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

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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.