Targeting angiopoietin-like protein 3 and Interleukin-1β alleviated liver and kidney injury through attenuation of lipotoxicity and normalization of glycolipid metabolism in db/db mice

Targeting angiopoietin-like protein 3 and Interleukin-1β alleviated liver and kidney injury through attenuation of lipotoxicity and normalization of glycolipid metabolism in db/db mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Targeting angiopoietin-like protein 3 and Interleukin-1β alleviated liver and kidney injury through attenuation of lipotoxicity and normalization of glycolipid metabolism in db/db mice Shuwen Xu, Longfei Wang, Zhanglian Cao, Xiaozhi Hu, Zihan Dou, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7930376/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Apr, 2026 Read the published version in Inflammation Research → Version 1 posted 8 You are reading this latest preprint version Abstract Objective Concomitant liver and kidney injury is a critical pathological feature of metabolic disorders, but current organ-specific therapies often fail to provide cross-protection. Lipotoxicity is a core mechanism linking damage in both organs. Therefore, this study aimed to investigate whether simultaneously targeting ANGPTL3 and IL-1β could attenuate lipotoxicity and thereby ameliorate concomitant liver and kidney injury. Methods A novel bispecific antibody (BsAb) targeting both ANGPTL3 and IL-1β was generated and characterized by SDS-PAGE, SEC-HPLC, thermal stability analysis, SPR and in vitro bioassay. Then, its protective effects were subsequently studied in the db/db mouse model and the underlying mechanisms were revealed by biochemical examinations, histopathological analysis, immunofluorescence (IF), ELISA, RNA-seq, Immunohistochemical (IHC). Results Administration of the BsAb in db/db mice effectively improved liver and kidney function with alleviated liver steatosis and inflammation, as well as reduced kidney glomerular injury. Furthermore, the treatment attenuated lipotoxicity in both organs and normalized the glycolipid metabolism including restored hepatic glycogen reserves and enhanced renal utilization of fatty acids. Conclusion The results demonstrate that the anti-ANGPTL3/IL-1β BsAb alleviates concomitant liver and kidney injury in db/db mice by attenuating lipotoxicity and normalizing glycolipid metabolism, which highlights a promising therapeutic approach for addressing multi-organ damage in metabolic disorders. ANGPTL3 IL-1β Bispecific antibody Liver and kidney injury Lipotoxicity Glucolipid metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Concomitant injury to liver and kidneys is a critical and increasingly recognized complication of metabolic disorders. The liver and kidneys, as vital metabolic organs, exhibit profound crosstalk that significantly amplifies injury in metabolic disorders and invariably leads to the compromise of one organ when the other is injured 1 . Metabolic dysfunction-associated steatotic liver disease (MASLD) and diabetic kidney disease (DKD) are common hepatic and renal manifestations of metabolic disorders 2 , 3 . A significant correlation between MASLD and DKD among individuals with diabetes was reported in a meta-analysis 4 . This intricate liver-kidney axis creates a vicious cycle, explaining why current therapeutic strategies targeting only one organ often exhibit limited cross-protection for the other and highlighting a critical need for strategies targeting their shared pathophysiology. Dyslipidemia is a central pathophysiological nexus linking liver and kidney injury in metabolic disorders. Abnormal lipid metabolism drives the ectopic lipid deposition within parenchymal cells, thereby promoting lipotoxicity in both liver and kidneys 5 , 6 . Angiopoietin-like protein 3 (ANGPTL3) is a member of angiopoietin-like proteins subfamily, which plays a critical role in the metabolism of triglyceride (TG)-rich lipoproteins through inhibition of lipoprotein lipase (LPL) 7 , 8 . Accumulating evidence indicates that elevated ANGPTL3 levels may promote lipotoxicity by dysregulating the lipid microenvironment, potentially contributing to the pathogenesis of organ damage 9 – 11 . In models of diabetes mellitus or nonalcoholic fatty liver disease, targeting ANGTPL3 demonstrated potential therapeutic benefits, including reduced serum TG and improved renal or hepatic function 12 – 14 . Therefore, ANGPTL3 may be a promising target for alleviating lipotoxicity-induced liver and kidney injury in metabolic disorders. Beyond lipid dysregulation, chronic inflammation constitutes a pivotal parallel mechanism driving organ injury in metabolic disorders 15 , 16 . Pro-inflammatory pathways are robustly activated in both DKD and MASLD, exacerbating the cellular stress and organ damage 17 , 18 . Notably, interleukin-1β (IL-1β), a pivotal cytokine in inflammatory response initiation, may play a central role in this process. Recent clinical studies have showed that elevated IL-1β levels positively correlated with MASLD severity and predicted the extent of MASLD improvement regardless of the intervention 19 . Moreover, Mendelian randomization analysis revealed that genetically predicted elevation in IL-1 receptor antagonist (IL-1ra) levels were inversely associated with the rate of estimated glomerular filtration rate decline, and clinical intervention with anakinra ameliorated renal function among patients with diabetic 20 , 21 . Critically IL-1β activation may interact with lipid-induced pathways, potentially forming a deleterious feedback loop that perpetuates injury 22 . This suggests that targeting IL-1β, alongside lipid regulators like ANGPTL3, could offer a more comprehensive therapeutic strategy for attenuating lipotoxicity by concurrently addressing inflammation and lipid deposition, thereby disrupting the intertwined mechanism of liver and kidney injury. In the present study, elevated levels of ANGPTL3 and IL-1β in plasma of db/db mice were found to positively correlated with hepatic and renal dysfunction, and then a novel anti-ANGPTL3/IL-1β BsAb was generated and its protective efficacy for liver and kidneys was determined. Our results demonstrated that the BsAb against ANGPTL3 and IL-1β improved hepatic and renal function and ameliorated pathological injury of liver and kidneys in db/db mice through attenuation of lipotoxicity and normalization of glycolipid metabolism, suggesting a promising approach to treat organ manifestation in metabolic disorders. 2. Materials and methods 2.1 Preparation of anti-ANGPTL3/IL-1β Bispecific antibody The genes encoding FD03 were the sequence of anti-ANGPTL3 nanobody C44 [13] fused with the IgG2-Fc domain. The genes encoding XOMA052 included the sequence of heavy and light chain of anti-IL-1β antibody XOMA052 23 . The genes encoding anti-ANGPTL3/IL-1β BsAb contained the sequences for the XOMA052 heavy and light chains, each having the C44 fused to its C-terminus through a flexible (Gly4Ser3) linker. The genes described above were constructed using Hieff Cloned Universal II One Step Cloning Kit (Yeason, Shanghai, China) and Gel Extraction Kit (SparkJade Biotechnology, Shandong, China). After the restriction digestion with XbaI and BamHI, the resulting fragments were inserted into the pTT5 plasmid. Subsequently, all plasmids were purified using plasmid extraction kit (CWBIO, Jiangsu, China). These purified plasmids were then transfected into Expi293F cells employing the polyethylenimine-based transfection reagent PEI MAX (Lablead, Beijing, China). Following a 7-day period after transfection, the cell culture supernatants were collected and purified using Protein A affinity chromatography. 2.2 SDS-PAGE analysis Electrophoretic separation was performed and the gels were subjected to staining with an ultrafast protein staining solution (MeilunBio, Dalian, China) and destained utilizing ultrapure water to enable clear visualization of protein bands. 2.3 SEC-HPLC analysis The monomeric purity of the protein samples was assessed using SEC-HPLC on an Agilent 1260 Infinity II SFC System equipped with a TOSOH TSKgel G3000WXL column. The mobile phase consisted of PBS buffer. Detection was carried out via ultraviolet absorbance at 280 nm. Quantification of monomer content was based on the peak area normalization approach. 2.4 Thermal stability analysis The Tm and Tagg were evaluated using the UNCLE platform. SYPRO Orange dye (Thermo Fisher Scientific, Oregon, USA), which binds to hydrophobic amino acid residues, was employed to monitor protein stability through fluorescence emission at 473 nm. All protein samples were prepared in PBS prior to analysis. The resulting fluorescence data were fitted to a sigmoidal curve using the integrated Uncle Analysis software. 2.5 SPR analysis The binding affinities of antibodies toward recombinant mouse ANGPTL3 (S17–T455) with a His-tag (ABclonal, Wuhan, China), mouse IL-1β (V118–S269)-His (ABclonal), human ANGPTL3-His (ABclonal), and human IL-1β-His (ABclonal) were evaluated using SPR technology on a BIAcore T200 instrument (GE Healthcare, NJ, USA). The KD were derived from the kinetic parameters using the BIAcore Evaluation Software. 2.6 Cell culture The MRC-5 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were maintained in MRC-5 complete growth medium under conditions of 37°C and 5% CO2. Expi293F cells (iCareab Biotechnology Co. Ltd., Suzhou, China) were grown in a blended medium of OPM-293 CD05 (OPM Biosciences, Shanghai, China) and SMM 293-Tii (Sino Biological Inc., Beijing, China), under conditions of 5% CO2 at 120rpm shaking speed and 37°C. 2.7 In vitro bioassay for determining ANGPTL3 inhibitory effects The inhibitory effects of FD03 and BsAb on ANGPTL3-induced suppression of LPL activity were evaluated using an in vitro cell-free assay system, as referenced 13 . Specifically, FD03, BsAb or a control antibody (Human-IgG2) were first mixed with murine ANGPTL3(S17-T206)-Fc at a concentration of 10µM, then incubated with 50nM LPL. Following the addition of 10µM Lipase Substrate, the reaction proceeded for 15 min at 25°C. Fluorescence intensity was measured at ex/em 529/600 nm. 2.8 In vitro bioassay for determining IL-1β inhibitory effects The ability of XOMA052 and BsAb to inhibit IL-1β-mediated stimulation of IL-6 secretion was assessed by MRC-5 assay 23 . After an overnight incubation of MRC-5 cells, supernatants were replaced with MRC-5 completed medium containing XOMA052 or the BsAb. Recombinant human IL-1β (ABclonal) was added to 82 pM. As a negative control, recombinant human IL-1β treated cells were incubated with 10 − 7 M control human-IgG2 antibody. Following 24h incubation, supernatants were assayed for IL-6 by ELISA kit (Multisciences, Hangzhou, China). 