Unraveling the influence of Indoxyl Sulfate and apixaban in drug metabolism and elimination: Is sex a major player?

doi:10.21203/rs.3.rs-4945804/v1

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Unraveling the influence of Indoxyl Sulfate and apixaban in drug metabolism and elimination: Is sex a major player?

https://doi.org/10.21203/rs.3.rs-4945804/v1

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Chronic Kidney Disease (CKD) is associated with heightened risk of thrombosis. Prescription of anticoagulants is key to manage it; however, CKD patients have shown an increased risk of bleeding under anticoagulation therapy compared to non-CKD patients. We hypothesized that the accumulation of uremic toxins, particularly of indoxyl sulfate (IS) could modify drug metabolism. Our intoxication model shows that higher doses of IS and apixaban accumulate in the plasma of female mice as a result of expression differences in efflux transporters and cytochromes in the liver, ileum and kidneys, when compared to males. Furthermore, we found that accumulation of apixaban in females contributes to increased bleeding. q-RT PCR analysis of liver samples revealed elevated Sult1a1 but reduced Abcg2 and Cyp3a11 in female mice, while in the kidneys the expression rates of Oat1 and Oat3 were respectively lower and higher than those observed in males, potentially affecting drug clearance. Whole proteomics liver analysis confirmed the previous mRNA results at the protein level and revealed that sex had a major influence in regulating both coagulation and drug metabolism pathways. Thus, our findings underline the need for inclusive clinical and preclinical trials to accurately reflect sex-specific metabolic variations, and to consider CKD-specific changes to optimize dosing, minimize side effects, and improve patient outcomes.

Health sciences/Nephrology

Health sciences/Diseases/Kidney diseases/Chronic kidney disease

Chronic kidney disease (CKD) can be defined as structural or functional abnormalities of the kidneys, regardless of presenting a decreased glomerular filtration rate (GFR), or as a GFR < 60 mL/min/1.73m² for ≥ 3 months irrespective of visible kidney damage [1]. CKD is a widespread condition with a prevalence between 9,1–13,4% [2]. Sex has been shown to affect both prevalence and disease progression: while women are more prone to develop CKD, men are more likely to reach end-stage kidney failure [3] and face a higher risk of death compared to women with CKD [4]. CKD is associated with several comorbidities such as hypertension, diabetes and most importantly cardiovascular disease (CVD) [5] - the main cause of death in developed countries [6]. Therefore, CKD should be viewed as a whole-body affliction and considered from a global perspective. Traditional cardiovascular risk factors are insufficient to explain the elevated rates of CVD, rising uremic toxins as a potential link between CKD and CVD outcomes. Uremic toxins are compounds excreted by the kidneys under physiological conditions. Nevertheless, they accumulate in the blood and tissues of patients with CKD due to inadequate renal clearance [7].

Haemostasis is dysregulated in patients with CKD. Indeed, an increased risk of thrombosis in the arterial and venous bed coexists with an enlarged bleeding risk, further exacerbated in more advanced stages of CKD [8] [9]. All the phases of the hemostasis cascade: primary hemostasis, coagulation and fibrinolysis, are impaired during CKD [10]. Although the prime cause of this imbalance is yet to be discerned, it is plausible that uremic toxins play a significant role in its development. Two major cardiovascular disorders, atrial fibrillation (AF) and venous thromboembolism (VTE), are associated with thrombotic events and require preventive and/or curative anticoagulation. Both conditions exhibit higher frequencies in CKD patients [11], [12]. Hence, correct coagulation management is vital to avoid stroke on AF or pulmonary embolism on VTE and haemorrhage in both conditions. Different anticoagulants have been used in these patients over the years. Warfarin, a vitamin K antagonist (VKA), is one of the most widely prescribed despite showing enhanced bleeding risk at later CKD stages, calciphylaxis and high discontinuation rates [13], [14]. Lately, apixaban, an oral direct factor Xa inhibitor, has gained increasing popularity as the preferred anticoagulation treatment for CKD patients, in both early and later stages [15]. This preference stems from two primary reasons: kidney clearance amounts only up to 27% [16] while having similar efficacy and a superior safety profile than warfarin [17]. Furthermore, amongst CKD patients, apixaban shows a reduction in bleeding risk when compared to VKA treatment. Nonetheless, bleeding risk is increased with the use of apixaban in patients with CKD in comparison with non-CKD patients [18]. Concerning its pharmacokinetics, apixaban is mainly metabolized by cytochrome 450 (CYP) 3A4 (CYP3A11 in mice) and to a lesser extent by CYP1A2 and excreted by efflux transporters such as P-glycoprotein (Pg-p) and breast cancer resistance protein (BCRP) (Fig. 1S) [16].

