(A) Representative RT-PCR demonstrating the presence/absence of GPR91 in various mouse kidney cell types. C, control mix with no cDNA; WK, whole kidney from WT GPR91^+/+ or KO GPR91^–/– mice. Same results were obtained in n = 6 different samples each. JGC, renin-producing JG cells. (B–E) Localization of GPR91 protein in rat kidney with immunohistochemistry. (B) Strong GPR91 labeling (red) was found in vascular endothelial cells, in both terminal afferent arteriole (Aff. art.) and glomerulus. One region of the glomerulus (indicated by a rectangle) is magnified in C and D for high-resolution colocalization studies. (C) Double labeling for rat endothelial cell marker RECA-1 (green) identified the endothelium of intraglomerular capillary loops (cl). (D) Overlay of GPR91 (red) and RECA-1 (green) images shows colocalization (yellow) of the 2 proteins in the same structures. DIC background is merged with fluorescence to show glomerular morphology. (E) Negative control using no GPR91 primary antibody. Nuclei are blue. Scale bar: 10 μm (B–E). (F) Elevations in GENC [Ca²⁺]_i levels from baseline (under normal glucose [NG], 5.5 mM) in response to high glucose level (25.5 mM) and TCA cycle inhibitors and intermediates. Succinate, 5 mM; malonate, 1 mM; fluorocitrate, 100 μM. *P < 0.001, compared with NG; n = 9 each. (G) Dose-response relationship of high glucose level– and succinate-induced elevations in GENC Ca²⁺ levels; n = 6 each. F/F₀, fluorescence (F) normalized to baseline (F₀). (H) Cell- and GPR91-specificity of the succinate-induced [Ca²⁺]_i response. HEK cells were used for biosensor experiments (see below). *P < 0.001, compared with baseline; n = 6 each. Cells were grown on coverslips to near confluency, loaded with Ca²⁺ sensitive fluorescent dye fura-2, and [Ca²⁺]_i was measured using a cuvette-based spectrofluorometer (Quantamaster-8; Photon Technology Inc.).