FAK调控代谢重编码促发DKD肾小管上皮细胞脂质沉积以及黄葵素干预效应的研究

Metabolic dysregulation can inhibit free fat oxidation, cause lipid deposition in the tubular epithelial cells (TECs), which has been shown to play a crucial role in the development and progression of diabetic kidney disease (DKD). Therefore reducing lipid deposition could be a target to treat DKD. In 2018, it was first reported that focal Adhesion Kinase (FAK) could control lipid oxidation in skeletal muscle. However, until now the role of FAK in kidney is unknown. Our previous research has shown that total flavones extracted from Abelmoschus manihot L.(Malvaceae) medic (TFA) has a definite effect on DKD, but unfortunately the mechanism has not been elucidated. We think that FAK can regulate lipid deposition in TECs and TFA can exert an effect on this pathway. The hypothesis will be verified by in vivo and in vitro study. Step one: to observe the activation of FAK and the lipid deposition in TECs in the db/db mice and the palmitic acid treated HK-2 cells; step two: to knock out/ high express FAK in HK-2 cells and the TECs in db/db mice by the CRISPR/Cas9 genome editing technique, then the role of FAK in the lipid deposition in TECs with DKD and the effect of TFA on this target will be studied in vivo and in vitro.

代谢障碍抑制脂肪酸氧化，引起肾小管脂质沉积，参与糖尿病肾脏病(diabetic kidney disease, DKD)发生发展，因此逆转脂质沉积是治疗DKD的靶点。局部粘着斑激酶(Focal Adhesion Kinase，FAK)调控骨骼肌脂肪酸氧化于2018年首次被提出，但它在肾脏中的作用尚不明确。课题组前期研究发现黄葵素对DKD疗效明确，但具体机制仍在研究中。我们认为FAK调控肾小管上皮细胞脂质沉积，而黄葵素可抑制FAK的活化发挥肾保护作用。本研究拟从体内外实验来验证该假说，第一步通过db/db小鼠及棕榈酸干预HK-2细胞来观察FAK的活化与肾小管脂质沉积的变化，及黄葵素的效应；第二步运用CRISPR/Cas9基因组编辑技术制备HK-2及db/db小鼠肾小管上皮细胞特异性FAK敲除/高表达模型来验证FAK在DKD肾小管上皮细胞脂质沉积中的作用，及黄葵素对该靶点的干预效应。

代谢障碍抑制脂肪酸氧化，引起肾小管脂质沉积，参与糖尿病肾脏病(diabetic kidney disease, DKD)发生发展，因此逆转脂质沉积是治疗DKD的靶点。局部粘着斑激酶(Focal Adhesion Kinase，FAK)调控骨骼肌脂肪酸氧化于2018年首次被提出，但它在肾脏中的作用尚不明确。课题组前期研究发现黄葵素对DKD疗效明确，但具体机制仍在研究中。我们认为FAK调控肾小管上皮细胞脂质沉积，而黄葵素可抑制FAK的活化发挥肾保护作用。本研究从体内外实验来验证该假说，第一步通过db/db小鼠及棕榈酸干预HK-2细胞来观察FAK的活化与肾小管脂质沉积的变化，及黄葵素的效应；第二步运用CRISPR/Cas9基因组编辑技术制备HK-2及db/db小鼠肾小管上皮细胞特异性FAK敲除/高表达模型来验证FAK在DKD肾小管上皮细胞脂质沉积中的作用，及黄葵素对该靶点的干预效应。.本研究结果显示db/db小鼠随着糖尿病病程加重，肾细胞凋亡增加，肾小管脂质沉积增多，肾脏纤维化明显，线粒体功能下降，伴随着FAK磷酸化上调，而中药黄葵素可显著逆转上述肾脏改变，降低FAK的磷酸化。同时，棕榈酸干预HK-2细胞引起细胞凋亡增加，细胞内脂质沉积明显，纤维化相关基因表达上调，FAK磷酸化上调，黄葵素可抑制上述改变。建立FAK敲除模型后，棕榈酸干预的细胞损伤效应可明显降低。因此本研究证实了FAK的活化参与DKD肾损伤机制，而中药黄葵素可通过干预该靶点发挥DKD肾保护作用。