Fig. 2: Inflammation response of high-GPR68-expressing monocytes in 5/6Nx mice.

a Double immunofluorescence labeling of GPR68 with MYL2, αSMA, and F4/80 in the cardiac ventricle of 5/6Nx mice. The scale bar indicates 20 μm. b The expression of Gpr68 mRNA in cardiac macrophages. After separation of F4/80⁺/CD11b⁺ cells into Ly6C⁺ and Ly6C⁻ populations, the mRNA levels of Gpr68 in each population were measured by RT-PCR. c Increase in the number of cardiac-infiltrated CD11b⁺/Ly6C⁺ cells in 5/6Nx mice. d Functional analysis of the genes whose expression was changed in the cardiac ventricle of 5/6Nx mice using the KEGG database. e The mRNA levels of Sele and Vcam1 in the cardiac ventricle of 5/6Nx mice. The mean value of the Sham group was set as 1.0. f Immunofluorescence labeling of VCAM1 (red) in the cardiac ventricle of Sham and 5/6Nx mice. The scale bar indicates 100 μm. g The number of TNFα- and IL-6-positive high-GPR68-expressing CD11b⁺/Ly6C⁺cells in the cardiac ventricle of Sham and 5/6Nx mice. h The ratio of high-GPR68-expressing monocytes in the blood of Sham and 5/6Nx mice at 4 or 8 weeks after nephrectomy. i The expression of Gpr68 mRNA in primary cultured monocytes after incubation in the media containing 10% serum collected from Sham and 5/6Nx mice. j Release of TNFα and IL-6 from LPS-stimulated monocytes. Primary cultured monocytes were incubated in the media containing 10% serum collected from Sham and 5/6Nx mice for 24 h. Thereafter, cells were incubated in the presence or absence of 5 µg/mL of LPS and 0.2 mM CuCl₂ under pH 7.8 or 6.3 conditions for 8 h. For i and j, monocytes were isolated from naive ICR mice and serum was collected from individual mice. For all panels, graphs show the mean ± SD of individual mice in independent experiments. Statistical significance was determined using two-way ANOVA with Tukey–Kramer post hoc tests (b, h, j) or two-tailed Student’s t-tests (c, e, g, i). Numbers and P-values are shown in each graph. Source data are provided as a Source Data file.