Adipocyte Extracellular Vesicles (Adevs) Promote a Proinflammatory and Profibrotic Profile in Human Renal and Endothelial Cells in Vitro

Adipocyte Extracellular Vesicles (Adevs) Promote a Proinflammatory and Profibrotic Profile in Human Renal and Endothelial Cells in Vitro | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Adipocyte Extracellular Vesicles (Adevs) Promote a Proinflammatory and Profibrotic Profile in Human Renal and Endothelial Cells in Vitro Cristian Carvajal, Pablo Carrión Valdés, María Paz Hernández Mejías, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5942263/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Feb, 2026 Read the published version in International Journal of Obesity → Version 1 posted 9 You are reading this latest preprint version Abstract In obesity,white adipose tissue (WAT) undergoes hypertrophic and hyperplastic changes that are driven by phenotypical changes in preadipocytes and adipocytes. WAT also causes a chronic inflammatory state that modifies gene expression and the secretome, including the shedding of adipose-derived extracellular vesicles (AdEVs) into the circulation, with these AdEVs affecting other cell types and potentially modifying their phenotypes. Aim: To evaluate the effects of AdEVs on renal and endothelial cells and their impacts on gene expression associated with inflammation, fibrosis, and endothelial function. Methods: Human SW872 adipocytes were differentiated from preadipocytes and further characterized. AdEVs were isolated via ultracentrifugation and characterized according to ISEV guidelines. AdEVs were added to either human renal (HCD) or endothelial cells (EA.hy926) for 24 hours. Adipokine, cytokine (IL-6 and IL-1B), fibrosis-related, NGAL, and eNOS gene expression wasevaluated via RT-qPCR and western blotting. Results: SW872 cells exhibited classical adipocyte morphologies and a significant accumulation of lipid droplets. Isolated AdEVs exhibited a donut-shaped morphology, a characteristic size, and both CD9 and TSG101 markers, which are compatible with EV features. Both renal and endothelial cells that were challenged with AdEVs were able to (1) incorporate AdEV-PKH67 (a fluorescent dye) and (2) induce high expression of IL-6 and IL-1B (p<0.05). A decrease in eNOS expression was detected in endothelial cells treated with AdEVs (p<0.05). Conclusion: AdEVs induced a proinflammatory profile in renal and endothelial cells (HCD and EA.hy926) in vitro , and a decrease of eNOS expression in EA.hy926 cells. These results support that EVs from mature adipocytes could increase the inflammation and vascular dysfunction in obesity. Health sciences/Endocrinology/Endocrine system and metabolic diseases/Obesity Health sciences/Medical research/Translational research Adipocyte adipocyte extracellular vesicles (AdEVs) cytokines eNOS Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Obesity has reached pandemic levels worldwide; moreover, according to the World Health Organization, approximately 39% of adults older than 18 years of age are overweight, and 13% are obese (1). Obesity is the result of an imbalance between energy intake and expenditure that leads to an increase in energy stored by adipocytes, which causes adipose hypertrophy and hyperplasia (2). The increasing mass of fat leads to a proinflammatory state and impaired adipokine secretion, as well as the development of different obesity-related comorbidities, such as insulin resistance, hypertension, type 2 diabetes, and chronic kidney disease (2, 3). Adipose tissue (AT) is an energy storage organ and a vital endocrine organ that regulates the functions of other tissues and organs by its secretome, which is composed of soluble factors, adipokines, cytokines, and extracellular vesicles (EVs) (4). EVs are a diverse group of nanoparticles that are released or shed by different types of tissues and cell types; additionally, they have been recently proposed as being key factors in intercellular communication (5). Small EVs are generated from endosomal trafficking and multivesicular bodies, with sizes ranging from 50–150 nm in diameter (5). Due to their bioactive cargo, EVs can play different functional roles in their target cells by inducing cellular transduction signals and/or metabolic changes (6). EVs from ATs that contain adipocyte proteins, miRNAs and adipokines could have paracrine and endocrine effects on various tissues, thereby affecting both physiological and pathophysiological processes (4, 7). Previous studies have demonstrated that patients with obesity have increased levels of circulatory EVs; thus, EVs may participate in the development of several comorbidities associated with obesity, such as T2DM and chronic inflammation (8, 9). Along with other researchers, we have highlighted the role of adEVs in metabolic disorders such as obesity and insulin resistance, whereby adEVs can regulate tissues in distant organs (10, 11). Additionally, regarding the role of adEVs in renal pathophysiology, recent studies both in vitro and in vivo have demonstrated crucial roles for these EVs in the development of renal diseases, whereby they contribute to changes in immunomodulation and thrombogenesis, as well as in the extracellular matrix (12, 13). Moreover, Liu et al. reported that plasma EVs from subjects with obesity participate in the activation of the renin-angiotensin-aldosterone axis and elicit renal tubular damage in renal tubule epithelial cells (12). Furthermore, the release of proinflammatory factors by renal cells induced by adEVs can trigger an inflammatory process that affects immune system cells, such as macrophages or lymphocytes, which can enhance the local inflammatory effect and alter the secretome (14). An increasing amount of evidence suggests that adipose-derived extracellular vesicles (AdEVs) interact with different tissues involved in obesity-related comorbidities and the generation of an inflammatory response (7, 15). This study aimed to evaluate the proinflammatory and profibrotic effects of AdEVs from the SW872 cell line (SW872-AdEVs) in endothelial and renal cells in vitro . Methodology Preadipocyte culture and differentiation The human SW872 preadipocyte line (obtained from ATCC) was grown in DMEM/F-12 high glucose (HG) medium (#12400024, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (#10437-028, Gibco, USA) and penicillin-streptomycin solution 100X (#30-002-Cl, Corning, USA). When preadipocytes reached 80% confluence, differentiation into adipocytes was induced using differentiation medium (DMEM/F-12 supplemented with 1% FBS (Thermo, USA) plus 10 µg/mL recombinant human insulin (Humulin HI0210), 1 µM dexamethasone (Sigma Aldrich), 0.5 mM isobutyl-methylxanthine (IBMX) (Sigma Aldrich), and 1 µM rosiglitazone (Cayman, USA)) for up to 10 days, and the medium was renewed every three days. Differentiation was confirmed via optical microscopy and Oil Red O staining. Determination of adipogenesis marker expression The expression of adipogenesis markers was determined via RT-qPCR by using primers for acetyl-CoA carboxylase (ACC), fatty acid-binding protein 4 (FABP4), fatty acid synthase (FASN), perilipin 1 (PLIN1) and peroxisome proliferator activated receptor gamma (PPARg). The sequences of the utilized primers are summarized in Supplementary data (Table S1 and Table S2). The protein expression of these markers was corroborated via western blotting by using the corresponding primary antibodies and the secondary anti-rabbit IgG antibody, which was HRP-linked from the “Adipogenesis Marker Antibody Sampler Kit” (1:2000; (C45B10) (C83B10) (2120) (C20G5) (D1D8) (C26H12) (7074) #12589; Cell Signaling, USA). The anti-B-actin rabbit mAb (1:2000; #4970; Cell Signaling Technology, USA) was used as a loading control. The relative expression of adipogenesis markers was measured on days 1, 3, 7 and 10 of the differentiation