Background

Juvenile-onset systemic lupus erythematosus

Juvenile-onset systemic lupus erythematosus (JSLE) is a rare, severe multi-systemic autoimmune disease, characterized by autoantibodies directed against nuclear antigens [1]. Up to 20 % of all patients with SLE will have an onset prior to adulthood, and childhood disease is associated with more serious organ involvement, a more aggressive disease course and the use of more immunosuppressive therapies, when compared to its adult onset counterpart [2].

JSLE has an estimated annual incidence of 0.36–0.9 per 100,000 children per year, with a significantly higher incidence in non-Caucasian children, especially Black, Hispanic and Asian ethnic populations [3]. The American College of Rheumatology (ACR) has formulated SLE classification criteria [4] which are used to classify the symptoms and signs of SLE in both adult and paediatric cases.

Over recent decades, the overall survival of patients with JSLE has improved considerably, with 10-year survival rates now approaching 90 % [5]. Despite medical advances, patients with JSLE have a significantly lower life expectancy than the general population, with a fourfold greater risk of death, and a 2.5-fold increased mortality risk when compared to adult-onset SLE disease [5].

Juvenile-onset lupus nephritis

Despite lupus being a rare cause of glomerulonephritis—accounting for only 1–2 % of all causes of end-stage renal disease (ESRD)—the incidence of ESRD in juvenile-onset lupus nephritis (LN) is high and ranges from 4 to 44 % [6, 7]. The largest unselected cohort of patients with JSLE identified 4 % of patients who had developed irreversible renal damage over a 5-year recruitment period, including one patient with established renal failure [8]. Variability in the reported rates of chronic renal damage in JSLE may depend on age, ethnicity, patient selection, and length of patient follow-up in the respective cohorts.

Irreversible renal damage is one of the most common long-term consequences seen in JSLE, and the presence of renal involvement in patients with JSLE is independently associated with a worse disease morbidity [9].

One of the major concerns is that prior to the onset of clinically detectable renal disease irreversible damage may be occurring in the kidney, contributing to future renal damage. Combined with this is the recurrent, fluctuating nature of LN.

Histological classification of lupus nephritis

At present, the renal biopsy is the gold standard in confirming the diagnosis of LN. Internationally standardized histological classification criteria allow important comparisons between individual patients and between international cohorts, as well as providing important prognostic information.

Histological analysis typically reveals mesangial expansion, hypercellularity, complement and immunoglobulin deposition and tubulointerstitial inflammation and, in more severe cases, glomerular scarring and crescent formation. The International Society of Nephrology–Royal Pathology Society Classification of Glomerulonephritis in Systemic Lupus Erythematosus (2003) classifies histological findings according to the extent of these changes [10]:

  • Class I: minimal mesangial lupus nephritis.

  • Class II: mesangial proliferative lupus nephritis, involving a mesangial pattern of disease with mesangial hypercellularity and matrix accumulation as a result of mesangial immune complex accumulation.

  • Class III: focal lupus nephritis where <50 % of the glomeruli have active and sclerotic lesions.

  • Class IV: diffuse segmental (IV-S) or diffuse global (IV-G) lupus nephritis where >50 % of the glomeruli will have fibrin necrosis and cellular crescents, and are divided into acute or chronic appearances.

  • Class V: membranous lupus nephritis sub-epithelial immunoglobulin deposits are seen along the basement membrane.

  • Class VI: advanced sclerosing lupus nephritis, >90 % of glomeruli are sclerosed with no residual functional ability.

Within the classification are subgroups of active (indicating may improve with treatment) and chronic (irreversible scarring) changes together with proliferative lesions, which can be segmental or global in nature. Global, active changes predict a worse renal outcome [11]. These revised classifications are important and hopefully will more accurately predict the eventual renal prognosis; however, the exact role of these revised criteria in paediatric LN is yet to be determined [12].

LN affects all compartments of the kidney, including the endothelial cells and tubular renal cells. The ISN-RPS guidance recommends that pathologists should indicate and grade (mild, moderate, severe) the presence of tubular atrophy, interstitial inflammation and fibrosis and the severity of any arteriosclerosis or other vascular lesions. However, the exact classification criteria are linked to glomerular lesions exclusively.

