RESUMO
Increased protein-bound uremic toxins (PBUTs) in patients with chronic kidney disease (CKD) are associated with cardiovascular diseases (CVDs); however, whether retention of PBUTs causes CVD remains unclear. Previous studies assessing the impacts of PBUTs on the vasculature have relied on 2D cell cultures lacking in vivo microenvironments. Here, we investigated the impact of various PBUTs (p-cresol (PC), indoxyl sulfate (IS), and p-cresyl sulfate (PCS)) on microvascular function using an organ-on-a-chip (OOC). Human umbilical vein endothelial cells were used to develop 3D vessels. Chronic exposure to PC resulted in significant vascular leakage compared with controls, whereas IS or PCS treatment did not alter the permeability of 3D vessels. Increased permeability induced by PC was correlated with derangement of cell adherens junction complex, vascular endothelial (VE)-cadherin and filamentous (F)-actin. Additionally, PC decreased endothelial viability in a concentration-dependent manner with a lower IC50 in 3D vessels than in 2D cultures. IS slightly decreased cell viability, while PCS did not affect viability. PC induced inflammatory responses by increasing monocyte adhesion to endothelial surfaces of 3D vessels and IL-6 production. In conclusion, this study leveraged an OOC to determine the diverse effects of PBUTs, demonstrating that PC accumulation is detrimental to ECs during kidney insufficiency.
Assuntos
Cresóis , Células Endoteliais da Veia Umbilical Humana , Inflamação , Humanos , Cresóis/metabolismo , Cresóis/toxicidade , Células Endoteliais da Veia Umbilical Humana/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Indicã/metabolismo , Indicã/toxicidade , Caderinas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Toxinas Urêmicas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Ésteres do Ácido Sulfúrico/metabolismoRESUMO
AIMS: Cardiovascular pathology is the main cause of death in chronic kidney disease (CKD) patients. CKD is associated with the accumulation of uremic toxins in the bloodstream, and indoxyl sulfate (IS) is one of the most abundant uremic toxins found in the blood of CKD patients. We conducted an in vitro study to assess the mechanisms underlying the IS-induced endothelial dysfunction that could lead to cardiovascular diseases. We also studied their extracellular vesicles (EVs) owing to their capacity to act as messengers that transmit signals through their cargo. MAIN METHODS: EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, flow cytometry, and tetraspanin expression. Cell lysates and isolated EVs were analyzed using liquid chromatography coupled with mass spectrometry, followed by Gene Set Enrichment Analysis to identify the altered pathways. KEY FINDINGS: Proteomic analysis of endothelial cells revealed that IS causes an increase in proteins related to adipogenesis, inflammation, and xenobiotic metabolism and a decrease in proliferation. Extracellular matrix elements, as well as proteins associated with myogenesis, response to UV irradiation, and inflammation, were found to be downregulated in IS-treated EVs. Fatty acid metabolism was also found to be increased along with adipogenesis and inflammation observed in cells. SIGNIFICANCE: The treatment of endothelial cells with IS increased the expression of proteins related to adipogenesis, inflammation, and xenobiotic metabolism and was less associated with proliferation. Furthermore, EVs from cells treated with IS may mediate endothelial dysfunction, since they present fewer extracellular matrix elements, myogenesis, inflammatory factors, and proteins downregulated in response to UV radiation.
