Animals
C57BL/6 mice(age 6-10weeks,weigh 20-30g, male) were purchased from the Shanghai Jiesijie Experimental Animal Co., Ltd. (Shanghai, China). All the animals were acclimatized in a room (12/12 h light/dark cycle; 25 ± 2°C) and allowed free access to diet and water. All animal experiments were approved by Medical Ethics Committee of Shanghai Jiaotong University, and were performed in strict accordance with approved guidelines.
Mice models
Mice were anesthetized with isoflurane and then randomly divided into control, CLP, HC and CLP+HC groups or NCsiRNA, NCsiRNA+CLP and TRPV4siRNA+CLP groups. The cecum was ligated with 4-0 suture and punctured with a 21-gauge needle. 0.2ml faeces was extruded from the puncture sites. All mice were received 1mL 0.9% normal saline subcutaneously after CLP for fluid resuscitation.
Cell culture
The MRGEC was purchased from Creative Bioarray (NY, USA). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM, 4.5 g/L glucose) (Life Technologies, USA) supplemented with 10% fetal bovine serum and antibiotics (100 KU/L penicillin and 100 mg/L streptomycin) in an incubator at 37°C with 5% CO2.
Western blotting
Protein was extracted from cultured cell lysates with RIPA lysis buffer with phosphatase inhibitor (Roche). The protein concentration was determined by BCA protein concentration assay kit. Equal amounts(30-50µg) proteins were subjected to 12% SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with 5% fat free milk at room temperature for 1 hour and incubated by following primary antibodys: ICAM1 (1:1000, Cell Signaling,
USA), VCAM1 (1:1000, Cell Signaling, USA), E-selectin (1:1000, Cell Signaling, USA), eNOS(1:1000), p-eNOS(1:1000, Cell Signaling, USA), NF-κB(1:1000, Cell Signaling,
USA), IκBα(1:1000, Cell Signaling, USA), p-NF-κB(1:1000, Cell Signaling, USA),IRF-3(1:1000, Beyotime, China),p-IRF-3(1:1000, Beyotime, China) at 4℃ overnight. On the second day, after a 1-hour incubation with HRP-conjugated secondary antibodies, the labeled proteins were detected by ECL(enhanced chemiluminescence). Image analysis was performed by Amersham Image680 Scanning machine and quantified intensity with Image J. The optical density was normalized to that of β-actin(1:1000, Beyotime, Haimen, China), which represented as relative optical density.
Renal histology
Kidney tissues of each group were harvested 24 hours after CLP. The samples were washed with PBS, fixed with 4% paraformaldehyde overnight, embedded in paraffin and cut into sections. Sections were stained with hematoxylin and eosin(HE). HE staining was semi-quantitatively graded under microscopy at 200× to evaluate tissue damage. The grader of the histology slides was examined blindly by a pathologist. The percentage of histological changes in the cortex and medulla was scored using a semi-quantitative scale designed to evaluate the degree of the necrosis in tubular and glomerular areas, tubular vacuolization and cast formation on a five point scale based on injury area of involvement as previous study(7). The scale is as follows: 0≤10%; 1=10-25%; 2=25-50%; 3=50-75%; and 4=75-100%.
Immunohistochemistry
The kidney tissues were fixed with 4% formalin, embedded in paraffin and cut into slices. The sections were incubated with the primary antibodies, ICAM1 (1:1000, Abcam, USA), (E-selectin, 1:100; Beyotime, Haimen, China). Every mouse kidney sample was stained in one slide, and ten viewing fields of the cortex in each slide at 200x magnification were randomly captured for next calculation. The positive cell count of one viewing field in glomerulus was calculated by Image J.
