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Lipid extractions were performed by slightly modifying the Matyash method. Thereafter, 75% ice-cold methanol (400 µL) containing 0.1% butylated hydroxytoluene was added in the kidney tissues and podocytes. After homogenizing the kidney tissues, podocytes were removed using stainless steel beads and TissueLyser (QIAGEN, Germany’s Helden). After adding 1 mL of methyl-tert-butyl ether with 0.1% butylated hydroxytoluene, the samples were shaken at 20 to 23°C for 1 h. In total, 250 µL of water was added and vortexed for 10 min. Thereafter, phase separation was performed via centrifugation at 14,000g for 15 min at 4°C. For targeted lipidomics analysis, the upper (220 µL) and lower (110 µL) phases were pooled and dried using N2 purge. , All lipid extractions using kidney tissues and epithelial and podocyte cells were performed by slightly modifying the Matyash method. Thereafter, 75% ice-cold methanol (400 µL) containing 0.1% butylated hydroxytoluene was added in the kidney tissues, epithelial cells, and podocytes. After homogenizing the kidney tissues, epithelial cells and podocytes were removed using stainless steel beads and TissueLyser (QIAGEN, Germany’s Helden). After adding 1 mL of methyl-tert-butyl ether with 0.1% butylated hydroxytoluene, the samples were shaken at 20 to 23°C for 1 h. In total, 250 µL of water was added and vortexed for 10 min. Thereafter, phase separation was performed via centrifugation at 14,000g for 15 min at 4°C. For targeted lipidomics analysis, the upper (550 µL) and lower (275 µL) phases were pooled and dried using N2 purge. , To analyze free fatty acid and cholesterol levels, lipid extractions were performed by slightly modifying the Matyash method. Thereafter, 75% ice-cold methanol (400 µL) containing 0.1% butylated hydroxytoluene was added in the kidney tissues, epithelial cell and podocytes. After homogenizing the kidney tissues, epithelial cell and podocytes were removed using stainless steel beads and TissueLyser (QIAGEN, Germany’s Helden). After adding 1 mL of methyl-tert-butyl ether with 0.1% butylated hydroxytoluene, the samples were shaken at 20 to 23°C for 1 h. In total, 250 µL of water was added and vortexed for 10 min. Thereafter, phase separation was performed via centrifugation at 14,000g for 15 min at 4°C. For targeted lipidomics analysis, the upper (110 μL) and lower (55 μL) phases were pooled and dried using N2 purge.

And then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS, LC-MS 8060, Shimadzu, Kyoto, Japan) with a Kinetex C18 column (100 x 2.1mm i.d., 2.6 μm particle size; Phenomenex, Torrance, CA, USA). Mobile phase A and B consisted of 10 mM ammonium acetate in water/methanol (1/9, v/v) and 10 mM ammonium acetate in methanol/isopropanol (1/1, v/v), respectively. The gradient elution condition was as follows: 30% B (0 min), 95% B (5 min), 95% B (15 min), 30% B (15.1 min) and 30% B (20 min). The sample injection volume was 5 μL and the flow rate was maintained at 0.2 mL/min. , All lipids were separated by Kinetex C18 columnn (100 x 2.1mm i.d., 2.6 μm particle size; Phenomenex, Torrance, CA, USA). The mobile phase consisted of 10 mM ammonium acetate in water/methanol (1/9, v/v) (A) and 10 mM ammonium acetate in methanol/isopropanol (1/1, v/v) (B) and the flow rate was 200 µL/min under gradient elution condition was set as follows: the gradient was started with 30% B and increased to 95% in 15 min and maintained with 95% for 5 min and decreased 30% for 5 min. Total analysis time was 25 minutes. , The derivatized samples were evaporated again, and then reconstitution using n-hexane which gas chromatography grade. And then analyzed by comprising a GC-MS(QP2010 Ultra Shimadzu, Japan) comprising an AOC-20i auto-sampler and gas-chromatography interfaced to a mass spectrometry. Rxi-5Sil MS Column (30 m x 0.25 mm, 0.25 ) was used to separate the samples and column flow was 1.3 mL/min. The samples were injected in 250 ℃ and split mode, with a ratio of 10:1 each. The temperature of the column was adjusted for 25 minutes:held at 150 ℃ for 1 minute and increased to 230 ℃ at 20 ℃/minute. And then increased to 280 ℃ at 5 ℃/minute, and finally increased to 320 ℃ at 20 ℃/minute. At the beginning of the analysis time, 6.5 minutes was solvent cut off, so the total analysis time was 7.0-25 minutes.

The mass operating conditions were as follows: desolvation temperature, 250 ℃; heat block temperature, 400 ℃; spray voltage 4 kV; drying gas (N2) flow rate, 10 L/min; collision gas, argon; nebulizing gas (N2) flow rate, 3 L/min; collision gas pressure, 230 kPa; and detector voltage, 1.66 kV. , General lipid such as phospholipid, neutral lipid, sphingolipid, sterol lipid and carnitine were assayed by using a Shimadzu LC-MS 8040 triple quadrupole mass spectrometry equipped with an electrospray ionization interface combined with a Nexera X2 LC system (Shimadzu, Kyoto, Japan). The mass operating conditions were as follows: desolvation temperature, 250 ℃; drying gas (N2) flow rate, 10 L/min; spray voltage. 4kV; collision gas, Ar; nebulizing gas (N2) flow rate, 3 L/min; detector voltage, 1.66 kV and collision gas pressure, 230 kPa. , The temperature of ion source and interface was 230 ℃, 250 ℃, and the electron voltage was 70 eV, respectively. We used full scan mode that the range was m/z 50-650, and selected ion monitoring (SIM).