Towards understanding the role of the negatively charged sugar sialic acid on embryonic development and kidney function

所有的动物细胞都被密集排列的聚糖覆盖，即糖萼，它由糖蛋白、糖脂和蛋白聚糖组成。糖缀合物位于细胞的外表面，在细胞相互作用中起关键作用，指导生理和病理过程。通常，非还原性末端（最外层）的糖，在大多数聚糖中是酸性的非纤维素唾液酸（Sia），决定着聚糖的性质。在唾液聚糖合成之前，Sia必须被激活为CMP-Sia。这一重要步骤是由核酶CMP-Sia合成酶（CMAS）催化的。在为当前应用奠定基础的研究中，我们发现CMAS基因缺失会导致e9前后小鼠的胚胎死亡，而CMAS [nls]小鼠中CMAS表达水平的整体降低会导致出生后3天内肾衰竭。虽然导致Cmas[-/-]小鼠胚胎死亡的原因尚不清楚，但Cmas[nls]小鼠的致死率可能归因于形成肾小球滤过屏障内脏层的足细胞的病理改变。重要的是，Cmas[nls]-小鼠表现出显著的肾素敲除表型特征，表明唾液化对于肾素作为裂隙隔膜结构成分的功能是必不可少的。几乎所有与蛋白尿相关的肾小球肾病都存在Sia缺失。因此，更好地了解唾液化糖缀合物的分子机制对于开发保护肾功能的治疗方法至关重要。本提案旨在了解唾液化如何影响(i)肾脏功能和（ii）小鼠胚胎发育。为了实现第一个目标，已经生成了两个足细胞特异性(P)小鼠模型，它们的P- CMAS表达减少（P- CMAS[-/-]）或减少（P- CMAS [nls]）。这些小鼠模拟肾病，如微小变化病，分别在出生后2个月或3个月内死亡。与Cmas[nls]-小鼠相似，该疾病的发展伴随着肾素上Sia的逐渐丧失。这些小鼠的存活时间较长，使它们成为体内研究Sia对狭缝隔膜组装和功能的影响、肾素作为支架和信号平台功能的意义、其对足细胞形态的贡献、以及足细胞与肾小体内其他细胞类型之间的运动和相互作用的理想工具。为了发现Sia在胚胎发育中的功能，我们将在Cmas[-/-]小鼠中评估导致胚胎致死的形态学改变的发生和质量。为了研究Sia在体内对外胚层的影响，我们将建立合适的小鼠模型。此外，为了进行生化研究，已经建立了所有基因型的胚胎干细胞系，并将产生胚外内胚层干细胞（XEN细胞）。

All animal cells are covered by a dense array of glycans, the glycocalyx, which is composed of glycoproteins, glycolipids, and proteoglycans. Localized at the outer face of the cell, glycoconjugates play a pivotal role in cellular interactions steering physiological as well as pathological processes. Frequently, the non-reducing end (outermost) sugar, in the majority of glycans the acidic nonulose sialic acid (Sia), is determining the nature of the glycans. Prior to the synthesis of sialo-glycans, Sia has to be activated to CMP-Sia. This essential step is catalysed by the nuclear enzyme CMP-Sia synthetase (CMAS). In studies building the basis for the current application we found that genetic depletion of CMAS causes embryonic lethality in mice around E 9, whereas an overall reduction of the CMAS expression level in Cmas[nls] mice results in kidney failure within 3 days after birth. While the circumstances leading to embryonic death in Cmas[-/-] mice are not yet understood, lethality in Cmas[nls] mice could be attributed to pathological changes in podocytes forming the visceral layer of the glomerular filtration barrier. Importantly, Cmas[nls]-mice exhibit significant phenotypic traits of nephrin knockouts, demonstrating that sialylation is indispensable for nephrins function as structural component of the slit diaphragm. A loss of Sia is found in almost all human glomerular nephropathies associated with proteinuria. Therefore, a better understanding of the molecular mechanisms involving sialylated glycoconjugates is essential for the development of therapies to preserve kidney function. This proposal aims at understanding how sialylation impacts (i) kidney function and (ii) mouse embryonic development. To achieve the first goal, two podocyte-specific (P) mouse models with either depleted (P-Cmas[-/-]) or reduced (P-Cmas[nls]) CMAS expression have already been generated. These mice mimic nephropathies such as minimal change disease and die within 2 or 3 months after birth, respectively. Similar to Cmas[nls]-mice, the development of the disease is accompanied by a progressive loss of Sia on nephrin. The longer survival of these mice makes them ideal tools for in vivo investigations on the impact of Sia on assembly and function of the slit diaphragm, the significance of nephrin functions as scaffold and as signalling platform, as well as its contribution to podocyte morphology, and on the movement and the interplay between podocytes and other cell types in the renal corpuscle. To discover the functions of Sia in embryonic development, the onset and quality of morphological changes that cause embryonic lethality will be evaluated in Cmas[-/-] mice. To address the impact of Sia exclusively on the epiblast in vivo, an appropriate mouse model will be established. Moreover, for biochemical investigations embryonic stem cell lines from all genotypes have already been established and extraembryonic endoderm stem cells (XEN cells) will be generated.