GRK4调控程序性坏死复合物RIPK3磷酸化在肾缺血再灌注损伤中的作用及机制研究

Renal ischemiareperfusion injury is an important cause of acute renal failure and other clinical kidney diseases, and proximal tubule necrosis is the main pathological basis. Previous studies have shown that programmed necrosis plays a key role in renal I/R injury，and the activation of RIPK3 phosphorylation in necrosomeis a key step in initiating necroptosis.However,the underlying mechanism forRIPK3 phosphorylation remains unclear. GRK, as a kinase of phosphorylation,extensively regulates a variety of substrates. Our preliminaryresearch showedthat GRK4 was abundant in proximal tubule cells, and the expression was elevatedafter renal I/R injury. GRK4 knockout mice attenuated renal I/R injury; overexpression of GRK4-A142V aggravated the renal I/Rdamage,along with RIPK3 phosphorylationactiviation.In addition, we also found that GRK4 and RIPK3 may co-exist, whose changes were significantly positivecorrelation.Therefore, we hypothesize that GRK4 is involved in renal I/R injuryby activating RIPK3 phosphorylation to initiate necroptosis. Inhibition of GRK4 may have an important protective effect on renal I/R injury. This study is to explore the detailed mechanism of GRK4 regulating RIPK3 phosphorylationmediated renal necroptosis, and to provide a noveltreatingtarget for renal I/R prevention.

肾缺血再灌注（I/R）损伤是导致急性肾功能衰竭等多种肾脏疾病的重要原因，近曲小管坏死是主要病理基础。研究显示程序性坏死在肾脏I/R损伤中发挥关键作用，坏死复合物中RIPK3磷酸化激活是启动程序性坏死的关键环节，但其中调控RIPK3磷酸化的机制尚不清楚。GRK是调节磷酸化的激酶，在机体病理生理过程中发挥重要作用。我们预实验发现：GRK4在近曲小管表达丰富，肾脏I/R损伤后表达升高；敲除GRK4可减轻损伤；过表达GRK4肾脏I/R损伤加重，伴RIPK3磷酸化活性增强；此外我们还发现GRK4与RIPK3存在共连接可能，两者变化呈正相关。我们推测：GRK4可能通过磷酸化激活RIPK3，启动程序性坏死致肾损伤；抑制GRK4对改善肾I/R损伤具有重要保护作用。本课题拟在肾I/R损伤动物和细胞实验基础上，探讨GRK4与RIPK3的激活在肾脏程序性坏死中作用机制，为临床肾脏保护提供关键分子靶点和新的思路。

临床上，肾缺血再灌注损伤具有较高的发病率和死亡率，其治疗目前仍是肾脏器官保护领域的重要难题。G蛋白偶联受体激酶(GRK)已被报道在原发性高血压和心肌梗死中发挥重要作用，但其在肾缺血再灌注损伤中的作用尚不明确。在本研究中，我们发现在肾脏GRK中，GRK4是肾缺血再灌注损伤中变化最显著的亚型。通过功能增加和功能丧失实验显示，GRK4过表达加重了急性肾缺血再灌注损伤，而小管特异性敲除GRK4可减轻缺血再灌注引起的肾功能障碍。程序性坏死是GRK4介导的肾小管细胞死亡的主要类型，因为GRK4过表达显著增加了RIPK1的表达和磷酸化，导致肾I/R损伤后RIPK3和MLKL磷酸化。通过免疫共沉淀、质谱结合siRNA筛选研究，我们发现STAT1是与GRK4直接结合的蛋白。肾脏缺血再灌注损伤候，STAT1与GRK4在肾小管细胞的细胞核中共定位明显增强。进一步发现，GRK4磷酸化了STAT1的S727位点，其失活突变可以有效逆转GRK4介导的RIPK1激活和肾小管细胞损伤。在转化方面，通过纳米颗粒药物递送肾靶向沉默GRK4，可以显著改善肾缺血再灌注损伤损伤。因此，这些发现提示GRK4可引起肾坏死并加重肾缺血再灌注损伤损伤，下调GRK4的表达可能为肾脏保护提供一种有前景的治疗策略。