Identification the Classification and Heterogeneity of Lymphatic Endothelial Cells, and Their Role in Mice with Kidney Fibrosis via Single-Cell RNA Sequencing Analysis

Background: We previously found that knockdown of lymphatic endothelial cells (LECs) delayed the progression of chronic kidney disease. However, due to the relatively low abundance of LECs, little is known about their classification and the heterogeneity in renal inflammation and subsequent fibrosis. Methods: We established a unilateral ureteral obstruction (UUO) model of mouse and isolated CD31+LYVE-1+ cells using CD31 magnetic bead enrichment and LYVE-1 flow sorting. We employed single-cell RNA sequencing to analyze the heterogeneity of LECs in mice with renal fibrosis. In order to selectively suppress proliferating lymphatic vessels, LYVE-1-TK transgenic mice were constructed. Results: LECs are classified into three types, including capillary lymphatic endothelial cells (Cap.LECs), pre-collecting lymphatic endothelial cells (Pre-col.LECs) and collecting lymphatic endothelial cells (Col.LECs). Single-cell sequencing analysis showed that Cap.LECs are characterized to participate in cell adhesion, angiogenesis and metal ion transport. Pre-col.LECs are primarily associated with ERK and WNT signaling as well as anion transport. Col.LECs are mainly focused on the regulation of antigen presentation, lipid transport, and immune cell migration. After UUO, all three types of renal LECs shows significant proliferation, and the activity of lymphangiogenesis related transcription factors, proliferation related genes, and lymphangiogenesis related receptors were upregulated. Pseudotime trajectory analysis showed that Pre-col.LECs had strong stem cell characteristics, and both Cap.LECs and Col.LECs were directly derived from the Pre-col.LECs. In the UUO group, the expression of the APP protein, secreted specifically by Pre-col.LECs and Col.LECs, was found to be upregulated. This protein interacts with the CD74 receptor expressed by NK cells and T cells, chemotactically attracting these cells and influencing their function. Knockdown of proliferative lymphatic vessels significantly reduced the number of Pre-col.LECs and Col.LECs in the UUO kidney, resulting in a significant decrease in the populations of T cells and NK T cells, thereby improving the inflammatory microenvironment and fibrosis. Conclusion: These findings provide a theoretical basis for future interventions targeting lymphangiogenesis in kidney diseases.