Indoxyl Sulfate-Induced Macrophage Toxicity and Therapeutic Strategies in Uremic Atherosclerosis.

Cardiovascular disease (CVD) frequently occurs in patients with chronic kidney disease (CKD), particularly those undergoing dialysis. The mechanisms behind this may be related to traditional risk factors and CKD-specific factors that accelerate atherosclerosis and vascular calcification in CKD patients. The accumulation of uremic toxins is a significant factor in CKD-related systemic disorders. Basic research suggests that indoxyl sulfate (IS), a small protein-bound uremic toxin, is associated with macrophage dysfunctions, including increased oxidative stress, exacerbation of chronic inflammation, and abnormalities in lipid metabolism. Strategies to mitigate the toxicity of IS include optimizing gut microbiota, intervening against the abnormality of intracellular signal transduction, and using blood purification therapy with higher efficiency. Further research is needed to examine whether lowering protein-bound uremic toxins through intervention leads to a reduction in CVD in patients with CKD.

Indoxyl Sulfate Aggravates Podocyte Damage through the TGF-ß1/Smad/ROS Signalling Pathway.

INTRODUCTION: Hyperglycaemia induces the production of a large quantity of reactive oxygen species (ROS) and activates the transforming growth factor ß1 (TGF-ß1)/Smad signalling pathway, which is the main initiating factor in the formation of diabetic nephropathy. Indoxyl sulphate (IS) is a protein-binding gut-derived uraemic toxin that localizes to podocytes, induces oxidative stress, and inflames podocytes. The involvement of podocyte damage in diabetic nephropathy through the TGF-ß1 signalling pathway is still unclear. METHODS: In this study, we cultured differentiated rat podocytes in vitro and measured the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA by quantitative real-time PCR (qRT-PCR) and Western blotting after siRNA-mediated TGF-ß1 silencing, TGF-ß1 overexpression, and the presence of the ROS inhibitor acetylcysteine. We detected the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, small mother against decapentaplegic (Smad)2/3, phosphorylated-Smad2/3 (p-Smad2/3), Smad7, NADPH oxidase 4 (NOX4), and ROS levels under high glucose (HG) and IS conditions. RESULTS: The results indicated that nephrin, synaptopodin, CD2AP, and SRGAP2a expressions were significantly upregulated, and α-SMA expression was significantly downregulated in the presence of HG under siRNA-mediated TGF-ß1 silencing or after the addition of acetylcysteine. However, in the presence of HG, the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a were significantly downregulated, and the expression of α-SMA was significantly upregulated with the overexpression of TGF-ß1. IS supplementation under HG conditions further significantly reduced the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a; altered the expressions of Smad2/3, p-Smad2/3, Smad7, and NOX4; and increased ROS production in podocytes. CONCLUSION: This study suggests that IS may modulate the expression of nephrin, synaptopodin, CD2AP, and SRGAP2a by regulating the ROS and TGF-ß1/Smad signalling pathways, providing new theoretical support for the treatment of diabetic nephropathy.

Roles of AhR/CYP1s signaling pathway mediated ROS production in uremic cardiomyopathy.

