N. Mckay

Serum microRNAs are altered in various stages of chronic kidney disease: a preliminary study

Background MicroRNAs (miRNAs) are innovative and informative blood-based biomarkers involved in numerous pathophysiological processes. In this study and based on our previous experimental data, we investigated miR-126, miR-143, miR-145, miR-155 and miR-223 as potential circulating biomarkers for the diagnosis and prognosis of patients with chronic kidney disease (CKD). The primary objective of this study was to assess the levels of miRNA expression at various stages of CKD. Methods RNA was extracted from serum, and RT-qPCR was performed for the five miRNAs and cel-miR-39 (internal control). Results Serum levels of miR-143, -145 and -223 were elevated in patients with CKD compared with healthy controls. They were further increased in chronic haemodialysis patients, but were below control levels in renal transplant recipients. In contrast, circulating levels of miR-126 and miR-155 levels, which were also elevated in CKD patients, were lower in the haemodialysis group and even lower in the transplant group. Four of the five miRNA species were correlated with estimated glomerular filtration rate, and three were correlated with circulating uraemic toxins. Conclusions This exploratory study suggests that specific miRNAs could be biomarkers for complications of CKD, justifying further studies to link changes of miRNA levels with outcomes in CKD patients.

Aryl hydrocarbon receptor is activated in patients and mice with chronic kidney disease

Patients with chronic kidney disease (CKD) are exposed to uremic toxins and have an increased risk of cardiovascular disease. Some uremic toxins, like indoxyl sulfate, are agonists of the transcription factor aryl hydrocarbon receptor (AHR). These toxins induce a vascular procoagulant phenotype. Here we investigated AHR activation in patients with CKD and in a murine model of CKD. We performed a prospective study in 116 patients with CKD stage 3 to 5D and measured the AHR-Activating Potential of serum by bioassay. Compared to sera from healthy controls, sera from CKD patients displayed a strong AHR-Activating Potential; strongly correlated with eGFR and with the indoxyl sulfate concentration. The expression of the AHR target genes Cyp1A1 and AHRR was up-regulated in whole blood from patients with CKD. Survival analyses revealed that cardiovascular events were more frequent in CKD patients with an AHR-Activating Potential above the median. In mice with 5/6 nephrectomy, there was an increased serum AHR-Activating Potential, and an induction of Cyp1a1 mRNA in the aorta and heart, absent in AhR-/- CKD mice. After serial indoxyl sulfate injections, we observed an increase in serum AHR-AP and in expression of Cyp1a1 mRNA in aorta and heart in WT mice, but not in AhR-/- mice. Thus, the AHR pathway is activated both in patients and mice with CKD. Hence, AHR activation could be a key mechanism involved in the deleterious cardiovascular effects observed in CKD.

Indoxyl Sulfate Upregulates Liver P-Glycoprotein Expression and Activity through Aryl Hydrocarbon Receptor Signaling

In patients with CKD, not only renal but also, nonrenal clearance of drugs is altered. Uremic toxins could modify the expression and/or activity of drug transporters in the liver. We tested whether the uremic toxin indoxyl sulfate (IS), an endogenous ligand of the transcription factor aryl hydrocarbon receptor, could change the expression of the following liver transporters involved in drug clearance: SLC10A1, SLC22A1, SLC22A7, SLC47A1, SLCO1B1, SLCO1B3, SLCO2B1, ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCC6, and ABCG2 We showed that IS increases the expression and activity of the efflux transporter P-glycoprotein (P-gp) encoded by ABCB1 in human hepatoma cells (HepG2) without modifying the expression of the other transporters. This effect depended on the aryl hydrocarbon receptor pathway. Presence of human albumin at physiologic concentration in the culture medium did not abolish the effect of IS. In two mouse models of CKD, the decline in renal function associated with the accumulation of IS in serum and the specific upregulation of Abcb1a in the liver. Additionally, among 109 heart or kidney transplant recipients with CKD, those with higher serum levels of IS needed higher doses of cyclosporin, a P-gp substrate, to obtain the cyclosporin target blood concentration. This need associated with serum levels of IS independent of renal function. These findings suggest that increased activity of P-gp could be responsible for increased hepatic cyclosporin clearance. Altogether, these results suggest that uremic toxins, such as IS, through effects on drug transporters, may modify the nonrenal clearance of drugs in patients with CKD.

