Inhibition of Renin Release, a Crucial Event in Homeostasis, is Mediated by Coordinated Calcium Oscillations within Juxtaglomerular Cell Clusters

Figure 1.. Angiotensin II (AngII) elicits robust and periodic Ca2+ oscillations propagating across juxtaglomerular cell clusters (JGCCs) expressing GCaMP6f in ex vivo kidneys. A, Schematic of Ren1c-GCaMP6f mouse. B, Immunofluorescence staining for renin in the kidney cortex of the Ren1c-GCaMP6f mice. The expression of GCaMP6f is colocalized with renin in the JG area. See also Figure S2. C, Schematic of ex vivo Ca2+ imaging using acutely prepared kidney slices from Ren1c-GCaMP6f mice. D, Time-series of captured images perfused with 3 nM AngII in 2 mM Ca2+-containing buffer. White arrows point to GCaMP6f-expressing JG cells. Orange-dashed lines encircle the individual JG cells adjacent to each other. Individual JG cells can be distinguished by cytosolic GCaMP6f blinking periodically (see also Video S1). E, Time-series of fluorescent intensity of GCaMP6f-expressing JG cells shown by the kymograph (top), indicating changes in Ca2+ activity across designated reslice lines (black arrows) spanning adjacent JG cells (gray arrows). Gray-dotted lines indicate the border of adjacent JG cells. Images at 0.1-second intervals (bottom) designated by a black square in the kymograph visualize oscillatory…

Figure 2.. Angiotensin II (AngII) dose-dependently elicits stereotypical Ca2+ spike patterns and suppresses renin secretion within juxtaglomerular cell clusters (JGCCs). A, Representative raster plots for Ca2+ spikes in JGCCs under treatment with 50 pM, 300 pM, 3 nM, and 300 nM AngII. B–E, Analysis for Ca2+ spikes in JGCCs of Ren1c-GCaMP6f mice imaged over 25 minutes under treatment with 50 pM (n=4 mice, 5 slices, 15 ROIs), 300 pM (n=5 mice, 5 slices, 22 ROIs), 3 nM (n=5 mice, 5 slices, 18 ROIs), and 300 nM AngII (n=5 mice, 5 slices, 17 ROIs). Each slice contains one JGCC analyzed. B, Histogram of spike intervals across all ROIs exhibit unimodal distribution across AngII concentrations. The threshold distinguishes between event periods (active phase comprising bursts) and quiescent periods (inactive inter-burst phase). See also Figure S3. C, Mean event periods in JGCCs showed no notable variances across AngII concentrations (One-way ANOVA). D, The number of bursts/min is dose-dependently increased, especially between 300 pM and 3 nM AngII (linear mixed model with Tukey’s multiple comparison test). Identical color circles represent ROIs from the same JGCCs. E, Mean burst duration (left) and…

Figure 3.. Angiotensin II (AngII)-elicited Ca2+ oscillations and renin secretion in juxtaglomerular cell clusters (JGCCs) depend on extracellular Ca2+, primarily via ORAI channels. A, A representative time-course of Ca2+ spikes/min in each ROI (n=3) within Ren1c-GCaMP6f mouse kidney slice (left). The green-shaded areas represent SEM. The right panel summarizes the effect of transiently removing and washing back extracellular Ca2+ on JGCCs (adjusted to Baseline, n=4 mice, 5 slices, n=16 ROIs, linear mixed model with Tukey’s multiple comparison test). B, The result of kidney slice renin ELISA with 2 mM Ca2+ and 0.1 mM Ca2+ + 1 mM ethyl-glycol tetraacetic acid (EGTA) buffer under No-AngII and 3 nM AngII (n=4 per concentration, two-way ANOVA; Šídák’s multiple comparison test). C. Bubble plots for gene expression profiles of L- and T-type voltage-dependent Ca2+ channels (VDCC) α1-subunits, TRPC and TRPV subfamilies, and ORAI/STIM complex in the renin lineage cell-related clusters. D, Schematic for pharmacological ion channel inhibition screening. The timeline (top) and target channels with inhibitors (bottom) are illustrated. E, The relative number of Ca2+ spikes/min (%) in captured JG cells…

Figure 4.. Endoplasmic reticulum (ER) Ca2+ stores and releases generate angiotensin II (AngII)-induced Ca2+ oscillatory signals in juxtaglomerular cell clusters (JGCCs). A, Bubble plots for gene expression profiles of ER channels indicate high gene expression of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA, Atp2a) and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R, Ip3r) genes in the JG clusters while showing low gene expression of ryanodine receptors (RyR, Ryr). B, Schematic for pharmacological ER channel inhibition and the inhibitors. SOCC; store-operated Ca2+ channels. C, The relative number of Ca2+ spikes/min (%) in captured JGCCs adjusted to Baseline (20 μM Cyclopiazonic acid (CPA), n=4 mice, 5 slices, 16 ROIs; 100 μM 2-Aminoethoxydiphenyl borate (2-APB), n=5 mice, 5 slices, 21 ROIs, 10 μM Dantrolene, n=5 mice, 5 slices, 20 ROIs, linear mixed model). Identical color circles represent ROIs from the same JGCCs. D–E, Representative time-courses of the number of Ca2+ spikes (left) and cytosolic fluorescence intensities (right) before and after perfusion of CPA (D, n=2 ROIs) or 2-APB (E, n=4 ROIs) under 3 nM AngII treatment. The data for treatment with the same volume of DMSO…

