Pro-calcifying analysis of uraemic serum from patients treated with medium cut-off membrane in a prospective, cross-over study

Abstract

Background: The retention of a large number of solutes that are normally excreted or metabolized by the kidney is responsible for the symptoms typical in uraemic patients. These molecules are defined as uraemic toxins and can be classified into three groups: small water-soluble molecules, middle molecules and protein-bound toxins. Recently, efforts were put towards developing dialysis membranes that allow the removal of large middle molecules without clinically relevant albumin loss. These membranes are the medium cut-off (MCO) membranes that allow the removal of middle molecules up to ∼50 000 Da.

Methods: We performed a prospective, open-label, controlled, cross-over pilot study comparing expanded haemodialysis (HDx) (novel MCO membrane Theranova 400) and conventional haemodialysis (HD) in 20 prevalent HD patients. Ten patients used conventional HD high-flux dialyser and 10 patients used HDx for 3 months; later the patients switched and received the other treatment for a further 3 months. We then analysed the pro-calcifying effect of uraemic serum in a model of high phosphate(Pi)-induced calcification in vascular smooth muscle cells (VSMCs).

Results: In this study, every patient was the control of himself and, interestingly, we found a tendency of less pro-calcifying potential from HDx-treated patients' serum compared with HD. Studying pathogenetic processes involved in high Pi-induced calcium deposition, we found that uraemic serum of HDx-treated patients induced less VSMC necrosis compared with uraemic serum of HD patients. Nevertheless, no differences were found between the different dialytic treatments in the serum potential to induce apoptosis and to modulate the expression of a panel of genes involved in VSMC simil-osteoblastic differentiation such as bone morphogenetic protein 2, runt-related transcription factor 2, osteocalcin, matrix Gla protein, osteopontin, elastin and collagen I α1. In an effort to characterize the difference in uraemic toxin profile during the two different dialytic treatments, we measured a panel of 10 uraemic toxins and 3 precursors, finding a significant increased removal during HDx of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, tryptophane and some of its metabolites, such as 3-indoxyl sulphate, indole 3-acetic acid and kynurenine.

Conclusions: These preliminary data are promising, although larger patients' groups are needed to better understand the effects of HDx on vascular calcification.

Keywords: necrosis; protein-bound uraemic toxins; uraemic serum; vascular calcification.

FIGURE 1:

Study design.

FIGURE 2:

Effect of HDx on human uraemic serum calcification potential in high Pi–induced VSMC calcification. Rat VSMCs were cultured with 10% human uraemic serum added in the calcification medium, harvested at three different time points (baseline, 3 and 6 months) following either HDx or HD and stimulated with 3.3 mM Pi for 3 days. (A) Calcium deposition was measured by destaining and normalized by cellular protein content. (B–E) Calcium deposition was visualized at the light microscopy level by Alizarin Red S staining. (A) Scatter plot representation of every patient’s uraemic serum calcification action in Group A (HDx->HD, left) and in Group B (HD->HDx, right) at three different time points. In Group B, HDx treatment induced a significant reduction of the uraemic sera calcification potential. (B) Group A and (C) Group B calcium deposition in 12- well plate cultured VSMCs stimulated with high Pi (red). Representative images showing the time course of serum calcification potential in a patient from (D) Group A and (E) Group B.The figure shows HDx dialytic treatment reduced the number of red calcific deposits [white arrows (D) 3 months and (E) 6 months]. In contrast, it is possible to detect necrosis as red non-specific staining around calcific red spots (arrowhead) and as a loss of cells during HD [(D) 6 months, black arrow]. Magnification ×200. Data are presented as mean ± standard error (*P < 0.05).

FIGURE 3:

Effect of HDx on human uraemic serum apoptotic and necrotic potential in high Pi–stimulated VSMCs. Rat VSMCs were cultured with 10% human uraemic serum added in the calcification medium, harvested at three different time points (baseline, 3 and 6 months) following either HDx or HD and stimulated with 3.3 mM Pi for 4 days. Apoptosis and necrosis were evaluated by histone-associated DNA fragment detection ELISA in cell lysate or culture medium, respectively. (A) HDx dialytic treatment did not modified high Pi–induced apoptosis that is similar in HDx and HD patients. (B) Sera from HDx patients showed a significant decreased serum necrotic potential in Group B (left panel). Data are presented as mean ± standard error (*P < 0.05).

FIGURE 4:

Effect of HDx on human uraemic serum potential to induce simil-osteoblastic differentiation in high Pi–stimulated VSMCs. Rat VSMCs were cultured with 10% human uraemic serum added in the calcification medium, harvested at three different time points (baseline, 3 and 6 months) following either HDx or HD and stimulated with 3.3 mM Pi for 4 days. mRNA expression was measured by reverse transcription PCR and expressed as relative expression. (A and B) Group A. (C and D) Group B. (A–C) HDx dialytic treatment did not induce any significant variation in the uraemic serum potential to modify BMP2 expression following high Pi stimulation compared with HD. (B and D) HDx dialytic treatment did not induce any significant variation in the uraemic serum potential to modify OC, RUNX2, OPN, MGP, elastin and CollIα1 expression following high Pi stimulation compared with HD. Data are presented as mean ± standard error (*P < 0.05).

FIGURE 5:

Serum content of protein-bound uraemic toxins following either HD or HDx. Protein-bound uraemic toxin levels were measured in serum by UPLC-MS/MS in Groups A and B at baseline, 3 and 6 months and represented as scatter plots of every patient. The percentage of variation is intended as the level variation compared with the level in the precedent time point. (A) Tryptophane, (B) kynurenine, (C) indole-3-acetic acid, (D) 3-IS and (E) CMPF. For all five protein-bound uraemic toxins represented, there was a significant decrease following HDx in Group B. 3-IS was significantly decreased by HDx in Group A. Data are presented as mean ± standard error (*P < 0.05).

FIGURE 6:

Serum content of alpha 1 acid glycoprotein and miRNA following either HD or HDx. In serum, alpha 1 acid glycoprotein was measured by (A) ELISA and miRNA in exosomes by reverse transcription PCR (B and C) in Groups A and B at baseline, 3 and 6 months and represented as scatter plots (alpha 1 acid glycoprotein) and histograms (miRNA) for every patient. (A) No significant effect of HDx on the percentage of variation in serum alpha 1 acid glycoprotein. No significant variation was induced by HDx dialytic treatment in exosome 125b and 145 miRNA levels in (B) Group A and (C) Group B. Data are presented as mean ± standard error.