CKD is associated with numerous alterations in drug pharmacodynamics and pharmacokinetics [19]. Among various mechanisms, activation of Aryl hydrocarbon receptor (AhR) by uremic toxins is proposed as an essential process to understand how CKD impairs drug metabolism [20]. AhR is a ligand-inducible transcription factor that regulates both nutrient metabolism and xeno- and endobiotic detoxification by inducing its target genes, amongst them CYP1A1, CYP1A2 and CYP1B1 (Fig. 1S) [21].

Amidst the AhR agonists’ we find Indoxyl sulfate (IS), a protein-bound, tryptophan-derived uremic toxin [22]. Tryptophan is an essential amino acid, commonly found in human diet, present in poultry, nuts, eggs and other daily consumed products. Tryptophanase-expressing bacteria metabolize tryptophan into indole, an intermediate metabolite which is absorbed in the gut and further processed in the liver by CYP2E1 transforming indole into indoxyl [23]. Indoxyl is then sulfonated by the Sulfotransferase 1A1 (SULT1A1) to produce IS [24]. Indole is a highly toxic compound [25] so the production of IS could be a mechanism to reduce its toxicity. In healthy individuals, IS is taken up by organic anion transporters (OAT) 1 and 3 (OAT1 and OAT3) in the proximal tubular cells and excreted through the urine (Fig. 1S) [26]. However, as CKD progresses, the impeded kidney function leads to tissue and plasma accumulation of IS, provoking deleterious effects [27].

In excess, IS is considered a pro-inflammatory, pro-oxidative and pro-coagulant molecule, which is also related to endothelial dysfunction [28]. Plasma levels of IS positively correlate with circulating tissue factor levels, whose concentration and activity increase as CKD advances. Furthermore, indolic uremic solutes increase tissue factor production in endothelial and peripheral blood mononuclear cells via AhR activation, stimulating the activation of the coagulation cascade [29]. When IS accumulates in the liver, it can activate the expression of efflux transporter: Pg-p, in hepatocytes also through AhR activation [30]. Pg-p is responsible for the efflux of neutral and cationic drugs many of which are substrates of CYP3A4 like apixaban [31]. An alteration in its expression and activity could therefore modify drug metabolism and pharmacokinetics [32].

Our goal is to test the hypothesis that accumulation of indoxyl sulfate can exert a modulatory effect on the expression rate of several metabolic enzymes and efflux transporters thereby altering apixaban metabolization and bleeding risk, and to observe possible sex-related differences.

Mice trial

C57BL/6J wild-type (WT) mice and Ahr^−/− mice (B6.129-Ahrtm1Bra/J) [33] were purchased from Jackson Laboratories and maintained as a breeding colony in the animal care facility at the Faculty of Pharmacy of Aix-Marseille Université. Genotypes were confirmed by polymerase chain reaction analysis of DNA from ear clippings obtained at 4 weeks of age (KAPA Mouse Genotyping Kit, KapaBiosystems). Mice were fed ad libitum with a regular diet (A04 standard, SAFE) during the whole trial. Both male and female mice were used in this study and comparisons were made between age-matched groups.

Six groups of mice per sex were created: Ahr ^−/−KCl, Ahr ^−/− IS, WT KCl, WT IS, WT KCl + Apixaban and WT IS + Apixaban.

The animal experiments were performed in compliance with the Directive 2010/63/EU of the European Parliament and were approved by the local Ethics Committee (“Comité d’Ethique en Expérimentation Animale de Marseille”, C2EA-14; ethics approval number: APAFIS#29595-2021020514483381 v4; approval date:04/16/2021).

All the animal experiments follow ARRIVE guidelines 2.0 [34].

Indoxyl Sulfate (IS) administration

At 10 weeks of age, mice drinking water was substituted by a 5% sucrose water solution with either 0.1% IS (Sigma-Aldrich, France) or KCl (Sigma-Aldrich, France) at the equivalent concentration as control treatment since IS is commercialised as a potassium salt. This solution was administered for 1-week prior to sacrifice.

