C-type lectin-like receptor (CLEC)-2, the ligand of podoplanin, induces morphological changes in podocytes

C-type lectin-like receptor (CLEC)-2, the ligand of podoplanin, induces morphological changes in podocytes | 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 C-type lectin-like receptor (CLEC)-2, the ligand of podoplanin, induces morphological changes in podocytes Keiko Tanaka, Masafumi Tanaka, Nobuo Watanabe, Masatoshi Ito, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1817977/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Dec, 2022 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Podoplanin (PDPN) is intensely expressed on the podocyte membrane in an evolutionally conserved manner. CLEC-2, the endogenous ligand of PDPN, is highly expressed in platelets and also exists in a soluble form in plasma. Normally, podocytes are sequestered from CLEC-2, but when the glomerular barrier is injured, podocytes gain access to CLEC-2. We tested the effects of CLEC-2 in podocytes in vitro and in vivo. Cultured podocytes treated with Fc-CLEC-2 demonstrated that CLEC-2 induced the dephosphorylation of moesin, which we found to be the major ERM in cultured podocytes. Podocytes treated with Fc-CLEC-2 also showed the dissociation of F-actin filaments from PDPN, F-actin degradation, detachment, and round morphology. Next, we perfused normal mouse kidney in vivo with FLAG-CLEC-2. CLEC-2 induced dephosphorylation of ezrin and widening of the foot processes of podocytes. Platelets were detected by immunostaining for CD41 in the urine of mice with podocyte injury, indicating that podocytes can encounter platelets when glomeruli are injured. Collectively, these observations suggest that when platelets leak through the injured glomeruli, CLEC-2 from the platelets acts on PDPN in podocytes and induces morphological change and detachment, which may further aggravate podocyte injury. Thus, PDPN on podocytes may work as a leaked-platelet sensor. Figures Figure 1 Figure 2 Figure 3 Introduction Podoplanin (PDPN) is a membranous mucin-type O -glycosylated glycoprotein, which is negatively charged by abundant sialic acid. PDPN is expressed on the surface of various types of cells, including kidney podocytes, alveolar epithelial cells, lymphatic endothelial cells, stromal fibroblastic reticular cells (FRCs) of lymph nodes 1 , and tumors 2 . Among these, PDPN is most intensely expressed on podocytes and its expression is evolutionally conserved 3 . PDPN was found to be the endogenous ligand of C-type lectin-like receptor 2 (CLEC-2) on platelets and is involved in platelet aggregation induced by tumor cells 4 , which facilitates invasion and metastasis of the tumor 2 , 5 , 6 . During developmental stages, CLEC-2 in platelets can interact with PDPN in lymphatic endothelial cells, and activated platelets facilitate blood-lymphatics vessel separation 7 . Platelet activation by PDPN also plays a critical role in the differentiation of alveolar duct myofibroblasts 8 . CLEC-2–PDPN interaction mediates bidirectional signaling. PDPN works as a ligand for CLEC-2 as shown above. In addition, binding PDPN with CLEC-2 influences PDPN-expressing cells. Binding PDPN with CLEC-2 attenuates actomyosin contractility in FRCs in the lymph node 9 , inhibits migration of lymphatic endothelial cells 10 , 11 , stimulates CCL5 secretion in FRC-like cells in bone marrow 12 , stimulates IGF-1 secretion from stromal cells 13 , and attenuates inflammatory responses in a subset of Th17 cells 14 . The role of PDPN in podocytes is not fully elucidated. Decreased expression of PDPN in podocytes is associated with foot process effacement, proteinuria, and decreased glomerular selective permeability in several animal models 15 – 17 . In biopsy specimens of patients with minimal change nephrotic syndrome, PDPN staining was decreased in the proteinuric state, and recovered when proteinuria was normalized 18 . These factors suggest that PDPN is important for maintenance of the normal function of podocytes. The cytoplasmic tail of PDPN interacts with ezrin, radixin, and moesin (ERM) proteins, which bind the actin cytoskeleton and regulate cell shape. It was reported that whole-body Pdpn -gene-disrupted mice showed no abnormal renal phenotypes 19 . Podocalyxin-NHERF2 complex and nephrin-ephrin-B1-NHERF2 complexes can bind and maintain ezrin-F-Actin complex 20 , 21 . This may be a reason for the lack of abnormal phenotype in podocytes of congenital Pdpn-deficient mice. However, suppression of PDPN by siRNA in cultured podocytes changed cell morphology from an elongated to a round shape along with a change in ezrin distribution 18 . Normally, podocytes are sequestered from platelets, but when the glomerular barrier is injured, podocytes gain access to CLEC-2 on platelets. In addition, a soluble form of CLEC-2 with molecular weight 25 kDa exists in human plasma 22 . We speculated that CLEC-2, the known ligand for PDPN, may have some biological impact on podocytes. This hypothesis is supported by previous reports that injection of antibodies against specific epitopes of PDPN caused transient proteinuria and foot process effacement in rats 23 . In the present study, we examined the effect of CLEC-2 in podocytes in vitro and in vivo . Results ・ Effects of CLEC-2 on in vitro podocytes. We first tested the effects of recombinant Fc-human CLEC-2 on cultured mouse podocytes. Although most proteins characteristic to podocytes, such as nephrin and podocin, are rapidly downregulated upon in vitro culture, PDPN staining was maintained in primary cultured podocytes with similar intensity to in vivo podocytes (Fig.1A). As reported previously 3 , a pull-down experiment showed that Fc-human CLEC-2 bound to mouse PDPN (S.Fig.1C). The majority of podocytes treated with Fc protein for 1 hour showed an elongated morphology with sharp protrusions and had numerous F-actin filaments. In contrast, podocytes treated with Fc-CLEC-2 showed a round cell shape without sharp protrusions and a decrease in F-Actin (Fig.1B). Furthermore, podocytes treated with Fc-CLEC-2 showed less adhesion to the collagen-1-coated plate within 1 hour than the Fc control cells (92.3 % vs. 100 %) (Fig.1C). Podocytes with Fc-CLEC-2 showed more migration than the Fc control (2.62 vs. 1.93 mm/24hr) (Fig.1D). We next studied the phosphorylation status of ERM, which links PDPN with F-actin, to reveal the intracellular signaling induced by CLEC-2. Western blot analysis revealed that treatment with Fc-CLEC-2 reduced the pERM/ERM ratio in cultured podocytes, indicating that CLEC-2 induced dephosphorylation of ERM proteins (Fig.1E, S.Fig.3). The most abundant ERM protein in in vivo podocytes is ezrin 24 . However, our Western blot analysis and quantitative PCR revealed that the most abundant ERM in cultured podocytes is moesin (S.Fig.2A and 2B). Immunostaining showed that moesin is intensely stained in the protrusions of Fc control podocytes, and that incubation with Fc-CLEC-2 markedly decreased moesin staining (Fig.1F). These results collectively indicate that CLEC-2 induced dephosphorylation of moesin, which caused dissociation of F-actin filaments from PDPN, F-actin degradation, and cell morphological change. ・ The effects of CLEC-2 on in vivo podocytes To test the effects of CLEC-2 on in vivo podocytes, we generated a new recombinant CLEC-2 protein with a smaller size, because the above Fc-CLEC-2 forms a tetramer with a size of about 240 kDa, which was not expected to reach podocytes through the normal glomerular barrier. The new mouse CLEC-2 protein, FLAG-CLEC-2, exists as a monomer in solution and the size is 30-35 kDa. We infused 5 mg/g body weight of FLAG-CLEC-2 into normal mice and excised the kidney 1 hour later. Immunostaining of FLAG confirmed that FLAG-CLEC-2 bound to podocytes (Fig.2A). However, when glomeruli were isolated from mice 1 hour after injection of FLAG-CLEC-2, FLAG was not detected by Western blot in the glomerular lysate. This implies that FLAG-CLEC-2 was not internalized to podocytes and that FLAG-CLEC-2 was detached