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Abstract: SA-PO644

High-Resolution Imaging Approaches to Assess Colocalization of Immunoglobulin-Component Chains in Glomerular Immunodeposits in IgA Nephropathy

Session Information

Category: Glomerular Diseases

  • 1302 Glomerular Diseases: Immunology and Inflammation

Authors

  • Novak, Lea, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Hall, Stacy D., University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Rizk, Dana, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Moldoveanu, Zina, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Julian, Bruce A., University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Haas, Mark, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Novak, Jan, University of Alabama at Birmingham, Birmingham, Alabama, United States
Background

Routine immunofluorescence microscopy of glomerular immunodeposits in IgA nephropathy (IgAN) reveals IgA with variable amounts of IgG and/or IgM and usually complement C3. We have developed a new high-resolution confocal-microscopy approach to detect immunoglobulins in biopsy specimens. In earlier studies, we found IgG in all IgAN kidney biopsies, including those IgG-negative by routine immunofluorescence microscopy. This improved visualization of immunoglobulins enables studies of colocalization of their component light chains. Here, we evaluated our imaging approach for assessment of colocalization of IgG with kappa and lambda light chains in IgAN kidney biopsies.

Methods

Frozen-tissue sections (4 μm) of remnant kidney biopsies from 20 IgAN patients (3 IgG-positive and 17 IgG-negative by routine immunofluorescence) were fixed, blocked, and stained sequentially with IgG-Fc nanobody, mouse monoclonal antibody (mAb) specific for human kappa light chain, mouse mAb specific for human lambda light chain, and a nuclear stain. Using a high-resolution confocal microscope (Nikon A1R/SIM), z-stack images were acquired (objective 60x). For each slide, 60 regions of interest were selected in areas with IgG staining. Using Advanced NIS analysis software, Pearson’s correlation coefficient (PCC) and Mander’s overlap coefficient (MOC) colocalization data were collected for each slide.

Results

IgG was detected in all 20 biopsies. Single-optical-plane images showed colocalization of IgG with kappa and lambda light chains; this finding was confirmed by line-intensity profiles. IgG was not colocalized with only one type of light chain. Colocalization for IgG with kappa or with lambda light chain exhibited variability by PCC and MOC analyses but colocalization for IgG with kappa versus IgG with lambda light chain did not significantly differ.

Conclusion

High-resolution confocal microscopy and the use of sensitive immunofluorescence staining are suitable for study of glomerular immunodeposits in IgAN. Colocalization of IgG with kappa and lambda light chains in all IgAN kidney biopsies is consistent with polyclonal origin of these pathogenic IgG autoantibodies.

Funding

  • NIDDK Support