Effect of GRK4 on renal gastrin receptor regulation in hypertension

ABSTRACT

Objective

To investigate whether GRK4 regulates the phosphorylation and function of renal CCKBR.

Methods

GRK4 A142V transgenic mice were used as an animal model of enhanced GRK4 activity, and siRNA was used to silence the GRK4 gene to investigate the regulatory effect of GRK4 on CCKBR phosphorylation and function. Finally, the co-localization and co-connection of GRK4 and CCKBR in RPT cells were observed by laser confocal microscopy and immunoprecipitation to explore the mechanism of GRK4 regulating CCKBR.

Results

Gastrin infusion significantly increased urinary flow and sodium excretion rates in GRK4 WT mice (P < .05). GRK4 siRNA did not affect CCKBR protein expression in WKY RPT cells and SHR RPT cells, but remarkably reduced CCKBR phosphorylation in WKY and SHR RPT cells (P < .05). The inhibitory effect of gastrin on Na⁺-K⁺ -ATPase activity in WKY RPT cells was further enhanced by the reduction of GRK4 expression (P < .05), while GRK4 siRNA restored the inhibitory effect of gastrin on Na⁺-K⁺ -ATPase activity in SHR RPT cells. Laser confocal and Co-immunoprecipitation results showed that GRK4 and CCKBR co-localized in cultured RPT cells’ cytoplasm.

Conclusion

GRK4 participates in the development of hypertension by regulating the phosphorylation of renal CCKBR leading to impaired CCKBR function and water and sodium retention. Knockdown of GRK4 restored the function of CCKBR. The enhanced co-connection between GRK4 and CCKBR may be an important reason for the hyperphosphorylation of GRK4 and CCKBR involved in the pathogenesis of hypertension.

KEYWORDS:

• Essential hypertension
• GRK4
• gastrin
• CCKBR
• Na⁺-K⁺ -ATPase

Introduction

Hypertension is an important risk factor for CVD and other diseases with adverse clinical outcomes, such as coronary heart disease, stroke, and heart failure (1). The complications of hypertension are associated with high mortality and disability rates, which have led to 7.6 million deaths per year worldwide (2,3). However, the pathogenesis of hypertension remains unclear. The imbalance of water and sodium metabolism is an important link in the process of hypertension. The kidneys and gastrointestinal tract play an important role in the metabolism of sodium and water. The kidney regulates the reabsorption of urinary sodium through the epithelial cells of the proximal convoluted tubules, and the gastrointestinal tract not only serves as a sodium absorption organ but also establishes a dialogue function with the kidney by secreting gastrointestinal hormones, which is called the “kidney-gastrointestinal sodium excretion axis” (4,5). Among many gastrointestinal hormones, gastrin has attracted our attention because of its large secretion. Our previous study found that gastrin exerts diuretic and natriuretic effects by acting on the renal gastrin receptor, also known as cholecystokinin receptor type B (CCKBR) (6). The gastrin-mediated urinary sodium excretion is impaired in hypertension (7), but the mechanism is not clear. In addition, gut-specific knockout of CCKBR was found to aggravate high-salt-induced blood pressure and sodium excretion in mice, suggesting that CCKBR may play a role in blood pressure control (8). GRK4 is highly expressed in blood pressure-regulating tissues and organs such as the kidney. The increase in its expression and activity occurred earlier than the increase in blood pressure. Approximately six variants of GRK4 exist in humans, and the presence of variants increases GRK4 activity. Among them, the incidence of variants A142V, A486V, and R65L is significantly related to the degree of blood pressure increase (9). A large number of animal studies have found that GRK4 transgenic mice have increased blood pressure and dysfunction of urinary sodium excretion (10). But whether GRK4 regulates the phosphorylation and function of renal CCKBR remains unclear. Therefore, in this study, GRK4 A142V transgenic mice will be used as an animal model of enhanced GRK4 activity, while silencing the GRK4 gene with siRNA, to investigate the regulatory effect of GRK4 on CCKBR phosphorylation and function.

