Decellularized Rat Tubules for tissue engineering

Decellularized Rat Tubules for tissue engineering | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Decellularized Rat Tubules for tissue engineering Qianfeng Jia, Kailin Li, Tongyan Liu, Feng Kong, Shengtian Zhao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6145935/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chronic kidney disease (CKD) remains a global health challenge, with tissue engineering strategies like decellularized scaffolds offering potential solutions for functional renal regeneration, yet hindered by the complexity of whole-organ recellularization. This study presents a microscale approach utilizing decellularized rat renal tubules to address these limitations. Renal tubules were microdissected from rat kidneys and decellularized with 0.5% sodium dodecyl sulfate (SDS), followed by structural and compositional characterization through immunofluorescence, transmission electron microscopy (TEM), DNA quantification, and collagen IV ELISA. Results demonstrated successful removal of cellular components while preserving tubular basement membranes and extracellular matrix (ECM) architecture. TEM confirmed ultrastructural integrity. This work establishes a reproducible method to generate acellular renal tubule scaffolds with native ECM properties, providing a critical platform for studying cell-ECM interactions, disease modeling, and drug screening, thereby advancing targeted renal tissue engineering applications. Decellularization Renal tubule Extracellular matrix Tissue engineering Chronic kidney disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Chronic kidney disease (CKD) has become a global public health problem with increasing incidence rates 1 . CKD progresses to end-stage renal failure(ESRD) if the kidney function further worsens. The effective therapy for ESKD includes dialysis or kidney transplantation. Dialysis may lead to some diseases and reduce quality of life. Kidney transplantation is the best treatment for ESRD. However, there is a great shortage of donor kidneys 2 . After kidney transplantation, there is a need for the continuous use of immunosuppressants 3 . With an increasing patients diagnosed with ESRD, tissue engineering and regenerative medicine strategies to reconstruct kidney structures and restore kidney function have been great popular research areas. Although modeling the kidney in vitro is challenging due to its complex structure and the intricate construct of many cell types, lots of tries have been made for kidney regeneration or mimicking kidney function, such as decellularized kidney scaffold strategy 4 , kidney organoid 5 , 6 , kidney chips 7 , 8 , etc. In recent years, stem-cell-derived kidney organoids have emerged as versatile research models to study kidney physiology. These models aim to mimic kidney function in vitro and to overcome the limitations of conventional research models and increase translational value of preclinical experiments 9 . But there are off-target cell types and no vasculatures in organoids, which leads to unmatured kidney function 10 . And there will be a long distance from clinical application. Organ-on-a-chip technology is a microscale engineering approach that enables reproduction of the microarchitecture and functions of human organs, which can model human diseases in vitro and perform drug screening. Although chip models for glomerulus 7 and renal tubules 11 have been reported, it is difficult to generate transplantable kidney, because they can only model partial kidney function and has no nature complicated kidney constructure. Kidney scaffold preserved not only 3D architecture, such as extracellular matrices (ECMs) and vascular networks, but also signaling molecules, such as growth factors and cytokines 4 , 12 . Some studies have demonstrated the efficacy of kidney scaffold in kidney regeneration. However, although the recelluarized kidney scaffolds provided excretory functions in rats, large amounts of cells are still needed for the recellularization process. In our previous studies, we obtained decellularized kidney scaffolds from rats, pigs and humans. However, after obtaining promising results with kidney decellularization, we realize that effectively advancing the recellularization of natural scaffolds is more complex than was previously hoped 13 – 15 . We found that the seeding cells were not well-distributed, and the cells proliferation and differentiation was poor. Therefore, we need smaller units to study the mechanisms of adhesion, proliferation and differentiation of stem cells in the kidneys. The aim of the present study was to establish renal units microdissection process. And we utilize the renal tubules obtained to construct acellular tubule scaffolds. This study will provide a research basis for the subsequent construction of regenerated nephrons, simulation of renal function, drug screening and so on. Results 2.1 Kidney procurement and Microdissection of renal tubules Kidney was retrieved and cut into slices(Fig. 1 A-C). Renal tubule segments were microdisected, including glomerulus, proximal tubule, distal tubule, etc. (Fig. 1 D-F). We can see the proximal tubule directly attached to the glomerulus and the straight part of proximal tubule. These parts of tubule were decellularized. Histology of the tubules showed preservation of tissue architecture and the complete cellular components(Fig. 2 ). Immunohistochemical staining confirmed the presence of key ECM components such as collagen IV in a physiologic distribution. Tubular basement membranes preserved well after microdissecton(Fig. 2 B).E-cadherin and LTL was expressed on the tubule, which indicated that tubular cell was intact(Fig. 2 C). Podocin was expressed in the glomerulus(Fig. 2 D). 