A rare case of Alport syndrome, atypical hemolytic uremic syndrome and Pauci-immune crescentic glomerulonephritis

Over the last several years, the causative role of the alternative complement pathway in atypical hemolytic uremic syndrome (aHUS) has been confirmed. Anti-complement therapy has been shown to be beneficial in aHUS. Pauci-immune crescentic glomerulonephritis (PCGN) lacks immune complex deposition and has not traditionally been associated with complement abnormalities. We present a patient with biopsy evidence of thrombotic microangiopathy (TMA), Alport syndrome (AS) and glomerular crescents and discuss potential pathogenic link.

Atypical hemolytic uremic syndrome (aHUS) is relatively rare, and studies over the last decade have vastly improved our understanding of the disease [5, 6]. In approximately 60% of cases, aHUS is linked to mutations in complement genes encoding both regulatory and activation proteins involved in the complement pathway. Disease results from either a loss-of-function mutation in a regulatory gene (eg. CFH, CFI, or CD46) or a gain-of-function mutation in an effector gene (eg. CFB or C3), resulting in alternative complement pathway dysregulation on cell surfaces. CFH mutation-associated aHUS, the most common form, comprises 20–30% of inherited disease [7]. In a subset of patients, similar to ours, combinations of gene abnormalities result in aHUS [5]. The disease penetrance is variable and several environmental triggers such as infections, pregnancy, respiratory illness and drugs likely precipitate overt aHUS. The precipitating factor in our patient is unclear. Only a third of aHUS patients have low serum C3 levels, usually in association with complement gene mutations. The biopsy findings of aHUS are those of TMA involving glomeruli and/or small arteries and arterioles.

The prevalence of alternative complement pathway dysregulation in disease appears to be greater than previously appreciated. Timmermans et al [8] examined complement abnormalities in nine patients with malignant hypertension-associated TMA, and found mutations in C3, CFI, CD46, or CFH genes in six. Complement activation in these patients was evidenced by elevated serum soluble C5b-9 levels and renal deposits of C3c and C5b-9. Co-localization of C4d deposits in vasculature and glomerular capillary walls indicates activation of classical and/or lectin pathway as well. Our patient did show systemic evidence of TMA (akin to malignant hypertension) with circulating schistocytes and elevated LDH levels. Chua et al [9] evaluated for C4d and C5b-9 deposits in 42 renal biopsies (both native and transplant) with TMA related to heterogeneous underlying diseases. C4d and C5b-9 deposits were quite common, although the pattern of staining was variable based on etiology. Our patient had deposits of both C4d and C5b-9, providing immunohistochemical support for complement pathway activation. Continuous alternative complement pathway abnormalities in aHUS patients result in endothelial dysfunction and formation of microvascular thrombi. Eculizumab is a humanized monoclonal IgG antibody that binds to complement protein C5, preventing cleavage into C5a and C5b. Blocking the formation of C5b interrupts this complement cascade and membrane attack complex (C5b-9)-mediated injury. Several studies have shown the safety and efficacy of eculizumab therapy in patients with aHUS [10].

Alport syndrome (AS), which usually manifests as a triad of hematuria, senisorineural hearing loss and ocular symptoms, is caused by the disruption of type IV collagen, a key component of the glomerular basement membrane. Specifically, mutations in the COL4A5, COL4A3 or COL4A4 genes, which encode the α3, α4, and α5 chains of collagen type IV respectively, cause the disease [11]. Over 500 mutations have been described, which result in disease with either X-linked (mutations in COL4A5; 85% of patients) or autosomal recessive inheritance (mutations in COL4A3 and COL4A4; 15% of patients) [12]. An autosomal dominant inheritance pattern has been reported in a minority of patients [12, 13].

A diagnosis of AS is generally determined by the family history, presence of visual and/or hearing dysfunction, and electron microscopy findings on kidney biopsy. The glomerular basement membrane changes are typically characterized by diffuse thickening and splitting of the lamina densa. Immunofluorescence in our patient revealed total lack of α3 and loss of α5 staining in glomerular basement membranes but preserved α5 staining in Bowman capsule and distal tubular basement membranes. Mutations in the COL4A3 (or COL4A4) gene results in the defective triple helix with demonstrable α5 protein loss only in those basement membranes (glomerulus) in which all three proteins are associated. [14] Based on these findings, we suspect that our patient has autosomal recessive AS.

Crescents are rarely reported in aHUS [15], but tend to be small and barely meet the criterion for a crescent [16]. It is also possible that the crescents in our patient are related to AS [17]. In reviewing 665 human Alport nephropathy biopsies, Ryu et al. [18] found glomerular crescents in 0.4% of biopsies, suggesting an extremely rare occurrence. In each of those biopsies, only 3–11% of glomeruli sampled had crescents. It has been proposed that plasma leakage due to glomerular vascular injury and glomerular basement membrane breaks might trigger parietal epithelial cell proliferation and crescent formation. Based on studies in Col4A3 –deficient mice, crescent formation occurs more frequently in late stage Alport glomerulopathy [18].