Methods

Animals

WKY and SHR rats, GRK4 A142V transgenic mice, and GRK4 WT transgenic mice used in this experiment were provided by the Institute of Laboratory Animal Studies, Chinese Academy of Medical Sciences. Mice were given a standard diet and fed in the same environment with alternating light and dark for 12 hours. All experiments were in accordance with the relevant regulations of experimental animal ethics.

Cells and siRNA transfection

Cell experiments were grouped as follows: WKY scramble, WKY GRK4 siRNA, SHR scramble, and SHR GRK4 siRNA. Synthetic GRK4 siRNA was synthesized from Guangzhou biological technology co., LTD.

GRK4 siRNA interference in WKY and SHR RPT cells: When the cells were cultured to about 50% density, the transfection culture was replaced and the cells were starved for 2 hours. The mixture of prepared Transfection medium, siRNA, and RNA iMAX Transfection Reagent was added to 6-well and 12-well plates in proportion, and gastrin was added after 24 hours of culture to continue for another 24 hours.

Na⁺-K⁺ -ATPase activity and immunofluorescence

Determination of Na⁺-K⁺ -ATPase activity in RPT cells: 1 mL of precooled saline was included in each well, and the cells were scraped and collected into a 1.5 mL centrifuge tube. Protein concentration was determined by the BCA method, and Na⁺-K⁺ -ATPase activity was determined according to the instructions of the Na⁺-K⁺ -ATPase activity assay kit.

Immunofluorescence: WKY and SHR RPT cells seeded on an easy slide were fixed with 4% paraformaldehyde for 10 min, blocked, and incubated with prepared dilutions of GRK4 or CCKBR primary antibody (1:100 dilution) overnight at 4°C. The prepared immunofluorescence secondary antibody (1:100 dilution) was added and incubated at 37°C for 1 hour. After DAPI staining, anti-fluorescence extraction was used to seal tablets, and laser confocal imaging was used to collect fluorescence images.

Urine flow rate and sodium excretion rate

The animals were anesthetized with a continuous infusion of sodium pentobarbital by a micropump, while the micropump continued to equilibrate with saline for 1 hour before the experiment began. The experiment was divided into three stages: control stage, gastrin stage (gastrin concentration 0.1, 0.5, 1.0 µg/kg/min), and recovery stage, each stage was 45 min. Normal saline was pumped into the control stage and recovery stage, and the pump rate of normal saline and gastrin was 40 µL/h. The urine flow rate was calculated from the collected urine at each stage, urinary sodium concentration was measured, and urinary sodium excretion rate was calculated as urine flow rate × urinary sodium concentration.

Western blot and immunoprecipitation

Tissues were dissociated by incubation in RIPA lysate for 30 min on ice and centrifugation at 15 000 g for 30 min at 4°C. One microliter of the supernatant was used to detect protein concentration to denature the protein. Protein immunoprecipitation required the addition of the corresponding protein antibody to incubate overnight before protein denaturation, the next day agarose beads were added for further 12 hours of incubation, and the protein was denatured after elution of the eluate. The proteins were separated by SDS-PAGE gel and electro-transferred to a nitrocellulose membrane, blocked in 5% skim milk, incubated with primary antibody overnight, and incubated with secondary antibody for 1 hour before exposure for data interpretation.

Statistical analysis

Statistical analysis was performed using the t-test for parametric data and the Mann-Whitney U test for non-parametric data. One-way analysis of variance (ANOVA) and the Bonferroni test or two-way ANOVA was used when there were more than two groups. The significance level was set as p < .05. All analyses were performed with SPSS V.17.0 (SPSS) software.

