at 37 °C for 2 h.
   Prepared the concentrated biotin conjugate antibody (30x) working
   solution. Repeated the aspiration/wash process, then added 100 μl of
   the working biotin conjugate antibody to each well and incubated at
   37 °C for 1 h. Prepared the streptavidin-HRP concentrated (30x) working
   solution. Repeated the aspiration/wash process, then added 100 μl of
   the working streptavidin-HRP to each well and incubated at 37 °C for
   1 h. Repeated the aspiration/wash process. Added 100 μl of TMB
   Substrate to each well, followed by a 15–20 min incubation at 37 °C in
   the dark. Added 50 μl of stop solution, then measured the optical
   density of each well within 5 min using a microplate reader at 450 and
   630 nm.

Determination of ATP content

   The ATP content in kidney tissue (25–30 mg) was determined using an
   Enhanced ATP Assay Kit (Beyotime, S0027) according to the
   manufacturer’s instructions. After complete tissue lysis, the lysate
   was centrifuged at 4 °C, 12,000 × g for 5 min, and the supernatant was
   collected for subsequent analysis. The ATP concentration was determined
   by preparing an ATP standard curve and measuring the concentration of
   the supernatant accordingly. Additionally, the protein concentration of
   the supernatant was measured using the BCA protein assay kit
   (Thermo-Fisher, 23227), and the ATP content was calculated per one µg
   of protein.

Serum creatinine (SCr) and blood urea nitrogen (BUN) analysis

   Venous blood was collected from mice in an EP tube containing
   anticoagulant, followed by centrifugation at 5000 rpm/min for 30 min to
   separate plasma. The upper serum was then obtained. Serum creatinine
   levels were analyzed using the Beckman Creatinine Analyzer II (DXC800;
   Beckman Coulter). Blood urea nitrogen was measured using a BUN
   Colorimetric Detection Kit (EIABUN; Invitrogen, Carlsbad, CA).

Periodic acid-Schiff (PAS) staining

   Kidney tissues obtained from mice were fixed in 4% paraformaldehyde,
   embedded in paraffin, and transversely sectioned to a thickness of 3 μm
   for renal pathology assessment. The sections were then subjected to
   dewaxing and rehydration using the following sequence: xylene I for
   10 min; xylene II for 10 min; 100% ethanol I for 3 min; 100% ethanol II
   for 3 min; 95% ethanol for 3 min; 80% ethanol for 3 min; 70% ethanol
   for 3 min; distilled water for 1 min.

   Renal sections underwent oxidation with 1% periodate at 37 °C for
   10 min, followed by rinsing with distilled water. Subsequently, the
   sections were incubated with Schiff dye at 42 °C for 40 min and rinsed
   with tap water for 10 min. Hematoxylin staining was carried out at
   37 °C for 3 min, followed by rinsing with a solution of 1% HCl and
   ethanol for 2 sec. After removing HCl, the sections were returned to a
   blue state for 1 min and rinsed with tap water for 10 min.

   Dehydration steps included 70% ethanol for 3 min, 80% ethanol for
   3 min, 90% ethanol for 3 min, 100% ethanol for 3 min, and xylene I, II,
   III for 3 min each. Finally, the renal sections were sealed with
   neutral resin. Photomicrographs of the renal sections were captured
   using an Olympus BX51 microscope (Olympus, Center Valley, PA, USA) and
   analyzed by a pathologist in a blinded manner. The tubular injury score
   was assigned as follows: 0 for normal histology, 1 for mild injury
   (5-25% injury), 2 for moderate injury (25-50% injury), 3 for severe
   injury (50–75% injury), and 4 for critical injury (more than 75%
   injury). At least 10 fields were examined and scored for each slide.

Immunohistochemical (IHC) analysis

   Mouse kidney tissues were fixed in 4% paraformaldehyde, embedded in
   paraffin, and transversely sectioned to a thickness of 3 μm for renal
   pathology slides. After deparaffinization, renal sections were washed
   three times with PBS and subjected to antigen retrieval by boiling in
   Citrate Antigen Retrieval Solution (Beyotime, P0083) for 20 min,
   followed by cooling to RT. Subsequent steps were conducted using the
   Universal Two-Step Test Kit (Mouse/Rabbit Enhanced Polymer test system)
   (ZSGB Bio, PV-9000) following the manufacturer’s instructions. The
   sections were incubated overnight at 4 °C with primary antibodies
   against SLC25A21 (Thermo Fisher Scientific, PA5-101672; RRID:
   AB_2851106), Mouse anti HA-tag mAb (ABclonal, AE008; RRID: AB_2770404)
   and 8-OHdG (Santa Cruz, sc-66036; RRID: AB_832272). Peroxidase
   conjugate localization was determined using a DAB kit (ZLI-9018,
   zsbio). Renal sections were photographed with an Olympus BX51
   microscope (Olympus, Center Valley, PA, USA), and the signals were
   analyzed using Image J software (RRID: SCR_003070).

Immunofluorescence staining

   Immunofluorescence staining was also performed on paraffin-embedded
   renal sections, following the same deparaffinization, antigen
   retrieval, and rehydration steps as the IHC staining process. The renal
   sections were incubated overnight at 4 °C with the primary antibody
   SLC25A21 Antibody (Thermo Fisher Scientific, PA5-101672), HA-Tag
   (C29F4) Rabbit mAb (Cell Signaling Technology, 3724S; RRID:
   AB_3662684), Aquaporin/AQP2 (E-2) (Santa Cruz, sc-515770; RRID:
   AB_2810957) and E-cadherin Antibody (G-10) (Santa Cruz, sc-8426; RRID:
   AB_626780). The next day, Alexa Fluor 488-conjugated Donkey anti-rabbit
   IgG (H + L) (ABclonal, AS035 RRID: AB_2768318) was used as a secondary
   antibody. Nuclei were counterstained with DAPI (Beyotime, P0131). Lotus
   tetragonolobus lectin (LTL) fluorescence (Vector Laboratories, ZG1023)
   was simultaneously incubated with the primary antibody. Images were
   acquired using an Olympus BX51 microscope (Olympus, Center Valley, PA,
   USA).

   For immunocytofluorescence, cells were seeded on polylysine-coated
   glass. Initially, 100 nM Mitotracker (Invitrogen, M7512) was incubated
   for 30 min at 37 °C. After treatment, the cells were fixed in cold
   acetone for 5 min, permeabilized with Triton X-100 (Beyotime, P0096)
   for 10 min, and blocked with 5% BSA for 1 hour at RT. The subsequent
   steps were identical to immunofluorescence staining in kidney tissues.

Apoptosis analysis

   Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
   (TUNEL) Assay: The assessment of apoptosis in renal tissue was
   determined using TUNEL BrightGreen Apoptosis Detection Kit (Vazyme
   Biotechnology, A112-01) according to the manufacturer’s instructions.
   Briefly, mouse kidney tissues were fixed in 4% paraformaldehyde,
   embedded in paraffin, and transversely sectioned to a thickness of 3 μm
   for renal pathology slides. After deparaffinization, renal sections
   were washed three times with PBS and incubated with the Proteinase K
   solution for 20 min. Added 100 µl 1× equilibration buffer and
   equilibrated at RT for 10–30 min. Prepared the TdT reaction mixture in
   the dark during equilibration. After equilibration, removed 1 ×
   equilibration buffer, and then dropped 50 μl of TdT reaction mixture on
   the sample. Incubated the plate at 37 °C for 60 min and washed twice
   with PBS, and incubated at RT for 5 min each time. Counterstained the
   sample in the dark with DAPI (Beyotime, P0131). Renal sections of TUNEL
   expression were photographed with an Olympus BX51 microscope (Olympus,
   Center Valley, PA, USA), and the signals were analyzed using Image J
   software (RRID: SCR_003070).

