Biomarkers in IgA nephropathy

Review

Biomarkers in IgA nephropathy

IgA nephropathy is the most common primary glomerulonephritis and presents with gross hematuria and upper respiratory infection, with slow progression to endstage renal disease in up to 50% of affected patients. Kidney biopsies are the gold standard method of diagnosis and frequently are not performed as the majority of individuals are asymptomatic. Thus, there is a need to discover and validate prognostic and predictive biomarkers that can be noninvasively obtained and are specific to this disease. Here we discuss the current state of research in this area and examine validated and clinically promising biofluid and tissue biomarkers of IgA nephropathy. Keywords: BIOmUIDsGLOMERULONEPHRITISsGLYCOMICSs)G!NEPHROPATHYsPREDICTIVE BIOMARKERsPROGNOSTICBIOMARKERsTISSUE

IgA nephropathy (or IgAN) is the most common primary glomerulonephritis in the developed world. Since kidney biopsies are commonly not performed in patients who likely have this disease, IgA nephropathy is underdiagnosed and its frequency is underestimated. The classic presentation of IgA nephropathy is gross hematuria associated with a viral-like illness. The majority of patients are asymptomatic and the diagnosis is suspected based on clinical presentation but currently can be confirmed only by obtaining an invasive and somewhat risky kidney biopsy. More importantly, IgA nephropathy is progressive in many patients and leads to end-stage renal disease in up to 50% of affected individuals, whereas 10–20% of patients show spontaneous remission [1–5] . Therefore, there is an unmet need to easily and successfully diagnose individuals and to determine their relative risk in order to prescribe the best course of treatment. Recently, histological and clinical predictors of progression have been discovered (such as the development of hypertension, proteinuria and eventual decrement in the glomerular filtration rate [GFR]), however, these are not reliable [6] and are of low specificity [6] .

10.2217/BMM.14.92 © 2014 Future Medicine Ltd

A number of therapies have been used in the treatment of progressive IgA nephropathy including fish oil, steroids and inhibitors of the renin-angiotensin systems, however, yet none has been an unmitigated success. In addition, there are currently no available accurate and/or easily accessible prognostic or predictive biomarkers. A prognostic biomarker refers to a biological or clinical trait that provides information on the outcome of the disease in an untreated individual, and therefore such biomarkers provide enormous benefit at diagnosis. A predictive biomarker is a biological or clinical trait that provides information of the severity and therefore the likelihood of the benefit of a given treatment. These biomarkers not only can help identify the responders in a population, but can also support targeted therapy. Due to the fact that there is a large cohort of undiagnosed or ‘latent’ IgAN in apparently healthy individuals [7] , the pathogenesis of this disease cannot be simply due to the presence of IgA deposition, but likely also requires a ‘second hit’ [8,9] . Because the clinicians’ initial index of suspicion for this disease is aroused by urinalysis which shows (microscopic or macroscopic) hematuria, it is logical that popula-

Biomark. Med. (2014) 8(10), 1263–1277

Vicki J Hwang1,2,‡, Arzu Ulu1,‡, Justin van Hoorebeke1,3 & Robert H Weiss*,1,2,3,4,5 1 $IVISIONOF.EPHROLOGY $EPARTMENTOF )NTERNAL-EDICINE 'ENOME"IOMEDICAL 3CIENCES"UILDING 2OOM 5NIVERSITY OF#ALIFORNIA $AVIS #! 53! )NTEGRATIVE'ENETICS'ENOMICS 'RADUATE'ROUP ,IFE3CIENCES 5NIVERSITYOF#ALIFORNIA $AVIS #! 53! -OLECULAR #ELLULAR)NTEGRATIVE 0HYSIOLOGY ,IFE3CIENCES 5NIVERSITY OF#ALIFORNIA $AVIS #! 53! 4 #ANCER#ENTER 5NIVERSITYOF#ALIFORNIA $AVIS #! 53! -EDICAL3ERVICE -ATHER6!-EDICAL #ENTER 3ACRAMENTO #! 53!

!UTHORFORCORRESPONDENCE 4EL &AX RHWEISS UCDAVISEDU ‡ !UTHORSCONTRIBUTEDEQUALLY

part of

ISSN 1752-0363

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Hwang, Ulu, van Hoorebeke & Weiss

tions and countries in which this is more frequent will appear to have a higher incidence of disease. When a patient is referred to the nephrology clinic with hematuria, macroscopic or microscopic, the normal workup involves an initial evaluation of the lower urinary tract with standard imaging techniques. Negative findings for hematuria-producing lesions leads to one of two courses of action for the clinician: perform a renal biopsy with its potential complications, or take a ‘wait and see’ attitude for potential decline (which occurs in 6–43% of affected individuals) [10,11] . Obviously, since response to any available or novel treatment would be enhanced by the earliest possible treatment, identification of those with true IgAN as well as those most likely to progress would be a major advancement in the field. Pathophysiology of IgA nephropathy

The recently worked out mechanism of IgAN relates to aberrant glycosylation of the IgA molecule (Figure 1) which, for reasons not yet clear, leads to its deposition in the glomerular mesangium. Such defective glycosylation leading to circulating (and potential urinary) IgAs or associated immune complexes, the mesangial trapping of these molecules, and the subsequent renal response to this injury: are all potential areas of biomarker pursuit. In this review, we evaluate the current status of three general areas of biomarker research in IgAN. We report on proteins, miRNAs and tissue biomarkers that can be found in body fluids and that may be used for disease diagnosis or predictors of progression. Although there has been some general progress in this field of research, translation to clinically validated biomarkers for detection and disease stratification has yet to be achieved. Biofluid protein biomarkers in IgAN While the renal biopsy maps immunoglobulin deposits of galactose-deficient IgA, IgG and IgM in the glomerular mesangium [8] , such a procedure is an invasive method with some risk and requires histological processing and pathological evaluation, therefore it would be ideal if this procedure could be avoided whenever possible and noninvasive biomarkers used instead. To date, hematuria is the most important indicator of IgAN activity, however, its specificity is low [12,13] . Therefore, there is an urgent and unmet need to identify noninvasive biofluid biomarkers for diagnosis and progression that might replace the biopsy requirement in IgA nephropathy. These biofluid protein biomarkers are compiled in Table 1.

