Autoimmune-mediated damage to the kidneys can be triggered by autoantibodies directed against renal proteins, as in the case of primary membranous nephropathy (MN) or Goodpasture syndrome, or may occur secondary as part of the wide-reaching effects of systemic autoimmune diseases such as vasculitis or systemic lupus erythematosus (SLE). The detection of specific autoantibodies in patient serum is of major importance for diagnosis and monitoring in both cases.

Organ specific autoantibodies in primary membranous nephropathy

Membranous nephropathy (MN) is a chronic disease characterized by the in-situ formation of immune complexes in the glomerular basement membranes, leading to the damage of the glomeruli and the disruption of the renal filter function. It results in proteinuria, frequently combined with hypoproteinemia, hypolipoproteinemia and the formation of edema (nephrotic syndrome). Most cases (70-80%) of MN belong to the idiopathic or primary form (pMN), while in 20-30% of patients another disease can be found that causes MN (secondary MN). Autoantibodies against phospholipase A2 receptor (PLA2R), which is expressed in podocyte cell membrane, are highly specific for pMN and are found in the serum of around 75% of patients at baseline. Two standardized assays are available to determine anti-PLA2R antibodies that are highly suitable for routine diagnostic purposes: a recombinant cell-based indirect immunofluorescence assay (RC-IFA) and an enzyme-linked immunosorbent assay (ELISA). In addition to differential diagnosis of primary and secondary MN, the ELISA-based quantification of PLA2R autoantibodies allows assessment of the disease activity and severity as well as prediction of the disease outcome (remission, relapse) and the risk of recurrence of MN after kidney transplantation. Studies analyzing the applicability of the two tests have been presented in a previous article of this blog.

Very recently, thrombospondin type-1 domain-containing 7A (THSD7A) was discovered as a second antigenic target in approx. 2.5 % to 5 % of patients with idiopathic MN. Importantly, these autoantibodies have exclusively been found in the anti-PLA2R-negative cohort. This suggests that these patients represent a distinct disease subgroup. No significant differences, however, have been found between the two patient cohorts except for a larger fraction of women in the anti-THSD7A-positive group.

Highly specific biomarker in Goodpasture syndrome: anti-GBM autoantibodies

The relevant antigenic target of autoantibodies against the glomerular basement membranes (GBM) is the non-collagenous 1 (NC1) domain of the alpha-3 chain of type IV collagen. The protein is mostly expressed in the lungs and kidneys, which is why symptoms of Goodpasture syndrome – progressive glomerulonephritis (GN) and lung hemosiderosis – are focused on these two organs. Anti-GBM are detected in more than 90% of patients with and in over 60% of patients without lung involvement. They can easily be detected by indirect immunofluorescence assays (IFA) using cryo-sections of primate kidney or by various monospecific assays based on the purified NC1 domain, e.g. ELISA.

Approximately a quarter of patients with anti-GBM antibodies also have anti-neutrophil cytoplasmic autoantibodies (ANCA). Therefore, it is recommended that anti-GBM and ANCA should be analyzed in parallel in patients with renal disease.

Glomerulonephritis and kidney failure in ANCA-associated vasculitis

ANCA are characteristic for a subgroup of vasculitides that manifests with inflammation of the small- and medium-sized blood vessels (ANCA-associated vasculitis, AAV). The kidneys are frequently affected by these systemic diseases, which may lead to acute kidney failure. Since the initial symptoms of AAV vary and are often unspecific, the serological determination of ANCA is an essential tool for the identification and differentiation of AAV.

The most important antigenic targets of ANCA are either proteinase 3 (PR3) or myeloperoxidase (MPO): PR3-ANCA are a sensitive and specific marker for Wegener’s granulomatosis, while MPO-ANCA occur in microscopic polyangiitis and Churg Strauss syndrome. An international consensus on ANCA testing recommends screening of patients with suspected vasculitis by means of indirect immunofluorescence assays (IFA) based on ethanol-fixed granulocytes and confirmation of positive findings by MPO- and PR3-ANCA specific assays. The EUROPLUS™ Granulocyte BIOCHIP Mosaics combine the conventional granulocyte substrates (ethanol and formalin fixation, HEp-2 cell + granulocytes) with single microdots of purified PR3 and MPO in one incubation field. It allows ANCA screening and confirmation with a single incubation and simultaneous exclusion of ANA interference by detecting antibody reactivities on HEp-2 cells.

A further major advance in ANCA testing is the development of an ELISA based on a novel PR3 diagnostic antigen, which reveals a particularly high sensitivity of 95% for PR3-ANCA at a specificity of 99%. The substrate consists of a mixture of human native (hn) PR3 and human  cell-expressed human recombinant (hr) designer PR3, exhibiting modified N- and C-terminal signal sequences as well as an inactivated enzymatic core. Compared to conventional Anti-PR3-ELISA this Anti-PR3-hn-hr ELISA is much more sensitive at a set specificity.

Antibodies against DNA and nucleosomes in lupus nephritis

Lupus nephritis is the term for renal inflammation in connection with SLE. Anti-nucleosome antibodies (ANuA) represent the first serological marker described in SLE with kidney involvement. They are particularly prevalent (79%) in patients with severe lupus nephritis requiring transplantation compared to less severe cases (18%) and SLE patients without nephritis (9%). While 1st generation test systems for the detection of ANuA demonstrated significant cross reactivities with sera from patients with progressive systemic sclerosis (PSS; specificity approx. 52%), ANuA ELISA of the 2nd generation reached specificities of up to 100% regarding blood donors and patients with PSS due to an innovative protocol for nucleosome purification.

Anti-dsDNA antibodies are found in 60-90% of SLE patients and represent the most established marker for the disease. An optimized test referred to as Anti-dsDNA-NcX ELISA has been developed based on a novel coating technology that applies the highly purified mononucleosomes used in the 2nd generation Anti-Nucleosomes ELISA as linker substance for the immobilization of dsDNA. The specific configuration of the Anti-dsDNA-NcXELISA provides a clear and authentic presentation of the major DNA epitopes, leading to a strong increase not only in sensitivity (67%) compared to conventional anti-dsDNA ELISA (42%), at a set specificity of 98%, but also in the detection of a considerable number of patient samples that are positive exclusively in the novel test system.

Mastroianni-Kirztajn et al., Front Immunol 2015, 6: 221.