Animals
Experimental procedures and protocols were approved by the Animal Ethics Committee of Keio University Tokyo, Japan, (approval number: 15015) and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). Female domestic pigs weighing approximately 20 kg and prenatal pigs were purchased from Sankyo Labo Service Corp., Inc., Tokyo, Japan.
Harvest of a porcine kidney and its decellularization
Porcine kidneys were harvested under general anesthesia. Briefly, pigs were sedated using an intramuscular injection cocktail comprising 0.02 mg kg–1 medetomidine and 1 mg kg–1 midazolam. After confirmation of sedation, pigs were intubated with a tracheal tube (outer diameter: 6.5 mm) and the anesthesia was maintained via 2% isoflurane inhalation with an endotracheal tube. Buprenorphine suppository (0.2 mg head–1) was rectally inserted for preoperative analgesia. The operating field was disinfected with 10% w/v povidone. A sterile drape was aseptically affixed to the abdomen. All surgeries were conducted under sterile conditions. Median incisions were performed with a scalpel, and the right and left renal arteries of the pigs were isolated and cannulated with 18 G indwelling needles. The renal arteries, renal veins, and ureters were cut to harvest the kidneys, and the pigs were euthanized by exsanguination. The harvested kidneys were either treated as healthy kidneys (sample 2 in Fig. 2g) or were stored over 24 h at −80 °C for decellularization. Before decellularization, the kidneys were stored at 4 °C for 18 h, then maintained at room temperature for 1 h. The kidneys were then perfused with phosphate-buffered saline (PBS; FUJIFILM Wako Pure Chemical Corp. Osaka, Japan) for 2 h (10 mL min–1) through the cannulated renal artery using a tubing pump (Masterflex L/S with Easy-Load Pump Head III; Yamato Scientific Co. Ltd., Tokyo, Japan) and tubes (Masterflex Tube L/S16; Yamato Scientific Co. Ltd.). Thereafter, samples were perfused with 0.5% SDS (FUJIFILM Wako Pure Chemical Corp.) for 4 h (10–20 mL min–1) for decellularization. Acellular kidneys were perfused with PBS for 3 h (20 mL min–1) for washing. They were sterilized using 25 kGy γ-irradiation (RADIA Industry Co. Ltd., Gunma, Japan), then were stored at 4 °C before use.
Protein analysis of the organ-derived extracellular matrix
The decellularized tissue soaked in PBS was subdivided with a scalpel. Ten milligrams of the subdivided tissue were digested in an aqueous solution (0.001% Trypsin, 10 mM Tris-HCl (pH: 8.8), 0.005% n-octyl glucopyranoside, 0.7 M guanidine hydrochloride) for 12 h. The digested peptides from the tissues were reduced using 5 mM tris(2-carboxyethyl) phosphine (TCEP) at 65 °C for 30 min, then alkylated using 10 mM iodoacetamide at 25 °C for 30 min, followed by purification using a column (MonoSpin C18; GL Science K.K., Tokyo, Japan). Purified peptides (500 ng) were analyzed using a liquid chromatogram (Easy-nLC 1200; Thermo Fisher Scientific K.K., Tokyo, Japan) coupled with a high-resolution quadrupole-orbitrap mass spectrometer (Q-Exactive HF-X; Thermo Fisher Scientific K.K.). The peptides were separated using a C18-separation column (3 μm, 100 µm inner diameter ×12 cm; Nikkyo Technos, Co. Ltd., Tokyo, Japan), where the mobile phases A and B consisted of 0.1% formic acid in water and 0.1% formic in 80% acetonitrile, respectively. The peptide solutions flowed at a constant rate of 300 nL min–1 in mobile phase B at a gradient of 5–40%. The eluent was directly introduced into the liquid chromatography-mass spectroscopy (LC-MS) instrument. MS spectra were recorded in a range of 380 to 1500 m/z. The resolution for MS/MS was set to 15,000 at 200 m/z. All data were analyzed using the Proteome Discoverer 2.2 (Thermo Fisher Scientific K.K.). All MS/MS spectra were searched based on protein sequences presented on the Sus scrofa protein database (NCBI: txid9823). The false discovery rate was set at 1% for the peptide-spectrum match. Peptide level quantitation was performed and label-free quantification was based on the signal intensity of precursors. Among the identified proteins, the proteins related to “extracellular” and “membrane” determined via Gene Ontology were extracted and are summarized in Supplementary Tables 1 and 2, where 100 proteins are shown in descending order of their detection.
