Dose-dependent exposure to indoxyl sulfate alters AHR signaling, sirtuins gene expression, oxidative DNA damage, and bone mineral status in rats

Indoxyl sulfate (IS), an agonist of aryl hydrocarbon receptors (AhR), can accumulate in patients with chronic kidney disease, but its direct effect on bone is not clear. The present study investigated the effect of chronic exposure to low (100 mg/kg b.w.; 100 IS) and high (200 mg/kg b.w.; 200 IS) dose of IS on bone AhR pathway, sirtuins (SIRTs) expression, oxidative DNA damage and bone mineral status in Wistar rats. The accumulation of IS was observed only in trabecular bone tissue in both doses. The differences were observed in the bone parameters, depending on the applied IS dose. The exposure to 100 IS increased AhR repressor (AhRR)-CYP1A2 gene expression, which was associated with SIRT-1, SIRT-3 and SIRT-7 expression. At the low dose group, the oxidative DNA damage marker was unchanged in the bone samples, and it was inversely related to the abovementioned SIRTs expression. In contrast, the exposure to 200 IS reduced the expression of AhRR, CYP1A, SIRT-3 and SIRT-7 genes compared to 100 IS. The level of oxidative DNA damage was higher in trabecular bone in 200 IS group. Femoral bone mineral density was decreased, and inverse relations were noticed between the level of trabecular oxidative DNA damage and parameters of bone mineral status. In conclusion, IS modulates AhR-depending signaling affecting SIRTs expression, oxidative DNA damage and bone mineral status in a dose dependent manner.


Preparation of bone tissue homogenates
The bone tissue was taken from the distal femoral epiphysis (trabecular bone) and femoral diaphysis (cortical bone) after 28 days of exposure to IS.The procedure of the preparation of 10% homogenates from bone tissue has been described in detail previously 25 .

Determination of IS in bone homogenates
Concentrations of free form of IS in the bone tissue homogenates were evaluated using high-performance liquid chromatography (HPLC) with fluorescence detection according to our modification 24 of the methods previously described by Al'Zhabi et al. 26 .Determination of IS was carried out at temperature of 24 °C.The chromatographic equipment was an Agilent 1200 series LC-system (Agilent Technologies, Boblingen, Germany) composed of G1322A degasser, G1311A quaternary pump, G1329A autosampler and G1330B thermostat for autosampler, HP1046A fluorescence detector (FLD).Deproteinated samples were prepared by adding 0.4 mL acetonitrile containing the methyl paraben (1 mg/mL) as internal standard into the 0.1 mL homogenate.The samples were vortexed, kept at 4 °C for 1 min, and then centrifuged for 30 min 14,000 g at 4 °C.Further, 1 µL of the supernatant was injected into high performance liquid chromatography (HPLC) system for analysis.The prepared samples were separated on column Phenomenex PEPTIDE 3.6mmXB-C18 4.6 × 250 mm (Phenomenex, Torrance, CA, USA).The column effluent was monitored by using programmable FLD.The optimized conditions were determined by recording fluorescence spectra with a stop-flow technique.Excitation and emission wavelengths were set at 280/375 nm.The output of the detector was connected to a single instrument LC ChemStation.The mobile phase was composed of acetate buffer (pH 4.5) containing 90% of acetonitrile and it was pumped at a flow-rate of 0.8 mL/min.

Oxidative status assay in bone homogenates
The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured in the femur homogenates with DNA damage ELISA kit from Enzo Life Sciences (ELS) AG, Lausen, Switzerland, according to the manufacturer's instructions.

Quantitative real-time polymerase chain reaction (qRT-PCR) assay
Total RNA was isolated from femoral bone using Thermo Scientific GeneJET RNA Purification Kit (Thermo Scientific, Vilnius, Lithuania) and a quantitative real-time polymerase chain reaction assay was performed as previously described 25 .Primers were designed using PRIMER-BLAST (http:// www.ncbi.nlm.nih.gov/ tools/ prime rblast) software.In the present study, the expression of sirtuins and AhR pathway genes were determined.The primer sequences used (50-30 forward, reverse) are included in the Table 1.All results were normalized to the endogenous reference glyceraldehyde 3-phosphate dehydrogenase (GADPH).The comparative cycle threshold method was used for relative quantification of gene expression.