2.9 Animal studies Four-week-old db/db mice (C57BLKS/J-Leprem2Cd479/Gpt) along with db/m were obtained from GemPharmatech Co., Ltd. (Nanjing, China). All mice were housed in SPF environment with a controlled 12-hour light/12-hour dark cycle and room temperature maintained at 20–24°C. The experimental were reviewed and approved by the Animal Ethics Committee of the School of Pharmacy, and all procedures complied with the relevant institutional guidelines and regulations. Animals were randomly divided into six groups: db/m group (treated with PBS, n = 8), db/db group (treated with PBS, n = 8), FD03 group (treated with 10mg/kg of FD03, n = 8), XOMA052 group (treated with 5mg/kg of XOMA052, n = 8), BsAb group (treated with 7 mg/kg of BsAb) and combo group (treated with 10mg/kg of FD03 and 5mg/kg of XOMA052, n = 8). All treatments were injected intraperitoneally once weekly over an 8-week period. 2.10 Biochemical examinations The hepatic or renal index was liver or kidney weight (mg) / body weight (g). Blood glucose, TG, total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine were detected by relevant assay kits (Jiancheng Bioengineering, Nanjing, China). Insulin (Enzyme-linked Biotechnology, Shanghai, China), microalbumin (Enzyme-linked Biotechnology), IL-1β (Jianglai biology, Shanghai, China), ANGPTL3 (Jianglai biology), TNF-α (ABclonal) and IL-6 (ABclonal) were measured with ELISA kits. TG and FA were detected by the enzymatic assay kit (Applygen, Beijing, China). Glycogen was detected by glycogen assay kit (Enzyme-linked Biotechnology). Homeostatic model assessment (HOMA-IR) and Triglycerides-Glucose (TyG) index for insulin resistance were calculated as follows: HOMA-IR = Blood glucose (mmol/L) ×Insulin (uU/mL)/22.5; TyG index = ln [Triglycerides (mg/dL) × Blood glucose (mg/dL) / 2]. 2.11 Insulin tolerance test (ITT) During the ITT, all mice received injection of insulin (1 U/kg; MedChemExpress, NJ, USA). Blood samples were subsequently collected from the tail vein at specified time intervals to measure blood glucose. 2.12 Histopathology Morphological alterations were examined using H&E staining, PAS staining, and Masson staining. Additionally, frozen sections of hepatic and renal tissue were subjected to Oil Red O staining for visualization of lipid droplets. All stained sections were imaged using a SlideView VS200 slide scanner (Olympus, Tokyo, Japan). The positively stained regions in PAS, Masson, and Oil Red O preparations were quantified and analyzed with ImageJ Pro software (NIH, Bethesda, USA) 24 , 25 . 2.13 Transcritomic analysis After the assessment of concentration, purity and integrity of total RNA, cDNA libraries were generated and subjected to sequencing on the Illumina NovaSeq 6000 platform. The resulting reads were aligned against the Ensembl mouse reference genome (assembly mm10/GRCm38). 2.14 Immunohistochemical analysis The expressions of insulin receptor (INSR) or insulin receptor substrate 2 (IRS-2) were determined by the immunohistochemical staining with INSR Rabbit pAb (Bioss, Beijing, China) or IRS-2 Rabbit mAb (Zen-bioscience, Chengdu, China). Micrograph acquisition was performed using SlideView VS200 system (Olympus). The proportion of tissue area exhibiting positive immunohistochemical staining for INSR or IRS-2 was quantitatively assessed using ImageJ Pro software (NIH). 2.15 Immunofluorescence Podocyte quantification in renal tissue sections was performed using immunofluorescence staining. Podocyte nuclei were identified based on the co-localization of Wilms' tumor 1 (WT1) and DAPI, visualized as a magenta fluorescence signal. The density of podocytes, expressed as the number per 1000 µm² of glomerular area, was quantified using ImageJ Pro software (NIH). 2.16 Statistical analysis The data results were processed with Graph Pad Prism 10 (San Diego) and displayed as mean ± SEM. Statistically significant differences were analyzed by one-way analysis of variance (ANOVA) or unpaired Student’s t-test. * P < 0.05 or ** P < 0.01were considered to be significant statistical difference. 3. Results 3.1 Concomitant Liver and Kidney Injury Correlated with Elevated ANGPTL3 and IL-1 β Levels in db/db Mice with Metabolic Disorders Concomitant liver and kidney injury was assessed in db/db mice using db/m mice as the control group (Fig. 1A). By 8 weeks of age, markedly higher body weight, blood glucose levels, and serum TG levels in db/db mice were observed (Fig. 1B). Histopathological damage in both liver and kidney tissues was also observed, evidenced by abnormal cellular morphology in H&E-stained sections (Fig. 1C). Impaired hepatic and renal function were further confirmed by significantly elevated ALT and albumin-to-creatinine ratio (ACR) levels, respectively (Fig. 1D). Critically, serum ELISA demonstrated notable elevations in both ANGPTL3 (8.716 ± 1.160 v.s. 3.641 ± 0.3946 ng/mL) and IL-1β (55.86 ± 5.699 v.s. 23.70 ± 1.903 pg/mL) levels in db/db mice (Fig. 1E and G). Furthermore, we found significant positive associations between serum ANGPTL3 levels and both ALT (R 2 =0.6502, P =0.0156) and ACR (R 2 =0.8376, P =0.0014) (Fig. 1F), as well as between serum IL-1β levels and both ALT (R 2 =0.6775, P =0.0121) and ACR (R 2 =0.7282, P =0.0070) (Fig. 1H). Collectively, these findings showed that db/db mice exhibited severe metabolic dysfunction accompanied by significant liver and kidney injury, and it is found that elevated circulating ANGPTL3 and IL-1β are potential targets strongly correlating with the severity of the damage. 3.2 Anti-ANGPTL3/IL-1 β BsAb was Generated and Characterized To simultaneously target ANGPTL3 and IL-1β, we developed a BsAb by fusing the anti-ANGPTL3 nanobody C44 13 to the C-terminus of both the heavy and light chains of the anti-IL-1β antibody XOMA052 23 , and C44-IgG2-Fc (FD03) and XOMA052 were employed as controls (Fig. 2A). SDS-PAGE and SEC-HPLC results determined that all samples corresponded to the expected size and were highly pure (Fig. 2B and C, Supplementary Material Fig. S1A and S1B). Compared with the FD03 or XOMA052 antibody, the BsAb exhibited appropriate thermal stability and aggregation stability, with its Tm1, Tagg266 and Tagg473 being 67.90 °C, 68.34 °C and 70.81 °C, respectively (Fig. 2D, Supplementary Material Fig. S1C and S1D). The kinetic parameter as well as the bioactivity were assessed in vitro afterwards. SPR analysis showed the binding affinity of the BsAb was 0.1780nM against mouse ANGPTL3 and 6.229nM against mouse IL-1β, respectively (Fig. 2E, Supplementary Material Fig. S1E). In the bioactivity assay, similarly to the control antibody FD03 or XOMA052, the BsAb effectively rescued LPL activity or inhibited IL-6 secretion (Fig. 2F and G). Taken together, these results indicate that anti-ANGPTL3/ IL-1β BsAb was effectively produced and maintained the intended bioactivities of both the anti-ANGPTL3 and anti-IL-1β components. 3.3 Anti-ANGPTL3/IL-1 β BsAb ameliorated renal and hepatic dysfunction as well as metabolic disturbance in db/db mice To determine the therapeutic effects of anti-ANGPTL3/IL-1β BsAb on liver and kidney injury in metabolic disorders, by 8 weeks of age, db/db mice were administered PBS, FD03, XOMA052, the BsAb or a combination of FD03 and XOMA052, while db/m mice received PBS alone for 8 consecutive weeks (Fig. 3A). By 16 weeks of age, significantly higher ALT and AST levels were found in db/db mice; ACR, BUN and serum creatinine levels were also increased compared with db/m control mice, demonstrating impaired hepatic and renal function. However, after the BsAb exposure, db/db mice exhibited significant reductions of ALT (80.44 ± 9.001 v.s. 210.9 ± 43.60 U/L), AST (62.95 ± 7.072 v.s. 138.2 ± 21.45 U/L) and liver index (Fig. 3B-D). Meanwhile, db/db mice also exhibited marked decrease of ACR (740.9 ± 74.84 v.s. 1282 ± 101.7 μg/mg) , BUN (9.375 ± 0.7392 v.s. 13.49 ± 1.010 mmol/L), serum creatinine (16.98 ± 2.712 v.s. 27.84 ± 3.604 μmol/L) and kidney index (Fig. 3E-H). Interestingly, we found that the hyperglycemia and hyperlipidemia in db/db mice were ameliorated, evidenced by markedly lower level of blood glucose (27.13 ± 1.134 v.s. 33.13 ± 1.815 mmol/L), serum TG (1.017 ± 0.07885 v.s. 1.415 ± 0.07341 mmol/L), serum TC and serum LDL-C (Fig. 3F and G, Supplementary Material Fig. S2A and S2B), with reduced food intake, water intake and urinary volume (Supplementary Material Fig. S3A-C). Thus, these results implied that the simultaneously targeting ANGPTL3 and IL-1β via the anti-ANGPTL3/IL-1β BsAb alleviated the hepatic and renal dysfunction, metabolic disturbance, and diabetic symptoms in db/db mice. 3.4 Anti-ANGPTL3/IL-1β BsAb attenuated hepatic steatosis, collagen deposition and inflammation in db /db mice To further corroborate these functional improvements at histological level, we next examined liver and kidney tissues by pathological staining and relevant analysis. First, the therapeutic efficacy of the BsAb in liver injury in db/db mice was examined. Consistent with impaired hepatic function, histopathological examinations using H&E and Masson staining revealed marked hepatic steatosis and enhanced collagen deposition compared to db/m control mice. After the BsAb treatment, ameliorated liver steatosis architecture and attenuated collagen deposition revealed by Masson-positive staining area were observed compared to db/db control mice (Fig. 4A and B). Meanwhile, quantitative analysis confirmed significantly reduction of Masson-positive area in the liver of BsAb-treated db/db mice (Fig. 4C). In addition, assessment of inflammatory cytokine expression in hepatic tissue revealed that BsAb treatment significantly reduced the levels of TNF-α and IL-6 in db/db mice (Fig. 4D and E). These findings indicated that the ANGPTL3/IL-1β BsAb treatment effectively alleviated important pathological features of liver injury, with attenuated hepatic steatosis, collagen deposition and inflammation in db/db mice. 3.5 Anti-ANGPTL3/IL-1β BsAb reduced glomerular matrix expansion, collagen deposition and podocytes loss in db/db mice Then, the therapeutic effects of the BsAb on kidney injury in db/db mice was evaluated by histological analysis. PAS staining demonstrated reduced glomerular matrix accumulation in the kidneys of BsAb-treated db/db mice, quantified by significantly decreased PAS-positive area (Fig. 5A). Masson staining further indicated reduced collagen deposition in the kidneys of db/db mice treated with the BsAb, quantified by a substantial reduction of Masson-positive area (Fig. 5B). Critically, immunofluorescence for Wilms’ tumor 1 (WT1) showed increased podocyte numbers per glomerulus, as revealed by the significantly higher WT1-positive cell counts per 1000 μm 2 glomerular area in the BsAb-treated mice compared to db/db control mice (Fig.5C). These results implied that the therapeutic efficacy of the anti-ANGPTL3/IL-1β BsAb was similarly evident in renal pathology of db/db mice, with reduced glomerular matrix expansion, collagen deposition and recovered podocyte numbers. 