SLC26A2 function in the kidney

硫酸盐是正常细胞功能所必需的。例如，它是促成正常软骨结构的蛋白聚糖分子的磺化所必需的。硫酸软骨转运蛋白（SLC26A2）的功能缺失突变是一组常染色体隐性软骨发育不良的原因。在这些疾病中，软骨细胞对硫酸盐的摄取受损导致蛋白多糖硫酸化减少。然而，SLC26A2不仅在软骨中表达，也在其他组织中表达，如肾脏。在那里，它特异性地定位于近端小管细胞的顶端（管腔）刷状边界膜。已知硫酸盐在肾脏，特别是近端小管中被自由过滤和广泛重吸收，但SLC26A2在肾脏硫酸盐运输中的确切功能尚未被探索。我们拟探讨SLC26A2的肾功能及其调控。在初步研究中，我们使用CRISPR/Cas9系统，在近端小管细胞模型OK（负鼠肾）细胞中敲除SLC26A2。我们建立了一个放射性试验，并证明SLC26A2敲除细胞中硫酸盐的细胞摄取减少。在这个项目的第一部分，我们的目标是进一步表征硫酸盐在OK细胞中的转运，包括转运体，它们的定位和相互作用，以及各种其他离子对硫酸盐转运的影响，重点是SLC26A2。Slc26a2基因敲除在小鼠中是致命的。因此，我们建议产生Slc26a2的近端小管特异性敲除，以确定Slc26a2在肾脏硫酸盐运输中的作用。我们将进行转运试验，比较敲除小鼠和对照组血液和尿液中的硫酸盐水平。最后，我们的目标是研究决定SLC26A2运输和顶膜插入的蛋白质结构域。我们将在小鼠模型中研究膳食硫酸盐对Slc26a2表达的影响。我们希望这些研究能为今后SLC26A2的转译研究奠定基础。

Sulfate is essential for normal cellular function. For example, it is required for the sulfation of proteoglycans, molecules that contribute to the structure of normal cartilage. Loss-of-function mutations in a cartilage sulfate transporter (SLC26A2) are the cause of a group of autosomal recessive chondrodysplasias. In these disorders, impaired sulfate uptake into chondrocytes leads to reduced proteoglycan sulfation. However, SLC26A2 is not only expressed in cartilage, but also in other tissues, such as the kidney. There, it specifically localizes to the apical (luminal) brush border membrane of proximal tubular cells. Sulfate is known to be freely filtered and extensively reabsorbed in the kidney, in particular the proximal tubule, but the exact function of SLC26A2 for renal sulfate transport has not been explored.We here propose to explore the renal function and regulation of SLC26A2. In preliminary studies, using the CRISPR/Cas9 system, we knocked out SLC26A2 in a cellular model of the proximal tubule, OK (opossum kidney) cells. We established a radioactive assay and demonstrated reduced cellular uptake of sulfate in SLC26A2 knockout cells.In the first part of this project, we aim to further characterize sulfate transport in OK cells regarding the transporters involved, their localization and interplay, as well as effects of various other ions on sulfate transport, with a focus on SLC26A2.Global Slc26a2 knockout is lethal in mice. We thus propose to generate a proximal tubule-specific knockout of Slc26a2 to define the role of Slc26a2 in renal sulfate transport. We will perform transport assays and compare sulfate levels in blood and urine between knockout mice and controls.Finally, we aim to study protein domains that determine SLC26A2 trafficking and apical membrane insertion. We will study the effect of dietary sulfate on Slc26a2 expression in the mouse model.We hope that these studies will form a basis for future translational studies on SLC26A2.