Indoxyl Sulfate Contributes to mTORC1-Induced Renal Fibrosis via The OAT/NADPH Oxidase/ROS Pathway

Activation of mTORC1 (mechanistic target of rapamycin complex 1) in renal tissue has been reported in chronic kidney disease (CKD)-induced renal fibrosis. However, the molecular mechanisms responsible for activating mTORC1 in CKD pathology are not well understood. The purpose of this study was to identify the uremic toxin involved in mTORC1-induced renal fibrosis. Among the seven protein-bound uremic toxins, only indoxyl sulfate (IS) caused significant activation of mTORC1 in human kidney 2 cells (HK-2 cells). This IS-induced mTORC1 activation was inhibited in the presence of an organic anion transporter inhibitor, a NADPH oxidase inhibitor, and an antioxidant. IS also induced epithelial–mesenchymal transition of tubular epithelial cells (HK-2 cells), differentiation of fibroblasts into myofibroblasts (NRK-49F cells), and inflammatory response of macrophages (THP-1 cells), which are associated with renal fibrosis, and these effects were inhibited in the presence of rapamycin (mTORC1 inhibitor). In in vivo experiments, IS overload was found to activate mTORC1 in the mouse kidney. The administration of AST-120 or rapamycin targeted to IS or mTORC1 ameliorated renal fibrosis in Adenine-induced CKD mice. The findings reported herein indicate that IS activates mTORC1, which then contributes to renal fibrosis. Therapeutic interventions targeting IS and mTORC1 could be effective against renal fibrosis in CKD.

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Early Elimination of Uremic Toxin Ameliorates AKI to CKD Transition

Clinical Science ◽

10.1042/cs20210858 ◽

2021 ◽

Author(s):

Jia Huang Chen ◽

Chia-Ter Chao ◽

Jenq-Wen Huang ◽

Kuan-Yu Hung ◽

Shing-Hwa Liu ◽

...

Keyword(s):

Er Stress ◽

Renal Fibrosis ◽

Epithelial Mesenchymal Transition ◽

Early Stage ◽

Ischemia Reperfusion ◽

Organic Anion ◽

Organic Anion Transporter ◽

Uremic Toxin ◽

Mesenchymal Transition ◽

Anion Transporter

Acute kidney injury (AKI)-related fibrosis is a major driver of chronic kidney disease (CKD) development. Aberrant kidney recovery after AKI is multifactorial and still unclear. The accumulation of indoxyl sulfate (IS), a protein-bound uremic toxin, has been identified as a detrimental factor of renal fibrosis. However, the mechanisms underlying IS-related aberrant kidney recovery after AKI is still unknown. The study aims to elucidate the effects of IS in the pathogenesis of AKI to CKD transition. Our results showed that serum IS started to accumulate associated with the downregulation of tubular organic anion transporter, but not observed in the small-molecule uremic toxins of the unilateral ischemia-reperfusion injury without a contralateral nephrectomy model(UIRI). Serum IS is positively correlated with renal fibrosis and ER stress-related protein expression induction in the UIRI with a contralateral nephrectomy model (UIRI+Nx). To evaluate the effects of IS in the AKI to CKD transition, we administered indole, a precursor of IS, at the early stage of UIRI. Our results demonstrated IS potentiates renal fibrosis, senescence-associated secretory phenotype (SASP), and activation of ER, which is attenuated by synergistic AST-120 administration. Furthermore, we clearly demonstrated that IS exposure potentiated hypoxia-reperfusion (H/R) induced G2/M cell cycle arrest, epithelial-mesenchymal transition, and aggravated ER stress induction in vitro. Finally, the ER chemical chaperon, 4-PBA, successfully reversed the above-mentioned AKI to CKD transition. Taken together, early IS elimination in the early stage of AKI is likely to be a useful strategy in the prevention or treatment of the AKI to CKD transition.

