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Indoxyl Sulfate and p-Cresyl Sulfate in Chronic Kidney Disease. Could These Toxins Modulate the Antioxidant Nrf2-Keap1 Pathway?
REVIEW
Indoxyl Sulfate and p-Cresyl Sulfate in Chronic Kidney Disease. Could These Toxins Modulate the Antioxidant Nrf2-Keap1 Pathway? Milena Barcza Stockler-Pinto, PhD,* Denis Fouque, PhD,† Christophe O. Soulage, PhD,‡ Marine Croze, PhD,‡ and Denise Mafra, PhD* Protein-bound uremic toxins (i.e., indoxyl sulfate or p-cresyl sulfate), produced by intestinal bacteria, are accumulated in the plasma of chronic kidney disease (CKD) patients. These toxins interact negatively with biological functions, having potent oxidative stressinducing effects and a pathological effect on cardiovascular disease. Recent research in CKD has shown that oxidative stress and inflammation can be compounded by impaired activation of the nuclear factor (erythroid-2-related factor)-2 (Nrf2)–Kelch-like ECH associating protein-1 (Keap1) pathway, a major cellular defense mechanism. However, to date, many questions arise regarding the role of this system in CKD. For example, protein-bound uremic toxins promote oxidative stress in CKD patients, but their putative effect on the Nrf2Keap1 system has yet to be examined in these patients. This review will focus on the putative relationship among protein-bound uremic toxins, oxidative stress, and a possible decreased expression of Nrf2 in CKD. Ó 2013 by the National Kidney Foundation, Inc. All rights reserved.
Introduction
N
UCLEAR FACTOR (ERYTHROID-2-RELATED factor)-2 (Nrf2) activates genes that encode phase II detoxifying enzymes and antioxidant enzymes, which play crucial roles in cellular defense by improving the removal of reactive oxygen species (ROS). Currently, the Nrf2– Kelch-like ECH associating protein-1 (Keap1) system has been recognized as a major cellular defense mechanism against oxidative stress. Indeed, Nrf2 modulates the antiinflammatory cascade through the inhibition of nuclear factor-kB (NF-kB) and regulates cellular antioxidant responses.1-5 Oxidative stress and inflammation are common features in patients with chronic kidney disease (CKD) at all stages of disease, particularly patients on hemodialysis (HD). They can be considered as major mediators of cardiovascular and other complications, and they could play a pivotal role in the progression of CKD.6-8 Given the importance of the oxidative insult, the activation of the Nrf2-Keap1 system should be further explored in CKD.9
* Cardiovascular Sciences Graduate Program, Federal University Fluminense, Niteroi-Rio de Janeiro, Brazil. † Department of Nephrology, Centre Hopitalier Lyon Sud, INSERM 1060, CENS, Universite de Lyon, France. ‡ Universite de Lyon, INSA de Lyon, CarMeN, INSERM U1060, Villeurbanne, France. Financial Disclosure: The authors declare that they have no relevant financial interests. Address correspondence to Milena Barcza Stockler-Pinto, PhD, Rua Tiradentes, 108, Bloco B, 703, Inga-Niteroi-Brazil. E-mail: [email protected] Ó 2013 by the National Kidney Foundation, Inc. All rights reserved. 1051-2276/$36.00 http://dx.doi.org/10.1053/j.jrn.2013.11.006
Journal of Renal Nutrition, Vol -, No - (-), 2013: pp 1-6
A recent review by Pedruzzi and colleagues10 demonstrated that impaired activation of the Nrf2-Keap1 system may aggravate oxidative stress and inflammation in CKD. Kim and Vaziri6 observed that despite severe oxidative stress and inflammation, which should have induced Nrf2 activation and consequent upregulation of antioxidant and detoxifying enzymes, the remnant kidney in 5/6 nephrectomized animals exhibited ‘‘paradoxical reduction’’ of Nrf2 activation and its downstream antioxidant systems. CKD patients have several risk factors for cardiovascular disease (CVD), and more recent studies have identified the imbalance of the intestinal microbiota as a new factor that may contribute to inflammation and CVD in these patients. Indeed, studies have reported a relationship between intestinal microbiota and some pathological conditions such as inflammation and oxidative stress. The uremic toxins produced by intestinal bacteria (such as indoxyl sulfate, p-cresyl sulfate, amines, ammonia) are eliminated by healthy kidneys; however, in CKD, they accumulate in plasma and cannot be efficiently removed by HD because of their tight binding to plasma proteins.11-15 Moreover, the proteinbound uremic toxins indoxyl sulfate and p-cresyl sulfate appear to promote oxidative stress. Watanabe and colleagues16 recently showed that p-cresyl sulfate causes renal tubular cell damage by inducing oxidative stress. Although knowledge has increased in mechanisms involved in the interaction between the gut microbiota and diseases, little information exists about the gut microbiota and their relationship with inflammation and oxidative stress in CKD. There are no available data regarding the relationship between protein-bound uremic toxins and decreased expression of Nrf2-Keap1. This review will emphasize the potential relationship between uremic toxins produced 1
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by gut microbiota and a possible decreased expression of Nrf2 in CKD patients.