Results

Comparison of renal GRK4 expression between WKY and SHR rats

To explore the relationship between the increased phosphorylation level of CCKBR and GRK4 in the kidney of SHR rats, we extracted mRNA and protein from the kidney tissues of WKY and SHR, respectively. The expression of GRK4 in the kidney of WKY and SHR rats was detected by QT-PCR and Western blot. The mRNA and protein expression levels of GRK4 in the kidney of SHR rats were significantly higher than those of WKY rats, and the mRNA level of SHR rats was twice that of WKY rats (Figure 1a), and the protein level was about 1.6 times that of WKY rats (Figure 1 B1, B2).

Figure 1. Renal GRK4 mRNA and protein expression in WKY and SHR rats. a, renal GRK4 mRNA expression in WKY and SHR rats. (P < 0.0001, compared with WKY; N = 5); b, renal GRK4 protein expression in WKY and SHR rats. (P < 0.0001, compared with WKY; N = 5); B1, protein levels of GRK4 were shown by Western blot. B1, statistical results for B1. Data are expressed as mean ± SE.

Effect of GRK4 on gastrin receptor activity and function

To investigate the relationship between GRK4 activity and gastrin receptor, we used GRK4 A142V transgenic mice with enhanced GRK4 activity to investigate the expression and phosphorylation of renal CCKBR. However, renal CCKBR phosphorylation was significantly higher in GRK4 A142V transgenic mice than in GRK4 WT mice (Figure 2 A1, A2). Urine output and urinary sodium excretion in the kidneys of GRK4 WT mice and GRK4 A142V mice were measured by systemic infusion of gastrin into the external jugular vein. The results revealed that gastrin infusion significantly increased urinary flow rate and urinary sodium excretion rate in GRK4 WT mice. Gastrin at a concentration of 0.5 µg/kg/min had a strong diuretic and natriuretic effect in a concentration-dependent manner. Gastrin infusion had no significant effect on the urinary flow rate or urinary sodium excretion rate in GRK4 A142V mice (Figure 2B1, B2).

Figure 2. Effect of GRK4 on gastrin receptor activity and function. a, renal CCKBR and p-CCKBR expression levels in GRK4 WT and A142V mice. A1, Protein levels of CCKBR and p-CCKBR in GRK4 WT and A142V mice were shown by Western blot. A2, Statistical results for A1. Data are expressed as mean ± SE. (P < 0.0001, compared with GRK4 WT; N = 6); B1, Effect of gastrin on renal urinary flow rate in GRK4 A142V mice. (P < 0.05, compared with GRK4 WT; N = 7); B2, effect of gastrin on renal urinary sodium in GRK4 A142V mice. (P < 0.05, compared with GRK4 WT; N = 6); data are expressed as mean ± SE.

Downregulation of GRK4 restores gastrin receptor function

To determine the effect of GRK4 on gastrin receptors, siRNA transfection of GRK4 into WKY RPT cells and SHR RPT cells revealed a significant reduction in the mRNA and protein expression levels of GRK4 (Figure 3A1, B1, B2). Interestingly, GRK4 siRNA did not affect CCKBR protein expression in WKY RPT cells and SHR RPT cells, but remarkably reduced CCKBR phosphorylation in WKY and SHR RPT cells (Figure 3B3, B4). The inhibitory effect of gastrin on Na⁺-K⁺-ATPase in the proximal tubule was reduced under hypertension. To further observe the effect of GRK4 siRNA on CCKBR-induced renal Na⁺-K⁺ -ATPase activity, we further examined Na⁺-K⁺ -ATPase activity in WKY and SHR RPT cells. The activity of Na⁺-K⁺ -ATPase in SHR RPT cells was higher than that in WKY RPT cells. When CCKBR was activated by gastrin (10⁻⁹mol/L, 15 min), the activity of Na⁺-K+ -ATPase in WKY RPT cells was significantly decreased, but this effect was lost in SHR RPT cells. The inhibitory effect of gastrin on Na⁺-K⁺ -ATPase activity in WKY RPT cells was further enhanced by the reduction of GRK4 expression, while GRK4 siRNA restored the inhibitory effect of gastrin on Na⁺-K⁺ -ATPase activity in SHR RPT cells (Figure 3c).

