The GWAS mentioned identified three different candidate loci that might impact on this pathway. They all have been identified on chromosome 6p21, are located on major histocompatibility complex (MHC) and are called: HLA-DRB1/DQB1, HLA-DPB1/DPB2 and TAP1/PSMB9.

A strength link was identified in a different HLA region that includes the HLA-DRB1-DQA1 genes[19]. The same region had been previously associated with several autoimmune diseases[20-27]. Another MHC locus has been found in the region that includes the HLA-DPA1, BPB1 and DPB2 genes.

Immunity mucosa-related: The clinical association of hematuria and infective episodes related to mucosal sites allowed to suspect that abnormalities in the IgA production might be responsible for the IgAN[28].

GWAS identified three loci involved in the mucosal pathogenesis of the IgAN. A locus is located on chromosome 17p13. This locus contains the gene TNFS 13 that codes a proliferation-inducing ligand (APRIL). APRIL might determine the proliferation of IgA-producing cells[29,30] and APRIL serum levels may be higher in subjects affected by IgAN[31].

A second locus on chromosome 22q12 affects the circulatory IgA load and the susceptibility to develop IgAN[19]. This locus includes several genes among which the genes LIF and OSM, that encode cytokines[32]. The cytokines encoded by these genes belong to the interleukin 6 (IL-6) family and influence the immunoregulation[33,34].

On the DEFA gene cluster located on the chromosome 8p23, another locus related to IgAN has been identified. It encodes the small peptides secreted by the mucosal cells with antimicrobial properties called α-defensin[35]. While α-defensin 1, 3 and 4 are secreted by neutrophil, α-defensin 5 and 6 are secreted into the gut by the Paneth cells.

On chromosome 1q32 is located the locus that contains the gene encoding complement factor H (CFH). GWAS found that the deletion of two CFH related genes (CFHR3 and CFHR1) is a protective genetic factor for IgAN[19].

Indeed, the deletion of CFHR3 and CFHR1 is associated with the lacking of their products and CFHR1 is a competitive antagonist of CFH in regulating the complement activity[36]. As a consequence, the association of elevated CFH levels with the absence or low levels of CFHR1 determines a strong inhibition of complement activation. In addition, the relationship between mesangial C3 deposits and different CFH, CFH and CFHR1 levels suggests that these proteins are related to the pathogenic IgA-IC deposition[37].

In addition to the above mentioned pathways, recently performing a GWAS in 20612 patients affected by IgAN, other relevant possible genes have been found; four in ITGAM-ITGAX, VAV3 and CARD 9 and two in the HLA-DQB1 and DEFA loci. Most loci carry genetic risk correlation with local intestinal pathogens, supporting the possibility that host pathogens might favor the IgAN in genetically predisposed patients[38]. All the candidate genes and their function are summarized in Table 1.

The IgA1 hinge region is located between the constant region domains CH1 and CH2 of the α 1 heavy chains. This region contains O-glycosylation sites composed of serine/threonine (Ser/Thr) and Proline residues. In normal condition only some of these sites are glycosylated[39,40]. A key role in the IgA O-glycosylation is exerted by core 1 β 1, 3 galactosyltransferase (C1β3Gal-T) and its molecular chaperone core 1-1-phosphateuridyltransferase (Gal-T)-specific chaperone (Cosmc). Patients affected by IgAN have more elevated blood levels of IgA1 with the O-glycans poorly galactosylated and having either GalNac as terminal molecule or GalNac containing the secretory J component (Figure 1)[41]. A recent study, using cell lines from a human B-lymphoma documented that the T 2 helper cytokine IL-4 may control the glycosylation of the IgA[42]. Indeed, the IL-4 stimulation decreased the messenger RNA (mRNA) levels of both core the enzyme (C1β3Gal-T) and its molecular chaperone. Studies on animals confirmed the relevance of cytokines on IgA glycosylation[43,44]. O-glycan specific lectin initially allowed identifying the defective IgA1 O-glycosylation in IgAN[45]. More recently, other techniques as the liquid chromatography and the 3D mass spectrometry have allowed us to an improved understanding of the O-glycosylation process and the molecules involved[46,47].

