During chronic kidney disease (CKD), there is a progressive accumulation of toxic solutes due to inadequate renal clearance. Here, the interaction between uremic toxins and two important efflux pumps, viz. multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP) was investigated. Membrane vesicles isolated from MRP4- or BCRP-overexpressing human embryonic kidney cells were used to study the impact of uremic toxins on substrate specific uptake. Furthermore, the concentrations of various uremic toxins were determined in plasma of CKD patients using high performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Our results show that hippuric acid, indoxyl sulfate and kynurenic acid inhibit MRP4-mediated [³H]-methotrexate ([³H]-MTX) uptake (calculated Ki values: 2.5 mM, 1 mM, 25 µM, respectively) and BCRP-mediated [³H]-estrone sulfate ([³H]-E1S) uptake (Ki values: 4 mM, 500 µM and 50 µM, respectively), whereas indole-3-acetic acid and phenylacetic acid reduce [³H]-MTX uptake by MRP4 only (Ki value: 2 mM and IC₅₀ value: 7 mM, respectively). In contrast, p-cresol, p-toluenesulfonic acid, putrescine, oxalate and quinolinic acid did not alter transport mediated by MRP4 or BCRP. In addition, our results show that hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid and phenylacetic acid accumulate in plasma of end-stage CKD patients with mean concentrations of 160 µM, 4 µM, 129 µM, 1 µM and 18 µM, respectively. Moreover, calculated Ki values are below the maximal plasma concentrations of the tested toxins. In conclusion, this study shows that several uremic toxins inhibit active transport by MRP4 and BCRP at clinically relevant concentrations.

Although there has been increasing interest in the use of high protein diets, little is known about dietary protein related changes in the mammalian metabolome. We investigated the influence of protein intake on selected tryptophan and phenolic compounds, derived from both endogenous and colonic microbial metabolism. Furthermore, potential inter-species metabolic differences were studied. For this purpose, 29 healthy subjects were allocated to a high (n = 14) or low protein diet (n = 15) for 2 weeks. In addition, 20 wild-type FVB mice were randomized to a high protein or control diet for 21 days. Plasma and urine samples were analyzed with liquid chromatography–mass spectrometry for measurement of tryptophan and phenolic metabolites. In human subjects, we observed significant changes in plasma level and urinary excretion of indoxyl sulfate (P 0.004 and P 0.001), and in urinary excretion of indoxyl glucuronide (P 0.01), kynurenic acid (P 0.006) and quinolinic acid (P 0.02). In mice, significant differences were noted in plasma tryptophan (P 0.03), indole-3-acetic acid (P 0.02), p-cresyl glucuronide (P 0.03), phenyl sulfate (P 0.004) and phenylacetic acid (P 0.01). Thus, dietary protein intake affects plasma levels and generation of various mammalian metabolites, suggesting an influence on both endogenous and colonic microbial metabolism. Metabolite changes are dissimilar between human subjects and mice, pointing to inter-species metabolic differences with respect to protein intake.

Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis‐inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25‐μM curcumin causes fibroblast apoptosis and that this could be inhibited by co‐administration of antioxidants N‐acetyl‐l‐cysteine (NAC), biliverdin or bilirubin, suggesting that reactive oxygen species (ROS) are involved. This is supported by our observation that 25‐μM curcumin caused the generation of ROS, which could be completely blocked by addition of NAC or bilirubin. Since biliverdin and bilirubin are downstream products of heme degradation by heme oxygenase (HO), it has been suggested that HO‐activity protects against curcumin‐induced apoptosis. Interestingly, exposure to curcumin maximally induced HO‐1 protein and HO‐activity at 10–15 μM, whereas, at a concentration of >20‐μM curcumin HO‐1‐expression and HO‐activity was negligible. NAC‐mediated inhibition of 25‐μM curcumin‐induced apoptosis was demonstrated to act in part via restored HO‐1‐induction, since the rescuing effect of NAC could be reduced by inhibiting HO‐activity. Moreover pre‐induction of HO‐1 using 5‐μM curcumin protected fibroblasts against 25‐μM curcumin‐induced apoptosis. On a functional level, fibroblast‐mediated collagen gel contraction, an in vitro wound contraction model, was completely prevented by 25‐μM curcumin, while this could be reversed by co‐incubation with NAC, an effect that was also partially HO‐mediated. In conclusion, curcumin treatment in high doses (>25 μM) may provide a novel way to modulate pathological scar formation through the induction of fibroblast apoptosis, while antioxidants, HO‐activity and its effector molecules act as a possible fine‐tuning regulator.