Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
Department of Pathology, Amsterdam Infection & Immunity, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
Cells. 2023 Dec 21;13(1):23. doi: 10.3390/cells13010023.
Chronic kidney disease often leads to kidney dysfunction due to renal fibrosis, regardless of the initial cause of kidney damage. Macrophages are crucial players in the progression of renal fibrosis as they stimulate inflammation, activate fibroblasts, and contribute to extracellular matrix deposition, influenced by their metabolic state. Nucleotide-binding domain and LRR-containing protein X (NLRX1) is an innate immune receptor independent of inflammasomes and is found in mitochondria, and it plays a role in immune responses and cell metabolism. The specific impact of NLRX1 on macrophages and its involvement in renal fibrosis is not fully understood.
To explore the specific role of NLRX1 in macrophages, bone-marrow-derived macrophages (BMDMs) extracted from wild-type (WT) and NLRX1 knockout (KO) mice were stimulated with pro-inflammatory and pro-fibrotic factors to induce M1 and M2 polarization in vitro. The expression levels of macrophage polarization markers (, , , and ), as well as the secretion of transforming growth factor β (TGFβ), were measured using RT-PCR and ELISA. Seahorse-based bioenergetics analysis was used to assess mitochondrial respiration in naïve and polarized BMDMs obtained from WT and NLRX1 KO mice. In vivo, WT and NLRX1 KO mice were subjected to unilateral ureter obstruction (UUO) surgery to induce renal fibrosis. Kidney injury, macrophage phenotypic profile, and fibrosis markers were assessed using RT-PCR. Histological staining (PASD and Sirius red) was used to quantify kidney injury and fibrosis.
Compared to the WT group, an increased gene expression of M2 markers-including and -and enhanced TGFβ secretion were found in naïve BMDMs extracted from NLRX1 KO mice, indicating functional polarization towards the pro-fibrotic M2 subtype. NLRX1 KO naïve macrophages also showed a significantly enhanced oxygen consumption rate compared to WT cells and increased basal respiration and maximal respiration capacities that equal the level of M2-polarized macrophages. In vivo, we found that NLRX1 KO mice presented enhanced M2 polarization markers together with enhanced tubular injury and fibrosis demonstrated by augmented TGFβ levels, fibronectin, and collagen accumulation.
Our findings highlight the unique role of NLRX1 in regulating the metabolism and function of macrophages, ultimately protecting against excessive renal injury and fibrosis in UUO.
慢性肾脏病常因肾纤维化导致肾功能障碍，而不论肾脏损伤的初始原因如何。巨噬细胞在肾纤维化的进展中起着至关重要的作用，因为它们刺激炎症、激活成纤维细胞，并促进细胞外基质的沉积，这受其代谢状态的影响。核苷酸结合域和富含亮氨酸重复序列的 X 蛋白（NLRX1）是一种独立于炎症小体的先天免疫受体，存在于线粒体中，在免疫反应和细胞代谢中发挥作用。NLRX1 对巨噬细胞的具体影响及其在肾纤维化中的作用尚不完全清楚。
为了探讨 NLRX1 在巨噬细胞中的特定作用，从野生型（WT）和 NLRX1 敲除（KO）小鼠中提取骨髓来源的巨噬细胞（BMDMs），并用促炎和促纤维化因子体外刺激诱导 M1 和 M2 极化。使用 RT-PCR 和 ELISA 测量巨噬细胞极化标志物（、、、和）的表达水平以及转化生长因子β（TGFβ）的分泌。使用 Seahorse 基于生物能量学分析评估来自 WT 和 NLRX1 KO 小鼠的幼稚和极化 BMDMs 的线粒体呼吸。在体内，WT 和 NLRX1 KO 小鼠接受单侧输尿管梗阻（UUO）手术以诱导肾纤维化。使用 RT-PCR 评估肾脏损伤、巨噬细胞表型特征和纤维化标志物。组织学染色（PASD 和 Sirius red）用于量化肾脏损伤和纤维化。
与 WT 组相比，在从 NLRX1 KO 小鼠中提取的幼稚 BMDMs 中，M2 标志物的基因表达增加，包括和，并增强 TGFβ 的分泌，表明其向促纤维化 M2 亚型的功能极化。与 WT 细胞相比，NLRX1 KO 幼稚巨噬细胞的耗氧率也显著增加，基础呼吸和最大呼吸能力增加，达到 M2 极化巨噬细胞的水平。在体内，我们发现 NLRX1 KO 小鼠表现出增强的 M2 极化标志物，同时 TGFβ 水平、纤连蛋白和胶原蛋白积累增加，表明肾小管损伤和纤维化增强。
我们的研究结果强调了 NLRX1 在调节巨噬细胞代谢和功能方面的独特作用，最终可防止 UUO 中过度的肾脏损伤和纤维化。