Assuntos
Células Endoteliais , Vesículas Extracelulares , Indicã , Proteômica , Insuficiência Renal Crônica , Indicã/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Insuficiência Renal Crônica/metabolismo , Proteômica/métodos , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteoma/metabolismoRESUMO
Sodium-phosphate transporter NPT4 (SLC17A3) is a membrane transporter for organic anionic compounds localized on the apical membranes of kidney proximal tubular epithelial cells and plays a role in the urinary excretion of organic anionic compounds. However, its physiological role has not been sufficiently elucidated because its substrate specificity is yet to be determined. The present study aimed to comprehensively explore the physiological substrates of NPT4 in newly developed Slc17a3-/- mice using a metabolomic approach. Metabolomic analysis showed that the plasma concentrations of 11 biological substances, including 3-indoxyl sulfate, were more than two-fold higher in Slc17a3-/- mice than in wild-type mice. Moreover, urinary excretion of 3-indoxyl sulfate was reduced in Slc17a3-/- mice compared to that in wild-type mice. The uptake of 3-indoxyl sulfate by NPT4-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. The calculated Km and Vmax values for NPT4-mediated 3-indoxyl sulfate uptake were 4.52 ± 1.18 mM and 1.45 ± 0.14 nmol/oocyte/90 min, respectively. In conclusion, the present study revealed that 3-indoxyl sulfate is a novel substrate of NPT4 based on the metabolomic analysis of Slc17a3-/- mice, suggesting that NPT4 regulates systemic exposure to 3-indoxyl sulfate by regulating its urinary excretion.
Assuntos
Indicã , Camundongos Knockout , Oócitos , Toxinas Urêmicas , Animais , Masculino , Camundongos , Indicã/metabolismo , Rim/metabolismo , Metabolômica/métodos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/genética , Toxinas Urêmicas/metabolismo , Xenopus laevisRESUMO
Although patients with chronic kidney disease (CKD) have a higher risk of colorectal cancer (CRC) aggravation, the connection between these two diseases is not well understood. Recent studies have shown that both CKD and CRC aggravation are closely related to an increased abundance of indole-producing Fusobacterium nucleatum in the gut. The indole absorbed from the gut is eventually metabolized to indoxyl sulfate in the liver. Since indoxyl sulfate is involved not only in accelerating CKD progression but also in the initiation and development of its associated complications, the present study aimed to clarify whether indoxyl sulfate induces the proliferation of CRC cells. This study found that indoxyl sulfate induced the proliferation of CRC-derived HCT-116 cells by activating the aryl hydrocarbon receptor (AhR) and the proto-oncogene Akt. The AhR antagonist CH223191 and Akt inhibitor MK2206 suppressed indoxyl sulfate-induced proliferation of HCT-116 cells. We also found that indoxyl sulfate upregulated epidermal growth factor receptor (EGFR) expression, which is associated with poor prognosis of CRC, whereas CH223191 and MK2206 repressed EGFR expression. Furthermore, indoxyl sulfate increased the sensitivity of CRC cells to EGF by upregulating EGFR expression. These findings suggest that indoxyl sulfate may be an important link between CKD and CRC aggravation.
Assuntos
Compostos Azo , Neoplasias Colorretais , Pirazóis , Insuficiência Renal Crônica , Humanos , Indicã/farmacologia , Indicã/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores ErbB/genética , Indóis , Proliferação de CélulasRESUMO
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.
Assuntos
Nefropatias , Toxinas Biológicas , Humanos , Toxinas Urêmicas , Indicã/toxicidade , Indicã/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Toxinas Biológicas/toxicidadeRESUMO
In the last decade, extensive attention has been paid to the uremic toxin indoxyl sulphate (IS) as an inducer of cardiac fibroblast (cFib) activation and cardiac fibrosis in chronic kidney disease. At cellular level, IS engages aryl hydrocarbon receptor (AhR) and regulates many biological functions. We analysed how AhR inhibition by CH-223191 (CH) and overexpression of non-functional (dominant negative, DN) nuclear factor-erythroid-2-related factor 2 (NRF2), a transcription factor recruited by AhR, modulate the response of neonatal mouse (nm) cFib to IS. We also evaluated nm-cardiomyocytes after incubation with the conditioned medium (CM) of IS±CH-treated nm-cFib. IS induced activation, collagen synthesis, TLR4 and-downstream-MCP-1, and the genes encoding angiotensinogen, angiotensin-converting enzyme, angiotensin type 1 receptor (AT1r) and neprilysin (Nepr) in nm-cFib. CH antagonized IS-initiated nm-cFib activation, but did not affect or even magnified the other features. IS promoted NRF2 nuclear translocation and expression the NRF2 target Nqo1. Both pre-incubation with CH and transfection of DN-NRF2 resulted in loss of NRF2 nuclear localization. Moreover, DN-NRF2 overexpression led to greater TLR4 and MCP-1 levels following exposure to IS. The CM of IS-primed nm-cFib and to a larger extent the CM of IS+CH-treated nm-cFib upregulated AT1r, Nepr and TNFα and myostatin genes in nm-cardiomyocytes. Hence, IS triggers pro-inflammatory activation of nm-cFib partly via AhR, and AhR-NRF2 counteract it. Strategies other than AhR inhibition are needed to target IS detrimental actions on cardiac cells.
