In diabetic patients, dyslipidemia frequently contributes to organ damage such as diabetic kidney disease (DKD). DKD is associated with excessive renal deposition of triacylglycerol (TAG) in lipid droplets (LD). In order to identify the changes in the lipidome of proximal tubules exposed to high levels of fatty acids and the role that LDs formation play in this context, we treated induced Renal Epithalial Cells (iRECS) with BSA, BSA-PA (Palmitic Acid), BSA-OA (Oleic Acid), BSA-PA/OA, BSA + DGAT1/2 inhibitors, BSA-PA + DGAT1/2 inhibitors and BSA-PA/OA + DGAT1/2 inhibitors. 

iRECs treated with BSA, BSA-PA, BSA-OA, BSA-PA/OA, BSA + DGAT1/2 inhibitors, BSA-PA + DGAT1/2 inhibitors and BSA-PA/OA + DGAT1/2 inhibitors  were subjected to shotgun lipidomics and quantified

Lipid extraction for mass spectrometry lipidomics 

Mass spectrometry-based lipid analysis was performed by Lipotype GmbH (Dresden, Germany) as described (Sampaio et al, 2011). Lipids were extracted using a two-step chloroform/methanol procedure (Ejsing et al, 2009). Samples were spiked with internal lipid standard mixture containing: cardiolipin 14:0/14:0/14:0/14:0 (CL), ceramide 18:1;2/17:0 (Cer), diacylglycerol 17:0/17:0 (DAG), hexosylceramide 18:1;2/12:0 (HexCer), lyso-phosphatidate 17:0 (LPA), lyso-phosphatidylcholine 12:0 (LPC), lyso-phosphatidylethanolamine 17:1 (LPE), lyso-phosphatidylglycerol 17:1 (LPG), lyso-phosphatidylinositol 17:1 (LPI), lyso-phosphatidylserine 17:1 (LPS), phosphatidate 17:0/17:0 (PhA), phosphatidylcholine 17:0/17:0 (PC), phosphatidylethanolamine 17:0/17:0 (PE), phosphatidylglycerol 17:0/17:0 (PG), phosphatidylinositol 16:0/16:0 (PI), phosphatidylserine 17:0/17:0 (PS), cholesterol ester 20:0 (CE), sphingomyelin 18:1;2/12:0;0 (SM), sulfatide d18:1;2/12:0;0 (Sulf), triacylglycerol 17:0/17:0/17:0 (TAG) and cholesterol D6 (Chol). After extraction, the organic phase was transferred to an infusion plate and dried in a speed vacuum concentrator. 1st step dry extract was re-suspended in 7.5 mM ammonium acetate in chloroform/methanol/propanol (1:2:4, V:V:V) and 2nd step dry extract in 33% ethanol solution of methylamine in chloroform/methanol (0.003:5:1; V:V:V). All liquid handling steps were performed using Hamilton Robotics STARlet robotic platform with the anti-droplet control feature for organic solvents pipetting. 

MS data acquisition 

Samples were analyzed by direct infusion on a QExactive mass spectrometer (Thermo Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). Samples were analyzed in both positive and negative ion modes with a resolution of Rm/z=200=280000 for MS and Rm/z=200=17500 for MSMS experiments, in a single acquisition. MSMS was triggered by an inclusion list encompassing corresponding MS mass ranges scanned in 1 Da increments (Surma et al, 2015). Both MS and MSMS data were combined to monitor CE, DAG and TAG ions as ammonium adducts; PC, PC O-, as acetate adducts; and CL, PA, PE, PE O-, PG, PI and PS as deprotonated anions. MS only was used to monitor LPA, LPE, LPE O-, LPI and LPS as deprotonated anions; Cer, HexCer, SM, LPC and LPC O- as acetate adducts and cholesterol as ammonium adduct of an acetylated derivative (Liebisch et al, 2006).

Data analysis and post-processing 

Data were analysed with in-house developed lipid identification software based on LipidXplorer (Herzog et al, 2012; Herzog et al, 2011). Data post-processing and normalization were performed using an in-house developed data management system. Only lipid identifications with a signal-to-noise ratio >5, and a signal intensity 5-fold higher than in corresponding blank samples were considered for further data analysis.