PURPOSE: Uremic cardiomyopathy (UCM) is the leading cause of chronic kidney disease (CKD) related mortality. Uremic toxins including indoxyl sulfate (IS) play important role during the progression of UCM. This study was to explore the underlying mechanism of IS related myocardial injury. METHODS: UCM rat model was established through five-sixths nephrectomy to evaluate its effects on blood pressure, cardiac impairment, and histological changes using echocardiography and histological analysis. Additionally, IS was administered to neonatal rat cardiomyocytes (NRCMs) and the human cardiomyocyte cell line AC16. DHE staining and peroxide-sensitive dye 2',7'-dichlorofluorescein diacetate (H2DCFDA) was conducted to assess the reactive oxygen species (ROS) production. Cardiomyocyte hypertrophy was estimated using wheat germ agglutinin (WGA) staining and immunofluorescence. Aryl hydrocarbon receptor (AhR) translocation was observed by immunofluorescence. The activation of AhR was evaluated by immunoblotting of cytochrome P450 1 s (CYP1s) and quantitative real-time PCR (RT-PCR) analysis of AHRR and PTGS2. Additionally, the pro-oxidative and pro-hypertrophic effects were evaluated using the AhR inhibitor CH-223191, the CYP1s inhibitor Alizarin and the ROS scavenger N-Acetylcysteine (NAC). RESULTS: UCM rat model was successfully established, and cardiac hypertrophy, accompanied by increased blood pressure, and myocardial fibrosis. Further research confirmed the activation of the AhR pathway in UCM rats including AhR translocation and downstream protein CYP1s expression, accompanied with increasing ROS production detected by DHE staining. In vitro experiment demonstrated a translocation of AhR triggered by IS, leading to significant increase of downstream gene expression. Subsequently study indicated a close relationship between the production of ROS and the activation of AhR/CYP1s, which was effectively blocked by applying AhR inhibitor, CYP1s inhibitor and siRNA against AhR. Moreover, the inhibition of AhR/CYP1s/ROS pathway collectively blocked the pro-hypertrophic effect of IS-mediated cardiomyopathy. CONCLUSION: This study provides evidence that the AhR/CYP1s pathway is activated in UCM rats, and this activation is correlated with the uremic toxin IS. In vitro studies indicate that IS can stimulate the AhR translocation in cardiomyocyte, triggering to the production of intracellular ROS via CYP1s. This process leads to prolonged oxidative stress stimulation and thus contributes to the progression of uremic toxin-mediated cardiomyopathy.

Uremic toxins mediate kidney diseases: the role of aryl hydrocarbon receptor.

Aryl hydrocarbon receptor (AhR) was originally identified as an environmental sensor that responds to pollutants. Subsequent research has revealed that AhR recognizes multiple exogenous and endogenous molecules, including uremic toxins retained in the body due to the decline in renal function. Therefore, AhR is also considered to be a uremic toxin receptor. As a ligand-activated transcriptional factor, the activation of AhR is involved in cell differentiation and senescence, lipid metabolism and fibrogenesis. The accumulation of uremic toxins in the body is hazardous to all tissues and organs. The identification of the endogenous uremic toxin receptor opens the door to investigating the precise role and molecular mechanism of tissue and organ damage induced by uremic toxins. This review focuses on summarizing recent findings on the role of AhR activation induced by uremic toxins in chronic kidney disease, diabetic nephropathy and acute kidney injury. Furthermore, potential clinical approaches to mitigate the effects of uremic toxins are explored herein, such as enhancing uremic toxin clearance through dialysis, reducing uremic toxin production through dietary interventions or microbial manipulation, and manipulating metabolic pathways induced by uremic toxins through controlling AhR signaling. This information may also shed light on the mechanism of uremic toxin-induced injury to other organs, and provide insights into clinical approaches to manipulate the accumulated uremic toxins.

Effects of the uremic toxin indoxyl sulfate on seizure activity, learning and brain oxidative stress parameters in mice.

Patients with end-stage renal disease often have neurological disorders, with a higher incidence of memory impairment or epilepsy than in the general population. Patients undergoing hemodialysis are particularly exposed to the biological effects of uremic toxins. Indoxyl sulfate (IS) is one of the most potent uremic toxins; however, its possible effects on seizure susceptibility or memory functions have yet to be elucidated. In the current study, we focused on investigating the possible convulsant and amnesic effects of IS in recognized animal models. The study was performed on adult male Swiss mice. IS and scopolamine (SCO) were administered intraperitoneally (i.p.), and pentylenetetrazole (PTZ) was injected subcutaneously (s.c.). All substances were given as single injections. Acute IS administration (400 mg/kg) led to its accumulation in the brain. IS at doses of 200 and 400 mg/kg decreased the PTZ convulsive threshold, and at the same doses, it did not significantly affect the threshold for electroconvulsions. IS (200 and 400 mg/kg) did not impair learning in the passive avoidance test and did not increase the SCO-induced memory impairment in this test. IS increased lipid peroxidation, decreased the level of reduced glutathione, and reduced the activity of superoxide dismutase and catalase in mouse brains. Exposure to IS did not significantly change the activity of acetylcholinesterase in the brain tissue. This study shows that acute exposure to IS induces oxidative stress in the brain and potentiates PTZ-induced seizures in mice. Further studies are needed to find out whether IS-induced oxidative stress may affect epileptic seizures and/or epileptogenesis.

Hyperoside ameliorates cisplatin-induced acute kidney injury by regulating the expression and function of Oat1.

