Although several studies have provided evidence for the various harmful effects of IS on the body, its direct effect on bone homeostasis is not clear. To the best of our knowledge, this is the first study, in which we measured the levels of IS in in trabecular and cortical bone after chronic exposure of rats to IS. The plasma level of IS reached in this model, published in our previous study24, reflected IS levels observed previously by us and others in CKD patients29,30. This should make the possibility to reliably explore the effect of IS on the processes occurring in the bone tissue of CKD patients. Importantly, our model enables us to observe impact of IS solely, by eliminating the influence of other toxins creating uremic milieu. Using our model, we prevented interferences from other toxins, which is impossible in models of CKD that allows to observe the results of effects of many uremic retention solutes.
Previously, we showed the significantly increased plasma levels of IS in control group exposed to chronic ingestion of IS in the dose of 100 and 200 mg/kg b.w. for examination the effect of this uremic toxin on the hemostatic system and arterial thrombosis24. In the present study, we used the femurs of these rats to investigate the effect of chronic exposure to this uremic toxin on IS-mediated signaling in bone, and its significance for bone mineralization. We observed the accumulation of IS only in trabecular, more metabolically active bone tissue of these rats. Interestingly, the levels of IS in this bone region were not dependent on the dose used (Fig. 1A). Because OATs mediate IS uptake into bone cells31,32, we measured the expression of OAT-1 and -3, which are known to be transporters with high affinity to IS27 in bone of studied rats. Obtained results showed practically inhibition of both transporters in 200 IS group (Fig. 1C,D). Similarly to our results, the reduced renal mRNA and protein expression of OAT-1/3 was previously found in rat adenine-induced CKD model33,34, suggesting that rats with renal failure can be regarded as „OAT-1/3 down-regulation model”. However, the kidney function of our animals that received IS was only slightly reduced compared to controls24, so we think that the high circulating IS could be rather responsible for such significant OATs suppression in our model. This is in agreement with the previous study of Enomoto et al.27, who examined the interactions of IS with OATs, using proximal tubule cells stably expressing these transporters.
AhR is a cytoplasmic receptor of several exogenous and endogenous molecules that can control bone homeostasis. It has been shown that the exposure to dioxin, which is the most widely known exogenous AhR ligand, resulted in imbalance of bone remodeling and mechanically weaker bones35. CKD is characterized by the accumulation of a mixture of uremic toxins, and IS, which belongs to tryptophan metabolites, represents both important uremic toxin and potent AhR ligand36,37. In the present study, we measured the expression of AhR pathway genes in bone of rats after chronic exposure to IS (Fig. 2), and we analyzed the associations between their expression (Table 2). We observed the dose-dependent differences in the activation of AhR pathway. The exposure to IS in the lower dose (100 IS) strongly increased AhRR gene expression, but practically did not affect CYP1A1 mRNA level. This is in line with observation of Schanz et al.38 that cells that highly express the AhRR display only mild CYP1A1 expression. Typically, once induced AhRR competes with the AhR for dimerization with ARNT, and AhRR-ARNT complex binds XRE but does not initiate transcription of AhR-target genes39. Interestingly, we noticed the increased CYP1A2 gene expression, which was strongly AhRR-dependent. In contrast to CYP1A1, which is a readily inducible AhR target gene encoding an important xenobiotic metabolizing enzyme, CYP1A2 is usually expressed constitutively and metabolizes some endogenous substances40. Thus, the elevated mRNA level of CYP1A2 in rats exposed to 100 IS may indirectly indicate an active metabolism of IS, and therefore manifest the protective properties of CYP1A2 in this group.
