Vascular Endothelium‐Derived ET‐1 Regulates The Renal T Cell‐Macrophage Interplay Under High Salt Diet

Abstract

The vasoactive peptide endothelin-1 (ET-1) is a key contributor to high salt-induced elevations of blood pressure and end-organ damage. In addition, ET-1 is known to be pro-inflammatory in the high salt setting. Although ET-1 was first described as produced by the vascular endothelium, we now know that there are other cellular sources, like kidney tubular cells or immune cells. However, the exact role of endothelium-derived ET-1 in regulating immune cell populations during high salt consumption remains unclear. We hypothesized that endothelium-derived ET-1 is critical in regulating the kidney inflammation that is associated with high salt consumption. Male vascular endothelial cell ET-1 knockout (VEET KO) and control floxed ET-1 mice were placed on 4.0% NaCl diet (HSD) for 3 weeks. Urine was collected for assessment of kidney injury, and the immunological status was evaluated in kidneys and spleen by flow cytometry analysis of live, singlet cells. After 3 weeks of HSD, VEET KO mice presented reduced urinary protein excretion compared to floxed ET-1 mice (floxed ET-1 vs. VEET KO: 8.59 ± 1.90 vs. 3.56 ± 0.87, pg/day; p=0.03; n=6/group), suggesting that vascular ET-1 is critical for developing high salt-induced kidney injury. Lack of endothelium-derived ET-1 resulted in a 53.9% reduction in the frequency of kidney TH17 cells (IL-17A+ CD4+ T cells; floxed ET-1 vs.VEET KO: 1.650.17 vs. 0.770.10, % IL-17A of CD4+; p=0.001; n=5-6/group) and a 77.9% reduction in kidney absolute TH17 cell count (3.10.5 vs. 0.850.22, x102 cells; p=0.008). Interestingly, the intra-renal frequency of T regulatory (Treg) cells was similar between the genotypes (floxed ET-1 vs. VEET KO: 12.81.58 vs. 12.31.29, %; p=0.78), but the absolute Treg cell number in the kidney was reduced in VEET KO mice (2.260.25 vs. 1.210.30, x103 cells, p=0.02); these findings suggest a potential role for vascular ET-1 in the regulation of HSD-induced Treg expansion/proliferation in this organ. In addition, VEET KO mice also presented significantly blunted frequency and absolute numbers of the innate-like IL-17A+ +TCR+ T cell population in the kidney (floxed ET-1 vs. VEET KO; % IL-17A of IL-17A+ + TCR+: 36.61.92 vs. 21.22.63, p=0.0009; absolute numbers: 2.510.41 vs. 1.020.39, x102, p=0.03). Examination of the kidney myeloid cell populations under HSD also revealed a reduced frequency of activated resident macrophages (F4/80hi CD64+: 25.14.52 vs. 10.51.44, %, p=0.01; F4/80hi CD86+: 85.32.38 vs. 78.131.69 %, p=0.03), while no differences were found in the activation of infiltrating macrophages (F4/80lo CD64+ and F4/80lo CD86+) in the kidney. Lymphoid and myeloid immune cell populations in the spleen were similar between genotypes. These data indicate a critical role for intra-renal endothelium-derived ET-1 in the cellular response of immune cell populations under HSD. More specifically, our data demonstrate that endothelium-derived ET-1 regulates the immune landscape during HSD consumption by activating pro-inflammatory TH17 and IL-17A+ +TCR+ T cells as well as resident macrophages specifically in the kidney.