Endothelin and RAS regulation of Ca 2 + - and NO-dependent signaling in human renal mesangial cells

BACKGROUND: Endothelin-1 (ET-1) is a potent regulatory peptide that acts on glomerular mesangial cells and has been implicated in altered glomerular hemodynamics and progression of chronic kidney disease (CKD). Components of the renin-angiotensin system (RAS), including Ang II and Ang III, are also present in the glomerulus and signal through receptors relevant to mesangial cells. Sparsentan is a first-in-class, dual endothelin A (ETA) and angiotensin II type 1 (AT1) receptor antagonist, provides a pharmacologic tool to examine potential interaction between these systems. This study investigates how ET-1 and RAS-related stimuli activate Ca 2 + and nitric oxide (NO) pathways in human renal mesangial cells and evaluates receptor-specific contributions using selective antagonists and sparsentan. METHODS: Primary human renal mesangial cells (HRMCs) were obtained from adult female non-transplantable human kidneys. To verify that responses were consistent across different HRMC sources, additional experiments were performed using commercially available adult male HRMCs (ScienCell, lot 20057). Live-cell confocal imaging was used to measure intracellular calcium (Fluo-8 AM) and NO (DAF-FM) following stimulation with ET-1, Ang II, or Ang III. Receptor contributions were evaluated using selective ETA (BQ123, 500 nM) and ETB (BQ788, 500 nM), or dual ETA/AT1 receptor antagonist, sparsentan (10 µM). RESULTS: ET-1 induced a concentration-dependent increase in intracellular Ca2+ in HRMCs, with high agonist sensitivity (Kd = 0.10±0.05 nM, R 2 = 0.9), determined using a Michaelis-Menten fit of maximum response amplitudes. ET-1, Ang II, and Ang III also evoked detectable NO production in HRMCs, indicating parallel engagement of NO-linked signaling pathways. Pre-treatment with receptor-selective antagonists demonstrated that both ETA and ETB contribute to the ET-1-evoked Ca2+ response. Inhibition of ETA markedly reduced the intracellular Ca2+ transient amplitude to ~75% reduction (1650±180 vs. 6668±488 a.u., BQ123 vs. vehicle, n=45-46 cells, p< 0.01). ETB blockade produced a smaller effect (~47% inhibition, 3508±158 vs. 6668±488 a.u., BQ788 vs. vehicle, n=40 cells, p< 0.01). The magnitude of ETA-dependent inhibition exceeded the ETB component by approximately 1.6-fold, indicating that ETA exerts the stronger functional contribution under these experimental conditions. These physiological differences were consistent with the gene expression profile from the human kidney scRNA-seq database (Susztak/KPMP). Mesangial cells exhibited one of the highest expression levels of EDNRA and EDNRB among nephron cell types, with slightly higher EDNRA than EDNRB expression (ETA/ETB ratio ≈1.3), aligning with the larger inhibitory effect observed during ETA blockade. Preincubation with sparsentan significantly reduced intracellular Ca2+ responses for both ET-1 and Ang III acute applications (~55 and ~70 % inhibition, respectively, p< 0.01). CONCLUSION: Our data demonstrates that both ETA and ETB receptors participate in ET-1-evoked calcium signaling in HRMCs, with ETA contributing to the larger functional component. Together, these findings provide mechanistic insight into endothelin-RAS interactions in mesangial cells and support the rationale for dual-pathway targeting as a potential strategy to modulate glomerular function in kidney disease. This abstract was presented at the American Physiology Summit 2026 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.