from glomeruli during the glomerular isolation procedure. To test this possibility, cultured podocytes were incubated with FLAG-CLEC-2 (10 mg/mL) at 37℃ and washed with an acidic buffer. FLAG staining in podocytes decreased after washing. When incubation was performed at 4℃, FLAG was stained in podocytes with similar intensity to those incubated at 37℃, and the staining was similarly decreased after washing with an acidic buffer (Fig.2B). These observations indicate that FLAG-CLEC-2 is not internalized into podocytes. Quantitative RT-PCR analysis of the glomerular RNA revealed that infusion of FLAG-CLEC-2 increased Serpine1 mRNA, a podocyte injury marker 25 , 3.49 (1.56-5.57)-fold compared to controls (Fig.2C). The Western blot of glomerular lysate revealed that FLAG-CLEC-2 decreased the pEzrin/Ezrin ratio 0.5 (0.3-0.94)-fold (Fig.2D, S.Fig.4), indicating that CLEC-2 induced dephosphorylation of ezrin, most abundant ERM in the in vivo podocytes. This was also confirmed by double immunostaining of pERM and podocalyxin (Fig.2E). In SEM images, widening of foot processes was observed in 18.5% of visual fields in the mice treated with FLAG-CLEC-2, contrasting with no such change in control mice, suggesting that CLEC-2 induced a change in the cytoskeleton in foot processes (Fig.2F). These findings indicate that CLEC-2 induced dephosphorylation of ezrin and concomitant widening of the foot processes of podocytes. ・ Urinary platelets excreted by mice with podocyte injury We tested the possibility that podocytes can encounter platelets when glomeruli are injured. For this purpose, we induced podocyte injury in NEP25 mice by injecting LMB2. Five days after the injection of LMB2 (5 ng/g body weight), NEP25 mice exhibited both urinary protein and urinary blood, indicating disruption of the glomerular barrier. Immunostaining of CD41 revealed that the urinary sediments at this time point contained CD41 positive platelets (Fig.3A). To further verify leakage of platelets, platelets were collected from wild-type mice, labeled with DiI, and injected into NEP25 mice 5 days after the injection of LMB2. DiI-labeled platelets were found in the urine collected from the injected mice (Fig.3B). Thus, platelets in the bloodstream can make contact with podocytes when the glomerular barrier is injured. Discussion The present study revealed that recombinant CLEC-2, the ligand of PDPN, induced significant morphological change, attenuated adhesion, and promoted migration in cultured podocytes. CLEC-2 causes dephosphorylation of ERM and decomposition of PDPN-ERM-F-actin complex. A previous report indicated that PDPN binds to ezrin, whose phosphorylated form tightly connects with F-actin in podocytes 18 . A similar phenomenon has been reported in the FRCs of lymph nodes. CLEC-2 on dendritic cells acts on the PDPN of FRCs and induces dephosphorylation of ERM, disconnection of ERM from the plasma membrane, and a reduction in actomyosin contractility, which elongates FRCs 26 . In both FRCs and podocytes, binding with CLEC-2 attenuates the basal function of PDPN. Similarly, CLEC-2 inhibits the basal function of PDPN in keratinocytes and lymphatic endothelial cells 11 , 27 , but in these cases the net effect of CLEC-2 is the inhibition of cell migration, which is opposite to that in podocytes. Transient exposure to recombinant FLAG-CLEC-2 protein in a short period (1 hour) induced a significant morphological change in intact in vivo podocytes although the effect was modest compared to those in cultured podocytes. The modest effect may be caused by the monomeric feature of FLAG-CLEC-2. In injured glomeruli, polymeric CLEC-2 on platelets may bind to PDPN on podocytes and exert greater impacts. Moreover, in injured glomeruli, nephrin and podocalyxin are rapidly and remarkably downregulated 28 , which bind to NHERF2 and stabilize ezrin-F-Actin complex in intact podocytes 20 , 21 . Therefore, CLEC-2 may have a more significant impact on injured podocytes than those of the perfusion study. CLEC-2 is a membrane-bound protein mainly expressed in platelets. In addition, CLEC-2 exists in the circulation as shed or microparticle-bound forms 29 . It was reported that the mean plasma concentration of these soluble forms of CLEC-2 was 59–100 pg/mL in healthy volunteers and was increased to 260–380 pg/ml in patients with platelet-activating diseases 22 . In some patients with thrombotic microangiopathy or disseminated intravascular coagulation, the concentration of soluble CLEC-2 exceeds 1000 pg/ml 30 – 32 . Because platelets are retained in inflamed glomerular capillaries 33 , the local concentration of soluble CLEC-2 may be higher in glomerular diseases. Nevertheless, these concentrations do not appear sufficient to induce morphological change in normal podocytes considering the high dose of the recombinant CLEC-2 (5 µg/g body weight) used in the present study. Platelets are smaller than erythrocytes, therefore they can pass through the damaged glomerular barrier in glomerular diseases with hematuria. In fact, urinary platelets were detected in glomerular diseases 34 – 37 . We also demonstrated that platelets are excreted into urine in our podocyte injury mouse model. We speculate that platelets may pass through the glomerular barrier and act on PDPN in podocytes in kidney diseases. Although urinary platelets have received almost no attention, they may reflect a distinct disease condition. Taken together, we propose that PDPN on podocytes works as a sensor of platelet CLEC-2, which is leaked through glomeruli after severe injury. Stimulation by CLEC-2 induces morphological change and detachment of podocytes, which appears to further aggravate podocyte injury. Considering that PDPN on podocytes and CLEC-2 on platelets are evolutionally conserved, this system may have some beneficial effects, such as facilitating the repair process. Further study is necessary to establish the role of PDPN on podocytes. Methods ・ Animal ethics All animal experiments were approved by the Animal Experimentation Committee of Tokai University School of Medicine. All animal experiments were performed in accordance with relevant guideline and regulations, and the study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). ・ Recombinant CLEC-2 proteins Fc-CLEC-2, a fusion of the Fc tag and C-terminal extracellular domain of human CLEC-2 (51-229), was prepared as previously reported 38 . Fc-CLEC-2 expression plasmid was transiently transfected in HEK293 cells using X-tremeGENE 9 DNA (Roche), and Fc-CLEC-2 protein was purified by protein A affinity chromatography (KANEKA KanCapA, Wako). SDS-PAGE and Coomassie Brilliant Blue (CBB) stain confirmed the expected size of bands (Fc; 30kDa, Fc-CLEC-2; 60kDa) (S.Fig.1A). Pull-down assay confirmed that mouse PDPN can bind Fc-CLEC-2 (S.Fig.1C). StepTagII-3FLAG-CLEC-2, a fusion of StrepTactin FLAG double tags and the C-terminal extracellular domain of mouse CLEC-2 (51-229), was generated in HEK293 cells transiently transfected with the expression plasmid using polyethyleneimine Max reagent (Polysciences, Inc.). The supernatant of cell lysate was added with biotin blocking solution (Biolock, iba Life Science), and StepTagII-FLAG-CLEC-2 protein (hereafter designated as FLAG-CLEC-2) was purified by a StrepTactin Sepharose column (StrepTrap HP, GE healthcare Life Sciences). Mass spectrometric analysis by the LCMS-IT-TOF (Shimadzu) confirmed that the purified protein contained peptides specific to mouse CLEC-2. SDS-PAGE and CBB stain showed double bands around 30-35kDa (S.Fig.1A). The deglycosylation by PNGase F (New England Biolabs) changed the two bands to a single band (S.Fig.1B). Pull-down assay confirmed that mouse PDPN can bind FLAG-CLEC-2 (S.Fig.1C). Blue Native PAGE showed that Fc and Fc-CLEC-2 exist as tetramers, and FLAG-CLEC-2 exists as a monomer in aqueous solution (S.Fig.1D). ・ Western blot analysis Cells were lysed in a lysis buffer containing 1% Triton X-100, 2mM CaCl2, 0.5mM PMSF, Complete (Roche), and 50mM Tris/HCl (pH7.4). For analysis of phosphorylated protein, 