2.1 Animal model and AnxA5 treatment
Male Sprague Dawley (SD) rat (8 weeks, 280–300 g) were purchased from Shanghai Lab Animal Research Center (Shanghai, China). Establish a bilateral renal ischemia-reperfusion injury model in rats. The bilateral renal pedicles of rats were exposed and clamped for 60 min to induce ischemia [21]. Then, the clamps were released. The animals were randomly divided into 6 groups, which consisted of the Control group, Sham group, IRI group, IRI + low AnxA5 (40 μg/kg) group, IRI + medium AnxA5 (200 μg/kg) group, and IRI + high AnxA5 (1000 μg/kg) group. Each group was assigned five rats. AnxA5 was purchased from Novoprotein (China). The AnxA5 treatment group injected AnxA5 through the tail vein before releasing the vascular clamp. Fresh blood and urine from all rats 1 day before IRI and 1, 3, 7 days after reperfusion was collected (Fig. 1 A). The animals were sacrificed 7 days later. Animals without clamping the renal pedicle were used as controls. The rats during the surgery were placed on a heating mat at 32℃. All surgical procedures are performed in a constant temperature operating room at 28℃.
2.2 Assessment of Renal Function
Blood was centrifuged by 20 min of centrifugation at 2000 ´g. Blood urea nitrogen (BUN), creatinine (Scr) and uric acid (UA) were measured with an automated analyzer (Shenzhen, China). Urine protein was detected using the Urinary protein quantification kit (Jiancheng, China).
2.3 Histopathological Analysis
Rat right kidney specimens were fixed in 4% paraformaldehyde for 24 h, graded with alcohol dehydration, and embedded in paraffin. Paraffin embedded tissue was cut into 4 μm thickness sections and performed hematoxylin and eosin (HE), periodic acid-silver metheramine (PASM), and Masson staining. Collagen deposition was scored semi-quantitatively by Image-Pro Plus version 6.0 (Medium Cybernetics, Bethesda, MD, USA).
2.4 Immunohistochemistry (IHC)
The 4 μm-thick sections of rat kidney tissues were deparaffinized and rehydrated with xylene and de-escalated ethanol. Antigen retrieval was conducted in sodium citrate buffer. To block endogenous peroxidase activity, the slices were incubated in 3% hydrogen peroxide in methanol for 20 min. After being washed with phosphate buffered saline (PBS), the sections were blocked in PBS containing 2% normal goat serum, 5% BSA, and 0.1% triton-X. The slices were then incubated with primary antibodies overnight at 4 ℃, followed by incubation with the secondary antibody (goat anti-rabbit, Abcam, UK) for 1 h at room temperature. DAB Kit was used to develop colors. The slices were counterstained with hematoxylin. The primary antibodies used in this study are listed below, including kidney injury molecule 1 (KIM-1) (NBP1-76701, 1:400, Novus), neutrophil gelatinase-associated lipocalin (NGAL) (ab216462, 1:1000, Abcam).
2.5 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining
Renal tissue sections were fixed in xylene, hydrated with a gradient of ethanol, and permeabilized with proteinase K for 30 min. Subsequently, samples were subjected to stain with TUNEL reagent (Roche,Switzerland) for 1 h at 37°C. The brown stained cell nuclei were identified as TUNEL-positive.
2.6 Cell culture and IRI treatment
Human proximal tubular cells (HK2) were purchased from American Type Culture Collection (ATCC, USA). HK2 cells were cultured in Dulbecco's modified eagle medium (DMEM, Gibco, USA) mixed with 10% fetal bovine serum (FBS, Gibco), and 1% penicillin-streptomycin (Sigma-Aldrich, USA) in an incubator of 5% CO2 at 37℃. All culture media should be changed every 2 d. For the IRI treatment, HK2 cells were cultured for 12 h under hypoxic conditions (1% O2, 94% N2, and 5% CO2) in medium without nutrients (glucose-free, serum-free) to induce hypoxic injury. Then, replace with fresh normal culture medium containing nutrients during reoxygenation, and the plates were moved to a cell incubator (5% CO2 and 95% air) for 24 h. The control group cells were cultured in complete medium in a conventional incubator (5% CO2 and 95% air), and the frequency of medium replacement was consistent with that of the experimental group cells [22].
2.7 Cell viability assay
HK2 cells were inoculated into 96-well plates at a density of 5×103 cells/well and cultured for 24 h in DMEM (supplemented with 10% FBS, 1% penicillin-streptomycin). Add AnxA5 according to grouping while changing the culture medium after reoxygenation. On the one hand, different concentrations of AnxA5 (0, 5, 50, 100 μg/ml) were used to treat normal HK2 cells for 24 h to determine the cytotoxicity of AnxA5. On the other hand, different doses of AnxA5 (0, 5, 50, 100 μg/ml) were used to treat HK2 cells after IRI treatment for 24 h to verify the protective effect of AnxA5. To verify whether the mutant M23 of AnxA5 has a protective effect, 50 μg/ml of normal AnxA5 and M23 were respectively used to treat HK2 cells after IRI for 24 h, followed by cell viability testing. A CCK-8 Assay Kit (Dojindo, Japan) was utilized to test cell viability. CCK-8 absorbance was tested at 450 nm in each well on a microplate reader.
