Biomarkers of lupus nephritis determined by serial urine proteomics

Abstract

Lupus nephritis is a frequent and serious complication of systemic lupus erythematosus (SLE), the treatment of which often requires the use of immunosuppressives that can have severe side effects. Here we determined the low-molecular weight proteome of serial lupus urine samples to uncover novel and predictive biomarkers of SLE renal flare. Urine from 25 flare cycles of 19 patients with WHO Class III, IV, and V SLE nephritis were obtained at baseline, pre-flare, flare and post-flare. Each sample was first fractionated to remove proteins larger than 30 kDa, then applied onto weak cation exchanger protein chips for analysis by SELDI-TOF mass spectrometry. We found 176 protein ions of which 27 were differentially expressed between specific flare intervals. On-chip peptide sequencing by integrated tandem mass spectrometry positively identified the 20 and 25 amino-acid isoforms of hepcidin, as well as fragments of alpha1-antitrypsin and albumin among the selected differentially expressed protein ions. Hepcidin 20 increased 4 months before renal flare and returned to baseline at renal flare, whereas hepcidin 25 decreased at renal flare and returned to baseline 4 months after the flare. These studies provide a beginning proteomic analysis aimed at predicting impending renal relapse, relapse severity, and the potential for recovery after SLE nephritis flare.

Figures

Figure 1

Analysis scheme for urine protein phenotyping of SLE nephritis flare cycle.

Figure 2

Urine SELDI spectra of class IV SLE nephritis flare cycles. A. The spectra of a whole flare cycle are presented between the 2000 and 10 000 Dalton region. The LMW urine proteome shows an overall increase in peaks between 2000 and 4000 Dalton as flare approaches, which then decrease during flare treatment. Peak intensity (relative protein abundance) is given on the y-axis. B. The spectra from 4 months pre-flare and flare of a Class IV GN patient showing that some protein ions decrease at the flare.

Figure 3

Expression of candidate urine biomarkers over time. The mean relative intensities of 4 differentially expressed protein ions (log-transformed data for M87) are plotted at several points of the renal flare cycle. Error bars indicate standard errors (A-D).

Figure 4

Urine Hepcidin Expression in SLE Nephritis. A. A typical urine SELDI spectrum in a class IV LN urine showing three hepcidin isoforms of 20, 22 and 25 at m/z of 2197, 2432 and 2798, respectively. B. Time course of hepcidin 20 and 25 expression (mean relative intensities) during the SLE nephritis flare cycle. Error bars indicate standard errors. C. On-chip CID fragmentation of peak 34, used to identify the ion as hepcidin 25. D. MS fragments of trypsin-digested hepcidin 25 standard. E. LC/MS/MS detection of the internal peptide of hepcidin 25 (y-ions are labeled) in the urine of an SLE nephritis patient.

Figure 4

Urine Hepcidin Expression in SLE Nephritis. A. A typical urine SELDI spectrum in a class IV LN urine showing three hepcidin isoforms of 20, 22 and 25 at m/z of 2197, 2432 and 2798, respectively. B. Time course of hepcidin 20 and 25 expression (mean relative intensities) during the SLE nephritis flare cycle. Error bars indicate standard errors. C. On-chip CID fragmentation of peak 34, used to identify the ion as hepcidin 25. D. MS fragments of trypsin-digested hepcidin 25 standard. E. LC/MS/MS detection of the internal peptide of hepcidin 25 (y-ions are labeled) in the urine of an SLE nephritis patient.

Figure 4

Urine Hepcidin Expression in SLE Nephritis. A. A typical urine SELDI spectrum in a class IV LN urine showing three hepcidin isoforms of 20, 22 and 25 at m/z of 2197, 2432 and 2798, respectively. B. Time course of hepcidin 20 and 25 expression (mean relative intensities) during the SLE nephritis flare cycle. Error bars indicate standard errors. C. On-chip CID fragmentation of peak 34, used to identify the ion as hepcidin 25. D. MS fragments of trypsin-digested hepcidin 25 standard. E. LC/MS/MS detection of the internal peptide of hepcidin 25 (y-ions are labeled) in the urine of an SLE nephritis patient.

Figure 5

Urine expression of A1AT and an albumin fragment during SLE renal flare. A. SELDI relative intensity of M17 (A1AT) and M26 (Albumin) between baseline and flare, and baseline and 4 month pre-flare. B. SELDI spectrum of M17 at flare; C. SELDI spectrum of M26 pre-flare.

Figure 6

Intrarenal expression of hepcidin. Immunohistochemical staining for hepcidin is shown for renal biopsy material from a normal kidney (Cont), and three patients with class IV SLE nephritis (SLE). The positive cells are infiltrating interstitial leukocytes.