Neutrophil-derived LL37 mediates IgA nephropathy via NETs-dependent mechanism

Neutrophil-derived LL37 mediates IgA nephropathy via NETs-dependent mechanism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Neutrophil-derived LL37 mediates IgA nephropathy via NETs-dependent mechanism Hong Jiang, Yang Chen, Anqi Zhang, Pingping Ren, Shi Feng, Chuchu Zhu, and 24 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6636772/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract IgAN is a common cause leading to end-stage renal disease. The deposition of pathogenic IgA in the glomerular mesangium is a critical step. However, the mechanisms of pathogenic IgA crossing the glomerular filtration barrier remain unclear. We hypothesized that neutrophil-derived LL37 is pathogenic and mediates IgAN through NETs-dependent mechanism. Serum LL37 were elevated in patients with active IgAN and correlated with poor long-term kidney survival (HR = 2.54). Functionally, LL37 is pathogenic in IgAN as mice lacking LL37 were protected from the development of severe IgAN by essentially reducing IgA and C3 deposition, mesangial cell proliferation, and proteinuria, which was reversed by neutrophils from WT but not from Cramp KO mice. Further study uncovered that LL37 were primarily derived from neutrophils as evidenced in Crampfl/flLy6gcre mice in which specific deletion of LL37 from neutrophils also suppressed the development of IgAN. With scRNA we found that LL37 acted via NETs to cause endothelials injury but increasing the permeability in mechanism, which was demonstrated by the addition of NETs, but not LL37-free NETs under high pIgA1 conditions in vivo and in vitro. LL37 is elevated in IgAN patients, derived from neutrophils, and plays a pathogenic role in IgAN via a NETs-dependent mechanism. Biological sciences/Immunology Health sciences/Diseases/Kidney diseases/Glomerular diseases/IgA nephropathy NETs LL37 IgA nephropathy glomerular endothelial pIgA deposition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis globally, leading to end-stage renal disease (ESRD) in 30%-40% of affected patients within 20 years of diagnosis 1 . While the exact cause of IgAN remains unclear, the multi-hit theory is widely accepted 2 . This theory suggests that IgAN begins with aberrant glycosylation of IgA1, which leads to the overproduction of galactose-deficient IgA1 (Gd-IgA1). Autoantibodies then bind to Gd-IgA1, forming nephritogenic immune complexes that deposit in the mesangium. This results in mesangial cell activation, inflammation, and subsequent renal damage 2 . Although pathogenic IgA deposition in the mesangium is a crucial event in IgAN pathogenesis, the mechanisms by which pathogenic IgA crosses the glomerular filtration barrier are not fully understood. Our previous studies showed elevated levels of the antimicrobial peptide LL37 in the plasma of IgAN patients, distinguishing them from healthy individuals 3 , 4 . LL37( CAMP ), a 37-amino acid peptide, is a major component of neutrophil tertiary granules and neutrophil extracellular traps (NETs), and CRAMP ( Cramp ) is the same in mice 5 , 6 . Clinical observations have also noted the presence of polymorphonuclear neutrophils in the glomerular capillaries of IgAN patients during episodes of macroscopic hematuria 7 . Furthermore, a high neutrophil-to-lymphocyte ratio (NLR) is an independent risk factor for ESRD in IgAN 8 . NETs have been implicated in the pathogenesis of several autoimmune diseases, including systemic lupus erythematosus (SLE), anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) 6 . Given that IgAN is, at least in part, an autoimmune disease and IgA can activate neutrophils 9 , it is crucial to explore the role of NETs in IgAN. Previous researches on LL37 focused on its antimicrobial role in urinary infections 10 , 11 . Recent studies have highlighted its immunomodulatory effects in ischemia/reperfusion-induced acute kidney injury 12 . In this study, we demonstrate that LL37 promotes the progression of IgAN by mediating NET-induced injury to glomerular endothelial cells, allowing the entry of pathogenic IgA into the mesangium. Methods Human subjects Patients diagnosed with IgAN between January 2012 and December 2018 at the Kidney Disease Center, First Affiliated Hospital, Zhejiang University (ZJU cohort), were followed up. Of these, 52 patients progressed to end-stage renal disease (ESRD) (IgAN-ESRD group), while 503 remained stable (IgAN-Stable group), defined as a less than 30% decline in eGFR during follow-up. The study adhered to the principles of the Declaration of Helsinki. It was approved by the Clinical Research Ethics Committee(Approval No. 2022 − 1087), with informed consent exemption. Additionally, 101 IgAN patients from Queen Mary Hospital, University of Hong Kong (HKU cohort), and 379 from RWTH Aachen University, Germany (Aachen cohort), underwent LL37 serological testing. Animals C57BL/6J and BALB/c nude mice were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. Cramp knockout (KO) mice were provided by Professor Jia Sun of Jiangnan University 13 . Cramp fl/fl Ly6g cre mice were generated by Cyagen Biosciences. Animal experiments were approved by the Animal Experiment Ethical Inspection (Approval No. 2022 − 1268). Mouse models The experimental IgAN model was induced as previously described 14 . Mice were sacrificed at week 9, and samples (24-hour urine, serum, and kidney) were collected. Polymeric IgA1 (pIgA1) was purified from the sera of IgAN patients, as previously described 15 , 16 . BALB/c nude mice received intravenous injections of 0.5 mg pIgA1 every 3–4 days for two weeks before being euthanized for sample collection 17 . Bone marrow transplantation and neutrphil adoptive transfer Six- to eight-week-old male wild-type (WT) or Cramp KO recipient mice were lethally irradiated (10 Gy) and subsequently received an intravenous injection of 2×10⁷ bone marrow cells from either WT or Cramp KO mice 4 hours later. Experiments were initiated four weeks after bone marrow reconstitution. Neutrophil adoptive transfer were taken as previously described 18 . ELISA Serum LL37 levels were measured using ELISA (Novus, NBP3-06932). Myeloperoxidase-DNA (MPO-DNA) complex levels were quantified as previously described 19 . Kidney histology Kidneys were harvested, fixed in 4% formalin overnight, dehydrated, and paraffin-embedded. Tissue sections were stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) for histopathological analysis. Immunohistochemistry and immunofluorescence study Paraffin-embedded and immunofluorescence sections were both taken as the usual protocol. The primary antibodies used were rabbit anti-human Cathelicidin (1:50, PA5-20513, Invitrogen); mouse anti-human CD16/FITC (1:100, sc-19620, Santa Cruz); goat anti-mouse IgA/FITC (1:200, Abcam, ab97234); goat anti-mouse IgM-Alexa Fluor 488 (1:1000, Abcam, ab150121); goat anti-mouse IgG-Alexa Fluor Plus 555 (1:500, Invitrogen, A32727); rat anti-mouse C3 (1:50, Abcam, ab11862); rat anti-mouse Ly6G-FITC (1:250, eBioscience, 11-9668-82); rabbit anti-Histone H3 (1:900, Abcam, ab5103); and mouse anti-human LL37 (1:50, Hycult Biotech, HM2070). Images were captured using an immunofluorescence microscope (Leica DM4000) or a confocal microscope (Leica TCS-SP8). Flow cytometry analysis Flow cytometry was performed on mouse blood and Peyer’s patches. The following antibodies were used: CD45.1 (A20), CD45.2 (104) from BioLegend, CD45 (30-F11), Ly6G (1A8) from eBioscience, mouse CD19 (Biolegend, 6D5) and CD138 (Biolegend, 281-2), anti-mouse IgA (Bethyl, A90-103F). Samples were analyzed on a BD FACS Canto II device (BD Bioscience), and data were processed using FlowJo software. Single-cell RNA sequencing The mice kidneys were isolated, followed by single-cell isolation and acquisition. The acquired single cell suspension at a concentration of 300–600 cells/µL was loaded onto the 10x Chromium Single Cell controller. Barcoding and cDNA synthesis were performed according to the manufacturer’s instructions. The cDNA libraries were constructed using Single Cell 3’ Library and Gel Bead Kit V3.1 and sequenced using an Illumina Novase6000 sequencer (performed by Capital Technology, Beijing). Downstream analyses were performed in R (4.1.1) using the Seurat package (3.0). To access technical variability between samples, an initial UMAP projection was generated using all 19,968 cells. The highly variable genes were selected using the Find Variable Features function. Clustering was performed using the Find Clusters function. Cell cultures HUVECs were purchased from Cell Bank of Chinese Academy of Sciences and cultured in DMEM(Sigma) with 10% FBS(Gibco)and 1% antibiotic solution (Solarbio). HL-60s and CAMP KO HL-60s were purchased from Cyagen Biosciences and cultured in IMDM(Gibco) supplemented with 15% FBS(Gibco) and 1% antibiotic solution (Solarbio). HL-60s and CAMP KO HL-60s were treated with 1.25% dimethyl sulfoxide for 6 days for differentiation to dHL-60 cells and CAMP KO dHL-60s. Western blot analysis HUVECs were lysed in radio immunoprecipitation assay buffer (Beyotime Biotechnology) supplemented with protease inhibitor cocktail (Beyotime Biotechnology). The primary antibodies against CD31 (1:1000, M1511-8, HUABIO), VE-Cadherin (1:1000, 2500S, CST) or β-actin (1:1000, sc-58673, Santa Cruz) were used. The target proteins were visualized using enhanced chemiluminescence substrate reagents (Millipore, USA). Semiquantitative analysis of western blot images was performed with ImageJ software. Apoptosis detection Cell death was evaluated by flow cytometry following annexin V-FITC/propidium iodide (PI) double staining according to the manufacturer’s protocol. The samples were analyzed by flow cytometry (BD Bioscience). Transendothelial albumin passage A transwell chamber with a membrane pore size of 0.4 µm (Corning) was used to evaluate transendothelial albumin passage. HUVECs were seeded in the upper chamber and treated with NETs for 24 h. Then, the medium in the upper chamber was replaced by serum-free medium containing 0.5 mg/mL FITC-labeled BSA (Solarbio, SF063), and the medium in the lower chamber was replaced with serum-free medium containing 0.5 mg/mL unlabeled BSA (Sigma-Aldrich). After 1 h, the upper chamber were removed from the wells, and fluorescence was determined at 495 nm excitation and 525 nm emission using a fluorescence microplate reader (TECAN). Neutrophil isolation, NETs induction and detection Neutrophils were isolated with a human neutrophil isolation kit (STEMCELL, 19666) or a mouse neutrophil isolation kit (STEMCELL, 19762) following the manufacturer’s instructions. NETs were quantified utilizing a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P11496) and verified through staining with Sytox green (Thermo Fisher Scientific, S7020). Statistical analysis Statistical analyses were conducted using SPSS, version 26.0. The figures were constructed by GraphPad Prism 9. Results LL37 is elevated in IgAN patients, is predominantly derived from neutrophils, and correlates with the severity of IgAN A total of 555 IgAN patients and 38 healthy volunteers were included in this study. Baseline clinicopathological characteristics are detailed in Supplemental Table 1. To investigate LL37 gene expression in IgAN, we first analyzed data from the Nephroseq renal transcriptomics database and found a significantly higher transcription levels of CAMP –the gene encoding LL37–in the peripheral blood of IgAN patients compared to healthy controls (Fig. 1A). In our cohort, serum LL37 levels were particularly elevated in IgAN-ESRD patients compared to IgAN-Stable patients and healthy controls ( p = 0.0078 and p = 0.0029, respectively, Fig. 1B). Correlation analysis showed serum LL37 levels were positively correlated with serum creatinine and UPCR ( p = 0.0011 and p = 0.0069, respectively, Fig. 1C and Fig. 1D). Patients were further stratified by LL37 levels into high and low groups. Those with higher LL37 exhibited greater urinary protein levels, elevated serum creatinine, reduced estimated glomerular filtration rate (eGFR), increased blood pressure, and worse Oxford Classification T scores (Table 1), indicating a significantly poorer long-term renal prognosis (HR = 2.60, p = 0.002, Fig. 1E). While LL37 can be produced by eosinophils and other cell types 20 , we also identified that patients with ESRD had higher neutrophil counts compared to those with stable conditions (Supplemental Table 2). Flow cytometry confirmed that LL37 expression was predominantly confined to neutrophils in IgAN patients, showing a threefold increase compared to healthy controls (Fig. 1F). Immunostaining demonstrated that LL37 was expressed primarily by CD16 + cells (Fig. 1G). Thus, neutrophils are a primary source of LL37 in patients with severe IgAN. LL37 is pathogenic in a mouse model of IgAN. We then examined the functional role of LL37 in the pathogenesis of IgAN in a mouse model of IgAN. As demonstrated in Fig. 2, we successfully induced a mouse mode of IgAN, showing mesangial cell proliferation, IgA and C3 deposition in the mesangial regions, and elevated proteinuria (Figs. 2A–2G, 2I), which was associated with upregulation of CRAMP, the murine homolog of LL37 (Fig. 2H). To investigate the role of LL37 in IgAN pathogenesis, we generated CRAMP-deficient ( Cramp −/− ) mice on a C57BL/6J background. We found that although CRAMP deficiency did not affect normal renal morphology compared to wild-type (WT) mice (Figs. 2B–2G), they did protect from the development of IgAN by largely reducing mesangial IgA, IgM, IgG, and C3 deposition, mesangial cell proliferation, and albuminuria post-IgAN induction (Figs. 2B–2G, 2I). However, renal function remained stable in both WT and Cramp −/− mice, as the disease was in its early stages (Figs. 2J, 2K). To identify the CRAMP-producing cells responsible for IgAN development and further confirm the role of LL37 in the development of IgAN, we performed BMT to reconstitute the immune systems with or without LL37-producing cells in recipient mice, followed by IgAN induction (Fig. 3A). Cramp −/− mice receiving BMT from WT donors developed typical IgAN with a marked IgA, IgM, IgG, and C3 deposition, mesangial cell proliferation, and albuminuria (Figs. 3B–3H), although renal function remained comparable between groups (Figs. 3I, 3J). In contrast, WT mice receiving BMT from Cramp −/− donors exhibited a significant inhibition of IgAN (Figs. 3B–3H). Taken together, these results indicate that LL37 produced by immune cells is pathogenic in IgAN. Neutrophil-derived LL37 is responsible for the development of mouse IgAN Because LL37 was primarily expressed by neutrophils in patients with IgAN (Fig. 1F, H) and BM-derived LL37 caused severe IgAN (Fig. 3), we further hypothesized that neutrophil-derived LL37 may play a key role in mouse IgAN. To test this, we first assessed neutrophil levels in IgAN mice. Like patients with IgAN (Fig. 1F, G), neutrophil counts were significantly elevated in IgAN mice compared to age-matched C57BL/6J controls ( p = 0.0001, Fig. 4A). Increased neutrophils were also observed during the acute inflammatory phase of IgAN (4 hours post-LPS administration in the sixth week, p = 0.0001, Fig. 4A). Although no overt neutrophil infiltration was observed in the kidneys of IgAN mice, neutrophils were found in the glomeruli and tubulointerstitium during acute inflammation (4h, 24h, 48h post-LPS administration, Fig. 4B). We also performed neutrophils adoptive transfer to recipient mice, followed by IgAN induction (Fig. 5A). Cramp −/− mice receiving neutrophils from WT donors developed typical IgAN with a marked IgA, IgM, IgG, and C3 deposition, mesangial cell proliferation, and albuminuria (Figs. 5B–5H), although renal function remained comparable between groups (Figs. 5I, 5J). In contrast, WT mice receiving neutrphils from Cramp −/− donors exhibited a significant inhibition of IgAN (Figs. 5B–5H). To confirm the role of neutrophil-derived CRAMP, we crossed Cramp fl/fl mice with Ly6g -Cre mice to generate neutrophil-specific LL37 knockout mice ( Cramp -NKO mice). Like global LL37 KO mice (Fig. 2), conditional knockout of Cramp from neutrophils largely reduced IgA, IgM, IgG, and C3 deposition, mesangial cell proliferation, and albuminuria (Fig. 6A–6G), although renal function remained unaffected (Figs. 6H, 6I). The neutrophils from WT mice with CRAMP expression could cause the IgAN symptoms in Cramp fl/fl Ly6g cre mice (Fig. 6B). These findings confirm that neutrophil-derived LL37 is essential for IgAN development in mice. Glomerular endothelial cells were key targets Single-cell RNA sequencing (scRNA-seq) was performed on kidneys from WT and Cramp −/− mice following IgAN induction (Fig. 7A). After rigorous quality control, we analyzed 10,472 cells from IgAN mice and 9,496 cells from controls. Clustering identified 12 distinct cell types, including renal parenchymal cells (proximal tubular cells, distal convoluted/loop of Henle cells, endothelial cells, podocytes, and mesangial cells) and immune cells (macrophages, T cells/NK cells, B cells, and neutrophils) (Figs. 7B, 7C). Compared to controls, IgAN mice displayed increased neutrophil and macrophage infiltration (Figs. 7D, 7E), supporting the role of neutrophils in IgAN pathogenesis via LL37. To further investigate changes in glomerular cells, we reclustered endothelial cells, podocytes, and mesangial cells, identifying eight cell clusters and six different cell types (Fig. 7F). The proportion of GECs significantly decreased in IgAN mice (Fig. 7G), indicating that GECs are likely the most affected cell type, consistent with recent scRNA-seq studies 21 . Immunohistochemical staining confirmed that CD31 + GECs were significantly reduced in IgAN patients and mice (Fig. 7H). Previous studies have highlighted GEC injury as a critical factor in IgAN pathogenesis 21–24 , so subsequent mechanistic studies focused on GEC damage. LL37 may interact with NETs to induce endothelial cell injury in vivo and in vitro As NETs are major carriers of LL37 and are known to cause endothelial dysfunction 6,9,19 , we hypothesized that LL37 exacerbates IgAN by amplifying NET-induced damage to GECs. We measured serum levels of MPO-DNA complexes, a marker for NETs, and found that these MO)-DNA complexes were elevated in IgAN-ESRD patients compared to IgAN-Stable patients ( p = 0.047, Fig. 8A). Correlation analysis showed MPO-DNA levels positively correlated with LL37 ( p = 0.0074, Fig. 8B). Furthermore, NET marker CitH3 colocalized with LL37 in IgAN glomeruli (Fig. 8C). Fluorescent staining (Sytox green) revealed that pIgA1 from IgAN patients induced NET formation, whereas pIgA1 from healthy controls did not (Fig. 8D). Purified pIgA1-induced NETs were cytotoxic to HUVECs in a dose-dependent manner, but LL37 alone had no direct effect (Fig. 8E). NET treatment reduced the endothelial integrity markers CD31 and VE-Cadherin (Fig. 8F) and induced cell death (Fig. 8G). To mimic the transendothelial migration of IgA-containing immune complexes, we conducted a transwell assay using albumin as a proxy. Results showed that NETs increased albumin passage across the endothelial barrier (Fig. 8H). Additionally, intravenous injection of pIgA1 from IgAN patients into immunodeficient mice resulted in stable IgA deposition and mesangial cell proliferation (Fig. 9A, 9B). Treatment with DNase I, which degrades NETs, alleviated both mesangial proliferation and IgA deposition in the IgAN model (Figs. 9C–9E). We next investigated how LL37 levels influence NET formation and their cytotoxicity to endothelial cells. LL37 was confirmed to be present in pIgA1-induced NETs (Fig. 10A). Neutrophils from IgAN patients generated more NETs compared to those from healthy donors (Fig. 10B). Similarly, neutrophils from Cramp KO mice showed reduced NET formation compared to WT under the same stimulation (Fig. 9B). Since neutrophils are terminally differentiated and unsuitable for RNA interference or gene editing, we used the HL-60 promyelocytic cell line. After inducing differentiation with 1.25% DMSO for six days (Supplemental Fig. 1), we generated CAMP KO HL-60 cells via CRISPR/Cas9. Both differentiated HL-60 (dHL-60) and CAMP KO HL-60 cells were stimulated with PMA to induce NET formation. Purified NETs and LL37-free NETs were then applied to HUVECs. Compared to standard NETs, treatment with LL37-free NETs significantly inhibited