Control and organ-derived matrix-sutured model
The pigs were allowed to fast for 12 h until surgery with ad libitum access to water. Surgeries were performed aseptically under general anesthesia according to a previously published method19. Midline incisions were performed, then the right renal arteries and veins were isolated. About one-third of each caudal kidney was resected, and bleeding was controlled with an electric device, such as a bipolar system. The open collecting systems were closed with bladed absorbable sutures (4-0 vicryl; Johnson & Johnson K.K., Tokyo, Japan). The organ-derived matrix was trimmed to a similar size as the resected kidney and was sutured onto the resected surface of the residual kidney with bladed absorbable sutures (4-0 vicryl) at approximately 1.5 cm intervals (Fig. 2a). As a control, the kidney treated with a similar resection and bleeding method was used without the suture of an organ-derived matrix (Fig. 2b). After abdominal closure, the pigs were awakened from anesthesia and were administered with an analgesic (0.2 mg buprenorphine head–1 day–1) and an antibiotic (20 mg kg–1 cefalexin (Keflex), Shionogi & Co. Ltd., Osaka, Japan) for 1 week. After 28 days, the pigs were sacrificed under general anesthesia, and their kidneys (samples 1, 3, and 4 in Fig. 2g) were harvested. The resection rate was calculated as follows: resection rate (%) = major axis of the resected kidney (mm) × 100 (%) / major axis of the native kidney (mm). After harvesting the kidneys, the major axes of both the control and matrix-sutured kidneys were also measured.
Histological analysis
After the harvested kidney samples were fixed with 4% paraformaldehyde (PFA) for 24 h, they were embedded in paraffin. They were then sliced into 4, 2, and 3 μm thick sections using a standard tissue processor. The first slice was stained with H&E (Sigma-Aldrich Co. LLC., MO, USA). The second was stained with Azan (Muto Pure Chemicals Co. Ltd., Tokyo, Japan). The third was used in immunohistochemical analysis. H&E and Azan staining were performed using standard protocols. Macro-scale montage images of H&E staining were created by merging low-magnification field images using Adobe Photoshop (Adobe Inc., CA, USA) and BZ-X800 viewer (KEYENCE Corp., Osaka, Japan).
Immunohistochemical analysis
The sample sections were deparaffinized and rehydrated with xylene, diluted ethanol aqueous solutions (100, 90, 80, and 70%), and distilled water. The antigens in each sample were retrieved with citrate buffer (Agilent Technologies, CA, USA) at 120 °C for 15 min. The samples were stored at room temperature for 30 min. Thereafter, samples were incubated with blocking solution (N102; NOF Corp., Tokyo, Japan) for 30 min at room temperature to inhibit nonspecific reactions. The blocked samples were incubated with a primary antibody solution at 4 °C overnight. Primary antibodies used were CD31 (50:1; Abcam, Cambridge, UK), nephrin (1,000:1; Abcam), AQP1 (500:1; Abcam), E-cadherin (50:1; Abcam), fibronectin (1,000:1; Abcam), Ki67 (50:1; BD Biosciences, NJ, USA), collagen type IV (500:1; Abcam), laminin (50:1; Abcam), WT-1 (10:1, Aviva Systems Biology, CA, USA), Sall1 (50:1, Biorbyt, Cambridge, UK), Six2 (200:1, Proteintech Group, Inc., IL, USA), collagen type I (50:1, Abcam), vimentin (100:1, Dako, CA, USA), αSMA (200:1, Sigma-Aldrich Co. LLC.), SLC12A3 (50:1, Bioss Antibodies, MA, USA), and AQP2 (50:1, Bioss Antibodies). After washing, secondary antibodies (DyLight 488, Alexa 488, or Alexa 568; 1,000:1, Abcam) were added and incubated for 1 h at room temperature. The slides were washed and mounted with a 4’,6-diamidino-2-phenylindole (DAPI) solution (Vector Laboratories, Inc., CA, USA). Images were acquired using a fluorescence microscope (EVOS Cell Imaging System; Thermo Fisher Scientific K.K., BZ-X810; KEYENCE Corp., ECLIPSE Ni, and DS-Ri2; Nikon Corp., Tokyo, Japan).
SEM
The kidney was harvested 28 days after the implantation of the organ-derived matrix. In the kidney, the parts of the matrix and residual kidney were fixed with 2.5% glutaraldehyde in HEPES for 24 h, then rinsed with HEPES. After the samples were subjected to 1% tannic acid at 4 °C for 2 h, they were fixed with 1% OsO4 in HEPES for 2 h. The fixed samples were dehydrated with a series of diluted ethanol aqueous solutions (50–100%), then substituted with t-butyl alcohol. After freeze-drying, they were osmium-coated. SEM images were obtained using a field emission-scanning electron microscope (SU6600; Hitachi High-Technologies Corp., Tokyo, Japan).