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Figure 1 Immunoglobulin A1 and its hinge region with O-linked glycans (white) and N-linked glycans (black). Under the aminoacids chain of the hinge region. Sites of attachment are in bold. IgA: Immunoglobulin A.

The origin of the poor galactosylated IgA1s is still a not resolved question. Several studies have documented a significant difference in IgA1s generated in the systemic compartment with respect to the IgA1s generated on the mucosal surface[48]. Mucosal IgAs are predominantly polymeric (pIgA), while systemic IgAs are monomeric. Interestingly, in the IgAN the pathogenic IgA immune complexes (IgA-IC) principally contain poor galactosylated pIgA with the J secretory component[49].

The high serum level of mucosal-type IgAs in the IgAN patients might suggest that mucosal sites are the origin of poor galactosylated IgA1s. In contrast, in IgAN either systemic pIgAs directed against antigens typical of the mucosa and systemic pIgA plasma cells in systemic sites have been described[50,51]. Hence the hypothesis that the overproduction in the serum of poor galactosylated IgA1 might be the result of the movement of mucosal IgA1 committed B cells from the mucosa to the systemic compartment. A mucosal B cell mis-homing to systemic sites is the most likely mechanism[52].

The abnormal activity of Toll-like receptors (TLRs) might be another factor that contributes to the increased response to mucosal antigens in IgAN. Indeed, the association of increased Toll-like receptor 4 in circulating cells and signs of renal diseases have been reported by several studies[53]. Other studies examining the single nucleotide polymorphism, found an association between the TLR-9 polymorphism and the IgAN progression. This suggests that the involvement of TLR-9/MyD88 might exert its effect over the progression of IgAN[54].

The synthesis of abnormally glycosylated IgAs is not “per se” enough to justify the mesangial lesions that characterize the IgAN. Indeed, comparing the IgA glycosylation of IgAs eluted from serum with those eluted from biopsy specimens we may observe that Gd-IgAs eluted from kidney biopsies are less glycosylated when compared to the glycosylation rate of serum IgAs from the same IgAN patients[55,56]. This fact highlights a GdIgA1 tropism for the mesangium which might contribute to explain the recurrence of IgA deposits on kidney allograft. Moreover, a study documented that in families affected by IgAN an abnormal glycosylation may be present both in IgAN patients and in asymptomatic relatives[57]. The latter observation confirms that the presence of abnormally glycosylated IgAs does not “per se” justify the mesangial lesions and that other factors should be associated.

Recent studies suggest that auto antibodies recognizing the abnormally glycosylated IgA1s are essential in the pathogenesis of the disease[58,59]. These findings document that IgAN is an autoimmune disease due to the mesangial deposition of immunocomplexes containing GdpIgA1. Other molecules such as sCD89, fibronectin, collagen and laminin are also found in IgA1 containing immune complexes, even if their role remains to be determined[60].

Circulatory auto antibodies (IgG and/or IgA) bind to Gd-IgA1s and form large pathogenetic immune complex[61]. A better understanding of these antibodies is provided from an elegant experiment that used Epstein Barr virus (EBV-immortalized) lymphocytes from subjects affected by IgAN[62]. These B cells are able to product IgG that bind Gd-IgA1 and a subsequent analysis of the cloned chains of these IgG auto antibodies identified as their unique feature the complementary-determining region (CDR) 3 of the heavy chains[61]. In particular the third portion in CD3 is typically serine in patients with IgAN. Whether bone marrow or mucosal tissues are at the origin of IgA1s in circulating immune complexes is still now a matter of controversy. The acute onset of the disease is often accompanied by a concurrent infection of the upper respiratory tract and in this site cells are present able to produce polymeric IgA1s, typical of the pathogen IgA[29]. However, in other studies, polymeric IgA1s and J chain producing cells have been found in the bone marrow of subjects affected by IgAN[63-65]. The aforementioned mis-homing with transposition of plasma cells from the mucosa to systemic sites might explain this finding[52].

