Administration Of Indoxyl Sulfate Research Articles

Metabolomics reveals that sulfotransferase 1 may regulate colchicine-induced liver injury

Colchicine is widely used to treat gouty arthritis for years. Previous studies showed that colchicine overdose can cause liver damage, yet the mechanism underlying its hepatotoxicity remains unclear. In this study, hepatotoxicity of colchicine was investigated in vivo. Metabolomic analysis of colchicine metabolites and endogenous metabolites was performed using Ultra High Performance Liquid Chromatography (UHPLC) – mass spectrometry (MS). Seventeen metabolites of colchicine were identified, including 3 novel sulfated metabolites. Meanwhile, endogenous sulfated metabolites were found to be decreased by colchicine. Colchicine might regulate sulfotransferase 1 (SULT1) through perixisome proliferation-activated receptor ɑ (PPARα), and inhibition of SULT1 reduced the levels of sulfated metabolites of colchicine. Inhibition of SULT1 aggravated colchicine-induced liver injury, whereas activation of SULT1 attenuated its liver injury. The supplementation of endogenous sulfated metabolites indoxyl sulfate (IS) or p-cresol sulfate (PCS) alleviated colchicine-induced liver injury through modulation of the CASPASE-1-gasdermin D (GSDMD) pathway. These results indicated that colchicine might cause hepatotoxicity through inhibition of SULT1 and decreased production of bioactive sulfated endogenous metabolites IS and PCS. Our results provided evidence for potential therapeutic targets and agents to prevent liver injury caused by colchicine. Targeting the SULT1 enzyme and administration of IS and PCS may be useful in alleviating colchicine hepatotoxicity.

Indoxyl Sulfate Promotes Metastatic Characteristics of Ovarian Cancer Cells via Aryl Hydrocarbon Receptor–Mediated Downregulation of the Mas Receptor

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.

Protective Roles of Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1) in Uremic Vascular Calcification.

Cellular phosphate transporters play critical roles in the pathogenesis of vascular calcification (VC) in chronic kidney disease (CKD). However, the mechanistic link between VC and xenotropic and polytropic receptor 1 (XPR1), a newly identified phosphate exporter, remains unknown. We developed a new mouse model with rapidly progressive uremic VC in C57BL/6 mice and examined the roles of XPR1. The combination of surgical heminephrectomy and 8weeks of feeding a customized warfarin and adenine-based diet induced extensive aortic VC in almost all mice. The XPR1 mRNA level in the aorta of CKD mice was significantly lower than those in control mice as early as week 2, when there was no apparent VC, which progressively declined thereafter. Dietary phosphate restriction increased XPR1 mRNA expression in the aorta but reduced aortic VC in CKD mice. In cultured vascular smooth muscle cells (VSMCs), a calcifying medium supplemented with high phosphate and calcium did not affect XPR1 mRNA expression. The XPR1 mRNA expression in cultured VCMCs was also unaffected by administration of indoxyl sulfate or calcitriol deficiency but was decreased by 1-34 parathyroid hormone or fibroblast growth factor 23 supplementation. Furthermore, XPR1 deletion in the cultured VSMCs exacerbated calcification of the extracellular matrix as well as the osteogenic phenotypic switch under the condition of calcifying medium. Our data suggest that XPR1 plays protective roles in the pathogenesis of VC and its decrease in the aorta may contribute to the progression of VC in CKD.

Intestinal secretion of indoxyl sulfate as a possible compensatory excretion pathway in chronic kidney disease.

Indoxyl sulfate (IS) is a protein-bound uremic toxin that progressively accumulates in plasma during chronic kidney disease (CKD), and its accumulation is associated with the progression of CKD. This study examined the intestinal secretion of IS using in situ single-pass intestinal perfusion in a rat model of renal insufficiency, MRP2- and BCRP-overexpressing Sf9 membrane vesicles, and Caco-2 cell monolayers. An in situ single-pass perfusion study in CKD model rats demonstrated that a small amount of IS is secreted into intestinal lumen after iv administration of IS, and the clearance increased AUC-dependently. An excess amount of IS (3mm) partially inhibited the MRP2- and BCRP-mediated uptake of specific fluorescent substrates, CDCF and Lucifer yellow, respectively, into the membrane vesicles, although IS was not taken up at a physiological concentration, 10 μm. In the Caco-2 cell monolayers, the IS transport was higher in the absorptive direction than in the secretory direction (p<0.05). p-Aminohippuric acid (PAH) strongly inhibited IS transport in both directions (absorptive, p=0.142; secretory, p<0.01). Given that the blood IS levels are much higher than those in the intestinal lumen, it is possible that this unknown PAH-sensitive system contributes to the intestinal IS secretion. Although in situ inhibition study is needed to confirm that this unknown transporter mediates the in vivo intestinal secretion of IS, we speculate that this unknown active efflux system works as a compensatory excretion pathway for excess organic anions such as IS especially in end-stage renal disease.