2.2 Decellularization of tubules We choose proximal tubule for decellularization. Tubule decellularization was carried out using the detergent method, by washing with 0.5% sodium dodecyl sulfate(SDS).After decellularization, we successfully removed cellular components including the cell membrane, intracellular organelles, and cellular debris from the tubules. During the process of decellularization, we recorded the dynamic changes of the tubules(Fig. 3 C-D).The decellularization protocol preserved tubular basement membranes, which was important for recellularization and renal function(Fig. 3 A-B). The ECM of the decellularized renal scaffold, was preserved as seen by the distribution of specific ECM proteins, collagen IV and fibronectin, which were similar to that in native rat kidney tissue (Fig. 3 A– 3 D). We removed approximately 95% of DNA in comparison to the native organ (Fig. 3 E), while still retaining total collagen levels similar to those in cadaveric kidney tissue (Fig. 3 F). Tubular basement membranes remained preserved, with dentate evaginations extending into the proximal tubular lumen(Fig. 4 B). Materials and Methods 4.1 Experimental animals Healthy male Wistar rats, aged 6–8 weeks and weighing 200-250g, were used as experimental animals. The rats were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Animal License No.: SYXK (Lu) 2020-0022). The experimental protocol was approved by the Research Ethics Committee of the Second Hospital of Shandong University (Approval No.: KYLL-2023-220). All methods were carried out in accordance with relevant guidelines and regulations, and all methods are reported in accordance with ARRIVE guidelines. 4.2 kidney procurement All animals were sacrificed by an overdose injection of pentobarbital (150 mg/kg), and cardiac arrest was confirmed via thoracotomy. A longitudinal abdominal incision was was then made, and we transected the renal artery. The 26 G intravenouscatheters were inserted into the renal artery to remove the blood with 10 ml PBS. The kidneys were then further perfused with 10 ml digestion solution containing 1 mg/ml of collagenase II(Solarbio, C8150). Then the kidneys were removed prepared for microdissection. 4.3 Microdissection We followed the previously published standard protocol for rat kidney tubule segment microdissection 16 , 17 . Kidneys were cut into thin slices and the medulla was removed. The samples were subsequently incubated with the same digestion solution at 37°C for 20 minutes. After the digestion, kidney tubule microdissection was performed under a LEICA MZ 10F stereomicroscope. Each tubule segment was distinguished by its characteristics as previously described 16 . 4.4 Decellularization of tubules The Decellularization process was performed by solution bath method. The microdissected tubules were bathed with 0.5% sodium dodecyl sulfate(SDS) solution. After 2 minutes, the tubules were decellularized. And in order to observe more clearly, 4,6-Diamino-2-phenylindole (DAPI) was used for nuclear counterstaining. We could obtain the dynamic process of decellularization. 4.5 Immunohistochemistry for tubules and decellularized tubules The microdissected tubules were transferred onto a small glass slide coated with 0.2% polylysine. Subsequently, the tubules were fixed with 4% paraformaldehyde, permeabilized using phosphate-buffered saline (PBS) containing 0.3% Triton X-100, and blocked with PBS supplemented with 10% normal horse serum. Following the blocking step with 10% normal horse serum in PBS, the slides were incubated with primary antibodies overnight at 4°C, followed by a 30-minute incubation with a biotinylated secondary antibody. The antibodies utilized in these studies included anti-E-Cadherin (Cell Signaling Technology, #14472), anti-NPHS2 (Abcam, ab229037), and anti-LTL (Vector Laboratories, B-1325). For immunostaining with biotinylated LTL, the Streptavidin/Biotin Blocking Kit (Vector Laboratories, #SP-2002) was employed in accordance with the manufacturer's instructions. Following three washes with PBS, the tissues were incubated with fluorescent secondary antibodies (Alexa Fluor® 488 conjugate and Alexa Fluor® 594 conjugate) for 45 minutes in the absence of light at room temperature. Subsequently, the nuclei were stained with DAPI for a duration of five minutes. Finally, the samples were mounted using an anti-fade mounting medium. 4.6 Detection of Residual DNA in Decellularized Scaffolds Both decellularized scaffolds and natural kidneys underwent freeze-drying utilizing a vacuum lyophilizer. Subsequently, DNA extraction was performed on these samples in accordance with the protocol specified by the DNA extraction kit (TIANGEN, DP304). The quantification of the extracted nucleic acids was conducted using a Nanodrop 2000c spectrophotometer. Comparative analysis of DNA elution efficiency was then carried out between the decellularized scaffolds and the natural kidneys. 4.7 Detection of Collagen IV Content in Decellularized Scaffolds Decellularized scaffolds and native kidney tissues were subjected to freeze-drying using a vacuum freeze dryer. Equivalent masses of decellularized scaffold and native kidney tissue were accurately weighed and homogenized in RIPA lysis buffer. Subsequently, the samples underwent sonication for a duration of 10 minutes and were then centrifuged at 12,000 rpm for 10 minutes. The resulting supernatants were collected for subsequent analysis. Quantification of collagen IV content in the samples was performed in accordance with the manufacturer's protocol for the ELISA kit(CUSABIO, CSB-E08883r). 