Identification and verification of novel therapeutic agents for diabetic kidney disease based on exosome-targeted high-throughput chemical screening

Identification and verification of novel therapeutic agents for diabetic kidney disease based on exosome-targeted high-throughput chemical screening | 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 Identification and verification of novel therapeutic agents for diabetic kidney disease based on exosome-targeted high-throughput chemical screening Daisuke Fujimoto, Shuro Umemoto, Teruhiko Mizumoto, Tomoko Kanki, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4010567/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Exosomes are important mediators of intercellular communication and play key roles in the regulation of pathophysiological processes. In diabetic kidney disease (DKD), it has been reported that macrophages recruited in the mesangial region may play pathogenic roles through inducing local inflammation in glomeruli. We focused on exosome-mediated crosstalk between mesangial cells (MC) and macrophages as a novel therapeutic target for DKD. Exosomes released from MC induced inflammation in macrophages and the effect was enhanced under high-glucose conditions. For discovering novel therapeutic agents which can inhibit such exosome-mediated mechanisms, drug repositioning is considered as aneffective tool. We established a unique screening strategy and screened agents to aim at maximizing their specificity and potency to inhibit exosomal mechanisms, along with minimizing their toxicity. We succeeded in identifying alvespimycin, an HSP90 inhibitor. Treatment of diabetic rats with alvespimycin significantly suppressed mesangial expansion, inflammatory gene activation including macrophage markers, and proteinuria. The inhibitory effect on exosome uptake was specific to alvespimycin compared with other known HSP90 inhibitors. MC-derived exosomes are crucial for inflammation by intercellular crosstalk between MC and macrophages in DKD, and alvespimycin effectively ameliorated the progression of DKD by suppressing exosome-mediated actions, suggesting that exosome-targeted agents can be a novel therapeutic strategy. Biological sciences/Drug discovery Health sciences/Diseases Health sciences/Nephrology exosomes drug screening diabetic kidney disease intraglomerular crosstalk mesangial cells macrophages Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Extracellular vesicles (EVs) are important mediators of intercellular communication and play a key role in the regulation of pathophysiological processes in various organs and cells. 1 Particular attention has been paid to intercellular crosstalk by exosomes, whose size is 30–100 nm. 2 The presence of exosomes in serum and urine has attracted attention not only in the field of oncology but also in the field of kidney diseases, because exosomes can influence the surrounding environment by propagating the properties of their mother cells to the neighboring cells and environment. 3 – 5 Diabetic kidney disease (DKD) is the most common cause of chronic kidney disease and end-stage renal disease, leading to a high risk of mortality. The pathophysiology of DKD is complicated and multifaceted, and yet to be fully elucidated. Furthermore, treatments to effectively prevent the progression of DKD are still quite limited, so that novel therapeutic strategies are strongly desired. It has been a general consensus that DKD progresses along with mesangial proliferation and matrix accumulation. Macrophages infiltrate into the mesangial areas upon mesangial activation. In particular, we already reported that endogenous ligands associated with inflammation are induced specifically in glomerular infiltrating macrophages, which may have a potential pathogenic role in DKD 6 and glomerulonephritis. 7 These findings suggest that, in DKD, there should be some yet undefined mechanisms that specifically activate infiltrating macrophages locally in a paracrine manner. Recently, a potential role of intraglomerular cellular crosstalk mediated by exosomes in DKD has been reported. Wu et al. reported that vascular endothelial cells exposed to hyperglycemia release exosomes and activate mesangial cells, leading to renal fibrosis. 8 Liu et al. showed that macrophage-derived exosomes promote activation of NLRP3 inflammasome and autophagy deficiency of mesangial cells in diabetic nephropathy. 9 However, the pathophysiological role of exosomes in cell-cell interaction between mesangial cells and macrophages still remains obscure, especially those from mesangial cells toward macrophages in the glomeruli. In the present study, we investigated the role of exosomes in the intercellular crosstalk between mesangial cells and macrophages in DKD. Furthermore, in order to explore a novel therapeutic strategy to intervene the exosomal actions between those cells and to deter the progression of DKD, we screened chemical agents that can specifically act on the behavior or action of exosomes. Exosomes include various kinds of proteins, messenger RNAs and microRNAs, 10 and multiple exosomal factors are assumed to be involved in the cell-cell crosstalk, suggesting that targeting a single factor might result in only partial effects. Therefore, we focused on finding chemical compounds that have effects on the primary actions on exosomes derived from mesangial cells, not pursuing particular factors present in the exosome as a target. Drug repositioning is a powerful and effective tool for discovering therapeutic chemical compounds in various kinds of disease. 11 , 12 Herein, we performed high-throughput chemical screening using a validated compound library of existing drugs to identify novel candidate agents, and verified their efficacy in DKD. Materials and Methods Methods of reporter analysis, cell culture, real-time quantitative RT-PCR, in vivo exosome-uptake study and histological analyses are described in Supplementary Methods. Exosome isolation Exosomes were collected from the medium using ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA) and MagCapture Exosome Isolation Kit PS (Wako, Osaka, Japan) in accordance with the manufacturer’s instruction. Sprague-Dawley rat glomerular mesangial cells (SDMCs) were maintained in DMEM/Nutrient Mixture F-12 Ham containing 5.6 mmol/L or 25 mmol/L glucose without fetal bovine serum for 24 h before isolation of exosomes. For osmotic adjustment, mannitol (Nacalai Tesque, Kyoto, Japan) was added to the 5.6 mmol/L medium (24.5 mM final concentration). For exosome preparation by the ExoQuick method, the cultured medium of SDMCs was centrifuged at 3,000 x g for 10 min to remove cellular debris. One-fifth of ExoQuick-TC was added to the supernatant and incubated overnight at 4°C. The suspension was centrifuged at 1,500 x g for 30 min. The supernatant was discarded, and the remaining pellet was subjected to another centrifugation at 1,500 x g for 5 min. The pellet was resuspended with PBS and used as exosomes. To isolate EVs by the MagCapture Exosome Isolation Kit PS, the cell culture medium was centrifuged at 300 x g for 30 min at 4°C to remove cells and debris. The supernatant was transferred into a new tube, and centrifuged at 1,200 x g for 20 min at 4°C. To remove large EVs, the supernatant was transferred again into a new tube, and centrifuged at 10,000 x g for 30 min at 4°C. Then, the sample was concentrated by using ultrafiltration unit (Vivaspin20, Sartorius, Gottingen, Germany), transferred to a new 1.5 mL microcentrifuge tube, and suspended in Exosome Binding Enhancer at a 1:500 volume. Well mixed samples were transferred into 1.5 mL Reaction Tube containing Exosome-Capture-immobilized beads, and the mixture was rotated overnight at 4°C. The beads were washed three times with 1 mL of washing buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.0005% Tween20, 2 mM CaCl 2 ), and the bound EVs were eluted with elution buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA). 13 An aliquot of the exosomal preparation was used for exosome counting by NanoSight NS300 system (Malvern Panalytical, Salisbury, UK) followed by normalization to the total cell number. Exosome protein content was quantified using BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). DiO labeling of exosomes To prepare labeled exosomes, we conducted fluorescent staining of the supernatant of cultured SDMCs with lipophilic green fluorescent dye 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO) and isolated exosomes derived from SDMCs by ExoQuick-TC. We added DiO (Vybrant DiO Cell-labeling Solution; Invitrogen, Carlsbad, CA, USA) to the collected supernatant at a final concentration of 0.5 µL/mL and incubated them for 10 min at 37°C protecting from light. Hereafter, the same procedure was performed as mentioned. Confocal microscopy and flow-cytometry For confocal microscopic analysis, RAW 264.7 mouse macrophages were stained with red fluorescent dye (CytoTrace Red CMTPX, AAT Bioquest Inc., Pleasanton, CA, USA) for 24 h, and culture medium is labeled with a blue water-soluble, cell-impermeant polar tracer (Cascade Blue hydrazide, Thermo Fisher Scientific). After incubation, DiO-labeled exosomes (DiO-exo) were added just before microscopic observation, and the uptake of DiO-exo in macrophages was evaluated. All images were scanned with confocal microscopy, FV3000 (Olympus, Tokyo, Japan). For flow cytometry (FCM), RAW 264.7 cells were seeded in six-well plates (5 x 10 5 /well) and grown overnight. Prior to treatment with DiO-exo, cells were washed with PBS, and then DiO-exo in 100 µL PBS/well were added and incubated at 37°C for 24 h. Cells stained directly with 1 µL/well DiO (1 µL/mL) served as a positive control, and unstained cells as a negative control. Pre-incubation was performed with 10 µg/mL of an endocytosis inhibitor, cytochalasin D (Cayman Chemical, MI, USA) for 30 min before adding exosomes. DiO-labeled exosome–treated cells were removed from plates by trypsin, centrifuged, and resuspended in 1 mL of PBS. FCM was performed by SH800S Cell Sorter (Sony Life Science, Tokyo, Japan). FCM data were analyzed with FlowJo V10 program (FlowJo LLC, Ashland, OR, USA). 