3.6 Anti-ANGPTL3/IL-1β BsAb attenuated lipotoxicity and normalized lipid metabolism in liver and kidneys of db/db mice. Having established the amelioration of hepatic and renal injury in db/db mice following the BsAb treatment, we next investigated the underlying metabolic alterations. Firstly, the lipid accumulation in liver and kidney was determined. Oil Red O staining and enzymatic triglyceride assay results exhibited prominent reduction of lipid droplets, TG and fatty acid (FA) content in liver of db/db mice treated with the BsAb (Fig. 6A-C). Parallel reduction of renal lipid accumulation was demonstrated, with diminished lipid droplets, hepatic TG and FA content in kidneys after the BsAb treatment (Fig. 6E-G). Next, the reprogramming of lipid metabolism in both liver and kidneys by the BsAb treatment was elucidated by transcriptomic profiling. In liver of the BsAb-treated db/db mice, mRNA levels of genes that mediated fatty acid synthesis (Fasn, Acara, Acss2), triglyceride synthesis (Agpat2, Agpat3, Dgat1), fatty acid transport (Cd36, Fabp1) and fatty acid β-oxidation (Cpt1a, Crat, Crot, Ppara) showed decreased expression (Fig. 6D). Conversely, renal analysis revealed distinct regulatory patterns: downregulation of lipid catabolic pathways was observed (Fig. 6H), with elevated mRNA levels of gene mediated lipolysis (Pnpla2, Lipe), fatty acid transport (Slc27a2, Cd36 Fabp3, Fabp4) and fatty acid β-oxidation (Cpt1a, Crat, Crot, Acox1). These results demonstrated that the anti-ANGPTL3/IL-1β BsAb attenuated lipotoxicity through reducing ectopic lipid accumulation and toxic lipid species, and reprogramed lipid metabolism in both liver and kidneys of db/db mice. 3.7 Anti-ANGPTL3/IL-1 β BsAb normalized glucose metabolism in liver and kidneys and improved insulin sensitivity in db/db mice Given the observed reduction of lipid accumulation and regulation of lipid metabolism, we further sought to determine whether the BsAb treatment could also ameliorate glucose metabolism. Transcriptomic profiling in liver demonstrated that gene Gys2 was up-regulated, which is related to glycogenesis, while Pygl was down-regulated, which is related to glycogenolysis. Additionally, genes related to gluconeogenesis in liver were down-regulated, including Pck1, Fbp1, G6pc and Gpd2 (Fig. 7A). Moreover, genes in relation with glycolysis in kidneys were down-regulated, including Pfkp, Pklr, Pkm, Hk1, Gpd1 and Gpd2 (Fig. 7B). Enhanced liver glycogen storage was confirmed afterwards, evidenced by intensified PAS staining and elevated quantitative glycogen content in the BsAb-treated mice (Fig. C and D). In addition to gene expression, insulin sensitivity is also a critical determinant of glucose metabolism 26-31 . Therefore, we evaluated the level of insulin resistance in db/db mice following the BsAb treatment. Although no significant change was observed in serum insulin levels (Supplementary Material Fig. S4A), BsAb treatment led to marked improvements in insulin resistance, as evidenced by a substantial reduction in the HOMA-IR (17.84 ± 3.535 vs. 56.02 ± 8.405) and a decreased TyG index compared with db/db control animals (Fig. 7E, Supplementary Material Fig. S4B), which are both surrogate markers to assess insulin resistance 32 . Furthermore, insulin tolerance test (ITT) was conducted and its quantitative analysis indicated that the BsAb treatment improved the efficiency of insulin in db/db mice (Fig. 7F). Insulin binds to the insulin receptor (INSR) and activated its function of phosphorylating substrates such as insulin receptor substrate-2 (IRS-2), which acts as a critical adaptor protein to activate downstream signaling pathways 33, 34 . The results of INSR and IRS-2 immunochemistry staining and quantitative analysis demonstrated a significant upregulation of INSR and IRS-2 expression in liver, indicating that the insulin signaling pathway was recovered in db/db mice after the BsAb treatment (Fig. 7G, Supplementary Material Fig. S5A). These findings suggested that the anti-ANGPTL3/IL-1β BsAb restored glucose homeostasis in liver and kidneys via transcriptional reprogramming, enhanced hepatic glycogen storage, and rescued insulin sensitivity in db/db mice. 4. Discussion The concurrent impairment of liver and kidney function has emerged as an important feature of the pathophysiology of metabolic diseases. This multifactorial crosstalk, often referred to as the “liver-kidney axis,” is driven by complex interactions, including lipid metabolism disorders and chronic low-grade inflammation. These common pathways create a vicious cycle of mutual organ dysfunction, where the deterioration of one organ often accelerates the decline of the other, thereby leading to the overall disease burden and elevating cardiovascular mortality 35 . Therefore, the clinical co-occurrence of MASLD and DKD in diabetic patients is not only a coincidence but also reflects a profound and interrelated pathological relationship. Although there is a large amount of literature on the pathogenesis of liver or kidney diseases, the complexity of organ crosstalk and its underlying mechanisms remain to be explored 36 , 37 . Clarifying the mechanisms that support this organ crosstalk remains a key objective in the field, offering significant hope for developing novel therapeutic strategies to address co-occurrence of liver and kidney complications. Lipotoxicity play a crucial role in the pathophysiology of liver and kidney injury in metabolic disorders 38 – 41 . Dysregulation of the lipid metabolism could lead to accumulation of harmful lipids, causing lipotoxicity. ANGPTL3 has been shown to modulate lipid metabolism by inhibiting LPL and targeting ANGPTL3 could lower TG and LDL-C levels, contributing to the treatment of hyperlipidemia, MASLD or atherosclerosis 7 , 14 , 42 . Moreover, lipotoxicity is intimately associated with chronic inflammation. Our preliminary study found that IL-1β was upregulated in db/db mice and its level was positively correlated with renal and hepatic dysfunction. Therefore, based on the hypothesis that simultaneously reducing lipid accumulation and inflammation would more effectively attenuate lipotoxicity and alleviate concomitant liver and kidney injury, we developed a BsAb targeting both ANGPTL3 and IL-1β. Treatment with this BsAb markedly ameliorated injury in liver and kidneys of db/db mice. Furthermore, the BsAb administration reduced lipotoxicity and regulated glycolipid metabolism in these organs, accompanied by lowered blood glucose levels and improved insulin sensitivity. The major concern we issued is that why reducing lipid accumulation and inflammation by the anti-ANGPTL3 and IL-1β BsAb had better therapeutic effects on ameliorating lipotoxicity compared with anti-ANGPTL3 treatment alone. During the process, it was found that targeting ANGPTL3 alone had limited therapeutic effect on hepatic collagen and inflammation. It was also reported that statin, which could lower cholesterol by suppression of the HMG-CoA, may lead to inflammation and fibrosis and fuel diabetic nephropathy progression in diabetic mice [5]. Plenty of studies have shown that the chronic inflammation induced by excessive lipid deposition, particularly within liver and kidneys, in turn, contributed to the insulin resistance and exacerbated the disturbances in glycolipid metabolism 43 – 49 . Taken together, these findings reminded that reducing lipid accumulation and inflammation cannot be separated to alleviate lipotoxicity, which explained why co-targeting lipid regulator ANGPTL3 and pro-inflammatory cytokine IL-1β better ameliorated lipotoxicity compared with targeting ANGPTL3 alone. Another important point that we focused on is that why the BsAb administration could normalize glycolipid metabolism in liver and kidneys. It was well documented that the lipotoxicity in the liver could impair hepatocyte functions, particularly in response to insulin signaling pathways, while reduction of lipotoxicity in liver could lead to amelioration of liver function and insulin sensitivity 50 – 53 . As the most important organ in glucose metabolism, the restoration of insulin sensitivity and hepatocyte functions in liver could trigger enhanced glycogenesis, reduced glycogenolysis and gluconeogenesis, which resulted in the increase of glycogen storage and decrease of blood glucose in db/db mice 54 , 55 . Besides, kidneys also play a critical part in systemic metabolism and are severely affected in diabetes, as DKD is one of the most significant complications. It was reported that kidneys originally experience elevated glucose utilization under hyperglycemic conditions in diabetes because of the exposure to abundant arterial blood flow, which may exacerbate the damage of kidneys 56 . Therefore, the management of blood glucose promoted the shift of energy substrate preference in kidneys from glucose to FA, with a significant upregulation in fatty acid oxidation (FAO) and downregulation in glycolysis. The restoration of FAO in kidneys was described to be beneficial for the treatment of fibrosis in DKD 41 . The elevated level of FAO also resulted in the reduction of lipid accumulation and contributed to attenuating lipotoxicity in kidneys, which was highly involved in the kidney diseases 57 – 59 . In summary, co-targeting ANGPTL3 and IL-1β by the BsAb rebuilt the glycolipid metabolism balance in both liver and kidneys through attenuating lipotoxicity, and thereby ameliorated liver and kidney injury in db/db mice. The significance of this work goes beyond the observed improvements in liver and kidney function and histology. This is the first study to explore metabolic crosstalk between the liver and kidneys in this situation, revealing that the reduction of lipotoxicity and improvement of glycolipid balance endow the two organs with mutual protection. By demonstrating that co-targeting ANGPTL3 and IL-1β can reduce lipotoxicity and normalize glycolipid metabolism in liver and kidneys, we have provided compelling evidence that disrupting the lipid disorder and inflammatory cycle is crucial for restoring metabolic balance throughout the body. Furthermore, this study suggested shifting the treatment approach from organ-specific to systemic interventions, which implies that concomitant liver and kidney injury is a systemic disease requiring a combined strategy. 