protocol. Isolation of adipocyte extracellular vesicles (AdEVs) AdEVs were obtained by a differential ultracentrifugation protocol. Briefly, preadipocytes from SW872 cells were grown to 80% confluence and subsequently differentiated for 7 days in a multilayer 875 cm 2 flask (#353144, Corning, USA). Afterwards, differentiated cells were cultured in exosome-free DMEM-F12 (supplemented with 1% FBS) HG medium, which was supplemented with a differentiation cocktail. The cells were maintained in that medium for 24 hours for EV release. After 24 hours, the culture medium was collected and centrifuged at 300 × g for 15 minutes (min) to remove the remaining cells. The supernatant was subsequently collected and centrifuged at 2000 × g for 30 min and at 12000 × g for 45 min to remove dead cells and cellular debris, respectively. The resulting supernatant was filtered through a 0.22 μm filter (EDLAB Sterile Syringe Filter CA 0.22 μm) and subsequently ultracentrifuged at 120000 × g for 70 min to precipitate the AdEVs. The resulting pellet was resuspended in PBS and centrifuged at 100000 × g for 70 min to obtain the AdEV pellet. The final pellet was resuspended in aliquots of 100 μL of filtered PBS and stored at -80 °C until analysis. Suspensions of AdEVs were characterized according to the guidelines of the International Society for Extracellular Vesicles (16) by nanoparticle tracking analysis, transmission electron microscopy, and western blot analysis. Nanoparticle tracking analysis (NTA) The concentrations and sizes of the AdEV suspensions were determined by NTA by using a NanoSight NS300 and NanoSight NTA 3.2 software (Malvern Instruments Ltd., Malvern, UK). Particles were tracked by passing a laser beam through the liquid sample, and the scattered light was detected and captured in three short videos of 20 second durations by using an sCMOS camera. The Brownian motion of the particles was determined on a frame-by-frame basis, and the distance traversed by the particles was used to determine the AdEV size and concentration using the Stokes-Einstein equation. Western bb lotting of exosome markers and adipogenic cargo The proteins CD9 and TSG101 were used as EV markers. PPARɣ, FASN and ADIPOQ were used as adipocyte markers. For western blotting, AdEVs were resuspended in RIPA buffer (Thermo Fisher Scientific Inc., IL, USA) to extract total protein, and the protein concentration was determined using the bicinchoninic acid (BCA) method. Protein lysates were separated through polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, blocked with 5% skim milk in PBS, and probed overnight with primary rabbit monoclonal anti-TSG101 (1:10000; Ab125011; Abcam, MS, USA) and rabbit monoclonal anti-CD9 (1:1000; (D801A) #13174; Cell Signaling, USA) antibodies. After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG-HRP (1:10000; Ab6939; Abcam, USA) for 1 hour at RT. Proteins were detected using chemiluminescence (ECL western blotting substrate reagent; Pierce, USA) with a ChemiDoc MP imaging system (Bio-Rad, CA, USA). Transmission electron microscopy (TEM) of AdEVs TEM was performed to verify the shapes and sizes of the obtained AdEVs. Briefly, AdEV suspensions were absorbed onto carbon-coated copper grids for 1 min. Samples were negatively stained with a 2% uranyl acetate solution. The grids were visualized with a Thermo Scientific Talos F200C G2 transmission electron microscope. Treatment of renal and endothelial cells with AdEVs To investigate the effects of AdEVs on the expression of inflammatory and fibrotic genes in both kidney and endothelial cells, both the HCD cell line (kidney) and the EA.hy926 cell line (endothelium) were exposed to AdEVs for 24 hours. Briefly, HCD cells were cultured in 6-well plates containing DMEM/F-12 low-glucose (LG) medium supplemented with 10% FBS, and EA.hy926 cells were cultured in 6-well plates containing IMDM supplemented with 10% FBS. Once the cells reached 80–90% confluence, the medium was changed to EV-free basal medium (DMEM/F-12 LG supplemented with 1% EV-free FBS or IMDM supplemented with 1% EV-free FBS) for 24 hours. Afterwards, AdEVs resuspended in PBS were added at a proportion of 1000 AdEVs/cell, and the cells were incubated for another 24 hours. For the control, the same volume of EV-free PBS was added. Subsequently, the cells were lysed using TRIzol reagent (#15596018; Invitrogen), and inflammatory, fibrotic, and endothelial markers were evaluated via RT-qPCR following the relative expression analysis by using the methodology of Livak et al. (17). The list of utilized primers is summarized in Supplementary data (Table S1 and Table S3). AdEV uptake assay AdEV pellets, which were isolated as previously described, were resuspended in PBS and stained with PKH67 green fluorescent dye (#MIDI67; Sigma-Aldrich, MO, USA). The negative control was an EV-free sample labeled with PKH67. After AdEV labeling, HCD and EA.hy926 cells were incubated with PKH67-fluorescent AdEVs (37000 AdEVs per cell) or a negative control for 24 hours. After incubation, the cells were rinsed twice with cold PBS and fixed with 4% paraformaldehyde at RT for 10 min. The cell nucleus was stained with DAPI, and the cells were mounted on glass slides with Vectashield Antifade mounting medium (#H-1200; Vector Laboratories, USA). The cells were visualized with an Olympus CKX41 epifluorescence microscope (USA). Statistical analysis Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA). The data are presented as the mean ± standard error. Mann-Whitney’s tests were performed for comparisons between two groups, while Kruskal-Wallis was used for comparisons of more than two groups. P < 0.05 was considered to be statistically significant. RESULTS 1. Characterization of SW872 cell adipogenesis based on cell histology and gene expression Morphological changes in SW872 preadipocytes were observed on days 3 to 10 of the differentiation protocol; specifically, the cells evolved from a conical stretched morphology to a rounded morphology, and the diameter of the lipid droplets increased (Fig. 1 A). At days 7 and 10, most of the cells exhibited a rounded shape and Oil Red O-positive labeling, which is distinctive of mature adipocytes (Supplementary Table 4 ). During adipogenic differentiation, the expression of the PPARg, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) genes significantly increased on the third day and then gradually decreased until day 10 (Fig. 1 B). In contrast, fatty acid-binding protein 4 (FABP4) and perilipin 1 (PLIN1) gene expression reached a maximal increase on day 10 (Fig. 1 B). Analysis of protein expression revealed that PPARg also significantly increased on the third day but then gradually decreased until day 10. In contrast, the relative abundances of the ACC and FASN proteins increased at days 7 and 10 (Fig. 1 C). 2. Characterization of AdEVs isolated from the SW872 cell culture The characterization of AdEVs from SW872 cells (SW872-AdEVs) revealed a mean concentration of 2.1x10 10 ± 3.1x10 8 AdEVs/mL, with a mean size of 128.3 ± 2.2 nm and a mode of 134.4 ± 0.4 nm (Fig. 2 A). Additionally, via TEM, we observed that AdEVs exhibited a classic donut-like morphology (Fig. 2 B); moreover, via western blotting, we identified the presence of the CD9 and TSG101 proteins, which are associated with endosomal biogenesis of EVs (Fig. 2 C). 3. SW872-AdEVs increase inflammatory and fibrotic gene expression in human collecting duct (HCD) cells The inflammatory and fibrotic effects of SW872-AdEVs on the human collecting duct (HCD) cell line were evaluated in vitro by changes in the gene expression of IL-6 and IL-1B (which are inflammatory markers); CTGF and FN1 (which are fibrosis markers); and NGAL (18). HCD-treated cells were incubated with AdEVs (1000 particles/cell) for 24 hours. We observed significant increases in gene expression of IL-6 (1.0 ± 0.1 vs. 2.5 ± 0.6 UR; p = 0.029), IL-1B (1.1 ± 0.1 vs. 2.9 ± 0.8 UR; p = 0.029), CTGF (1.0 ± 0.1 vs. 2.2 ± 0.6 UR; p = 0.031) and NGAL (1.0 ± 0.1 vs. 2.1 ± 0.5 UR; p = 0.04) when comparing vehicle versus treatment with adEVs. We did not observe statistically significant changes in FN1 expression (1.0 ± 0.2 vs. 1.3 ± 0.2 UR, p = 0.3) (Fig. 2 A). 