Limited Effect of Intravenously Administered Indoxyl Sulfate, a Uremic Toxin, on the Hepatic Transport of Pravastatin in Normal Rats

Indoxyl sulfate (IS) is a typical uremic toxin that extensively accumulates in the plasma of patients with seriously impaired renal function. This study seeks to clarify whether IS exerts a potent modulating effect on the hepatic transport of pravastatin, which is a substrate of both organic anion transporting peptides (OATPs) and multidrug resistance-associated protein (Mrp) 2 in rats. When IS is administered intravenously to the normal rats at a dose of 120 μmol/kg; plasma IS levels are approximately 600 μM after 2 min and 100 μM after 120 min. In rats with acute renal failure (ARF) induced by cisplatin, the area under the curve (AUC) was more than 2.5-fold greater compared with that in the normal rats, indicating that IS accumulates in ARF rats. Intravenously administered pravastatin almost disappeared from the plasma by 60 min post-administration and approximately 55% of dose was excreted in the bile within 60 min. This result suggested that pravastatin was efficiently taken up from the sinusoid into hepatocytes via rat OATPs on the sinusoidal membrane and preferentially transported in the bile mediated by Mrp2 on the canalicular membrane. IS administered intravenously at a dose of 120 μmol/kg caused neither an increase in plasma pravastatin levels nor a decrease in its biliary excretion. In conclusion, the present results demonstrate that single intravenous administration of IS does not interfere with the hepatic transport of pravastatin directly in vivo, which is at variance with the results of previous in vitro studies.

MPGES-1-Derived PGE2 Contributes to Indoxyl Sulfate-Induced Mesangial Cell Proliferation

Background/Aims: We previously reported that indoxyl sulfate (IS) could cause mesangial cell (MC) proliferation via a cyclooxygenase (COX)-2-dependent mechanism. However, the specific prostaglandin contributing to COX-2 effect on IS-induced MC proliferation remained unknown. Thus, the present study was undertaken to examine the role of microsomal prostaglandin E synthase-1 (mPGES-1)-derived Prostaglandin E2 (PGE₂) in IS-induced MC proliferation. Methods: IS was administered to the MCs with or without mPGES-1 siRNA pretreatment to induce the MC proliferation which was determined by cell cycle analysis, DNA synthesis, and the expressions of cyclins. In another experimental setting, PGE₂ was applied to the MCs to examine its direct effect on MC proliferation, as well as the regulation of prostaglandin E receptors (EPs) by qRT-PCR. Results: With the administration of IS, mPGES-1(not mPGES-2 and cytosolic PGES) was significantly upregulated at both protein and mRNA levels in line with a promoted MC proliferation. Interestingly, silencing mPGES-1 reduced cell number in S and G2 phases and blocked the upregulation of cyclin A2 and cyclin D1 in parallel with blunted PGE₂ release after IS treatment, indicating that mPGES-1-derived PGE₂ could contribute to MC proliferation. Furthermore, we confirmed that exogenous PGE₂ could directly trigger the proliferative response in MCs. Lastly, we observed a selective upregulation of EP2 after PGE2 treatment and enhanced phosphorylation of NF-κB following IS administration in MCs, suggesting the potential involvements of EP2 and NF-κB in this pathological process. Conclusion: mPGES-1-derived PGE₂ contributed to IS-induced mesangial cell proliferation.

Upregulation of nuclear factor-κB activity mediates CYP24 expression and reactive oxygen species production in indoxyl sulfate-induced chronic kidney disease.