4.8 Transmission Electron Microscopy Examination of Renal Tubules and Decellularized Tubules A piece of approximately 1mm³ renal parenchyma was microscopically separated to obtain renal tubules, washed with PBS, and transferred to a 1.5ml centrifuge tube. After centrifugation at 1000g for 5 minutes, the renal tubules were observed to precipitate in clumps and were immediately placed in 3% glutaraldehyde fixative (pH 7.4) for 2 hours. Approximately 1mm³ of renal parenchyma from the same kidney was microscopically separated to obtain renal tubules, which were treated with 0.5% SDS for decellularization in a culture dish, followed by PBS washing. The decellularized tubule scaffolds were transferred to a 1.5ml centrifuge tube, centrifuged at 1000g for 5 minutes, and observed to precipitate in clumps, then immediately placed in 3% glutaraldehyde fixative (pH 7.4) for 2 hours. Following standard TEM sample preparation methods, rinsing, 1% osmium tetroxide fixation, rinsing, dehydration, infiltration, and Epon812 embedding were performed sequentially. After semi-thin sectioning, ultra-thin sections of 70-100nm were made using an LKB-V type ultramicrotome. Lead citrate and uranyl acetate were used for electron staining, and observations were made using a JEOL-1200E transmission electron microscope, with recordings done by MORADA-G2. Discussion CKD have been a great burden for the public health. Many researches have been performed for CKD, such as kidney regeneration, disease modeling, or drug screening. Although there remain many difficulties, we report a new approach for investigating the mechanism of recellularization process, disease modeling or drug screening. In our study, we microdissected the kidney tubules and obtained glomerulus and tubules. Then we decellularized the proximal tubules and got the decellularized tubules. And after immunization, we confirmed that the cells were removed and tubule membrane was retained. Many fast and efficient procedures for kidney decellularization have been proved to be effective 12 , 18 . And our previous studies also proved that 0.5% SDS could lead to the decellularization of the kidney 19 . These findings allow us to maintain vascular structure and ECM composition, which is very important for implantable regenerated kidney. But effective cell seeding of entire organ scaffolds is actually a major task 20 . Some studies were performed in the directions with the recellularization of rat kidney scaffolds 4 , 21 . However, small amount of cells were observed in the renal units in these studies. Our previous studies also proved this point 13 – 15 , 19 . Low cells attachment and uneven distribution was observed in the nephron. The major reason for the limited seeding efficiency might come from the complicated kidney structure and reduced hydraulic permeability of the tubular, or vascular membranes after losing some cellular components of the scaffold 22 . And billions cells would be necessary to construct a whole kidney, which would occlude the vascular or tubular lumen when the infusion would be conducted in a short time period. So our study demonstrated that small scale tubules can be decellularized. The research is of great significance. Firstly, at the microscopic nephron level, a decellularized renal tubular scaffold was constructed, capable of supporting cell-based regeneration of renal tubules. The study may investigate the adhesion, proliferation, and differentiation patterns of stem cells within the decellularized scaffold. Compared with the whole kidney scaffold, the scale of the tubular was very small, which just needs few cells to fill the tubular cavity. So we could observe the change of stem cells more conveniently. And this may help us to find new ways for whole kidney scaffold recellularization. Secondly, recellularized renal tubules allows for disease modeling and drug screening. In previous studies about tubule microdissection, most of them were focused on their transport activity or RNA expression 23 , 24 . And compared with current renal tubular chips and tubuloids, the decellularized renal tubular scaffold preserves the natural membrane structure of renal tubules as well as their 3D architecture, thereby maximizing the simulation of renal tubular function. Although tubuloids are obtained from primary adult cells, immaturity can be a limitation for certain applications and research questions. And they need more stimuli to reach near human kidney expression levels 25 , 26 . A fundamental advance in tubule on-a-chip technology is a microfluidic device that provides a mechanical stimulus and fluid shear stress, which can enable the reproduction of physiological and transepithelial activities 11 , 27 . But they still can not mimic the natural tubule membrane and 3D structure, which may affect the effect of researches. So we obtained the decelluarized tubules, which may construct new models for tubule regeneration, disease modeling or drug screening. But there are still some limitations in our study. Firstly, in our study, we didn’t perform recelluarization study. In fact, we made some experiments combing decellularized tubules and proximal tubule epithelial cells to coculture. Although the cells can live well, which means the decellularized tubules have good compatity, the cells could not attach with the tubules spontaneously. So in our future studies, we will make a perfusion device to perform cell seeding. Secondly, it is difficult to transfer the decellularized tubules, which was very fragile and stick. So in next studies, we will find new ways to transfer and fix the decellularized tubules. In summary, we report a method to decellularize the tubules. And our decellularization protocol achieved complete tubule cell removal and successfully preserved the architecture of the extracellular matrix. Although there are some limitations, this method may useful in whole kidney regeneration, disease modeling and drug screening. Declarations Ethics approval and consent to participate The experimental protocol was reviewed and approved by the Scientific Research Ethics Committee of the Second Hospital