The relationship of indoxyl sulfate and p-cresyl sulfate with target cardiovascular proteins in hemodialysis patients.

Protein-bound uremic toxins (Indoxyl sulfate [IS] and p-cresyl sulfate [PCS]) are both associated with cardiovascular (CV) and all-cause mortality in subjects with chronic kidney disease (CKD). Possible mechanisms have not been elucidated. In hemodialysis patients, we investigated the relationship between the free form of IS and PCS and 181 CV-related proteins. First, IS or PCS concentrations were checked, and high levels were associated with an increased risk of acute coronary syndrome (ACS) in 333 stable HD patients. CV proteins were further quantified by a proximity extension assay. We examined associations between the free form protein-bound uremic toxins and the quantified proteins with correction for multiple testing in the discovery process. In the second step, the independent association was evaluated by multivariable-adjusted models. We rank the CV proteins related to protein-bound uremic toxins by bootstrapped confidence intervals and ascending p-value. Six proteins (signaling lymphocytic activation molecule family member 5, complement component C1q receptor, C-C motif chemokine 15 [CCL15], bleomycin hydrolase, perlecan, and cluster of differentiation 166 antigen) were negatively associated with IS. Fibroblast growth factor 23 [FGF23] was the only CV protein positively associated with IS. Three proteins (complement component C1q receptor, CCL15, and interleukin-1 receptor-like 2) were negatively associated with PCS. Similar findings were obtained after adjusting for classical CV risk factors. However, only higher levels of FGF23 was related to increased risk of ACS. In conclusion, IS and PCS were associated with several CV-related proteins involved in endothelial barrier function, complement system, cell adhesion, phosphate homeostasis, and inflammation. Multiplex proteomics seems to be a promising way to discover novel pathophysiology of the uremic toxin.

Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells.

Introduction: The aim of this study was to determine the role of Notch in indoxyl sulfate (IS)-induced vascular calcification (VC). Materials and methods: VC and expression of Notch-related and osteogenic molecules were examined in Dahl salt-sensitive (DS), DS hypertensive (DH), and DH IS-treated rats (DH+IS). The effects of IS on expression of Notch receptors, apoptotic activity, and calcification were examined in cultured aortic smooth muscle cells (SMCs). Results: Medial calcification was noted only in aortas and coronary arteries of DH+IS rats. Notch1, Notch3, and Hes-1 were expressed in aortic SMCs of all rats, but only weakly in the central areas of the media and around the calcified lesions in DH+IS rats. RT-PCR and western blotting of DH+IS rat aortas showed downregulation of Notch ligands, Notch1 and Notch3, downstream transcriptional factors, and SM22, and conversely, overexpression of osteogenic markers. Expression of Notch1 and Notch3 in aortic SMCs was highest in incubation under 500 µM IS for 24hrs, and then decreased time- and dose-dependently. Coupled with this decrease, IS increased caspase 3/7 activity and TUNEL-positive aortic SMCs. In addition, pharmacological Notch signal inhibition with DAPT induced apoptosis in aortic SMCs. ZVAD, a caspase inhibitor abrogated IS-induced and DAPT-induced in vitro vascular calcification. Knockdown of Notch1 and Notch3 cooperatively increased expression of osteogenic transcriptional factors and decreased expression of SM22. Conclusion: Our results suggested that IS-induced VC is mediated through suppression of Notch activity in aortic SMCs, induction of osteogenic differentiation and apoptosis.

Indoxyl sulfate induces intestinal barrier injury through IRF1-DRP1 axis-mediated mitophagy impairment.