Assuntos
Indicã , Transdução de Sinais , Camundongos , Animais , Indicã/farmacologia , Indicã/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Receptor 4 Toll-Like/genética , Fibroblastos/metabolismoRESUMO
Asperulosidic acid is a bioactive iridoid isolated from Hedyotis diffusa Willd. with anti-inflammatory and renal protective effects. However, its mechanism on renal interstitial fibrosis has not been elucidated yet. The present study aims to explore whether asperulosidic acid could retard renal fibrosis by reducing the circulating indoxyl sulfate (IS), which is a uremic toxin and accelerates chronic kidney disease progression by inducing renal fibrosis. In this paper, a unilateral ureteral obstruction (UUO) model of Balb/C mice was established. After the mice were orally administered with asperulosidic acid (14 and 28 mg/kg) for two weeks, blood, liver and kidney were collected for biochemical, histological, qPCR and Western blot analyses. Asperulosidic acid administration markedly reduced the serum IS level and significantly alleviated the histological changes in glomerular sclerosis and renal interstitial fibrosis. It is noteworthy that the mRNA and protein levels of the organic anion transporter 1 (OAT1), OAT3 and hepatocyte nuclear factor 1α (HNF1α) in the kidney were significantly increased, while the mRNA expressions of cytochrome P450 2e1 (Cyp2e1) and sulfotransferase 1a1 (Sult1a1) in the liver were not altered after asperulosidic acid administration. These results reveal that asperulosidic acid could accelerate the renal excretion of IS by up-regulating OATs via HNF1α in UUO mice, thereby alleviating renal fibrosis, but did not significantly affect its production in the liver, which might provide important information for the development of asperulosidic acid.
Assuntos
Nefropatias , Transportadores de Ânions Orgânicos , Insuficiência Renal Crônica , Obstrução Ureteral , Camundongos , Animais , Obstrução Ureteral/tratamento farmacológico , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Indicã/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Nefropatias/tratamento farmacológico , Nefropatias/etiologia , Nefropatias/metabolismo , Rim , Insuficiência Renal Crônica/metabolismo , Fibrose , RNA Mensageiro/metabolismoRESUMO
Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min-1 mg protein-1 but not as good as estrone-3-sulfate; OAT1 translocates pCS less efficiently. The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules.
Assuntos
Insuficiência Renal Crônica , Toxinas Biológicas , Humanos , Toxinas Urêmicas , Indicã/metabolismo , Cromatografia Líquida , Células HEK293 , Espectrometria de Massas em Tandem , Insuficiência Renal Crônica/metabolismo , Cresóis/metabolismo , Toxinas Biológicas/metabolismo , Membrana Celular/metabolismo , Transportadores de Ânions Orgânicos Sódio-IndependentesRESUMO
Indoxyl sulfate is a microbially derived uremic toxin that accumulates in late-stage chronic kidney disease and contributes to both renal and cardiovascular toxicity. Indoxyl sulfate is generated by the metabolism of indole, a compound created solely by gut microbial tryptophanases. Here, we characterize the landscape of tryptophanase enzymes in the human gut microbiome and find remarkable structural and functional similarities across diverse taxa. We leverage this homology through a medicinal chemistry campaign to create a potent pan-inhibitor, (3S) ALG-05, and validate its action as a transition-state analog. (3S) ALG-05 successfully reduces indole production in microbial culture and displays minimal toxicity against microbial and mammalian cells. Mice treated with (3S) ALG-05 show reduced cecal indole and serum indoxyl sulfate levels with minimal changes in other tryptophan-metabolizing pathways. These studies present a non-bactericidal pan-inhibitor of gut microbial tryptophanases with potential promise for reducing indoxyl sulfate in chronic kidney disease.