The indications for undertaking a renal biopsy in cases of LN remain controversial, particularly in childhood disease, as clinical indicators fail to reliably predict the histological findings [13]. Despite its diagnostic and prognostic strengths, the renal biopsy is an invasive procedure with the risk of complications. In children, the procedure frequently requires sedation, which increases the risk of complications and demands the need for observation. Protocol renal biopsies, performed following a period of intensive induction treatment, can be extremely informative in determining treatment response, but they are not routinely conducted, especially in children [14].

At the time of histological assessment, around 50 % of children with LN will have class IV glomerulonephritis, which is the most severe histological category, carrying the worst renal prognosis [15]. In a series of children with LN, treatment response, histological classification and ethnicity were both independently related to a poor renal outcome [15], and a multi-national study of JSLE patients demonstrated 5-year renal damage rates of 22 % [16].

Management

The management of LN relies heavily on the use of corticosteroids, together with systemic immunosuppression. The development of steroid-sparing regimens is the focus of many current adult-orientated SLE clinical trials; however, the burden of corticosteroids on children with JSLE is likely to remain for some time. In a study undertaken by Brunner et al., higher levels of corticosteroid were used in JSLE (around 90 %) when directly compared to adult-onset SLE, where only 37 % of adult patients ever required steroid therapy [17]. A suggested treatment plan for histologically proven class III and IV LN in children has been devised by the Childhood Arthritis Rheumatology Research Alliance (CARRA) using consensus methodology [18]. Severe cases of LN rely on the use of cytotoxic immunosuppressive treatment, such as cyclophosphamide, a drug associated with serious long-term side effects, including malignancy and infertility. Mycophenolate mofetil is being increasingly used and has been shown to have good efficacy in achieving remission in JSLE [19]. For mild to moderate renal involvement, azathioprine is used. Hydroxychloroquine is recommended for all patients unless contraindicated as it has renal-protective effects [20]. Rituximab, a therapy with steroid-sparing potential, is used in JSLE, with the clinical impression that it has a key role in second-line management despite varying evidence supporting its use in clinical trials [21]. However, open-label studies on the use of rituximab as a steroid-sparing therapy have been successful in adults [22] and children with LN [23], and further clinical trials are currently planned.

The high prevalence of patients experiencing nephritis in the course of JSLE and the significance of its consequences on overall life expectancy and treatment modalities represent the enormity of this problem. The renal biopsy continues to play an essential role in this condition, and disease activity biomarkers with better accuracy and more acceptability to the paediatric age group are needed.

Clinical presentation

The clinical presentation of LN is varied, ranging from mild urine abnormalities to nephrotic or nephritic syndrome. LN typically enters remission following treatment but may flare at any point over the patient’s lifetime, with around one-third of patients experiencing a renal flare over an 8-year period [24]. The extent of proteinuria at disease onset and a failure to respond to treatment are both independently related to eventual renal damage and long-term outcome [24]. In disease that has progressed to ESRD requiring kidney transplantation, asymptomatic subclinical disease recurrence is seen in the renal graft despite immunosuppressive therapy [25].

Standard laboratory markers of disease activity

Proteinuria

Proteinuria is currently the principal urinary biomarker used in clinical practice when screening for renal involvement. Proteinuria quantification is incorporated in both of the commonly used SLE disease activity tools, namely, the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and the British Isles Lupus Assessment Group (BILAG) scoring tool, both of which act as measures of overall disease activity and include a renal subsection [26, 27]. Methods employed for collecting and analysing proteinuria in clinical practice vary greatly, ranging from urine dipstick analysis to 24-h urine collection, and both disease activity tools accommodate for this. Urine dipstick analysis testing can be useful in screening for the absence of proteinuria; however, its specificity is poor and quantification of proteinuria using dipstick is inaccurate, and so more formal microscopic and protein quantification is recommended in routine JSLE monitoring [28]. Although timed 24-h urine collection remains the most accurate method of quantifying protein loss from the kidney, in clinical practice a prolonged urine collection is cumbersome and impractical, particularly in a childhood population, leading to the more frequent use of spot urine measurements. Early morning urine albumin to creatinine ratios have been adopted as the most useful and practical method of quantifying protein loss in juvenile-onset LN as they correlate well with 24-h loss [29]. Spot measurements correlate well in cases of moderate to heavy 24-h protein loss, but their ability to accurately quantify protein loss in patients with microscopic proteinuria has been questioned [30]. With the establishment of irreversible renal damage in LN, which can cause chronic renal protein loss, proteinuria does not necessarily correlate with the active or acute histological changes seen in LN and therefore is a poor indicator of underlying disease severity or activity [31].