of Shandong University (Approval No.: KYLL-2023-220). Consent for publication Not applicable. Availability of data and materials The original contributions presented in the study are included in the article. Competing interests The authors declare no competing interests. Funding This research was supported by Shandong Postdoctoral Science foundation(SDCX-ZG-202400062), National Natural Science Foundation of China (82370706), the Training Fund of the Second Hospital of Shandong University (No. 2022YP94). Authors’ contributions Guan Yong and Zhao Shengtian conceived the project. Yang Xianzhen and Li Kailin designed and performed the experiments. Jia Qianfeng also performed the experiments. Liu Tongyan analyzed data collected from the experiments. Guan Yong wrote the manuscript. Kong Feng supervised the project. All authors read and approved the final manuscript. Acknowledgements Not applicable. References Hill NR, Fatoba ST, Oke JL, et al. Global Prevalence of Chronic Kidney Disease - A Systematic Review and Meta-Analysis. 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Embryonic stem cells proliferate and differentiate when seeded into kidney scaffolds. J Am Soc Nephrol Nov. 2009;20(11):2338–47. 10.1681/ASN.2008111196 . Hsu CY, Chi PL, Chen HY, et al. Kidney bioengineering by using decellularized kidney scaffold and renal progenitor cells. Tissue Cell Feb. 2022;74:101699. 10.1016/j.tice.2021.101699 . Cheng CJ, Nizar JM, Dai DF, Huang CL. Transport activity regulates mitochondrial bioenergetics and biogenesis in renal tubules. FASEB J May. 2024;31(10):e23703. 10.1096/fj.202400358RR . Limbutara K, Chou CL, Knepper MA. Quantitative Proteomics of All 14 Renal Tubule Segments in Rat. J Am Soc Nephrol Jun. 2020;31(6):1255–66. 10.1681/ASN.2020010071 . Olde Hanhof CJA, Dilmen E, Yousef Yengej FA, et al. Differentiated mouse kidney tubuloids as a novel in vitro model to study collecting duct physiology. Front Cell Dev Biol. 2023;11:1086823. 10.3389/fcell.2023.1086823 . Lindoso RS, Yousef Yengej FA, Voellmy F, et al. Differentiated kidney tubular cell-derived extracellular vesicles enhance maturation of tubuloids. J Nanobiotechnol Jul. 2022;15(1):326. 10.1186/s12951-022-01506-6 . Vriend J, Peters JGP, Nieskens TTG, et al. Flow stimulates drug transport in a human kidney proximal tubule-on-a-chip independent of primary cilia. Biochim Biophys Acta Gen Subj Jan. 2020;1864(1):129433. 10.1016/j.bbagen.2019.129433 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6145935","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440428083,"identity":"6678147c-1df1-4a3f-820c-4f08db8cfed5","order_by":0,"name":"Qianfeng Jia","email":"","orcid":"","institution":"Department of Urology, Yantai Affiliated Hospital of Binzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qianfeng","middleName":"","lastName":"Jia","suffix":""},{"id":440428084,"identity":"f81d2fc6-a83b-4ec0-9d65-df3dfbe66fe8","order_by":1,"name":"Kailin Li","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine, First Clinical Medical College","correspondingAuthor":false,"prefix":"","firstName":"Kailin","middleName":"","lastName":"Li","suffix":""},{"id":440428085,"identity":"cf225b5d-c9a0-42d4-8001-6a962d4c0253","order_by":2,"name":"Tongyan Liu","email":"","orcid":"","institution":"Department of Ultrasound, The Second Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Tongyan","middleName":"","lastName":"Liu","suffix":""},{"id":440428086,"identity":"e2132a45-a6f7-4772-a656-fcefed1cb9e2","order_by":3,"name":"Feng Kong","email":"","orcid":"","institution":"Shandong Provincial Engineering Laboratory of Urologic Tissue Reconstruction","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Kong","suffix":""},{"id":440428087,"identity":"97fe43c9-b751-44b9-84e1-f9c3e6a4c046","order_by":4,"name":"Shengtian Zhao","email":"","orcid":"","institution":"Department of Urology, Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Shengtian","middleName":"","lastName":"Zhao","suffix":""},{"id":440428088,"identity":"72b49417-0594-4520-b464-d37ce26f173b","order_by":5,"name":"Xianzhen Yang","email":"","orcid":"","institution":"Department of Urology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xianzhen","middleName":"","lastName":"Yang","suffix":""},{"id":440428089,"identity":"18b503fc-e72e-4acd-8c4d-f88567122d56","order_by":6,"name":"Yong Guan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYHCDBMYHCRU1pGlhNnhw5hhpWtgkH7YwE1YnH5H87DFPxR27+e05ZhWJDWwM/O3dCXi1GN5IMzfmOfMsecOZN2Y3EnfIMEicObsBv5YZCWbSvG2Hkw0kcoBazrAxGEjkEtKS/g2sRX5GjllBYhszYS3yQMNBWuwYbuSYMRClxYDnTZnknDOHEwzOPCuWSDhzjIegX+Tb07dJvKk4bC/fnrzx44+KGjn+9l4CthxgYGDiYWBIbIAK8OBVDrYFqJTxBwODPUGVo2AUjIJRMHIBANOpSvpjS5hhAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Urology, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yong","middleName":"","lastName":"Guan","suffix":""}],"badges":[],"createdAt":"2025-03-03 11:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6145935/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6145935/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80324894,"identity":"35d997e3-5555-4c12-bdd0-e5a9dfb7098c","added_by":"auto","created_at":"2025-04-10 14:09:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1199822,"visible":true,"origin":"","legend":"\u003cp\u003eMicrodissection of renal tubules. A. Kidneys were cut into thin slices B. The medulla was removed. C. The copula was cut into 1mm\u003csup\u003e3\u003c/sup\u003e slices. D-E. The microdissected tubules. F. The microdissected glomerulus.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6145935/v1/27a69a638169538c8ad4cc62.png"},{"id":80324895,"identity":"a802f8ad-9bb1-48fc-9fc4-c0bd4ba2e3dd","added_by":"auto","created_at":"2025-04-10 14:09:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":667495,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence micrographs of the tubule. The figure showed the tubules structure was completely preserved. Collagen IV(red) showed the membrane was intact.