Assuntos
Microbioma Gastrointestinal , Insuficiência Renal Crônica , Humanos , Camundongos , Animais , Indicã/farmacologia , Indicã/metabolismo , Triptofanase , Microbioma Gastrointestinal/fisiologia , Indóis/farmacologia , Indóis/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Mamíferos/metabolismoRESUMO
Rates of pregnancy-related acute kidney injury (PR-AKI) have increased in the U.S over the past two decades, but how PR-AKI affects the blood-brain barrier (BBB) is understudied. AKI is associated with increased amounts of uremic toxins, like indoxyl sulfate (I.S), whose chronic administration leads to BBB and cognitive changes. This study's objective was to determine if (1) PR-AKI increases I.S and (2) if administration of I.S during pregnancy elicits renal injury and/or increases BBB permeability. From gestational day (GD) 11 to GD19, Sprague Dawley rats were given either 100 or 200 mg/kg body-weight dose of I.S. PR-AKI was induced on GD18 via 45 min bilateral renal ischemic reperfusion surgery. On GD18, metabolic cage metrics and metabolic waste was collected and on GD19 blood pressure, and BBB permeability (by Evan's Blue infusion) were measured. I.S and creatinine were measured in both urine and circulation, respectively. One-way ANOVA or student t-tests were performed using GraphPad Prism with a p < 0.05 significance. I.S and PR-AKI led to oliguria. I.S administration led to increased BBB permeability compared to normal pregnant and PR-AKI animals. These results suggest that I.S administration during pregnancy leads to increased BBB permeability and evidence of renal injury comparable to PR-AKI animals.
Assuntos
Injúria Renal Aguda , Barreira Hematoencefálica , Ratos , Gravidez , Animais , Feminino , Barreira Hematoencefálica/metabolismo , Ratos Sprague-Dawley , Indicã/metabolismo , Rim/metabolismo , PermeabilidadeRESUMO
Renal fibrosis is the final manifestation of chronic kidney disease (CKD); its prevention is vital for controlling CKD progression. Indoxyl sulfate (IS), a typical sulfate-conjugated uremic solute, is produced in the liver via the enzyme sulfotransferase (SULT) 1A1 and accumulates significantly during CKD. We investigated the toxicopathological role of IS in renal fibrosis using Sult1a1-KO mice and the underlying mechanisms. The unilateral ureteral obstruction (UUO) model was created; kidney IS concentrations, inflammation, and renal fibrosis were assessed on day 14. After UUO treatment, inflammation and renal fibrosis were exacerbated in WT mice, with an accumulation of IS in the kidney. However, they were significantly suppressed in Sult1a1-KO mice. CD206+ expression was upregulated, and ß-catenin expression was downregulated in Sult1a1-KO mice. To confirm the impact of erythropoietin (EPO) on renal fibrosis, we evaluated the time-dependent expression of EPO. In Sult1a1-KO mice, EPO mRNA expression was improved considerably; UUO-induced renal fibrosis was further attenuated by recombinant human erythropoietin (rhEPO). Thus, UUO-induced renal fibrosis was alleviated in Sult1a1-KO mice with a decreased accumulation of IS. Our findings confirmed the pathological role of IS in renal fibrosis and identified SULT1A1 as a new therapeutic target enzyme for preventing and attenuating renal fibrosis.
