Clinical Immunology (2015) 161, 2330

available at www.sciencedirect.com

Clinical Immunology
www.elsevier.com/locate/yclim

Proteomics and autoimmune kidney disease

Brad H. Rovin a,, Jon B. Klein b
a

Nephrology Division, The Ohio State University Wexner Medical Center, Columbus, OH, United States
Nephrology Division, The University of Louisville School of Medicine, Robley Rex VA Medical Center, Louisville, KY,
United States
b

Received 21 April 2015; accepted with revision 27 April 2015

Available online 13 May 2015
KEYWORDS
Proteomics

Abstract Proteomics has long been considered an ideal platform, and urine an ideal source for
biomarker discovery in human autoimmune kidney diseases. A number of studies have examined
the urine proteome to identify biomarkers of disease activity, kidney pathology, and response to
therapy. Increasingly, proteomic studies of kidney disease have expanded to include blood,
circulating cells and kidney tissue. Recently the clinical potential of renal proteomics has been
realized through a handful of investigations whose results appear to be applicable to patient
care. In this review, approaches to the proteomic evaluation of autoimmune kidney diseases will
be considered in the context of developing clinically useful disease biomarkers.
2015 Elsevier Inc. All rights reserved.

1. Introduction
Several omic techniques are now available for the investigation of human disease. These include the broad categories of
genomics, proteomics, metabolomics, and lipidomics. Within
each category is the possibility of focusing down on more
specific subgroups within an ome, such as peptides or phosphoproteins within the proteome. All of these techniques provide
the tools to examine genes, proteins, metabolites or lipids in
an unbiased fashion to uncover relationships of analytes to
disease that may not be intuitive. Theoretically an agnostic
omic approach may give a more complete understanding of
disease than investigations in which analytes are chosen
and studied on the basis of pre-conceived ideas of disease
pathogenesis, which may be flawed or incomplete.
Corresponding author at: Nephrology Division, Ground Floor, 395
West 12th Avenue Columbus, OH 43210, United States.
E-mail address: [email protected] (B.H. Rovin).

http://dx.doi.org/10.1016/j.clim.2015.04.021
1521-6616/ 2015 Elsevier Inc. All rights reserved.

With respect to kidney diseases, and specifically autoimmune

kidney diseases, the majority of studies thus far have focused on
genomics and proteomics. Proteomics has been used to
characterize proteins in the blood, urine, and renal tissue of
patients with autoimmune kidney diseases. The goal of most of
these studies has been to identify biomarkers of disease
pathology, activity, and response to treatment, and to understand disease pathogenesis to uncover potential novel therapeutic approaches. This review will cover recent developments
in the application of proteomics to autoimmune kidney disease
with an emphasis on the translation of proteomics to the clinic.

2. Proteomic techniques for the investigation

of autoimmune kidney disease
The instrumentation and informatic tools used to study the
proteome have evolved significantly over time. In the
early days of proteomics two-dimensional polyacrylamide

24
gel electrophoresis (2D-PAGE) was used to separate proteins
based on their molecular size and isoelectric point. Proteins
from urine or the kidney were resolved and individual spots of
differential intensity were cut from the gel and analyzed by
mass spectrometry. In many studies, Matrix Assisted Laser
Desorption Ionization Mass Spectrometry (MALDI-MS) was used
to identify proteins that distinguished normal from diseased
tissue. Although several investigations applied 2D-PAGE to the
urine proteome [1], it quickly became apparent that the
resolving power of 2D-PAGE, especially for membrane proteins
and highly basic proteins, was limited. Furthermore, MALDI-MS
was ineffective for very low abundance proteins, and these
early reports generally identified and quantitated only 30 to 40
urine proteins. The separation of proteins improved rapidly with
the development of nanoscale reversed phase liquid chromatography (RPLC), and this rapidly replaced 2D-PAGE. When RPLC
was coupled to mass spectrometers using nanoelectrospray
ionization (ESI) sources, the number of proteins that could be
resolved, identified and quantitated in the urine and kidney
increased considerably, and several hundred proteins were
detectable in urine [2,3]. Also essential to improved protein
identification was the development of reliable protein search
algorithms that permitted the design of high-throughput search
engines. Subsequent advances in mass spectrometers using ion
traps and particularly orbitraps have dramatically increased the
ability to detect proteins in tissue and biological fluids [4,5]. In
conjunction with strategies to enrich individual sub-proteomes
into fractions, these new approaches now permit the identification of several thousand proteins in human urine [6].