Keap1-Nrf2 System Nrf2 is expressed in a wide range of tissues and cell types and belongs to a subset of basic leucine zipper proteins sharing a conserved structural domain that is designated as a Cap-N-Collar. The cytosolic inhibitor Keap1 retains Nrf2 in the cytoplasm.17,18 Keap1 contains several reactive cysteine residues that serve as sensors of the intracellular redox state. Oxidative or covalent modification of thiols in some of these cysteine residues leads to conformational changes in Keap1 that result in disruption of 1 of the 2 Keap1 interactions with Nrf2. By limiting the proteasomal degradation of Nrf2, this process results in accumulation of the synthesized Nrf2 and its translocation to the nucleus.10,19 The dissociation of Nrf2 from Keap1 and its translocation to the nucleus binds it to regulatory sequences that are termed antioxidant response elements (AREs; or electrophile response elements), which are located in the promoter region of genes encoding the antioxidant and phase II detoxifying enzymes, such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1 as well as those that increase glutathione biosynthesis.20 The Nrf2-Keap1 system is currently recognized as a major cellular defense mechanism against oxidative stress. Many genes regulated by the Nrf2/ARE pathway play important roles in modulating the redox signals involved in the expression of genes related to chronic inflammation. Nrf2-AREs might inhibit the production or expression of proinflammatory mediators, including cytokines, chemokines, cell adhesion molecules, matrix metalloproteinase9, cyclooxygenase-2, and inducible nitric oxide synthase (iNOS). It is likely that the cytoprotective function of genes targeted by Nrf2 may cooperatively regulate the innate immune response and repress the induction of proinflammatory genes.1,21-23 The important role of Nrf2 in reducing inflammation has been linked to its ability to antagonize NF-kB.24,25 Using a Nrf2-null mouse, Mao and colleagues26observed increased NF-kB activation, tumor necrosis factor-a production, and matrix metalloproteinase-9 expression after spinal cord injury compared with the wild-type controls. In another study, the Nrf2 knockout mouse exhibited more NF-kB activation; more inflammatory cytokine tumor necrosis factor-a, interleukin-1b, and interleukin-6 production; and more intercellular adhesion molecule-1 expression in the brain after traumatic brain injury compared with their wild-type counterparts.25 All of these studies clearly suggest that the function of Nrf2 can be of crucial importance in some diseases (e.g., cancer; diabetes; neurodegenerative, cardiovascular, and pulmonary complications; and CKD) in which oxidative stress causes Nrf2 disorder.27-32
Figure 1. Nuclear factor (erythroid-2-related factor)-2 levels from hemodialysis patients and healthy individuals (P , .005).
