Extracellular Signal-Regulated Kinase is Activated in Podocytes from Patients with Diabetic Nephropathy

Extracellular Signal-Regulated Kinase is Activated in Podocytes from Patients with Diabetic Nephropathy | 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 Short Report Extracellular Signal-Regulated Kinase is Activated in Podocytes from Patients with Diabetic Nephropathy Aoi Yamashiro, Yasushi Satoh, Naoki Oshima, Shogo Endo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4495845/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jul, 2024 Read the published version in Human Cell → Version 1 posted 5 You are reading this latest preprint version Abstract In recent decades, the global prevalence of diabetes has provided a warning of chronic complications. Diabetic nephropathy (DN) is a serious complication of both type 1 and type 2 diabetes that affects approximately 35% of diabetic individuals. DN is the main cause of end-stage kidney disease, in which the kidneys can no longer function on their own. Podocytes in the glomerulus play a critical role in regulating glomerular permeability, and podocyte injury is the main cause of DN. Therefore, an increasing number of studies have focused on podocyte injury in DN, and interventions targeting podocyte injury have emerged as potential therapeutic strategies against DN. Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family that plays critical roles in intracellular signal transduction. In human patients with DN, phosphorylated ERK (pERK), the active form of ERK, is increased in the glomerulus. However, information on the expression of pERK, specifically in podocytes in DN, is limited. Meanwhile, high glucose induces ERK activation in immortalized podocyte cell lines, suggesting the involvement of podocytic ERK in DN. We performed an immunohistochemical study to investigate whether podocytic pERK levels increase in patientswith DN. In comparison to healthy controls, patients with DN showed significantly increased pERK expression levels in cells that were positive for the podocyte-specific marker Wilms’ tumor-1 (DN: 51.3 ± 13.1% vs. Control: 7.3 ± 1.6%, p = 0.0158, t -test, n = 4 for each group). This suggests that ERK activation in podocytes is involved in the pathogenesis of DN. Diabetic nephropathy (DN) Extracellular signal-regulated kinase (ERK) Podocyte Figures Figure 1 Figure 2 Introduction Diabetic nephropathy (DN) remains an incurable disease and is the most common cause of end-stage kidney disease, which necessitates renal replacement therapy. However, the mechanisms underlying the pathogenesis of DN are largely unknown, and a detailed understanding of these mechanisms is critical for developing therapeutic strategies for DN. DN is typically characterized by hyperglycemia-induced podocyte injury [ 1 ]. Podocytes are highly specialized epithelial cells in the glomerulus that form the glomerular ultrafiltration barrier in conjunction with capillary endothelial cells and the glomerular basement membrane (GBM). Podocyte injury includes hypertrophy, loss of podocyte foot processes, reduced density, and apoptosis, which result in an abnormal glomerular filtration rate, proteinuria, and high blood creatinine levels. Since podocytes are terminally differentiated, they exhibit a limited ability to divide and cannot be readily replaced when injured [ 2 , 3 ]. Once lost, podocytes in patients with DN have a reduced capacity for repair and regeneration. Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family that plays critical roles in intracellular signal transduction. ERK in podocytes is activated under DN conditions, as described below. Since individual cell types in the glomeruli have highly specialized functions and structures, defining the roles of phosphorylated-ERK (pERK) specifically in podocytes is critical to understanding the pathophysiology of DN. Some in vitro studies using cultured podocyte cell lines have suggested that the podocytic ERK pathway is involved in DN. Hoshi et al. demonstrated that high glucose led to cell hypertrophy via ERK activation in a cultured mouse podocyte cell line [ 1 ]. Mariappan et al. showed that high glucose induced ribosomal biogenesis through ERK activation in a cultured rat podocyte cell line [ 4 ]. Lei et al. showed that high glucose induced cell hypertrophy via ERK activation in a cultured human podocyte cell line [ 5 ]. However, these cultured podocyte cell lines, which have limited podocyte characteristics, do not completely mimic podocytes in vivo [ 6 ]. In addition to the in vitro studies described above, Haneda et al. [ 7 ] reported that ERK was activated mainly in the glomeruli in a rat diabetic model through western blotting, although the individual cell types were not defined. In human DN, Yu et al. [ 8 ] showed an increase in pERK levels in kidney biopsy specimens from DN patients using western blotting and immunohistochemistry. Sakai et al. [ 9 ] also reported that the number of glomerular pERK-positive cells in DN patients increased in accordance with the severity of glomerular lesions and extent of proteinuria through immunohistochemistry. However, the characteristics of pERK specifically in podocytes were not fully elucidated in these studies since the podocyte count was quite difficult to determine due to the highly complex structure of podocytes and glomeruli [ 10 – 16 ]. Because of the complex processes and interdigitations between cells superimposed on the complexity of the glomerular structure, it is difficult to identify where one podocyte begins and another ends [ 10 ]. Thus, podocytes cannot be reliably identified using an ultrastructural definition of podocytes based on their relationship to the GBM. To avoid such difficulties, immunohistochemistry using reliable podocyte markers is required. Wilms’ tumor-1 (WT-1) is a reliable podocyte marker [ 17 , 18 ] that is positive for podocytes, whereas mesangial cells and endothelial cells are WT-1 negative [ 10 ]. In this study, we performed immunohistochemistry using WT-1 as a podocyte marker to investigate whether ERK activation specifically increases in podocytes in human DN. Materials and Methods Ethics approval This study was approved by the Ethics Committee of National Defense Medical College (Approval No. 4961). Patients were guaranteed the opportunity to opt out. Sampling Among the patients who underwent percutaneous needle kidney biopsy at the National Defense Medical College Hospital, Department of Nephrology and Endocrinology, between November 2017 and December 