Proteins that are associated with inflammation & complement component pathways

Some abundant proteins that are elevated in IgAN appear to be in the extracellular matrix–receptor interaction, inflammatory complement and coagulation pathways. In their global proteomics analyses, Kalantari et al. [14] reported that urine samples from 13 patients with IgAN at different stages of the disease correlate well with the Oxford classification, particularly for endocapillary hypercellularity. In this study, 18 out of the 232 proteins identified with mass spectroscopy in IgAN patients showed significant changes in abundance, among which 11 were suggested to be prognostic biomarkers. These protein biomarker candidates include afamin (α-albumin), apolipoprotein A1 (Apo A1), vitamin D binding protein, apolipoprotein A-IV and ceruloplasmin. These proteins are associated with biological processes including the acute inflammatory response, steroid metabolism, ion transport, cell adhesion and activation of complement and coagulation pathways. Many cytokines that are excreted in the urine of IgAN patients (e.g., IL-1-β, MCP-1, IL-17, IFN-γ, IL-10) have also been reported to predict progression of the disease. However, these results are awaiting validation and are further confounded by the use of steroids and immunosuppressants in some of the patients [15] . Lastly, complement components C3a and C5a have been shown to correlate well with renal injury in IgAN [16] ; however, these markers have broad pro-inflammatory roles in many autoimmune diseases and thus are not specific to IgAN. Urinary mannose binding lectin

Mannose binding lectin (MBL) is one of the proteins that are involved in complement component system activation and has been associated with IgAN pathogenesis [17,18] . Urinary levels of MBL were found to be increased in IgAN patients when compared with healthy controls. In addition, the urinary MBL levels were higher in individuals in advanced stages of the disease, suggesting that MBL can also serve as a predictive biomarker for IgAN. Lastly, urinary MBL also correlates well with histopathological changes and changes in renal function, such as increased proteinuria and decreased estimated GFR (eGFR) and elevated serum C4 levels. While such correlation strengthens the validity of this biomarker, concerns remain as to whether this protein is specific to IgAN or may also be increased in other immune-mediated glomerular disease. Urinary LG3 fragment of endorepellin

Urinary biomarkers

In extant biofluid investigations for IgAN biomarkers, urine proteomics has been the most prevalent approach (Table 1) .

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Another promising predictive biomarker is the urinary LG3 fragment of endorepellin, which resides in the C-terminal domain of the perlecan glycoprotein. Using a proteomics approach (2DE and Western and

future science group


O-glycans Hinge region

GalNAc

GAL

GalNAc

GAL

SA

GalNAc

GAL

SA

Review

Ser/Thr

Normal IgA1

IgA1 SA

GalNAc

GalNAc

Gd-IgA1-specific IgG

SA SA

SA

Galactose-deficient IgA1

Deposits of immune complexes in glomerular mesangium

Gd-IgA1-specific IgA

Figure 1. IgA glycosylation changes and associated antibodies in IgAN. The hinge region of the IgA1 molecule encompasses the amino acids serine and threonine, which have hydroxyl functional groups that are O-glycosylated in the hinge region of the IgA1. The first glycan that is attached to these residues is GalNAc (N-acetylgalactosamine) (light blue). Following GalNAc, a galactose (GAL) molecule (red) is added as the next step in the glycosylation of IgA1. A sialic acid (SA) (yellow) molecule can be added in two different positions to the GAL or GalNAc. However, when the IgA1 lacks the GAL, as it happens in IgAN patients, SA binds to the GalNAc instead, which results in GAL deficient (Gd)-IgA1. When the levels of Gd-IgA1 increase in circulation of IgAN patients, antibodies against this galactose-deficient IgA1 are produced. The anti-Gd-IgA1-specific IgAs or IgGs form immune circulation complexes with the Gd-IgA1. Such immune complexes accumulate in the glomerular mesangium and activate the complement component system, cytokine release and mesangial proliferation leading to glomerular injury. CKD: Chronic kidney disease; GFR: Glomerular filtration rate; MCP: Monocyte attractant protein.

ELISA to quantify), Surin et al. [19] showed decreased levels of LG3 in IgAN patients when compared with controls (Table 1) . While urinary LG3 was decreased in most of the IgAN patients and showed an inverse correlation with the GFR, some patients had increased LG3 levels that correlated with lower renal function (as measured by lower eGFR). Regardless of the variation, the correlation between decreased levels of LG3 and GFR was only present in IgAN and not in other glomerulonephritis. These results need to be validated and further supporting data are needed to show associations with the biopsy data. In addition to the LG3 fragment, α-1-antitrypsin and α-1-β-glycoprotein were also increased in IgAN individuals in this study.