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6145935/v1/7df7b612c1e37a8793d4ebe2.png"},{"id":80325521,"identity":"49de7f8e-767c-467b-8f8c-339db2b555d9","added_by":"auto","created_at":"2025-04-10 14:17:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":419153,"visible":true,"origin":"","legend":"\u003cp\u003eDecellularization of Renal Tubules A-B. Bright field showing complete removal of cellular components while retaining the tubular architecture. C-D. Dynamic changes during decellularization: C. before treatment and D. after 0.5% SDS treatment. E. Quantification of residual DNA in decellularized scaffolds compared to native kidney tissue. F. Collagen IV content in decellularized scaffolds and native tissue\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6145935/v1/324040e1ff46368367f5376c.png"},{"id":80324902,"identity":"97182e3b-b936-4bd2-b24b-3b8a3ad6306f","added_by":"auto","created_at":"2025-04-10 14:09:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":700892,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission Electron Microscopy (TEM) Analysis of Renal Tubules A. TEM image of a native renal tubule, showing intact cellular structures and basement membrane. B. TEM image of a decellularized tubule, demonstrating the preservation of the basement membrane (BM) and ECM components.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6145935/v1/996029fae31dd51e032214b5.png"},{"id":81997581,"identity":"d625cdc2-56e9-4b4d-9c9f-e39300c69292","added_by":"auto","created_at":"2025-05-05 18:31:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4444713,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6145935/v1/8850aa6d-5931-4a74-a97c-b0dc24ee2252.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Decellularized Rat Tubules for tissue engineering","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic kidney disease (CKD) has become a global public health problem with increasing incidence rates\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. CKD progresses to end-stage renal failure(ESRD) if the kidney function further worsens. The effective therapy for ESKD includes dialysis or kidney transplantation. Dialysis may lead to some diseases and reduce quality of life. Kidney transplantation is the best treatment for ESRD. However, there is a great shortage of donor kidneys\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. After kidney transplantation, there is a need for the continuous use of immunosuppressants\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. With an increasing patients diagnosed with ESRD, tissue engineering and regenerative medicine strategies to reconstruct kidney structures and restore kidney function have been great popular research areas.\u003c/p\u003e \u003cp\u003eAlthough modeling the kidney in vitro is challenging due to its complex structure and the intricate construct of many cell types, lots of tries have been made for kidney regeneration or mimicking kidney function, such as decellularized kidney scaffold strategy\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, kidney organoid\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, kidney chips\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, etc. In recent years, stem-cell-derived kidney organoids have emerged as versatile research models to study kidney physiology. These models aim to mimic kidney function in vitro and to overcome the limitations of conventional research models and increase translational value of preclinical experiments\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. But there are off-target cell types and no vasculatures in organoids, which leads to unmatured kidney function\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. And there will be a long distance from clinical application. Organ-on-a-chip technology is a microscale engineering approach that enables reproduction of the microarchitecture and functions of human organs, which can model human diseases in vitro and perform drug screening. Although chip models for glomerulus\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and renal tubules\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e have been reported, it is difficult to generate transplantable kidney, because they can only model partial kidney function and has no nature complicated kidney constructure.\u003c/p\u003e \u003cp\u003eKidney scaffold preserved not only 3D architecture, such as extracellular matrices (ECMs) and vascular networks, but also signaling molecules, such as growth factors and cytokines\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Some studies have demonstrated the efficacy of kidney scaffold in kidney regeneration. However, although the recelluarized kidney scaffolds provided excretory functions in rats, large amounts of cells are still needed for the recellularization process. In our previous studies, we obtained decellularized kidney scaffolds from rats, pigs and humans. However, after obtaining promising results with kidney decellularization, we realize that effectively advancing the recellularization of natural scaffolds is more complex than was previously hoped\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. We found that the seeding cells were not well-distributed, and the cells proliferation and differentiation was poor. Therefore, we need smaller units to study the mechanisms of adhesion, proliferation and differentiation of stem cells in the kidneys.\u003c/p\u003e \u003cp\u003eThe aim of the present study was to establish renal units microdissection process. And we utilize the renal tubules obtained to construct acellular tubule scaffolds. This study will provide a research basis for the subsequent construction of regenerated nephrons, simulation of renal function, drug screening and so on.