Effects of Uremic Toxins from the Gut Microbiota on Bone: A Brief Look at Chronic Kidney Disease

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1 bs_bs_banner Therapeutic Apheresis and Dialysis 2015; 19(5): doi: / Review Effects of Uremic Toxins from the Gut Microbiota on Bone: A Brief Look at Chronic Kidney Disease Ana Paula Black, 1 Ludmila FMFCardozo, 2 * and Denise Mafra 1,2 1 Graduate Program in Medical Sciences, and 2 Graduate Program in Cardiovascular Sciences, Universidade Federal Fluminense (UFF), Niterói-RJ, Brazil Abstract: Patients with chronic kidney disease (CKD) frequently have mineral and bone disorders (CKD-MBD) that are caused by several mechanisms. Recent research has suggested that uremic toxins from the gut such as p-cresyl sulfate (PCS) and indoxyl sulfate (IS) could also be involved in the development of bone disease in patients with CKD. IS and PCS are produced by microbiota in the gut, carried into the plasma bound to serum albumin, and are normally excreted into the urine. However, in patients with CKD, there is an accumulation of high levels of these uremic toxins. The exact mechanisms of action of uremic toxins in bone disease remain unclear. The purpose of this brief review is to discuss the link between uremic toxins (IS and PCS) and bone mineral disease in chronic kidney disease. Key Words: Bone mineral disease, Chronic kidney disease, Uremic toxins. Chronic kidney disease (CKD) is a worldwide public health problem with growing prevalence and adverse health effects, including cardiovascular disease and mineral and bone disorder (MBD). It causes decreased quality of life, morbidity and mortality in these patients (1 3). Patients with CKD may develop osteomalacia, adynamic bone, hyperparathyroidism, vascular calcification or a combination of these, which may be associated with an increased risk of fractures (4,5). Bone disease inhibits osteoclast differentiation and function and induces osteoblast resistance to parathyroid hormone (PTH) leading to osteodystrophy (6,7). Disorders in the metabolism of vitamin D and minerals, such as hypocalcemia, hyperphosphatemia, hyperphosphaturia, and abnormalities in the strength of bone determined by bone mass and bone quality are also linked to CKD-MBD (8,9). There are many uremic toxins such as phosphate, leptin, proinflammatory cytokines, advanced protein oxidation products and advanced glycation products Received August 2014; revised January Address correspondence and reprint requests to Mrs Ludmila FMF Cardozo, Post doctoral, Estrada Caetano Monteiro 391 Niterói-RJ, , Brazil. ludmila.cardozo@gmail.com that may affect skeletal remodeling by interfering with osteoblast and osteoclast function in CKD patients. In addition, it has recently been suggested that uremic toxins from the gut microbiota can also contribute to the development of CKD-MBD (10). Kazama et al. (5) proposed the term uremic osteoporosis to describe one of the main causes of the increase in the risk of hip fracture in patients with CKD, considering that uremic toxins induce the deterioration of bone elastic mechanical properties causing bone fragility. However, the exact mechanisms of action of uremic toxins in bone disease remain unclear (11). Uremic toxins have been defined by the European Uremic Toxin Work Group as measurable, quantifiable solutes that exert negative biological effects and which are not excreted by the failing kidneys (12). The retention of these toxins is implicated in uremic syndrome and might be a missing link that explains the persistently high mortality rates in CKD (13). Uremic toxins, such as p-cresyl sulfate (PCS) and indoxyl sulfate (IS), are prototypic protein-bound molecules that share various similarities, such as their production by intestinal microbiota, strong albumin binding capacity, which impairs its efficient removal from the bloodstream due to the difficulty 436

