Podocyte injury damages podocytes in chimeric organoids

Podocyte injury damages podocytes in chimeric organoids | 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 Article Podocyte injury damages podocytes in chimeric organoids Tomohiro Udagawa, Toshikazu Araoka, Kenji Osafune, Taiji Matsusaka This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7373933/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 We have previously shown that injury to a subset of podocytes can trigger secondary damage in neighboring podocytes, but whether this phenomenon depends on direct intercellular interaction is unknown. To address this, kidney organoids were generated from nephron progenitor cells of two mouse lines: one expressing a receptor for a podocyte-specific immunotoxin and another expressing a tagged ribosomal protein. In chimeric organoids containing a mosaic of these podocyte types, immunotoxin exposure selectively injured the targeted podocytes and also induced secondary injury in adjacent, non-targeted podocytes. This was evidenced by reduced podocin staining and decreased expression of podocyte-specific genes in the non-targeted podocytes. The bystander effect was absent when organoids of each type were cultured separately but in close proximity, indicating that direct cell-to-cell contact within the same glomerular structure is required. These findings show that podocyte injury can propagate locally within kidney organoids, independent of glomerular filtration or other glomerular cell types, and suggest that local podocyte interactions may contribute to the progression of chronic kidney disease. Biological sciences/Cell biology Health sciences/Nephrology Podocytes organoids cell-cell communication bystander effect immunotoxin transgenic mice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Podocyte injury is a critical initiating event in the development and progression of glomerulosclerosis 1 , 2 . In our previous study, we developed a transgenic mouse model in which podocytes express human CD25 (hCD25), allowing selective injury by the hCD25-targeted immunotoxin LMB2 3 . In hCD25-expressing podocytes, LMB2 inhibits protein synthesis and induces apoptotic caspase activation 4 . Despite its rapid clearance from circulation, a single injection of LMB2 results in progressive kidney injury over several weeks. Similar progression has been observed in other models of podocyte injury 5 . In human and animal models, segmental sclerosis lesions are typically localized to a few regions per glomerulus, despite the widespread exposure of podocytes to injurious stimuli. This finding indicates that podocyte injury may spread locally and autonomously within the glomerulus. Thus, we previously generated chimeric and mosaic mouse models in which only a subset of podocytes express hCD25 6,7 . As expected, hCD25(+) podocytes were selectively injured following LMB2 injection. Surprisingly, adjacent hCD25-negative podocytes were also affected, showing reduced nephrin and podocin expression levels and increased desmin staining, despite being incapable of directly binding to LMB2. Transcriptomic analysis revealed similar changes in hCD25-positive and hCD25-negative podocytes, including decreased nephrin and increased desmin expression levels. Ultimately, most podocytes were lost, and the mice developed global glomerulosclerosis. These findings indicate that injured podocytes can indirectly harm neighboring healthy podocytes, thereby establishing a self-amplifying cycle of damage 8 . A similar secondary injury has been observed in other in vivo models 9 , 10 . However, this phenomenon could not be reproduced in vitro using traditional podocyte culture systems 7 . In conventional 2D cultures, podocytes rapidly lose expression of key differentiation markers such as nephrin and podocin. By contrast, kidney organoids generated from pluripotent stem cells or nephron progenitor cells (NPCs) contain more physiologically relevant, differentiated podocytes. The culture-dependent purification (CDP) method, which was developed by Li and Araoka, enables the expansion and differentiation of NPCs from mice 11 . Following brief stimulation with Wnt and FGF signals, progenitor cell aggregates differentiate into kidney organoids containing podocytes in glomerulus-like structures and tubular cells in tubular structures. In the present study, NPCs were isolated from NEP25 mice and from mice lacking hCD25 to generate chimeric kidney organoids. Using this in vitro system, whether podocyte injury could propagate from LMB2-targeted podocytes to neighboring, untargeted podocytes was determined. Our findings indicate that podocyte-to-podocyte injury signaling occurs in kidney organoids, thereby confirming that podocyte injury is autonomous and self-propagating. Materials and Methods Mice All animal experimental procedures were approved by the Animal Experimentation Committee of Tokai University School of Medicine and were conducted and reported in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and the ARRIVE guidelines ( https://arriveguidelines.org ). Three transgenic mouse lines were used in this study, namely, NEP25 (carrying Nphs1-hCD25 ), Nephrin-Cre ( Nphs1-Cre ), and Ribotag ( Rpl22 tm1.1Psam ). NEP25 3 and Nephrin-Cre 5 , 12 lines were established in our laboratory and maintained in C57BL/6N genetic background. Ribotag line (IMSR_JAX:011029) was obtained from the Jackson Laboratory and maintained by crossing Nephrin-Cre line 13 . NEP25 mice expressed hCD25 selectively in podocytes, enabling the induction of selective podocyte injury by the administration of the hCD25-targeted immunotoxin LMB2 (a gift of Ira Pastan) 3 . Nephrin-Cre/Ribotag mice expressed the ribosomal protein L22 (Rpl22) tagged with a hemagglutinin (HA) epitope specifically in podocytes 13 . To generate NPC lines, mice carrying the combinations of Nphs1-hCD25 (0 or 1 copy), Nphs1-Cre (0 or 1 copy), and Rpl22 tm1.1Psam (2 copies) were intercrossed, and embryos were harvested at embryonic day 12.5. The primers used for genotyping are listed in Table S1 . Organoid Culture NPC lines were established using the CDP method with modifications 4 , 11 . In brief, the kidneys were isolated from each embryo, and 1 × 10⁴ cells were seeded in NPSR medium on laminin-coated 12-well plates. The FGF-2 and 2-mercaptoethanol concentrations were increased to 300 ng/mL and 0.1 mM, respectively. Simultaneously, the presence of Nphs1-Cre and Nphs1-hCD25 transgenes was determined by real-time PCR analysis of genomic DNA from embryonic tail biopsies. Cells derived from embryos carrying Nphs1-hCD25 or Nphs1-Cre were selected for further purification. After 4 days of culture, floating cell aggregates were collected, dissociated, and recultured in 96-well U-bottom low-attachment plates with NPSR medium. Aggregates were passaged every 4 days. Cell lines that are capable of sustained proliferation formed spherical aggregates over 10 passages. The cells from passages 4–16 were used for organoid generation. For differentiation, aggregates with a diameter of ~ 1 mm were transferred to Transwell membranes and cultured in KR5-CF medium containing 4.5 µM CHIR99021 and 300 ng/mL FGF-2 for 2 days and then in KR5 medium