L-AKI model

Male C57BL/6 mice (8 weeks old, n = 6–10 in each group) were purchased from KOATECH (South Korea) and intraperitoneally injected with LPS (10 mg/kg, cat. L2630, Sigma-Aldrich, St. Louis, MO, USA) for L-AKI model, which was reconstituted with distilled PBS. The mice, divided into four groups based on the time elapsed since LPS injection (0, 6, 12, and 24 h), were sacrificed corresponding to their respective time points post-injection. For the LPS + Stattic-treated group, the STAT3 inhibitor (5 mg/kg, 10 mg/kg) (Stattic; 6-nitrobenzo[b]thiophene-1,1-dixodide, cat. S7947, Sigma-Aldrich) was injected 1 h before LPS induction in the peritoneal region, and the mice were sacrificed 6 h after LPS injection. The control group consisted of vehicle-treated sham control mice. Blood samples were collected at sacrifice via an abdominal aortic puncture to evaluate renal function. All animal studies were performed under the guidance of the Institutional Animal Care and Use Committee (IACUC: 23–0055-S1A0) of Seoul National University Hospital.

L-CKD model

In the L-CKD model, mice received intraperitoneal injections of LPS (1 mg/kg, Sigma-Aldrich) and Stattic (5 mg/kg, 10 mg/kg, Sigma-Aldrich) every 2 days, following a similar administration schedule as used in the L-AKI model. All animals were sacrificed after 14 days in the L-CKD model [14].

Assessment of kidney function

Body weights and blood samples were obtained at 0, 6, 12, and 24 h after LPS injection. Blood urea nitrogen (BUN) (mg/dL) and creatinine (mg/dL) concentrations were measured by determining the rate of the modified Jaffe reaction using an autoanalyzer (HITACHI7180, Hitachi Chemical Industries) [22].

Flow cytometry

Intrarenal mononuclear cells were isolated from mouse kidneys, and homogenates were obtained using a Stomacher H 80 Biomaster (Seward, Worthing, Sussex, UK). Single-cell suspensions were obtained by passing the tissue through a 40-μm cell strainer. Kidneys were resuspended in 40% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and overlaid on 80% Percoll. After being centrifuged for 30 min at 3000 rpm and 25 °C, renal mononuclear cells were isolated from the interface and washed in PBS [23]. Cells were diluted in 100 μL chilled FACS buffer (1X Hank’s Balanced Salt Solution; cat. 14025–092, Gibco, Billings, MT, USA) with 0.5% BSA and 0.05% sodium azide and incubated with mouse Fc block (cat. 5531–42, BD Biosciences, Franklin Lakes, NJ, USA) for 10 min. For surface marker staining, the cells were incubated with 1:100 dilutions of CD45-BV785 (cat. 103149; BioLegend, San Diego, CA, USA), CD11b-FITC (cat. 101206, BioLegend), and F4/80-PerCP Cy5.5 (cat. 157318; BioLegend) for 30 min at 25 °C. Finally, the cells were washed and resuspended in FACS buffer. For intracellular staining, the cells were stained with CD206-BV605 (cat. 141721, BioLegend) and phospho-STAT3 (Tyr 705)-APC (cat. 17–9033-42, Invitrogen, Waltham, MA, USA) using eBioscience™ IC Fixation Buffer (cat. 00–8222-49, Invitrogen) according to the manufacturer’s protocol. Stained cells were analyzed using a BD LSRFortessa™ device with BD FACSDiva™ software (BD Biosciences). Data were processed using FlowJo (v10.8.1., BD Biosciences).

Protein purification and Western blot analysis

Kidney proteins from LPS-treated mice were isolated using RIPA buffer (cat. RC2002-050–00, Biosesang, Yongin, Korea; 150 mM NaCl; 100 mM Na3VO4; 50 mM Tris; HCL, pH 7.3; 0.1 mM EDTA 1% (vol/vol) sodium deoxycholate; 1% (vol/vol) Triton X-100; and 0.2% NaF) with protease inhibitor (GeneDEPOT, Katy, TX, USA). Kidney tissue lysates were obtained using steel beads in RIPA buffer with a TissueLyser instrument (Qiagen, Hilden, Germany) set at 30 strokes/s for 5 min. BCA assay was used to measure unknown protein concentrations. Protein lysates were electrophoresed in glycine-sodium dodecyl sulfate buffer and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) on ice. The membrane was blocked for 1 h using blocking solution and probed with antibodies against pSTAT3 (cat. 9145L, Cell Signaling Technology, Danvers, MA, USA), STAT3 (cat. 9132L, Cell Signaling Technology), NGAL (cat. Sc-515876, Santa Cruz Biotechnology, Dallas, TX, USA), β-actin (cat. A1978, Sigma-Aldrich), ICAM-1 (cat. A00171, Boster Bio, Pleasanton, CA, USA), GAPDH (cat. 2118, Cell Signaling Technology), IL-6 (cat. CAB14687, Assay Genie, Dublin, Ireland), KIM-1 (cat. Ab233720, Abcam, Cambridge, UK), and Fibronectin (cat. Ab2413, Abcam). For the secondary antibodies, anti-rabbit IgG (cat. 7074S, Cell Signaling Technologies) and anti-mouse IgG (cat. 7076S, Cell Signaling Technologies) were used. Targeted proteins were detected using ImageQuant™ Las 4000 mini (GE HealthCare, Chicago, IL, USA) and analyzed using the ImageJ software (ImageJ v. 1.52; Wayne Rasband, National Institutes of Health).

