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Rheum Dis Clin North Am. Author manuscript; available in PMC 2016 August 11.
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Published in final edited form as:

Rheum Dis Clin North Am. 2013 November ; 39(4): 833–853. doi:10.1016/j.rdc.2013.05.001.

Biomarkers and Updates on Pediatrics Lupus Nephritis

Michael Bennett, PhDa and Hermine I. Brunner, MD, MScb,*
aDivision of Nephrology and Hypertension, Cincinnati Children’s Hospital Medical Center,
University of Cincinnati, MC 7022, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
bDivision
of Rheumatology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati,
MC 4010, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
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Keywords
Biomarkers; Lupus nephritis; Systemic lupus erythematosus; Childhood-onset SLE; Treatment

INTRODUCTION
Systemic lupus erythematosus (SLE) is a multiorgan autoimmune disease with increasing
mortality that often targets young women and children of United States minorities.
Childhood-onset SLE (cSLE)1 has manifestations similar to those of SLE in adults, but
earlier disease onset is accompanied by more severe multiorgan involvement, including
lupus nephritis (LN) in up to 80% of pediatric patients. Treatment of LN in children
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continues to lack support from large randomized clinical trials. Instead, medication regimens
for pediatric LN are deduced from studies in adult SLE and pediatric solid-organ transplants,
or are based on consensus reached by associations of health care providers.

The criterion standard for the diagnosis and monitoring of LN remains histologic evidence
from a kidney biopsy. Conversely, to reduce cost and avoid invasive procedures, monitoring
of LN in clinics is achieved by measures that consider changes in certain blood and urine
tests. Because such traditional testing for LN has limited responsiveness to change, it is ill
suited to capture worsening or improvement of LN in a timely manner. Recently, promising
LN biomarkers have been discovered that accurately reflect LN activity and chronicity as
seen on kidney biopsy, and can forecast LN flares. In the future, such biomarkers are
expected to facilitate the monitoring of LN in daily clinical care and the conduct of research
studies in support of evidence-based therapies for LN in children.
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EPIDEMIOLOGY, COURSE, AND ECONOMIC IMPACT

Given the phenotypic differences of cSLE around the world, the prevalence of kidney
involvement with cSLE likely also varies with racial background and environmental
exposures. The incidence of SLE is thought to have increased 10-fold during the preceding
50 years in industrialized Western countries,2 which could indicate that cSLE in general, and

*
Corresponding author. [email protected].
Conflict of Interest: Nil.
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LN in children in particular, also are becoming more frequent. Using information available
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in administrative databases and an algorithm that correctly identifies 80% of LN cases,

Hiraki and colleagues3 report that in the United States 37% of children with cSLE and who
are enrolled in Medicaid have renal disease. Based on this study, the risk of developing LN
is independent of gender but is higher among teens than younger children. Compared with
Caucasians, Asians have almost 5 times and African Americans a nearly 3 times higher risks
of developing LN. Overall, the annual incidence of LN is 0.72 cases per 100,000 children in
the United States. This figure may be a conservative estimate of the frequency of LN, as
higher estimates are reported by population-based studies from tertiary pediatric
rheumatology centers and a recent meta-analysis.4,5

Recent 5-year renal survival rates in children with cSLE have ranged from 77% to 93%,6–8
with marked improvement over the preceding decades.9 Nonetheless, adults with LN have
an 8-times higher mortality and children with LN a 19-times higher risk of dying compared
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with age-matched general populations.6,7,10 The poor prognosis of children with end-stage
renal disease from LN is particularly troublesome. There is 22% mortality during the 5-year
period since the initiation of renal replacement therapy, with cardiopulmonary compromise
and infections accounting for 47% of all causes of death.6

Associated with the higher mortality is the need of more intensive therapy for LN in
children. Among the almost 7400 cSLE-related hospitalizations in the United States in 2006,
57% noted the presence of LN11 with an average charge of $43,100 per admission.11 Based
on this and an earlier study, LN accounts for 11% to 28% of cSLE-associated medical costs
in the United States.12 Taken together, the cost of therapy for LN in children likely exceeds
$350 million annually in the United States.3,11–13
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DIAGNOSIS OF LN AND CLASSIFICATION

Kidney biopsies are required to establish the diagnosis of LN. Despite considerable variation
in practice, there is consensus that reproducible daily proteinuria of at least 0.5 g, especially
in the setting of an active urinary sediment, warrants a kidney biopsy in a child with cSLE
who has not yet been diagnosed with LN.14,15 Although clinically relevant biopsy findings
are more common in the presence of significant proteinuria, the current approach results in
at least 50% of newly diagnosed patients already being found to have proliferative LN,
rendering them at a higher risk of end-stage renal disease.16–19 A lower threshold for
performing a kidney biopsy arguably is warranted in cSLE patients, including those with
persistent isolated glomerular hematuria and new-onset low-grade proteinuria.

