Chronic kidney disease leads to microglial potassium efflux and inflammasome activation in the brain

Chronic kidney disease leads to microglial potassium efflux and inflammasome activation in the brain | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chronic kidney disease leads to microglial potassium efflux and inflammasome activation in the brain silke zimmermann, Akash Mathew, Olga Bondareva, Ahmed Elwakiel, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2511118/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Cognitive impairment is common in peripheral diseases such as chronic kidney disease (CKD). Kidney transplantation reverses cognitive impairment, indicating that cognitive impairment driven by CKD is therapeutically amendable. Yet, we lack mechanistic insights allowing targeted therapies. Using a combination of mouse models (including mice with neuron-specific IL-1R1 deficiency), single cell analyses (single nuclei RNA sequencing and single cell thallium automethallography), human samples and in vitro experiments we demonstrate that microglia activation impairs neuronal potassium homeostasis and impairs cognition in CKD. CKD conditions disrupt the barrier of brain endothelial cells in vitro and the blood-brain barrier in vivo , establishing that brain cells are exposed to uremic conditions. Exposure to uremic conditions impairs calcium homeostasis in microglia, enhances microglial potassium (K + ) efflux via the calcium-dependent channel K Ca 3.1, and induces p38-MAPK associated IL-1β maturation in microglia. Restoring K + homeostasis in microglia using a K Ca 3.1-specific inhibitor (TRAM34) improves CKD-triggered cognitive impairment. Likewise, inhibition of the IL-1β receptor 1 (IL-1R1) using anakinra or genetically abolishing neuronal IL-1R1 expression in neurons prevent CKD-mediated reduced neuronal potassium turnover and CKD-induced impaired cognition. Thus, in CKD mice impaired cognition can be ameliorated by either preventing microglia activation or inhibiting IL-1R-signaling in neurons. These data suggest that potassium efflux from microglia triggers their activation, which promotes microglia IL-1β release and IL-1R1-mediated neuronal dysfunction in CKD. This study provides new mechanistic insight into cognitive impairment in association with CKD and identifies possible new therapeutic approaches. Biological sciences/Immunology/Inflammation/Inflammasome Biological sciences/Cell biology Biological sciences/Immunology/Cytokines/Interleukins Chronic kidney disease cognitive impairment potassium flux cytokines microglia-neuron crosstalk Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files SupplFile.pdf Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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-2511118","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":172598515,"identity":"6f06219d-ef3f-4e13-841f-521e3830a4de","order_by":0,"name":"silke zimmermann","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIie2PMUvEMBiGUwq32JJNAjfkL+Q4OB1O/SvfcaiLg6ObgsMtJ13v0H/gEigEBIcPBKcW15QsFcFN6G0dBE2cHGKpm2Ce6Rveh+99CQkE/iLxtxtrJOeZu+C0rwJIovWFU0Tfl06R6K4OhS+2DuXmXvGd4eIZoZjG46fbWVMLwuk2epXoMlXV6tWM7m4KgaCPBhP9ljNbbLS+Bv+UOFUmQRNJfWK3NA/JRJfSKSCMXxk45R3NgdTHtVU+2HhV5m2XkjiFoJlJDa4YCkGvVOcXZpVqiWZui1mlmAPTqdoFwX7cwrNS6RbNni320jSP+0CzMq/asymnQ7/ie/yVZH3jDoq/SQcCgcA/4BMcKHAbcILByAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9644-8907","institution":"university clinic Leipzig","correspondingAuthor":true,"prefix":"","firstName":"silke","middleName":"","lastName":"zimmermann","suffix":""},{"id":172598516,"identity":"a9a83cdf-f134-4924-b111-0ae53a118434","order_by":1,"name":"Akash Mathew","email":"","orcid":"","institution":"Universitätsklinikum 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Germany","correspondingAuthor":false,"prefix":"","firstName":"Jürgen","middleName":"","lastName":"Goldschmidt","suffix":""},{"id":172598532,"identity":"855dd062-c499-4afa-b14b-3e29b965d8c5","order_by":17,"name":"Bilal Sheikh","email":"","orcid":"","institution":"Helmholtz Institute for Metabolic, Obesity and Vascular Research (Hl-MAG) of the Helmholtz Center