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Diagnostics
Urinary Protein Patterns as a Diagnostic Tool for Patients with IgA Nephropathy
a report by
Marion Haubitz,
1
Harald Mischak,
2
Bruce A Julian
3
and Jan Novak
3
1. Department of Nephrology, Hannover Medical School; 2. Mosaiques Diagnostics and Therapeutics AG, Hannover Medical School;
3. University of Alabama, Birmingham
Immunoglubulin A (IgA) nephropathy (IgAN) is the most common type of Furthermore, identification of an array of urinary protein biomarkers will also
primary glomerulonephritis worldwide.
1
In Europe it is found in 20%, and advance our understanding of the pathogenesis of the renal diseases.
in Asia in up to 50%, of all biopsy-proved patients with glomerulonephritis.
Clinical features and prognosis vary. In general, patients develop painless Proteomic Approaches to Identify Urinary
microhaematuria and/or macrohaematuria and proteinuria, which is usually Biomarkers of Renal Diseases
below the nephrotic range. Therefore, many patients are unaware of the It has been speculated that urinary proteins and polypeptides reflect the
disease, and discovery of the clinical manifestations of IgAN is often a nature of renal diseases. Using urine to search for potential biomarkers
chance finding. However, up to one-third of patients develop end-stage offers several advantages. It is easily accessible, obtained by non-invasive
renal disease (ESRD) within 20 years of diagnosis and, consequently, require means and available in large quantities, and urinary polypeptides display the
dialysis or transplantation. After transplantation, IgAN histologically recurs ‘status’ of the kidney. Furthermore, polypeptides in urine samples do not
in up to 60% of allografts.
2
degrade appreciably when stored for as long as six hours at room
temperature (or for several months at -20°C).
9,10
Urinary proteins can be
Currently, the diagnosis of IgAN can be established only from examination analysed directly or after a separation process. For fractionation and
of cortical renal tissue. Typical histological features include mesangio- detection, 2D gel electrophoresis, protein arrays, chromatography, mass
proliferative glomerulonephritis with predominant or co-dominant IgA spectrometry (MS) and other methods have been used. For polypeptides
deposits in the mesangium, often with C3 and IgG or IgM, or both. The smaller than 10kDa, MS-based technologies have generated the best results.
differential diagnosis includes several glomerulonephritides and
nephrosclerosis due to hypertension. Unfortunately, renal biopsy is Two MS-based technologies that are commonly used for analysis of clinical
complicated by serious bleeding in about 2% of patients.
3
Therefore, many samples are surface-enhanced laser desorption ionisation MS (SELDI MS)
affected individuals are not identified early and monitoring of the disease and capillary electrophoresis-MS(CE-MS). SELDI MS combines a relatively
in the latter stages is compromised.
4
Thus, there is a need for a non-invasive selective capture of proteins with direct detection by MS. Urinary samples
diagnostic/screening test for the detection of subclinical IgAN, the are applied to protein chips with different surfaces to bind a subset of
estimation of the degree of activity during follow-up and assessment of the proteins. The bound proteins are then ionised and desorbed (knocked off
efficacy of treatment. However, there has been no valid, alternative non- the surface), and the ions are identified by time-of-flight (TOF) MS. SELDI MS
invasive test with sufficient sensitivity and specificity.
5
For example, while is easy to use and requires only a small amount of sample. However, this
urinary levels of interleukin (IL)-6 are elevated in patients with IgAN, technique has several disadvantages. SELDI MS profiles are much more
patients with other glomerulonephritides also exhibit this finding. A similar sparse due to failure to retain many proteins and peptides in the selective
situation has been described in the analysis of other cytokines/chemokines capture step, and the mass spectra tend to have relatively low resolution and
such as chemotactic peptide-1 (MCP-1) and IL-8, both correlating with mass accuracy.
11
Moreover, the relative signal depends on the concentration
tubulointerstitial damage.
6,7
Unfortunately, a reliable differentiation of the analyte and confounding variables. This results in highly diverse
between IgAN patients and controls was precluded by the large overlap in spectra from identical samples analysed at different concentrations. CE
cytokine excretion between the groups.
6,8
coupled online to an electrospray ionisation TOF (CE-ESI-TOF) mass
spectrometer permits rapid identification of the polypeptide pattern in urine
New diagnostic approaches may rely more heavily on the establishment of a in a single step,
12
with more than 1,000 polypeptides detected per sample.
panel of markers, whereby the search for a single protein that distinguishes For CE, samples are first desalted and concentrated using reversed-phase
a specific disease (and also can monitor its progression) could be abandoned. chromatography. The eluted polypeptides are lyophilised and re-suspended
in high-performance liquid chromatography (HPLC)-grade water shortly
before use, normalising the total protein content in the samples. The CE is
Marion Haubitz is a Professor of Nephrology at the
coupled online via a sheath-flow-interface to ESI-TOF mass spectrometer;
University of Hannover, Germany. She has conducted several
clinical trials and is a member of the European Vasculitis
the total CE-MS run requires less than 60 minutes. The vast amount of data
Group. Beside her work in systemic autoimmune diseases, from this analysis is then evaluated using specially designed software.
Dr Haubitz’s interest is focused on biomarkers of vascular
diseases and urinary proteomics as a diagnostic and
prognostic tool in renal disease. She received a Dorothea-
Proteomics in Patients with IgA Nephropathy
Erxleben fellowship and was appointed as Professor of
Very few investigators have focused on the application of urinary
Internal Medicine and Nephrology in 2001. She obtained
proteomics for patients with IgAN.
13–16
Park et al. used 2D gel
her MD from the Justus-Liebig-University of Giessen.
electrophoresis
13
and matrix-assisted laser desorption ionisation (MALDI)
E:
haubitz.marion@mh-hannover.de
TOF MS to identify proteins to establish a urinary proteomic map in 13
12 © TOUCH BRIEFINGS 2007
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