3. Proteomic analysis of biological fluids in the

study of kidney disease
The earliest applications of proteomics focused on the urine
proteome as urine is easily accessible and changes in its protein
complement may reflect alterations in renal protein expression.
In some studies, investigators focused on optimizing the number
of proteins identified in normal urine so as to develop a
database for future studies [1,7]. However, it quickly became
apparent that the study of urine with proteomic techniques
faced a number of barriers including variability in pH, solute
content and concentration that may occur as a result of changes
in hydration, diurnal variations and medication. In normal urine,
the dynamic range of protein expression is several logs due to
the abundance of uromodulin or TammHorsfall protein (THP).
Modern mass spectrometers use an approach termed datadependent acquisition of ions that allows only a fixed number of
precursor ions to be scanned in a single survey. In this setting,
highly abundant proteins like THP compete for ionization with
less abundant, and potentially interesting peptides so they are
undetectable, thereby limiting the sensitivity of the analysis.
Affinity antibodies to highly abundant proteins have been used
to compress the dynamic range of protein expression and
allow detection and quantitation of low abundance proteins and
peptides. Alternatively, THP can be removed by changing urine
pH. The removal of highly abundant proteins with affinity antibodies is particularly necessary when studying urine with
nephrotic range proteinuria.
The same broad dynamic range of protein expression is
present in plasma and must be addressed in the study of
renal disease. Removal of abundant plasma proteins by

B.H. Rovin, J.B. Klein

affinity antibodies is a common approach in this setting, as
well [8]. However, an alternative approach is to use mass
spectrometry to study the low molecular weight peptides in
plasma that represent intact protein degradation products.
In that setting the peptides are separated from highly abundant
proteins by size exclusion filters [9]. The presence of these
small molecular weight peptides in both plasma and urine,
termed the peptidome, points out the complexity of biological
fluids that must be appreciated in their proteomic analysis.
Urine, in particular, has both peptidomic and proteomic components. Additionally, urine contains small microvesicles including exosomes and apoptotic bodies that are derived from
all nephron segments [10]. Exosomes contain proteins, peptides, RNA and metabolites that may reflect changes in renal
function and pathology. Thus, these multiple constituents of
urine may each serve as a source of information regarding how
the kidney alters its behavior during normal physiologic
changes and disease. In order to capture as much information
as possible, a workflow to process urine and retain its multiple
constituents is outlined in Fig. 1. Even with this workflow some
urine components will be lost during the separation steps.
Evaluation of the renal proteome can complement and
extend the information obtained from the urine and serum
proteomes. Differences between the proteomes of normal
and affected renal parenchyma provide direct insights into
disease-relevant mechanisms. Kidney injury in autoimmune
disease is often initiated through glomerular immune complex accumulation, however the ultimate fate of the kidneys
is determined mainly by tubulointerstitial damage [11]. The
urine proteome is an integration of the whole kidney
proteome; it cannot distinguish events occurring in the
glomerular compartment from events occurring in the
tubulointerstitial compartment. Using laser-capture microdissection (Fig. 2), the proteomes of the glomeruli and
the tubulointerstitium can be evaluated separately [12].
Additionally, differentially-expressed kidney proteins can
focus urine biomarker searches to specific targets improving efficiency in developing non-invasive urine biomarkers
of disease. Using only urine proteomics, such targets may be
missed because of masking by more abundant, non-informative
proteins. Parenchymal proteomics also identifies proteins that
are not expressed or under-expressed in disease. It is much
more difficult to be certain of the absence of a protein in a urine
specimen given all the potential variables (e.g. osmolarity, pH,
concentration, albumin) present in this heterogeneous fluid
that could impair identification of unknown proteins.
Recently, we developed a proteomics workflow specifically for quantitative liquid chromatography-tandem mass
spectrometry (LCMS/MS) analysis of laser-captured isolates
from frozen and formalin-fixed paraffin-embedded (FFPE)
tissue sections [13,14]. It is now feasible to perform proteomic
analyses using the small amounts of tissue available from
clinical kidney biopsies. An example of the data generated by
this technique is shown in Fig. 3. Two points of general
importance may be taken from these data. First, despite
labeling a patient as having Class IV or Class V LN based on
clinical and pathological findings, examination of the kidney
tissue at the molecular level shows different patterns of
protein expression within an LN class. This is likely due to
timing of the kidney biopsy during the disease course, and also
to the intrinsic heterogeneity of all autoimmune kidney
diseases. Proteomics and other omics offer platforms for

