Quantitative planar array screen of 1000 proteins uncovers novel urinary protein biomarkers of lupus nephritis.

OBJECTIVE: The goal of these studies is to discover novel urinary biomarkers of lupus nephritis (LN). METHODS: Urine from systemic lupus erythematosus (SLE) patients was interrogated for 1000 proteins using a novel, quantitative planar protein microarray. Hits were validated in an independent SLE cohort with inactive, active non-renal (ANR) and active renal (AR) patients, in a cohort with concurrent renal biopsies, and in a longitudinal cohort. Single-cell renal RNA sequencing data from LN kidneys were examined to deduce the cellular origin of each biomarker. RESULTS: Screening of 1000 proteins revealed 64 proteins to be significantly elevated in SLE urine, of which 17 were ELISA validated in independent cohorts. Urine Angptl4 (area under the curve (AUC)=0.96), L-selectin (AUC=0.86), TPP1 (AUC=0.84), transforming growth factor-ß1 (TGFß1) (AUC=0.78), thrombospondin-1 (AUC=0.73), FOLR2 (AUC=0.72), platelet-derived growth factor receptor-ß (AUC=0.67) and PRX2 (AUC=0.65) distinguished AR from ANR SLE, outperforming anti-dsDNA, C3 and C4, in terms of specificity, sensitivity and positive predictive value. In multivariate regression analysis, urine Angptl4, L-selectin, TPP1 and TGFß1 were highly associated with disease activity, even after correction for demographic variables. In SLE patients with serial follow-up, urine L-selectin (followed by urine Angptl4 and TGFß1) were best at tracking concurrent or pending disease flares. Importantly, several proteins elevated in LN urine were also expressed within the kidneys in LN, either within resident renal cells or infiltrating immune cells, based on single-cell RNA sequencing analysis. CONCLUSION: Unbiased planar array screening of 1000 proteins has led to the discovery of urine Angptl4, L-selectin and TGFß1 as potential biomarker candidates for tracking disease activity in LN.

Alteration of Angiopoietin-Like Protein 4 Levels in Serum or Urine Correlate with Different Biochemical Markers in Hyperlipidemia-Related Proteinuria.

Angiopoietin-like protein 4 (ANGPTL4) is widely known as a key regulator of lipid metabolism. We investigated the relationship between ANGPTL4 expression in serum or urine and blood lipid or urine protein levels of patients with hyperlipidemia- (HL-) related proteinuria. Sixty-eight patients with HL-related proteinuria (HL-Pro group), 68 patients with HL without proteinuria (HL-NPro group), 46 patients with non-HL-related proteinuria (NHL-Pro group), and 50 healthy control (Con) subjects were selected. There were no significant differences in serum ANGPTL4 levels between the Con group (36.82 ± 17.03 ng/ml) and the HL-Pro group (27.94 (18.90, 53.72) ng/ml). Additionally, the serum ANGPTL4 levels in the HL-Pro group were significantly lower than those in the HL-NPro group (53.32 ± 24.01 ng/ml) (P < 0.001). The urine ANGPTL4/Cr levels in the HL-Pro group (52.01 (45.25, 79.79) µg/g) were significantly higher than those in the HL-NPro group (9.96 (8.35, 12.43) ng/ml) (P < 0.05). A significant alteration in urine ANGPTL4/Cr levels was observed in the NHL-Pro group (69.41 ± 55.36 µg/g) and the Con group (10.08 ± 2.38 µg/g) as well. There was no correlation between serum and urine ANGPTL4 levels of the four groups (P > 0.05). Serum ANGPTL4 levels (HL-Pro/HL-NPro group) were positively correlated with total cholesterol (TC) and triglyceride (TG) levels in hyperlipidemia patients. However, there was no correlation between urinary ANGPTL4 levels and TC or TG (P > 0.05). Urine ANGPTL4 levels were positively correlated with 24hUPro in patients with renal impairment (HL-Pro/NHL-Pro group). To summarize, ANGPTL4 may be considered an accurate predictor of proteinuria with HL. Notably, serum or urine ANGPTL4 levels indicated the degree of proteinuria or hyperlipidemia, respectively, in HL patients.