5. Conclusions This study demonstrated that simultaneously blocking ANGPTL3 and IL-1β could attenuate lipotoxicity, regulate glycolipid metabolism, and thereby ameliorate liver and kidney injury in db/db mice. Moreover, we also explored the metabolic crosstalk between liver and kidneys and found that attenuating lipotoxicity and improving the balance of glycolipid metabolism could provide cross-protection for the liver and kidneys. These findings offered new ideas for the treatment of concomitant organ injury associated with metabolic disorders in the future. Declarations Author Contribution Shuwen Xu designed the experiment, conducted the experiment, written the manuscript and reviewed the manuscript. Longfei Wang, Zhanglian Cao and Xiaozhi Hu designed the experiment. Zihan Dou, Yuanzhen Zhang, Xianhan Jiang, Tao Wu and Zhuojin Li conducted the experiment and processed the data. Yanyang Nan, An Zhu, Yu Bai, Ziqian Zou, Xuyao Zhang and Xian Zeng reviewed the manuscript. Haidong He, Dianwen Ju, Shaofei Wang and Jiajun Fan designed the experiment, reviewed the manuscipt and provided funding support. Acknowledgements This work was supported by grants from National Key Research and Development Program of China (2023YFC3404000), National Key Research and Development Program of China (2023YFC3503400) and the National Nature Science Foundation of China (82371781) References Yang M, Luo S, Yang J, Chen W, He L, Liu D, et al. Crosstalk between the liver and kidney in diabetic nephropathy. Eur J Pharmacol. 2022;931:175219. Ali MK, Pearson-Stuttard J, Selvin E, Gregg EW. Interpreting global trends in type 2 diabetes complications and mortality. Diabetologia. 2022;65:3–13. Alfieri CM, Molinari P, Cinque F, Vettoretti S, Cespiati A, Bignamini D, et al. What Not to Overlook in the Management of Patients with Type 2 Diabetes Mellitus: The Nephrological and Hepatological Perspectives. Int J Mol Sci. 2024;25:7728. Mantovani A, Petracca G, Beatrice G, Csermely A, Lonardo A, Schattenberg JM, et al. 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18:18:47","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":247229,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/7271c0ada665013db124daed.png"},{"id":96209939,"identity":"47a7bba1-a5a9-4bb1-81e8-7af46dbc1746","added_by":"auto","created_at":"2025-11-18 18:18:47","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146730,"visible":true,"origin":"","legend":"","description":"","filename":"e686ac587f0544708814f5f6e3e595931structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/e4ec987e1df4d5d526139502.xml"},{"id":96251882,"identity":"6ec26388-0b6c-4525-a588-a7363274b1f4","added_by":"auto","created_at":"2025-11-19 07:40:09","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161065,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/184911bd02a79c3c2d20e590.html"},{"id":96253195,"identity":"389fbb6a-91a6-4c66-a336-47e3d8cfd47e","added_by":"auto","created_at":"2025-11-19 07:42:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":99276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of metabolic dysfunction, liver and kidney injury, and elevated ANGPTL3/IL-1β in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003edb/db\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mice \u003c/strong\u003e(A) Schematic diagram of the 8-week study in \u003cem\u003edb/db\u003c/em\u003e mice and \u003cem\u003edb/m\u003c/em\u003e mice. (B) Quantitative analysis of body weight, blood glucose, serum TG (n=8). (C) H\u0026amp;E images of liver and kidney samples. (D) Quantitative analysis of ALT and ACR (n=8). (E) Quantitative analysis of serum ANGPTL3 levels (n=8). (F) Pearson correlation between serum ANGPTL3 and both ALT and ACR (n=8). (G) Quantitative analysis of serum IL-1β levels (n=8). (H) Pearson correlation between serum IL-1βand both ALT and ACR (n=8). Data in (B, D, E and G) are means ± SEM; * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/3803cb00b2002ab1ea5a9c79.png"},{"id":96209917,"identity":"886bb925-1d7f-4622-8b0e-2d43d7494594","added_by":"auto","created_at":"2025-11-18 18:18:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":103050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-ANGPTL3/IL-1β BsAb was designed, expressed and purified; its properties and biological activity were then characterized\u003c/strong\u003e(A) Structural representation of FD03, XOMA052 and anti-ANGPTL3/IL-1β BsAb. (B) Non-reduced and reduced SDS-PAGE analysis of antibodies. (C) SEC-HPLC analysis of the BsAb. (RT: retention time). (D) Thermal stability analysis of the BsAb. (Tm: melting temperature; Tagg: aggregation temperature) (E) Affinity analysis of the BsAb for mouse ANGPTL3 and mouse IL-1βmeasured by SPR. (F) The neutralization by FD03 and the BsAb on ANGPTL3-induced the inhibition of LPL activity (n=3). (G) The inhibition by XOMA052 and the BsAb on IL-6 release induced by IL-1βin MRC-5 cells (n=3). Data in (F) are means ± SEM; * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/4323b57dbcf2bf72f730ccc6.png"},{"id":96209918,"identity":"89d3c7e3-7561-4bae-ab0f-a17d1f5eed35","added_by":"auto","created_at":"2025-11-18 18:18:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe therapeutic effects of the BsAb in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003edb/db\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mice \u003c/strong\u003e(A) Schematic illustration for experimental design of anti-ANGPTL3/IL-1β BsAb in\u003cem\u003e db/db\u003c/em\u003e mice. (B-J) Quantitative analysis of ALT (B), AST(C), liver index (D), ACR (E), BUN (F), serum creatinine (G), kidney index (H), blood glucose(I) and serum TG (J) (n=8). Data in (B-J) are means ± SEM; * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/c6bc41a7fffc2c8f2ffc987e.png"},{"id":96209921,"identity":"70c41431-49f5-4897-ad87-b850f7e9ba16","added_by":"auto","created_at":"2025-11-18 18:18:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":350140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHepatic structural and inflammatory ameliorations following the BsAb treatment in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003edb/db\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mice \u003c/strong\u003e(A) H\u0026amp;E staining images of liver samples. (B) Masson staining images of liver samples. (C) Quantitative analysis of Masson-positive staining (n=8). (D-E) Quantitative analysis of TNF-α(D) and IL-6 (E) in liver tissue (n=6). Data in (C-E) are means ± SEM; * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/362345236ce2f4692b14ef6e.png"},{"id":96209933,"identity":"1a1a2d4d-8a89-4f96-bd53-dde0611b55ab","added_by":"auto","created_at":"2025-11-18 18:18:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":292513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRenal histopathological improvement following the BsAb treatment in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003edb/db\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mice \u003c/strong\u003e(A) PAS staining images of kidney samples and quantitative analysis of glomerular mesangial index (n=8). (B) Masson staining images of kidney samples and quantitative analysis of Masson-positive staining (n=8). (C) Immunofluorescence images of WT1 of kidney samples and quantitative analysis of WT1-positive cells in glomerulus (n=8). Data in (A-C) are means ± SEM; * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/bb7febf4ac4520d60af2f88e.png"},{"id":96209937,"identity":"d31fdf6f-839a-494d-ab7a-6d066a5a66ee","added_by":"auto","created_at":"2025-11-18 18:18:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":303228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAttenuated lipotoxicity and normalized lipid metabolism in liver and kidneys of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003edb/db\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mice after the BsAb exposure\u003c/strong\u003e (A) Oil red O staining images of liver samples and quantitative analysis of oil red O-positive staining (n=8). (B-C) Quantitative analysis of TG (B) and FA (C) in liver tissues (n=6). (D) Heatmap of differentially expression genes related to FA synthesis, TG synthesis, FA transport and FA β-oxidation in liver of mice in \u003cem\u003edb/db\u003c/em\u003e group and BsAb group (n=3). (E) Oil red O staining images of kidney samples and quantitative analysis of oil red O-positive staining (n=8). (F-G) Quantitative analysis of TG (F) and FA (G) in kidney tissues (n=6). (H) Heatmap of differentially expression genes related to lipolysis, FA transport and FA β-oxidation in kidneys of mice in \u003cem\u003edb/db\u003c/em\u003e group and BsAb group (n=3). Data in (B, C, F, G) are means ± SEM; * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/59414ee65fd97eb86da2a6c0.png"},{"id":96250694,"identity":"dc44daf2-7d07-4d0e-8bb4-8044c69d27c8","added_by":"auto","created_at":"2025-11-19 07:38:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":247229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNormalized glucose metabolism in liver and kidneys of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003edb/db\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mice and rescued insulin sensitivity after the BsAb exposure \u003c/strong\u003e(A) Heatmap of differentially expression genes related to glycogenesis, glycogenolysis and gluconeogenesis in liver of mice in \u003cem\u003edb/db\u003c/em\u003e group and BsAb group (n=3) (B) Heatmap of differentially expression genes related to glycolysis in kidneys of mice in \u003cem\u003edb/db\u003c/em\u003e group and BsAb group (n=3). (C) PAS staining images of liver samples. (D) Quantitative analysis of glycogen content in liver tissues (n=6). (E) Quantitative analysis of HOMA-IR (n=8). (F) Insulin tolerance test (ITT) and the quantitative analysis of AUC of ITT (n=8). (G) Immunohistochemical staining images of IRS-2 of liver samples and quantitative analysis of IRS-2-positive staining (n=8). Data in (C-F) are means ± SEM; * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/34c2c4df40e790900c328626.png"},{"id":107352671,"identity":"e4d04c95-3590-43f0-9b56-9be03fdcec00","added_by":"auto","created_at":"2026-04-20 16:14:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2609107,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/38b7a74a-9bf1-48fa-a03b-0716f68be83e.pdf"},{"id":96209930,"identity":"427a2ab7-cdaf-405a-9261-067168911362","added_by":"auto","created_at":"2025-11-18 18:18:47","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":12358431,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7930376/v1/3a3661d6e5f421002737420d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting angiopoietin-like protein 3 and Interleukin-1β alleviated liver and kidney injury through attenuation of lipotoxicity and normalization of glycolipid metabolism in db/db mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eConcomitant injury to liver and kidneys is a critical and increasingly recognized complication of metabolic disorders. The liver and kidneys, as vital metabolic organs, exhibit profound crosstalk that significantly amplifies injury in metabolic disorders and invariably leads to the compromise of one organ when the other is injured\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Metabolic dysfunction-associated steatotic liver disease (MASLD) and diabetic kidney disease (DKD) are common hepatic and renal manifestations of metabolic disorders\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. A significant correlation between MASLD and DKD among individuals with diabetes was reported in a meta-analysis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This intricate liver-kidney axis creates a vicious cycle, explaining why current therapeutic strategies targeting only one organ often exhibit limited cross-protection for the other and highlighting a critical need for strategies targeting their shared pathophysiology.