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Acute Exposure to Indoxyl Sulfate Impairs Endothelium-Dependent Vasorelaxation in Rat Aorta

International Journal of Molecular Sciences ◽

10.3390/ijms20020338 ◽

2019 ◽

Vol 20 (2) ◽

pp. 338 ◽

Cited By ~ 9

Author(s):

Takayuki Matsumoto ◽

Keisuke Takayanagi ◽

Mihoka Kojima ◽

Kumiko Taguchi ◽

Tsuneo Kobayashi

Keyword(s):

Oxidative Stress ◽

Gut Microbiota ◽

Nadph Oxidase ◽

Vascular Function ◽

Organic Anion ◽

Organic Anion Transporter ◽

Indoxyl Sulfate ◽

Transient Receptor Potential Vanilloid ◽

Ionophore A23187 ◽

Anion Transporter

Gut microbiota are emerging as potential contributors to the regulation of host homeostasis. Dysbiosis of the gut microbiota associated with increased intestinal permeability facilitates the passage of endotoxins and other microbial products, including indoxyl sulfate in the circulation. Although an emerging body of evidence has suggested that indoxyl sulfate is a key substance for the development of chronic kidney disease, few studies have investigated the direct association of indoxyl sulfate with vascular function. We hypothesized that indoxyl sulfate adversely affects vascular function. Aortas isolated from male Wistar rat were examined in the presence or absence of indoxyl sulfate to assess the vascular function, including vasorelaxation and vasocontraction. Indoxyl sulfate (vs. vehicle) (1) decreased vasorelaxation induced by acetylcholine (ACh) but not by sodium nitroprusside; (2) had no significant alterations of noradrenaline-induced vasocontraction in the absence and presence of endothelium; (3) decreased adenylyl cyclase activator (forskolin)-induced vasorelaxation, while such a difference was eliminated by endothelial denudation; and (4) decreased vasorelaxations induced by calcium ionophore (A23187) and transient receptor potential vanilloid 4 agonist (GSK1016790A). The indoxyl sulfate-induced decrease in the vasorelaxations induced by ACh and A23187 increased by cell-permeant superoxide dismutase or by organic anion transporter inhibitor. However, apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, had no effects on vasorelaxations induced by ACh, A23187, forskolin, and GSK1016790A in the presence of indoxyl sulfate. These results suggest that indoxyl sulfate directly affects the vascular function, particularly, endothelium-dependent vasorelaxation, and this effect may be attributable to increased oxidative stress after cell transportion via organic anion transporter, and such increased oxidative stress may not be attributable to activation of NADPH oxidase activation.

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Remote sensing and signaling in kidney proximal tubules stimulates gut microbiome-derived organic anion secretion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1821809116 ◽

2019 ◽

Vol 116 (32) ◽

pp. 16105-16110 ◽

Cited By ~ 18

Author(s):

Jitske Jansen ◽

Katja Jansen ◽

Ellen Neven ◽

Ruben Poesen ◽

Amr Othman ◽

...

Keyword(s):

Proximal Tubule ◽

Gut Microbiome ◽

Organic Anion ◽

Organic Anion Transporter ◽

Indoxyl Sulfate ◽

Regulation Mechanism ◽

Uremic Toxin ◽

Proximal Tubule Cell ◽

Anion Secretion ◽

Anion Transporter

Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

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Role of blood-brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain

Journal of Neurochemistry ◽

10.1046/j.1471-4159.2002.01108.x ◽

2002 ◽

Vol 83 (1) ◽

pp. 57-66 ◽

Cited By ~ 139

Author(s):

Sumio Ohtsuki ◽

Hiroshi Asaba ◽

Hitomi Takanaga ◽

Tsuneo Deguchi ◽

Ken-ichi Hosoya ◽

...