2 Effects of Uremic Toxins on Bone 437 in crossing the dialysis membrane, substantial renal metabolism, low dialytic clearance and their role in the development of cardiovascular disease and mortality in patients with CKD (14,15). Recent research has addressed their activities in the development of bone disease (16) and their elimination could be an option for controlling low-turnover bone disease in patients with CKD (3). There are few studies about the effects of uremic toxins from the gut microbiota on BMD in CKD patients. Due to the high prevalence of CKD-MBD and the need to understand the mechanisms of action of uremic toxins in order to propose intervention measures, this review aims to discuss the role of the uremic toxins IS and PCS in the development of mineral and bone disorders in chronic kidney disease. METABOLISM AND TRANSPORT PATHWAYS OF UREMIC TOXINS FROM THE GUT MICROBIOTA Indoxyl sulfate Indoxyl sulfate (IS) is derived from dietary protein; a part of the protein-derived tryptophan is metabolized into indole by tryptophanase in the large intestine by bacteria such as Escherichia coli. Indole is absorbed into the blood from the intestine and is metabolized into IS in the liver; approximately 90% is bound to serum albumin in the plasma (17 20). IS is normally excreted into the urine through tubular secretion and partially by glomerular filtration. Organic anion transporters (OATs) localized in the basolateral membrane of renal proximal tubular cells, such as OAT1 (proximal) and OAT3 (proximal and distal), play an important role in the transcellular transport of IS (19,21). Indeed, researchers observed gene expression of OAT3 in the tibia of rats, in the primary osteoblast cultures from mouse calvariae and in the MC3T3-E1 cell line (3,22,23). Individuals with normal renal function eliminate nearly all IS, which results in serum levels of IS close to zero. Whereas, values increase progressively in patients with renal compromise and may reach 1.0 mg/dl, causing endogenous intoxication (24). p-cresyl sulfate p-cresyl sulfate (PCS) is the sulfated conjugate of p-cresol, which occurs via the action of sulfotransferases in the submucosal tissue of the small intestine (25). It is one of the phenols that results from the catabolism of the amino acids tyrosine and phenylalanine from dietary proteins by putrefactive bacteria of the gut microbiota (26 28). These amino acids are metabolized into 4-hydroxyphenylacetic acid, which is decarboxylated to p-cresol. In the intestinal mucosa, a cytosolic sulfotransferase metabolizes p-cresol to p-cresyl sulfate, and this form is present in the circulation (29). The clearance of PCS seems to depend on intact kidney function and belongs to the group of colon-derived albumin-bound uremic toxins (30). Their serum levels are nearly 30 times higher in patients with progressive renal dysfunction than in healthy subjects. The ratio is even higher (100 or more) for the free fraction. Both OAT1 and OAT3 are involved in the renal uptake of PCS, with a greater contribution by OAT3 (27). It is known that the accumulation of high levels of PCS and IS are correlated with chronic kidney disease progression and cardiovascular mortality, and recently, they were correlated with the development of bone abnormalities in CKD (16). INVOLVEMENT OF PROTEIN-BOUND UREMIC TOXINS FROM THE GUT MICROBIOTA IN THE DEVELOPMENT OF MINERAL AND BONE DISORDERS IN CHRONIC KIDNEY DISEASE: EXPERIMENTAL AND CLINICAL STUDIES Uremic toxins such as PCS and IS may be involved in the regulation of bone-mineral metabolism and altering the chemical composition of bone; they may also account for the increased prevalence of fractures in patients with CKD (4,16,22). However, most studies are experimental and the mechanisms of uremic toxins in this bone disorder have not been elucidated. Pre-treatment of primary mouse osteoblasts with to 0.5 mmol/l of PCS for 24 h decreased PTH-induced camp production, cell viability by augmenting DNA fragmentation and decreasing cell proliferation, and inhibited PTHr mrna expression level. PCS to 0.25 to 0.5 mmol/l increased the intracellular production of reactive oxygen species (ROS). IS in higher concentrations (0.5 mmol/l) acted just as PCS, but it did not reduce camp production and PTHr mrna expression. Also, PCS damages osteoblastic cells by increasing production and activating antibodies specific for phosphorylation by different mechanisms of IS. PCSinduced oxidative stress through the activation of JNK/p38 MAPK phosphorylation in osteoblastic cells while IS activated ERK 1/ 2. The addition of the antioxidant N-acetylcysteine in osteoblast cells treated with PCS in low concentrations did not inhibit DNA fragmentation and activation of JNK or p38 MAPK phosphorylation, suggesting that factors other than ROS production may be involved Ther Apher Dial, Vol. 19, No. 5, 2015