   Annexin V-FITC/PI staining: The assessment of apoptosis in the renal
   tubular cells was determined using Annexin V-FITC Apoptosis Detection
   Kit (BD Biosciences, 556547) according to the manufacturer’s
   instructions. After transfection with Slc25a21 plasmids and empty
   vector following cisplatin treatment, the cells were washed with PBS
   twice and then double-stained with FITC and PI. Analyzed by flow
   cytometry within 1 h.

Mitochondria and cytosol separation

   Mitochondria were isolated from mouse kidney tissues using the
   Minute^TM Mitochondria Isolation Kit for Mammalian Cells and Tissues
   (Invent, MP-007) following the manufacturer’s instructions. Briefly,
   kidney tissues were homogenized in 250 μl buffer A, centrifuged at
   16,000 × g for 30 s. The filter was discarded, and the pellet was
   resuspended, followed by centrifugation at 700 × g for 1 min. The
   resulting supernatant was carefully transferred to a fresh 1.5 ml tube
   and then centrifuged at 16,000 × g for 30 min. The obtained supernatant
   is the cytosol for downstream detection. For mitochondria isolation,
   after the initial centrifugation at 700 × g for 1 min, 300 μl of buffer
   B was added to the tube, followed by centrifugation at 16,000 × g for
   30 min. The supernatant was discarded, and the pellet was resuspended
   in 200 μl of buffer B, followed by centrifugation at 8000 × g for
   5 min. The resulting supernatant was transferred to a fresh 2.0 ml
   tube; 1.6 ml of cold PBS was added, and the mixture was centrifuged at
   16,000 × g for 30 min. The supernatant was discarded, and the pellet,
   representing isolated mitochondria, was retained for further detection.

Untargeted metabolomics analysis

   This assay was carried out in PANOMIX company, Suzhou, China.
   Accurately weighed an appropriate amount of kidney tissues from mice,
   added 200 μl 50% acetonitrile solution containing
   2-Amino-3-(2-chloro-phenyl)-propionic acid, prepared for LC-MS
   detection. The LC analysis was conducted on a Vanquish UHPLC System
   (Thermo Fisher Scientific, USA). For LC-ESI ( + )-MS analysis, the
   mobile phases comprised 0.1% formic acid in acetonitrile (v/v) and 0.1%
   formic acid in water (v/v). Mass spectrometric detection of metabolites
   was performed on Orbitrap Exploris 120 (Thermo Fisher Scientific, USA)
   with ESI ion source. Simultaneous MS1 and MS/MS (Full MS-ddMS2 mode,
   data-dependent MS/MS) acquisition was employed. Raw data were initially
   converted to mzXML format using MS Convert in ProteoWizard software
   package (v3.0.8789) and processed with XCMS for feature detection,
   retention time correction, and alignment. Ropls software was employed
   for all multivariate data analyses and modeling. Prior to the actual
   principal component analysis (PCA), data matrices were pre-processed
   using the Pareto scaling. Orthogonal partial least squares discriminant
   analysis (OPLS-DA) employing the software SIMCA (v. 13.0, 2011,
   Umetrics, Umea, Sweden) was performed. OPLS-DA facilitated the
   identification of different metabolites through variable importance on
   projection (VIP) analysis. VIP > 1 were statistically significant
   metabolites. The identified differential metabolites were subjected to
   pathway analysis by MetaboAnalyst 5.0.

Detection of levels of 2-oxoadipate and TCA cycle intermediates (citrate,
fumarate and malate) in the mitochondria and cytoplasm

   This assay was carried out in BIOTREE accompany, Shanghai, China.
   Mitochondria and cytoplasm were isolated from mouse kidney tissues
   using the Minute^TM Mitochondria Isolation Kit for Mammalian Cells and
   Tissues (Invent, MP-007) following the manufacturer’s instructions,
   respectively. After the extraction of metabolites from mitochondria and
   cytoplasm, and preparation of standard solutions, all samples and
   calibration solutions were detected by HPIC-MS/MS. The HPIC separation
   was performed using a Dionex ICS-6000 HPIC System (Thermo Fisher
   Scientific, USA). This assay utilized a Triple Quad 6500+ System (AB
   SCIEX) equipped with an electrospray ionization (ESI) interface. The
   injection volume was set at 5 μl. For each target compound, the ion
   pair with the highest signal intensity in terms of the parent
   ion-daughter ion was chosen for qualitative analysis. The Y-axis of the
   calibration curve represented the peak area of the target compound,
   while the X-axis represented the concentration of the target compound
   (µmol/l). The concentration of the target metabolite per milligram of
   kidney tissue (µmol/l/mg): C[Final metabolite] =C[Target
   metabolite]×V[Final extraction]/V[Injection]. The ratio of 2-oxoadipate
   and TCA cycle intermediates (citrate, fumarate and malate) between the
   mitochondria and the cytoplasm was calculated by dividing the
   mitochondrial concentrations by the respective cytoplasmic
   concentrations.

Datasets used for the analysis

   Three bulk-RNA seq datasets were used in this study. Bulk-RNA seq data
   ([78]GSE30718) [[79]28] contained 26 AKI patients and 11 healthy
   individuals. The [80]GSE30122 [[81]29] were derived from human renal
   tubular or glomerular SLC25A21 expression from 12 and 10 healthy
   subjects, respectively. It also contained the information of glomerular
   filtration rate (GFR) for each sample. The [82]GSE145085 [[83]30] was
   derived from human iPSC-derived kidney organoids treated with/without
   cisplatin. In addition, we used a mouse kidney single-cell RNA
   sequencing dataset ([84]GSE197266) [[85]31], including 2
   cisplatin-induced AKI mice and 2 normal control mice. The original cell
   annotation provided by authors was used for downstream analysis.

Statistical analysis

   All data are presented as means ± standard error of the mean (SEM)
   using GraphPad Prism 10.0 (GraphPad Software, RRID:SCR_002798). One-way
   analysis of variance (ANOVA) or unpaired Student’s t test (2-tailed)
   were employed to test for significant differences as indicated.
   P < 0.05 was considered statistically significant, unless otherwise
   indicated.

Results

Decreased renal expression of SLC25A21 is associated with AKI and
cisplatin-induced kidney damage

   To evaluate the association between SLC25A21 and AKI, we first examined
   the expression of SLC25A21 in the human kidney (Fig. [86]1A).
   Immunohistochemistry (IHC) staining shows a clear distribution of
   SLC25A21 in normal kidneys, especially within the renal tubules, and
   SLC25A21 was significantly diminished in AKI kidneys (P < 0.05, Fig.
   [87]1A). Bulk-RNA sequencing analysis confirmed the reduction in renal
   SLC25A21 expression in AKI compared with controls (P < 0.05, Fig.
   [88]1B) [[89]28]. A significant reduction in SLC25A21 mRNA expression
   was further detected in cisplatin-treated kidney organoids compared to
   normal controls (Figure [90]S1A) [[91]30]. Moreover, the tubular
   expression of SLC25A21 in the kidney was positively correlated with the
   levels of glomerular filtration rate (GFR) (r = 0.608; P = 0.036, Fig.
   [92]1C), while glomerular SLC25A21 expression was not correlated with
   GFR levels (Fig. [93]S1B) [[94]29].

Fig. 1. Decreased renal expression of SLC25A21 is associated with AKI and
cisplatin-induced kidney damage.

