A Novel Mouse Model of Idiopathic Nephrotic Syndrome Induced by Immunization with the Podocyte Protein Crb2.

BACKGROUND: The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process. METHODS: C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody. RESULTS: Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which colocalized with Crb2 at the podocyte foot process. A subset of mice developed features resembling FSGS after 18 weeks. In glomeruli of immunized mice and in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution. CONCLUSION: The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunologic pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.

A novel site-specific chemical conjugation of IgG antibodies by affinity peptide for immunoassays.

Recently, there has been an increasing interest in site-specific modifications of antibodies used in immunoassays for disease diagnosis and as antibody therapeutics, such as antibody-drug conjugates. Previously, we established a site-specific chemical conjugation system using an IgG-Fc binding chemical conjugation affinity peptide (CCAP). CCAP could be used only for the modification of human IgG owing to the lack of affinity of CCAP to rodent IgG molecules. In this study, novel CCAP reagents are proposed, which can be used for both human and mouse IgG, based on the Staphylococcus aureus protein A domain-derived affinity peptides Z34C and Z33. Compared with the activity of a conventional randomly modified antibody, mouse IgG modified using this method had favourable features in two immunoassays, demonstrating the advantages of the proposed CCAP method in preserving antibody functionality during conjugation.

Serum podocalyxin levels correlate with carotid intima media thickness, implicating its role as a novel biomarker for atherosclerosis.

Podocalyxin is a cell surface sialomucin, which is expressed in not only glomerular podocytes but also vascular endothelial cells. Urinary podocalyxin is used as a marker for glomerular disease. However, there are no reports describing serum podocalyxin (s-Podxl) levels. Therefore, the association between s-Podxl levels and clinical parameters were examined with 52 patients. s-Podxl level was evaluated using enzyme-linked immunosorbent assay. The median s-Podxl level was 14.2 ng/dL (interquartile range: 10.8-22.2 ng/dL). There were significant correlations (correlation coefficient: r > 0.2) of s-Podxl levels with carotid intima media thickness (IMT) (r = 0.30, p = 0.0307). Multiple logistic regression analysis showed that s-Podxl levels remained significantly associated with carotid IMT > 1 mm (OR: 1.15; 95% CI 1.02-1.31, p = 0.026) after adjustments for traditional cardiovascular risk factors such as age, sex, current smoking status, hypertension, dyslipidemias, and diabetes. In conclusion, s-Podxl is independently associated with carotid IMT and might be used as a novel biomarker for cardiovascular disease.