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Kidney procurement and Microdissection of renal tubules\u003c/h2\u003e \u003cp\u003eKidney was retrieved and cut into slices(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). Renal tubule segments were microdisected, including glomerulus, proximal tubule, distal tubule, etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F). We can see the proximal tubule directly attached to the glomerulus and the straight part of proximal tubule. These parts of tubule were decellularized. Histology of the tubules showed preservation of tissue architecture and the complete cellular components(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Immunohistochemical staining confirmed the presence of key ECM components such as collagen IV in a physiologic distribution. Tubular basement membranes preserved well after microdissecton(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).E-cadherin and LTL was expressed on the tubule, which indicated that tubular cell was intact(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Podocin was expressed in the glomerulus(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Decellularization of tubules\u003c/h2\u003e \u003cp\u003eWe choose proximal tubule for decellularization. Tubule decellularization was carried out using the detergent method, by washing with 0.5% sodium dodecyl sulfate(SDS).After decellularization, we successfully removed cellular components including the cell membrane, intracellular organelles, and cellular debris from the tubules. During the process of decellularization, we recorded the dynamic changes of the tubules(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D).The decellularization protocol preserved tubular basement membranes, which was important for recellularization and renal function(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). The ECM of the decellularized renal scaffold, was preserved as seen by the distribution of specific ECM proteins, collagen IV and fibronectin, which were similar to that in native rat kidney tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We removed approximately 95% of DNA in comparison to the native organ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), while still retaining total collagen levels similar to those in cadaveric kidney tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Tubular basement membranes remained preserved, with dentate evaginations extending into the proximal tubular lumen(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Experimental animals\u003c/h2\u003e \u003cp\u003eHealthy male Wistar rats, aged 6\u0026ndash;8 weeks and weighing 200-250g, were used as experimental animals. The rats were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Animal License No.: SYXK (Lu) 2020-0022). The experimental protocol was approved by the Research Ethics Committee of the Second Hospital of Shandong University (Approval No.: KYLL-2023-220). All methods were carried out in accordance with relevant guidelines and regulations, and all methods are reported in accordance with ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.2 kidney procurement\u003c/h2\u003e \u003cp\u003eAll animals were sacrificed by an overdose injection of pentobarbital (150 mg/kg), and cardiac arrest was confirmed via thoracotomy. A longitudinal abdominal incision was was then made, and we transected the renal artery. The 26 G intravenouscatheters were inserted into the renal artery to remove the blood with 10 ml PBS. The kidneys were then further perfused with 10 ml digestion solution containing 1 mg/ml of collagenase II(Solarbio, C8150). Then the kidneys were removed prepared for microdissection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Microdissection\u003c/h2\u003e \u003cp\u003eWe followed the previously published standard protocol for rat kidney tubule segment microdissection\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Kidneys were cut into thin slices and the medulla was removed. The samples were subsequently incubated with the same digestion solution at 37\u0026deg;C for 20 minutes. After the digestion, kidney tubule microdissection was performed under a LEICA MZ 10F stereomicroscope. Each tubule segment was distinguished by its characteristics as previously described\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Decellularization of tubules\u003c/h2\u003e \u003cp\u003eThe Decellularization process was performed by solution bath method. The microdissected tubules were bathed with 0.5% sodium dodecyl sulfate(SDS) solution. After 2 minutes, the tubules were decellularized. And in order to observe more clearly, 4,6-Diamino-2-phenylindole (DAPI) was used for nuclear counterstaining. We could obtain the dynamic process of decellularization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Immunohistochemistry for tubules and decellularized tubules\u003c/h2\u003e \u003cp\u003eThe microdissected tubules were transferred onto a small glass slide coated with 0.2% polylysine. Subsequently, the tubules were fixed with 4% paraformaldehyde, permeabilized using phosphate-buffered saline (PBS) containing 0.3% Triton X-100, and blocked with PBS supplemented with 10% normal horse serum. Following the blocking step with 10% normal horse serum in PBS, the slides were incubated with primary antibodies overnight at 4\u0026deg;C, followed by a 30-minute incubation with a biotinylated secondary antibody. The antibodies utilized in these studies included anti-E-Cadherin (Cell Signaling Technology, #14472), anti-NPHS2 (Abcam, ab229037), and anti-LTL (Vector Laboratories, B-1325). For immunostaining with biotinylated LTL, the Streptavidin/Biotin Blocking Kit (Vector