Cisplatin is a widely used chemotherapeutic agent to treat solid tumours in clinics. However, cisplatin-induced acute kidney injury (AKI) limits its clinical application. This study investigated the effect of hyperoside (a flavonol glycoside compound) on regulating AKI.The model of cisplatin-induced AKI was established, and hyperoside was preadministered to investigate its effect on improving kidney injury.Hyperoside ameliorated renal pathological damage, reduced the accumulation of SCr, BUN, Kim-1 and indoxyl sulphate in vivo, increased the excretion of indoxyl sulphate into the urine, and upregulated the expression of renal organic anion transporter 1 (Oat1). Moreover, evaluation of rat kidney slices demonstrated that hyperoside promoted the uptake of PAH (p-aminohippurate, the Oat1 substrate), which was confirmed by transient over-expression of OAT1 in HEK-293T cells. Additionally, hyperoside upregulated the mRNA expression of Oat1 upstream regulators hepatocyte nuclear factor-1α (HNF-1α) and pregnane X receptor (PXR).These findings indicated hyperoside could protect against cisplatin-induced AKI by promoting indoxyl sulphate excretion through regulating the expression and function of Oat1, suggesting hyperoside may offer a potential tactic for cisplatin-induced AKI treatment.

Emodin Inhibits the Indoxyl Sulfate-Induced trans-Differentiation of Vascular Smooth Muscle Cells through Upregulating Thrombospondin-1.

BACKGROUND: Indoxyl sulfate (IS) is a protein-bound uremic toxin with vascular toxicity. The primary cause of death in uremic patients on maintenance hemodialysis is vascular disease, and it had been reported that vascular smooth muscle cells (VSMCs) trans-differentiation (VT) plays a vital role in the context of vascular diseases, but the underlying mechanisms remain obscure. Thrombospondin-1 (TSP-1) participates in vascular calcification by keeping the balance of extracellular matrix, but its role in IS-induced VT is unclear. METHODS: In this study, clinical specimens, animal models, and in vitro VSMCs were used to investigate the role of TSP-1 in IS induced VT and the potential therapeutic methods. RESULTS: We found that TSP-1 was significantly decreased in arterial samples from uremic patients, animal models, and in VSMCs after IS treatment. Downregulation of TSP-1 sufficiently induced the trans-differentiation genotypes of VSMCs. CONCLUSION: Emodin, the main monomer extracted from rhubarb, could alleviate IS-induced VT in vitro by upregulating TSP-1. Taken together, IS induces VT by downregulating TSP-1. Emodin might be a candidate drug to alleviate VT under IS treatment.

Uremic Toxins Induce THP-1 Monocyte Endothelial Adhesion and Migration through Specific miRNA Expression.

Atherosclerosis is initiated by the activation of endothelial cells that allows monocyte adhesion and transmigration through the vascular wall. The accumulation of uremic toxins such as indoxyl sulphate (IS) and p-cresol (PC) has been associated with atherosclerosis. Currently, miRNAs play a crucial role in the regulation of monocyte activation, adhesion, and trans-endothelial migration. The aim of the present study is to evaluate the effect of IS and PC on monocyte adhesion and migration processes in monocytes co-cultured with endothelial cells as well as to determine the underlying mechanisms. The incubation of HUVECs and THP-1 cells with both IS and PC toxins resulted in an increased migratory capacity of THP-1 cells. Furthermore, the exposure of THP-1 cells to both uremic toxins resulted in the upregulation of BMP-2 and miRNAs-126-3p, -146b-5p, and -223-3p, as well as the activation of nuclear factor kappa B (NF-κB) and a decrease in its inhibitor IĸB. Uremic toxins, such as IS and PC, enhance the migratory and adhesion capacity of THP-1 cells to the vascular endothelium. These toxins, particularly PC, contribute significantly to uremia-associated vascular disease by increasing in THP-1 cells the expression of BMP-2, NF-κB, and key miRNAs associated with the development of atherosclerotic vascular diseases.

Indoxyl sulfate induces apoptosis in mononuclear blood cells via mitochondrial pathway.