Immunohistochemistry (IHC) and histological analysis

The kidney tissue sections were fixed in 10% formalin and embedded in paraffin overnight. Then, 4 μm-thick sections of the kidneys were deparaffinized with xylene and rehydrated with ethanol. Briefly, the kidney sections were microwaved with sodium citrate buffer for antigen retrieval. Hydrogen peroxide (3%) was used to block endogenous peroxidase activity after dilution in methyl alcohol. The sections were probed with antibodies against F4/80 (cat. 70076 s, Cell Signaling Technology), ICAM-1 (cat. A00171, Boster Bio), NGAL (cat. Sc-515876, Santa Cruz Biotechnology), and pSTAT3 (cat. 9145L, Cell Signaling Technology). Dako Envision+ System-HRP-labeled polymer anti-rabbit (cat. K4003, Agilent DAKO, Santa Clara, CA, USA) and anti-mouse (cat. K4001, Agilent DAKO) antibodies were added and incubated for 1 h at 25 °C. Periodic Acid-Schiff (PAS) staining was conducted to evaluate the tubular injury scores based on the percentage of injured area, with cell nuclei counterstained using Mayer’s hematoxylin (Sigma-Aldrich) [24, 25]. The injury scores were determined as follows: 0 for no damage; 1 for an injured area of 1–10%; 2 for 11–25%; 3 for 26–75%; and 4 for an injured area of 75% or greater [26,27,28,29]. Sirius red (cat. Ab150681, Abcam) staining was used to evaluate the fibrotic changes in L-CKD kidney. For IHC, the stained area was examined and quantified using a Leica inverted microscope (Leica Camera, Wetzlar, Germany) and the LAS-4000 program (Leica Camera).

RNA isolation and real-time qPCR

Total RNA was isolated from mouse kidney tissues using TRIzol reagent (Thermo Fischer Scientific, Waltham, MA, USA) following the manufacturer’s protocol. cDNA obtained from the total RNA of mouse kidney tissues was amplified using the ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). The gene expression levels were detected using EvaGreen qPCR Mastermix (Applied Biological Materials, Richmond, Canada), and real-time PCR was performed on the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with the following PCR conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 20 s. Relative quantification was conducted using the comparative CT (ΔΔCT) method, and each target gene expression level was normalized relative to GAPDH expression. The sequence of qPCR primers used is listed in Table 1.

Table 1 Primer sequences for real-time qPCRProcessing of sequencing data

Total RNA from the kidneys of L-AKI mice was sequenced and analyzed by EBIOGEN, Inc. (Seoul Korea). A TapeStation 4000 system (Agilent Technologies, Amstelveen, Netherlands) was used to assess RNA quality. Briefly, an RNA sequencing (RNA-seq) library was generated using a CORALL RNA-seq V2 Library Prep Kit (LEXOGEN GmbH, Vienna, Austria). mRNA was isolated using a Poly (A) RNA Selection Kit (LEXOGEN GmbH). To obtain high-throughput sequencing results, paired-end 100 bp sequencing was carried out using a NovaSeq 6000 (Illumina, San Diego, CA, USA). The Excel-based ExDEGA software from EBIOGEN was employed to identify differentially expressed genes (DEGs) and gene ontologies [30]. KEGG pathway enrichment analysis was performed using the enrichKEGG function, and GO enrichment analysis was conducted using the enrichGO function from the clusterprofiler package (v. 4.6.2) [31]. These analyses were used to identify significant pathways and GO profiles associated with DEGs.

Cell culture

The murine macrophage cell line RAW264.7 (TIB-71) and the proximal tubule epithelial cell line OK (CRL-1840) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). RAW264.7 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, cat. L0103, BioWest, Nuaillé, France), and OK cells were cultured in Minimum Essential Medium Eagle (MEM, cat. M4655, Sigma-Aldrich). Both media were supplemented with 10% fetal bovine serum (FBS, cat. S1480, BioWest) and 1% penicillin/streptomycin (cat. 15,140–122, Gibco), and the cells were maintained at 37℃ with 5% CO2.

Establishment of an in vitro model for LPS-induced inflammation

To evaluate the effect of LPS-induced inflammation, RAW264.7 cells were treated with LPS (cat. L2630, Sigma-Aldrich) in a time-dependent (0.5 μg/mL LPS for 6, 12, and 24 h) or dose-dependent manner (0.25 μg/mL and 0.5 μg/mL). For the STAT3 inhibition, Stattic (1 μM or 2 μM, cat. S7947, Sigma-Aldrich) was administered to the cells 1 h before LPS induction. The expression of pSTAT3 and iNOS in RAW264.7 cells were then observed using FACS analysis.

Co-culture system of macrophages and tubular epithelial cells

RAW264.7 cells and OK cells were pretreated with Stattic (1, 2 μM) 1 h before being exposed to 0.5 μg/mL LPS for 12 h. After incubation, the cells were collected for further experiments to observe the expression of IL-6, KIM-1, pSTAT3, and STAT3. For the co-culture system, OK cells (5 × 105) were plated and cultured in MEM media a day before co-culture. Furthermore, to confirm macrophage-driven inflammation, RAW264.7 cells (1.5 × 106), stimulated with or without LPS for 12 h were then added to the OK cells. OK cells were treated with Stattic (2 μM) either 1 h before or at the start of co-culture to evaluate the effects of pre/concomitant-treatment. After 12 h of co-culture, RAW264.7 cells were isolated with the MagniSort™ mouse CD11b positive selection kit (cat. 8802–6860-74, Thermo Fisher Scientific) following the manufacturer’s protocol. Subsequently, OK cell lysates were analyzed by western blot to assess the protein levels of KIM-1.

Statistical analysis

All results are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed using an unpaired two-tailed Student’s t-test in GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA). In KEGG and GO enrichment analyses, the p-values were calculated using the R packages enrichGO and enrichKEGG. Each experiment was independently repeated three times. A p-value less than 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).