4. AdEVs affect IL-6 and eNOS expression in EA.hy926 endothelial cells Similar to HCD cells, we evaluated the inflammatory and fibrotic effects of SW872-AdEVs on the EA.hy926 endothelial cell line by gene expression analysis. We evaluated the gene expression of IL-6 and IL-1B (inflammatory markers), as well as CTGF and FN1 (fibrosis markers), with eNOS expression being used as a proxy for endothelial function. We observed an increase in gene expression of IL-6 expression (1.0 ± 0.1 vs. 2.0 ± 0.5 UR; p = 0.03) and a nonsignificant increase in IL-1B expression (1.0 ± 0.1 vs. 2.2 ± 0.8 UR; p = 0.15) when comparing vehicle versus treatment with adEVs. Nonsignificant changes were observed in both CTGF (1.1 ± 0.1 vs. 0.9 ± 0.1 UR; p = 0.06) and FN1 (1.0 ± 0.2 vs. 0.9 ± 0.2 UR; p = 0.41). Furthermore, eNOS expression was significantly decreased in cells treated with AdEVs (1.0 ± 0.1 vs. 0.7 ± 0.1 UR; p = 0.02) (Fig. 3 A). 5. AdEVs can be internalized into HCD and EA.hy926 cells The internalization of AdEVs in both HCD and EA.hy926 cells was evaluated by EV labeling with the PKH67 green fluorescent dye. After 12 hours of incubation, fluorescently labeled AdEVs were observed in the subcellular compartments of both cell lines (Fig. 2 B and Fig. 3 B). DISCUSSION Our study revealed that AdEVS from SW872 adipocytes (SW872-AdEVs) changed the gene expression of cytokines, fibrosis-related genes, and eNOS in renal (HCD) and endothelial (EA.hy926) cultured cells, which suggests an interesting association of AdEVs with renal epithelial and endothelial cells in the context of obesity (9). We observed that the incubation of SW872-AdEVs from mature adipocytes for 24 hours with both renal cells and endothelial cells increased the mRNA expression of the proinflammatory cytokines IL-6 and IL-1B. Both cytokines are associated with inflammatory processes. Additionally, we observed a significant increase in the expression of NGAL in renal epithelial cells treated with SW872-AdEVs. NGAL (or LCN2) is a lipocalin that is currently considered to be an inflammatory marker commonly associated with acute kidney injury, and it is also associated with activation of the mineralocorticoid receptor (MR) pathway (19, 20). These results suggest that SW872-AdEVs can modify the phenotypes of target cells via the activation of proinflammatory pathways, including the MR pathway. With respect to the fibrotic responses of target cells that are challenged with SW872-AdEVs, we observed changes in CTGF gene expression only in HCD renal cells, which is interesting because it can be a proxy for the profibrotic phenotype (12, 13, 21). Taken together, these results in HCD cells support a novel link between AdEVs and epithelial renal cells, with increased inflammation and fibrosis-related gene expression being observed (22, 23). Hence, we encourage the performance of further studies aimed at detecting fibrotic signs in renal and vascular fibroblasts to confirm the impact of SW872-AdEVs. With respect to endothelial cells, we did not detect changes in fibrosis-related gene expression; however, we detected a significant decrease in relative eNOS expression when these cells were challenged with AdEVs-SW872. Reduced eNOS expression is associated with endothelial dysfunction, since eNOS is a key factor in the vasodilation response in the endothelium (24–27). Previous reports on eNOS and EVs have shown that under pathological conditions, EVs from different sources are able to impair vasorelaxation via the inhibition of eNOS, a reduction in NO bioavailability, and the activation of ERK signaling (28, 29). In contrast, EVs from adipose-derived stem cells (ADSCs) improve eNOS signaling associated with the overexpression of GLO-1, which is a protein associated with reducing oxidative stress (30). Both results suggest that the donor adipose cell type and the cellular microenvironment (such as environments with low/high oxidative stress or low/high proinflammatory mediator levels, among other conditions) are key factors in establishing the EV cargo in AdEVs and further affecting target cells (30, 31). We also evaluated the internalization of fluorescent SW872-AdEVs labeled with PKH67 dye in target cells. We demonstrated that AdEV-SW872 was efficiently internalized by HCD renal and EA.hy.926 endothelial cells. Several studies have shown that EVs carry interleukins as part of their cargo (32) and induce the secretion of different proinflammatory interleukins (such as IL-1B, IL-6, IL-8 and TNF-α) in target cells (32–34). Therefore, the specific cargo transported within adEVs, such as interleukins and miRNAs, can change or mediate the phenotypes of target cells (31). EV-miRNAs can act as biomarkers, distant gene expression repressors, and potential therapeutic agents (10, 35). Moreover, miRNAs can travel to neighboring and distant cells via the circulation, thereby mediating cell-to-cell communication by targeting mRNAs and ultimately causing changes in the expression of target genes (11, 36). Thomou et al. reported that adipose tissue is the major source of circulating exosomal miRNAs and functions as a gene regulator in distant tissues (37). These results encourage the performance of a transcriptomic assay of AdEVs to identify specific mRNAs and miRNAs within AdEVs that can exert an impact on the phenotypes of target cells. However, several limitations are associated with this study, which are mainly related to the dose and timing of AdEV challenge in target cells; these factors can cause biases in specific gene expression changes. Additionally, AdEV challenge is an experimental model that simplifies the overall impact of the secretome and other inflammatory factors (such as TNF-α) that are present in the AT microenvironment. Further studies are needed to evaluate the effects of AdEVs linked to proinflammatory conditions in donor and/or target cells. In summary, adipose extracellular vesicles (AdEVs) from SW872 adipocytes were efficiently isolated by UCF by using a novel isolation protocol. We observed that SW872-AdEVs induced a proinflammatory profile in renal and endothelial cells (HCD and EA.hy926) in vitro , and a decrease of eNOS expression in EA.hy926 cells. These results support that EVs from mature adipocytes could increase the inflammation and vascular dysfunction in obesity. Declarations Declaration of interest. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review. Funding statements. This study was supported by the following grants: ANID-CONICYT FONDECYT 1212006 & 11251675; ICM-ANID ICN2021_045; SOCHED 2024-06 and CETREN-UC 2024–2025. Authors' contributions. P.C. and MP-H. contributed equally to this work, both designed the study, collected, analyzed and interpreted of the data, contributed to the statistics of the data, wrote the first draft of the manuscript, contributed to discussion, and reviewed the manuscript. J.P. contributed to data analysis and reviewed the manuscript. A.T-C analyzed and interpreted the statistical data, contributed to discussion, and reviewed the manuscript. AV and A.S-B contributed to the development of in vitro analyses and reviewed the manuscript. RFB and CEF contributed to discussion and reviewed the manuscript. CAC designed the study, analyzed and interpreted of the data, reviewed to the statistics of the data, contributed to discussion, and reviewed the manuscript. All reviewed the manuscript and authors approved the final version. References Camino T, Lago-Baameiro N, Pardo M. Extracellular Vesicles as Carriers of Adipokines and Their Role in Obesity. Biomedicines. 