When interpreting a kidney biopsy specimen it is important to ensure that an adequate

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sample with sufficient numbers of glomeruli is available, namely, a minimum of 8 glomeruli

that can be examined under light microscopy.20,21 The International Society of Nephrology/
Renal Pathology Society (ISN/RPS) Classification replaced in 2004 the previously used
World Health Organization (WHO) Classification for LN.20 The ISN/RPS Classification is
based on light microscopy, rather than electron microscopy, as a tool for interpreting LN
histology, even though it has been shown that electron microscopy greatly enhances the
interpretation and classification of kidney biopsies.20

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The ISN/RPS Classification was introduced to standardize and clarify the interpretation of
LN histology findings.20 Six classes of LN are described with focus on changes concerning
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the renal glomeruli, and the National Institutes of Health (NIH) Histology Score is often
used to quantify the degree of LN activity and chronicity (Table 1).22 The maximum score of
the NIH-AI (activity index) and the NIH-CI (chronicity index) is 24 and 12, respectively,
because scores from “(fibro)cellular crescents” and “fibrinoid necrosis/karyorrhexis” are
given a weight of 2 in the NIH-AI (see Table 1). Pathologic changes of the kidney
interstitium, are not well considered in the ISN/RPS Classification, although they are
considered critical for the course of LN.23 However, it is recommended to report the extent,
severity, and type of tubulointerstitial (tubular atrophy, interstitial inflammation, and
fibrosis) and vascular disease (vascular deposits, thrombi, vasculitis, sclerosis).20

RISK FACTORS TO POOR OUTCOME OF LN

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Clinical research has identified, albeit inconsistently, several risk factors for poor LN
outcome3,6,16,24–33; these include male gender, non-Caucasian race, nonadherence to
treatment, presence of antiphospholipid or anti-dsDNA antibodies, persistent
hypocomplementemia or proteinuria, nephrotic syndrome at presentation, failure to
adequately respond to therapy by 3 months,34 flare of LN,35 or diagnosis with proliferative
LN, especially in the setting of a high degree of histologic activity and damage. Given the
multitude of the proposed risk factors for LN, close monitoring of any child patient with LN
seems to be warranted in achieving the best possible control of LN.15,34

MONITORING OF LN IN CLINICAL CARE

There are no studies that directly compare the clinical features of the various classes of LN
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between children and adults with SLE. However, the presentation of children with LN varies
considerably, ranging from mild abnormalities on urinalysis, to anasarca caused by marked
proteinuria, to posterior reversible encephalopathy owing to uncontrolled hypertension with
nephritic syndrome.36

Proteinuria
Abnormally elevated excretions of albumin and total protein in the urine are highly sensitive
indicators of glomerular disease. Albumin is a small-sized molecule, and one of the first
proteins able to pass through the kidneys. The value of monitoring microalbuminuria for the
early diagnosis of LN has not been well established, and mesangial LN can be present
without proteinuria.37 A prompt and significant decrease in proteinuria after 3 and 6 months
of therapy is an important prognostic factor for good long-term renal outcome.38 Proteinuria
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furthers the development of tubulointerstitial inflammation and injury, and thereby a decline
in renal function in the long term.39

Traditionally proteinuria is quantified by a 24-hour urine collection. Conversely, and despite

its common use, urine dipstick is poorly suited to quantify the degree of proteinuria.40 There
is now sufficient evidence that the protein-to-creatinine ratio in a random urine specimen,
best from first morning urine,41–43 is adequate to estimate daily proteinuria in cSLE.

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Whether 12-hour overnight urine collection is more accurate than estimation of proteinuria
using spot urine will need further study.44
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Urine Sediment
The presence of cellular casts on urine-sediment examinations, for example, the microscopic
examination of the cellular components at casts seen in centrifuged urine, is supportive of
glomerulonephritis. Accuracy of urine-sediment interpretation requires timely processing of
the urine, as lysis of leukocytes and erythrocytes occurs even within the first hour after
collection, especially when low specific gravity and high urine pH are present. Presence of
mucus in the urine can entrap both cells and casts, and sometimes repeated assessment of
urine sediment is necessary to detect cellular casts.45

Glomerular Filtration Rate

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The reference method for assessing the “true” glomerular filtration rate (GFR) is to measure
the renal clearance of inulin, ethylenediaminetetraacetic acid, and iohexol; that is, markers
freely filtered through the glomerulus, neither secreted nor reabsorbed by the tubule.
Because such techniques are complex and costly to perform, alternative means to estimate
the GFR in a clinical setting have been developed.46 In pediatrics, the 2009 modification of
the Schwartz Formula and serum cystatin C–based methods seem reasonably accurate and
easy to use in a clinical setting (Table 2).46 Despite its appeal, the use of serum cystatin C to
estimate the GFR of patients with LN will need further evaluation, as levels of cystatin C
seem positively correlated with general SLE activity, even in the absence of LN or changes
in renal function.47,48

