Defining the interaction of platelets, neutrophil extracellular traps and thrombo-inflammation in diabetic kidney disease

糖尿病肾病(DKD)是终末期肾病的主要原因。这使得DKD成为糖尿病患者发病率和死亡率的主要原因，并增加了医疗保健系统的负担。肾小球内皮功能障碍和相关的凝血激活与肾小球滤过屏障(GFB)破坏和DKD进展有关。虽然已经描述了GFB上的可溶性凝血调节剂独立于止血的动态平衡功能，但缺乏将血小板激活、血栓炎症和肾小球内皮功能障碍联系起来的机制研究。血栓炎症机制的特征是血小板与天然免疫细胞，如中性粒细胞的相互作用。此外，DKD的严重程度与肾脏中免疫细胞的聚集有关。中性粒细胞可通过形成中性粒细胞胞外陷阱而引起炎症。在我们的前期工作中，我们在糖尿病小鼠的肾小球中检测到了Net。在体内，血小板加重了葡萄糖诱导的肾小球内皮细胞和肾小球的网络形成，这与NLRP3炎症小体激活增加和内皮功能受损(eNOS、KLF2、KLF4、TM减少)有关。更多的数据表明，网络的形成不仅导致组蛋白瓜氨酸化，而且还导致内皮蛋白的瓜氨酸化。基于这些初步数据，我们假设血小板与中性粒细胞相互作用，促进Net的形成、积聚和稳定，从而加剧DKD的不孕症炎症(NLRP3炎症体)并损害内皮功能。为了解决这一假说，我们提出了以下目标：(1)明确DKD中血小板相关网络形成的调节、机制相关性和翻译重要性；(2)建立NLRP3炎症体与DKD中血小板和网络介导的不孕症炎症的相关性；(3)表征内皮KLF-2/4-血栓调节蛋白轴在血小板网络依赖性内皮功能障碍和肾血栓炎症中的机制相关性；(4)表征蛋白瓜氨酸化在内皮功能障碍和肾脏血栓炎症中的作用；这些研究将为DKD的肾小球血栓炎症机制提供新的见解，并评估血小板和/或净抑制在DKD中预防或减轻肾小球内皮功能障碍的相关性。因此，这些将为GFB的永久化炎症机制提供洞察力，这可能是治疗靶点。

Diabetic kidney disease (DKD) is a major cause of end-stage renal disease. This makes DKD a major cause of morbidity and mortality in diabetic patients and increases the burden on the healthcare system. Glomerular endothelial dysfunction and associated coagulation activation have been linked with glomerular filtrations barrier (GFB) disruption and DKD progression. While a homeostatic function of soluble coagulation regulators at the GFB independent of hemostasis has been described, mechanistic studies linking platelet activation, thrombo-inflammation and glomerular endothelial dysfunction are missing. Thrombo-inflammatory mechanisms are characterized by an interaction of platelets with innate immune cells, such as neutrophils. Additionally, DKD severity is associated with accumulation of immune cells in the kidney. Neutrophils can induce inflammation by formation of neutrophil extracellular traps. In our preliminary work we detected NETs in glomeruli of diabetic mice. Platelets exacerbate glucose-induced NET-formation on glomerular endothelial cells and in glomeruli in vivo, which is associated with increased NLRP3 inflammasome activation and impaired endothelial function (reduction of eNOS, KLF2, KLF4, TM). Additional data suggest that NET formation not only leads to histone citrullination, but also to citrullination of endothelial proteins. Based on these preliminary data, we hypothesize that platelets interact with neutrophils, promoting formation, accumulation, and stabilization of NETs which exacerbate renal sterile inflammation (NLRP3 inflammasome) and impairs endothelial function in DKD. In order to address this hypothesis, we propose the following aims: (1) Defining the regulation, mechanistic relevance, and translational importance of platelet associated NET-formation in DKD; (2) Establish the relevance of the NLRP3 inflammasome for platelet and NET mediated sterile inflammation in DKD; (3) Characterizing the mechanistic relevance of the endothelial Klf-2/4-thrombomodulin axis in platelet-NET-dependent endothelial dysfunction and renal thrombo-inflammation; (4) Characterizing the role of protein citrullination for endothelial dysfunction and renal thrombo-inflammation; these studies will provide new insights into the mechanisms of glomerular thrombo-inflammation in DKD and evaluate the relevance of platelet and/or NET inhibition to prevent or reduce glomerular endothelial dysfunction in DKD. These will thus provide insights into perpetuating inflammatory mechanisms at the GFB, which may be therapeutically targeted.