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

慢性肾脏病(CKD)影响着大约10%的世界人口，是全球主要的公共卫生负担。在CKD的一个亚型中，肾小球滤器受损导致尿中血浆蛋白大量丢失，从而导致低蛋白血症、全身性浮肿和肾病综合征的临床表现。如果无法治疗，这种疾病不可避免地会进展为终末期肾功能衰竭，需要肾脏替代治疗或移植才能存活。人类基因如果突变，会导致单基因形式的肾病综合征，这为其发病机制提供了新的见解，并确定肾小球足细胞是主要的损害部位。足细胞是终末分化的细胞，再生能力非常有限。因此，受损的足细胞无法补充，超过20%的足细胞丢失会导致不可逆转的肾小球硬化。在遗传学研究中，我们发现编码进化高度保守的Keops复合体的4个亚单位的LAGE3、OSGEP、TP53RK或TPRKB突变是31个无关家系中激素抵抗型肾病综合征合并小头畸形的新的单基因原因。Keops复合体介导tRNA的一种重要的转录后修饰，称为t6A修饰，对核糖体蛋白质翻译的准确性和效率至关重要。我们的初步数据显示，敲除Keops复合体的不同基因会导致永生化人足细胞的内质网应激和细胞凋亡。我们推测这两种机制在KeOPS相关肾病综合征的发病机制中起作用。我们的第一个目标是深入分析不同Keops基因敲除后足细胞损伤的分子机制。特别是，我们将分析Keops相关的内质网应激对其他与足细胞功能高度相关的信号通路的影响，以及对足细胞裂隙-横隔膜蛋白翻译后处理的影响。此外，我们将研究这些细胞中容易出错的蛋白质翻译如何影响细胞能量平衡，以及是否像在其他疾病中看到的那样，错误折叠的蛋白质积累。下一步，我们将测试不同的药理策略在Keops基因敲除的足细胞中的有效性，并建立用于高通量药物筛选的荧光内质网应激报告足细胞系。为了确定Keops复合体在成熟的终末分化的足细胞和肾小球发育过程中的功能，我们将为Osgep基因产生足细胞特异的敲除小鼠。利用这些小鼠，我们将确定KeOPS相关肾小球疾病的相关病理生理特征，进行蛋白质组学分析，以分析KeOPS复杂功能障碍对不同年龄足细胞蛋白质组的影响，并测试体内的治疗干预措施。

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.