Proteomics and autoimmune kidney disease

25

Figure 1 Workflow for investigating the urine proteome. Urine is initially clarified of cells and debris by routine centrifugation. The
acquired cellular sediments are stored for future studies. The clarified urine is then subjected to ultracentrifugation across sucrose
density gradients to isolate exosomes and microparticles. The exosome and microparticle-depleted urine is then processed using size
fractionating filters that partition the urine into fractions containing proteins and peptides either less than or greater than 4 kDa. The
low molecular weight fraction is then stored for peptidomic and metabolomics studies. If proceeding to proteomic analysis the
proteins N 4 kDa are stripped from the ultrafiltration membrane and the analyte is concentrated and de-salted before analysis by mass
spectrometry. Immunodepletion can be used to remove the most highly abundant proteins from urine and improve resolution for
low-abundance proteins.

understanding a disease in individuals and thereby personalizing therapies. Secondly, the tubulointerstitial compartment
from some of the LN patients shows a marked decline in
enzymes important for mitochondrial metabolism. It is
intriguing to speculate that individuals with autoimmune
kidney disease who develop chronic kidney injury do so
because tubular metabolism is disrupted and energy cannot
be generated properly for recovery. This mechanism could be
tested, and may suggest a way to prevent chronic kidney
disease therapeutically.
FFPE biopsies have several advantages over frozen tissue,
including storage and transportation logistics, and excellent
preservation of kidney histology which is important for
laser-capture microdissection. Approximately 800010,000
cells (roughly 23 g of protein) are sufficient for proteomic
analysis [14]. Applying these requirements to clinical kidney

biopsies translates into about 70 glomerular cross-sections

and 1.52 million m2 of tubulointerstitium from 10 m-thick
tissue sections. Analyzing this quantity of kidney tissue on
an early-generation mass spectrometer yielded 662 and 607
proteins from the glomerular and tubulointerstitial compartments, respectively [14]. Using state-of-the-art technology (1dimensional liquid chromatography coupled to a high-resolution
LTQ-Orbitrap Elite mass spectrometer), 1399 proteins from
the glomerular compartment and 1742 proteins from the
tubulointerstitial compartment were identified from FFPE
biopsies of patients with idiopathic immunoglobulin-A (IgA)
nephropathy (unpublished data). In this analysis the false
discovery rate was estimated at b 1% using a decoy database
generated from the UniprotKB Homo sapiens reference
proteome. Many tissue proteins from autoimmune kidney
diseases are also found in the urine, supporting the idea that

Figure 2 Laser-capture microdissection of a kidney biopsy section. A. Hematoxylin and eosin-stained section of a kidney biopsy
from a patient with lupus nephritis. B. Hematoxylin-stained section of the biopsy before laser dissection with the glomeruli to be
captured outlined. C. Hematoxylin-stained section of the biopsy before laser dissection with the tubulointerstitial areas to be
captured outlined. D. The same tissue section as in (B) after the glomeruli have been laser-dissected and captured. E. The same tissue
section as in C after the tubulointerstitial areas have been laser-dissected and captured.