3 438 AP Black et al. in reducing viability through activation of JNK or p38mapk. It may play a critical role in impairing bone metabolism in patients with CKD (16). Nii-Kono et al. (23) have shown that the addition of IS in primary osteoblast cultures from mouse calvariae suppressed PTH-stimulated intracellular camp production and decreased PTHr expression only higher concentrations (2 mmol/l) for 24 h. However, pretreatment for 48 h with 0.5 mmol/l of IS also decreased PTHr gene expression. IS increased production of ROS and inhibited cell proliferation in a dose-dependent manner. These results indicate that IS taken up by osteoblasts via OAT3 in these cells increases oxidative stress, which impairs osteoblast function and downregulates PTHr expression. Researchers believe that IS that accumulates in the blood due to renal dysfunction is at least one of the factors that induces skeletal resistance to PTH. In monocyte/macrophage cellular models, Mozar et al. (6) observed that IS inhibited osteoclast differentiation, bone-resorbing activity and specific antibodies against phosphorylation in a dose-dependent manner, and these effects were enhanced in the presence of inorganic phosphate. They suggest that IS not only inhibits osteoblast function, but it also has an inhibitory effect on osteoclast function, and thus could affect bone remodeling in patients with CKD. In another study, the direct effect of IS on osteoblast differentiation and apoptosis in the MC3T3-E1 cell line was evaluated. IS induced apoptosis via caspase activity and increased production of ROS. Moreover, IS inhibited the expression of mrna type 1 collagen and osteonectin and alkaline phosphatase activity. The inhibition of the OAT expression, mainly OAT3, improved cell viability and suppressed the production of ROS. These results suggest that IS may be a bone toxin being transported via OAT, which causes oxidative stress to inhibit osteoblast differentiation (3). Results of a study by Idziak et al. (31) demonstrated that p-cresol and IS are cytotoxic and induce functional incompetence of human bone marrowderived mesenchymal stem cells in vitro, without inducing cell apoptosis. The activity of cytoplasmic enzyme-lactate dehydrogenase in toxins-treated cell media, which correlates with cell membrane damage, was significantly increased, suggesting cell necrosis. Therefore, uremia impaired the viability of cells. In conclusion, renal osteodystrophy might be caused by impaired production of osteoblasts from the accumulation of uremic toxins. Few researchers have investigated the relationship between uremic toxins and CKD-MBD in animals or humans, with most studies conducted in vitro. Clinical research conducted by Goto et al. (32) examined the relationship between IS and biochemical markers of bone turnover in 47 hemodialysis patients, suggesting that IS may relate skeletal resistance to PTH in uremia. IS was also correlated negatively with alkaline phosphatase and bone-specific alkaline phosphatase, independent of intact PTH. In a cross-sectional study that enrolled 80 stable non-dialysis patients with CKD stage 3 to 5, it was observed that only IS, and not PCS, correlated independently with fibroblast growth factor 23 (FGF23) that is a regulator of mineral metabolism, with direct effects on renal inorganic phosphate reabsorption, 1,25-D3 vitamin synthesis and PTH secretion and may be an independent factor involved in the regulation of bone-mineral metabolism. However, the researchers did not explain this fact (7). Some researchers have investigated the effect of the use of AST-120, an oral charcoal adsorbent of hydrophobic uremic substances including IS in the gastrointestinal tract, in bone turnover for reduction of uremic toxin levels in patients with CKD (22,33,34). Iwasaki et al. (22) analyzed the effect of AST-120 on bone turnover in male Sprague Dawley rats with renal dysfunction and observed that administration of AST-120 inhibited the accumulation of serum IS, ameliorated bone formation and reversed the downregulation of osteoblast-related genes. AST-120 was also effective in protecting against the development of low bone turnover in renal failure, which may be a result of the prevention of uremic toxin accumulation in the blood. In 2013, Iwasaki et al. (35) examined the effects of accumulated uremic toxins on bone properties in male Sprague Dawley rats that underwent thyroparathyroidectomy and nephrectomy (TPTx- Nx) or TPTx alone. Reduced bone storage modulus and cortical bone crystallinity, increased enzymatic crosslink ratio, pentosidine : matrix ratio, mineral/ matrix ratio and carbonate substitution in TPTx-Nx rats was observed. There was no difference in bone mineral density and histomorphometric parameters between the groups. Administration of AST-120 abolished the effects of TPTx-Nx and decreased the serum IS concentration. The accumulated uremic toxins, including IS, appear to alter the chemical composition of bone, which can increase the prevalence of hip fracture among patients with CKD. These data suggest that uremic toxins may contribute to the development of bone abnormalities in CKD that seem to present a gut microbiota imbalance. In fact, uremia per se can profoundly modify the gut microbiota composition. According to Vaziri Ther Apher Dial, Vol. 19, No. 5, 2015