Rationale: The dysfunctional gut-kidney axis forms a vicious circle, which eventually becomes a catalyst for the progression of chronic kidney disease (CKD) and occurrence of related complications. However, the pathogenic factors of CKD-associated intestinal dysfunction and its mechanism remain elusive. Methods: We first identified the protein-bound uremic toxin indoxyl sulfate (IS) as a possible contributor to intestinal barrier injury. Transepithelial electrical resistance, permeability assay and transmission electron microscopy were carried out to evaluate the damaging effect of IS on intestinal barrier in intestinal epithelial cells, IS-injected mice and CKD mice. In vitro and in vivo experiments were performed to investigate the role of IS in intestinal barrier injury and the underlying mechanism. Finally, CKD mice treated with AST-120 (an oral adsorbent for IS) and gene knockout mice were used to verify the mechanism and to explore possible interventions for IS-induced intestinal barrier injury. Results: Transepithelial electrical resistance and the expressions of tight junction-related genes were significantly suppressed by IS in intestinal epithelial cells. In vitro experiments demonstrated that IS inhibited the expression of dynamin-related protein 1 (DRP1) and mitophagic flux, whereas DRP1 overexpression attenuated IS-induced mitophagic inhibition and intestinal epithelial cell damage. Furthermore, IS suppressed DRP1 by upregulating the expression of interferon regulatory factor 1 (IRF1), and IRF1 could directly bind to the promoter region of DRP1. Additionally, the decreased expression of DRP1 and autophagosome-encapsulated mitochondria were observed in the intestinal tissues of CKD patients. Administration of AST-120 or genetic knockout of IRF1 attenuated IS-induced DRP1 reduction, mitophagic impairment and intestinal barrier injury in mice. Conclusions: These findings suggest that reducing IS accumulation or targeting the IRF1-DRP1 axis may be a promising therapeutic strategy for alleviating CKD-associated intestinal dysfunction.

Klotho downregulation contributes to myocardial damage of cardiorenal syndrome in sepsis.

Klotho is a type of single­pass transmembrane protein that is important for the proper function of numerous organs. The aim of the present study was to investigate the role of Klotho in sepsis­associated myocardial damage. In the present study, reverse transcription­quantitative PCR, western blotting and ELISA were conducted to examine the expression levels of function genes, and flow cytometry was performed to detect cell apoptosis and reactive oxygen species. The present study demonstrated that Klotho expression was significantly downregulated in septic mice and that the myocardial function of septic mice improved after treatment with exogenous Klotho protein. It further demonstrated that indoxyl sulfate inhibited the expression of Klotho protein. In addition, decreased Klotho protein further led to activation of the reactive oxygen species­p38 mitogen­activated protein kinase signaling pathway, finally resulting in myocardial damage. In conclusion, Klotho protein may be a key regulator in the myocardial damage of cardiorenal syndrome in sepsis. It also has a potential to be a therapeutic target for sepsis­associated myocardial damage in the future.

1,25(OH)2 D3 attenuates indoxyl sulfate-induced epithelial-to-mesenchymal cell transition via inactivation of PI3K/Akt/ß-catenin signaling in renal tubular epithelial cells.

OBJECTIVES: Indoxyl sulfate (IS), a uremic toxin, has been shown to promote the epithelial-to-mesenchymal transition (EMT) of human proximal tubular cells and to accelerate the progression of chronic kidney disease (CKD). Despite the well-known protective role of 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] in EMT, the effect of 1,25(OH)2 D3 on IS-induced EMT in human proximal tubular epithelial cells and the underlying mechanism remain unclear. The aim of this study was to determine whether IS (0-1 mM) dose-dependently inhibited the protein expression of E-cadherin and increased the protein expression of alpha-smooth muscle actin, N-cadherin, and fibronectin. METHODS: This study investigated the molecular mechanism by which 1,25(OH)2 D3 attenuates IS-induced EMT. HK-2 human renal tubular epithelial cells was used as the study model, and the MTT assay, Western Blotting, siRNA knockdown technique were used to explore the effects of 1,25(OH)2 D3 on EMT in the presence of IS. RESULTS: Pretreatment with 1,25(OH)2 D3 inhibited the IS-induced EMT-associated protein expression in HK-2 cells. IS induced the phosphorylation of Akt (S473) and ß-catenin (S552) and subsequently increased the nuclear accumulation of ß-catenin. Pretreatment with 1,25(OH)2 D3 and LY294002 (phosphoinositide 3-kinase [PIK3] inhibitor) significantly inhibited the IS-induced phosphorylation of Akt and ß-catenin, nuclear ß-catenin accumulation, and EMT-associated protein expression. CONCLUSIONS: Results from the present study revealed that the anti-EMT effect of 1,25(OH)2 D3 is likely through inhibition of the PI3K/Akt/ß-catenin pathway, which leads to down-regulation of IS-driven EMT-associated protein expression in HK-2 human renal tubular epithelial cells.