\u003c/p\u003e\u003cp\u003eDyslipidemia is a central pathophysiological nexus linking liver and kidney injury in metabolic disorders. Abnormal lipid metabolism drives the ectopic lipid deposition within parenchymal cells, thereby promoting lipotoxicity in both liver and kidneys\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Angiopoietin-like protein 3 (ANGPTL3) is a member of angiopoietin-like proteins subfamily, which plays a critical role in the metabolism of triglyceride (TG)-rich lipoproteins through inhibition of lipoprotein lipase (LPL)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Accumulating evidence indicates that elevated ANGPTL3 levels may promote lipotoxicity by dysregulating the lipid microenvironment, potentially contributing to the pathogenesis of organ damage\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In models of diabetes mellitus or nonalcoholic fatty liver disease, targeting ANGTPL3 demonstrated potential therapeutic benefits, including reduced serum TG and improved renal or hepatic function\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, ANGPTL3 may be a promising target for alleviating lipotoxicity-induced liver and kidney injury in metabolic disorders.\u003c/p\u003e\u003cp\u003eBeyond lipid dysregulation, chronic inflammation constitutes a pivotal parallel mechanism driving organ injury in metabolic disorders\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Pro-inflammatory pathways are robustly activated in both DKD and MASLD, exacerbating the cellular stress and organ damage\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Notably, interleukin-1β (IL-1β), a pivotal cytokine in inflammatory response initiation, may play a central role in this process. Recent clinical studies have showed that elevated IL-1β levels positively correlated with MASLD severity and predicted the extent of MASLD improvement regardless of the intervention\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Moreover, Mendelian randomization analysis revealed that genetically predicted elevation in IL-1 receptor antagonist (IL-1ra) levels were inversely associated with the rate of estimated glomerular filtration rate decline, and clinical intervention with anakinra ameliorated renal function among patients with diabetic\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Critically IL-1β activation may interact with lipid-induced pathways, potentially forming a deleterious feedback loop that perpetuates injury\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This suggests that targeting IL-1β, alongside lipid regulators like ANGPTL3, could offer a more comprehensive therapeutic strategy for attenuating lipotoxicity by concurrently addressing inflammation and lipid deposition, thereby disrupting the intertwined mechanism of liver and kidney injury.\u003c/p\u003e\u003cp\u003eIn the present study, elevated levels of ANGPTL3 and IL-1β in plasma of \u003cem\u003edb/db\u003c/em\u003e mice were found to positively correlated with hepatic and renal dysfunction, and then a novel anti-ANGPTL3/IL-1β BsAb was generated and its protective efficacy for liver and kidneys was determined. Our results demonstrated that the BsAb against ANGPTL3 and IL-1β improved hepatic and renal function and ameliorated pathological injury of liver and kidneys in \u003cem\u003edb/db\u003c/em\u003e mice through attenuation of lipotoxicity and normalization of glycolipid metabolism, suggesting a promising approach to treat organ manifestation in metabolic disorders.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Preparation of anti-ANGPTL3/IL-1β Bispecific antibody\u003c/h2\u003e\u003cp\u003eThe genes encoding FD03 were the sequence of anti-ANGPTL3 nanobody C44 [13] fused with the IgG2-Fc domain. The genes encoding XOMA052 included the sequence of heavy and light chain of anti-IL-1β antibody XOMA052\u003csup\u003e23\u003c/sup\u003e. The genes encoding anti-ANGPTL3/IL-1β BsAb contained the sequences for the XOMA052 heavy and light chains, each having the C44 fused to its C-terminus through a flexible (Gly4Ser3) linker.\u003c/p\u003e\u003cp\u003eThe genes described above were constructed using Hieff Cloned Universal II One Step Cloning Kit (Yeason, Shanghai, China) and Gel Extraction Kit (SparkJade Biotechnology, Shandong, China). After the restriction digestion with XbaI and BamHI, the resulting fragments were inserted into the pTT5 plasmid. Subsequently, all plasmids were purified using plasmid extraction kit (CWBIO, Jiangsu, China). These purified plasmids were then transfected into Expi293F cells employing the polyethylenimine-based transfection reagent PEI MAX (Lablead, Beijing, China). Following a 7-day period after transfection, the cell culture supernatants were collected and purified using Protein A affinity chromatography.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 SDS-PAGE analysis\u003c/h2\u003e\u003cp\u003eElectrophoretic separation was performed and the gels were subjected to staining with an ultrafast protein staining solution (MeilunBio, Dalian, China) and destained utilizing ultrapure water to enable clear visualization of protein bands.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 SEC-HPLC analysis\u003c/h2\u003e\u003cp\u003eThe monomeric purity of the protein samples was assessed using SEC-HPLC on an Agilent 1260 Infinity II SFC System equipped with a TOSOH TSKgel G3000WXL column. The mobile phase consisted of PBS buffer. Detection was carried out via ultraviolet absorbance at 280 nm. Quantification of monomer content was based on the peak area normalization approach.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Thermal stability analysis\u003c/h2\u003e\u003cp\u003eThe Tm and Tagg were evaluated using the UNCLE platform. SYPRO Orange dye (Thermo Fisher Scientific, Oregon, USA), which binds to hydrophobic amino acid residues, was employed to monitor protein stability through fluorescence emission at 473 nm. All protein samples were prepared in PBS prior to analysis. The resulting fluorescence data were fitted to a sigmoidal curve using the integrated Uncle Analysis software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 SPR analysis\u003c/h2\u003e\u003cp\u003eThe binding affinities of antibodies toward recombinant mouse ANGPTL3 (S17\u0026ndash;T455) with a His-tag (ABclonal, Wuhan, China), mouse IL-1β (V118\u0026ndash;S269)-His (ABclonal), human ANGPTL3-His (ABclonal), and human IL-1β-His (ABclonal) were evaluated using SPR technology on a BIAcore T200 instrument (GE Healthcare, NJ, USA). The KD were derived from the kinetic parameters using the BIAcore Evaluation Software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Cell culture\u003c/h2\u003e\u003cp\u003eThe MRC-5 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were maintained in MRC-5 complete growth medium under conditions of 37\u0026deg;C and 5% CO2.\u003c/p\u003e\u003cp\u003eExpi293F cells (iCareab Biotechnology Co. Ltd., Suzhou, China) were grown in a blended medium of OPM-293 CD05 (OPM Biosciences, Shanghai, China) and SMM 293-Tii (Sino Biological Inc., Beijing, China), under conditions of 5% CO2 at 120rpm shaking speed and 37\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 In vitro bioassay for determining ANGPTL3 inhibitory effects\u003c/h2\u003e\u003cp\u003eThe inhibitory effects of FD03 and BsAb on ANGPTL3-induced suppression of LPL activity were evaluated using an \u003cem\u003ein vitro\u003c/em\u003e cell-free assay system, as referenced\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Specifically, FD03, BsAb or a control antibody (Human-IgG2) were first mixed with murine ANGPTL3(S17-T206)-Fc at a concentration of 10\u0026micro;M, then incubated with 50nM LPL. Following the addition of 10\u0026micro;M Lipase Substrate, the reaction proceeded for 15 min at 25\u0026deg;C. Fluorescence intensity was measured at ex/em 529/600 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 In vitro bioassay for determining IL-1β inhibitory effects\u003c/h2\u003e\u003cp\u003eThe ability of XOMA052 and BsAb to inhibit IL-1β-mediated stimulation of IL-6 secretion was assessed by MRC-5 assay\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. After an overnight incubation of MRC-5 cells, supernatants were replaced with MRC-5 completed medium containing XOMA052 or the BsAb. Recombinant human IL-1β (ABclonal) was added to 82 pM. As a negative control, recombinant human IL-1β treated cells were incubated with 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003eM control human-IgG2 antibody. Following 24h incubation, supernatants were assayed for IL-6 by ELISA kit (Multisciences, Hangzhou, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Animal studies\u003c/h2\u003e\u003cp\u003eFour-week-old \u003cem\u003edb/db\u003c/em\u003e mice (C57BLKS/J-Leprem2Cd479/Gpt) along with \u003cem\u003edb/m\u003c/em\u003e were obtained from GemPharmatech Co., Ltd. (Nanjing, China). All mice were housed in SPF environment with a controlled 12-hour light/12-hour dark cycle and room temperature maintained at 20\u0026ndash;24\u0026deg;C. The experimental were reviewed and approved by the Animal Ethics Committee of the School of Pharmacy, and all procedures complied with the relevant institutional guidelines and regulations.