Assuntos
Indicã , Rim , Insuficiência Renal Crônica , Animais , Humanos , Camundongos , Modelos Animais de Doenças , Eritropoetina/metabolismo , Fibrose , Indicã/metabolismo , Inflamação/metabolismo , Rim/patologia , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismo , Obstrução Ureteral/metabolismoRESUMO
Atherosclerosis (AS), a chronic vascular inflammatory disease, has become a main focus of attention worldwide for its chronic progressing disease course and serious complications in the later period. Nevertheless, explanations for the exact molecular mechanisms of AS initiation and development remain to be an unsolved problem. The classic pathogenesis theories, such as lipid percolation and deposition, endothelium injury, inflammation and immune damage, provide the foundation for discovering the new key molecules or signaling mechanisms. Recently, indoxyl sulfate (IS), one of non-free uremia toxins, has been noticeable for its multiple atherogenic effects. IS exists at high concentration in plasma for its great albumin binding rate. Patients with uremia have markedly elevated serum levels of IS due both to the deterioration of renal function and to the high binding affinity of IS to albumin. Nowadays, elevated incidence of circulatory disease among patients with renal dysfunction indicates correlation of uremic toxins with cardiovascular damage. In this review, the atherogenic effects of IS and the underlying mechanisms are summarized with emphasis on several key pathological events associated with AS developments, such as vascular endothelium dysfunction, arterial medial lesions, vascular oxidative stress, excessive inflammatory responses, calcification, thrombosis and foam cell formation. Although recent studies have proved the great correlation between IS and AS, deciphering cellular and pathophysiological signaling by confirming key factors involved in IS-mediated atherosclerosis development may enable identification of novel therapeutic targets.
Assuntos
Aterosclerose , Doenças Cardiovasculares , Insuficiência Renal Crônica , Uremia , Humanos , Indicã/metabolismo , Doença Crônica , Albuminas , Insuficiência Renal Crônica/metabolismoRESUMO
Objective: Acyl-CoA-binding protein (ACBP)/diazepam-binding inhibitor has lately been described as an endocrine factor affecting food intake and lipid metabolism. ACBP is dysregulated in catabolic/malnutrition states like sepsis or systemic inflammation. However, regulation of ACBP has not been investigated in conditions with impaired kidney function, so far. Design/methods: Serum ACBP concentrations were investigated by enzyme-linked immunosorbent assay i) in a cohort of 60 individuals with kidney failure (KF) on chronic haemodialysis and compared to 60 individuals with a preserved kidney function; and ii) in a human model of acute kidney dysfunction (AKD). In addition, mACBP mRNA expression was assessed in two CKD mouse models and in two distinct groups of non-CKD mice. Further, mRNA expression of mACBP was measured in vitro in isolated, differentiated mouse adipocytes - brown and white - after exposure to the uremic agent indoxyl sulfate. Results: Median [interquartile range] serum ACBP was almost 20-fold increased in KF (514.0 [339.3] µg/l) compared to subjects without KF (26.1 [39.1] µg/l) (p<0.001). eGFR was the most important, inverse predictor of circulating ACBP in multivariate analysis (standardized ß=-0.839; p<0.001). Furthermore, AKD increased ACBP concentrations almost 3-fold (p<0.001). Increased ACBP levels were not caused by augmented mACBP mRNA expression in different tissues of CKD mice in vivo or in indoxyl sulfate-treated adipocytes in vitro. Conclusions: Circulating ACBP inversely associates with renal function, most likely through renal retention of the cytokine. Future studies need to investigate ACBP physiology in malnutrition-related disease states, such as CKD, and to adjust for markers of renal function.