Transporter-mediated interaction of indican and methotrexate in rats.

Indican (indoxyl-ß-D-glucoside) is present in several Chinese herbs e.g. Isatis indigotica, Polygonum tinctorium and Polygonum perfoliatum. The major metabolite of indican was indoxyl sulfate (IS), an uremic toxin which was a known substrate/inhibitor of organic anion transporter (OAT) 1, OAT 3 and multidrug resistance-associated protein (MRP) 4. Methotrexate (MTX), an important immunosuppressant with narrow therapeutic window, is a substrate of OAT 1, 2, 3, 4 and MRP 1, 2, 3, 4. We hypothesized that IS, the major metabolite of oral indican, might inhibit the renal excretion of MTX mediated by OAT 1, OAT 3 and MRP 4. Therefore, this study investigated the effect of oral indican on the pharmacokinetics of MTX. Rats were orally given MTX with and without indican (20.0 and 40.0 mg/kg) in a parallel design. The serum MTX concentration was determined by a fluorescence polarization immunoassay. For mechanism clarification, phenolsulfonphthalein (PSP, 5.0 mg/kg), a probe substrate of OAT 1, OAT 3, MRP 2 and MRP 4, was intravenously given to rats with and without a intravenous bolus of IS (10.0 mg/kg) to measure the effect of IS on the elimination of PSP. The results indicated that 20.0 and 40.0 mg/kg of oral indican significantly increased the area under concentration-time curve0-t (AUC0-t) of MTX by 231% and 259%, prolonged the mean residence time (MRT) by 223% and 204%, respectively. Furthermore, intravenous IS significantly increased the AUC0-t of PSP by 204% and decreased the Cl by 68%. In conclusion, oral indican increased the systemic exposure and MRT of MTX through inhibition on multiple anion transporters including OAT 1, OAT 3 and MRP 4 by the major metabolite IS.

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.

The Uremic Toxin Indoxyl-3-Sulfate Induces CYP1A2 In Primary Human Hepatocytes.

Chronic kidney disease (CKD) generally impacts clearance of renally eliminated drugs but growing evidence shows that it can influence clearance of hepatically eliminated drugs and a complete mechanistic understanding of this phenomenon is still lacking. CKD leads to accumulation of uremic toxins, including indoxyl- 3-sulfate (3-INDS) and indole-3-acetic acid (3-IAA). OBJECTIVE: In this study, we evaluated the potential of 3-INDS and 3-IAA (10, 30 and 100 µM) to induce liver cytochrome P450 (CYP) enzymes CYP1A2, 2B6 and 3A4/5 using cultured primary human hepatocytes following once daily treatment for 3 days. RESULTS: 3-INDS potently induced CYP1A2 mRNA and enzyme activity in a dose-dependent manner but did not induce CYP2B6 or 3A4. At 100 µM, a concentration observed in humans under uremic conditions, 3-INDS increased CYP1A2 mRNA and activity by 93% and 292% respectively when compared with prototypical inducer omeprazole. However, 3-IAA did not induce CYP1A2, 2B6 or 3A4. CONCLUSION: These results suggest that the uremic toxin, 3-INDS, is a potent CYP1A2 inducer and lends valuable mechanistic basis for how kidney disease can affect hepatic metabolism.

Activation of aryl hydrocarbon receptor mediates suppression of hypoxia-inducible factor-dependent erythropoietin expression by indoxyl sulfate.