26

B.H. Rovin, J.B. Klein

Tubulointerstitium
Normal
Normal
Normal
LN Class IV
DN
LN Class V
LN Class IV
LN Class IV
LN Class V
DN

Glomeruli

Normal
Normal
Normal
LN Class IV
DN
LN Class V
LN Class IV
LN Class IV
LN Class V
DN

Normal
Normal
Normal
LN Class IV
DN
LN Class V
LN Class IV
LN Class IV
LN Class V
DN

Glomeruli

PCK2

Vimentin
Hemoglobin
PUC-IgG1
IGK Protein
25kDa protein
PUC-IgG3

C3
C3
C3

PCK2
GLYAT
Ubiquinone
Glud1

HbD globin

C9

Complement

Vitronectin

Desmin
Histone H3

C4B
C4B
C4A
C4A
C4A
C4B

SHC-1
PUC-IgG1
PUC-IgG2
PUC-IgG1
PUC-IgG3
PUC-IgG4

Immunoglobulins

ACAT1
ALDH4A1
Funarate Hydratase
Acyl-CoA dehydrogenase
Choline dehydrogenase
MMSADH
HIBCH
Pyruvate Carboxylase
Acyl-CoA Synthetase
Delta-dienoyl CoA Isomerase

Mitochondrial Proteins

Figure 3 Differential kidney protein expression in lupus nephritis. Kidney biopsies from normal kidneys, lupus nephritis Class IV or V
kidneys, and diabetic nephropathy kidneys were laser-microdissected into glomerular and tubulointerstitial compartments. The
kidney compartments were analyzed for global protein expression by LCMS/MS and heat maps showing relative protein expression
among the samples are shown. The glomerular heat maps are focused on complement proteins and immunoglobulins. It is not
surprising that complement and immunoglobulins are generally increased in LN and not in normal kidney tissue. It is interesting that
complement components are also increased in diabetic nephropathy. The interstitium shows a down-regulation of mitochondrial
metabolic enzymes in most of the LN biopsies. It is notable that individuals with the same class of LN (compare the three Class IV LN
biopsies) can have markedly different glomerular and interstitial proteomes.

tissue proteomics can focus the search for urine biomarkers.

In a global proteomic screen of urine from 15 lupus nephritis
(LN) patients we identified 1126 proteins and compared this
list to the list of differentially-expressed proteins between
control and Class IV LN tissue (n = 3 each). This showed that
65% of proteins from the glomerular compartment and 54%
of the proteins from the tubulointerstitial compartment
were found in the LN urines (unpublished data).
Proteomic analysis of laser-captured glomeruli from
kidney biopsies by LCMS/MS is currently in use clinically
to identify the composition of proteins in glomerular diseases
with organized deposits, such as amyloidosis, fibrillary and
immunotactoid glomerulonephritis, and membranoproliferative
glomerulonephritis [1517]. Although these are not strictly
autoimmune kidney diseases, proteomics has crossed the
threshold into the clinical nephropathology laboratory as
a diagnostic tool, suggesting it will not be long before
proteomics will be applied to the diagnosis and evaluation
of other types of kidney disease, including autoimmune
diseases.

4. Proteomics in the identification of

autoantibody targets in glomerulonephritis
One of the most successful and clinically relevant applications of proteomics to autoimmune kidney diseases has been
in the identification of the target antigens of autoantibodies
in a number of glomerular diseases. This is best exemplified
by the discovery of the phospholipase A2 receptor as the

dominant antigen in primary membranous nephropathy. In

these studies, sera from patients with primary membranous
nephropathy were used to immunoblot normal glomerular
proteins separated by PAGE. The gel bands corresponding to
bands of sera reactivity on the immunoblots were excised and
analyzed by mass spectrometry. This identified eighteen
proteins of relatively high abundance in the 185 kDa region
corresponding to sera reactivity. Subsequent experiments
identified one of these eighteen proteins, the M-type phospholipase A2 receptor, as the renal target antigen of antibodies
present in 70% of primary membranous nephropathy patients
[18]. In recent follow-up experiments, the same proteomic
approach was used to identify the thrombospondin type-1
domain-containing 7A protein as a membranous nephropathy
target antigen in 10% of patients who did not produce antibodies to the phospholipase A2 receptor [19].
Similar approaches have been used to look for antigens in
anti-neutrophil cytoplasmic antibody-associated vasculitis
(AAV) and LN [17,20]. In AAV, the proteome of human
umbilical vein endothelial cells (HUVEC) was separated into
component proteins by 2D-PAGE and blotted with serum
from patients with either microscopic polyangiitis (MPA) or
granulomatosis with polyangiitis (GPA) to look for antiendothelial cell reactivity. This study identified HUVEC
antigens that distinguished MPA from GPA patients, including
-enolase, vimentin, lamin-A/C, far upstream binding protein
2, and protein disulfide-isomerase A3 precursor. Although
these data provide evidence that the pathogenesis of MPA and
GPA may be different, at present the data are not developed
enough to suggest different approaches to therapy for these