SHORTCOMINGS OF TRADITIONAL MEASURES OF LN

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Whereas blood urea nitrogen and creatinine often stay in the normal range in cSLE, even if
with profound histologic pathology, the urinary sediment and urinalysis are generally
abnormal in untreated LN. Conversely, in pretreated patients only minor abnormalities on
urinalysis, including mild proteinuria or hematuria, may be present in patients with severely
active biopsy-proven LN. This finding is supported by the research of Christopher-Stine and
colleagues,49 who reviewed 25 LN patients undergoing serial kidney biopsies. At diagnosis
proteinuria, hematuria, hypoalbuminemia, and hypertension were all associated with a worse
LN class. By contrast, none of these parameters correlated with the LN class on follow-up
biopsy, raising the possibility that normal urinalyses do not necessarily ensure the absence of
active LN.49

With LN, there is a balance between complement activation via the classical pathway, which
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facilitates the removal of immune complexes, and activation of the alternative pathway,
which promotes kidney injury.50 The literature is inconsistent at best as to whether the
concentration of complement and anti-dsDNA antibodies can serve as useful markers of
concurrent SLE activity or future flares.51 In 98 patients who experienced 146 flares, Ho and
colleagues52 showed that hypocomplementemia and anti-dsDNA antibodies accompanied
SLE relapse in only 54% and 27% of patients, respectively.

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Research in adults with LN suggests that less than 25% of LN patients with low C3, C4, or
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anti-dsDNA levels have a concurrent flare of LN, and only 50% of LN flares are preceded
by a drop in the levels of C3 and C4 or an increase in anti-dsDNA antibodies,
respectively.51,53 In other words, these tests are not much better than the flip of a coin in
helping clinicians anticipate LN flare. These reports from adults with LN have been
confirmed in children with LN.54

Like the immunologic markers C3, C4, and anti-dsDNA antibodies that are traditionally
used to assess the course of LN, kidney biopsies have their pitfalls. In a recent study, 5
experienced nephropathologists rated 126 renal biopsy specimens of 87 patients with
proliferative LN.55 These experts demonstrated significant variation in agreement when
rating the various histologic aspects of biopsy specimens as part of the ISN/RPS
Classification. Excellent agreement (>60%) was reached only for the number of glomeruli
seen in the biopsy, the overall activity index score, and the presence of proliferative features.
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Conversely, agreement was less than 40% (interclass correlation coefficient <0.4) for the
presence of mesangial proliferation, tubular necrosis, and, notably, the overall ISN-RPS
class designation.55

The aforementioned shortcomings of kidney biopsies, as well as the limitations of currently

available urine and blood laboratory tests, support a need for potent biomarkers to help
accurately diagnose LN and to determine the response of LN to therapy in a clinical setting.

BIOMARKERS AND ASSESSMENT OF THEIR QUALITY

In its simplest definition, a biomarker is anything that can be measured to extract
information about a biological state or process. The NIH Biomarkers Definitions Working
Group has defined a biological marker (biomarker) as “A characteristic that is objectively
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measured and evaluated as an indicator of normal biological processes, pathogenic

processes, or pharmacologic responses to a therapeutic intervention.”56 Biomarkers are the
essential tools for the implementation of personalized medicine. The biomarker development
process, also sometimes referred to as biomarker qualification, has typically been divided
into 5 phases,57 as shown in Table 3. In recent years, the ready availability of powerful tools
to scan both the genome and the proteome of an organism have revolutionized and greatly
accelerated biomarker discovery.

For biomarker discovery, microarrays are used to screen messenger RNA (mRNA) levels.
This approach has yielded several biomarkers of kidney disease, such as neutrophil
gelatinase-associated lipocalin (NGAL). Microarrays can be combined with other
techniques, such as laser-capture microdissection, to target specific areas of diseased tissue
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to give mechanistic clues not possible just a decade ago. Even with this level of specificity, a
daunting amount of biomarker candidates will be identified with these approaches, and the
usefulness of such candidates must be sifted through for relevance. Another shortcoming of
transcriptomic profiling approaches is that direct measurement in biological fluids is not
possible and that mRNA levels do not always correlate with protein levels or enzyme
activity. Hence, larger validation studies are necessary that measure protein levels to confirm
the biological relevance of mRNA biomarkers.

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Focusing on peptides and actual proteins, proteomics allow one to go beyond simple
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translation of mRNA into protein. Instead protein regulation, posttranslational modifications

such as glycosylation and methylation, and even disease-specific fragmentation of proteins
are assessed. Proteomic techniques are capable of identifying and quantifying proteins and
peptides in exceedingly large numbers.58 The urinary proteome itself is quite large, with
laboratories having identified more than 1500 proteins to date.59,60 The blood proteome is
even larger, with more than 3000 nonredundant proteins identified in the plasma alone.61–63
Adding the proteome of the cellular component of blood will yield thousands more.64,65 To
this end, we have entered what has been termed an “open loop,”66 or unbiased, approach to
biomarker discovery, in stark contrast to the hypothesis-driven approach of our past. With
such a vast pool of potential biomarkers from readily available, noninvasive sources, one
must take care to plan and design the proper experimental approach to ensure parsimony.

