Uremia Impacts VE-Cadherin and ZO-1 Expression in Human Endothelial Cell-to-Cell Junctions

Abstract

Endothelial dysfunction in uremia can result in cell-to-cell junction loss and increased permeability, contributing to cardiovascular diseases (CVD) development. This study evaluated the impact of the uremic milieu on endothelial morphology and cell junction's proteins. We evaluated (i) serum levels of inflammatory biomarkers in a cohort of chronic kidney disease (CKD) patients and the expression of VE-cadherin and Zonula Occludens-1 (ZO-1) junction proteins on endothelial cells (ECs) of arteries removed from CKD patients during renal transplant; (ii) ECs morphology in vitro under different uremic conditions, and (iii) the impact of uremic toxins p-cresyl sulfate (PCS), indoxyl sulfate (IS), and inorganic phosphate (Pi) as well as of total uremic serum on VE-cadherin and ZO-1 gene and protein expression in cultured ECs. We found that the uremic arteries had lost their intact and continuous endothelial morphology, with a reduction in VE-cadherin and ZO-1 expression. In cultured ECs, both VE-cadherin and ZO-1 protein expression decreased, mainly after exposure to Pi and uremic serum groups. VE-cadherin mRNA expression was reduced while ZO-1 was increased after exposure to PCS, IS, Pi, and uremic serum. Our findings show that uremia alters cell-to-cell junctions leading to an increased endothelial damage. This gives a new perspective regarding the pathophysiological role of uremia in intercellular junctions and opens new avenues to improve cardiovascular outcomes in CKD patients.

Keywords: chronic kidney disease; intercellular junctions; uremic toxins.

Figure 1

Uremic toxins’ serum concentrations and correlation with eGFR. Right panel. Box plots of the p-cresyl sulfate (PCS) (a), indoxyl sulfate (IS) (c) and inorganic phosphate (Pi) (e) serum concentrations in patients’ according to their chronic kidney disease (CKD) stages. Left panel. Correlation between PCS (b), IS (d), Pi (f) serum concentrations and eGFR in CKD patients (**** P < 0.0001, ρ = −0.59; *** P < 0.0001, ρ = −0.70; ** P < 0.001, ρ = −0.37, respectively).

Figure 2

VE-cadherin and Zonula Occludens-1 (ZO-1) protein expressions in renal arteries. VE-cadherin immunolabeling in renal artery of (a,b) donor (control) and (c,d) recipient (CKD patient). ZO-1 immunolabeling in renal artery of (e,f) donor (control) and (g,h) recipient (CKD patient). Magnifications: 100× (a,c,e,g) and 400× (b,d,f,h). Arrowheads indicate intact endothelial cell monolayer. Arrows indicate loss of endothelial monolayer’s integrity. Positive immunoreaction was observed as a brown precipitate. Photos shown are representative of all the analyses.

Figure 3

Effect of PCS, IS and Pi on cell viability. Cells were incubated with the various concentrations of uremic toxins or serum for 24 h. Cell viability was assessed using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. Control (non-treated cells); PCS normal (PCSn) and IS normal (ISn) at normal concentrations; PCS uremic (PCSu) and IS uremic (ISu) at minimum uremic concentrations; PCS maximum uremic (PCSm) and IS Maximum uremic (Ism) at maximal uremic concentrations; inorganic phosphate at 2 mM (Pi2), 3 mM (Pi3), and 4 mM (Pi4). Results are expressed as % of control (non-treated cells). **** P < 0.0001 for PCSm, Ism, and Pi4 vs. control.

Figure 4

Effect of uremic toxins and uremic pools on cell permeability. Endothelial cells were seeded at high density and cultured until confluency. Cells were then exposed to the various concentrations of uremic toxins or 10% of uremic pools. Fluorescein isothiocyanate (FITC)-conjugated dextran (4 kDa, final concentration 2.5 mg/mL) was added to the upper compartment of the inserts for 20 min. Aliquots were then taken from the lower compartment and fluorescence read. Permeability is expressed as % of control (non-treated cells) and represents mean ± SEM (standard mean error) of 12 determinations. * P < 0.05 Pi3 vs. control; ** P < 0.01 GI vs. control; *** P < 0.001 GII and GIII vs. control. Below are shown the crystal violet staining of the cells in the corresponding inserts.

Figure 5

Morphological and ultrastructural analyses of endothelial intercellular junctions by scanning electronic microscopy (SEM) Non-treated cells were used as negative controls (a–c). 4% dimethyl sulfoxide (DMSO)-treated cells were used as positive controls (d–f). Cells were treated with PCSm (g–i), ISm (j–l), Pi3 (m–o), GI (p–r), GII (s–u), and GIII (v–x) for 24 h. Magnifications of the images: a,d,g,j,m,p,s (1000×), b,e,h,k,n,q,t,w (6000×), c,f,i,l,o,r,u,x (15,000×).

Figure 6

Effect of a uremic environment on F-actin protein expression. After treatment with either uremic toxins or uremic pools, F-actin protein expression was visualized using ActinGreen™ 488 ReadyProbes^® (green). DAPI (4′,6-diamidino-2-phenylindole) was used to label the nuclei (blue). Control (non-treated cells), Magnification 600×.

Figure 7

Uremic environment impacts endothelial cell adherent junctions and VE-cadherin expression. Control (non-treated cells). Effect of uremic toxins and uremic pools on VE-cadherin, p120 and vinculin gene expression: VE-cadherin-** P < 0.01 for ISm, Pi3, GI, GII, and GIII vs. Control (a); p120-** P < 0.01 for PCSm and Pi3 vs. Control (b); vinculin-* P < 0.05 for PCSm and GI vs. Control and ** P < 0.01 for ISm vs. Control (c). Effect of uremic toxins and uremic pools on VE-cadherin protein expression: by immunoblotting: Actin was used as protein loading control. Lower panel: A representative immunoblot of 8 experiments. Upper panel: Quantification of the bands (d), by immunofluorescence staining: Magnification 600× (e) and by flow cytometry: Fluorescence intensity was evaluated by flow cytometry and quantified using Flowing 2 software. Dots represent median fluorescence intensity (MFI) compared to control (non-treated cells; dashed lines) in each experiments (n = 6). Bars represent median of all values for each group (f).

Figure 8

Uremic environment differently modulates ZO-1 gene and protein expression. Control (non-treated cells). Effect of uremic toxins and uremic pools on ZO-1 gene expression. ** P < 0.01 for PCSm, ISm, GI, GII, and GIII vs. Control (a); Effect of uremic toxins and uremic pools on ZO-1 protein expression by Western blotting: Actin was used as a loading control. Lower panel: A representative immunoblot of 8 experiments. Upper panel: Quantification of the bands. * P < 0.05 for Pi3, GII and GIII vs. Control; (b), by immunofluorescence: After treatment, cells were labeled for ZO-1 (green) and nuclei (blue) (magnification 600×) (c) and by flow cytometry: Fluorescence intensity was evaluated by flow cytometry and quantified using Flowing 2 software. Dots represent median fluorescence intensity (MFI) as compared to Control (dashed lines) in each experiment (n = 6). Bars represent median of all values for each group (d).