Blood specimens

Patients were recruited from the First Hospital of Jilin University, China. All clinical records were obtained with the approval of the Ethics Committee. Clinical records were divided into three groups; 69 cases with ci0, 63 cases with ci1, and 19 cases with ci2/3. Targeted lipidomic detection used blood samples from seven patients with ci0, six patients with ci1, and one patient with ci2. There was a partial overlap in the above samples for different projects.

Rat kidney transplantation model

Sprague–Dawley rats (180-200 g) were used as donors and receptors. Prior to operation, anesthesia was administered to the mice using isoflurane. For the donor, heparinized saline (at a concentration of 125 U/mL) was injected, and the blood supply was halted using styptic clips. The left renal vein was removed before perfusion. The left renal vein, ureter, and artery were ligated after perfusion was complete. Heparinized saline (0 ~ 4 °C) was used as a preserving fluid for the separated left kidney. For the recipient, the left kidney, renal artery, renal vein, and ureter were separated and cut off. The renal artery, vein, and ureter were then anastomosed between the donor and recipient before the blood supply was restored. Bladder anastomoses were created to avoid ureteral obstruction, which may lead to unsuccessful ureter anastomosis. For postoperative management, 5 mL saline and ketoprofen (5 mg/kg) were injected into the abdominal cavity before the abdomen was closed. After renal transplantation, penicillin was administered for 3 days and cyclosporin A for 1 week. The animals were included in the study if they underwent successful renal transplantation, defined by improved blood supply of renal veins and arteries.

Immunofluorescence (IF) staining

The sections were dewaxed and washed with water conventionally. Thermal antigen retrieval with citrate for 5 min at 95 °C was performed. Then 5% BSA was used to block the sections for 30 min before being incubated with the primary antibodies, including antibodies against acyl-CoA oxidase 1 (ACOX1) (DF12046, Affinity Biosciences, Ohio, USA) and α-smooth muscle actin (α-SMA) (ab7817, Abcam, Cambridge, UK), in PBS containing 1% BSA, and the sections were incubated overnight at 4 °C. The sections were washed thrice before being incubated with the different fluorescent dye–conjugated secondary antibodies for 1 h. After washing the sections three times with PBS, Lotus Tetragonolobus Lectin (LTL) (FL-1321–2, Vector, California, US) was used for incubation for 30 min. The sections were then washed thrice before the cell nuclei were stained with DAPI for 10 min. After washing them again thrice, the sections were sealed with antifade mounting medium (P0128, Beyotime, Shanghai, China). The sections with stained tissues were mounted onto a confocal microscope and images were captured.

Hematoxylin & eosin (HE)

Sections were dewaxed twice with xylene for 30 min, then dipped into a concentration gradient of ethyl alcohol for 5 min. They were then washed with distilled water and subjected to 5 min of hematoxylin staining, differentiated with acid alcohol, blued with ammonia water, and subjected to a 10 min eosin staining procedure. The sections were dehydrated with a gradient of 70%, 80%, 95% I, 95% II, 95% III, 100% I, and 100% II ethyl alcohol. The samples were immersed twice in xylene for a duration of 10 min before they were sealed with neutral resins.

Masson

The sections underwent a range of procedures, including dewaxing, staining with hematoxylin and ponceau-acid fuchsin solution, washing with 2% glacial acetic acid, differentiation with 1% phosphomolybdic acid, staining with aniline blue, dehydrating, and sealed with neutral resin.

Targeted lipidomics

A standard solution was prepared. Fatty acid calibrators at concentrations of 10–40000 ng/mL were prepared by mixing the stock solutions of individual fatty acids with a fatty acid-free matrix. The samples were resuspended and homogenized in liquid nitrogen with 300 µL of a mixture of isopropanol and acetonitrile (1:1) containing internal standards, which were mixed. The samples were subsequently underwent centrifugation at 12,000 g for a duration of 10 min. The 2 µL supernatant was extracted and injected into the LC–MS/MS system. The fatty acids were quantified using a UHPLC-MS/MS system. The concentration series of the standard solutions were detected using LC–MS. The ratio of standard to internal standard concentrations was determined using the x-axis, whereas the y-axis, denoting the peak area of the standard to internal standard, was designated as the ordinate to assess the linearity of the standard solution. Each metabolite was allowed to reach a correlation coefficient (r) > 0.99. The limit of quantification was determined using the signal-to-noise ratio method.

Immunohistochemistry (IHC)

The sections were dewaxed and washed with water conventionally. Thermal antigen retrieval with citrate for 5 min at 95 °C was performed. An ultra-sensitive ™ SP IHC kit procured from MXB biotechnologies (KIT-7710, Fujian, China) was used for IHC detection. Tissues then underwent the following treatment steps: incubation with Reagent I for 15 min, washed thrice with PBS for 5 min, and incubation with Reagent II for 15 min. The tissues were then incubated with dilute specific antibodies, including Fibrillin 1 1:200 (AF0429, Affinity Biosciences, Ohio, USA), MMP7 1:4000 (AF0218, Affinity Biosciences, Ohio, USA), and collagen IV 1:400 (AF0510, Affinity Biosciences, Ohio, USA), at 4 °C overnight. The next day, the tissues were subjected to three washes with PBS and were then incubated with Reagent III. Subsequently, the tissues were washed again with PBS, incubated with Reagent IV, and then washed three more times with PBS. Subsequently, 3,3-diaminobenzidine (DAB) staining was performed until tissues were stained with brown or red, then terminated with water. In this step, one antibody performed same dyeing time. Cell nuclei were visualized by staining the sections with hematoxylin. To retain the sample, the sections were dehydrated and preserved using neutral resin.

Total cholesterol (TC) and triacylglycerol (TG) detection in rat blood

Serum was collected from rats with renal transplantation. TC (A111-1–1) and TG (A110-1–1) detection kits were obtained from Nanjing Jiancheng Bioengineering Institute, located in Nanjing, China. Serum (2.5 μL) or standard substance (TG 2.39 mmol/L, TC 6.15 mmol/L) was mixed with working solution in a 96-well plate and subsequently incubated at 37 °C for 10 min. Absorbance was measured at a wavelength of 500 nm. TC and TG concentrations were counted using the subsequent equations: \(\mathrm\;(\mathrm/\mathrm L)\:=\:(\mathrm-\mathrm/\mathrm-\mathrm)\:\times\:2.39\;\mathrm/\mathrm L;\;\mathrm\;(\mathrm/\mathrm L)\:=\:(\mathrm-\mathrm/\mathrm-\mathrm)\:\times\:6.15\;\mathrm/\mathrm L\).

Statistical analysis

GraphPad Prism 9, developed by GraphPad Software in California (USA), was utilized to perform statistical analyses in this study. The data have been provided as means ± standard errors of the means. Statistical significance was determined using Student’s t-tests, where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 signify statistical significance.