Lupus nephritis is closely associated with in vivo autoantibody-binding to glomerular membrane-associated electron-dense structures (EDS). The biochemical nature and cellular origin of EDS are controversial, and definitive characterisation needs to be performed.
By using the terminal transferase biotin-dUTP nick end-labelling (TUNEL) assay at the electron microscopic level, we have traced extracellular chromatin within the glomerular basement membranes of nephritic (NZBxNZW)F1 mice. The TUNEL assay was subsequently used in combination with standard immune electron microscopy (IEM). To analyse why chromatin particles associate with membranes, we determined the affinity of nucleosomes and DNA for glomerular laminin, collagen IV and the mesangial matrix proteoglycan perlecan by surface plasmon resonance.
This intra-assay colocalisation TUNEL IEM demonstrated that autoantibodies fully colocalised with extracellular TUNEL-positive chromatin observed as EDS in glomerular membranes, similar to results obtained by the same technique applied to human lupus nephritis. Most importantly, these data validate the murine variant of lupus nephritis as a model to study origin of extracellular chromatin as a key element in human lupus nephritis. Kinetic analyses demonstrated that nucleosomes had a high affinity for collagen IV and laminin, but not for perlecan.
Collectively, these results provide firm evidence that dominant target structures for nephritogenic autoantibodies are constituted by TUNEL-positive chromatin associated with glomerular capillary and mesangial matrix membranes at high affinity.