Correlations between Plasma Levels of Anionic Uremic Toxins and Clinical Parameters in Hemodialysis Patients.

When the kidney is seriously impaired, various uremic toxins (UTs) accumulate in the body, often exerting unfavorable effects on physiological functions and drug pharmacokinetics. To prevent this, it is important to determine plasma UT levels accurately in chronic kidney disease patients. Although attempts to predict plasma UT levels using biomarkers have been made, the correlation between UT levels and the markers is not yet fully understood. In this study, we assessed the correlations among plasma levels of indoxyl sulfate (IS), indoleacetic acid (IA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) in 20 hemodialysis patients and evaluated the relationship between the plasma levels of UTs and clinical parameters, such as serum creatinine (Scr), blood urea nitrogen, and estimated glomerular filtration rate (eGFR), with special focus on IS. There were no correlations among the plasma levels of the three UTs before and immediately after hemodialysis. However, a significant correlation was observed between plasma IS levels and Scr before hemodialysis (r=0.643, p=0.002), with the correlation becoming much stronger when using the data obtained immediately after hemodialysis (r=0.744, p<0.001). Further, plasma IS levels showed a significant negative correlation with eGFR (r=-0.558, p=0.011). However, no correlations were observed for IA or CMPF. The results obtained from this study suggest that plasma IS levels can be predicted from Scr values, although the precise mechanism behind the correlation remains to be clarified.

[Plasma concentrations of anionic uremic toxins in hemodialysis patients and their effects on protein binding of pravastatin].

Uremic toxins (UTs) accumulate in the body of hemodialysis patients. UTs often exert unfavorable effects on patients and cause significant interactions with clinically relevant drugs. In this study, we assayed plasma concentrations of three typical anionic UTs, indoxyl sulfate (IS), 3-indoleacetic acid (IA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), in 20 hemodialysis patients and 5 healthy volunteers. Moreover, the effects of these anionic UTs on the binding of pravastatin to human serum albumin (HSA) were also evaluated. CMPF concentrations in the plasma of patients were unchanged before and after dialysis (63.0 ± 6.3 µM and 65.1 ± 6.7 µM, respectively), and these values were about 5-fold greater compared with those in healthy volunteers. Although dialysis decreased the plasma IS concentration from 157.9 ± 19.9 µM to 103.8 ± 13.3 µM, the value after hemodialysis was still ca. 27-fold greater than that in healthy volunteers. IA concentrations before and after hemodialysis were almost identical to those in healthy volunteers. There were no significant differences in the plasma concentrations of the three anionic UTs between male and female patients. The magnitude of protein binding was in the order CMPF>IS>IA, indicating that hemodialysis clearance of these anionic UTs was dependent on their protein binding capacities. The ability of IS to reduce pravastatin binding to HSA was much greater than that of CMPF. The present results suggest that statins bind to site II on HSA, and that their binding is modulated by IS when elevated in hemodialysis patients.

Fluorescence resonance energy transfer-based assay for DNA-binding protein tagged by green fluorescent protein.

Specific interaction between green fluorescent protein (GFP)-tagged human alpha- or gamma-enolase(97-242) (alpha or gammaENO(97-242)) and the rhodamine-labeled DNA fragment containing the c-myc P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a "real-time FRET assay." The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO(97-242) and the c-myc P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (R(as)) of ENO(97-242) mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.