Animals

The study conformed to the APS Guidelines for the Care and Use of Animals and was approved by the Animal Ethics Committee of the University of Namur. The experiments were conducted on C57Bl/6J male and female mice (Janvier Labs, Le Genest Saint-Isle, France) that were housed in cages with free access to food and water. The mice were maintained at 35–40% relative humidity and a temperature of 20–23 °C with a 12:12 h light–dark cycle. Over a 16-week period, 8-week-old mice (n = 6 per sex and per treatment) were randomized to either a LFD (10% of total calories from fat; D12450J, Research Diets, New Brunswick, NJ, USA) or HFD (60% of total calories from fat; D12492, Research Diets, New Brunswick, NJ, USA). Body weight (BW) was measured every 4 weeks and 24-h urine collection was performed at the end of the study using metabolic cages. Fasting blood glucose concentration (One Touche Vita, LifeScan Inc., Milpitas, CA) was measured from the tail vein. At week 16, mice were anesthetized after an overnight fast with a solution of ketamine (Nimatek®, Eurovet Animal Health, Blabel, The Netherlands, 80 mg/kg b.w.) and medetomidine (Domitor®, Orion Pharma, Espoo, Finland, 0.5 mg/kg b.w.), and blood was collected by intracardiac puncture. Blood samples were collected and centrifuged at a high speed for 20 min at 4 °C. The plasma was collected and stored at − 80 °C until further use. The kidneys, liver, and heart were removed, weighed, and reported to tibia length in order to normalize the data, as previously reported [14]. A portion of the collected organs was fixed in Duboscq-Brazil solution, while the remaining tissues were frozen in liquid nitrogen and stored at − 80 °C for further analysis.

Urine collection and urinary markers analyses

After 16 weeks of feeding, mice were placed for 24-h urine collection in metabolic cages that provide ad libitum access to food and water and allow refrigeration of urine samples. Urine was subsequently stored at − 20 °C. Urinary albumin and creatinine levels were measured using a mouse Albuwell ELISA kit and Creatinine Companion kit (Exocell, Philadelphia, PA, USA). Total proteinuria was quantified using the Bradford method based on the absorbance of the Coomassie Brilliant Blue dye. As an index of oxidative stress, urine samples were analyzed for hydrogen peroxide using the Amplex red assay (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. All urinary marker values were normalized to the urinary creatinine concentration.

Biochemical assays

Plasma leptin and adiponectin (full-length form of adiponectin) concentrations were determined by ELISA (Mouse/rat leptin immunoassay Quantikine ELISA, Mouse adiponectin/Acrp30 Immunoassay Quantikine ELISA, R&D Systems Europe, Abingdon, UK). Plasma insulin levels were determined by ELISA using the rat/mouse insulin ELISA kit (Merck, Darmstadt, Germany). The homeostasis model assessment (HOMA-IR) for the insulin resistance index was determined using a calculator available from the Oxford Center for Diabetes, Endocrinology, and Metabolism (https://www.dtu.ox.ac.uk/homacalculator/). Colorimetric enzymatic tests were performed to measure plasma cholesterol levels (Diasys, Diagnostic System, Holzheim, Germany) and plasma non-esterified fatty acid (NEFA) levels (Wako Pure Chemical Industries, Ltd., Osaka, Japan), following the manufacturer’s instructions.

Histology and morphological analyses

Five-μm paraffin-embedded kidney sections were stained with Periodic Acid Schiff (PAS), Hemalun, and Luxol Fast Blue to assess morphological alterations. Morphometry of kidney sections was performed as previously reported [13]. Briefly, the frequency of tubules containing vacuolated cells was evaluated using a semi-quantitative single-blind analysis. To standardize the evaluation procedure, an additional lens engraved with a square grid was inserted into one of the microscope’s eyepieces. For each paraffin section, 10 square fields (0.084 mm2/field) were observed at × 400 magnification. Ten randomly selected areas of each cortex kidney section were analyzed using the ImageJ software. Paraffin-embedded liver sections were stained with hematoxylin and eosin and steatosis was graded as described by Ryu et al. [39].

Immunohistochemistry

Five-μm paraffin-embedded kidney sections were dewaxed and rehydrated, followed by microwave pre-treatment in 1 mM EDTA buffer to unmask antigens present in the renal tissue. Endogenous peroxidase activity was removed by incubation with 3% H2O2 for 10 min and blocking with 10% normal goat serum. Sections were incubated with a primary antibody against LAMP-1 (Abcam, Cambridge, UK) overnight at 4 °C. After rinsing in TBS, slides were exposed for 30 min with SignalStain® Boost IHC Detection Reagent (Cell Signaling, Danvers, MA, USA), and bound peroxidase activity was detected using a DAB kit (Agilent DAKO, Heverlee, Belgium). Counterstaining was performed using Hemalun and Luxol Fast Blue. The evaluation of the relative positive area was performed on one section per experimental animal. For each section, ten square fields (0.084 mm2/field) were observed at 400 × magnification in each renal zone. The relative area occupied by positive staining was expressed as a percentage.

Quantitative real-time polymerase chain reaction (PCR)

Frozen kidney cortex was homogenized and total RNA was extracted using TRIzol (Sigma-Aldrich, St. Louis, MO, USA) and treated with DNAse (Promega, Madison, WI, USA). The total RNA concentration was measured using a NanoDrop spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific, Waltham, MA, USA). Transcript-specific primers were generated based on the mouse sequences from GenBank. The NCBI Primer BLAST was used to ensure the specificity of the primers for each target. All primer pairs were analyzed for their dissociation curves and melting temperatures. Real-time quantitative PCR was performed to quantify the mRNA levels of AdiporR1, Lamp1, Cathepsin D, p62, CerS2, CerS5, CerS6, Acer2, Acer3, and 18S as housekeeping gene (Table 1). Briefly, 2 μg of total RNA was reverse-transcribed using MLV reverse transcriptase (Promega, Madison, WI, USA) for 1 h at 70 °C. Quantitative PCR amplification was performed using SYBR Green Master Mix (Roche, Belgium) and Prism 7300 Real-Time PCR Detection System (Applied Biosystems, CA, USA). Mean fold changes were calculated by averaging duplicate measurements for each gene. The relative gene expression was calculated using the 2−ΔΔCT method.