endothelial cell injury by preventing the loss of endothelial markers CD31 and VE-Cadherin (Fig. 10C) and cell death (Fig. 10D). These findings suggest that LL37 expression contributes to NET formation and cytotoxicity to endothelial cells. Discussion In our study, we found that serum levels of LL37 were elevated in patients with active IgAN, which was associated with a significant increase in urinary protein, serum creatinine, blood pressure, and higher T scores, and correlated with poor long-term kidney survival and a loss of glomerular endothelial cells. Functionally, LL37 is pathogenic in IgAN as mice lacking LL37 were protected from the development of severe IgAN by largely reducing IgA and C3 deposition, mesangial cell proliferation, and proteinuria, which was reversed by BMT from WT but not from Cramp KO mice. Further study uncovered that LL37 were primarily derived from neutrophils as evidenced in Cramp fl/fl Ly6 g cre mice in which specific deletion of LL37 from neutrophils also suppressed the development of IgAN. Mechanistically, we found that LL37 acted via NETs to cause endothelial cell injury by reducing CD31 expression but increasing the permeability, which was demonstrated by the addition of NETs, but not LL37-free NETs to cause endothelial damage under high pIgA1 conditions in vivo and in vitro . The most critical finding in the present study is the discovery of the pathogenic role of LL37, particularly neutrophil-derived LL37, in IgAN. Our clinical data showed increased CAMP expression in PBMCs from the renal transcriptomics database Nephroseq (GSE14795, Fig. 1 A). ELISA analysis also found that serum levels of LL37 increased in IgAN patients from the ZJU cohort, which was further confirmed in the HKU and Aachen cohorts, particularly in newly diagnosed patients, but not in prolonged treatment. Neutrophils, the first line of immune defense 25 , are highly reactive but sensitive to external factors like drugs and diseases 26 , making them prone to instability 27 . This may explain why LL37 has early diagnostic potential but is limited in real-time monitoring during treatment. Using flow cytometry, immunofluorescence, and scRNA sequencing, we confirmed neutrophils as the primary source of LL37. Neutrophils produce three times more LL37 than other cells, consistent with previous studies 20 . Thus, LL37-expressing neutrophils are a crucial contributor to IgAN pathogenesis. We validated the role of LL37/CRAMP using various genotypes and IgAN mouse models. WT mice exhibited IgAN features following CCL4/LPS induction, while Cramp KO mice and Cramp fl/fl Ly6g cre mice showed reduced IgAN symptoms. Bone marrow transplants confirmed that Cramp KO neutrophils did not induce IgAN in WT mice, but the neutrophils from WT mice with CRAMP expression caused severe IgAN symptoms in Cramp fl/fl Ly6g cre mice. Additionally, nude mice injected with pIgA1 displayed increased LL37/CRAMP expression, emphasizing neutrophil-derived LL37's specific role in IgAN pathogenesis. To investigate the relationship between LL37/CRAMP and pathogenic IgA, we analyzed IgAN mice, which had a higher frequency of plasma cells and IgA + plasma cells in Peyer’s patches compared to age-matched controls. However, Cramp knockout did not affect this increase (Supplemental Fig. 2A–2D). In IgAN patients, serum LL37 levels did not significantly correlate with Gd-IgA1 or total IgA (Supplemental Fig. 2E and 2F), indicating that LL37 may not involve the "first hit" of IgAN pathogenesis. Then, we verified the possible mechanisms of LL37 mediates IgAN via NETs. Neutrophils play a pivotal role in the pathogenesis of various diseases 28 – 30 . Initially, LL37 was studied for its antimicrobial properties in urinary infections 10 , 11 , but recent research identified its immunomodulatory role in ischemia/reperfusion-induced acute kidney injury 12 . The role of NETs in diseases like diabetes and IgAN has also been increasingly recognized 31 – 33 . Our findings from scRNA sequencing and immunohistochemistry revealed decreased glomerular endothelial cells in IgAN. This reduction was linked to NETs amplified by LL37/CRAMP, as confirmed by MPO-DNA analysis in IgAN patients and ds-DNA analysis in IgAN mouse models. It is possible that NET-induced endothelial injury may enhance albumin passage through the endothelial barrier post-NET stimulation by LL37. Reduced NET formation and endothelial apoptosis in CAMP KO HL-60 cells support the notion that LL37-amplified NETs promote glomerular endothelial injury, facilitating mesangial IgA deposition in IgAN. There are several shortcomings in this study. First, the CCL4/LPS-induced IgAN mouse model may not fully represent the IgAN in patients clinically, although it is suitable for analyzing LL37/CRAMP genotypes and NET stimulation. The use of nude mice injected with pIgA1 may be more appreciative. Second, HUVECs were used in place of glomerular endothelial cells, which may not fully represent the bioactivities of glomerular endothelial cells. As a new target, LL37 can reduce the pathological IgA deposition in the mesangial area by attenuating the damage to endothelial cells and the glomerular filtration barrier. It is unlikely that mono-therapy will be effective for all patients with IgAN. In the future, multi-specific targeted therapy may be used for IgAN to alleviate and control disease progression, improve disease prognosis, and minimize adverse drug reactions. Nevertheless, our findings provide new insights into IgAN pathogenesis and suggest that NET-targeted therapies may have clinical value in the future. Declarations Acknowledgements Funding This work was supported by the Huadong Medicine Joint Funds of the Zhejiang Provincial Natural Science Foundation of China under Grant No. LHDMZ23H100001, and National Natural Science Foundation of China (82070729, 81170697, 81470938, 82100738, 82471713), and 20243BBI91026. Conflict of Interest The authors declare no competing interests. References Lai, K. N. et al. IgA nephropathy. Nat Rev Dis Primers 2 , 16001 (2016). Suzuki, H. et al. The pathophysiology of IgA nephropathy. J Am Soc Nephrol 22 , 1795–1803 (2011). Lu, Y. et al. 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Neutrophil trapping and nexocytosis, mast cell-mediated processes for inflammatory signal relay. Cell 187, 5316-5335.e28 (2024). Table 1 Table 1. Baseline clinicopathologic characteristics of IgAN patients grouped by LL37 concentration Low LL37 Concentration ( n = 50) High LL37 Concentration （ n = 50 ） p value Male, n (%) 20 (40.00) 22 (44.00) 0.69 Age (yr), median (IQR) 34.00 (28.75–43.75) 33.00 (27.75–43.25) 0.78 UPCR (g/g), median (IQR) 0.94 (0.50–1.88) 2.23 (0.82–3.71) 0.017 Serum creatinine (μM), median (IQR) 80.50 (64.50–118.75) 135.50 (75.25–194.25) 0.004 eGFR (mL/min per 1.73 m 2 ), median (IQR) 85.08 (61.06–110.87) 45.71 (30.26–91.06) 0.002 SBP (mmHg), median (IQR) 123.50 (116.00–138.75) 138.00 (124.00–158.50) 0.011 DBP (mmHg), median (IQR) 78.50 (70.75–89.00) 88.50 (76.25–104.25) 0.004 Oxford classification, n (%) M1 8 (16.00) 13 (26.53) 0.20 E1 2 (4.00) 4 (8.16) 0.44 S1 44 (88.00） 41 (83.67） 0.54 T1-T2 11 (22.00) 25 (51.02) 0.003 C1-C2 24 (48.00) 20 (40.81) 0.47 IQR, interquartile range; UPCR, urine protein to creatinine ratio; NA, not available; eGFR: estimated glomerular filtration rate; SBP: systolic blood pressure; DBP: diastolic blood pressure; M1, mesangial hypercellularity; E1, endocapillary hypercellularity; S1, segmental glomerulosclerosis; T, tubular atrophy/interstitial fibrosis; T1, T 26%–50%; T2, T >50%; C0, no crescents; C1, crescents in 25% of glomeruli. Additional Declarations There is no conflict of interest Supplementary Files Graphicalabstract.pdf Graphical abstract Supplementaryinformation.pdf SUPPLEMENTAL MATERIAL Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6636772","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":469615176,"identity":"4c056c1c-f845-4e67-8d63-89ddd16f62f2","order_by":0,"name":"Hong 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Tang","email":"","orcid":"","institution":"Nephrology, The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Sydney","middleName":"C.W.","lastName":"Tang","suffix":""},{"id":469615203,"identity":"93cb3374-15e6-45da-9ad3-4190641ca796","order_by":27,"name":"Claudia Seikrit","email":"","orcid":"","institution":"RWTH Aachen University","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Seikrit","suffix":""},{"id":469615204,"identity":"dda91627-c20a-47b3-8062-06ced9af3a01","order_by":28,"name":"Jürgen Floege","email":"","orcid":"","institution":"RWTH Aachen University","correspondingAuthor":false,"prefix":"","firstName":"Jürgen","middleName":"","lastName":"Floege","suffix":""},{"id":469615205,"identity":"b7474d8f-1486-4a96-ae32-0ffcee8775cd","order_by":29,"name":"Jiang-Hua Chen","email":"","orcid":"","institution":"Kidney Disease Center, Zhejiang University, Hangzhou, CN","correspondingAuthor":false,"prefix":"","firstName":"Jiang-Hua","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-05-10 21:15:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6636772/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6636772/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84695680,"identity":"796915dd-f7da-4da5-b6a9-19cae1f65dca","added_by":"auto","created_at":"2025-06-16 10:38:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1402814,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLL37 levels are elevated in IgAN patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) \u003cem\u003ecamp\u003c/em\u003e gene transcription in peripheral blood from healthy controls and IgAN patients. (B) Serum LL37 levels in healthy controls, IgAN-Stable, and IgAN-ESRD patients. (C) Spearman correlation of serum LL37 with UPCR. (D) Spearman correlation of serum LL37 with serum creatinine. (E) Kaplan-Meier renal survival curves by median serum LL37 level. (F) LL37 expression in peripheral blood immune cells detected by flow cytometry. (G) LL37 immunostaining in kidney tissues from IgAN patients and living donors. Scale bar, 50 μm.