2.1 Patients and samples
Twenty patients with T2DM and biopsy-proven DKD, diagnosed from January 2017 to December 2017 in Peking University First Hospital35, were enrolled in this study. T2DM was defined according to the criteria proposed by the American Diabetes Association in 201736. None of the patients had coexisting non-diabetes-related renal disease. DKD was defined as previously described35. Healthy control kidney samples were obtained from healthy kidney poles of individuals (n = 10) receiving tumor nephrectomies without diabetes or other kidney diseases. All healthy control kidney samples were confirmed by pathological examinations including immunofluorescence, light microscopy, and electron microscopy. Clinical data of the patients at the time of renal biopsy was systematically recorded. Biopsies were scored independently by two experienced pathologists respectively. Interstitial fibrosis and tubular atrophy (IFTA) scores were assessed semi-quantitatively based on the proportion of the tubulointerstitial compartment affected (0, none; 1, < 25%; 2, 25–50%; 3, > 50%)35. The investigation was conducted according to the Declaration of Helsinki and was approved by the Ethics Committee of Peking University First Hospital (2017 − 1280). Written informed consent was obtained from each participant at renal biopsy.
2.2 Animal experimentation
Wild-type and db/db mice (male, 8 weeks old) inbred on C57BLKS/J background were purchased from Gempharmatech Co., Ltd (Nanjing, China). The mice were randomly allocated into three groups after one-week acclimatization: a wild-type group (m/m, n = 7), a db/db group receiving 1 mg/kg/day dapagliflozin (AstraZeneca Pharmaceuticals LP, solving in 0.5% methylcellulose) treatment (db/db-Dapa, n = 7) and a db/db group receiving solvent as a vehicle (db/db-Veh, n = 6). During the 13-week treatment, body weight was monitored every week. Fasting blood glucose (FBG) was obtained at week0, 4, 8, and 13. The 24-h urine was collected using the metabolic cage to measure urinary albumin (E99-134; Bethyl Laboratories, Montgomery, TX, USA) and creatinine (C011; Nanjing Jiancheng, Nanjing, China) at week0, 4, and 13. Urinary HO1 levels were measured at week0 and 13 (ab205524; Abcam, Cambridge, MA, USA). Kidney tissues were collected before the time of euthanization and preserved for examination. All animal experiments were approved by the Laboratory Animal Ethics Committee of Peking University First Hospital (J202138).
2.3 Renal histology
Staining of kidney sections was performed using periodic acid Schiff (PAS; K1433; BioVision, CA, USA). Glomerular area was measured by tracing around the perimeter of the glomerular tuft. The mesangial matrix expansion area was assessed from the images of glomeruli and presented as a proportion of PAS-stained per glomerular cross-sectional area. The tubulointerstitial injury index was determined by assessing the extent and severity of tubular dilation, atrophy, and loss of tubular cells. Twenty images of a kidney section (magnification ×400) were scored as follows: 0 for no injury, 1 for < 25%, 2 for 25–50%, 3 for 50–75%, and 4 for > 75% tubulointerstitial injury35. Quantitation analyses were performed on Image-Pro Plus software V.6.0 (Media Cybernetics, Bethesda, MD).
2.4 Immunohistochemistry (IHC)
Formalin-fixed paraffin-embedded kidney tissue sections were blocked by 3% bovine serum albumin (BSA, A1933; Sigma-Aldrich, St Louis, MO, USA) after heat-induced epitope retrieval, and stained with anti-HO1 antibody (ab52947; Abcam), anti-TFRC antibody (ab84036; Abcam), or anti-GPX4 antibody (ab125066; Abcam) overnight at 4° C, respectively. Subsequently, an HRP-DAB system (PV-9002; ZLI-9018; ZSBIO, Beijing, China) was used for color development. Twenty images (magnification ×400) of each section were assessed by Image-Pro Plus.
2.5 Transmission electron microscopy
The renal cortical tissues of mice were fixed in 3% glutaraldehyde and further sample handling was performed by the Laboratory of Electron Microscopy, Peking University First Hospital. Imaging was performed by a Hitachi HT7800 transmission electron microscope (Hitachi, Japan). The measurement of glomerular basement membrane (GBM) thickness and mitochondria analysis of renal tubular epithelial cells was processed using Image-pro plus.
