All animal protocols were approved by the Animal Ethics Committee of the Chinese PLA General Hospital and Military Medical College. Eight- to twelve-week-old male C57BL/6 mice were obtained from the Si Bei Fu Laboratory Animal Company (Beijing, China). Male C57BL/6-TgN (ACTb-EGFP) transgenic mice expressing GFP were obtained from the Model Animal Research Center of Chinese Nanjing University (Nanjing, China). Animals were housed in a temperature-controlled room (22°C±1°C) with a 12-hour light-dark cycle and were given free access to food and water.
Parabiosis was performed based on the methods developed by Donskoy and Goldschneider . The shared circulation created between the two mice was verified in our previously study [23, 24]. Briefly, after anaesthetisation (intraperitoneal injection of 1% pentobarbital sodium at a dose of 30 mg/kg) and sterilisation, the skin and subcutaneous tissue of two mice were cut to expose the subcutaneous muscle. For each pair, the chest and back muscles were isolated in the donor mouse and sutured to the chest and back muscles of the recipient mouse. Then, the edges of the skin were sutured. The details of this procedure are described in Figure S1.
After 3 weeks of parabiosis, the RM model was induced as previously reported. The mice were deprived of water for 24 hours and then administered diluted glycerol (50% v/v in sterile saline) in each hindlimb muscle at a dose of 8 mL/kg following mild sedation with pentobarbital. Blood samples and kidney tissue were collected for further processing 48 hours after the induction of RM.
The mice were divided into three groups: the sham group with sterile saline administration; the RM group with glycerol administration; and the parabiosis + RM group. Three weeks after the parabiosis model was established, the recipient mouse was administered glycerol, and this mouse was defined as the P_RM_R. The other mouse in the parabiosis model supplied exogenous biological renal support and was defined as the P_RM_S.
Verification of the establishment of shared circulation in parabiotic mice
GFP-expressing mice and wild-type mice were used for parabiosis. The successful establishment of shared blood circulation was proven by three methods from our previous studies : (1) a peripheral blood smear test for GFP detection; (2) flow cytometry for the measurement of the GFP+ cell ratio; and (3) small animal in vivo imaging. For further details on the methods, see the supplementary section (Figure S2).
Serum biochemistry analysis
Serum samples were collected and centrifuged at 3000 rpm for 10 minutes and stored at -80°C before analysis. Serum creatinine (Cr) and blood urea nitrogen (BUN) were analysed with an autoanalyser (Cobas 8000; Roche, Mannheim, Germany).
Histopathologic examination and scoring
All tissue sections were independently evaluated by two investigators (XDG and QH) in a blinded manner. Mouse kidneys were ﬁxed in 4% paraformaldehyde, embedded in parafﬁn, sectioned at a thickness of 3 μm and stained with periodic acid–Schiff (PAS). Renal tubular injury was scored by counting the percentage of tubules that displayed cellular necrosis, the loss of the brush border, cast formation and tubule dilatation. Zero represents normal histology and 1 to 5 represent ≤ 10%, ≤ 25%, ≤ 50%, ≤ 75%, and >75%, respectively .
Measurements of SOD, GSH, MDA, CK and PC contents in kidney samples
A 10% homogenate was prepared from the kidney and centrifuged at 730 g at 4°C for 15 minutes to obtain the supernatant. Total superoxide dismutase (SOD), glutathione peroxidase (GSH), malondialdehyde (MDA), creatine kinase (CK) and protein carbonyl (PC) contents were measured with commercial kits according to the manufacturer’s instructions (Beijing Jinenlai Biochemistry Co., Beijing, China).
Measurements of MYO, CK, NGAL, TNF-α, SAA1 and SAA2 contents in serum samples
Mouse plasma was centrifuged at 730 g at 4°C for 10 minutes. Then, the upper serum layer was collected. The contents of MYO, CK, NGAL, TNF-α, SAA1 and SAA2 were measured with commercial kits according to the manufacturer’s instructions (Jinenlai Biochemistry Co., Beijing, China).
