Fig 2: Cropped Western Blot image showing the presence of nNOS in the human IG. a Rat CC as control; b and c human IG tissue lysates from two cases (10, 11) on 8% SDS-polyacrilamide gel. The detected band shows the molecular weight of 160 kDa expected for nNOS.The blot retains at least six band width below the nNOS band. Lanes that were not contiguous in the experiment have a clear separation (vertical white space)

Fig 3: Camera lucida drawings of nNOS-immunopositive neurons in the human IG. a Three nNOS positive neurons (ovoidal, 1 and 3; fusiform, 2), one of them showing a long dendrite (Den). b Outline figure of CC and overlying IG. The distribution of nNOS-positive neurons in IG is shown (I); two regions (II, III; arrows) are enlarged below in II and III, showing the distribution of nNOS-positive neurons close to pial arterioles (PA). Calibration bars: a 10 µm; b I, 1 cm; b II, III, 100 µm

Fig 4: Impaired renin secretion upon adrenergic stimulation.a Slices of kidneys dissected from 2- to 3-month-old mice of the indicated genotypes were incubated with the indicated chemicals for 30 min and cryosections of these kidney slices were subjected to immunofluorescence staining with an anti-renin antibody (green), an anti-nNOS antibody (red), and DAPI (blue). Arrowheads indicate renin-positive cells. Bar, 20 µm. A representative result from three independent experiments with similar results are shown. b The percentages of glomeruli with adjacent renin-positive cells are shown as means ± SEM (in both genotype, n = 6 (mock), 6 (Isoproterenol), 4 (PGE2), and 2 (db-cAMP)). The p-values were determined by two-way ANOVA with Holm-Sidak post hoc tests. ***p < 0.001. c 2- to 3-month-old mice of the indicated genotypes were intraperitoneally administered with 10 mg/kg isoproterenol, and plasma collected the day before and 30 min after drug administration were subjected to renin activity measurement (n = 4 for Trpm6+/+; Six2-Cre, and n = 8 for Trpm6fl/fl; Six2-Cre). Data are shown as means ± SEM. The p-values were determined by two-way ANOVA with Holm-Sidak post hoc tests. *p < 0.05.

Fig 5: Distribution of nNOS-immunopositive (nNOSIP) neurons in the rat corpus callosum from lateral to medial. Stereotaxic coordinates according to the atlas of Paxinos and Watson (1982) on bottom left side. Calibration bar: 1 mm