Effects of urea on macrophages and T cells

Abstract

Urea has been known to be inert. There are only a few reports on the potential biological effects of urea, including inhibition of the arginine transport in vascular endothelial cells and inhibition of glycolysis in pancreatic β-cells. In this study, I investigated whether high levels of extracellular urea influence macrophages and found that pathological concentrations of urea attenuated arginase-1 induction by IL-4 in murine macrophages in a dose-dependent manner at both transcriptional and translational levels, indicating that urea inhibits M2 polarization of macrophages. Other genes involved in M2 polarization of macrophages, such as Fizz1 and Ym1, were also significantly downregulated by urea. To examine the effect of urea on M2 macrophage polarization in vivo, IL-4/anti-IL-4 antibody complex was injected into control and urea-fed mice. Peritoneal macrophages from urea-fed mice showed lower arginase-1 expression than did macrophages from control mice. To elucidate the mechanism underlying the inhibitory effects of urea on macrophages, bulk RNA sequencing was performed using IL-4–treated bone marrow-derived macrophages (BMDMs) treated with different concentrations of urea. Gene set enrichment analysis (GSEA) of the RNA-seq data revealed that urea downregulated gene sets related to mTOR signaling, glycolysis, and oxidative phosphorylation. We confirmed that the phosphorylation of mTOR downstream molecules, such as S6 and AKT, was attenuated by urea. In addition, Seahorse real-time metabolic analysis revealed diminished extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in urea-treated M2 BMDMs. Furthermore, LC-MS analysis of the intracellular metabolites associated with the urea cycle and the TCA cycle revealed that urea reduces the ratio of α-ketoglutarate/succinate which is known to be upregulated in M2 polarization. In line with these results, the urea did not further inhibit M2 polarization when M2 polarization of BMDMs were suppressed by treatment with pan-mTOR inhibitor. These results suggest that urea inhibits M2 polarization by attenuation of mTOR signaling in macrophages. Based on a previous report that urea induces carbamylation of mTOR molecules and impairs mTOR signaling in neurons, I tested whether mTOR was also carbamylated in urea-treated macrophages. However, urea did not enhance the carbamylation of mTOR molecules in M2 BMDMs. As observed in macrophages, phosphorylation of mTOR downstream molecules was also downregulated in CD8+ T cells. Urea also attenuated the activation and proliferation of CD8+ T cells in vitro and in vivo. Based on these results, I suggest that urea attenuates M2 macrophage polarization and effector differentiation of CD8+ T cells via inhibition of mTOR, and is no longer an inert molecule for macrophages and T cells.