Indoxyl sulfate (IS) is a representative uremic toxin that accumulates in the blood of patients with chronic kidney disease (CKD). In addition to the involvement in the progression of CKD, a recent report indicates that IS suppresses hypoxia-inducible factor (HIF)-dependent erythropoietin (EPO) production, suggesting that IS may also contribute to the progression of renal anemia. In this report, we provide evidence that aryl hydrocarbon receptor (AhR) mediates IS-induced suppression of HIF activation and subsequent EPO production. In HepG2 cells, IS at concentrations similar to the blood levels in CKD patients suppressed hypoxia- or cobalt chloride-induced EPO mRNA expression and transcriptional activation of HIF. IS also induced AhR activation, and AhR blockade resulted in abolishment of IS-induced suppression of HIF activation. The HIF transcription factor is a heterodimeric complex composed of HIF-α subunits (HIF-1α and HIF-2α) and AhR nuclear translocator (ARNT). IS suppressed nuclear accumulation of the HIF-α-ARNT complex accompanied by an increase of the AhR-ARNT complex in the nucleus, implying the involvement of interactions among AhR, HIF-α, and ARNT in the suppression mechanism. In rats, oral administration of indole, a metabolic precursor of IS, inhibited bleeding-induced elevation of renal EPO mRNA expression and plasma EPO concentration and strongly induced AhR activation in the liver and renal cortex tissues. Collectively, this study is the first to elucidate the detailed mechanism by which AhR plays an indispensable role in the suppression of HIF activation by IS. Hence, IS-induced activation of AhR may be a potential therapeutic target for treating renal anemia.

Nitration of indoxyl sulfate facilitates its cytotoxicity in human renal proximal tubular cells via expression of heme oxygenase-1.

Peroxynitrite, the reaction product of superoxide [Formula: see text] and nitric oxide (NO), nitrates tyrosine residues, unsaturated fatty acids, cyclic guanosine monophosphate and other phenolics. We report herein that indoxyl sulfate (IS) is also nitrated by peroxynitrite in vitro and forms 2-nitro-IS, as determined from spectral characteristics and (1)H-NMR. IS is one of the very important uremic toxins that accelerate the progression of chronic kidney disease via various mechanisms. However, cell viability experiments with human proximal tubular cells show that the cytotoxicity of 2-nitro-IS is several-fold higher than that of IS. The explanation for this finding seems to be that 2-nitro-IS induces a much more pronounced generation of intracellular reactive oxygen species (ROS) than IS. Results with inhibitors revealed that an organic anion transporter, several intracellular enzymes and nonprotein-bound iron ions are reasons for this finding. Most importantly, however, as detected by immunofluorescence and Western blotting, 2-nitro-IS induces the expression of heme oxygenase-1 and thereby the formation of ROS; most probably through the Fenton reaction. The final result of the increased amounts of ROS is death of the kidney cells. Thus, nitration of uremic toxins by peroxynitrite may help us to understand the initiation and progress of chronic kidney diseases.

Klotho Protects Against Indoxyl Sulphate-Induced Myocardial Hypertrophy.

Left ventricular hypertrophy (LVH) is a common complication in patients with CKD and an independent risk factor for death. Changes in the levels of uremic solutes or Klotho have been reported to be related to CKD, whereas the relationships between these factors and CKD-associated LVH remain unclear. Here, we investigated the interaction between Klotho and indoxyl sulfate (IS), a typical uremic solute, in CKD-associated LVH. In a survey of 86 patients with CKD, a negative relationship was found between serum levels of IS and Klotho (r=-0.59, P<0.001). Furthermore, serum levels of IS and Klotho were independently associated with LVH (for IS: r=0.69, P<0.001; for Klotho: r=-0.49, P<0.001). In normal mice, intraperitoneal injection of IS for 8 weeks induced LVH accompanied by substantial downregulation of renal Klotho. Notably, IS-induced LVH was more severe in heterozygous Klotho-deficient (kl/+) mice. In vitro, treatment with Klotho strongly inhibited IS-induced cardiomyocyte hypertrophy by blocking oxidative stress and inhibiting p38 and extracellular signal-regulated protein kinase 1/2 signaling pathways. In a mouse model of CKD-associated LVH, the renal expression of Klotho was lower and the level of serum IS was higher than in healthy controls. Moreover, treatment of CKD mice with Klotho protein significantly restrained the development of LVH. Taken together, these results suggest that Klotho is an endogenous protector against IS-induced LVH, and the imbalance between Klotho and IS may contribute to the development of LVH in CKD.

Protein-bound uremic toxins induce tissue remodeling by targeting the EGF receptor.