Immunofluorescence staining
Mice were anesthetized with isoflurane and perfused through the left ventricle with normal saline followed by 4% paraformaldehyde. After perfusion, kidney tissues were dissected and cut
into sections at 10 µm with Leica freezing microtome. Slices were washed with 0.01 M of PBS and blocked in 2% BSA for 1 hours at room temperature. After blocking, slices were incubated with primary antibody: anti-CD68(1:100, Abcam, USA ), anti-NF-κB(1:100, Cell Signaling, USA), anti-IRF3(1:100, Beyotime, China) followed by the secondary antibody (1:200, Beyotime,China). The nuclei were stained with DAPI. Finally, the fluorescence was detected using light microscopy and fluorescence microscopy. MRGECs were seeded onto circular coverslips and fixed with 4% formaldehyde for 10 min, followed by permeabilization with 0.1% Triton X-100 in PBS. The cells were blocked with 2% BSA at room temperature for 60 min and then incubated with primary anti-VE-cadherin(1:100, R&D systems Inc.,MN, USA), anti-NF-κB(1:100, Cell Signaling, USA), anti-IRF3(1:100, Beyotime, China) at 4°C overnight. Following incubation, samples were washed with PBS and incubated with secondary antibody. The coverslips were mounted using 90% glycerol in PBS, and the fluorescence was detected with light microscopy and fluorescence microscopy.
NO detection
The levels cellular NO were measured by a commercial NO assay kit (Beyotime, Haimen, China), according to the manufacturer’s instructions. Briefly, the cells were lysed and centrifuged. RLU/OD was detected on 540nm by spectrum detection. The NO contents were calculated by constrast with the standard curve.
ROS detection
The levels of kidney and cellular ROS production were evaluated using standardized methods by ROS assay kit, according to the manufacturer’s instructions(Beyotime, Haimen, China). Production of ROS was evaluated by changes in the fluorescence intensity resulted from oxidation of the intracellular fluoroprobe 5- (and -6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA, Molecular Probes, Eugene, OR, USA). ROS contents were observed or detected by confocal or fluorescence microscopy.
The measurement of blood urea and creatine.
Mice blood were collected before kidney harvest and kept at room temperature for 2 hours. Serum was isolated by 3500g for 10 minutes centrifugation. The serum was detected by ELISA blood urean and creatine assay kit(Westang Biotech, Shanghai,China) according to the manufacturer’s instruction.
Doppler hemodynamics
The PeriCam PSI Zoom System was used to collect the blood flow perfusion data. Kidney blood flow was detected by PeriCam PSI Zoom 24 hours after CLP. The mice were anesthetized by isoflurane. Then kidneys were exposed and kidney blood flow was detected by PeriCam PSI Zoom.
Ca2+ image
Cytoplasmic calcium concentration was monitored using Zeiss 710 confocal confocal laser system. Briefly, cells were loaded with 10 mol/L Fluo-4/AM. Ca2+ stores was depleted by treating MRGEC with 4 mol/L thapsigargin for 6 to 8 minutes in a Ca2+-free physiological saline (0Ca2+-PSS), which contained (in mmol/L): 140 NaCl, 5 KCl, 1 MgCl2, 10 glucose, 0.2 EGTA, 5 Hepes, pH 7.4. Ca2+ influx was initiated by applying1 mmol/L extracellular Ca2+. The cells were pretreated with/without HC for 1 hour before experiments. Changes in [Ca2+]i were displayed as a ratio of fluorescence relative to the intensity before the application of extracellular Ca2+ (F1/F0).
siRNA transfection
siRNA and negative control were synthesized by GenePharma (Shanghai, China),siRNA sequences were as bellows: 5’-AAGACUUGUUCACGAAGAAAU-3’; 5’-UUCUUCGUGAACAAGUCUUUG-3’. siRNA transfection in vitro was performed by lipo8000(Beyotime, Shanghai, China), according to the manufactural instructions. The siRNA transfection in vivo was performed as follows: The TRPV4 siRNA or scrambled siRNA (5 mg/kg) was diluted in 50 uL of EntransterTM-in vivo RNA transfection reagent (Engreen Biosystem Co., Ltd., Beijing, China) and 10% glucose mixture according to the manufacturer’s instructions. A volume of 50 uL siRNA or siTRPV4 mix was continuously subjected to mice by intravenous injection 3 days before CLP according to the reagent’s instructions
Data analysis
All data are represented as mean ± SD. Statistical analysis between two groups was performed using student t test, and one-way ANOVA was used to compare the significance among three or more groups. Bonferroni method was used to evaluate the significance conservatively. The calculations and data processing were performed using Sigmaplot 14.0.