Effect of the sGC activator BAY 60-2770 on human proximal tubular (HK-2) cells under hypoxia and after TGF-ß1 treatment

Increasing cGMP levels by modulating soluble guanylyl cyclase (sGC) can be renoprotective. While sGC stimulators can modulate cGMP levels, their application is limited as they require native sGC to function. sGC activators on the other hand can function with heme-free sGC. Therefore, we tested the renoprotective effects of the sGC activator BAY 60-2770 that also functions with oxidized sGC in an acute kidney injury (AKI) model with transition to chronic kidney disease (CKD). In order to study the effects of BAY 60-2770 on gene expression in tubular cells, we used the proximal tubular cell line HK-2 as a model. To mimic the ischemia and fibrosis observed in vivo, we subjected HK-2 cells to hypoxia and TGF-ß1 treatment, respectively. RNASeq analysis showed that BAY 60-2770 had a significant influence on the expression of genes involved in fibrosis, inflammation and tissue damage after both hypoxia and TGF-ß1 treatments. Overall design: HK-2 cells were treated with BAY 60-2770 or solvent control and incubated for 24h either under hypoxia (0.3% oxygen) or normoxia. In another set of experiments, HK-2 cells were treated with TGF-ß1 alone or in combination with BAY 60-2770 and incubated for 24h under normoxia. Analysis of RNA-seq data was performed using DESeq2 with a multifactor design within two experiments, comparing both treatment effects and sGC activator status to the respective controls.

通过调控可溶性鸟苷酸环化酶（soluble guanylyl cyclase, sGC）升高环磷酸鸟苷（cyclic guanosine monophosphate, cGMP）水平可发挥肾脏保护作用。尽管sGC激动剂可调控cGMP水平，但其应用受到限制，原因是该类药物需依赖天然状态的sGC方可发挥功能。而sGC激活剂则可在缺乏血红素的sGC环境中发挥作用。因此，我们针对可与氧化型sGC结合发挥功能的sGC激活剂BAY 60-2770，在可进展为慢性肾脏病（chronic kidney disease, CKD）的急性肾损伤（acute kidney injury, AKI）模型中，测试其肾脏保护效应。

为探究BAY 60-2770对肾小管细胞基因表达的调控作用，我们选用近端肾小管细胞系HK-2作为实验模型。为模拟体内观察到的缺血与纤维化过程，我们分别对HK-2细胞施加缺氧处理与转化生长因子-β1（transforming growth factor-β1, TGF-β1）刺激。

RNA测序（RNA sequencing, RNASeq）分析结果显示，在缺氧及TGF-β1刺激后，BAY 60-2770均可对纤维化、炎症及组织损伤相关基因的表达产生显著调控作用。

实验整体设计：
第一组实验：将HK-2细胞分别置于缺氧（氧浓度0.3%）或常氧条件下，用BAY 60-2770或溶剂对照处理，孵育24小时。第二组实验：将HK-2细胞单独用TGF-β1处理，或联合BAY 60-2770处理，于常氧环境下孵育24小时。RNA测序数据分析采用DESeq2工具，基于两项实验的多因素设计，分别比较处理效应及sGC激活剂干预状态与各自对应对照组的差异。