Indole 3-acetic acid, indoxyl sulfate and paracresyl-sulfate do not influence anemia parameters in hemodialysis patients

BackgroundThe main reason for anemia in renal failure patients is the insufficient erythropoietin production by the kidneys. Beside erythropoietin deficiency, in vitro studies have incriminated uremic toxins in the pathophysiology of anemia but clinical data are sparse. In order to assess if indole 3-acetic acid (IAA), indoxyl sulfate (IS), and paracresyl sulfate (PCS) -three protein bound uremic toxins- are clinically implicated in end-stage renal disease anemia we studied the correlation between IAA, IS and PCS plasmatic concentrations with hemoglobin and Erythropoietin Stimulating Agents (ESA) use in hemodialysis patients.MethodsBetween June and July 2014, we conducted an observational cross sectional study in two hemodialysis center. Three statistical approaches were conducted. First, we compared patients treated with ESA and those not treated. Second, we performed linear regression models between IAA, IS, and PCS plasma concentrations and hemoglobin, the ESA dose over hemoglobin ratio (ESA/Hemoglobin) or the ESA resistance index (ERI). Third, we used a polytomous logistic regression model to compare groups of patients with no/low/high ESA dose and low/high hemoglobin statuses.ResultsOverall, 240 patients were included in the study. Mean agexa0±xa0SD was 67.6xa0±xa016.0xa0years, 55.4% were men and 42.5% had diabetes mellitus.When compared with ESA treated patients, patients with no ESA had higher hemoglobin (mean 11.4xa0±xa01.1 versus 10.6xa0±xa01.2xa0g/dL; p <0.001), higher transferrin saturation (TSAT, 31.1xa0±xa016.3% versus 23.1xa0±xa011.5%; pxa0<xa00.001), less frequentlyxa0an IV iron prescription (52.1 versus 65.7%, pxa0=xa00.04) and were more frequently treated with hemodiafiltration (53.5 versus 36.7%). In univariate analysis, IAA, IS or PCS plasma concentrations did not differ between the two groups.In the linear model, IAA plasma concentration was not associated with hemoglobin, but was negatively associated with ESA/Hb (pxa0=xa00.02; Rxa0=xa00.18) and with the ERI (pxa0=xa00.03; Rxa0=xa00.17). IS was associated with none of the three anemia parameters. PCS was positively associated with hemoglobin (pxa0=xa00.03; Rxa0=xa00.14), but negatively with ESA/Hb (pxa0=xa00.03; Rxa0=xa00.17) and the ERI (pxa0=xa00.02; Rxa0=xa00.19). In multivariate analysis, the association of IAA concentration with ESA/Hb or ERI was not statistically significant, neither was the association of PCS with ESA/Hb or ERI. Identically, in the subgroup of 76 patients with no inflammation (CRP <5xa0mg/L) and no iron deficiency (TSAT >20%) linear regression between IAA, IS or PCS and any anemia parameter did not reach significance.In the third model, univariate analysis showed no intergroup significant differences for IAA and IS. Regarding PCS, the Low Hb/High ESA group had lower concentrations. However, when we compared PCS with the other significant characteristics of the five groups to the Low Hb/high ESA (our reference group), the polytomous logistic regression model didn’t show any significant difference for PCS.ConclusionsIn our study, using three different statistical models, we were unable to show any correlation between IAA, IS and PCS plasmatic concentrations and any anemia parameter in hemodialysis patients. Indolic uremic toxins and PCS have no or a very low effect on anemia parameters.

Mechanisms of tissue factor induction by the uremic toxin indole-3 acetic acid through aryl hydrocarbon receptor/nuclear factor-kappa B signaling pathway in human endothelial cells

Chronic kidney disease (CKD) is associated with high risk of thrombosis. Indole-3 acetic acid (IAA), an indolic uremic toxin, induces the expression of tissue factor (TF) in human umbilical vein endothelial cells (HUVEC) via the transcription factor aryl hydrocarbon receptor (AhR). This study aimed to understand the signaling pathways involved in AhR-mediated TF induction by IAA. We incubated human endothelial cells with IAA at 50xa0µM, the maximal concentration found in patients with CKD. IAA induced TF expression in different types of human endothelial cells: umbilical vein (HUVEC), aortic (HAoEC), and cardiac-derived microvascular (HMVEC-C). Using AhR inhibition and chromatin immunoprecipitation experiments, we showed that TF induction by IAA in HUVEC was controlled by AhR and that AhR did not bind to the TF promoter. The analysis of TF promoter activity using luciferase reporter plasmids showed that the NF-κB site was essential in TF induction by IAA. In addition, TF induction by IAA was drastically decreased by an inhibitor of the NF-κB pathway. IAA induced the nuclear translocation of NF-κB p50 subunit, which was decreased by AhR and p38MAPK inhibition. Finally, in a cohort of 92 CKD patients on hemodialysis, circulating TF was independently related to serum IAA in multivariate analysis. In conclusion, TF up-regulation by IAA in human endothelial cells involves a non-genomic AhR/p38 MAPK/NF-κB pathway. The understanding of signal transduction pathways related to AhR thrombotic/inflammatory pathway is of interest to find therapeutic targets to reduce TF expression and thrombotic risk in patients with CKD.