Urine and Kidney Lipid Profiling

Urine was collected from nonobese and obese mice and centrifuged at 1,000g for 10 minutes to remove cellular debris. Lysosome-enriched subcellular fractions were isolated from kidneys using a modified version of a method described previously (66 (link)). Kidneys were homogenized with pestles in 1 mL of subcellular fractionation buffer (HEPES 20 mM, sucrose 250 mM, KCl 10 mM, MgCl₂ 1.5 mM, EDTA 1 mM, EGTA 1 mM, dithiothreitol 8 mM, pH adjusted to 7.5 with NaOH). Debris and nuclei were pelleted at 750g for 12 minutes. The supernatant was centrifuged at 10,000g for 35 minutes to pellet the lysosome-enriched fraction. The pellet was washed once with subcellular fractionation buffer. Lipid extraction from urine and the lysosome-enriched fraction was performed using the Bligh and Dyer method with minor modifications (67 (link)). BMP, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, monoacylglycerol, diacylglycerol, triacylglycerol, cholesterol, ceramide, hexose ceramide, lactosylceramide, and sphingomyelin were analyzed by supercritical fluid chromatography (SFC) (Nexera UC system, Shimadzu; equipped with an ACQUITY UPC² Torus diethylamine [DEA] column: 3.0 mm inner diameter [i.d.] × 100 mm, 1.7 μm particle size, Waters) and triple quadrupole mass spectrometry (TQMS; LCMS-8060, Shimadzu) (DEA-SFC/MS/MS) in multiple reaction monitoring (MRM) mode (68 (link)). Fatty acids and cholesterylester were analyzed using an SFC (Shimadzu) with an ACQUITY UPC² HSS C18 SB column (3.0 mm i.d. × 100 mm, 1.8 μm particle size, Waters) coupled with a TQMS (Shimadzu) (C18-SFC/MS/MS) in MRM mode (69 (link)). The amount of each lipid species was normalized either to the urine creatinine concentration, measured using a QuantiChrom Creatinine Assay Kit (DICT-500) (BioAssay Systems), or to kidney weight.