alone. LMB2 (20 nM) was added on day 6, and the culture was continued for 2–4 days. Then, the organoids were subjected to histological and polysome analyses. In selected experiments, organoids were cotreated with 100 µM carbenoxolone disodium (CBX, gap junction inhibitor), 1 µM SAR7334 (TRPC6 inhibitor), 1 µM A83-01 (TGF-β receptor inhibitor), or 2 µM IKK-16 (NF-κB inhibitor) alongside LMB2. Histological Analysis Organoids were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 2 µm for PAS staining and immunohistochemistry. Some organoids were fixed, permeabilized with 0.1% Triton X-100 in PBS, and stained with fluorescent markers for WT1 and Lotus tetragonolobus lectin (LTL). The details of the primary antibodies and lectins are provided in Table S2. In addition, CanGet signal amplification solutions (Toyobo, Osaka, Japan) were used to detect HA, WT1, podocalyxin, and podocin. Brightfield images were acquired using an Axioplan 2 microscope (Zeiss), and immunofluorescent images were obtained using a BZ-X710 microscope (Keyence). RNA Analysis Approximately 14–16 organoids were harvested after treatment with TrypLE containing cycloheximide (100 µg/mL). Then, the samples were homogenized in 220 µL of lysis buffer (50 mM Tris [pH 7.4], 100 mM KCl, 12 mM MgCl₂, 1% Nonidet P-40, 1 mM DTT, 200 U/mL RNasin, 1 mg/mL heparin, 100 µg/mL cycloheximide, and 1% protease inhibitor cocktail) and vortexed for 1 min. After centrifugation (10,000 rpm, 10 min, 4°C), HA-tagged ribosomes were immunoprecipitated (IP) using an anti-HA.11 antibody (clone 16B12) as previously described. RNA was extracted from IP polysomes using the RNeasy Micro Kit (QIAGEN), and RNA was extracted from unbound fractions using the RNeasy Mini Kit (QIAGEN). Quantitative RT-PCR was performed using the ΔΔCT method, with Actb as the internal control. The primer and probe information is shown in Tables S3 and S4. Statistical Analysis Log-transformed RNA ratios (IP polysomes vs. the supernatant, and IP with LMB2 vs. IP without LMB2) were analyzed by one-sample t-tests, with comparison to log (1) = 0. Holm’s method was used to adjust the p -values. Relative hCD25 mRNA levels were compared using unpaired t-tests. All statistical analyses were performed using EZR version 4.5.0, and p -value < 0.05 was considered statistically significant. AI tools ChatGPT-4 was used to check and correct grammatical errors in the manuscript. Results Generation of Kidney Organoids from NEP25 and Ribotag Mice Using the CDP method, NPCs were obtained from three types of transgenic embryos, namely, (1) NEP25 (carrying Nphs1-hCD25 ), (2) Ribotag ( Nphs1-Cre ), and (3) NEP25/Ribotag ( Nphs1-hCD25/Nphs1-Cre ). The NPC aggregates were cultured on Transwell membranes and exposed to CHIR99021 and FGF-2 to initiate differentiation. Although these organoids did not fully recapitulate the kidney’s architecture because of the absence of endothelial, interstitial, and ureteric bud-derived cells, they developed glomerulus-like and tubular structures. Immunostaining confirmed the presence of differentiated podocytes and proximal tubular cells, as evidenced by the expression of WT1, nephrin, podocalyxin, podocin, and LTL (Figs. 1 and 2 ). In addition, NEP25/Ribotag organoids coexpressed hCD25 and HA in podocytes, mirroring the parental transgenic mice. LMB2-induced Selective Podocyte Injury in NEP25 Organoids Treatment of NEP25 and NEP25/Ribotag organoids with LMB2 resulted in podocyte-specific injury. Fluorescent imaging revealed reduced WT1 staining and preserved LTL staining 4 days after treatment (Fig. 2 ). In NEP25/Ribotag organoids, PAS and immunostaining showed the loss of podocyte clusters, with only scattered HA-positive cells between tubular structures. The expression level of WT1, nephrin, and podocin was markedly reduced, while megalin and LTL signals persisted (Fig. S1 ). At 2 days posttreatment, residual HA-positive clusters remained WT1 positive, but they showed decreased nephrin and podocin staining (Fig. S2). HA immunoprecipitation enabled the isolation of podocyte-specific polysomes. In untreated organoids, Nphs1 , Nphs2 , Wt1 , and hCD25 mRNAs were enriched in IP samples, whereas Lrp2 (megalin) was diluted (Fig. 3 a). LMB2-treated organoids showed undetectable Nphs2 , reduced Nphs1 and Wt1 expression levels, and increased injury-related transcript expression, including Cxcl1 , Gadd45b , Egr1 , and P2rx7 , which is consistent with injured podocyte profiles in NEP25 mice (Fig. 3 b). Podocyte Injury Propagates in Chimeric Organoids Chimeric organoids were generated by combining NEP25 and Ribotag NPCs at a 1:1 ratio to assess the potential intercellular propagation of podocyte injury. Podocytes in these organoids expressed either hCD25 or HA but not both (Fig. 4 ). Two days post-LMB2 treatment, the expression of hCD25 was lost, but HA staining persisted. Despite the survival of HA-positive podocytes, podocin staining was nearly absent (Fig. 5 ). By contrast, the coculture of separate NEP25 and Ribotag organoids on the same Transwell showed that podocin loss occurred only in NEP25 organoids (Fig. S3), indicating that the indirect effect requires close cell-cell contact. In chimeric organoids, HA-immunoprecipitated RNA represents transcripts from hCD25-negative (LMB2-unexposed) podocytes. The mRNA levels of hCD25 were significantly lower in chimeric IP samples than in NEP25/Ribotag organoids (Fig. 3 c). Following LMB2 treatment, these indirectly affected podocytes displayed reduced Nphs1 , Nphs2 , and Wt1 expression levels and increased Gadd45b expression level (Fig. 3 d). We hypothesized that intercellular signaling through gap junctions might mediate the indirect injury. RT-PCR revealed a high expression level of Gja3 (Cx46), Gja5 (Cx40), and Gjc1 (Cx45) but a low expression level of Gja1 (Cx43) in the podocytes of mosaic mice and organoids (Fig. S5). Treatment with CBX, which is a gap junction inhibitor, failed to prevent the loss of podocin in chimeric organoids (Fig. 6 ). Additional treatments with SAR7334 (TRPC6 inhibitor), A83-01 (TGF-β inhibitor), or IKK-16 (NF-κB inhibitor) also failed to block the injury (Fig. S4). Discussion We had hypothesized that indirect podocyte injury occurs in kidney organoids. We found that primary injury to a subset of podocytes can lead to secondary damage in neighboring podocytes in chimeric kidney organoids containing immunotoxin-sensitive and -resistant podocytes. These findings indicate that podocyte injury can propagate to neighboring podocytes independent of glomerular filtration or interaction with other glomerular cell types. Indirect injury, which is also referred to as bystander killing, is well recognized in nervous tissue damage models 14 . However, our initial attempt to recapitulate this effect in conventional cultured podocytes was unsuccessful. Factors unique to the in vivo environment, such as the physical forces of glomerular filtration, macromolecular leakage into the Bowman’s space, or interactions with other types of glomerular cells, may contribute to the propagation of podocyte injury. Our previous ultrastructural analysis of NEP25 mice revealed that podocytes detach from the glomerular basement membrane in clusters, depending on glomerular