Abstract: SA-PO339
Generating a Mouse Model of Membranous Nephropathy (MN)
Session Information
- Glomerular Diseases: Immunology and Inflammation - III
October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1202 Glomerular Diseases: Immunology and Inflammation
Authors
- Rhoden, Samuel J., University of Manchester, Manchester, United Kingdom
- Lausecker, Franziska, University of Manchester, Manchester, United Kingdom
- Fresquet, Maryline, University of Manchester, Manchester, United Kingdom
- Adamson, Antony, University of Manchester, Manchester, United Kingdom
- Jowitt, Thomas A., University of Manchester, Manchester, United Kingdom
- Brenchley, Paul E., Manchester Royal Infirmary, Manchester, United Kingdom
- Lennon, Rachel, University of Manchester, Manchester, United Kingdom
Background
75% of patients with MN develop autoantibodies against PLA2R, which is expressed on podocytes. Autoantibody binding causes immune complex deposition in the glomerular basement membrane (GBM), podocyte effacement and the clinical phenotypes of proteinuria and nephrotic syndrome. The lack of an in vivo model for MN, due to rodents expressing minimal PLA2R on podocytes, has impeded the investigation of disease mechanisms and novel therapeutics. The aims of this study were to 1) develop a podocyte-specific PLA2R knock-in mouse and 2) induce MN through passive transfer of anti-PLA2R antibodies.
Methods
A transgene was created for insertion of a flag-tagged PLA2R fragment (NC3) containing the transmembrane and the first 5 extracellular domains, including the immunodominant epitope of PLA2R. Podocyte restricted expression of NC3 is driven by the NPHS2 promoter. Transgene positive pups were bred with BL6/J mice to spawn heterozygous knock-in mice. Kidneys, glomeruli and podocytes were isolated from subsequent offspring for analysis by reverse transcription PCR (RT-PCR), targeted locus amplification (TLA) sequencing, western blotting (WB), and immunohistochemistry (IHC). Induction of MN in PLA2R knock-in mice will be achieved by intravenous injection of human anti-PLA2R and analysed for glomerular deposition, proteinuria and complement activation.
Results
TLA sequencing confirmed successful in-frame insertion of the transgene in PLA2R knock-in mice. RT-PCR and WB analysis of kidneys and glomeruli demonstrated the expression of NC3 mRNA and protein in PLA2R knock-in mice. IHC of kidney sections using human anti-PLA2R antibodies indicated strong podocyte staining of NC3 in the glomerulus of PLA2R knock-in mice. PLA2R knock-in mice demonstrated no differences to wild type mice in regards to proteinuria, weight and histology under basal conditions. We anticipate that injection of MN patient IgG will induce the MN phenotype in PLA2R knock-in mice.
Conclusion
We have successfully developed a PLA2R knock-in mouse. Expression of NC3 did not induce a phenotype under basal conditions. Current work focusses on the induction of MN using patient IgG. This mouse model gives exciting prospects for validating the pathogenicity of anti-PLA2R antibodies and the role of immune complex formation, complement activation and GBM thickening in causing proteinuria.