The rats treated with higher dose of IS (200 IS) revealed the significant decrease in the expression of AhRR and both CYP1A genes, whereas the expression of AhR-ARNT complex tended to increase. In this group, AhR mRNA (but not AhRR) was directly associated with CYP1A2 gene expression, and AhR-ARNT-AhRR-CYP1A2 axis was positively linked with OAT-1 gene expression. These results suggest that higher dose chronically administered IS can disrupt the classical negative regulatory loop, in which AhR ligand could be rapidly degraded. As a consequence, the persistent overstimulation of AhR can be harmful for physiological processes in bone of these animals, as have been previously described by us and other authors41,42. Another interesting observation resulting from this study was that plasma and bone IS can diversely regulate CYP1A genes expression, as plasma IS levels were related to CYP1A1, whereas trabecular IS concentrations affected CYP1A2 gene expression in each of the studied group.
SIRTs are NAD + cofactor-dependent histone deacetylases (class III–HDAC), which take part in many processes regulating biological functions of cells, like cell cycle, cellular metabolism, mitochondrial function, protection against oxidative stress and inflammation43,44. SIRTs are present in many different organs including bones, but until now, little is known about the role of SIRTs in maintaining bone health11,45. We measured the expression of four SIRTs in bone of rats exposed to IS, namely SIRT-1, SIRT-2, SIRT-3 and SIRT-7. These SIRTs differ from each other in subcellular localization and function: SIRT-1 and SIRT-7 are nuclear SIRTs, and they are responsible for modulation of gene expression and DNA repair46,47, SIRT-2 is mostly found in the cytoplasm, and it plays an essential role in oxidative stress protection13, whereas SIRT-3 is located in the mitochondria and contribute to the regulation of ATP production, antioxidant defenses and energy metabolism48. However, there are data that SIRT-1, -2 and -3 can migrate between organelles43.
In 100 IS group we observed increased expression of SIRT-2, and slight but not significant elevation of the rest of SIRTs, which were related to CYP1A2 gene expression, and majority of them (except for SIRT-2) were also associated with AhRR mRNA levels. The exposure of animals to higher dose of IS resulting in diverse regulation of SIRTs expression. While SIRT-3 and SIRT-7 mRNA levels were significantly reduced in this group compared to 100 IS and controls, SIRT-1 expression remained constant, and SIRT-2 gradually increased (Fig. 3). Interestingly, the disrupted association between CYP1A2 and AhRR resulted in reduced expression of SIRT-3 and SIRT-7, whereas the direct relation between CYP1A2 and AhR augmented expression of SIRT-1, and particularly SIRT-2 gene.
In the available literature, there are no data so far on the regulation of SIRTs by IS-dependent components of the AhR pathway in bone. Nonetheless, the previous study of Szychowski et al.20 showed that exposure of mouse neurons to triclosan increased the expression of the SIRT-1 and SIRT-3 proteins in response to AhR stimulation. On the other hand, Diani-Moore et al.21 found that SIRT-1 activity in cultured hepatocytes of chicken embryos was reduced as a result of activation of the AhR receptor by dioxin. Similarly, the activation of AhR decreased SIRT-3 activity in transgenic mice with constitutively active AhR22. Koizumi et al.23 proved that IS-AhR pathway reduced NAD + content and SIRT-1 activity in human umbilical vein endothelial cells (HUVECs), inducing endothelial senescence. In our previous study, performed on these same animals, we show for the first time that chronic exposure to IS led to reduced aortic contents of SIRT-1 and SIRT-324. Thus, the results of the present and previous studies indicated the existence of interaction between AhR and SIRTs, which seem to be depend on the type, concentration and duration of exposure to the used AhR agonist, as well as the subcellular localization of the SIRTs.
Recently, the abundant evidence shows that CKD is pro-oxidative state49 and the excessive generation of ROS are considered to be major mediators of numerous physiological complications in CKD patients, including CKD-MBD development50. Among the various metabolites that accumulate in the plasma of CKD patients, IS—a typical uremic solute, has been shown as an inducer of oxidative stress, modifying the balance between pro- and antioxidant mechanisms both in vitro condition51,52,53, and in patients with CKD30. Although most literature reports emphasize the pro-oxidative nature of IS, it has been also reported that IS can balance the oxidative stress in CKD, based on its physiological concentration in serum54,55. In the presence of concentrations of less than 10 µM in HUVECs, IS showed radical scavenging ability against superoxide generation in lipopolysaccharide-stimulated neutrophils54. It has been also shown that IS serves as an endogenous antioxidant to eliminate superoxides in the blood, protecting endothelial cells from oxidative damage under physiological conditions55.