**\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/f6e9d4971814aa9484482eea.png"},{"id":84695670,"identity":"967feaa6-9d9b-4ac9-9ee9-f20c832f1d25","added_by":"auto","created_at":"2025-06-16 10:38:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1888920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRAMP deficiency reduces glomerular lesions and albuminuria in murine IgAN models. CRAMP deficient (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCramp\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e) mice and WT mice were subjected to IgAN model construction.\u003c/strong\u003e (A) IgAN model construction schedule. (B) HE, PAS staining, and immunostaining of kidney tissues. (C) Mesangial matrix fraction quantification. (D-G) Semi-quantification of IgA, IgM, IgG, and C3 immunofluorescence intensity. (H) Serum CRAMP levels by ELISA (\u003cem\u003en \u003c/em\u003e= 5–10). (I) UACR in WT and \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice with or without IgAN model construction. (J-K) Serum creatinine and BUN levels. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001; ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/6be05d86c9c7ae6cda54b358.png"},{"id":84696922,"identity":"91f1efc3-27d7-4392-ae72-474b5e2e17fb","added_by":"auto","created_at":"2025-06-16 10:46:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2560473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBone marrow-derived CRAMP is involved in the development of mice.\u003c/strong\u003e CRAMP deficient (\u003cem\u003eCramp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e) mice and WT mice were subjected to bone marrow transplantation followed by IgAN model construction. (A) Bone marrow transplantation and IgAN model construction schedule. (B) HE, PAS staining, and immunostaining of kidneys. (C) Mesangial matrix fraction quantification. (D–G) Semi-quantification of IgA, IgM, IgG, and C3 immunofluorescence intensity. (H–I) Serum BUN and creatinine levels. (J) UACR in mice (\u003cem\u003en\u003c/em\u003e = 4–5). (K) Serum CRAMP levels by ELISA. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01; ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figures3.png","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/93633358564ea94d26aae7c4.png"},{"id":84695678,"identity":"0c5cc2cf-3f4f-4ef2-8b04-270bd1fc1410","added_by":"auto","created_at":"2025-06-16 10:38:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":493899,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeutrophil increase in mouse IgAN.\u003c/strong\u003e (A) The percentage of CD45\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e neutrophils in the peripheral blood of controls, IgAN, and 4 hours after LPS injection during disease model construction. ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001. (B) Ly6G immunostaining in kidney sections during IgAN model construction. Scale bar, 50 μm.\u003c/p\u003e","description":"","filename":"Figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/9cb24015dd4b6ba257ef3d19.png"},{"id":84695688,"identity":"e61f31d2-9c9b-4661-b67f-d2748f87e07b","added_by":"auto","created_at":"2025-06-16 10:38:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2655214,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRAMP derived from neutrophils was involved in the development of mouse IgAN.\u003c/strong\u003e CRAMP deficient (\u003cem\u003eCramp\u003c/em\u003e-/-) mice and WT mice were subjected to neutrophils adoptive transfer followed by IgAN model construction. (A) The schedule for neutrophils adoptive transfer and subsequent IgAN model construction. (B) Representative images of HE-stained, PAS-stained and immunostained kidneys. (C) Quantification of mesangial matrix fraction per mouse. (D-G) Semi-quantification of the intensity of immunofluorescence for IgA, IgM, IgG and C3 per mouse. (H and I) Serum BUN and creatinine levels in mice. (J) UACR in mice (n = 3). (K) Serum CRAMP levels by ELISA. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/595afa1a82307fca29903a67.png"},{"id":84695681,"identity":"d4a3ad3c-909e-44b4-96a9-1b22f0fe5c3a","added_by":"auto","created_at":"2025-06-16 10:38:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":909177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeutrophil-specific \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCramp\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockout alleviates glomerular lesions and albuminuria in mouse IgAN.\u003c/strong\u003e (A)Neutrophil-specific ablation of CRAMP (\u003cem\u003eCramp\u003c/em\u003e-NKO) mice and WT mice were subjected to IgAN model construction. (B) HE, PAS staining, and immunostaining of kidney tissues. (C) Mesangial matrix fraction quantification. (D-G) Semi-quantification of IgA, IgM, IgG, and C3 immunofluorescence intensity. (H) UACR in mice (\u003cem\u003en \u003c/em\u003e= 3-5). (I-J) Serum creatinine and BUN levels. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001; ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figures6.png","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/7c57df6ecc9adef349ac2ce4.png"},{"id":84696924,"identity":"e6a4c9d3-0939-4b85-8fdf-0ec276fa3b90","added_by":"auto","created_at":"2025-06-16 10:46:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1785770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlomerular endothelial cell injury observed in both mouse IgAN and human IgAN.\u003c/strong\u003e (A) Overview of the mouse kidney single-cell transcriptomics analysis workflow. After albuminuria analysis, mice were sacrificed, kidneys were isolated, followed by single-cell isolation and acquisition. RNA quality was analyzed, cDNA libraries were generated and sequenced, followed by quality control and data analysis. (B) Uniform Manifold Approximation and Projection (UMAP) analysis of 12 cell clusters. (C) Dot plot of the expression levels of representative marker genes across clusters. (D) Differences in cell percentage of renal parenchymal cells and immune cells in \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice and WT mice subjected to IgAN model construction. (E) Differences in cell proportion of immune cells in \u003cem\u003ecramp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice and WT mice subjected to IgAN model construction. The asterisk indicated elevated neutrophils. (F) Intrinsic glomerular cells (endothelial cells, podocytes, and mesangial cells) were reclustered into 8 clusters. (G) Differences in cell percentage of intrinsic glomerular cells in \u003cem\u003ecramp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice and WT mice subjected to IgAN model construction. The asterisk indicated reduced glomerular endothelial cells. (H) Representative CD31 immunohistochemistry images of human and mouse kidneys and quantification of CD31\u003csup\u003e+\u003c/sup\u003e glomerular endothelial cells. Five glomeruli were analyzed per kidney.\u003c/p\u003e\n\u003cp\u003ePT, proximal tubular cells; Macro, macrophages; T/NK, T cells, and natural killer cells; DCT/LOH, distal convoluted tubular cells and loop of Henle cells; Endo, endothelial cells; Pod, podocytes; CD collecting duct cells; lymph, lymphocyte; Mes, mesangial cells; Neu, neutrophils. GEC, glomerular endothelial cells; PEC, parietal epithelial cells; Efferent, endothelial cells from the efferent arterioles; Afferent, endothelial cells from the afferent arterioles; Proliferating, proliferating cells; HC, healthy control; NC, normal control.\u003c/p\u003e","description":"","filename":"Figures7.png","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/dde4051ffcdc192ef473d012.png"},{"id":84695687,"identity":"f3dd53a6-9ffa-42e6-a6e0-36e8e73c3394","added_by":"auto","created_at":"2025-06-16 10:38:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1472977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIgA-induced NETs injure endothelial cells in IgAN.\u003c/strong\u003e (A) Serum MPO-DNA complexes in IgAN-Stable and IgAN-ESRD patients. (B) Spearman correlation of serum MPO-DNA and LL37 levels. (C) NETs immunostaining in kidney tissues from IgAN patients and living donors. (D) Fluorescent Sytox green staining of NETs induced by pIgA1. (E) Cell viability measured by CCK-8 assay. (F) CD31 and VE-cadherin expressions in HUVECs by Western blot. (G) Flow cytometry of cell death (annexin V-FITC/PI). (H) NET-induced increase in transendothelial albumin passage. Scale bar, 50 μm.\u003c/p\u003e","description":"","filename":"Figures8.png","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/5e9c51a6b4bb0146a0560c9f.png"},{"id":84695689,"identity":"fd35012c-e043-4d98-a280-e048f6546dc1","added_by":"auto","created_at":"2025-06-16 10:38:11","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1538890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNET degradation reduces IgA deposition and albuminuria induced by pIgA injection in nude mice.\u003c/strong\u003e (A) pIgA1 injection schedule. (B) HE, PAS staining, and immunostaining of kidneys. (C) Serum dsDNA levels. (D) Mesangial matrix fraction quantification (\u003cem\u003en\u003c/em\u003e = 5-6). (E) Semi-quantification of IgA intensity. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figures9.png","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/b214590671127aab3af80947.png"},{"id":84695692,"identity":"f115e7e9-104e-4ac5-9a56-d9c242ce6795","added_by":"auto","created_at":"2025-06-16 10:38:11","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1323646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLL37-free NETs cause less endothelial injury.\u003c/strong\u003e (A) Immunostaining of MPO and LL37 in pIgA1-induced NETs. Scale bar, 50μm. (B) Representative images of the staining of Sytox green in neutrophils isolated from healthy controls or IgAN patients, \u003cem\u003ecramp\u003c/em\u003eWT, or KO neutrophils when treated with PMA, and the fluorescence intensities as measured using a microplate reader (\u003cem\u003en\u003c/em\u003e= 3). Scale bar, 50μm. (C) The expressions of CD31 and VE-cadherin in HUVECs determined by Western blot. (D) Cell death as measured by flow cytometry for annexin V-FITC/PI staining. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figures10.png","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/d31333081394abc914fc287d.png"},{"id":90970930,"identity":"1db24983-0465-41a4-bf4f-0a772b7f9778","added_by":"auto","created_at":"2025-09-10 07:39:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20799705,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/a8be5297-7cfe-4487-8b86-9ec800675b94.pdf"},{"id":84696921,"identity":"1192a718-8e66-48a6-8469-9049ef8950a5","added_by":"auto","created_at":"2025-06-16 10:46:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":64675,"visible":true,"origin":"","legend":"Graphical abstract","description":"","filename":"Graphicalabstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/c06320a32fd1dd4163a786a2.pdf"},{"id":84695679,"identity":"ac1390e6-95cb-44dd-b70f-7927369494c9","added_by":"auto","created_at":"2025-06-16 10:38:11","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":534283,"visible":true,"origin":"","legend":"SUPPLEMENTAL MATERIAL","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6636772/v1/f6d4e3a3a1f5100061aa442d.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Neutrophil-derived LL37 mediates IgA nephropathy via NETs-dependent mechanism","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIgA nephropathy (IgAN) is the most common form of primary glomerulonephritis globally, leading to end-stage renal disease (ESRD) in 30%-40% of affected patients within 20 years of diagnosis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. While the exact cause of IgAN remains unclear, the multi-hit theory is widely accepted\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This theory suggests that IgAN begins with aberrant glycosylation of IgA1, which leads to the overproduction of galactose-deficient IgA1 (Gd-IgA1). Autoantibodies then bind to Gd-IgA1, forming nephritogenic immune complexes that deposit in the mesangium. This results in mesangial cell activation, inflammation, and subsequent renal damage\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough pathogenic IgA deposition in the mesangium is a crucial event in IgAN pathogenesis, the mechanisms by which pathogenic IgA crosses the glomerular filtration barrier are not fully understood.\u003c/p\u003e \u003cp\u003eOur previous studies showed elevated levels of the antimicrobial peptide LL37 in the plasma of IgAN patients, distinguishing them from healthy individuals\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. LL37(\u003cem\u003eCAMP\u003c/em\u003e), a 37-amino acid peptide, is a major component of neutrophil tertiary granules and neutrophil extracellular traps (NETs), and CRAMP (\u003cem\u003eCramp\u003c/em\u003e) is the same in mice\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Clinical observations have also noted the presence of polymorphonuclear neutrophils in the glomerular capillaries of IgAN patients during episodes of macroscopic hematuria\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Furthermore, a high neutrophil-to-lymphocyte ratio (NLR) is an independent risk factor for ESRD in IgAN\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNETs have been implicated in the pathogenesis of several autoimmune diseases, including systemic lupus erythematosus (SLE), anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Given that IgAN is, at least in part, an autoimmune disease and IgA can activate neutrophils\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, it is crucial to explore the role of NETs in IgAN.\u003c/p\u003e \u003cp\u003ePrevious researches on LL37 focused on its antimicrobial role in urinary infections\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Recent studies have highlighted its immunomodulatory effects in ischemia/reperfusion-induced acute kidney injury\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In this study, we demonstrate that LL37 promotes the progression of IgAN by mediating NET-induced injury to glomerular endothelial cells, allowing the entry of pathogenic IgA into the mesangium.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman subjects\u003c/h2\u003e \u003cp\u003ePatients diagnosed with IgAN between January 2012 and December 2018 at the Kidney Disease Center, First Affiliated Hospital, Zhejiang University (ZJU cohort), were followed up. Of these, 52 patients progressed to end-stage renal disease (ESRD) (IgAN-ESRD group), while 503 remained stable (IgAN-Stable group), defined as a less than 30% decline in eGFR during follow-up. The study adhered to the principles of the Declaration of Helsinki. It was approved by the Clinical Research Ethics Committee(Approval No. 2022\u0026thinsp;\u0026minus;\u0026thinsp;1087), with informed consent exemption.\u003c/p\u003e \u003cp\u003eAdditionally, 101 IgAN patients from Queen Mary Hospital, University of Hong Kong (HKU cohort), and 379 from RWTH Aachen University, Germany (Aachen cohort), underwent LL37 serological testing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eC57BL/6J and BALB/c nude mice were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. \u003cem\u003eCramp\u003c/em\u003e knockout (KO) mice were provided by Professor Jia Sun of Jiangnan University\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLy6g\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e mice were generated by Cyagen Biosciences. Animal experiments were approved by the Animal Experiment Ethical Inspection (Approval No. 2022\u0026thinsp;\u0026minus;\u0026thinsp;1268).\u003c/p\u003e\n\u003ch3\u003eMouse models\u003c/h3\u003e\n\u003cp\u003eThe experimental IgAN model was induced as previously described\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Mice were sacrificed at week 9, and samples (24-hour urine, serum, and kidney) were collected.\u003c/p\u003e \u003cp\u003ePolymeric IgA1 (pIgA1) was purified from the sera of IgAN patients, as previously described\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. BALB/c nude mice received intravenous injections of 0.5 mg pIgA1 every 3\u0026ndash;4 days for two weeks before being euthanized for sample collection\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eBone marrow transplantation and neutrphil adoptive transfer\u003c/h3\u003e\n\u003cp\u003eSix- to eight-week-old male wild-type (WT) or \u003cem\u003eCramp\u003c/em\u003e KO recipient mice were lethally irradiated (10 Gy) and subsequently received an intravenous injection of 2\u0026times;10⁷ bone marrow cells from either WT or \u003cem\u003eCramp\u003c/em\u003e KO mice 4 hours later. Experiments were initiated four weeks after bone marrow reconstitution. Neutrophil adoptive transfer were taken as previously described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eSerum LL37 levels were measured using ELISA (Novus, NBP3-06932). Myeloperoxidase-DNA (MPO-DNA) complex levels were quantified as previously described\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKidney histology\u003c/h2\u003e \u003cp\u003eKidneys were harvested, fixed in 4% formalin overnight, dehydrated, and paraffin-embedded. Tissue sections were stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) for histopathological analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry and immunofluorescence study\u003c/h3\u003e\n\u003cp\u003eParaffin-embedded and immunofluorescence sections were both taken as the usual protocol. The primary antibodies used were rabbit anti-human Cathelicidin (1:50, PA5-20513, Invitrogen); mouse anti-human CD16/FITC (1:100, sc-19620, Santa Cruz); goat anti-mouse IgA/FITC (1:200, Abcam, ab97234); goat anti-mouse IgM-Alexa Fluor 488 (1:1000, Abcam, ab150121); goat anti-mouse IgG-Alexa Fluor Plus 555 (1:500, Invitrogen, A32727); rat anti-mouse C3 (1:50, Abcam, ab11862); rat anti-mouse Ly6G-FITC (1:250, eBioscience, 11-9668-82); rabbit anti-Histone H3 (1:900, Abcam, ab5103); and mouse anti-human LL37 (1:50, Hycult Biotech, HM2070). Images were captured using an immunofluorescence microscope (Leica DM4000) or a confocal microscope (Leica TCS-SP8).\u003c/p\u003e\n\u003ch3\u003eFlow cytometry analysis\u003c/h3\u003e\n\u003cp\u003eFlow cytometry was performed on mouse blood and Peyer\u0026rsquo;s patches. The following antibodies were used: CD45.1 (A20), CD45.2 (104) from BioLegend, CD45 (30-F11), Ly6G (1A8) from eBioscience, mouse CD19 (Biolegend, 6D5) and CD138 (Biolegend, 281-2), anti-mouse IgA (Bethyl, A90-103F). Samples were analyzed on a BD FACS Canto II device (BD Bioscience), and data were processed using FlowJo software.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSingle-cell RNA sequencing\u003c/h2\u003e \u003cp\u003eThe mice kidneys were isolated, followed by single-cell isolation and acquisition. The acquired single cell suspension at a concentration of 300\u0026ndash;600 cells/\u0026micro;L was loaded onto the 10x Chromium Single Cell controller. Barcoding and cDNA synthesis were performed according to the manufacturer\u0026rsquo;s instructions. The cDNA libraries were constructed using Single Cell 3\u0026rsquo; Library and Gel Bead Kit V3.1 and sequenced using an Illumina Novase6000 sequencer (performed by Capital Technology, Beijing). Downstream analyses were performed in R (4.1.1) using the Seurat package (3.0). To access technical variability between samples, an initial UMAP projection was generated using all 19,968 cells. The highly variable genes were selected using the Find Variable Features function. Clustering was performed using the Find Clusters function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell cultures\u003c/h2\u003e \u003cp\u003eHUVECs were purchased from Cell Bank of Chinese Academy of Sciences and cultured in DMEM(Sigma) with 10% FBS(Gibco)and 1% antibiotic solution (Solarbio). HL-60s and \u003cem\u003eCAMP\u003c/em\u003e KO HL-60s were purchased from Cyagen Biosciences and cultured in IMDM(Gibco) supplemented with 15% FBS(Gibco) and 1% antibiotic solution (Solarbio). HL-60s and \u003cem\u003eCAMP\u003c/em\u003e KO HL-60s were treated with 1.25% dimethyl sulfoxide for 6 days for differentiation to dHL-60 cells and \u003cem\u003eCAMP\u003c/em\u003e KO dHL-60s.