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

RRBP1 genetic variants are associated with blood pressure in the SAPPHIRe study

To identify genetic loci that influenced blood pressure, we performed a genome-wide linkage scan of 1144 participants from 360 nuclear families in the SAPPHIRe cohort and quantitatively mapped a trait locus located on chromosome 20 between 14.7–18.3 Mb. This region was further refined by performing a sliding window analysis to identify haplotypes associated with blood pressure. A 7-SNP haplotype numbered H2:2,211,121 (haplotype frequency: 16.1%) of the RRBP1 gene containing the SNP rs6080761 was associated with lower systolic blood pressure (Z =  − 3.90, haplotype-specific P = 9.6 × 10−5, global P = 5.29 × 10−3), lower diastolic blood pressure (Z =  − 3.796, haplotype-specific P = 1.47 × 10−5, global P = 0.02), and lower mean blood pressure (Z =  − 4.135, haplotype-specific P = 3.6 × 10−5, global P = 5.12 × 10−3) (Table 1). In the International Consortium for Blood Pressure study (ICBP pha003588) that included 132,671 participants and found that rs6080761 was associated with diastolic blood pressure (P = 0.01) and mean blood pressure (P = 0.02). Similarly, in the National Heart, Lung, and Blood Institute (NHLBI, pha003048) Family Heart Study, rs6080761 was associated with diastolic blood pressure (P = 0.01) and mean blood pressure (P = 0.03) among 2,756 participants. This SNP was also associated with systolic blood pressure (P = 0.03) among 1,538 Caucasians in the Genetic Epidemiology Network of Arteriopathy study (GENOA pha00309.1).

Table 1 FBAT association analysis of RRBP1 7-SNP (rs7272683, rs2236255, rs6034875, rs6080761, rs6080765, rs8120179, rs3790308) haplotypes with blood pressure Rrbp1 knockout mice exhibit lower blood pressure and are prone to sudden death

We created Rrbp1-KO mice by knocking out the regions from exons 4 to exons 24 of the Rrbp1 gene to determine how RRBP1 regulates blood pressure (Additional file 1: Fig. S1A and B). Immunoblots revealed that RRBP1 is primarily expressed in the intestine, liver, kidney, and pancreas of mice (Additional file 1: Fig. S1C). The Rrbp1-WT and Rrbp1-KO mice showed no significant differences in morphology, anatomy, body composition, body weight, or abdominal organ weight (Additional file 1: Fig. S2).

Rrbp1-WT, Rrbp1-heterozygous knockout (HE), and Rrbp1-KO mice had systolic pressure of 112.00 ± 2.94, 103.80 ± 2.12, and 92.60 ± 2.42 mmHg, respectively (P-for-trend < 1 × 10−3) (Fig. 1A). The three groups' diastolic blood pressures were 61.53 ± 2.16, 57.53 ± 1.36, and 48.94 ± 2.31 mmHg, respectively (P-for-trend < 0.01) (Fig. 1B). Furthermore, Rrbp1-KO mice were prone to sudden death. Figure 1C shows the Kaplan–Meier survival curves of Rrbp1-WT, Rrbp1-HE, and Rrbp1-KO mice. Rrbp1-KO mice died at a higher rate than Rrbp1-WT mice (hazard ratio [HR]: 2.45, 95% confidence interval [CI]: 1.55–3.88, P < 1 × 10−4) (Fig. 1C). The median survival time was 481 days for Rrbp1-KO mice, 772 days for Rrbp1-HE mice, and 793 days for Rrbp1-WT mice. The cardiac rhythms of moribund Rrbp1-KO and Rrbp1-WT mice were monitored using surface ECG to further investigate the causes of sudden death in Rrbp1-KO mice. The terminal ECG of Rrbp1-KO mice (Fig. 1D) was consistent with severe hyperkalemia, as shown by flattened P waves, widened QRS complexes, and a tall T wave, followed by asystole [27]. The ECGs of Rrbp1-WT mice of the same age are shown as references.

Fig. 1figure 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 with Mann–Whitney test. Data are represented as mean ± SEM. ns, no significance; *P < 0.05