The presence of extensive crescents seen in our patient also suggests co-existent ANCA negative (antineutrophil cytoplasmic antibody anti-PR3 and anti-MPO) pauci-immune crescentic glomerulonephritis (PCGN). Review of the literature demonstrates a growing appreciation for the role of complement in PCGN [19, 20]. Complement plays a key role in murine ANCA-associated PCGN induced by injection of anti-MPO antibodies [21]. The alternative complement pathway has been shown to be critical in the pathogenesis as mice deficient in factor B or C3 fail to develop PCGN [21]. Anaphylatoxin C5a and the neutrophil C5a receptor mediate an amplification loop for ANCA-induced neutrophil activation that triggers the complement cascade in the serum. C5a, a potent chemoattractant for neutrophils, monocytes and macrophages, also increases the membrane expression of ANCA on neutrophils, leading to reactive oxygen species generation and necrotizing vasculitis [22].

Despite the general impression, a high percentage of patients with ANCA-associated PCGN have immunofluorescence and ultrastructural evidence of immune deposits. These immunoglobulin and complement deposits are typically of low intensity [23]. Recently, Chen et al. examined 112 biopsies from patients from ANCA-associated PCGN [24]. In one third of the patients, glomerular C3c deposits were detected and this feature was associated with greater proteinuria and worse renal function at presentation. Other components of alternative complement pathway activation such as factor Bb, C3d and properdin deposits were also demonstrable in the kidney biopsies with ANCA-associated vasculitis [25‐27] .The presence of C3d and properdin deposits correlated with more severe crescentic disease [27]. Other investigators have sought evidence of alternative pathway activation and complement consumption in serum and urine. Low serum C3 level at diagnosis of renal ANCA vasculitis is associated with lower renal function at presentation and worse renal and patient survivals [28]. Circulating serum factor Bb is elevated in active ANCA vasculitis and may in fact be a biomarker for disease activity [29]. Alternative complement pathway activation products Bb, C3a, and C5a and common terminal pathway product soluble C5b-9 were all found to be significantly elevated in the urine of patients with active compared to remission stage of ANCA PCGN [25]. There may also be activation of classic and lectin complement pathways, but the overall evidence suggests that they do not play a critical pathogenic role [25, 27, 30].

Given the involvement of alternative complement pathway in both ANCA disease and aHUS, it seems quite plausible that these diseases occur concurrently, as has been rarely reported [31]. Manenti et al. studied 46 patients with ANCA vasculitis, and eight of 30 patients who underwent biopsy (27%) had histologic signs of TMA [32]. This subgroup with histological evidence of TMA had a dramatically worse death-censored renal survival than patients without TMA. Interestingly, serum C3 levels were low in more than a third of these patients with ANCA vasculitis. Identifying a pathogenic role for complement in PCGN disease has significant therapeutic implications. Initiation of anti-complement therapy (eculizumab, anti-C5) immediately after a diagnosis of aHUS is associated with better renal outcome [33]. There can be a potential beneficial role of anti-complement therapy in PCGN disease as well [34, 35]. Recently, Sathe et al. [36] reported a pediatric patient with biopsy proven ANCA disease who developed aHUS 6 months later, as evidenced by low serum C3 and circulating anti-factor H antibodies. No genetic studies were performed. Complete renal recovery was achieved following treatment with intravenous rituximab, steroids, cyclophosphamide, and plasmapheresis.

Overall, it seems that TMA in ANCA disease may be under recognized both clinically and on biopsy. The identification of at risk genetic variants and polymorphisms in complement genes in our patient with both PCGN and TMA lends further support for shared pathogenic pathway in these two histologically distinct entities. Ours is a rather unusual case with a third diagnosis of AS. Glomerular crescents can rarely be seen in AS, and there are rare reports of TMA in an AS patient [37, 38]). A 16 year old patient presented with nephrotic syndrome and the biopsy revealed chronic TMA and a diagnosis of X-linked AS was subsequently made based on whole-exome sequencing. So, while a PCGN superimposed on TMA is a distinct possibility, crescent formation may have been triggered or accentuated by both glomerular injury caused by aHUS and the compromised basement membrane integrity due to AS.

In conclusion, we report a case of AS patient with concurrent aHUS and glomerular crescents. Genetic analysis revealed at risk variants and polymorphisms in alternative complement pathway genes. We hypothesize that this genetic predisposition in this unfortunate patient led to precipitation of aHUS and potentially PCGN in the context of an unidentified environmental trigger.