Abstract
BackgroundChronic 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.MethodsIn this study, we applied 1H-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.ResultsProtein 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.ConclusionsThis study demonstrates the importance of sample preparation techniques in the identification of uremic retention solutes using 1H-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.
Highlights
The kidneys play an important role in maintaining total body homeostasis by facilitating the urinary secretion of both endogenous and exogenous waste products
It is well documented that uremic toxins accumulate in dialysis patients and several biomarkers of Chronic kidney disease (CKD) have been identified [2,3,4,5]; yet, less is known about the retention of possible toxic solutes in other patients with a compromised kidney function
Our results revealed that ultrafiltration and acetonitrile extraction are complementary deproteinization techniques and both are required as sample preparation methods for the proper detection of uremic retention solutes using 1H-nuclear magnetic resonance (NMR) spectroscopy
Summary
The kidneys play an important role in maintaining total body homeostasis by facilitating the urinary secretion of both endogenous and exogenous waste products. Chronic kidney disease (CKD) affects approximately 10% of the adult population in developed countries. In half of these patients the diagnosis of CKD is based on the presence of a reduced kidney function (chronic renal failure; CRF). In CKD patients adequate renal clearance is compromised resulting in the accumulation of a plethora of uremic solutes [1]. It is well documented that uremic toxins accumulate in dialysis patients and several biomarkers of CKD have been identified [2,3,4,5]; yet, less is known about the retention of possible toxic solutes in other patients with a compromised kidney function. Uremic plasma is a complex mixture hampering accurate determination of uremic toxin levels and the identification of novel uremic solutes
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