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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.


Effects of RNA interference on G protein–coupled receptor kinase type 4 (GRK4) expression in spontaneously hypertensive rat (SHR) renal proximal tubule (RPT) cells. A, GRK4 mRNA expression in SHR RPT cells treated with three GRK4 siRNAs (n=4, *P<0.05 vs control). NC indicates normal control. B, GRK4 protein expression in the #1 GRK4 small interfering RNA (siRNA)–treated SHR PRT cells (n=4, *P<0.05 vs control).
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jah31805-fig-0001: Effects of RNA interference on G protein–coupled receptor kinase type 4 (GRK4) expression in spontaneously hypertensive rat (SHR) renal proximal tubule (RPT) cells. A, GRK4 mRNA expression in SHR RPT cells treated with three GRK4 siRNAs (n=4, *P<0.05 vs control). NC indicates normal control. B, GRK4 protein expression in the #1 GRK4 small interfering RNA (siRNA)–treated SHR PRT cells (n=4, *P<0.05 vs control).

Mentions: To achieve the expected gene silencing effect of the RNA interference (RNAi), we designed 3 GRK4 siRNAs and then screened for the best. The results revealed that all 3 of the GRK4 siRNAs led to decreased GRK4 mRNA expression in the SHR RPT cells (Figure 1A). However, the #1 GRK4 siRNA had the best effect on GRK4 expression compared with the others (Figure 1B). Therefore, we chose the #1 GRK4 siRNA for the subsequent experiments.


Downregulation of Renal G Protein – Coupled Receptor Kinase Type 4 Expression via Ultrasound ‐ Targeted Microbubble Destruction Lowers Blood Pressure in Spontaneously Hypertensive Rats
Effects of RNA interference on G protein–coupled receptor kinase type 4 (GRK4) expression in spontaneously hypertensive rat (SHR) renal proximal tubule (RPT) cells. A, GRK4 mRNA expression in SHR RPT cells treated with three GRK4 siRNAs (n=4, *P<0.05 vs control). NC indicates normal control. B, GRK4 protein expression in the #1 GRK4 small interfering RNA (siRNA)–treated SHR PRT cells (n=4, *P<0.05 vs control).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121504&req=5

jah31805-fig-0001: Effects of RNA interference on G protein–coupled receptor kinase type 4 (GRK4) expression in spontaneously hypertensive rat (SHR) renal proximal tubule (RPT) cells. A, GRK4 mRNA expression in SHR RPT cells treated with three GRK4 siRNAs (n=4, *P<0.05 vs control). NC indicates normal control. B, GRK4 protein expression in the #1 GRK4 small interfering RNA (siRNA)–treated SHR PRT cells (n=4, *P<0.05 vs control).
Mentions: To achieve the expected gene silencing effect of the RNA interference (RNAi), we designed 3 GRK4 siRNAs and then screened for the best. The results revealed that all 3 of the GRK4 siRNAs led to decreased GRK4 mRNA expression in the SHR RPT cells (Figure 1A). However, the #1 GRK4 siRNA had the best effect on GRK4 expression compared with the others (Figure 1B). Therefore, we chose the #1 GRK4 siRNA for the subsequent experiments.

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.