Preliminary data from our group study in Brazil confirm a reduced expression of Nrf2 in peripheral blood mononuclear cells from 20 HD patients with a real-time reverse transcriptase polymerase chain reaction technique when compared with those from 11 healthy individuals (Fig. 1). Oxidative stress in CKD patients on HD has been reported as a result of overproduction of ROS. In fact, the activation of transcription of NF-kB by ROS triggers the transcription of many inflammatory genes coding for cytokines and adhesion molecules; furthermore, the ROS production also increased DNA damage. All of these factors are associated with vascular dysfunction, inflammation, and atherosclerosis in maintenance dialysis patients.6,33 The activation of Nrf2 in CKD should be important in response to the presence of ROS or some uremic toxins. A recent and elegant review by Saito34 described the Nrf2 activations and enhancers as promising candidate therapeutic agents for the treatment of CKD. According to Saito, the suppressive effects of Nrf2 activation on the inflammation reaction via NF-kB inhibition are likely to be much more important for preventing the progression of CKD. Some bioactive compounds present in many foods may activate the Keap1-Nrf2 system. Recent studies showed that bardoxolone methyl, polyphenols, resveratrol, curcumin, catechins, sulforaphane, allicin, lycopene, cinnamaldehyde, vitamin E, and coffee could alter the transcription of Nrf2. These food components may act at different levels: translocation of Nrf2 to the nucleus, thereby encode the detoxifying enzymes of phase II and/or inhibit NF-kB.35-48 However, to the best of our knowledge, there is currently no study evaluating the effect of uremic toxins on the Keap1-Nrf2 system in patients with CKD.
UREMIC TOXINS AND NRF2-KEAP1 HYPOTHESIS IN CKD
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Figure 2. Putative mechanism of Nrf2 and proteinbound uremic toxins. AREs, antioxidant response elements; Keap1, Kelch-like ECH associating protein-1; NF-kB, nuclear factor-kB; Nrf2, nuclear factor (erythroid-2-related factor)-2; OAT, organic anion transporter; ROS, reactive oxygen species.
Gut Microbiota As a Major Source of Protein-Bound Uremic Toxins: Linking Oxidative Stress in CKD Many toxins produced by intestinal bacteria (indoxyl sulfate, p-cresyl sulfate) are eliminated by healthy kidneys; thus, in CKD, they accumulate in plasma. These toxins cannot be efficiently removed by dialysis because of a tight binding to plasma proteins.
Indoxyl Sulfate Indoxyl sulfate is an organic anion metabolized in the liver from indole, which is produced by the intestinal bacteria as a metabolite of tryptophan derived from dietary protein.49 It is known that indoxyl sulfate plays an important role in the progression of renal damage by inducing an inflammatory reaction and enhancing expression of profibrotic cytokines.50 Recent studies have shown that the serum indoxyl sulfate level is a valuable marker of CVD and mortality in CKD patients.51,52 Indoxyl sulfate is 1 of the most studied uremic toxins. The mechanism by which indoxyl sulfate induces toxicity has recently been clarified. The intracellular accumulation of indoxyl sulfate via organic anion transporters and the subsequent increased production of ROS play a key role in the toxicity of indoxyl sulfate.27,53-55 Gelasco and colleagues56 showed that indoxyl sulfate stimulates the production of intracellular and extracellular ROS in rat mesangial cells through a pathway that likely involves NADPH oxidase. According to Masai and colleagues,57 indoxyl sulfate increases NADPH-oxidase-derived ROS (i.e., superoxide, O22), which, in turn, activates the mitogen-activated protein kinase/NF-kB pathway in human umbilical vein endothelial cells. Thus, these findings indicate
that NADPH oxidase plays an important role in indoxylsulfate-induced ROS production. It is remarkable to note that in proximal tubular cells indoxyl sulfate promotes activation (phosphorylation) of NF-kB p65 through ROS, followed by p53 expression.58 A recent publication by Bolati and colleagues59 found that indoxyl sulfate downregulated Nrf2 expression in HK-2 cells and that downregulation was alleviated by an inhibitor of NF-kB (pyrrolide dithiocarbomate).