   [95]Fig. 1
   [96]Open in a new tab

   A Representative immunohistochemistry images of SLC25A21 from human
   kidney biopsy samples with AKI (left) and semi-quantitative IOD
   analysis of SLC25A21 expression (right). The details of AKI patients
   (n = 4) were described in Table [97]S1, and normal samples from
   para-kidney carcinoma kidney tissues (n = 5) were served as controls.
   Arrows indicates renal tubules. IOD, integral optical density. Scale
   bars, 50 µm. B The mRNA levels of SLC25A21 in the kidney from both AKI
   patients and healthy controls (data were derived from [98]GSE30718)
   [[99]28]. C Pearson correlation between tubular SLC25A21 expression and
   glomerular filtration rate (GFR) (derived from [100]GSE30122)
   [[101]29]. D Representative Periodic Acid-Schiff (PAS) staining images
   from cisplatin-induced AKI and control mice, illustrating tubular
   dilatation and atrophy (upper). Scale bars, 50 µm. Representative
   immunohistochemistry images of SLC25A21 from cisplatin-induced AKI and
   control mice (down). Scale bars, 50 µm. E The expression of Slc25a21 in
   the kidneys from cisplatin-induced AKI and control mice. Representative
   western blotting for Slc25a21 protein levels (upper); Slc25a21 mRNA
   expression changes were determined by RT-qPCR (down). n = 10, each
   group, and values are reported as mean ± SEM. F Pearson correlation
   analysis between Slc25a21 mRNA expression relative to control in the
   kidney and Serum creatinine (SCr, upper), as well as Blood Urea
   Nitrogen (BUN, down) in cisplatin-induced AKI mice (n = 10). In each
   case, data were presented as means ± SEM and statistical significance
   is assessed by the unpaired Student’s t-test. * indicates P < 0.05;
   *****indicates P < 0.0001 compared between two groups. See also Fig.
   [102]S1.

   We constructed cisplatin-induced AKI mice models, which showed kidney
   dysfunction with severe tubular dilatation and epithelial cells
   degeneration (Fig. [103]1Dupper). Cisplatin administration also
   resulted in pronounced cell apoptosis, necroptosis, and increased
   inflammatory response in cisplatin-injured kidneys compared to controls
   (Fig. [104]S1C-I). Furthermore, cisplatin-injured kidneys presented a
   significant down-regulation of SLC25A21 expression in both protein and
   mRNA levels (Fig. [105]1Ddown and Fig. [106]S1J; Fig. [107]1E). The
   protein and mRNA level of Slc25a21 was negatively correlated with
   kidney function parameters (Serum Creatinine (SCr) and Blood Urea
   Nitrogen (BUN)) in the cisplatin-induced AKI mouse models (Fig.
   [108]1F, Fig. [109]S1K).

AAV9-sustaining Slc25a21 renal expression attenuated cisplatin-induced AKI

   To elucidate the role of Slc25a21 in the kidney, we used the AAV9
   system with kidney multi-point in situ injection in the renal cortex
   (Slc25a21^AAV9, Fig. [110]2A). Compared with AAV9-scramble (a scramble
   sequence) controls (NC^AAV9), AAV9-Slc25a21 delivery enhanced Slc25a21
   expression in renal tubules, tracked through IHC staining with
   SLC25A21-targeted HA (Fig. [111]2A, Fig. [112]S2A), and partially
   restored the expression of Slc25a21 in AKI kidneys (Fig. [113]S2B, C).
   Furthermore, Slc25a21^AAV9 cisplatin-induced AKI mice showed decreased
   tubular dilatation and epithelial cells atrophy, which was associated
   with a lower tubular injury score (Fig. [114]2B). These mice also
   exhibited significantly decreased expression of kidney injury molecule
   1 (KIM1) and Neutrophil gelatinase associated lipocalin (NGAL) (Fig.
   [115]2C, Fig. [116]S2D), lower kidney to body weight ratio, and
   decreased levels of SCr and BUN (Fig. [117]2D), suggesting a functional
   improvement in tubular injury and kidney damage with respect to NC^AAV9
   AKI mice. Both levels of SCr and BUN were strongly negatively
   correlated with the expression of Slc25a21 in cisplatin-induced AKI
   mouse kidneys following AAV application (Fig. [118]S2E), with
   correlation coefficients of r = –0.92 (P = 0.0004) and r = –0.76
   (P = 0.0176), respectively. These findings suggest that maintaining the
   expression of Slc25a21 is associated with protection against
   cisplatin-induced kidney dysfunction.

Fig. 2. AAV9-sustaining Slc25a21 renal expression attenuated
cisplatin-induced AKI.

   [119]Fig. 2
   [120]Open in a new tab

   A Experimental schematic of mice receiving AAV9-Slc2521 (Slc25a21^AAV9)
   or AAV9-scramble (NC^AAV9) in situ injection in the kidneys, followed
   by cisplatin treatment (25 mg/kg) for three days to induce AKI (upper).
   Representative immunohistochemistry images of Slc25a21-targeted HA from
   Slc25a21^AAV9 and NC^AAV9 mice (down). Scale bars, 50 µm. B
   Representative Periodic Acid-Schiff (PAS) staining image in
   Slc25a21^AAV9 and NC^AAV9 mice following cisplatin or saline injection.
   left: Representative PAS staining for AKI lesions in the kidneys. Scale
   bars, 50 µm. right: semi-quantitative analysis of tubular injury score
   in the kidneys. n = 10, each group and values are reported as
   mean ± SEM. C Representative western blotting for KIM1 and NGAL protein
   levels in Slc25a21^AAV9 and NC^AAV9 mice after cisplatin or saline
   injection. D The ratio of kidney to body weight and concentrations of
   Serum SCr and BUN in Slc25a21^AAV9 and NC^AAV9 mice after cisplatin or
   saline injection. n = 10, each group, and values are reported as
   mean ± SEM. E Representative immune images for TUNEL expression in
   Slc25a21^AAV9 and NC^AAV9 mice after cisplatin or saline injection
   (left), with quantification of TUNEL-positive cells per field (right).
   200× field images were recorded to calculate the mean for each mouse
   kidney, represented with one dot. n = 5, each group and values are
   reported as mean ± SEM. Scale bars, 50 µm. F Representative western
   blotting for BAX and Cleaved-Caspase 3 (C-Caspase 3) expression level
   in the kidneys from both Slc25a21^AAV9 and NC^AAV9 mice after cisplatin
   or saline injection. G Representative western blotting for RIPK1 and
   RIPK3 expression level in the kidneys from both Slc25a21^AAV9 and
   NC^AAV9 mice after cisplatin or saline injection. H Expression of
   inflammatory markers (Il-1β, Il-6, Il-18, Tnfα and Mcp1), determined by
   RT-qPCR, in the kidneys from Slc25a21^AAV9 and NC^AAV9 mice after
   cisplatin or saline injection. n = 9–10, each group, and values are
   reported as mean ± SEM. In each case, data were presented as
   means ± SEM and statistical significance is assessed by One-way ANOVA
   analysis of variance of Tukey’s multiple comparisons test. * indicates
   P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; ****
   indicates P < 0.0001; NS indicates not significant. See also Fig.
   [121]S2, [122]S3.