7 Screening of compounds inhibiting exosome-mediated mechanisms Compound screening was conducted using a chemical library containing 3,267 compounds from Drug Discovery Initiative at the University of Tokyo. The protocol was approved prior to the initiation of this study (approved by JP23ama121053, Project No. 0202). The screening strategy consisted of 5 steps. Briefly, in Step 1, small molecules (2 mM) in dimethyl sulfoxide (DMSO) solution from library plates were added to cultured THP-1-Dual Cells in 96 well plates, and secreted embryonic alkaline phosphatase (SEAP)–reporter activities were monitored (see Supplementary Methods). Compounds inhibiting the NF-kB activity induced by mesangial cell–derived exosomes (MC-exo) over 40% were selected and proceeded to the next step. In Step 2, steroidal compounds with an apparent NF-kB inhibitory action were excluded. Step 3 was composed of two strategies: in Step 3A, compounds exhibiting concentration-dependent NF-kB inhibition were selected, in which final compound concentrations were 0.2 µM, 1 µM, and 5 µM; in Step 3B, compounds showing a high toxicity were excluded, defined as the cell survival rate < 60% determined by Cell Count Reagent SF (Nacalai Tesque). The top 160 compounds that passed both Steps 3A and 3B proceeded further. In Step 4, compounds with a higher specificity to exosome-mediated inflammation compared to lipopolysaccharide (LPS)-mediated inflammation were chosen; the criteria were set as the ratio of the inhibition rate against exosomes to that against LPS to be > 1.5. Finally, in Step 5, compounds that inhibited exosome uptake in THP-1 cells were selected, using DiO-exo and FCM analysis. Animal experiments Among the compounds thus selected, we focused on one compound, alvespimycin (Alv, also known as 17-dimethylaminoethylamino-17-demethoxygeldanamycin, 17-DMAG; InvivoGen, San Diego, CA, USA) as a candidate inhibitor of exosome-mediated mechanisms in vivo . Experiments were conducted using eight-week-old male Sprague-Dawley rats (Japan SLC, Shizuoka, Japan), dividing into the following four groups: 1) wild type (WT) + vehicle, 2) WT + Alv, 3) streptozotocin (STZ) + vehicle, and 4) STZ + Alv. Rats were housed in a room maintained at constant temperature, humidity, and light cycle (12:12-h light-dark) with free access to food and water. They were maintained maximum 2 individuals in one cage. After allowing rats to adapt to their environment for 1 week, STZ group and STZ + Alv group rats were injected with STZ (50 mg/kg body weight in citrate buffer, pH 4.0; Sigma-Aldrich, St. Louis, MO, USA) from the tail vein after 16 h of fasting to develop insulin-dependent diabetes. All rats survived, and their blood glucose level increased over 280 mg/dL a week after STZ administration. Three weeks after STZ administration, Alv was administered intravenously via the tail vein twice a week at a dose of 0.5 mg/kg. After 6 weeks of treatment with vehicle (200 µL saline) or Alv, 16h-fasted rats were anesthetized and sacrificed. Blood, urine and kidney samples were collected. Urine albumin and creatinine levels were measured by using an immunoturbidimetric method (Oriental Yeast, Shiga, Japan). In all animal experiments, we complied with the ARRIVE guidelines. Ethics All animal procedures were conducted in accordance with the guidelines for care and use of laboratory animals approved by Kumamoto University (No. 29–115). Data deposition The original complete datasets are openly available in repository figshare at https://figshare.com/articles/dataset/Original_raw_data_of_each_step_of_the_drug_screening_/25018745 . Statistics Data are expressed as means ± SEM. Differences between multiple groups were assessed by ANOVA with Tukey’s test using Graphpad Prism (Graphpad Software). Comparison between two groups was carried out by unpaired Student’s t test. Statistical significance was defined as p < 0.05. Role of the funding source Funders had no input on study design, data collection, data analyses, interpretation, or writing of report. Results Quality assessment of mesangial cell–derived exosomes and evaluation of their activity on NF-kB signaling in macrophages It is crucial to assure the quality of extracted exosomes, MC-exo, which are the target of drug screening. Hence, first, quality evaluation of the extracted exosomes was performed. We compared the most widely used precipitation method using ExoQuick with that using MagCapture, which has been reported to obtain relatively purer exosomes. The MagCapture method is an extraction technique based on the binding of phosphatidylserine and Tim4. 14 Although exosomes by ExoQuick contained larger particles compared to those by MagCapture (Fig. 1 a), particle counts gated in diameters within 30–120 nm were equivalent in the both isolation methods (Fig. 1 b). Next, the effects of MC-exo upon NF-kB signaling were evaluated by SEAP-reporter assay in THP-1 cells. Exosomes extracted from mesangial cells induced the NF-kB activity in a dose-dependent manner with both techniques (Fig. 1 c). According to these results, we employed the ExoQuick method throughout the following all experiments. Exosomes derived from mesangial cells are endocytosed by macrophages in vitro and in vivo In order to examine whether macrophages uptake prepared exosomes, we evaluated the localization of fluorescence-labeled MC-exo stained with lipophilic green fluorescent dye DiO in tracer-labeled macrophages. Confocal microscopy findings showed that DiO-exo were colocalized with cell-impermeant polar tracer, Cascade blue in macrophages, indicating endocytosis (Fig. 2 a). These uptakes were obviously suppressed by Cytochalasin D, an endocytosis inhibitor (Fig. 2 b). We also examined the exosome uptake in animal models with high-glucose condition. Non-diabetic control and STZ-mice were administered with DiO-exo by tail-vein injection and sacrificed at 1 h from injection. FCM analysis of peripheral blood was conducted and the number of DiO-positive monocytes was evaluated. The ratio of DiO-positive macrophages to all macrophages was significantly higher in STZ mice than non-diabetic mice (Fig. 2 c). Thus, exosomes released from mesangial cells could be uptaken through endocytosis by macrophages both in vitro and in vivo . Exosomes derived from high glucose–conditioned mesangial cells augment inflammation in macrophages Particle counts determined by NanoSight showed no significant changes in exosomes prepared from mesangial cells with either high-glucose (HG-exo) or low-glucose (LG-exo) conditions (Fig. 3 a). However, as to the induction of inflammatory response, corrected by particle counts, HG-exo exhibited more NF-kB activation than LG-exo in macrophages, leading to the higher upregulation of TNF-a and IL-1b expressions (Fig. 3 b and c). These findings suggest that the high-glucose condition affects the characteristics of the exosomes rather than their quantity. Establishment of a novel screening strategy for exosome-targeting drugs Next, we designed a high-throughput drug screening strategy using a validated compound library, in order to explore the compounds that could specifically and effectively inhibit exosome-induced inflammation (Fig. 4 ). Before starting the screening, the study was approved by the Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS project headed by Dr. Hirotatsu Kojima, Drug Discovery Initiative, the University of Tokyo; Registration No. 03625), and the validated compound library was provided. Step 1 was to select compounds which showed an inhibitory effect on NF-kB activation. Compounds whose inhibition rate of NF-kB pathway induction by MC-exo was over 40% were chosen (Fig. 5 a). Step 2 was to exclude known anti-inflammatory compounds such as steroids. Through these steps, candidate drugs were narrowed down to 399 compounds. Step 3 was to evaluate dose-dependent effects (Fig. 5 b), as well as the effects on cell viability using a WST assay (Fig. 5 c). Compounds exerting cytotoxicity defined by cell viability lower than 60% were excluded (Fig. 5 c). Step 4 was to evaluate the specificity to exosomal actions. We examined the inhibitory effect on inflammation induced by MC-exo as compared to that by LPS as a non-specific control. The compounds were selected to predominantly inhibit exosome-induced inflammation compared to LPS (an inhibition rate of MC-exo/LPS > 1.5; Fig. 5 d). For the final step, we conducted FCM analysis to evaluate an inhibitory effect on DiO-exo uptake in macrophages. Then, we integrated the results with the NF-kB inhibition rate and created a scatter plot (Fig. 5 e). After completing all stages of the screening process, 25 compounds were shortlisted as final candidates, which have both NF-kB inhibitory and exosome uptake inhibitory effects (Table 1 ). Among them, it is noteworthy that there were four compounds with a category of heat shock protein 90 (HSP90) inhibitors, of which alvespimycin (Alv) was identified to show the highest rate of inhibition of exosome uptake into cells, with an NF-kB suppression rate of nearly 80% (Fig. 5 e and Table 1 ). Therefore, we finally decided to examine the effects of Alv in vivo . The original complete datasets are openly available in repository figshare at https://figshare.com/articles/dataset/Original_raw_data_of_each_step_of_the_drug_screening_/25018745 . Table 1 Table of final shortlisted 25 compounds and their functions. Compounds Functions T-139403 5-HT1B/1D serotonin receptor antagonist T-139571 Peptide inhibitor of aminopeptidases T-140671 T-type Ca2 + channel blocker T-180698 Anti-Serum Amyloid A antibody T-196308 Antimalarial, Heme polymerase inhibitor T-196379 Antifungal, Inhibitor of mitochondrial electron transport T-196595 Antibacterial, Bacterial DNA damage T-196831 Mucolytic, Bronchitis T-207120 Anti-cancer effects T-207163 Inhibitor of P-glycoprotein and MRP1 T-207191 Potent inhibitor of snake venom PLA2 T-208251 p210Bcr/Abl kinase inhibitor T-210562 XPO1 inhibitor T-210608 Tyrosine kinase inhibitor Alvespimycin HSP90 inhibitor T-210751 HSP90 inhibitor T-210633 Potent Topoisomerase II inhibitor T-196893 Antifungal, Antibacterial T-196571 Antibacterial T-210623 HSP90 inhibitor T-207208 Specific inhibitor of eIF2α phosphatase T-196778 Naturally occurring flavonoid T-210582 HSP90 inhibitor T-207192 ATP synthase inhibitor Alvespimycin alleviates diabetic kidney disease in rats The average area of the mesangial region of the 10 glomeruli increased in the STZ group compared to the WT group, which was significantly improved by administration of Alv (Fig. 6 a and b, Supplementary Fig. 1a and b). In addition, Alv treatment significantly ameliorated the exacerbation of albuminuria in diabetic rats (Fig. 6 c). It is noteworthy that expression of macrophage markers (CD68 and CD11b) was markedly suppressed by Alv administration in the glomeruli of diabetic rats together with reduction in inflammatory genes (Fig. 6 d). These results suggested that Alv can mitigate diabetic renal lesions, associated with less macrophage infiltration and subsequent inflammation in glomeruli. Alvespimycin exerts a specific inhibitory effect on exosome-mediated mechanisms We compared the effects of exosome-uptake inhibition among 5 HSP90 inhibitors including Alv and pimitespib (Fig. 7 ), which is a recently launched HSP90 inhibitor in the clinical practice as an anti-cancer drug. 15 Alv showed an obvious inhibitory effect on exosome uptake (Fig. 7 a). Furthermore, the results showed that Alv was the only compound exerting a significant inhibitory effect on exosome uptake among HSP90 inhibitors (Fig. 7 b). These results suggested that the exosome inhibitory effect of Alv may not be a class effect of HSP90 inhibitors, but rather an Alv-specific action. Discussion Although there have been great advances in the treatment of diabetes and its complications including sodium-glucose co-transporter 2 inhibitors, glucagon-like peptide-1 receptor agonists and mineralocorticoid receptor blockers, DKD is still a leading cause of end-stage renal disease in most countries, so that novel therapeutic strategies for DKD are urgently sought. Pathophysiology of DKD is complicated and yet to be fully elucidated; nonetheless, the pathogenic roles of macrophages, both resident and infiltrating, have been reported as the main immune cells to prepare a local inflammatory milieu in DKD. 16 , 17 High-glucose conditions promote expressions of several adhesion molecules and inflammatory cytokines locally, which can activate and recruit macrophages, leading to fibrosis and sclerosis in the kidney. 15 , 16 We previously reported that macrophages could infiltrate in the glomeruli, thereby inducing local inflammation through intraglomerular crosstalk with mesangial cells in DKD. 6 Thus, it has been postulated that such local cell-cell communication should be involved in various pathophysiology in the kidney as well as key pathogenic mechanisms in the glomeruli. 6 , 7 , 16 – 18 Among local mediators proposed so far, exosomes have been thought to be crucial paracrine mediators of cell-cell crosstalk and assumed to be involved in several disease progression. 2 – 5 They contain various kinds of proteins and nucleic acids such as messenger RNAs and microRNAs, 1 , 19 and their roles have been investigated extensively in the oncology field not only as diagnostic biomarkers but also as therapeutic targets in clinical practice. 20 – 22 In the field of kidney disease, it has been suggested that urinary exosomes could serve as diagnostic markers of acute kidney injury as well as glomerular disease such as focal segmental glomerulosclerosis. 23 Moreover, exosomes may modify water, electrolyte and acid-base transport in the renal tubules, thereby modulating kidney pathophysiology. 