High K+ intake drastically increases sudden death in Rrbp1-KO mice

Rrbp1-KO mice were given a high-K+ diet and water for a month to further investigate the causes of the high sudden death rate (Fig. 1E); they were later found to be markedly prone to sudden death (Fig. 1F). The cardiac rhythms of 12–16-week-old mice were monitored by telemetry ECG with and without high K+ intake to determine if the death of Rrbp1-KO mice under high K+ intake was related to life-threatening arrhythmia (Fig. 1G). Under normal dietary conditions, ECGs revealed no differences in the RR interval, heart rate, PR interval, P duration, QRS interval, QT interval, corrected QT interval, T amplitude, and P amplitude (Fig. 1H–P). However, Rrbp1-KO mice showed longer PR intervals, P durations, QRS intervals, QT intervals, and corrected QT intervals thanRrbp1-WT mice (0.044 ± 0.008 versus 0.034 ± 0.003 s; P < 0.05; 0.014 ± 0.003 versus 0.010 ± 0.001 secs; P < 0.05; 0.017 ± 0.007 versus 0.011 ± 0.001 secs; P < 0.05; 0.036 ± 0.005 versus 0.029 ± 0.005 secs; P < 0.05; 0.105 ± 0.014 versus 0.093 ± 0.016 secs; P < 0.05, respectively) (Fig. 1J–N) at 2 days after high K+ intake. There was no difference in the RR interval, heart rate, T amplitude, and P amplitude of Rrbp1-KO and WT mice after two days of high K+ intake, but the P values still showed a similar trend. Compared to Rrbp1-WT mice, the T waves of two Rrbp1-KO mice in the high K+ intake group peaked, which indicates severe hyperkalemia. Figure 1Q shows representative ECG waveforms.

Rrbp1-KO mice feature volume depletion and hyporeninemic hypoaldosteronism

After the normal diet, the cardiac output of Rrbp1-KO mice was lower than that of Rrbp1-WT mice according to echocardiography. (12.99 ± 1.70 versus 15.63 ± 1.83 ml/min; P = 0.01) (Additional file 1: Fig. S3A). Correspondingly, the stroke volume and left ventricular volume in diastole of Rrbp1-KO mice were also lower than those of Rrbp1-WT mice (43.65 ± 5.55 versus 59.28 ± 11.01 μl; P < 0.01; and 69.66 ± 8.51 versus 92.26 ± 21.56 μl, respectively; P = 0.02) (Additional file 1: Fig. S3B, S3C). There were no significant differences in left ventricular mass, left ventricular posterior wall thickness, interventricular septum thickness in diastole, relative wall thickness, left ventricular fractional shortening, or left ventricular ejection fraction (Additional file 1: Fig. S3D-3I). Cardiac output and stroke volume were decreased in Rrbp1-KO mice, while their wall thickness and contractility did not change.

Plasma renin, angiotensinogen, angiotensin-I, angiotensin-II, and aldosterone were measured to assess the role of the RAAS axis in volume-related lower blood pressure and hyperkalemia. Rrbp1-KO mice had significantly higher plasma angiotensinogen levels than Rrbp1-WT mice (49.72 ± 12.53 versus 40.72 ± 7.20 µg/ml; P = 1 × 10−4) (Fig. 2A) but lower plasma renin (28.21 ± 2.47 versus 29.84 ± 2.60 ng/ml; P = 5.9 × 10−3) (Fig. 2B), angiotensin-I (172.7 ± 63.5 versus 251.0 ± 141.4 pg/ml; P = 1.6 × 10−3) (Fig. 2C), angiotensin-II (343.2 ± 203.8 versus 526.9 ± 229.5 pg/ml; P = 1.7 × 10−3) (Fig. 2D), and aldosterone (812.1 ± 486.1 versus 1697.0 ± 651.2 pg/ml; P < 1 × 10−4) (Fig. 2E).The plasma renin activity (PRA) was also significantly lower in knockout mice (Fig. 2F) (P < 0.01). The expression of serum/glucocorticoid regulated kinase 1 (SGK1) as an early aldosterone-induced protein significantly decreased in kidney homogenates from Rrbp1-KO mice (Fig. 2G, H); SGK1 is an aldosterone-responsive protein that modulates the expression and function of various renal ion channels such as epithelial Na+ channel ENaC and renal K+ channel ROMK to regulate sodium reabsorption and K+ secretion [28]. RT-qPCR revealed no difference in levels of Scnn1a encoding ENaC-α protein expression, but lower levels of Scnn1b, Scnn1g, and Kcnj1 encoding ENaC-β, ENaC-γ, and ROMK were found in Rrbp1-KO mice compared to wild-type controls (Fig. 2I). H&E staining, adrenal gland weight, and ACTH (adrenocorticotropic hormone) stimulation test were performed to characterize the adrenal gland in Rrbp1-KO mice (Additional file 1: Fig. S4). There were no obvious lesions or weight changes in adrenal glands from Rrbp1-WT and Rrbp1-KO mice. The basal plasma level of corticosterone was lower in Rrbp1-KO mice, suggesting that the lower plasma angiotensin-II level in Rrbp1-KO reduced corticosterone levels in plasma. After ACTH stimulation, there was no significant difference in plasma levels of corticosterone in Rrbp1-WT and Rrbp1-KO mice. The basal plasma potassium level was higher in Rrbp1-KO mice compared with Rrbp1-WT mice (6.77 ± 0.14 versus 6.28 ± 0.11 mM; P < 0.01). The basal plasma potassium value was approximately 6.28 mM in Rrbp1-WT mice rather than 4–5 mM in normal C57BL/6 mice, which could be attributed to the mixed genetic background of the C57BL6/129J mice. In addition, the basal fractional excretion of potassium was significantly lower in Rrbp1-KO mice compared to Rrbp1-WT mice (11.52 ± 0.843 versus 16.44 ± 0.997; P < 1 × 10−3) (Table 2). Moreover, Rrbp1-KO mice still had lower plasma renin (18.12 ± 1.08 versus 19.51 ± 1.601 ng/ml; P = 0.02) (Fig. 2K), angiotensin-II (855.7 ± 310.8 versus 1365 ± 803.5 pg/ml; P = 0.02) (Fig. 2L), and aldosterone (3361 ± 826.2 versus 4397 ± 1370 pg/ml; P = 0.04) levels than Rrbp1-WT mice (Fig. 2M) after 2 days of high K+ intake (Fig. 2J). Consistently, plasma K+ concentration significantly increased in Rrbp1-KO mice compared to Rrbp1-WT mice (6.77 ± 0.14 versus 6.28 ± 0.11 mmol/L; P < 0.01). Two days after high K+ intake, Rrbp1-KO mice had higher serum potassium levels (8.53 ± 0.38 versus 7.23 ± 0.27 mmol/L; P = 0.0086), decreased transtubular potassium gradient (TTKG) (14.62 ± 0.79 versus 18.44 ± 1.04; P = 5.8 × 10−3), and lower urine fractional excretion of potassium (43.14 ± 4% versus 64.84 ± 6.7%; P = 8.1 × 10−3) than Rrbp1-WT mice (Table 3), indicating that Rrbp1-KO mice developed hyporeninemic hypoaldosteronism with hyperkalemia.