p-Cresol p-Cresol is produced in the gut from the metabolism of tyrosine by the putrefactive bacteria of the gut microbiota. It undergoes conjugation with a sulfate group through the action of sulfotransferases in the submucosal tissue of the small intestine, and it is present in the plasma in the form of p-cresyl sulfate.11,60,61 p-Cresol is known to exert various deleterious effects, including endothelial dysfunction and the suppression of respiratory burst action in the blood, and it contributes to the progression of CKD. p-Cresyl sulfate has also been found to be associated with the occurrence of CVD and elevated mortality in CKD patients.14,62-64 Watanabe and colleagues16 showed that p-cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by enhancing NADPH oxidase activity. However, the mechanism by which p-cresyl sulfate induces its toxicity has not been thoroughly clarified. Furthermore, researchers recently showed that p-cresyl sulfate interferes with intracellular insulin signaling pathways and triggers insulin resistance in mice and cultured muscle cells.65 In addition, this protein-bound uremic toxin induces adipocyte dysfunction, causing a redistribution of ectopic lipids and lipotoxicity. In skeletal muscle,
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p-cresyl sulfate interferes directly with insulin signaling via the activation of extracellular signal-regulated kinase-1/2 phosphorylation and inhibitory serine phosphorylation of insulin receptor substrate-1.66 In summary, these studies show increased ROS production and NF-kB activation by protein-bound uremic toxins; however, there are no available data regarding the relationship between these microbiota toxins and Nrf2 activation. In addition, the use of pre- and probiotics should be considered in CKD patients to reduce the uremic toxins and oxidative stress. Prebiotics are nondigestible compounds that beneficially modify the composition and function of the intestinal microbiota. Probiotics are live microorganisms that contribute to the balance of the gut and, when administered in adequate amounts, confer benefits.67 Indeed, studies have shown that the use of pre- and probiotics modulates the intestinal microbiota68,69 and thus reduces the uremic toxins, such as p-cresol.70
Conclusion and Perspectives: Putative Role of Nrf2 The protein-bound uremic toxins are known to promote oxidative stress; however, the Nrf2/keap1 system, a major regulator of antioxidant response, is altered in CKD patients. Therefore, the question that arises is whether protein-bound uremic toxins could contribute to Nrf2keap1 pathway dysfunction in CKD (Fig. 2). Therefore, we urge scientists to perform experiments to demonstrate evidence to such a link because it could be pivotal to understanding the oxidative damages associated with CKD. At this moment, many questions regarding the activation of the Nrf2/Keap1 system in CKD need to be answered. More studies are needed to understand how this system and its mechanisms behave in CKD.
Practical Application This review shows an important aspect from the viewpoint of a practical application because we may improve the condition of oxidative stress, inflammation, and the risk of CVD in CKD patients by regulating the imbalance of the intestinal microbiota.
Acknowledgments
The authors thank Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nıvel Superior (CAPES) and Comite Franc¸ais d’Evaluation de la Cooperation Universitaire avec le Bresil (COFECUB).
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46. Huang TC, Chung YL, Wu ML, Chuang SM. Cinnamaldehyde enhances Nrf2 nuclear translocation to upregulate phase II detoxifying enzyme expression in HepG2 cells. J Agric Food Chem. 2011;59: 5164-5171. 47. Huang Y, Khor TO, Shu L, et al. A g-tocopherol-rich mixture of tocopherols maintains Nrf2 expression in prostate tumors of TRAMP mice via epigenetic inhibition of CpG methylation. J Nutr. 2012;142: 818-823. 48. Boettler U, Sommerfeld K, Volz N, et al. Coffee constituents as modulators of Nrf2 nuclear translocation and ARE (EpRE)-dependent gene expression. J Nutr Biochem. 2011;22:426-440. 49. Yu M, Kim YJ, Kang DH. Indoxyl sulfate-induced endothelial dysfunction in patients with chronic kidney disease via an induction of oxidative stress. Clin J Am Soc Nephrol. 2011;6:30-39. 