Elevated blood pressure with anti-VEGFR3 treatment was paralleled by increased skin ClC content but not with increased skin Na+ content. homeostatic and blood pressureCregulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3Cmediated modification of cutaneous lymphatic capillary function. Introduction Mechanisms causing salt-sensitive hypertension are imperfectly defined (1). Guyton et al. attributed long-term blood pressure regulation to the kidney, arguing that blood flow, auto-regulation, and pressure natriuresis control blood pressure (2, 3). This model suggests a close relationship among total body Na+, total body volume, and blood pressure. It assumes isosmolarity of body fluids among the bodily compartments (2). Along with others (4C7), we (8C14) showed AGN 205327 earlier that electrolytes are distributed in a more complex 3-compartment model, in which intravascular and AGN 205327 the interstitial fluids do not equilibrate as readily as believed (15, 16). We underscored the importance of Na+ binding to negatively charged proteoglycans in the skin, the largest organ with the most extracellular space (8, 11). We suggested that, in addition to renal control, local extrarenal regulatory mechanisms for electrolyte clearance of interstitial fluid are operative to maintain extracellular electrolyte clearance and blood pressure. We postulated that electrolyte accumulation in the skin occurs in excess of water and causes local hypertonicity. Mononuclear phagocyte system (MPS) cells respond to osmotic stress via the transcription factor tonicity-responsive enhancer-binding protein (TONEBP) that provokes a tissue-specific, MPS-driven, regulatory response (15, 16). MPS cells infiltrate the salt-overloaded interstitium, initiate TONEBP-driven VEGFC expression, and restructure the interstitial lymphatic capillary network, while increasing eNOS expression in blood vessels. Blocking this MPS-driven regulatory process leads to reduced cutaneous lymphatic capillary density, skin electrolyte accumulation, reduced eNOS expression in blood vessels, and increased blood pressure. The findings suggest that immune cells are regulators of internal environment and blood pressure homeostasis (15, 16). Our model implies that the local skin microenvironment is hypertonic to plasma, that MPS cells dictate regulatory events via TONEBP, and that skin VEGFC is important for systemic blood pressure control. It was unclear whether MPS cells influence blood pressure via VEGFC/VEGFR3Cdriven lymphatic electrolyte clearance or perhaps by VEGFC/VEGFR2Cdriven modulation of eNOS expression. Furthermore, the relationship between Na+ and ClC disposition in the microenvironment was also ill defined. Here, we show that selective depletion of TONEBP in MPS cells, blockade of VEGFR3 with antibody leaving VEGFR2 intact, and deletion of VEGFC signaling in skin all disrupt cutaneous lymphatic capillary architecture and result in predominantly ClC accumulation in the skin, which is paralleled by salt-sensitive hypertension. Finally, we AGN 205327 document with several independent methods the hypertonic electrolyte concentrations of the interstitial microenvironment in the skin. These findings strengthen our proposal of a third, locally regulated, skin fluid compartment relevant to systemic blood pressure regulation. Results Eliminating TONEBP in MPS cells reduces skin ClC clearance and causes salt-sensitive hypertension. To understand the role of TONEBP in MPS cells in modulating lymphatic density and skin electrolyte storage, we investigated the TONEBP/VEGFC regulatory axis in mice with MPS cellCspecific conditional gene deletion (mice). We first harvested macrophages from mice (without TONEBP deficiency) and from Rabbit polyclonal to HEPH mice (with TONEBP deficiency). We exposed the cells to standard cell culture medium, to NaCl-mediated osmotic stress, or urea-mediated hyperosmolality (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI60113DS1). Increasing NaCl, which is an effective osmolyte, caused increased TONEBP expression in macrophages without TONEBP deficiency. In contrast, primary bone marrow macrophages from mice showed reduced TONEBP.