2.8 Lactate dehydrogenase (LDH) release assay
Membrane permeability was evaluated using the LDH Cytotoxicity Assay Kit (Beyotime, China). HK2 cells were inoculated into 96 well plates and cultured normally for 24 h. After IRI treatment, the cells were treated with different doses of AnxA5 (0, 5, 50, 100 μg/ml) for 24 h. When verifying the effect of Ca2+ on AnxA5, AnxA5 was used to treat cells under reoxygenation conditions for 6 h, and the supernatant of different treatment groups was taken for LDH detection. When comparing the effects of normal AnxA5 and M23 on LDH release rate, corresponding tests were performed on HK2 cells after 24 h of treatment. The LDH level in the supernatant was measured according to the instructions of the kit. Then measure the absorbance at 490nm using a microplate reader.
2.9 Flow cytometry analysis
Cell apoptosis was assessed by flow cytometry using the Apoptosis and Necrosis Detection Kit with YO-PRO-1 and PI (Beyotime, China) according to the manufacturer's instructions. In short, HK2 cells were reoxygenated for 24 h after hypoxia, during which the experimental group was treated with different concentrations of AnxA5 (0, 5, 50, 100 μg/ml). After reaching the experimental endpoint, cells from different intervention groups were collected and washed twice with PBS. Then, cells were stained with 499 μL binding buffer containing 0.5 μL YO-PRO-1 and 0.5 μL PI under dark and room temperature conditions for 20 min. The apoptotic cells were detected with a FACS flow cytometer (BD, Germany). YO-PRO-1 single positive cells are apoptotic cells, while PI positive cells are necrotic cells.
Further validate the effect of AnxA5 on early cell membrane permeability and its binding form with damaged cells, perform fluorescence staining on cells treated with different concentrations of AnxA5 (0, 5, 50, 100 μg/ml) for 2 h. Annexin V-PE (Beyotime, China) was used for detecting the binding of AnxA5 to cell membranes, and cells were stained with 194.5μl binding buffers containing 5 μL Annexin V-PE and 0.5 μL YO-PRO-1. After incubating at room temperature in the dark for 20 min, the FACS flow cytometer was used for detection.
2.10 Real-time Quantitative PCR (RT-qPCR) analysis
In accordance with the manufacturer's instructions, the total RNA was isolated by using TRIzol reagent. cDNA was synthesized with 1 mg of total RNA. RT-qPCR uses a reaction volume of 20 μl and was carried out on QuantStudio™ 6 Flex system (Thermo Fisher Scientific, MA, USA) using SYBR Green qPCR master mix. The conditions of RT-qPCR were as follows: 95°C for 5 min, 15 s 95 °C, 1 min 60 °C, a total of 40 cycles. The cycle threshold (Ct) value was collected and normalized to the GAPDH level. The mRNA level relative to each target gene was calculated using the 2−ΔΔCt method. The Total RNA Extraction Reagent, Color Reverse Transcription Kit, 2 × Color SYBR Green qPCR Master Mix were purchased from EZBioscience (Suzhou, China). Full list of primers is listed below, including KIM-1 (Forward primer 5‘- ATAGTCTACTGACGGCCAA-3’, Reverse primer 5‘- GGCTGCTAAATGAAACACT-3’), NGAL (Forward primer 5‘- GTGAGCACCAACTACAACCAG-3’, Reverse primer 5‘- AGCTCCTTGGTTCTCCCGTA-3’), GAPDH (Forward primer 5‘- CACCATCTTCCAGGAGCGAG-3’, Reverse primer 5‘- TGATGACCCTTTTGGCTCCC-3’).
2.11 AnxA5 binding assay
The control group cells were cultured normally, while the experimental group HK2 cells were cultured for 24 h after hypoxia and reoxygenation. After washing the cells twice with PBS, incubate cells with 195 μL binding buffers containing 5 μL Annexin V-FITC (Beyotime, China) under dark room temperature conditions for 20 min. After incubation, clean the cells again with PBS and reserve 0.5 mL of PBS in the well plate to prevent the cells from drying out. Observe the sample under an inverted fluorescence microscope (ZEISS, Germany).
2.12 Statistical Analysis
In vitro experiments were repeated at least three times. Data were expressed as the mean ± SEM. One-way ANOVA was used for multiple comparisons using SPSS version 22.0 (SPSS, Inc., IL, USA). Single groups were compared by an unpaired two-tailed t-test. P < 0.05 was considered significant.