Disease mechanisms of podocyte injury caused by mutations in genes encoding proteins of the tRNA modifying KEOPS complex.

由编码 tRNA 修饰 KEOPS 复合体蛋白的基因突变引起的足细胞损伤的疾病机制。

• 批准号: 391152220
• 负责人: Privatdozentin Dr. Daniela Anne Braun
• 金额: --
• 依托单位: Medizinische Klinik D: Allg. Innere Medizin und Notaufnahme sowie Nieren- und Hochdruckkrankheiten und Rheumatologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/391152220?language=en
• 关键词: Disease; mechanisms; podocyte; injury; caused

Chronic kidney disease (CKD), affecting approximately 10% of the world population, is a major global public health burden. In a subset of CKD, damage to the renal glomerular filter causes substantial loss of plasma proteins in the urine, thus resulting in hypoalbuminemia, generalized edema, and the clinical presentation of nephrotic syndrome. If refractory to treatment, the disease inevitably progresses to end-stage renal failure requiring renal replacement therapy or transplantation for survival. The identification of human genes that, if mutated, cause monogenic forms of nephrotic syndrome has provided novel insights into its pathogenesis and has identified glomerular podocytes, specialized epithelial cells, as the primary site of damage. Podocytes are terminally differentiated cells with very limited regenerative capacity. Consequently, injured podocytes cannot be replenished and loss of more than 20% of podocytes causes irreversible glomerulosclerosis. In genetic studies, we identified mutations in LAGE3, OSGEP, TP53RK, or TPRKB encoding the 4 subunits of the evolutionarily highly conserved KEOPS complex as novel monogenic causes of steroid-resistant nephrotic syndrome with microcephaly in 31 unrelated families. The KEOPS complex mediates an essential posttranscriptional modification of tRNA, known as t6A modification that crucial for accuracy and efficiency of protein translation at the ribosome. Our preliminary data show that knockdown of different genes of the KEOPS complex causes ER stress and apoptosis in immortalized human podocytes. We hypothesize that these two mechanisms contribute to the pathogenesis of KEOPS-related nephrotic syndrome. Our first objective is to perform an in-depth analysis of the molecular mechanisms of podocyte injury following knockdown of different KEOPS genes. In particular, we will analyze the impact of KEOPS-related ER stress on other cross-talking signaling pathways with high relevance for podocyte function as well as on posttranslational processing of podocyte slit-diaphragm proteins. Furthermore, we will study how error-prone protein translation in these cells affects the cellular energy balance and whether, as seen in other diseases, misfolded proteins accumulate. In a next step, we will test different pharmacological strategies for their efficacy in podocytes with KEOPS gene knockdown and establish a fluorescent ER stress reporter podocyte cell line for high-throughput drug-screening. In order to determine the function of the KEOPS complex in mature, terminally differentiated podocytes and during glomerular development, we will generate a podocyte-specific knockout mouse for the gene Osgep. Using these mice, we will determine relevant pathophysiological features of KEOPS-related glomerular disease, perform proteomics to analyze the impact of KEOPS complex dysfunction on the podocyte proteome at different ages, and test therapeutic interventions in vivo.

慢性肾脏疾病（CKD）影响着约10%的世界人口，是全球主要的公共卫生负担。在CKD的一个子集中，肾小球滤过器的损伤导致尿液中血浆蛋白的大量损失，从而导致低白蛋白血症、全身性水肿和肾病综合征的临床表现。如果治疗无效，疾病不可避免地进展为终末期肾衰竭，需要肾脏替代治疗或移植才能生存。人类基因的鉴定，如果突变，导致单基因形式的肾病综合征提供了新的见解，其发病机制，并确定肾小球足细胞，专门的上皮细胞，作为主要的损害部位。足细胞是具有非常有限的再生能力的终末分化细胞。因此，受损的足细胞不能得到补充，超过20%的足细胞损失会导致不可逆的肾小球硬化。在遗传学研究中，我们发现LAGE 3、OSGEP、TP 53 RK或TPRKB编码进化上高度保守的KEOPS复合物的4个亚基的突变是31个无关家族中激素耐药肾病综合征伴小头畸形的新单基因原因。KEOPS复合物介导tRNA的一种重要的转录后修饰，称为t6A修饰，这对核糖体上蛋白质翻译的准确性和效率至关重要。我们的初步数据表明，敲低不同基因的KEOPS复合物导致ER应激和细胞凋亡的永生化人足细胞。我们假设这两种机制有助于KEOPS相关肾病综合征的发病机制。我们的第一个目标是进行深入的分析足细胞损伤的分子机制后，敲低不同的KEOPS基因。特别是，我们将分析KEOPS相关的ER应激对其他与足细胞功能高度相关的串扰信号通路以及对足细胞裂膜蛋白的翻译后加工的影响。此外，我们还将研究这些细胞中易出错的蛋白质翻译如何影响细胞能量平衡，以及是否像其他疾病中那样，错误折叠的蛋白质积累。在下一步中，我们将测试不同的药理学策略在KEOPS基因敲低的足细胞中的功效，并建立荧光ER应激报告足细胞系用于高通量药物筛选。为了确定KEOPS复合物在成熟的终末分化足细胞和肾小球发育过程中的功能，我们将产生一个足细胞特异性基因Osgep敲除小鼠。使用这些小鼠，我们将确定KEOPS相关肾小球疾病的相关病理生理学特征，进行蛋白质组学分析KEOPS复杂功能障碍对不同年龄足细胞蛋白质组的影响，并在体内测试治疗干预措施。