Recently Barratt et al[66,67] postulated the so called hypothesis of the “right antibodies in the wrong place at the wrong time”. According this hypothesis, the “right” antigen represented by the mucosal derived Gd-IgA is in the systemic compartment that is the wrong place. Later on, when a large quantity of Gd-IgA1 is in circulation, a large quantity of the right antibody anti Gd-IgA1 is secreted at the wrong time.

The stimuli leading to the production of these auto antibodies remains to be explained.

A hypothesis might be that these antibodies are secreted against pathogen cell surface GalNac-containing glycoconiugates cross-reacting with GdIgA1, realizing a molecular mimicry[68]. A prevalence of IgA1 autoantibody response[61] anti GdIgA1 may justify the fact that some patients have only IgA1 antibody in the glomeruli[69].

In conclusion, a portion of the IgA1 molecules secreted by the plasma cells in patients affected by IgAN is Gal-deficient and is identified by the anti-glycan IgG (or IgA1) antibodies[70]. The formed IC, due to their size, cannot reach and bind the asialoglycoprotein receptor (ASGP-R) in the liver, and be metabolized. Moreover, the terminal GalNac residues, which might interact with the ASGP-R, are covered by specific antibodies that prevent such interaction[71]. As a consequence, a larger fraction of the IgA-IC may reach the glomerular capillaries overlying the mesangium.

Summarizing the two steps above described in the IgA pathogenesis, in the first time a high quantity of under-galactosylated IgA1 is present in the blood. At this regard several points remain to be clarified. The plasma cell defect is inherited or acquired? In addition, to justify the systemic presence of GdIgA1 high quantity, a plasma cell mis-homing from mucosa to systemic sites is needed or not?

In the second step we have the IgG auto antibodies production anti the GdIgA1. At this regard several questions remain to be answered.

Are these antibodies the result of a molecular mimicry triggered by infections?

In addition, are these antibodies the result of a Toll-like receptor polymorphism?

Finally, is there a genetically determination induced by somatic mutations in the IgG heavy chains?

Circulating anti-glycan auto antibodies recognize Gd-IgA1 and pathogenic immune complexes are formed as a consequence. IgA1s must be within an immune complex to activate the mesangial cell proliferation; indeed not complexed GdIgA1s do not stimulate the proliferation of mesangial cells[72-74]. Additional components from serum need to be present to form stimulatory complex[72].

Several models have been proposed to explain the IgA1 immune complex depositions on the mesangial cells. Immune complexes containing IgA1/IgG or IgA could directly deposit on mesangial cell[61,62,75]. Another hypothesis is that poor glycosylated IgA1 might be present in the mesangial area as lanthanic deposits and later newly generated anti-glycan antibodies might bind and realize immune complexes in situ capable to activate mesangial cells[3] (Figures 2 and 3). In addition, a further theory is that self-aggregated Gd-IgA1s might deposit or bind to specific receptors in the mesangial area realizing “planted” depositions that are not pathogenic “per se”. When an exposure to similar environmentally derived antigens occurs, an auto-antibody production and the involvement of several mediators cascade lead to the disease[76,77]. Further studies led to identify the relevance of IgA receptors (IgA-R) in the deposits. Several IgA-Rs have been recognized[78-80]. In the IgAN pathogenesis, two IgA-R expressing cells have been principally involved: (1) mesangial cells which have been documented to be implicated in kidney injury; and (2) myeloid cells (essentially kidney infiltrating macrophages) which have been documented to modulate the extent of the inflammatory response. Studies from several groups have ruled out that the mesangial expression of receptors as ASGP-R, CD 89 and pIgAR might have a role[81]. In addition, although on the mesangial cells is located the Fc alpha mu receptor (Fcα/µR), neither IgM nor the recombinant Fcα/µR inhibit the IgA1 binding to mesangium[82]. The transferrin receptor (TfR1 or CD71) has been identified as the mesangial IgA1 receptor implicated and the pIgA1 binding to TfR1 induces mesangial cells activation[83,84]. Moreover, TfR1 co-localizes with deposited IgA in the IgAN biopsies[85]. The same group documented that both glycosylation and size of IgA1 are relevant factors for the TfR-IgA1 interaction. This probably is the first step of the IgAN injury[86]. Finally, as an alternative hypothesis, has been proposed that the soluble form of the Fcα receptor (sCD89) might “per se” generate complexes with Gd-IgA1[87]. The formation of circulating Gal deficient pIgA1 immune complexes (IgA1-CIC) induces an alteration in the interaction between IgA and CD89 that are found in the mesangial deposits and is implicated in the diseases exacerbation through the activation of pro-inflammatory cytokines and the secretion of chemokines[88,89].