4 Effects of Uremic Toxins on Bone 439 FIG. 1. Possible mechanisms of action of uremic toxins from gut microbiota that may modify bone metabolism in chronic kidney disease (CKD) patients. et al. (36) nephrectomized rats showed reduction of the microbial community variability caused, at least in part, by uremia. Furthermore, CKD patients must submit to dietary restrictions that also contribute to the alteration of the microbial flora. Evidence suggests that protein absorption is impaired in uremia and the decreased ratio of available carbohydrate to nitrogen may increase the proliferation of proteolytic species and generate potentially toxic protein fermentation metabolites, such as phenols and indols, which contribute to the increase of the uremic toxins (36,37). Then, more studies are needed to elucidate the mechanisms of action of uremic toxins in bone disease progression in uremia and propose effective interventions for patients with CKD. Researchers have proposed nutritional and pharmacological interventions to minimize the serum and urinary concentrations of these uremic toxins. Small interventional studies have shown that the use of low protein diet, prebiotics, probiotics, symbiotic or the use of medications such as AST-120 can reduce the uremic toxin levels and consequently may reduce the risk of bone abnormalities in CKD patients (3,18,38). CONCLUSION Bone metabolism seems to be modified by uremic toxins from the gut such as indoxyl sulfate and p-cresyl sulfate that are accumulated in the blood due to renal failure. The mechanisms of action of uremic toxins need to be better elucidated, but some researchers have found a positive correlation in the suppression parathyroid hormone-stimulated intracellular camp production, decreased PTHr expression, and induction of oxidative stress which increases the intracellular production of reactive oxygen species (Fig. 1). However, the exact mechanisms remain largely unknown and require further study but studies have indicated that CKD patients could benefit from strategies such as low protein diet, prebiotics, probiotics, symbiotic or the use of medications to reduce the levels of uremic toxins mitigating the effects of their accumulation in the body. Conflict of interest: all authors have no conflicts of interest. REFERENCES 1. Moe S, Drueke T, Cunningham J et al. Definition, evaluation, and classification of renal osteodystrophy: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int 2006;69: Fujii H, Nakai K, Fukagawa M. Role of oxidative stress and indoxyl sulfate in progression of cardiovascular disease in chronic kidney disease. Ther Apher Dial 2011;15: Kim Y, Kwak K, Gil H, Song H, Hong S. Indoxyl sulfate promotes apoptosis in cultured osteoblast cells. BMC Pharmacol Toxicol 2013;14: Jamal SA, Hayden LA, Beyene J. Low bone mineral density and fractures in long-term hemodialysis patients: a metaanalysis. Am J Kidney Dis 2007;49: Kazama JJ, Iwasaki Y, Fukagawa M. Uremic osteoporosis. Kidney Int Suppl 2013;3: Mozar A, Louvet L, Godin C et al. Indoxyl sulphate inhibits osteoclast differentiation and function. Nephrol Dial Transplant 2012;27: Lin C, Pan C, Chuang C et al. Association of indoxyl sulfate with fibroblast growth factor 23 in patients with advanced chronic kidney disease. Am J Med Sci 2014;347: Abe M, Okada K, Soma M. Mineral metabolic abnormalities and mortality in dialysis patients. Nutrients 2013;5: Iwasaki Y, Kazama JJ, Yamato H, Fukagawa M. Changes in chemical composition of cortical bone associated with bone fragility in rat model with chronic kidney disease. Bone 2011;48: Tanaka H, Komaba H, Koizumi M, Kakuta T, Fukagawa M. Role of uremic toxins and oxidative stress in the development of chronic kidney disease mineral and bone disorder. J Ren Nutr 2012;22: Ther Apher Dial, Vol. 19, No. 5, 2015

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