Animals

The Cre/loxP recombination system was used to generate ASIC1afl/fl/CDH16cremice. Mice that were maintained on the C57BL/6 background with the ASIC1a floxed allele were crossed with mice that expressed Cre recombinase under the cadherin 16 promoter (CDH16Cre). The ASIC1afl/fl and wild-type alleles were detected using the following primers: 5’- GTGTTGTTTGCTTCTGGCCG-3’ and 5’- CAGATGACAGTTTGCAGGCC-3’, which generated a 442 bp product in the floxed allele and a 281 bp product in the wild-type allele. The CDH16-Cre transgene was detected using primers 5’- GCAGATCTGGCTCTCCAAAG-3’ and 5’- AGGCAAATTTTGGTGTACGG-3’, which amplified a 420 bp fragment. The internal positive control was detected using the primers 5’- CAAATGTTGCTTGTCTGGTG-3’ and 5’- GTCAGTCGAGTGCACAGTTT-3’, which amplified a 200 bp fragment. C57BL/6 wild-type (WT) mice and floxed without CDH16-Cre (ASIC1afl/fl) mice served as the control group for tubule-specific ASIC1a deletion (ASIC1afl/fl/CDH16cre) mice. Mice were housed in an acclimatized room and allowed free access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Fudan University.

Renal IRI model in mouse

Renal ischemia AKI was induced in 8-week-old male mice [14]. Briefly, following treatment with 4% phenobarbitone (10 mL kg−1 body weight), bilateral renal pedicles were exposed and clamped to induce 35 min of ischemia. The atraumatic clamps were then released. The sham-operated group underwent the same procedure without clamping. The body temperature of mice was maintained at 37 °C. The kidneys and blood were harvested after 24 h reperfusion.

Renal function

Blood was collected by cardiac puncture. Serum creatinine levels were determined using a Quantichrom Creatinine Assay kit (BioAssay Systems, Hayward, CA, USA).

Histopathological examinations

Paraffin-embedded 5-μm kidney sections were stained with periodic acid-Schiff (PAS). Histological injury scores were evaluated by light microscopy in a blinded manner. Severity was determined in a semi-quantitative manner by the percentage of tubules manifesting epithelial necrosis, loss of the brush border, cast formation, and tubular dilation. A five-point scale was used, as follows: 0, no injury; 1, less than 25%; 2, 25–50%; 3, 50–75%; and 4, more than 75%.

Real-time quantitative PCR

Total RNA was extracted with TRIzol reagent. First-strand cDNA was synthesized by reverse transcription. Quantitative PCR was performed with SYBR-green. Primers are described in Table 1. Relative levels of mRNA expression were normalized to GAPDH expression for each gene.

Table 1 Primer sets used for real‐time PCRDetermination of ROS

Dihydroethidium (DHE) staining was used to characterize reactive oxygen species (ROS) production. Cryosections were incubated with 10 μM DHE (red fluorescence) at 37 °C for 30 min followed by staining with DAPI (blue fluorescence), and then observed by confocal microscopy. For quantification, the fluorescent density was determined.

Flow cytometry

Kidneys were collected to determine the neutrophil infiltration by flow cytometry. Briefly, kidney tissue was homogenized after digestion with collagenase/DNase I. Cell suspensions were incubated with fluorescently-labeled antibodies against Ly6G and CD45. Flow cytometry was then performed. Neutrophils were identified as Ly6G+ CD45+ cells. The result was expressed as fraction of Ly6G+ CD45+ cells among total CD45+ cells.

Cell culture and treatments

The human kidney proximal tubular epithelial cell line (HK-2, ATCC) was incubated in a humidified atmosphere containing 5% CO2 at 37 °C. For hypoxia/reoxygenation (H/R), cells were cultured in incubator containing 1% O2, 94% N2, and 5% CO2 for 6 h and subsequently cultured in normoxic conditions with 21% O2 for 1 h. For the acidosis treatment, the pH of the medium was adjusted using an appropriate amount of HCl. Psalmotoxin-1 (PcTx-1; peptide institute, Japan) and BAY 11-7082 (Beyotime, Shanghai, China) were dissolved and diluted in ddH2O. The cells were treated with PcTx-1 or BAY11-7082 for 1 h prior to H/R or acidosis.

Cell viability

HK-2 cells were seeded in 96-well plates at a density of 5 × 103 cells per well. After treatment, the cells were incubated with the CCK-8 reagent (Dojindo, Kumamoto, Japan). The absorbance of CCK-8 was measured at 450 nm wavelength using a microplate reader.

Western blotting

The protein samples were separated and transferred to a PVDF membrane for incubation with primary antibodies against NLRP3 (1:1000), ASC (1:400), caspase-1 (1:1000), GSDMD-N (1:1000), IL-1β (1:800), phosphorylated NF-κB p65 (p-NF-κB p65, 1:1000), NF-κB p65 (1:1000), and GAPDH (1:4000) at 4 °C overnight. Then the membranes were incubated with secondary antibodies. Protein band intensities were quantified using densitometry. All values were normalized to GAPDH and expressed as fold change relative to the control. Detailed information on the primary antibodies is given in Table 2.

Table 2 List of primary antibodiesTUNEL assay

Apoptosis was detected using a TUNEL apoptosis assay kit (Beyotime, Shanghai, China). Briefly, 5-μm kidney sections were deparaffinized and rehydrated, or HK-2 cells were fixed. The samples were incubated with the TUNEL (green fluorescence) reagent mixture for 1 h at 37 °C, and the nuclei were stained with DAPI (blue fluorescence). We calculated six view fields per specimen under magnification × 400 in a blinded manner to the treatment. Cells expressing both green and blue fluorescence were considered as apoptotic cells. The number of apoptotic cells in each field was counted and the result was expressed as Number/VF.

Immunohistochemistry

For NLRP3 immunohistochemistry, 5-μm kidney sections were incubated with antibody. Then the reactions were developed using an avidin–biotin-HRP complex immunodetection kit and photographed under a light microscope. Relative area (positive staining/background) of positive stained cells were calculated in six random sections from each animal under magnification × 200. The result was expressed as the percentage of positive area.

Immunofluorescence

Immunofluorescence double staining was performed on frozen sections as previously described [14]. Briefly, slices were incubated with guinea pig anti-ASIC1a antibody (1:50) and rabbit anti-aquaporin 1 (AQP1, 1:100). Secondary antibodies were Alexa Fluor 488-conjugated donkey anti-guinea pig IgG (1:100) and Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:100). Visualization was under a confocal microscope.

For NF-κB p65 immunofluorescence, HK-2 cells were fixed, permeabilized and then blocked. The cells were incubated with rabbit anti-NF-κB p65 (1:1000), followed by Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:200), and counterstained with DAPI. Images were acquired using an inverted fluorescence microscope. Detailed information on the primary antibodies is given in Table 2.

Statistical analysis

All values are expressed as mean ± standard deviation (SD) and were analyzed using SPSS. Data were analyzed by one-way ANOVA or two-way ANOVA with subsequent post hoc Student and Newman-Keul’s tests where applicable. Statistical significance was set at P < 0.05.