Munich, Leipzig, Germany","correspondingAuthor":false,"prefix":"","firstName":"Bilal","middleName":"","lastName":"Sheikh","suffix":""},{"id":172598533,"identity":"14e82745-c277-4215-a8f1-12f120eacb13","order_by":18,"name":"Berend Isermann","email":"","orcid":"","institution":"Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University Hospital, Leipzig, Germany","correspondingAuthor":false,"prefix":"","firstName":"Berend","middleName":"","lastName":"Isermann","suffix":""}],"badges":[],"createdAt":"2023-01-24 16:21:06","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-2511118/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-2511118/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57870834,"identity":"ae6f8328-868e-44ed-9846-a6b9f665c5a1","added_by":"auto","created_at":"2024-06-06 17:42:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":265551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCKD impairs cognition in association with increased microglial potassium efflux\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Scheme of experimental approach (5/6 nephrectomy, Nx) to induce chronic kidney disease (CKD) in mice. Nonspatial object recognition, NSOR; radial arm maze, RAM.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb-d\u003c/strong\u003e) Cognition, as determined by NSOR (b; the results are reported as the percentage of time spent with the new unknown object) and RAM (c; the results are reported as working memory errors, WMEs) tests in wild-type sham-operated control (sham) and CKD mice. (d) Bar graph showing activity of the mice in cm/s in the NSOR.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee, f\u003c/strong\u003e) Pathway analyses (\u003cstrong\u003ee\u003c/strong\u003e, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, log10 of p values (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05) obtained after correcting for multiple testing by Benjamini‒Hochberg) and heatmap (\u003cstrong\u003ef\u003c/strong\u003e) of Alzheimer’s disease (AD)-associated differentially expressed genes (DEGs) in neurons of brains of CKD versus sham mice (snRNA-seq analyses, n=3 per condition).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg\u003c/strong\u003e) Top enriched terms obtained from Gene Ontology (GO) terms of biological process pathway analyses based on DEGs within the neuronal cluster of CKD mice compared to sham-operated mice (n=3). The log10 values of \u003cem\u003ep\u003c/em\u003e values (p\u0026lt; 0.05) obtained after correcting for multiple testing (Benjamini‒Hochberg correction) are shown. Red bars: biological process associated with learning and synaptic formation or neuron and axon biology.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eh\u003c/strong\u003e) Example images (left) and bar graph (mean ± SEM, each dot represents number of positive cells in one field of view; data from 4 different mice per group) with dot plot (right) summarizing thallium-positive (Tl) cells in \u003cem\u003eex vivo\u003c/em\u003e thallium autometallography (AMG).\u003c/p\u003e\n\u003cp\u003eStatistics (B-D, H): \u003cem\u003eP\u003c/em\u003e values: two-tailed Student’s t test; scale bar (h): 500 µm.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2511118/v2/dd125d69eb22c12f3ca33831.jpg"},{"id":57871192,"identity":"b109992a-74c3-4af8-8463-4080df094dd5","added_by":"auto","created_at":"2024-06-06 17:50:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":125924,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlood- brain barrier disruption in chronic kidney disease\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eScheme of the \u003cem\u003ein vitro\u003c/em\u003e blood- brain barrier model:\u003cstrong\u003e \u003c/strong\u003eBoyden chamber with brain endothelial cells (b.End3, cell-type 1) seeded on the insert surface. Dextran (4.4 kDa TRITC-conjugated) was added to the upper chamber.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Bar graph (mean ± SEM) summarizing the amount of dextran in the lower chamber of the Boyden chamber. \u003cem\u003eP\u003c/em\u003e values: two-tailed Student’s t test.