G蛋白耦联受体激酶4变异体R65L在盐敏感性高血压发生中的作用及机制

Role of the G protein-coupled receptor kinase 4 R65L variant in salt-sensitive hypertension

    摘要
  • 摘要:
    目的  探讨G蛋白耦联受体激酶4（GRK4）γ变异体R65L在盐敏感性高血压发生中的作用及机制。
    方法  通过成簇规律间隔短回文重复序列关联蛋白 9（CRISPR-Cas9）基因编辑技术将人源性野生型（WT）及R65L变异体GRK4γ基因敲入大鼠基因组，构建人源化GRK4 γ WT及GRK4γ R65L基因敲入大鼠，并分别研究GRK4γ R65L变异体对基础血压及肾脏尿钠排泄功能、4%高盐饮食后血压及肾脏尿钠排泄功能的影响，检测肾脏尿钠排泄关键调控受体多巴胺D1受体（D1R）的表达及磷酸化水平变化情况。
    结果  通过CRISPR-Cas9技术构建了hGRK4γ WT及hGRK4γ R65L基因敲入大鼠模型。正常盐饮食（0.4%氯化钠）条件下，hGRK4γ R65L大鼠与hGRK4γ WT大鼠及野生型SD大鼠在基础血压、尿量及尿钠排泄方面差异均无统计学意义基础尿钠：（1.678 ± 0.336）比（1.730 ± 0.295）比（1.678 ± 0.328） mmol/kg，F = 0.06273，P = 0.9394、尿量：（41.03 ± 6.08）比（38.68 ± 7.03）比（43.57 ± 6.56） mL/kg，F = 1.178，P = 0.327。给予高盐饮食（4%氯化钠）诱导后，与hGRK4γ WT及野生型SD大鼠相比，hGRK4γ R65L大鼠表现出显著的血压升高，伴随尿钠排泄能力显著降低（7.156 ± 0.685）比（6.680 ± 1.235）比（3.722 ± 0.706）mmol/kg，F = 25.00，P＜0.05。肾脏动脉灌注结果显示，hGRK4γ R65L大鼠肾脏多巴胺D1R介导的利尿利钠作用显著下降，提示肾脏多巴胺D1R功能受损。D1R蛋白表达水平未发生明显变化，而D1R蛋白磷酸化水平升高。
    结论  GRK4γ R65L变异体导致肾脏尿钠排泄功能降低和盐敏感性高血压发生，GRK4γ R65L介导的肾脏D1R过度磷酸化是潜在分子机制。

     

    Abstract:
    Objective  To investigate the role and mechanism of the G protein-coupled receptor kinase 4γ (GRK4γ) variant R65L in the development of salt-sensitive hypertension.
    Methods Humanized GRK4γ wild-type (WT) and GRK4γ R65L gene knock-in rats were generated using Clustered regularly interspaced short palindromic repeats-associated protein 9（CRISPR-Cas9）gene editing technology to introduce the human GRK4γ WT or R65L variant into the rat genome. The effects of the GRK4γ R65L variant on basal blood pressure and renal sodium excretion, as well as on blood pressure and renal sodium excretion after a high-salt diet (4% NaCl), were investigated. Changes in the expression and phosphorylation levels of the key renal natriuretic regulatory receptor, dopamine D1 receptor (D1R), were detected.
    Results In this study, humanized GRK4γ WT and GRK4γ R65L gene knock-in rats were successfully constructed using CRISPR-Cas9 technology. Under normal salt diet (0.4% NaCl), no difference was observed in basal blood pressure, urine volume, or urinary sodium excretion among wild-type SD rats, hGRK4γ WT or hGRK4γ R65L rats (baseline urinary sodium: 1.678 ± 0.336 vs. 1.730 ± 0.295 vs. 1.678 ± 0.328 mmol/kg, F = 0.063, P = 0.939; urine volume: 41.03 ± 6.08 vs. 38.68 ± 7.03vs. 43.57 ± 6.56 mL/kg, F = 1.178, P = 0.327). After administration of a high-salt diet (4% NaCl), compared with hGRK4γ WT and wild-type SD rats, hGRK4γ R65L rats exhibited significantly elevated blood pressure and a marked reduction in urinary sodium excretion ( 7.156 ± 0.685 vs. 6.680 ± 1.235 vs. 3.722 ± 0.706 mmol/kg, F = 25.00, P ＜ 0.05). Renal artery perfusion experiments further revealed a significant impairment in diuresis and natriuresis mediated by the renal dopamine D1R in hGRK4γ R65L rats. While the protein expression level of D1R remained unchanged, its phosphorylation level was significantly increased, indicating dysfunction of renal D1R.
    Conclusions The GRK4γ R65L variant leads to impaired renal sodium excretion and salt-sensitive hypertension. Excessive phosphorylation of renal D1R induced by GRK4γ R65L may be the potential mechanism.

     

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