Glycosphingolipid Levels in Urine Extracellular Vesicles Enhance Prediction of Therapeutic Response in Lupus Nephritis

Brian Troyer, Jessalyn Rodgers, Bethany J Wolf, James C Oates, Richard R Drake, Tamara K Nowling, Brian Troyer, Jessalyn Rodgers, Bethany J Wolf, James C Oates, Richard R Drake, Tamara K Nowling

Abstract

The development of nephritis increases the risk of morbidity and mortality in systemic lupus erythematosus (SLE) patients. While standard induction therapies, such as mycophenolate mofetil (MMF) induce clinical remission (i.e., complete response) in approximately 50% of SLE patients with nephritis, many patients fail to respond. Therapeutic response is often not assessed until 6-12 months after beginning treatment. Those patients that fail to respond to treatment continue to accumulate organ damage, thus, there is a critical need to predict which patients will fail therapy before beginning treatment, allowing physicians to optimize therapy. Our previous studies demonstrated elevated urine, but not serum, glycosphingolipids (GSLs) in SLE patients with nephritis compared to SLE patients without nephritis, suggesting the urine GSLs were derived from the kidney. In this study, we measured the GSLs hexosylceramide and lactosylceramide in extracellular vesicles isolated from longitudinal urine samples of LN patients that were treated with MMF for 12 months. GSL levels were significantly elevated in the baseline samples (prior to treatment) of non-responders compared to complete responders. While a few other proteins measured in the whole urine were higher in non-responders at baseline, only GSLs demonstrated a significant ability to discriminate treatment response in lupus nephritis patients.

Keywords: biomarker; extracellular vesicle; glycosphingolipid; lupus nephritis.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in study design; in collection, analyses, or interpretation of data; in writing the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Total HexCers and LacCers are significantly higher in baseline urine EVs of patients that did not respond to therapy. EVs were isolated from urine samples of LN patients that met the clinical criteria of a non-responder (NR) or a complete responder (CR) after 12 months of treatment with MMF. Total of all chain lengths of HexCers (A) and LacCers (B) were quantified in EVs from urine collected prior to treatment (baseline) and at 3 months (3 Mos) and 12 months (12 Mos) post-treatment. HexCers and LacCers were normalized to urine creatinine (UCr) levels measured in the urine samples from which the EVs were isolated. p-values were calculated as described in the Methods and adjusted for multiple comparisons using Bonferonni correction. Adjusted p-values are provided on the graph.
Figure 2
Figure 2
Gelsolin is significantly higher in baseline urine of patients that did not respond to therapy. Gelsolin was quantified in urine EVs (A) and whole urine (B) for all available LN patients (see Figure S1). (C) Galectin-3 binding protein was measured in whole urine of the same patients. All measures were normalized to UCr. p-values were calculated as described in the Methods and Bonferonni adjusted p-values are provided.

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