Hemodialysis removes uremic toxins that alter the biological actions of endothelial cells

Abstract

Chronic kidney disease is linked to systemic inflammation and to an increased risk of ischemic heart disease and atherosclerosis. Endothelial dysfunction associates with hypertension and vascular disease in the presence of chronic kidney disease but the mechanisms that regulate the activation of the endothelium at the early stages of the disease, before systemic inflammation is established remain obscure. In the present study we investigated the effect of serum derived from patients with chronic kidney disease either before or after hemodialysis on the activation of human endothelial cells in vitro, as an attempt to define the overall effect of uremic toxins at the early stages of endothelial dysfunction. Our results argue that uremic toxins alter the biological actions of endothelial cells and the remodelling of the extracellular matrix before signs of systemic inflammatory responses are observed. This study further elucidates the early events of endothelial dysfunction during toxic uremia conditions allowing more complete understanding of the molecular events as well as their sequence during progressive renal failure.

Figure 1. Effect of pre- or post-HD serum on proliferation, apoptosis, migration and wound healing activities of HUVEC.

(A): HUVEC were incubated in medium supplemented with 5%, 10%, 20% pre- or post-HD serum and 48 h later their number was estimated by crystal violet. (B): HUVEC were incubated in medium supplemented with 20% pre- or post-HD serum and 48 or 72 h later the number of apoptotic cells was measured by FACS. (C): HUVEC were incubated in microchemotaxis chambers in culture medium supplemented with 5%, 10%, 20% pre- or post-HD serum and 4 h later the number of cells that migrated through the filter was quantified. (D) Endothelial monolayers were scratched, incubated in culture medium supplemented with 20% pre- or post-HD serum and 24 h later representative images of the plates were taken. Data are expressed as mean ± SEM of three independent experiments. ^*, ^** and ^*** represent p<0.05, p<0.01 and p<0.001 respectively.

Figure 2. Effect of pre- or post-HD serum on MMP-2, -9 expression.

(A): HUVEC were incubated in medium supplemented with 5%, 10%, 20% pre- or post- HD serum. 4 h later the medium was replaced by minimal medium and 20 h later the supernatants were analyzed for MMP-2 and MMP-9 activity by zymography. (B): HUVEC were incubated with culture medium supplemented with 20% pre- or post- HD serum. 4 h later the medium was replaced by minimal medium and 8 or 20 h later the supernatants were analyzed for MMP-2 and MMP-9 activity by zymography. (C): HUVEC were incubated in culture medium supplemented with 20% pre- or post- HD serum. 6, 12, or 24 h later total RNA was extracted from the cells, RT-PCR reactions were performed using specific primers for MMP-2, MMP-9 or GAPDH mRNAs, the PCR products were analyzed in agarose gels and quantified. Data are expressed as mean ± SEM of three independent experiments.. ^*, ^** and ^*** represent p<0.05, p<0.01 and p<0.001 respectively.

Figure 3. Effect of pre- or post-HD serum on the expression of TIMP-1 and TIMP-2.

(A): HUVEC were incubated in culture medium supplemented with 20% pre- or post- HD serum. 4 h later the medium was replaced by minimal medium and 8 h or 20 h later the supernatants were analyzed for TIMP-1 and TIMP-2 proteins by SDS-PAGE. (B): HUVEC were incubated with culture medium supplemented with 20% pre- or post- HD serum. 6, 12, or 24 h later total RNA was extracted from the cells, RT-PCR reactions were performed using specific primers for TIMP-1, TIMP-2 or GAPDH mRNAs, the PCR products were analyzed in agarose gels and quantified. Data are expressed as mean ± SEM of three independent experiments. ^* and ^** represent p<0.05 and p<0.01 respectively.

Figure 4. Effect of pre- or post-HD serum on the expression of collagen-IV and elastin.

HUVEC were incubated with culture medium supplemented with 20% pre- or post- HD serum. 6, 12, or 24 h later total RNA was extracted from the cells, RT-PCR reactions were performed using specific primers for Collagen IV, Elastin or GAPDH mRNAs, the PCR products were analyzed in agarose gels and quantified. Data are expressed as mean ± SEM of three independent experiments. ^*, ^** and ^*** represent p<0.05, p<0.01 and p<0.001 respectively.