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Kidney allograft failure due to chronic injury/rejection remains the main cause of graft loss in renal transplant recipients (RTR). Here, we investigated whether specific biomarkers of extracellular matrix (ECM) turnover are associated with allograft function and chronic kidney disease (CKD) stage in RTR. Seventy-eight patients who attended the University Medical Center Groningen for a routine check-up after kidney transplantation were enrolled in the study. Plasma and/or 24h-urine samples were collected and specific matrix-metalloproteinase-generated neo-epitope fragments of collagens were measured by enzyme-linked immunosorbent assay. Our results demonstrated that urinary levels of C3M, a marker for collagen type III degradation, correlated with estimated glomerular filtration rate (eGFR; r = 0.58, p

To date, over 150 possible uremic solutes have been listed, but their role in the progression of CKD is largely unknown. Here, the association between a selected panel of uremic solutes and progression in CKD patients was investigated.Patients from the MASTERPLAN study, a randomized controlled trial in CKD patients with a creatinine clearance between 20 and 70 ml/min per 1.73m2, were selected based on their rate of eGFR decline during the first five years of follow-up. They were categorized as rapid (decline >5 ml/min per year) or slow progressors. Concentrations of eleven uremic solutes were obtained at baseline and after one year of follow-up. Logistic regression was used to compare the odds for rapid to slow progression by uremic solute concentrations at baseline. Variability in uremic solute levels was assessed using scatter plots, and limits of variability were calculated.In total, 40 rapidly and 40 slowly progressing patients were included. Uremic solutes were elevated in all patients compared to reference values for healthy persons. The serum levels of uremic solutes were not associated with rapid progression. Moreover, we observed substantial variability in solute levels over time.Elevated concentrations of uremic solutes measured in this study did not explain differences in rate of eGFR decline in CKD patients, possibly due to lack of power as a result of the small sample size, substantial between patient variability, and variability in solute concentrations over time. The etiology of intra-individual variation in uremic solute levels remains to be elucidated.

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.

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

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 [(3)H]-methotrexate ([(3)H]-MTX) uptake (calculated Ki values: 2.5 mM, 1 mM, 25 µM, respectively) and BCRP-mediated [(3)H]-estrone sulfate ([(3)H]-E1S) uptake (Ki values: 4 mM, 500 µM and 50 µM, respectively), whereas indole-3-acetic acid and phenylacetic acid reduce [(3)H]-MTX uptake by MRP4 only (Ki value: 2 mM and IC(50) 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.

BACKGROUND: We previously identified curcumin as a potent inducer of fibroblast apoptosis, which could be used to treat hypertrophic scar formation. Here we investigated the underlying mechanism of this process. PRINCIPAL FINDINGS: Curcumin-induced apoptosis could not be blocked by caspase-inhibitors and we could not detect any caspase-3/7 activity. Curcumin predominantly induced mitochondria-mediated ROS formation and stimulated the expression of the redox-sensitive pro-apoptotic factor p53. Inhibition of the pro-apoptotic signaling enzyme glycogen synthase kinase-3beta (GSK-3beta) blocked curcumin-induced apoptosis. Apoptosis was associated with high molecular weight DNA damage, a possible indicator of apoptosis-inducing factor (AIF) activity. Indeed, curcumin caused nuclear translocation of AIF, which could be blocked by the antioxidant N-acetyl cysteine. We next investigated how AIF is effluxed from mitochondria in more detail. The permeability transition pore complex (PTPC), of which the voltage-dependent anion channel (VDAC) is a component, could be involved since the VDAC-inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) efficiently blocked AIF translocation. However, PTPC is not involved in AIF release since cyclosporine A, a specific inhibitor of the complex did not block apoptosis. Alternatively, the pro-apoptotic protein Bax could have formed mitochondrial channels and interacted with VDAC. Curcumin caused mitochondrial translocation of Bax, which was blocked by DIDS, suggesting a Bax-VDAC interaction. Interestingly, ceramide channels can also release apoptogenic factors from mitochondria and we found that addition of ceramide induced caspase-independent apoptosis. Surprisingly, this process could also be blocked by DIDS, suggesting the concerted action of Bax, VDAC and ceramide in the efflux of AIF from the mitochondrion. CONCLUSIONS: Curcumin-induced fibroblast apoptosis is totally caspase-independent and relies on the mitochondrial formation of ROS and the subsequent nuclear translocation of AIF, which is released from a mitochondrial pore that involves VDAC, Bax and possibly ceramides. The composition of the AIF-releasing channel seems to be much more complex than previously thought.

