The primary aim of this chapter was to assess the hypothesis that
urinary biomarkers can distinguish patients with active LN. In this cohort of JSLE patients, urinary concentrations of MCP-1 and AGP were able to distinguish patients with JSLE active renal disease (peer reviewed
Appendix 22). Surprisingly, on cross sectional evaluation, no difference in the
urine concentrations of IP10 or NGAL in patients with active LN was
demonstrated.
Previously, Marks et al have demonstrated that expression of MCP-1 within
the glomerulus and the urine was associated with histological class III and IV
LN in a cohort of children with JSLE (280). This present study has confirmed
that MCP-1 distinguishes patients with active LN when compared to patients
with inactive renal disease using a disease activity tool as a monitoring
standard. Within the kidney, MCP-1 can be secreted from the glomerular
podocyte in response to pro-inflammatory stimuli including TNF-α, and MCP-1 knockout mice have no inflammatory cell infiltrate typical of LN (343). As anti-
MCP-1 therapies reduce the T cell expression of IFN-γ (344) it can be suggested that MCP-1 has a key role in LN.
This cross sectional analysis is the first to investigate the use of AGP as a
urinary biomarker in JSLE, demonstrating significantly elevated
concentrations in patients with active renal disease. This cytokine was first
linked to LN by mass spectrometry along with several other markers of iron
regulation (290). AGP acts to protect the glomerulus and, similar to MCP-1, is
activated by a network of plasma cytokines like we have described in our
cohort, including IL-1β, TNF-α and IL-6 (345). Although urine AGP was significantly increased in our active renal cohort, its role as a specific LN urine
systemically active non-renal JSLE. Further analysis using larger cohorts and
matched global disease activity would assist in determining this.
Previous studies reported an increased IP10 mRNA expression within the
glomerulus and the urinary sediment and indicated its potential role in disease
pathogenesis (283, 290, 292). In contrast to these studies, this study did not
find significantly elevated urinary IP10 protein concentrations in children with
active LN. The urinary sediment, containing numerous cell types including
leucocytes, glomerular and tubular cells, can make gene expression assays
difficult to relate directly to the kidney. Inability to detect elevated IP10 may be
due to protein degradation despite high gene expression described in
previous studies, differences in childhood cytokine expression, too early a
marker so as not to capture on a cross sectional basis, differences in patient
cohort characteristics, or the assay employed for use. Whilst this study did not
detect significantly raised IP10 protein concentrations, a strongly significant
correlation between IP10 and the other biomarker concentrations was
demonstrated, supporting its role in the pathogenesis of LN.
The final urine protein that was assessed was NGAL. Preclinical studies have
found that the renal tissue in murine models of LN overexpress NGAL in
response to pathogenic anti-DNA antibodies, and NGAL knock-out mice have
a less marked renal pathology and improved outcome in autoimmune
nephritis, suggesting an important role for NGAL in the pathogenesis of LN
role in LN by facilitating recruitment of inflammatory cells to the kidney
through the up regulation of pro-inflammatory mediators (275). Despite the
strong evidence for its role in LN, surprisingly a significantly elevated
concentration of urine NGAL in active renal JSLE on this cross sectional
analysis was not evident. Only one study has reported a similar lack of
association (347). This lack of association may be that urine NGAL is peaking
earlier than can be clinically detected using the pBILAG2004 and
subsequently normalising at the time of disease flare or may be that this
present cohort was too small or too diverse to provide it justification. To date,
urine NGAL is the only single biomarker to stand robust longitudinal cohort
investigation in other paediatric specific cohorts (278, 279) and therefore the
next step in determining whether urinary NGAL is a robust biomarker in active
renal JSLE requires longitudinal validation.
Finally, in each of the urine markers assessed, the JSLE patients without
active renal disease had similar concentrations of the urinary biomarkers to
healthy controls, suggesting that biomarkers return to a baseline comparable
to healthy children in the absence of renal inflammation.
The secondary aim of this chapter sought to demonstrate whether patients
with JSLE active renal disease had differing plasma cytokine profiles when
compared to patients with JSLE non-active renal disease. Using a selected
panel of cytokines in a small group of JSLE patients, we have shown a
trend toward increased concentration of IFN-γ, TNF-α, IL-6 and VEGF in patients with active LN.
Despite a small, heterogeneous group, within this cohort of JSLE patients with
active renal disease, there was a tendency for an increased systemic
expression of IL-13, IFN-γ, IL-6, TNF-α, and VEGF. In agreement with findings from this study, previous studies have also described a significant
overexpression of IFN-γ in SLE patients who primarily have renal manifestations (308, 313). Additionally, others have described the IFN-γ over-
expression and a specific IFN-γ genotype in LN patients that can be associated with the amount of leucocyte infiltration and the differing
histological classes of LN (170, 303, 305). IFN-γ therefore has a leading role
in the onset of LN. The polarisation of peripheral cells towards a Th1
phenotype, and thus IFN-γ over-expression, in LN is regulated by the presence of IL-12 and IL-18 – two cytokines seen at higher expression in LN
(304, 308, 311). TLRs located within the kidney are activated and up-
regulated in LN (348), this may lead to the increase in pro-inflammatory
cytokines, IFN-γ, TNF-α and IL-6, that are shown in this study and is in agreement with studies observing TNF-α genetic polymorphisms in patients with LN (26, 349).
Surprisingly, this study identified a statistically significant increase in the
expression of the Th2 cytokine IL-13. The glomerular epithelial cells express
transepithelial electrical resistance (350). It may therefore have a role in
changing the electrical potential of the glomerular basement membrane during
active LN.
Increased plasma VEGF that was noted in this study, has been previously
described in LN patients (332). Interestingly, IL1β is able to increase the
expression of VEGF, both of which can arise from the glomerular epithelial
cells and have a key role in maintaining glomerular permeability (351). VEGF
alters vessel wall permeability facilitating inflammatory cell influx into the
glomerulus. The increase in IL-13 and VEGF deserve further attention; both
are likely to have fundamental roles in the onset of proteinuria.
Using a cohort to JSLE patients recruited using the UK JSLE cohort study this
chapter has identified key differences in the inflammatory profile of the
plasma. In addition, a group of urinary biomarkers of active LN have been
identified that may have potential in providing a useful adjunct to determining
renal involvement in current clinical practice. These can be assessed using
longitudinal validation (Chapter 4).
Limitations of this work include the cross sectional nature of the study, the
small cohort size however recruitment was conducted at two different UK
sites. The JSLE active renal group had a significantly increased disease
results. Regardless of these limitations the increased understanding of these
biomarkers allows further exploration into their clinical utility.
3.7.
Summary
Childhood-onset SLE is associated with a more active disease course and
preponderance for LN. LN is difficult to diagnose clinically as current markers
occur later in the disease process, during which time irreversible damage may
occur within the renal parenchyma. The renal biopsy is the gold standard in
diagnosing and classifying LN but disease activity tools are used for
monitoring purposes. Understanding the systemic pathophysiology of LN and
identifying early non-invasive methods of detecting impending renal flares are
particularly attractive, especially in a childhood population. Early inflammatory
systemic processes, including the elevated systemic IFN-γ and TNF-α expression in LN that have been described in this study, and the urine
biomarkers, especially MCP-1, can provide important insight for further
investigating the underlying pathophysiology of active LN. In addition they
may be useful as biomarkers at a given time point or over time and may
ultimately lead to the identification of more targeted therapies.