filtration. This detachment was preceded by the expansion of the subpodocyte space, forming pseudocysts 15 . These findings indicate that initially uninjured podocytes adjacent to damaged ones may also be lifted and detached by the pseudocyst. Such hemodynamic forces may contribute to secondary podocyte damage because glomerular filtration exacerbates podocyte injury 16 . Although the involvement of these in vivo factors cannot be excluded, this study showed that secondary podocyte injury can occur independently of glomerular filtration. Our findings also indicate that other glomerular cell types, namely endothelial and mesangial cells, are not necessary for this process. The rapid downregulation of podocin expression level in hCD25-negative podocytes within chimeric organoids following LMB2 treatment indicates a mechanism involving direct cell-cell communication. Such communication may be mediated via the interaction of membrane proteins, paracrine factors, extracellular vesicles, gap junctions, or tunneling nanotubes (TNTs) 17 . Gap junctions mediate the intercellular transfer of small molecules, including ions, inositol 1,4,5-trisphosphate, nitric oxide, reactive oxygen species, and 2′3′-cGAMP, which is the second messenger in the STING pathway 18 , 19 . Connexins (Cxs) are gap junction proteins that facilitate intercellular organelle transfer and contribute to TNT formation 18 , 20 . Many studies have implicated the role of Cxs in bystander killing in radiation-induced and ischemic brain injury models 14 , 17 , 21 . Injured podocytes have been reported to express Cx43 and Cx45 22,23 , and our previous microarray analyses revealed that mouse podocytes express Gja3 (encoding Cx46) and Gja5 (Cx40) 13 . Our quantitative RT-PCR analysis revealed that Gja3 was abundantly expressed at baseline and downregulated following LMB2 treatment in the podocytes of mosaic mice and organoids. The expression of Gja1 (Cx43) was minimal; however, low levels of Gja5 and Gjc1 (Cx45) were detected after injury in the podocytes of mosaic mice (Fig. S5). The role of gap junctions was tested by treating chimeric organoids with carbenoxolone, which is a gap junction inhibitor. Compared with previous studies in neural tissue 14 , carbenoxolone did not prevent the loss of podocin in indirectly affected podocytes, indicating that gap junctions may not be the principal mediators of bystander injury in this model. TNTs represent another direct way of intercellular communication. These membranous structures can bridge distant cells and transfer a wide range of cargos, from ions to organelles 24 . Barutta et al. reported that podocytes express M-Sec ( Tnfaip2 ), which is a key regulator of TNT formation 25 , 26 . In vitro , adriamycin-exposed podocytes formed TNTs in a Tnfaip2 -dependent manner. Furthermore, Tnfaip2 -knockout mice with a BALB/c background spontaneously developed FSGS with associated mitochondrial dysfunction. In the C57BL/6 background, Tnfaip2 -deficient mice displayed no baseline renal abnormalities but showed exaggerated podocyte injury, lysosomal alterations, and impaired autophagy following streptozotocin-induced diabetes 27 . These findings indicated that TNTs may play a protective role by transferring organelles from healthy to injured podocytes. However, given their bidirectional nature, TNTs could mediate injury propagation. In our current model, LMB2 directly injured approximately 50% of podocytes in chimeric organoids. Under such conditions, injury signals were transmitted from hCD25-positive to hCD25-negative podocytes via TNTs. Although TNT involvement was not directly assessed, RT-PCR analysis revealed that Tnfaip2 mRNA was expressed in the podocytes of mosaic mice and organoids. LMB2 downregulated the expression level of Tnfaip2 in hCD25-positive podocytes from NEP25/Ribotag organoids but not in those from mosaic mice (Fig. S6). Despite the remarkable results, this study has limitations. First, the molecular mechanism underlying the indirect injury could not be elucidated. Second, the direct injury caused by LMB2 may not generally represent podocyte injury in actual kidney diseases. Our findings indicate that podocytes directly injured by LMB2 can induce secondary damage to neighboring, initially intact podocytes within chimeric organoids, despite the absence of glomerular filtration or other glomerular cell types. Therefore, direct cell-cell communication plays a central role in the intraglomerular spread of injury. Further investigation is necessary to identify the molecular mechanism underlying this indirect podocyte injury in the chimeric organoid and mosaic mouse models. Such mechanisms may also contribute to injury amplification in various glomerular diseases, forming a vicious cycle that leads to global glomerulosclerosis. Declarations Data availability: All data generated or analyzed during this study are included in this published article and the supplementary files. Acknowledgments: We acknowledge Shiho Kuroiwa, Chie Sakurai, and the Support Center for Medical Research and Education of Tokai University for their excellent technical assistance and Yukiko Tanaka for administrative assistance. We also thank Dr. Ira Pastan for his years of collaboration and for providing LMB2. Parts of this study were presented in abstract form at the annual meeting of the American Society of Nephrology in 2019 and 2021. Funding: This work was supported by a Grant-in-Aid for Scientific Research (18H02827 and 23K21437). Authors’ contributions: TM and UT conceived and designed the research, performed the experiments, analyzed the data, and wrote the manuscript. AT and OK supplied the materials and key technical advice for organoid generation and contributed to figure generation. All authors approved the final version of the manuscript. Competing interests: The authors declare no competing interests. References Altintas, M. M. et al. 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Ichikawa, I., Ma, J., Motojima, M. & Matsusaka, T. Podocyte damage damages podocytes: Autonomous vicious cycle that drives local spread of glomerular sclerosis. Curr Opin Nephrol Hypertens 14 , 205–210 (2005). Patek, C. E. et al. Murine Denys-Drash syndrome: Evidence of podocyte de-differentiation and systemic mediation of glomerulosclerosis. Human Molecular Genetics vol. 12 2379–2394 Preprint at https://doi.org/10.1093/hmg/ddg240 (2003). Mollet, G. et al. Podocin inactivation in mature kidneys causes focal segmental glomerulosclerosis and nephrotic syndrome. Journal of the American Society of Nephrology 20 , 2181–2189 (2009). Li, Z. et al. 3D Culture Supports Long-Term Expansion of Mouse and Human Nephrogenic Progenitors. Cell Stem Cell 19 , 516–529 (2016). Asano, T. et al. Permanent genetic tagging of podocytes: Fate of injured podocytes in a mouse model of glomerular sclerosis. Journal of the American Society of Nephrology 16 , 2257–2262 (2005). Okabe, M. et al. Global polysome analysis of normal and injured podocytes. Am J Physiol Renal Physiol 316 , F241–F252 (2019). Contreras, J. E. et al. Role of connexin-based gap junction channels and hemichannels in ischemia-induced cell death in nervous tissue. Brain Res Rev 47 , 290–303 (2004). Saga, N., Sakamoto, K., Matsusaka, T. & Nagata, M. Glomerular filtrate affects the dynamics of podocyte detachment in a model of diffuse toxic podocytopathy. Kidney Int 99 , 1149–1161 (2021). Matsusaka, T. et al. Glomerular sclerosis is prevented during urinary tract obstruction due to podocyte protection. Am J Physiol Renal Physiol 300 , 792–800 (2011). Kandouz, M. Cell Death, by Any Other Name…. Cells vol. 13 Preprint at https://doi.org/10.3390/cells13040325 (2024). Lucaciu, S. A., Leighton, S. E., Hauser, A., Yee, R. & Laird, D. W. Diversity in connexin biology. Journal of Biological Chemistry vol. 299 Preprint at https://doi.org/10.1016/j.jbc.2023.105263 (2023). Xie, W. & Patel, D. J. Structure-based mechanisms of 2′3′-cGAMP intercellular transport in the cGAS–STING immune pathway. Trends Immunol 44 , 450–467 (2023). Norris, R. P. Transfer of mitochondria and endosomes between cells by gap junction internalization. Traffic 22 , 174–179 (2021). Ramadan, R., Baatout, S., Aerts, A. & Leybaert, L. The role of connexin proteins and their channels in radiation-induced atherosclerosis. Cellular and Molecular Life Sciences vol. 78 3087–3103 Preprint at https://doi.org/10.1007/s00018-020-03716-3 (2021). Kosovic, I. et al. Spatio-temporal patterning of different connexins in developing and postnatal human kidneys and in nephrotic syndrome of the Finnish type (CNF). Sci Rep 10 , (2020). Yaoita, E. et al. Up-regulation of connexin43 in glomerular podocytes in response to injury. American Journal of Pathology 161 , 1597–1606 (2002). Zurzolo, C. Tunneling nanotubes: Reshaping connectivity. Current Opinion in Cell Biology vol. 71 139–147 Preprint at https://doi.org/10.1016/j.ceb.2021.03.003 (2021). Ohno, H., Hase, K. & Kimura, S. M-Sec: Emerging secrets of tunneling nanotube formation. Commun Integr Biol 3 , 231–233 (2010). Barutta, F. et al. Protective role of the M-sec-tunneling nanotube system in podocytes. Journal of the American Society of Nephrology 32 , 1114–1130 (2021). Barutta, F. et al. Protective effect of the tunneling nanotube-TNFAIP2/M-sec system on podocyte autophagy in diabetic nephropathy. Autophagy 19 , 505–524 (2023). Additional Declarations No competing interests reported. Supplementary Files Supplements.pdf 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. 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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-7373933","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":514183908,"identity":"d1bb9901-d044-4d2c-989f-f28b3bb70934","order_by":0,"name":"Tomohiro Udagawa","email":"","orcid":"","institution":"Tokai University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tomohiro","middleName":"","lastName":"Udagawa","suffix":""},{"id":514183909,"identity":"cba46753-2450-4b59-8991-0704bb2a83cb","order_by":1,"name":"Toshikazu Araoka","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Toshikazu","middleName":"","lastName":"Araoka","suffix":""},{"id":514183910,"identity":"f8e1cd5f-feb9-4a08-8ffe-3dd4a26c0b8d","order_by":2,"name":"Kenji Osafune","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Osafune","suffix":""},{"id":514183911,"identity":"12bd01b9-55c6-49b6-88ea-07404250dc07","order_by":3,"name":"Taiji Matsusaka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYFACxgaDBAYbBgYeBoYDED4BDTwQLWkSpGgBg8MSMBZhYM9+uKHgYc75On6eww8PMNTYMTDPJmAND09ig0HittsSkr1tBgcYjiUzMM45QMhhUC0G5xmAWtgOMDDOSCCghf8hSMs5oBb2DwcY/hGjRQJsywEJg7M9BgcY24jRcgNsS7LkzJ4zBQcS+5J5CPqFvT/9meHPbXb8/Dzpmz98+GYnZ0goxICAzQDOBDqJx3AGQR0MzA9QuPIShLWMglEwCkbByAIA9xpFKX76zAIAAAAASUVORK5CYII=","orcid":"","institution":"Tokai University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Taiji","middleName":"","lastName":"Matsusaka","suffix":""}],"badges":[],"createdAt":"2025-08-14 12:38:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7373933/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7373933/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91500526,"identity":"f0f5fc65-fa8a-4493-abdb-1d30d4cac7c6","added_by":"auto","created_at":"2025-09-17 07:23:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2708076,"visible":true,"origin":"","legend":"\u003cp\u003eKidney organoids were generated from NEP25/Ribotag nephron progenitor cells. The organoids contain glomerulus-like clusters (a, PAS) stained for nephrin (b), HA (c), hCD25 (d), WT1 (e), and Podocalyxin1 (f). They also contain tubular structures stained for LTL (g). Images were taken from equivalent visual fields of the serial sections.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7373933/v1/c68771c5fa42ae83bacc9a75.png"},{"id":91500533,"identity":"b851768e-285c-4a75-bd3f-998e1c7b1832","added_by":"auto","created_at":"2025-09-17 07:23:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2724948,"visible":true,"origin":"","legend":"\u003cp\u003eLMB2-induced selective podocyte injury in NEP25/Ribotag organoids. In control organoids (without LMB2), WT1 and LTL are strongly expressed (a and b). After 4 days of LMB2 treatment, the expression level of WT1 is markedly reduced, whereas LTL remains unaffected (h and i). In the absence of LMB2, podocytes form clusters, and they are stained for nephrin, WT1, HA, and podocin (c–g). Following LMB2 treatment, these clusters disappear, and staining for nephrin, WT1, HA, and podocin is markedly reduced (j–n). Panels (c–f) and (j–m) represent the equivalent visual fields of the serial sections.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7373933/v1/eb7814150ed3c02f59fd734b.png"},{"id":91501456,"identity":"58ea00b0-e890-4e8f-845b-d879107f3121","added_by":"auto","created_at":"2025-09-17 07:31:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":291384,"visible":true,"origin":"","legend":"\u003cp\u003eRNA analyses in kidney organoids. (a) Ratio of podocyte polysomal RNA (IP) to total organoid RNA (SUP) in NEP25/Ribotag organoids (n = 6). \u003cem\u003eNphs1\u003c/em\u003e, \u003cem\u003eNphs2\u003c/em\u003e, \u003cem\u003eWt1\u003c/em\u003e, and \u003cem\u003ehCD25\u003c/em\u003e were enriched in podocyte polysomes (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), whereas \u003cem\u003eLrp2\u003c/em\u003e was diluted (\u003cem\u003ep\u003c/em\u003e = 0.03). (b) Ratio of LMB2-treated to control polysomal RNA in NEP25/Ribotag organoids (n = 6). LMB2 markedly reduced the expression level of \u003cem\u003eNphs1\u003c/em\u003eand \u003cem\u003eWt1\u003c/em\u003e and increased the expression level of \u003cem\u003eGadd45b\u003c/em\u003e, \u003cem\u003eP2rx7\u003c/em\u003e, \u003cem\u003eCxcl1\u003c/em\u003e, and \u003cem\u003eEgr\u003c/em\u003e1 (all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). \u003cem\u003eNphs2\u003c/em\u003e was undetectable in all LMB2-treated samples. (c) Relative abundance of \u003cem\u003ehCD25\u003c/em\u003ein IP samples from chimeric (n = 13) versus NEP25/Ribotag (n = 6) organoids (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01). (d) Ratio of LMB2-treated to control polysomal RNA derived from hCD25(−) podocytes in chimeric organoids. LMB2 reduced the expression level of \u003cem\u003eNphs1\u003c/em\u003e, \u003cem\u003eNphs2\u003c/em\u003e, and \u003cem\u003eWt1\u003c/em\u003e and increased the expression level of \u003cem\u003eGadd45b\u003c/em\u003e(all \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01). The expression level of \u003cem\u003eP2rx7\u003c/em\u003e remained unchanged.