   Slc25a21^AAV9 mice exhibited decreased cell apoptosis in the kidneys
   following cisplatin injection compared to NC^AAV9 AKI mice, as shown by
   the lower TUNEL-positive cells (Fig. [123]2E) and expression of BAX and
   Cleaved-Caspase 3 (Fig. [124]2F, Fig. [125]S2F). In addition to cell
   apoptosis, we also evaluated the necroptosis in cisplatin-induced AKI.
   The protein expression of necroptosis markers RIPK1 and RIPK3 was
   significantly reduced in Slc25a21^AAV9 AKI mice compared to NC^AAV9 AKI
   mice (Fig. [126]2G, Fig. [127]S2G). The inflammatory response was also
   attenuated in the Slc25a21^AAV9 AKI kidney, which had lower renal
   expression of Interleukin-1 (IL-1β), Interleukin-6 (IL-6),
   Interleukin-18 (IL-18), Tumor necrosis factor α (TNFα) and Monocyte
   chemoattractant protein 1 (MCP1) than NC^AAV9 mice after cisplatin
   treatment (Fig. [128]2H), which was mirrored by a reduction in
   circulating IL-18 and TNFα levels (Fig. [129]S2H). We used another
   systemic delivery method of Slc25a21 by high-pressure tail-vein
   injection of plasmids (Slc25a21^DNA), which confirmed that maintaining
   Slc25a21 expression attenuates cisplatin-induced renal injury and
   dysfunction, as well as tubular apoptosis, necroptosis, and
   inflammatory response (Fig. [130]S3). Thus, preservation of Slc25a21
   expression protects against cisplatin-induced AKI in mice.

Rescuing Slc25a21 ameliorates cisplatin-induced acute tubular injury in vitro

   Our data suggested that renal overexpression of Slc25a21 mitigates
   tubular injury in AKI (Fig. [131]2B); we therefore investigated whether
   renal tubular cells were directly regulated by Slc25a21. We first
   showed the distribution of Slc25a21 in the kidney and analyzed a public
   single-cell RNA sequencing (ScRNA-seq) dataset derived from
   cisplatin-induced AKI kidneys (Fig. [132]3A) [[133]31]. Slc25a21 was
   mainly enriched in the tubular cells, including proximal tubules (PT),
   distal convoluted tubule cells (DCT) and Loop of Henle cells (LOH).
   Among these cell-types, Slc25a21 was primarily expressed in the PT cell
   compartment. Compared with controls, Slc25a21 expression was
   dramatically decreased in PT derived from AKI kidneys (Fig. [134]3B).
   The immunofluorescence co-staining Lotus tetragonolobus lectin (LTL, a
   marker for proximal renal tubules), Aquaporin 2 (AQP2, a marker for
   collecting ducts) and E-cadherin for renal tubules with SLC25A21,
   further confirmed the renal PT localization of SLC25A21. SLC25A21 is
   expressed in some tubular cells (Fig. [135]3Cupper), mostly in the LTL
   positive cells (proximal tubular cells), but rarely in the AQP2
   positive tubules (collecting ducts). In addition, following AAV9
   administration exogenous SLC25A21 presents a similar distribution with
   native protein of SLC25A21 (Fig. [136]S4A).

Fig. 3. Rescuing Slc25a21 ameliorates cisplatin-induced acute tubular injury
in vitro.

   [137]Fig. 3
   [138]Open in a new tab

   A Uniform manifold approximation and projection (UMAP) plots of
   cisplatin-induced AKI datasets based on single-cell RNA sequencing
   showing 13 subclusters of kidney tissue ([139]GSE197266) [[140]31]. B
   The expression of Slc25a21 in each subcluster in cisplatin-induced AKI
   and control mice (left) and Slc25a21 was mainly expressed in proximal
   renal tubular cells (right). T cells T lymphocytes, B cells B
   lymphocytes, EC endothelial cells, DC dendritic cells, PT proximal
   renal tubular cells, LOH Loop of Henle cells, DCT Distal convoluted
   tubule cells, CDIC collecting duct-intercalated cells, CDPC: collecting
   duct-principal cells, Pod podocyte, Fib fibroblasts. C Representative
   immunofluorescence image depicting co-staining of SLC25A21 with
   E-cadherin, Lotus tetragonolobus lectin (LTL), AQP2 in the human
   kidneys. Green presents tubular makers; red presents SLC25A21; blue
   presents DAPI. Scale bars, 50 µm. D Experimental schematic of
   Slc25a21-OE or normal control (NC) plasmids transfection into the TKPTS
   cells for 24 h, exposed by cisplatin (10 µM) for 24 h (upper).
   Representative western blotting for Slc25a21 and Slc25a21-tagged HA
   protein levels (down). E Representative flow cytometry images of
   cellular apoptosis in Slc25a21-OE and NC cells after cisplatin (10 µM)
   treatment for 24 h (left). Relative apoptotic cells (including
   apoptotic and even dead cells, right). n = 9, each group, and values
   are reported as mean ± SEM. F Representative western blotting for
   Slc25a21-tagged HA, BAX and C-Caspase 3 protein expression in
   Slc25a21-OE and NC cells after cisplatin (10 µM) treatment for 24 h.
   C-Caspase 3: Cleaved Caspase 3. G Representative western blotting for
   MLKL, RIPK1 and RIPK3 protein expression in Slc25a21-OE and NC cells
   after cisplatin (10 µM) treatment for 24 h. H Expression of
   inflammatory markers (Il-6 and Tnfα) in TKPTS cells, determined by
   RT-qPCR, in Slc25a21-OE and NC cells after cisplatin (10 µM) treatment
   for 24 h. n = 8–9, each group, and values are reported as mean ± SEM.
   In each case, data were presented as means ± SEM and statistical
   significance is assessed by One-way ANOVA analysis of variance of
   Tukey’s multiple comparisons test. * indicates P < 0.05; *** indicates
   P < 0.001; **** indicates P < 0.0001; NS indicates not significant. See
   also Fig. [141]S4.

   Using an in vitro AKI model of kidney proximal tubular epithelial cells
   (TKPTS), we showed a dose- and time-dependent reduction in Slc25a21
   expression in tubular epithelial cells after cisplatin treatment (Fig.
   [142]S4B, C). To further explore the role of Scl25a21 in tubular cells,
   we increased the expression of Slc25a21 in these cisplatin-induced
   TKPTS cells through transfection with Slc25a21 plasmids (Slc25a21-OE,
   Fig. [143]3D, Fig. [144]S4D). Rescue of Slc25a21 expression
   significantly increased tubular cell viability following cisplatin
   treatment (Fig. [145]S4E). Compared to normal control (NC) cells, we
   also observed a decreased proportion of apoptotic cells (Fig. [146]3E)
   and reduced expression of apoptosis markers (e.g. BAX and C-Caspase 3),
   as well as necroptosis markers (e.g. MLKL, RIPK1 and RIPK3) after
   cisplatin induction (Fig. [147]3F, G, Fig. [148]S4F, G). The mRNA
   expression of inflammatory markers, Il-6 and Tnfα, was also reduced
   upon Slc25a21 plasmids transfection (Fig. [149]3H). Taken together,
   these data show that tubular cells are a major cell type in the kidney
   regulated by Slc25a21 during AKI, and Slc25a21 directly modulates
   cellular apoptosis, necroptosis and inflammatory response following
   cisplatin-induced acute tubular injury.

Slc25a21 is important for preserving mitochondrial homeostasis during AKI

   Consistent with previous reports that identified Slc25a21 as a
   mitochondrial carrier in yeast and human cells [[150]23, [151]24], we
   showed the mitochondrial sub-cellular localization of Slc25a21 in the
   tubular cells (TKPTS) by Mitotracker (Fig. [152]4A). Cisplatin-treated
   tubular cells showed reduced Slc25a21 expression in the mitochondria
   (Fig. [153]S5A). We then investigated the potential mitochondrial
   function of Slc25a21 in tubular epithelial cell, as well as its role in
   AKI.

Fig. 4. Slc25a21 is important for preserving mitochondrial homeostasis during
AKI.