The consequence of chronic kidney disease is the accumulation of metabolic products called uremic toxins in the body. Indoxyl sulfate (IS) is a toxin with a high affinity for proteins. This study focuses on the deleterious effect of IS, especially apoptosis induction, in mononuclear blood cells (MNCs). Thus, in MNCs treated with IS at three different concentrations for 24 h, the survival, mitochondrial potential, caspases activity and expression, Bcl-2 and Bax protein expression, DNA damage, and PARP degradation were estimated. The study showed a decrease in survival and mitochondrial potential of MNCs treated with IS compared to the control. IS increased the activity of caspase 2-, 3-, 9-, and the expression of caspase 3-, and 9- in MNCs but does not affect the activity of caspase 6- and 8. The treatment of MNCs with IS also increased DNA damage and degradation of PARP. Indoxyl sulfate significantly influences the expression of Bcl-2 and Bax proteins. Indoxyl sulfate induces the programmed death of MNCs through the intrinsic mitochondrial apoptotic pathway. The observed cellular changes are mostly dose-dependent.

Effects of resveratrol on inflammation and oxidative stress induced by the uremic toxin indoxyl sulfate in Murine macrophage-like RAW 264.7.

Indoxyl sulfate (IS) is a uremic toxin produced by the gut microbiota that commonly accumulates in patients with chronic kidney disease (CKD) and can be harmful. Resveratrol is a polyphenol with properties that attenuate oxidative stress and inflammation. This study aims to evaluate the effect of resveratrol against the damage caused by IS in RAW 264.7 murine macrophages. Cells were treated with 0, 250, 500 and 1000 µmol/L of IS, in the presence of 50 µmol/L of resveratrol. The mRNA and protein expressions of erythroid-related nuclear factor 2 (Nrf2) and nuclear factor kappa-B (NF-κB) were measured using rt-PCR and Western blot analysis, respectively. Malondialdehyde (MDA) and reactive oxygen species (ROS) levels were also analyzed. As a result, it was demonstrated that resveratrol induces the activation of the Nrf2 pathway that enhances cytoprotective response. IS upregulated the NF-κB expression and downregulated the Nrf2 expression. In contrast, resveratrol treatment significantly reduced the MDA and ROS production and inhibited the IS-induced expression of NF-κB in macrophage-like RAW 264.7. In conclusion, resveratrol can mitigate inflammation and oxidative stress caused by uremic toxins produced by the gut microbiota, such as IS.

The Uremic Toxin Indoxyl Sulfate Accelerates Senescence in Kidney Proximal Tubule Cells.

Kidney fibrosis is the common final pathway of nearly all chronic and progressive nephropathies. One cause may be the accumulation of senescent cells that secrete factors (senescence associated secretory phenotype, SASP) promoting fibrosis and inflammation. It has been suggested that uremic toxins, such as indoxyl sulfate (IS), play a role in this. Here, we investigated whether IS accelerates senescence in conditionally immortalized proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1), thereby promoting kidney fibrosis. Cell viability results suggested that the tolerance of ciPTEC-OAT1 against IS increased in a time-dependent manner at the same dose of IS. This was accompanied by SA-ß-gal staining, confirming the accumulation of senescent cells, as well as an upregulation of p21 and downregulation of laminB1 at different time points, accompanied by an upregulation in the SASP factors IL-1ß, IL-6 and IL-8. RNA-sequencing and transcriptome analysis revealed that IS accelerates senescence, and that cell cycle appears to be the most relevant factor during the process. IS accelerates senescence via TNF-α and NF-ĸB signalling early on, and the epithelial-mesenchymal transition process at later time points. In conclusion, our results suggest that IS accelerates cellular senescence in proximal tubule epithelial cells.

Curcumin enhances p-cresyl sulfate-induced cytotoxic effects on renal tubular cells.

Indoxyl sulfate (IS) and p-cresyl sulfate (PCS), protein-bound uremic toxins, can induce oxidative stress and cause renal disease progression. However, the different cytotoxic effects on renal cells between IS and PCS are not stated. Due to uremic toxins are generally found in CKD patients, the mechanisms of uremic toxins-induced renal injury are required to study. Curcumin has anti-oxidant, anti-inflammatory and anti-apoptotic effects which may be potential used to protect against renal damage. In contrast, curcumin also exert cytotoxic effects on various cells. In addition, curcumin may reduce or enhance cytotoxicity combined with different chemicals treatments. However, whether curcumin may influence uremic toxins-induced renal injury is unclear. The goal of this study is to compare the different cytotoxic effects on renal cells between IS and PCS treatment, as well as the synergistic or antagonistic effects by combination treatments with curcumin and PCS. Our experimental result shows the PCS exerts a stronger antiproliferative effect on renal tubular cells than IS treatment. In addition, our study firstly demonstrates that curcumin enhances PCS-induced cell cytotoxicity through caspase-dependent apoptotic pathway and cell cycle alteration.