Despite these limitations, proteinuria has a proven relationship with the eventual renal outcome. Alternative methods of estimating 24-h protein loss, such as 6- or 12-h timed collections are under investigation, but are as yet to be in routine use in patients with LN [32].

Glomerular filtration rate

The glomerular filtration rate (GFR) is the most useful and standardized indicator for defining and classifying the extent of chronic kidney disease in a range of conditions, including lupus glomerulonephritis. Determining a precise measure of the GFR is timely, costly and unsuitable for routine monitoring in JSLE. The Schwartz formula based on body habitus (height) and creatinine concentration provides an acceptable estimation of GFR [33]. It is worth noting that estimations of renal function based on creatinine may be imprecise in cases of active lupus where patients may have experienced weight loss and have a reduced muscle bulk. Despite this, the estimated GFR is important in standardizing the classification of chronic kidney disease, and a decrease in GFR at diagnosis is associated with a worse renal outcome [34].

Urinary sediment

Urinary sediment is the centrifuged deposit of cells and contains red cells, white cells or urinary casts in the urine collection, all of which are then suitable for microscopic evaluation. It is increasingly being used to guide new biomarker discovery in many renal diseases and has played a significant role in directing many of the current biomarker studies in LN. The presence or absence of ‘active’ urinary sediment is incorporated into the two main JSLE disease activity tools (SLEDAI, BILAG), with ‘active’ sediment generally being accepted as >5 cells per high power field (HPF) in the absence of an infective organism [26, 27]. The urine sediment is a useful measure of disease activity and has facilitated the understanding of inflammatory substances present in active LN. However, it should be noted that reports have been published on patients with active LN who had a normal urine sediment, and so its role as a solitary indicator may be limited [35].

Serum complement levels

Reduced serum complement levels with organ deposition of complement are typical of this disease. Reduced serum complement protein levels have been used as a marker of active lupus disease flares for over 30 years, with cross-sectional studies demonstrating their relationship with overall disease activity [36]. More recently, the reduction of complement 3 and 4 (C3, C4, respectively) at diagnosis of LN has been linked to a worse overall mortality, but no associated difference in overall renal survival has been found [37]. On a cross-sectional basis, varying reports exist regarding the ability of C3 or C4 to significantly identify patients with active LN. Longitudinal studies highlight the limitations of serum complement proteins in SLE. A 6-year longitudinal study assessing complement levels in predicting LN disease flares demonstrated excellent negative predictive values—normal results are reassuring—but highlighted the need to combine these laboratory markers with others for a more precise estimation of flares as they may alter with non-renal lupus activity [38]. Complement proteins C3 and C4 clearly have significant strengths as biomarkers, including a role in disease pathogenesis, helping to distinguish JSLE glomerulonephritis from other renal diseases, good negative predictive value and a relationship with overall lupus disease activity; in addition, they form an important part of disease activity monitoring tools. Used in isolation, however, they act as weak indicators of LN in lupus cohorts, and given the higher proportion of JSLE patients who will have inherited complement deficiencies they may not be suitable for childhood-specific disease [39].

Anti-complement 1q antibodies

Complement 1q (C1q)is a subunit of the C1 enzyme complex that activates the serum complement system and normally allows a site for attachment of antibody-associated immune complexes. Although rare, complete deficiency of C1q fails to control the release of interferon-alpha by plasma dendritic cells. C1q has a key role in JSLE pathogenesis, as inherited C1q deficiency is the strongest single genetic factor identified to predispose to early-onset JSLE [40]. Patients who do develop C1q-associated JSLE have a more severe disease course with a predominance of cutaneous and renal manifestations. Antibodies to C1q (anti-C1q) are detected in the blood of patients with lupus in around 30–50 % of cases [41]. Elevated anti-C1q titres are strongly associated with renal involvement in lupus, and they can be extremely beneficial in disease monitoring [42]; for these reasons they are considered blood biomarkers of disease activity. In unselected cohorts, however, the sensitivity of anti-C1q as a marker of renal disease activity is generally around 40–60 %, with a good specificity of >80 % [43]. One drawback may be the association they have with global disease activity, as seen in large prospective unselected cohorts and the high rate of C1q deficiency seen in JSLE [44].

