Background/Purpose: Lupus nephritis (LN) is a chronic, immune complex-mediated glomerulonephritis affecting up to 60% of patients with lupus. Progression of the most aggressive forms of LN (proliferative classes III and IV) to end-stage renal failure is ~20% and another ~40% develop chronic kidney disease despite aggressive treatment. Patients that fail to respond continue to accumulate organ damage. Thus, there is a critical need for noninvasive biomarkers to predict therapeutic response. We previously demonstrated that the glycosphingolipid (GSL) lactosylceramide (LacCer) is elevated in the urine of lupus patients, and preliminary data suggests that GSLs in extracellular vesicles (EVs) in urine are higher prior to treatment in patients that do not respond to treatment. Thus, we compared levels of GSLs in urine EVs in longitudinal samples from LN patients that completely responded (CR) or did not respond (NR) to MMF treatment after one year of treatment.

Methods: EVs were isolated from longitudinal (baseline, and 3 or 12 months post-treatment) urine samples obtained from 59 LN patients (29 CR, 30 NR). All patients met ACR classification for SLE with biopsy-confirmed nephritis (93% with class III or IV LN). Criteria for CR included UPr:UCr < 0.5, normal sCr, inactive urine sediment, UPr:UCr reduced >75%, and eGFR increased >25%. NR criteria included persistent UPr:UCr >3, decreased UPr:UCr < 25%, eGFR decreased >25%, and failure to taper glucocorticoid therapy to < 10 mg/day. GSLs were quantified in the urine EV samples by SFC/MS/MS. Galectin-3 binding protein (G3BP) and gelsolin, identified in a small proteomics screen, were quantified in the urine EV samples or total urine using independent ELISAs. A panel of cytokines and chemokines were measured in total urine samples by a multiplex laser bead array.

Results: All chain lengths of Hexosylceramides (HexCers) were significantly higher (p< 0.001) in the baseline EV samples from NR patients (total HexCers, 48.6 +56.9 pmol/ml/mg UCr) compared to CR patients (total HexCers, 11.7 +13.4 pmol/ml/mg UCr). All chain lengths of LacCers were significantly higher (p< 0.001) in the baseline EV samples from NR patients (total LacCers, 13.5 +10.9 pmol/ml/mg UCr) compared to CR patients (total LacCers, 3.5 +4.1 pmol/ml/mg UCr). Gelsolin levels in total urine, but not in urine EVs, were significantly different (p=0.003) between NR (574.8 +473.3 ng/mg UCr) and CR (178.1 +177.9 ng/mg UCr) at baseline. Total urine G3BP levels were significantly higher (p=0.039) in NR (282.4 +421.4 ng/mg UCr) compared to CR (115.2 +146.4 ng/mg UCr). Of the 23 cytokines/chemokines measured in total urine, 18 were detected and all 18 were significantly or showed a trends of higher levels in NR compared to CR.

Conclusion: These results suggest that in general, patients that failed to respond to therapy likely had worse disease prior to starting therapy. Urine EV levels of HexCers and LacCers, along with urinary levels of a panel of proinflammatory factors and clinical measures may be useful in identifying LN patients prior to beginning treatment that are unlikely to respond to conventional therapies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/glycosphingolipids-potential-urine-biomarkers-of-therapeutic-response-in-lupus-nephritis/