Chronic kidney disease (CKD) is associated with an inflammation-mediated process, and the vitamin D (3) catabolizing enzyme, CYP24, is frequently overexpressed in CKD, where it may play a crucial role in kidney disease. Herein, in this study, we investigated CYP24, reactive oxygen species (ROS), and inflammatory responses in an indoxyl sulfate (IS)-induced CKD model to elucidate the role of CYP24 in CKD. Our results showed that IS upregulates proinflammatory cytokine, CYP24 and nuclear factor-κB (NF-κB) expression in human renal proximal tubule epithelial cells. In addition, IS treatment increased ROS production and simultaneously upregulated CYP24 expression and NF-κB translocation. Moreover, the IS-induced upregulation of CYP24 expression was alleviated by an inhibitor of NF-κB, as well as a siRNA specific to NF-κB p65. Furthermore, the renal cortex of DN (Dahl salt-resistant normotensive) + IS, DH (Dahl salt-sensitive hypertensive), and DH + IS rats showed increased expression of NF-κB p65, CYP24, 8-hydroxydeoxyguanosine (8-OHdG), a marker of ROS and macrophage infiltration compared with DN rats. These results provide evidence that administration of IS in human renal tubular epithelial cells upregulates NF-κB, which leads to increase CYP24 expression and ROS production. They also suggest that suppressing NF-κB signalling is promising for the development into a strategy for CKD treatment.

Indoxyl sulfate potentiates skeletal muscle atrophy by inducing the oxidative stress-mediated expression of myostatin and atrogin-1.

Skeletal muscle atrophy, referred to as sarcopenia, is often observed in chronic kidney disease (CKD) patients, especially in patients who are undergoing hemodialysis. The purpose of this study was to determine whether uremic toxins are involved in CKD-related skeletal muscle atrophy. Among six protein-bound uremic toxins, indole containing compounds, indoxyl sulfate (IS) significantly inhibited proliferation and myotube formation in C2C12 myoblast cells. IS increased the factors related to skeletal muscle breakdown, such as reactive oxygen species (ROS) and inflammatory cytokines (TNF-α, IL-6 and TGF-β1) in C2C12 cells. IS also enhanced the production of muscle atrophy-related genes, myostatin and atrogin-1. These effects induced by IS were suppressed in the presence of an antioxidant or inhibitors of the organic anion transporter and aryl hydrocarbon receptor. The administered IS was distributed to skeletal muscle and induced superoxide production in half-nephrectomized (1/2 Nx) mice. The chronic administration of IS significantly reduced the body weights accompanied by skeletal muscle weight loss. Similar to the in vitro data, IS induced the expression of myostatin and atrogin-1 in addition to increasing the production of inflammatory cytokines by enhancing oxidative stress in skeletal muscle. These data suggest that IS has the potential to accelerate skeletal muscle atrophy by inducing oxidative stress-mediated myostatin and atrogin-1 expression.

Indoxyl Sulfate Down-Regulates SLCO4C1 Transporter through Up-Regulation of GATA3

The accumulated uremic toxins inhibit the expression of various renal transporters and this inhibition may further reduce renal function and subsequently cause the accumulation of uremic toxins. However, the precise mechanism of the nephrotoxicity of uremic toxins on renal transport has been poorly understood. Here we report that indoxyl sulfate, one of the potent uremic toxins, directly suppresses the renal-specific organic anion transporter SLCO4C1 expression through a transcription factor GATA3. The promoter region of SLCO4C1 gene has several GATA motifs, and indoxyl sulfate up-regulated GATA3 mRNA and subsequently down-regulated SLCO4C1 mRNA. Overexpression of GATA3 significantly reduced SLCO4C1 expression, and silencing of GATA3 increased SLCO4C1 expression vice versa. Administration of indoxyl sulfate in rats reduced renal expression of slco4c1 and under this condition, plasma level of guanidinosuccinate, one of the preferable substrates of slco4c1, was significantly increased without changing plasma creatinine. Furthermore, in 5/6 nephrectomized rats, treatment with oral adsorbent AST-120 significantly decreased plasma indoxyl sulfate level and conversely increased the expression of slco4c1, following the reduction of plasma level of guanidinosuccinate. These data suggest that the removal of indoxyl sulfate and blocking its signal pathway may help to restore the SLCO4C1-mediated renal excretion of uremic toxins in CKD.

