Circulating MicroRNAs Are Not Eliminated by Hemodialysis

BACKGROUND: Circulating microRNAs are stably detectable in serum/plasma and other body fluids. In patients with acute kidney injury on dialysis therapy changes of miRNA patterns had been detected. It remains unclear if and how the dialysis procedure itself affects circulating microRNA level. METHODS: We quantified miR-21 and miR-210 by quantitative RT-PCR in plasma of patients with acute kidney injury requiring dialysis and measured pre- and post-dialyser miRNA levels as well as their amount in the collected spent dialysate. Single treatments using the following filters were studied: F60 S (1.3 m(2), Molecular Weight Cut Off (MWCO): 30 kDa, n = 8), AV 1000 S (1.8 m(2), MWCO: 30 kDa, n = 6) and EMiC 2 (1.8 m(2), MWCO: 40 kDa, n = 6). RESULTS: Circulating levels of miR-21 or -210 do not differ between pre- and post-dialyzer blood samples independently of the used filter surface and pore size: miR-21: F60S: p = 0.35, AV 1000 S p = 1.0, EMiC2 p = 1.0; miR-210: F60S: p = 0.91, AV 1000 S p = 0.09, EMiC2 p = 0.31. Correspondingly, only traces of both miRNAs could be found in the collected spent dialysate and ultrafiltrate. CONCLUSIONS: In patients with acute kidney injury circulating microRNAs are not removed by dialysis. As only traces of miR-21 and -210 are detected in dialysate and ultrafiltrate, microRNAs in the circulation are likely to be transported by larger structures such as proteins and/or microvesicles. As miRNAs are not affected by dialysis they might be more robust biomarkers of acute kidney injury

Structure and function of the Genius 75/90 Dialysis System and sample collection.

<p>Patient’s blood runs countercurrent to dialysate and enters the dialysis circuit from a Shaldon catheter. The “arterial”, i.e. predialyser blood line (red) passes the filter and blood flows back to the patient via the “venous”, i.e. post-dialyser blood line (blue). Fresh dialysate is prepared individually for every patient’s conditions and stored in a 75 or 90 liter tank. A tube leads the fresh dialysate (light-blue) to the dialysis filter. Exchange of substances and filtration is mediated by an osmotic and pressure gradient and is supported by counter-flow principle. Spent dialysate (grey) flows through the outlet dialysate line back into the tank. Phase formation prevents the mixture of fresh and spent dialysate. An ultrafiltration pump transports the excess filtrate and maintains the pressure gradient of the system. Samples are taken at the marked locations.</p

Levels of circulating miR-21 and -210 in different sample types with the F60S dialysate filter.

<p>Ten independent dialysis sessions in eight different patients were analyzed for the F60S filter system. MiR-21 and miR-210 levels are not significantly different between pre-dialyser and corresponding post-dialyser blood lines (<u>miR-21:</u> p = 0.35; <u>miR-210:</u> p = 0.91). Also the difference between spent dialysate and ultrafiltrate is not of statistical significance (<u>miR-21:</u> p = 0.80; <u>miR-210:</u> p = 0.74). However, averaged values of blood side versus spent dialysate reached high statistical significance for both microRNAs with p-values of <0.0001. Data are represented as mean and standard error of the mean.</p

Comparison of influence of two different filter properties on circulating microRNA levels.

<p>Six patients received at least one dialysis session with each of the two filter systems AV 1000 S and EMiC 2. Circulating levels of miR-21 and -210 were not differently altered by the two different dialyser properties. (<u>miR-21:</u> AV1000S pre-dialyser vs. EMiC 2 predialyser p = 0.59, post-dialyser p = 0.70, dialysate p = 0.94, ultrafiltrate p = 0.39; <u>miR-210:</u> pre-dialyser p = 0.02, post-dialyser p = 0.82, dialysate p = 0.59, ultrafiltrate p = 0.31) Only pre-dialyser line showed a significant alteration, which is not of relevance for our main conclusion. Data are represented as mean and standard error of the mean.</p