10mM NaF, 1mM Na 3 VO 4 , and 5mM Na 4 P 2 O 7 were added. The homogenates were centrifuged at 15,000 rpm to remove the insoluble fraction. Each protein sample was separated by SDS-PAGE and transferred onto a PVDF membrane. The protein-blotted membranes were incubated with 1:1000 diluted primary antibodies overnight at 4℃ and then incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. The density of the positive bands was quantified by image analysis with CS Analyzer 3.0 (ATTO). The following primary antibodies were used: pERM (Cell Signaling, #3726), ERM (Cell Signaling, #3142), and b-tubulin (Cell Signaling, #2128). ・ Pull-down assay Podocyte lysate containing 1% Triton X-100 was mixed with Fc-CLEC-2 or FLAG-CLEC-2 for 1hr at 4℃, and the samples were incubated with KANEKA KanCapA (Wako) or Strep-Tactin Superflow plus (Qiagen), respectively, for 1hr at 4℃. After removing the supernatant, the beads were washed three times with cell lysis buffer and then incubated in SDS sample buffer. The supernatants were subjected to SDS-PAGE followed by Western blot with anti-PDPN antibody. ・I solation of glomeruli and primary podocyte culture Glomeruli were harvested using the bead method as previously reported 28 . They were cultured on a collagen-1-coated dish for 7 days in DMEM/F12 medium containing 5% FCS and 0.5% ITS-A. Outgrowing cells were detached and passaged after removing residual beads and glomeruli. Cells were used for in vitro experiments 1 to 7 days after the first to third passages. Cultured podocytes were treated with Fc or Fc-CLEC-2 (10 ug/mL). ・ Adhesion assay Primary cultured podocytes were seeded at a density of 30,000/cm 2 in collagen-1-coated 96-well plates. After 1 of hour incubation at 37℃ with Fc or Fc-CLEC-2 in 0.5% FCS medium, all wells were washed several times with PBS. The number of attached cells was quantified by Cell Counting Kit-8 (Dojindo Laboratories). ・ Migration assay Primary cultured podocytes were seeded at a density of 30,000/cm 2 within O-rings on collagen-1-coated dishes and cultured to reach 90-100% confluency. After the O-rings were removed, the cells were allowed to migrate for 24 hours at 37℃ in 5% FCS medium with Fc or Fc-CLEC-2. The longest migration length was measured. ・ Perfusion of mouse kidney with recombinant CLEC-2 protein C57BL/6 mice (4-6 months of age, approximately 12-19g body weight) were used for the experiments. Under anesthesia, the celiac and superior mesenteric arteries were transiently occluded with clips. The kidneys were perfused with 300 ml of PBS or PBS containing 5 mg/g body weight of FLAG-CLEC-2 through a catheter placed in the abdominal aorta at a distal site and then the clips were removed. After 1 hour, kidneys were perfused with PBS, harvested, and analyzed by electron microscopy and immunohistochemistry. In some experiments, glomeruli were collected and used for PCR and Western blot analyses. ・ Quantitative RT-PCR Total RNA was extracted from isolated glomeruli with an RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. Single-stranded cDNA was prepared from 100ng of RNA using TaqMan Reverse Transcription Reagents (ThermoFisher). A TaqMan primer probe set (Thermo Fisher) was used for Gapdh . For other genes, the following primers were used: Serpine1 , 5’-AGGATCGAGGTAAACGAGAGC-3’ and 5’-GCGGGCTGAGATGACAAA-3’; Moesin , 5’-TCTTATGCCGTCCAGTCTAAGT-3’ and 5’-GGTCCTTGTTGAGTTTGTGCT -3’; Ezrin , 5’-CAATCAACGTCCGGGTGAC-3’ and 5’-GCCAATCGTCTTTACCACCTGA-3’. Relative amounts of mRNA were determined using the delta-delta CT method. ・ Immunostaining and F-actin staining Primary cultured podocytes were seeded at a density of 5000/cm 2 on glass-based dishes 1 to 2 days before staining. For PDPN and moesin staining, cells were fixed in acetone. For F-actin staining, cells were fixed in 4% paraformaldehyde (PFA) and permeabilized in 0.1% Triton X-100/PBS. After blocking, they were incubated with Alexa Fluor 594-phalloidin (Invitrogen, diluted at 1/100). For FLAG staining, frozen kidney sections were fixed in acetone. For double staining of pERM and podocalyxin, kidneys were fixed in trichloroacetic acid (TCA) for 1 hour, followed by 4% PFA/PBS for 1 hour before preparing frozen blocks with OCT to preserve the phosphorylation of ERM. Information regarding antibodies is shown in Table 1. Table 1. Antibodies for immunostaining Antibody Company Cat. number Dilution or concentration PDPN (Gift from Dr. Umetsu) (PMab-1) 0.2 mg/ml Moesin Cell signaling 3150 1/150 FLAG Cell signaling 2368 1/800 pERM Cell signaling 3726 1/400 Podocalyxin R&D MAB1556 1/100 ・ Electron microscopy Kidneys were fixed by perfusion with 4% PFA and then immersed in 2.5% glutaraldehyde for 30min. Subsequent preparation of SEM was performed using standard methods. We randomly selected 8-10 glomeruli in each mouse and 3 images at X8000 magnification were captured for evaluation of foot processes. The number of images containing foot processes that were more than twice as thick as normal ones were counted. ・ Urinary platelets in mice with podocyte injury To induce podocyte injury, NEP25 mice were injected with 5 ng/g body weight of LMB2. Five days after the LMB2 injection, urine was collected and centrifuged at 500g. The sediment was washed several times with PBS containing EGTA 1mM and PGE1 0.25μM and stained with anti-CD41 antibody (Biolegend, #133901, 1:100). ・ Injection of DiI-labeled platelet solution into NEP25 mice Platelets were isolated from the blood of C57BL/6 mice 39 and stained with Vybrant DiI (Thermo Fisher scientific). A separate set of NEP25 mice were injected with LMB2. Five days later, DiI-labeled platelets were injected. Urinary sediment was inspected with confocal microscopy (ZEISS, LSM-880). ・ Statistical Analyses The results are expressed as the median and interquartile range (IQR). P values of <0.05 were considered to indicate statistical significance. Differences between groups were analyzed using the Mann–Whitney U‐test for continuous data. Statistical analyses were performed using the JMP software program (version 11, SAS Institute Inc.; Cary, NC, USA). Declarations Data availability All data generated or analyzed during this study are included in this published article. Acknowledgments This work was supported by JSPS KAKENHI Grant Numbers JP20K17257. We acknowledge Ms. Shiho Imai, Ms. Chie Sakurai and the Support Center for Medical Research and Education of Tokai University for excellent technical assistance and Ms. Yukiko Tanaka for administrative assistance. Author contributions KT and TM were involved in the conception and design of the experiments. KT performed the experiments. MT, NW, and MI were contributed to generating recombinant proteins. IP provided the LMB2 and related insights. KT, MK and TM analyzed the data and interpreted the results. 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Kidney international 95 , 363–374, doi: 10.1016/j.kint.2018.08.042 (2019). Shirato, I., Tomino, Y. & Koide, H. Detection of 'activated platelets' in the urinary sediments using a scanning electron microscope in patients with IgA nephropathy. Am J Nephrol 10 , 186–190, doi: 10.1159/000168079 (1990). Tomino, Y. et al. Detection of activated platelets in urinary sediments by immunofluorescence using monoclonal antibody to human platelet GMP-140 in patients with IgA nephropathy. J Clin Lab Anal 7 , 329–333, doi: 10.1002/jcla.1860070606 (1993). Kamitsuji, H., Nakajima, M., Kawahara, S. & Nishimura, T. Detection of activated platelets in urine by double immunofluorescence in children with IgA nephropathy. Nephron 78 , 162–167, doi: 10.1159/000044905 (1998). Taira, K., Hewitson, T. D. & Kincaid-Smith, P. Urinary platelet factor four (Pf4) levels in mesangial IgA glomerulonephritis and thin basement membrane disease. Clinical nephrology 37 , 8–13 (1992). Watanabe, N. et al. A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction. PLoS One 14 , e0222331, doi: 10.1371/journal.pone.0222331 (2019). Im, J. H. & Muschel, R. J. Protocol for Murine/Mouse Platelets Isolation and Their Reintroduction in vivo. Bio