24 Despite these investigations, unveiling the role of exosomes in the intraglomerular crosstalk and progression of glomerular lesions including DKD has remained unchallenged yet. In this study, we focused on the functional role of exosomes in the kidney, hypothesizing them as crucial mediators between mesangial cells and macrophages in diabetic glomeruli. The results of our study showed that MC-exo were uptaken in macrophages and induced inflammatory responses in vitro and in vivo , where the findings were more augmented under high-glucose than low-glucose conditions. These results suggested that exosomes upon high-glucose conditions can affect macrophages and induce local inflammation in vivo . Based on these findings, we next tried to explore the substances that can interfere such exosome-mediated mechanisms by novel screening strategy. Drug repositioning is a powerful and effective tool for searching novel therapeutic chemical compounds. It could facilitate the discovery of novel mechanisms of action for existing drugs, thus potentially reducing clinical trial steps, the cost and time for drug development. In regard to the nephrology field, there have been a couple of promising preclinical studies so far. Hamano et al. reported the potential efficacy of diphenhydramine against cisplatin-induced kidney injury. 25 As for the treatment of autosomal dominant polycystic kidney disease, several drug candidates are listed, targeting cAMP signaling, somatostatin receptors, mTOR1 signaling, and so forth. 26 , 27 Such candidates and strategies should provide novel therapeutic options in clinical practice in the near future. In this study, we screened a validated compound library of over 3,000 chemical compounds from Drug Discovery Initiative and established a unique multi-step assay to efficiently dig up substances that inhibit exosome-mediated mechanisms. As a result, several candidate agents to potentially exhibit a therapeutic effect on DKD were identified. In the final candidate agents, several HSP90 inhibitors, antibacterials, P-glycoprotein inhibitors, and mitochondrial function modulators were listed. Of note, among them, four HSP90 inhibitors including Alv were shortlisted. HSP90 inhibitors have been investigated as therapeutic drugs for cancers, and pimitespib has been launched in market as the first HSP90 inhibitor for gastrointestinal stromal tumor. 28 Besides, there are some reports showing that HSP90 inhibitors can attenuate atherosclerotic vascular and renal complications, mainly by attenuating stress-induced inflammation. 29 – 31 Nevertheless, their effects against exosome-mediated actions have never been addressed. Our study revealed for the first time that an HSP90 inhibitor Alv could inhibit the uptake of MC-exo into macrophages and relevant inflammatory responses. Administration of Alv in diabetic rats effectively suppressed local inflammation in glomeruli, leading to amelioration of diabetic glomerular lesions (Fig. 6 ). As for the mechanisms, HSP90 contained within exosomes or expressed on the surface of exosomes may mediate membrane-deforming function and promote exosome release. 32 However, our results revealed that the inhibitory effect on exosome uptake of Alv was somewhat confined to Alv and not evident in other HSP90 inhibitors examined, suggesting that such inhibitory effect was not related to HSP90 inhibition, but rather an Alv-specific action. Further investigations are no doubt necessary to explore the precise mechanisms for this inhibition. There are several concerns and limitations to this study. HSP90 is a chaperone protein that plays essential roles in many cellular processes including protein folding, cell cycle control and intracellular signaling pathways. 33 Thus far, HSP90 inhibitors have been investigated as potential anti-cancer therapeutic drugs. The first generation HSP90 inhibitor, geldanamycin, was abandoned for clinical usage due to its hepatotoxicity. 34 Thereafter, several derivatives including Alv have been developed to reduce their toxicity; however, we should carefully check its potential toxicity for future clinical use. Pimitespib, the first agent applied to clinical practice, is alerted to the risks of night blindness, bleeding tendency and diarrhea. 15 Actually, in our experiment, a few rats receiving Alv showed loose stool, suggesting an adverse event common to HSP90 inhibitors. Second, we showed its renoprotective effect for only 6 weeks of treatment, and did not investigate the long-term effect of Alv in DKD. Third, we administered Alv intraperitoneally, but for future clinical use, orally active agents should no doubt be required. Further studies are needed to verify the effects, examine the safety and potency, and optimize drug design, route, and dose of administration in the near future. And finally, all of the animal experiments were conducted using male rodents. Therefore, the results may not be able to be applied to all the genders as they are. In conclusion, by establishing a unique multi-step screening assay, we successfully discovered a novel agent targeting exosomes from existing chemical compounds. Alv identified by drug screening can effectively ameliorate the progression of DKD by suppressing exosome-mediated actions, suggesting that exosome-targeted agents can be a novel therapeutic strategy for DKD. Declarations Acknowledgments This research was supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP23ama121053 (support number 0202). This work was also financially supported in part by research grants from JSPS KAKENHI (Grant Numbers 22K16223 to DF; 19K08729 and 22K08311 to TM; 17K09706 and 20K08611 to MM; 19K08728 and 22K08357 to T. Kuwabara), Strategic Grants from the Center for Metabolic Regulation of Healthy Aging, Kumamoto University Faculty of Life Sciences (Grant Number 09021407 to DF) and Kumamoto University Hospital Young Researcher Activation Project (Grant Number R4-6 and R5-3 to DF). We gratefully acknowledge Dr. Daisuke Nakano (independent researcher, currently working at Laboratory for Pharmacology, Asahi-Kasei Pharma Co., Ltd., Tokyo, Japan) for technical counselling about confocal exosome imaging. This study was conducted fully independent of the project of Asahi-Kasei Pharma Co., Ltd. We also appreciate Ms. Hikari Shibuta, Kazumi Saito and Naoko Hirano for technical assistance, and Ms. Noriko Nakagawa and Miki Horikiri for secretarial assistance. Author Contributions D.F., S.U., H.K., and T. Kuwabara. designed the study. D.F., S.U., R.D. and J.Z. performed the experiments. D.F., T. Kuwabara and M.M. drafted the manuscript. D.F., S.U., T.M., T. Kanki, Y.H., Y.N., Y.K., Y.I., M.A., M.M. and T. Kuwabara. interpreted the results. All authors approved the final version of the manuscript. Data availability statement All data are available in the main text or the Supplementary materials. Original raw data of each step of the drug screening supporting the findings of this study are openly available in repository figshare at https://figshare.com/articles/dataset/Original_raw_data_of_each_step_of_the_drug_screening_/25018745. Additional Information Declaration of competing interests All the authors declared no competing interests. References Tkach M, Thery C. Communication by Extracellular Vesicles: Where We Are and Where We Need to Go. Cell. 164, 1226–32 (2016). Isaac R, Reis FCG, Ying W, Olefsky JM. Exosomes as mediators of intercellular crosstalk in metabolism. Cell Metab. 33, 1744–62 (2021). Zhang W. et al. Extracellular vesicles in diagnosis and therapy of kidney diseases. Am J Physiol Renal Physiol. 311, F844-F51 (2016). Hessvik NP, Llorente A. Current knowledge on exosome biogenesis and release. Cell Mol Life Sci. 75, 193–208 (2018). Medeiros T, Myette RL, Almeida JR, Silva AA, Burger D. Extracellular Vesicles: Cell-Derived Biomarkers of Glomerular and Tubular Injury. Cell Physiol Biochem. 54, 88–109 (2020). Kuwabara T. et al. Exacerbation of diabetic nephropathy by hyperlipidaemia is mediated by Toll-like receptor 4 in mice. Diabetologia. 55, 2256–66 (2012). Hata Y. et al. Ablation of Myeloid Cell MRP8 Ameliorates Nephrotoxic Serum-induced Glomerulonephritis by Affecting Macrophage Characterization through Intraglomerular Crosstalk. Sci Rep. 10, 3056 (2020). Wu XM, Gao YB, Cui FQ, Zhang N. Exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells to promote renal fibrosis. Biol Open. 5, 484–91 (2016). Liu Y. et al. Macrophage-derived exosomes promote activation of NLRP3 inflammasome and autophagy deficiency of mesangial cells in diabetic nephropathy. Life Sci. 330, 121991 (2023). Waldenstrom A, Genneback N, Hellman U, Ronquist G. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells. PLoS One. 7, e34653 (2012). Minami Y. et al. Liver lipophagy ameliorates nonalcoholic steatohepatitis through extracellular lipid secretion. Nat Commun. 14, 4084 (2023). Nishigaya Y. et al. Discovery of Novel Substrate-Competitive Lysine Methyltransferase G9a Inhibitors as Anticancer Agents. J Med Chem. 66, 4059–85 (2023). Nakai W. et al. A novel affinity-based method for the isolation of highly purified extracellular vesicles. Sci Rep. 6, 33935 (2016). Miyanishi M. et al. Identification of Tim4 as a phosphatidylserine receptor. Nature. 450, 435–9 (2007). Kurokawa Y. et al. Pimitespib in patients with advanced gastrointestinal stromal tumor (CHAPTER-GIST-301): a randomized, double-blind, placebo-controlled phase III trial. Ann Oncol. 33, 959–67 (2022). Tang SCW, Yiu WH. Innate immunity in diabetic kidney disease. Nat Rev Nephrol. 16, 206–22 (2020). Li HD et al. Roles and crosstalks of macrophages in diabetic nephropathy. Front Immunol. 13, 1015142 (2022). Fujimoto D. et al. Suppressed ER-associated degradation by intraglomerular cross talk between mesangial cells and podocytes causes podocyte injury in diabetic kidney disease. FASEB J. 34, 15577–90 (2020). Valadi H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 9, 654–9 (2007). Marleau AM, Chen CS, Joyce JA, Tullis RH. Exosome removal as a therapeutic adjuvant in cancer. J Transl Med. 10, 134 (2012). Mohammed HR. et al. Exosomes: From Garbage Bins to Promising Therapeutic Targets. Int J Mol Sci. 18, 538 (2017). Xu Z, Zeng S, Gong Z, Yan Y. Exosome-based immunotherapy: a promising approach for cancer treatment. Mol Cancer. 19, 160 (2020). Erdbrugger U, Le TH. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers? J Am Soc Nephrol. 27, 12–26 (2016). Rudolphi CF, Blijdorp CJ, van Willigenburg H, Salih M, Hoorn EJ. Urinary extracellular vesicles and tubular transport. Nephrol Dial Transplant. 38, 1583–90 (2023). Hamano H. et al. Diphenhydramine may be a preventive medicine against cisplatin-induced kidney toxicity. Kidney Int. 99, 885–99 (2021). Zhou JX, Torres VE. Drug repurposing in autosomal dominant polycystic kidney disease. Kidney Int . 103, 859 – 71 (2023). Mae SI. et al. Human iPSC-derived renal collecting duct organoid model cystogenesis in ADPKD. Cell Rep. 42, 113431 (2023). Hoy SM. Pimitespib: First Approval. Drugs. 82, 1413–8 (2022). Madrigal-Matute J. et al. Heat shock protein 90 inhibitors attenuate inflammatory responses in atherosclerosis. Cardiovasc Res. 86, 330–7 (2010). Lazaro I. et al. Targeting HSP90 Ameliorates Nephropathy and Atherosclerosis Through Suppression of NF-kappaB and STAT Signaling Pathways in Diabetic Mice. Diabetes. 