Cytokine-Driven Glycosphingolipid Metabolism and Calcium Dysregulation in Renal Mesangial Cells

Background: Lupus nephritis is characterized by the deposition of immune complexes in glomeruli, leading to inflammation, tissue damage, and progressive loss of kidney function. Glycosphingolipids (GSLs), including hexosylceramides (HexCers) and other glycolipids composed of one or more sugar residues attached to ceramide, such as lactosylceramides (LacCers) and gangliosides, are essential components of cell membranes. These molecules play pivotal roles in cell signaling, vesicle trafficking, and cellular stress responses. Dysregulated GSL metabolism has been implicated in various inflammatory and autoimmune diseases, including kidney disorders; however, its contribution to renal mesangial cell dysfunction remains largely unexplored. This study examines how cytokine-driven alterations in GSL metabolism influence calcium homeostasis, endoplasmic reticulum (ER) stress, and inflammatory signaling in mesangial cells, providing valuable insights into potential therapeutic strategies for inflammatory kidney diseases. Methods: Primary human renal mesangial cells (hRMCs) were treated with cytokines (25 ng/mL IL-1β, 100 ng/mL TNF-α, 100 ng/mL IFN-α, and 100 ng/mL IFN-γ) for 24 hours, with or without eliglustat, a glucosylceramide synthase inhibitor. Cytosolic calcium levels and ER calcium store capacity (which influences cellular processes, including the early stages of glycosphingolipid synthesis) were assessed using confocal imaging of hRMCs loaded with the Fluo8-AM calcium indicator, with ER calcium release measured following thapsigargin (TG) stimulation. GSL levels were quantified via mass spectrometry. IL-6 secretion, cell proliferation, and gene expression were evaluated using ELISA, alamarBlue, and RT-qPCR, respectively. Additionally, RNA sequencing was conducted to investigate transcriptional pathways activated by cytokine treatment at 24, 48, and 72 hours. Results: Cytokine treatment caused a significant increase in basal cytosolic calcium concentrations, with a ~20% elevation compared to controls (N=4, n≥57 cells per group, p<0.001). TG application revealed a cytokine-induced reduction in ER calcium store capacity in hRMCs, which was markedly restored by eliglustat treatment (N=4, n≥44 cells per group, p<0.001 for female-derived cells; N=2, n≥22 cells per group, p<0.001 for male-derived cells). Exposure to cytokines (25 ng/mL IL-1β for 24, 48, and 72 hours) significantly increased IL-6 secretion, upregulated stress-response genes—including ER stress-associated CXCL8 and CCL2—and led to the accumulation of HexCers in both male and female cells. Notably, eliglustat pretreatment (20 ng/mL) effectively attenuated HexCer accumulation. Conclusions: Our results indicate that dysregulated GSL metabolism plays a critical role in driving inflammatory responses, ER stress, and calcium dysregulation in renal cells. These processes are further exacerbated by cytokines, which act as central mediators of inflammation and contribute to the pathological changes observed in lupus nephritis. Moreover, variations in calcium signaling and ER stress responses were identified as key factors influencing disease progression, underscoring the importance of further investigating these mechanisms to advance our understanding and therapeutic strategies for lupus nephritis. Supported by the Office of the Assistant Secretary of Defense for Health Affairs W81XWH-22-1-0330 (to TKN); MUSC College of Medicine-Program Project Grant (to OP). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.