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Representative immunofluorescence images of murine brain endothelial cells (b.End3) stained for phalloidin (PHA, green) prior to the experiment or following exposure to plasma from healthy controls (C Pl) or plasma from patients with CKD (CKD Pl); DAPI: nuclear counterstain. Scale bar: 100 µm.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Experimental scheme and timeline of Evans Blue extravasation test.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Bar graph (mean ± SEM) summarizing the relative absorbance of Evans Blue in brain tissue of sham or CKD mice. \u003cem\u003eP\u003c/em\u003e values: two-tailed Student’s t test.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2511118/v2/ce4b8d454e6814ee583bab65.jpg"},{"id":57870522,"identity":"2eaa26a4-f643-49ae-aa67-9114f733127c","added_by":"auto","created_at":"2024-06-06 17:34:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":223945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroglia activation in CKD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea-c\u003c/strong\u003e) Representative images of murine microglia exposed to control plasma (C Pl) or CKD plasma and stained for allograft inflammatory factor 1 (IBA1, DAPI nuclear counterstain, blue,\u003cstrong\u003e a\u003c/strong\u003e) and bar graphs with dot plot summarizing the corrected total cell IBA1 fluorescence (CTCF, \u003cstrong\u003eB\u003c/strong\u003e) and soma width (\u003cstrong\u003ec\u003c/strong\u003e). Scale bar: 50 µm; dots summarize mean ± standard error of the mean (SEM); each dot represents the analysis of one field of view of in total 3 biological replicates. \u003cem\u003eP\u003c/em\u003e values: one-way ANOVA with Dunnett-post hoc comparison.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed-g\u003c/strong\u003e) Representative images of IBA1 immunohistochemistry in the brains of control and CKD mice (\u003cstrong\u003ed\u003c/strong\u003e, Scale bar: 50 µm) and (\u003cstrong\u003ed-g\u003c/strong\u003e) bar graphs (mean ± SEM, each dot represents analysis of one field of view of in total 3 independent experiments) representing normalized process length (\u003cstrong\u003ee\u003c/strong\u003e) and soma width (\u003cstrong\u003ef\u003c/strong\u003e) and CTCF of IBA1 fluorescence (\u003cstrong\u003eg\u003c/strong\u003e) of murine microglia. Two-tailed Student’s t test was used.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eh-j\u003c/strong\u003e) Representative images of IBA1-immunofluorescence in brains of control and CKD patients (\u003cstrong\u003eh\u003c/strong\u003e) and bar graphs summarizing results of microglia process length (\u003cstrong\u003ei\u003c/strong\u003e) and soma size (\u003cstrong\u003ej\u003c/strong\u003e). Each dot represents one field of view of in total 4 patients.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ek-n\u003c/strong\u003e) Representative immunoblots of caspase-1 (Casp1, \u003cstrong\u003ek\u003c/strong\u003e) and IL-1β (\u003cstrong\u003el\u003c/strong\u003e) in brain lysates (prefrontal cortex) from sham and CKD mice. Total (T) and cleaved (Cl) forms are shown. Bar graphs (mean ± SEM, each dot represents one brain) summarizing immunoblot results of cleaved caspase 1 (\u003cstrong\u003em\u003c/strong\u003e, Cl. Casp1) and IL-1β (\u003cstrong\u003en, \u003c/strong\u003eCl. Casp1) in brain lysates of sham and CKD mice. Two-tailed Student’s t test was used.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eo, p\u003c/strong\u003e) Representative immunoblot of IL-1β (\u003cstrong\u003eo\u003c/strong\u003e) and bar graph summarizing results (\u003cstrong\u003ep\u003c/strong\u003e, each dot represents one biological sample) in SIM-A9 cells treated with control medium (C), control plasma (C Pl) or CKD plasma (CKD Pl); \u003cem\u003eP \u003c/em\u003evalues were determined using Mann-Whitney test.