Aryl Hydrocarbon Receptor Inhibition Restores Indoxyl Sulfate-Mediated Endothelial Dysfunction in Rat Aortic Rings Ⅱ

3. Discussion

Increased CYP1A1 and ROS production induced by AhR activation plays an integral role in the impairment of endothelium-dependent vasodilatation [24,25]. The results of the present study suggest that blockade of the AhR with CH223191 alleviated IS-mediated prooxidative CYP1A1 gene expression and ROS production in the aorta and preserved eNOS expression and the endothelium-dependent vasodilator response to ACh. Impairment of endothelium-dependent vasodilatation following IS exposure has previously been observed in the clinical setting in CKD patients, furthermore, lowering IS levels in the circulation with AST-120 improved FMD in CKD patients [8] and improved endothelial response in the microvasculature of end-stage renal disease patients using iontophoresis [26]. Similarly, in animal models of CKD, treatment with AST-120 restored endothelium-dependent relaxation in aortic rings [11]. Together, these findings indicate that IS is involved in the impairment of endothelial-dependent function in microvascular and conduit vessels. A potential limitation to this study is that experiments were performed in the absence of plasma proteins such as albumin, hence the concentration of free IS used for these acute studies may be higher than that observed in CKD patients, however the purpose of the current study was to investigate mechanisms of IS-induced vascular function 

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The vascular endothelium regulates vessel tone in conduit and resistance arteries via several factors with nitric oxide (NO) thought to be the main mechanism affected by uremia [8,10,11,26–28]. Previous studies have demonstrated reduced endothelial nitric oxide synthase (eNOS) and NO production in IS-stimulated endothelial cells is associated with increased ROS through NADPH oxidase activation, as well as inhibition of antioxidant systems [8,10,14,15]. Furthermore, CKD rats treated with AST-120 reported a restoration in NO and eNOS expression, and lower serum IS levels [29]. In addition, genetic deletion of AhR reduces pulse wave velocity compared to wild type mice, indicating enhanced vessel elasticity, which is accompanied by an increase in NO content and eNOS activity [30]. Results from these previous studies are in agreement with those observed here, where an increase in endothelial NOX4, nitrotyrosine and superoxide expression and a concomitant reduction in eNOS was observed following IS stimulation. Moreover these changes in the endothelium were prevented in the presence of CH223191. Previous studies have examined endothelium-independent relaxation in the aorta in the presence of IS using the NO donor sodium nitroprusside; however, no change in response was observed after even after four days incubation with 1mM IS when compared to control conditions [11], hence this relaxation was not investigated in the current study.

Activation of AhR and its downstream pathway have been reported to play a role in endothelium-dependent vascular dysfunction, hypertension, and cardiac hypertrophy inducing CYP1A1 expression and increasing ROS in the vasculature and heart [24,25]. Studies in CYP1A1 knock-out mice reported that AhR activation prevented the increase in superoxide production in these tissues and restored endothelial relaxation [25]. Moreover, AhR knockout mice, or pharmacological inhibition of the receptor with α-naphthoflavone (α-NF), also a partial agonist at the AhR, in diabetic nephropathy resulted in reduced oxidative stress [31]. In line with these observations, studies of AhR activation of human umbilical vein endothelial cells (HUVECs) have similarly demonstrated an elevation in IS-induced oxidative stress with concomitant reduction in NO, and increased CYP1A1 expression, which were inhibited by AhR inhibition, or prevented from entering the cell by inhibition of the organic anion transporter probenecid [8,15].

Increased oxidative stress can be caused by an imbalance between pro- and anti-oxidative states. On examining AhR induced Nrf2-induced activation of the ARE, we found no change in the expression of anti-oxidant enzymes. Previously, Bolati et al. have shown that increased IS down-regulates Nrf2 gene expression in HK2 cells, furthermore in a rat model of CKD with elevated serum IS, reduced Nrf2 and NQO1 protein expression were attenuated following treatment with AST-120 [32]. Our results suggest that the phase II of the genomic pathway may not yet be activated considering the acute 4 h exposure of

IS used in this study. Future evaluation of these anti-oxidant mechanisms are warranted utilizing a longer time course of IS-induced AhR activation.

