ER ribosomal-binding protein 1 regulates blood pressure and potassium homeostasis by modulating intracellular renin trafficking

Figures

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  Fig. 1. Phenotypes of Rrbp1-KO mice. A Mean value of systolic blood pressure and B diastolic blood pressure in mice. Data represent mean ± SEM, n = 12–49 per group. C Kaplan–Meier and log-rank survival analysis of Rrbp1-WT, Rrbp1-HE and Rrbp1-KO mice. D Representative electrocardiogram (ECG) of Rrbp1-KO and Rrbp1-WT mice. The ECG waveform of #1 and #2 Rrbp1-WT mice and the ECG waveform of terminal rhythms from #3 and #4 Rrbp1-KO mice. E Protocol for assessing the survival rate of mice under high K+ intake for 30 days. F Kaplan–Meier survival curve and log-rank survival analysis of Rrbp1-WT and Rrbp1-KO mice under high K+ intake for 30 days. G Protocol for telemetry ECG recording. H–P Quantitation of telemetry electrocardiogram (ECG) parameters in Rrbp1-KO and Rrbp1-WTmice with or without high K+ intake. RR interval; heart rate; PR interval; P duration; QRS duration; QT interval; correct QT interval; T amplitude; P amplitude (n = 6–7 per group). Representative telemetric ECG waveform of mice two days before and after high K.+ intake of Rrbp1-WT and Rrbp1-KO mice (Q). WT, wild-type; KO, knock-out. Data in (A) and (B) were analyzed with ordinary one-way ANOVA; data in (H)–(P) were analyzed…

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  Fig. 2. Rrbp1-KO mice show hyporeninemic hypoaldosteronism. A–F Plasma angiotensinogen, renin, Ang-I, Ang-II, aldosterone levels, and plasma renin activity (PRA) in mice. G Western blot analysis of SGK1 protein expression in kidneys harvested from Rrbp1-WT and Rrbp1-KO mice. H Quantification of the immunoblot in (G). I mRNA levels of Scnn1a, Scnn1b, Scnn1g, and Kcnj1 in mice kidney measured using quantitative RT-PCR. Data were analyzed using the 2-ΔΔCt method with GAPDH as the reference gene (n = 28–35 per group). J Protocol for blood and urine test for mice that underwent high K+ intake. K–M Plasma renin, Ang-II, and aldosterone levels in mice that underwent high K+ intake for 48 h. N Study protocol for recording survival rate of mice that underwent high K.+ intake for 30 days with 0, 2.5, 10 mg/kg fludrocortisone treatment (FC) and control mice, respectively. O Kaplan–Meier survival curve and log-rank analysis of Rrbp1-KO mice rescued with 0, 2.5, 10 mg/kg fludrocortisone treatment (FC) and control mice, respectively. WT, wild-type; KO, knock-out. Data in A–F, I, and K–M were analyzed with an unpaired, two-tailed Student’s t-test; data in H were analyzed using the Mann–Whitney test.…

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  Fig. 3. RRBP1 deficiency decreases renin transportation and secretion. A, B Representative renin immunohistochemical staining of kidneys of Rrbp1-WT and Rrbp1-KO mice. C Quantification of stain intensity in kidneys from Rrbp1-WT and Rrbp1-KO mice (scale bar = 20 μm). D Intracellular mRNA levels of RRBP1 of scramble-control and sh-RRBP1 knockdown Calu-6 cells were measured using quantitative RT-PCR. Data were analyzed using the 2-ΔΔCt method with GAPDH as the reference gene (n = 4 per group). E–H Representative immunogold staining of renin by transmission electron microscopy (TEM) in control and RRBP1 knockdown Calu-6 cells. Dark red arrow indicates the nanogold particle (scale bar = 0.5 μm). Nu, nucleus; IC, intracellular; PM, plasma membrane. I Quantification of intracellular renin particles by TEM in control and Rrbp1 knockdown Calu-6 cells. J Quantification of intracellular renin particles with a distance of more and less than 1000 nm from plasma membranes. K Protocol for collecting cell lysates and supernatant of control and RRBP1 knockdown Calu-6 cells. L Western blot analysis of RRBP1, renin protein expression in RRBP1-knockdown Calu-6 cells, and supernatant. M Protocol for…

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  Fig. 4. RRBP1 deficiency increases renin accumulation in ER. A Protocol to stimulate renin trafficking in control and RRBP1 knockdown cells. B–E Representative confocal microscopy images of control and RRBP1 knockdown Calu-6 cells showing renin (green), calnexin (red), and DAPI (blue) after 50 µM of forskolin induction for 0, 60, 120, and 180 min. F Overlap coefficients of renin (green) and calnexin (red) in control and RRBP1 knockdown Calu-6 cells after 50 µM of forskolin induction for 0, 60, 120, and 180 min (n = 8 per group). Data in (F) were analyzed with an unpaired, two-tailed Student’s t-test. Data are represented as mean ± SEM. ns, no significance; *P < 0.05

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  Fig. 5. RRBP1 deficiency reduces renin transport from ER to Golgi apparatus. A–D Representative confocal microscopy images of control and RRBP1 knockdown Calu-6 cells showing renin (green), GOLIM4 (red), and DAPI (blue) after 50 µM of forskolin induction for 0, 60, 120, and 180 min. E Overlap coefficients of renin (green) and GOLIM4 (red) in control and RRBP1 knockdown of Calu-6 cells after 50 µM of forskolin induction for 0, 60, 120, and 180 min (n = 8 per group). Data in (E) were analyzed with an unpaired, two-tailed Student’s t-test. Data are represented as mean ± SEM. *P < 0.05; ***P < 0.001

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  Fig. 6. Schematic of the mechanism through which RRBP1 modulates renin trafficking and secretion. RRBP1 deficiency causes hyporeninemic hypoaldosteronism, lower blood pressure, and hyperkalemia