There are universal characteristics important for any biomarker: (1) they should be
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noninvasive, easily measured, inexpensive, and produce rapid results; (2) they should be
from readily available sources, such as blood or urine; (3) they should have a high
sensitivity, allowing early detection, and no overlap in values between diseased patients and
healthy controls; (4) they should have a high specificity, being greatly upregulated (or
downregulated) specifically in the diseased samples and unaffected by comorbid conditions;
(5) their levels should vary rapidly in response to treatment; (6) their levels should aid in risk
stratification and possess prognostic value in terms of real outcomes; and (7) they should be
biologically plausible and provide insight into the underlying disease mechanism.56,57

The most readily available sources of biomarkers are urine and blood. Urine is an excellent
source of biomarkers produced in the kidney,67 and thus may give better mechanistic insight
into specific renal abnormalities. Urine is less complex than serum, and thus is easier to
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screen for potential biomarkers. Urinary biomarker studies typically adjust for urine
creatinine to account for differences in urine concentration resulting from hydration status
and medications such as diuretics. However, the utility of urine creatinine in biomarker
correction has been questioned because of its variable excretion throughout the day and its
dependence on normal renal function.

Serum biomarkers are considered more stable, as they are less prone than urine biomarkers
to bacterial contamination. However, serum biomarkers are more likely to represent a
systemic response to disease, rather than an organ response. There are exceptions, such as
the troponins in cardiac disease. The real problem with serum as a source of biomarkers lies
in the discovery phase. Serum has a wide range of protein concentrations across several
orders of magnitude, with a small number of proteins (such as albumin) accounting for a
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large percentage of the volume; this can be akin to trying to spot a single strand of cotton in
a large tapestry. Although assays do exist to remove these high-abundance proteins from
serum, many potential biomarkers have been shown to bind to albumin.68 Thus, albumin
depletion to help identify relevant biomarkers risks erroneous removal of proteins relevant to
LN.

The sensitivity and specificity of a biomarker go hand in hand. The receiver-operating

characteristic (ROC) curve is a binary classification test, based on the sensitivity and

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specificity of a biomarker at certain cutoff points. ROC curves are often used to determine
the clinical diagnostic value of a biomarker.57,69 The area under the ROC curve (AUCROC)
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is a common statistic derived from ROC curves. An AUCROC of 1.0 represents a perfect
biomarker, whereas an AUCROC of 0.5 is a result that is no better than expected by chance.
An AUCROC of 0.75 or greater is generally considered a good biomarker while an AUCROC
of 0.90 is considered an excellent biomarker.57 However, even a sensitive biomarker with
what experimentally would be considered an excellent specificity of 90% would still yield a
false-positive rate of 10%, which may be unacceptably high for clinical use as a stand-alone
marker.66 As a result, the best approach clinically may be to find multiple biomarkers that
can be combined as part of a panel to achieve even higher specificity.

TYPES OF LN BIOMARKERS
Traditional measures of LN have limited responsiveness to change, and are unsuited to
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capture worsening or improvement of LN in a timely manner. This lack of early response

measures to verify the effectiveness of LN therapies hinders clinical care, requires clinical
trials of new medications for LN to study large populations and follow them over several
years, and increases the risk of negative trials. In addition, traditional measures of kidney
function, such as creatinine clearance or protein-to-creatinine ratio, reflect significant loss of
kidney function such that major renal damage can occur before it is detected by these
traditional methods. Thus, novel biomarkers that can rapidly detect lupus renal involvement
and severity, predict flares, and monitor treatment response and disease progression are
greatly needed, and have been the subject of intense research.

The advent of new technologies to rapidly screen the genome and proteome over the last few
decades has led to an explosion in the identification of novel biomarkers for many disease
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states. Ann immense number of biomarkers has been investigated in recent years, far too
many to discuss in this article. The authors therefore focus the discussion on the most
promising investigational biomarkers for LN discovered over the last several years.

Urine MCP-1
Monocyte chemoattractant protein-1 (MCP-1) is a leukocyte chemotactic protein involved in
the mediation of inflammation and renal injury in LN.70 Animal models of LN have
demonstrated direct involvement of MCP-1 in renal abnormality, as blockade of MCP-1
through the use of an antagonist or an RNA oligonucleotide specifically designed to bind to
and sequester MCP-1 (also known as a spiegelmer) led to marked improvement in LN and
lupus-like inflammatory skin lesions.71,72 Several cross-sectional studies have demonstrated
that urine MCP-1 levels are concurrently higher in those patients with active LN than with
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nonactive LN.73–75 The AUCROC of MCP-1 for distinguishing active LN from inactive LN76
or nonrenal flares is 0.76.77 Urine MCP-1 also seems to have promise in helping to
distinguish certain classes of LN. Urine MCP-1 levels are significantly higher with ISN/RPS
Classes III and IV than with other classes of LN (P = .01).78,79 Both children and adults with
Class IV LN have the highest glomerular expression of MCP-1.46 There are some differing
findings regarding the potential of urine MCP-1 to predict renal flares. A study by Rovin and
colleagues73 reported increases in urine MCP-1 as early as 2 to 4 months before the clinical

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diagnosis of a renal flare. However, a similar study by Tian and colleagues,80 while
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demonstrating elevated MCP-1 during renal flares, did not find MCP-1 levels to be an
independent predictor of flare.