Bone mineral status determination
Densitometry analysis of left femurs was performed using dual-energy x-ray absorptiometry (DXA) scans (Horizon QDR Series X-ray Bone Densitometer, Hologic Inc., Bedford, MA, USA), and analyzed with the specific small animal software.For each scanned femur the results of the bone mineral area (BMA, cm 2 ), bone mineral content (BMC, mg), and areal bone mineral density (BMD, mg/cm 2 ) were obtained.The variation coefficient of BMC and BMD measurements were less than 4%.

Statistical analysis
Shapiro-Wilk's test of normality was used for data distribution analysis.The normally distributed data were presented as mean ± SD.The non-Gaussian data were expressed as median (interquartile range; IQR).Comparison between parametric data was made by one-way analysis of variance (ANOVA), and significant differences between the groups were assessed using Tukey's post-hoc test at p < 0.05.The Kruskal-Wallis with Dunn's test was used for nonparametric data at p < 0.05.The correlations were calculated by Spearman's rank correlation analysis.p-value < 0.05 was considered statistically significant.Graphic design presentation of results was prepared using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA) or Statistica ver.10 computer software (StatSoft, Tulsa, OK, USA).

The concentration of IS in bones regions and the expression of IS transporters in bone
As shown in Fig. 1A, chronic exposure to IS in both doses resulted in increased IS levels in trabecular regions of bones compared to CON group (p < 0.001).The median concentrations of IS were 2.5 µmol/g (IQR 1.36-9.12) in 100 IS group and 2.5 µmol/g (IQR 1.71-9.56) in 200 IS group.There were no differences in IS levels in cortical regions of bones (Fig. 1B).The only downward trend was observed in 100 IS group (p = 0.063; Fig. 1B).We did not notice any correlation between plasma IS levels and bone IS concentrations.
The expression of organic anion transporters OAT-1 and OAT-3 genes, which represented the transporters with high affinity to IS 27 was non-significantly decreased at a lower dose of IS, and was strongly inhibited at a dose of 200 mg IS/kg b.w.compared to CON and 100 IS groups (p < 0.001, Fig. 1C,D).Despite such strong inhibition of OATs expression in 200 IS group, there was no direct association between plasma IS level and the expression of OAT-1 (R = − 0.354, NS) and OAT-3 (R = − 0.293, NS) genes.

The impact of IS on AhR pathway genes expression in bone
The different effects of IS on the expression of the AhR pathway genes in bone were noticed in experimental groups (Fig. 2).In 100 IS group, the mRNA level of AhRR was tenfold higher, and CYP1A2 was 1.8-fold higher compared to CON group.The administration of high doses of IS resulted in increased expression of AhR compared to CON (p = 0.029) and 100 IS (p = 0.043) groups.. While, the expression of AhR repressor (AhRR), CYP1A1 and CYP1A2-the key target genes activated by the genomic AhR pathway was significantly inhibited in 200 IS group in comparison with 100 IS group (p < 0.001, p < 0.001 and p = 0.005, respectively) .The analysis of associations existed between AhR pathway genes revealed inverse relations between CYP1A1 and the expression of OAT-3, ARNT and AhRR in the group exposed to lower IS dose.In turn, the strong positive correlation was observed between CYP1A2 and AhRR expression in this group (Table 2, left side).In the 200 IS group, a significant positive association was present between OAT-1 and analyzed genes that belong to the AhR pathway, except for CYP1A1.Moreover, all these genes were positively related each to others (Table 2, right side).
Interestingly, we noticed that plasma IS levels were positively related to bone CYP1A1 expression in CON group (R = 0.601, p = 0.017) and 200 IS group (R = 0.538, p = 0.047), whereas trabecular IS concentrations were significantly associated with CYP1A2 gene expression only in CON group (R = 0.535, p = 0.033).