   [154]Fig. 4
   [155]Open in a new tab

   A Representative immunofluorescence image of co-staining of SLC25A21
   with Mitotracker revealed the mitochondrial localization of SLC25A21 in
   TKPTS cells. Green presents SLC25A21; red presents Mitotracker; blue
   presents DAPI. Scale bars, 20 µm. B The rate of oxygen consumption was
   assessed using the Seahorse XF-96 Extracellular Flux Analyzer in
   Slc25a21-OE and normal control (NC) cells after cisplatin (10 µM)
   treatment for 24 h (left). OLIGO oligomycin, FCCP Carbonyl cyanide
   4-(trifluoromethoxy)phenylhydrazone, ROT rotenone, AA antimycin A.
   Oxygen consumption rate for basal and maximal respiration was analyzed
   (right). n = 6, each group, and values are reported as mean ± SEM. C
   Relative ATP production in the kidneys of Slc25a21^AAV9 and NC^AAV9
   mice after cisplatin or saline addition. n = 6, each group, and values
   are reported as mean ± SEM. D Heatmap showing mRNA expression of the 13
   mitochondrially encoded genes in Slc25a21^AAV9 and NC^AAV9 mice after
   cisplatin or saline addition, determined by RT-qPCR (n = 10). Data are
   presented as Z-score. *indicates Slc25a21^AAV9 vs NC^AAV9, #indicates
   Slc25a21^AAV9+Cisplatin vs NC^AAV9+Cisplatin. E Representative western
   blotting for protein expression levels of enzymes involved in
   mitochondrial OXPHOS in the kidneys of Slc25a21^AAV9 and NC^AAV9 mice
   after cisplatin or saline addition. NDUFB8 NADH dehydrogenase
   (ubiquinone) 1 beta subcomplex subunit 8, ND1 NADH-ubiquinone
   oxidoreductase chain 1, SDHB Succinate dehydrogenase (ubiquinone)
   iron-sulfur subunit, CYTB Cytochrome b; UQCRC2 Cytochrome b-c1 complex
   subunit 2, COX1 Cytochrome c oxidase subunit 1, COX IV cytochrome c
   oxidase subunit 4 isoform 1, ATP5A1 ATP synthase subunit alpha, ATP6
   ATP synthase subunit a. F Representative western blotting for PGC1α and
   TFAM protein expression levels of enzymes associated with mitochondrial
   biogenesis in the kidneys of Slc25a21^AAV9 and NC^AAV9 mice after
   cisplatin or saline addition. G The mtDNA copy number in the kidneys of
   Slc25a21^AAV9 and NC^AAV9 mice after cisplatin or saline addition
   measured by qRT-PCR. n = 8–10, each group, and values are reported as
   mean ± SEM. H Representative western blotting for Porin and CypD
   protein expression levels of enzymes associated with mitochondrial
   integrity in the kidneys of Slc25a21^AAV9 and NC^AAV9 mice after
   cisplatin or saline addition. CypD: cyclophilin D. I Relative fold
   change of MitoSOX detected in Slc25a21-OE and NC cells after cisplatin
   (10 µM) treatment for 24 h. n = 3, each group, and values are reported
   as mean ± SEM. J Representative immunohistochemistry images of 8-OHdG
   expression in the kidneys of Slc25a21^AAV9 and NC^AAV9 mice after
   cisplatin or saline addition (left) and semi-quantitative IOD analysis
   of 8-OHdG expression (right). n = 7–9, each group, and values are
   reported as mean ± SEM. Scale bars, 50 µm. IOD integral optical
   density. In each case, data were presented as means ± SEM and
   statistical significance is assessed by One-way ANOVA analysis of
   variance of Tukey’s multiple comparisons test. * indicates P < 0.05; **
   indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001;
   # indicates P < 0.05; ## indicates P < 0.01; ### indicates P < 0.001;
   #### indicates P < 0.0001; NS indicates not significant. See also Fig.
   [156]S5.

   Maintaining Slc25a21 expression was associated with preserved
   mitochondrial respiratory function in Slc25a21-OE tubular cells upon
   cisplatin treatment, when compared with NC cells (Fig. [157]4B).
   AAV9-Slc25a21 administration in the kidney partially restored impaired
   ATP production in cisplatin-induced AKI kidneys (Fig. [158]4C).
   Together with the respiratory dysfunction during AKI, there was a
   decrease in the renal expression of 13 mitochondrial genes involved in
   the oxidative phosphorylation (OXPHOS) complex, impacting all OXPHOS
   subunits (Fig. [159]4D, E, Fig. [160]S5B). Following cisplatin
   treatment, maintaining Slc25a21 expression preserved the expression of
   these OXPHOS genes at both mRNA and protein levels (Fig. [161]4D, E,
   Fig. [162]S5B). Similarly, expression of these mitochondrial OXPHOS
   genes was restored in AKI kidney by high-pressure tail-vein injection
   of Slc25a21 plasmids (Fig. [163]S5C, D) and by transfection of Slc25a21
   plasmids in acutely injured TKPTS cell lines (Fig. [164]S5E).

   In addition to respiratory ATP synthesis, mitochondrial biogenesis and
   integrity are also important for mitochondrial homeostasis. Peroxisome
   proliferator-activated receptor gamma co-activator-1 alpha (PGC1α) and
   mitochondrial transcription factor A (TFAM) are two key proteins
   involved in mitochondrial biogenesis pathway [[165]13, [166]32]. The
   expression of PGC1α and TFAM in the kidney is significantly suppressed
   by cisplatin injection, and rescued by Slc25a21 expression induced with
   renal AAV9 administration (Fig. [167]4F, Fig. [168]S5F). The reduction
   in mitochondrial DNA (mtDNA) copy number in AKI kidneys was also
   ameliorated by maintaining Slc25a21 expression (Fig. [169]4G). Porin
   and cyclophilin D (CypD) are components of the mitochondrial outer
   membrane and mediators of the mitochondrial permeability transition
   pore, which play a significant role in maintaining mitochondrial
   integrity. The expression levels of Porin and CypD in the kidneys were
   reduced following cisplatin-induced AKI, which was rescued by
   AAV9-Slc25a21 injection (Fig. [170]4H, Fig. [171]S5G).

   Additionally, the disturbance of mitochondrial homeostasis was
   accompanied by the release of reactive oxygen species (ROS) in AKI
   (Fig. [172]4I-J). MitoSOX staining showed that mitochondrial ROS levels
   in TKPTS cells were significantly increased following cisplatin
   treatment, and in vitro rescue of Slc25a21 suppressed oxidative stress
   in these tubular cells (Fig. [173]4I). The levels of 8-OHdG, a
   biomarker of oxidative DNA damage, were increases in AKI kidneys, and
   alleviated by AAV9-Slc25a21 intervention (Fig. [174]4J).