Similar results were found by Chan and colleagues81 when examining chemokine mRNA
from urine sediment of LN patients. MCP-1 mRNA levels were elevated during active LN in
comparison with inactive LN and healthy controls. However, in this study urine MCP-1
mRNA levels were found not to be useful predictors of LN flares. It should be noted that the
best use for MCP-1 as it relates to SLE is as part of a broader panel of markers, as elevated
urine MCP-1 can also signal chronic fibrosis82,83 and has presented in other glomerular
disorders.84 Thus a combinatorial approach may lead to additional specificity for LN.

Urine NGAL
NGAL is expressed in several cell types, including neutrophils, specific epithelia, and renal
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tubular cells. NGAL is markedly upregulated in the distal tubules in response to many types
of kidney injury. It has garnered significant attention as a promising early marker for acute
kidney injury,85–91 but recent studies have also shed light on NGAL’s potential as a
biomarker for chronic kidney disease, such as diabetic nephropathy92,93 and focal segmental
glomerulosclerosis,94 as well as LN.95,96 Two cross-sectional studies investigated NGAL as
a biomarker for LN in pediatric patients95 and adults.97 In children, elevated urine NGAL
levels had a high sensitivity and specificity for active biopsy-proven LN (AUCROC 0.94). In
adults the specificity was still high (91%), but sensitivity was lower (50%) for LN. This
thread is a common one in biomarker studies, as adults typically have more concurrent
confounding physiologic conditions, which leads to higher variability in biomarker
measurements. NGAL was not correlated with extrarenal SLE disease activity in either
population. More recent longitudinal studies in the pediatric population have shown that
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urine NGAL as well as plasma NGAL levels are significantly higher in SLE patients than
those with juvenile idiopathic arthritis (JIA) or healthy controls, unrelated to physiologic
factors such as height, weight, and age.98 Levels of urine NGAL, but not plasma NGAL,
correlated well with LN activity scores.96,98 Urine NGAL rose 3 to 6 months before
worsening renal disease activity, demonstrating value in predicting flares.96,98 One study
demonstrated a lesser, though significant, increase in plasma NGAL as early as 3 months
before flare.96 In addition, in patients with a biopsy, urine NGAL levels were greater in
patients with diffuse proliferative than membranous nephropathy, indicating, along with
MCP-1, the possible use of NGAL in a panel to distinguish LN classes.98 Similar to MCP-1,
urine NGAL is not specific to LN and thus must be used in a context-specific setting.

Hepcidin
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Hepcidin is a small peptide hormone mainly produced in the liver, and has a role in iron
homeostastis. Hepcidin is upregulated in response to high iron levels and inflammation, and
decreases during anemia and iron deficiency. Proteomic evaluation by surface-enhanced
laser desorption-ionization time-of-flight mass spectrometry (SELDI) revealed the 25- and
20-amino-acid isoforms of hepcidin as potential biomarkers for LN.79 Zhang and
colleagues79 prospectively analyzed 24 LN flare cycles in 19 patients, and demonstrated an
increase in hepcidin-20 4 months before flare, which then decreased to baseline levels by 4

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months after flare. An opposing pattern was discovered for hepcidin-25, which decreased
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during renal activity then returned to baseline along with hepcidin-20 after flare. It will be
interesting in future studies to evaluate the physiologic role of hepcidin in LN because it is
regulated in part by inflammatory cytokines, such as interferon-α and interleukin (IL)-6,
which are known to play a role in modulating tissue damage in SLE,99,100 and have been
shown experimentally to induce monocyte expression of hepcidin in vitro.101 It has been
speculated that monocyte infiltration of the kidney may be the source of urine hepcidin in
LN.

Urine Protein Signature

Also using SELDI, Suzuki and colleagues102 discovered and subsequently validated54 a
protein signature that identified active LN in children. After removal of 4 albumin fragments
from the signature, the panel included transferrin (Tf), orosomucoid (or α-1 acid
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glycoprotein [AGP]), ceruloplasmin (CP), and lipocalin-type prostaglandin D synthase (L-

PDGS, or β-trace protein). Using enzyme-linked immunosorbent assay or
immunonephelometry, all 4 proteins were found to be significantly higher in patients with
active LN than in those with nonrenal SLE or JIA controls. Urine L-PDGS, AGP, and Tf all
increased as early as 3 months before renal flare, but Tf did so most consistently,
demonstrating increased sensitivity to renal changes in SLE in comparison with L-PDGS or
AGP. Urine CP did not demonstrate the ability to predict flares. Combining this panel with
other markers such as NGAL and MCP-1 may demonstrate enhanced predictive and
diagnostic value in comparison with individual markers alone.

