Abstract：
BACKGROUND: Receptor-interacting protein kinase (RIPK)3 is an essential molecule for necroptosis and its role in kidney fibrosis has been investigated using various kidney injury models. However, the relevance and the underlying mechanisms of RIPK3 to podocyte injury in albuminuric diabetic kidney disease (DKD) remain unclear. Here, we investigated the role of RIPK3 in glomerular injury of DKD.
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.
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.
摘要：
背景：受体相互作用蛋白激酶（RIPK）3是坏死性凋亡的重要分子，已使用各种肾损伤模型研究了其在肾纤维化中的作用。然而,RIPK3与白蛋白尿型糖尿病肾病(DKD)足细胞损伤的相关性和潜在机制尚不清楚.这里,我们研究了RIPK3在DKD肾小球损伤中的作用。
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。
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。