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eHUVECs were lysed in radio immunoprecipitation assay buffer (Beyotime Biotechnology) supplemented with protease inhibitor cocktail (Beyotime Biotechnology). The primary antibodies against CD31 (1:1000, M1511-8, HUABIO), VE-Cadherin (1:1000, 2500S, CST) or β-actin (1:1000, sc-58673, Santa Cruz) were used. The target proteins were visualized using enhanced chemiluminescence substrate reagents (Millipore, USA). Semiquantitative analysis of western blot images was performed with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis detection\u003c/h2\u003e \u003cp\u003eCell death was evaluated by flow cytometry following annexin V-FITC/propidium iodide (PI) double staining according to the manufacturer\u0026rsquo;s protocol. The samples were analyzed by flow cytometry (BD Bioscience).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTransendothelial albumin passage\u003c/h2\u003e \u003cp\u003eA transwell chamber with a membrane pore size of 0.4 \u0026micro;m (Corning) was used to evaluate transendothelial albumin passage. HUVECs were seeded in the upper chamber and treated with NETs for 24 h. Then, the medium in the upper chamber was replaced by serum-free medium containing 0.5 mg/mL FITC-labeled BSA (Solarbio, SF063), and the medium in the lower chamber was replaced with serum-free medium containing 0.5 mg/mL unlabeled BSA (Sigma-Aldrich). After 1 h, the upper chamber were removed from the wells, and fluorescence was determined at 495 nm excitation and 525 nm emission using a fluorescence microplate reader (TECAN).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNeutrophil isolation, NETs induction and detection\u003c/h2\u003e \u003cp\u003eNeutrophils were isolated with a human neutrophil isolation kit (STEMCELL, 19666) or a mouse neutrophil isolation kit (STEMCELL, 19762) following the manufacturer\u0026rsquo;s instructions. NETs were quantified utilizing a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P11496) and verified through staining with Sytox green (Thermo Fisher Scientific, S7020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using SPSS, version 26.0. The figures were constructed by GraphPad Prism 9.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLL37 is elevated in IgAN patients, is predominantly derived from neutrophils, and correlates with the severity of IgAN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 555 IgAN patients and 38 healthy volunteers were included in this study. Baseline clinicopathological characteristics are detailed in Supplemental Table\u0026nbsp;1.\u003c/p\u003e\n\u003cp\u003eTo investigate LL37 gene expression in IgAN, we first analyzed data from the Nephroseq renal transcriptomics database and found a significantly higher transcription levels of \u003cem\u003eCAMP\u003c/em\u003e–the gene encoding LL37–in the peripheral blood of IgAN patients compared to healthy controls (Fig.\u0026nbsp;1A). In our cohort, serum LL37 levels were particularly elevated in IgAN-ESRD patients compared to IgAN-Stable patients and healthy controls (\u003cem\u003ep\u003c/em\u003e = 0.0078 and \u003cem\u003ep\u003c/em\u003e = 0.0029, respectively, Fig.\u0026nbsp;1B). Correlation analysis showed serum LL37 levels were positively correlated with serum creatinine and UPCR (\u003cem\u003ep\u003c/em\u003e = 0.0011 and \u003cem\u003ep\u003c/em\u003e = 0.0069, respectively, Fig.\u0026nbsp;1C and Fig.\u0026nbsp;1D).\u003c/p\u003e\n\u003cp\u003ePatients were further stratified by LL37 levels into high and low groups. Those with higher LL37 exhibited greater urinary protein levels, elevated serum creatinine, reduced estimated glomerular filtration rate (eGFR), increased blood pressure, and worse Oxford Classification T scores (Table 1), indicating a significantly poorer long-term renal prognosis (HR = 2.60, \u003cem\u003ep\u003c/em\u003e = 0.002, Fig. 1E).\u003c/p\u003e\n\u003cp\u003eWhile LL37 can be produced by eosinophils and other cell types\u003csup\u003e20\u003c/sup\u003e, we also identified that patients with ESRD had higher neutrophil counts compared to those with stable conditions (Supplemental Table\u0026nbsp;2). Flow cytometry confirmed that LL37 expression was predominantly confined to neutrophils in IgAN patients, showing a threefold increase compared to healthy controls (Fig.\u0026nbsp;1F). Immunostaining demonstrated that LL37 was expressed primarily by CD16 + cells (Fig.\u0026nbsp;1G). Thus, neutrophils are a primary source of LL37 in patients with severe IgAN.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLL37 is pathogenic in a mouse model of IgAN.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then examined the functional role of LL37 in the pathogenesis of IgAN in a mouse model of IgAN. As demonstrated in Fig.\u0026nbsp;2, we successfully induced a mouse mode of IgAN, showing mesangial cell proliferation, IgA and C3 deposition in the mesangial regions, and elevated proteinuria (Figs.\u0026nbsp;2A–2G, 2I), which was associated with upregulation of CRAMP, the murine homolog of LL37 (Fig.\u0026nbsp;2H). To investigate the role of LL37 in IgAN pathogenesis, we generated CRAMP-deficient (\u003cem\u003eCramp\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e) mice on a C57BL/6J background. We found that although CRAMP deficiency did not affect normal renal morphology compared to wild-type (WT) mice (Figs.\u0026nbsp;2B–2G), they did protect from the development of IgAN by largely reducing mesangial IgA, IgM, IgG, and C3 deposition, mesangial cell proliferation, and albuminuria post-IgAN induction (Figs.\u0026nbsp;2B–2G, 2I). However, renal function remained stable in both WT and \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e mice, as the disease was in its early stages (Figs.\u0026nbsp;2J, 2K).\u003c/p\u003e\n\u003cp\u003eTo identify the CRAMP-producing cells responsible for IgAN development and further confirm the role of LL37 in the development of IgAN, we performed BMT to reconstitute the immune systems with or without LL37-producing cells in recipient mice, followed by IgAN induction (Fig.\u0026nbsp;3A). \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e mice receiving BMT from WT donors developed typical IgAN with a marked IgA, IgM, IgG, and C3 deposition, mesangial cell proliferation, and albuminuria (Figs.\u0026nbsp;3B–3H), although renal function remained comparable between groups (Figs.\u0026nbsp;3I, 3J). In contrast, WT mice receiving BMT from \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e donors exhibited a significant inhibition of IgAN (Figs.\u0026nbsp;3B–3H). Taken together, these results indicate that LL37 produced by immune cells is pathogenic in IgAN.\u003c/p\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003eNeutrophil-derived LL37 is responsible for the development of mouse IgAN\u003c/h2\u003e\n \u003cp\u003eBecause LL37 was primarily expressed by neutrophils in patients with IgAN (Fig.\u0026nbsp;1F, H) and BM-derived LL37 caused severe IgAN (Fig.\u0026nbsp;3), we further hypothesized that neutrophil-derived LL37 may play a key role in mouse IgAN. To test this, we first assessed neutrophil levels in IgAN mice. Like patients with IgAN (Fig.\u0026nbsp;1F, G), neutrophil counts were significantly elevated in IgAN mice compared to age-matched C57BL/6J controls (\u003cem\u003ep\u003c/em\u003e = 0.0001, Fig.\u0026nbsp;4A). Increased neutrophils were also observed during the acute inflammatory phase of IgAN (4 hours post-LPS administration in the sixth week, \u003cem\u003ep\u003c/em\u003e = 0.0001, Fig.\u0026nbsp;4A). Although no overt neutrophil infiltration was observed in the kidneys of IgAN mice, neutrophils were found in the glomeruli and tubulointerstitium during acute inflammation (4h, 24h, 48h post-LPS administration, Fig.\u0026nbsp;4B).\u003c/p\u003e\n \u003cp\u003eWe also performed neutrophils adoptive transfer to recipient mice, followed by IgAN induction (Fig.\u0026nbsp;5A). \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e mice receiving neutrophils from WT donors developed typical IgAN with a marked IgA, IgM, IgG, and C3 deposition, mesangial cell proliferation, and albuminuria (Figs.\u0026nbsp;5B–5H), although renal function remained comparable between groups (Figs.\u0026nbsp;5I, 5J). In contrast, WT mice receiving neutrphils from \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e donors exhibited a significant inhibition of IgAN (Figs.\u0026nbsp;5B–5H).\u003c/p\u003e\n \u003cp\u003eTo confirm the role of neutrophil-derived CRAMP, we crossed \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e mice with \u003cem\u003eLy6g\u003c/em\u003e-Cre mice to generate neutrophil-specific LL37 knockout mice (\u003cem\u003eCramp\u003c/em\u003e-NKO mice). Like global LL37 KO mice (Fig.\u0026nbsp;2), conditional knockout of \u003cem\u003eCramp\u003c/em\u003e from neutrophils largely reduced IgA, IgM, IgG, and C3 deposition, mesangial cell proliferation, and albuminuria (Fig.\u0026nbsp;6A–6G), although renal function remained unaffected (Figs.\u0026nbsp;6H, 6I). The neutrophils from WT mice with CRAMP expression could cause the IgAN symptoms in \u003cem\u003eCramp\u003c/em\u003e \u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLy6g\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e mice (Fig.\u0026nbsp;6B). These findings confirm that neutrophil-derived LL37 is essential for IgAN development in mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003eGlomerular endothelial cells were key targets\u003c/h2\u003e\n \u003cp\u003eSingle-cell RNA sequencing (scRNA-seq) was performed on kidneys from WT and \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e mice following IgAN induction (Fig.\u0026nbsp;7A). After rigorous quality control, we analyzed 10,472 cells from IgAN mice and 9,496 cells from controls. Clustering identified 12 distinct cell types, including renal parenchymal cells (proximal tubular cells, distal convoluted/loop of Henle cells, endothelial cells, podocytes, and mesangial cells) and immune cells (macrophages, T cells/NK cells, B cells, and neutrophils) (Figs.\u0026nbsp;7B, 7C). Compared to controls, IgAN mice displayed increased neutrophil and macrophage infiltration (Figs.\u0026nbsp;7D, 7E), supporting the role of neutrophils in IgAN pathogenesis via LL37.\u003c/p\u003e\n \u003cp\u003eTo further investigate changes in glomerular cells, we reclustered endothelial cells, podocytes, and mesangial cells, identifying eight cell clusters and six different cell types (Fig.