\u003c/p\u003e\u003cp\u003eAnimals were randomly divided into six groups: \u003cem\u003edb/m\u003c/em\u003e group (treated with PBS, n\u0026thinsp;=\u0026thinsp;8), \u003cem\u003edb/db\u003c/em\u003e group (treated with PBS, n\u0026thinsp;=\u0026thinsp;8), FD03 group (treated with 10mg/kg of FD03, n\u0026thinsp;=\u0026thinsp;8), XOMA052 group (treated with 5mg/kg of XOMA052, n\u0026thinsp;=\u0026thinsp;8), BsAb group (treated with 7 mg/kg of BsAb) and combo group (treated with 10mg/kg of FD03 and 5mg/kg of XOMA052, n\u0026thinsp;=\u0026thinsp;8). All treatments were injected intraperitoneally once weekly over an 8-week period.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Biochemical examinations\u003c/h2\u003e\u003cp\u003eThe hepatic or renal index was liver or kidney weight (mg) / body weight (g). Blood glucose, TG, total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine were detected by relevant assay kits (Jiancheng Bioengineering, Nanjing, China). Insulin (Enzyme-linked Biotechnology, Shanghai, China), microalbumin (Enzyme-linked Biotechnology), IL-1β (Jianglai biology, Shanghai, China), ANGPTL3 (Jianglai biology), TNF-α (ABclonal) and IL-6 (ABclonal) were measured with ELISA kits. TG and FA were detected by the enzymatic assay kit (Applygen, Beijing, China). Glycogen was detected by glycogen assay kit (Enzyme-linked Biotechnology). Homeostatic model assessment (HOMA-IR) and Triglycerides-Glucose (TyG) index for insulin resistance were calculated as follows: HOMA-IR\u0026thinsp;=\u0026thinsp;Blood glucose (mmol/L) \u0026times;Insulin (uU/mL)/22.5; TyG index\u0026thinsp;=\u0026thinsp;ln [Triglycerides (mg/dL) \u0026times; Blood glucose (mg/dL) / 2].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Insulin tolerance test (ITT)\u003c/h2\u003e\u003cp\u003eDuring the ITT, all mice received injection of insulin (1 U/kg; MedChemExpress, NJ, USA). Blood samples were subsequently collected from the tail vein at specified time intervals to measure blood glucose.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Histopathology\u003c/h2\u003e\u003cp\u003eMorphological alterations were examined using H\u0026amp;E staining, PAS staining, and Masson staining. Additionally, frozen sections of hepatic and renal tissue were subjected to Oil Red O staining for visualization of lipid droplets. All stained sections were imaged using a SlideView VS200 slide scanner (Olympus, Tokyo, Japan). The positively stained regions in PAS, Masson, and Oil Red O preparations were quantified and analyzed with ImageJ Pro software (NIH, Bethesda, USA)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Transcritomic analysis\u003c/h2\u003e\u003cp\u003eAfter the assessment of concentration, purity and integrity of total RNA, cDNA libraries were generated and subjected to sequencing on the Illumina NovaSeq 6000 platform. The resulting reads were aligned against the Ensembl mouse reference genome (assembly mm10/GRCm38).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Immunohistochemical analysis\u003c/h2\u003e\u003cp\u003eThe expressions of insulin receptor (INSR) or insulin receptor substrate 2 (IRS-2) were determined by the immunohistochemical staining with INSR Rabbit pAb (Bioss, Beijing, China) or IRS-2 Rabbit mAb (Zen-bioscience, Chengdu, China). Micrograph acquisition was performed using SlideView VS200 system (Olympus). The proportion of tissue area exhibiting positive immunohistochemical staining for INSR or IRS-2 was quantitatively assessed using ImageJ Pro software (NIH).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Immunofluorescence\u003c/h2\u003e\u003cp\u003ePodocyte quantification in renal tissue sections was performed using immunofluorescence staining. Podocyte nuclei were identified based on the co-localization of Wilms' tumor 1 (WT1) and DAPI, visualized as a magenta fluorescence signal. The density of podocytes, expressed as the number per 1000 \u0026micro;m\u0026sup2; of glomerular area, was quantified using ImageJ Pro software (NIH).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.16 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe data results were processed with Graph Pad Prism 10 (San Diego) and displayed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistically significant differences were analyzed by one-way analysis of variance (ANOVA) or unpaired Student\u0026rsquo;s t-test. *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01were considered to be significant statistical difference.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Concomitant Liver and Kidney Injury Correlated with Elevated ANGPTL3 and IL-1\u003c/strong\u003e\u003cstrong\u003e\u0026beta;\u003c/strong\u003e\u003cstrong\u003e Levels in db/db Mice with Metabolic Disorders\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eConcomitant liver and kidney injury was assessed in \u003cem\u003edb/db\u003c/em\u003e mice using \u003cem\u003edb/m\u003c/em\u003e mice as the control group (Fig. 1A). By 8 weeks of age, markedly higher body weight, blood glucose levels, and serum TG levels in \u003cem\u003edb/db\u003c/em\u003e mice were observed (Fig. 1B). Histopathological damage in both liver and kidney tissues was also observed, evidenced by abnormal cellular morphology in H\u0026amp;E-stained sections (Fig. 1C). Impaired hepatic and renal function were further confirmed by significantly elevated ALT and albumin-to-creatinine ratio (ACR) levels, respectively (Fig. 1D). Critically, serum ELISA demonstrated notable elevations in both ANGPTL3 (8.716 \u0026plusmn; 1.160 v.s. 3.641 \u0026plusmn; 0.3946 ng/mL) and IL-1\u0026beta; (55.86 \u0026plusmn; 5.699 v.s. 23.70 \u0026plusmn; 1.903 pg/mL) levels in \u003cem\u003edb/db\u003c/em\u003e mice (Fig. 1E and G). Furthermore, we found significant positive associations between serum ANGPTL3 levels and both ALT (R\u003csup\u003e2\u003c/sup\u003e=0.6502, \u003cem\u003eP\u003c/em\u003e=0.0156) and ACR (R\u003csup\u003e2\u003c/sup\u003e=0.8376, \u003cem\u003eP\u003c/em\u003e=0.0014) (Fig. 1F), as well as between serum IL-1\u0026beta; levels and both ALT (R\u003csup\u003e2\u003c/sup\u003e=0.6775, \u003cem\u003eP\u003c/em\u003e=0.0121) and ACR (R\u003csup\u003e2\u003c/sup\u003e=0.7282, \u003cem\u003eP\u003c/em\u003e=0.0070) (Fig. 1H). \u003c/p\u003e\n\u003cp\u003eCollectively, these findings showed that \u003cem\u003edb/db\u003c/em\u003e mice exhibited severe metabolic dysfunction accompanied by significant liver and kidney injury, and it is found that elevated circulating ANGPTL3 and IL-1\u0026beta; are potential targets strongly correlating with the severity of the damage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Anti-ANGPTL3/IL-1\u003c/strong\u003e\u003cstrong\u003e\u0026beta;\u003c/strong\u003e\u003cstrong\u003e BsAb was Generated and Characterized\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo simultaneously target ANGPTL3 and IL-1\u0026beta;, we developed a BsAb by fusing the anti-ANGPTL3 nanobody C44\u003csup\u003e13\u003c/sup\u003e to the C-terminus of both the heavy and light chains of the anti-IL-1\u0026beta; antibody XOMA052\u003csup\u003e23\u003c/sup\u003e, and C44-IgG2-Fc (FD03) and XOMA052 were employed as controls (Fig. 2A). SDS-PAGE and SEC-HPLC results determined that all samples corresponded to the expected size and were highly pure (Fig. 2B and C, Supplementary Material Fig. S1A and S1B). Compared with the FD03 or XOMA052 antibody, the BsAb exhibited appropriate thermal stability and aggregation stability, with its Tm1, Tagg266 and Tagg473 being 67.90 \u0026deg;C, 68.34 \u0026deg;C and 70.81 \u0026deg;C, respectively (Fig. 2D, Supplementary Material Fig. S1C and S1D). \u003c/p\u003e\n\u003cp\u003eThe kinetic parameter as well as the bioactivity were assessed \u003cem\u003ein vitro\u003c/em\u003e afterwards. SPR analysis showed the binding affinity of the BsAb was 0.1780nM against mouse ANGPTL3 and 6.229nM against mouse IL-1\u0026beta;, respectively (Fig. 2E, Supplementary Material Fig. S1E). In the bioactivity assay, similarly to the control antibody FD03 or XOMA052, the BsAb effectively rescued LPL activity or inhibited IL-6 secretion (Fig. 2F and G). \u003c/p\u003e\n\u003cp\u003eTaken together, these results indicate that anti-ANGPTL3/ IL-1\u0026beta; BsAb was effectively produced and maintained the intended bioactivities of both the anti-ANGPTL3 and anti-IL-1\u0026beta; components.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Anti-ANGPTL3/IL-1\u003c/strong\u003e\u003cstrong\u003e\u0026beta;\u003c/strong\u003e\u003cstrong\u003e BsAb ameliorated renal and hepatic dysfunction as well as metabolic disturbance in db/db mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the therapeutic effects of anti-ANGPTL3/IL-1\u0026beta; BsAb on liver and kidney injury in metabolic disorders, by 8 weeks of age, \u003cem\u003edb/db\u003c/em\u003e mice were administered PBS, FD03, XOMA052, the BsAb or a combination of FD03 and XOMA052, while \u003cem\u003edb/m\u003c/em\u003e mice received PBS alone for 8 consecutive weeks (Fig. 3A). By 16 weeks of age, significantly higher ALT and AST levels were found in\u003cem\u003e db/db\u003c/em\u003e mice; ACR, BUN and serum creatinine levels were also increased compared with\u003cem\u003e db/m\u003c/em\u003e control mice, demonstrating impaired hepatic and renal function. However, after the BsAb exposure, \u003cem\u003edb/db\u003c/em\u003e mice exhibited significant reductions of ALT (80.44 \u0026plusmn; 9.001 v.s. 210.9 \u0026plusmn; 43.60 U/L), AST (62.95 \u0026plusmn; 7.072 v.s. 138.2 \u0026plusmn; 21.45 U/L) and liver index (Fig. 3B-D). Meanwhile, \u003cem\u003edb/db\u003c/em\u003e mice also exhibited marked decrease of ACR (740.9 \u0026plusmn; 74.84 v.s. 1282 \u0026plusmn; 101.7 \u0026mu;g/mg) , BUN (9.375 \u0026plusmn; 0.7392 v.s. 13.49 \u0026plusmn; 1.010 mmol/L), serum creatinine (16.98 \u0026plusmn; 2.712 v.s. 27.84 \u0026plusmn; 3.604 \u0026mu;mol/L) and kidney index (Fig. 3E-H). \u003c/p\u003e\n\u003cp\u003eInterestingly, we found that the hyperglycemia and hyperlipidemia in \u003cem\u003edb/db\u003c/em\u003e mice were ameliorated, evidenced by markedly lower level of blood glucose (27.13 \u0026plusmn; 1.134 v.s. 33.13 \u0026plusmn; 1.815 mmol/L), serum TG (1.017 \u0026plusmn; 0.07885 v.s. 1.415 \u0026plusmn; 0.07341 mmol/L), serum TC and serum LDL-C (Fig. 3F and G, Supplementary Material Fig. S2A and S2B), with reduced food intake, water intake and urinary volume (Supplementary Material Fig. S3A-C). \u003c/p\u003e\n\u003cp\u003eThus, these results implied that the simultaneously targeting ANGPTL3 and IL-1\u0026beta; via the anti-ANGPTL3/IL-1\u0026beta; BsAb alleviated the hepatic and renal dysfunction, metabolic disturbance, and diabetic symptoms in \u003cem\u003edb/db\u003c/em\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Anti-ANGPTL3/IL-1\u0026beta; BsAb attenuated hepatic steatosis, collagen deposition and inflammation in db /db mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eTo further corroborate these functional improvements at histological level, we next examined liver and kidney tissues by pathological staining and relevant analysis. First, the therapeutic efficacy of the BsAb in liver injury in \u003cem\u003edb/db\u003c/em\u003e mice was examined. Consistent with impaired hepatic function, histopathological examinations using H\u0026amp;E and Masson staining revealed marked hepatic steatosis and enhanced collagen deposition compared to \u003cem\u003edb/m \u003c/em\u003econtrol mice. After the BsAb treatment, ameliorated liver steatosis architecture and attenuated collagen deposition revealed by Masson-positive staining area were observed compared to \u003cem\u003edb/db\u003c/em\u003e control mice (Fig. 4A and B). Meanwhile, quantitative analysis confirmed significantly reduction of Masson-positive area in the liver of BsAb-treated \u003cem\u003edb/db\u003c/em\u003e mice (Fig. 4C). In addition, assessment of inflammatory cytokine expression in hepatic tissue revealed that BsAb treatment significantly reduced the levels of TNF-\u0026alpha; and IL-6 in \u003cem\u003edb/db\u003c/em\u003e mice (Fig. 4D and E).