Succinate- and GPR91-induced endothelial cytosolic Ca²⁺ [Ca²⁺]_i signaling and NO production were studied using the microperfused mouse afferent arteriole–attached glomerulus preparation and confocal fluorescence microscopy with fluo-4/fura red ratiometric Ca²⁺ (A) and DAF-FM imaging (B), respectively. Arrows point at glomerulus and afferent arteriole endothelial cells in situ. Scale bar: 20 μm. (C) Summary of the high glucose– (25.5 mM) and succinate- (5 mM) induced normalized changes in endothelial [Ca²⁺]_i and NO production in WT GPR91^+/+ or KO GPR91^–/– kidney tissue. *P < 0.05, WT (^+/+) vs. KO (^–/–); n = 6 each. (D) Dose-response relationship of succinate-induced elevations in PGE₂ production and release from cultured GENCs, measured using a PGE₂ biosensor. Specially engineered biosensor cells, HEK293 cells, expressing the Ca²⁺-coupled PGE₂ receptor EP1 were loaded with fluo-4 and positioned next to GENCs in culture. Effects of succinate on GENC PGE₂ production were measured based on the biosensor cell Ca²⁺ signal, since upon PGE₂-binding these biosensor cells produce a Ca²⁺ response detected by fluorescence imaging. The cyclooxygenase inhibitor indomethacin (50 μM) and EP1 receptor blocker SC-51322 (10 μM) in presence of 5-mM succinate both served as negative controls for PGE₂ specificity. Normalized changes in HEK293-EP1 biosensor cell fluo-4 intensity are shown and served as an index of PGE₂ release. n = 6 for each dose.