Afamin, leucine-rich α-2 glycoprotein, ceruloplasmin, α-1 microglobulin, hemopexin, apolipoprotein-1, complement C3, vitamin D binding protein, apolipoprotein A-IV, β-2 microglobulin and retinol binding protein 4. Among the low MW proteins identified in IgAN, altered biology of hemopexin is also seen in minimal change disease [20] , suggesting that this protein may not be specific to IgAN. Another potential biomarker, apolipoprotein A-1, which is also considered a biomarker in chronic kidney disease (CKD) stage 5, has also been evaluated in IgAN, and is therefore more of a predictive biomarker of injury in IgAN. In addition, a higher ApoB/apolipoprotein A-1 ratio has been found to indicate an increased risk of developing end-stage renal disease (ESRD) in IgAN patients [21] .

Low-molecular-weight proteins

Among urinary biomarkers, some low-molecular-weight (MW) proteins stand out as prognostic and predictive biomarkers. Such low MW proteins in the urine include


Afamin (α-albumin)

Using nontargeted proteomics, the glycoprotein, afamin, was found to be abundant in the urine of IgAN

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Table 1. Most commonly studied protein biofluid biomarkers in IgA nephropathy. Study (year)

Biomarker

Cohort

Kalantari et al. (2013)

Afamin (α-albumin)

13

Urine

↓

Apolipoprotein A-1

13

Urine

↑

Vitamin D-binding protein

13

Urine

↑

Apolipoprotein A-IV

13

Urine

↑

Leucine-rich-α -2-glycoprotein

13

Urine

↓

Ceruloplasmin

13

Urine

↓

Control (n) IgAN (n)

Sample Changes in Clinical pearls matrix levels

α-1-macroglobulin

13

Urine

↑

Hemopexin

13

Urine

↑

Complement C3

↓ GFR, good correlation with advanced disease stage based on the Oxford’s classification

Comments

Ref.

Prognostic; may be specific for IgAN

[14]

Predictive; specificity may be for other immune-mediated glomerulonephritis as well as IgAN

13

Urine

↑

β-2-microglobulin

13

Urine

↑

Retinol-binding protein 4

13

Urine

↑

43

Urine

↓

↓ % renal fibrillar collagen Inverse correlation with GFR

Prognostic

[19]

50

Serum

↑

↑ Proteinuria, ↑ hematuria

Prognostic

[8,29,

Gd-IgA1 specific IgA: ↓ proteinuria, ↓ mesangial IgAN deposits

Prognostic; good specificity when evaluated together with serum levels of Gd-IgA1

[26]

Biomarker for therapeutic response

[35]

Surin et al. Laminin G-like (2013) 3 fragment of endorepellin

30

Suzuki et al. (2009, 2014), also see Hastings et al. (2010)

Galactose-deficient IgA1 (Gd-IgA1)

Yanagawa et al. (2014)

Gd-IgA1-specific antibodies

Iwatani et al. (2012)

Hinge region 30 healthy 7 O-glycan controls composition (hinge peptide from IgA)

106 healthy controls; 79 CKD controls

135

Serum

↑ Serum IgA ↑Serum Gd-IgA1 ↑ Gd-IgA1 specific IgG ↑ Gd-IgA1 specific IgA

Serum

↓

34]

#+$#HRONICKIDNEYDISEASE'&2'LOMERULARlLTRATIONRATE-#0-ONOCYTEATTRACTANTPROTEIN

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Review

Table 1. Most commonly studied protein biofluid biomarkers in IgA nephropathy (cont.). Study (year)

Biomarker

Liu et al. (2012)

Mannose-binding lectin

Stangou et al. (2013)

Cytokines

Cohort Control (n) IgAN (n) 50 healthy 162 controls

53

Sample Changes in Clinical pearls matrix levels Urine

↑

Urine

↑ IL-1β, ↑ Proteinuria, MCP-1, ↑ creatinine, ↑ IL-17, IFN-γ, GFR IL-6 and IL-10

↑ Proteinuria, ↑ hematuria, ↓ GFR, ↑ arterial hypertension, ↑ creatinine, ↑ histopathological changes

Comments

Ref.

Predictive

[18]

Biomarker for therapeutic response

[15]

#+$#HRONICKIDNEYDISEASE'&2'LOMERULARlLTRATIONRATE-#0-ONOCYTEATTRACTANTPROTEIN

patients [14] . Afamin is a member of the albumin family of proteins that is abundant in the liver and kidney. It has a vitamin E binding property and its tissue levels are decreased in hepatocellular carcinoma and ovarian cancer, suggesting that lower levels of Afamin may be associated with increased proliferation [22] . However, involvement of this protein in pathogenesis of IgAN has not been further evaluated; serum levels of this protein have also been shown to be increased in cancer [23] . Leucine-rich α-2 glycoproteins

Leucine-rich α-2 glycoproteins have been found to be abundant in different stages of IgAN when evaluated in the urine of such patients, however, its specificity to IgAN is diminished as it is also induced by inflammation. Two studies, one of which reports data from a pediatric IgAN case with Hodgkin’s disease, have similar positive associations with IgAN regarding leucine-rich α-2 glycoproteins [14,24] . Ceruloplasmin

Ceruloplasmin has been identified as a potential biomarker in two recent studies. While one study compares urinary proteins between IgAN patients and healthy or CKD controls [14] , the other study compares IgAN with thin basement membrane nephropathy, a disease which has a better clinical outcome than IgAN [25] . Urine and urinary exosomes have been evaluated in these studies. While aminopeptidase N, nasorin precursor, ceruloplasmin and α-1-antitrypsin were identified between IgAN and thin basement membrane nephropathy, only ceruloplasmin showed up in both studies. Although not specific to IgAN, other urinary biomarkers include increased levels of EGF and FGF, loss of podocytes in the urine, glycan-specific IgG antibod-


ies along with proteinuria, advanced oxidative protein products and acute kidney injury molecule [8] . Biomarkers in serum & circulation