2022, four patients with a pathological diagnosis of DN were evaluated retrospectively. For the healthy control group, kidney biopsy specimens from living kidney transplant donors were used. Donor specimens were collected during renal transplant surgery, immediately after restarting blood flow to the kidneys in recipients. All donors met the living renal transplant donor guidelines of the Japan Society for Transplantation and the Japanese Society for Clinical Renal Transplantation. The pathological diagnoses of all specimens from donors showed no kidney problems (equivalent to Category 1 of Banff Classification 2019). Detailed information on each participant is presented in Table 1 . Table 1 Participant Characteristics Case No. Pathological Diagnosis Age (years) Sex UPCR (g/gCr) eGFR (mL/min/1.73m 2 ) CCr (mL/min) 1 DN 84 Male 5.63 54.4 no data available 2 DN 58 Male 7.34 71.3 no data available 3 DN 58 Male 5.06 38.5 no data available 4 DN 81 Female 11.30 24.8 no data available 5 Control 49 Female bdl 92.1 93.2 6 Control 33 Female bdl 85.6 87.2 7 Control 50 Male bdl 62.4 98.9 8 Control 61 Male bdl 72.2 93.4 UPCR, urinary protein creatinine ratio; bdl, below detection limit eGFR, estimated glomerular filtration rate; CCr, Creatinine clearance Immunofluorescence staining Kidney specimens fixed with 10% formalin were embedded in paraffin and sectioned (3-µm thickness). After deparaffinization, rehydration, and heat-induced antigen retrieval, sections were incubated with blocking reagent (Nacalai Tesque, Inc., Kyoto, Japan), and then, incubated with primary antibodies at 4℃ overnight. As primary antibodies, we used rabbit anti-pERK (Cell Signaling Technology, Danvers, MA, USA) and mouse anti-WT-1 (abcam, Cambridge, UK) antibodies. The mouse anti-WT-1 antibody was used to stain the cytoplasm and nuclei of podocytes. Then, Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 546-conjugated anti-rabbit IgG secondary antibodies (Invitrogen, Carlsbad, CA, USA) were incubated for 60 min at room temperature. Finally, nuclear counterstaining was performed using mounting medium with 4’,6-diamidino-2-phenylindole (DAPI)-fluoromount-G® (SouthernBiotech, Birmingham, Alabama, USA). For quantitative evaluation, 5 glomeruli from each specimen were photographed using a Nikon C2 confocal laser microscope (Nikon, Tokyo, Japan), and the percentage of pERK-positive cells per WT-1-positive cells in each glomerulus was determined. Statistical analysis Results are expressed as the mean ± SE. Student's t -test was used to evaluate significant differences between the patient and control groups. P values of < 0.05 were considered to indicate statistical significance. Results Immunofluorescence staining was performed to examine the pERK expression specifically in podocytes. In the glomeruli of the DN group, we observed remarkable co-staining for WT-1 and pERK (Fig. 1 ). In contrast, the glomeruli of the control group contained only a few pERK-positive podocytes (Fig. 1 ). The analysis of the percentages of pERK-positive cells per WT-1-positive cells indicate that the pERK expression in podocytes from patients with DN significantly increased in comparison to the control group (Fig. 2 , DN; 51.3 ± 13.1% vs. control; 7.3 ± 1.6%, p = 0.0158, t -test, n = 4 for each group). Discussion Our results showed a higher positive rate of co-localization of pERK and podocyte markers in the glomeruli of the DN group in comparison to the control group. This suggests that the activation of the ERK pathway is involved in the mechanisms of podocyte injury in DN, which is consistent with previous studies. As described above, high glucose led to cell hypertrophy via ERK activation in immortalized podocyte cell lines, which partially mimics podocytes in vivo [ 1 , 4 , 5 ]. In contrast to our study and other studies, Fujita et al. [ 19 ] reported that pERK was expressed in renal tubular cells but not in glomeruli of diabetic rat models 3 weeks after induction of diabetes with a single injection of streptozotocin based on immunohistochemistry. The most reasonable explanation for this dissociation is that ERK activation in podocyte injury might occur in the limited time window during the chronic course of DN in this animal model. In this context, Liu et al. reported that pERK showed a marked increase in the limited time window of podocyte injury, albeit in a rat model of puromycin aminonucleoside (PAN) nephropathy rather than a model of DN [ 20 ]. In this study, western blotting showed that pERK was significantly increased in the rat glomeruli on day 10 after PAN induction (before the appearance of significant podocyte apoptosis), and that the levels of pERK subsequently decreased and returned to basal levels before day 15 [ 20 ]. They also performed an experiment using mouse cultured podocyte cells, indicating that the pERK expression increased 2 min after PAN induction and was sustained until 60 min. It was possible that Fujita et al. did not observe pERK expression 3 weeks after the injection of streptozotocin since the time window for the pERK expression was closed. However, the progress in DN patients in the present study was unclear and we do not have information about the time course of the podocytic pERK expression in the progress of human DN. Thus, it is too early to conclude absolutely that ERK activation was prominent in all diabetic individuals in the present study because the time window for the pERK expression was open. It is also possible that the progress of pathology and ERK activation may differ between animal models and human DN. Numerous factors within the context of diabetes have been implicated in the pathogenesis of podocyte injury in DN. Hoshi et al. demonstrated that high glucose induced the upregulation of vascular endothelial growth factor (VEGF) via activation of the protein kinase C (PKC)/ERK pathway in a differentiated mouse podocyte cell line [ 1 ]. Podocytes are the major sites for the production of VEGF, which increases glomerular permeability [ 1 ]. Antibodies against VEGF were also reported to inhibit hyperfiltration, albuminuria, and glomerular hypertrophy in a rat diabetes model [ 21 ]. Lei et al. showed that high glucose induced podocyte injury via PKC-a mediating epidermal growth factor receptor (EGFR) ubiquitination, endocytosis from the cell surface, and subsequent ERK activation in an immortalized human podocyte cell line [ 5 ]. Mariappan et al. showed that pERK is increased in the renal cortex of rodent models of type 1 and type 2 diabetes [ 4 ]. They also showed