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7373933/v1/3abca297f63d754374a5d957.png"},{"id":91501451,"identity":"7ef8f598-7442-4aa5-bc39-0c4bfd0d3f2c","added_by":"auto","created_at":"2025-09-17 07:31:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1716017,"visible":true,"origin":"","legend":"\u003cp\u003eChimeric organoids. NEP25 organoids express hCD25 (a) but not HA (b). Ribotag organoids express HA (d) but not hCD25 (c). Chimeric organoids coexpress hCD25 and HA (e, f). High-magnification imaging reveals the mutually exclusive expression of hCD25 (brown) and HA (gray) among podocytes (g).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7373933/v1/bb17dda53349292c58469318.png"},{"id":91500540,"identity":"9cf498dc-aeca-4dfd-a08b-d0cdf3aedaad","added_by":"auto","created_at":"2025-09-17 07:23:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1266056,"visible":true,"origin":"","legend":"\u003cp\u003eIndirect podocyte injury in chimeric organoids. In control organoids (without LMB2), hCD25, HA, and podocin are robustly expressed (a–c). After LMB2 treatment, hCD25 expression is completely lost (d), and although HA(+) podocytes persist (e), podocin staining is markedly reduced (f). Panels (a–c) and (d–f) show the equivalent fields of the serial sections.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7373933/v1/5b623973877c6d8162c8fa23.png"},{"id":91500547,"identity":"b8ffc3c8-ac70-44a6-8dc1-c29a679dc97b","added_by":"auto","created_at":"2025-09-17 07:23:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":910298,"visible":true,"origin":"","legend":"\u003cp\u003eCarbenoxolone disodium (CBX), which is a gap junction inhibitor, does not prevent the LMB2-induced suppression of podocin. After 3 days of LMB2 treatment, podocin staining is markedly diminished in chimeric organoids (b) despite the presence of HA staining in adjacent sections (a). Cotreatment with CBX does not rescue podocin expression (d) despite persistent HA staining (c). Panels (a–b) and (c–d) are derived from the equivalent visual fields of the serial sections.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7373933/v1/5883f47adceaacab108d81bc.png"},{"id":93559199,"identity":"3513f55e-db26-4765-b724-221eb9c05bc2","added_by":"auto","created_at":"2025-10-15 07:17:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11444600,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7373933/v1/f7e38dd7-7b0f-40a1-a2f4-856cabe9744d.pdf"},{"id":91500541,"identity":"03a8ef6d-40b2-48e2-a5ff-0274ac20f13d","added_by":"auto","created_at":"2025-09-17 07:23:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1790233,"visible":true,"origin":"","legend":"","description":"","filename":"Supplements.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7373933/v1/8de629b9185fd0e555bde4a3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Podocyte injury damages podocytes in chimeric organoids","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePodocyte injury is a critical initiating event in the development and progression of glomerulosclerosis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In our previous study, we developed a transgenic mouse model in which podocytes express human CD25 (hCD25), allowing selective injury by the hCD25-targeted immunotoxin LMB2 \u003csup\u003e3\u003c/sup\u003e. In hCD25-expressing podocytes, LMB2 inhibits protein synthesis and induces apoptotic caspase activation \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Despite its rapid clearance from circulation, a single injection of LMB2 results in progressive kidney injury over several weeks. Similar progression has been observed in other models of podocyte injury \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In human and animal models, segmental sclerosis lesions are typically localized to a few regions per glomerulus, despite the widespread exposure of podocytes to injurious stimuli. This finding indicates that podocyte injury may spread locally and autonomously within the glomerulus.\u003c/p\u003e\u003cp\u003eThus, we previously generated chimeric and mosaic mouse models in which only a subset of podocytes express hCD25 \u003csup\u003e6,7\u003c/sup\u003e. As expected, hCD25(+) podocytes were selectively injured following LMB2 injection. Surprisingly, adjacent hCD25-negative podocytes were also affected, showing reduced nephrin and podocin expression levels and increased desmin staining, despite being incapable of directly binding to LMB2. Transcriptomic analysis revealed similar changes in hCD25-positive and hCD25-negative podocytes, including decreased nephrin and increased desmin expression levels. Ultimately, most podocytes were lost, and the mice developed global glomerulosclerosis. These findings indicate that injured podocytes can indirectly harm neighboring healthy podocytes, thereby establishing a self-amplifying cycle of damage \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. A similar secondary injury has been observed in other \u003cem\u003ein vivo\u003c/em\u003e models \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, this phenomenon could not be reproduced \u003cem\u003ein vitro\u003c/em\u003e using traditional podocyte culture systems \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn conventional 2D cultures, podocytes rapidly lose expression of key differentiation markers such as nephrin and podocin. By contrast, kidney organoids generated from pluripotent stem cells or nephron progenitor cells (NPCs) contain more physiologically relevant, differentiated podocytes. The culture-dependent purification (CDP) method, which was developed by Li and Araoka, enables the expansion and differentiation of NPCs from mice \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Following brief stimulation with Wnt and FGF signals, progenitor cell aggregates differentiate into kidney organoids containing podocytes in glomerulus-like structures and tubular cells in tubular structures.\u003c/p\u003e\u003cp\u003eIn the present study, NPCs were isolated from NEP25 mice and from mice lacking hCD25 to generate chimeric kidney organoids. Using this \u003cem\u003ein vitro\u003c/em\u003e system, whether podocyte injury could propagate from LMB2-targeted podocytes to neighboring, untargeted podocytes was determined. Our findings indicate that podocyte-to-podocyte injury signaling occurs in kidney organoids, thereby confirming that podocyte injury is autonomous and self-propagating.