Slc25a21 facilitates mitochondrial 2-oxoadipate transport and metabolism

   Given that one of the main functions of Slc25a21 is to transport
   2-oxodicarboxylate across inner mitochondrial membrane into
   mitochondria [[175]23], we speculate that Slc25a21 might regulate the
   progression of AKI by mediating the influx of 2-oxoadipate into tubular
   cells. To test this hypothesis, we first assessed the concentration of
   2-oxoadipate in AKI kidneys. Cisplatin treatment significantly
   increased the whole levels of 2-oxoadipate in AKI kidneys, which was
   suppressed by the rescue of Slc25a21 through AAV9 administration (Fig.
   [176]5A). Importantly, high levels of 2-oxoadipate in the kidney
   positively correlated with AKI-induced kidney dysfunction (Fig.
   [177]5B) and pro-inflammatory response (Fig. [178]5C). Moreover, in
   vitro exposure to moderate levels of 2-oxoadipate led to increased cell
   viability in TKPTS cells (Fig. [179]5D), whereas high 2-oxoadipate
   concentration (500 nM) resulted in the increased cellular apoptosis and
   decreased cell viability (Fig. [180]5D, E). Notably, sustaining
   Slc25a21 expression significantly attenuated apoptosis in TKPTS cells
   induced by 2-oxoadipate at high concentration. Specifically, increased
   2-oxoadipate levels in cisplatin-induced kidneys were detected mainly
   in the cytoplasm of tubular cells, and associated with a lower
   mitochondrial to cytoplasmic ratio (Fig. [181]5F). However, maintaining
   the level of Slc25a21 by AAV9 introduction reversed the mitochondrial
   to cytoplasmic ratio of 2-oxoadipate (Fig. [182]5F). These findings
   demonstrate that decreased Slc25a21 expression in AKI hindered the
   mitochondrial transport of 2-oxoadipate, leading to its cytoplasmic
   accumulation and resulting in acute kidney injury.

Fig. 5. Slc25a21 facilitates mitochondrial 2-oxoadipate transport and
metabolism.

   [183]Fig. 5
   [184]Open in a new tab

   A Assessment of 2-oxoadipate level in the kidneys of Slc25a21^AAV9 and
   NC^AAV9 mice after cisplatin or saline addition, determined by
   HPLC-MSMS. n = 6, each group, and values are reported as mean ± SEM. B
   Pearson correlation analysis between 2-oxoadipate intensity and the
   concentrations of SCr as well as BUN in the kidneys of Slc25a21^AAV9
   and NC^AAV9 mice after cisplatin addition (n = 12). C Pearson
   correlation analysis between 2-oxoadipate intensity and the
   concentrations of TNFα as well as IL-18 in the kidneys of Slc25a21^AAV9
   and NC^AAV9 mice after cisplatin addition (n = 12). D Relative cell
   viability detected by TKPTS cells exposed to a different gradient of
   2-oxoadipate concentrations for 24 h. n = 9–12, each group, and values
   are reported as mean ± SEM. * indicates 2-oxoadipate-treated TKPTS
   cells vs normal control cells. E Representative flow cytometry images
   of cellular apoptosis measured in Slc25a21-OE and normal control (NC)
   cells after 2-oxoadipate (500 nM) addition for 24 h (left) and relative
   apoptotic cells to control (including apoptotic and even dead cells,
   right). n = 9, each group, and values are reported as mean ± SEM. F The
   intensity of 2-oxoadipate in the cytoplasm (left) and the ratio of
   2-oxoadipate between the mitochondria and the cytoplasm in
   Slc25a21^AAV9 and NC^AAV9 mice after cisplatin or saline addition,
   determined by HPIC-MSMS (right). n = 3, each group, and values are
   reported as mean ± SEM. G, H The expression of enzymes related to
   2-oxoadipate metabolism in the kidneys of Slc25a21^AAV9 and NC^AAV9
   mice after cisplatin or saline addition. G mRNA expression changes were
   determined by RT-qPCR. n = 8–10, each group, and values are reported as
   mean ± SEM. Dhtkd1: Dehydrogenase E1 and transketolase domain
   containing 1; Gcdh: Glutaryl-CoA dehydrogenase; Echs1: Enoyl-CoA
   hydratase, short chain 1; Hadh: Hydroxyacyl-CoA dehydrogenase; Acaa2:
   Acetyl-CoA Acyltransferase 2. (H) Representative western blotting for
   representative protein (ACAA2 and DHTKD1) levels. I The levels of
   metabolites (crotonyl-CoA and acetyl-CoA) from 2-oxoadipate metabolism
   were detected by HPLC-MSMS in the kidneys of Slc25a21^AAV9 and NC^AAV9
   mice after cisplatin or saline addition. n = 6, each group, and values
   are reported as mean ± SEM. J Schematic of 2-oxoadipate conservation
   into acetyl-CoA summarizing the metabolic changes observed
   Slc25a21^AAV9 following cisplatin administration compared to normal
   controls in AKI. K Heatmap visualizing differentially expressed
   metabolites relevant to 2-oxoadipate in Slc25a21^AAV9 and NC^AAV9 mice
   after cisplatin or saline addition, determined by HPLC-MSMS (left,
   n = 6). Data are presented as Z-score. *indicates Slc25a21^AAV9 vs
   NC^AAV9, #indicates Slc25a21^AAV9+Cisplatin vs NC^AAV9+Cisplatin.
   Schematic of tryptophan metabolism summarizing the metabolic changes
   observed Slc25a21^AAV9 following cisplatin administration compared to
   normal controls in AKI (right). L Pearson correlation analysis between
   metabolites relevant to 2-oxoadipate (Kynurenic acid,
   3-Hydroxykynurenine, Xanthurenic acid, Quinolinic acid and Picolinic
   acid) and the concentrations of SCr, BUN, TNFα as well as IL-18 in
   Slc25a21^AAV9 and NC^AAV9 mice after cisplatin addition (n = 12).
   *indicates P values of Pearson correlation. In each case, data were
   presented as means ± SEM and statistical significance is assessed by
   One-way ANOVA analysis of variance of Tukey’s multiple comparisons
   test. * indicates P < 0.05; ** indicates P < 0.01; *** indicates
   P < 0.001; **** indicates P < 0.0001; # indicates P < 0.05; ##
   indicates P < 0.01; ### indicates P < 0.001; #### indicates P < 0.0001;
   NS indicates not significant. See also Fig. [185]S6.

   2-oxoadipate can be also converted into acetyl-CoA after entering the
   mitochondrial matrix [[186]23]. During cisplatin-induced AKI, the
   expression of representative enzymes responsible for converting
   2-oxoadipate to acetyl-CoA was significantly reduced (Fig. [187]5G),
   which would be consistent with the reduction in mitochondrial transport
   of 2-oxoadipate under these conditions (Fig. [188]5A, F). We observed
   that the levels of these enzymes were increased following Slc25a21
   administration via AAV9, such as DHTKD1 and ACAA2 (Fig. [189]5G, H,
   Fig. [190]S6A). Consequently, AAV9-Slc25a21 administration also
   increased the levels of acetyl-CoA in AKI kidneys compared to AKI
   controls, together with crotonyl-CoA, another intermediary metabolite
   derived from the conversion of 2-oxoadipate into acetyl-CoA (Fig.
   [191]5I). The regulation of Slc25a21 on 2-oxoadipate conversation was
   confirmed in a separate in vivo AKI kidney model (following
   high-pressure tail-vein injection of Slc25a21 plasmids), and by in
   vitro acutely injured TKPTS cells (Fig. [192]S6B–D). Thus, these data
   would be consistent with a mechanism where sustaining Slc25a21
   expression rescue the metabolic pathway of 2-oxoadipate following its
   mitochondrial transport (Fig. [193]5J).

   We also investigated potential differences in the metabolite influx
   between AAV9-Slc25a21 and AAV9-NC treated-AKI kidneys, and found that
   several metabolites involved in tryptophan metabolism were regulated by
   Slc25a21 (Fig. [194]5K). AKI triggers the upregulation of these
   metabolites, which was reversed by Slc25a21. The perturbation of these
   metabolites was shown to be associated with renal dysfunction and
   inflammatory response in AKI progression (Fig. [195]5L). Since
   2-oxoadipate is an intermediate in the metabolism of tryptophan
   [[196]23, [197]25], these findings suggest a role of Slc25a21 in
   regulating the metabolic pathway that contributes to 2-oxoadipate
   production.