that high glucose induced ribosomal biogenesis through ERK activation along with augmented phosphorylation of upstream binding factor (UBF), an rDNA transcription factor, in a cultured rat podocyte cell line [ 4 ]. Increased ribosomal biogenesis facilitate an increase in global protein synthesis and synthesis of matrix protein, which contributes to renal hypertrophy and matrix protein accumulation in DN [ 4 ]. In addition to podocytes, ERKs in several types of cells in the kidney may have various roles in the pathology of DN. Using western blotting, Yu et al. showed that pERK was increased in kidney biopsy specimens from DN patients and db/db mice, which led to inflammation in tubular epithelial cells via activation of nuclear factor (NF)-κB [ 8 ]. Fujita et al. demonstrated that hyperglycemia induced ERK activation and increased the expression of transforming growth factor (TGF) in a pig renal proximal tubular epithelial cell line [ 19 ]. Moreover, these changes were partially abolished by pharmacological inhibition of ERK [ 19 ]. Isono et al. reported that ERK activation was observed in mouse mesangial cells cultured in highglucose conditions, which activated the TGF-β system and induced the overproduction of matrix proteins [ 22 ]. In this study, we used WT-1 as a podocyte marker. WT-1 is a marker of normal mature differentiated podocytes and even podocytes with abnormal foot processes [ 10 ]. Since no data suggest that podocytes in the common forms of glomerulosclerosis (e.g., diabetes and hypertension) are WT-1 negative [ 10 ], our use of WT-1 as a marker is considered reasonable. We showed that ERK is activated in the podocytes of patients with DN. Based on the critical role of glomerular permeability in podocytes, ERK activation in podocytes may be a novel therapeutic target for improving the prognosis of DN. However, the roles of ERK in podocyte injury is still poorly understood. It seems likely that the levels of ERK activation in the 4 DN patients examined in this study were not correlated with age, kidney function, or urinary protein excretion (Fig. 2 ). Furthermore, the upstream and downstream factors of ERK in DN patients remain unclear. Further studies are needed to elucidate the role of ERK in the pathogenesis of podocyte injury. ERK is ubiquitously expressed in several cells in the glomerulus, and ERK activation is observed not only in podocytes but also in various resident glomerular cells, including endothelial, mesangial, and tubular epithelial cells in DN models [ 1 , 4 , 5 , 8 , 9 , 19 , 22 ]. These results, along with our own, suggest that the ERK pathway might be associated with DN through various pathways in various cells. Thus, the regulatory mechanisms of the ERK pathway in the pathology of DN are complicated, and the roles of ERK might differ among cell types in DN. Furthermore, the roles for podocyte injury might differ among ERK isomers, although we could not distinguish the phosphorylation between ERK1 and 2 (ERK isomers) in this study. Thus, investigation using the cell-type specific gene manipulation methods is warranted. Declarations Acknowledgments We express our gratitude to Drs. Susumu Matsukuma and Keiichi Ito for their support in preparing specimens. Funding This research was funded by the National Defense Medical College. Conflicts of interest and Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval and Informed consent This study was approved by the Ethics Committee of National Defense Medical College (Approval No. 4961). Patients were guaranteed the opportunity to opt out. References Hoshi S, Nomoto K, Kuromitsu J, Tomari S, Nagata M. High glucose induced VEGF expression via PKC and ERK in glomerular podocytes. Biochemical and biophysical research communications. 2002;290(1):177-84. doi:10.1006/bbrc.2001.6138. Kriz W, Gretz N, Lemley KV. Progression of glomerular diseases: is the podocyte the culprit? Kidney Int. 1998;54(3):687-97. doi:10.1046/j.1523-1755.1998.00044.x. Nagata M, Nakayama K, Terada Y, Hoshi S, Watanabe T. Cell cycle regulation and differentiation in the human podocyte lineage. Am J Pathol. 1998;153(5):1511-20. doi:10.1016/s0002-9440(10)65739-2. Mariappan MM, D'Silva K, Lee MJ, Sataranatarajan K, Barnes JL, Choudhury GG et al. Ribosomal biogenesis induction by high glucose requires activation of upstream binding factor in kidney glomerular epithelial cells. American journal of physiology Renal physiology. 2011;300(1):F219-30. doi:10.1152/ajprenal.00207.2010. Lei CT, Wei YH, Tang H, Wen Q, Ye C, Zhang C et al. PKC-α Triggers EGFR Ubiquitination, Endocytosis and ERK Activation in Podocytes Stimulated with High Glucose. Cell Physiol Biochem. 2017;42(1):281-94. doi:10.1159/000477329. Veissi S, Smeets B, van den Heuvel LP, Schreuder MF, Jansen J. Nephrotic syndrome in a dish: recent developments in modeling in vitro. Pediatr Nephrol. 2020;35(8):1363-72. doi:10.1007/s00467-019-4203-8. Haneda M, Araki S, Togawa M, Sugimoto T, Isono M, Kikkawa R. Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions. Diabetes. 1997;46(5):847-53. doi:10.2337/diab.46.5.847. Yu C, Li Z, Nie C, Chang L, Jiang T. Targeting Src homology phosphatase 2 ameliorates mouse diabetic nephropathy by attenuating ERK/NF-κB pathway-mediated renal inflammation. Cell communication and signaling : CCS. 2023;21(1):362. doi:10.1186/s12964-023-01394-9. Sakai N, Wada T, Furuichi K, Iwata Y, Yoshimoto K, Kitagawa K et al. Involvement of extracellular signal-regulated kinase and p38 in human diabetic nephropathy. Am J Kidney Dis. 2005;45(1):54-65. doi:10.1053/j.ajkd.2004.08.039. Sanden SK, Wiggins JE, Goyal M, Riggs LK, Wiggins RC. Evaluation of a thick and thin section method for estimation of podocyte number, glomerular volume, and glomerular volume per podocyte in rat kidney with Wilms' tumor-1 protein used as a podocyte nuclear marker. Journal of the American Society of Nephrology : JASN. 2003;14(10):2484-93. doi:10.1097/01.asn.0000089829.45296.7c. Kriz W, Lemley KV. The role of the podocyte in glomerulosclerosis. Curr Opin Nephrol Hypertens. 1999;8(4):489-97. doi:10.1097/00041552-199907000-00014. Kim YH, Goyal M, Kurnit D, Wharram B, Wiggins J, Holzman L et al. Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat. Kidney Int. 2001;60(3):957-68. doi:10.1046/j.1523-1755.2001.060003957.x. Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG et al. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest. 1997;99(2):342-8. doi:10.1172/jci119163. Meyer TW, Bennett PH, Nelson RG. Podocyte number predicts long-term urinary albumin excretion in Pima Indians with Type II diabetes and microalbuminuria. Diabetologia. 