Laboratories, #SP-2002) was employed in accordance with the manufacturer's instructions. Following three washes with PBS, the tissues were incubated with fluorescent secondary antibodies (Alexa Fluor\u0026reg; 488 conjugate and Alexa Fluor\u0026reg; 594 conjugate) for 45 minutes in the absence of light at room temperature. Subsequently, the nuclei were stained with DAPI for a duration of five minutes. Finally, the samples were mounted using an anti-fade mounting medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Detection of Residual DNA in Decellularized Scaffolds\u003c/h2\u003e \u003cp\u003eBoth decellularized scaffolds and natural kidneys underwent freeze-drying utilizing a vacuum lyophilizer. Subsequently, DNA extraction was performed on these samples in accordance with the protocol specified by the DNA extraction kit (TIANGEN, DP304). The quantification of the extracted nucleic acids was conducted using a Nanodrop 2000c spectrophotometer. Comparative analysis of DNA elution efficiency was then carried out between the decellularized scaffolds and the natural kidneys.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Detection of Collagen IV Content in Decellularized Scaffolds\u003c/h2\u003e \u003cp\u003eDecellularized scaffolds and native kidney tissues were subjected to freeze-drying using a vacuum freeze dryer. Equivalent masses of decellularized scaffold and native kidney tissue were accurately weighed and homogenized in RIPA lysis buffer. Subsequently, the samples underwent sonication for a duration of 10 minutes and were then centrifuged at 12,000 rpm for 10 minutes. The resulting supernatants were collected for subsequent analysis. Quantification of collagen IV content in the samples was performed in accordance with the manufacturer's protocol for the ELISA kit(CUSABIO, CSB-E08883r).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Transmission Electron Microscopy Examination of Renal Tubules and Decellularized Tubules\u003c/h2\u003e \u003cp\u003eA piece of approximately 1mm\u0026sup3; renal parenchyma was microscopically separated to obtain renal tubules, washed with PBS, and transferred to a 1.5ml centrifuge tube. After centrifugation at 1000g for 5 minutes, the renal tubules were observed to precipitate in clumps and were immediately placed in 3% glutaraldehyde fixative (pH 7.4) for 2 hours. Approximately 1mm\u0026sup3; of renal parenchyma from the same kidney was microscopically separated to obtain renal tubules, which were treated with 0.5% SDS for decellularization in a culture dish, followed by PBS washing. The decellularized tubule scaffolds were transferred to a 1.5ml centrifuge tube, centrifuged at 1000g for 5 minutes, and observed to precipitate in clumps, then immediately placed in 3% glutaraldehyde fixative (pH 7.4) for 2 hours. Following standard TEM sample preparation methods, rinsing, 1% osmium tetroxide fixation, rinsing, dehydration, infiltration, and Epon812 embedding were performed sequentially. After semi-thin sectioning, ultra-thin sections of 70-100nm were made using an LKB-V type ultramicrotome. Lead citrate and uranyl acetate were used for electron staining, and observations were made using a JEOL-1200E transmission electron microscope, with recordings done by MORADA-G2.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCKD have been a great burden for the public health. Many researches have been performed for CKD, such as kidney regeneration, disease modeling, or drug screening. Although there remain many difficulties, we report a new approach for investigating the mechanism of recellularization process, disease modeling or drug screening. In our study, we microdissected the kidney tubules and obtained glomerulus and tubules. Then we decellularized the proximal tubules and got the decellularized tubules. And after immunization, we confirmed that the cells were removed and tubule membrane was retained.\u003c/p\u003e \u003cp\u003eMany fast and efficient procedures for kidney decellularization have been proved to be effective\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. And our previous studies also proved that 0.5% SDS could lead to the decellularization of the kidney\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These findings allow us to maintain vascular structure and ECM composition, which is very important for implantable regenerated kidney. But effective cell seeding of entire organ scaffolds is actually a major task\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Some studies were performed in the directions with the recellularization of rat kidney scaffolds\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, small amount of cells were observed in the renal units in these studies. Our previous studies also proved this point\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Low cells attachment and uneven distribution was observed in the nephron. The major reason for the limited seeding efficiency might come from the complicated kidney structure and reduced hydraulic permeability of the tubular, or vascular membranes after losing some cellular components of the scaffold\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. And billions cells would be necessary to construct a whole kidney, which would occlude the vascular or tubular lumen when the infusion would be conducted in a short time period.