Malate assay kit

The knee joints, excluding the excessive surrounding tissues, were collected into individual Eppendorf tubes containing 1 mL of sterile PBS, followed by homogenization with TissueLyser II (Qiagen, Hilden, Germany). Afterward, the samples were centrifuged (11,000 rpm for 10 min at 4°C), and the supernatant was processed following metabolite extraction and sample preparation for assessment of metabolite expression. Lactate, fumarate, and malate were measured by using lactate assay kit (catalog no. MAK064; Sigma, Germany), fumarate assay kit (catalog no. MAK060, Sigma, Germany), and malate assay kit (catalog no. MAK067, Sigma, Germany) according to the manufacturer’s instructions.

ADSCs (2 × 10⁶ in quantity) transfected with shNC or shBcat1#1 were used for intracellular metabolite concentration measurement. Each metabolite level was determined using the following assay kits: BCAA detection kit (BioVision, K564‐100, α‐ketoglutarate assay kit (Sigma‐Aldrich, MAK054), glutamate assay kit (Sigma‐Aldrich, MAK004), citrate assay kit (Sigma‐Aldrich, MAK057), fumarate assay kit (Sigma‐Aldrich, MAK060), succinate assay kit (Sigma‐Aldrich, MAK184), and malate assay kit (Sigma‐Aldrich, MAK196). BCKA concentration was measured by high‐performance liquid chromatography (HPLC) as previously described.^[42] Intracellular metabolite levels were normalized to the ADSCs transfected with shNC.

JB58 and JB485 were cultured under AKI conditions for 3.5h when the culture optical density at 600 nm was recorded and a one mL culture aliquot was collected. The cells were separated from the supernatant by centrifugation and the supernatant and cell pellet retained. Cell lysates were generated by resuspending the cell pellet in one mL of ddH₂O before subjecting the cells to three freeze-thaw cycles followed by sonication. Aliquots of the culture supernatant and cell lysates were then assayed for malate using the Malate Assay Kit (Sigma) according to the manufacturer’s directions and normalized by optical density. The reported results are the means ± standard deviation from six biological replicates, with each biological replicate generated from two technical replicates.