Assuntos
Inibidor da Ligação a Diazepam , Desnutrição , Camundongos , Humanos , Animais , Indicã/metabolismo , Proteínas de Transporte/genética , Rim/metabolismo , Diazepam/metabolismo , RNA Mensageiro/metabolismo , Desnutrição/metabolismoRESUMO
In this study, we investigated the impact of the uremic toxin indoxyl sulfate on macrophages and tubular epithelial cells and its role in modulating the response to lipopolysaccharide (LPS). Indoxyl sulfate accumulates in the blood of patients with chronic kidney disease (CKD) and is a predictor of overall and cardiovascular morbidity/mortality. To simulate the uremic condition, primary macrophages and tubular epithelial cells were incubated with indoxyl sulfate at low concentrations as well as concentrations found in uremic patients, both alone and upon LPS challenge. The results showed that indoxyl sulfate alone induced the release of reactive oxygen species and low-grade inflammation in macrophages. Moreover, combined with LPS (proinflammatory conditions), indoxyl sulfate significantly increased TNF-α, CCL2, and IL-10 release but did not significantly affect the polarization of macrophages. Pre-treatment with indoxyl sulfate following LPS challenge induced the expression of aryl hydrocarbon receptor (Ahr) and NADPH oxidase 4 (Nox4) which generate reactive oxygen species (ROS). Further, experiments with tubular epithelial cells revealed that indoxyl sulfate might induce senescence in parenchymal cells and therefore participate in the progression of inflammaging. In conclusion, this study provides evidence that indoxyl sulfate provokes low-grade inflammation, modulates macrophage function, and enhances the inflammatory response associated with LPS. Finally, indoxyl sulfate signaling contributes to the senescence of tubular epithelial cells during injury.
Assuntos
Indicã , Toxinas Urêmicas , Humanos , Indicã/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Lipopolissacarídeos/toxicidade , Lipopolissacarídeos/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Células Epiteliais/metabolismoRESUMO
The aryl hydrocarbon receptor (AhR) is a decisive regulatory ligand-dependent transcription factor. It binds highly diverse ligands, which can be categorized as either endogenous or exogenous. Ligand binding activates AhR, which can adjust inflammatory responses by modulating immune cells such as dendritic cells (DCs). However, how different AhR ligand classes impact the phenotype and function of human monocyte-derived DCs (hMoDCs) has not been extensively studied in a comparative manner. We, therefore, tested the effect of the representative compounds Benzo(a)pyrene (BP), 6-formylindolo[3,2-b]carbazole (FICZ), and Indoxyl 3-sulfate (I3S) on DC biology. Thereby, we reveal that BP significantly induces a tolerogenic response in lipopolysaccharide-matured DCs, which is not apparent to the same extent when using FICZ or I3S. While all three ligand classes activate AhR-dependent pathways, BP especially induces the expression of negative immune regulators, and subsequently strongly subverts the T cell stimulatory capacity of DCs. Using the CRISPR/Cas9 strategy we also prove that the regulatory effect of BP is strictly AhR-dependent. These findings imply that AhR ligands contribute differently to DC responses and incite further studies to uncover the mechanisms and molecules which are involved in the induction of different phenotypes and functions in DCs upon AhR activation.
Assuntos
Regulação da Expressão Gênica , Receptores de Hidrocarboneto Arílico , Humanos , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Ligantes , Carbazóis/farmacologia , Carbazóis/metabolismo , Indicã/metabolismo , Células Dendríticas , BiologiaRESUMO
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.