To address whether AhR induction by IS at bone level may led to generation of oxidative stress, we measured 8-OHdG, which is recognized marker of oxidative DNA damage28 in homogenates from both bone regions. As has been presented on Fig. 4, the levels of 8-OHdG was decreased in trabecular bone tissue in 100 IS group compared to 200 IS group, and tended to be lower even in comparison with controls. The association observed between 8-OHdG and CYP1A1 (but not CYP1A2) in this group confirmed that CYP1A1 activation is responsible for ROS generation56,57. In contrast, the inverse association was observed between trabecular 8-OHdG concentration and AhR-CYP1A2 axis in rats received the higher dose of IS. These results suggest that in bone of animals exposed to lower dose of IS, the negative regulatory loop between ARNT-AhRR is able to protect the cells against CYP1A1-dependent ROS generation. On the other hand, a protective effect of CYP1A2 on ROS formation has been demonstrated58. In the current study, we observed the inverse associations between 8-OHdG levels and SIRTs expression, which were particularly seen in cortical bone (Table 4), where the increase in the oxidative DNA damage marker was not observed (Fig. 4B). As has been reviewed by Singh et al.59, SIRTs constitute an integral part of cellular defense against ROS formation, and each of them has a distinct subcellular localization, which allows rapidly sensing and responding to changes in subcellular ROS within the cellular organelles, like mitochondria, nucleus, and cytoplasm. In 100 IS group, we noticed the strong relations between nuclear SIRT-1 and SIRT-7, as well as between them and mitochondrial SIRT-3, suggesting that these SIRTs may interact closely to coregulate the levels of oxidative DNA damage In 200 IS group, the strong association was only seen between SIRT-2 and SIRT-7, and these SIRTs seem to be the major contributors of protection against oxidative DNA damage in both bone regions (Table 4, right side). Based on the above results, we created working hypothesis that after exposure to low dose of IS, the protective capacity of CYP1A2-SIRTs axis might exceed CYP1A1-dependent ROS production. However, after exposure to higher dose of IS, in the conditions of continual IS-mediated AhR activation, this protective mechanism can be disrupted, leading to increased oxidative DNA damage in trabecular bone region.
The last aim of the present study was to establish, if this specific intracellular target of IS could have the physiological relevance in relation to bone health in CKD. Osteoporosis and reduced bone mineral density (BMD) are the common complications related to uremia60. BMD measurements by Dual energy X-ray absorptiometry (DXA) are currently recommended for assessment of bone status in the CKD population by Kidney Disease Improving Global Outcomes (KDIGO) guidelines, as low BMD can predict fracture risk61. In the present study, the parameters of femoral mineral status, like bone mineral area (BMA) and bone mineral content (BMC) were unchanged in all studied groups, but BMD values of rats exposed to the higher dose of IS were reduced compared to healthy animals (Fig. 5). Interestingly, there was no clear, direct association between the parameters of bone mineral status and SIRTs, as well as AhR pathway gene expression in rats receiving IS. However, the strong, negative impact of IS content in trabecular bone was noticed in relation to BMA, BMC and BMD values in 100 IS group (Fig. 6A–C). In rats exposed to IS at the dose of 200 mg/kg b.w., the level of oxidative DNA damage was inversely associated with these parameters, especially with BMD (Fig. 6D–F). These results showed that even a little content of IS in bone tissue, through activation of AhR system in situ, is able unfavorable affecting bone mineral status. Moreover, these results confirmed our hypothesis that protective effect of AhR-dependent induction of SIRTs expression after exposure to low dose of IS can effectively balance oxidative DNA damage, counteracting BMD reduction.
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