Fig. 2figure 2

Rrbp1-KO mice show hyporeninemic hypoaldosteronism. AF 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. KM 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 AF, I, and KM were analyzed with an unpaired, two-tailed Student’s t-test; data in H were analyzed using the Mann–Whitney test. Data are represented as mean ± SEM. PRA, plasma renin activity; RFU, relative fluorescence units; ns, no significance; *P < 0.05; ** P < 0.01; ***P < 0.001; **** P < 0.0001

Table 2 Plasma and urine electrolyte levels in Rrbp1-WT and Rrbp1-KO miceTable 3 Plasma and urine electrolyte levels in Rrbp1-WT and Rrbp1-KO mice under high K+ intakeFludrocortisone rescues high K+ load-induced sudden death in Rrbp1-KO mice

To confirm the relationship between high K+ load-induced sudden death of Rrbp1-KO mice and suppression of the RAAS system, mice were rescued by fludrocortisone, a synthetic mineralocorticoid (0, 2.5, 10 mg/kg intraperitoneal injection once every two days) for 30 days (Fig. 2N). Fludrocortisone improved the survival of Rrbp1-KO mice dose-dependently (Fig. 2O). Accordingly, there were no significant differences in levels of blood Na+, K+, Cl− or excretion of urinary potassium measured two days after high K+ intake with or without 100 mg/kg fludrocortisone treatment (Table 4).

Table 4 Plasma and urine electrolyte levels in Rrbp1-WT and Rrbp1-KO mice under high K+ intake for 48 h with and without fludrocortisone treatmentDeficiency of RRBP1 increases intracellular renin level and decreases renin secretion in vivo and vitro

Immunostaining was used to measure renin expression in the kidneys of Rrbp1-KO and Rrbp1-WT mice because Rrbp1-KO mice showed low PRA (Fig. 3A, B). Unexpectedly, the intensity of renin stain was significantly higher in Rrbp1-KO than in Rrbp1-WT mice (15.35 ± 2.93 versus 14.51 ± 1.62 arbitrary units; P < 0.05) in the kidneys (Fig. 3C).

Fig. 3figure 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). EH 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 forskolin-induced renin production in control and RRBP1 knockdown cells. N Western blot analysis of RRBP1, renin, and ADCY6 protein expression in RRBP1-knockdown Calu-6 cells and supernatant. Lanes 1–3 represent cells with DMSO control treatment. Lane 4–6 represents cells induced with 50 μM forskolin. WT, wild-type; KO, knock-out. Data in C, E, J were analyzed with an unpaired, two-tailed Student’s t-test; data in (D) were analyzed using a Mann–Whitney test. Data were represented as mean ± SEM. ns, no significance; *P < 0.05; ***P < 0.001

To clarify how RRBP1 affects renin distribution, RRBP1 was knocked down in cultured Calu-6 cells (Fig. 3D), a human renin-producing cell line, which were stained with immunogold-labeled anti-renin antibody and examined by transmission electron microscopy (TEM) (Fig. 3E–H). The total intracellular renin particles increased in RRBP1-knockdown Calu-6 cells relative to scramble controls (P < 0.05) (Fig. 3I). TEM data showed that renin particles were more distant from the plasma membrane (> 1000 nm) in RRBP1-knockdown cells compared to controls (Fig. 3J). These results indicated that more renin particles accumulated intracellularly, and less renin was transported to the plasma membrane and secreted by RRBP1-knockdown cells.