2023;11(2):422. 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Additional Declarations There is NO conflict of interest to disclose Supplementary Files SUPPLEMENTARYDATA.pdf Cite Share Download PDF Status: Published Journal Publication published 23 Feb, 2026 Read the published version in International Journal of Obesity → Version 1 posted Editorial decision: revise 28 Mar, 2025 Review # 2 received at journal 25 Mar, 2025 Review # 1 received at journal 03 Mar, 2025 Reviewer # 2 agreed at journal 02 Mar, 2025 Reviewer # 1 agreed at journal 18 Feb, 2025 Reviewers invited by journal 06 Feb, 2025 Submission checks completed at journal 03 Feb, 2025 First submitted to journal 01 Feb, 2025 Editor assigned by journal 01 Feb, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5942263","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":412145680,"identity":"c8ff277c-b652-41d8-a3fa-b295f1a8eb0c","order_by":0,"name":"Cristian Carvajal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYPACNgZ+MF1AihbJBhBtQIo9BgeI1cLffvjZZ54avmjjG8kPGH4Qo0XiTJrxbJ5jbLnbbqQZMPYQo8VAgsGYcQYbSEsOAzNRDjOQYP/MOOMfW+7mGcRr4TFm+NjGlrtBglgtEmdyihk+9rHlzjjzzOAgUX7hbz++mSHh27Hc/vbkhw9+VBChBQqOgckDxGtgYKghRfEoGAWjYBSMNAAA06owBSeNQ/8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0668-412X","institution":"Pontificia Universidad Catolica de Chile","correspondingAuthor":true,"prefix":"","firstName":"Cristian","middleName":"","lastName":"Carvajal","suffix":""},{"id":412145681,"identity":"8907cd3d-54a9-4829-8672-c24f4f923609","order_by":1,"name":"Pablo Carrión Valdés","email":"","orcid":"","institution":"Pontificia Universidad Catolica de Chile","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"Carrión","lastName":"Valdés","suffix":""},{"id":412145682,"identity":"e9154df7-0560-442f-91e0-f8566d6a8c00","order_by":2,"name":"María Paz Hernández Mejías","email":"","orcid":"","institution":"Pontificia Universidad Catolica de Chile","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Paz Hernández","lastName":"Mejías","suffix":""},{"id":412145683,"identity":"ecd559fc-f435-4d28-ae9f-fc1247d778e1","order_by":3,"name":"Jorge Pérez","email":"","orcid":"","institution":"Pontificia Universidad Catolica de Chile","correspondingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"","lastName":"Pérez","suffix":""},{"id":412145684,"identity":"a81771c1-b92b-45c8-8486-107e70ac941f","order_by":4,"name":"Alejandra Tapia-Castillo","email":"","orcid":"","institution":"Pontificia Universidad Catolica de Chile","correspondingAuthor":false,"prefix":"","firstName":"Alejandra","middleName":"","lastName":"Tapia-Castillo","suffix":""},{"id":412145685,"identity":"529aad90-4897-4d80-b230-0bc2d24f5167","order_by":5,"name":"Andrea Vecchiola","email":"","orcid":"","institution":"Pontificia Universidad Catolica de Chile","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Vecchiola","suffix":""},{"id":412145686,"identity":"941ac219-ea2b-4c5d-b73b-82ebf53bdf4f","order_by":6,"name":"Alejandra Sandoval-Bórquez","email":"","orcid":"","institution":"Pontificia Universidad Católica de Valparaiso","correspondingAuthor":false,"prefix":"","firstName":"Alejandra","middleName":"","lastName":"Sandoval-Bórquez","suffix":""},{"id":412145687,"identity":"7e27fd78-9f88-4445-b14b-b4ac98c68263","order_by":7,"name":"Rene Baudrand","email":"","orcid":"","institution":"Pontificia Universidad Catolica de Chile","correspondingAuthor":false,"prefix":"","firstName":"Rene","middleName":"","lastName":"Baudrand","suffix":""},{"id":412145688,"identity":"29516996-6e5e-43e0-b880-ae7ea479ee3e","order_by":8,"name":"Carlos Fardella","email":"","orcid":"","institution":"Pontificia Universidad Catolica de Chile","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Fardella","suffix":""}],"badges":[],"createdAt":"2025-02-01 14:35:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5942263/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5942263/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41366-026-02033-2","type":"published","date":"2026-02-23T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75906667,"identity":"3d895dd8-2526-484e-9c71-ce843e6be8bd","added_by":"auto","created_at":"2025-02-10 12:03:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1543816,"visible":true,"origin":"","legend":"\u003cp\u003eAdipogenic differentiation of SW872 cells. Data are presented as mean ± SEM, and Kruskal-Wallis analysis was used with p\u0026lt;0.05 to be statistically significant. N=3.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5942263/v1/0bf350e05262ccdb7c3ef083.png"},{"id":75906668,"identity":"c2a22c3d-7221-4124-948d-9c36a32bab9c","added_by":"auto","created_at":"2025-02-10 12:03:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":631505,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization and quantification of adipocyte-derived EVs. (A) Representative size distribution plot of AdEVs obtained via a NanoSight NS300 instrument.(B) Identification of AdEVs via transmission electron microscopy (TEM) (indicated by red arrows). (C) western blot analysis of the classic extracellular vesicle markers TSG101 and CD9.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5942263/v1/b46a86977ebf7409ae8bd092.png"},{"id":75907906,"identity":"f039a19c-04f4-4139-836c-48c452710256","added_by":"auto","created_at":"2025-02-10 12:11:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":671904,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Effects on gene expression in HCD cells after treatment with AdEVs for 24 hours. (B) Uptake of AdEVs by HCD cells was examined by exposing target cells to PKH67-labeled AdEVs followed by fluorescence microscopy (IL-6: interleukin 6; IL-1B: interleukin 1B; CTFG: connective tissue growth factor; FN1: fibronectin 1; NGAL: neutrophil gelatinase-associated lipocalin). Data are presented as mean ± SEM, and Mann-Whitney analysis was used with p\u0026lt;0.05 considered to be statistically significant. N=3.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5942263/v1/81c1e5eaac6dc15f38633050.png"},{"id":75906670,"identity":"523ac2d3-50fb-49d8-92be-6303a39024cf","added_by":"auto","created_at":"2025-02-10 12:03:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1210039,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Effects on gene expression in EA.hy926 cells after treatment with AdEVs for 24 hours. (B) Uptake of AdEVs by EA.hy926 cells was examined by exposing the target cells to PKH67-labeled AdEVs followed by fluorescence microscopy (IL-6: interleukin 6; IL-1B: interleukin 1B; CTFG: connective tissue growth factor; FN1: fibronectin 1; eNOS: endothelial nitric oxide synthase). Data are presented as mean ± SEM, and Mann-Whitney analysis was used with p\u0026lt;0.05 considered to be statistically significant. N=3.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5942263/v1/6aafad972c28b6c550652a59.png"},{"id":103301447,"identity":"c30d7ae3-8916-4f41-b23e-934bfe953082","added_by":"auto","created_at":"2026-02-24 08:13:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5010945,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5942263/v1/7e771985-ee23-48f3-a766-c3303c0d6222.pdf"},{"id":75907907,"identity":"0a429347-14cd-493a-b281-6f504c741a63","added_by":"auto","created_at":"2025-02-10 12:11:20","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":76163,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SUPPLEMENTARYDATA.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5942263/v1/7a7107ec842a8c03c5efcdd4.