Nishad Rajkishor, Meshram Prajakta, Singh Ashish Kumar, Reddy G Bhanuprakash, Pasupulati Anil Kumar
Biochemistry, University of Hyderabad, Hyderabad, India.
Biochemistry, National Institute of Nutrition, Hyderabad, India.
BMJ Open Diabetes Res Care. 2020 Jun;8(1). doi: 10.1136/bmjdrc-2020-001203.
Advanced glycation end-products (AGEs) are implicated in the pathogenesis of diabetic nephropathy (DN). Previous studies have shown that AGEs contribute to glomerulosclerosis and proteinuria. Podocytes, terminally differentiated epithelial cells of the glomerulus and the critical component of the glomerular filtration barrier, express the receptor for AGEs (RAGE). Podocytes are susceptible to severe injury during DN. In this study, we investigated the mechanism by which AGEs contribute to podocyte injury.
Glucose-derived AGEs were prepared in vitro. Reactivation of Notch signaling was examined in AGE-treated human podocytes (in vitro) and glomeruli from AGE-injected mice (in vivo) by quantitative reverse transcription-PCR, western blot analysis, ELISA and immunohistochemical staining. Further, the effects of AGEs on epithelial to mesenchymal transition (EMT) of podocytes and expression of fibrotic markers were evaluated.
Using human podocytes and a mouse model, we demonstrated that AGEs activate Notch1 signaling in podocytes and provoke EMT. Inhibition of RAGE and Notch1 by FPS-ZM1 (N-Benzyl-4-chloro-N-cyclohexylbenzamide) and DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester), respectively, abrogates AGE-induced Notch activation and EMT. Inhibition of RAGE and Notch1 prevents AGE-induced glomerular fibrosis, thickening of the glomerular basement membrane, foot process effacement, and proteinuria. Furthermore, kidney biopsy sections from people with DN revealed the accumulation of AGEs in the glomerulus with elevated RAGE expression and activated Notch signaling.
The data suggest that AGEs activate Notch signaling in the glomerular podocytes. Pharmacological inhibition of Notch signaling by DAPT ameliorates AGE-induced podocytopathy and fibrosis. Our observations suggest that AGE-induced Notch reactivation in mature podocytes could be a novel mechanism in glomerular disease and thus could represent a novel therapeutic target.
晚期糖基化终末产物（AGEs）与糖尿病肾病（DN）的发病机制有关。先前的研究表明，AGEs会导致肾小球硬化和蛋白尿。足细胞是肾小球终末分化的上皮细胞，也是肾小球滤过屏障的关键组成部分，表达AGEs受体（RAGE）。在DN过程中，足细胞易受严重损伤。在本研究中，我们探究了AGEs导致足细胞损伤的机制。
体外制备葡萄糖衍生的AGEs。通过定量逆转录聚合酶链反应、蛋白质免疫印迹分析、酶联免疫吸附测定和免疫组织化学染色，检测AGE处理的人足细胞（体外）和注射AGE的小鼠肾小球（体内）中Notch信号的重新激活。此外，评估了AGEs对足细胞上皮-间质转化（EMT）和纤维化标志物表达的影响。
利用人足细胞和小鼠模型，我们证明AGEs激活足细胞中的Notch1信号并引发EMT。分别用FPS-ZM1（N-苄基-4-氯-N-环己基苯甲酰胺）和DAPT（N-[N-(3,5-二氟苯乙酰基)-L-丙氨酰基]-S-苯基甘氨酸叔丁酯）抑制RAGE和Notch1，可消除AGE诱导的Notch激活和EMT。抑制RAGE和Notch1可预防AGE诱导的肾小球纤维化、肾小球基底膜增厚、足突消失和蛋白尿。此外，DN患者的肾活检切片显示肾小球中AGEs积聚，RAGE表达升高且Notch信号激活。
数据表明AGEs激活肾小球足细胞中的Notch信号。DAPT对Notch信号的药理学抑制可改善AGE诱导的足细胞病变和纤维化。我们的观察结果表明，成熟足细胞中AGE诱导的Notch重新激活可能是肾小球疾病的一种新机制，因此可能代表一个新的治疗靶点。
