Background/Purpose: Non-invasive biomarkers of lupus nephritis (LN) damage are needed to guide treatment decisions and determine risk for kidney failure. Urinary proteomics has advanced as a tool for novel biomarker discovery in recent years. Specifically, Isobaric Tags for Relative and Absolute Quantification (iTRAQ) is an advanced proteomics technique that quantifies and compares protein expression among samples by mass spectrometry in a single experiment. We aimed to use iTRAQ for discovery of candidate urine biomarkers (CUBMs) for LN chronicity in a pediatric lupus cohort and then further pursued validation of CUBMs.

Methods: For the initial discovery cohort, urine was collected from children with LN (n=21) at the time of kidney biopsy. LN damage was characterized as per the NIH chronicity index (NIH CI, score range: 0-12) and categorized as none (0), low (1), moderate (2), or high (≥3). iTRAQ experiments compared protein composition in four urine samples from different LN damage categories, respectively. The relative expression of differentially excreted proteins was tested for significant differences using a log-rank test. We then generated heat maps and performed network analysis to identify proteins associated with LN damage, also considering concurrent histological LN activity (NIH activity index scores). Upon identification of CUBMs from the discovery cohort, specific commercial ELISAs were performed on urine samples from children with varying levels of LN chronicity in a separate validation cohort (n=41). Analysis of Variance was performed to detect statistical differences between each of the CUBMs and LN chronicity, with adjustment for clinical LN activity.

Results: Overall, iTRAQ detected 112 proteins from the urine sample sets of the discovery cohort, and 51 proteins were quantifiable in all replicate sample runs. Initial log-rank test revealed 4 differentially expressed proteins with p-values <0.05. Further evaluation by heat map and network analysis led to identification of 7 CUBMs for LN chronicity: Afamin (AFM), Immunoglobulin Heavy Constant Alpha 1 (IGHA1), Alpha-1-Antichymotrypsin (SerpinA3), Transthyretin (TTR), Retinol Binding Protein 4 (RBP4), Alpha-1-Acid Glycoprotein, Type 2 (ORM2) and Transferrin (TF). In the validation cohort, only SerpinA3 was found to be different based on degree of LN chronicity after adjustment for concurrent LN activity. SerpinA3 levels were found to increase with higher degrees of LN chronicity.

Conclusion: Using advanced proteomic techniques followed by confirmation using specific ELISAs, we identified SerpinA3 as a potential urine biomarker to help quantify the degree of tissue damage from LN. Elevated levels of SerpinA3, a known inhibitor of neutrophil cathepsin G and angiotensin II production, could serve as both a danger signal and protective mechanism from further kidney damage. Further validation of SerpinA3 as an LN damage biomarker in an independent cohort is needed to determine its ability to guide treatment and predict prognosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/applying-urine-proteomics-for-discovery-of-lupus-nephritis-damage-biomarkers-in-a-pediatric-cohort/