Complement Component C4d

C4d is a breakdown product of the activated complement factor C4b, a critical component of
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the C5 convertase. In a controversial pilot study using an alternative approach, Batal and
colleagues103 evaluated cellular deposition of the immune complex C4d on circulating
erythrocytes, reticulocytes, and platelets as a potential biomarker for LN activity. Previous
studies had linked peritubular capillary and glomerular staining of C4d with severity of LN
and development of renal thrombotic microangiopathy, respectively.104,105 The investigators
found higher circulating levels of erythrocyte-bound C4d (EC4d) and reticulocyte-bound
C4d (RC4d) in LN patients than in both nonrenal SLE patients and patients with renal
disease without SLE. Moreover, EC4d levels correlated with the NIH renal activity index.
There has been some level of skepticism106 regarding the ability of these markers to
distinguish renal from nonrenal SLE, as higher levels can also observed in SLE patients
without LN,107,108 and there have been no scientific findings to date that dispute the results.
An additional study lends credence to the finding in this study, indicating higher levels of
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certain C4d-positive circulating T cells in LN patients than in those without LN.109 Further
prospective investigations of circulating C4d are needed for it to rise to the levels of the
previously discussed biomarkers for LN, but the novel approach warranted mention in this
review.

TWEAK
Tumor necrosis factor–like weak inducer of apoptosis (TWEAK) is a member of the tumor
necrosis factor (TNF) superfamily, and is involved in modulating cell survival and induction

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of several proinflammatory chemokines through its receptor fibroblast growth factor–

inducible protein 14 (Fn14).110 In human kidney, TWEAK acts on multiple Fn14-expressing
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cells types, including podocytes, tubular cells, and mesangial cells, and is responsible for
induction of several mediators of inflammation, including MCP-1, interferon-γ–inducible
protein 10 (IP-10), intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1
(VCAM-1), matrix metalloproteinases 1 and 9, and macrophage inflammatory protein
α.111,112 During periods of inflammation, Fn14 expression is upregulated, which lends itself
to enhancing a positive feedback loop. The major source of TWEAK in LN is thought to be
infiltrating monocytes and macrophages. Cross-sectionally, urinary TWEAK levels are
significantly higher in active LN; levels are significantly higher in patients with LN flare
than in those with stable disease.113,114 In a multicenter longitudinal analysis, Schwartz and
colleagues115 discovered that whereas urinary TWEAK levels peaked at the height of renal
flare, urinary TWEAK was significantly elevated 4 to 6 months before and following renal
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flare. Performance of urinary TWEAK in distinguishing LN patients from SLE patients

without kidney involvement was better than that of anti-dsDNA levels and complement C3
or C4 levels. The study also demonstrated a strong association between urinary TWEAK
levels and LN activity over time. Conversely, serum levels of TWEAK were not associated
with LN activity. TWEAK is intriguing as a biomarker for LN, and has a biologically
plausible role in LN pathology.

Other Chemokines, Receptors, and Adhesion Molecules

Space does not permit in-depth discussion of all biomarkers under investigation for LN, but
several cytokines, chemokines, and their receptors deserve some mention. Chemokine C-X-
C motif ligand 10 (CXCL10, also known as IP-10) and its receptor CXCR3 promote T-cell
migration to areas of inflammation and are upregulated in SLE.116,117 CXCL10 and CXCR3
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mRNA levels collected from urine sediment were highly specific for identifying Class IV
LN (AUCROC 0.89 for CXCL10 and 0.79 for CXCR3), and also demonstrated reduction in
response to successful treatment signified by clinical remission.118 FOXP3 (forkhead box
P3) mRNA collected from urine sediment of LN patients has been found to be significantly
higher in LN patients,119 despite FOXP3 levels in regulatory T cells having been found to be
lower in patients with active lupus than in healthy controls.120 Research has also shown that
a reduced number of circulating FOXP3+ T cells and serum transforming growth factor β
levels inversely correlated with LN activity as measured by SLE disease activity index renal
domain score (P = .0013 and 0.0005, respectively).109 Collection of mRNA from urine
sediment presents several technical difficulties, such as stability, which may limit the clinical
utility of urine mRNAs as biomarkers. So although there may be a link between FOXP3 and
LN, additional study must be completed to solidify its role and usefulness as a biomarker for
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LN.

VCAM-1 demonstrates reliability as an indicator of renal disease activity in LN. VCAM-1

has been shown to be induced in mice by inflammatory cytokines such as IL-1 and TNF.121
VCAM-1 plays a role in tethering leukocytes, which are drawn to sites of inflammation, to
endothelial cells.122 Urinary VCAM-1 has been shown in several studies of human disease
to be strongly correlated with LN activity and severity77,123,124 in LN. Serum levels of
VCAM have previously been shown to correlate with the severity of LN, being highest in

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 Bennett and Brunner Page 11