Western blot analysis
Frozen kidney tissues (100 mg) and 1 mL of RIPA buffer (Thermo Fisher Scientific, Inc.) were homogenised at 12000 g at 4°C for 30 minutes to obtain the supernatant. Equal amounts of proteins were obtained from each sample and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membranes and probed with primary antibodies against the following proteins at 4°C overnight: beta-actin (0061R, 1:2000; Beijing Biosynthesis Biotechnology Co., Beijing, China), cleaved caspase-3 (9664; 1:500; Cell Signaling Technology, Boston, USA), Bcl2 (2876, 1:1000; Cell Signaling Technology), cyclin D1 (60186; 1:1000; Proteintech), and cyclin E1 (11554; 1:1000; Proteintech). The blots were probed with horseradish peroxidase-conjugated IgG (sc-2096 and sc-2963, 1:1000; Santa Cruz Biotechnology, USA). Immunoreactive bands were visualised by enhanced chemiluminescence, and blot signals were analysed by the image analysis software ImageJ 1.52.
Kidney tissue was fixed in 10% formalin overnight, dehydrated, embedded in paraffin, cut into 3-μm-thick sections, and placed on a numbered polylysine-coated glass slide. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL)-positive cells, which were stained brown, were counted under 200x magnification. Nuclei were stained with haematoxylin to observe the characteristics of TUNEL-positive cells. Six to eight fields per section and two to three sections per kidney were examined in each experiment. We calculated the percentage of TUNEL-positive cells relative to the total number of renal tubular cells as the apoptosis rate. The TUNEL assay was performed according to the manufacturer’s instructions (Merck Millipore, Billerica, MA, USA).
Immunohistochemical staining for the detection of CD3 and proliferating cell nuclear antigen (PCNA) in renal tissue was performed on formaldehyde-fixed and paraffin-embedded tissues using the avidin-biotin-immunoperoxidase method. As in our previous study [16, 23], we performed antigen retrieval from sections by microwaving them for 10 minutes in 10 mM sodium citrate buffer (pH 6.0), incubating them in 3% hydrogen peroxide for 30 minutes, and placing them in 1.5% normal goat serum for 40 minutes. Then, the sections were incubated in anti-CD3 (5690, 1:100; Abcam) or anti-PCNA antibodies (Abcam, 18197, 1:1000) overnight at 4°C, followed by immersion in biotin-conjugated goat anti-rabbit IgG for 40 minutes and finally in streptavidin-conjugated peroxidase for 30 minutes. The sections were observed under a microscope.
Immunofluorescence staining for the detection of complement 3 (C3) in renal tissue was performed on formaldehyde-fixed and paraffin-embedded tissues. Slides were air-dried, fixed with methanol/acetone for 10 minutes, and treated with a FITC-conjugated anti-C3 antibody (21337; 1:500; Proteintech) at room temperature for 1 hour. Nuclei were counterstained by DAPI. Five fields on each of three slides per animal were randomly selected for visualisation, and analysis was performed using ImageJ software.
Proteomic sample extraction and LC-MS/MS analysis
Peripheral blood and kidney tissue were collected and digested with trypsin. After isobaric tags for relative and absolute quantitation (iTRAQ) labelling, the tryptic peptides were fractionated with high pH reverse-phase high performance liquid chromatography (HPLC) using a Thermo BetaSil C18 column (5-μm particles, 10-mm ID, and 250-mm length), and then the peptides were subjected to a nanospray ionisation (NSI) source followed by tandem mass spectrometry (MS/MS) in a Q ExactiveTM Plus (Thermo) coupled online to an ultra performance liquid chromatography (UPLC) platform. The resulting MS/MS data were processed using the MaxQuant search engine (v.18.104.22.168). The details of those procedures are provided in the supplementary materials (Supplementary File).
Bioinformatics analysis was conducted with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, functional enrichment analysis and clustering analysis. The details of the software and analysis parameters are provided in the supplementary materials (Supplementary File). A 1.3-fold up- or downregulation was chosen to identify significant protein over- or underexpression, respectively, with a P value lower than 0.05.
All data were analysed using R 3.6.1 software. Data are expressed as the mean and standard deviation (SD). Statistical significance was determined using two-way analysis of variance (ANOVA) or Student’s t test. Statistical graphs were produced with GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). A threshold of P < 0.05 was defined as statistically significant.