Protoc 7 , e2132, doi: 10.21769/BioProtoc.2132 (2017). Additional Declarations No competing interests reported. Supplementary Files Supplimentaryfigures.pdf Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2022 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 20 Sep, 2022 Reviews received at journal 06 Aug, 2022 Reviewers agreed at journal 27 Jul, 2022 Reviewers invited by journal 25 Jul, 2022 Editor assigned by journal 20 Jul, 2022 Editor invited by journal 12 Jul, 2022 Submission checks completed at journal 12 Jul, 2022 First submitted to journal 02 Jul, 2022 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-1817977","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":120461168,"identity":"7897841f-6617-4cc6-ae13-1335eba53e82","order_by":0,"name":"Keiko Tanaka","email":"","orcid":"","institution":"Tokai University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Keiko","middleName":"","lastName":"Tanaka","suffix":""},{"id":120461170,"identity":"13b02fe8-b6bd-4041-ac12-f3b8fb4fa939","order_by":1,"name":"Masafumi Tanaka","email":"","orcid":"","institution":"Tokai University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masafumi","middleName":"","lastName":"Tanaka","suffix":""},{"id":120461171,"identity":"1703aa03-4ebc-461e-9e6c-3ba2a828d229","order_by":2,"name":"Nobuo Watanabe","email":"","orcid":"","institution":"Tokai University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nobuo","middleName":"","lastName":"Watanabe","suffix":""},{"id":120461172,"identity":"e038f2f5-791c-450f-9f5c-84c2d753f936","order_by":3,"name":"Masatoshi Ito","email":"","orcid":"","institution":"Tokai University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masatoshi","middleName":"","lastName":"Ito","suffix":""},{"id":120461173,"identity":"89fe681b-b49c-43b3-b0d1-d2ae167e3af6","order_by":4,"name":"Ira Pastan","email":"","orcid":"","institution":"National Cancer Institute, NIH","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ira","middleName":"","lastName":"Pastan","suffix":""},{"id":120461174,"identity":"72508498-259f-4f12-856c-23dbd36e2f25","order_by":5,"name":"Masahiro Koizumi","email":"","orcid":"","institution":"Tokai University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Koizumi","suffix":""},{"id":120461175,"identity":"8c9914dc-a2bd-4fa4-9de9-a25e8ca6d5e2","order_by":6,"name":"Taiji Matsusaka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie2QsWrDMBCGzwjs5cCrAgG9gkyXQkv8KjIBe+nQMdAhKgF7MeRtMhsE7uIma0uWeMns0d0q223aQWrXQvUN4jjp478TgMPxJ/EkF3CLQ/HR8QHEWFRGAScl/VLwd2VEfe/4P48VB0ret3eHOZPkseu1Gwd50p1gwcB7NsYgJpInuyPyytvMSq0g1ooKWEYS9sI82KcCXk70hOuSZlIrRD9vuFEJ20HZI5OTAkizTS9gbVfomFIhVBclrXWKsisvY8oSuRp24RlgU6fXgj9FuWWXYJudo7fdImZF0Xb96gaCIr967VcPLKTmH9P4UzwZjsskuvBpYzGAnCwXYWlTHA6H43/xDpOrV6b9HshjAAAAAElFTkSuQmCC","orcid":"","institution":"Tokai University School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Taiji","middleName":"","lastName":"Matsusaka","suffix":""}],"badges":[],"createdAt":"2022-07-02 09:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1817977/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1817977/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-022-26456-9","type":"published","date":"2022-12-26T18:07:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":24094829,"identity":"7d0d1c1f-ee24-49d1-9294-3f97765c5405","added_by":"auto","created_at":"2022-07-20 14:53:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":252565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e study with Fc and Fc-CLEC-2.\u003c/p\u003e\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;Representative images of kidney sections and cultured podocytes, stained with podoplanin (PDPN). PDPN is intensely stained both in \u003cem\u003ein vivo\u003c/em\u003e podocytes and cultured podocytes. Scale bar: 50μm\u003c/p\u003e\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;Representative images of cultured podocytes, stained with phalloidin after 1 hour incubation at 37℃ with Fc or Fc-CLEC-2. Podocytes incubated with Fc-CLEC-2 showed a round shape with degradation of F-actin, while those with Fc showed an elongated morphology with numerous F-actin filaments. Scale bar: 100μm\u003c/p\u003e\u003cp\u003eC.\u0026nbsp;Adhesion assay. The percentage of attached podocytes was evaluated after 1 hour incubation at 37℃ with Fc or Fc-CLEC-2. Podocytes incubated with Fc-CLEC-2 showed less attachment than those with Fc.\u003c/p\u003e\u003cp\u003eD.\u0026nbsp;Migration assay. The distance of migration of podocytes was measured while incubated at 37℃ with Fc or Fc-CLEC-2 for 24 hours. Podocytes incubated with Fc-CLEC-2 showed greater migration than those with Fc.\u003c/p\u003e\u003cp\u003eE.\u0026nbsp;Western blot analysis for ERM and pERM after treatment of Fc or Fc-CLEC-2 at 37℃ for 1 hour. The pERM/ERM ratio was 0.47-fold decreased in podocytes with Fc-CLEC-2, compared to those with Fc. The images of full-length blots are shown in Supplementary figure 3. Ezrin: 81kDa, Moesin: 75kDa, β-tubulin 55kDa.\u003c/p\u003e\u003cp\u003eF.\u0026nbsp;Representative images of cultured podocytes, stained with moesin after 1 hour incubation at 37℃ with Fc or Fc-CLEC-2. Fc increased the number of protrusions that were positive for moesin staining, and Fc-CLEC-2 markedly decreased them. Scale bar: 50μm\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1817977/v1/23c113b7cd722d271ce44594.jpg"},{"id":24094827,"identity":"67bc8f21-363a-48f8-86bd-dc12fd926023","added_by":"auto","created_at":"2022-07-20 14:53:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":445815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e study with FLAG-CLEC-2 protein\u003c/p\u003e\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;Immunostaining for FLAG in kidneys with or without infusion of FLAG-CLEC-2. After infusion of FLAG-CLEC-2, glomeruli were intensely stained for FLAG in a podocyte pattern. Scale bar: 50μm\u003c/p\u003e\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;Internalization assay using cultured podocytes. Representative images of podocytes stained for FLAG, after 3 hour incubation at 37℃ or 4℃ with or without subsequent washing with a stripping buffer. At either temperature, FLAG staining diminished after washing, indicating that FLAG-CLEC-2 was not internalized within the cells. Scale bar: 100μm\u003c/p\u003e\u003cp\u003eC. Serpine1 mRNA in the glomeruli (relative amount). Serpine1 mRNA expression was 3.49-fold increased in the mice with FLAG-CLEC-2 infusion.\u003c/p\u003e\u003cp\u003eD. Western blot analysis of glomerular lysate for ERM. Phosphorylation of ezrin (upper band; 81kDa), the major ERM in \u003cem\u003ein vivo\u003c/em\u003e podocytes, was 0.50-fold decreased by FLAG-CLEC-2. The images of full-length blots are shown in Supplementary figure 4. Ezrin: 81kDa, Moesin: 75kDa, β-tubulin 55kDa.\u003c/p\u003e\u003cp\u003eE. Dephosphorylation of ERM in podocytes by FLAG-CLEC-2 perfusion.\u003c/p\u003e\u003cp\u003eDouble immunostaining showed an intense signal for pERM (red) in podocytes labeled by podocalyxin staining (green) in control mice (upper panels). In the mice perfused with FLAG-CLEC-2, some podocytes lack pERM staining. Scale bar: 50μm.\u003c/p\u003e\u003cp\u003eF. SEM images of the foot processes. Podocytes perfused with FLAG-CLEC-2 exhibited widening of foot processes (arrows) in 18.5% of visual fields. Original magnification: ×8000. Scale bar: 2μm.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1817977/v1/78355d801026096d2817f973.jpg"},{"id":24095471,"identity":"ef081d7c-1ba9-46ab-9c52-16409d081782","added_by":"auto","created_at":"2022-07-20 14:58:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24971,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of urinary platelets in podocyte injured mice.\u003c/p\u003e\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;Confocal microscopy of Cy3-CD41 platelets in the urine.\u003c/p\u003e\u003cp\u003e\u0026nbsp;Immunostaining for CD41 showed platelets (arrows) were detected in the urine 5 days after LMB2 injection. \u003c/p\u003e\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;Confocal microscopy of DiI-platelets in the urine.