Research on serum biomarkers of IgAN has focused mostly on circulating galactose-deficient IgA1 (GdIgA1) containing immune complexes, autoantibodies formed against Gd-IgA1-specific IgA or IgG, and O-glycosylation patterns in the hinge region of IgA. Galactose-deficient IgA1 & Gd-IgA1-specific immune complexes

A recent study compared serum biomarkers between IgAN patients and healthy or CKD controls [26] . This study reported that serum levels of Gd-IgA1-specific IgA and IgG are predictive of IgAN progression, which can be differentiated from healthy controls as well as patients with CKD. In a subgroup of IgAN patients, serum levels of Gd-IgA1-specific IgG, but not GdIgA1-specific IgA correlated well with the high serum levels of Gd-IgA1, suggesting the surprising concept that there is a more significant role of Gd-IgA1-specific IgG than IgA in the pathogenesis of IgAN. Despite such an association, finding a serum biomarker is complex due to the fact that IgAN patients with normal serum levels of Gd-IgA1 also display elevated levels of Gd-IgA1-specific autoantibodies. Furthermore, this conclusion was supported by the finding that IgA1 levels are variable among IgAN patients. The GdIgA1-specific IgG was reported to have high prognostic sensitivity and specificity in IgAN patients as compared with healthy as well as CKD controls. However, the specificity lags when it comes to differentiating between IgAN patients and other immune-mediated renal disease. In addition, while this study included a sufficient number of patients, the IgAN patients


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and CKD controls were not aged-matched and were mostly Asian, thus diminishing the generalizability of these results. Overall, this study provides an excellent comparison of immune biomarkers of IgAN between IgAN patients and CKD/healthy controls and offers the Gd-Ig A1-specific IgG together with serum IgA levels as a good candidate biomarker in IgAN. Another study also supported these findings as they evaluated Gd-IgA1 in IgAN patients and found correlations between hematuria and proteinuria and changes in serum Gd-IgA1 and IgA/IgG immune complexes [27] . A multiple biomarker approach suggested that TGF-β1 is a prognostic biomarker when used in combination with Gd-IgA1 in IgAN [28] . Elevated levels of Gd-IgA1 in IgAN pathogenesis have recently been reviewed and validated in other studies, indicating that it is associated with disease progression [29–31] . Hinge-region O-glycosylation patterns in IgA1

The recently worked out mechanism of IgAN relates to aberrant glycosylation of the IgA molecule (Figure 1) which results in several areas of potential biomarker pursuits. Besides the serum levels of Gd-IgA1, some studies have focused on the O-glycosylation patterns of the hinge region on IgA1, and suggest that such patterns are associated with the accumulation of these immunoglobulins in the glomerular mesangium [32,33] . The circulating Gd-IgA1 might be present with a terminal N-acetyl galactose amine or are sialylated. It has been suggested that cytokines, specifically IL-6 and IL-4, affect the galactose-deficient sites in the IgA1 molecule [34] . In addition, intervention with tonsillectomy and corticosteroid IV was shown to improve aberrant O-glycosylation in IgAN patients in remission [35] ; however, the correlation of these changes to renal function has not been evaluated. Although future glycomics studies are warranted (including a glycomics study currently in progress in the authors’ laboratory), the studies published thus far suggest an important role of O-glycosylation patterns in the pathogenesis of IgAN. Serum C4a desArg & serum IgA/C3 ratio

Levels of serum C4a desArg were found to be increased in IgAN patients when compared with healthy controls [36] . While serum C4a desArg is suggested to be a prognostic biomarker, this finding needs to be evaluated in other glomerulonephritis to isolate it from IgAN. Serum IgA/C3 ratio has been suggested as a prognostic biomarker in several studies from 2003 to date [13,37,38] . Other plasma biomarkers

In a recent study, biopsy-proven IgAN patients were supplemented either with fish oil or corn oil (control). This study showed that inflammatory mediators of

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the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) pathways (two major omega-3 fatty acids found in fish or fish oil) might be involved in improving proteinuria in IgAN patients [39] . While this finding is relevant to the inflammation and complement components in IgAN, future metabolomics studies are necessary to identify new biomarkers through comparisons of the urinary or serum metabolome of IgAN patients with healthy controls as well as control individuals with other, non-IgA, glomerulonephritides. In addition to novel biomarkers that are recently identified, urinary soluble transferrin receptor is promising in the diagnosis and progression of IgAN; however it appears to be a biomarker for Henoch-Schonlein purpura nephritis as well [40] . Biofluid microRNA (miRNA) biomarkers in IgAN

miRNAs are small regulatory RNAs that regulate gene expression by post-transcriptional effects and are involved in many biological processes [41] (Figure 2) . miRNAs have been found to be directly involved in IgAN and hence several miRNAs have been evaluated and found to be dysregulated in serum, blood or urine in individuals with IgAN. Here we will discuss their potential as biomarkers for this disease (Table 2) . miR-148b