Indoxyl sulfate and p-cresol sulfate have been suggested to induce kidney tissue remodeling. This study aimed to clarify the molecular mechanisms underlying this tissue remodeling using cultured human proximal renal tubular cells and half-nephrectomized mice treated with indoxyl sulfate or p-cresol sulfate as study models. Molecular docking results suggested that indoxyl sulfate and p-cresol sulfate dock on a putative interdomain pocket of the extracellular EGF receptor. In vitro spectrophotometric analysis revealed that the presence of a synthetic EGF receptor peptide significantly decreased the spectrophotometric absorption of indoxyl sulfate and p-cresol sulfate. In cultured cells, indoxyl sulfate and p-cresol sulfate activated the EGF receptor and downstream signaling by enhancing receptor dimerization, and increased expression of matrix metalloproteinases 2 and 9 in an EGF receptor-dependent manner. Treatment of mice with indoxyl sulfate or p-cresol sulfate significantly activated the renal EGF receptor and increased the tubulointerstitial expression of matrix metalloproteinases 2 and 9. In conclusion, indoxyl sulfate and p-cresol sulfate may induce kidney tissue remodeling through direct binding and activation of the renal EGF receptor.

Indoxyl sulfate-induced activation of (pro)renin receptor promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.

UNLABELLED: Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease (CVD). (Pro)renin receptor (PRR) is activated in the kidney of CKD. The present study aimed to determine the role of indoxyl sulfate (IS), a uremic toxin, in PRR activation in rat aorta and human aortic smooth muscle cells (HASMCs). We examined the expression of PRR and renin/prorenin in rat aorta using immunohistochemistry. Both CKD rats and IS-administrated rats showed elevated expression of PRR and renin/prorenin in aorta compared with normal rats. IS upregulated the expression of PRR and prorenin in HASMCs. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed IS-induced expression of PRR and prorenin in HASMCs. Knock down of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR) and nuclear factor-κB p65 (NF-κB p65) with small interfering RNAs inhibited IS-induced expression of PRR and prorenin in HASMCs. Knock down of PRR inhibited cell proliferation and tissue factor expression induced by not only prorenin but also IS in HASMCs. CONCLUSION: IS stimulates aortic expression of PRR and renin/prorenin through OAT3-mediated uptake, production of reactive oxygen species, and activation of AhR and NF-κB p65 in vascular smooth muscle cells. IS-induced activation of PRR promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.

Indoxyl sulfate downregulates expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells.

UNLABELLED: Renin-angiotensin system (RAS) plays a pivotal role in chronic kidney disease (CKD). Angiotensin converting enzyme-related carboxypeptidase 2 (ACE2)/angiotensin (Ang)-(1-7)/Mas receptor axis counteracts the deleterious actions of Ang II. ACE2 exerts its actions by cleaving Ang II into Ang-(1-7) which activates Mas receptor. This study aimed to determine if the expression of Mas receptor is altered in the kidneys of CKD rats, and if indoxyl sulfate (IS), a uremic toxin, affects the expression of Mas receptor in rat kidneys and cultured human proximal tubular cells (HK-2 cells). The expression of Mas receptor was examined in the kidneys of CKD and AST-120-treated CKD rats using immunohistochemistry. Further, the effects of IS on Mas receptor expression in the kidneys of normotensive and hypertensive rats were examined. The effects of IS on the expression of Mas receptor and phosphorylation of endothelial nitric oxide synthase (eNOS) in HK-2 cells were examined using immunoblotting. CKD rats showed reduced renal expression of Mas receptor, while AST-120 restored its expression. Administration of IS downregulated Mas receptor expression in the kidneys of normotensive and hypertensive rats. IS downregulated Mas receptor expression in HK-2 cells in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR), and signal transducer and activator of transcription 3 (Stat3) inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. N-acetylcysteine, an antioxidant, also inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. Ang-(1-7) attenuated IS-induced transforming growth factor-ß1 (TGF-ß1) expression. CONCLUSION: Mas receptor expression is reduced in the kidneys of CKD rats. IS downregulates renal expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells. IS-induced downregulation of Mas receptor might be involved in upregulation of TGF-ß1 in proximal tubular cells.