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eAll animal experimental procedures were approved by the Animal Experimentation Committee of Tokai University School of Medicine and were conducted and reported in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThree transgenic mouse lines were used in this study, namely, NEP25 (carrying \u003cem\u003eNphs1-hCD25\u003c/em\u003e), Nephrin-Cre (\u003cem\u003eNphs1-Cre\u003c/em\u003e), and Ribotag (\u003cem\u003eRpl22\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1.1Psam\u003c/em\u003e\u003c/sup\u003e). NEP25\u003csup\u003e3\u003c/sup\u003e and Nephrin-Cre\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e lines were established in our laboratory and maintained in C57BL/6N genetic background. Ribotag line (IMSR_JAX:011029) was obtained from the Jackson Laboratory and maintained by crossing Nephrin-Cre line\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. NEP25 mice expressed hCD25 selectively in podocytes, enabling the induction of selective podocyte injury by the administration of the hCD25-targeted immunotoxin LMB2 (a gift of Ira Pastan) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Nephrin-Cre/Ribotag mice expressed the ribosomal protein L22 (Rpl22) tagged with a hemagglutinin (HA) epitope specifically in podocytes \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. To generate NPC lines, mice carrying the combinations of \u003cem\u003eNphs1-hCD25\u003c/em\u003e (0 or 1 copy), \u003cem\u003eNphs1-Cre\u003c/em\u003e (0 or 1 copy), and \u003cem\u003eRpl22\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1.1Psam\u003c/em\u003e\u003c/sup\u003e (2 copies) were intercrossed, and embryos were harvested at embryonic day 12.5. The primers used for genotyping are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eOrganoid Culture\u003c/h3\u003e\n\u003cp\u003eNPC lines were established using the CDP method with modifications \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In brief, the kidneys were isolated from each embryo, and 1 \u0026times; 10⁴ cells were seeded in NPSR medium on laminin-coated 12-well plates. The FGF-2 and 2-mercaptoethanol concentrations were increased to 300 ng/mL and 0.1 mM, respectively.\u003c/p\u003e\u003cp\u003eSimultaneously, the presence of \u003cem\u003eNphs1-Cre\u003c/em\u003e and \u003cem\u003eNphs1-hCD25\u003c/em\u003e transgenes was determined by real-time PCR analysis of genomic DNA from embryonic tail biopsies. Cells derived from embryos carrying \u003cem\u003eNphs1-hCD25\u003c/em\u003e or \u003cem\u003eNphs1-Cre\u003c/em\u003e were selected for further purification. After 4 days of culture, floating cell aggregates were collected, dissociated, and recultured in 96-well U-bottom low-attachment plates with NPSR medium. Aggregates were passaged every 4 days. Cell lines that are capable of sustained proliferation formed spherical aggregates over 10 passages. The cells from passages 4\u0026ndash;16 were used for organoid generation.\u003c/p\u003e\u003cp\u003eFor differentiation, aggregates with a diameter of ~\u0026thinsp;1 mm were transferred to Transwell membranes and cultured in KR5-CF medium containing 4.5 \u0026micro;M CHIR99021 and 300 ng/mL FGF-2 for 2 days and then in KR5 medium alone. LMB2 (20 nM) was added on day 6, and the culture was continued for 2\u0026ndash;4 days. Then, the organoids were subjected to histological and polysome analyses. In selected experiments, organoids were cotreated with 100 \u0026micro;M carbenoxolone disodium (CBX, gap junction inhibitor), 1 \u0026micro;M SAR7334 (TRPC6 inhibitor), 1 \u0026micro;M A83-01 (TGF-β receptor inhibitor), or 2 \u0026micro;M IKK-16 (NF-κB inhibitor) alongside LMB2.\u003c/p\u003e\n\u003ch3\u003eHistological Analysis\u003c/h3\u003e\n\u003cp\u003eOrganoids were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 2 \u0026micro;m for PAS staining and immunohistochemistry. Some organoids were fixed, permeabilized with 0.1% Triton X-100 in PBS, and stained with fluorescent markers for WT1 and Lotus tetragonolobus lectin (LTL). The details of the primary antibodies and lectins are provided in Table S2. In addition, CanGet signal amplification solutions (Toyobo, Osaka, Japan) were used to detect HA, WT1, podocalyxin, and podocin.\u003c/p\u003e\u003cp\u003eBrightfield images were acquired using an Axioplan 2 microscope (Zeiss), and immunofluorescent images were obtained using a BZ-X710 microscope (Keyence).\u003c/p\u003e\n\u003ch3\u003eRNA Analysis\u003c/h3\u003e\n\u003cp\u003eApproximately 14\u0026ndash;16 organoids were harvested after treatment with TrypLE containing cycloheximide (100 \u0026micro;g/mL). Then, the samples were homogenized in 220 \u0026micro;L of lysis buffer (50 mM Tris [pH 7.4], 100 mM KCl, 12 mM MgCl₂, 1% Nonidet P-40, 1 mM DTT, 200 U/mL RNasin, 1 mg/mL heparin, 100 \u0026micro;g/mL cycloheximide, and 1% protease inhibitor cocktail) and vortexed for 1 min. After centrifugation (10,000 rpm, 10 min, 4\u0026deg;C), HA-tagged ribosomes were immunoprecipitated (IP) using an anti-HA.11 antibody (clone 16B12) as previously described. RNA was extracted from IP polysomes using the RNeasy Micro Kit (QIAGEN), and RNA was extracted from unbound fractions using the RNeasy Mini Kit (QIAGEN). Quantitative RT-PCR was performed using the ΔΔCT method, with \u003cem\u003eActb\u003c/em\u003e as the internal control. The primer and probe information is shown in Tables S3 and S4.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eLog-transformed RNA ratios (IP polysomes vs. the supernatant, and IP with LMB2 vs. IP without LMB2) were analyzed by one-sample t-tests, with comparison to log (1)\u0026thinsp;=\u0026thinsp;0. Holm\u0026rsquo;s method was used to adjust the \u003cem\u003ep\u003c/em\u003e-values. Relative \u003cem\u003ehCD25\u003c/em\u003e mRNA levels were compared using unpaired t-tests. All statistical analyses were performed using EZR version 4.5.0, and \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAI tools\u003c/h2\u003e\u003cp\u003eChatGPT-4 was used to check and correct grammatical errors in the manuscript.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eGeneration of Kidney Organoids from NEP25 and Ribotag Mice\u003c/h2\u003e\u003cp\u003eUsing the CDP method, NPCs were obtained from three types of transgenic embryos, namely, (1) NEP25 (carrying \u003cem\u003eNphs1-hCD25\u003c/em\u003e), (2) Ribotag (\u003cem\u003eNphs1-Cre\u003c/em\u003e), and (3) NEP25/Ribotag (\u003cem\u003eNphs1-hCD25/Nphs1-Cre\u003c/em\u003e). The NPC aggregates were cultured on Transwell membranes and exposed to CHIR99021 and FGF-2 to initiate differentiation. Although these organoids did not fully recapitulate the kidney\u0026rsquo;s architecture because of the absence of endothelial, interstitial, and ureteric bud-derived cells, they developed glomerulus-like and tubular structures. Immunostaining confirmed the presence of differentiated podocytes and proximal tubular cells, as evidenced by the expression of WT1, nephrin, podocalyxin, podocin, and LTL (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, NEP25/Ribotag organoids coexpressed hCD25 and HA in podocytes, mirroring the parental transgenic mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLMB2-induced Selective Podocyte Injury in NEP25 Organoids\u003c/h2\u003e\u003cp\u003eTreatment of NEP25 and NEP25/Ribotag organoids with LMB2 resulted in podocyte-specific injury. Fluorescent imaging revealed reduced WT1 staining and preserved LTL staining 4 days after treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In NEP25/Ribotag organoids, PAS and immunostaining showed the loss of podocyte clusters, with only scattered HA-positive cells between tubular structures. The expression level of WT1, nephrin, and podocin was markedly reduced, while megalin and LTL signals persisted (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). At 2 days posttreatment, residual HA-positive clusters remained WT1 positive, but they showed decreased nephrin and podocin staining (Fig. S2).