Figure 3. Downregulation of GRK4 restores gastrin receptor function. A, GRK4 mRNA expression in WKY and SHR cells in the presence of siRNA silencing GRK4, in which empty vector was added as a control. B1, protein levels of GRK4, CCKBR and p-CCKBR in WKY and SHR RPT cells. B2-B4 statistical results for B1. Data are expressed as Mean ± SE. (P < 0.0001, compared with scramble; N = 3); C, alterations of Na⁺-K⁺ -ATPase activity in WKY and SHR cells in response to gastrin stimulation after silencing GRK4 by siRNA. (p < 0.0001, compared with scramble; N = 3).

GRK4 directly interacts with CCKBR in RPT cells from WKY and SHRs

To investigate the mechanism by which GRK4 regulates CCKBR, co-immunoprecipitation and confocal laser scanning microscopy were used to observe the co-connection between GRK4 and CCKBR. Laser confocal results showed that GRK4 and CCKBR were co-localized in the cytoplasm of cultured RPT cells (Figure 4a). Co-immunoprecipitation showed that the co-ligation of GRK4 with CCKBR was significantly increased in SHR RPT cells compared with WKY RPT cells (Figure 4 B1, B2).

Figure 4. Interaction between CCKBR and GRK4 in WKY and SHR RPT cells. a, colocalization of CCKBR and GRK4 in RPT cells from WKY and SHRs. Colocalization appears as yellow after merging the images of Alexa Fluor 546-tagged CCKBR (red) and Alexa Fluor 488-tagged GRK4 (green). B1, Co-immunoprecipitation of GRK4 and CCKBR in RPT cells from WKY and SHRs.B2, statistical results for B1. (P < .0001, compared with WKY, N = 4). Data are expressed as mean ± SE.