In acute kidney injury, indoxyl sulfate impairs human endothelial progenitor cells: modulation by statin

Renal ischemia rapidly mobilizes endothelial progenitor cells (EPCs), which provides renoprotection in acute kidney injury (AKI). Indoxyl sulfate (IS) is a protein-binding uremic toxin with a potential role in endothelial injury. In this study, we examined the effects and mechanisms of action of IS on EPCs in AKI. Forty-one consecutive patients (26 male; age, 70.1±14.1years) diagnosed with AKI according to the AKIN criteria were enrolled. The AKI patients had higher serum IS levels than patients with normal kidney function (1.35±0.94×10(-4)M vs. 0.02±0.02×10(-4)M, P<0.01). IS levels were negatively correlated to the number of double-labeled (CD34(+)KDR(+)) circulating EPCs (P<0.001). After IS stimulation, the cells displayed decreased expression of phosphorylated endothelial nitric oxide (NO) synthase, vascular cell adhesion molecule-1, increased reactive oxygen species, decreased proliferative capacity, increased senescence and autophagy, as well as decreased migration and angiogenesis. These effects of IS on EPCs were reversed by atorvastatin. Further, exogenous administration of IS significantly reduced EPC number in Tie2-GFP transgenic mice and attenuated NO signaling in aortic and kidney arteriolar endothelium after kidney ischemia-reperfusion injury in mice, and these effects were restored by atorvastatin. Our results are the first to demonstrate that circulating IS is elevated in AKI and has direct effects on EPCs via NO-dependent mechanisms both in vitro and in vivo. Targeting the IS-mediated pathways by NO-releasing statins such as atorvastatin may preempt disordered vascular wall pathology, and represent a novel EPC-rescued approach to impaired neovascularization after AKI.

Indoxyl sulfate promotes vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A through oxidative stress

We previously demonstrated that indoxyl sulfate (IS), a uremic toxin, induces aortic calcification in hypertensive rats and induces oxidative stress and the expression of osteoblast-specific proteins in vascular smooth muscle cells. This study aimed to clarify whether IS stimulates senescence of cultured human aortic smooth muscle cells (HASMCs) and aorta in Dahl salt-sensitive hypertensive rats and whether AST-120, an oral sorbent, prevents senescence of aorta in subtotally nephrectomized uremic rats. IS increased the mRNA expression of p53 and p21 in HASMCs, whereas it did not change that of p16 and retinoblastoma protein (pRb). The IS-induced expression of p53 and p21 was suppressed by N-acetylcysteine, an antioxidant. IS promoted protein expression of p53, p21, and senescence-associated β-galactosidase (SA-β-gal) activity in HASMCs, and N-acetylcysteine and pifithrin-α,p-nitro, a p53 inhibitor, blocked these effects. IS upregulated prelamin A, a hallmark of vascular smooth muscle cell senescence, and downregulated FACE1/Zempste24 protein expression in HASMCs, and N-acetylcysteine suppressed these effects. Administration of IS to hypertensive rats increased expression of SA-β-gal, p53, p21, prelamin A, and oxidative stress markers such as 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in the cells embedded in the calcification area of arcuate aorta. Further, the uremic rat model showed positive staining for SA-β-gal, p53, p21, prelamin A, 8-OHdG, and MDA in the cells embedded in the calcification area of arcuate aorta, whereas AST-120 reduced the expression of these biomarkers. Taken together, IS accelerates vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A and downregulation of FACE1 through oxidative stress.

Indoxyl sulfate, a uremic toxin, promotes cell senescence in aorta of hypertensive rats

We demonstrated that administration of indoxyl sulfate, a uremic toxin, promotes aortic calcification in hypertensive rats. This study aimed to clarify if indoxyl sulfate could contribute to cell senescence in the aorta of hypertensive rats. The rat groups consisted of (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS). After 32 weeks, their arcuate aortas were excised for histological and immunohistochemical analysis. Cell senescence was evaluated by immunohistochemistry of senescence-associated β-galactosidase (SA-β-gal), and senescence-related proteins such as p16 INK4a, p21 WAF1/CIP1, p53 and retinoblastoma protein (Rb). Both DH and DH + IS rats showed significantly higher systolic blood pressure than DN and DN + IS rats, respectively. Serum indoxyl sulfate levels were significantly higher in DN + IS and DH + IS rats than in DN and DH rats, respectively. In aorta, DH rats showed significantly increased aortic calcification and wall thickness, and increased expression of SA-β-gal, p16 INK4a, p21 WAF1/CIP1, p53 and Rb in the calcification area of arcuate aorta as compared with DN rats. More notably, DH + IS rats showed significantly increased aortic calcification and wall thickness, and significantly increased expression of SA-β-gal, p16 INK4a, p21 WAF1/CIP1, p53 and Rb in the cells embedded in the calcification area as compared with DH rats. In conclusion, indoxyl sulfate promotes cell senescence with aortic calcification and expression of senescence-related proteins in hypertensive rats.