\u003c/p\u003e\u003cp\u003eHA immunoprecipitation enabled the isolation of podocyte-specific polysomes. In untreated organoids, \u003cem\u003eNphs1\u003c/em\u003e, \u003cem\u003eNphs2\u003c/em\u003e, \u003cem\u003eWt1\u003c/em\u003e, and \u003cem\u003ehCD25\u003c/em\u003e mRNAs were enriched in IP samples, whereas \u003cem\u003eLrp2\u003c/em\u003e (megalin) was diluted (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). LMB2-treated organoids showed undetectable \u003cem\u003eNphs2\u003c/em\u003e, reduced \u003cem\u003eNphs1\u003c/em\u003e and \u003cem\u003eWt1\u003c/em\u003e expression levels, and increased injury-related transcript expression, including \u003cem\u003eCxcl1\u003c/em\u003e, \u003cem\u003eGadd45b\u003c/em\u003e, \u003cem\u003eEgr1\u003c/em\u003e, and \u003cem\u003eP2rx7\u003c/em\u003e, which is consistent with injured podocyte profiles in NEP25 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePodocyte Injury Propagates in Chimeric Organoids\u003c/h2\u003e\u003cp\u003eChimeric organoids were generated by combining NEP25 and Ribotag NPCs at a 1:1 ratio to assess the potential intercellular propagation of podocyte injury. Podocytes in these organoids expressed either hCD25 or HA but not both (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Two days post-LMB2 treatment, the expression of hCD25 was lost, but HA staining persisted. Despite the survival of HA-positive podocytes, podocin staining was nearly absent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBy contrast, the coculture of separate NEP25 and Ribotag organoids on the same Transwell showed that podocin loss occurred only in NEP25 organoids (Fig. S3), indicating that the indirect effect requires close cell-cell contact.\u003c/p\u003e\u003cp\u003eIn chimeric organoids, HA-immunoprecipitated RNA represents transcripts from hCD25-negative (LMB2-unexposed) podocytes. The mRNA levels of \u003cem\u003ehCD25\u003c/em\u003e were significantly lower in chimeric IP samples than in NEP25/Ribotag organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Following LMB2 treatment, these indirectly affected podocytes displayed reduced \u003cem\u003eNphs1\u003c/em\u003e, \u003cem\u003eNphs2\u003c/em\u003e, and \u003cem\u003eWt1\u003c/em\u003e expression levels and increased \u003cem\u003eGadd45b\u003c/em\u003e expression level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eWe hypothesized that intercellular signaling through gap junctions might mediate the indirect injury. RT-PCR revealed a high expression level of \u003cem\u003eGja3\u003c/em\u003e (Cx46), \u003cem\u003eGja5\u003c/em\u003e (Cx40), and \u003cem\u003eGjc1\u003c/em\u003e (Cx45) but a low expression level of \u003cem\u003eGja1\u003c/em\u003e (Cx43) in the podocytes of mosaic mice and organoids (Fig. S5). Treatment with CBX, which is a gap junction inhibitor, failed to prevent the loss of podocin in chimeric organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Additional treatments with SAR7334 (TRPC6 inhibitor), A83-01 (TGF-β inhibitor), or IKK-16 (NF-κB inhibitor) also failed to block the injury (Fig. S4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe had hypothesized that indirect podocyte injury occurs in kidney organoids. We found that primary injury to a subset of podocytes can lead to secondary damage in neighboring podocytes in chimeric kidney organoids containing immunotoxin-sensitive and -resistant podocytes. These findings indicate that podocyte injury can propagate to neighboring podocytes independent of glomerular filtration or interaction with other glomerular cell types.\u003c/p\u003e\u003cp\u003eIndirect injury, which is also referred to as bystander killing, is well recognized in nervous tissue damage models \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, our initial attempt to recapitulate this effect in conventional cultured podocytes was unsuccessful. Factors unique to the \u003cem\u003ein vivo\u003c/em\u003e environment, such as the physical forces of glomerular filtration, macromolecular leakage into the Bowman\u0026rsquo;s space, or interactions with other types of glomerular cells, may contribute to the propagation of podocyte injury. Our previous ultrastructural analysis of NEP25 mice revealed that podocytes detach from the glomerular basement membrane in clusters, depending on glomerular filtration. This detachment was preceded by the expansion of the subpodocyte space, forming pseudocysts \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These findings indicate that initially uninjured podocytes adjacent to damaged ones may also be lifted and detached by the pseudocyst. Such hemodynamic forces may contribute to secondary podocyte damage because glomerular filtration exacerbates podocyte injury \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough the involvement of these \u003cem\u003ein vivo\u003c/em\u003e factors cannot be excluded, this study showed that secondary podocyte injury can occur independently of glomerular filtration. Our findings also indicate that other glomerular cell types, namely endothelial and mesangial cells, are not necessary for this process. The rapid downregulation of podocin expression level in hCD25-negative podocytes within chimeric organoids following LMB2 treatment indicates a mechanism involving direct cell-cell communication. Such communication may be mediated via the interaction of membrane proteins, paracrine factors, extracellular vesicles, gap junctions, or tunneling nanotubes (TNTs) \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGap junctions mediate the intercellular transfer of small molecules, including ions, inositol 1,4,5-trisphosphate, nitric oxide, reactive oxygen species, and 2\u0026prime;3\u0026prime;-cGAMP, which is the second messenger in the STING pathway \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Connexins (Cxs) are gap junction proteins that facilitate intercellular organelle transfer and contribute to TNT formation \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Many studies have implicated the role of Cxs in bystander killing in radiation-induced and ischemic brain injury models \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Injured podocytes have been reported to express Cx43 and Cx45 \u003csup\u003e22,23\u003c/sup\u003e, and our previous microarray analyses revealed that mouse podocytes express \u003cem\u003eGja3\u003c/em\u003e (encoding Cx46) and \u003cem\u003eGja5\u003c/em\u003e (Cx40) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Our quantitative RT-PCR analysis revealed that \u003cem\u003eGja3\u003c/em\u003e was abundantly expressed at baseline and downregulated following LMB2 treatment in the podocytes of mosaic mice and organoids. The expression of \u003cem\u003eGja1\u003c/em\u003e (Cx43) was minimal; however, low levels of Gja5 and Gjc1 (Cx45) were detected after injury in the podocytes of mosaic mice (Fig. S5). The role of gap junctions was tested by treating chimeric organoids with carbenoxolone, which is a gap junction inhibitor. Compared with previous studies in neural tissue \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, carbenoxolone did not prevent the loss of podocin in indirectly affected podocytes, indicating that gap junctions may not be the principal mediators of bystander injury in this model.