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Figure 2 Schematic representation for the possible pathways involved in the generation of a circulating immune complex pathogenesis for the immunoglobulin A nephropathy. CIC: Circulating immuno complex; Gd IgA1: Galactose deficient immunoglobulin A1.

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Figure 3 Schematic representation for the possible pathways involved in an in situ pathogenesis for the IgA nephropathy. IgA: Immunoglobulin A; Gd-IgA1: Galactose deficient immunoglobulin A1; ANTI GalNac: ANTI N-acetylgalactosamine.

The renal damage after mesangial cell deposition of immune complexes may be distinguished into three phases: (1) mesangial cell activation; (2) podocyte injury; and (3) tubulo-interstitial scarring. All these kinds of renal injuries may be mediated by different pathways as the complement activation, the innate immunity activation, and the non-complement mediators of IgA nephropathy (Figure 4).

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Figure 4 Pathways to glomerular damage and tubulointerstitial injury in Immunoglobulin A nephropathy. Deposition of IgA-ICs in the mesangium leads to activation of mesangial cells, triggering mesangial cell proliferation and release of proinflammatory and profibrotic mediators. Podocyte loss accentuates glomerular scarring and filtered mesangial cell-derived mediators and IgA-ICs stimulate PTEC to adopt a proinflammatory and profibrotic phenotype, which in turn drives tubulointerstitial scarring. IgA: Immunoglobulin A; PTEC: Proximal tubule epithelial cells.

Mesangial cells are activated by IgAs and may transform into inflammatory and fibrotic cells after the exposure to IgA-IC. Indeed, the mesangial cells binding to IgA-IC containing poorly galactosylated IgA1 triggers the proliferation and the programmed death of the mesangial cells. In addition a reduced synthesis of vascular endothelial growth factor (VEGF), an abnormal integrin production and an abnormal production of extracellular matrix increase the renal damage[86,90-92].

The role of complement in the activation of the mesangial cells in IgAN has been recently reviewed by Maillard et al[93]. In IgAN, polymeric or aggregated IgA1s, principally Gd-IgA1s, may stimulate the alternative complement pathway, determine the C3 deposition and the production of the complement terminal complex (C5b-C9)[94-97]. Similarly the same pathways might be locally stimulated on the mesangial cells by polymeric Gd-IgA1s as well as by the secretory IgAs (SIgA)[97]. In addition, the complement involvement by the alternative and the mannan binding lectin (MBL) pathways may be activated by the polymeric IgA1, thus participating to the pathogenesis of the IgAN and characterizing a more severe disease.

Components of the innate immunity are similarly involved in the pathogenesis of the IgAN. Some studies have documented that the TLRs are able to induce the IgA production by the B cells[98]. It has also been documented a link between the TLRs stimulation, the overproduction of Gd-IgA1s, the more aggressive aspects of IgAN and the activation of the enzyme and molecules involved in the IgA glycosylation[54,99,100].