\u003c/p\u003e\n\u003cp\u003eThese findings indicated that the ANGPTL3/IL-1\u0026beta; BsAb treatment effectively alleviated important pathological features of liver injury, with attenuated hepatic steatosis, collagen deposition and inflammation in \u003cem\u003edb/db\u003c/em\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Anti-ANGPTL3/IL-1\u0026beta; BsAb reduced glomerular matrix expansion, collagen deposition and podocytes loss in db/db mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eThen, the therapeutic effects of the BsAb on kidney injury in \u003cem\u003edb/db\u003c/em\u003e mice was evaluated by histological analysis. PAS staining demonstrated reduced glomerular matrix accumulation in the kidneys of BsAb-treated \u003cem\u003edb/db\u003c/em\u003e mice, quantified by significantly decreased PAS-positive area (Fig. 5A). Masson staining further indicated reduced collagen deposition in the kidneys of \u003cem\u003edb/db\u003c/em\u003e mice treated with the BsAb, quantified by a substantial reduction of Masson-positive area (Fig. 5B). Critically, immunofluorescence for Wilms\u0026rsquo; tumor 1 (WT1) showed increased podocyte numbers per glomerulus, as revealed by the significantly higher WT1-positive cell counts per 1000 \u0026mu;m\u003csup\u003e2\u003c/sup\u003e glomerular area in the BsAb-treated mice compared to \u003cem\u003edb/db\u003c/em\u003e control mice (Fig.5C). \u003c/p\u003e\n\u003cp\u003eThese results implied that the therapeutic efficacy of the anti-ANGPTL3/IL-1\u0026beta; BsAb was similarly evident in renal pathology of \u003cem\u003edb/db\u003c/em\u003e mice, with reduced glomerular matrix expansion, collagen deposition and recovered podocyte numbers. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Anti-ANGPTL3/IL-1\u0026beta; BsAb attenuated lipotoxicity and normalized lipid metabolism in liver and kidneys of db/db mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHaving established the amelioration of hepatic and renal injury in \u003cem\u003edb/db\u003c/em\u003e mice following the BsAb treatment, we next investigated the underlying metabolic alterations. Firstly, the lipid accumulation in liver and kidney was determined. Oil Red O staining and enzymatic triglyceride assay results exhibited prominent reduction of lipid droplets, TG and fatty acid (FA) content in liver of \u003cem\u003edb/db \u003c/em\u003emice treated with the BsAb (Fig. 6A-C). Parallel reduction of renal lipid accumulation was demonstrated, with diminished lipid droplets, hepatic TG and FA content in kidneys after the BsAb treatment (Fig. 6E-G). \u003c/p\u003e\n\u003cp\u003eNext, the reprogramming of lipid metabolism in both liver and kidneys by the BsAb treatment was elucidated by transcriptomic profiling. In liver of the BsAb-treated \u003cem\u003edb/db\u003c/em\u003e mice, mRNA levels of genes that mediated fatty acid synthesis (Fasn, Acara, Acss2), triglyceride synthesis (Agpat2, Agpat3, Dgat1), fatty acid transport (Cd36, Fabp1) and fatty acid \u0026beta;-oxidation (Cpt1a, Crat, Crot, Ppara) showed decreased expression (Fig. 6D). Conversely, renal analysis revealed distinct regulatory patterns: downregulation of lipid catabolic pathways was observed (Fig. 6H), with elevated mRNA levels of gene mediated lipolysis (Pnpla2, Lipe), fatty acid transport (Slc27a2, Cd36 Fabp3, Fabp4) and fatty acid \u0026beta;-oxidation (Cpt1a, Crat, Crot, Acox1).\u003c/p\u003e\n\u003cp\u003eThese results demonstrated that the anti-ANGPTL3/IL-1\u0026beta; BsAb attenuated lipotoxicity through reducing ectopic lipid accumulation and toxic lipid species, and reprogramed lipid metabolism in both liver and kidneys of \u003cem\u003edb/db\u003c/em\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Anti-ANGPTL3/IL-1\u003c/strong\u003e\u003cstrong\u003e\u0026beta;\u003c/strong\u003e\u003cstrong\u003e BsAb normalized glucose metabolism in liver and kidneys and improved insulin sensitivity in db/db mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGiven the observed reduction of lipid accumulation and regulation of lipid metabolism, we further sought to determine whether the BsAb treatment could also ameliorate glucose metabolism. Transcriptomic profiling in liver demonstrated that gene Gys2 was up-regulated, which is related to glycogenesis, while Pygl was down-regulated, which is related to glycogenolysis. Additionally, genes related to gluconeogenesis in liver were down-regulated, including Pck1, Fbp1, G6pc and Gpd2 (Fig. 7A). Moreover, genes in relation with glycolysis in kidneys were down-regulated, including Pfkp, Pklr, Pkm, Hk1, Gpd1 and Gpd2 (Fig. 7B). Enhanced liver glycogen storage was confirmed afterwards, evidenced by intensified PAS staining and elevated quantitative glycogen content in the BsAb-treated mice (Fig. C and D).\u003c/p\u003e\n\u003cp\u003eIn addition to gene expression, insulin sensitivity is also a critical determinant of glucose metabolism\u003csup\u003e26-31\u003c/sup\u003e. Therefore, we evaluated the level of insulin resistance in db/db mice following the BsAb treatment. Although no significant change was observed in serum insulin levels (Supplementary Material Fig. S4A), BsAb treatment led to marked improvements in insulin resistance, as evidenced by a substantial reduction in the HOMA-IR (17.84 \u0026plusmn; 3.535 vs. 56.02 \u0026plusmn; 8.405) and a decreased TyG index compared with db/db control animals (Fig. 7E, Supplementary Material Fig. S4B), which are both surrogate markers to assess insulin resistance\u003csup\u003e32\u003c/sup\u003e. Furthermore, insulin tolerance test (ITT) was conducted and its quantitative analysis indicated that the BsAb treatment improved the efficiency of insulin in \u003cem\u003edb/db\u003c/em\u003e mice (Fig. 7F). Insulin binds to the insulin receptor (INSR) and activated its function of phosphorylating substrates such as insulin receptor substrate-2 (IRS-2), which acts as a critical adaptor protein to activate downstream signaling pathways\u003csup\u003e33, 34\u003c/sup\u003e. The results of INSR and IRS-2 immunochemistry staining and quantitative analysis demonstrated a significant upregulation of INSR and IRS-2 expression in liver, indicating that the insulin signaling pathway was recovered in \u003cem\u003edb/db\u003c/em\u003e mice after the BsAb treatment (Fig. 7G, Supplementary Material Fig. S5A).\u003c/p\u003e\n\u003cp\u003eThese findings suggested that the anti-ANGPTL3/IL-1\u0026beta; BsAb restored glucose homeostasis in liver and kidneys via transcriptional reprogramming, enhanced hepatic glycogen storage, and rescued insulin sensitivity in \u003cem\u003edb/db\u003c/em\u003e mice. \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe concurrent impairment of liver and kidney function has emerged as an important feature of the pathophysiology of metabolic diseases. This multifactorial crosstalk, often referred to as the \u0026ldquo;liver-kidney axis,\u0026rdquo; is driven by complex interactions, including lipid metabolism disorders and chronic low-grade inflammation. These common pathways create a vicious cycle of mutual organ dysfunction, where the deterioration of one organ often accelerates the decline of the other, thereby leading to the overall disease burden and elevating cardiovascular mortality\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Therefore, the clinical co-occurrence of MASLD and DKD in diabetic patients is not only a coincidence but also reflects a profound and interrelated pathological relationship. Although there is a large amount of literature on the pathogenesis of liver or kidney diseases, the complexity of organ crosstalk and its underlying mechanisms remain to be explored\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Clarifying the mechanisms that support this organ crosstalk remains a key objective in the field, offering significant hope for developing novel therapeutic strategies to address co-occurrence of liver and kidney complications.\u003c/p\u003e\u003cp\u003eLipotoxicity play a crucial role in the pathophysiology of liver and kidney injury in metabolic disorders\u003csup\u003e\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Dysregulation of the lipid metabolism could lead to accumulation of harmful lipids, causing lipotoxicity. ANGPTL3 has been shown to modulate lipid metabolism by inhibiting LPL and targeting ANGPTL3 could lower TG and LDL-C levels, contributing to the treatment of hyperlipidemia, MASLD or atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Moreover, lipotoxicity is intimately associated with chronic inflammation. Our preliminary study found that IL-1β was upregulated in \u003cem\u003edb/db\u003c/em\u003e mice and its level was positively correlated with renal and hepatic dysfunction. Therefore, based on the hypothesis that simultaneously reducing lipid accumulation and inflammation would more effectively attenuate lipotoxicity and alleviate concomitant liver and kidney injury, we developed a BsAb targeting both ANGPTL3 and IL-1β. Treatment with this BsAb markedly ameliorated injury in liver and kidneys of \u003cem\u003edb/db\u003c/em\u003e mice. Furthermore, the BsAb administration reduced lipotoxicity and regulated glycolipid metabolism in these organs, accompanied by lowered blood glucose levels and improved insulin sensitivity.