50. Miyazaki T, Ise M, Hirata M, et al. Indoxyl sulfate stimulates renal synthesis of transforming growth factor-beta 1 and progression of renal failure. Kidney Int Suppl. 1997;63:S211-S214. 51. Shimazu S, Hirashiki A, Okumura T, et al. Association between indoxyl sulfate and cardiac dysfunction and prognosis in patients with dilated cardiomyopathy. Circ J. 2013;77:390-396. 52. Lin CJ, Liu HL, Pan CF, et al. Indoxyl sulfate predicts cardiovascular disease and renal function deterioration in advanced chronic kidney disease. Arch Med Res. 2012;43:451-456. 53. Shimizu H, Bolati D, Adijiang A, et al. Senescence and dysfunction of proximal tubular cells are associated with activated p53 expression by indoxyl sulfate. Am J Physiol Cell Physiol. 2010;299:C1110-C1117. 54. Dou L, Jourde-Chiche N, Faure V, et al. The uremic solute indoxyl sulfate induces oxidative stress in endothelial cells. J Thromb Haemost. 2007;5: 1302-1308. 55. Muteliefu G, Enomoto A, Niwa T. Indoxyl sulfate promotes proliferation of human aortic smooth muscle cells by inducing oxidative stress. J Ren Nutr. 2009;19:29-32. 56. Gelasco AK, Raymond JR. Indoxyl sulfate induces complex redox alterations in mesangial cells. Am J Physiol Ren Physiol. 2006;290:F1551F1558. 57. Masai N, Tatebe J, Yoshino G, Morita T. Indoxyl sulfate stimulates monocyte chemoattractant protein-1 expression in human umbilical vein endothelial cells by inducing oxidative stress through activation of the NADPH oxidase-nuclear factor-kB pathway. Circ J. 2010;74:22162224. 58. Shimizu H, Bolati D, Adijiang A, et al. NF-kB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells. Am J Physiol Cell Physiol. 2011;301:1201-1212. 59. Bolati D, Shimizu H, Yisireyili M, Nishijima F, Niwa T. Indoxyl sulfate, a uremic toxin, downregulates renal expression of Nrf2 through activation of NF-kB. BMC Nephrol. 2013;14:56. 60. Martinez AW, Recht NS, Hostetter TH, Meyer TW. Removal of p-cresol sulfate by hemodialysis. J Am Soc Nephrol. 2005;16:3430-3436. 61. Curtius HC, Mettler M, Ettlinger L. Study of the intestinal tyrosine metabolism using stable isotopes and gas chromatography-mass spectrometry. J Chromatogr. 1976;126:569-580. 62. Meijers BK, Bammens B, De Moor B, Verbeke K, Vanrenterghem Y, Evenepoel P. Free p-cresol is associated with cardiovascular disease in hemodialysis patients. Kidney Int. 2008;73:1174-1180. 63. Bammens B, Evenepoel P, Keuleers H, Verbeke K, Vanrenterghem Y. Free serum concentrations of the protein-bound retention solute p-cresol predict mortality in hemodialysis patients. Kidney Int. 2006;69:1081-1087. 64. Liabeuf S, Barreto DV, Barreto FC, et al. European Uraemic Toxin Work Group (EUTox): free p-cresylsulphate is a predictor of mortality in patients at different stages of chronic kidney disease. Nephrol Dial Transplant. 2010;25:1183-1191. 65. Koppe L, Pillon NJ, Vella RE, et al. p-Cresyl sulfate promotes insulin resistance associated with CKD. J Am Soc Nephrol. 2013;24:88-99.
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Indoxyl Sulfate and p-Cresyl Sulfate in Chronic Kidney Disease. Could These Toxins Modulate the Antioxidant Nrf2-Keap1 Pathway? Milena Barcza Stockler-Pinto, PhD,* Denis Fouque, PhD,† Christophe O. Soulage, PhD,‡ Marine Croze, PhD,‡ and Denise Mafra, PhD* Protein-bound uremic toxins (i.e., indoxyl sulfate or p-cresyl sulfate), produced by intestinal bacteria, are accumulated in the plasma of chronic kidney disease (CKD) patients. These toxins interact negatively with biological functions, having potent oxidative stressinducing effects and a pathological effect on cardiovascular disease. Recent research in CKD has shown that oxidative stress and inflammation can be compounded by impaired activation of the nuclear factor (erythroid-2-related factor)-2 (Nrf2)–Kelch-like ECH associating protein-1 (Keap1) pathway, a major cellular defense mechanism. However, to date, many questions arise regarding the role of this system in CKD. For example, protein-bound uremic toxins promote oxidative stress in CKD patients, but their putative effect on the Nrf2Keap1 system has yet to be examined in these patients. This review will focus on the putative relationship among protein-bound uremic toxins, oxidative stress, and a possible decreased expression of Nrf2 in CKD. Ó 2013 by the National Kidney Foundation, Inc. All rights reserved.