64, 3600–13 (2015). Ding X. et al. Extracellular Hsp90alpha, which participates in vascular inflammation, is a novel serum predictor of atherosclerosis in type 2 diabetes. BMJ Open Diabetes Res Care. 10, e002579 (2022). Lauwers E. et al. Hsp90 Mediates Membrane Deformation and Exosome Release. Mol Cell. 71, 689–702 e9 (2018). Jackson SE. Hsp90: structure and function. Top Curr Chem. 328, 155–240 (2013). Samuni Y. et al. Reactive oxygen species mediate hepatotoxicity induced by the Hsp90 inhibitor geldanamycin and its analogs. Free Radic Biol Med. 48, 1559–63 (2010). Additional Declarations No competing interests reported. Supplementary Files 240301SupplementaryMethodSciRep.docx Cite Share Download PDF Status: Published Journal Publication published 25 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 06 Oct, 2024 Reviews received at journal 05 Oct, 2024 Reviewers agreed at journal 24 Sep, 2024 Reviews received at journal 03 Apr, 2024 Reviewers agreed at journal 25 Mar, 2024 Reviewers agreed at journal 21 Mar, 2024 Reviewers invited by journal 20 Mar, 2024 Editor assigned by journal 20 Mar, 2024 Editor invited by journal 20 Mar, 2024 Submission checks completed at journal 20 Mar, 2024 First submitted to journal 03 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4010567","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":282219773,"identity":"756d4801-7011-4e6e-8085-c0f223deed44","order_by":0,"name":"Daisuke Fujimoto","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Daisuke","middleName":"","lastName":"Fujimoto","suffix":""},{"id":282219774,"identity":"d046f3da-be61-48a6-98c1-ad65087aeaeb","order_by":1,"name":"Shuro Umemoto","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shuro","middleName":"","lastName":"Umemoto","suffix":""},{"id":282219775,"identity":"294b65fa-4fc1-453b-af03-f4caeb7bd34b","order_by":2,"name":"Teruhiko Mizumoto","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Teruhiko","middleName":"","lastName":"Mizumoto","suffix":""},{"id":282219776,"identity":"509f0678-e29d-4177-ac97-d17ae7805979","order_by":3,"name":"Tomoko Kanki","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tomoko","middleName":"","lastName":"Kanki","suffix":""},{"id":282219777,"identity":"ad2c0b0b-da7f-45c4-b32a-611b4b1018d4","order_by":4,"name":"Yusuke Hata","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Hata","suffix":""},{"id":282219778,"identity":"04edfdc8-555f-42a6-96ec-2f76681ce20d","order_by":5,"name":"Yoshihiko Nishiguchi","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yoshihiko","middleName":"","lastName":"Nishiguchi","suffix":""},{"id":282219779,"identity":"56b17619-59ba-4c55-a542-2ee7a5f839fc","order_by":6,"name":"Ryosuke Date","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ryosuke","middleName":"","lastName":"Date","suffix":""},{"id":282219780,"identity":"6714a715-f9e5-449b-8b2f-40407206e5a1","order_by":7,"name":"Jingxuan Zhang","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jingxuan","middleName":"","lastName":"Zhang","suffix":""},{"id":282219781,"identity":"43b2256f-da40-402d-9df2-2553cba63b35","order_by":8,"name":"Yutaka Kakizoe","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yutaka","middleName":"","lastName":"Kakizoe","suffix":""},{"id":282219782,"identity":"1ec66737-0aef-4a13-9640-5c28408aaea3","order_by":9,"name":"Yuichiro Izumi","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yuichiro","middleName":"","lastName":"Izumi","suffix":""},{"id":282219783,"identity":"b8869c77-aac7-498b-bc01-6993011080ce","order_by":10,"name":"Masataka Adachi","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Masataka","middleName":"","lastName":"Adachi","suffix":""},{"id":282219784,"identity":"fa1c5bd0-187b-45ff-839b-f23e8ec1d679","order_by":11,"name":"Hirotatsu Kojima","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Hirotatsu","middleName":"","lastName":"Kojima","suffix":""},{"id":282219785,"identity":"2fe360d6-87c0-42c1-8bd8-af38163613c0","order_by":12,"name":"Masashi Mukoyama","email":"","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Masashi","middleName":"","lastName":"Mukoyama","suffix":""},{"id":282219786,"identity":"9dd4ebea-ca90-4e20-bf54-5f570dd0891d","order_by":13,"name":"Takashige Kuwabara","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYBACCQYGZjCDnwdNhhmnFjaonGQP3AxitRicQdOCE0jOb35s8OOPjbzxmTOmG37m2NTxM/AYMPyoYWA3x6FFmo3NOLG3Lc1w29kes5u929IkJBt4DBh7jjEwWzZg1yLHxmB8gLfhMOO28zxmN3i3HZYwuP/GgIG3gYHZ4AAuLeyfD/75899+cz+P2c2/IC0HgLb8xaNFmo3HOJmH7UDiBt4es9u8UC3M+GyRbMspNpZtS06eceZY2W3ZbWmSMxvYCg7LHJPA6ReJw8c3S775Y2fb35O87ebbbTb8/AzMGx++qbFJxhVi2AHQSRLJBiRpAQE70rWMglEwCkbBMAUA91RT4fpbOJIAAAAASUVORK5CYII=","orcid":"","institution":"Kumamoto University Graduate School of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Takashige","middleName":"","lastName":"Kuwabara","suffix":""}],"badges":[],"createdAt":"2024-03-04 05:00:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4010567/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4010567/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-98894-0","type":"published","date":"2025-04-25T15:57:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53249171,"identity":"3e8fcf7d-37b3-4783-bcc2-434eba51c692","added_by":"auto","created_at":"2024-03-22 12:18:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of mesangial cell-derived exosomes upon macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Evaluation of extracted exosomes from mesangial cells by nanoparticle tracking analysis.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Evaluation of extracted exosomes from mesangial cells by nanoparticle counts. (particle size 30 - 120 nm, n=5)\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) NF-kB activity in macrophages stimulated with mesangial cell-derived exosomes isolated by polymer method; ExoQuick and phosphatidylserine affinity method; MagCapture (n=8).\u003c/p\u003e\n\u003cp\u003eGraph data are mean ± s.e.m. N.S., not significant; EQ, ExoQuick; M-Cap, MagCapture. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus control.\u003c/p\u003e","description":"","filename":"fIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-4010567/v1/16a9cd656c4fde8854971277.png"},{"id":53248799,"identity":"6ce73a1b-b45a-42df-83fa-4705d4768b0e","added_by":"auto","created_at":"2024-03-22 12:10:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":760021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExosomes derived from mesangial cells are endocytosed by macrophages \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Representative images of RAW264.7 macrophage uptaking DiO-labeled exosomes. DiO-exo were extracted from DiO-labeled mesangial cells. Macrophages are stained with red fluorescent dye (CytoTrace Red CMTPX) and culture medium is labeled with blue water-soluble, cell-impermeant polar tracer (Cascade blue hydrazide).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Positivity of DiO-exo in RAW264.7 macrophages evaluated by FCM was reduced by cytochalasin D, an endocytosis inhibitor. N.C., untreated negative control; P.C., DiO-exo treated positive control; CytoD, DiO-exo and cytochalasin D-treated.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Percentages of DiO-positive macrophages / all macrophages in the peripheral blood of non-STZ and STZ-mice were evaluated by FCM at an hour after DiO-exo injection (n=4-6).\u003c/p\u003e\n\u003cp\u003eGraph data are mean ± s.e.m. Mf, macrophage; STZ, streptozotocin. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-4010567/v1/47bbcda7911c3c3d58d3a6ac.png"},{"id":53249172,"identity":"789383b9-f94e-41f9-b671-5c295f7803af","added_by":"auto","created_at":"2024-03-22 12:18:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of exosomes isolated from high-glucose and low-glucose conditioned-mesangial cells upon macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Particle counts of exosomes derived from high-glucose (triangles, HG-exo) and low-glucose (squares, LG-exo) conditioned-mesangial cells (n=6). (\u003cstrong\u003eb\u003c/strong\u003e) NF-kB activation evaluated by SEAP-reporter in macrophages stimulated with HG-exo or LG-exo (n=4-6). (\u003cstrong\u003ec\u003c/strong\u003e) Expressions of TNF-a and IL-1bmRNA by real-time PCR in macrophages stimulated with HG-exo and LG-exo (n=5-6). Graph data are mean ± s.e.m. N.S., not significant; low glucose: 5.6 mM, high glucose: 25 mM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-4010567/v1/4c6677e6ec303412d25affc1.png"},{"id":53248804,"identity":"5d4153a5-55ce-4321-8b85-dd162f3360c8","added_by":"auto","created_at":"2024-03-22 12:10:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":403938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExosome-targeted strategy for exploring novel drug candidates for diabetic kidney disease.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchema of the part of our drug screening assay. Exosomes extracted from the supernatant of cultured mesangial cells were added to macrophages, and reacted with each compound. Flowchart of high-throughput drug screening strategy targeting exosome is shown. From the 3267 compounds in the validated library, candidate drugs are selected through the indicated 5 steps. LPS, lipopolysaccharide.\u003c/p\u003e","description":"","filename":"fIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-4010567/v1/a5b4d5484d89f192b86799a9.png"},{"id":53248807,"identity":"13808a75-ac1b-4af7-83e1-daf81f3cebfd","added_by":"auto","created_at":"2024-03-22 12:10:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResults of each step of the drug screening (Step 1 - 5).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Step 1. Compounds whose inhibition rate of NF-kB pathway induction by MC-exo ≧40% were selected. (Grey bars; 453 compounds)\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Step 3A. Compounds which showed dose-dependent NF-kB inhibition rate were selected. Representative compounds are indicated. Whole data are listed in the Supplementary Table.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Step 3B. Compounds which showed cell viability higher than 60 % after treatment were selected. (Grey bars; 182 compounds)\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Step 4. Compounds which specifically inhibit exosome-induced inflammation compared to nonspecific inflammation such as LPS. (Inhibition ratio [exosome / LPS] \u0026gt;1.5) Grey bars; 44 compounds. LPS, lipopolysaccharide.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Step 5. Final step was to evaluate exosome inhibitory effect and integrated with the results of NF-kB inhibition rate. Open circles: HSP90-inhibitory effect-exerting compounds, arrowhead: Alvespimycin.\u003c/p\u003e","description":"","filename":"fIG5.png","url":"https://assets-eu.researchsquare.com/files/rs-4010567/v1/64b62bb40729f4caf1acc4ba.png"},{"id":53248805,"identity":"e719b4ec-1ac1-421a-9b5e-1d214604baee","added_by":"auto","created_at":"2024-03-22 12:10:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1447130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtective effects of Alvespimycin on renal pathogenesis in STZ-induced diabetic rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Representative images of PAS-stained kidney sections from nondiabetic control with vehicle (non-STZ+Veh), nondiabetic control treated with alvespimycin (non-STZ+Alv), STZ rats treated with vehicle (STZ+Veh) and STZ rats treated with alvespimycin (STZ+Alv) for 6 weeks. Scale bars = 20 mm. (\u003cstrong\u003eb\u003c/strong\u003e) Evaluation of the average area of the mesangial region of the randomly selected 10 glomeruli. (\u003cstrong\u003ec\u003c/strong\u003e) Evaluation of urinary protein level between the four groups. (n=4) (\u003cstrong\u003ed\u003c/strong\u003e) mRNA expression of inflammatory genes (IL-1b, TNFa) and macrophage markers (CD68, CD11b) in the kidney glomeruli. (n=4-7) STZ, streptozotocin; Alv, alvespimycin; ACR, albumin creatinine ratio. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fIG6.png","url":"https://assets-eu.researchsquare.com/files/rs-4010567/v1/e0ac1c4df81c8b1bc971d2e1.png"},{"id":53248803,"identity":"67c060b3-d306-4dca-9124-18737de1dd3d","added_by":"auto","created_at":"2024-03-22 12:10:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":79907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlowcytometric evaluation of the effect of alvespimycin on exosome uptake inhibition.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Alvespimycin inhibits DiO-stained exosome uptake in macrophages. N.C., untreated negative control; P.C., DiO-exo treated positive control; CytoD: DiO-exo and cytochalasin D-treated; Alv, DiO-exo and alvespimycin-treated.