Anti-double-stranded DNA antibodies

Antibodies against the double-stranded DNA (anti-dsDNA) nuclear components of cells are the classical marker of lupus and form an important component of the diagnostic process, assisting with classification and differential diagnosis of other causes of glomerulonephritis. The determination of anti-dsDNA is recommended in the regular monitoring of all patients with lupus, and the presence of anti-dsDNA has been found to precede the onset of lupus symptoms by up to 5 years [45]. The sensitivity of anti-dsDNA in detecting renal flares is around 50 % and in a similar manner to other anti-nucleosome antibodies, the specificity is good at >80 % [46]. Recent studies have combined anti-C1q and anti-dsDNA antibody testing to produce a stronger predictive value of renal disease flares, but still at the cost of suboptimal sensitivity [43].

Other laboratory markers

Other autoantibodies, such as anti-alpha actinin antibodies and many novel blood biomarkers, have been assessed in relation to lupus renal disease activity. A detailed discussion of these markers is beyond the scope of this review.

In conclusion, conventional laboratory markers provide a useful adjunct in the clinical diagnosis of LN, and negative results may be very informative. Combining standard markers with novel markers may produce acceptable sensitivity and specificity levels to act as a tool for screening renal inflammation in juvenile lupus. Overall, as individual biomarkers of renal disease activity, standard markers of disease activity each perform poorly, and there is a need for novel non-invasive urinary biomarkers of LN.

What is a biomarker?

A biomarker is defined as a ‘characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention’ [47]. In clinical practice many tests of physiological measures are used as disease biomarkers, such as the use of glycated haemoglobin as an indicator of glycaemic control in diabetic disease monitoring. The National Institute of Health (NIH) Definition Working Group divided biomarkers into either those that act as markers of the natural history of a disease and correlate longitudinally with known clinical indices, or those that capture the effects of a therapeutic intervention in accordance with its mechanism of action [47]. For the purposes of this review looking at urine biomarkers of LN disease activity, we concentrate on biomarkers that correlate with the natural history of the disease.

The potential impact of validating robust biomarkers in LN is enormous. As an example, in other conditions biomarkers can be potential early indicators of disease traits, such as raised immunoreactive trypsin in neonatal cystic fibrosis, and can reflect the disease prognostic stage. They may also identify patients at high risk of developing disease and may act as prognostic biomarkers enabling a prediction on the likely outcome and response to intervention.

The ideal biomarker has many similar characteristics: it needs to be accurate, show good sensitivity (identifying disease when it is present) and good specificity (excluding disease when it is not present), be relatively non-invasive and be reliably reproducible. Following initial identification, biomarkers must demonstrate evidence of robust validation using large, longitudinal, prospective studies to demonstrate a high predictive ability of the disease in question. For use in clinical practice, they should also demonstrate cost-effectiveness.

The principle objective of identifying an effective biomarker in LN should be to augment—rather than necessarily replace—current conventional clinical observations.

Biomarker development

A biomarker must go through several phases of development, including planning, discovery, validation and commercialization (see Fig. 1). In reality, many potential biomarkers remain in the planning and discovery phase, with few undergoing robust clinical validation. Biomarker discovery usually begins with the identification of a proposed biomarker through techniques such as proteomics, metabolomics and transcriptomics. This is generally followed by cross-sectional cohort analysis of the clinical utility of the proposed biomarker to predict a firm outcome—preferably the disease ‘gold standard’. The suspected biomarker needs to demonstrate a sound sensitivity and specificity using receiver-operating characteristic (ROC) curves to determine the optimal biomarker concentration that achieves the most reliable interpretation. Following cross-sectional assessment, proposed biomarkers need to demonstrate an ongoing relationship with standard disease markers, with fluctuations in disease activity and/or with interventional processes over a defined longitudinal period. In reality, very few tests are ideal biomarkers in any one disease at every point in time.