Introduction
N
UCLEAR FACTOR (ERYTHROID-2-RELATED factor)-2 (Nrf2) activates genes that encode phase II detoxifying enzymes and antioxidant enzymes, which play crucial roles in cellular defense by improving the removal of reactive oxygen species (ROS). Currently, the Nrf2– Kelch-like ECH associating protein-1 (Keap1) system has been recognized as a major cellular defense mechanism against oxidative stress. Indeed, Nrf2 modulates the antiinflammatory cascade through the inhibition of nuclear factor-kB (NF-kB) and regulates cellular antioxidant responses.1-5 Oxidative stress and inflammation are common features in patients with chronic kidney disease (CKD) at all stages of disease, particularly patients on hemodialysis (HD). They can be considered as major mediators of cardiovascular and other complications, and they could play a pivotal role in the progression of CKD.6-8 Given the importance of the oxidative insult, the activation of the Nrf2-Keap1 system should be further explored in CKD.9
* Cardiovascular Sciences Graduate Program, Federal University Fluminense, Niteroi-Rio de Janeiro, Brazil. † Department of Nephrology, Centre Hopitalier Lyon Sud, INSERM 1060, CENS, Universite de Lyon, France. ‡ Universite de Lyon, INSA de Lyon, CarMeN, INSERM U1060, Villeurbanne, France. Financial Disclosure: The authors declare that they have no relevant financial interests. Address correspondence to Milena Barcza Stockler-Pinto, PhD, Rua Tiradentes, 108, Bloco B, 703, Inga-Niteroi-Brazil. E-mail: [email protected] Ó 2013 by the National Kidney Foundation, Inc. All rights reserved. 1051-2276/$36.00 http://dx.doi.org/10.1053/j.jrn.2013.11.006
Journal of Renal Nutrition, Vol -, No - (-), 2013: pp 1-6
A recent review by Pedruzzi and colleagues10 demonstrated that impaired activation of the Nrf2-Keap1 system may aggravate oxidative stress and inflammation in CKD. Kim and Vaziri6 observed that despite severe oxidative stress and inflammation, which should have induced Nrf2 activation and consequent upregulation of antioxidant and detoxifying enzymes, the remnant kidney in 5/6 nephrectomized animals exhibited ‘‘paradoxical reduction’’ of Nrf2 activation and its downstream antioxidant systems. CKD patients have several risk factors for cardiovascular disease (CVD), and more recent studies have identified the imbalance of the intestinal microbiota as a new factor that may contribute to inflammation and CVD in these patients. Indeed, studies have reported a relationship between intestinal microbiota and some pathological conditions such as inflammation and oxidative stress. The uremic toxins produced by intestinal bacteria (such as indoxyl sulfate, p-cresyl sulfate, amines, ammonia) are eliminated by healthy kidneys; however, in CKD, they accumulate in plasma and cannot be efficiently removed by HD because of their tight binding to plasma proteins.11-15 Moreover, the proteinbound uremic toxins indoxyl sulfate and p-cresyl sulfate appear to promote oxidative stress. Watanabe and colleagues16 recently showed that p-cresyl sulfate causes renal tubular cell damage by inducing oxidative stress. Although knowledge has increased in mechanisms involved in the interaction between the gut microbiota and diseases, little information exists about the gut microbiota and their relationship with inflammation and oxidative stress in CKD. There are no available data regarding the relationship between protein-bound uremic toxins and decreased expression of Nrf2-Keap1. This review will emphasize the potential relationship between uremic toxins produced 1
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by gut microbiota and a possible decreased expression of Nrf2 in CKD patients.