The effect of chronic exposure to exogenous IS on SIRTs expression in bone
Among the studied SIRTs, only the expression of the SIRT-2 gene was significantly elevated in rats chronically exposed to both doses of IS (p < 0.001) compared to controls.In turn, SIRT-3 and SIRT-7 gene expressions were significantly lowered in 200 IS group compared to CON and 100 IS group (p < 0.001, and p < 0.01, respectively).We did not notice significant changes in SIRT-1 mRNA levels in both experimental groups (Fig. 3A-D).
In CON group, we only found inverse association between SIRT-7 gene expression and CYP1A2 mRNA levels (R = − 0.541, p = 0.030).Table 2.The associations between genes involved in AhR-dependent pathway in bone of healthy rats after chronic exposure to indoxyl sulphate.AhR-aryl hydrocarbon receptor; AhRR-aryl hydrocarbon receptor repressor; ARNT-aryl hydrocarbon receptor nuclear translocator; 100 IS-group receiving indoxyl sulphate in the dose of 100 mg/kg b.w./day; 200 IS-group receiving indoxyl sulphate in the dose of 200 mg/kg b.w./day; OAT-1-organic anion transporter 1; OAT-3-organic anion transporter 3. Bold values mean that correlation was statistically significant.Tendencies are in italic.The nonspecific DNA repair enzymes excise DNA lesions to release deoxynucleotides, which are enzymatically hydrolyzed to stable deoxynucleotides.Among numerous types of oxidative DNA damage, the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a ubiquitous marker of oxidative DNA damage 27 .As shown in Fig. 4A,  In the group of rats administered with lower dose of IS the cortical 8-OHdG level was inversely associated with SIRT-1 and SIRT-7 gene expression, whereas 8-OHdG concentration in trabecular bone region positively correlated with CYP1A1 gene expression.Moreover, this oxidative DNA damage marker was inversely related to ARNT mRNA levels in trabecular bone region (Table 4, left side).In 200 IS rats, the inverse association was found between trabecular 8-OHdG levels and AhR, CYP1A2 gene expression, and the tendency to inverse correlation existed between 8-OHdG levels and SIRT-2 (R = − 0.495, p = 0.085), SIRT-7 gene expression (R = − 0.478, p = 0.098).In cortical part of bone, we noticed inverse relations between 8-OHdG levels and ARNT as well as tendency with SIRT-2 expression (R = − 0.516, p = 0.058).Table 3.The interrelationships between bone expression of SIRTs genes and AhR pathway genes in bone of rats after chronic exposure to IS. AhR-aryl hydrocarbon receptor; AhRR-aryl hydrocarbon receptor repressor; ARNT-aryl hydrocarbon receptor nuclear translocator; 100 IS-group receiving indoxyl sulphate in the dose of 100 mg/kg b.w./day; 200 IS-group receiving indoxyl sulphate in the dose of 200 mg/kg b.w./day; OAT-1-organic anion transporter 1; SIRT-1-sirtuin 1; SIRT-2-sirtuin 2; SIRT-3-sirtuin 3; SIRT-7-sirtuin 7. Bold values mean that correlation was statistically significant.Tendencies are in italic.The analysis of relationships between bone mineral status and bone IS levels, activation of AhR pathway, SIRTs expression and bone oxidative status revealed that in the group of 100 IS the trabecular level of IS was strongly and inversely associated with BMA, BMC and BMD (Fig. 6, panels A-C).In contrary, in group of 200 IS, the parameters of bone mineral status, particularly BMC and BMD were inversely associated with trabecular oxidative status, representing by 8-OHdG levels (Fig. 6, panels D-F).There was no direct link between bone mineral status and SIRTs expression in both experimental groups, except of the weak association between SIRT-7 and bone mineral area (R = 0.490, p = 0.054) in 100 IS group.

Discussion
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 study 24 , reflected IS levels observed previously by us and others in CKD patients 29,30 .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 thrombosis 24 .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 cells 31,32 , we measured the expression of OAT-1 and -3, which are known to be transporters with high affinity to IS 27 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 model 33,34 , 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 controls 24 , 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 bones 35 .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 ligand 36,37 .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 genes 39 .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 substances 40 .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 authors 41,42 .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 inflammation 43,44 .SIRTs are present in many different organs including bones, but until now, little is known about the role of SIRTs in maintaining bone health 11,45 .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 repair 46,47 , SIRT-2 is mostly found in the cytoplasm, and it plays an essential role in oxidative stress protection 13 , whereas SIRT-3 is located in the mitochondria and contribute to the regulation of ATP production, antioxidant defenses and energy metabolism 48 .However, there are data that SIRT-1, -2 and -3 can migrate between organelles 43 .
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. 21found 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 AhR 22 .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-3 24 .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 state 49 and the excessive generation of ROS are considered to be major mediators of numerous physiological complications in CKD patients, including CKD-MBD development 50 .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 condition [51][52][53] , and in patients with CKD 30 .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 serum 54,55 .In the presence of concentrations of less than 10 µM in HUVECs, IS showed radical scavenging ability against superoxide generation in lipopolysaccharide-stimulated neutrophils 54 .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 conditions 55 .
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 damage 28 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 generation 56,57 .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 demonstrated 58 .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 uremia 60 .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 risk 61 .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.