Apixaban administration

To study the impact of IS on Apixaban metabolism, mice that were following the aforementioned procedure received an Apixaban (Eliquis®, Bristol-Meyers Squibb/Pzizer EEIG) solution (0.16 mg/mL). A 5 mg pill was solubilised in 0.6 mL of DMSO (SIGMA® Life Science) and diluted to the 50th in filtered tap water. Gavage was performed twice a day (morning-afternoon) with a volume of 8 µL per g of weight, starting 48 hours before sacrifice. The last dose was given 4 hours prior the sacrifice.

Tail Bleeding Time, blood and tissue collection

At 11 weeks, mice were anesthetized (ketamine 125 mg/kg, xylazine 12.5 mg/kg, atropine 0.25 mg/kg; intraperitoneally (i.p.) and placed on a hot plate maintained at 37°C. A fragment 5 mm away from the tip of the tail was excised with a razor blade. The tail was immediately immersed in a saline solution at 37°C. The time to establish cessation of bleeding (no rebleeding within 30 seconds) was visually determined. If necessary, bleeding was stopped at 5 minutes, mice that bled beyond this endpoint were counted as 5 minutes. Immediately after, blood samples (500 µL) were obtained from blood drawn via cardiac puncture. 60 µL of citrate (0,109 mM, BD Vacutainer®, Becton, Dickinson and Company) were added to the blood extracts to avoid coagulation. Mice were sacrificed by cervical dislocation under general anesthesia, before liver, ileum, and kidney samples were extracted. Organs were bathed in PBS to reduce blood contamination and frozen either in an RNAprotect Tissue Reagent (Qiagen) and kept at -20°C for RNA analysis or Snap frozen in liquid Nitrogen for protein conservation, then kept at -80°C

Obtention of platelet-poor plasma (PPP)

Whole blood was centrifuged at 2500 G for 15 minutes and then at 10000 G for 10 minutes to obtain platelet-poor plasma. Aliquots of PPP were stored at -80°C.

Indoxyl Sulfate plasma concentrations

IS was measured in PFP plasma by high-performance liquid chromatography (HPLC) using a reversed-phase column, an ion-pairing mobile phase, and an isocratic flow, as previously described [35].

Apixaban plasma concentrations

Analyses evaluating apixaban levels were performed in PFP diluted ½ in a pool of normal human plasma and quantified by LC-MS [36].

Organ dissection, RNA Extraction and RT-qPCR Analysis of mRNA Expression

Mice kidneys, liver and ileum that were kept in the RNAprotect Tissue Reagent (Qiagen), were thawed on ice, immersed in QIAzol Lysis Reagent (Qiagen) and ground with TissueRuptor system (Qiagen). RNA was purified by chloroform extraction and isopropanol precipitation. Reverse transcription (RT) was performed on 500ng of total RNA using the PrimeScript™ RT Reagent Kit (Takara®), followed by quantitative polymerase chain reaction (qPCR) on 25 ng of cDNA using the Taqman® Universal Master Mix II, no UNG (Applied biosystems™ by Thermo Fisher Scientific) reaction mixture. Taqman® gene expression assays (Applied biosystems™ by Thermo Fisher Scientific) were used as probes to quantify the mRNA expression of the following genes: Abcb1a, Abcb1b, Abcg2, Cyp1a1, Cyp1a2, Cyp3a11, Cyp1b1, Cyp2e1, Sult1a1, Scl22a6 and Scl22a8 (Table 1SM, Supplementary methods).

All PCR reactions were performed using the Applied Biosystems™ StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). The data was acquired by StepOne™software v2.3 (©2012 Life Technologies Corporation) and Relative Quantification (RQ) Application Module on Thermo Fisher Connect Platform (Thermo Fisher Scientific).