\u0026nbsp;7F). The proportion of GECs significantly decreased in IgAN mice (Fig.\u0026nbsp;7G), indicating that GECs are likely the most affected cell type, consistent with recent scRNA-seq studies\u003csup\u003e21\u003c/sup\u003e. Immunohistochemical staining confirmed that CD31 + GECs were significantly reduced in IgAN patients and mice (Fig.\u0026nbsp;7H). Previous studies have highlighted GEC injury as a critical factor in IgAN pathogenesis\u003csup\u003e21–24\u003c/sup\u003e, so subsequent mechanistic studies focused on GEC damage.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eLL37 may interact with NETs to induce endothelial cell injury\u003c/strong\u003e \u003cstrong\u003ein vivo\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAs NETs are major carriers of LL37 and are known to cause endothelial dysfunction\u003csup\u003e6,9,19\u003c/sup\u003e, we hypothesized that LL37 exacerbates IgAN by amplifying NET-induced damage to GECs. We measured serum levels of MPO-DNA complexes, a marker for NETs, and found that these MO)-DNA complexes were elevated in IgAN-ESRD patients compared to IgAN-Stable patients (\u003cem\u003ep\u003c/em\u003e = 0.047, Fig.\u0026nbsp;8A). Correlation analysis showed MPO-DNA levels positively correlated with LL37 (\u003cem\u003ep\u003c/em\u003e = 0.0074, Fig.\u0026nbsp;8B). Furthermore, NET marker CitH3 colocalized with LL37 in IgAN glomeruli (Fig.\u0026nbsp;8C).\u003c/p\u003e\n \u003cp\u003eFluorescent staining (Sytox green) revealed that pIgA1 from IgAN patients induced NET formation, whereas pIgA1 from healthy controls did not (Fig.\u0026nbsp;8D). Purified pIgA1-induced NETs were cytotoxic to HUVECs in a dose-dependent manner, but LL37 alone had no direct effect (Fig.\u0026nbsp;8E). NET treatment reduced the endothelial integrity markers CD31 and VE-Cadherin (Fig.\u0026nbsp;8F) and induced cell death (Fig.\u0026nbsp;8G). To mimic the transendothelial migration of IgA-containing immune complexes, we conducted a transwell assay using albumin as a proxy. Results showed that NETs increased albumin passage across the endothelial barrier (Fig.\u0026nbsp;8H).\u003c/p\u003e\n \u003cp\u003eAdditionally, intravenous injection of pIgA1 from IgAN patients into immunodeficient mice resulted in stable IgA deposition and mesangial cell proliferation (Fig.\u0026nbsp;9A, 9B). Treatment with DNase I, which degrades NETs, alleviated both mesangial proliferation and IgA deposition in the IgAN model (Figs.\u0026nbsp;9C–9E).\u003c/p\u003e\n \u003cp\u003eWe next investigated how LL37 levels influence NET formation and their cytotoxicity to endothelial cells. LL37 was confirmed to be present in pIgA1-induced NETs (Fig.\u0026nbsp;10A). Neutrophils from IgAN patients generated more NETs compared to those from healthy donors (Fig.\u0026nbsp;10B). Similarly, neutrophils from \u003cem\u003eCramp\u003c/em\u003e KO mice showed reduced NET formation compared to WT under the same stimulation (Fig.\u0026nbsp;9B).\u003c/p\u003e\n \u003cp\u003eSince neutrophils are terminally differentiated and unsuitable for RNA interference or gene editing, we used the HL-60 promyelocytic cell line. After inducing differentiation with 1.25% DMSO for six days (Supplemental Fig.\u0026nbsp;1), we generated \u003cem\u003eCAMP\u003c/em\u003e KO HL-60 cells via CRISPR/Cas9. Both differentiated HL-60 (dHL-60) and \u003cem\u003eCAMP\u003c/em\u003e KO HL-60 cells were stimulated with PMA to induce NET formation. Purified NETs and LL37-free NETs were then applied to HUVECs. Compared to standard NETs, treatment with LL37-free NETs significantly inhibited endothelial cell injury by preventing the loss of endothelial markers CD31 and VE-Cadherin (Fig.\u0026nbsp;10C) and cell death (Fig.\u0026nbsp;10D). These findings suggest that LL37 expression contributes to NET formation and cytotoxicity to endothelial cells.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our study, we found that serum levels of LL37 were elevated in patients with active IgAN, which was associated with a significant increase in urinary protein, serum creatinine, blood pressure, and higher T scores, and correlated with poor long-term kidney survival and a loss of glomerular endothelial cells. Functionally, LL37 is pathogenic in IgAN as mice lacking LL37 were protected from the development of severe IgAN by largely reducing IgA and C3 deposition, mesangial cell proliferation, and proteinuria, which was reversed by BMT from WT but not from \u003cem\u003eCramp\u003c/em\u003e KO mice. Further study uncovered that LL37 were primarily derived from neutrophils as evidenced in \u003cem\u003eCramp\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLy6\u003c/em\u003eg\u003csup\u003ecre\u003c/sup\u003e mice in which specific deletion of LL37 from neutrophils also suppressed the development of IgAN. Mechanistically, we found that LL37 acted via NETs to cause endothelial cell injury by reducing CD31 expression but increasing the permeability, which was demonstrated by the addition of NETs, but not LL37-free NETs to cause endothelial damage under high pIgA1 conditions \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe most critical finding in the present study is the discovery of the pathogenic role of LL37, particularly neutrophil-derived LL37, in IgAN. Our clinical data showed increased \u003cem\u003eCAMP\u003c/em\u003e expression in PBMCs from the renal transcriptomics database Nephroseq (GSE14795, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). ELISA analysis also found that serum levels of LL37 increased in IgAN patients from the ZJU cohort, which was further confirmed in the HKU and Aachen cohorts, particularly in newly diagnosed patients, but not in prolonged treatment. Neutrophils, the first line of immune defense\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, are highly reactive but sensitive to external factors like drugs and diseases\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, making them prone to instability\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This may explain why LL37 has early diagnostic potential but is limited in real-time monitoring during treatment.\u003c/p\u003e \u003cp\u003eUsing flow cytometry, immunofluorescence, and scRNA sequencing, we confirmed neutrophils as the primary source of LL37. Neutrophils produce three times more LL37 than other cells, consistent with previous studies\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Thus, LL37-expressing neutrophils are a crucial contributor to IgAN pathogenesis.\u003c/p\u003e \u003cp\u003eWe validated the role of LL37/CRAMP using various genotypes and IgAN mouse models. WT mice exhibited IgAN features following CCL4/LPS induction, while \u003cem\u003eCramp\u003c/em\u003e KO mice and \u003cem\u003eCramp\u003c/em\u003e \u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLy6g\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e mice showed reduced IgAN symptoms. Bone marrow transplants confirmed that \u003cem\u003eCramp\u003c/em\u003e KO neutrophils did not induce IgAN in WT mice, but the neutrophils from WT mice with CRAMP expression caused severe IgAN symptoms in \u003cem\u003eCramp\u003c/em\u003e \u003csup\u003efl/fl\u003c/sup\u003e\u003cem\u003eLy6g\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e mice. Additionally, nude mice injected with pIgA1 displayed increased LL37/CRAMP expression, emphasizing neutrophil-derived LL37's specific role in IgAN pathogenesis.\u003c/p\u003e \u003cp\u003eTo investigate the relationship between LL37/CRAMP and pathogenic IgA, we analyzed IgAN mice, which had a higher frequency of plasma cells and IgA\u0026thinsp;+\u0026thinsp;plasma cells in Peyer\u0026rsquo;s patches compared to age-matched controls. However, \u003cem\u003eCramp\u003c/em\u003e knockout did not affect this increase (Supplemental Fig.\u0026nbsp;2A\u0026ndash;2D). In IgAN patients, serum LL37 levels did not significantly correlate with Gd-IgA1 or total IgA (Supplemental Fig.\u0026nbsp;2E and 2F), indicating that LL37 may not involve the \"first hit\" of IgAN pathogenesis.\u003c/p\u003e \u003cp\u003eThen, we verified the possible mechanisms of LL37 mediates IgAN via NETs. Neutrophils play a pivotal role in the pathogenesis of various diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Initially, LL37 was studied for its antimicrobial properties in urinary infections\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, but recent research identified its immunomodulatory role in ischemia/reperfusion-induced acute kidney injury\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The role of NETs in diseases like diabetes and IgAN has also been increasingly recognized\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Our findings from scRNA sequencing and immunohistochemistry revealed decreased glomerular endothelial cells in IgAN. This reduction was linked to NETs amplified by LL37/CRAMP, as confirmed by MPO-DNA analysis in IgAN patients and ds-DNA analysis in IgAN mouse models. It is possible that NET-induced endothelial injury may enhance albumin passage through the endothelial barrier post-NET stimulation by LL37. Reduced NET formation and endothelial apoptosis in \u003cem\u003eCAMP\u003c/em\u003e KO HL-60 cells support the notion that LL37-amplified NETs promote glomerular endothelial injury, facilitating mesangial IgA deposition in IgAN.\u003c/p\u003e \u003cp\u003eThere are several shortcomings in this study. First, the CCL4/LPS-induced IgAN mouse model may not fully represent the IgAN in patients clinically, although it is suitable for analyzing LL37/CRAMP genotypes and NET stimulation. The use of nude mice injected with pIgA1 may be more appreciative. Second, HUVECs were used in place of glomerular endothelial cells, which may not fully represent the bioactivities of glomerular endothelial cells.\u003c/p\u003e \u003cp\u003eAs a new target, LL37 can reduce the pathological IgA deposition in the mesangial area by attenuating the damage to endothelial cells and the glomerular filtration barrier. It is unlikely that mono-therapy will be effective for all patients with IgAN. In the future, multi-specific targeted therapy may be used for IgAN to alleviate and control disease progression, improve disease prognosis, and minimize adverse drug reactions.