Fig. 1
figure 1

The process of biomarker validation

Full size image

In LN, the validation of a novel biomarker should be compared against the current gold standard, which is histological classification. However, there are currently no standardized guidelines on when to conduct a renal biopsy in JSLE. In childhood disease, biopsy is only conducted if there is a strong clinical suspicion of renal involvement and is rarely routinely repeated following the initial diagnosis and classification. Longitudinal validation of a novel biomarker may therefore require the use of an alternative ‘gold standard’, such as non-invasive disease activity tools. The SLEDAI or the BILAG scoring tools can provide a non-invasive multi-system measure of disease activity, and both include aspects of the renal system [26, 48]. These tools are validated for use in monitoring lupus, but were not specifically designed for renal flares in isolation, which may limit their ability to mimic the gold standard. However, they do take into account several aspects of LN, including proteinuria, urinary sediment, hypertension and histological classification, and at the present time they are the only alternative standard available.

Urine as a biomarker of glomerulonephritis

Validated, non-invasive biomarkers in a childhood population are particularly attractive for clinical use, with urine being readily available and easy to process. Childhood-specific biomarker validation studies are important as urine cytokine and proteome profiles may differ according to age [49]. A significant advantage of the paediatric age group is that it represents a population with few co-morbidities; as such, identified biomarkers may provide direct insight into renal pathophysiology, with urine sampling often reflecting local renal inflammation. Variations in urine concentration throughout the daytime mean that biomarkers may require correction for the urine creatinine concentration. A useful urine biomarker should represent direct renal injury and inflammation and not just increased permeability of the basement membrane with protein loss in the urine. Commercialization, with point of care testing, needs to be produced in collaboration with industrial partners for those biomarkers that demonstrate sound longitudinal assessment to standardize and facilitate ease of testing.

A perfect biomarker in JSLE nephritis would be able to identify the presence of renal involvement, improve as the condition responds to treatment and anticipate subsequent disease flares.

Promising biomarkers in JSLE nephritis

A number of potential LN biomarkers have been identified in recent years. Most of these potential biomarkers focused on distinguish JSLE patients who have nephritis, while some focused on identify the histological classification; only rarely were potential biomarkers identified that guide prognosis. One of the first and informative studies looking at urinary biomarkers in adults with LN was that by Rovin’s group, who found a strong association of urinary monocyte chemoattractant protein 1 (MCP1) with LN and a lack of relationship of interleukin (IL) 8 with renal disease [50]. Since then, proteomic and transcriptomic studies have utilized the mass screening of micro-proteins, genes and metabolites, respectively, in cohorts of patients with LN to guide many of the cross-sectional and longitudinal validation studies. The most promising biomarkers are those that have demonstrated a robust relationship with disease activity over a longitudinal period in childhood-specific cohorts.

Longitudinally validated biomarkers in a paediatric cohort

Urine neutrophil gelatinase-associated lipocalin

The most promising urine biomarker in predicting flares in children with LN to date is neutrophil gelatinase-associated lipocalin (NGAL). NGAL is a member of the lipocalin family of proteins and is responsible for the growth and differentiation of epithelial cells, including renal tubular epithelial cells [51]. It is known to exert bacteriostatic potential due to interference with bacterial siderophore-mediated iron acquisition. It is up-regulated by the kidney in response to renal injury and has been investigated as a useful non-invasive marker of acute kidney injury in a number of conditions, including predicting the need for dialysis in cases of diarrhoea-associated haemolytic uraemic syndrome and renal graft rejection [52–54].

Preclinical studies have found that renal tissue in murine models of LN overexpresses NGAL in response to pathogenic anti-dsDNA antibodies and, more recently, NGAL knock-out mice have a less marked renal pathology and improved outcome in autoimmune nephritis, suggesting an important role of NGAL in pathogenesis [55]. In contrast to other sepsis-related studies, NGAL is believed to play a detrimental role in LN by inducing apoptosis in mesangial cells and facilitating recruitment of inflammatory cells to the kidney through the up-regulation of pro-inflammatory mediators [56].

