Albumin-bound kynurenic acid is an appropriate endogenous biomarker for assessment of the renal tubular OATs-MRP4 channel.

Renal tubular secretion mediated by organic anion transporters (OATs) and the multidrug resistance-associated protein 4 (MRP4) is an important means of drug and toxin excretion. Unfortunately, there are no biomarkers to evaluate their function. The aim of this study was to identify and characterize an endogenous biomarker of the renal tubular OATs-MRP4 channel. Twenty-six uremic toxins were selected as candidate compounds, of which kynurenic acid was identified as a potential biomarker by assessing the protein-binding ratio and the uptake in OAT1-, OAT3-, and MRP4-overexpressing cell lines. OAT1/3 and MRP4 mediated the transcellular vectorial transport of kynurenic acid in vitro. Serum kynurenic acid concentration was dramatically increased in rats treated with a rat OAT1/3 (rOAT1/3) inhibitor and in rOAT1/3 double knockout (rOAT1/3-/-) rats, and the renal concentrations were markedly elevated by the rat MRP4 (rMRP4) inhibitor. Kynurenic acid was not filtered at the glomerulus (99% of albumin binding), and was specifically secreted in renal tubules through the OAT1/3-MRP4 channel with an appropriate affinity (Km) (496.7 µM and 382.2 µM for OAT1 and OAT3, respectively) and renal clearance half-life (t1/2) in vivo (3.7 ± 0.7 h). There is a strong correlation in area under the plasma drug concentration-time curve (AUC0-t) between cefmetazole and kynurenic acid, but not with creatinine, after inhibition of rOATs. In addition, the phase of increased kynurenic acid level is earlier than that of creatinine in acute kidney injury process. These results suggest that albumin-bound kynurenic acid is an appropriate endogenous biomarker for adjusting the dosage of drugs secreted by this channel or predicting kidney injury.

Gentamicin alleviates cholestatic liver injury by decreasing gut microbiota-associated bile salt hydrolase activity in rats.

Intrahepatic cholestasis lacks effective therapeutic drugs. The gut microbiota-associated bile salt hydrolases (BSH) may be a potential therapeutic target. In this study, oral administration of gentamicin (GEN) decreased the serum and hepatic levels of total bile acid in 17α-ethynylestradiol (EE)-induced cholestatic male rats, significantly improved the serum levels of hepatic biomarkers and reversed the histopathological changes in the liver. In healthy male rats, the serum and hepatic levels of total bile acid were also decreased by GEN, the ratio of primary to secondary bile acids, and conjugated to unconjugated bile acids was significantly increased, and the urinary excretion of total bile acid was elevated. 16S rDNA sequencing of the ileal contents revealed that GEN treatment substantially reduced the abundance of Lactobacillus and Bacteroides both of which expressed BSH. Consistently, BSH activity analysis by the generation of d5-chenodeoxycholic acid from d5-taurochenodeoxycholic acid in situ showed BSH was significantly inhibited in the ileal contents of rats treated with GEN. This finding led to an increased proportion of hydrophilic conjugated bile acids and facilitated the urinary excretion of total bile acids, thereby decreasing serum and hepatic total bile acids and reversing liver injury related to cholestasis. Our results provide important evidence that BSH can be a potential drug target for treating cholestasis.

The utility of endogenous glycochenodeoxycholate-3-sulfate and 4ß-hydroxycholesterol to evaluate the hepatic disposition of atorvastatin in rats.

The liver is an important organ for drugs disposition, and thus how to accurately evaluate hepatic clearance is essential for proper drug dosing. However, there are many limitations in drug dosage adjustment based on liver function and pharmacogenomic testing. In this study, we evaluated the ability of endogenous glycochenodeoxycholate-3-sulfate (GCDCA-S) and 4ß-hydroxycholesterol (4ß-HC) plasma levels to evaluate organic anion-transporting polypeptide (Oatps)-mediated hepatic uptake and Cyp3a-meidated metabolism of atorvastatin (ATV) in rats. The concentration of ATV and its metabolites, 2-OH ATV and 4-OH ATV, was markedly increased after a single injection of rifampicin (RIF), an inhibitor of Oatps. Concurrently, plasma GCDCA-S levels were also elevated. After a single injection of the Cyp3a inhibitor ketoconazole (KTZ), plasma ATV concentrations were significantly increased and 2-OH ATV concentrations were decreased, consistent with the metabolism of ATV by Cyp3a. However, plasma 4ß-HC was not affected by KTZ treatment despite it being a Cyp3a metabolite of cholesterol. After repeated oral administration of RIF, plasma concentrations of ATV, 2-OH ATV and 4-OH ATV were markedly increased and the hepatic uptake ratio of ATV and GCDCA-S was decreased. KTZ did not affect plasma concentrations of ATV, 2-OH ATV and 4-OH ATV, but significantly decreased the metabolic ratio of total and 4-OH ATV. However, the plasma level and hepatic metabolism of 4ß-HC were not changed by KTZ. The inhibition of hepatic uptake of GCDCA-S by RIF was fully reversed after a 7-d washout of RIF. Plasma concentration and hepatic uptake ratio of GCDCA-S were correlated with the plasma level and hepatic uptake of ATV in rats with ANIT-induced liver injury, respectively. These results demonstrate that plasma GCDCA-S is a sensitive probe for the assessment of Oatps-mediated hepatic uptake of ATV. However, Cyp3a-mediated metabolism of ATV was not predicted by plasma 4ß-HC levels in rats.

An LC-MS/MS analytical method for the determination of uremic toxins in patients with end-stage renal disease.

End-stage renal disease (ESRD) is the last stage of chronic kidney disease, characterized by the progressive accumulation of uremic toxins (UTs). Hemodialysis is the standard approach to remove UTs from the body. Creatinine and urea levels are important indices of hemodialysis effectiveness, but the utility of those markers to estimate the removal of UTs, especially protein-binding UTs is limited. We developed an LC-MS/MS method for the quantification of UTs and to provide markers for evaluating hemodialysis effectiveness. These substances were extracted from serum samples after acetonitrile precipitation of protein and then separated on a HILIC column. The flow rate was 0.6 mL/min with a run time of 8.0 min for the negative ion mode and positive ion mode each. In this study 26 UTs were determined in normal subjects and in patients with ESRD before and after hemodialysis; serum levels were significantly higher in patients with ESRD than in subjects with normal renal function. A significant decrease in a variety of serum UTs were observed in patients after dialysis treatment, but no change in the levels of orotic acid, CMPF, kynurenic acid, p-cresol sulfate, phenyl-ß-d-glucuronide, 4-ethylphenyl sulfate and 3-indolyl-ß-d-glucopyranoside was found. These results show that some UTs could not be completely removed by hemodialysis. In addition, some biomarkers of different types of UTs are proposed for evaluating hemodialysis effectiveness.