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Alvespimycin exerts significantly higher inhibitory effect of exosome-uptake than other HSP90 inhibitors. T-210623, T-210751 and T-210582 are HSP90 inhibitors shortlisted in the final candidates of the screening. (n=4-6) Alv, alvespimycin; CytoD, cytochalasinD; Pim, pimitespib. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fIG7.png","url":"https://assets-eu.researchsquare.com/files/rs-4010567/v1/d59b03c11e7f9958781f46c6.png"},{"id":81569859,"identity":"f0511685-21aa-4717-a910-1f0da8f71b90","added_by":"auto","created_at":"2025-04-28 16:12:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4150277,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4010567/v1/d4ce79b3-d32c-47fc-9577-74b50e75c180.pdf"},{"id":53249364,"identity":"11640cf0-9d49-476f-83de-69fa59c1eda0","added_by":"auto","created_at":"2024-03-22 12:26:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30262,"visible":true,"origin":"","legend":"","description":"","filename":"240301SupplementaryMethodSciRep.docx","url":"https://assets-eu.researchsquare.com/files/rs-4010567/v1/eda3a1d67932cab5e3a6b168.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification and verification of novel therapeutic agents for diabetic kidney disease based on exosome-targeted high-throughput chemical screening","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExtracellular vesicles (EVs) are important mediators of intercellular communication and play a key role in the regulation of pathophysiological processes in various organs and cells.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Particular attention has been paid to intercellular crosstalk by exosomes, whose size is 30\u0026ndash;100 nm.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The presence of exosomes in serum and urine has attracted attention not only in the field of oncology but also in the field of kidney diseases, because exosomes can influence the surrounding environment by propagating the properties of their mother cells to the neighboring cells and environment.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDiabetic kidney disease (DKD) is the most common cause of chronic kidney disease and end-stage renal disease, leading to a high risk of mortality. The pathophysiology of DKD is complicated and multifaceted, and yet to be fully elucidated. Furthermore, treatments to effectively prevent the progression of DKD are still quite limited, so that novel therapeutic strategies are strongly desired. It has been a general consensus that DKD progresses along with mesangial proliferation and matrix accumulation. Macrophages infiltrate into the mesangial areas upon mesangial activation. In particular, we already reported that endogenous ligands associated with inflammation are induced specifically in glomerular infiltrating macrophages, which may have a potential pathogenic role in DKD\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and glomerulonephritis.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e These findings suggest that, in DKD, there should be some yet undefined mechanisms that specifically activate infiltrating macrophages locally in a paracrine manner.\u003c/p\u003e \u003cp\u003eRecently, a potential role of intraglomerular cellular crosstalk mediated by exosomes in DKD has been reported. Wu \u003cem\u003eet al.\u003c/em\u003e reported that vascular endothelial cells exposed to hyperglycemia release exosomes and activate mesangial cells, leading to renal fibrosis.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Liu \u003cem\u003eet al.\u003c/em\u003e showed that macrophage-derived exosomes promote activation of NLRP3 inflammasome and autophagy deficiency of mesangial cells in diabetic nephropathy.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e However, the pathophysiological role of exosomes in cell-cell interaction between mesangial cells and macrophages still remains obscure, especially those from mesangial cells toward macrophages in the glomeruli.\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the role of exosomes in the intercellular crosstalk between mesangial cells and macrophages in DKD. Furthermore, in order to explore a novel therapeutic strategy to intervene the exosomal actions between those cells and to deter the progression of DKD, we screened chemical agents that can specifically act on the behavior or action of exosomes. Exosomes include various kinds of proteins, messenger RNAs and microRNAs,\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and multiple exosomal factors are assumed to be involved in the cell-cell crosstalk, suggesting that targeting a single factor might result in only partial effects. Therefore, we focused on finding chemical compounds that have effects on the primary actions on exosomes derived from mesangial cells, not pursuing particular factors present in the exosome as a target. Drug repositioning is a powerful and effective tool for discovering therapeutic chemical compounds in various kinds of disease.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Herein, we performed high-throughput chemical screening using a validated compound library of existing drugs to identify novel candidate agents, and verified their efficacy in DKD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eMethods of reporter analysis, cell culture, real-time quantitative RT-PCR, \u003cem\u003ein vivo\u003c/em\u003e exosome-uptake study and histological analyses are described in Supplementary Methods.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExosome isolation\u003c/h2\u003e \u003cp\u003e Exosomes were collected from the medium using ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA) and MagCapture Exosome Isolation Kit PS (Wako, Osaka, Japan) in accordance with the manufacturer\u0026rsquo;s instruction. Sprague-Dawley rat glomerular mesangial cells (SDMCs) were maintained in DMEM/Nutrient Mixture F-12 Ham containing 5.6 mmol/L or 25 mmol/L glucose without fetal bovine serum for 24 h before isolation of exosomes. For osmotic adjustment, mannitol (Nacalai Tesque, Kyoto, Japan) was added to the 5.6 mmol/L medium (24.5 mM final concentration). For exosome preparation by the ExoQuick method, the cultured medium of SDMCs was centrifuged at 3,000 x g for 10 min to remove cellular debris. One-fifth of ExoQuick-TC was added to the supernatant and incubated overnight at 4\u0026deg;C. The suspension was centrifuged at 1,500 x g for 30 min. The supernatant was discarded, and the remaining pellet was subjected to another centrifugation at 1,500 x g for 5 min. The pellet was resuspended with PBS and used as exosomes.\u003c/p\u003e \u003cp\u003eTo isolate EVs by the MagCapture Exosome Isolation Kit PS, the cell culture medium was centrifuged at 300 x g for 30 min at 4\u0026deg;C to remove cells and debris. The supernatant was transferred into a new tube, and centrifuged at 1,200 x g for 20 min at 4\u0026deg;C. To remove large EVs, the supernatant was transferred again into a new tube, and centrifuged at 10,000 x g for 30 min at 4\u0026deg;C. Then, the sample was concentrated by using ultrafiltration unit (Vivaspin20, Sartorius, Gottingen, Germany), transferred to a new 1.5 mL microcentrifuge tube, and suspended in Exosome Binding Enhancer at a 1:500 volume. Well mixed samples were transferred into 1.5 mL Reaction Tube containing Exosome-Capture-immobilized beads, and the mixture was rotated overnight at 4\u0026deg;C. The beads were washed three times with 1 mL of washing buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.0005% Tween20, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e), and the bound EVs were eluted with elution buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA).\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e An aliquot of the exosomal preparation was used for exosome counting by NanoSight NS300 system (Malvern Panalytical, Salisbury, UK) followed by normalization to the total cell number. Exosome protein content was quantified using BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDiO labeling of exosomes\u003c/h2\u003e \u003cp\u003eTo prepare labeled exosomes, we conducted fluorescent staining of the supernatant of cultured SDMCs with lipophilic green fluorescent dye 3,3\u0026rsquo;-dioctadecyloxacarbocyanine perchlorate (DiO) and isolated exosomes derived from SDMCs by ExoQuick-TC. We added DiO (Vybrant DiO Cell-labeling Solution; Invitrogen, Carlsbad, CA, USA) to the collected supernatant at a final concentration of 0.5 \u0026micro;L/mL and incubated them for 10 min at 37\u0026deg;C protecting from light. Hereafter, the same procedure was performed as mentioned.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eConfocal microscopy and flow-cytometry\u003c/h2\u003e \u003cp\u003eFor confocal microscopic analysis, RAW 264.7 mouse macrophages were stained with red fluorescent dye (CytoTrace Red CMTPX, AAT Bioquest Inc., Pleasanton, CA, USA) for 24 h, and culture medium is labeled with a blue water-soluble, cell-impermeant polar tracer (Cascade Blue hydrazide, Thermo Fisher Scientific). After incubation, DiO-labeled exosomes (DiO-exo) were added just before microscopic observation, and the uptake of DiO-exo in macrophages was evaluated. All images were scanned with confocal microscopy, FV3000 (Olympus, Tokyo, Japan).\u003c/p\u003e \u003cp\u003eFor flow cytometry (FCM), RAW 264.7 cells were seeded in six-well plates (5 x 10\u003csup\u003e5\u003c/sup\u003e/well) and grown overnight. Prior to treatment with DiO-exo, cells were washed with PBS, and then DiO-exo in 100 \u0026micro;L PBS/well were added and incubated at 37\u0026deg;C for 24 h. Cells stained directly with 1 \u0026micro;L/well DiO (1 \u0026micro;L/mL) served as a positive control, and unstained cells as a negative control. Pre-incubation was performed with 10 \u0026micro;g/mL of an endocytosis inhibitor, cytochalasin D (Cayman Chemical, MI, USA) for 30 min before adding exosomes. DiO-labeled exosome\u0026ndash;treated cells were removed from plates by trypsin, centrifuged, and resuspended in 1 mL of PBS. FCM was performed by SH800S Cell Sorter (Sony Life Science, Tokyo, Japan). FCM data were analyzed with FlowJo V10 program (FlowJo LLC, Ashland, OR, USA).\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eScreening of compounds inhibiting exosome-mediated mechanisms\u003c/h2\u003e \u003cp\u003eCompound screening was conducted using a chemical library containing 3,267 compounds from Drug Discovery Initiative at the University of Tokyo. The protocol was approved prior to the initiation of this study (approved by JP23ama121053, Project No. 0202).