ANCA-Associated Kidney Disease and Vasculitis

Clinicopathologic Classification

The recognition of AAV as a clinical entity predated the discovery of ANCAs. Hence, AAV was initially classified based on clinical manifestations and pathologic findings. Three subtypes of AAV have been historically defined: (1) granulomatosis with polyangiitis (previously known as Wegener’s granulomatosis ), (2) microscopic polyangiitis, and (3) eosinophilic granulomatosis with polyangiitis (previously known as Churg-Strauss syndrome ). All forms of AAV cause necrotizing small-vessel vasculitis and the same pathologic lesion in the kidney: a pauci-immune, necrotizing (and often crescentic) glomerulonephritis.

Granulomatosis with polyangiitis (GPA) is distinguished from MPA by the presence of extravascular granulomatous inflammation. Common clinical features of GPA include sinusitis, destructive nasal lesions, cavitary lung masses, and necrotizing glomerulonephritis. GPA is more commonly associated with PR3-ANCA than MPO-ANCA. Microscopic polyangiitis (MPA) is characterized by the absence of granulomatous inflammation and often presents with pauci-immune necrotizing glomerulonephritis and pulmonary hemorrhage. A subset of patients with isolated pauci-immune necrotizing vasculitis and no extrarenal manifestations (i.e., renal-limited vasculitis) is sometimes designated as MPA. MPA is more commonly associated with MPO-ANCA than PR3-ANCA.

Eosinophilic granulomatosis with polyangiitis (EGPA) is distinguished from other forms of AAV by the presence of asthma and eosinophilia in addition to extravascular granulomatous inflammation. Only approximately 40% of cases of EGPA are associated with ANCA, with MPO-ANCA being more common than PR3-ANCA. Clinical manifestations differ between ANCA-positive and ANCA-negative patients, and these subsets likely have a different underlying pathogenesis. Patients with EGPA and ANCA positivity are more likely to have vasculitic manifestations including glomerulonephritis and mononeuritis multiplex.

A clinicopathologic definition of disease can be limited in that granulomatous inflammation may be missed by sampling error or may not be present in the organ biopsied. Therefore, the decision to designate a patient with AAV as GPA or MPA is often inferred on the basis of clinical findings rather than confirmed by histopathology. This subjectivity can lead to variation across individual centers and specialties.

Serologic Classification

Another system for classifying AAV is based on ANCA serotype (MPO vs. PR3) rather than clinicopathologic features. Classification by ANCA serotype has several advantages including an inherent link to disease pathogenesis and a superior ability to predict clinically important outcomes. In genome-wide association studies, genetic variants associated with AAV correlate more strongly with ANCA serotype (MPO vs. PR3) than clinical phenotype (GPA vs. MPA). Certain disease manifestations are also closely linked with ANCA specificity. For example, renal-limited vasculitis and ANCA-associated interstitial lung disease predominantly occur in the setting of MPO-ANCA. Drug-associated AAV is uniformly associated with MPO-ANCA positivity, with dual positivity for MPO- and PR3-ANCA present in some cases, but essentially never PR3-ANCA alone.

ANCA serotype is also a predictor of clinically relevant outcomes, particularly relapse. In multiple retrospective series and clinical trials, PR3-ANCA consistently associates with a greater probability of relapse than MPO-ANCA. Moreover, ANCA serotype is a better predictor of relapse in most studies than the clinicopathologic designation of MPA or GPA. Clinical trials now routinely stratify randomization by ANCA serotype, and some future clinical trials are likely to enroll only patients with a single ANCA specificity.

The Pathogenic Role of ANCAs

There is substantial evidence that ANCAs are pathogenic and not merely biomarkers of AAV. Under normal conditions, the MPO and PR3 enzymes are sequestered in the cytoplasm of neutrophils in azurophilic (primary) granules. Priming of neutrophils by proinflammatory stimuli, such as tumor necrosis factor and complement factor C5a, leads to translocation of MPO and PR3 to the cell surface, where they can interact with circulating ANCAs. It has been hypothesized that infection can precipitate disease flare by leading to the priming of neutrophils in a patient with preexisting circulating ANCA.

In vitro, both MPO- and PR3-ANCA are able to activate primed neutrophils via binding of the target antigen at or near the cell surface and signaling through the Fcγ receptor. Activation of neutrophils by ANCA leads to the release of reactive oxygen species and lytic enzymes that mediate tissue damage. More recently, it has been appreciated that ANCA activation can promote the generation of neutrophil extracellular traps (NETs). NETosis is a unique form of cell death, whereby decondensed chromatin and associated granule proteins (including MPO and PR3) are released into the extracellular space, forming a web-like structure that can trap and kill invading pathogens. Components of NETs, predominantly histones and matrix metalloproteases, are toxic to the vascular endothelium and are thought to contribute to tissue injury in AAV. Moreover, NET formation increases exposure to MPO and PR3 antigens, which may further promote autoimmunity.