Indoxyl sulfate enhances angiotensin II signaling through upregulation of epidermal growth factor receptor expression in vascular smooth muscle cells.

AIMS: Indoxyl sulfate, a uremic toxin, is considered a risk factor for arteriosclerosis in patients with chronic kidney disease (CKD). We previously reported the actions of indoxyl sulfate including crosstalk with platelet-derived growth factor (PDGF) signaling in vascular smooth muscle cells (VSMCs). The present study examines whether indoxyl sulfate enhances angiotensin II (Ang II) signaling because serum levels of Ang II are elevated in patients with CKD. MAIN METHODS: The effect of indoxyl sulfate and Ang II on phosphorylation of ERK and epidermal growth factor receptor (EGFR), and migration were determined using VSMCs. The expression of EGFR was determined using not only VSMCs but also artery of normal, uremic, and indoxyl sulfate-administrated uremic rats. KEY FINDINGS: Ang II-dependent phosphorylation of ERK and EGFR, and migration of VSMCs were augmented by a prior 24-h incubation with indoxyl sulfate even in the absence of indoxyl sulfate during Ang II stimulation. The expression of EGFR was increased in indoxyl sulfate-stimulated cultured VSMCs. In arterial VSMCs of rats, serum levels of indoxyl sulfate reflected the expression level of EGFR. The upregulated EGFR expression by indoxyl sulfate was suppressed by the antioxidant, N-acetylcysteine. An EGFR inhibitor, AG1478, repressed the enhancement of Ang II-induced cellular effects by indoxyl sulfate. Taken together, these findings indicate that indoxyl sulfate enhances Ang II signaling through reactive oxygen species-induced EGFR expression. SIGNIFICANCE: The actions of indoxyl sulfate including crosstalk with Ang II signaling may be closely involved in the pathogenesis of CKD associated with arteriosclerosis.

Uremic toxin indoxyl sulfate inhibits human vascular smooth muscle cell proliferation.

Uremic syndrome is attributed to the progressive retention of a large number of compounds, such as indoxyl sulfate, which under physiological conditions are excreted by the kidneys. Previous in vitro studies have demonstrated that uremic indoxyl sulfate concentrations induce a weak increase in the proliferation of both rat and human vascular aortic smooth muscle cells (hVASMC) after short term exposition to the toxin (i.e. 24 h). In the present study, we evaluated indoxyl sulfate effects on the proliferation of hVASMC at three different concentrations after long-term exposure (seven days). In contrast to previously published studies, we observed a dose-dependent and significant inhibitory effect of this toxin on hVASMC proliferation. We also demonstrated that indoxyl sulfate inhibits epidermal growth factor-induced hVASMC proliferation after long-term exposure. Indoxyl sulfate effects were associated with a dose-dependent induction of intracellular reactive oxygen species and up-regulation of p21 and p27 protein expression. Chronic exposure to indoxyl sulfate produces a significant inhibitory effect on hVASMC proliferation. The relevance of these findings must be evaluated by further studies, particularly in an in vivo setting.

Treatment with pravastatin attenuates oxidative stress and protects osteoblast cell viability from indoxyl sulfate.

Chronic kidney disease has a high level of oxidative stress, a phenomenon that is induced, at least in part, by the accumulation of uremic toxins. Several reports have revealed that indoxyl sulfate, one of the uremic toxins, accelerates oxidative stress in chronic kidney disease. On the other hand, it is also well known that statins have pleiotropic effects; however, it still remains unclear whether statins suppress osteoblastic cell dysfunction or cytotoxicity induced by uremic toxins. To elucidate whether statins ameliorate osteoblast dysfunction induced by uremic toxins, we conducted an in vitro study using primary cultured osteoblastic cells from mouse calvariae. Indoxyl sulfate induced reactive oxygen species production and reduced cell viability in osteoblastic cells in a dose dependent manner. The addition of pravastatin suppressed reactive oxygen species production and ameliorated cell viability. These data suggest that pravastatin attenuates oxidative stress and protects osteoblastic cell viability from indoxyl sulfate.

Does indoxyl sulfate, a uraemic toxin, have direct effects on cardiac fibroblasts and myocytes?

