IgG4-related disease (IgG4-RD) is a recently recognized clinical entity characterized by a dense lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells with fibrosis affecting several organs.
The kidney is one of the most frequently affected organs in IgG4-RD in which tubulointerstitial nephritis (TIN) is the most representative disorder although a variety of glomerular lesions can sometimes overlap with TIN, such as membranous nephropathy (MN).
We previously described a 54-year-old male patient with IgG4-related disease manifesting as pancreatitis, Mikulicz disease that later developed nephrotic-range proteinuria, and MN.
The patient was negative for circulating anti-phospholipase A2 receptor (PLA2R) antibodies. Conversely, we found IgG3 reactivity against superoxide dismutase 2 (SOD2), previously identified as an idiopathic MN autoimmune.
In the renal tissue IgG3 and IgG4 deposited within the patient’s glomeruli.
Patients with IgG4-RD, particularly when associated with pancreatitis, have circulating IgG4 subclass antibodies that recognize carbonic anhydrase II (CAII).
Aims of the study
- To establish whether IgG4 anti-CAII are involved in the development of MN in IgG4-RD;
- To identify the cellular mechanisms by which IgG4 anti-CAII could lead to SOD2 enrichment on the podocyte plasma membrane;
- To recapitulate the pathogenetic chain of events in other IgG4-related disease patients.
The patient with IgG4-RD and MN was admitted to the Nephrology Unit of the Ospedali Riuniti, Bergamo, Italy. Additional four IgG4-RD patients with TIN (IgG4-RD1, IgG4-RD2 and IgG4-RD4) or without renal involvement (IgG4-RD3) were from the Faculty of Medicine, Fukuoka University, Japan). Eight renal biopsies from an uninvolved portion of kidney collected from tumor nephrectomy specimens were used as controls.
IgG subclasses reactivity in patients’ sera was assessed by Western blot analysis. IgG-subclass specificity was evaluated using an anti-human IgG4- or IgG3-HRP antibody (Life Technologies).
IgG purification from patients sera was performed through affinity chromatography using Affi-Prep-ProteinA (Bio-Rad Laboratories).
Conditionally immortalized human podocytes (kindly provided by Prof. M.A. Saleem, Children’s Renal Unit and Academic Renal Unit, University of Bristol, Southmead Hospital, Bristol) were differentiated for 12 days.
Podocytes intracellular pH was assessed by fluorimetric analysis of BCECF-AMAM (Molecular Probes, Invitrogen). Acetozolamide was used as positive control.
MitoTracker and JC-1 AM (Molecular Probes, Invitrogen) were used to monitor podocyte mitochondrial mass and membrane potential, respectively. H2O2 was used as positive control.
Mitochondrial ROS were measured using MitoSOXTM Red AM (Molecular Probes, Invitrogen), a live-cell-permeant mitochondrial superoxide indicator. H2O2 was used as positive control.
Here we proposed a two-stage model (Figure 6E) in which IgG4 binding to CAII alters pH leading to mitochondrial dysfunction and SOD2externalization. At a later stage SOD2 serves as a neoantigen for the binding of complement-fixing IgG3-subtype antibodies, contributing to the MN lesion likely favoured by individual genetic predisposition.