Background
IgA nephropathy (IgAN) is the most prevalent primary chronic glomerular disease worldwide [
1], and although benign in many cases, it is reported that up to 30–50% will slowly progress to end stage renal failure [
2,
3]. The pathogenesis of IgAN involves mesangial deposition of immune-complexes containing galactose deficient IgA1 that leads to mesangial cell activation and initiation of glomerular injury [
4]. Mesangial proliferation is scored in the Oxford classification of IgAN and has been shown to predict progressive disease [
5]. Activated mesangial cells secrete inflammatory mediators and components of the extracellular matrix, and mesangial hypercellularity is thus morphologically often associated with increase of extracellular matrix (ECM).
The glomerular extracellular matrix is a dynamic structure which acts both as structural support for the cells and as an active component in cell signaling [
6]. Lennon et al. previously described the protein composition of the glomerular ECM, identifying 144 structural and regulatory ECM proteins [
7]. Recently, Hobeika et al. [
8] expanded the list by including their identified proteins from microdissected glomerular tissue and comparing with proteins described in the Matrisome project database - a curated database of ECM structural and associated proteins [
9],
http://matrisomeproject.mit.edu/. IgAN is a disease characterized by glomerular ECM expansion, but the glomerular proteomic changes have not been investigated in detail.
In the present study we microdissected glomerular tissue from 25 patients with IgAN and 15 patients with normal findings on kidney biopsy (controls) in order to investigate glomerular ECM proteins which either had been defined by Lennon et al. [
7] or by the Matrisome project database. Our hypothesis was that composition of glomerular ECM would be changed in IgAN as compared to controls and that some ECM proteins would also be associated with progressive clinical course in IgAN. An improved understanding of these changes may be important for a better understanding of the glomerular damage in IgAN.
Discussion
In the present study we have investigated the glomerular extracellular matrix (ECM) proteome in IgAN and quantitatively compared this to the proteome of glomeruli from patients with normal findings on kidney biopsy. We made several interesting findings. First, a high number of ECM associated proteins showed altered abundance in IgAN as compared to controls, several of these were related to inflammation, immune response and fibrosis development. Second, several structural ECM proteins had increased abundance in IgAN compared with controls and the protein with the strongest difference in abundance was periostin. Periostin was also more abundant in patients with progressive IgAN as compared to patients with non-progressive IgAN. Third, basement membrane proteins were increased in IgAN. Overall, our study suggests that glomerular ECM changes in IgAN have strong similarities to changes seen in fibrosis development in general.
In IgAN mesangial IgA deposition and formation of immune complexes leads to mesangial cell proliferation, the release of proinflammatory mediators by mesangial cells and matrix expansion [
14]. In our study, most of the ECM associated proteins which had higher abundance in IgAN compared to controls were proteins involved/associated with the immune response and inflammation (eg. azurocidin, myeloperoxidase, neutrophil elastase, cathepsin G, annexin A1, protein S100-A9 etc). Most of these proteins have been described as present in polymorphonuclear leukocytes [
15‐
17], and some may also act as a chemoattractants and activators of monocytes and macrophages [
18]. To our knowledge the roles of these proteins have not been described in IgAN, but may mark underlying mechanisms of glomerular damage. Inflammation is for example known to be a propagator of fibrosis development in general [
19].
Several structural ECM proteins, such as vitronectin, extracellular matrix protein 1, fibulin-5 and fibrinogen were significantly more abundant in IgAN than in controls. We had not microdissected glomeruli with more than minimal sclerosis and our findings thus most likely illustrate the proteomic changes in mesangial expansion or early glomerular sclerosis. Several proteins that have been shown to be involved in fibrosis development were shown to be more abundant in IgAN compared with controls, such as periostin [
20], serpin H1 [
21], MMP2 [
22] and MMP9 [
22,
23]. Periostin was also significantly more abundant in IgAN patients who progressed to ESRD as compared to IgAN patients who did not progress. In the kidney, periostin has been implicated in progression of hypertensive nephropathy [
24] and it was increased in glomeruli of patients with progressive proteinuric disease [
25]. A recently published study showed that periostin is induced by proinflammatory factors, mainly NFκB in a model of chronic renal disease, and that inhibition of periostin can be used as a therapeutic strategy to slow down renal disease progression [
26]. We are not sure why the findings of increased abundance of periostin in progressive IgAN vs non-progressive IgAN from the proteomic analysis were not seen using immunohistochemistry analyses. We believe that the most likely explanation is that mass spectrometry quantification is more precise than quantification by immunohistochemistry.
