Initial validation of a novel protein biomarker panel for active pediatric lupus nephritis

Abstract

Lupus nephritis (LN) is among the main determinants of poor prognosis in systemic lupus erythematosus (SLE). The objective of this study was to 1) isolate and identify proteins contained in the LN urinary protein signature (PS) of children with SLE; 2) assess the usefulness of the PS proteins for detecting activity of LN over time. Using surface-enhanced or matrix-assisted laser desorption/ionization time of flight mass spectrometry, the proteins contained in the LN urinary PS were identified. They were transferrin (Tf), ceruloplasmin (Cp), alpha1-acid-glycoprotein (AGP), lipocalin-type prostaglandin-D synthetase (L-PGDS), albumin, and albumin-related fragments. Serial plasma and urine samples were analyzed using immunonephelometry or ELISA in 98 children with SLE (78% African American) and 30 controls with juvenile idiopathic arthritis. All urinary PS proteins were significantly higher with active vs. inactive LN or in patients without LN (all p < 0.005), and their combined area under the receiver operating characteristic curve was 0.85. As early as 3 mo before a clinical diagnosis of worsening LN, significant increases of urinary Tf, AGP (both p < 0.0001), and L-PGDS (p < 0.01) occurred, indicating that these PS proteins are biomarkers of LN activity and may help anticipate the future course of LN.

Figure 1. Plasma concentrations of the PS-proteins

Values are means and SE in mg/dL. Significant differences are based on Tukey post-hoc testing. The histograms show the level of the PS-proteins, Tf (A), Cp (B), AGP (C) and L-PGDS (D). SLE patients with active lupus nephritis (LN), inactive LN or without LN are compared with groups were defined by the SLEDAI. Twenty children with active and 10 with inactive JIA served as controls. Significant differences between groups are indicated as follows: *= P<0.02; **= P<0.01; ¶= P<0.001; §= P<0.0002.

Figure 2. Urinary concentration of the PS-proteins

Values are means and SE. Significant differences are based on Tukey post-hoc testing. The histograms show urinary concentrations of Tf (A), Cp (B), AGP (C) and L-PGDS (D) for the groups defined as Figure 1. Uncorrected PS-protein levels (per mL or dL of urine) are depicted. Significant differences between groups are indicated as follows: * = P<0.004; **= P<0.002; ¶= P<0.00001.

Figure 3. Urinary concentration of the PS-proteins

Values are means and SE. Significant differences are based on Tukey post-hoc testing. The histograms show urinary concentrations of Tf (A), Cp (B), AGP (C) and L-PGDS (D) for the groups defined as Figure 1. PS-protein excretion standardized by urine creatinine (mg/mL urine) is shown. Significant differences between groups are indicated as follows: *= P<0.0005; **= P<0.0001; §= P<0.05; ¶ = P<0.001.

Figure 4. Urinary concentration of the PS-proteins

Values are means and SE. Significant differences are based on Tukey post-hoc testing. The histograms show urinary concentrations of Tf (A), Cp (B), AGP (C) and L-PGDS (D) for the groups defined as Figure 1. PS-protein excretion standardized by nonselective proteinuria is depicted with significant differences between groups indicated as follows: * = P<0.05; ** = P<0.005; § = P<0.02; ¶ = P<0.009.

Figure 5. Changes of the PS-proteins in relationship to future changes in LN activity

Values are geometric means of uncorrected urinary levels of Tf (A), Cp (B), AGP (C) and L-PGDS (D) at months -6, -3 and 0, respectively. Month 0 is the time point when the clinical diagnosis of the course of LN is made and months -3 corresponds to the timepoint of 3 months prior to the clinical diagnosis of the LN flare. ‘Improved LN’ describes the course of LN with decreasing renal SLEDAI scores; ‘worse LN’ describes the course of LN associated with an increase of the renal SLEDAI scores; ‘stable active LN’ describes patients with stable renal SLEDAI scores > 0; and ‘inactive LN’ describes the course of continuously inactive LN (renal SLEDAI = 0). Significant differences in the levels between two consecutive visits are indicated in the plots as follows. § = P< 0.009; ¶ = P<0.0001; * = P<0.001. The above defined LN courses are depicted as follows: Improved LN, squares; Worsened LN, circles; Stable active LN, triangles; Inactive LN, diamonds.