TCA cycle enriched in the metabolism regulated by Slc25a21 in AKI

   To further understand the regulation of Slc25a2 in metabolic profile
   during AKI, we profiled the global metabolomic changes associated with
   Slc25a21 and detected 497 metabolites in cisplatin-induced AKI kidneys
   (Fig. [198]6A). Principal component analysis (PCA) identified 100
   metabolites with variable importance in projection (VIP) scores greater
   than 1, which can separate Slc25a21 AAV9-treated from control AKI
   kidneys (Fig. [199]6A and Table [200]S3). Kyoto Encyclopedia of Genes
   and Genomes (KEGG) pathway enrichment analysis revealed that these
   metabolites were significantly associated with energy metabolism
   pathways, such as the tricarboxylic acid cycle (TCA cycle) and pentose
   phosphate pathway etc. (Fig. [201]6B).

Fig. 6. TCA cycle enriched in the metabolism regulated by Slc25a21 in AKI.

   [202]Fig. 6
   [203]Open in a new tab

   A Principal components analysis (PCA) score depicting metabolite
   profiles in Slc25a21^AAV9 and NC^AAV9 mice after cisplatin or saline
   administration (n = 6). B KEGG enrichment analysis of significantly
   different metabolites (VIP > 1) between Slc25a21^AAV9 and NC^AAV9 mice
   following cisplatin addition analyzed by MetaboAnalyst 5.0. The P value
   of displayed pathway in the figure is less than 0.05 and calculated
   using MetaboAnalyst 5.0. C Heatmap showing differentially expressed
   metabolites in the TCA cycle in Slc25a21^AAV9 and NC^AAV9 mice
   following cisplatin or saline addition (n = 6), determined by
   HPLC-MSMS. Data are presented as Z-score. *indicates Slc25a21^AAV9 vs
   NC^AAV9, #indicates Slc25a21^AAV9+Cisplatin vs NC^AAV9+Cisplatin. D, E
   The expression of enzymes associated with the TCA cycle in the kidneys
   of Slc25a21^AAV9 and NC^AAV9 mice following cisplatin or saline
   addition. D mRNA expression changes were determined by RT-qPCR.
   n = 9–10, each group, and values are reported as mean ± SEM. Cs:
   Citrate synthase; Aco2: Aconitase 2; Idh2: Isocitrate dehydrogenase;
   Ogdh: Oxoglutarate dehydrogenase; Sucla2: Succinate-CoA ligase
   ADP-forming subunit β; Fh1: Fumarate hydratase 1; Mdh2: Malate
   dehydrogenase 2; Sdha: Succinate dehydrogenase flavoprotein subunit. E
   Representative western blotting for representative protein (ACO2, OGDH,
   SUCLG2 and CS) levels. F The levels of citrate, fumarate and malate in
   the cytoplasm and the ratio of citrate, fumarate and malate between the
   mitochondria and the cytoplasm in Slc25a21^AAV9 and NC^AAV9 mice
   following cisplatin or saline addition, determined by HPIC-MSMS. n = 3,
   each group, and values are reported as mean ± SEM. Schematic of TCA
   cycle summarizing the metabolic changes observed Slc25a21^AAV9
   following cisplatin treatment compared to normal controls in AKI
   (middle). G Schematic of proposed mechanism of maintaining Slc25a21
   mitigated renal tubular injury induced by cisplatin. Preservation of
   Slc25a21 expression facilitated the TCA cycle by enhancing 2-oxoadipate
   transportation and metabolism, improved mitochondrial homeostasis along
   with elevated ATP synthesis and reduced ROS production, as well as
   supported mitochondrial biogenesis and integrity, thereby ameliorating
   cellular apoptosis, necroptosis and inflammatory response. Red
   indicates up-regulation; blue indicates down-regulation. This figure
   was created by BioRender website. In each case, data were presented as
   means ± SEM and statistical significance is assessed by One-way ANOVA
   analysis of variance of Tukey’s multiple comparisons test. * indicates
   P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; ****
   indicates P < 0.0001; # indicates P < 0.05; ## indicates P < 0.01; ###
   indicates P < 0.001; #### indicates P < 0.0001; NS indicates not
   significant. See also Fig. [204]S7.

   We focused on the TCA cycle, which occurs in the mitochondria and
   provides energy in aerobic conditions together with acetyl-CoA
   [[205]33]. The TCA cycle is also the major pathway for interconversion
   of 4- and 6-carbon compounds in the cell, including citrate, fumarate
   and oxaloacetate [[206]34]. In keeping with the observed changes in TCA
   cycle metabolites (Fig. [207]6C), the gene expression of representative
   enzymes participating in the TCA cycle in AKI kidney was increased in
   Slc25a21^AAV9 compared with controls (NC^AAV9) (Fig. [208]6D, E, Fig.
   [209]S7A). Particularly, a significant increase in the levels of
   representative metabolites of TCA cycles, citrate, fumarate and malate,
   was observed in the cytoplasm during AKI; however, maintaining Slc25a21
   expression increased the metabolites ratio of mitochondria to cytoplasm
   (Fig. [210]6F). The regulation of Slc25a21 on representative enzymes
   involved in TCA cycle was confirmed in a separate in vivo AKI kidney
   model following high-pressure tail-vein injection of Slc25a21 plasmids,
   and in vitro using acutely injured TKPTS cells (Fig. [211]S7B–D).

   Here, we first demonstrated the localization of Slc25a21 in renal
   tubular mitochondria. We then showed a reduction in Slc25a21 expression
   in AKI-affected kidneys in both humans and mice, which results in
   compromised mitochondrial transport of 2-oxoadipate. The resultant
   cytoplasmic accumulation of 2-oxoadipate and relevant metabolic changes
   will directly or indirectly lead to the disturbance of mitochondrial
   homeostasis, mainly presenting decreased respiratory ATP generation, as
   well as compromised mitochondrial biogenesis, integrity, and an
   increase in ROS. Consequently, these alterations trigger tubular
   apoptosis, necroptosis as well as inflammatory responses, and
   ultimately, kidney dysfunction of AKI. Importantly, maintaining
   Slc25a21 expression preserves mitochondrial homeostasis and metabolic
   balance in tubular cells, which protects against the progression of AKI
   (Fig. [212]6G).

Discussion

   In this study, we report the association of Slc25a21 in renal tubular
   cells with the progression of cisplatin-induced AKI. Cisplatin
   suppresses Slc25a21 expression in tubular cells, leading to a
   disturbance in mitochondrial homeostasis and subsequent cell apoptosis,
   necroptosis as well as inflammation, contributing to AKI progression.
   Mechanistically, the reduction of Slc25a21 hampers the mitochondrial
   transport of 2-oxoadipate, which results in metabolic disturbance and
   mitochondrial energy alterations. We propose that maintaining Slc25a21
   could be a new approach for metabolic intervention, aiming to rescue
   mitochondrial homeostasis and protect against tubular injury, and then
   prevent the progression of AKI.