Important proteins in matrix metabolism, MMP-9 and MMP-2 were also significantly more abundant in IgAN patients compared with controls. MMP-9 and MMP-2 are the most abundant intrarenal metalloproteinases [
27] and although it was initially thought that they were mainly implicated in collagen degradation, MMP-2 has been shown to have a pro-inflammatory effect by acting on mesangial cells [
28]. Both MMP-2 [
29] and MMP-9 [
30] are involved in the renal tubular cell epithelial–mesenchymal transition (EMT) and through that promoting fibrosis. Future studies should investigate the roles of these pathways in progressive glomerular sclerosis in IgAN.
In a recent study by Liu et al., genes highly expressed in mesangial cells discriminated better IgAN patients from control patients than genes highly expressed in podocytes and the study thus argued for a strong involvement of mesangial cells in IgAN [
31]. Few studies have investigated the proteomic composition of mesangial matrix in IgAN [
32], and to our knowledge no previous studies have used modern proteomic approaches. As discussed above, the proteomic changes of the glomerular ECM in our study have strong similarities with those of fibrosis development in general. Mesangial matrix expansion has by many been regarded as a step towards glomerular sclerosis, as was suggested in a review paper by Fogo in 1999 [
33], but there seem to be a paucity in data on the underlying mechanisms of this process. Our data and the Liu paper indicate important mechanisms of this process that should be analyzed further.
Several proteins classically described as basement membrane proteins were more abundant in IgAN compared to controls, for example collagen alpha-1 (IV) chain, fibronectin, laminin subunit beta-1, nidogen 1, etc. We could not find studies of thickened GBM in IgAN and previous studies reported rather thinning of GMB in patients with IgAN [
34,
35] Using immunostaining Masuda et al. [
35] showed reduced α5 (IV) collagen and increased α2 collagen as well as structural changes of α5 (IV) collagen in patients with IgAN. They reported thinning, irregular thickening, small gaps and double contour of GBM examined by
transmission electron microscopy [
35]. By visually inspecting glomerular staining for our proteins in the Human Protein Atlas we did however observe that although these proteins showed clear linear staining of the basement membrane, they also showed positive mesangial staining. We thereby believe that our findings represent changes in the mesangial matrix rather than in the glomerular basement membrane.
The most important strengths of the present study are the large number of quantified proteins from microdissected glomerular tissue. It is also a strength that we included IgAN patients with moderate risk of progressive disease (based on classical prognostic factors), a cohort highly relevant for the clinical nephrologists and that we could separate progressive from non-progressive patients. From a clinical perspective, the number of patients might seem low, but as compared to other proteomic studies this is not the case and we would argue that the similar clinical characteristics of the patients outweigh this limitation. As control group, we used patients with normal kidney biopsy (indication of biopsy was hematuria, proteinuria or reduced eGFR). An extra control group of patients with similar eGFR and another glomerulonephritis would have added more information regarding the particularly proteomic changes that occur in IgAN versus chronic kidney disease in general. It would however have been difficult to know which particular disease to choose for such a comparison as patients with hypertensive nephropathy, FSGS, or lupus nephritis, will also differ from control in several aspects and would have their own proteomic changes. In our opinion, patients with normal kidney biopsy was a good option as control group. As IgAN is an chronic kidney disease we believe that there are many similarities with CKD in general.
Although our control group was defined with GFR over 60 ml/min/1.73m2, it is important to mention that only one patient had GFR under 90 ml/min/1.73m2 and although the IgAN group was defined as GFR over 45 ml/min/1.73 m 2, only one patient had GFR under 60 ml/min/1.73 m 2.
The most common indication for biopsy was hematuria (11 patients) or proteinuria (2 patients). This patients did not have known hypertension, diabetes or malignancy and the kidney biopsies were described as normal. It is important to keep in mind that although almost 200 extracellular matrix proteins could be relatively quantified, the exact localization and role of these proteins could not be described. It is for example possible that some of the proteins are deposited intracellularly or have altered levels of activity as compared to normal, we do however not believe this to be the case for the majority of proteins.
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