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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

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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

Hefei Huang et al. J Am Heart Assoc.

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.

Keywords: G protein–coupled receptor kinase type 4; blood pressure; kidney; ultrasound‐targeted microbubble destruction.

Figures

Figure 1
Figure 1
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).
Figure 2
Figure 2
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).
Figure 3
Figure 3
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).
Figure 4
Figure 4
Effects of ultrasound‐targeted microbubble destruction (UTMD)–mediated G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA delivery on blood pressure. UTMD‐mediated GRK4 small interfering RNA delivery decreased systolic blood pressure (SBP) (A) and mean arterial pressure (MAP) (B) in spontaneously hypertensive rats (n=5, *P<0.05 vs control).
Figure 5
Figure 5
Effects of ultrasound‐targeted microbubble destruction (UTMD)–mediated G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) delivery on sodium excretion. A and B, Sodium excretion (A) and urine volume (B) of spontaneously hypertensive rats (SHRs) were recorded after UTMD treatment (n=5, *P<0.05 vs control). C and D, Renal function (C, serum urea nitrogen [BUN]; D, serum creatinine) of the SHRs after treatment with UTMD‐mediated GRK4 siRNA for 20 days. E, Representative photographs of Masson trichrome staining of the UTMD‐mediated GRK4 siRNA deliveries in the kidneys of SHRs compared with controls. a, Glomerulosclerosis was evident in the SHR controls. b, The ×20 amplification of the black frame of figure (a). c, Significantly decreased fibrotic areas in the UTMD‐mediated GRK4 siRNA deliveries in the SHRs. d, The amplified part in the black frame of figure (c). F, The renal glomerular sclerosis indices for Masson staining (n=5, *P<0.05 vs control).
Figure 6
Figure 6
Effects of ultrasound‐targeted microbubble destruction (UTMD)–mediated G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) delivery on dopamine type 1 receptor (D1R) phosphorylation and D1R‐mediated natriuresis. A and B, Effects of UTMD‐mediated GRK4 siRNA delivery for 20 days on renal D1R expression (A) and phosphorylation (B) in spontaneously hypertensive rats (SHRs) (n=5, *P<0.05 vs control). C and D, Effects of fenoldopam, a D1‐like receptor agonist, on sodium excretion and urine volumes in the UTMD (20 days)‐treated SHRs (n=5, *P<0.05 vs control).
Figure 7
Figure 7
Schematic representation of the effects of ultrasound‐targeted microbubble destruction (UTMD)–mediated G protein–coupled receptor kinase type 4 (GRK4) small interfering RNA (siRNA) delivery on blood pressure in spontaneously hypertensive rats (SHRs).

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