Keyword(s):

Blood Brain Barrier ◽

Organic Anion ◽

Organic Anion Transporter ◽

Indoxyl Sulfate ◽

Uremic Toxin ◽

Anion Transporter ◽

Metabolite Clearance ◽

Organic Anion Transporter 3 ◽

The Brain

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Molecular Mechanisms for Species Differences in Organic Anion Transporter 1, OAT1: Implications for Renal Drug Toxicity

Molecular Pharmacology ◽

10.1124/mol.117.111153 ◽

2018 ◽

Vol 94 (1) ◽

pp. 689-699 ◽

Cited By ~ 15

Author(s):

Ling Zou ◽

Adrian Stecula ◽

Anshul Gupta ◽

Bhagwat Prasad ◽

Huan-Chieh Chien ◽

...

Keyword(s):

Drug Toxicity ◽

Molecular Mechanisms ◽

Species Differences ◽

Organic Anion ◽

Organic Anion Transporter ◽

Anion Transporter

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Adaptive responses of renal organic anion transporter 3 (OAT3) during cholestasis

AJP Renal Physiology ◽

10.1152/ajprenal.00139.2008 ◽

2008 ◽

Vol 295 (1) ◽

pp. F247-F252 ◽

Cited By ~ 29

Author(s):

Jiarong Chen ◽

Tomohiro Terada ◽

Ken Ogasawara ◽

Toshiya Katsura ◽

Ken-ichi Inui

Keyword(s):

Bile Acids ◽

Cholic Acid ◽

Molecular Mechanisms ◽

Competitive Inhibition ◽

Organic Anion ◽

Organic Anion Transporter ◽

Specific Substrate ◽

Renal Secretion ◽

Sd Rats ◽

Anion Transporter

During cholestasis, bile acids are mainly excreted into the urine, but adaptive renal responses to cholestasis, especially molecular mechanisms for renal secretion of bile acids, have not been well understood. Organic anion transporters (OAT1 and OAT3) are responsible for membrane transport of anionic compounds at the renal basolateral membranes. In the present study, we investigated the pathophysiological roles of OAT1 and OAT3 in terms of renal handling of bile acids. The Eisai hyperbilirubinemic rats (EHBR), mutant rats without multidrug resistance-associated protein 2, showed higher serum and urinary concentrations of bile acids, compared with Sprague-Dawley (SD) rats (wild type). The protein expression level of rat OAT3 was significantly increased in EHBR compared with SD rats, whereas the expression of rat OAT1 was unchanged. The transport activities of rat and human OAT3, but not OAT1, were markedly inhibited by various bile acids such as chenodeoxycholic acid and cholic acid. Cholic acid, glycocholic acid, and taurocholic acid, which mainly increased during cholestasis, are transported by OAT3. The plasma concentration of β-lactam antibiotic cefotiam, a specific substrate for OAT3, was more increased in EHBR than in SD rats despite upregulation of OAT3 protein. This may be due to the competitive inhibition of cefotiam transport by bile acids via OAT3. In conclusion, the present study clearly demonstrated that OAT3 is responsible for renal secretion of bile acids during cholestasis and that the pharmacokinetic profile of OAT3 substrates may be affected by cholestasis.

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Indoxyl Sulfate Contributes to Adipose Tissue Inflammation through the Activation of NADPH Oxidase

Toxins ◽

10.3390/toxins12080502 ◽

2020 ◽

Vol 12 (8) ◽

pp. 502

Author(s):

Shoma Tanaka ◽

Hiroshi Watanabe ◽

Takehiro Nakano ◽

Tadashi Imafuku ◽

Hiromasa Kato ◽

...