Mesangial injury: IgA1-ICs containing secretory IgA with a high sialic acid content and anionic IgA-IC stimulate mesangial cells, may stimulate the p42/p44 mitogen activated protein kinase, activator protein 1, and NF-kB signal transduction. Similarly other chemokines and cytokines are up-regulated, among which the IL-6, the transforming growth factor β (TGFβ), the tumor necrosis factor α (TNFα) and the monocyte chemo attractant protein (MCP-1)[101-103].

The platelet derived growth factors (PDGFs) are among the growth factors most involved in the mesangial cell proliferation[104]. The PDGFs are five potent mitogens and chemoattractants that play important roles in the mesangio-proliferative diseases, principally in the IgA nephropathy. The PDGF-BB and PDGF receptors (PDGFR-β) are over expressed in the experimental mesangio-proliferative nephritis and in the human IgAN[104].

The pathogen IgA1s are also able to activate the renin-angiotensin-aldosterone system (RAAS) intrarenally[105]. This system too is involved in the IgAN injury.

Finally a protective affect against mesangial cell activation by IgA-IC is exerted by bone morphogenetic protein 7 (BMP-7)[106]. BMP-7 suppresses TNFα induced synthesis of proinflammatory cytokines, and has an anti-fibrotic effect through antagonism of the cellular actions of TGF β[107,108].

Podocyte injury: Podocyte necrosis and detachment from the glomerular basal membrane has been reported in the IgAN and the degree of podocytopenia is closely related to the increasing severity of glomerular lesions[109]. Nephrin is a key component of the podocyte slit diaphragm and is essential for the maintenance of an intact glomerular filtration barrier. Interestingly, nephrin mRNA and extracellular nephrin expression are reduced in the IgAN[110,111]. In addition, evidence from in vitro studies suggests that the podocyte injury in the IgAN is likely to be mediated by both the mesangial cell derived soluble mediators and by the direct contact with filtered IgAs[112,113].

It has also been documented that IgA1-IC in IgAN patients might up regulate the production of CXCL1 and TGF beta from the mesangial cells. CXCL1 and TGF beta might exert a synergistic effect upon the podocytes, inducing podocyte dysfunction and podocyte death[114].

Tubulointerstitial scarring: It has until recently been thought that the mechanisms of the subsequent tubulointerstitial injury were generic and common to all forms of chronic kidney diseases. Recent studies have documented that specific mechanisms are operating in the IgAN.

Among the factors that are up regulated after stimulation of mesangial cells, the TGFβ is the most involved in generating fibrotic tissue related to the cell damage. It is generated by growth factors involved in the connective tissue generation[115,116]. In addition to producing fibrotic tissue, TGFβ also acts favoring the transformation of the tubular cells into a fibrotic phenotype.

With an increasing damage to the perm selective barrier, increasing amounts of high-molecular weight IgA-ICs enter the urine. In IgAN these IgA-ICs are enriched by GdIgA1s that reflects their localization within the mesangium[117]. Therefore the proximal tubule epithelial cells (PTEC) are constantly exposed to filtered IgA-ICs because of the impairment of the glomerular size barrier.

Recent studies suggest that there may be a direct and specific interaction between filtered IgA-ICs, mesangial cell-derived mediators, and PTEC. Indeed, when the mesangial cells are activated by the IgAs, they release mediators, which subsequently lead to up-regulation of angiotensin II production, inflammatory changes and apoptosis of PTECs[118]. Similarly, the mesangial cell derived TNFα is known to activate the tubular cells inducing pro-inflammatory mediators, thus establishing an IgAs specific glomerular-tubular cross-talk[119]. There is also convincing in vitro evidence that the filtered mesangial cell-derived mediators also may determine a proinflammatory and profibrotic transformation of PTEC[118-121]. As a consequence, an IgA specific pathogenetic effects might exist, which further accelerate the progressive lost of renal function. In addition, a recent study documents that[122] IgAN might be associated to the Epithelial-mesenchimal transition and the apoptosis of renal tubular epithelial cells favoring the renal scarring.