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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.


Microbubbles with 5‐Carboxyfluorescein (5‐FAM)–labeled G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) observed under fluorescence microscopy. Microbubbles conjugated with 5‐FAM–labeled GRK4 siRNA were observed under bright‐field microscopy (A) and fluorescence microscopy (B). Only the microbubbles conjugated with 5‐FAM–labeled GRK4 siRNA exhibited green fluorescence. Blank microbubbles under bright‐field microscopy (C). No fluorescence was observed in the blank microbubbles under fluorescence microscopy (×400) (D).
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jah31805-fig-0002: Microbubbles with 5‐Carboxyfluorescein (5‐FAM)–labeled G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) observed under fluorescence microscopy. Microbubbles conjugated with 5‐FAM–labeled GRK4 siRNA were observed under bright‐field microscopy (A) and fluorescence microscopy (B). Only the microbubbles conjugated with 5‐FAM–labeled GRK4 siRNA exhibited green fluorescence. Blank microbubbles under bright‐field microscopy (C). No fluorescence was observed in the blank microbubbles under fluorescence microscopy (×400) (D).

Mentions: Images obtained under bright‐field microscopy revealed that the microbubbles with 5‐FAM‐labeled GRK4 siRNA were homogeneous in size and distributed evenly with no significant aggregation (Figure 2A). Fluorescence microscopy confirmed that the siRNA labeling approach was effective, and only the microbubbles conjugated with the 5‐FAM‐labeled GRK4 siRNA exhibited green fluorescence (Figure 2B). No fluorescence was observed from the control microbubbles (Figure 2C and 2D).


Downregulation of Renal G Protein – Coupled Receptor Kinase Type 4 Expression via Ultrasound ‐ Targeted Microbubble Destruction Lowers Blood Pressure in Spontaneously Hypertensive Rats
Microbubbles with 5‐Carboxyfluorescein (5‐FAM)–labeled G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) observed under fluorescence microscopy. Microbubbles conjugated with 5‐FAM–labeled GRK4 siRNA were observed under bright‐field microscopy (A) and fluorescence microscopy (B). Only the microbubbles conjugated with 5‐FAM–labeled GRK4 siRNA exhibited green fluorescence. Blank microbubbles under bright‐field microscopy (C). No fluorescence was observed in the blank microbubbles under fluorescence microscopy (×400) (D).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121504&req=5

jah31805-fig-0002: Microbubbles with 5‐Carboxyfluorescein (5‐FAM)–labeled G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) observed under fluorescence microscopy. Microbubbles conjugated with 5‐FAM–labeled GRK4 siRNA were observed under bright‐field microscopy (A) and fluorescence microscopy (B). Only the microbubbles conjugated with 5‐FAM–labeled GRK4 siRNA exhibited green fluorescence. Blank microbubbles under bright‐field microscopy (C). No fluorescence was observed in the blank microbubbles under fluorescence microscopy (×400) (D).
Mentions: Images obtained under bright‐field microscopy revealed that the microbubbles with 5‐FAM‐labeled GRK4 siRNA were homogeneous in size and distributed evenly with no significant aggregation (Figure 2A). Fluorescence microscopy confirmed that the siRNA labeling approach was effective, and only the microbubbles conjugated with the 5‐FAM‐labeled GRK4 siRNA exhibited green fluorescence (Figure 2B). No fluorescence was observed from the control microbubbles (Figure 2C and 2D).

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.