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2511118/v2/fa3faf127721df5aa77b0ab3.jpg"},{"id":57870527,"identity":"ebad95a9-9d69-41ef-8090-3e350227928a","added_by":"auto","created_at":"2024-06-06 17:34:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":249146,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePriming of microglia in CKD depends on potassium efflux\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea, b\u003c/strong\u003e) Representative images (\u003cstrong\u003ea\u003c/strong\u003e) and bar graph with dot plot summarizing the corrected total cell density (CTCD, \u003cstrong\u003eb\u003c/strong\u003e) of thallium autometallographically stained murine microglia (SIM-A9) exposed to control plasma or CKD plasma with or without pretreatment with triarylmethane-34 (TRAM34, T34; control: vehicle DMSO, DS). Scale bar: 50 µm; \u003cem\u003ep\u003c/em\u003e values were determined using one-way ANOVA with Dunnett’s post hoc comparison.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) K\u003csub\u003eCa\u003c/sub\u003e3.1 expression levels in in snRNAseq data of murine brain (n=3). AC=astrocytes, MC=microglia cell, N=neurons, OC=oligodendrocytes, VC=vascular cells.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed, e\u003c/strong\u003e) Exemplary immunofluorescence images (\u003cstrong\u003ed\u003c/strong\u003e) of Cal520 (yellow), DAPI (nuclear counterstain, blue), and phalloidin (PHA, purple) stained microglia and bar graphs with dot plot (mean ± SEM, each dot represents the analysis of one field of view of 3 independent experiments) summarizing microglia calcium content (represented as AU, \u003cstrong\u003ee\u003c/strong\u003e). Microglia were cultured with control plasma (C Pl) or chronic kidney disease plasma (CKD Pl) following preloading with Cal520 for 45 min. Scale bar: 50 µm. \u003cem\u003eP\u003c/em\u003e values were determined using two-tailed Student’s t test.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef-h\u003c/strong\u003e) Exemplary immunofluorescence images (\u003cstrong\u003ef\u003c/strong\u003e) of allograft inflammatory factor 1 (IBA1, red), DAPI (nuclear counterstain, blue), and phalloidin (PHA, white) stained microglia and bar graphs with dot plot (mean ± SEM, each dot represents the analysis of one field of view of 3 independent experiments) summarizing microglia cell soma width (\u003cstrong\u003eg\u003c/strong\u003e) and IBA1 staining intensity (represented as corrected total cell fluorescence, CTCF, \u003cstrong\u003eh\u003c/strong\u003e). Microglia were cultured with control plasma (C Pl) or chronic kidney disease plasma (CKD Pl) without (vehicle control, dimethyl sulfoxide, DS) or with Triarylmethane-34 (TRAM34, T34) pretreatment. Scale bar: 50 µm. \u003cem\u003eP\u003c/em\u003e values were determined using one-way ANOVA with Dunnett-post hoc comparison.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ei, j)\u003c/strong\u003e Example images (\u003cstrong\u003ei\u003c/strong\u003e) and bar graph with dot plot summarizing the corrected total cell density (CTCD, \u003cstrong\u003ej\u003c/strong\u003e) of thallium-autometallographically stained murine microglial (SIM-A9) exposed to control plasma or CKD plasma with or without pretreatment with triarylmethane-34 (TRAM34, T34; control: vehicle DMSO, DS). Scale bar: 50 µm; \u003cem\u003ep\u003c/em\u003e values were determined using one-way ANOVA with Dunnett’s post hoc comparison.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2511118/v2/4f6bf7753c5a732cee67dff3.jpg"},{"id":57870529,"identity":"1f3beb52-3a14-4542-8c7e-60aa118733d8","added_by":"auto","created_at":"2024-06-06 17:34:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":70100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRAM34 leads to reduced IL-1β cleavage in microglia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Example of cytoimmunofluorescence images. Microglia were exposed to normal medium (control, C) or to plasma obtained from control (C Pl) or CKD (CKD Pl) patients and stained for p38 (green) and IBA1 (red; DAPI nuclear counterstain, blue).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Representative western blot of phospho-p38, total p38, pro-Il-1β, and cleaved Il-1β (loading control: β-actin) in microglial exposed to C Pl or CKD Pl with T34 or without T34 (solvent only, DS).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec-e\u003c/strong\u003e) Bar graphs (mean ± SEM, each dot represents one biological replicate) summarizing levels of pro-Il-1β, cleaved IL-1β (cl.Il1β) and phospho-p38 in microglia exposed to control or uremic plasma without or with T34 treatment. \u003cem\u003eP\u003c/em\u003e values: one-way ANOVA with Dunnett-post hoc comparison.