Cross-sectional cohort trials have demonstrated the strength of NGAL in relation to LN flares. One of the first of these studies was conducted by Brunner et al. and involved 35 JSLE patients who were compared against eight children with juvenile idiopathic arthritis (JIA) [57]. These investigators found a significantly higher level of urinary NGAL in the JSLE patients and a trend towards increased urinary NGAL in those JSLE patients with biopsy-proven LN and a high renal disease activity score [57]. Since then, a large prospective cross-sectional lupus cohort study has demonstrated the ability of NGAL to distinguish lupus patients with and without nephritis [58].

In the next step of validation, longitudinal studies have been conducted using urinary NGAL as a biomarker of active LN, remarkably led by childhood cohorts. Brunner et al. selected a cohort of 85 JSLE patients, of whom 52 had >1 follow-up visit, and compared these patients to 30 JIA and 50 healthy control patients [59]. In this study, the investigators found a significant increase in urinary NGAL, but not plasma NGAL, in those with worsening renal disease activity [59]. The second longitudinal cohort study involved 111 JSLE children, some of whom were included in the above study, seen on at least three follow-up visits. The investigators concluded that urinary NGAL is an excellent candidate as a predictive biomarker for worsening of childhood-onset SLE renal disease activity [60]. Since then, SLE longitudinal studies have been published confirming these results in adult populations [55].

The limitations of urine NGAL include its inability to obviously discriminate between histological classes of LN, its fluctuations with global JSLE flares and its non-specificity for JSLE, with rises seen in the previously mentioned conditions and also in acute urinary tract infections [61]. The non-specificity of urine NGAL for LN should not distract from its obvious strengths, as distinguishing patients from the aforementioned other renal diagnoses should be straightforward. Additionally, all patients with lupus should routinely have urine microscopy and culture performed to evaluate the urine sediment and to exclude urine infection. To date, urine NGAL is the only single biomarker to stand robust longitudinal cohort investigation in paediatric-specific cohorts, and its future is promising. The next step in developing urinary NGAL as a robust biomarker in LN requires validation in other large JSLE and adult lupus cohorts, to determine optimal thresholds and, if reliable, collaboration with industry in designing high-throughput real time testing platforms. Identifying complementary biomarkers that may further strengthen NGAL as a biomarker of LN may provide a more standardized approach to the investigation and management of this condition.

Cross-sectional biomarkers in a paediatric cohort

Urine monocyte chemoattractant protein-1

monocyte chemoattractant protein-1 is involved in a cytokine network acting to recruit inflammatory cells to infiltrate the kidney in LN. MCP1 is expressed by the mesangial, podocyte and monocyte cells in response to pro-inflammatory signals, such as tumor necrosis factor-alpha (TNF-α). These inflammatory cells and substances subsequently mediate tissue injury and can lead to renal dysfunction [62]. Additionally, MCP1 binding has been shown to reduce the expression of nephrin, an important marker in preserving kidney cell function, and antagonists to MCP1 prevent renal disease progression in murine models [63]. Marks et al. demonstrated that the presence of MCP1 within the glomerulus correlates with a poor renal prognosis in childhood LN and can distinguish more severe histological classes of the renal disease [64].

Early studies analyzing MCP1 as a biomarker, involving adults with SLE, demonstrated its correlation with histological disease, and short-term follow up showed improvement in urinary MCP1 levels in quiescent disease [65]. Since then, several cross-sectional studies have confirmed these findings using larger adult SLE cohorts [66], and a longitudinal cohort showed its strength in predicting renal flares up to 4 months prior to clinical detection [50].

In a cross-sectional childhood cohort, urinary concentrations of MCP1 correlated with histological diagnosis of lupus glomerulonephritis [49], and more recently urinary concentrations of MCP1 have been shown to relate to the renal component of the BILAG disease activity tool [67].

Limitations of urinary MCP include its association with other inflammatory and autoimmune renal conditions, including diabetic nephropathy; however, given its success in cross-sectional analysis and in adult studies, paediatric-specific, longitudinal cohort validation is required.

Urine transforming growth factor-beta

Transforming growth factor-beta (TGF-β) has a leading role in the control and onset of autoimmunity. It has been shown to contribute to early mesangial proliferation and crescent formation in several causes of glomerulonephritis [68]. Reduced serum TGF-β concentrations are typical of lupus, while raised urine TGF-β levels have been found in adult gene expression assays at the time of active disease and a reduction seen in treatment responders [69]. An elevation in urinary TGF beta was also seen in a cross-sectional paediatric cohort involving 32 JSLE patients with LN[70].