\u003c/p\u003e \u003cp\u003eSo our study demonstrated that small scale tubules can be decellularized. The research is of great significance. Firstly, at the microscopic nephron level, a decellularized renal tubular scaffold was constructed, capable of supporting cell-based regeneration of renal tubules. The study may investigate the adhesion, proliferation, and differentiation patterns of stem cells within the decellularized scaffold. Compared with the whole kidney scaffold, the scale of the tubular was very small, which just needs few cells to fill the tubular cavity. So we could observe the change of stem cells more conveniently. And this may help us to find new ways for whole kidney scaffold recellularization. Secondly, recellularized renal tubules allows for disease modeling and drug screening. In previous studies about tubule microdissection, most of them were focused on their transport activity or RNA expression\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. And compared with current renal tubular chips and tubuloids, the decellularized renal tubular scaffold preserves the natural membrane structure of renal tubules as well as their 3D architecture, thereby maximizing the simulation of renal tubular function. Although tubuloids are obtained from primary adult cells, immaturity can be a limitation for certain applications and research questions. And they need more stimuli to reach near human kidney expression levels\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. A fundamental advance in tubule on-a-chip technology is a microfluidic device that provides a mechanical stimulus and fluid shear stress, which can enable the reproduction of physiological and transepithelial activities\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. But they still can not mimic the natural tubule membrane and 3D structure, which may affect the effect of researches.\u003c/p\u003e \u003cp\u003eSo we obtained the decelluarized tubules, which may construct new models for tubule regeneration, disease modeling or drug screening. But there are still some limitations in our study. Firstly, in our study, we didn\u0026rsquo;t perform recelluarization study. In fact, we made some experiments combing decellularized tubules and proximal tubule epithelial cells to coculture. Although the cells can live well, which means the decellularized tubules have good compatity, the cells could not attach with the tubules spontaneously. So in our future studies, we will make a perfusion device to perform cell seeding. Secondly, it is difficult to transfer the decellularized tubules, which was very fragile and stick. So in next studies, we will find new ways to transfer and fix the decellularized tubules.\u003c/p\u003e \u003cp\u003eIn summary, we report a method to decellularize the tubules. And our decellularization protocol achieved complete tubule cell removal and successfully preserved the architecture of the extracellular matrix. Although there are some limitations, this method may useful in whole kidney regeneration, disease modeling and drug screening.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocol was reviewed and approved by the Scientific Research Ethics Committee of the Second Hospital of Shandong University (Approval No.: KYLL-2023-220).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Shandong Postdoctoral Science foundation(SDCX-ZG-202400062), National Natural Science Foundation of China (82370706), the Training Fund of the Second Hospital of Shandong University (No. 2022YP94).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuan Yong and Zhao Shengtian conceived the project. Yang Xianzhen and Li Kailin designed and performed the experiments. Jia Qianfeng also performed the experiments. Liu Tongyan analyzed data collected from the experiments. Guan Yong wrote the manuscript. Kong Feng supervised the project. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHill NR, Fatoba ST, Oke JL, et al. Global Prevalence of Chronic Kidney Disease - A Systematic Review and Meta-Analysis. 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Biochim Biophys Acta Gen Subj Jan. 2020;1864(1):129433. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbagen.2019.129433\u003c/span\u003e\u003cspan address=\"10.1016/j.bbagen.2019.129433\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Decellularization, Renal tubule, Extracellular matrix, Tissue engineering, Chronic kidney disease","lastPublishedDoi":"10.21203/rs.3.rs-6145935/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6145935/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic kidney disease (CKD) remains a global health challenge, with tissue engineering strategies like decellularized scaffolds offering potential solutions for functional renal regeneration, yet hindered by the complexity of whole-organ recellularization. This study presents a microscale approach utilizing decellularized rat renal tubules to address these limitations. Renal tubules were microdissected from rat kidneys and decellularized with 0.5% sodium dodecyl sulfate (SDS), followed by structural and compositional characterization through immunofluorescence, transmission electron microscopy (TEM), DNA quantification, and collagen IV ELISA. Results demonstrated successful removal of cellular components while preserving tubular basement membranes and extracellular matrix (ECM) architecture. TEM confirmed ultrastructural integrity. This work establishes a reproducible method to generate acellular renal tubule scaffolds with native ECM properties, providing a critical platform for studying cell-ECM interactions, disease modeling, and drug screening, thereby advancing targeted renal tissue engineering applications.\u003c/p\u003e","manuscriptTitle":"Decellularized Rat Tubules for tissue engineering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 14:08:59","doi":"10.21203/rs.3.rs-6145935/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"637c0446-2b16-4615-a8b2-3c5726b0567f","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-05T18:23:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-10 14:08:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6145935","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6145935","identity":"rs-6145935","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}