Table 1 Primer sequences for RT-qPCR analysis of mRNA expressionWestern blot analysis

Proteins were extracted from renal cortex tissues using Cell Lysis Buffer (Cell Signaling, Danvers, MA, USA) with phosphatase and protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C followed by centrifugation at 14,000 × g for 15 min at 4 °C. Protein concentrations were quantified by Pierce BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and then 20 µg of total lysate were separated by SDS-PAGE 12% and transferred onto nitrocellulose membranes. Following blocking step in 5% BSA for 1 h, the membranes were incubated with primary antibodies against phosphorylated AMPK (P-AMPK), AMPK (Cell Signaling, Danvers, MA, USA), AdipoR1 (Abcam, Cambridge, UK) or β-actin (Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4 °C and then with secondary antibodies (Li-Cor Biosciences, Lincoln, NE, USA) for 1 h at room temperature. Antibodies were diluted in Odyssey Blocking Buffer TBS containing 0.1% Tween20. Proteins were visualized and quantified using the Odyssey® imaging system (Li-Cor Biosciences, Lincoln, NE, USA).

Lipidomic analysis

Lipid extraction Tissue lysates containing 10 μg of DNA were homogenized in 700 μl of water with an handheld sonicator and were mixed with 800 μl HCl(1 M):CH3OH 1:8 (v/v), 900 μl CHCl3, 200 μg/ml of the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT; Sigma-Aldrich, St. Louis, MO, USA) and 3 μl of SPLASH® LIPIDOMIX® Mass Spec Standard (#330707, Avanti Polar Lipids, Birmingham, AL, USA). After vortex and centrifugation, the lower organic fraction was collected and evaporated using a Savant Speedvac spd111v (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature and the remaining lipid pellet was stored at − 20 °C under argon.

Mass spectrometry (MS) Lipid pellets were reconstituted in 100% ethanol and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with hybrid triple quadrupole/linear ion trap mass spectrometer (6500 + QTRAP system; AB SCIEX). Chromatographic separation was performed on a XBridge amide column (150 mm × 4.6 mm, 3.5 μm; Waters) maintained at 35 °C using mobile phase A [1 mM ammonium acetate in water–acetonitrile 5:95 (v/v)] and mobile phase B [1 mM ammonium acetate in water–acetonitrile 50:50 (v/v)] in the following gradient: (0–6 min: 0% B → 6% B; 6–10 min: 6% B → 25% B; 10–11 min: 25% B → 98% B; 11–13 min: 98% B → 100% B; 13–19 min: 100% B; 19–24 min: 0% B) at a flow rate of 0.7 ml/min which was increased to 1.5 ml/min from 13 min onwards. Sphingomyelins (SM), cholesterol esters (CE) and ceramides (CER) were measured in positive ion mode with a precursor scan of 184.1, 369.4, 264.4, respectively. Triacylglycerides (TG), diacylglycerides (DG) and monoacylglycerides (MG) were measured in positive ion mode with a neutral loss scan for one of the fatty acyl moieties. Phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phosphatidylinositols (PI) and phosphatidylserines (PS) were measured in negative ion mode by fatty acyl fragment ions. Lipid quantification was performed by scheduled multiple reactions monitoring, the transitions being based on the neutral losses or the typical product ions as described above. The instrument parameters were as follows: Curtain Gas = 35 psi; Collision Gas = 8 a.u. (medium); IonSpray Voltage = 5500 V and − 4500 V; Temperature = 550 °C; Ion Source Gas 1 = 50 psi; Ion Source Gas 2 = 60 psi; Declustering Potential = 60 V and − 80 V; Entrance Potential = 10 V and − 10 V; Collision Cell Exit Potential = 15 V and − 15 V.

The following fatty acyl moieties were taken into account for the lipidomic analysis: 14:0, 14:1, 16:0, 16:1, 16:2, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:4, 22:5 and 22:6 except for TGs which considered: 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 20:5, 22:2, 22:3, 22:4, 22:5, 22:6.

Data analysis Peak integration was performed with the MultiQuant™ software version 3.0.3. Lipid species signals were corrected for isotopic contributions (calculated with Python Molmass 2019.1.1), quantified based on internal standard signals and adheres to the guidelines of the Lipidomics Standards Initiative (LSI) (level 2 type quantification as defined by the LSI).

Statistical analysis

The results are presented as the mean ± SEM. The level of statistical significance was set at p < 0.05. Analyses were performed using Prism GraphPad Software version 6 (San Diego, CA, USA). Differences were analyzed using two-way ANOVA to examine the overall effects of diet, sex, and interaction (see detailed statistical analysis in Table 2). In cases of significant ANOVA effects, post hoc comparisons were performed using Tukey’s multiple comparisons test to determine the significance between groups when appropriate. For lipidomic data, multiple t-tests with p-values corrected for multiple comparisons using the Bonferroni method were performed within each sex-matched group to analyze the FA profile for each lipid class. Partial least squares discriminant analysis (PLS-DA) and heat map visualization were performed using MetaboAnalyst 4.0 (http://www.metaboanalyst.ca).

Table 2 Results from two-way ANOVA statistical analysis