Indole-3 acetic acid (IAA) is another AhR agonist and PBUT, which is progressively increased in the circulation of CKD patients and predictive of mortality and major adverse cardiovascular events, serum levels of this molecule were correlated with markers of inflammation and oxidative [33]. In addition, IAA stimulation of HUVECs resulted in increased CYP1A1 and cyclo-oxgenase-2 (COX-2) gene expression and increased COX-2 protein and ROS production. In the presence of CH223191, these changes in inflammation and oxidative stress were significantly reduced or abolished [33].

IS-induced inflammatory responses are known to result in the expression of adhesion molecules inflammatory cytokines, and chemokines [11,15,34] that contributes to the pathogenesis of vascular dysfunction. CH223191 has been reported to inhibit IS-induced monocyte chemo-attractant protein-1 (MCP-1) expression in HUVECs [15], thus attenuating vascular inflammation. In addition, siAhR-transfected HUVECs suppressed IS-enhanced vascular inflammation (mediated by TNFα), leukocyte adhesion, and E-selectin gene expression [34]. Furthermore, IS exposure (1 mM) of aortic rings isolated from healthy animals for up to four days, not only reduced endothelium-dependent relaxation, but also reduced CD31 staining and increased vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) expression in the aorta [11]. By the same token, aortae harvested from CKD mice at ten weeks showed reduced endothelium-dependent relaxation, this was further reduced when incubated with IS. These changes were accompanied by a reduction in CD31 staining and increased ICAM-1 and VCAM-1 staining. Moreover treatment of CKD animals with AST-120 improved endothelium-dependent relaxation and reduced adhesion marker expression toward control levels [11]. Our results examining the short term effect of IS with and without AhR inhibition on VCAM-1 expression is consistent with previous findings outlined above. Interestingly at 4 h following IS stimulation TNFα gene expression appeared to be on the rise, a finding that we have previously demonstrated at 18 h in human aortic endothelial cells, THP-1 cells, rat cardiac myocytes and fibroblasts, and rat renal mesangial cells [35–37]. Others have similarly reported increased TNFα expression in monocyte and macrophage cell lines [38,39]. In macrophages, this is thought to involve cross talk between AhR, nuclear factor κB (a central mediator in inflammation), and suppressor of cytokine signaling 2 [39]. However, the direct inhibitory effect of CH223191 on IS-induced TNFα gene expression in the vasculature, to our knowledge, has not previously been reported and this may be a new mechanism for future investigation into IS-induced vascular inflammation.

AhR is ubiquitously expressed [40], suggesting that its inhibition may play a role in attenuating adverse effects in many tissues associated with CKD. We have previously shown that IS induces cardiac and renal collagen synthesis in cardiac fibroblasts and renal mesangial cells as well as myocyte hypertrophy in cardiac myocytes and inflammatory responses in THP-1 cells [35]. Furthermore, in models of CKD and myocardial infarction where serum IS and cardiac and renal fibrosis is increased, AST-120 treatment was shown to reduce these effects [41–43]. Ichii et al. (2014) demonstrated chronic IS administration for eight weeks resulted in progressive kidney and vascular disease, observed by increased fibrosis, tubular atrophy, urinary albumin/creatinine ratio, as well as podocyte injury with concomitant increased in CYP1A1 expression, indicative of AhR activation [44]. In the diabetic kidney, AhR is thought to play a role in fibrosis, inflammation, and oxidative stress, increasing expression of trichrome staining, α-smooth muscle actin, fibronectin, serum prostaglandin E2 (PGE2), serum advanced glycation end products, COX-2, F4/80 macrophage infiltration, NOX activity, and the marker of oxidative damage 8-hydroxydeoxyguanosine. Increased expression of these markers was prevented in diabetic AhR knockout mice and diabetic wild type mice treated with the AhR antagonist α-NF [31]. In addition, in vitro studies by Lee et al. (2016) showed that whilst stimulation of rat and mouse mesangial cells and human kidney tubular cells with N-ε-carboxymethyl-lysine (CML), a major advanced glycation end product, increased expression of AhR, collagen IV, connective tissue growth factor,fibronectin, and PGE2, this expression was prevented in cells transfected with shRNA-AhR, or pharmacologically treated with α-NF [31]. 

