Peritoneal Dialysis Fluid and Some of Its Components Potentiate Fibrocyte Differentiation

Abstract

Long-term peritoneal dialysis (PD) often results in the development of peritoneal fibrosis. In many other fibrosing diseases, monocytes enter the fibrotic lesion and differentiate into fibroblast-like cells called fibrocytes. We find that peritoneal tissue from short-term PD patients contains few fibrocytes, while fibrocytes are readily observed in the peritoneal membrane of long-term PD patients. The PD fluid Dianeal (Baxter Healthcare Corporation, Deerfield, IL, USA) contains dextrose, a number of electrolytes including sodium chloride, and sodium lactate. We find that PD fluid potentiates human fibrocyte differentiation in vitro and implicates sodium lactate in this potentiation. The plasma protein serum amyloid P (SAP) inhibits fibrocyte differentiation. Peritoneal dialysis fluid and sodium chloride decrease the ability of human SAP to inhibit human fibrocyte differentiation in vitro Together, these results suggest that PD fluid contributes to the development of peritoneal fibrosis by potentiating fibrocyte differentiation.

Keywords: Peritoneal dialysis; fibrocytes; fibrosis; lactate.

Figure 1 —

Long-term PD is associated with an increase in fibrocytes. A) Sections from the peritoneum of patients on short-term and long-term PD were stained with picrosirius red, and the stain was quantified. Bar is 200 μM. * indicates p < 0.05 (t-test). Peritoneal sections from B) short-term and C) long-term PD patients were stained for the indicated markers. All images are representative of biopsy samples from at least 3 different patients of each type. Arrows indicate cells with significant overlap in staining with CD45RO and α-SMA. Bar is 10 μm. PD = peritoneal dialysis; α-SMA = α-smooth muscle actin.

Figure 2 —

Peritoneal dialysis fluid and some of its components potentiate the differentiation of fibrocytes. Peripheral blood mononuclear cells were incubated with the indicated concentrations of A) PD fluid or B–F) PD fluid components added to RPMI. G) Purified monocytes were incubated with sodium lactate at the indicated concentrations. After 5 days, the cells were stained and counted. Values are mean±SEM, ≥3. *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001 compared to control (1-way ANOVA, Dunnett's test). The absence of an error bar indicates that the error was smaller than the plot symbol. PD = peritoneal dialysis; SEM = standard error of the mean; NaCl = sodium chloride; MgCl₂ = magnesium chloride; CaCl₂ = calcium chloride; NaLactate = sodium lactate.

Figure 3 —

The effect of PD fluid components on total cells in the assay after fixation. Fixed cells from Figure 2 were stained with DAPI and counted. The percent of adhered cells compared to buffer alone is shown for the indicated components. Values are mean±SEM, n=3. The absence of an error bar indicates that the error was smaller than the plot symbol. PD = peritoneal dialysis; SEM = standard error of the mean; NaCl = sodium chloride; MgCl₂ = magnesium chloride; CaCl₂ = calcium chloride; NaLactate = sodium lactate.

Figure 4 —

Peritoneal dialysis fluid, sodium chloride, and sodium lactate increase the expression of collagen. After a 5-day incubation, plated cells were stained for collagen I. A) Forward and side scatter properties of 5-day cultured cells. P1 outlined in red indicates a population that we previously observed to contain fibrocytes (31). Histograms show the fluorescent intensities of rabbit IgG control (black line) and collagen I (red line) for the P1 cells after incubation in B) media alone, C) 12.5% PD fluid, D) 12.5 mM sodium chloride, or E) 5 mM sodium lactate. F) The total number of collagen positive cells was calculated from the percentage of positive cells within the region P1. Values are mean±SEM, n≥3. * = p<0.05; ** = p<0.01 (t-test); PD = peritoneal dialysis; SEM = standard error of the mean; SSC = side scatter; FSC = forward scatter; IgG = immunoglobulin G; FL = fluorescent intensity.

Figure 5 —

Peritoneal dialysis fluid and sodium chloride, but not sodium lactate, interfere with the ability of SAP to inhibit fibrocyte differentiation. Human PBMC were incubated in the presence or absence of 12.5% PD fluid, 12.5 mM sodium chloride, or 5 mM sodium lactate and the indicated concentrations of human SAP for 5 days. The number of fibrocytes were counted. Values are mean±SEM, n=3. Curves are fits to sigmoidal dose-response with variable Hill coefficients. SAP = serum amyloid P; PBMC = peripheral blood mononuclear cells; PD = peritoneal dialysis; SEM = standard error of the mean; NaLac = sodium lactate; NaCl = sodium chloride.