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"\u003cp\u003eAdipocyte Extracellular Vesicles (Adevs) Promote a Proinflammatory and Profibrotic Profile in Human Renal and Endothelial Cells in Vitro\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eObesity has reached pandemic levels worldwide; moreover, according to the World Health Organization, approximately 39% of adults older than 18 years of age are overweight, and 13% are obese (1). Obesity is the result of an imbalance between energy intake and expenditure that leads to an increase in energy stored by adipocytes, which causes adipose hypertrophy and hyperplasia (2). The increasing mass of fat leads to a proinflammatory state and impaired adipokine secretion, as well as the development of different obesity-related comorbidities, such as insulin resistance, hypertension, type 2 diabetes, and chronic kidney disease \u0026nbsp;(2, 3).\u003c/p\u003e\n\u003cp\u003eAdipose tissue (AT) is an energy storage organ and a vital endocrine organ that regulates the functions of other tissues and organs by its secretome, which is composed of soluble factors, adipokines, cytokines, and extracellular vesicles (EVs) (4). EVs are\u0026nbsp;a diverse group of nanoparticles that are released or shed by different types of tissues and cell types; additionally, they have been recently proposed as being key factors in intercellular communication\u0026nbsp;(5).\u0026nbsp;Small\u0026nbsp;EVs are generated from endosomal trafficking and multivesicular\u0026nbsp;bodies, with sizes ranging from\u0026nbsp;50\u0026ndash;150\u0026nbsp;nm\u0026nbsp;in\u0026nbsp;diameter\u0026nbsp;(5). Due to their bioactive cargo, EVs can\u0026nbsp;play\u0026nbsp;different functional roles in their target\u0026nbsp;cells\u0026nbsp;by inducing\u0026nbsp;cellular transduction signals and/or metabolic changes\u0026nbsp;(6). EVs from\u0026nbsp;ATs\u0026nbsp;that contain adipocyte proteins,\u0026nbsp;miRNAs\u0026nbsp;and adipokines could have paracrine and endocrine effects on various tissues,\u0026nbsp;thereby affecting both physiological and pathophysiological processes\u0026nbsp;(4, 7).\u003c/p\u003e\n\u003cp\u003ePrevious studies have demonstrated that patients with obesity have increased levels of circulatory EVs; thus, EVs may participate in the development of several comorbidities associated with obesity, such as T2DM and chronic inflammation (8, 9). Along with other researchers, we have highlighted the role of adEVs in metabolic disorders such as obesity and insulin resistance, whereby adEVs can regulate tissues in distant organs (10, 11). Additionally, regarding the role of adEVs in renal pathophysiology, recent studies both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e have demonstrated crucial roles for these EVs in the development of renal diseases, whereby they contribute to changes in immunomodulation and thrombogenesis, as well as in the extracellular matrix (12, 13). Moreover, Liu et al. reported that plasma EVs from subjects with obesity participate in the activation of the renin-angiotensin-aldosterone axis and elicit renal tubular damage in renal tubule epithelial cells (12). Furthermore, the release of proinflammatory factors by renal cells induced by adEVs can trigger an inflammatory process that affects immune system cells, such as macrophages or lymphocytes, which can enhance the local inflammatory effect and alter the secretome (14).\u003c/p\u003e\n\u003cp\u003eAn increasing amount of evidence suggests that adipose-derived extracellular vesicles (AdEVs) interact with different tissues involved in obesity-related comorbidities and the generation of an inflammatory response (7, 15). This study aimed to evaluate the proinflammatory and profibrotic effects of AdEVs from the SW872 cell line (SW872-AdEVs) in endothelial and renal cells\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methodology","content":"\u003col\u003e\n \u003cli\u003e\u003cstrong\u003ePreadipocyte culture and differentiation\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe human SW872 preadipocyte line (obtained from ATCC) was grown in DMEM/F-12 high glucose (HG) medium (#12400024, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (#10437-028, Gibco, USA) and penicillin-streptomycin solution 100X (#30-002-Cl, Corning, USA). When preadipocytes reached 80% confluence, differentiation into adipocytes was induced using differentiation medium (DMEM/F-12 supplemented with 1% FBS (Thermo, USA) plus 10 \u0026micro;g/mL recombinant human insulin (Humulin HI0210), 1 \u0026micro;M dexamethasone (Sigma Aldrich), 0.5 mM isobutyl-methylxanthine (IBMX) (Sigma Aldrich), and 1 \u0026micro;M rosiglitazone (Cayman, USA)) for up to 10 days, and the medium was renewed every three days. Differentiation was confirmed via optical microscopy and Oil Red O staining.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003e\u003cstrong\u003eDetermination of adipogenesis\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emarker\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;expression\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe expression of adipogenesis markers was determined via RT-qPCR by using primers for acetyl-CoA carboxylase (ACC), fatty acid-binding protein 4 (FABP4), fatty acid synthase (FASN), perilipin 1 (PLIN1) and peroxisome proliferator activated receptor gamma (PPARg). The sequences of the utilized primers are summarized in Supplementary data (Table S1 and Table S2).\u003c/p\u003e\n\u003cp\u003eThe protein expression of these markers was corroborated via western blotting by using the corresponding primary antibodies and the secondary anti-rabbit IgG antibody, which was HRP-linked from the \u0026ldquo;Adipogenesis Marker Antibody Sampler Kit\u0026rdquo; (1:2000; (C45B10) (C83B10) (2120) (C20G5) (D1D8) (C26H12) (7074) #12589; Cell Signaling, USA). The anti-B-actin rabbit mAb (1:2000; #4970; Cell Signaling Technology, USA) was used as a loading control. The relative expression of adipogenesis markers was measured on days 1, 3, 7 and 10 of the differentiation protocol.\u0026nbsp;\u003c/p\u003e\n\u003col start=\"3\"\u003e\n \u003cli\u003e\u003cstrong\u003eIsolation of adipocyte extracellular vesicles (AdEVs)\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAdEVs were obtained by a differential ultracentrifugation protocol. Briefly, preadipocytes from SW872 cells were grown to 80% confluence and subsequently differentiated for 7 days in a multilayer 875 cm\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eflask (#353144, Corning, USA). Afterwards, differentiated cells were cultured in exosome-free DMEM-F12 (supplemented with 1% FBS) HG medium, which was supplemented with a differentiation cocktail. The cells were maintained in that medium for 24 hours for EV release. After 24 hours, the culture medium was collected and centrifuged at 300 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 minutes (min) to remove the remaining cells. The supernatant was subsequently collected and centrifuged at 2000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 30 min and at 12000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 45 min to remove dead cells and cellular debris, respectively. The resulting supernatant was filtered through a 0.22 \u0026mu;m filter (EDLAB Sterile Syringe Filter CA 0.22 \u0026mu;m) and subsequently ultracentrifuged at 120000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 70 min to precipitate the AdEVs. The resulting pellet was resuspended in PBS and centrifuged at 100000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 70 min to obtain the AdEV pellet. The final pellet was resuspended in aliquots of 100 \u0026mu;L of filtered PBS and stored at -80 \u0026deg;C until analysis. Suspensions of AdEVs were characterized according to the guidelines of the International Society for Extracellular Vesicles (16) by nanoparticle tracking analysis, transmission electron microscopy, and western blot analysis.