ROS and PDFG-β receptors are critically involved in indoxyl sulfate actions that promote vascular smooth muscle cell proliferation and migration

Patients with chronic renal failure are at greater risk of developing atherosclerosis than healthy individuals, and recent data suggest that the putative uremic toxin indoxyl sulfate (IS) promotes the pathogenesis of atherosclerosis. The present study examined the effects of IS on vascular smooth muscle cells (VSMCs) with respect to reactive oxygen species (ROS), platelet-derived growth factor (PDGF) receptors, and mitogen-activated protein kinases (MAPKs). IS induced the migration and proliferation of VSMCs and synergistically enhanced their PDGF-induced migration as well as proliferation. The effects of PDGF were promoted after a 24-h incubation with IS despite the absence of IS during PDGF stimulation. Intracellular ROS levels were increased in the presence of IS, and PDGF-dependent ROS production was augmented by a prior 24-h incubation with IS even in the absence of IS during PDGF stimulation. These data suggest that IS increases the sensitivity of VSMCs to PDGF. IS also phosphorylated PDGF-beta-receptors and upregulated PDGF-beta receptor but not alpha-receptor protein expression in the absence of exogenous PDGF. The NADPH oxidase inhibitor diphenylene iodonium blocked IS-dependent increase in receptor expression. Administration of IS to nephrectomized rats also elevated receptor protein expression in arterial VSMCs. Inhibitors of NADPH oxidase, PDGF-beta receptors, extracellular-regulated protein kinase (ERK), and p38 MAPK all inhibited IS-induced VSMCs migration and proliferation. Taken together, these findings indicate that IS induces the migration as well as proliferation of VSMCs through PDGF-beta receptors and that ROS generation is critically involved in this process, which promotes the development of atherosclerosis.

Indoxyl Sulfate Reduces Superoxide Scavenging Activity in the Kidneys of Normal and Uremic Rats

Background/Aim: Indoxyl sulfate (IS) is a uremic toxin that accelerates the progression of chronic renal failure (CRF). This study aimed at determining whether IS impairs antioxidative systems (redox status) in the kidney. Methods: IS was orally administered to normal and subtotally nephrectomized (three fourths and five sixths) rats (CRF rats) for 2 weeks. By use of in vivo and ex vivo electron spin resonance spectroscopy, the kidney redox status was evaluated using carbamoyl-PROXYL as a radical spin probe in living rats, and the kidney superoxide scavenging activity was measured. Immunohistochemistry of superoxide dismutase (SOD) in the kidney was performed. Results: Administration of IS increased serum and kidney levels of IS and serum creatinine and decreased creatinine clearance. CRF rats showed reduced spin reduction rate, prolonged half-life of the spin probe, and reduced superoxide scavenging activity and SOD-positive areas in the kidney as compared with normal rats. Administration of IS further reduced radical spin reduction rate, prolonged half-life of the spin probe, and reduced superoxide scavenging activity and SOD-positive areas in the kidneys. Conclusions: Administration of IS reduced superoxide scavenging activity in the kidneys of normal and CRF rats. Thus, the nephrotoxicity of IS may be induced by impairing the antioxidative systems in the kidney.