1999;42(11):1341-4. doi:10.1007/s001250051447. White KE, Bilous RW, Marshall SM, El Nahas M, Remuzzi G, Piras G et al. Podocyte number in normotensive type 1 diabetic patients with albuminuria. Diabetes. 2002;51(10):3083-9. doi:10.2337/diabetes.51.10.3083. Lemley KV, Lafayette RA, Safai M, Derby G, Blouch K, Squarer A et al. Podocytopenia and disease severity in IgA nephropathy. Kidney Int. 2002;61(4):1475-85. doi:10.1046/j.1523-1755.2002.00269.x. Mundlos S, Pelletier J, Darveau A, Bachmann M, Winterpacht A, Zabel B. Nuclear localization of the protein encoded by the Wilms' tumor gene WT1 in embryonic and adult tissues. Development. 1993;119(4):1329-41. doi:10.1242/dev.119.4.1329. Barisoni L, Kriz W, Mundel P, D'Agati V. The dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy. Journal of the American Society of Nephrology : JASN. 1999;10(1):51-61. doi:10.1681/asn.V10151. Fujita H, Omori S, Ishikura K, Hida M, Awazu M. ERK and p38 mediate high-glucose-induced hypertrophy and TGF-beta expression in renal tubular cells. American journal of physiology Renal physiology. 2004;286(1):F120-6. doi:10.1152/ajprenal.00351.2002. Liu S, Ding J, Fan Q, Zhang H. The activation of extracellular signal-regulated kinase is responsible for podocyte injury. Mol Biol Rep. 2010;37(5):2477-84. doi:10.1007/s11033-009-9761-6. Vriese AS, Tilton RG, Elger M, Stephan CC, Kriz W, Lameire NH. Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. Journal of the American Society of Nephrology : JASN. 2001;12(5):993-1000. doi:10.1681/asn.V125993. Isono M, Cruz MCI, Chen S, Hong SW, Ziyadeh FN. Extracellular signal-regulated kinase mediates stimulation of TGF-beta1 and matrix by high glucose in mesangial cells. Journal of the American Society of Nephrology : JASN. 2000;11(12):2222-30. doi:10.1681/asn.v11122222. Cite Share Download PDF Status: Published Journal Publication published 25 Jul, 2024 Read the published version in Human Cell → Version 1 posted Editorial decision: Major Revisions Needed 04 Jul, 2024 Reviewers agreed at journal 02 Jun, 2024 Reviewers invited by journal 31 May, 2024 Editor assigned by journal 30 May, 2024 First submitted to journal 29 May, 2024 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4495845","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":309257338,"identity":"c996ac18-7e93-40a6-b4e1-af9fcf9c52a0","order_by":0,"name":"Aoi Yamashiro","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYPCCAzwMDMwHGHigXAMitbAlkKYFiHkM4FrwAv4ZyU83/PhzR8a8vefbg7c5dgz87QcYigvwaJG4kWZ2s7ftGY/MmbPbDeduS2aQOJPAYDwDnzU3Esxu8DYc5pGQyN0mzbsN6MIbDAzG+FwofyP9280/f0Bacp6BtcgT0mJwI8fsNg8bWAsbWIsBIS2GZ96U3ZZtA2rhOWYmCfQLj+GZxAa8fpE7nr7t5ps/h+0l2JufSbzdZicnd/zwMWN8IcYgkIDKBzqJsc0Ynw4G/gOYYsyP8WoZBaNgFIyCkQYAxnNOGo1FnNkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6848-3676","institution":"National Defense Medical College: Boei Ika Daigakko","correspondingAuthor":true,"prefix":"","firstName":"Aoi","middleName":"","lastName":"Yamashiro","suffix":""},{"id":309257339,"identity":"e1035f10-07d3-4c2d-bf65-72b59b5ea981","order_by":1,"name":"Yasushi Satoh","email":"","orcid":"","institution":"National Defense Medical College: Boei Ika Daigakko","correspondingAuthor":false,"prefix":"","firstName":"Yasushi","middleName":"","lastName":"Satoh","suffix":""},{"id":309257340,"identity":"4380543a-d4d8-4598-bd1b-0fbec0fcd2bb","order_by":2,"name":"Naoki Oshima","email":"","orcid":"","institution":"National Defense Medical College: Boei Ika Daigakko","correspondingAuthor":false,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Oshima","suffix":""},{"id":309257341,"identity":"d6b6707d-3541-4f2a-a81c-99e282eb407f","order_by":3,"name":"Shogo Endo","email":"","orcid":"","institution":"Tokyo Metropolitan Institute of Gerontology: Tokyo-to Kenko Choju Iryo Center","correspondingAuthor":false,"prefix":"","firstName":"Shogo","middleName":"","lastName":"Endo","suffix":""}],"badges":[],"createdAt":"2024-05-29 09:19:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4495845/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4495845/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13577-024-01108-4","type":"published","date":"2024-07-25T16:16:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58386109,"identity":"a98698d8-64a3-432e-8ba7-763f08e84ca5","added_by":"auto","created_at":"2024-06-14 18:43:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2408923,"visible":true,"origin":"","legend":"\u003cp\u003eCo-staining of WT-1 and pERK revealed increased expression levels in podocytes from patients with diabetic nephropathy (DN)\u003c/p\u003e\n\u003cp\u003e(a) Representative images of immunofluorescence staining of WT-1, pERK, and DAPI in patients with DN (upper column) and the control group (lower column). Note that the antibody to WT-1 stains the cytoplasm and nuclei of podocytes. Scale bar; 50 µm. (b) Corresponding high-power images of the boxed area indicated in (a). White arrows indicate colocalization of WT-1 and pERK. Scale bar; 10 µm.\u003c/p\u003e","description":"","filename":"Fig1..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4495845/v1/46f17072c831e03f34c807f0.jpg"},{"id":58386108,"identity":"28ddcd32-c0b3-421b-8fc3-8bcd73991ffd","added_by":"auto","created_at":"2024-06-14 18:43:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1039145,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical analysis of pERK-positive podocytes (%) in glomeruli\u003c/p\u003e\n\u003cp\u003eResults are shown as the mean ± SE. Differences were determined by Student’s \u003cem\u003et\u003c/em\u003e-test. Numbers next to bars indicate the Case No. in Table1.\u003c/p\u003e","description":"","filename":"Fig2..