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003col start=\"4\"\u003e\n \u003cli\u003e\u003cstrong\u003eNanoparticle\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etracking analysis\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(NTA)\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe concentrations and sizes of the AdEV suspensions were determined by NTA by using a NanoSight NS300 and NanoSight NTA 3.2 software (Malvern Instruments Ltd., Malvern, UK). Particles were tracked by passing a laser beam through the liquid sample, and the scattered light was detected and captured in three short videos of 20 second durations by using an sCMOS camera. The Brownian motion of the particles was determined on a frame-by-frame basis, and the distance traversed by the particles was used to determine the AdEV size and concentration using the Stokes-Einstein equation.\u0026nbsp;\u003c/p\u003e\n\u003col start=\"5\"\u003e\n \u003cli\u003e\u003cstrong\u003eWestern bb\u003c/strong\u003e\u003cstrong\u003elotting\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of exosome markers and adipogenic cargo\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe proteins CD9 and TSG101 were used as EV markers. PPARɣ, FASN and ADIPOQ were used as adipocyte markers. For western blotting, AdEVs were resuspended in RIPA buffer (Thermo Fisher Scientific Inc., IL, USA) to extract total\u0026nbsp;protein,\u0026nbsp;and\u0026nbsp;the\u0026nbsp;protein concentration was determined using the bicinchoninic acid (BCA) method. Protein lysates were separated through polyacrylamide gel electrophoresis,\u0026nbsp;transferred\u0026nbsp;to\u0026nbsp;nitrocellulose\u0026nbsp;membranes,\u0026nbsp;blocked with 5% skim milk\u0026nbsp;in PBS,\u0026nbsp;and probed overnight with primary rabbit monoclonal anti-TSG101 (1:10000; Ab125011; Abcam, MS, USA) and rabbit monoclonal anti-CD9 (1:1000; (D801A) #13174; Cell Signaling, USA)\u0026nbsp;antibodies. After washing,\u0026nbsp;the\u0026nbsp;membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG-HRP (1:10000; Ab6939;\u0026nbsp;Abcam, USA) for 1 hour at RT. Proteins were detected using chemiluminescence (ECL western blotting substrate reagent; Pierce, USA)\u0026nbsp;with\u0026nbsp;a\u0026nbsp;ChemiDoc\u0026nbsp;MP imaging system (Bio-Rad,\u0026nbsp;CA, USA).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003col start=\"6\"\u003e\n \u003cli\u003e\u003cstrong\u003eTransmission\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eelectron microscopy\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(TEM) of AdEVs\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eTEM was performed to verify the shapes and sizes of the obtained AdEVs. Briefly, AdEV suspensions were absorbed onto carbon-coated copper grids for 1 min. Samples were negatively stained with a 2% uranyl acetate solution. The grids were visualized with a Thermo Scientific Talos F200C G2 transmission electron microscope.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003col start=\"7\"\u003e\n \u003cli\u003e\u003cstrong\u003eTreatment of renal and endothelial cells with AdEVs\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eTo investigate the effects of AdEVs on the expression of inflammatory and fibrotic genes in both kidney and endothelial cells, both the HCD cell line (kidney) and the EA.hy926 cell line (endothelium) were exposed to AdEVs for 24 hours. Briefly, HCD cells were cultured in 6-well plates containing DMEM/F-12 low-glucose (LG) medium supplemented with 10% FBS, and EA.hy926 cells were cultured in 6-well plates containing IMDM supplemented with 10% FBS. Once the cells reached 80\u0026ndash;90% confluence, the medium was changed to EV-free basal medium (DMEM/F-12 LG supplemented with 1% EV-free FBS or IMDM supplemented with 1% EV-free FBS) for 24 hours. Afterwards, AdEVs resuspended in PBS were added at a proportion of 1000 AdEVs/cell, and the cells were incubated for another 24 hours. For the control, the same volume of EV-free PBS was added. Subsequently, the cells were lysed using TRIzol reagent (#15596018; Invitrogen), and inflammatory, fibrotic, and endothelial markers were evaluated via RT-qPCR following the relative expression analysis by using the methodology of Livak et al. (17). The list of utilized primers is summarized in Supplementary data (Table S1 and Table S3).\u0026nbsp;\u003c/p\u003e\n\u003col start=\"8\"\u003e\n \u003cli\u003e\u003cstrong\u003eAdEV\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;uptake assay\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAdEV pellets, which were isolated as previously described, were resuspended in PBS and stained with PKH67 green fluorescent dye (#MIDI67; Sigma-Aldrich, MO, USA). The negative control was an EV-free sample labeled with PKH67. After AdEV labeling, HCD and EA.hy926 cells were incubated with PKH67-fluorescent AdEVs (37000 AdEVs per cell) or a negative control for 24 hours. After incubation, the cells were rinsed twice with cold PBS and fixed with 4% paraformaldehyde at RT for 10 min. The cell nucleus was stained with DAPI, and the cells were mounted on glass slides with Vectashield Antifade mounting medium\u003cs\u003e\u0026nbsp;\u003c/s\u003e(#H-1200; Vector Laboratories, USA). The cells were visualized with an Olympus CKX41 epifluorescence microscope (USA).\u0026nbsp;\u003c/p\u003e\n\u003col start=\"9\"\u003e\n \u003cli\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA). The data are presented as the mean \u0026plusmn; standard error. Mann-Whitney\u0026rsquo;s tests were performed for comparisons between two groups, while Kruskal-Wallis was used for comparisons of more than two groups. P \u0026lt; 0.05 was considered to be statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e1. Characterization of SW872 cell adipogenesis based on cell histology and gene expression\u003c/h2\u003e \u003cp\u003eMorphological changes in SW872 preadipocytes were observed on days 3 to 10 of the differentiation protocol; specifically, the cells evolved from a conical stretched morphology to a rounded morphology, and the diameter of the lipid droplets increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). At days 7 and 10, most of the cells exhibited a rounded shape and Oil Red O-positive labeling, which is distinctive of mature adipocytes (Supplementary \u003cb\u003eTable\u0026nbsp;4\u003c/b\u003e). During adipogenic differentiation, the expression of the PPARg, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) genes significantly increased on the third day and then gradually decreased until day 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In contrast, fatty acid-binding protein 4 (FABP4) and perilipin 1 (PLIN1) gene expression reached a maximal increase on day 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Analysis of protein expression revealed that PPARg also significantly increased on the third day but then gradually decreased until day 10. In contrast, the relative abundances of the ACC and FASN proteins increased at days 7 and 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2. Characterization of AdEVs isolated from the SW872 cell culture\u003c/h2\u003e \u003cp\u003eThe characterization of AdEVs from SW872 cells (SW872-AdEVs) revealed a mean concentration of 2.1x10\u003csup\u003e10\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1x10\u003csup\u003e8\u003c/sup\u003e AdEVs/mL, with a mean size of 128.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 nm and a mode of 134.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, via TEM, we observed that AdEVs exhibited a classic donut-like morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB); moreover, via western blotting, we identified the presence of the CD9 and TSG101 proteins, which are associated with endosomal biogenesis of EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3. SW872-AdEVs increase inflammatory and fibrotic gene expression in human collecting duct (HCD) cells\u003c/h2\u003e \u003cp\u003eThe inflammatory and fibrotic effects of SW872-AdEVs on the human collecting duct (HCD) cell line were evaluated \u003cem\u003ein vitro\u003c/em\u003e by changes in the gene expression of IL-6 and IL-1B (which are inflammatory markers); CTGF and FN1 (which are fibrosis markers); and NGAL (18). HCD-treated cells were incubated with AdEVs (1000 particles/cell) for 24 hours. We observed significant increases in gene expression of IL-6 (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs. 