Proteomics and autoimmune kidney disease

AAV subtypes. Furthermore, patients with AAV and renal
involvement were not examined separately, so no conclusions
can be drawn as to why only some patients have renal disease.
Finally, HUVECs display relatively non-specific endothelial
antigens. It would be interesting to repeat these experiments
using cells from organs likely to be affected in AAV, such as
glomerular capillary endothelial cells. Conceivably, autoantibodies against glomerular endothelial cell antigens could
be of use in predicting who will develop kidney injury. Some
experiments were done using glomerular capillary endothelial
cells and sera from patients with mixed connective tissue
disease, and an antibody to human voltage-dependent anion
selective channel 1 was identified, but its role/relationship in
the glomerular injury of MCTD is not established [21].
The identification of autoantigens in LN kidneys was accomplished by eluting immunoglobulins from laser-captured
glomeruli and assessing their reactivity to podocyte proteins
separated by 2D-PAGE. This yielded several potential antigens,
but antibodies to two antigens, annexin AI and -enolase, were
found in the glomeruli of all classes of LN and in all LN sera,
indicating a potentially fundamental role in the pathogenesis of
LN. These antibodies were isotyped as IgG2. Interestingly,
anti--enolase was also found in glomeruli of patients with
idiopathic membranous nephropathy, but was isotyped as IgG4
[20]. Antibodies to annexin AI and -enolase did not bind to
nuclear material, and eluted anti-DNA antibodies did not bind
to -enolase. Intraperitoneal administration of hybridomas
that produced anti--enolase in BALB/C mice led to the
appearance of proteinuria in 25% of the mice; 66% of SCID mice
injected with these hybridomas developed crescentic glomerulonephritis. These findings renew the controversy of the role
of endogenous glomerular antigens versus planted nuclear
antigens in the development of LN. However, although serum
levels of anti--enolase were higher in LN patients then
patients with non-renal SLE, there was moderate overlap, so it
remains to be seen if these antibodies predict and/or mediate
the development of LN in SLE.

5. Proteomics and biomarkers of autoimmune

kidney diseases
Thus far, the main focus of proteomic investigations of
autoimmune kidney diseases has been to identify disease
biomarkers. In this context disease biomarkers have been
sought that can be used diagnostically, prognostically, and
to better understand the mechanisms of kidney injury. The
approaches to biomarker discovery in autoimmune kidney
disease will be reviewed with the intention of exemplifying
important issues in this field. It should be pointed out that
none of the biomarkers discussed below have been validated
in independent, appropriately-sized patient cohorts, a
ubiquitous problem that has limited the clinical qualification
of most reported biomarkers.
Autoimmune kidney diseases are usually diagnosed by
kidney biopsy. The possibility of having non-invasive diagnostic biomarkers that could substitute for the biopsy is
appealing. The general approach to diagnostic biomarker
development has been to identify patterns of differentiallyexpressed urine proteins characteristic of a particular autoimmune kidney disease. As an example, capillary-electrophoresis
coupled to mass spectrometry was used to characterize the