Discussion

The pathogenesis of hypertension is affected by environmental factors, genetic factors, and a variety of neurohumoral factors (11). The humoral factors involved in maintaining the stability of blood pressure include angiotensin, dopamine, antidiuretic hormone, and natriuretic peptide (12). Their receptors all belong to GPCR and are regulated by GRKs. GRKs regulate these GPCR-mediated biological effects by increasing receptor phosphorylation and internalization (13). GRKs include multiple subtypes that differ in their tissue distribution and physiological roles. Among them, GRK1, 7 belongs to the rhodopsin family and is mainly expressed in the retina (14,15), while GRK2,3,5,6 (16–18) belongs to the β-adrenergic receptor kinase family. Although it is widely expressed in various organs and tissues, its changes are often secondary to hypertension (19). GRK4 is only expressed in the kidney, heart, testis, brain, and myometrium, and is most closely related to hypertension (20). However, the gene location of GRK4 is closely related to essential hypertension, and the functional changes of GRK4 precede the development of essential hypertension. Studies have shown that a variety of GRK4 variants can lead to enhanced GRK4 activity and are closely related to the occurrence of hypertension (21). A large number of studies have shown that the probability of the occurrence of the GRK4 variant is significantly related to the degree of elevated blood pressure, and the accuracy of predicting hypertension with the GRK4 variant is more than 70% in African Ghanaian and 94% in Japanese (22,23). A large number of animal studies have also found that GRK4 transgenic mice have increased blood pressure and dysfunction of urinary sodium excretion (24). Further studies have found that the increased renal GRK4 activity in SHR rats leads to a significant increase in the protein phosphorylation of dopamine D1R, and the reduction of GRK4 expression can reduce the phosphorylation of dopamine D1R, and finally improve the D1R-mediated urinary sodium excretion (25). In addition, studies have shown that increased GRK4 activity can also lead to enhanced renal AT1R function, reduce renal water and sodium excretion, and eventually participate in the formation of hypertension (26). Like D1R and AT1R, renal CCKBR receptors are also GPCR, suggesting that renal CCKBR receptor function and phosphorylation may be regulated by renal GRK4. Our study found that renal GRK4 expression was significantly higher in SHR rats than in WKY rats, consistent with the results of previous studies. In the present study, we used GRK4 A142V transgenic mice as an animal model of enhanced GRK4 activity and administered single-kidney perfusion of gastrin. We found that GRK4 A142V transgenic mice had loss of gastrin diuretic and natriuretic function and significantly higher renal CCKBR phosphorylation than wild-type mice, similar to the results obtained in SHR rats. These results suggest that GRK4 may be an upstream regulator of renal CCKBR hyperphosphorylation that impairs diuretic and natriuretic function. To further clarify the regulation of GRK4 on renal CCKBR, we used siRNA to silence the GRK4 gene in RPT cells and then intervened with gastrin to observe the effect of GRK4 knockdown on CCKBR-mediated Na⁺-K⁺ -ATPase activity and CCKBR phosphorylation. Consistent with previous studies, gastrin showed a strong inhibitory effect on Na⁺-K⁺ -ATPase activity in RPT cells derived from WKY rats. However, in SHR-derived RPT cells, the inhibitory effect of gastrin on Na⁺-K⁺ -ATPase activity was lost, which corresponds to the results of gastrin loss of renal natriuretic function in SHR rats. Knockdown of GRK4 significantly restored the inhibitory effect of gastrin on Na⁺-K⁺ -ATPase activity and the phosphorylation level of CCKBR, indicating that intervention of GRK4 restored the function of gastrin receptors and the diuretic and natriuretic effect of gastrin on the kidney. Further study on the regulatory mechanism of GRK4 on CCKBR can not only enrich the pathogenesis theory of hypertension but also play an important role in the prevention and treatment of hypertension. Finally, to clarify the mechanism by which GRK4 regulates CCKBR, we observed the co-localization of GRK4 and CCKBR in RPT cells by immunofluorescence staining, and the co-connection of GRK4 and CCKBR was observed by Co-IP and Western blot. The results showed that GRK4 and CCKBR were co-localized in RPT cells, and Co-IP and Western blot results showed that GRK4 and CCKBR were co-connected at a higher level in SHR-derived RPT cells than in WKY-derived RPT cells, indicating that in addition to the direct interaction between GRK4 and CCKBR, the expression of CCKBR in SHR-derived RPT cells was significantly higher than that in WKY-derived RPT cells. It may be an important way for it to regulate CCKBR. From these results, we can see that GRK4 has a regulatory effect on the phosphorylation and function of renal CCKBR through direct interaction, and this regulatory effect is abnormally enhanced with the increase of the expression of GRK4 and the increase of the combination of the two in the state of hypertension, thereby damaging the inhibitory effect of CCKBR on water and sodium reabsorption, leading to water and sodium retention and the development of hypertension. However, this study still has some limitations. First, we were unable to knock out renal GRK4 in vivo and observe the regulatory effect of GRK4 intervention on blood pressure. Second, we only confirmed the direct interaction between GRK4 and CCKBR. More work is still needed to further reveal the mechanism of action of the stomach-kidney axis and its role in hypertension.

Conclusion

GRK4 regulates the phosphorylation of renal CCKBR, leading to impaired function of CCKBR, causing water and sodium retention and participating in the formation of hypertension. The intervention of GRK4 restored the function of CCKBR. The enhanced co-connection between GRK4 and CCKBR may be an important reason for the hyperphosphorylation of GRK4 and CCKBR involved in the pathogenesis of hypertension.

Acknowledgments

All study participants are thanked for their contributions to this work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10641963.2023.2245580

Additional information

Funding

The work was supported by the National Natural Science Foundation of China [81900792, 82000476]; Chongqing natural science foundation [cstc2020jcyj-msxmX0091].