\u003c/p\u003e \u003cp\u003eThe screening strategy consisted of 5 steps. Briefly, in Step 1, small molecules (2 mM) in dimethyl sulfoxide (DMSO) solution from library plates were added to cultured THP-1-Dual Cells in 96 well plates, and secreted embryonic alkaline phosphatase (SEAP)\u0026ndash;reporter activities were monitored (see Supplementary Methods). Compounds inhibiting the NF-kB activity induced by mesangial cell\u0026ndash;derived exosomes (MC-exo) over 40% were selected and proceeded to the next step. In Step 2, steroidal compounds with an apparent NF-kB inhibitory action were excluded. Step 3 was composed of two strategies: in Step 3A, compounds exhibiting concentration-dependent NF-kB inhibition were selected, in which final compound concentrations were 0.2 \u0026micro;M, 1 \u0026micro;M, and 5 \u0026micro;M; in Step 3B, compounds showing a high toxicity were excluded, defined as the cell survival rate\u0026thinsp;\u0026lt;\u0026thinsp;60% determined by Cell Count Reagent SF (Nacalai Tesque). The top 160 compounds that passed both Steps 3A and 3B proceeded further. In Step 4, compounds with a higher specificity to exosome-mediated inflammation compared to lipopolysaccharide (LPS)-mediated inflammation were chosen; the criteria were set as the ratio of the inhibition rate against exosomes to that against LPS to be \u0026gt;\u0026thinsp;1.5. Finally, in Step 5, compounds that inhibited exosome uptake in THP-1 cells were selected, using DiO-exo and FCM analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003eAmong the compounds thus selected, we focused on one compound, alvespimycin (Alv, also known as 17-dimethylaminoethylamino-17-demethoxygeldanamycin, 17-DMAG; InvivoGen, San Diego, CA, USA) as a candidate inhibitor of exosome-mediated mechanisms \u003cem\u003ein vivo\u003c/em\u003e. Experiments were conducted using eight-week-old male Sprague-Dawley rats (Japan SLC, Shizuoka, Japan), dividing into the following four groups: 1) wild type (WT)\u0026thinsp;+\u0026thinsp;vehicle, 2) WT\u0026thinsp;+\u0026thinsp;Alv, 3) streptozotocin (STZ)\u0026thinsp;+\u0026thinsp;vehicle, and 4) STZ\u0026thinsp;+\u0026thinsp;Alv. Rats were housed in a room maintained at constant temperature, humidity, and light cycle (12:12-h light-dark) with free access to food and water. They were maintained maximum 2 individuals in one cage. After allowing rats to adapt to their environment for 1 week, STZ group and STZ\u0026thinsp;+\u0026thinsp;Alv group rats were injected with STZ (50 mg/kg body weight in citrate buffer, pH 4.0; Sigma-Aldrich, St. Louis, MO, USA) from the tail vein after 16 h of fasting to develop insulin-dependent diabetes. All rats survived, and their blood glucose level increased over 280 mg/dL a week after STZ administration. Three weeks after STZ administration, Alv was administered intravenously via the tail vein twice a week at a dose of 0.5 mg/kg. After 6 weeks of treatment with vehicle (200 \u0026micro;L saline) or Alv, 16h-fasted rats were anesthetized and sacrificed. Blood, urine and kidney samples were collected. Urine albumin and creatinine levels were measured by using an immunoturbidimetric method (Oriental Yeast, Shiga, Japan). In all animal experiments, we complied with the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEthics\u003c/h2\u003e \u003cp\u003e All animal procedures were conducted in accordance with the guidelines for care and use of laboratory animals approved by Kumamoto University (No. 29\u0026ndash;115).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData deposition\u003c/h2\u003e \u003cp\u003eThe original complete datasets are openly available in repository figshare at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://figshare.com/articles/dataset/Original_raw_data_of_each_step_of_the_drug_screening_/25018745\u003c/span\u003e\u003cspan address=\"https://figshare.com/articles/dataset/Original_raw_data_of_each_step_of_the_drug_screening_/25018745\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eData are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Differences between multiple groups were assessed by ANOVA with Tukey\u0026rsquo;s test using Graphpad Prism (Graphpad Software). Comparison between two groups was carried out by unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRole of the funding source\u003c/h2\u003e \u003cp\u003eFunders had no input on study design, data collection, data analyses, interpretation, or writing of report.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eQuality assessment of mesangial cell\u0026ndash;derived exosomes and evaluation of their activity on NF-kB signaling in macrophages\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt is crucial to assure the quality of extracted exosomes, MC-exo, which are the target of drug screening. Hence, first, quality evaluation of the extracted exosomes was performed. We compared the most widely used precipitation method using ExoQuick with that using MagCapture, which has been reported to obtain relatively purer exosomes. The MagCapture method is an extraction technique based on the binding of phosphatidylserine and Tim4.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Although exosomes by ExoQuick contained larger particles compared to those by MagCapture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), particle counts gated in diameters within 30\u0026ndash;120 nm were equivalent in the both isolation methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, the effects of MC-exo upon NF-kB signaling were evaluated by SEAP-reporter assay in THP-1 cells. Exosomes extracted from mesangial cells induced the NF-kB activity in a dose-dependent manner with both techniques (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). According to these results, we employed the ExoQuick method throughout the following all experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExosomes derived from mesangial cells are endocytosed by macrophages\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to examine whether macrophages uptake prepared exosomes, we evaluated the localization of fluorescence-labeled MC-exo stained with lipophilic green fluorescent dye DiO in tracer-labeled macrophages. Confocal microscopy findings showed that DiO-exo were colocalized with cell-impermeant polar tracer, Cascade blue in macrophages, indicating endocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). These uptakes were obviously suppressed by Cytochalasin D, an endocytosis inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also examined the exosome uptake in animal models with high-glucose condition. Non-diabetic control and STZ-mice were administered with DiO-exo by tail-vein injection and sacrificed at 1 h from injection. FCM analysis of peripheral blood was conducted and the number of DiO-positive monocytes was evaluated. The ratio of DiO-positive macrophages to all macrophages was significantly higher in STZ mice than non-diabetic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Thus, exosomes released from mesangial cells could be uptaken through endocytosis by macrophages both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExosomes derived from high glucose\u0026ndash;conditioned mesangial cells augment inflammation in macrophages\u003c/h2\u003e \u003cp\u003eParticle counts determined by NanoSight showed no significant changes in exosomes prepared from mesangial cells with either high-glucose (HG-exo) or low-glucose (LG-exo) conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). However, as to the induction of inflammatory response, corrected by particle counts, HG-exo exhibited more NF-kB activation than LG-exo in macrophages, leading to the higher upregulation of TNF-a and IL-1b expressions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and c). These findings suggest that the high-glucose condition affects the characteristics of the exosomes rather than their quantity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of a novel screening strategy for exosome-targeting drugs\u003c/h2\u003e \u003cp\u003eNext, we designed a high-throughput drug screening strategy using a validated compound library, in order to explore the compounds that could specifically and effectively inhibit exosome-induced inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Before starting the screening, the study was approved by the Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS project headed by Dr. Hirotatsu Kojima, Drug Discovery Initiative, the University of Tokyo; Registration No. 03625), and the validated compound library was provided.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStep 1 was to select compounds which showed an inhibitory effect on NF-kB activation. Compounds whose inhibition rate of NF-kB pathway induction by MC-exo was over 40% were chosen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Step 2 was to exclude known anti-inflammatory compounds such as steroids. Through these steps, candidate drugs were narrowed down to 399 compounds. Step 3 was to evaluate dose-dependent effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), as well as the effects on cell viability using a WST assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Compounds exerting cytotoxicity defined by cell viability lower than 60% were excluded (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Step 4 was to evaluate the specificity to exosomal actions. We examined the inhibitory effect on inflammation induced by MC-exo as compared to that by LPS as a non-specific control. The compounds were selected to predominantly inhibit exosome-induced inflammation compared to LPS (an inhibition rate of MC-exo/LPS\u0026thinsp;\u0026gt;\u0026thinsp;1.5; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). For the final step, we conducted FCM analysis to evaluate an inhibitory effect on DiO-exo uptake in macrophages. Then, we integrated the results with the NF-kB inhibition rate and created a scatter plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). After completing all stages of the screening process, 25 compounds were shortlisted as final candidates, which have both NF-kB inhibitory and exosome uptake inhibitory effects (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among them, it is noteworthy that there were four compounds with a category of heat shock protein 90 (HSP90) inhibitors, of which alvespimycin (Alv) was identified to show the highest rate of inhibition of exosome uptake into cells, with an NF-kB suppression rate of nearly 80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, we finally decided to examine the effects of Alv \u003cem\u003ein vivo\u003c/em\u003e. The original complete datasets are openly available in repository figshare at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://figshare.com/articles/dataset/Original_raw_data_of_each_step_of_the_drug_screening_/25018745\u003c/span\u003e\u003cspan address=\"https://figshare.com/articles/dataset/Original_raw_data_of_each_step_of_the_drug_screening_/25018745\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTable of final shortlisted 25 compounds and their functions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFunctions\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-139403\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5-HT1B/1D serotonin receptor antagonist\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-139571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeptide inhibitor of aminopeptidases\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-140671\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT-type Ca2\u0026thinsp;+\u0026thinsp;channel blocker\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-180698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnti-Serum Amyloid A antibody\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-196308\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntimalarial, Heme polymerase inhibitor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-196379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntifungal, Inhibitor of mitochondrial electron transport\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-196595\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibacterial, Bacterial DNA damage\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-196831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMucolytic, Bronchitis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-207120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnti-cancer effects\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-207163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInhibitor of P-glycoprotein and MRP1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-207191\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePotent inhibitor of snake venom PLA2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-208251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep210Bcr/Abl kinase inhibitor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-210562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXPO1 inhibitor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-210608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTyrosine kinase inhibitor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlvespimycin\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHSP90 inhibitor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-210751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHSP90 inhibitor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-210633\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePotent Topoisomerase II inhibitor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-196893\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntifungal, Antibacterial\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-196571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibacterial\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-210623\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHSP90 inhibitor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-207208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecific inhibitor of eIF2α phosphatase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-196778\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaturally occurring flavonoid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-210582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHSP90 inhibitor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-207192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATP synthase inhibitor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAlvespimycin alleviates diabetic kidney disease in rats\u003c/h2\u003e \u003cp\u003eThe average area of the mesangial region of the 10 glomeruli increased in the STZ group compared to the WT group, which was significantly improved by administration of Alv (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and b, Supplementary Fig.\u0026nbsp;1a and b). In addition, Alv treatment significantly ameliorated the exacerbation of albuminuria in diabetic rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). It is noteworthy that expression of macrophage markers (CD68 and CD11b) was markedly suppressed by Alv administration in the glomeruli of diabetic rats together with reduction in inflammatory genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). These results suggested that Alv can mitigate diabetic renal lesions, associated with less macrophage infiltration and subsequent inflammation in glomeruli.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAlvespimycin exerts a specific inhibitory effect on exosome-mediated mechanisms\u003c/h2\u003e \u003cp\u003eWe compared the effects of exosome-uptake inhibition among 5 HSP90 inhibitors including Alv and pimitespib (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), which is a recently launched HSP90 inhibitor in the clinical practice as an anti-cancer drug.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Alv showed an obvious inhibitory effect on exosome uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Furthermore, the results showed that Alv was the only compound exerting a significant inhibitory effect on exosome uptake among HSP90 inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). These results suggested that the exosome inhibitory effect of Alv may not be a class effect of HSP90 inhibitors, but rather an Alv-specific action.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough there have been great advances in the treatment of diabetes and its complications including sodium-glucose co-transporter 2 inhibitors, glucagon-like peptide-1 receptor agonists and mineralocorticoid receptor blockers, DKD is still a leading cause of end-stage renal disease in most countries, so that novel therapeutic strategies for DKD are urgently sought. Pathophysiology of DKD is complicated and yet to be fully elucidated; nonetheless, the pathogenic roles of macrophages, both resident and infiltrating, have been reported as the main immune cells to prepare a local inflammatory milieu in DKD.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e High-glucose conditions promote expressions of several adhesion molecules and inflammatory cytokines locally, which can activate and recruit macrophages, leading to fibrosis and sclerosis in the kidney.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e We previously reported that macrophages could infiltrate in the glomeruli, thereby inducing local inflammation through intraglomerular crosstalk with mesangial cells in DKD.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Thus, it has been postulated that such local cell-cell communication should be involved in various pathophysiology in the kidney as well as key pathogenic mechanisms in the glomeruli.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAmong local mediators proposed so far, exosomes have been thought to be crucial paracrine mediators of cell-cell crosstalk and assumed to be involved in several disease progression.\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e They contain various kinds of proteins and nucleic acids such as messenger RNAs and microRNAs,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and their roles have been investigated extensively in the oncology field not only as diagnostic biomarkers but also as therapeutic targets in clinical practice.\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e In the field of kidney disease, it has been suggested that urinary exosomes could serve as diagnostic markers of acute kidney injury as well as glomerular disease such as focal segmental glomerulosclerosis.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Moreover, exosomes may modify water, electrolyte and acid-base transport in the renal tubules, thereby modulating kidney pathophysiology.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Despite these investigations, unveiling the role of exosomes in the intraglomerular crosstalk and progression of glomerular lesions including DKD has remained unchallenged yet.\u003c/p\u003e \u003cp\u003eIn this study, we focused on the functional role of exosomes in the kidney, hypothesizing them as crucial mediators between mesangial cells and macrophages in diabetic glomeruli. The results of our study showed that MC-exo were uptaken in macrophages and induced inflammatory responses \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, where the findings were more augmented under high-glucose than low-glucose conditions. These results suggested that exosomes upon high-glucose conditions can affect macrophages and induce local inflammation \u003cem\u003ein vivo\u003c/em\u003e. Based on these findings, we next tried to explore the substances that can interfere such exosome-mediated mechanisms by novel screening strategy.\u003c/p\u003e \u003cp\u003eDrug repositioning is a powerful and effective tool for searching novel therapeutic chemical compounds. It could facilitate the discovery of novel mechanisms of action for existing drugs, thus potentially reducing clinical trial steps, the cost and time for drug development. In regard to the nephrology field, there have been a couple of promising preclinical studies so far. Hamano \u003cem\u003eet al.\u003c/em\u003e reported the potential efficacy of diphenhydramine against cisplatin-induced kidney injury.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e As for the treatment of autosomal dominant polycystic kidney disease, several drug candidates are listed, targeting cAMP signaling, somatostatin receptors, mTOR1 signaling, and so forth.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Such candidates and strategies should provide novel therapeutic options in clinical practice in the near future.\u003c/p\u003e \u003cp\u003eIn this study, we screened a validated compound library of over 3,000 chemical compounds from Drug Discovery Initiative and established a unique multi-step assay to efficiently dig up substances that inhibit exosome-mediated mechanisms. As a result, several candidate agents to potentially exhibit a therapeutic effect on DKD were identified. In the final candidate agents, several HSP90 inhibitors, antibacterials, P-glycoprotein inhibitors, and mitochondrial function modulators were listed. Of note, among them, four HSP90 inhibitors including Alv were shortlisted. HSP90 inhibitors have been investigated as therapeutic drugs for cancers, and pimitespib has been launched in market as the first HSP90 inhibitor for gastrointestinal stromal tumor.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Besides, there are some reports showing that HSP90 inhibitors can attenuate atherosclerotic vascular and renal complications, mainly by attenuating stress-induced inflammation.\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Nevertheless, their effects against exosome-mediated actions have never been addressed. Our study revealed for the first time that an HSP90 inhibitor Alv could inhibit the uptake of MC-exo into macrophages and relevant inflammatory responses. Administration of Alv in diabetic rats effectively suppressed local inflammation in glomeruli, leading to amelioration of diabetic glomerular lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). As for the mechanisms, HSP90 contained within exosomes or expressed on the surface of exosomes may mediate membrane-deforming function and promote exosome release.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e However, our results revealed that the inhibitory effect on exosome uptake of Alv was somewhat confined to Alv and not evident in other HSP90 inhibitors examined, suggesting that such inhibitory effect was not related to HSP90 inhibition, but rather an Alv-specific action. Further investigations are no doubt necessary to explore the precise mechanisms for this inhibition.\u003c/p\u003e \u003cp\u003eThere are several concerns and limitations to this study. HSP90 is a chaperone protein that plays essential roles in many cellular processes including protein folding, cell cycle control and intracellular signaling pathways.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Thus far, HSP90 inhibitors have been investigated as potential anti-cancer therapeutic drugs. The first generation HSP90 inhibitor, geldanamycin, was abandoned for clinical usage due to its hepatotoxicity.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Thereafter, several derivatives including Alv have been developed to reduce their toxicity; however, we should carefully check its potential toxicity for future clinical use. Pimitespib, the first agent applied to clinical practice, is alerted to the risks of night blindness, bleeding tendency and diarrhea.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Actually, in our experiment, a few rats receiving Alv showed loose stool, suggesting an adverse event common to HSP90 inhibitors. Second, we showed its renoprotective effect for only 6 weeks of treatment, and did not investigate the long-term effect of Alv in DKD. Third, we administered Alv intraperitoneally, but for future clinical use, orally active agents should no doubt be required. Further studies are needed to verify the effects, examine the safety and potency, and optimize drug design, route, and dose of administration in the near future. And finally, all of the animal experiments were conducted using male rodents. Therefore, the results may not be able to be applied to all the genders as they are.