It is clear AhR plays an important role in the pathology of cardiovascular and renal disease in the setting of CKD, offering a new possibility for therapeutic intervention. With results from the EPPIC trials reporting a neutral outcome with AST-120 [23], other strategies are required to mitigate the damaging effects of PBUTs in CKD. Given the abundance of data indicating the benefit of AhR inhibition, in vivo, in vitro, or ex vivo, whether genetic or pharmacological, an in vivo proof of principle study examining CH223191 in a clinically relevant model of CKD that shows efficacy would be required to progress this class of drug to the next stage. 

In conclusion, the present study demonstrates that IS-mediated impairment of endothelial-dependent vasodilatation is associated with induced oxidative stress, involving an increase in CYP1A1 expression. Reversal of these effects by inhibition of the AhR with CH223191 may provide therapeutic utility in maintaining endothelial function in the CKD population. Potentially, AhR antagonists may be used as an adjunct therapy in addition to standard-of-care treatment in CKD patients and attenuate the progression of CVD risk in this population. 

4. Materials and Methods

4.1. Vascular Reactivity 

Regarding the effect of IS in the absence and presence of the AhR antagonist, CH223191 was assessed for endothelial function. Briefly, 10–14 week old male Sprague-Dawley rats were anaesthetized with Lethbarb (Troy Laboratories, Australia) and the descending thoracic aorta harvested as described previously [36]. Aortae were cleaned and dissected into four 5 mm rings and carefully placed between 2 horizontal stainless steel supports with one end connected to a micrometer for adjusting tension. Tissues were submerged into oxygenated tissue baths containing Krebs-Henseleit buffer (119 mM NaCl, 4.7 mM KCl, 1.17 mM MgSO4.7H2O, 25 mM NaHCO3, 1.18 mM KH2PO4, 11 mM D-glucose, 0.03 mM EDTA, 2.0 mM CaCl2) [11], and randomly allocated to the following conditions: (1) Control; (2) IS (300 µM); (3) IS + CH223191 (1 µM); (4) IS + CH223191 (10 µM). Tissues were stabilized under 2 g of baseline tension and washed 5 times with Krebs buffer every 5 min. Viability and contractile responses were assessed with (80 mmol/L) and allowed to reach maximum contraction prior to a 3 × 5 min washout period with buffer. 

Tissues placed in Krebs-Henseleit buffer were preincubated with vehicle (0.1% DMSO for conditions 1 and 2) or the AhR antagonist CH223191 (1 or 10 µM for conditions 3 and 4, respectively) for 2 h followed by a 1 h incubation with IS (300 µM, conditions 2–4, the control condition received 120 µL of distilled water). Aortic rings were then preconstricted with phenylephrine (PE, 30 µM), where upon attaining maximum constriction endothelium-dependent vascular function was assessed by a dose-response relaxation curve to acetylcholine (ACh; 1 nM–30 µM). The change in tension was obtained via isometric transducers (FT03C, Grass Instruments, Quincy, MA, USA), amplified (Unicor Instruments, Box Hill, Vic, Australia) and recorded using data acquisition hardware (PowerLab 4sp, ADInstruments, Castle Hill, NSW, Australia) and Chart software (ADInstruments v5.6). The resulting relaxation with each dose of ACh was expressed as a percentage reduction of the maximum contraction obtained with phenylephrine for each condition. Animal experiments were approved by the Animal Ethics Committee of St. Vincent's Hospital, AEC 006/17. 

