All animal experimentation was conducted in accordance with the guidelines of the institutional Animal Care and Use Committee of the National Institutes of Health at Nanjing Medical University. Male CD-1 mice aged 6–8 weeks were purchased from Viral River Laboratory (Beijing, China). Animals were housed in the animal facilities in Nanjing Medical University with a 12 h: 12 h light–dark cycle and free access to food and water. A diabetic status was induced by intraperitoneal injection of 40 mg/kg of streptozotocin (STZ, S0130, Sigma Aldrich, St. Louis, MO, USA) for 3 consecutive days. Fasted blood glucose level was determined 2 weeks later, and diabetic status was established by the manifestation of polyuria, weight loss, and fasted blood glucose level greater than 16.7 mmol/l. The diabetic mice were randomly assigned into four groups: diabetic mice treated with vehicle, losartan (10 mg kg−1 d−1, HY-17512A, MCE, Monmouth Junction, NJ, USA), dapagliflozin (1 mg kg−1 d−1, PharmaBlock Science, Nanjing, China), and dapagliflozin (5 mg kg−1 d−1). Losartan and dapagliflozin were administrated by oral gavage. Mice were killed after 12 weeks of treatment. Serum and urine were collected, and kidneys were harvested for further analysis. No blinding was done during the treatment.
The human study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Ethics Committees of Nanjing Medical University for Medical Experiments. Written informed consent was obtained from every enrolled individual. Renal biopsy samples and urine samples were collected from individuals of non-diabetes and diabetes from Center for Kidney Disease of Second Affiliated Hospital of Nanjing Medical University. Individuals were clinically diagnosed with or without diabetes according to the World Health Organization criteria and further confirmed with or without DKD by pathologic evaluation of their kidney biopsy samples. Characterization of the enrolled individuals is shown in Supplementary Table S1.
Cell culture and treatment
Primary tubular epithelial cells (PTCs) were cultured under sterile conditions from collagenase-digested cortical fragments of kidneys isolated from mice by a modification of previously described methods25,26. PTCs were cultured in DMEM (no glucose, 11966, Gibco, Grand Island, NY, USA) and F-12 (11765, Gibco) medium (1:1) containing 5 μg/mL of insulin, 2.75 μg/mL of transferrin, 3.35 ng/mL of selenium (41400, Invitrogen, Grand Island, NY, USA), 40 ng/mL of hydrocortisone (614157, Sigma Aldrich), 10 ng/mL of recombinant mouse epidermal growth factor (2028-EG-200, R&D Systems, Minneapolis, MN, USA), and 1% vol./vol. antibiotic solution containing 10,000 U/mL of penicillin and 0.1 mg/mL of streptomycin (15140, Sigma Aldrich). The glucose concentration in cell culture medium was 5.5 mmol/l.
PTCs were seeded on six-well culture plates to 60–70% confluence in complete medium for 16 h, and changed to serum-free medium. Cells were then exposed to the 30 mmol/l of D-glucose (G8270, Sigma Aldrich). Because of the osmotic pressure of high glucose, control cells were treated with D-mannitol (M4125, Sigma Aldrich). The glucose decline in medium is the difference between the initial and terminal glucose concentrations in the cell culture medium. Molidustat (10 μmol/l, S8138, Selleckchem, Houston, TX, USA) or dapagliflozin (2 μmol/l, PharmaBlock Science) treatment was given for the indicated time periods. Dapagliflozin was given 30 min before glucose. PTCs were transiently transfected with negative control siRNA or HIF-1α siRNA (Ibsbio, Shanghai, China) using Lipofectamine RNAiMAX transfection reagent (13778, Invitrogen) according to the manufacturer’s instructions. After transfection for 24 h, PTCs were then exposed to the high glucose. The sequences of siRNA were as follows: HIF-1α: sense 5′-GAU GGA AGC ACU AGA CAA AGU-3′; anti-sense 5′-UUU GUC UAG UGC UUC CAU CAG-3′. Negative control (N.C.): sense 5′-UUC UCC GAA CGU GUC ACG UTT-3′; anti-sense 5′-ACG UGA CAC GUU CGG AGA ATT-3′.
Western blot analysis
Cells were lysed in 1 × SDS sample buffer. Kidney tissue was homogenized by a polytron homogenizer (Brinkmann Instruments) in RIPA lysis buffer on ice. The supernatants were collected after centrifugation at 13,000 × g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay. An equal amount of protein was loaded into 10% or 15% wt/vol. SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. The primary antibodies were as follows: anti-PPARα (ab24509, Abcam, Cambridge, MA, USA), anti-CPT1α (ab128568, Abcam), anti-ACADL (ab196655, Abcam), anti-HK2 (ab76959, Abcam), anti-LDH (3558, Cell Signaling Technology, Danvers, MA, USA), anti-PDK1 (ab110025, Abcam), anti-HIF-1α (ab1, Abcam), anti-PHD2 (4835, Cell Signaling Technology), and anti-tubulin (T6074, Sigma Aldrich). Western blot was performed at least three times independently. Chemiluminescence is applied for detecting proteins on western blot membranes. The enhanced chemiluminescent ECL substrate (32209, Thermofisher Scientific, Carlsbad, CA, USA) enables immunodetection of horseradish peroxidase (HRP)-conjugated secondary antibodies using an imaging system. Quantification was performed by measurement of the intensity of the signals with the aid of Image J software package.