Urine N-acetyl-beta-D-glucosaminidase and urine retinol binding protein

Tubular interstitial disease is an important part of the pathophysiology in LN. The tubular markers N-acetyl-beta-D-glucosaminidase (NAG) and retinol binding protein (RBP) were measured in the urine of a cohort of 21 JSLE patients, demonstrating their ability to distinguish between those with and without nephritis. Over a period of 12 months after biomarker evaluation, two non-nephritis patients with initially elevated RBP developed LN, suggesting the potential of RBP to predict impending flares prior to the onset of proteinuria [71]. An adult cross-sectional cohort (n = 24 SLE patients) has also shown how urine NAG levels correlated with proteinuria and renal severity and also distinguished all SLE patients from healthy participants or those with rheumatoid arthritis [72]. RBP has been investigated in small adult cohorts, where they demonstrated a relationship with nephritis and potential as an early marker [73]. Elevated urinary NAG is seen in other disorders, including vesicoureteric reflux and urinary tract infections [74], and RBP levels can increase in drug-induced toxicity [75]. The role of increased RBP levels in JSLE deserves further investigation in larger, longitudinal cohorts, as does a better understanding of their pathophysiological role in LN.

Acute phase proteins and carrier proteins

Acute inflammatory response proteins released from the liver have been identified to have a role in lupus glomerulonephritis. Proteomic studies have highlighted the potential for alpha 1 acid glycoprotein (AGP) and transferrin as early biomarkers in active LN, confirmed in a cohort of 98 children with JSLE where AGP identified active LN patients [76]. Our group has recently replicated these findings with AGP in a cohort of 52 JSLE patients [67]. These proteins require further scientific investigation into their role in the pathogenesis of LN and, if found supportive, longitudinal validation in larger cohorts.

Interferon-inducible protein 10

Interferon-inducible protein 10 is a cytokine up-regulated in response to interferon and is frequently present along with the above-mentioned potential biomarkers. Previous reports have shown an elevation in urinary IP10 expression in LN, and large adult cohort studies have shown that urinary levels of IP10 protein rise preceding a renal flare and correlate with renal disease activity [77]. IP10 receptor expression has been found not to be not significantly raised in the renal histology of patients with active LN [78], and protein expression of urinary IP10 in children with active LN failed to demonstrate a significant difference [67]. Urinary IP10 was, however, positively correlated with other inflammatory biomarkers, such as MCP1 and AGP, showing that despite its poor potential as a clinical biomarker, it must contribute to the pathogenesis of LN.

Adult SLE longitudinal studies

Urine tumor necrosis factor-like inducer of apoptosis

Urine tumor necrosis factor-like inducer of apoptosis (TWEAK) is a cytokine and a member of the TNF-ligand superfamily. Its role is to up-regulate inflammatory mediators, induce cell death and apoptosis and enable cell survival [79]. Stimulation of TWEAK will lead to the expression of pro-inflammatory mediators, such as MCP1 and IP10, both of which are linked to LN pathogenesis. TWEAK is up-regulated in glomerular and tubular renal tissue in active LN [78].

A longitudinal multi-center analysis found a good correlation between urinary TWEAK levels and renal flares in adult-onset SLE [80]. To date, no paediatric-specific cohort studies have been conducted, but these are required.

Adult SLE cross-sectional studies

Intracellular and vascular cell adhesion molecules

Cellular adhesion molecules have a role in the migration of lymphocytes to areas of inflammation and hence a role in autoimmune conditions. Intracellular adhesion molecule 1 (ICAM) has been identified in the glomerular histology of patients with active LN [81] and associated with serum TNF-α levels. ICAM and vascular cell adhesion molecule 1 (VCAM-1) have been shown in small cross-sectional adult cohorts to correlate with histological LN [82], but correlations with clinical disease activity tools have failed to confirm this finding [83]. Whilst there is insufficient evidence, the role of ICAM and VCAM-1 as biomarkers in LN may be limited, but their role in the pathogenesis deserves further attention.