Strong evidence for the pathogenicity of ANCAs is also derived from animal models of AAV. In a classic experiment, passive transfer of MPO-ANCA into a recombinase-activating gene 2 (RAG-2)-deficient mouse, which lacks functioning B and T cells, was sufficient to reproduce the lesion of pauci-immune necrotizing glomerulonephritis seen in man. The pathogenic role of MPO-ANCA was substantiated in a natural human model, whereby placental transfer of MPO-ANCA from a mother with AAV resulted in pulmonary hemorrhage and RPGN in the neonate.

Given the central role of ANCA in the pathogenesis of AAV, it has been difficult to reconcile the observation that some patients with clinicopathologic features consistent with AAV do not have detectable ANCA. At least in some cases, this may be due to assay sensitivity. It has been demonstrated that ANCAs directed against specific epitopes on the MPO molecule are not detectable using commercially available assays. In other cases, particularly in those with predominantly granulomatous manifestations of disease, alternative pathogenic mechanisms not involving ANCA may predominate.

Development of ANCAs

The underlying mechanisms culminating in the production of autoantibodies targeting MPO or PR3 are not fully known; however, genetic studies and specific exposures that have been linked with ANCA production provide some insight. Antigens are presented to the adaptive immune system via human leukocyte antigen (HLA) class II molecules expressed on antigen-presenting cells. Given the central role of antigen presentation in the development of the adaptive immune response, it is not surprising that certain HLA variants have been linked to the development of autoimmunity. Indeed, specific HLA associations exist for a wide array of autoimmune diseases, including AAV. In two separate genome-wide association studies, the development of MPO-ANCA vasculitis was associated with HLA-DQ while PR3-ANCA vasculitis was associated with HLA-DP.

In addition to HLA variants, factors influencing the autoantigens themselves are also associated with the development of ANCAs. Variants in SERPINA1, the gene encoding alpha-1 antitrypsin, have been associated with the development of PR3-ANCA. Alpha-1 antitrypsin is a protease inhibitor that targets PR3, and genetic polymorphisms that result in attenuated activity of the protein translate into reduced PR3 clearance. This finding is consistent with the observation that patients with alpha-1 antitrypsin deficiency are at increased risk for developing PR3-ANCA. A variant in the gene encoding PR3 itself, which leads to increased PR3 expression, has also been linked to the development of PR3-ANCA in genome-wide association studies.

ANCA production can also be stimulated by a number of medications including propylthiouracil, methimazole, hydralazine, and minocycline. The exact mechanisms remain unclear, although a number of plausible hypotheses have been proposed. Patients on long-term propylthiouracil develop seropositivity for MPO-ANCA at a rate of close to 30% in some series, although only a small fraction of these patients develop clinical vasculitis. Propylthiouracil accumulates in neutrophils, where it interacts with MPO, thereby resulting in structural alterations that may increase antigenicity. In addition, propylthiouracil promotes the development of NETs that are resistant to degradation by DNase I, resulting in extended durations of antigen exposure.

Hydralazine has also been demonstrated to induce NETosis. Moreover, in addition to its role as a vasodilator, hydralazine is an inhibitor of DNA methylation. This property may prevent the epigenetic silencing of certain genes, including MPO and PR3 , which are important in the development of ANCA. Indeed, patients with active AAV exhibit hypomethylation at the MPO or PR3 genes relative to healthy controls and patients with AAV who are in remission.

Role of Complement

Patients with AAV generally exhibit normal levels of serum complement and a paucity of C3 and C1q deposition on kidney biopsy. These observations have obscured the important role of complement in the pathogenesis of AAV. The alternative complement pathway, however, has recently been identified as a central and necessary mediator of the inflammatory cascade in AAV.

Experiments conducted with animal models provide the framework for the current understanding of complement in AAV. In a passive anti-MPO IgG transfer model, C4 knockout mice developed pauci-immune necrotizing and crescentic glomerulonephritis akin to that observed with wild-type mice, indicating ANCA-mediated injury can occur independently of the classical and alternative lectin pathways. Conversely, knockout of complement factor B, which is necessary for alternative pathway activation, resulted in complete abrogation of disease activity. Subsequent experiments revealed C5a, and not the membrane attack complex, is the key mediator of disease activity. Indeed, treatment of mice with a C5a receptor antagonist attenuated MPO-ANCA-induced injury in a dose-dependent fashion.

Studies conducted in vitro demonstrate that neutrophil activation by MPO-ANCA or PR3-ANCA results in the release of factors that activate the alternative complement pathway. The generated C5a then recruits and primes additional neutrophils, resulting in increased autoantigen migration to the cell surface and, hence, a greater propensity for interaction with ANCA. This interplay results in a positive feedback loop that propagates the inflammatory cascade in AAV ( Fig. 17.1 ). Given the pivotal role of C5a, new therapies targeting this pathway are currently under investigation.