An Oral Adsorbent, AST-120 Protects Against the Progression of Oxidative Stress by Reducing the Accumulation of Indoxyl Sulfate in the Systemic Circulation in Renal Failure

The effect of AST-120, an oral adsorbent, on oxidative stress in the systemic circulation in chronic renal failure (CRF) was examined and the potential role of indoxyl sulfate (IS), an uremic toxin adsorbed by AST-120, in inducing the formation of reactive oxygen species (ROS) in the vascular system was studied, in vitro and in vivo. The level of oxidized albumin, a marker for oxidative stress in the systemic circulation was determined by HPLC, as previously reported. The mRNA levels of TGF-beta (1) and Oat1 were measured by quantitative RT-PCR. The IS induced ROS generation in cultured human umbilical vein endothelial cells (HUVECs) was estimated using a fluorescence microplate reader. An increase in the ratio of oxidized to unoxidized albumin was determined using 5/6 nephrectomized rats (CRF rats) compared to a control group. The ratio was significantly reduced in the group that received AST-120 of 4 weeks, suggesting that AST-120 inhibits oxidative stress in CRF. An anti-oxidative effect of AST-120 was also observed in CRF rats with a similar renal function. The ratio of oxidized albumin was correlated with serum IS levels in vivo. The same relationship was also observed in CRF rats with the continued administration of IS. In addition, IS dramatically increased the generation of ROS in both a dose- and time- dependent manner in HUVEC, suggesting that accumulated IS may play an important role in enhancing intravascular oxidative stress. We propose that AST-120 reduces IS concentrations in the blood that induces ROS production in endothelial cells, thereby inhibiting the subsequent occurrence of oxidative stress in the systemic circulation in renal failure.

Role of organic anion transporters in the tubular transport of indoxyl sulfate and the induction of its nephrotoxicity.

In uremic patients, various uremic toxins are accumulated and exert various biologic effects on uremia. Indoxyl sulfate (IS) is one of uremic toxins that is derived from dietary protein, and serum levels of IS are markedly increased in both uremic rats and patients. It has been previously reported that the accumulation of IS promotes the progression of chronic renal failure (CRF). This study demonstrates the role of rat organic anion transporters (rOATs) in the transport of IS and the induction of its nephrotoxicity. The administration of IS to 5/6-nephrectomized rats caused a faster progression of CRF, and immunohistochemistry revealed that IS was detected in the proximal and distal tubules where rOAT1 (proximal tubules) and/or rOAT3 (proximal and distal tubules) were also shown to be localized. In in vitro study, the proximal tubular cells derived from mouse that stably express rOAT1 (S2 rOAT1) and rOAT3 (S2 rOAT3) were established. IS inhibited organic anion uptake by S2 rOAT1 and S2 rOAT3, and the Ki values were 34.2 and 74.4 microM, respectively. Compared with mock, S2 rOAT1 and S2 rOAT3 exhibited higher levels of IS uptake, which was inhibited by probenecid and cilastatin, organic anion transport inhibitors. The addition of IS induced a decrease in the viability of S2 rOAT1 and S2 rOAT3 as compared with the mock, which was rescued by probenecid. These results suggest that rOAT1 and rOAT3 play an important role in the transcellular transport of IS and the induction of its nephrotoxicity.

Myenteric plexus in Hirschsprung's disease.

ABSTRACT. In uremic patients, various uremic toxins are accumulated and exert various biologic effects on uremia. Indoxyl sulfate (IS) is one of uremic toxins that is derived from dietary protein, and serum levels of IS are markedly increased in both uremic rats and patients. It has been previously reported that the accumulation of IS promotes the progression of chronic renal failure (CRF). This study demonstrates the role of rat organic anion transporters (rOATs) in the transport of IS and the induction of its nephrotoxicity. The administration of IS to 5/6-nephrectomized rats caused a faster progression of CRF, and immunohistochemistry revealed that IS was detected in the proximal and distal tubules where rOAT1 (proximal tubules) and/or rOAT3 (proximal and distal tubules) were also shown to be localized. In <i>in vitro</i> study, the proximal tubular cells derived from mouse that stably express rOAT1 (S2 rOAT1) and rOAT3 (S2 rOAT3) were established. IS inhibited organic anion uptake by S2 rOAT1 and S2 rOAT3, and the Ki values were 34.2 and 74.4 μM, respectively. Compared with mock, S2 rOAT1 and S2 rOAT3 exhibited higher levels of IS uptake, which was inhibited by probenecid and cilastatin, organic anion transport inhibitors. The addition of IS induced a decrease in the viability of S2 rOAT1 and S2 rOAT3 as compared with the mock, which was rescued by probenecid. These results suggest that rOAT1 and rOAT3 play an important role in the transcellular transport of IS and the induction of its nephrotoxicity.