Ristov Marie-Christin, Lange Tim, Artelt Nadine, Nath Neetika, Kuss Andreas W, Gehrig Jochen, Lindenmeyer Maja, Cohen Clemens D, Gul Sheraz, Endlich Karlhans, Völker Uwe, Endlich Nicole
Institute of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany.
Institute of Bioinformatics, University Medicine Greifswald, Greifswald, Germany.
Front Cell Dev Biol. 2022 May 16;10:838086. doi: 10.3389/fcell.2022.838086. eCollection 2022.
Chronic kidney disease (CKD) is a major public health burden affecting more than 500 million people worldwide. Podocytopathies are the main cause for the majority of CKD cases due to pathogenic morphological as well as molecular biological alterations of postmitotic podocytes. Podocyte de-differentiation is associated with foot process effacement subsequently leading to proteinuria. Since currently no curative drugs are available, high throughput screening methods using a small number of animals are a promising and essential tool to identify potential drugs against CKD in the near future. Our study presents the implementation of the already established mouse Glom as a semi-automated high-throughput screening method-shGlom-allowing the analysis of several hundreds of FDA-verified compounds in combination with downstream pathway analysis like transcriptomic and proteomic analyses from the same samples, using a small number of animals. In an initial prescreening we have identified vitamin D3 and its analog calcipotriol to be protective on podocytes. Furthermore, by using RT-qPCR, Western blot, and RNA sequencing, we found that mRNA and protein expression of nephrin, the vitamin D receptor and specific podocyte markers were significantly up-regulated due to vitamin D3- and calcipotriol-treatment. In contrast, kidney injury markers were significantly down-regulated. Additionally, we found that vitamin D3 and calcipotriol have had neither influence on the expression of the miR-21 and miR-30a nor on miR-125a/b, a miRNA described to regulate the vitamin D receptor. In summary, we advanced the established mouse Glom to a semi-automated high-throughput assay and combined it with downstream analysis techniques by using only a minimum number of animals. Hereby, we identified the vitamin D signaling pathway as podocyte protective and to be counteracting their de-differentiation.
慢性肾脏病（CKD）是一项重大的公共卫生负担，全球有超过5亿人受其影响。足细胞病是大多数CKD病例的主要病因，这是由于有丝分裂后足细胞发生了致病性形态学以及分子生物学改变。足细胞去分化与足突消失相关，进而导致蛋白尿。由于目前尚无治愈性药物，使用少量动物的高通量筛选方法是在不久的将来识别抗CKD潜在药物的一种有前景且必不可少的工具。我们的研究展示了已建立的小鼠肾小球（Glom）作为一种半自动化高通量筛选方法——短发夹RNA介导的肾小球（shGlom）——的应用，该方法允许使用少量动物，结合下游通路分析，如对同一批样品进行转录组学和蛋白质组学分析，来分析数百种经美国食品药品监督管理局（FDA）验证的化合物。在初步预筛选中，我们已确定维生素D3及其类似物卡泊三醇对足细胞具有保护作用。此外，通过使用逆转录定量聚合酶链反应（RT-qPCR）、蛋白质免疫印迹法（Western blot）以及RNA测序，我们发现，由于维生素D3和卡泊三醇处理，nephrin、维生素D受体及特定足细胞标志物的mRNA和蛋白质表达显著上调。相比之下，肾损伤标志物显著下调。此外，我们发现维生素D3和卡泊三醇对miR-21和miR-30a的表达均无影响，对miR-125a/b也无影响，miR-125a/b是一种被描述为可调节维生素D受体的微小RNA（miRNA）。总之，我们将已建立的小鼠Glom推进为一种半自动化高通量检测方法，并仅使用最少数量的动物将其与下游分析技术相结合。据此，我们确定维生素D信号通路对足细胞具有保护作用，并可对抗其去分化。
