Kidney involvement in patients with systemic lupus erythematosus (SLE) is a serious complication that often requires aggressive immunosuppressive therapy. Although current therapeutics are generally effective in controlling renal SLE, the morbidity associated with treatment can be severe (1
). Therapy of SLE nephritis would be more effective, and treatment toxicities mitigated, if the onset, severity, or responsiveness of SLE renal flare could be predicted, and treatment modified accordingly for individual patients. Supporting this concept, renal survival is improved with early treatment and rapid induction of remission in lupus nephritis (2
). At present there are no biomarkers that reliably predict the onset of renal flare, flare phenotype, or how flare will respond to treatment (4
). We postulated that proteomic analysis of serial urine samples taken before, during, and after lupus nephritis flares could screen for proteins or peptides that are differentially expressed during the initiation, maintenance, and resolution phases of the flare cycle. Some of these proteins could be clinically useful biomarkers for lupus nephritis. For example, a urine protein that changes between disease quiescence and a time point before flare is clinically evident could be a forecaster of impending renal flare. Urine proteins that are differentially-expressed during flare may be markers of flare severity and could have pathogenic or therapeutic implications. Finally, urine proteins that are differentially expressed during flare resolution may be biomarkers of prognosis or response to therapy.
The urine proteome has been studied by two main techniques. One technique involves 2-dimensional gel electrophoresis (2DE) to separate proteins by isoelectric point and size, followed by protein identification using mass spectrometry (MS) (7
). The most significant disadvantage of 2DE for detecting candidate biomarkers is the limited resolution of low-molecular weight (LMW), low-abundance proteins (11
). The LMW proteome is especially important for biomarker discovery in SLE, because it contains biologic mediators (cytokines, chemokines, growth factors) that are expected to be involved in disease pathogenesis. Some of these mediators are produced in the kidney and may reflect organ-specific injury. Nonetheless, 2DE followed by mass spectrometry was used to detect urine proteins that discriminate between ISN/RPS lupus nephritis classes (12
). These putative biomarkers were serum glycoproteins mostly larger than 20 000 Daltons (Da).
The second major technique used in urine proteomics is surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). This involves spotting urine samples on a solid-phase matrix (protein chip) that has specific binding properties. Because the surface chemistry of the protein chip influences the number and types of proteins that bind, several different chip types must be used to completely characterize a sample’s proteome. Protein spectra generated after laser ionization of the spotted samples (13
) are characterized by their mass to charge (m/z) ratio. Relative abundance can be estimated by protein ion peak height (intensity). The advantage of SELDI-TOF-MS in SLE nephritis is sensitivity for detection of LMW proteins, especially below 20 000 Daltons (14
). For urine protein analysis, SELDI-TOF-MS is accurate to within 0.01% of the molecular mass, and has a sensitivity of 0.1 fmole (16
). There are two main drawbacks associated with SELDI-TOF-MS. Most importantly, it cannot directly identify specific proteins, but rather patterns of protein expression (17
). Although patterns can provide biomarker information, protein identities are required to advance the understanding of disease pathogenesis and to develop novel therapeutics. The identities of specific SELDI-derived candidate protein ions can be determined after chromatographic enrichment and protein sequencing by capillary liquid chromatography-nanospray tandem mass spectrometry (LC/MS/MS), or by on-chip protein sequencing using an LC/MS/MS system integrated with the SELDI-TOF mass spectrometer (9
A second drawback of SELDI-TOF-MS is the reproducibility of relative protein abundance. Although SELDI-derived protein masses are consistent (intra- and interassay coefficients of variation of 0.07%), intra- and interassay coefficients of variation for protein peak intensities are on the order of 20%, and in the urine have ranged from 8-30% (17
). SELDI-TOF-MS is thus a semi-quantitative, high-throughput screening technique, and SELDI-derived candidate biomarkers need to be validated by another method.
SELDI-TOF-MS has been used for urine biomarker discovery in urological malignancies (20
), kidney transplant rejection (22
), intravenous contrast nephropathy (16
), urolithiasis (15
), ischemic acute kidney injury (15
), and SLE nephritis (24
). Five potential polypeptide biomarkers were identified for transitional cell carcinoma of the bladder, and one of them was subsequently shown to be a member of the defensin family (20
). In acute allograft rejection there was no concordance of the results of three studies that used protein ion expression patterns to define biomarkers (22
). One SELDI-TOF-MS evaluation of acute rejection produced a list of peaks that were later identified using microcapillary LC/MS/MS as proteolytic products of β2
-microglobulin, presumably reflecting damage to renal proximal tubular cells (26
). Only two SLE nephritis studies used the SELDI platform to examine the urine proteome. One reported two protein ions at m/z 3340 and 3980 that together distinguished active nephritis from inactive nephritis with 92% sensitivity and specificity, but the actual peptides were not identified (24
). The other investigation found 8 protein ions that correlated with renal disease activity (27
). The majority of these were larger than 20 kDa and mainly unidentified, but one appeared to be albumin (27
In the current study SELDI-TOF-MS was used to screen the LMW proteome of prospectively acquired, serial urine samples from a cohort of SLE patients with known renal involvement, to demonstrate the feasibility of candidate biomarker discovery for specific phases of the lupus nephritis flare cycle, according to the scheme outlined in . Twenty seven peptide ions showed statistically significant differential expression over the flare cycle. Selected protein ions were characterized further by on-chip peptide sequencing and were identified as hepcidin, α1-antitrypsin (A1AT) and a fragment of albumin. These data show that urine proteomic screening with SELDI-TOF-MS followed by MS peptide sequencing can positively identify peptides that can then be validated as lupus nephritis biomarkers.
Analysis scheme for urine protein phenotyping of SLE nephritis flare cycle.