In humans, normal IgA1 glycosylation includes addition of galactose by the enzyme core 1, B1,3-galactosyltransferase 1 (C1GALT1) [42] . IgAN is thought to be caused by mesangial aggregation of aberrantly glycosylated IgA1 antibodies that are galactose deficient [43–45] . Serino et al. found higher expression of miR148b in peripheral blood mononuclear cells (PBMCs) in two independent cohorts of individuals with IgAN using microarray and qRT-PCR analyses [46] . The expression level of miR-148b was further validated on a larger, independent cohort of individuals with IgAN (n = 50 IgAN; n = 50 controls). Bioinformatic analyses suggested that C1GALT1 is a target and indeed, increasing the amount of endogenous miR-148b in PBMCs isolated from healthy individuals resulted in a threefold reduction in endogenous C1GALT1 mRNA levels (p < 0.03) and a 2.2-fold reduction in C1GALT1 protein levels (p < 0.002). Additionally, transfecting synthetic hairpin miRNA148b inhibitors in PBMCs isolated from individuals with IgAN led to a threefold increase in endogenous C1GALT1 mRNA levels (p < 0.01) and a 1.7-fold increase in endogenous C1GALT1 protein production (p < 0.01). This relationship was further validated by transfecting human B lymphoma DAKIKI cells (cells that produce IgA1) with either an miR-148b mimic or inhibitor. Cells transfected with the miR-148b mimic



Review

miRNA gene Exportin-5

Drosha

miRNA duplex

Premature miRNA Dicer

Primary miRNA

Mature miRNA

Nucleus

Cytoplasm

Initiation block of 40S and 60 S ribosomes

mRNA degradation

Proteolysis degradation of nascent peptide

Transcriptional repression

Stalled elongation or termination of translation

Translational activation

Prevention of ribosome assembly Figure 2. Methods of miRNA gene regulation. miRNAs are transcribed in the nucleus into primary miRNAs with distinctive stem loop structures. Drosha then cleaves the primary miRNA by the base of the stem loop, generating premature miRNAs with a 3’ overhang. They are exported out of the nucleus into the cytoplasm by Exportin-5 and then Dicer cleaves both strands by the terminal loop to reveal the 22 nt long miRNA duplex. The duplex unwinds and the mature miRNA can regulate gene expression in a variety of ways.

led to a significant increase in galactose-deficient IgA1 levels and those transfected with miR-148b inhibitor showed a significant reduction in galactose-deficient IgA1 levels. Lastly, although miR-148b expression was found in patients with other renal diseases such as membranoproliferative GN type 1, focal segmental glomerulosclerosis and Henoch-Schonlein purpura nephritis, the highest upregulation was found in individuals with IgAN, suggesting that its expression is likely to be specific to IgAN [46] . Out of all miRNA biomarkers published in the literature for IgAN, miR-148b has the most potential as a clinical biomarker as it was validated in several different cohorts and platforms and has been shown to be involved in regulating C1GALT1, a key player in the pathogenesis of IgAN [46] . PBMCs do not produce IgA, thus the exact degree of miR-148b upregulation and potential effect of inhibitors is unknown. However, PBMCs and major IgA producing cells both originate from bone marrow, suggesting that upregulation of miR-148b found in blood is likely to be related and that the degree of dysregulation can correlate with disease. Interestingly, Inversin (INVS) and phosphatase and tensin homolog (PTEN), putative target genes,


have been found to be downregulated in two independent IgAN cohorts, further suggesting that miR-148b is indeed dysregulated in individuals with IgAN and that its quantification may be used to diagnose IgAN [46,47] . Overall, the data for miR-148b are very suggestive; however, further validation studies are required to confirm the extent of miR-148b upregulation as one study found a downregulation of miR-148b in biopsy tissue from individuals with IgAN [48] . Additionally, it would be ideal to measure miR-148b expression in cells that synthesize IgA1. miR-223

Bao et al. profiled miRNAs in cultured glomerular endothelial cells (GEnCs) treated with conditioned medium from human mesangial cells (HMC) prepared with polymeric IgA (pIgA) from patients with glomerular endothelial proliferation (EP) [49] . They found decreased expression of miR-223 when compared with cells treated with IgA-HMC medium prepared with pIgA from patients without EP or the medium prepared with monomeric IgA from any source. This change was validated by qRT-PCR. This miRNA is promising as it is only specific in cells treated with medium prepared


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Table 2. Most commonly studied biofluid miRNA biomarkers in IgA nephropathy. Study (year)

Biomarker

Sample matrix

Changes Clinical pearls in levels

Comments

50 healthy 50 controls

PBMC

↑

‐

Prognostic; likely specific to IgAN ↑ C1GALT1

miR-188-5p 10 healthy 10 controls

PBMC

↑

‐

Prognostic

miR-361-3p

↑

‐

miR-8863p

↑

‐

Let-7b

↑

‐

Let 7d

↑

‐

miR-223

GEnCs treated with IgA-HMC medium prepared with pIgA

↓

‐

Urine

↑

↑ GFR, ↓ glomerular Predictive; ↓ Vimentin scarring, ↑ proteinuria

miR-200a

↓

↓ Proteinuria

miR-200b

↓

↑ GFR, ↓ proteinuria Predictive; ↓ ZEB2; ↓ Vimentin

↑

↑ Proteinuria

Predictive; ↑ RANTES; ↓ IL-1β; ↓ IL-6; ↓ TNF-α

↑

↑ Proteinuria

Predictive; ↑ FOXP3; ↑ RANTES; ↓ IL-1β; ↓ TNF-α;

↓

↑ GFR, ↓ proteinuria Predictive; ↓ SMAD3

miR-29c

↓

↑ GFR, ↓ proteinuria

miR-93

↑

↓ Glomerulosclerosis

miR-21

↓

↑ GFR

↑

‐

Serino miR-148b et al. (2012)

Bao et al. (2013)

Cohort Control (n) IgAN (n)

Wang et al. miR-429 (2010)

Wang et al. miR-146a (2011)


13 healthy controls

13 healthy controls

43

43

Urine

miR-155

Wang et al. miR-29b (2012)

Szeto et al. (2012)

miR-17

13 healthy controls

17 DOS controls; 22 HTN controls

43

17

Urine

Urine

Ref.