Real-time quantitative-PCR analysis
Quantitative polymerase chain reaction (Q-PCR) was performed using an Applied Biosystems 7300 Sequence Detection system. The total RNA of tissues was prepared using a TRIzol isolation system according to the instructions by the manufacturer (Invitrogen). The first strand of cDNA and subsequent real-time quantification were performed according to the instructions by the manufacturer (Thermofisher Scientific). All reactions were run in triplicate. The CT data were determined using default threshold settings, and the mean CT was calculated from the triplicate PCRs. The ratio of mRNA was calculated by using the equation 2−ΔCT, in which ΔCT = CTtreatment–CTcontrol. Sequences of primer pairs are shown in Supplementary Table S2.
Lactate and glucose measurement
Lactate concentration of urine, kidney tissue and cell supernatant were measured using Lactate Colorimetric Assay Kit (K607-100, Biovision, Milpitas, CA, USA) according to the manufacturer’s instructions. According to the manufacturer, the detection range of lactate with this kit was above 0.04 nmol/μl. Urinary lactate level was normalized with the urine creatinine level. Glucose concentration in cell supernatant or mouse urine was measured using Glucose Colorimetric Assay Kit (K606-100; Biovision).
Urinary creatinine was determined by using a QuantiChrom creatinine assay kit according to the protocol (DICT-500; BioAssay Systems, Hayward, CA, USA). Quantikine Elisa kits were used for measurement of urinary albumin (E90-1134, Bethyl, Montgomery, TX, USA) and urinary Neutrophil gelatinase-associated lipocalin (NGAL) (MLCN20, R&D Systems) according to the manufacturer’s instructions.
Kidney tissue minced into small pieces was homogenized in NP40 Assay Reagent, and triacylglycerol (TG) was measured using quantification kits (10010303, Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions.
Neutraformalin (10% vol./vol.)-fixed kidney samples were kept at 4 °C overnight. The samples were then paraffin-embedded and sectioned at 3 μm in thickness for hematoxylin and eosin (H&E), periodic Acid Schiff (PAS), Masson and Sirius Red staining. Slides were viewed with a Nikon Eclipse 80i microscope equipped with a digital camera (DS-Ri1, Nikon, Shanghai, China). For determination of glomerular tuft area and fractional mesangial area (FMA, %), at least ten randomly chosen fields under the microscope were evaluated for each mouse with Image J software, and an average score was calculated.
Paraffin-embedded kidney sections were deparaffinized, hydrated, antigen retrieved, and endogenous peroxidase activity was quenched by 3% vol./vol. H2O2. Sections were then blocked with 10% vol./vol. normal donkey serum, followed by incubation with anti-PPARα (ab24509, Abcam), anti-CPT1α (ab128568, Abcam), anti-HIF-1α (NB100-123, Novus, New York, NY, USA), anti-HK2 (ab76959, Abcam), or anti-phospho-LDH (8176S, Cell Signaling Technology) overnight at 4 °C. After wash, sections were incubation with secondary antibody for 1 h, followed by incubation with avidin–biotin complex reagents for 1 h at room temperature before being subjected to substrate 3-amino-9-ethylcarbazole (Vector Laboratories, Burlingame, CA, USA). The percentage of positive cells to the selected field was analyzed using Image Pro Plus 6.0 software. An average percentage for each section was calculated. At least ten randomly chosen fields under the microscope were evaluated for each sample, and an average score was calculated.
Cells cultured on coverslips were washed twice with cold PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20 °C. After extensive washings with PBS, the cells were blocked with 0.1% vol./vol. Triton X-100 and 2% vol./vol. normal donkey serum in PBS buffer for 40 min at room temperature and then incubated with anti-HIF-1α antibodies (NB100-123, Novus), followed by staining with FITC-conjugated secondary antibodies. Cells were double stained with DAPI to visualize the nuclei.
Lipid droplets staining
Lipid droplets were viewed by BODIPY or oil red O staining. Briefly, freshly prepared kidney tissues were OCT-embedded and sectioned at 3 μm for BODIPY (D3922, Thermofisher Scientific) staining, which was diluted in DMSO at a concentration of 1 mg/ml according to the manufacturer’s instructions. After stained with BODIPY, slides were immunostained with laminin (ab11575, Abcam) and DAPI. Oil Red O was performed according to the manufacturer’s protocol (O0625, Sigma Aldrich) on 9-μm thickness sections of fixed or frozen kidney tissue and cells cultured on coverslips. The kidney sections and cells were rinsed in distilled water and isopropanol, and stained for 15 min in the Oil Red O working solution at room temperature, then rinsed again for 1 min in 60% vol./vol. isopropanol and returned to distilled water. The slides were counterstained with hematoxylin for 1 min. The percentage of positive area to the selected field was analyzed using Image Pro Plus 6.0 software. An average positive area for each section was calculated. At least ten randomly chosen fields under the microscope were evaluated for each sample, and an average score was calculated.
Quantitative determination of collagen in kidney tissue
Paraffin-embedded kidney tissues were stained with Sirius red F3BA and Food green FCF (Sigma Aldrich) overnight. After washing with PBS buffer, the dye was eluted from tissue sections with 0.1 N sodium hydroxide methanol. Absorbance at 540 and 605 nm was determined for Sirius red F3BA and Food green FCF-binding protein, respectively. This assay provides a simple, relative measurement of the ratio of collagen to total protein, which is expressed as micrograms per milligram of total protein.
Statistical evaluation was carried out using the one-way ANOVA followed by the Tukey post test using GraphPad Prism (GraphPad 7.00 Software). A p < 0.05 value was considered to be significant. Error bars indicate SD.