METHODS: We analyzed RIPK3 expression levels in the kidneys of patients with biopsy-proven DKD and animal models of DKD. Additionally, to confirm the clinical significance of circulating RIPK3, RIPK3 was measured by ELISA in plasma obtained from a prospective observational cohort of patients with type 2 diabetes, and estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR), which are indicators of renal function, were followed up during the observation period. To investigate the role of RIPK3 in glomerular damage in DKD, we induced a DKD model using a high-fat diet in Ripk3 knockout and wild-type mice. To assess whether mitochondrial dysfunction and albuminuria in DKD take a Ripk3-dependent pathway, we used single-cell RNA sequencing of kidney cortex and immortalized podocytes treated with high glucose or overexpressing RIPK3.
RESULTS: RIPK3 expression was increased in podocytes of diabetic glomeruli with increased albuminuria and decreased podocyte numbers. Plasma RIPK3 levels were significantly elevated in albuminuric diabetic patients than in non-diabetic controls (p = 0.002) and non-albuminuric diabetic patients (p = 0.046). The participants in the highest tertile of plasma RIPK3 had a higher incidence of renal progression (hazard ratio [HR] 2.29 [1.05-4.98]) and incident chronic kidney disease (HR 4.08 [1.10-15.13]). Ripk3 knockout improved albuminuria, podocyte loss, and renal ultrastructure in DKD mice. Increased mitochondrial fragmentation, upregulated mitochondrial fission-related proteins such as phosphoglycerate mutase family member 5 (PGAM5) and dynamin-related protein 1 (Drp1), and mitochondrial ROS were decreased in podocytes of Ripk3 knockout DKD mice. In cultured podocytes, RIPK3 inhibition attenuated mitochondrial fission and mitochondrial dysfunction by decreasing p-mixed lineage kinase domain-like protein (MLKL), PGAM5, and p-Drp1 S616 and mitochondrial translocation of Drp1.
CONCLUSIONS: The study demonstrates that RIPK3 reflects deterioration of renal function of DKD. In addition, RIPK3 induces diabetic podocytopathy by regulating mitochondrial fission via PGAM5-Drp1 signaling through MLKL. Inhibition of RIPK3 might be a promising therapeutic option for treating DKD.
方法:我们分析了活检证实的DKD患者和DKD动物模型的肾脏中RIPK3的表达水平。此外,为了证实循环RIPK3的临床意义,通过ELISA从2型糖尿病患者的前瞻性观察队列中获得的血浆中测量RIPK3,和估计的肾小球滤过率(eGFR)和尿白蛋白-肌酐比值(UACR),它们是肾功能的指标,在观察期间进行了随访。探讨RIPK3在DKD肾小球损伤中的作用。我们在Ripk3基因敲除和野生型小鼠中使用高脂饮食诱导了DKD模型。为了评估DKD中的线粒体功能障碍和蛋白尿是否采用Ripk3依赖性途径,我们对高糖或过表达RIPK3的肾皮质和永生化足细胞进行了单细胞RNA测序.
结果:RIPK3在糖尿病肾小球足细胞中的表达增加,蛋白尿增加,足细胞数量减少。蛋白尿型糖尿病患者的血浆RIPK3水平明显高于非糖尿病对照组(p=0.002)和非蛋白尿型糖尿病患者(p=0.046)。血浆RIPK3最高三元组的参与者肾脏进展(危险比[HR]2.29[1.05-4.98])和慢性肾病(HR4.08[1.10-15.13])的发生率较高。Ripk3敲除改善蛋白尿,足细胞丢失,DKD小鼠肾脏超微结构。线粒体碎片化增加,上调线粒体裂变相关蛋白,如磷酸甘油酸变位酶家族成员5(PGAM5)和动力蛋白相关蛋白1(Drp1),Ripk3敲除DKD小鼠足细胞中线粒体ROS降低。在培养的足细胞中,RIPK3抑制通过减少p-混合谱系激酶结构域样蛋白(MLKL)减弱线粒体裂变和线粒体功能障碍,PGAM5和p-Drp1S616以及Drp1的线粒体易位。
结论:研究表明RIPK3反映了DKD肾功能的恶化。此外,RIPK3通过MLKL通过PGAM5-Drp1信号调节线粒体裂变诱导糖尿病足细胞病。抑制RIPK3可能是治疗DKD的有希望的治疗选择。