Next, renin levels were measured in RRBP1-knockdown cells to explore how RRBP1 affects renin secretion. Indeed, less renin was secreted into the culture medium of RRBP1-knockdown cells, whereas more renin accumulated in those cells (Fig. 3K, L).

Renin secretion is controlled by the cyclic adenosine monophosphate (cAMP) signaling in response to various external stimuli [10]. However, the expression level of adenylyl cyclase 6 (ADCY6), the major enzyme responsible for producing intracellular cAMP [10, 29], was not significantly different in Calu-6 cells (Additional file 1: Fig. S5A).

Cells were induced with forskolin, an adenylyl cyclase activator, to enhance intracellular cAMP levels and therefore test if enhanced intracellular cAMP levels can rescue renin secretion during RRBP1 deficiency. After forskolin induction, intracellular cAMP levels became comparable to those of controls (Additional file 1: Fig. S5B). Correspondingly, forskolin induction reversed the decreased renin secretion into the culture medium and the accumulation of renin within RRBP1-knockdown cells (Fig. 3M and N). However, the level of secreted renin detected in the culture medium was still lower in RRBP1-knockdown cells #2 compared to those that of control cells and RRBP1-knockdown cells #1, which may be attributed to the significantly lower knockdown efficiency of knockdown cells #1 compared with #2 in Fig. 3N after forskolin induction. Additionally, the level of secreted renin in sh-RRBP1#1 cells with very low knockdown efficiency was comparable to that of control cells after forskolin treatment (Fig. 3N). Despite the difference in knockdown efficiency, these findings, however, indicate that renin secretion is strongly related to RRBP1 expression. These findings suggest that renin secretion is strongly related to RRBP1 expression; RRBP1 deficiency still reduces renin secretion despite increased intracellular cAMP.

Deficiency of RRBP1 causes retention of renin in the endoplasmic reticulum

After forskolin induction, cells were immune-stained with anti-renin, anti-RRBP1, anti-calnexin (an ER marker), and anti-GOLIM4 (a Golgi apparatus marker) to investigate intracellular trafficking of renin in RRBP1-knockdown and control cells (Fig. 4A and Additional file 1: Fig. S6). At 0, 60, 120, and 180 min post-induction, confocal microscopy revealed that renin was retained in the ER (Fig. 4B–E) and not transported to the Golgi apparatus (Fig. 5A-D) upon forskolin induction in RRBP1-knockdown Calu-6 cells. However, renin was transported to the Golgi apparatus upon forskolin induction in scramble controls. To quantify the intracellular trafficking of renin, we calculated the fluorescent signal intensity of renin overlapped with calnexin as well as with GOLIM4. Initially, the overlapped signal of renin with calnexin did not differ between RRBP1-knockdown cells and controls. However, after forskolin induction for 120 and 180 min, the overlapped fluorescent signal between renin and calnexin of RRBP1-knockdown cells was higher than that of control cells (0.875 ± 0.021 versus 0.850 ± 0.009; P = 0.02) and (0.898 ± 0.021 versus 0.845 ± 0.029; P = 1 × 10−3) (Fig. 4F). On the contrary, the overlapped fluorescent signal between renin and GOLIM4 of RRBP1-knockdown cells was consistently lower compared to that of control cells after forskolin induction at 0 (0.559 ± 0.024 versus 0.600 ± 0.026; P = 8 × 10−3), 60 (0.641 ± 0.053 versus 0.731 ± 0.027; P < 1 × 10−3), 120 (0.691 ± 0.055 versus 0.739 ± 0.006; P = 0.04), and 180 (0.711 ± 0.030 versus 0.744 ± 0.025; P = 0.03) minutes, respectively (Fig. 5E). These findings indicated that the renin was retained in the ER of RRBP1-knockdown cells. Accordingly, renin required more time to leave the ER and enter the Golgi apparatus in the RRBP1-knockdown cells after stimulation. Ultimately, these data show that RRBP1 regulates renin trafficking between the ER and the Golgi apparatus as well as renin secretion. RRBP1 deficiency causes hyporeninemic hypoaldosteronism and hyperkalemia (Fig. 6).

Fig. 4figure 4

RRBP1 deficiency increases renin accumulation in ER. A Protocol to stimulate renin trafficking in control and RRBP1 knockdown cells. BE 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

Fig. 5figure 5

RRBP1 deficiency reduces renin transport from ER to Golgi apparatus. AD 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

Fig. 6figure 6

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

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