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Downregulation of Renal G Protein – Coupled Receptor Kinase Type 4 Expression via Ultrasound ‐ Targeted Microbubble Destruction Lowers Blood Pressure in Spontaneously Hypertensive Rats

View Article: PubMed Central - PubMed

ABSTRACT

Background: G protein–coupled receptor kinase type 4 (GRK4) plays a vital role in the long‐term control of blood pressure (BP) and sodium excretion by regulating renal G protein–coupled receptor phosphorylation, including dopamine type 1 receptor (D1R). Ultrasound‐targeted microbubble destruction (UTMD) is a promising method for gene delivery. Whether this method can deliver GRK4 small interfering RNA (siRNA) and lower BP is not known.

Methods and results: BP, 24‐hour sodium excretion, and urine volume were measured after UTMD‐targeted GRK4 siRNA delivery to the kidney in spontaneously hypertensive rats. The expression levels of GRK4 and D1R were determined by immunoblotting. The phosphorylation of D1R was investigated using immunoprecipitation. The present study revealed that UTMD‐mediated renal GRK4 siRNA delivery efficiently reduced GRK4 expression and lowered BP in spontaneously hypertensive rats, accompanied by increased sodium excretion. The increased sodium excretion might be accounted for by the UTMD regulation of D1R phosphorylation and function in spontaneously hypertensive rats. Further analysis showed that, although UTMD had no effect on D1R expression, it reduced D1R phosphorylation in spontaneously hypertensive rats kidneys and consequently increased D1R‐mediated natriuresis and diuresis.

Conclusions: Taken together, these study results indicate that UTMD‐targeted GRK4 siRNA delivery to the kidney effectively reduces D1R phosphorylation by inhibiting renal GRK4 expression, improving D1R‐mediated natriuresis and diuresis, and lowering BP, which may provide a promising novel strategy for gene therapy for hypertension.

No MeSH data available.


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Inhibitory effects of ultrasound‐targeted microbubble destruction (UTMD)–mediated G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) delivery in renal GRK4 expression. A, Ultrasound image of the kidney of a spontaneously hypertensive rat (SHR) (42 frames per s, mechanical index=0.9, frequency=7.00 MHz). B‐mode scans, gray‐scale mapping. The images were obtained at 0 seconds (a), 15 seconds (b), and 5 minutes (c) after the injection of the microbubbles. B and C, Renal GRK4 mRNA and protein expression levels after treatment with UTMD‐mediated GRK4 siRNA delivery for 20 days in SHRs (n=5, *P<0.05 vs control). D and E, Effects of UTMD‐mediated GRK4 siRNA delivery on GRK4 expression in the heart and mesenteric arteries of SHRs after treatment with UTMD‐mediated GRK4 siRNA delivery for 20 days (n=5).
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jah31805-fig-0003: Inhibitory effects of ultrasound‐targeted microbubble destruction (UTMD)–mediated G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) delivery in renal GRK4 expression. A, Ultrasound image of the kidney of a spontaneously hypertensive rat (SHR) (42 frames per s, mechanical index=0.9, frequency=7.00 MHz). B‐mode scans, gray‐scale mapping. The images were obtained at 0 seconds (a), 15 seconds (b), and 5 minutes (c) after the injection of the microbubbles. B and C, Renal GRK4 mRNA and protein expression levels after treatment with UTMD‐mediated GRK4 siRNA delivery for 20 days in SHRs (n=5, *P<0.05 vs control). D and E, Effects of UTMD‐mediated GRK4 siRNA delivery on GRK4 expression in the heart and mesenteric arteries of SHRs after treatment with UTMD‐mediated GRK4 siRNA delivery for 20 days (n=5).

Mentions: Figure 3A‐a demonstrates a renal ultrasound image in one SHR. Renal opacification and subsequent microbubble destruction were observed under ultrasonic irradiation, which indicated that the renal perfusion was sufficient (Figure 3A‐b and 3A‐c). After treatment with UTMD for 20 days, both the renal GRK4 mRNA and protein expression levels were found to be markedly decreased (Figure 3B and 3C). To exhibit the specificity of the UTMD, we also checked the GRK4 expression in the heart and arteries, which resulted in UTMD‐mediated GRK4 siRNA delivery that had no effect on GRK4 expression in those tissues (Figure 3D and 3E).