Predictive; may be specific to IgAN ↑ Importin α 4, ↑ Importin α 5

Prognostic; specific to CKD

[46]

[49]

[50]

[51]

[52]

[53]

#+$#HRONICKIDNEYDISEASE'EN#'LOMERULARENDOTHELIALCELL'&2'LOMERULARlLTRATIONRATE(-#(UMANMESANGIALCELL)G!.)G!NEPROPATHYMI2MI2.! 0"-#0ERIPHERALBLOODMONONUCLEARCELLP)G!0OLYMERIC)G!

from patients with EP and pIgA. However, no change in miR-223 expression was found in GEnCs treated with IgA-HMC medium prepared with pIgA from patients without EP and thus miR-223 may be a better biomarker for EP in general rather than for IgAN [49] . Other miRNAs

Several other miRNAs have been found to be significantly dysregulated in IgAN and have been suggested to be fluid biomarkers for the disease. Serino et al. per-

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formed the first global evaluation of miRNA expression in PBMCs of individuals with IgAN and found higher expression of miR-188–5p, miR-361–3p, miR886–3p, let-7b and let-7d in two independent cohorts of individuals with IgAN using microarray and qRTPCR analyses [46] . The expression levels of the miRNAs were quite low and sample sizes were small, thus further studies are required for validation of these miRNAs before they can be considered markers of IgAN.



Review

Table 3. Most commonly studied histological tissue biomarkers in IgA nephropathy. Study (year) Tissue biomarker

Sample matrix


Comments

Glomerulus

↑

↑ Proteinuria, ↑ TA/IF score†

Predictive; specific to IgAN and nephrotic syndrome [79]

[56]

Biglycan

↑

↑ Proteinuria, ↑ TA/IF score†

Perlecan

↑

↓ creatinine clearance, ↑ Proteinuria, ↓ progression, ↓ TA/IF score

Syndecan-1

↓

NDST

↑ Predictive; specific to IgAN and diabetic nephropathy [80]

[60]

Ebefor et al. Decorin (2011)

Cohort Control (n) IgAN (n) 14 normal adjacent tissue

19

↓

↑ Proteinuria, ↑ creatinine clearance

Hill et al. (2011)

Nephrin

Lai et al. (2009)

Synaptopod 15 healthy 22 in controls

Lai et al. (2009)

Podocin


Podocytes

↓

↑ Proteinuria, ↓ creatinine clearance

Predictive; specific to IgAN

[61]

Lai et al. (2009)

Ezrin


Podocytes

↓

↑ Proteinuria, ↓ creatinine clearance


[61]

Hill et al. (2011)

GLEPP-1

Podocytes

↓

Predictive; specific ↑ Proteinuria, to IgAN and pre↑ pathological eclampsia [81] grade of IgAN, ↑ serum creatinine, ↓ serum albumin, ↓ creatinine clearance

Tian et al. (2007) Kodama et al. (2013)

128

11 normal adjacent tissue Nuclear dendrin

Asanuma et al. (2007) Qiu et al. (2004)

128

Bcl-2

Podocytes

Ref.

↓

↓

51

14 129 (healthy mice)

129 (mice)

10

51

[61]

Podocytes

Podocytes

[60]

[62]

↑


[65]

↑

Glomerulonephritis

[66]

↓

↑ Proteinuria, Predictive; specific to ↑ serum creatinine, IgAN ↓ creatinine clearance, ↑ proteinuria

[67]

4!)&SCORE/XFORDCLASSIlCATIONFORDIAGNOSING)G!NEPHROPATHY!HIGHSCOREINDICATESPOORPROGNOSIS )G!.)G!NEPROPATHY

†

Wang et al. found a decrease in expression of urinary miR-200a, miR-200b, miR-429, miR-29b, miR-29c, and miR-21 and increased expression of urinary miR-146a, miR-155, and miR-93 in IgAN. They report several cor-


relations between specific miRNA and several disease measures such as proteinuria, GFR and other urinary proteins [50–52] . These studies are important as they point toward a correlation between disease symptoms and


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miRNA expression. However, some of the correlations are weak and further studies are required for validation. Szeto et al. found increased expression of urinary miR-17; however, the connection to IgAN is ambiguous. The sample cohort was small and it is not clear if any correlations to disease measures are specific to IgAN or to CKD in general [53] . Histological & immunological markers of IgA nephropathy

The ‘gold standard’ for diagnosing IgA nephropathy is biopsy of renal tissue. Standard criteria include evaluating mesangial deposition of IgA, IgG, an antibody specific for galactose-deficient IgA1 and complement protein (C3) accumulation in renal tissue [54] . The most common histological and immunological tissue biomarkers for IgAN are shown in Tables 3 & 4. Basement membrane specific biomarkers