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4495845/v1/6e3af826d62ce674c19a1bed.jpg"},{"id":61596595,"identity":"06d9b040-5021-4536-a48a-56ac4a84cfe0","added_by":"auto","created_at":"2024-08-01 17:28:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3779190,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4495845/v1/7bdb2540-b332-44bc-afde-1efb56d39706.pdf"}],"financialInterests":"","formattedTitle":"Extracellular Signal-Regulated Kinase is Activated in Podocytes from Patients with Diabetic Nephropathy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic nephropathy (DN) remains an incurable disease and is the most common cause of end-stage kidney disease, which necessitates renal replacement therapy. However, the mechanisms underlying the pathogenesis of DN are largely unknown, and a detailed understanding of these mechanisms is critical for developing therapeutic strategies for DN. DN is typically characterized by hyperglycemia-induced podocyte injury [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Podocytes are highly specialized epithelial cells in the glomerulus that form the glomerular ultrafiltration barrier in conjunction with capillary endothelial cells and the glomerular basement membrane (GBM). Podocyte injury includes hypertrophy, loss of podocyte foot processes, reduced density, and apoptosis, which result in an abnormal glomerular filtration rate, proteinuria, and high blood creatinine levels. Since podocytes are terminally differentiated, they exhibit a limited ability to divide and cannot be readily replaced when injured [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Once lost, podocytes in patients with DN have a reduced capacity for repair and regeneration.\u003c/p\u003e \u003cp\u003eExtracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family that plays critical roles in intracellular signal transduction. ERK in podocytes is activated under DN conditions, as described below. Since individual cell types in the glomeruli have highly specialized functions and structures, defining the roles of phosphorylated-ERK (pERK) specifically in podocytes is critical to understanding the pathophysiology of DN. Some \u003cem\u003ein vitro\u003c/em\u003e studies using cultured podocyte cell lines have suggested that the podocytic ERK pathway is involved in DN. Hoshi et al. demonstrated that high glucose led to cell hypertrophy via ERK activation in a cultured mouse podocyte cell line [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mariappan et al. showed that high glucose induced ribosomal biogenesis through ERK activation in a cultured rat podocyte cell line [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Lei et al. showed that high glucose induced cell hypertrophy via ERK activation in a cultured human podocyte cell line [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, these cultured podocyte cell lines, which have limited podocyte characteristics, do not completely mimic podocytes \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition to the \u003cem\u003ein vitro\u003c/em\u003e studies described above, Haneda et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] reported that ERK was activated mainly in the glomeruli in a rat diabetic model through western blotting, although the individual cell types were not defined. In human DN, Yu et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] showed an increase in pERK levels in kidney biopsy specimens from DN patients using western blotting and immunohistochemistry. Sakai et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] also reported that the number of glomerular pERK-positive cells in DN patients increased in accordance with the severity of glomerular lesions and extent of proteinuria through immunohistochemistry. However, the characteristics of pERK specifically in podocytes were not fully elucidated in these studies since the podocyte count was quite difficult to determine due to the highly complex structure of podocytes and glomeruli [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Because of the complex processes and interdigitations between cells superimposed on the complexity of the glomerular structure, it is difficult to identify where one podocyte begins and another ends [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, podocytes cannot be reliably identified using an ultrastructural definition of podocytes based on their relationship to the GBM. To avoid such difficulties, immunohistochemistry using reliable podocyte markers is required. Wilms\u0026rsquo; tumor-1 (WT-1) is a reliable podocyte marker [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] that is positive for podocytes, whereas mesangial cells and endothelial cells are WT-1 negative [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this study, we performed immunohistochemistry using WT-1 as a podocyte marker to investigate whether ERK activation specifically increases in podocytes in human DN.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003eThis study was approved by the Ethics Committee of National Defense Medical College (Approval No. 4961). Patients were guaranteed the opportunity to opt out.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eSampling\u003c/p\u003e \u003cp\u003eAmong the patients who underwent percutaneous needle kidney biopsy at the National Defense Medical College Hospital, Department of Nephrology and Endocrinology, between November 2017 and December 2022, four patients with a pathological diagnosis of DN were evaluated retrospectively. For the healthy control group, kidney biopsy specimens from living kidney transplant donors were used. Donor specimens were collected during renal transplant surgery, immediately after restarting blood flow to the kidneys in recipients. All donors met the living renal transplant donor guidelines of the Japan Society for Transplantation and the Japanese Society for Clinical Renal Transplantation. The pathological diagnoses of all specimens from donors showed no kidney problems (equivalent to Category 1 of Banff Classification 2019). Detailed information on each participant is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParticipant Characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePathological Diagnosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003cp\u003e(years)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUPCR\u003c/p\u003e \u003cp\u003e(g/gCr)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eeGFR\u003c/p\u003e \u003cp\u003e(mL/min/1.73m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCCr\u003c/p\u003e \u003cp\u003e(mL/min)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e54.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eno data available\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eno data available\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eno data available\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eno data available\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ebdl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e92.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e93.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ebdl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e85.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ebdl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e62.