\u003c/p\u003e\u003cp\u003eThe major concern we issued is that why reducing lipid accumulation and inflammation by the anti-ANGPTL3 and IL-1β BsAb had better therapeutic effects on ameliorating lipotoxicity compared with anti-ANGPTL3 treatment alone. During the process, it was found that targeting ANGPTL3 alone had limited therapeutic effect on hepatic collagen and inflammation. It was also reported that statin, which could lower cholesterol by suppression of the HMG-CoA, may lead to inflammation and fibrosis and fuel diabetic nephropathy progression in diabetic mice [5]. Plenty of studies have shown that the chronic inflammation induced by excessive lipid deposition, particularly within liver and kidneys, in turn, contributed to the insulin resistance and exacerbated the disturbances in glycolipid metabolism\u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45 CR46 CR47 CR48\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Taken together, these findings reminded that reducing lipid accumulation and inflammation cannot be separated to alleviate lipotoxicity, which explained why co-targeting lipid regulator ANGPTL3 and pro-inflammatory cytokine IL-1β better ameliorated lipotoxicity compared with targeting ANGPTL3 alone.\u003c/p\u003e\u003cp\u003eAnother important point that we focused on is that why the BsAb administration could normalize glycolipid metabolism in liver and kidneys. It was well documented that the lipotoxicity in the liver could impair hepatocyte functions, particularly in response to insulin signaling pathways, while reduction of lipotoxicity in liver could lead to amelioration of liver function and insulin sensitivity\u003csup\u003e\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. As the most important organ in glucose metabolism, the restoration of insulin sensitivity and hepatocyte functions in liver could trigger enhanced glycogenesis, reduced glycogenolysis and gluconeogenesis, which resulted in the increase of glycogen storage and decrease of blood glucose in \u003cem\u003edb/db\u003c/em\u003e mice\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Besides, kidneys also play a critical part in systemic metabolism and are severely affected in diabetes, as DKD is one of the most significant complications. It was reported that kidneys originally experience elevated glucose utilization under hyperglycemic conditions in diabetes because of the exposure to abundant arterial blood flow, which may exacerbate the damage of kidneys\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Therefore, the management of blood glucose promoted the shift of energy substrate preference in kidneys from glucose to FA, with a significant upregulation in fatty acid oxidation (FAO) and downregulation in glycolysis. The restoration of FAO in kidneys was described to be beneficial for the treatment of fibrosis in DKD\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The elevated level of FAO also resulted in the reduction of lipid accumulation and contributed to attenuating lipotoxicity in kidneys, which was highly involved in the kidney diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In summary, co-targeting ANGPTL3 and IL-1β by the BsAb rebuilt the glycolipid metabolism balance in both liver and kidneys through attenuating lipotoxicity, and thereby ameliorated liver and kidney injury in \u003cem\u003edb/db\u003c/em\u003e mice.\u003c/p\u003e\u003cp\u003eThe significance of this work goes beyond the observed improvements in liver and kidney function and histology. This is the first study to explore metabolic crosstalk between the liver and kidneys in this situation, revealing that the reduction of lipotoxicity and improvement of glycolipid balance endow the two organs with mutual protection. By demonstrating that co-targeting ANGPTL3 and IL-1β can reduce lipotoxicity and normalize glycolipid metabolism in liver and kidneys, we have provided compelling evidence that disrupting the lipid disorder and inflammatory cycle is crucial for restoring metabolic balance throughout the body. Furthermore, this study suggested shifting the treatment approach from organ-specific to systemic interventions, which implies that concomitant liver and kidney injury is a systemic disease requiring a combined strategy.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study demonstrated that simultaneously blocking ANGPTL3 and IL-1β could attenuate lipotoxicity, regulate glycolipid metabolism, and thereby ameliorate liver and kidney injury in \u003cem\u003edb/db\u003c/em\u003e mice. Moreover, we also explored the metabolic crosstalk between liver and kidneys and found that attenuating lipotoxicity and improving the balance of glycolipid metabolism could provide cross-protection for the liver and kidneys. These findings offered new ideas for the treatment of concomitant organ injury associated with metabolic disorders in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eShuwen Xu designed the experiment, conducted the experiment, written the manuscript and reviewed the manuscript. Longfei Wang, Zhanglian Cao and Xiaozhi Hu designed the experiment. Zihan Dou, Yuanzhen Zhang, Xianhan Jiang, Tao Wu and Zhuojin Li conducted the experiment and processed the data. Yanyang Nan, An Zhu, Yu Bai, Ziqian Zou, Xuyao Zhang and Xian Zeng reviewed the manuscript. Haidong He, Dianwen Ju, Shaofei Wang and Jiajun Fan designed the experiment, reviewed the manuscipt and provided funding support.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work was supported by grants from National Key Research and Development Program of China (2023YFC3404000), National Key Research and Development Program of China (2023YFC3503400) and the National Nature Science Foundation of China (82371781)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYang M, Luo S, Yang J, Chen W, He L, Liu D, et al. Crosstalk between the liver and kidney in diabetic nephropathy. 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FEBS J. 2017;284:1070\u0026ndash;81.\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":"inflammation-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inre","sideBox":"Learn more about [Inflammation Research](http://link.springer.com/journal/11)","snPcode":"11","submissionUrl":"https://submission.nature.com/new-submission/11/3","title":"Inflammation Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ANGPTL3, IL-1β, Bispecific antibody, Liver and kidney injury, Lipotoxicity, Glucolipid metabolism","lastPublishedDoi":"10.21203/rs.3.rs-7930376/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7930376/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eConcomitant liver and kidney injury is a critical pathological feature of metabolic disorders, but current organ-specific therapies often fail to provide cross-protection. Lipotoxicity is a core mechanism linking damage in both organs. Therefore, this study aimed to investigate whether simultaneously targeting ANGPTL3 and IL-1β could attenuate lipotoxicity and thereby ameliorate concomitant liver and kidney injury.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA novel bispecific antibody (BsAb) targeting both ANGPTL3 and IL-1β was generated and characterized by SDS-PAGE, SEC-HPLC, thermal stability analysis, SPR and \u003cem\u003ein vitro\u003c/em\u003e bioassay. Then, its protective effects were subsequently studied in the \u003cem\u003edb/db\u003c/em\u003e mouse model and the underlying mechanisms were revealed by biochemical examinations, histopathological analysis, immunofluorescence (IF), ELISA, RNA-seq, Immunohistochemical (IHC).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAdministration of the BsAb in \u003cem\u003edb/db\u003c/em\u003e mice effectively improved liver and kidney function with alleviated liver steatosis and inflammation, as well as reduced kidney glomerular injury. Furthermore, the treatment attenuated lipotoxicity in both organs and normalized the glycolipid metabolism including restored hepatic glycogen reserves and enhanced renal utilization of fatty acids.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe results demonstrate that the anti-ANGPTL3/IL-1β BsAb alleviates concomitant liver and kidney injury in \u003cem\u003edb/db\u003c/em\u003e mice by attenuating lipotoxicity and normalizing glycolipid metabolism, which highlights a promising therapeutic approach for addressing multi-organ damage in metabolic disorders.\u003c/p\u003e","manuscriptTitle":"Targeting angiopoietin-like protein 3 and Interleukin-1β alleviated liver and kidney injury through attenuation of lipotoxicity and normalization of glycolipid metabolism in db/db mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 18:18:41","doi":"10.21203/rs.3.rs-7930376/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-23T19:57:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T07:08:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272371291184122831770449946164522099110","date":"2025-11-29T12:21:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72631783099683638588193825512253764343","date":"2025-11-12T14:23:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-09T18:43:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-30T20:19:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-30T20:18:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation Research","date":"2025-10-23T08:48:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inre","sideBox":"Learn more about [Inflammation Research](http://link.springer.com/journal/11)","snPcode":"11","submissionUrl":"https://submission.nature.com/new-submission/11/3","title":"Inflammation Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"03229223-fd0a-4935-ad60-f1ce8fa9cbde","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:14:02+00:00","versionOfRecord":{"articleIdentity":"rs-7930376","link":"https://doi.org/10.1007/s00011-026-02218-5","journal":{"identity":"inflammation-research","isVorOnly":false,"title":"Inflammation Research"},"publishedOn":"2026-04-17 15:58:15","publishedOnDateReadable":"April 17th, 2026"},"versionCreatedAt":"2025-11-18 18:18:41","video":"","vorDoi":"10.1007/s00011-026-02218-5","vorDoiUrl":"https://doi.org/10.1007/s00011-026-02218-5","workflowStages":[]},"version":"v1","identity":"rs-7930376","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7930376","identity":"rs-7930376","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}