Downregulation of Renal G Protein – Coupled Receptor Kinase Type 4 Expression via Ultrasound ‐ Targeted Microbubble Destruction Lowers Blood Pressure in Spontaneously Hypertensive Rats
Inhibitory effects of ultrasound‐targeted microbubble destruction (UTMD)–mediated G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) delivery in renal GRK4 expression. A, Ultrasound image of the kidney of a spontaneously hypertensive rat (SHR) (42 frames per s, mechanical index=0.9, frequency=7.00 MHz). B‐mode scans, gray‐scale mapping. The images were obtained at 0 seconds (a), 15 seconds (b), and 5 minutes (c) after the injection of the microbubbles. B and C, Renal GRK4 mRNA and protein expression levels after treatment with UTMD‐mediated GRK4 siRNA delivery for 20 days in SHRs (n=5, *P<0.05 vs control). D and E, Effects of UTMD‐mediated GRK4 siRNA delivery on GRK4 expression in the heart and mesenteric arteries of SHRs after treatment with UTMD‐mediated GRK4 siRNA delivery for 20 days (n=5).
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jah31805-fig-0003: Inhibitory effects of ultrasound‐targeted microbubble destruction (UTMD)–mediated G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) delivery in renal GRK4 expression. A, Ultrasound image of the kidney of a spontaneously hypertensive rat (SHR) (42 frames per s, mechanical index=0.9, frequency=7.00 MHz). B‐mode scans, gray‐scale mapping. The images were obtained at 0 seconds (a), 15 seconds (b), and 5 minutes (c) after the injection of the microbubbles. B and C, Renal GRK4 mRNA and protein expression levels after treatment with UTMD‐mediated GRK4 siRNA delivery for 20 days in SHRs (n=5, *P<0.05 vs control). D and E, Effects of UTMD‐mediated GRK4 siRNA delivery on GRK4 expression in the heart and mesenteric arteries of SHRs after treatment with UTMD‐mediated GRK4 siRNA delivery for 20 days (n=5).
Mentions: Figure 3A‐a demonstrates a renal ultrasound image in one SHR. Renal opacification and subsequent microbubble destruction were observed under ultrasonic irradiation, which indicated that the renal perfusion was sufficient (Figure 3A‐b and 3A‐c). After treatment with UTMD for 20 days, both the renal GRK4 mRNA and protein expression levels were found to be markedly decreased (Figure 3B and 3C). To exhibit the specificity of the UTMD, we also checked the GRK4 expression in the heart and arteries, which resulted in UTMD‐mediated GRK4 siRNA delivery that had no effect on GRK4 expression in those tissues (Figure 3D and 3E).

View Article: PubMed Central - PubMed

ABSTRACT

Background: G protein&ndash;coupled receptor kinase type 4 (GRK4) plays a vital role in the long&#8208;term control of blood pressure (BP) and sodium excretion by regulating renal G protein&ndash;coupled receptor phosphorylation, including dopamine type 1 receptor (D1R). Ultrasound&#8208;targeted microbubble destruction (UTMD) is a promising method for gene delivery. Whether this method can deliver GRK4 small interfering RNA (siRNA) and lower BP is not known.

Methods and results: BP, 24&#8208;hour sodium excretion, and urine volume were measured after UTMD&#8208;targeted GRK4 siRNA delivery to the kidney in spontaneously hypertensive rats. The expression levels of GRK4 and D1R were determined by immunoblotting. The phosphorylation of D1R was investigated using immunoprecipitation. The present study revealed that UTMD&#8208;mediated renal GRK4 siRNA delivery efficiently reduced GRK4 expression and lowered BP in spontaneously hypertensive rats, accompanied by increased sodium excretion. The increased sodium excretion might be accounted for by the UTMD regulation of D1R phosphorylation and function in spontaneously hypertensive rats. Further analysis showed that, although UTMD had no effect on D1R expression, it reduced D1R phosphorylation in spontaneously hypertensive rats kidneys and consequently increased D1R&#8208;mediated natriuresis and diuresis.

Conclusions: Taken together, these study results indicate that UTMD&#8208;targeted GRK4 siRNA delivery to the kidney effectively reduces D1R phosphorylation by inhibiting renal GRK4 expression, improving D1R&#8208;mediated natriuresis and diuresis, and lowering BP, which may provide a promising novel strategy for gene therapy for hypertension.

No MeSH data available.


Related in: MedlinePlus