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Decellularization of Canine Kidney for Three-Dimensional Organ Regeneration

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Decellularization of Canine Kidney for Three-Dimensional Organ Regeneration Veterinary World, EISSN: 2231-0916 RESEARCH ARTICLE Available at www.veterinaryworld.org/Vol.13/March-2020/9.pdf Open Access Decellularization of canine kidney for three-dimensional organ regeneration Kazuki Tajima1,2, Kohei Kuroda2, Yuya Otaka1, Rie Kinoshita2, Mizuki Kita1, Toshifumi Oyamada3 and Kazutaka Kanai1 1. Department of Small Animal Internal Medicine II, School of Veterinary Medicine, Kitasato University, Towada, Japan; 2. Department of Surgery, Keio University School of Medicine, Shinjuku, Japan; 3. Department of Veterinary Pathology, School of Veterinary Medicine, Kitasato University, Towada, Japan. Corresponding author: Kazutaka Kanai, e-mail: [email protected] Co-authors: KT: [email protected], KKu: [email protected], YO: [email protected], RK: [email protected], MK: [email protected], TO: [email protected] Received: 27-11-2019, Accepted: 04-02-2020, Published online: 12-03-2020 doi: www.doi.org/10.14202/vetworld.2020.452-457 How to cite this article: Tajima K, Kuroda K, Otaka Y, Kinoshita R, Kita M, Oyamada T, Kanai K (2020) Decellularization of canine kidney for three-dimensional organ regeneration, Veterinary World, 13(3): 452-457. Abstract Background and Aim: Kidney regeneration is required for dogs with end-stage renal failure. Decellularization is one of the bioengineering techniques, which involves the removal of all tissue cells and cellular components and conservation of the extracellular matrix (ECM). Studies in rats have shown that decellularized kidney has regenerative potential; however, there are no reports on renal decellularization in dogs. Here, we showed the decellularization of the canine kidney. Materials and Methods: The renal artery of the cadaveric canine kidney was cannulated and the whole kidney was frozen at −80°C. After completely thawing, it was perfused with physiological saline and sodium dodecyl sulfate (0.5%, 6 h) through the cannulated renal artery to achieve decellularization. To assess the efficiency of the decellularization protocol, histological and immunohistochemical analysis of decellularized kidney was performed. Results: The results of hematoxylin and eosin (H and E) staining revealed that the decellularized canine kidney had no apparent cellular components. In addition, 4’,6-diamidino-2-phenylindole (DAPI) staining showed no visible nuclear components within the whole decellularized kidney. Therefore, both H and E and DAPI staining showed decellularization of the canine kidney. Our decellularization protocol also preserved the basement membrane of glomerulus, shown by periodic acid methenamine silver, periodic acid–Schiff, fibronectin, and collagen type IV stain. Conclusion: Our decellularization protocol could eliminate cellular components and remaining native ECM structures of canine kidney. These results could promote further research into canine kidney regeneration, which may be the first small step to regenerate the canine kidney waiting for renal transplantation. Keywords: bioengineering, dog, extracellular matrix, kidney, regeneration. Introduction Therefore, organ regeneration could be possible Kidney disease is an important problem in dogs therapeutics for animals with renal failure. and cats. Chronic kidney disease (CKD) is the most Decellularization is a unique procedure with common kidney disease in dogs and cats, and the esti- a potential ability to overcome organ failure [8,9]. mated prevalence of CKD, according to one study in It involves the removal of all cells while preserving the UK, was 0.37% in dogs [1]. CKD is irreversible extracellular matrix (ECM) [10-12]. These organ-spe- and progressive [2-5], which may eventually lead to cific ECM proteins contribute to cell migration, a terminal loss of renal function. Apart from renal proliferation, and differentiation [13-15]. Hence, transplantation, there is no treatment to improve the these properties enable the decellularized scaffold renal function of patients with end-stage renal fail- of the organ to be a niche for the reseeded cells [8]. ure [6]. Some reports have indicated that old age and This bioscaffold also preserves the three-dimensional severe azotemia may increase mortality rates after structure; therefore, organ regeneration could be renal transplantation [6,7]. Hence, renal transplanta- achieved by recellularization of the original or imma- tion in the early stages is desired to improve progno- ture cells [8]. sis. However, the fundamental shortage of donors has Our group previously reported the decellulariza- tion of organs including the liver, heart, and