Basement membrane-specific heparin sulfate proteoglycans (HSPGs) necessary for glomerular filtration include perlecan and syndecan-1. Compared with normal tissue, injury caused by IgAN leads to the regulation of HSPGs and are involved in endothelial growth and regeneration [55] ; there is an 87% upregulation of perlecan and a 72.1% downregulation of syndecan-1. [56] . Specifically upon tissue injury, syndecan-1 ectodomains are cleaved by heparanases, which activate bFGF [55] . The bFGFs have been shown to reduce functional and structural damage in chronic kidney disease [57] . Furthermore, N-deacetylase/N-sulfotransferase 1 (NDST1), an enzyme responsible for reducing perlecan and other proteoglycans, was increased by 37.6% in IgAN [58] . Clinically, glomerular expression of perlecan mRNA correlated with a decrease in creatinine clearance and an increase in proteinuria measured over a 24-h period, suggesting that it may be used as a predictive indicator for IgAN progression [56] . Podocyte-specific biomarkers

Podocyte injury and detachment, which is associated with many nephropathies, has become a valuable diagnostic marker although it lacks specificity for glomerular disease. Loss of podocyte-specific markers, such as nephrin, podocin, ezrin, GLEPP-1 and synaptopodin, which form lipid rafts on podocytes that contribute to the slit diaphragm [59] , have been associated with proteinuria and diminished creatinine clearance [60–62] . Although the importance of such markers in IgAN is unknown, Lai et al. added IgAN conditioned media from mesangial cells to podocytes [61] and demonstrated paracrine-mediated suppression of these podocyte-specific markers. Furthermore, Xu et al. showed that podocyte loss or flattening, which is seen in IgAN

1272

Biomark. Med. (2014) 8(10)

and negatively affects glomerular permselectivity, is inversely proportional to nephrin mRNA expression and may be indicative of IgAN progression [63,64] . It has been suggested that translocation of dendrin (a protein whose function is still unclear) into the nucleus results in apoptosis and loss of podocytes [65,66] . In addition to the translocation of dendrin, downregulation of anti-apoptotic Bcl-2 in podocytes has been associated with elevated serum creatinine, a decline in creatinine clearance, and an increase in proteinuria in IgAN [67] . Ultimately, the loss of podocytes results in a breakdown of the filtration barrier, which leads to a dysregulation of homeostasis and therefore ESRD associated with IgAN progression. Biomarkers in the mesangial matrix

A hallmark of IgAN is mesangial cell and matrix expansion. Zhou et al. studied matrix alterations associated with IgAN injury and found that decorin and its analog biglycan – pericellular matrix proteoglycans involved in fibrillogenesis and the regulation of the cell cycle [68] – were increased by 45.1 and 136.6%, respectively [56] . Recognizing this injury, MBL has been proposed to attack damaged and/or apoptotic renal cells, which results in its accumulation in renal tissue and expansion of the mesangial matrix [69] . Immunofluorescence studies on 131 IgAN patients yielded positive staining of MBL in 35% of patients [69] . Clinically, MBL deposition results in more severe proteinuria, decreased renal function, lower levels of serum albumin, a greater probability of hypertension and an overall poorer prognosis [69] . Promising IgA receptors supporting IgA deposition

A study using IgAN-like renal disease in transgenic mice, found that CD89, an Fc-α receptor involved in inflammation, was overexpressed [70] . Upon further examination, CD89 was found to complex with IgA and facilitate its deposition in the mesangium [70,71] . This is further supported by the fact that no detectable renal dysfunction occurs upon IgA1 deposition in the absence of CD89 [70] , helping to clarify why IgAN patients may or may not express signs of glomerulonephritis. Furthermore, CD89 expression was found to correlate with levels of proteinuria and hematuria [72] . Another immune receptor, CD71, was upregulated in proliferating HMCs in IgAN [73] . CD71 is a transferrin receptor that binds IgA1. Studies show that IgA binding creates a positive feedback loop which causes overexpression of CD71 [74,75] . Furthermore, CD89–CD71 interaction was found to favor IgA deposition and activate mesangial cells, suggesting a direct cause for IgAN development. However, the relationship between CD71 levels and severity of IgAN has not been established.



Review

Table 4. Most commonly studied immunological tissue biomarkers in IgA nephropathy. Study (year)

Immune biomarker

Liu et al. (2013)

MBL

Zhou et al. FCGR2B (2013) rs12118043 and rs1954174

Cohort

Sample matrix


Comments

Ref.

131

Mesangial matrix

↑

↑ Proteinuria, ↓ renal function, ↑ serum albumin ↑ Hypertension


[69]

1200

Macrophages, neutrophils, natural killer cells, mast cells

↑

↑ Proteinuria, ↑ hematuria

Prognostic and predictive; specific to IgAN

[68]

B cells

↑

↑ Serum creatinine, ↓ eGFR

Mesangial matrix

↑

↑ Macrophage Predictive; infiltration, specific to ↑ proteinuria, IgAN ↑ hematuria, ↑ serum creatinine levels

[72]

↑


[73]

↑ Proteinuria, Predictive; ↑ hematuria, ↑ serum specific to creatinine level, IgAN ↑Severity

[49]

↑ Proteinuria

[53]

Control (n) IgAN (n)

900 healthy controls

FCRLB rs4657093 Launay et al. (2000)

CD89


Moura et al. (2001)

CD71

5 normal adjacent tissue

Bao et al. (2014)

miR-223


↓ Circulating endothelial cells

Szeto et al. (2012)

miR-29c


Intrarenal and urinary

5 (normal Mesangial cells adjacent tissue)

↓

Predictive; specific to IgAN and renal interstitial fibrosis [82]