Keyword(s):

Oxidative Stress ◽

Adipose Tissue ◽

Nadph Oxidase ◽

Organic Anion ◽

Indoxyl Sulfate ◽

Epididymal Adipose Tissue ◽

Ros Production ◽

Adipose Tissue Inflammation ◽

Tissue Inflammation ◽

Anion Transporter

Adipose tissue inflammation appears to be a risk factor for the progression of chronic kidney disease (CKD), but the effect of CKD on adipose tissue inflammation is poorly understood. The purpose of this study was to clarify the involvement of uremic toxins (indoxyl sulfate (IS), 3-indoleacetic acid, p-cresyl sulfate and kynurenic acid) on CKD-induced adipose tissue inflammation. IS induces monocyte chemoattractant protein-1 (MCP-1) expression and reactive oxygen species (ROS) production in the differentiated 3T3L-1 adipocyte. An organic anion transporter (OAT) inhibitor, an NADPH oxidase inhibitor or an antioxidant suppresses the IS-induced MCP-1 expression and ROS production, suggesting the OAT/NADPH oxidase/ROS pathway is involved in the action of IS. Co-culturing 3T3L-1 adipocytes and mouse macrophage cells showed incubating adipocytes with IS increased macrophage infiltration. An IS-overload in healthy mice increased IS levels, oxidative stress and MCP-1 expression in epididymal adipose tissue compared to unloaded mice. Using 5/6-nephrectomized mice, the administration of AST-120 suppressed oxidative stress and the expression of MCP-1, F4/80 and TNF-α in epididymal adipose tissue. These collective data suggest IS could be a therapeutic target for the CKD-related inflammatory response in adipose tissue, and that AST-120 could be useful for the treatment of IS-induced adipose tissue inflammation.

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Renal secretion of hydrochlorothiazide involves organic anion transporter 1/3, organic cation transporter 2, and multidrug and toxin extrusion protein 2-K

AJP Renal Physiology ◽

10.1152/ajprenal.00141.2019 ◽

2019 ◽

Vol 317 (4) ◽

pp. F805-F814

Author(s):

Jia Yin ◽

David J. Wagner ◽

Bhagwat Prasad ◽

Nina Isoherranen ◽

Kenneth E. Thummel ◽

...

Keyword(s):

Molecular Mechanisms ◽

Organic Cation ◽

Organic Anion ◽

Organic Cation Transporter ◽

Tubular Secretion ◽

Organic Anion Transporter ◽

Anion Transporters ◽

Anion Transporter ◽

Organic Cation Transporter 2 ◽

Toxin Extrusion

Hydrochlorothiazide (HCTZ) is the most widely used thiazide diuretic for the treatment of hypertension either alone or in combination with other antihypertensives. HCTZ is mainly cleared by the kidney via tubular secretion, but the underlying molecular mechanisms are unclear. Using cells stably expressing major renal organic anion and cation transporters [human organic anion transporter 1 (hOAT1), human organic anion transporter 3 (hOAT3), human organic cation transporter 2 (hOCT2), human multidrug and toxin extrusion 1 (hMATE1), and human multidrug and toxin extrusion 2-K (hMATE2-K)], we found that HCTZ interacted with both organic cation and anion transporters. Uptake experiments further showed that HCTZ is transported by hOAT1, hOAT3, hOCT2, and hMATE2-K but not by hMATE1. Detailed kinetic analysis coupled with quantification of membrane transporter proteins by targeted proteomics revealed that HCTZ is an excellent substrate for hOAT1 and hOAT3. The apparent affinities ( Km) for hOAT1 and hOAT3 were 112 ± 8 and 134 ± 13 μM, respectively, and the calculated turnover numbers ( kcat) were 2.48 and 0.79 s−1, respectively. On the other hand, hOCT2 and hMATE2-K showed much lower affinity for HCTZ. The calculated transport efficiency ( kcat/ Km) at the single transporter level followed the rank order of hOAT1> hOAT3 > hOCT2 and hMATE2-K, suggesting a major role of organic anion transporters in tubular secretion of HCTZ. In vitro inhibition experiments further suggested that HCTZ is not a clinically relevant inhibitor for hOAT1 or hOAT3. However, strong in vivo inhibitors of hOAT1/3 may alter renal secretion of HCTZ. Together, our study elucidated the molecular mechanisms underlying renal handling of HCTZ and revealed potential pathways involved in the disposition and drug-drug interactions for this important antihypertensive drug in the kidney.

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