pancreas limited renal transplantation in veterinary medicine. in pigs, and these organs could achieve recellulariza- Copyright: Tajima, et al. Open Access. This article is distributed under tion [15-18]. In addition, some reports indicated that the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which the possibility of renal regeneration in rats using the permits unrestricted use, distribution, and reproduction in any decellularized kidney; this bioengineering technique medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons also showed a potential for urine production in the recel- license, and indicate if changes were made. The Creative Commons lularized kidney [14,19]. Furthermore, some groups Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this have reported large-scale kidney decellularization in article, unless otherwise stated. animals such as pigs and rhesus monkeys [14,20,21]. Veterinary World, EISSN: 2231-0916 452 Available at www.veterinaryworld.org/Vol.13/March-2020/9.pdf These reports showed that the recellularized organ can cadaveric and decellularized kidneys were immediately potentially regenerate the functional organ. immersed and fixed in 4% paraformaldehyde in phos- The protocol of whole-organ decellularization phate-buffered saline (PBS) for 24 h at 4°C. The fixed relies on the animal species and organ type. Various samples were embedded in paraffin and cut into 4 μm decellularization methods have been reported in the sections. Subsequently, they were deparaffinized in kidney [14,20,21]. Although freezing is widely used xylene, rehydrated in water, and stained with hema- for physical decellularization, it could effectively dis- toxylin and eosin (H and E) or mounted with mount- rupt the cellular membrane and induce cell lysis [11]. ing medium 4’,6-diamidino-2-phenylindole (DAPI) In addition, sodium dodecyl sulfate (SDS) has been (Vector Laboratories, Inc., USA). Using four random reported as one of the most effective chemicals for microscopic photographs of the cadaveric and decellu- decellularization, due to its ability to remove cellular larized kidney, the DAPI-positive area was measured components and remnants [11]. Hence, the combination by ImageJ software (National Institute of Health, of physical and chemical decellularization protocols is Bethesda, USA). In addition, the decellularized kidney used for many organs [16-18]. However, decellulariza- sections also stained with periodic acid methenamine tion of the canine kidney has not been reported. silver (PAM) and periodic acid–Schiff (PAS) by stan- In this study, we performed canine kidney decel- dard protocol [22] and observed by microscopy. lularization using a cadaveric kidney. To the best of Immunohistochemical analysis our knowledge, this is the first report on canine kidney Paraffin-embedded sections of decellularized decellularization. kidney were also used for immunohistochemical anal- Materials and Methods ysis including fibronectin (Merck K.K., Tokyo, Japan) Ethical approval and collagen type IV (LifeSpan Biosciences, Inc., This experimental procedure to use canine Washington, USA). In brief, the slides were rehydrated cadaveric kidneys from euthanized dogs was approved in the same way as histological analysis. To perform by the Animal Care and Use Committee of Kitasato diaminobenzidine staining, the tissue was incubated University (Approval No. 19-006). with H2O2 for 30 min for removing endogenous stain- ing. Antigen retrieval was performed by microwave Harvest and storage of canine kidney for 20 min, and non-specific binding of antibodies was Kidneys of beagle dogs (n=3) euthanized for blocked by normal goat serum (Agilent Technologies reasons not related to this study were obtained. The Japan, Ltd., Tokyo, Japan). Then, the slides were incu- cadaveric kidneys were harvested within 12 h post-eu- bated with anti-fibronectin antibody (1:100) for over- thanasia. To harvest the cadaveric kidneys, all pro- night at 4°C. After washing with PBS, the EnVision cedures were performed under sterile conditions. A labeled polymer-horseradish peroxidase system midline incision was made to open the retroperito- (Agilent Technologies Japan, Ltd.) was used as the sec- neum. The renal artery, renal vein, and ureter were ondary antibody. Peroxidase activity was detected with isolated. The renal artery was cannulated with an 18G diaminobenzidine substrate (Agilent Technologies indwelling needle and the renal vein and ureter were Japan, Ltd.). Alternatively, collagen type IV was cut before harvesting the kidney from the canine’s detected by immunofluorescence staining. Sections body. The harvested kidneys (approximately 5-7 cm were treated for heat-mediated antigen retrieval in in the major axis) were
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