\u003c/p\u003e \u003cp\u003eIn conclusion, by establishing a unique multi-step screening assay, we successfully discovered a novel agent targeting exosomes from existing chemical compounds. Alv identified by drug screening can effectively ameliorate the progression of DKD by suppressing exosome-mediated actions, suggesting that exosome-targeted agents can be a novel therapeutic strategy for DKD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP23ama121053 (support number 0202). This work was also financially supported in part by research grants from JSPS KAKENHI (Grant Numbers 22K16223 to DF; 19K08729 and 22K08311 to TM; 17K09706 and 20K08611 to MM; 19K08728 and 22K08357 to T. Kuwabara), Strategic Grants from the Center for Metabolic Regulation of Healthy Aging, Kumamoto University Faculty of Life Sciences (Grant Number 09021407 to DF) and Kumamoto University Hospital Young Researcher Activation Project (Grant Number R4-6 and R5-3 to DF). We gratefully acknowledge Dr. Daisuke Nakano (independent researcher, currently working at Laboratory for Pharmacology, Asahi-Kasei Pharma Co., Ltd., Tokyo, Japan) for technical counselling about confocal exosome imaging. This study was conducted fully independent of the project of Asahi-Kasei Pharma Co., Ltd. We also appreciate Ms. Hikari Shibuta, Kazumi Saito and Naoko Hirano for technical assistance, and Ms. Noriko Nakagawa and Miki Horikiri for secretarial assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.F., S.U., H.K., and T. Kuwabara. designed the study. D.F., S.U., R.D. and J.Z. performed the experiments. D.F., T. Kuwabara and M.M. drafted the manuscript. D.F., S.U., T.M., T. Kanki, Y.H., Y.N., Y.K., Y.I., M.A., M.M. and T. Kuwabara. interpreted the results. All authors approved the final version of the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the Supplementary materials. Original raw data of each step of the drug screening supporting the findings of this study are openly available in repository figshare at https://figshare.com/articles/dataset/Original_raw_data_of_each_step_of_the_drug_screening_/25018745. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeclaration of competing interests\u003c/p\u003e\n\u003cp\u003eAll the authors declared no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTkach M, Thery C. Communication by Extracellular Vesicles: Where We Are and Where We Need to Go. Cell. 164, 1226\u0026ndash;32 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIsaac R, Reis FCG, Ying W, Olefsky JM. Exosomes as mediators of intercellular crosstalk in metabolism. Cell Metab. 33, 1744\u0026ndash;62 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W. et al. Extracellular vesicles in diagnosis and therapy of kidney diseases. Am J Physiol Renal Physiol. 311, F844-F51 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHessvik NP, Llorente A. Current knowledge on exosome biogenesis and release. Cell Mol Life Sci. 75, 193\u0026ndash;208 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedeiros T, Myette RL, Almeida JR, Silva AA, Burger D. Extracellular Vesicles: Cell-Derived Biomarkers of Glomerular and Tubular Injury. Cell Physiol Biochem. 54, 88\u0026ndash;109 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuwabara T. et al. Exacerbation of diabetic nephropathy by hyperlipidaemia is mediated by Toll-like receptor 4 in mice. Diabetologia. 55, 2256\u0026ndash;66 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHata Y. et al. Ablation of Myeloid Cell MRP8 Ameliorates Nephrotoxic Serum-induced Glomerulonephritis by Affecting Macrophage Characterization through Intraglomerular Crosstalk. Sci Rep. 10, 3056 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu XM, Gao YB, Cui FQ, Zhang N. Exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells to promote renal fibrosis. Biol Open. 5, 484\u0026ndash;91 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y. et al. Macrophage-derived exosomes promote activation of NLRP3 inflammasome and autophagy deficiency of mesangial cells in diabetic nephropathy. Life Sci. 330, 121991 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaldenstrom A, Genneback N, Hellman U, Ronquist G. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells. PLoS One. 7, e34653 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinami Y. et al. Liver lipophagy ameliorates nonalcoholic steatohepatitis through extracellular lipid secretion. Nat Commun. 14, 4084 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishigaya Y. et al. Discovery of Novel Substrate-Competitive Lysine Methyltransferase G9a Inhibitors as Anticancer Agents. J Med Chem. 66, 4059\u0026ndash;85 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakai W. et al. A novel affinity-based method for the isolation of highly purified extracellular vesicles. Sci Rep. 6, 33935 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyanishi M. et al. Identification of Tim4 as a phosphatidylserine receptor. Nature. 450, 435\u0026ndash;9 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurokawa Y. et al. Pimitespib in patients with advanced gastrointestinal stromal tumor (CHAPTER-GIST-301): a randomized, double-blind, placebo-controlled phase III trial. Ann Oncol. 33, 959\u0026ndash;67 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang SCW, Yiu WH. Innate immunity in diabetic kidney disease. Nat Rev Nephrol. 16, 206\u0026ndash;22 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi HD et al. Roles and crosstalks of macrophages in diabetic nephropathy. Front Immunol. 13, 1015142 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujimoto D. et al. Suppressed ER-associated degradation by intraglomerular cross talk between mesangial cells and podocytes causes podocyte injury in diabetic kidney disease. FASEB J. 34, 15577\u0026ndash;90 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValadi H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 9, 654\u0026ndash;9 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarleau AM, Chen CS, Joyce JA, Tullis RH. Exosome removal as a therapeutic adjuvant in cancer. J Transl Med. 10, 134 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammed HR. et al. Exosomes: From Garbage Bins to Promising Therapeutic Targets. Int J Mol Sci. 18, 538 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Z, Zeng S, Gong Z, Yan Y. Exosome-based immunotherapy: a promising approach for cancer treatment. Mol Cancer. 19, 160 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErdbrugger U, Le TH. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers? J Am Soc Nephrol. 27, 12\u0026ndash;26 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRudolphi CF, Blijdorp CJ, van Willigenburg H, Salih M, Hoorn EJ. Urinary extracellular vesicles and tubular transport. Nephrol Dial Transplant. 38, 1583\u0026ndash;90 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamano H. et al. Diphenhydramine may be a preventive medicine against cisplatin-induced kidney toxicity. Kidney Int. 99, 885\u0026ndash;99 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou JX, Torres VE. Drug repurposing in autosomal dominant polycystic kidney disease. \u003cem\u003eKidney Int\u003c/em\u003e. 103, 859\u0026thinsp;\u0026ndash;\u0026thinsp;71 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMae SI. et al. Human iPSC-derived renal collecting duct organoid model cystogenesis in ADPKD. Cell Rep. 42, 113431 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoy SM. Pimitespib: First Approval. Drugs. 82, 1413\u0026ndash;8 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadrigal-Matute J. et al. Heat shock protein 90 inhibitors attenuate inflammatory responses in atherosclerosis. Cardiovasc Res. 86, 330\u0026ndash;7 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLazaro I. et al. Targeting HSP90 Ameliorates Nephropathy and Atherosclerosis Through Suppression of NF-kappaB and STAT Signaling Pathways in Diabetic Mice. Diabetes. 64, 3600\u0026ndash;13 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing X. et al. Extracellular Hsp90alpha, which participates in vascular inflammation, is a novel serum predictor of atherosclerosis in type 2 diabetes. BMJ Open Diabetes Res Care. 10, e002579 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLauwers E. et al. Hsp90 Mediates Membrane Deformation and Exosome Release. Mol Cell. 71, 689\u0026ndash;702 e9 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJackson SE. Hsp90: structure and function. Top Curr Chem. 328, 155\u0026ndash;240 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamuni Y. et al. Reactive oxygen species mediate hepatotoxicity induced by the Hsp90 inhibitor geldanamycin and its analogs. Free Radic Biol Med. 48, 1559\u0026ndash;63 (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"exosomes, drug screening, diabetic kidney disease, intraglomerular crosstalk, mesangial cells, macrophages","lastPublishedDoi":"10.21203/rs.3.rs-4010567/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4010567/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExosomes are important mediators of intercellular communication and play key roles in the regulation of pathophysiological processes. In diabetic kidney disease (DKD), it has been reported that macrophages recruited in the mesangial region may play pathogenic roles through inducing local inflammation in glomeruli. We focused on exosome-mediated crosstalk between mesangial cells (MC) and macrophages as a novel therapeutic target for DKD. Exosomes released from MC induced inflammation in macrophages and the effect was enhanced under high-glucose conditions. For discovering novel therapeutic agents which can inhibit such exosome-mediated mechanisms, drug repositioning is considered as aneffective tool. We established a unique screening strategy and screened agents to aim at maximizing their specificity and potency to inhibit exosomal mechanisms, along with minimizing their toxicity. We succeeded in identifying alvespimycin, an HSP90 inhibitor. Treatment of diabetic rats with alvespimycin significantly suppressed mesangial expansion, inflammatory gene activation including macrophage markers, and proteinuria. The inhibitory effect on exosome uptake was specific to alvespimycin compared with other known HSP90 inhibitors. MC-derived exosomes are crucial for inflammation by intercellular crosstalk between MC and macrophages in DKD, and alvespimycin effectively ameliorated the progression of DKD by suppressing exosome-mediated actions, suggesting that exosome-targeted agents can be a novel therapeutic strategy.\u003c/p\u003e","manuscriptTitle":"Identification and verification of novel therapeutic agents for diabetic kidney disease based on exosome-targeted high-throughput chemical screening","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 12:10:38","doi":"10.21203/rs.3.rs-4010567/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-07T01:37:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-05T14:09:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336516199345234574254142015683379212162","date":"2024-09-24T04:31:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-03T12:18:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"665e2dd2-08ca-433e-a3c3-a6942e631962","date":"2024-03-25T10:06:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21816513-d650-4f54-9d0a-84b137f43f37","date":"2024-03-21T10:56:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-20T15:09:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-20T13:03:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-20T07:28:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-20T07:25:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-03-04T04:54:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7a3c3e5-496e-4551-a4cd-64288aa4a5ea","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29716128,"name":"Biological sciences/Drug discovery"},{"id":29716129,"name":"Health sciences/Diseases"},{"id":29716130,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2025-04-28T16:06:05+00:00","versionOfRecord":{"articleIdentity":"rs-4010567","link":"https://doi.org/10.1038/s41598-025-98894-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-04-25 15:57:52","publishedOnDateReadable":"April 25th, 2025"},"versionCreatedAt":"2024-03-22 12:10:38","video":"","vorDoi":"10.1038/s41598-025-98894-0","vorDoiUrl":"https://doi.org/10.1038/s41598-025-98894-0","workflowStages":[]},"version":"v1","identity":"rs-4010567","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4010567","identity":"rs-4010567","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}