Optimized metabolomic approach to identify uremic solutes in plasma of stage 3-4 chronic kidney disease patients.
BACKGROUND
Chronic kidney disease (CKD) is characterized by the progressive accumulation of various potential toxic solutes. Furthermore, uremic plasma is a complex mixture hampering accurate determination of uremic toxin levels and the identification of novel uremic solutes.


METHODS
In this study, we applied (1)H-nuclear magnetic resonance (NMR) spectroscopy, following three distinct deproteinization strategies, to determine differences in the plasma metabolic status of stage 3-4 CKD patients and healthy controls. Moreover, the human renal proximal tubule cell line (ciPTEC) was used to study the influence of newly indentified uremic solutes on renal phenotype and functionality.


RESULTS
Protein removal via ultrafiltration and acetonitrile precipitation are complementary techniques and both are required to obtain a clear metabolome profile. This new approach, revealed that a total of 14 metabolites were elevated in uremic plasma. In addition to confirming the retention of several previously identified uremic toxins, including p-cresyl sulphate, two novel uremic retentions solutes were detected, namely dimethyl sulphone (DMSO2) and 2-hydroxyisobutyric acid (2-HIBA). Our results show that these metabolites accumulate in non-dialysis CKD patients from 9±7 µM (control) to 51±29 µM and from 7 (0-9) µM (control) to 32±15 µM, respectively. Furthermore, exposure of ciPTEC to clinically relevant concentrations of both solutes resulted in an increased protein expression of the mesenchymal marker vimentin with more than 10% (p<0.05). Moreover, the loss of epithelial characteristics significantly correlated with a loss of glucuronidation activity (Pearson r = -0.63; p<0.05). In addition, both solutes did not affect cell viability nor mitochondrial activity.


CONCLUSIONS
This study demonstrates the importance of sample preparation techniques in the identification of uremic retention solutes using (1)H-NMR spectroscopy, and provide insight into the negative impact of DMSO2 and 2-HIBA on ciPTEC, which could aid in understanding the progressive nature of renal disease.
Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Henricus A M
HA
Mutsaers
Udo F H
UF
Engelke
Martijn J G
MJ
Wilmer
Jack F M
JF
Wetzels
Ron A
RA
Wevers
Lambertus P
LP
van den Heuvel
Joost G
JG
Hoenderop
Rosalinde
R
Masereeuw
D000328:Adult; D015415:Biomarkers / Q000097:blood*; D016022:Case-Control Studies; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007687:Kidney Tubules, Proximal; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D008875:Middle Aged; D006435:Renal Dialysis; D051436:Renal Insufficiency, Chronic / Q000150:complications / Q000378:metabolism* / Q000473:pathology; D014118:Toxins, Biological / Q000097:blood*; D014511:Uremia / Q000097:blood / Q000175:diagnosis* / Q000209:etiology
D015415:Biomarkers; D014118:Toxins, Biological