Chronic kidney disease (CKD) is a major global health concern and renal fibrosis is an integral part of the pathophysiological mechanism underlying disease progression. In CKD patients, the majority of metabolic pathways are in disarray and perturbations in enzyme activity most likely contribute to the wide variety of comorbidities observed in these patients. To illustrate, catabolism of tryptophan by indoleamine 2,3-dioxygenase (IDO) gives rise to numerous biologically active metabolites implicated in CKD progression. Here, we evaluated the effect of antagonizing IDO on renal fibrogenesis. To this end, we antagonized IDO using 1-methyl-D-tryptophan (1-MT) and BMS-98620 in TGF-β-treated murine precision-cut kidney slices (mPCKS) and in mice subjected to unilateral ureteral obstruction (UUO). The fibrotic response was evaluated on both the gene and protein level using qPCR and western blotting. Our results demonstrated that treatment with 1-MT or BMS-985205 markedly reduced TGF-β-mediated fibrosis in mPCKS, as seen by a decreased expression of collagen type 1, fibronectin, and α-smooth muscle actin. Moreover, IDO protein expression clearly increased following UUO, however, treatment of UUO mice with either 1-MT or BMS-986205 did not significantly affect the gene and protein expression of the tested fibrosis markers. However, both inhibitors significantly reduced the renal deposition of collagen in UUO mice as shown by Sirius red and trichrome staining. In conclusion, this study demonstrates that IDO antagonism effectively mitigates fibrogenesis in mPCKS and reduces renal collagen accumulation in UUO mice. These findings warrant further research into the clinical application of IDO inhibitors for the treatment of renal fibrosis.

Animal models are a valuable tool in preclinical research. However, limited predictivity of human biological responses in the conventional models has stimulated the search for reliable preclinical tools that show translational robustness. Here, we used precision-cut kidney slices (PCKS) as a model of renal fibrosis and investigated its predictive capacity for screening the effects of anti-fibrotics. Murine and human PCKS were exposed to TGFβ or PDGF pathway inhibitors with established anti-fibrotic efficacy. For each treatment modality, we evaluated whether it affected: (1) culture-induced collagen type I gene expression and interstitial accumulation; (2) expression of markers of TGFβ and PDGF signaling; and (3) expression of inflammatory markers. We summarized the outcomes of published in vivo animal and human studies testing the three inhibitors in renal fibrosis, and drew a parallel to the PCKS data. We showed that the responses of murine PCKS to anti-fibrotics highly corresponded with the known in vivo responses observed in various animal models of renal fibrosis. Moreover, our results suggested that human PCKS can be used to predict drug efficacy in clinical trials. In conclusion, our study demonstrated that the PCKS model is a powerful predictive tool for ex vivo screening of putative drugs for renal fibrosis.

Abstract Objective Renal fibrosis is one of the main pathophysiological processes underlying the progression of chronic kidney disease and kidney allograft failure. In the past decades, overwhelming efforts have been undertaken to find druggable targets for the treatment of renal fibrosis, mainly using cell- and animal models. However, the latter often do not adequately reflect human pathogenesis, obtained results differ per strain within a given species, and the models are associated with considerable discomfort for the animals. Therefore, the objective of this study is to implement the 3Rs in renal fibrosis research by establishing an animal-free drug screening platform for renal fibrosis based on human precision-cut kidney slices (PCKS) and by limiting the use of reagents that are associated with significant animal welfare concerns. Results Using Western blotting and gene expression arrays, we show that transforming growth factor-β (TGF-β) induced fibrosis in human PCKS. In addition, our results demonstrated that butaprost, SC-19220 and tamoxifen – all putative anti-fibrotic compounds – altered TGF-β-induced pro-fibrotic gene expression in human PCKS. Moreover, we observed that all compounds modulated fairly distinct sets of genes, however they all impacted TGF-β/SMAD signaling. In conclusion, this study revealed that it is feasible to use an animal-free approach to test drug efficacy and elucidate mechanisms of action.

Renal fibrosis is characterized by progressive accumulation of extracellular matrix (ECM) proteins, resulting in loss of organ function and eventually requiring renal replacement therapy. Unfortunately, no efficacious treatment options are available to halt renal fibrosis and translational models to test pharmacological agents are not always representative. Here, we evaluated murine precision-cut kidney slices (mPCKS) as a promising ex vivo model of renal fibrosis in which pathophysiology as well as therapeutics can be studied. Unique to this model is the use of rodent as well as human renal tissue, further closing the gap between animal models and clinical trials. Kidneys from C57BL/6 mice were used to prepare mPCKS and slices were incubated up to 96h. Viability, morphology, gene expression of fibrosis markers (Col1a1, Acta2, Serpinh1, Fn1, and Pai-1), inflammatory markers (Il1b, Il6, Cxcl1), and protein expression (collagen type 1, α-smooth muscle actin, HSP47) were determined. Furthermore, to understand the role of the transforming-growth factor β (TGF-β) pathway in mPCKS, slices were incubated with a TGF-β receptor inhibitor (LY2109761) for 48 h. Firstly, viability and morphology revealed an optimal incubation period of 48 h. Secondly, we demonstrated an early inflammatory response in mPCKS, which was accompanied by subsequent spontaneous fibrogenesis. Finally, LY2109761 showed great antifibrotic capacity in mPCKS by decreasing fibrosis markers on mRNA level as well as by reducing HSP47 protein expression. To conclude, we here present an ex vivo model of renal fibrosis, which can be used to further unravel the mechanisms of renal fibrogenesis and to screen antifibrotic therapy efficacy.