Anti-ANXA1 monoclonal antibody, clone 3G2

Hypercalciuria is associated with kidney stone formation and impaired renal function. However, responses of renal tubular cells upon exposure to high-calcium environment remain largely unknown. We thus performed a proteomic analysis of altered proteins in renal tubular cells induced by high-calcium and evaluated functional significance of these changes. MDCK cells were maintained with or without 20 mM CaCl2 for 72 h. Cellular proteins were then analyzed by two-dimensional electrophoresis (2-DE) (n = 5 gels derived from 5 independent culture flasks per group). Spot matching and quantitative intensity analysis revealed 20 protein spots (from a total of 700) that were differentially expressed between the two groups. These altered proteins were then identified by Q-TOF-MS and MS/MS analyses, including those involved in calcium binding, protein synthesis, carbohydrate metabolism, lipid metabolism, cell proliferation, mitosis regulation, apoptosis, cell migration, oxidative stress, and ion transport. Protein network analysis and functional validation revealed that high-calcium-exposed cells had 36.5% increase in calcium oxalate monohydrate (COM) crystal-binding capacity. This functional change was consistent to the expression data in which annexin A1 (ANXA1), a membrane-associated calcium-binding protein, was markedly increased on the apical surface of high-calcium-exposed cells. Pretreatment with anti-ANXA1 antibody could neutralize this increasing crystal-binding capacity. Moreover, high-calcium exposure caused defects in cell proliferation and wound healing. These expression and functional data demonstrate the enhanced crystal-binding capacity but impaired cell proliferation and wound healing in renal tubular cells induced by high-calcium. Taken together, these phenomena may contribute, at least in part, to the pathogenic mechanisms of hypercalciuria-induced nephrolithiasis and impaired renal function. Our in vitro study offers several candidates for further targeted functional studies to confirm their relevance in hypercalciuria and kidney stone disease in vivo.

Rationale: Giant-cell arteritis (GCA) is a large-vessel vasculitis characterized by immune cell infiltration, yet the potential involvement of neutrophils has rarely been studied. Objective: We investigated whether alterations in neutrophil reactivity occurred in the pathogenesis of GCA or during its clinical management with a canonical glucocorticoid dose regimen during a 6-month period. Methods and Results: Blood samples were taken within 48 hours of therapy commencement and at weeks 1, 4, and 24 after glucocorticoid dose. Flow cytometric analysis revealed 3 distinct neutrophil populations and phenotypes. Within 48 hours of steroid treatment, neutrophils displayed an AnxA1(hi)CD62L(lo)CD11b(hi) phenotype, whereas week 1 neutrophils were AnxA1(hi)CD62L(lo)CD11b(lo) and displayed minimal adhesion to endothelial monolayers under flow, and week 24 (ie, lowest glucocorticoid dose) neutrophils were AnxA1(hi)CD62L(hi)CD11b(hi) with increased endothelial adhesion under flow. Week 24 plasma analyses showed high levels of C-X-C motif chemokine ligand 5, interleukin (IL) 8, IL-17, and IL-6. Importantly, comparison of week 1 and week 24 samples revealed a suppressive neutrophil effect on T-cell proliferation at the former time point only. Finally, in vitro incubation of naive neutrophils with concentrations of IL-6 and IL-17 quantified in GCA plasma at weeks 1 and 24 replicated this differential modulation of lymphocyte proliferation. Conclusions: This translational study highlights a novel clinical manifestation of GCA, with evidence for a neutrophil component and an escaped proinflammatory phenotype when glucocorticoid therapy is tapered. These results indicate potential involvement of neutrophils in GCA pathogenesis.