Effects of the uremic toxin indoxyl sulphate on human microvascular endothelial cells.

Indoxyl sulphate (IS) is a uremic toxin accumulating in the plasma of chronic kidney disease (CKD) patients. IS accumulation induces side effects in the kidneys, bones and cardiovascular system. Most studies assessed IS effects on cell lines by testing higher concentrations than those measured in CKD patients. Differently, we exposed a human microvascular endothelial cell line (HMEC-1) to the IS concentrations measured in the plasma of healthy subjects (physiological) or CKD patients (pathological). Pathological concentrations reduced cell proliferation rate but did not increase long-term oxidative stress level. Indeed, total protein thiols decreased only after 24 h of exposure in parallel with an increased Nrf-2 protein expression. IS induced actin cytoskeleton rearrangement with formation of stress fibres. Proteomic analysis supported this hypothesis as many deregulated proteins are related to actin filaments organization or involved in the endothelial to mesenchymal transition. Interestingly, two proteins directly linked to cardiovascular diseases (CVD) in in vitro and in vivo studies underwent deregulation: COP9 signalosome complex subunit 9 and thrombomodulin. Future experiments will be needed to investigate the role of these proteins and the signalling pathways in which they are involved to clarify the possible link between CKD and CVD.

Uremic toxin indoxyl sulfate induces dysfunction of vascular smooth muscle cells via integrin-ß1/ERK signaling pathway.

BACKGROUND: Protein-bound uremic toxins (PBUTs) are reported to be one of the major culprits in chronic kidney disease-cardiovascular disease (CKD-CVD) development, yet its mechanism is not fully clear. Our previous study confirmed elevated expression of integrin-ß1 (ITGß1) in vascular smooth muscle cells of uremic patients. Thus, this study aimed to explore the relationship between PBUTs and ITGß1 in uremic vasculature injury. METHODS: Human umbilical vein smooth muscle cells (HUVSMCs) and endothelial cells (HUVECs) were treated with two representative PUBTs, indoxyl sulfate (IS) and p-cresyl sulfate (PC). Both cells were measured for the expression of ITGß1 and downstream signaling pathways and assayed for proliferation, migration, adhesion and apoptosis. RESULTS: The IS treatments were observed with significantly up-regulated ITGß1 in HUVSMCs but not in HUVECs, while PC did not induce ITGß1 alteration in either HUVSMCs or HUVECs. Furthermore, overexpression of ITGß1 revealed activated downstream signal-regulated kinase (ERK) signaling pathway with promoted focal adhesion, migration, proliferation but no apoptosis in HUVSMCs by IS. These functional and pathway alterations could be significantly suppressed by RNA interference of ITGß1. More importantly, the application of ERK1/2 inhibitor significantly suppressed the focal adhesion, migration and proliferation of HUVSMCs. CONCLUSION: We first demonstrated that ITGß1/ERK signaling pathway mediated abnormal focal adhesion, migration and proliferation of vascular smooth muscle cells stimulated by IS. ITGß1/ERK signaling may serve as a novel therapeutic target for CKD-CVD.

Co-toxicity of Endotoxin and Indoxyl Sulfate, Gut-Derived Bacterial Metabolites, to Vascular Endothelial Cells in Coronary Arterial Disease Accompanied by Gut Dysbiosis.

Gut dysbiosis, alongside a high-fat diet and cigarette smoking, is considered one of the factors promoting coronary arterial disease (CAD) development. The present study aimed to research whether gut dysbiosis can increase bacterial metabolites concentration in the blood of CAD patients and what impact these metabolites can exert on endothelial cells. The gut microbiomes of 15 age-matched CAD patients and healthy controls were analyzed by 16S rRNA sequencing analysis. The in vitro impact of LPS and indoxyl sulfate at concentrations present in patients' sera on endothelial cells was investigated. 16S rRNA sequencing analysis revealed gut dysbiosis in CAD patients, further confirmed by elevated LPS and indoxyl sulfate levels in patients' sera. CAD was associated with depletion of Bacteroidetes and Alistipes. LPS and indoxyl sulfate demonstrated co-toxicity to endothelial cells inducing reactive oxygen species, E-selectin, and monocyte chemoattractant protein-1 (MCP-1) production. Moreover, both of these metabolites promoted thrombogenicity of endothelial cells confirmed by monocyte adherence. The co-toxicity of LPS and indoxyl sulfate was associated with harmful effects on endothelial cells, strongly suggesting that gut dysbiosis-associated increased intestinal permeability can initiate or promote endothelial inflammation and atherosclerosis progression.