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e98.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ebdl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e72.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e93.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eUPCR, urinary protein creatinine ratio; bdl, below detection limit\u003c/p\u003e \u003cp\u003eeGFR, estimated glomerular filtration rate; CCr, Creatinine clearance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence staining\u003c/p\u003e \u003cp\u003eKidney specimens fixed with 10% formalin were embedded in paraffin and sectioned (3-\u0026micro;m thickness). After deparaffinization, rehydration, and heat-induced antigen retrieval, sections were incubated with blocking reagent (Nacalai Tesque, Inc., Kyoto, Japan), and then, incubated with primary antibodies at 4℃ overnight. As primary antibodies, we used rabbit anti-pERK (Cell Signaling Technology, Danvers, MA, USA) and mouse anti-WT-1 (abcam, Cambridge, UK) antibodies. The mouse anti-WT-1 antibody was used to stain the cytoplasm and nuclei of podocytes. Then, Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 546-conjugated anti-rabbit IgG secondary antibodies (Invitrogen, Carlsbad, CA, USA) were incubated for 60 min at room temperature. Finally, nuclear counterstaining was performed using mounting medium with 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI)-fluoromount-G\u0026reg; (SouthernBiotech, Birmingham, Alabama, USA). For quantitative evaluation, 5 glomeruli from each specimen were photographed using a Nikon C2 confocal laser microscope (Nikon, Tokyo, Japan), and the percentage of pERK-positive cells per WT-1-positive cells in each glomerulus was determined.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eResults are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. Student's \u003cem\u003et\u003c/em\u003e-test was used to evaluate significant differences between the patient and control groups. \u003cem\u003eP\u003c/em\u003e values of \u0026lt;\u0026thinsp;0.05 were considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eImmunofluorescence staining was performed to examine the pERK expression specifically in podocytes. In the glomeruli of the DN group, we observed remarkable co-staining for WT-1 and pERK (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the glomeruli of the control group contained only a few pERK-positive podocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The analysis of the percentages of pERK-positive cells per WT-1-positive cells indicate that the pERK expression in podocytes from patients with DN significantly increased in comparison to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, DN; 51.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.1% vs. control; 7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0158, \u003cem\u003et\u003c/em\u003e-test, n\u0026thinsp;=\u0026thinsp;4 for each group).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e Our results showed a higher positive rate of co-localization of pERK and podocyte markers in the glomeruli of the DN group in comparison to the control group. This suggests that the activation of the ERK pathway is involved in the mechanisms of podocyte injury in DN, which is consistent with previous studies. As described above, high glucose led to cell hypertrophy via ERK activation in immortalized podocyte cell lines, which partially mimics podocytes \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In contrast to our study and other studies, Fujita et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] reported that pERK was expressed in renal tubular cells but not in glomeruli of diabetic rat models 3 weeks after induction of diabetes with a single injection of streptozotocin based on immunohistochemistry. The most reasonable explanation for this dissociation is that ERK activation in podocyte injury might occur in the limited time window during the chronic course of DN in this animal model. In this context, Liu et al. reported that pERK showed a marked increase in the limited time window of podocyte injury, albeit in a rat model of puromycin aminonucleoside (PAN) nephropathy rather than a model of DN [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, western blotting showed that pERK was significantly increased in the rat glomeruli on day 10 after PAN induction (before the appearance of significant podocyte apoptosis), and that the levels of pERK subsequently decreased and returned to basal levels before day 15 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. They also performed an experiment using mouse cultured podocyte cells, indicating that the pERK expression increased 2 min after PAN induction and was sustained until 60 min. It was possible that Fujita et al. did not observe pERK expression 3 weeks after the injection of streptozotocin since the time window for the pERK expression was closed. However, the progress in DN patients in the present study was unclear and we do not have information about the time course of the podocytic pERK expression in the progress of human DN. Thus, it is too early to conclude absolutely that ERK activation was prominent in all diabetic individuals in the present study because the time window for the pERK expression was open. It is also possible that the progress of pathology and ERK activation may differ between animal models and human DN.\u003c/p\u003e \u003cp\u003eNumerous factors within the context of diabetes have been implicated in the pathogenesis of podocyte injury in DN. Hoshi et al. demonstrated that high glucose induced the upregulation of vascular endothelial growth factor (VEGF) via activation of the protein kinase C (PKC)/ERK pathway in a differentiated mouse podocyte cell line [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Podocytes are the major sites for the production of VEGF, which increases glomerular permeability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Antibodies against VEGF were also reported to inhibit hyperfiltration, albuminuria, and glomerular hypertrophy in a rat diabetes model [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Lei et al. showed that high glucose induced podocyte injury via PKC-a mediating epidermal growth factor receptor (EGFR) ubiquitination, endocytosis from the cell surface, and subsequent ERK activation in an immortalized human podocyte cell line [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Mariappan et al. showed that