27
pattern of proteins/peptides in urine from patients with IgA and
primary membranous nephropathy [22]. A panel of 28 peptides
based on the mass spectrometric pattern was sufficient to
distinguish these two glomerular diseases with a sensitivity of
77% and a specificity of 100%. In other studies using capillary
electrophoresis coupled to mass spectrometry, urine peptide
panels were able to discriminate between IgAN and diabetic
nephropathy, minimal change disease, or focal segmental
glomerulosclerosis (FSGS) with high sensitivity and specificity.
Importantly, the identity of many of the peptides within these
individual panels was not determined. The mass spectrometric
patterns were sufficient for the panels to serve as effective
biomarkers. However to find novel therapeutic targets and to
elucidate disease pathogenesis, knowing a protein's identification is important.
A point to be emphasized from this IgAN study is that
diagnostic accuracy was achieved with a panel of proteins/
peptides, not a single biomarker. This concept was extended
in another study that also used changes in the urine proteome
to differentiate between LN, primary membranous nephropathy, diabetic nephropathy and FSGS. The relationship of the
number of differentially-expressed proteins in a biomarker
panel to the diagnostic sensitivity and accuracy of the panel
was determined [23]. Sensitivity was maintained above 70%
until there were fewer than 5 proteins, but accuracy fell
toward 50% below 20 proteins. It is therefore unlikely that
single biomarkers will be able to reach sufficient discriminatory power for clinical use, so focusing on biomarker panels is
appropriate.
Another important diagnostic dilemma that has been
addressed by biomarker studies is how to predict that a kidney
disease will flare before the flare is clinically apparent. A
biomarker of impending flare would open these diseases up to
the possibility of flare prevention rather than flare treatment.
Early intervention may limit inflammatory injury and prevent
chronic kidney damage. To address this question we used
SELDI-TOF mass spectrometry to analyze the low-molecular
weight (b 30 kilo-daltons) urine proteome of urine samples
collected from patients before, during and after LN flare [24].
Among other differentially-expressed proteins during the LN
flare cycle, these studies identified the 20 amino-acid form of
hepcidin as a potential flare predictor, because it increased
4 months pre-flare and returned to baseline levels at flare.
Given the dynamic changes of urine hepcidin expression over
the LN flare cycle, it is unlikely that this potential biomarker
would have been identified or correctly interpreted by
examining only cross-sectional urine samples (Fig. 4A, B).
This limitation probably applies to most other putative biomarkers of autoimmune kidney diseases, however to date the
majority of proteomic studies have been cross-sectional.
The importance of longitudinal studies is emphasized by
two recent investigations that identified potential biomarkers of chronic kidney damage in LN and IgAN [25,26].
Chronic kidney disease worsens prognosis, not only because
patients can progress to end-stage kidney disease requiring
renal replacement therapy, but also because there is
increased risk of cardiovascular morbidity in patients with
chronic kidney disease. Biomarkers of chronic kidney
damage and progressive decline in kidney function are
therefore high priority targets. Such biomarkers could
identify patients in whom anti-fibrotic and kidney protective measures are needed.

28

B.H. Rovin, J.B. Klein

Urine from patients with LN at or within 6 months of renal

biopsy was evaluated with an antibody microarray that
focused on cytokines [25]. Angiostatin, an anti-angiogenic
fragment of plasminogen was found to be greatly increased
in LN urine compared to urine from healthy volunteers.
Angiostatin correlated positively with serum creatinine and
chronicity on the kidney biopsy, suggesting it may be a
marker of progressive kidney failure.
The proteome of urine obtained from IgAN patients at
kidney biopsy was investigated by 2D-PAGE and differentiallyexpressed proteins were identified by mass spectrometry [26].
One interesting protein was significantly decreased in most of
the IgA urines compared to urine from healthy individuals. This
protein was identified as the LG3 fragment of endorepellin,
which is a cleavage product of perlecan. Like angiostatin, LG3
has anti-angiogenic effects. Also like angiostatin, LG3 levels
correlated inversely with glomerular filtration rate, and
positively with increased fibrosis on kidney biopsy.
The observation that anti-angiogenic cytokines may be
markers for, and/or involved in progressive parenchymal
damage in two different autoimmune kidney diseases is
compelling and suggests that angiogenesis may be important

in preserving kidney tissue after inflammatory injury. However, before these cytokines can be accepted as biomarkers of
prognosis, or used to develop targeted therapeutics, they
need to be measured prospectively in patients to determine if
they correlate with the development of chronic kidney disease
(Fig. 4C).