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2511118/v2/41be8eea72781d3a66ce737b.jpg"},{"id":57870525,"identity":"2aaa9c0d-efa9-48b6-80f6-41e2532b58c6","added_by":"auto","created_at":"2024-06-06 17:34:08","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":303554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRAM34 ameliorates microglial potassium efflux and behavior in CKD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Scheme summarizing experimental \u003cem\u003ein vivo\u003c/em\u003e approach. post CKD induction, a subgroup of mice received TRAM34 for further 6 weeks, followed by non-spatial object recognition (NSOR) and radial arm maze (RAM) test and tissue harvest.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb-d\u003c/strong\u003e) Example images (b) and bar graph (mean ± SEM, each dot represents number of positive cells in one field of view; data from 4 different mice per group) with dot plot (c, d) summarizing thallium-positive (Tl) cells in \u003cem\u003eex vivo\u003c/em\u003e thallium autometallography (AMG) from sham-operated control and CKD mice treated with vehicle DMSO or T34. Each dot represents the count of the respective cell type in one field of view; \u003cem\u003ep\u003c/em\u003e values were determined using one-way ANOVA with Dunnett’s post hoc comparison; scale bar (b): 500 µm. Arrows depict microglia (yellow) or neurons (red).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee-g\u003c/strong\u003e) Cognition, as determined by NSOR (\u003cstrong\u003ee\u003c/strong\u003e; the results are reported as the percentage of time spent with the new unknown object) and RAM (\u003cstrong\u003ef\u003c/strong\u003e; the results are reported as working memory errors, WMEs) tests in sham-operated control (sham) and CKD mice treated with vehicle (DS) or T34, while the overall activity in the non-spatial object recognition test was comparable (\u003cstrong\u003eg\u003c/strong\u003e). The results are represented as bar graphs, with each dot representing one mouse; \u003cem\u003ep\u003c/em\u003e values were determined using one-way ANOVA with Dunnett’s post hoc comparison.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2511118/v2/56d343d04b8e99f2d1f7a846.jpg"},{"id":57870526,"identity":"bc4551cb-f361-4ebc-9aac-71c27951cc5b","added_by":"auto","created_at":"2024-06-06 17:34:08","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":214565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUremia-induced reduced neuronal potassium turnover depends on microglia-neuron crosstalk via IL-1R1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea, b\u003c/strong\u003e) Representative images (\u003cstrong\u003ea\u003c/strong\u003e) and bar graph with dot plot (\u003cstrong\u003eb\u003c/strong\u003e, each dot presents the corrected total cell density (CTCD) of stained cells in one field of view) summarizing the results of thallium autometallography in neuronal cells. Cells were exposed to control plasma (C Pl) or CKD plasma (CKD Pl). Scale bar: 50 µm. \u003cem\u003eP\u003c/em\u003e values were determined using one-way ANOVA with Dunnett’s post hoc comparison.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Scheme summarizing the \u003cem\u003ein vitro\u003c/em\u003e experimental approach: CM is generated by culture of microglia with control (C) or CKD plasma followed by washing the cells with PBS and adding standard medium. This medium is obtained after 12\u0026nbsp;h and used as conditioned medium (CM\u003csup\u003eC\u003c/sup\u003e or CM\u003csup\u003eCKD\u003c/sup\u003e) for further experiments with neuronal cells.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed, e\u003c/strong\u003e) Representative images (\u003cstrong\u003ed\u003c/strong\u003e) and bar graph with dot plot (\u003cstrong\u003ee, \u003c/strong\u003eeach dot presents the corrected total cell density (CTCD) of stained cells in one field of view) summarizing the results of thallium autometallography in neuronal cells. Cells were exposed to conditioned medium (CM) from microglial (mCM) pretreated with control plasma (C Pl) or CKD plasma (CKD Pl) with or without anakinra (Ankr; control: PBS solvent). Scale bar: 50 µm. \u003cem\u003eP\u003c/em\u003e values were determined using one-way ANOVA with Dunnett’s post hoc comparison.