WHO Class III and IV, versus inactive or mild nephritis (WHO Class I or II),125 and levels
diminished with treatment. Singh and colleagues126 compared urine levels of VCAM-1,
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MCP-1, and CXCL16 (another potential LN biomarker) with pathologic features of LN on

biopsy collected concurrently with the urine sample. Urine VCAM and MCP-1 were highly
predictive of LN when compared with healthy controls (AUCROC 0.92 and 0.89,
respectively). Surprisingly, urine MCP-1 was also significantly higher in African American
subjects than in persons of other ethnic origins. Of the 3 markers, urine VCAM-1 was most
highly correlated with LN activity, with none shown by CXCL16. CXCL16 and urine
VCAM-1 were significantly higher in patients with WHO Class IV LN compared with other
Classes, as determined by concurrent biopsy analysis. It should be noted that this association
with Class IV proliferative nephritis may not be specific to pathology, but a may be a result
of these patients having a high degree of renal disease activity. These findings provide a
great deal of support for urine VCAM-1 as a biomarker for LN, but these studies have all
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been cross-sectional. Longitudinal studies are needed to determine the utility of VCAM-1 in
monitoring disease progression and detecting flares. It should also be noted that, like NGAL
and MCP-1, elevated VCAM-1 is not exclusive to LN. Increased levels of VCAM-1 have
been found in other glomerular diseases such as membranous nephropathy and focal
segmental glomerulosclerosis.126

CURRENT TREATMENT OF LUPUS NEPHRITIS IN CHILDREN

The novel biomarkers introduced in the preceding sections are not used to support efficacy
in clinical trials at present, although validation studies are ongoing to achieve biomarker
qualification by regulatory bodies. Qualification would allow for the use of biomarkers in
clinical care and research.127,128 In addition, there is no known biomarker at present that a
priori would support the choice of therapeutics for the treatment of LN. However, it seems
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reasonable to assume that novel biomarkers will become available for clinical use within the
next 5 to 7 years.

No medication has likely improved the prognosis of LN more than systemic glucocorticoids
(GC), especially if combined with immunosuppressive medications. Nonetheless, use of GC
is a concern, given the often devastating short-term and long-term side effects. There is a
lack of systematic studies in support of the most appropriate dose of GC in patients with LN.
Based on consensus among pediatric rheumatologists in the United States, three GC dosing
regimens for the treatment of proliferative LN in children have been proposed,129 but data
are lacking to determine which regimen is the most appropriate for a given patient. Of note,
the Joint European League Against Rheumatism and the American College of
Rheumatology consider much lower GC exposure sufficient for mainly adults with LN.14,130
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Unless commanded by cSLE activity in other organ systems, hydroxychloroquine and GC

are considered sufficient for the treatment of ISN/RPS Class I and, often, Class II LN.14,131
For proliferative LN Class III or IV with or without membranous features, treatment with
cyclophosphamide or mycophenolate mofetil (MMF) for induction therapy, and maintenance
therapy using MMF or azathioprine are proposed.14,129 Based on a Cochrane review of
studies of adults with LN,132,133 compared with intravenous cyclophosphamide, MMF was
as effective in achieving stable kidney function and complete remission of proteinuria. No

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 Bennett and Brunner Page 12

differences in mortality or major infections were observed. In maintenance therapy, the risk
Author Manuscript

of LN flare was significantly higher with azathioprine or cyclophosphamide compared with

MMF. Based on small studies, children and adolescents have a response to MMF and
cyclophosphamide similar to that of adults with LN.134 Whether MMF is as effective in
children as it is in adults135 or whether cyclophosphamide might have a better risk/benefit
profile in children than in adults owing to lower frequency of clinically relevant ovarian
injury and lower risk of nonadherence is not supported by high-level scientific evidence.129
In addition, the pediatric correlate of the “Euro Lupus Regimen” for the dosing of
intravenous cyclophosphamide has not been developed or systematically studied.14

There is mounting evidence that individualized dosing of MMF based on pharmacokinetic

profiling will increase the likelihood of achieving remission of LN.136,137 Target exposure
between 60 and 90 mg/h/L is more often associated with LN improvement, with the highest
exposures being reserved for the most severe cases because of the increased frequency of
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adverse effects.136 Given high interindividual differences, weight-based or body-surface–

based dosing of MMF does not suffice to reliably achieve such a target exposure.138

Pure membranous lupus glomerulonephritis (ISN/RPS Class V) seems rarely the initially
diagnosed type of LN, and typically the other forms of LN develop into Class V over time.
Treatment of Class V probably should not differ from that of idiopathic membranous
nephropathy. Depending on the degree of proteinuria, only angiotensin-inhibiting
medications, or GC with MMF or other immunosuppressives are the preferred initial
therapy.139

Despite favorable reports mostly from observational studies,140–144 the clinical trial of the
anti-CD20 antibody rituximab (Rituxan, Mabthera) failed to show clinically relevant
improvement of LN.145 The anti–B-lymphocyte stimulator antibody belimumab (Benlysta)
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has recently been approved for the treatment of active SLE,146,147 but its benefit or
detrimental effects on LN will require further study.

There are currently several ongoing studies of LN, some including younger patients, which
explore the efficacy of various combination therapies of GC with regimens including various
combinations of cyclophosphamide, cyclosporin, azathioprine, tacrolimus, MMF,
fludarabine, azathioprine, rituximab, abatacept, etanercept, and leflunomide, as well as
mesenchymal stem cells. It is hoped that these studies consider the genetic differences of
patients and include potent LN biomarkers when assessing the benefits of these therapies
under investigation.