\u003c/p\u003e\u003cp\u003e\u0026nbsp;DiI labeled platelets (arrows) were detected in the urine of mice injected with DiI platelets 5 days after LMB2 injection. Scale bar: 10μm.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1817977/v1/6d17badfdfed498ee4628f40.jpg"},{"id":44715467,"identity":"1955bd55-fdd4-4a8c-ae37-d69c169e717a","added_by":"auto","created_at":"2023-10-16 18:14:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":610133,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1817977/v1/30c83961-8c56-4d70-b252-26a8c15e2d15.pdf"},{"id":24094830,"identity":"a2556a10-e767-4402-ad19-925a4cce2f7d","added_by":"auto","created_at":"2022-07-20 14:53:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":319477,"visible":true,"origin":"","legend":"","description":"","filename":"Supplimentaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1817977/v1/fe75a6415397131289fd6314.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"C-type lectin-like receptor (CLEC)-2, the ligand of podoplanin, induces morphological changes in podocytes","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePodoplanin (PDPN) is a membranous mucin-type \u003cem\u003eO\u003c/em\u003e-glycosylated glycoprotein, which is negatively charged by abundant sialic acid. PDPN is expressed on the surface of various types of cells, including kidney podocytes, alveolar epithelial cells, lymphatic endothelial cells, stromal fibroblastic reticular cells (FRCs) of lymph nodes \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and tumors \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Among these, PDPN is most intensely expressed on podocytes and its expression is evolutionally conserved \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePDPN was found to be the endogenous ligand of C-type lectin-like receptor 2 (CLEC-2) on platelets and is involved in platelet aggregation induced by tumor cells \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, which facilitates invasion and metastasis of the tumor \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. During developmental stages, CLEC-2 in platelets can interact with PDPN in lymphatic endothelial cells, and activated platelets facilitate blood-lymphatics vessel separation \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Platelet activation by PDPN also plays a critical role in the differentiation of alveolar duct myofibroblasts \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCLEC-2\u0026ndash;PDPN interaction mediates bidirectional signaling. PDPN works as a ligand for CLEC-2 as shown above. In addition, binding PDPN with CLEC-2 influences PDPN-expressing cells. Binding PDPN with CLEC-2 attenuates actomyosin contractility in FRCs in the lymph node \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, inhibits migration of lymphatic endothelial cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, stimulates CCL5 secretion in FRC-like cells in bone marrow \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, stimulates IGF-1 secretion from stromal cells \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and attenuates inflammatory responses in a subset of Th17 cells \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe role of PDPN in podocytes is not fully elucidated. Decreased expression of PDPN in podocytes is associated with foot process effacement, proteinuria, and decreased glomerular selective permeability in several animal models \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In biopsy specimens of patients with minimal change nephrotic syndrome, PDPN staining was decreased in the proteinuric state, and recovered when proteinuria was normalized \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These factors suggest that PDPN is important for maintenance of the normal function of podocytes. The cytoplasmic tail of PDPN interacts with ezrin, radixin, and moesin (ERM) proteins, which bind the actin cytoskeleton and regulate cell shape. It was reported that whole-body \u003cem\u003ePdpn\u003c/em\u003e-gene-disrupted mice showed no abnormal renal phenotypes \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Podocalyxin-NHERF2 complex and nephrin-ephrin-B1-NHERF2 complexes can bind and maintain ezrin-F-Actin complex \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This may be a reason for the lack of abnormal phenotype in podocytes of congenital Pdpn-deficient mice. However, suppression of PDPN by siRNA in cultured podocytes changed cell morphology from an elongated to a round shape along with a change in ezrin distribution \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNormally, podocytes are sequestered from platelets, but when the glomerular barrier is injured, podocytes gain access to CLEC-2 on platelets. In addition, a soluble form of CLEC-2 with molecular weight 25 kDa exists in human plasma \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. We speculated that CLEC-2, the known ligand for PDPN, may have some biological impact on podocytes. This hypothesis is supported by previous reports that injection of antibodies against specific epitopes of PDPN caused transient proteinuria and foot process effacement in rats \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In the present study, we examined the effect of CLEC-2 in podocytes \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eEffects of CLEC-2 on\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e podocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first tested the effects of recombinant Fc-human CLEC-2 on cultured mouse podocytes. Although most proteins characteristic to podocytes, such as nephrin and podocin, are rapidly downregulated upon \u003cem\u003ein vitro\u003c/em\u003e culture, PDPN staining was maintained in primary cultured podocytes with similar intensity to \u003cem\u003ein vivo\u003c/em\u003e podocytes (Fig.1A). As reported previously \u003csup\u003e3\u003c/sup\u003e, a pull-down experiment showed that Fc-human CLEC-2 bound to mouse PDPN (S.Fig.1C). The majority of podocytes treated with Fc protein for 1 hour showed an elongated morphology with sharp protrusions and had numerous F-actin filaments. In contrast, podocytes treated with Fc-CLEC-2 showed a round cell shape without sharp protrusions and a decrease in F-Actin (Fig.1B). Furthermore, podocytes treated with Fc-CLEC-2 showed less adhesion to the collagen-1-coated plate within 1 hour than the Fc control cells (92.3 % vs. 100 %) (Fig.1C). Podocytes with Fc-CLEC-2 showed more migration than the Fc control (2.62 vs. 1.93 mm/24hr) (Fig.1D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next studied the phosphorylation status of ERM, which links PDPN with F-actin, to reveal the intracellular signaling induced by CLEC-2. Western blot analysis revealed that treatment with Fc-CLEC-2 reduced the pERM/ERM ratio in cultured podocytes, indicating that CLEC-2 induced dephosphorylation of ERM proteins (Fig.1E, S.Fig.3). The most abundant ERM protein in \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003epodocytes is ezrin \u003csup\u003e24\u003c/sup\u003e. However, our Western blot analysis and quantitative PCR revealed that the most abundant ERM in cultured podocytes is moesin (S.Fig.2A and 2B). Immunostaining showed that moesin is intensely stained in the protrusions of Fc control podocytes, and that incubation with Fc-CLEC-2 markedly decreased moesin staining (Fig.1F). These results collectively indicate that CLEC-2 induced dephosphorylation of moesin, which caused dissociation of F-actin filaments from PDPN, F-actin degradation, and cell morphological change.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eThe effects of CLEC-2 on\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e podocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the effects of CLEC-2 on \u003cem\u003ein vivo\u003c/em\u003e podocytes, we generated a new recombinant CLEC-2 protein with a smaller size, because the above Fc-CLEC-2 forms a tetramer with a size of about 240 kDa, which was not expected to reach podocytes through the normal glomerular barrier. The new mouse CLEC-2 protein, FLAG-CLEC-2, exists as a monomer in solution and the size is 30-35 kDa.