Cytokine-driven glycosphingolipid metabolism modulates endoplasmic reticulum calcium homeostasis in primary human renal mesangial cells

Glycosphingolipids (GSLs), including hexosylceramides (HexCers), lactosylceramides (LacCers), and gangliosides composed of one or more sugar residues attached to ceramide, are essential components of cell membranes. Dysregulated GSL metabolism has been implicated in various inflammatory and autoimmune diseases, including lupus nephritis; however, its contribution to renal cell dysfunction remains largely unexplored. In this study, we demonstrate that in primary human renal mesangial cells (hRMCs), proinflammatory cytokines relevant to lupus elicit significant upregulation and secretion of inflammatory mediators that parallel intracellular and extracellular accumulation of HexCers and elevated cytosolic calcium (Ca2+) levels. The increase in cytosolic Ca2+ was attributed to a decrease in endoplasmic reticulum (ER) Ca2+ store capacity. Pharmacological inhibition of GSL synthesis with eliglustat significantly reduced HexCers levels and restored ER Ca2+ stores, but did not impact cytokine-induced cytokine/chemokine secretion or cell viability/proliferation. Together, these data suggest that elevated GSL synthesis modulates cytokine-induced ER Ca²? dysregulation in mesangial cells and may play a role in the pathogenesis of lupus nephritis. Overall design: hRMCs (lots 12445, 17544, and 20018) were treated in quadruplicate wells in 6-well plates with vehicle, 25 ng/ml IL-1b, or 25 ng/ml IL-1b +20 nM eliglustat for 24, 48, or 72 hrs. Media was collected for ELISAs and GSL quantification, cell viability was measured by the alamar blue assay, and cells scraped into DPBS and replicate wells combined. The cell suspension was split into two tubes, centrifuged, and DPBS removed. Half the cells were used for GSL quantification, and the other half were used for RNA isolation.