AIMS: Indoxyl sulfate (IS) is a uraemic toxin found at high concentration in patients with chronic kidney disease (CKD) co-morbid with chronic heart failure (CHF). The aim of this study was to determine direct effects of IS on cardiac cells as well as the pro-inflammatory effect of IS. METHODS AND RESULTS: Indoxyl sulfate significantly increased neonatal rat cardiac fibroblast collagen synthesis (by 145.7% vs. control, P < 0.05) and myocyte hypertrophy (by 134.5% vs. control, P < 0.001) as determined by (3)H-proline or (3)H-leucine incorporation, respectively. Indoxyl sulfate stimulated tumour necrosis factor-alpha, interleukin-6 (IL-6), and IL-1beta mRNA expression in THP-1 cells as quantified by RT-PCR. Both p38 (RWJ-67657) and MEK1/2 (U0126) inhibitors suppressed all these effects by IS. Furthermore, western blot analysis showed that IS activated mitogen-activated protein kinase (MAPK) (p38, p42/44) and nuclear factor-kappa B (NFkappaB) pathways. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that IS exerted its effects without affecting cell viability. CONCLUSION: This study has, for the first time, demonstrated that IS has pro-fibrotic, pro-hypertrophic, and pro-inflammatory effects, indicating that IS might play an important role in adverse cardiac remodelling mediated via activation of the p38 MAPK, p42/44 MAPK, and NFkappaB pathways. Targeting reduction of IS and/or the pathways it activates may represent a novel therapeutic approach to the management of CHF with concomitant CKD.

Indoxyl sulfate, a uremic toxin, is biotransformed from indoxyl-beta-D-glucoside (indican) in rats.

Indican (Indoxyl-beta-D-glucoside) is present in many Chinese herbs such as Isatis indigotica, Clerodendrum crytophyllum, Glehnia littoralis, Polygonum tinctorium and P. perfoliatum. This study aims to investigate whether indoxyl sulfate, a uremic toxin, would be biotransformed from indican in rats. Indican was administered intravenously and orally to Sprague-Dawley rats. The blood samples were withdrawn via cardiopuncture at specific time points and the serum concentrations of indican and indoxyl sulfate were assayed by HPLC method. The results showed that indican was rapidly and extensively metabolized to indoxyl sulfate either given intravenously or orally. Indoxyl sulfate showed markedly higher systemic exposure than indican. Because indoxyl sulfate is a harmful uremic toxin, we suggest that the content of indican in the aforementioned medicinal plants be quantitated and well controlled to ensure the safety for clinical use.

Indoxyl sulfate induces complex redox alterations in mesangial cells.

Indoxyl sulfate is a protein metabolite that is concentrated in the serum of patients with chronic renal insufficiency. It also is a uremic toxin that has been implicated in the progression of chronic renal disease in rodent models. We have shown previously that mesangial cell redox status is related to activation of mitogen-activated protein kinases and cell proliferation, which are factors related to glomerular damage. We used three methods to examine the ability of indoxyl sulfate to alter mesangial cell redox as a possible mechanism for its toxicity. Indoxyl sulfate increases mesangial cell reduction rate in a concentration-dependent manner as demonstrated by redox microphysiometry. Alterations occurred at concentrations as low as 100 microM, with more marked alterations occurring at higher concentrations associated with human renal failure. We demonstrated that indoxyl sulfate induces the production of intracellular reactive oxygen species (ROS) in mesangial cells (EC50 = 550 microM) by using the ROS-sensitive fluorescent dye CM-DCF. ROS generation was only partially (approximately 50%) inhibited by the NADPH oxidase inhibitor diphenylene iodinium at low (< or = 300 microM) indoxyl sulfate concentrations. Diphenylene iodinium was without effect at higher concentrations of indoxyl sulfate. We also used electron paramagnetic spin resonance spectroscopy with extracellular and intracellular spin traps to show that indoxyl sulfate increases extracellular SOD-sensitive O2-* production and intracellular hydroxyl radical production that may derive from an initial O2-* burst. These results document that indoxyl sulfate, when applied to renal mesangial cells at pathological concentrations, induces rapid and complex changes in mesangial cell redox.