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We infused 5\u0026nbsp;mg/g body weight of FLAG-CLEC-2 into normal mice and excised the kidney 1 hour later. Immunostaining of FLAG confirmed that FLAG-CLEC-2 bound to podocytes (Fig.2A). However, when glomeruli were isolated from mice 1 hour after injection of FLAG-CLEC-2, FLAG was not detected by Western blot in the glomerular lysate. This implies that FLAG-CLEC-2 was not internalized to podocytes and that FLAG-CLEC-2 was detached from glomeruli during the glomerular isolation procedure. To test this possibility, cultured podocytes were incubated with FLAG-CLEC-2 (10\u0026nbsp;mg/mL) at 37℃ and washed with an acidic buffer. FLAG staining in podocytes decreased after washing. When incubation was performed at 4℃, FLAG was stained in podocytes with similar intensity to those incubated at 37℃, and the staining was similarly decreased after washing with an acidic buffer (Fig.2B). These observations indicate that FLAG-CLEC-2 is not internalized into podocytes.\u003c/p\u003e\n\u003cp\u003eQuantitative RT-PCR analysis of the glomerular RNA revealed that infusion of FLAG-CLEC-2 increased Serpine1 mRNA, a podocyte injury marker\u0026nbsp;\u003csup\u003e25\u003c/sup\u003e, 3.49 (1.56-5.57)-fold compared to controls (Fig.2C). The Western blot of glomerular lysate revealed that FLAG-CLEC-2 decreased the pEzrin/Ezrin ratio 0.5 (0.3-0.94)-fold (Fig.2D, S.Fig.4), indicating that CLEC-2 induced dephosphorylation of ezrin, most abundant ERM in the \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003epodocytes. This was also confirmed by double immunostaining of pERM and podocalyxin (Fig.2E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn SEM images, widening of foot processes was observed in 18.5% of visual fields in the mice treated with FLAG-CLEC-2, contrasting with no such change in control mice, suggesting that CLEC-2 induced a change in the cytoskeleton in foot processes (Fig.2F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese\u003cem\u003e\u0026nbsp;\u003c/em\u003efindings indicate that CLEC-2 induced dephosphorylation of ezrin and concomitant widening of the foot processes of podocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eUrinary platelets excreted by mice with podocyte injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe tested the possibility that podocytes can encounter platelets when glomeruli are injured. For this purpose, we induced podocyte injury in NEP25 mice by injecting LMB2. Five days after the injection of LMB2 (5 ng/g body weight), NEP25 mice exhibited both urinary protein and urinary blood, indicating disruption of the glomerular barrier. Immunostaining of CD41 revealed that the urinary sediments at this time point contained CD41 positive platelets (Fig.3A). To further verify leakage of platelets, platelets were collected from wild-type mice, labeled with DiI, and injected into NEP25 mice 5 days after the injection of LMB2. DiI-labeled platelets were found in the urine collected from the injected mice (Fig.3B). Thus, platelets in the bloodstream can make contact with podocytes when the glomerular barrier is injured.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study revealed that recombinant CLEC-2, the ligand of PDPN, induced significant morphological change, attenuated adhesion, and promoted migration in cultured podocytes. CLEC-2 causes dephosphorylation of ERM and decomposition of PDPN-ERM-F-actin complex. A previous report indicated that PDPN binds to ezrin, whose phosphorylated form tightly connects with F-actin in podocytes \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. A similar phenomenon has been reported in the FRCs of lymph nodes. CLEC-2 on dendritic cells acts on the PDPN of FRCs and induces dephosphorylation of ERM, disconnection of ERM from the plasma membrane, and a reduction in actomyosin contractility, which elongates FRCs \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In both FRCs and podocytes, binding with CLEC-2 attenuates the basal function of PDPN. Similarly, CLEC-2 inhibits the basal function of PDPN in keratinocytes and lymphatic endothelial cells \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, but in these cases the net effect of CLEC-2 is the inhibition of cell migration, which is opposite to that in podocytes.\u003c/p\u003e \u003cp\u003eTransient exposure to recombinant FLAG-CLEC-2 protein in a short period (1 hour) induced a significant morphological change in intact \u003cem\u003ein vivo\u003c/em\u003e podocytes although the effect was modest compared to those in cultured podocytes. The modest effect may be caused by the monomeric feature of FLAG-CLEC-2. In injured glomeruli, polymeric CLEC-2 on platelets may bind to PDPN on podocytes and exert greater impacts. Moreover, in injured glomeruli, nephrin and podocalyxin are rapidly and remarkably downregulated \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, which bind to NHERF2 and stabilize ezrin-F-Actin complex in intact podocytes\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Therefore, CLEC-2 may have a more significant impact on injured podocytes than those of the perfusion study.\u003c/p\u003e \u003cp\u003eCLEC-2 is a membrane-bound protein mainly expressed in platelets. In addition, CLEC-2 exists in the circulation as shed or microparticle-bound forms \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. It was reported that the mean plasma concentration of these soluble forms of CLEC-2 was 59\u0026ndash;100 pg/mL in healthy volunteers and was increased to 260\u0026ndash;380 pg/ml in patients with platelet-activating diseases \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In some patients with thrombotic microangiopathy or disseminated intravascular coagulation, the concentration of soluble CLEC-2 exceeds 1000 pg/ml \u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Because platelets are retained in inflamed glomerular capillaries \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, the local concentration of soluble CLEC-2 may be higher in glomerular diseases. Nevertheless, these concentrations do not appear sufficient to induce morphological change in normal podocytes considering the high dose of the recombinant CLEC-2 (5 \u0026micro;g/g body weight) used in the present study.\u003c/p\u003e \u003cp\u003ePlatelets are smaller than erythrocytes, therefore they can pass through the damaged glomerular barrier in glomerular diseases with hematuria. In fact, urinary platelets were detected in glomerular diseases \u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. We also demonstrated that platelets are excreted into urine in our podocyte injury mouse model. We speculate that platelets may pass through the glomerular barrier and act on PDPN in podocytes in kidney diseases. Although urinary platelets have received almost no attention, they may reflect a distinct disease condition.\u003c/p\u003e \u003cp\u003eTaken together, we propose that PDPN on podocytes works as a sensor of platelet CLEC-2, which is leaked through glomeruli after severe injury. Stimulation by CLEC-2 induces morphological change and detachment of podocytes, which appears to further aggravate podocyte injury. Considering that PDPN on podocytes and CLEC-2 on platelets are evolutionally conserved, this system may have some beneficial effects, such as facilitating the repair process. Further study is necessary to establish the role of PDPN on podocytes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eAnimal ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Animal Experimentation Committee of Tokai University School of Medicine. All animal experiments were performed in accordance with relevant guideline and regulations, and the study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eRecombinant CLEC-2 proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFc-CLEC-2, a fusion of the Fc tag and C-terminal extracellular domain of human CLEC-2 (51-229), was prepared as previously reported\u0026nbsp;\u003csup\u003e38\u003c/sup\u003e. Fc-CLEC-2 expression plasmid was transiently transfected in HEK293 cells using X-tremeGENE 9 DNA (Roche), and Fc-CLEC-2 protein was purified by protein A affinity chromatography (KANEKA KanCapA, Wako). SDS-PAGE and Coomassie Brilliant Blue (CBB) stain confirmed the expected size of bands (Fc; 30kDa, Fc-CLEC-2; 60kDa) (S.Fig.1A). Pull-down assay confirmed that mouse PDPN can bind Fc-CLEC-2 (S.Fig.1C).