pERK is increased in the renal cortex of rodent models of type 1 and type 2 diabetes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. They also showed that high glucose induced ribosomal biogenesis through ERK activation along with augmented phosphorylation of upstream binding factor (UBF), an rDNA transcription factor, in a cultured rat podocyte cell line [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Increased ribosomal biogenesis facilitate an increase in global protein synthesis and synthesis of matrix protein, which contributes to renal hypertrophy and matrix protein accumulation in DN [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to podocytes, ERKs in several types of cells in the kidney may have various roles in the pathology of DN. Using western blotting, Yu et al. showed that pERK was increased in kidney biopsy specimens from DN patients and \u003cem\u003edb/db\u003c/em\u003e mice, which led to inflammation in tubular epithelial cells via activation of nuclear factor (NF)-κB [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Fujita et al. demonstrated that hyperglycemia induced ERK activation and increased the expression of transforming growth factor (TGF) in a pig renal proximal tubular epithelial cell line [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, these changes were partially abolished by pharmacological inhibition of ERK [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Isono et al. reported that ERK activation was observed in mouse mesangial cells cultured in highglucose conditions, which activated the TGF-β system and induced the overproduction of matrix proteins [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we used WT-1 as a podocyte marker. WT-1 is a marker of normal mature differentiated podocytes and even podocytes with abnormal foot processes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Since no data suggest that podocytes in the common forms of glomerulosclerosis (e.g., diabetes and hypertension) are WT-1 negative [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], our use of WT-1 as a marker is considered reasonable.\u003c/p\u003e \u003cp\u003eWe showed that ERK is activated in the podocytes of patients with DN. Based on the critical role of glomerular permeability in podocytes, ERK activation in podocytes may be a novel therapeutic target for improving the prognosis of DN. However, the roles of ERK in podocyte injury is still poorly understood. It seems likely that the levels of ERK activation in the 4 DN patients examined in this study were not correlated with age, kidney function, or urinary protein excretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, the upstream and downstream factors of ERK in DN patients remain unclear. Further studies are needed to elucidate the role of ERK in the pathogenesis of podocyte injury. ERK is ubiquitously expressed in several cells in the glomerulus, and ERK activation is observed not only in podocytes but also in various resident glomerular cells, including endothelial, mesangial, and tubular epithelial cells in DN models [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These results, along with our own, suggest that the ERK pathway might be associated with DN through various pathways in various cells. Thus, the regulatory mechanisms of the ERK pathway in the pathology of DN are complicated, and the roles of ERK might differ among cell types in DN. Furthermore, the roles for podocyte injury might differ among ERK isomers, although we could not distinguish the phosphorylation between ERK1 and 2 (ERK isomers) in this study. Thus, investigation using the cell-type specific gene manipulation methods is warranted.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our gratitude to Drs. Susumu Matsukuma and Keiichi Ito for their support in preparing specimens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Defense Medical College.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest and Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and Informed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of National Defense Medical College (Approval No. 4961). Patients were guaranteed the opportunity to opt out.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHoshi S, Nomoto K, Kuromitsu J, Tomari S, Nagata M. High glucose induced VEGF expression via PKC and ERK in glomerular podocytes. Biochemical and biophysical research communications. 2002;290(1):177-84. doi:10.1006/bbrc.2001.6138.\u003c/li\u003e\n\u003cli\u003eKriz W, Gretz N, Lemley KV. Progression of glomerular diseases: is the podocyte the culprit? Kidney Int. 1998;54(3):687-97. doi:10.1046/j.1523-1755.1998.00044.x.\u003c/li\u003e\n\u003cli\u003eNagata M, Nakayama K, Terada Y, Hoshi S, Watanabe T. Cell cycle regulation and differentiation in the human podocyte lineage. Am J Pathol. 1998;153(5):1511-20. doi:10.1016/s0002-9440(10)65739-2.\u003c/li\u003e\n\u003cli\u003eMariappan MM, D\u0026apos;Silva K, Lee MJ, Sataranatarajan K, Barnes JL, Choudhury GG et al. Ribosomal biogenesis induction by high glucose requires activation of upstream binding factor in kidney glomerular epithelial cells. American journal of physiology Renal physiology. 2011;300(1):F219-30. doi:10.1152/ajprenal.00207.2010.\u003c/li\u003e\n\u003cli\u003eLei CT, Wei YH, Tang H, Wen Q, Ye C, Zhang C et al. PKC-\u0026alpha; Triggers EGFR Ubiquitination, Endocytosis and ERK Activation in Podocytes Stimulated with High Glucose. Cell Physiol Biochem. 2017;42(1):281-94. doi:10.1159/000477329.\u003c/li\u003e\n\u003cli\u003eVeissi S, Smeets B, van den Heuvel LP, Schreuder MF, Jansen J. Nephrotic syndrome in a dish: recent developments in modeling in vitro. Pediatr Nephrol. 2020;35(8):1363-72. doi:10.1007/s00467-019-4203-8.\u003c/li\u003e\n\u003cli\u003eHaneda M, Araki S, Togawa M, Sugimoto T, Isono M, Kikkawa R. Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions. Diabetes. 1997;46(5):847-53. doi:10.2337/diab.46.5.847.\u003c/li\u003e\n\u003cli\u003eYu C, Li Z, Nie C, Chang L, Jiang T. Targeting Src homology phosphatase 2 ameliorates mouse diabetic nephropathy by attenuating ERK/NF-\u0026kappa;B pathway-mediated renal inflammation. Cell communication and signaling : CCS. 2023;21(1):362. doi:10.1186/s12964-023-01394-9.\u003c/li\u003e\n\u003cli\u003eSakai N, Wada T, Furuichi K, Iwata Y, Yoshimoto K, Kitagawa K et al. Involvement of extracellular signal-regulated kinase and p38 in human diabetic nephropathy. Am J Kidney Dis. 2005;45(1):54-65. doi:10.1053/j.ajkd.2004.08.039.