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.

Figure 1. Addition of NaCl to Fibrolife or RPMI based-medium increases fibrocyte differentiation.

Human PBMCs were cultured for 5 days in serum-free medium in the presence of the indicated concentrations of additional NaCl in either Fibrolife (A) or RPMI (B) medium. Results are expressed as mean ± SEM, n = 8. * indicates statistical significance with p<0.05, ** indicates P<0.01, and *** indicates p<0.001 by ANOVA compared to the no additional NaCl control.

Figure 2. The elongated cells are fibrocytes.

PBMCs were cultured in Fibrolife medium for five days in the presence or absence of 25 mM additional NaCl. Cells were fixed and stained for the indicated markers. (A) The elongated spindle-shaped cells in the wells stained positive for CD13, CD45, CD68, Prolyl 4-hydroxylase (P-4-H), Pro-Collagen I (Pro-Col I) and had no apparent staining for the fibroblast marker CD90. Results are expressed as mean ± SEM, n = 3. (B) Cells were stained with the indicated antibodies. Positive staining was identified by red for alkaline phosphatase staining and brown for peroxidase staining. Cells were counterstained blue with hematoxylin to identify nuclei. Size bar is 50 µm.

Figure 3. The effect of NaCl on adherent cells after 5 days.

PBMCs were incubated with the indicated concentrations of added NaCl, and the number of adherent cells was counted after 5 days. Counts were then normalized to the control with no added NaCl (0). Values are mean ± SEM, n = 3. *** indicates statistical significance by ANOVA compared to the control (p<0.001).

Figure 4. NaCl does not influence the adhesion of PBMCs to plastic, plasma fibronectin or collagen I.

PBMCs were incubated on uncoated polystyrene plates, fibronectin coated plates, or bovine collagen I coated plates for one hour in the indicated concentrations of additional NaCl. The non-adhered cells were then rinsed off and the number of adherent cells were counted. Values are mean ± SEM, n = 4.

Figure 5. NaCl directly potentiates the differentiation of monocytes into fibrocytes.

CD14⁺ monocytes were incubated with the indicated additional concentrations of NaCl. After 5 days, fibrocytes were counted. Values are mean ± SEM, n = 3. * indicates p<0.05 compared to control (ANOVA). The absence of error bars indicate that error was smaller than the plot symbol.

Figure 6. The presence of additional NaCl during PBMC adhesion increases fibrocyte differentiation.

PBMCs were incubated with additional NaCl, before, during, before, and after their adhesion to tissue culture treated plates. Values are mean ± SEM, n = 6. *** indicates p<0.001 compared to control (t-test). The absence of an error bar indicates that the error was smaller than the line thickness.

Figure 7. The presence of additional NaCl during CD14⁺ monocyte adhesion increases fibrocyte formation.

Isolated monocytes were incubated with additional NaCl before, during, and after their adhesion to tissue culture treated plates. Values are mean ± SEM, n = 4, ** indicates p<0.01, and *** indicates p<0.001 compared to control (ANOVA).

Figure 8. Incubation of PBMCs with additional NaCl does not influence the cell-surface levels of several adhesion molecules.

PBMCs were incubated in the presence or absence of 25 mM additional NaCl for 1 hour at 37°C and then stained for the indicated adhesion molecules. Monocytes were identified by their forward and side scatter characteristics and the expression of CD14. Flow cytometry plots are representative results from three separate donors. Histograms represent fluorescence intensity of the indicated marker in control cells (Black line) and NaCl treated Cells (Red line).

Figure 9. Sodium nitrate and potassium chloride potentiate fibrocyte differentiation.

PBMCs were cultured with the indicated concentrations of additional (A) sodium chloride, sodium nitrate, or sodium gluconate, or (B) potassium chloride. Values are mean ± SEM, n = 6. * indicates p<0.05, and ** indicated p<0.01 compared to control by ANOVA.

Figure 10. NaCl interferes with the ability of hSAP to inhibit fibrocyte differentiation.

PBMCs were incubated with or without additional 25 mM NaCl with the indicated concentrations of human SAP (hSAP). After 5 days, fibrocytes were counted. Values are mean ± SEM, n = 5. * indicates p<0.05 compared to SAP alone control (dashed line) by ANOVA. The absence of error bars indicate that error was smaller than the plot symbol. Lines are sigmoidal dose-response curves with variable Hill coefficient fit to the data.