\u003c/p\u003e\n\u003cp\u003eStepTagII-3FLAG-CLEC-2, a fusion of StrepTactin FLAG double tags and the C-terminal extracellular domain of mouse CLEC-2 (51-229), was generated in HEK293 cells transiently transfected with the expression plasmid using polyethyleneimine Max reagent (Polysciences, Inc.). The supernatant of cell lysate was added with biotin blocking solution (Biolock, iba Life Science), and StepTagII-FLAG-CLEC-2 protein (hereafter designated as FLAG-CLEC-2) was purified by a StrepTactin Sepharose column (StrepTrap HP, GE healthcare Life Sciences). Mass spectrometric analysis by the LCMS-IT-TOF (Shimadzu) confirmed that the purified protein contained peptides specific to mouse CLEC-2. SDS-PAGE and CBB stain showed double bands around 30-35kDa (S.Fig.1A). The deglycosylation by PNGase F (New England Biolabs) changed the two bands to a single band (S.Fig.1B). Pull-down assay confirmed that mouse PDPN can bind FLAG-CLEC-2 (S.Fig.1C).\u003c/p\u003e\n\u003cp\u003eBlue Native PAGE showed that Fc and Fc-CLEC-2 exist as tetramers, and FLAG-CLEC-2 exists as a monomer in aqueous solution (S.Fig.1D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed in a lysis buffer containing 1% Triton X-100, 2mM CaCl2, 0.5mM PMSF, Complete (Roche), and 50mM Tris/HCl (pH7.4). For analysis of phosphorylated protein, 10mM NaF, 1mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, and 5mM Na\u003csub\u003e4\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e were added. The homogenates were centrifuged at 15,000 rpm to remove the insoluble fraction. Each protein sample was separated by SDS-PAGE and transferred onto a PVDF membrane. The protein-blotted membranes were incubated with 1:1000 diluted primary antibodies overnight at 4℃ and then incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. The density of the positive bands was quantified by image analysis with CS Analyzer 3.0 (ATTO). The following primary antibodies were used: pERM (Cell Signaling, #3726), ERM (Cell Signaling, #3142), and b-tubulin (Cell Signaling, #2128).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003ePull-down assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePodocyte lysate containing 1% Triton X-100 was mixed with Fc-CLEC-2 or FLAG-CLEC-2 for 1hr at 4℃, and the samples were incubated with KANEKA KanCapA (Wako) or Strep-Tactin Superflow plus (Qiagen), respectively, for 1hr at 4℃. After removing the supernatant, the beads were washed three times with cell lysis buffer and then incubated in SDS sample buffer. The supernatants were subjected to SDS-PAGE followed by Western blot with anti-PDPN antibody.\u003c/p\u003e\n\u003cp\u003e・I\u003cstrong\u003esolation of glomeruli and primary podocyte culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGlomeruli were harvested using the bead method as previously reported \u003csup\u003e28\u003c/sup\u003e. They were cultured on a collagen-1-coated dish for 7 days in DMEM/F12 medium containing 5% FCS and 0.5% ITS-A. Outgrowing cells were detached and passaged after removing residual beads and glomeruli. Cells were used for \u003cem\u003ein vitro\u003c/em\u003e experiments 1 to 7 days after the first to third passages. Cultured podocytes were treated with Fc or Fc-CLEC-2 (10 ug/mL).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e・\u003cstrong\u003eAdhesion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary cultured podocytes were seeded at a density of 30,000/cm\u003csup\u003e2\u003c/sup\u003e in collagen-1-coated 96-well plates. After 1 of hour incubation at 37℃ with Fc or Fc-CLEC-2 in 0.5% FCS medium, all wells were washed several times with PBS. The number of attached cells was quantified by Cell Counting Kit-8 (Dojindo Laboratories).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e・\u003cstrong\u003eMigration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary cultured podocytes were seeded at a density of 30,000/cm\u003csup\u003e2\u003c/sup\u003e within O-rings on collagen-1-coated dishes and cultured to reach 90-100% confluency. After the O-rings were removed, the cells were allowed to migrate for 24 hours at 37℃ in 5% FCS medium with Fc or Fc-CLEC-2. The longest migration length was measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003ePerfusion of mouse kidney with recombinant CLEC-2 protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6 mice (4-6 months of age, approximately 12-19g body weight) were used for the experiments. Under anesthesia, the celiac and superior mesenteric arteries were transiently occluded with clips. The kidneys were perfused with 300 ml of PBS or PBS containing 5 mg/g body weight of FLAG-CLEC-2 through a catheter placed in the abdominal aorta at a distal site and then the clips were removed. After 1 hour, kidneys were perfused with PBS, harvested, and analyzed by electron microscopy and immunohistochemistry. In some experiments, glomeruli were collected and used for PCR and Western blot analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eQuantitative RT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from isolated glomeruli with an RNeasy Plus Mini Kit (Qiagen) according to the manufacturer\u0026rsquo;s instructions. Single-stranded cDNA was prepared from 100ng of RNA using TaqMan Reverse Transcription Reagents (ThermoFisher). A TaqMan primer probe set (Thermo Fisher) was used for\u003cem\u003e\u0026nbsp;Gapdh\u003c/em\u003e. For other genes, the following primers were used: \u003cem\u003eSerpine1\u003c/em\u003e, 5\u0026rsquo;-AGGATCGAGGTAAACGAGAGC-3\u0026rsquo; and 5\u0026rsquo;-GCGGGCTGAGATGACAAA-3\u0026rsquo;; \u003cem\u003eMoesin\u003c/em\u003e, 5\u0026rsquo;-TCTTATGCCGTCCAGTCTAAGT-3\u0026rsquo; and 5\u0026rsquo;-GGTCCTTGTTGAGTTTGTGCT -3\u0026rsquo;; \u003cem\u003eEzrin\u003c/em\u003e, 5\u0026rsquo;-CAATCAACGTCCGGGTGAC-3\u0026rsquo; and 5\u0026rsquo;-GCCAATCGTCTTTACCACCTGA-3\u0026rsquo;. Relative amounts of mRNA were determined using the delta-delta CT method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eImmunostaining and F-actin staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary cultured podocytes were seeded at a density of 5000/cm\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eon glass-based dishes 1 to 2 days before staining. For PDPN and moesin staining, cells were fixed in acetone. For F-actin staining, cells were fixed in 4% paraformaldehyde (PFA) and permeabilized in 0.1% Triton X-100/PBS. After blocking, they were incubated with Alexa Fluor 594-phalloidin (Invitrogen, diluted at 1/100). For FLAG staining, frozen kidney sections were fixed in acetone. For double staining of pERM and podocalyxin, kidneys were fixed in trichloroacetic acid (TCA) for 1 hour, followed by 4% PFA/PBS for 1 hour before preparing frozen blocks with OCT to preserve the phosphorylation of ERM. Information regarding antibodies is shown in Table 1.