Uremic toxins activate CREB/ATF1 in endothelial cells related to chronic kidney disease.

Uremic toxins, such as p-cresyl sulfate (PCS) and indoxyl sulfate (IS), contribute to endothelial dysfunction in chronic kidney disease (CKD). This process is mediated by several cellular pathways, but it is unclear whether cAMP-responsive element-binding protein (CREB) and activating transcription factor 1 (ATF1) participate in endothelial dysfunction in uremic conditions despite playing roles in inflammatory modulation. This study aimed to evaluate the expression, activation, and transcriptional activity of CREB/ATF1 in endothelial cells exposed to PCS, IS, and uremic serum (US). In vitro, ATF1 protein levels were increased by PCS and IS, whereas CREB levels were enhanced only by IS. Activation through CREB-Ser133 and ATF1-Ser63 phosphorylation was induced by PCS, IS, and US. We evaluated the CREB/ATF1 transcriptional activity by analyzing the expression of their target genes, including ICAM1, PTGS2, NOX1, and SLC22A6, which are related to endothelial dysfunction through their roles in vascular inflammation, oxidative stress, and cellular uptake of PCS and IS. The expression of ICAM1, PTGS2 and NOX1 genes was increased by PCS, IS, and US, whereas that of SLC22A6 was induced only by IS. KG-501, a CREB inhibitor, restored the inductive effects of PCS on ICAM1, PTGS2, and NOX1 expression; IS on ICAM1, PTGS2 and SLC22A6 expression; and US on NOX1 expression. The presence of CREB and ATF1 was observed in healthy arteries and in arteries of patients with CKD, which were structurally damaged. These findings suggest that CREB/ATF1 is activated by uremic toxins and may play a relevant role in endothelial dysfunction in CKD.

Indoxyl Sulfate Activates NLRP3 Inflammasome to Induce Cardiac Contractile Dysfunction Accompanied by Myocardial Fibrosis and Hypertrophy.

In patients with chronic kidney diseases (CKD), high serum indoxyl sulfate (IS) levels correlate with cardiac fibrosis and hypertrophy and thus a critical risk factor for heart failure. The aim of this study was to determine the effects of IS on cardiac function and inflammasome pathway in a rat model of CKD. We assessed the physiological and pathological changes and measured biomarkers of fibrosis and hypertrophy in the hearts of Dahl salt-sensitive (DS), DS hypertensive (DH), and DH IS-treated rats (DH + IS). Low left ventricular (LV) ejection fraction, LV dilatation, and advanced myocardial fibrosis and hypertrophy were observed in DH + IS, which resemble changes found in uremic cardiomyopathy. These changes were independent of renal function and blood pressure. RT-PCR and western blotting analysis showed upregulation of fibrosis and hypertrophy-related biomarkers and adhesion molecules in the hearts of DH + IS rats. IS activated aryl hydrocarbon receptor (AHR) pathway, nuclear factor kappa B p65 (NF-κB p65), and inflammasome in the myocardium of DH + IS rat. Moreover, IS upregulated the expression of critical NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and increased production of IL-1ß and IL-18. Finally, IS upregulated various inflammatory cytokines, such as MCP-1, TNF-α, IL-6, and TGFß1, in the myocardium. Our results suggested that IS induced cardiac fibrosis and hypertrophy and impaired LV function through activation of cardiac NLRP3 inflammasome via the AHR/NF-κB pathway.

MicroRNA-mediated vascular intercellular communication is altered in chronic kidney disease.

