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A novel chronic ANCA associated vasculitis model suitable for targeting reveals matrisomal changes

ABSTRACT: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are severe inflammatory disorders that often lead to rapid and irreversible organ failure. Rapid progressive glomerulonephritis (RPGN) is a particularly frequent renal manifestation and often leads to extensive glomerular scarring and interstitial fibrosis, resulting in chronic kidney disease and end stage renal failure. Current management options of AAV and its sequelae are limited and common therapies have serious side effects that impair quality of life. A better understanding of the deleterious scarring process of AAV may help to identify novel mechanisms and potential targets for therapy. However, robust murine models of scarring RPGN are still lacking. Here, we present a novel murine model of severe RPGN that recapitulates both acute injury and the subsequent glomerular and interstitial scarring that is based on combined administration of antibodies against the glomerular basement membrane (GBM) and myeloperoxidase (MPO), and bacterial lipopolysaccharides (LPS). Renal injury presented with severe hematuria, glomerular necrosis and crescent formation at 12 days, and consequent glomerular scarring 29 days after initial treatment. We observed increased expression of matrisomal components such as collagens, fibronectin, tenascin-C, in accordance with human AAV as deduced from analysis of gene expression microarray data and tissue staining. Moreover, we observed tissue infiltration by neutrophils, macrophages, T cells and myofibroblasts upon injury. Inhibition of CXCR4 using AMD3100 led to histological and molecular changes in injury with reduced chemokine expression and lower immune cells activation. Using mass-spectrometric proteome analysis, we provide a comprehensive overview of molecular and cellular changes in our AAV model. Altogether, we demonstrate a novel RPGN model that enables the study of matrisomal changes both in disease and upon intervention, as exemplified via CXCR4 inhibition, which could prove to be a viable pharmacological tool and provide matrix as novel targeting opportunity.

INSTRUMENT(S): Exactive Plus

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Oliver Schilling

PROVIDER: MSV000088028 | MassIVE | Fri Aug 27 08:04:00 BST 2021

SECONDARY ACCESSION(S): PXD028173

REPOSITORIES: MassIVE

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Project description:Background: Uromodulin (Tamm-Horsfall) protein is secreted by the thick ascending limb into urine and crosses the basolateral membrane into the kidney interstitium and blood. Uromodulin may engage the kidney’s innate immune system with pro and anti-inflammatory effects described. Mutations in the UMOD gene are a cause of autosomal dominant tubulointerstitial kidney disease (ADTKD) due to protein misfolding and endoplasmic reticulum stress. We identified a family with a UMOD mutation (C106F) that leads to glomerular and interstitial inflammation atypical for ADTKD. Methods: To determine if the observed phenotype is due to mutant uromodulin induced changes in the innate or adaptive immune system, we developed a transgenic (tg) mouse with a homologous cysteine to phenylalanine mutation (C105F) in the UMOD gene using CRISPR-Cas9 gene editing. Results: LLC-PK1 cells expressing wt and mutant protein had increased basolateral secretion of protein compared with cells expressing wt or mutant protein alone. EM examination of mutant medium showed protein aggregates in contrast to filaments in wt medium. Immunoprecipitation of plasma uromodulin from mutant mice demonstrated aggregate formation not seen in wt mice. Tg/+ mice displayed increased F4/80+ mononuclear phagocyte (MP) and podocyte inflammasome activity at baseline. Tg/+ mice developed glomerular and interstitial matrix deposition, myofibroblast proliferation and increased Creatinine with aging. Following ischemia-reperfusion injury, tg/+ mice had no increase in inflammasome activation over baseline in contrast to wt littermates and displayed improved tubular repair and renal function at 7 days. Differential gene expression analysis by RNA sequencing of kidney MP revealed activation of the ATF4 mediated stress response pathway and genes associated with the development of tissue fibrosis. Conclusions: The C106F mutation results in glomerular and interstitial inflammatory kidney disease due to interaction of aggregated misfolded protein with mononuclear phagocytic cells resulting in ER stress response. The resulting alteration in the immune system leads to improved kidney repair following acute injury but fibrosis and chronic renal failure with aging.

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A novel chronic ANCA associated vasculitis model suitable for targeting reveals matrisomal changes

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