2.6 Cell culture
HK-2 human kidney proximal tubular cells (American Type Culture Collection, Rockville, MD) were cultured in DMEM nutrient mix F12 (11330-032; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (10099141; Gibco, Australia), 1% penicillin-streptomycin (V900929; Sigma-Aldrich) at 37℃, 95% humidity, and 5% CO2. Cells were seeded in 6, 12 or 96-well plates, and cells were exposed to different treatments for 24, 48 or 72h, including 30 mM D-glucose (high glucose; HG) (G8270, Sigma-Aldrich) or D-mannitol (Man) (M4125, Sigma-Aldrich), 200 µM palmitic acid (high fat; HF) (P0500, Sigma-Aldrich) or BSA (A9576, Sigma-Aldrich), 20 µM dapagliflozin (Dapa) (HY-10450; MedChemExpress (MCE), NJ, USA), 20 nM PX-478 (HY-10231; MCE), 80 µM hemin (HY-19424; MCE), 5 µM zinc protoporphyrin (Znpp) (HY-101193; MCE), 1 mM dimethyloxallyl glycine (DMOG) (HY-B0988; MCE), 50 µM deferoxamine mesylate (DFO) (HY-B0988; MCE), 2 µM erastin (HY-15763; MCE), 5 µM RSL3 (HY-100218A; MCE) and 1 µM ferrostatin-1 (Fer-1) (HY-100579; MCE), respectively.
2.7 Cell viability assay
Cell viability was measured using Cell Counting Kit-8 (CCK8-100; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions.
2.8 Iron and malondialdehyde (MDA) assay
Intracellular ferrous iron (Fe2+) was assessed by fluorescent probe FerroOrange (F374; Dojindo) via fluorescence-activated cell sorter (FACS) analysis using BD FACSVerse (BD biosciences, NJ, USA), while total iron concentration was measured by Iron detection kit (TC1015; Leagene, Bejing, China). MDA concentrations were determined by the Micro Malondialdehyde Assay Kit (BC0025; Solarbio, Beijing, China).
2.9 Quantification of lipid peroxidation
HK-2 cells were incubated with 2.5 µM C11-BODIPY581/591 (D3861; Thermofisher Scientific, MA, USA) for 45 min at 37℃. Then cells were collected and washed once with PBS followed by FACS. Fluorescence intensity was analyzed using FlowJo_V7 software.
2.10 Assessment of mitochondrial activity
Mitochondrial activity in HK-2 cells was assessed (C1035; Beyotime, Beijing, China) via FACS analysis or confocal laser scanning microscope. The fluorescence intensity of FACS was analyzed using FlowJo_V7 software.
2.11 Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted (DP441; Tiangen, Beijing, China) and reverse transcribed into cDNA (4374966; Applied Biosystems, MA, USA). The qRT-PCR analysis was carried out in an ABI Prism 7500 sequence detection system using SYBR Green Master Mix (A25742; Applied Biosystems). Primers are listed in Table S1.
2.12 Western blot
Total protein was extracted, and nucleoprotein and cytoplasmic proteins from HK-2 cells were isolated (P0027; Beyotime). Protein was separated using 10% SDS-PAGE and transferred to PVDF membranes, which were probed with primary antibodies against HIF-1α (10006421; Cayman, USA), TFRC (13-6800; Thermo Scientific) GPX4 (ab125066; Abcam) and HO1 (10701-1-AP; Proteintech, CHI, USA). β-actin (sc-47778; Santa Cruz Biotechnology, CA, USA) and histone 3 (4499T; Cell Signaling Technology, MA, USA) were used as internal controls. Protein was visualized on autoradiographic film using an ECL Plus Western blot detection system (GE Healthcare).
2.13 Bioinformatic analysis
The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Raw data were processed using Trimmomatic37. After removing the low-quality reads, clean data were mapped to the reference genome using hisat238. FPKM39 value was calculated using cufflinks40, and the read counts were obtained by htseq-count41. Differentially expressed genes (DEGs) were identified using DESeq42 R package, and p < 0.05 and fold change > 2 or fold change < 0.5 was set as the threshold for significantly differential expression. KEGG43 pathway enrichment analysis of DEGs was performed using the Cluster Profiler R package.
2.14 Statistical analysis
Normally distributed data were presented as mean ± standard deviation (SD), and non-normally distributed data were presented as median and interquartile range (IQR). Groups were compared using unpaired two-tailed Student’s t-tests or one-way analysis of variance (ANOVA) as appropriate. Pearson or Spearman correlation analysis was used to evaluate the association between immunohistochemistry staining intensity (IOD/area) and clinicopathological parameters as appropriate. A p value less than 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). All the results were plotted using GraphPad Prism 8 software.