Background/Purpose: SLE primarily afflicts women and many SLE patients develop nephritis, a serious complication of lupus. Identification of biomarkers and the pathogenic mechanisms underlying LN is crucial to better understand sex bias and disease progression. Therefore, we interrogated glycosphingolipid (GSL) metabolism and the N-glycome with respect to disease and biologic sex in LN patient samples. We also evaluated response of human primary renal mesangial cells (hRMCs) with respect to disease and biologic sex.

Methods: Urine and serum were collected from 20 healthy control (HC) subjects and 20 LN patients who met the ACR criteria for active disease. Ten males and 10 females were included in each group. N-linked glycans attached to proteins (N-glycans) were measured by matrix-assisted laser desorption/ionization quadrupole time of flight (MALDI-QTOF) and the GSL lactosylceramide (LacCer) was quantified by Supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS). Responses of male- and female-derived hRMCs to treatment with sera from each LN or HC subject were assessed by measuring intracellular calcium (Ca²⁺) using fluor8 indicator, cytokine secretion by ELISA, GSLs levels by SFC-MS/MS, and N-glycans by MALDI-QTOF. Associations between N-glycans with LN status and biologic sex were evaluated using linear mixed models.P-values were adjusted using False Discover Rate with < 0.05 considered meaningful.

Results: All major LacCer species and total LacCers were significantly elevated and 72 urine N-glycans were significantly altered in LN patients compared to HCs. In particular, there was a significant increase in the N-glycans associated with pro-inflammatory IgGs and a decrease in N-glycans associated with anti-inflammatory IgGs in the urine of LN patients. The increase in urine LacCers (LN vs HC) was 2-3 fold higher in males, and three urine N-glycans differed significantly between the sexes. Three individual N-glycans provided perfect separation of LN and HC (AUC of 1.0) when added to a model that included only total LacCers and biologic sex. In the serum, 2 of the major LacCer species and 21 N-glycans were significantly altered in LN compared to HC. In vitro, no differences were observed in the responses of hRMCs to female- vs male-derived sera. However, the female-derived hRMCs exhibited significantly higher Ca²⁺ flux and cytokine secretion compared to male-derived hRMCs in response to LN sera. The female-derived hRMCs expressed higher levels of GSLs than the male-derived hRMCs.

Conclusion: Urine LacCers levels and changes in the N-glycome may serve as robust biomarkers of LN and likely reflect renal disease activity. The larger increase in LacCers in LN males compared to LN females may underscore worse renal disease in males once tolerance is broken. The significantly higher levels of LacCers observed in the female-derived hRMCs may partly explain the heightened pathologic response. Thus, elevated GSL metabolism in females may poise renal cells to be more sensitive to or respond more robustly to inflammatory stimuli.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/association-of-biologic-sex-with-glycosphingolipids-and-the-n-glycome-in-lupus-nephritis-and-renal-mesangial-cell-function/