Urine proteomic, transcriptomic and metabolomic studies

Mass screening of urine substances in cohorts of lupus patients has provided important direction for identifying novel urine biomarkers [84]. Proteomic studies have described similar changes in glomerular permeability proteins released into the urine in differing causes of glomerulonephritis resulting in heavy proteinuria [85], including LN, and a urine proteomic profile specific for childhood LN has been identified consisting of eight key proteins of differing molecular size [86]. Transcriptomic analysis of blood and urine sediment has identified interferon inducible (Th1 induced) chemokines as leading agents in the pathogenesis of LN [87, 88]. Other identified urine proteins or genes discovered in active LN through these methods include IL 10, chemokine ligand 16, hepcidin and liver fatty acid binding protein [84, 89].

Others—preclinical phase

Vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) has shown an increased expression in the urine sediment in patients with class IV LN, and reduced glomerular VEGF levels are associated with worsening LN disease [90]. Edelbauer et al. recently described the occurrence of soluble VEGF receptor 1 expression in the urine of patients with LN [91]. Its involvement in the pathophysiology of the disease is interesting.

Urine endothelin 1

Raised urinary concentrations of endothelin 1 (ET-1) were identified in a cohort of chronic kidney disease patients, where ET-1 was associated with renal disease in the subgroup of patients with SLE and levels altered according to treatment response [92]. Further investigation may be feasible.

Urinary forkhead box p3 protein

Forkhead box p3 protein (FOXP3) is a protein involved in immune responses and controls regulatory T cells within the immune system. Urine FOXP3 gene expression is elevated in patients with active LN and has been linked with a poor response to treatment [93]; its presence in the urine and its exact relationship with disease have yet to be determined.

Urine osteoprotegerin

Urine osteoprotegerin (OPG) forms part of the TNF superfamily and acts to inhibit nuclear factor kappa beta (NF-kB), an essential transcription factor for immune-related genes and a key regulator of inflammation. Large adult cohort studies have assessed urine OPG, as part of a range of cytokines, and found a correlation between urine OPG concentrations and clinical LN disease activity [66]. Further investigation into its role within the kidney and its relationship with JSLE may be warranted.

Combining biomarkers

Novel mathematical modeling has begun to allow several of these promising biomarkers to be combined, producing excellent non-invasive measures of histological disease activity and chronicity in LN. Brunner et al. have described the most promising combination of urine protein biomarkers for the identification of disease activity as being MCP1, AGP, caeruloplasmin and the protein:creatinine ratio, and for chronic renal changes as either NGAL, creatinine clearance and MCP1 or MCP1, AGP, creatinine clearance and C4 levels [94]. International validation of these combinations of biomarkers is required.

A summary of the promising biomarkers described in JSLE-associated nephritis and their stage in the clinical validation process are given in Table 1.

Table 1 A summary of key urine biomarkers in lupus nephritis and their current stage in the validation process
Full size table

Conclusion

Novel urine biomarkers in LN are much needed, especially in juvenile-onset disease where renal biopsy is often impractical, and standardized indications for biopsy are not yet available. The identification of disease biomarkers in LN, such as those achieved in proteomic and transcriptomic studies, can also provide direct insight into the disease process and can guide further translational research.

Promising urine biomarkers include urinary NGAL, MCP1 and TGF-β, for which further longitudinal paediatric validation studies are needed. Following methodological processes from mass identification, cross-sectional cohort testing and robust longitudinal assessment, the next step will then be to move towards commercialization, with the ultimate aim of improving the long-term renal outcome for our patients. Many more urine substances show promise as biomarkers in adult cohort studies, such as urinary TWEAK, although only a very few have been validated over a longitudinal period in paediatric cohorts.

Other potential biomarkers share many similarities in their involvement as pro-inflammatory mediators and their role in effecting inflammatory cell entry to the glomerulus, core mechanisms in the underlying pathophysiology of LN.

Individual biomarkers are unlikely to be robust enough to provide a direct relationship to disease activity in isolation, thereby reflecting the complexity of the pathophysiological process in lupus glomerulonephritis. Combining a few key markers, such as those described by Brunner et al. [94], should be the focus of future validation studies.

A move toward biomarker-driven individualized patient management would enable earlier disease detection and a more accurate prediction of impending renal flares, permit prompt treatment interventions and hopefully improve overall renal and patient survival.

The advancement of urinary biomarkers through robust validation is still a key priority in JSLE research.