背景:糖尿病肾病(DN)是终末期肾脏疾病的主要原因,目前尚无特异性有效的治疗药物。足细胞损伤是DN蛋白尿的有害特征和主要原因。我们之前报道了汤神配方(TSF),一种中草药,对DN有治疗作用。然而,潜在的机制仍然模糊。
目的:本研究旨在探讨TSF对足细胞凋亡的保护作用及其可能的机制。
方法:使用雄性KKAy糖尿病小鼠在小鼠模型中评估TSF的作用,以及晚期糖基化终产物刺激的原代小鼠足细胞。转录因子EB(TFEB)敲低原代足细胞用于机理研究。进行了体内和体外研究,并使用透射电子显微镜评估了结果,免疫荧光染色,和西方印迹。
结果:TSF治疗可减轻足细胞凋亡和结构损伤,蛋白尿减少,减轻KKAy小鼠的肾功能障碍。值得注意的是,两次提取的TSF比三次提取的TSF显示出更显著的蛋白尿减少。TSF治疗显著改变了DN小鼠足细胞中自噬生物标志物p62和LC3的积累以及异常的自噬通量。与体内结果一致,TSF可防止暴露于AGEs的原代足细胞凋亡并激活自噬。然而,自噬-溶酶体抑制剂氯喹抵消了TSF的抗凋亡能力。我们发现TSF增加了糖尿病足细胞中TFEB的核易位,从而上调其几个自噬靶基因的转录。TSF对TFEB的药理激活加速了自噬体向自溶酶体的转化和溶酶体的生物发生,进一步增强自噬通量。相反,TFEB敲低消除了TSF对AGEs刺激的原代足细胞自噬的有利作用。
结论:这些发现表明,TSF似乎通过TFEB介导的自噬-溶酶体系统减弱DN的足细胞凋亡并促进自噬。因此,TSF可以是DN的治疗候选物。
BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease and currently there are no specific and effective drugs for its treatment. Podocyte injury is a detrimental feature and the major cause of albuminuria in DN. We previously reported Tangshen Formula (TSF), a Chinese herbal medicine, has shown therapeutic effects on DN. However, the underlying mechanisms remain obscure.
OBJECTIVE: This study aimed to explore the protective effect of TSF on podocyte apoptosis in DN and elucidate the potential mechanism.
METHODS: The effects of TSF were assessed in a murine model using male KKAy diabetic mice, as well as in advanced glycation end products-stimulated primary mice podocytes. Transcription factor EB (TFEB) knockdown primary podocytes were employed for mechanistic studies. In vivo and in vitro studies were performed and results assessed using transmission electron microscopy, immunofluorescence staining, and western blotting.
RESULTS: TSF treatment alleviated podocyte apoptosis and structural impairment, decreased albuminuria, and mitigated renal dysfunction in KKAy mice. Notably, TSF extracted twice showed a more significant reduction in proteinuria than TSF extracted three times. Accumulation of autophagic biomarkers p62 and LC3, and aberrant autophagic flux in podocytes of DN mice were significantly altered by TSF therapy. Consistent with the in vivo results, TSF prevented the apoptosis of primary podocytes exposed to AGEs and activated autophagy. However, the anti-apoptosis capacity of TSF was countered by the autophagy-lysosome inhibitor chloroquine. We found that TSF increased the nuclear translocation of TFEB in diabetic podocytes, and thus upregulated transcription of its several autophagic target genes. Pharmacological activation of TFEB by TSF accelerated the conversion of autophagosome to autolysosome and lysosomal biogenesis, further augmented autophagic flux. Conversely, TFEB knockdown negated the favorable effects of TSF on autophagy in AGEs-stimulated primary podocytes.
CONCLUSIONS: These findings indicate TSF appears to attenuate podocyte apoptosis and promote autophagy in DN via the TFEB-mediated autophagy-lysosome system. Thus, TSF may be a therapeutic candidate for DN.