Assuntos
Indicã , Toxinas Urêmicas , Humanos , Indicã/toxicidade , Indicã/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais Proximais/metabolismo , FibroseRESUMO
Although platinum-combination chemotherapy shows a high response rate at the primary site, epithelial ovarian cancer (EOC) treatment remains challenging because of tumor recurrence and metastasis. Recent studies have revealed that chemotherapy paradoxically promotes cancer cell survival, proliferation, and metastasis, although the reason for this remains unclear. The underlying molecular mechanisms that contribute to chemotherapy-induced metastasis need to be elucidated to establish effective therapeutic strategies. Acute kidney injury is a known side effect of cisplatin treatment, and kidney dysfunction results in the accumulation of uremic toxins in the serum. The present study aimed to investigate whether indoxyl sulfate (IS), a representative uremic toxin, affects the pathophysiology of EOC. In this study, IS reduced the expression of Mas receptor (MasR) in cultured human EOC cells. Both knockdown of the aryl hydrocarbon receptor (AhR), which is an intracellular IS receptor, and inhibition of AhR function suppressed IS-mediated downregulation of MasR in SK-OV-3 cells. IS induced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in an AhR-dependent manner. Inhibition of the STAT3 pathway or reactive oxygen species production suppressed the IS-mediated reduction of MasR. IS stimulated cell migration and invasion of SK-OV-3 cells in an AhR-dependent manner. Cisplatin-nephropathy model mice exhibited elevated levels of serum IS accompanied by elevated levels of blood urea nitrogen and serum creatinine. Furthermore, intraperitoneal administration of IS in mice promoted tumor growth and metastasis. Finally, we found that the MasR agonist Ang-(1-7) suppressed the IS-mediated effects on cell proliferation, migration, and invasion of SK-OV-3 cells. However, the knockdown of MasR expression by specific small interfering RNA in the absence of IS resulted in only minimal promotion of cell migration and invasion. These findings demonstrate that IS promotes malignancy in ovarian cancer via AhR-mediated downregulation of MasR function, whereas Ang-(1-7) attenuates this effect, thereby suggesting that Ang-(1-7) could provide a future treatment strategy for this cancer type.
Assuntos
Indicã , Neoplasias Ovarianas , Camundongos , Humanos , Animais , Feminino , Indicã/farmacologia , Indicã/metabolismo , Regulação para Baixo , Receptores de Hidrocarboneto Arílico/metabolismo , Cisplatino/farmacologiaRESUMO
BACKGROUND: Calcific aortic stenosis (CAS) is more prevalent, occurs earlier, progresses faster and has worse outcomes in patients with chronic kidney disease (CKD). The uremic toxin indoxyl sulfate (IS) is powerful predictor of cardiovascular mortality in these patients and a strong promoter of ectopic calcification whose role in CAS remains poorly studied. The objective of this study was to evaluate whether IS influences the mineralization of primary human valvular interstitial cells (hVICs) from the aortic valve. METHODS: Primary hVICs were exposed to increasing concentrations of IS in osteogenic medium (OM). The hVICs' osteogenic transition was monitored by qRT-PCRs for BMP2 and RUNX2 mRNA. Cell mineralization was assayed using the o-cresolphthalein complexone method. Inflammation was assessed by monitoring NF-κB activation using Western blots as well as IL-1ß, IL-6 and TNF-α secretion by ELISAs. Small interfering RNA (siRNA) approaches enabled us to determine which signaling pathways were involved. RESULTS: Indoxyl-sulfate increased OM-induced hVICs osteogenic transition and calcification in a concentration-dependent manner. This effect was blocked by silencing the receptor for IS (the aryl hydrocarbon receptor, AhR). Exposure to IS promoted p65 phosphorylation, the blockade of which inhibited IS-induced mineralization. Exposure to IS promoted IL-6 secretion by hVICs, a phenomenon blocked by silencing AhR or p65. Incubation with an anti-IL-6 antibody neutralized IS's pro-calcific effects. CONCLUSION: IS promotes hVIC mineralization through AhR-dependent activation of the NF-κB pathway and the subsequent release of IL-6. Further research should seek to determine whether targeting inflammatory pathways can reduce the onset and progression of CKD-related CAS.