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef\u003c/strong\u003e) Representative immunoblot images of neuronal cells incubated with CM from microglia treated with normal medium (control, C), C Pl or CKD Pl without Ankr (CKD Pl + PBS) or with anakinra (CKD Pl + Ankr). Immunoblots for phospho-Ca\u003csup\u003e2+\u003c/sup\u003e/calmodulin-dependent protein kinase II (phospho-CAMKII) and phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor (phospho-IĸBα) and respective total proteins are shown.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg, h\u003c/strong\u003e) Quantification of immunoblots (bar graph, mean ± SEM. Each dot represents one biological replicate) showing expression of phospho-CAMKII (\u003cstrong\u003eg\u003c/strong\u003e, phospho-Ca\u003csup\u003e2+\u003c/sup\u003e/calmodulin-dependent protein kinase II) and phospho-IκBα (\u003cstrong\u003eh\u003c/strong\u003e), in neuronal cells. \u003cem\u003eP\u003c/em\u003e values: one-way ANOVA with Dunnett-post hoc comparison.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2511118/v2/8fd242c7426753e8d7e96b5f.jpg"},{"id":57870528,"identity":"1239020f-1618-4f7d-8c30-36ab09c02685","added_by":"auto","created_at":"2024-06-06 17:34:08","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":319042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal IL-1R-expression is required for uremia-induced cognitive impairment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Scheme of conditional knock out of IL-1R1 in neuronal cells \u003cem\u003ein vivo\u003c/em\u003e by crossing Il1R1\u003csup\u003eLoxP\u003c/sup\u003e mice to Emx1\u003csup\u003eCre\u003c/sup\u003e mice, yiedling Il1R\u003csup\u003eΔN\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Experimental scheme showing 5-day injection period of tamoxifen, followed by two-step nephrectomy surgery (Nx), followed by \u003cem\u003eex vivo\u003c/em\u003e analysis or behavioral tests.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec-e\u003c/strong\u003e) Example images (c) and bar graph (mean ± SEM, each dot represents number of positive cells in one field of view; data from 4 different mice per group) with dot plot (d, e) summarizing thallium-positive (Tl) cells in \u003cem\u003eex vivo\u003c/em\u003e thallium autometallography (AMG) in the brains of IL-1R1 control (IL1R\u003csup\u003eCont\u003c/sup\u003e) and IL1R\u003csup\u003eΔN\u003c/sup\u003e CKD sham-operated control (sham) and CKD mice. Each dot represents the count of the respective cell type in one field of view; \u003cem\u003ep\u003c/em\u003e values were determined using one-way ANOVA with Dunnett’s post hoc comparison; scale bar (c): 500 µm. Arrows depict microglia (yellow) or neurons (red).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef, g\u003c/strong\u003e) Cognition, as determined by nonspatial object recognition (NSOR, \u003cstrong\u003ef\u003c/strong\u003e; the results are reported as the percentage of time spent with the new unknown object) and radial arm maze (RAM, \u003cstrong\u003eg\u003c/strong\u003e; the results are reported as working memory errors, WMEs) tests summarized as bar graphs with dot plot (each dot representing one mouse) in IL-1R1 control (IL1R\u003csup\u003eCont\u003c/sup\u003e) and IL1R\u003csup\u003eΔN\u003c/sup\u003e CKD sham-operated control (sham) and CKD mice. The data shown represent the mean ± SEM.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eh, i\u003c/strong\u003e) Cognition, as determined by NSOR (left; the results are reported as the percentage of time spent with the new unknown object) and RAM (right; the results are reported as working memory errors, WMEs) tests, in wild-type sham-operated control (sham) and CKD mice with or without treatment with anakinra (Ankr).