   Mitochondrial homeostasis refers to the balance and regulation of
   mitochondrial number, function, and quality within cells [[213]35]. The
   kidney is one of the most metabolically active organs in the human body
   and requires substantial mitochondrial homeostasis to perform its
   proper functions [[214]13]. Several kidney diseases are reported to be
   associated with disruptions in mitochondrial homeostasis [[215]36,
   [216]37], with mitochondrial dysfunction being a major hallmark that
   affects the production of adenosine triphosphate (ATP) and leads to
   renal dysfunction [[217]38]. Specifically, the proximal tubules are
   highly sensitive to energy fluctuations, as the first exhibiting signs
   of injury compared to other renal cell types [[218]39]. The disruption
   of mitochondrial homeostasis is also a critical component in the
   pathogenesis of AKI [[219]32, [220]40, [221]41], presenting damaged
   OXPHOS complex and mitochondrial respiration, impaired mitochondrial
   biogenesis and integrity, a deficit in cellular energy production and
   leading to tubular cell apoptosis and necroptosis [[222]42–[223]44].
   Therapeutically, the modulation of homeostatic mechanisms was reported
   to protect against mitochondrial dysfunction in AKI [[224]45–[225]50].
   Formoterol, as a potent, highly specific, and long-acting β2-adrenergic
   agonist, is shown to induce renal mitochondrial biogenesis. After
   injury has occurred, Formoterol works by restoring mitochondrial
   function and accelerating the recovery of renal function
   [[226]51–[227]53]. Mitochondria-targeted peptide (MTP) was designed to
   target the inner mitochondrial membrane, and reduce attenuated acute
   mitochondrial dysfunction and cell apoptosis in the kidneys [[228]54].
   Here, we provide proof of principle that maintaining Slc25a21 can be
   considered as a potential therapeutic approach to ameliorate AKI by
   restoring mitochondrial homeostasis. This approach counteracts
   cisplatin-induced mitochondrial dysfunction, improves cell respiration
   and ATP synthesis in tubular cells, and supports mitochondrial
   biogenesis and structural integrity.

   Mitochondrial homeostasis is widely recognized as involving the
   metabolic cascades of energy substrates, ensuring the availability of
   sufficient energy (ATP) [[229]55–[230]57]. Aerobic respiration is the
   primary method of ATP production in tubular cells, starting with the
   conversion of glucose into pyruvate through glycolysis. Pyruvate is
   then transformed into acetyl-CoA, a crucial substrate for the TCA
   cycle, which facilitates ATP synthesis [[231]58]. In addition to
   glucose, fatty acids are also major substrates for energy metabolism.
   The process of breaking down fatty acids via β-oxidation maximizes the
   production of acetyl-CoA through further oxidation [[232]59]. The
   chemical reaction that initiates each “turn” of the TCA cycle is the
   condensation of the metabolite oxaloacetate (OAA) with acetyl-CoA to
   generate citrate. Subsequent reactions oxidize citrate to produce ATP
   molecules, depending on the specific enzymes involved at each step
   [[233]33].

   Acetate is a key metabolite for normal kidney development. Reductions
   in acetyl-CoA metabolism hinder normal kidney development, whereas
   supplementation stimulates it [[234]60]. AKI induces progressive
   pyruvate depletion in the kidneys, while exogenous pyruvate
   administration leads to modest ATP increases and offer protection
   against kidney injury [[235]61]. In addition, modulating the pyruvate
   carriers and decreasing mitochondrial pyruvate uptake is a central
   adaptive response following AKI, which enhances alternative metabolic
   pathways such as the pentose phosphate pathway and attenuates AKI
   severity [[236]62]. During AKI, the intermediate metabolites and key
   enzymes related to citrate metabolism in the TCA cycle are consequently
   impacted, coupled with oxidative phosphorylation-driven ATP production.
   Slc25a21 is also an energy metabolic carrier. It transports
   2-oxoadipate produced by the catabolism of cytosolic lysine (as well as
   tryptophan and hydroxylysine) into the mitochondrial matrix, where
   2-oxoadipate is decarboxylated and further metabolized to provide a
   source of acetyl-CoA [[237]23, [238]63]. Similar to other pathways
   modulating acetyl-CoA, sustaining Slc25a21 further enhanced the TCA
   cycle, presenting increased the expression of key enzymes, such as
   Citrate synthase (Cs) and Oxoglutarate dehydrogenase (Ogdh), and
   increased levels of citrate and malate in the mitochondria. Although
   the detailed functional and regulatory interplay between cytosolic
   lysine and the metabolism of glucose and fatty acids in coordinating
   energy supply is not fully understood [[239]64], key metabolites such
   as acetyl-CoA, pyruvate, and 2-oxoadipate have proven to be crucial in
   maintaining mitochondrial homeostasis.

   Furthermore, the Slc25a21-mediated mitochondrial transport of
   2-oxoadipate is linked to tryptophan metabolic flux, as 2-oxoadipate is
   product of tryptophan metabolism. The maintenance of Slc25a21 leads to
   a strong decrease in the levels of the several metabolites produced by
   tryptophan metabolism, including Kynurenic acid, 3-Hydroxykynurenine,
   and Picolinic acid. However, dysregulation of tryptophan metabolism and
   elevated levels of these intermediates, have been observed in plasma
   and kidney tissues during AKI [[240]65–[241]71]. These metabolites have
   also been implicated in the inflammatory response in kidneys and
   further aggravated kidney dysfunction [[242]72–[243]76]. In addition,
   overproduction of ROS, the by-products of abnormal OXPHOS, is reported
   contribute to renal inflammation [[244]77, [245]78]. Maintaining
   Slc25a21 restores mitochondrial homeostasis, and simultaneously reduces
   excessive ROS production. This reduction is beneficial for alleviating
   inflammatory responses in the injured kidneys. Thus, our data suggest a
   role of Slc25a21 in modulating inflammation during cisplatin-induced
   AKI.

   Some limitations of our study should be highlighted. With respect to
   tubular cells as the main target of cisplatin-induced kidney injury and
   Slc25a21 being predominantly enriched in proximal tubular cells, in our
   study we have focused on renal tubular cells. However, our model of
   AAV9 system used multi-point in situ injection in the renal cortex to
   maintain the expression of Slc25a21, and non-specific promotor may lead
   to expression of exogenous Slc25a21 in multiple renal cells. Although
   the exogenous Slc25a21 following AAV9 application was mainly expressed
   in the PT cells (Figure [246]S4A), we could not exclude the role of
   Slc25a21 in other cells which also contribute to AKI progression.
   Moreover, the mechanism by which cisplatin modulates Slc25a21
   expression in renal tubular cells is not explored in this study. Liu et
   al. [[247]79]. reported that lncRNA SLC25A21-AS1 regulates the
   stability of SLC25A21 mRNA in the cytoplasm. Additionally, the
   transcriptional regulation of another Slc25 family member, SLC25A13,
   was shown to involve USF1 as a main factor and FOXA2 as an activator in
   liver cells [[248]80]. Understanding the genetic and epigenetic
   regulation of Slc25a21 expression in kidneys might provide more direct
   evidence of Slc25a21 as a target for cisplatin-induced AKI.

   In conclusion, our study suggests novel therapeutic strategies for AKI
   by maintaining Slc25a21 expression to restore mitochondrial metabolic
   homeostasis. This strategy might effectively counteract tubular
   apoptosis, necroptosis and decrease inflammatory response, thereby
   mitigating AKI progression. Importantly, Slc25a21 ameliorates metabolic
   energy deficits in AKI by mediating the mitochondrial transport of
   2-oxoadipate, supporting the concept that modulation of fuel
   metabolites is crucial for maintaining mitochondrial homeostasis in
   healthy kidney. Thus, the findings presented in our study provide new
   insights into mitochondrial metabolism in AKI, offering a promising
   perspective for interventions involving fuel metabolite supplementation
   and transportation.

Supplementary information

   [249]Supplemental Material Table S1^ (9.8KB, xlsx)
   [250]Supplemental Material Table S2^ (12.2KB, xlsx)
   [251]Supplemental Material Table S3^ (28.6KB, xlsx)
   [252]41419_2024_7231_MOESM4_ESM.docx^ (15.3MB, docx)

   Supplemental Material-supplementary information
   [253]41419_2024_7231_MOESM5_ESM.pdf^ (37MB, pdf)

   Supplemental Material Original western blots

Acknowledgements