Figure 5.. Gap junction (GJ) inhibition suppresses intracellular and intercellular Ca2+ activity within the juxtaglomerular cell clusters (JGCCs). A, Bubble plots for gene expression profiles of GJ indicate diverse and highly expressed gene expression profiles of GJ proteins, including Connexin43 (Cx43, Gja1), Cx37 (Gja4), Cx40 (Gja5), and Cx45 (Gjc1) in JG cells and their adjacent cells. B, Representative kymographs show the time-series of fluorescent intensity under 3 nM angiotensin II (AngII) and 3 nM AngII + 100 μM carbenoxolone (CBX) perfusion using Ren1c-GCaMP6f mouse kidney slice. Black arrows designate reslice lines. Black-dashed lines encircle the individual JG cells adjacent to each other. Gray arrows indicate the spanning Ca2+ activity across the border of adjacent JG cells (gray-dashed lines). See also Video S2. Scale bar, 20 μm. C, The relative number of Ca2+ spikes/min (%) in captured JGCCs under 3 nM AngII treatment with 100 μM CBX (n=4 mice, 5 slices, n=16 ROIs, linear mixed model). Identical color circles represent ROIs from the same JGCCs. D, The kidney slice renin ELISA results with or without 100 μM CBX under 3 nM and no AngII (n=4 per concentration, 2-month-old male…

Figure 6.. In vivo juxtaglomerular cell clusters (JGCCs) generate coordinated intracellular and intercellular Ca2+ oscillations within the native clusters. A, Schematic of in vivo Ca2+ imaging of JGCCs in Ren1c-GCaMP6f mice. B, Captured fluorescence volume series of GCaMP6f exhibit robust oscillatory signals propagate across the JGCCs within the cluster. (29.989 Hz). A yellow-dashed square line indicates a JGCC, shown on the right in representative images at 10-image intervals. See also Video S3. C, Schematic strategy for in vivo Ca2+ spike analysis. Multi-page TIFF images captured in 3 minutes were used to trace the fluorescence intensity for the spatiotemporal JGCCs, following the ex vivo Ca2+ imaging strategy shown in Figure 1F. D, The ridgeline plots (left) for Ca2+ spikes patterns across all ROIs in five JGCCs captured by in vivo Ca2+ imaging under basal state (green, n=4 mice, 5 JGCCs, 17 ROIs) and ex vivo Ca2+ imaging treated with each AngII concentration (gray; 50 pM, blue; 300 pM, orange; 3 nM, red; 300 nM. Mean event periods (right) showed no significant difference between in vivo Ca2+ imaging and ex vivo in all AngII treated groups (Mann-Whitney U test). E, Mean burst duration…

Figure 7.. Angiotensin II (AngII) enhances Ca2+ oscillatory activity within the native juxtaglomerular cell clusters (JGCCs) and inhibits acute renin secretion in vivo. A–B, Representative images of GCaMP6f signals from JGCCs in Ren1c-GCaMP6f mice before (Baseline) and 30 seconds after bolus AngII (100 ng/g) intraperitoneal administration (A) and the time-series of GCaMP6f intensities (B) in the cropped JGCC (dashed-yellow lines). White arrows represent JGCCs. Scale bar, 20 μm. See also Video 4S. C, A mean GCaMP6f intensity was significantly increased 30 seconds after AngII administration (n=5 mice, 8 JGCCs, linear mixed model). Mean GCaMP6f F/F0 of 300 multipage TIFFs before and 30 seconds after bolus AngII injection were compared. Identical color circles represent distinct JGCCs captured within the same mouse. D, Schematic of acute in vivo renin secretion assay. E–F, plasma renin levels before and after surgical procedures under continuous administration with 0 (saline vehicle), 10, and 100 ng/g/min AngII (E, n=7, 6, and 7 Ren1c-GCaMP6f mice, respectively, paired t-test) and the ratio adjusted to vehicle (F, One-way ANOVA with Tukey’s multi-comparison test). Figure S5 describes carotid…

Figure 8.. Overview of Angiotensin II (AngII)-elicited Ca2+ dynamics within juxtaglomerular cell clusters (JGCCs). AngII induces robust and sustained Ca2+-oscillations within their native JGCC structure and suppresses renin secretion. Coordinated Ca2+ signaling is mediated by intracellular Ca2+ release at the endoplasmic reticulum (ER) supplied through ORAI channels and intercellular gap junctions that propagate signaling molecules. AA; afferent arteriole, IP3R; 1,4,5-trisphosphate (IP3) receptor, SERCA; sarcoplasmic/endoplasmic reticulum Ca2+-ATPase.