\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2511118/v2/b958e17afc5281eca6708046.jpg"},{"id":57871568,"identity":"078c23d1-31dc-4160-afe2-cfe6d491c70b","added_by":"auto","created_at":"2024-06-06 18:06:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2498540,"visible":true,"origin":"","legend":"","description":"","filename":"240524CKDbrain1KidIntcorrectedVersionclean.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2511118/v2_covered_ef1b1a76-db70-4d62-b1f4-7533fe4023be.pdf"},{"id":57870530,"identity":"79b6873a-c764-418d-93db-0ed9b2bcd7e6","added_by":"auto","created_at":"2024-06-06 17:34:08","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2637079,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2511118/v2/16608c5974745660c0811369.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eChronic kidney disease leads to microglial potassium efflux and inflammasome activation in the brain\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chronic kidney disease, cognitive impairment, potassium flux, cytokines, microglia-neuron crosstalk","lastPublishedDoi":"10.21203/rs.3.rs-2511118/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2511118/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCognitive impairment is common in peripheral diseases such as chronic kidney disease (CKD). Kidney transplantation reverses cognitive impairment, indicating that cognitive impairment driven by CKD is therapeutically amendable. Yet, we lack mechanistic insights allowing targeted therapies. Using a combination of mouse models (including mice with neuron-specific IL-1R1 deficiency), single cell analyses (single nuclei RNA sequencing and single cell thallium automethallography), human samples and \u003cem\u003ein vitro\u003c/em\u003e experiments we demonstrate that microglia activation impairs neuronal potassium homeostasis and impairs cognition in CKD. CKD conditions disrupt the barrier of brain endothelial cells \u003cem\u003ein vitro\u003c/em\u003e and the blood-brain barrier \u003cem\u003ein vivo\u003c/em\u003e, establishing that brain cells are exposed to uremic conditions. Exposure to uremic conditions impairs calcium homeostasis in microglia, enhances microglial potassium (K\u003csup\u003e+\u003c/sup\u003e) efflux via the calcium-dependent channel K\u003csub\u003eCa\u003c/sub\u003e3.1, and induces p38-MAPK associated IL-1β maturation in microglia. Restoring K\u003csup\u003e+\u003c/sup\u003e homeostasis in microglia using a K\u003csub\u003eCa\u003c/sub\u003e3.1-specific inhibitor (TRAM34) improves CKD-triggered cognitive impairment. Likewise, inhibition of the IL-1β receptor 1 (IL-1R1) using anakinra or genetically abolishing neuronal IL-1R1 expression in neurons prevent CKD-mediated reduced neuronal potassium turnover and CKD-induced impaired cognition. Thus, in CKD mice impaired cognition can be ameliorated by either preventing microglia activation or inhibiting IL-1R-signaling in neurons. These data suggest that potassium efflux from microglia triggers their activation, which promotes microglia IL-1β release and IL-1R1-mediated neuronal dysfunction in CKD. This study provides new mechanistic insight into cognitive impairment in association with CKD and identifies possible new therapeutic approaches.\u003c/p\u003e","manuscriptTitle":"Chronic kidney disease leads to microglial potassium efflux and inflammasome activation in the brain","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2024-06-06 17:34:03","doi":"10.21203/rs.3.rs-2511118/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2023-02-01 21:38:29","doi":"10.21203/rs.3.rs-2511118/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a56226b3-8292-4e80-99c2-f78bf288fc99","owner":[],"postedDate":"June 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32921526,"name":"Biological sciences/Immunology/Inflammation/Inflammasome"},{"id":32921527,"name":"Biological sciences/Cell biology"},{"id":32921528,"name":"Biological sciences/Immunology/Cytokines/Interleukins"}],"tags":[],"updatedAt":"2023-03-01T15:45:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-06 17:34:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-2511118","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2511118","identity":"rs-2511118","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}