NaCl Potentiates Human Fibrocyte Differentiation

Abstract

Excessive NaCl intake is associated with a variety of fibrosing diseases such as renal and cardiac fibrosis. This association has been attributed to increased blood pressure as the result of high NaCl intake. However, studies in patients with high NaCl intake and fibrosis reveal a connection between NaCl intake and fibrosis that is independent of blood pressure. We find that increasing the extracellular concentration of NaCl to levels that may occur in human blood after high-salt intake can potentiate, in serum-free culture conditions, the differentiation of freshly-isolated human monocytes into fibroblast-like cells called fibrocytes. NaCl affects the monocytes directly during their adhesion. Potassium chloride and sodium nitrate also potentiate fibrocyte differentiation. The plasma protein Serum Amyloid P (SAP) inhibits fibrocyte differentiation. High levels of extracellular NaCl change the SAP Hill coefficient from 1.7 to 0.8, and cause a four-fold increase in the concentration of SAP needed to inhibit fibrocyte differentiation by 95%. Together, our data suggest that NaCl potentiates fibrocyte differentiation. NaCl-increased fibrocyte differentiation may thus contribute to NaCl-increased renal and cardiac fibrosis.

A Brief Exposure to Tryptase or Thrombin Potentiates Fibrocyte Differentiation in the Presence of Serum or Serum Amyloid P

“…The cells were then stained and scored for fibrocyte formation. In the absence of added proteases, we observed 388 to 1120 fibrocytes per 10 5 PBMCs from the different donors, similar to what we have previously observed (11, 13, 14, 48). Because of this variability, fibrocyte numbers were thus normalized to protease-free controls.…”

“…Peripheral blood mononuclear cells (PBMC) and monocytes were isolated as previously described (13), and cultured as previously described (48), in either serum-free or protein-free media. Protein-free media (PFM) was Fibrolife basal media (Lifeline Cell Technology, Walkersville, MD) supplemented with 10 mM HEPES (Sigma, St. Louis, MO), 1× non-essential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 2 mM glutamine (Lonza, Basel, Switzerland), 100 U/ml penicillin and 100 µg/ml streptomycin (Lonza).…”

TNF-α–stimulated fibroblasts secrete lumican to promote fibrocyte differentiation

“…We and others have previously shown that ∼1-2% of PBMC or 10-20% of monocytes can readily differentiate into fibrocytes (12,13,39). Because of this variability, for each donor, fibrocyte numbers were normalized to serum-free controls.…”

Serum Amyloid P Contained in Alveolar Fluid From Patients With Acute Respiratory Distress Syndrome Mediates the Inhibition of Monocyte Differentiation into Fibrocyte

“…Fibrocytes were then identified and counted based on their elongated spindle-shaped morphology as previously described (6,20,26,27), in five different 500-μm-diameter fields of view per well and expressed as a percentage of total cells, or as a percentage of the fibrocyte count in the saline basal control in experiments comparing BALF stimulations. .…”

“…Unexpectedly, we observed that ARDS BALF dramatically and reproducibly inhibited monocyte-tofibrocyte differentiation in vitro. As sodium chloride could modulate fibrocyte differentiation (27), we were particularly careful to compare the effect of ARDS BALF to that provided by the same volume of 0.9% saline solution as a control condition, thus excluding a role of the vehicle and suggesting a specific role for alveolar fluid components.…”