\u003c/p\u003e\u003cp\u003eTNTs represent another direct way of intercellular communication. These membranous structures can bridge distant cells and transfer a wide range of cargos, from ions to organelles \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Barutta et al. reported that podocytes express M-Sec (\u003cem\u003eTnfaip2\u003c/em\u003e), which is a key regulator of TNT formation \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e, adriamycin-exposed podocytes formed TNTs in a \u003cem\u003eTnfaip2\u003c/em\u003e-dependent manner. Furthermore, \u003cem\u003eTnfaip2\u003c/em\u003e-knockout mice with a BALB/c background spontaneously developed FSGS with associated mitochondrial dysfunction. In the C57BL/6 background, \u003cem\u003eTnfaip2\u003c/em\u003e-deficient mice displayed no baseline renal abnormalities but showed exaggerated podocyte injury, lysosomal alterations, and impaired autophagy following streptozotocin-induced diabetes \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. These findings indicated that TNTs may play a protective role by transferring organelles from healthy to injured podocytes. However, given their bidirectional nature, TNTs could mediate injury propagation. In our current model, LMB2 directly injured approximately 50% of podocytes in chimeric organoids. Under such conditions, injury signals were transmitted from hCD25-positive to hCD25-negative podocytes via TNTs. Although TNT involvement was not directly assessed, RT-PCR analysis revealed that \u003cem\u003eTnfaip2\u003c/em\u003e mRNA was expressed in the podocytes of mosaic mice and organoids. LMB2 downregulated the expression level of \u003cem\u003eTnfaip2\u003c/em\u003e in hCD25-positive podocytes from NEP25/Ribotag organoids but not in those from mosaic mice (Fig. S6).\u003c/p\u003e\u003cp\u003eDespite the remarkable results, this study has limitations. First, the molecular mechanism underlying the indirect injury could not be elucidated. Second, the direct injury caused by LMB2 may not generally represent podocyte injury in actual kidney diseases.\u003c/p\u003e\u003cp\u003eOur findings indicate that podocytes directly injured by LMB2 can induce secondary damage to neighboring, initially intact podocytes within chimeric organoids, despite the absence of glomerular filtration or other glomerular cell types. Therefore, direct cell-cell communication plays a central role in the intraglomerular spread of injury. Further investigation is necessary to identify the molecular mechanism underlying this indirect podocyte injury in the chimeric organoid and mosaic mouse models. Such mechanisms may also contribute to injury amplification in various glomerular diseases, forming a vicious cycle that leads to global glomerulosclerosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAll data generated or analyzed during this study are included in this published article and the supplementary files.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We acknowledge Shiho Kuroiwa, Chie Sakurai, and the Support Center for Medical Research and Education of Tokai University for their excellent technical assistance and Yukiko Tanaka for administrative assistance. We also thank Dr. Ira Pastan for his years of collaboration and for providing LMB2. Parts of this study were presented in abstract form at the annual meeting of the American Society of Nephrology in 2019 and 2021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by a Grant-in-Aid for Scientific Research (18H02827 and 23K21437).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u0026nbsp;\u003c/strong\u003eTM and UT conceived and designed the research, performed the experiments, analyzed the data, and wrote the manuscript. AT and OK supplied the materials and key technical advice for organoid generation and contributed to figure generation. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAltintas, M. 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M-Sec: Emerging secrets of tunneling nanotube formation. \u003cem\u003eCommun Integr Biol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 231\u0026ndash;233 (2010).\u003c/li\u003e\n\u003cli\u003eBarutta, F. \u003cem\u003eet al.\u003c/em\u003e Protective role of the M-sec-tunneling nanotube system in podocytes. \u003cem\u003eJournal of the American Society of Nephrology\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 1114\u0026ndash;1130 (2021).\u003c/li\u003e\n\u003cli\u003eBarutta, F. \u003cem\u003eet al.\u003c/em\u003e Protective effect of the tunneling nanotube-TNFAIP2/M-sec system on podocyte autophagy in diabetic nephropathy. \u003cem\u003eAutophagy\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 505\u0026ndash;524 (2023).\u003c/li\u003e\n\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":"Podocytes, organoids, cell-cell communication, bystander effect, immunotoxin, transgenic mice","lastPublishedDoi":"10.21203/rs.3.rs-7373933/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7373933/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe have previously shown that injury to a subset of podocytes can trigger secondary damage in neighboring podocytes, but whether this phenomenon depends on direct intercellular interaction is unknown. To address this, kidney organoids were generated from nephron progenitor cells of two mouse lines: one expressing a receptor for a podocyte-specific immunotoxin and another expressing a tagged ribosomal protein. In chimeric organoids containing a mosaic of these podocyte types, immunotoxin exposure selectively injured the targeted podocytes and also induced secondary injury in adjacent, non-targeted podocytes. This was evidenced by reduced podocin staining and decreased expression of podocyte-specific genes in the non-targeted podocytes. The bystander effect was absent when organoids of each type were cultured separately but in close proximity, indicating that direct cell-to-cell contact within the same glomerular structure is required. These findings show that podocyte injury can propagate locally within kidney organoids, independent of glomerular filtration or other glomerular cell types, and suggest that local podocyte interactions may contribute to the progression of chronic kidney disease.\u003c/p\u003e","manuscriptTitle":"Podocyte injury damages podocytes in chimeric organoids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 07:23:10","doi":"10.21203/rs.3.rs-7373933/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":"4ccea62a-512e-4f4e-941a-c14d2f15e752","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54628184,"name":"Biological sciences/Cell biology"},{"id":54628185,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2025-10-15T07:09:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 07:23:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7373933","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7373933","identity":"rs-7373933","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}