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 UR; p\u0026thinsp;=\u0026thinsp;0.029), IL-1B (1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs. 2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 UR; p\u0026thinsp;=\u0026thinsp;0.029), CTGF (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs. 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 UR; p\u0026thinsp;=\u0026thinsp;0.031) and NGAL (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs. 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 UR; p\u0026thinsp;=\u0026thinsp;0.04) when comparing vehicle versus treatment with adEVs. We did not observe statistically significant changes in FN1 expression (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 vs. 1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 UR, p\u0026thinsp;=\u0026thinsp;0.3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4. AdEVs affect IL-6 and eNOS expression in EA.hy926 endothelial cells\u003c/h2\u003e \u003cp\u003eSimilar to HCD cells, we evaluated the inflammatory and fibrotic effects of SW872-AdEVs on the EA.hy926 endothelial cell line by gene expression analysis. We evaluated the gene expression of IL-6 and IL-1B (inflammatory markers), as well as CTGF and FN1 (fibrosis markers), with eNOS expression being used as a proxy for endothelial function. We observed an increase in gene expression of IL-6 expression (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs. 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 UR; p\u0026thinsp;=\u0026thinsp;0.03) and a nonsignificant increase in IL-1B expression (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs. 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 UR; p\u0026thinsp;=\u0026thinsp;0.15) when comparing vehicle versus treatment with adEVs. Nonsignificant changes were observed in both CTGF (1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs. 0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 UR; p\u0026thinsp;=\u0026thinsp;0.06) and FN1 (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 vs. 0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 UR; p\u0026thinsp;=\u0026thinsp;0.41). Furthermore, eNOS expression was significantly decreased in cells treated with AdEVs (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs. 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 UR; p\u0026thinsp;=\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5. AdEVs can be internalized into HCD and EA.hy926 cells\u003c/h2\u003e \u003cp\u003eThe internalization of AdEVs in both HCD and EA.hy926 cells was evaluated by EV labeling with the PKH67 green fluorescent dye. After 12 hours of incubation, fluorescently labeled AdEVs were observed in the subcellular compartments of both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study revealed that AdEVS from SW872 adipocytes (SW872-AdEVs) changed the gene expression of cytokines, fibrosis-related genes, and eNOS in renal (HCD) and endothelial (EA.hy926) cultured cells, which suggests an interesting association of AdEVs with renal epithelial and endothelial cells in the context of obesity (9).\u003c/p\u003e \u003cp\u003eWe observed that the incubation of SW872-AdEVs from mature adipocytes for 24 hours with both renal cells and endothelial cells increased the mRNA expression of the proinflammatory cytokines IL-6 and IL-1B. Both cytokines are associated with inflammatory processes. Additionally, we observed a significant increase in the expression of NGAL in renal epithelial cells treated with SW872-AdEVs. NGAL (or LCN2) is a lipocalin that is currently considered to be an inflammatory marker commonly associated with acute kidney injury, and it is also associated with activation of the mineralocorticoid receptor (MR) pathway (19, 20). These results suggest that SW872-AdEVs can modify the phenotypes of target cells via the activation of proinflammatory pathways, including the MR pathway.\u003c/p\u003e \u003cp\u003eWith respect to the fibrotic responses of target cells that are challenged with SW872-AdEVs, we observed changes in CTGF gene expression only in HCD renal cells, which is interesting because it can be a proxy for the profibrotic phenotype (12, 13, 21). Taken together, these results in HCD cells support a novel link between AdEVs and epithelial renal cells, with increased inflammation and fibrosis-related gene expression being observed (22, 23). Hence, we encourage the performance of further studies aimed at detecting fibrotic signs in renal and vascular fibroblasts to confirm the impact of SW872-AdEVs.\u003c/p\u003e \u003cp\u003eWith respect to endothelial cells, we did not detect changes in fibrosis-related gene expression; however, we detected a significant decrease in relative eNOS expression when these cells were challenged with AdEVs-SW872. Reduced eNOS expression is associated with endothelial dysfunction, since eNOS is a key factor in the vasodilation response in the endothelium (24\u0026ndash;27). Previous reports on eNOS and EVs have shown that under pathological conditions, EVs from different sources are able to impair vasorelaxation via the inhibition of eNOS, a reduction in NO bioavailability, and the activation of ERK signaling (28, 29). In contrast, EVs from adipose-derived stem cells (ADSCs) improve eNOS signaling associated with the overexpression of GLO-1, which is a protein associated with reducing oxidative stress (30). Both results suggest that the donor adipose cell type and the cellular microenvironment (such as environments with low/high oxidative stress or low/high proinflammatory mediator levels, among other conditions) are key factors in establishing the EV cargo in AdEVs and further affecting target cells (30, 31).\u003c/p\u003e \u003cp\u003eWe also evaluated the internalization of fluorescent SW872-AdEVs labeled with PKH67 dye in target cells. We demonstrated that AdEV-SW872 was efficiently internalized by HCD renal and EA.hy.926 endothelial cells. Several studies have shown that EVs carry interleukins as part of their cargo (32) and induce the secretion of different proinflammatory interleukins (such as IL-1B, IL-6, IL-8 and TNF-α) in target cells (32\u0026ndash;34). Therefore, the specific cargo transported within adEVs, such as interleukins and miRNAs, can change or mediate the phenotypes of target cells (31). EV-miRNAs can act as biomarkers, distant gene expression repressors, and potential therapeutic agents (10, 35). Moreover, miRNAs can travel to neighboring and distant cells via the circulation, thereby mediating cell-to-cell communication by targeting mRNAs and ultimately causing changes in the expression of target genes (11, 36). Thomou et al. reported that adipose tissue is the major source of circulating exosomal miRNAs and functions as a gene regulator in distant tissues (37). These results encourage the performance of a transcriptomic assay of AdEVs to identify specific mRNAs and miRNAs within AdEVs that can exert an impact on the phenotypes of target cells.\u003c/p\u003e \u003cp\u003eHowever, several limitations are associated with this study, which are mainly related to the dose and timing of AdEV challenge in target cells; these factors can cause biases in specific gene expression changes. Additionally, AdEV challenge is an experimental model that simplifies the overall impact of the secretome and other inflammatory factors (such as TNF-α) that are present in the AT microenvironment. Further studies are needed to evaluate the effects of AdEVs linked to proinflammatory conditions in donor and/or target cells.