Keap1-Nrf2 System Nrf2 is expressed in a wide range of tissues and cell types and belongs to a subset of basic leucine zipper proteins sharing a conserved structural domain that is designated as a Cap-N-Collar. The cytosolic inhibitor Keap1 retains Nrf2 in the cytoplasm.17,18 Keap1 contains several reactive cysteine residues that serve as sensors of the intracellular redox state. Oxidative or covalent modification of thiols in some of these cysteine residues leads to conformational changes in Keap1 that result in disruption of 1 of the 2 Keap1 interactions with Nrf2. By limiting the proteasomal degradation of Nrf2, this process results in accumulation of the synthesized Nrf2 and its translocation to the nucleus.10,19 The dissociation of Nrf2 from Keap1 and its translocation to the nucleus binds it to regulatory sequences that are termed antioxidant response elements (AREs; or electrophile response elements), which are located in the promoter region of genes encoding the antioxidant and phase II detoxifying enzymes, such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1 as well as those that increase glutathione biosynthesis.20 The Nrf2-Keap1 system is currently recognized as a major cellular defense mechanism against oxidative stress. Many genes regulated by the Nrf2/ARE pathway play important roles in modulating the redox signals involved in the expression of genes related to chronic inflammation. Nrf2-AREs might inhibit the production or expression of proinflammatory mediators, including cytokines, chemokines, cell adhesion molecules, matrix metalloproteinase9, cyclooxygenase-2, and inducible nitric oxide synthase (iNOS). It is likely that the cytoprotective function of genes targeted by Nrf2 may cooperatively regulate the innate immune response and repress the induction of proinflammatory genes.1,21-23 The important role of Nrf2 in reducing inflammation has been linked to its ability to antagonize NF-kB.24,25 Using a Nrf2-null mouse, Mao and colleagues26observed increased NF-kB activation, tumor necrosis factor-a production, and matrix metalloproteinase-9 expression after spinal cord injury compared with the wild-type controls. In another study, the Nrf2 knockout mouse exhibited more NF-kB activation; more inflammatory cytokine tumor necrosis factor-a, interleukin-1b, and interleukin-6 production; and more intercellular adhesion molecule-1 expression in the brain after traumatic brain injury compared with their wild-type counterparts.25 All of these studies clearly suggest that the function of Nrf2 can be of crucial importance in some diseases (e.g., cancer; diabetes; neurodegenerative, cardiovascular, and pulmonary complications; and CKD) in which oxidative stress causes Nrf2 disorder.27-32
Figure 1. Nuclear factor (erythroid-2-related factor)-2 levels from hemodialysis patients and healthy individuals (P , .005).
Preliminary data from our group study in Brazil confirm a reduced expression of Nrf2 in peripheral blood mononuclear cells from 20 HD patients with a real-time reverse transcriptase polymerase chain reaction technique when compared with those from 11 healthy individuals (Fig. 1). Oxidative stress in CKD patients on HD has been reported as a result of overproduction of ROS. In fact, the activation of transcription of NF-kB by ROS triggers the transcription of many inflammatory genes coding for cytokines and adhesion molecules; furthermore, the ROS production also increased DNA damage. All of these factors are associated with vascular dysfunction, inflammation, and atherosclerosis in maintenance dialysis patients.6,33 The activation of Nrf2 in CKD should be important in response to the presence of ROS or some uremic toxins. A recent and elegant review by Saito34 described the Nrf2 activations and enhancers as promising candidate therapeutic agents for the treatment of CKD. According to Saito, the suppressive effects of Nrf2 activation on the inflammation reaction via NF-kB inhibition are likely to be much more important for preventing the progression of CKD. Some bioactive compounds present in many foods may activate the Keap1-Nrf2 system. Recent studies showed that bardoxolone methyl, polyphenols, resveratrol, curcumin, catechins, sulforaphane, allicin, lycopene, cinnamaldehyde, vitamin E, and coffee could alter the transcription of Nrf2. These food components may act at different levels: translocation of Nrf2 to the nucleus, thereby encode the detoxifying enzymes of phase II and/or inhibit NF-kB.35-48 However, to the best of our knowledge, there is currently no study evaluating the effect of uremic toxins on the Keap1-Nrf2 system in patients with CKD.
UREMIC TOXINS AND NRF2-KEAP1 HYPOTHESIS IN CKD
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Figure 2. Putative mechanism of Nrf2 and proteinbound uremic toxins. AREs, antioxidant response elements; Keap1, Kelch-like ECH associating protein-1; NF-kB, nuclear factor-kB; Nrf2, nuclear factor (erythroid-2-related factor)-2; OAT, organic anion transporter; ROS, reactive oxygen species.
Gut Microbiota As a Major Source of Protein-Bound Uremic Toxins: Linking Oxidative Stress in CKD Many toxins produced by intestinal bacteria (indoxyl sulfate, p-cresyl sulfate) are eliminated by healthy kidneys; thus, in CKD, they accumulate in plasma. These toxins cannot be efficiently removed by dialysis because of a tight binding to plasma proteins.