Conclusions
Recently, some studies suggested that IS and other uremic toxins may contribute to the loss of bone quantity and quality in CKD 36 but majority of these works were conducted in bone cell culture models with high micromolar IS concentration, or in animal CKD models, so their physiological relevance remain uncertain.In contrast, our data clearly demonstrate that chronic exposure of control group to IS can modulate AhR-depending biological signaling at bone level, affecting SIRTs expression, oxidative DNA damage level and bone mineral status.The results presented here describe for the first time a specific, dose-dependent intracellular target of IS in bone, on the basis of which we propose the mechanism (Fig. 7), showing both protective, as well as detrimental nature of IS in CKD-related osteoporosis.Because the patients with CKD are continuously exposed to high IS levels 30 , it would be expected that their bone AhR should be fully and sustained activated, that might predispose these patients to bone loss and risk of fracture.

Figure 1 .
Figure 1.IS levels and the expression of IS transporters (OATs) in bone tissue of rats after chronic exposure to IS.Data are shown as the mean ± SD (C,D) or median (A,B) depending on their distribution.CON-control group; IS-indoxyl sulfate; 100 IS-group receiving indoxyl sulfate in the dose of 100 mg/kg b.w./day; 200 IS-group receiving indoxyl sulfate in the dose of 200 mg/kg b.w./day; OAT-1-organic anion transporter 1; OAT-3-organic anion transporter 3. *** p < 0.001 compared to control; ### p < 0.001 compared to 100-IS group.

Figure 2 .
Figure 2. The expression of AhR pathway genes in bone tissue of rats after chronic exposure to IS.Data are shown as the mean ± SD.AhR-aryl hydrocarbon receptor; ; AhRR-aryl hydrocarbon receptor repressor; ARNT-aryl hydrocarbon receptor nuclear translocator; CON-control group; IS-indoxyl sulfate; 100 ISgroup receiving indoxyl sulfate in the dose of 100 mg/kg b.w./day; 200 IS-group receiving indoxyl sulfate in the dose of 200 mg/kg b.w./day.* p < 0.05 compared to control; *** p < 0.001 compared to control; # p < 0.05 compared to 100-IS group; ## p < 0.01 compared to 100 IS group; ### p < 0.001 compared to 100 IS group.
free ROS are produced as a result of normal metabolism, but their overproduction can impair DNA.
https://doi.org/10.1038/s41598-024-53164-3www.nature.com/scientificreports/The effect of chronic exposure to exogenous IS on bone mineral status As shown in Fig. 5, there were no statistically significant differences between bone mineral area (BMA) [panel A] and bone mineral content (BMC) [panel B] between the all analyzed group.The values of bone mineral density (BMD) in 200 IS group were decreased compared to controls (p = 0.047) [panel C].

Figure 5 .
Figure 5.The bone mineral status of rats after chronic exposure to IS.Data are shown as the mean ± SD.BMA-bone mineral area; BMC-bone mineral content; BMD-bone mineral density; CON-control group; 100 IS-group receiving indoxyl sulfate in the dose of 100 mg/kg b.w./day; 200 IS-group receiving indoxyl sulfate in the dose of 200 mg/kg b.w./day; * p < 0.05 compared to control.

Figure 6 .
Figure 6.The effect of bone IS and bone oxidative status on mineral parameters of femurs in rats exposed to lower (A-C) and higher (D-F) dose of exogenous IS. 8-OHdG-8-hydroxy-2'-deoxyguanosine; BMA-bone mineral area; BMC-bone mineral content; BMD-bone mineral density; IS-indoxyl sulfate.

Figure 7 .
Figure 7.The proposed mechanism of both protective (left side) as well as detrimental (right side) action of IS in CKD-related osteoporosis.AhR-aryl hydrocarbon receptor; AhRR-aryl hydrocarbon receptor repressor; ARNT-aryl hydrocarbon receptor nuclear translocator; BMD-bone mineral density; SIRT-1-sirtuin 1; SIRT-2-sirtuin 2; SIRT-3-sirtuin 3; SIRT-7-sirtuin 7. Legend: The low dose of IS, trough the activation of AhR/AhRR/CYP1A2 axis and SIRTs expression, can effectively balance the oxidative DNA damage in bone tissue, maintaining proper bone mineral status.At the high dose of IS this protective mechanism is interrupted, resulting in increase of bone oxidative DNA damage that leads to reduced BMD.