\u003c/p\u003e\n\n\u003cp\u003eTable 1. Antibodies for immunostaining\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003eAntibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"32.098765432098766%\"\u003e\n \u003cp\u003eCompany\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.691358024691358%\"\u003e\n \u003cp\u003eCat. number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.280423280423282%\"\u003e\n \u003cp\u003eDilution or concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003ePDPN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"32.098765432098766%\"\u003e\n \u003cp\u003e(Gift from Dr. Umetsu)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.691358024691358%\"\u003e\n \u003cp\u003e(PMab-1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.280423280423282%\"\u003e\n \u003cp\u003e0.2\u0026nbsp;mg/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003eMoesin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"32.098765432098766%\"\u003e\n \u003cp\u003eCell signaling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.691358024691358%\"\u003e\n \u003cp\u003e3150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.280423280423282%\"\u003e\n \u003cp\u003e1/150\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003eFLAG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"32.098765432098766%\"\u003e\n \u003cp\u003eCell signaling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.691358024691358%\"\u003e\n \u003cp\u003e2368\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.280423280423282%\"\u003e\n \u003cp\u003e1/800\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003epERM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"32.098765432098766%\"\u003e\n \u003cp\u003eCell signaling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.691358024691358%\"\u003e\n \u003cp\u003e3726\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.280423280423282%\"\u003e\n \u003cp\u003e1/400\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003ePodocalyxin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"32.098765432098766%\"\u003e\n \u003cp\u003eR\u0026amp;D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.691358024691358%\"\u003e\n \u003cp\u003eMAB1556\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.280423280423282%\"\u003e\n \u003cp\u003e1/100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eElectron microscopy\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Kidneys were fixed by perfusion with 4% PFA and then immersed in 2.5% glutaraldehyde for 30min. Subsequent preparation of SEM was performed using standard methods. We randomly selected 8-10 glomeruli in each mouse and 3 images at X8000 magnification were captured for evaluation of foot processes. The number of images containing foot processes that were more than twice as thick as normal ones were counted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eUrinary platelets in mice with podocyte injury\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo induce podocyte injury, NEP25 mice were injected with 5 ng/g body weight of LMB2. Five days after the LMB2 injection, urine was collected and centrifuged at 500g. The sediment was washed several times with PBS containing EGTA 1mM and PGE1 0.25\u0026mu;M and stained with anti-CD41 antibody (Biolegend, #133901, 1:100).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e・\u003cstrong\u003eInjection of DiI-labeled platelet solution into NEP25 mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlatelets were isolated from the blood of C57BL/6 mice \u003csup\u003e39\u003c/sup\u003e and stained with Vybrant DiI (Thermo Fisher scientific). A separate set of NEP25 mice were injected with LMB2. Five days later, DiI-labeled platelets were injected. Urinary sediment was inspected with confocal microscopy (ZEISS, LSM-880).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e・\u003c/strong\u003e\u003cstrong\u003eStatistical Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results are expressed as the median and interquartile range (IQR). \u003cem\u003eP\u003c/em\u003e values of \u0026lt;0.05 were considered to indicate statistical significance. Differences between groups were analyzed using the Mann\u0026ndash;Whitney U‐test for continuous data. Statistical analyses were performed using the JMP software program (version 11, SAS Institute Inc.; Cary, NC, USA).\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI Grant Numbers JP20K17257. We acknowledge Ms. Shiho Imai, Ms. Chie Sakurai and the Support Center for Medical Research and Education of Tokai University for excellent technical assistance and Ms. Yukiko Tanaka for administrative assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKT and TM were involved in the conception and design of the experiments. KT performed the experiments. MT, NW, and MI were contributed to generating recombinant proteins. IP provided the LMB2 and related insights. KT, MK and TM analyzed the data and interpreted the results. KT prepared the figures and drafted the manuscript, and TM critically revised the manuscript. All authors reviewed and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSuzuki-Inoue, K., Osada, M. \u0026amp; Ozaki, Y. 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Bio Protoc \u003cb\u003e7\u003c/b\u003e, e2132, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21769/BioProtoc.2132\u003c/span\u003e\u003cspan address=\"10.21769/BioProtoc.2132\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1817977/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1817977/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePodoplanin (PDPN) is intensely expressed on the podocyte membrane in an evolutionally conserved manner. CLEC-2, the endogenous ligand of PDPN, is highly expressed in platelets and also exists in a soluble form in plasma. Normally, podocytes are sequestered from CLEC-2, but when the glomerular barrier is injured, podocytes gain access to CLEC-2. We tested the effects of CLEC-2 in podocytes \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eCultured podocytes treated with Fc-CLEC-2 demonstrated that CLEC-2 induced the dephosphorylation of moesin, which we found to be the major ERM in cultured podocytes. Podocytes treated with Fc-CLEC-2 also showed the dissociation of F-actin filaments from PDPN, F-actin degradation, detachment, and round morphology. Next, we perfused normal mouse kidney \u003cem\u003ein vivo\u003c/em\u003e with FLAG-CLEC-2. CLEC-2 induced dephosphorylation of ezrin and widening of the foot processes of podocytes. Platelets were detected by immunostaining for CD41 in the urine of mice with podocyte injury, indicating that podocytes can encounter platelets when glomeruli are injured.\u003c/p\u003e \u003cp\u003eCollectively, these observations suggest that when platelets leak through the injured glomeruli, CLEC-2 from the platelets acts on PDPN in podocytes and induces morphological change and detachment, which may further aggravate podocyte injury. Thus, PDPN on podocytes may work as a leaked-platelet sensor.\u003c/p\u003e","manuscriptTitle":"C-type lectin-like receptor (CLEC)-2, the ligand of podoplanin, induces morphological changes in podocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-20 14:53:43","doi":"10.21203/rs.3.rs-1817977/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-09-20T12:17:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-08-06T14:19:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f00b7517-2135-406b-9041-bb43b2c3ecc8","date":"2022-07-28T03:05:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-25T09:49:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-20T08:21:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-07-12T10:20:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-12T10:09:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-07-02T09:03:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c9515bdb-8fb4-41b2-98b7-6f4f76d033df","owner":[],"postedDate":"July 20th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T18:13:38+00:00","versionOfRecord":{"articleIdentity":"rs-1817977","link":"https://doi.org/10.1038/s41598-022-26456-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2022-12-26 18:07:20","publishedOnDateReadable":"December 26th, 2022"},"versionCreatedAt":"2022-07-20 14:53:43","video":"","vorDoi":"10.1038/s41598-022-26456-9","vorDoiUrl":"https://doi.org/10.1038/s41598-022-26456-9","workflowStages":[]},"version":"v1","identity":"rs-1817977","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1817977","identity":"rs-1817977","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}