TEM
Similar to SEM experiments, samples were harvested. The samples were fixed with 2.5% glutaraldehyde in HEPES for 24 h. After the samples were rinsed with HEPES, they were subjected to 1% tannic acid at 4 °C for 1–2 h. The samples were then fixed with 1% OsO4 in HEPES for 2 h. The fixed samples were dehydrated with a series of diluted ethanol aqueous solutions (50–100%). They were substituted overnight with epoxy resin (Okenshoji Co., Ltd., Tokyo, Japan) graded with n-butyl glycidyl ether. On the following day, the samples were embedded into 100% epoxy resin at 4 °C for 48 h, followed by polymerization at 60 °C for 72 h. Ultrathin sections at 70 nm thickness were prepared on copper grids (Veco Specimen Grids; Nisshin-EM, Tokyo, Japan) using an ultramicrotome (Leica UC7; Leica Biosystems, Wetziar, Germany) and stained with uranyl acetate and lead citrate each for 10 min. TEM images were obtained under 100 kV using JEM-1400Plus (JEOL Ltd., Tokyo, Japan).
CT, angiography, and urography
Plain- and contrast-enhanced abdominal CT scans (Optima CT660 Advance; GE Healthcare Japan Corp., Tokyo, Japan) were performed 28 days after the implantation of the organ-derived matrix. Scanning was performed under general anesthesia using the following parameters: 120 kV, 150 mA, 40 mm beam collimation, 1 s rotation time, and 1.25 mm slice thickness. According to a phase-contrast injection protocol, a power injector (Stellant CT Injection System; Nihon Medrad K.K., Osaka, Japan) was used. Iopamidol (Iopamiron 300 injectable; Bayer Yakuhin Ltd., Osaka, Japan) was administered intravenously at 600 mg iodine per kg body weight for 15–20 s. Scanning was performed before (plain) and 10, 20, 120, and 180 s after administration of the contrast agent. All data were obtained in Digital Communications in Medicine (DICOM) format and were analyzed using OsiriX Lite ver. 10.0.2 (Pixmeo Sàrl, Bernex, Switzerland). Cross-sectional color images were obtained using the Color Look-Up Tables (CLUT) function with the French setting. Regions of interest (ROI) for the renal cortex, renal pelvis, and implanted organ-derived matrix were used to calculate the mean and standard deviation (SD) values of the CT numbers (Houncefield unit, HU). Each ROI was set manually.
Angiography and urography were performed using mobile C-arm equipment (ARCADIS Avantic; SIEMENS Healthcare K.K. Tokyo, Japan). An iodinated intravenous contrast agent (Omnipaque 350; Daiichi Sankyo Co. Ltd., Tokyo, Japan) was injected through the renal artery and ureter.
Gene expression
After harvest, the kidney samples were immediately immersed in RNAlater stabilization solution (Thermo Fisher Scientific K.K.) for gene expression analysis. Samples were stored at −20 °C until total RNA extraction. Each sample was homogenized, then total RNA was isolated with a commercially available column-based purification kit (RNeasy Mini; QIAGEN K.K., Tokyo, Japan). RNA was measured using NanoDrop spectrophotometer (Thermo Fisher Scientific K.K.). Complementary DNA (cDNA) was synthesized from 1 μg total RNA per sample with PrimeScript RT Master Mix (TaKaRa Bio Inc., Shiga, Japan) and QuantiTect Reverse Transcription Kit (QIAGEN K.K.). Gene expression was analyzed using a commercially available mRNA array kit (RT2 Profiler PCR array; QIAGEN K.K.) combined with RT2 SYBR Green qPCR Mastermix (QIAGEN K.K.) or PowerUP SYBR Green Mastermix (Thermo Fisher Scientific K.K.) using ViiA7 real-time PCR system (Thermo Fisher Scientific K.K.). The PCR array contained primers for genes encoding ECMs, cellular adhesion molecules, inflammatory cytokines, chemokines, growth factors, and signal transduction proteins. The PCR array data were analyzed using the QIAGEN Data Analysis Center website (https://www.qiagen.com/ca/shop/genes-and-pathways/data-analysis-center-overview-page/). A clustergram for the genes was established automatically on the website.
Statistics
Data were processed using GraphPad PRISM v. 7.0d for Mac (GraphPad Software Inc., CA, USA). Differences between pairs of groups, including resection rate (n = 4) and size of the sacrificed kidney (n = 4), were analyzed using the Mann-Whitney unpaired t-test. The resection rate and size of the sacrificed kidneys were determined from distinct samples. Two-sided statistical tests were performed in all analyses and significance was set at P < 0.05.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