4.2. Gene Expression Studies 

Thoracic aortic rings from separate animals were isolated as described above and incubated with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with antibiotics (1%) in 5% CO2 at 37 ◦C. Under conditions 1–4 described above, aortic rings were incubated with CH223191 for 2 h followed by the addition of IS for 4 h. RNA was extracted using the Trizol extraction method according to manufacturer's instructions, followed by DNase treatment and reverse transcription. PCR was performed using SYBR green and sequence-specific primers for rat CYP1A1, Nrf2, NQO1, GSTA1/2, TNFα, and VCAM-1 (see Supplemental Table S1 for primer sequences), using a QuantStudio 7-Flex Real-Time PCR system (Applied Biosystems). Analysis of the relative change in CYP1A1 mRNA expression was conducted using the comparative ∆∆Ct method using the house-keeping gene 18S. 

4.3. Immunohistochemistry 

At the conclusion of the vascular reactivity experiments, aortic rings were immediately fixed in 10% neutral buffered formalin overnight, processed in an automatic tissue processor and paraffin-embedded the following day for immunohistochemistry. LV tissue sections (4 µm) from each experiment were stained for RECA-1 (BioRad #MCA970GA, 1/200 dilution), eNOS (BD Biosciences #610296, 1/200 dilution), NOX4 (Abcam #133303, 1/150 dilution), and nitrotyrosine (Merck #06-284, 1/100 dilution), and detected with diaminobenzimide as previously described [45]. Stained slides were digitally scanned using the Aperio Scanscope scanner with Scanscope Console software (v8, Aperio Technologies, Vista, CA, USA) and quantitated for positive luminal staining as a percentage of total lumen perimeter using Imagescope software (v 11.1.2.760, Aperio Technologies). 

4.4. Assessment of Reactive Oxygen Species

Reactive oxygen species (ROS) were detected in aortic tissue obtained from the functional studies. Tissue sections were stained with dihydroethidium (DHE, 2 µM) for 30 min at 37 ◦C [46]. A confocal microscope (Nikon A1R, Nikon Instruments Inc., Melville NY, USA) was used to visualize red-fluorescent staining with an emission spectrum of 610 nm at ×20 magnification. The percentage area of DHE staining for was determined using ImageJ (NIH, Bethesda, MD, USA). 

4.5. Statistical Methods 

Effects on endothelial function were compared by a two-way repeated measures' analysis of variance (ANOVA) with Tukey's post-hoc comparison to determine the effect of percentage relaxation to each ACh dose. Maximum relaxation (Rmax) and pEC50 values for ACh were obtained by non-linear regression followed by a one-way repeated measures ANOVA with Tukey post-hoc comparison. This was used to determine the statistical significance of maximum relaxation (Rmax) and pEC50 values for ACh. Similarly, gene expression, and immunohistochemical and DHE staining was compared by a one-way ANOVA with Tukey's post-hoc comparison. 

Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxins14020100/s1, Figure S1: Effect of CH223191 alone on vascular reactivity in isolated rat aortic rings; Figure S2: Effect of IS and AhR inhibition on Nrf2 and antioxidant gene expression Table S1: Nucleotide sequences of primers used for gene quantification by real time PCR. 

Author Contributions: Conceptualization, A.R.K.; methodology, C.N. and A.R.K.; validation, C.N., A.J.E., and A.R.K.; formal analysis, C.N. and A.R.K.; investigation, C.N.; resources, A.R.K. and D.J.K.; data curation, C.N. and A.R.K.; writing-original draft preparation, C.N.; writing-review and editing, C.N., A.J.E., D.J.K. and A.R.K.; visualization, C.N. and A.R.K.; supervision, A.J.E. and A.R.K.; project administration, A.J.E. and D.J.K.; funding acquisition, D.J.K. All authors have read and agreed to the published version of the manuscript. 

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