Assuntos
Estenose da Valva Aórtica , Calcinose , Humanos , Valva Aórtica/metabolismo , NF-kappa B/metabolismo , Estenose da Valva Aórtica/metabolismo , Interleucina-6/farmacologia , Indicã/farmacologia , Indicã/metabolismo , Osteogênese , Receptores de Hidrocarboneto Arílico/metabolismo , Calcinose/metabolismo , Células Cultivadas , Diferenciação Celular , RNA Interferente Pequeno/metabolismo , Sulfatos/metabolismo , Sulfatos/farmacologiaRESUMO
Kidney transplantation (KTx) is the preferred form of renal replacement therapy in chronic kidney disease (CKD) patients, owing to increased quality of life and reduced mortality when compared to chronic dialysis. Risk of cardiovascular disease is reduced after KTx; however, it is still a leading cause of death in this patient population. Thus, we aimed to investigate whether functional properties of the vasculature differed two years post-KTx (postKTx) compared to baseline (time of KTx). Using the EndoPAT device in 27 CKD patients undergoing living-donor KTx, we found that vessel stiffness significantly improved while endothelial function worsened postKTx vs. baseline. Furthermore, baseline serum indoxyl sulphate (IS), but not p-cresyl sulphate, was independently negatively associated with reactive hyperemia index, a marker of endothelial function, and independently positively associated with P-selectin postKTx. Finally, to better understand the functional effects of IS in vessels, we incubated human resistance arteries with IS overnight and performed wire myography experiments ex vivo. IS-incubated arteries showed reduced bradykinin-mediated endothelium-dependent relaxation compared to controls via reduced nitric oxide (NO) contribution. Endothelium-independent relaxation in response to NO donor sodium nitroprusside was similar between IS and control groups. Together, our data suggest that IS promotes worsened endothelial dysfunction postKTx, which may contribute to the sustained CVD risk.
Assuntos
Indicã , Transplante de Rim , Insuficiência Renal Crônica , Doenças Vasculares , Humanos , Doenças Cardiovasculares , Endotélio Vascular/metabolismo , Indicã/metabolismo , Transplante de Rim/efeitos adversos , Nitroprussiato/farmacologia , Qualidade de Vida , Insuficiência Renal Crônica/terapia , Doenças Vasculares/metabolismo , Doenças Vasculares/patologiaRESUMO
INTRODUCTION: In the advanced stage of chronic kidney disease (CKD), electrolytes, fluids, and metabolic wastes including various uremic toxins, accumulate at high concentrations in the patients' blood. Hemodialysis (HD) is the conventional procedure used worldwide to remove metabolic wastes. The creatinine and urea levels have been routinely monitored to estimate kidney function and effectiveness of the HD process. This study, first from in Indian perspective, aimed at the identification and quantification of major uremic toxins in CKD patients on maintenance HD (PRE-HD), and compared with the healthy controls (HC) as well as after HD (POST-HD). OBJECTIVES: The study mainly focused on the identification of major uremic toxins in Indian perspective and the quantitative analysis of indoxyl sulfate and p-cresol sulfate (routinely targeted uremic toxins), and phenyl sulfate, catechol sulfate, and guaiacol sulfate (targeted for the first time), apart from creatinine and urea in PRE-HD, POST-HD, and HC groups. METHODS: Blood samples were collected from 90 HD patients (both PRE-HD and POST-HD), and 74 HCs. The plasma samples were subjected to direct ESI-HRMS and LC/HRMS for untargeted metabolomics and LC-MS/MS for quantitative analysis. RESULTS: Various known uremic toxins, and a few new and unknown peaks were detected in PRE-HD patients. The p-cresol sulfate and indoxyl sulfate were dominant in PRE-HD, the concentrations of phenyl sulfate, catechol sulfate, and guaiacol sulfate were about 50% of that of indoxyl sulfate. Statistical evaluation on the levels of targeted uremic toxins in PRE-HD, POST-HD, and HC groups showed a significant difference among the three groups. The dialytic clearance of indoxyl sulfate and p-cresol sulfate was found to be < 35%, while that of the other three sulfates was 50-58%. CONCLUSION: LC-MS/MS method was developed and validated to evaluate five major uremic toxins in CKD patients on HD. The levels of the targeted uremic toxins could be used to assess kidney function and the effectiveness of HD.