Indoxyl Sulfate Indoxyl sulfate is an organic anion metabolized in the liver from indole, which is produced by the intestinal bacteria as a metabolite of tryptophan derived from dietary protein.49 It is known that indoxyl sulfate plays an important role in the progression of renal damage by inducing an inflammatory reaction and enhancing expression of profibrotic cytokines.50 Recent studies have shown that the serum indoxyl sulfate level is a valuable marker of CVD and mortality in CKD patients.51,52 Indoxyl sulfate is 1 of the most studied uremic toxins. The mechanism by which indoxyl sulfate induces toxicity has recently been clarified. The intracellular accumulation of indoxyl sulfate via organic anion transporters and the subsequent increased production of ROS play a key role in the toxicity of indoxyl sulfate.27,53-55 Gelasco and colleagues56 showed that indoxyl sulfate stimulates the production of intracellular and extracellular ROS in rat mesangial cells through a pathway that likely involves NADPH oxidase. According to Masai and colleagues,57 indoxyl sulfate increases NADPH-oxidase-derived ROS (i.e., superoxide, O22), which, in turn, activates the mitogen-activated protein kinase/NF-kB pathway in human umbilical vein endothelial cells. Thus, these findings indicate
that NADPH oxidase plays an important role in indoxylsulfate-induced ROS production. It is remarkable to note that in proximal tubular cells indoxyl sulfate promotes activation (phosphorylation) of NF-kB p65 through ROS, followed by p53 expression.58 A recent publication by Bolati and colleagues59 found that indoxyl sulfate downregulated Nrf2 expression in HK-2 cells and that downregulation was alleviated by an inhibitor of NF-kB (pyrrolide dithiocarbomate).
p-Cresol p-Cresol is produced in the gut from the metabolism of tyrosine by the putrefactive bacteria of the gut microbiota. It undergoes conjugation with a sulfate group through the action of sulfotransferases in the submucosal tissue of the small intestine, and it is present in the plasma in the form of p-cresyl sulfate.11,60,61 p-Cresol is known to exert various deleterious effects, including endothelial dysfunction and the suppression of respiratory burst action in the blood, and it contributes to the progression of CKD. p-Cresyl sulfate has also been found to be associated with the occurrence of CVD and elevated mortality in CKD patients.14,62-64 Watanabe and colleagues16 showed that p-cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by enhancing NADPH oxidase activity. However, the mechanism by which p-cresyl sulfate induces its toxicity has not been thoroughly clarified. Furthermore, researchers recently showed that p-cresyl sulfate interferes with intracellular insulin signaling pathways and triggers insulin resistance in mice and cultured muscle cells.65 In addition, this protein-bound uremic toxin induces adipocyte dysfunction, causing a redistribution of ectopic lipids and lipotoxicity. In skeletal muscle,
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p-cresyl sulfate interferes directly with insulin signaling via the activation of extracellular signal-regulated kinase-1/2 phosphorylation and inhibitory serine phosphorylation of insulin receptor substrate-1.66 In summary, these studies show increased ROS production and NF-kB activation by protein-bound uremic toxins; however, there are no available data regarding the relationship between these microbiota toxins and Nrf2 activation. In addition, the use of pre- and probiotics should be considered in CKD patients to reduce the uremic toxins and oxidative stress. Prebiotics are nondigestible compounds that beneficially modify the composition and function of the intestinal microbiota. Probiotics are live microorganisms that contribute to the balance of the gut and, when administered in adequate amounts, confer benefits.67 Indeed, studies have shown that the use of pre- and probiotics modulates the intestinal microbiota68,69 and thus reduces the uremic toxins, such as p-cresol.70
Conclusion and Perspectives: Putative Role of Nrf2 The protein-bound uremic toxins are known to promote oxidative stress; however, the Nrf2/keap1 system, a major regulator of antioxidant response, is altered in CKD patients. Therefore, the question that arises is whether protein-bound uremic toxins could contribute to Nrf2keap1 pathway dysfunction in CKD (Fig. 2). Therefore, we urge scientists to perform experiments to demonstrate evidence to such a link because it could be pivotal to understanding the oxidative damages associated with CKD. At this moment, many questions regarding the activation of the Nrf2/Keap1 system in CKD need to be answered. More studies are needed to understand how this system and its mechanisms behave in CKD.
Practical Application This review shows an important aspect from the viewpoint of a practical application because we may improve the condition of oxidative stress, inflammation, and the risk of CVD in CKD patients by regulating the imbalance of the intestinal microbiota.
Acknowledgments
The authors thank Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nıvel Superior (CAPES) and Comite Franc¸ais d’Evaluation de la Cooperation Universitaire avec le Bresil (COFECUB).
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