It is plausible to assume that the use of novel biomarkers will yield better stratification of
Author Manuscript

patient populations for the purpose of clinical trials, and enable researchers to determine the
response to LN therapy earlier and more accurately. This approach would necessitate smaller
sample sizes for clinical trials, and ultimately make possible adequately powered studies in
children with LN.

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 Bennett and Brunner Page 13

Acknowledgments
Author Manuscript

Funding Sources: H.I.B. is supported by research grants from the National Institutes of Health, including
2P60AR047784, U01AR059509 and UL1 TR000077-04; M.B. is supported by a research grant from the National
Institutes of Health, P50 DK096418 and two translational research grants from the Cincinnati Children’s Hospital
Medical Center/University of Cincinnati Joint Center for Clinical and Translational Science and Training.

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KEY POINTS
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• Lupus nephritis is frequently diagnosed in children with systemic lupus

erythematosus and warrants close medical attention to avoid
progression to end-stage renal disease.

• Diagnosis of lupus nephritis requires at present a kidney biopsy.

• Current laboratory tests used to monitor lupus nephritis lack accuracy,

making appropriate management difficult.

• Novel urine biomarkers hold promise for improving the approach to the
surveillance of lupus nephritis and interpretation of patient response to
therapy.

• Despite the lack of adequately powered clinical trials, standardized

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approaches to the therapy for children and adolescents with lupus

nephritis are now available.
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Table 1

Classification and interpretation of lupus nephritis biopsy findings

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ISN/RPS Lupus Nephritis Classification Criteria

Class I Minimal mesangial lupus nephritis

Class II Mesangial proliferative lupus nephritis

Class III Focal lupus nephritisa

Class IV Diffuse segmental (IV-S) or global (IV-G) lupus nephritisb

Class V Membranous lupus nephritisc

Class VI Advanced sclerosing lupus nephritis

NIH Activity and Chronicity Indexd

Active Lesions Chronic Lesions

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1. Endocapillary hypercellularity, with or without leukocyte infiltration and with substantial 1. Glomerular sclerosis (segmental, global)
luminal reduction

2. Karyorrhexis (fibrinoid necrosis)e 2. Fibrous adhesions

3. Rupture of glomerular basement membrane 3. Fibrous crescents

4. Crescents (cellular or fibrocellular)e 4. Tubular atrophy

5. Subendothelial deposits identifiable by light microscopy (wireloops)

6. Intraluminal immune aggregates (hyaline thrombi)

NIH Activity Index 0–24 NIH Chronicity Index 0–12

a
Indicates the proportion of glomeruli with active and with sclerotic lesions.
b
Indicates the proportion of glomeruli with fibrinoid necrosis and cellular crescents.
c
Class V may occur in combination with class III or IV, in which case both will be diagnosed.
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d
Each item scored from 0 to 3 depending on degree of involvement: 0 = no lesions; 1 = <25% of glomeruli; 2 = 25%–50% of glomeruli; 3 = >50%
of glomeruli.
e
These items scores have a weight of 2.
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Table 2

Estimation of GFR (eGFR) in children in comparison with the reference standard of inulin clearance (iGFR)
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Comparators Modified Schwartz Formulaa Le Briconb

eGFR = 36.5 × Height (cm)/Cr eGFR 5 (78/Cys) + 4

eGFR means ± SD (mL/min per 1.73 m2) 109 ± 44c 99 ± 26

iGFR-eGFR means ± SD (mL/min per 1.73 m2) −8 ± 29 2 ± 19

Accuracy 10% (%)d 38 46

Accuracy 30% (%) 84 90

Accuracy 50% (%) 96 98

Correlation between eGFR and iGFR 0.779e 0.784e

a
Schwartz GJ, Muñoz A, Schneider MF, et al. New equations to estimate GFR in children with CKD. J Am Soc Nephrol 2009;20:629–37.
b
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Le Bricon T, Thervet E, Froissart M, et al. Plasma cystatin C is superior to 24-h creatinine clearance and plasma creatinine for estimation of
glomerular filtration rate 3 months after kidney transplantation. Clin Chem 2000;46;1206–7.
c
P<.05, Wilcoxon paired test, in comparison with inulin clearance.
d
Interpretation: 38% of the patient’s eGFR is within 10% of the reference standard, ie, inulin clearance.
e
P<.001; Spearman correlation coefficient.
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Table 3

Phases of biomarker discovery, translation, and validation

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Phase Terminology Action Steps

Phase 1 Preclinical discovery Discover biomarkers in tissues or body fluids
Confirm and prioritize promising candidates

Phase 2 Assay development Develop and optimize clinically useful assay

Test on existing samples of established disease

Phase 3 Retrospective study Test biomarker in completed clinical trial

Test if biomarker detects the disease early
Evaluate sensitivity, specificity, receiver-operating characteristic

Phase 4 Prospective screening Use biomarker to screen population

Identify extent and characteristics of disease
Identify false referral rate

Phase 5 Disease control Determine impact of screening on reducing disease burden

From Devarajan P. Proteomics for biomarker discovery in acute kidney injury. Semin Nephrol 2007;27(6):637–51; with permission.
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