6. Looking toward the future

The use of proteomic approaches to identify auto-antibodies is
now clearly established with the identification of phospholipase A2 receptor and the thrombospondin type-1 domaincontaining 7A protein auto-antibodies in primary membranous
nephropathy. In a similar manner, proteomic analysis of
kidney proteins isolated by laser capture micro-dissection
appears to be established and has great potential to identify
proteins relevant to renal pathology. Further advances in
identifying pathology-related proteins and auto-antigens will
likely require development of mass spectrometry methods
that extend the ability of the instrument to identify more
proteins in experimental specimens and to identify proteins

A
Biomarker Level

LN
Healthy

Health

Pre-Flare

Flare

Post-Flare

Remission

Biomarker Level

Health

Pre-Flare

Flare

Post-Flare Remission

Pre-Flare

Flare

Biomarker Level

C
CKD

Remission

Health

Pre-Flare

Flare

Post-Flare

Remission or CKD

Figure 4 Hypothetical urine biomarker patterns in lupus nephritis. Several possible scenarios (not exhaustive) are presented to
illustrate how a biomarker level may change over the course of an LN flare cycle. A. In this scenario a putative urine protein
biomarker is found to be either higher or lower in LN than in healthy controls. If this protein was measured only at flare in a
cross-sectional study the protein may be considered a biomarker of LN flare. However, when examined over time in serial urine
samples it is seen that the biomarker does not change with the flare cycle, and levels were different even before LN flare. The
biomarker may thus indicate an intrinsic abnormality of SLE as opposed to indicating active LN. B. In this scenario, the putative urine
biomarker increases before flare, peaks during flare, and declines toward baseline as the patient is treated and remits. This pattern is
repeated in the next flare cycle. If the urine was sampled only during flare, the biomarker may be interpreted as a marker of active
LN. Following the biomarker levels in serial urine samples demonstrates a consistent increase before each flare, and the biomarker
may thus be a predictor of impending LN flare. C. In this scenario the levels of a putative urine protein biomarker are shown for two
separate patients. One patient remits after treatment and the other patient develops chronic kidney disease (CKD). During the
evolution of CKD, the biomarker level increases during flare and treatment, whereas the biomarker level falls over time in the patient
who responds to treatment. These biomarker patterns suggest that the biomarker may indicate and contribute to the development of
chronic kidney injury.

Proteomics and autoimmune kidney disease

with high confidence in the absence of validated antibodies
for ELISA or immunoblot assays. Recent developments in mass
spectrometers and associated software permit a greater
number of proteins to be identified in a specimen. As
discussed previously, mass spectrometers have used an
approach termed data-dependent acquisition of ions that
allows only a fixed number of precursor ions to be scanned
in a single survey. In this setting, highly abundant proteins
displace less abundant and potentially interesting peptide
ions from the scan, and thereby limit the sensitivity of the
analysis. A new approach uses data-independent acquisition of ion characteristics that can, in theory, interrogate
a specimen for the identity and quantity of any protein of
interest. This approach uses extensive peptide fragment ion
libraries and compares them to the observed peptide maps.
This more open-ended approach to mass spectra analysis
has been termed SWATH-MS after the swaths used to
designate the ion scanning windows of the mass spectrometer.
SWATH-MS depends upon the reproducibility of high-resolution
spectra and the recent availability of proteome-wide spectral
libraries [27].
A second recent advance in proteomics is the use of
targeted analysis termed selected reaction monitoring (SRM),
also known as multiple reaction monitoring (MRM). In SRM,
selected precursor fragments of peptides of interest are
monitored. The selected peptide fragments and their resultant
ions are determined by either ion scanning, repository data or
rules that govern enzymatic proteolysis of protein amino acid
sequences. When coupled with isotopic labeling of peptides,
SRM can be quantitative for individual proteins down to the
sub-femtomole level. Thus, SRM assays can be developed that
are quantitative for proteins for which validated antibodies
have not been developed and can serve as an alternative to
antibody based assays such as ELISA. SRM assays are currently
being used to quantitate small molecular weight peptides that
may reflect flares of autoimmune renal disease.
These techniques are expected to accelerate the pace at
which proteomic findings achieve clinical applicability in the
diagnosis and management of autoimmune kidney diseases.

29

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[9]

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[11]

[12]

[13]

[14]

[15]

[16]

[17]

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