E'&2%STIMATEDGLOMERULARlLTRATIONRATE)G!.)G!NEPROPATHY

Nucleic acid biomarkers

Zhou et al. sought to study IgAN-associated gene polymorphisms in a population of 2100 Han Chinese. They found single nucleotide polymorphisms (SNPs) rs12118043, rs1954174 and rs4657093, within the gene loci FCGR2B, FCRLA, and FCRLB, (respectively) that independently associated with IgAN [68] . Physiologically, Fc gamma receptors (FCγRs) and Fc receptorlike proteins (FcRLs) couple the recognition of antigens by IgG1 antibodies to cellular responses in innate and humoral immunity, respectively [76] . Clinically, there exists a correlation between proteinuria, hematuria and the expression of FCGR2B rs12118043. Furthermore, a correlation between FCRLB rs4657093 expression and levels of serum creatinine and eGFR has also been established [68] . This indicates that these variants not only impact IgAN susceptibility, but also its severity. Tissue microRNAs

Gene regulation is an important part of disease prevention. Critical components of gene regulation


include miRNAs. As described in an earlier section, many miRNAs may be important biofluid markers for IgAN. In circulating endothelial cells, Bao et al. discovered that levels of miR-223 inversely correlate with levels of proteinuria, hematuria and directly correlate with creatinine clearance [49] . Furthermore, low intrarenal concentrations of miR-29c were found to be an indicator of late-stage disease progression as it [77] inversely correlated with the severity of proteinuria [52,78] . A genome-wide sequencing effort of miRNAs in renal specimens from six individuals with IgAN and six controls observed 85 miRNAs that were differentially expressed between the two groups that included miR-133, miR-17, miR-106 and miR-185 [48] . Interestingly, there was downregulated expression for miR- 429, miR-200a, miR-200b, miR-29b and miR-29c found in tissue and biofluid in separate studies, suggesting that this difference in expression may be real [48,50,52] . Only miR-17 was found to be upregulated in biofluid and downregulated in tissue [48,53] .


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Conclusion IgA nephropathy is a prevalent disease that currently suffers from the lack of specific biomarkers for disease presence as well as activity. Many patients progress to CKD/ESRD but, it is not infrequent for others to show spontaneous remission. Since renal biopsy (the current gold standard) and many putative therapies (e.g., tonsillectomy) are far from benign, it is important to be able to differentiate such patients. There has been some progress in biofluid markers for both of these issues, especially proteins encompassing Gd-IgA-specific IgAs and IgGs. In addition, the emerging field of biofluid miRNAs, yielding miR-148b, is also showing promise. Further validation of all of these markers in appropriate well-controlled patient populations will soon lead to clinically validated markers that can be translated to the nephrology clinic. Future perspective Given the current level of interest in, and rapidity of, research in the pathogenesis and potential therapy of

IgA nephropathy, it is highly likely that a serum biomarker for this disease will be discovered and fully validated in the next 5–10 years. The most likely marker will relate to the presence in serum of the aberrantly glycosylated IgA and/or an immune response to the same. It is also possible that, given the evolving field of miRNAs, that a miRNA marker may be identified. Prognostic biomarkers will likely lag in relation to predictive biomarkers, in light of the time it takes to establish the outcome of patients in this slowly progressive disease. Financial & competing interests disclosure 4HE AUTHORS HAVE NO RELEVANT AFlLIATIONS OR lNANCIAL INVOLVEMENT WITH ANY ORGANIZATION OR ENTITY WITH A lNANCIAL INTEREST IN OR lNANCIAL CONmICT WITH THE SUBJECT MATTER OR MATERIALSDISCUSSEDINTHEMANUSCRIPT4HISINCLUDESEMPLOYMENT CONSULTANCIES HONORARIA STOCKOWNERSHIPOROPTIONS EXPERTTESTIMONY GRANTSORPATENTSRECEIVEDORPENDINGOR ROYALTIES .OWRITINGASSISTANCEWASUTILIZEDINTHEPRODUCTIONOFTHIS MANUSCRIPT

Executive summary IgA nephropathy is a common glomerular disease with no clinically useful biofluid markers s Renal biopsy is the current gold standard diagnosis. s Patients with unexplained gross or microscopic hematuria can be expected to have IgA nephropathy (IgAN). s End-stage renal disease occurs in up to 50% of IgAN patients, but 10–20% show spontaneous remission.

Protein biomarkers s Among the urinary and serum biomarkers of IgAN, Gd-IgA1 and Gd-IgA1-specific IgG appear to be helpful biomarkers to differentiate between patients with IgAN and healthy controls and those with chronic kidney disease. s The emerging field of glycomics may yield novel markers based on the fact that IgAN is caused by deranged O-glycosylation at the hinge region of the IgA1 molecule.

miRNA biomarkers s The identification of miRNAs as fluid biomarkers is becoming increasingly important as many miRNAs have emerged as key players in the pathogenesis and progression of IgAN. s miR-148b is the most promising candidate as it was found to be upregulated in blood of IgAN patients.

Histologic markers of IgAN s Renal biopsy is currently the only diagnostic instrument for IgAN in widespread clinical use. s The standard criteria for diagnosing IgAN includes evaluating mesangial deposition of IgA, IgG, an antibody specific for galactose-deficient IgA1 and complement protein (C3) accumulation in renal tissue. s Basement membrane specific heparin sulfate proteoglycans (HSPG) may prove promising for prognostication. s Novel mesangial matrix markers and SNPs are still being investigated and may add to the armamentarium for tissue IgAN diagnosis.

Conclusions s IgAN is a common disease that lacks noninvasive biomarkers. s IgAN can lead to progressive renal disease, can be stable, or can spontaneously remit so specific biomarkers are essential for appropriate management of these patients. s New candidate proteins and nucleic acids are being studied and may yield new therapeutic approaches.

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