\u003c/p\u003e \u003cp\u003eNevertheless, our findings provide new insights into IgAN pathogenesis and suggest that NET-targeted therapies may have clinical value in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Huadong Medicine Joint Funds of the Zhejiang Provincial Natural Science Foundation of China under Grant No. LHDMZ23H100001, and National Natural Science Foundation of China (82070729, 81170697, 81470938, 82100738, 82471713), and 20243BBI91026.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLai, K. 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C., Pena, O. M. \u0026amp; Hancock, R. E. W. Host defense peptides: front-line immunomodulators. \u003cem\u003eTrends Immunol\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 443\u0026ndash;450 (2014).\u003c/li\u003e\n\u003cli\u003eZambrano, S. \u003cem\u003eet al.\u003c/em\u003e Molecular insights into the early stage of glomerular injury in IgA nephropathy using single-cell RNA sequencing. \u003cem\u003eKidney Int\u003c/em\u003e \u003cstrong\u003e101\u003c/strong\u003e, 752\u0026ndash;765 (2022).\u003c/li\u003e\n\u003cli\u003eMakita, Y. \u003cem\u003eet al.\u003c/em\u003e Glomerular deposition of galactose-deficient IgA1-containing immune complexes via glomerular endothelial cell injuries. \u003cem\u003eNephrol Dial Transplant\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 1629\u0026ndash;1636 (2022).\u003c/li\u003e\n\u003cli\u003eKusano, T. \u003cem\u003eet al.\u003c/em\u003e Endothelial cell injury in acute and chronic glomerular lesions in patients with IgA nephropathy. \u003cem\u003eHum Pathol\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 135\u0026ndash;144 (2016).\u003c/li\u003e\n\u003cli\u003eYang, Y.-C., Fu, H., Zhang, B. \u0026amp; Wu, Y.-B. Interleukin-6 Downregulates the Expression of Vascular Endothelial-Cadherin and Increases Permeability in Renal Glomerular Endothelial Cells via the Trans-Signaling Pathway. \u003cem\u003eInflammation\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 2544\u0026ndash;2558 (2022).\u003c/li\u003e\n\u003cli\u003eZhu, Y. P. \u003cem\u003eet al.\u003c/em\u003e NET formation is a default epigenetic program controlled by PAD4 in apoptotic neutrophils. \u003cem\u003eScience Advances\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, eadj1397 (2023).\u003c/li\u003e\n\u003cli\u003eNg, M. S. F. \u003cem\u003eet al.\u003c/em\u003e Deterministic reprogramming of neutrophils within tumors. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e383\u003c/strong\u003e, eadf6493 (2024).\u003c/li\u003e\n\u003cli\u003eLiu, Y. \u003cem\u003eet al.\u003c/em\u003e The RNA m6A demethylase ALKBH5 drives emergency granulopoiesis and neutrophil mobilization by upregulating G-CSFR expression. \u003cem\u003eCell Mol Immunol\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 6\u0026ndash;18 (2024).\u003c/li\u003e\n\u003cli\u003eHe, M. \u003cem\u003eet al.\u003c/em\u003e Serum amyloid A promotes glycolysis of neutrophils during PD-1 blockade resistance in hepatocellular carcinoma. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1754 (2024).\u003c/li\u003e\n\u003cli\u003eHidalgo, A., Chilvers, E. R., Summers, C. \u0026amp; Koenderman, L. The Neutrophil Life Cycle. \u003cem\u003eTrends in Immunology\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 584\u0026ndash;597 (2019).\u003c/li\u003e\n\u003cli\u003eNg, L. G., Ostuni, R. \u0026amp; Hidalgo, A. Heterogeneity of neutrophils. \u003cem\u003eNat Rev Immunol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 255\u0026ndash;265 (2019).\u003c/li\u003e\n\u003cli\u003eWong, S. L. \u003cem\u003eet al.\u003c/em\u003e Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 815\u0026ndash;819 (2015).\u003c/li\u003e\n\u003cli\u003eWang, Y. \u003cem\u003eet al.\u003c/em\u003e GSDMD-dependent neutrophil extracellular traps promote macrophage-to-myofibroblast transition and renal fibrosis in obstructive nephropathy. \u003cem\u003eCell Death Dis\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1\u0026ndash;15 (2022).\u003c/li\u003e\n\u003cli\u003eMihlan, M. \u003cem\u003eet al.\u003c/em\u003e Neutrophil trapping and nexocytosis, mast cell-mediated processes for inflammatory signal relay. \u003cem\u003eCell 187, 5316-5335.e28 (2024).\u003c/em\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1. Baseline clinicopathologic characteristics of IgAN patients grouped by LL37 concentration\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"634\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow LL37 Concentration\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 50)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh LL37 Concentration\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003en\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;= 50\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eMale, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e20 (40.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e22 (44.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eAge (yr), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e34.00 (28.75\u0026ndash;43.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e33.00 (27.75\u0026ndash;43.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eUPCR (g/g), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e0.94 (0.50\u0026ndash;1.88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e2.23 (0.82\u0026ndash;3.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eSerum creatinine (\u0026mu;M), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e80.50 (64.50\u0026ndash;118.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e135.50 (75.25\u0026ndash;194.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eeGFR (mL/min per 1.73 m\u003csup\u003e2\u003c/sup\u003e), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e85.08 (61.06\u0026ndash;110.87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e45.71 (30.26\u0026ndash;91.06)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eSBP (mmHg), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e123.50 (116.00\u0026ndash;138.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e138.00 (124.00\u0026ndash;158.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eDBP (mmHg), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e78.50 (70.75\u0026ndash;89.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e88.50 (76.25\u0026ndash;104.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eOxford classification, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e8 (16.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e13 (26.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e2 (4.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e4 (8.16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e44 (88.00）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e41 (83.67）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eT1-T2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e11 (22.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e25 (51.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eC1-C2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e24 (48.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e20 (40.81)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp; IQR, interquartile range; UPCR, urine protein to creatinine ratio; NA, not available; eGFR: estimated glomerular filtration rate; SBP: systolic blood pressure; DBP: diastolic blood pressure; M1, mesangial hypercellularity; E1, endocapillary hypercellularity; S1, segmental glomerulosclerosis; T, tubular atrophy/interstitial fibrosis; T1, T 26%\u0026ndash;50%; T2, T \u0026gt;50%; C0, no crescents; C1, crescents in \u0026lt;25% of glomeruli; C2, crescents in \u0026gt;25% of glomeruli.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NETs, LL37, IgA nephropathy, glomerular endothelial, pIgA deposition","lastPublishedDoi":"10.21203/rs.3.rs-6636772/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6636772/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIgAN is a common cause leading to end-stage renal disease. The deposition of pathogenic IgA in the glomerular mesangium is a critical step. However, the mechanisms of pathogenic IgA crossing the glomerular filtration barrier remain unclear. We hypothesized that neutrophil-derived LL37 is pathogenic and mediates IgAN through NETs-dependent mechanism. Serum LL37 were elevated in patients with active IgAN and correlated with poor long-term kidney survival (HR\u0026thinsp;=\u0026thinsp;2.54). Functionally, LL37 is pathogenic in IgAN as mice lacking LL37 were protected from the development of severe IgAN by essentially reducing IgA and C3 deposition, mesangial cell proliferation, and proteinuria, which was reversed by neutrophils from WT but not from Cramp KO mice. Further study uncovered that LL37 were primarily derived from neutrophils as evidenced in Crampfl/flLy6gcre mice in which specific deletion of LL37 from neutrophils also suppressed the development of IgAN. With scRNA we found that LL37 acted via NETs to cause endothelials injury but increasing the permeability in mechanism, which was demonstrated by the addition of NETs, but not LL37-free NETs under high pIgA1 conditions in vivo and in vitro. LL37 is elevated in IgAN patients, derived from neutrophils, and plays a pathogenic role in IgAN via a NETs-dependent mechanism.\u003c/p\u003e","manuscriptTitle":"Neutrophil-derived LL37 mediates IgA nephropathy via NETs-dependent mechanism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 10:38:06","doi":"10.21203/rs.3.rs-6636772/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4da44ec1-7ee7-486a-9963-cca7fa8cbd5a","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49862169,"name":"Biological sciences/Immunology"},{"id":49862170,"name":"Health sciences/Diseases/Kidney diseases/Glomerular diseases/IgA nephropathy"}],"tags":[],"updatedAt":"2025-09-10T07:31:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-16 10:38:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6636772","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6636772","identity":"rs-6636772","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}