\u003c/li\u003e\n\u003cli\u003eSanden SK, Wiggins JE, Goyal M, Riggs LK, Wiggins RC. Evaluation of a thick and thin section method for estimation of podocyte number, glomerular volume, and glomerular volume per podocyte in rat kidney with Wilms\u0026apos; tumor-1 protein used as a podocyte nuclear marker. Journal of the American Society of Nephrology : JASN. 2003;14(10):2484-93. doi:10.1097/01.asn.0000089829.45296.7c.\u003c/li\u003e\n\u003cli\u003eKriz W, Lemley KV. The role of the podocyte in glomerulosclerosis. Curr Opin Nephrol Hypertens. 1999;8(4):489-97. doi:10.1097/00041552-199907000-00014.\u003c/li\u003e\n\u003cli\u003eKim YH, Goyal M, Kurnit D, Wharram B, Wiggins J, Holzman L et al. Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat. Kidney Int. 2001;60(3):957-68. doi:10.1046/j.1523-1755.2001.060003957.x.\u003c/li\u003e\n\u003cli\u003ePagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG et al. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest. 1997;99(2):342-8. doi:10.1172/jci119163.\u003c/li\u003e\n\u003cli\u003eMeyer TW, Bennett PH, Nelson RG. Podocyte number predicts long-term urinary albumin excretion in Pima Indians with Type II diabetes and microalbuminuria. Diabetologia. 1999;42(11):1341-4. doi:10.1007/s001250051447.\u003c/li\u003e\n\u003cli\u003eWhite KE, Bilous RW, Marshall SM, El Nahas M, Remuzzi G, Piras G et al. Podocyte number in normotensive type 1 diabetic patients with albuminuria. Diabetes. 2002;51(10):3083-9. doi:10.2337/diabetes.51.10.3083.\u003c/li\u003e\n\u003cli\u003eLemley KV, Lafayette RA, Safai M, Derby G, Blouch K, Squarer A et al. Podocytopenia and disease severity in IgA nephropathy. Kidney Int. 2002;61(4):1475-85. doi:10.1046/j.1523-1755.2002.00269.x.\u003c/li\u003e\n\u003cli\u003eMundlos S, Pelletier J, Darveau A, Bachmann M, Winterpacht A, Zabel B. Nuclear localization of the protein encoded by the Wilms\u0026apos; tumor gene WT1 in embryonic and adult tissues. Development. 1993;119(4):1329-41. doi:10.1242/dev.119.4.1329.\u003c/li\u003e\n\u003cli\u003eBarisoni L, Kriz W, Mundel P, D\u0026apos;Agati V. The dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy. Journal of the American Society of Nephrology : JASN. 1999;10(1):51-61. doi:10.1681/asn.V10151.\u003c/li\u003e\n\u003cli\u003eFujita H, Omori S, Ishikura K, Hida M, Awazu M. ERK and p38 mediate high-glucose-induced hypertrophy and TGF-beta expression in renal tubular cells. American journal of physiology Renal physiology. 2004;286(1):F120-6. doi:10.1152/ajprenal.00351.2002.\u003c/li\u003e\n\u003cli\u003eLiu S, Ding J, Fan Q, Zhang H. The activation of extracellular signal-regulated kinase is responsible for podocyte injury. Mol Biol Rep. 2010;37(5):2477-84. doi:10.1007/s11033-009-9761-6.\u003c/li\u003e\n\u003cli\u003eVriese AS, Tilton RG, Elger M, Stephan CC, Kriz W, Lameire NH. Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. Journal of the American Society of Nephrology : JASN. 2001;12(5):993-1000. doi:10.1681/asn.V125993.\u003c/li\u003e\n\u003cli\u003eIsono M, Cruz MCI, Chen S, Hong SW, Ziyadeh FN. Extracellular signal-regulated kinase mediates stimulation of TGF-beta1 and matrix by high glucose in mesangial cells. Journal of the American Society of Nephrology : JASN. 2000;11(12):2222-30. doi:10.1681/asn.v11122222.\u003c/li\u003e\n\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Diabetic nephropathy (DN), Extracellular signal-regulated kinase (ERK), Podocyte","lastPublishedDoi":"10.21203/rs.3.rs-4495845/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4495845/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn recent decades, the global prevalence of diabetes has provided a warning of chronic complications. Diabetic nephropathy (DN) is a serious complication of both type 1 and type 2 diabetes that affects approximately 35% of diabetic individuals. DN is the main cause of end-stage kidney disease, in which the kidneys can no longer function on their own. Podocytes in the glomerulus play a critical role in regulating glomerular permeability, and podocyte injury is the main cause of DN. Therefore, an increasing number of studies have focused on podocyte injury in DN, and interventions targeting podocyte injury have emerged as potential therapeutic strategies against DN. Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family that plays critical roles in intracellular signal transduction. In human patients with DN, phosphorylated ERK (pERK), the active form of ERK, is increased in the glomerulus. However, information on the expression of pERK, specifically in podocytes in DN, is limited. Meanwhile, high glucose induces ERK activation in immortalized podocyte cell lines, suggesting the involvement of podocytic ERK in DN. We performed an immunohistochemical study to investigate whether podocytic pERK levels increase in patientswith DN. In comparison to healthy controls, patients with DN showed significantly increased pERK expression levels in cells that were positive for the podocyte-specific marker Wilms’ tumor-1 (DN: 51.3 ± 13.1% vs. Control: 7.3 ± 1.6%, \u003cem\u003ep \u003c/em\u003e= 0.0158, \u003cem\u003et\u003c/em\u003e-test, n = 4 for each group). This suggests that ERK activation in podocytes is involved in the pathogenesis of DN.\u003c/p\u003e","manuscriptTitle":"Extracellular Signal-Regulated Kinase is Activated in Podocytes from Patients with Diabetic Nephropathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 18:43:07","doi":"10.21203/rs.3.rs-4495845/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-07-05T03:15:35+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-06-02T08:06:44+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-31T22:27:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-30T15:49:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2024-05-29T05:18:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"544afa29-4bc5-4345-89ee-529a3794735e","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:13:12+00:00","versionOfRecord":{"articleIdentity":"rs-4495845","link":"https://doi.org/10.1007/s13577-024-01108-4","journal":{"identity":"human-cell","isVorOnly":false,"title":"Human Cell"},"publishedOn":"2024-07-25 16:16:57","publishedOnDateReadable":"July 25th, 2024"},"versionCreatedAt":"2024-06-14 18:43:07","video":"","vorDoi":"10.1007/s13577-024-01108-4","vorDoiUrl":"https://doi.org/10.1007/s13577-024-01108-4","workflowStages":[]},"version":"v1","identity":"rs-4495845","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4495845","identity":"rs-4495845","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}