AIMS: Chronic kidney disease (CKD) is an independent risk factor for the development of coronary artery disease (CAD). For both, CKD and CAD, the intercellular transfer of microRNAs (miRs) through extracellular vesicles (EVs) is an important factor of disease development. Whether the combination of CAD and CKD affects endothelial function through cellular crosstalk of EV-incorporated miRs is still unknown. METHODS AND RESULTS: Out of 172 screened CAD patients, 31 patients with CAD + CKD were identified and matched with 31 CAD patients without CKD. Additionally, 13 controls without CAD and CKD were included. Large EVs from CAD + CKD patients contained significantly lower levels of the vasculo-protective miR-130a-3p and miR-126-3p compared to CAD patients and controls. Flow cytometric analysis of plasma-derived EVs revealed significantly higher numbers of endothelial cell-derived EVs in CAD and CAD + CKD patients compared to controls. EVs from CAD + CKD patients impaired target human coronary artery endothelial cell (HCAEC) proliferation upon incubation in vitro. Consistent with the clinical data, treatment with the uraemia toxin indoxyl sulfate (IS)-reduced miR-130a-3p levels in HCAEC-derived EVs. EVs from IS-treated donor HCAECs-reduced proliferation and re-endothelialization in EV-recipient cells and induced an anti-angiogenic gene expression profile. In a mouse-experiment, intravenous treatment with EVs from IS-treated endothelial cells significantly impaired endothelial regeneration. On the molecular level, we found that IS leads to an up-regulation of the heterogenous nuclear ribonucleoprotein U (hnRNPU), which retains miR-130a-3p in the cell leading to reduced vesicular miR-130a-3p export and impaired EV-recipient cell proliferation. CONCLUSION: Our findings suggest that EV-miR-mediated vascular intercellular communication is altered in patients with CAD and CKD, promoting CKD-induced endothelial dysfunction.

The effect of dihydroceramide desaturase 1 inhibition on endothelial impairment induced by indoxyl sulfate.

Protein-bound uremic toxins (PBUTs) have adverse effects on vascular function, which is imperative in the progression of cardiovascular and renal diseases. The role of sphingolipids in PBUT-mediated vasculo-endothelial pathophysiology is unclear. This study assessed the therapeutic potential of dihydroceramide desaturase 1 (Des1) inhibition, the last enzyme involved in de novo ceramide synthesis, to mitigate the vascular effects of the PBUT indoxyl sulfate (IS). Rat aortic rings were isolated and vascular reactivity was assessed in organ bath experiments followed by immunohistochemical analyses. Furthermore, cultured human aortic endothelial cells were assessed for phenotypic and mechanistic changes. Inhibition of Des1 by a selective inhibitor CIN038 (0.1 to 0.3 µM) improved IS-induced impairment of vasorelaxation and modulated immunoreactivity of oxidative stress markers. Des1 inhibition also reversed IS-induced reduction in endothelial cell migration (1.0 µM) by promoting the expression of angiogenic cytokines and reducing inflammatory and oxidative stress markers. These effects were associated with a reduction of TIMP1 and the restoration of Akt phosphorylation. In conclusion, Des1 inhibition improved vascular relaxation and endothelial cell migration impaired by IS overload. Therefore, Des1 may be a suitable intracellular target to mitigate PBUT-induced adverse vascular effects.

Toxic Effects of Indoxyl Sulfate on Osteoclastogenesis and Osteoblastogenesis.

Uremic toxins, such as indoxyl sulfate (IS) and kynurenine, accumulate in the blood in the event of kidney failure and contribute to further bone damage. To maintain the homeostasis of the skeletal system, bone remodeling is a persistent process of bone formation and bone resorption that depends on a dynamic balance of osteoblasts and osteoclasts. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates the toxic effects of uremic toxins. IS is an endogenous AhR ligand and is metabolized from tryptophan. In osteoclastogenesis, IS affects the expression of the osteoclast precursor nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) through AhR signaling. It is possible to increase osteoclast differentiation with short-term and low-dose IS exposure and to decrease differentiation with long-term and/or high-dose IS exposure. Coincidentally, during osteoblastogenesis, through the AhR signaling pathway, IS inhibits the phosphorylation of ERK, and p38 reduces the expression of the transcription factor 2 (Runx2), disturbing osteoblastogenesis. The AhR antagonist resveratrol has a protective effect on the IS/AhR pathway. Therefore, it is necessary to understand the multifaceted role of AhR in CKD, as knowledge of these transcription signals could provide a safe and effective method to prevent and treat CKD mineral bone disease.