\u003c/p\u003e \u003cp\u003eIn summary, adipose extracellular vesicles (AdEVs) from SW872 adipocytes were efficiently isolated by UCF by using a novel isolation protocol. We observed that SW872-AdEVs induced a proinflammatory profile in renal and endothelial cells (HCD and EA.hy926) \u003cem\u003ein vitro\u003c/em\u003e, and a decrease of eNOS expression in EA.hy926 cells. These results support that EVs from mature adipocytes could increase the inflammation and vascular dysfunction in obesity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of interest.\u003c/h2\u003e \u003cp\u003eThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding statements.\u003c/h2\u003e \u003cp\u003eThis study was supported by the following grants: ANID-CONICYT FONDECYT 1212006 \u0026amp; 11251675; ICM-ANID ICN2021_045; SOCHED 2024-06 and CETREN-UC 2024\u0026ndash;2025.\u003c/p\u003e\u003ch2\u003eAuthors' contributions.\u003c/h2\u003e \u003cp\u003eP.C. and MP-H. contributed equally to this work, both designed the study, collected, analyzed and interpreted of the data, contributed to the statistics of the data, wrote the first draft of the manuscript, contributed to discussion, and reviewed the manuscript. J.P. contributed to data analysis and reviewed the manuscript. A.T-C analyzed and interpreted the statistical data, contributed to discussion, and reviewed the manuscript. AV and A.S-B contributed to the development of in vitro analyses and reviewed the manuscript. RFB and CEF contributed to discussion and reviewed the manuscript. CAC designed the study, analyzed and interpreted of the data, reviewed to the statistics of the data, contributed to discussion, and reviewed the manuscript. All reviewed the manuscript and authors approved the final version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCamino T, Lago-Baameiro N, Pardo M. Extracellular Vesicles as Carriers of Adipokines and Their Role in Obesity. Biomedicines. 2023;11(2):422.\u003c/li\u003e\n\u003cli\u003eBalistreri CR, Caruso C, Candore G. The role of adipose tissue and adipokines in obesity-related inflammatory diseases. 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Int J Mol Sci. 2017;18(6).\u003c/li\u003e\n\u003cli\u003eBardi GT, Smith MA, Hood JL. Melanoma exosomes promote mixed M1 and M2 macrophage polarization. Cytokine. 2018;105:63-72.\u003c/li\u003e\n\u003cli\u003eLim WQ, Michelle Luk KH, Lee KY, Nurul N, Loh SJ, Yeow ZX, et al. Small Extracellular Vesicles\u0026apos; miRNAs: Biomarkers and Therapeutics for Neurodegenerative Diseases. Pharmaceutics. 2023;15(4).\u003c/li\u003e\n\u003cli\u003eZhang S, Cheng Z, Wang Y, Han T. The Risks of miRNA Therapeutics: In a Drug Target Perspective. Drug Des Devel Ther. 2021;15:721-33.\u003c/li\u003e\n\u003cli\u003eThomou T, Mori MA, Dreyfuss JM, Konishi M, Sakaguchi M, Wolfrum C, et al. Adipose-derived circulating miRNAs regulate gene expression in other tissues. Nature. 2017;542(7642):450-5.\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-obesity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ijo","sideBox":"Learn more about [International Journal of Obesity](http://www.nature.com/ijo/)","snPcode":"41366","submissionUrl":"https://mts-ijo.nature.com/cgi-bin/main.plex","title":"International Journal of Obesity","twitterHandle":"@intjobesity","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Adipocyte, adipocyte extracellular vesicles (AdEVs), cytokines, eNOS","lastPublishedDoi":"10.21203/rs.3.rs-5942263/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5942263/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn obesity,white adipose tissue (WAT) undergoes hypertrophic and hyperplastic changes that are driven by phenotypical changes in preadipocytes and adipocytes. WAT also causes a chronic inflammatory state that modifies gene expression and the secretome, including the shedding of adipose-derived extracellular vesicles (AdEVs) into the circulation, with these AdEVs affecting other cell types and potentially modifying their phenotypes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAim: \u003c/strong\u003eTo evaluate the effects of AdEVs on renal and endothelial cells and their impacts on gene expression associated with inflammation, fibrosis, and endothelial function.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eHuman SW872 adipocytes were differentiated from preadipocytes and further characterized. AdEVs were isolated via ultracentrifugation and characterized according to ISEV guidelines. AdEVs were added to either human renal (HCD) or endothelial cells (EA.hy926) for 24 hours. Adipokine, cytokine (IL-6 and IL-1B), fibrosis-related, NGAL, and eNOS gene expression wasevaluated via RT-qPCR and western blotting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eSW872 cells exhibited classical adipocyte morphologies and a significant accumulation of lipid droplets. Isolated AdEVs exhibited a donut-shaped morphology, a characteristic size, and both CD9 and TSG101 markers, which are compatible with EV features. Both renal and endothelial cells that were challenged with AdEVs were able to (1) incorporate AdEV-PKH67 (a fluorescent dye) and (2) induce high expression of IL-6 and IL-1B (p\u0026lt;0.05). A decrease in eNOS expression was detected in endothelial cells treated with AdEVs (p\u0026lt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003eAdEVs induced a proinflammatory profile in renal and endothelial cells (HCD and EA.hy926) \u003cem\u003ein vitro\u003c/em\u003e, and a decrease of eNOS expression in EA.hy926 cells. These results support that EVs from mature adipocytes could increase the inflammation and vascular dysfunction in obesity.\u003c/p\u003e","manuscriptTitle":"Adipocyte Extracellular Vesicles (Adevs) Promote a Proinflammatory and Profibrotic Profile in Human Renal and Endothelial Cells in Vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-10 12:03:15","doi":"10.21203/rs.3.rs-5942263/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-03-28T11:09:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-03-25T18:49:32+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-03-03T17:55:03+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-03-02T15:36:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-02-18T08:45:08+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-02-06T14:43:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-03T12:08:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Obesity","date":"2025-02-01T14:34:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-01T14:34:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-obesity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ijo","sideBox":"Learn more about [International Journal of Obesity](http://www.nature.com/ijo/)","snPcode":"41366","submissionUrl":"https://mts-ijo.nature.com/cgi-bin/main.plex","title":"International Journal of Obesity","twitterHandle":"@intjobesity","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1eeb31f5-2297-4f29-862e-8012cfd28116","owner":[],"postedDate":"February 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44102801,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Obesity"},{"id":44102802,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2026-02-24T08:12:41+00:00","versionOfRecord":{"articleIdentity":"rs-5942263","link":"https://doi.org/10.1038/s41366-026-02033-2","journal":{"identity":"international-journal-of-obesity","isVorOnly":false,"title":"International Journal of Obesity"},"publishedOn":"2026-02-23 05:00:00","publishedOnDateReadable":"February 23rd, 2026"},"versionCreatedAt":"2025-02-10 12:03:15","video":"","vorDoi":"10.1038/s41366-026-02033-2","vorDoiUrl":"https://doi.org/10.1038/s41366-026-02033-2","workflowStages":[]},"version":"v1","identity":"rs-5942263","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5942263","identity":"rs-5942263","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}