Target gene mRNA expression levels were normalized based on the Gusb expression (Reference gene) of each sample using equation shown below [37]. Normalized mRNA levels were determined by dividing the N target value, by the N target value of the smallest quantifiable amount of target gene mRNA (target gene Ct value = 35), where ∆Ct is the Ct value of the target gene minus the Ct value of the Gusb gene.

$$\:ARNm\:expression\:levels\:normalised=\frac{N\:target}{N\:target\:smallest}=\frac{{2}^{-\varDelta\:CT\:(gene-Gusb)}}{{2}^{-\varDelta\:CT\:(35-Gusb)}}$$

Protein Extraction and Whole proteome analysis of liver samples

Approximately 10 mg of tissue was combined with 150 µL 1x SDT buffer. Samples were disrupted by glass beads in the sonicator Bioruptor® Pico (Diagenode). 50 µg of protein were loaded onto the FASP (Filter assisted sample preparation) membrane, 30 kDa cut-off filters were used. Protein reduction (DTT) and alkylation (iodoacetamide) were performed and samples were incubated with trypsin for 18h at 37°C. The peptide mixture was cleaned-up using ethylacetate extraction to remove potential residual SDS prior to LC-MS analyses. LC-MS/MS analyses were done using nanoElute (Bruker) online connected to timsTOF Pro system (Bruker): 90 min long linear LC gradient. MS and MS/MS spectra were recorded in a time of flight (TOF) analyzer using data-independent acquisition (DIA) mode utilizing trapped ion mobility (TIMS) in the precursor m/z range of 400–1000. All peptide mixtures were analyzed separately.

DIA LC-MS data processing was done using DIA-NN application (version 1.8): https://github.com/vdemichev/DiaNN). Library free search mode was used using the following protein databases:

cRAP contaminant database (version 170518)
- based on http://www.thegpm.org/crap/
- 111 protein sequences in total
UniProtKB_Mouse (version from 16.6.2021)
taxonomy: Mus musculus
taxon ID: 10090
download link: ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/reference_proteomes/Eukaryota/UP000000589/UP000000589_10090.fasta.gz
22,001 protein sequences in total

Match between runs (MBR) used across the whole dataset. Database search results were set to follow these false discovery rate (FDR) thresholds, precursor level: 1% FDR and protein group level: 1% FDR. The default protein inference algorithm implemented in DIA-NN was used to construct the protein groups list utilizing proteotypic (i.e. peptides unique for the given protein within the whole protein database) and non-proteotypic peptides.

LC-MS data evaluation: More than 6000 proteins were identified across all 60 samples. Proteins with no proteotypic peptides, no UniProtKB ID to gene symbol match (Omnipath[38]), or with more than 90% of missing values across samples were discarded. Samples: F WT IS 4, F WT IS 6 and the most prominent M KO IS 6, were considered outliers and not considered in the posterior analysis due to a higher number of missing values across features compared to the other samples, and abnormal PCA separation. Variance stabilizing normalization (Vsn) [39] was used to normalize the filtered dataset, followed by row-mean imputation of missing values. Moderated t-test using LIMMA package in R [40] was used to perform differential expression analysis, P values adjustment on multiple hypothesis testing was done using Benjamini & Hochberg method. Comparisons are mentioned as subtractions of two compared sample types as it was done on log2-transformed data. The comparisons performed were the following: M WT KCl – F WT KCl, M WT IS - F WT IS, M KO KCl - F KO KCl, and M KO IS - F KO IS. Volcano plots for the selected comparisons were created to determine differentially expressed proteins (FDR: 0.5 and FC: ±1.5). Pathway enrichment of differentially expressed proteins with cutoff values of Uncorrected P value < 0.05 and logFC > 0.5 or logFC < -0.5, was performed using univariate linear models (ulm) in DecoupR [41] and the MsigDB hallmark database for Mus Musculus [42].

Statistical Analysis

Data was presented as scatter plots (median +/- interquartile range). Statistical analyses were performed using GraphPad Prism version 9.1.2. (GraphPad Software Inc, San Diego, CA, USA). Statistical tests for non-parametric data were chosen since the number of mice per group was insufficient to assume a Gaussian distribution. Mann–Whitney U test was used for comparisons between two groups, P values ≤ 0.05 were considered significant. For comparisons between more than two groups, Kruskal–Wallis test followed by a Dunn’s test for comparisons between more than two groups was used. Spearman correlation coefficient (r) was used to measure linear correlation between two variables.

IS and apixaban accumulate in WT female mice

Apixaban accumulation in female mice is related to sex-dependent differences in mRNA expression of efflux transporters and metabolic enzymes

To further investigate the possible cause of apixaban accumulation in female plasma, we analyzed the sex-dependent expression of efflux transporters Abcb1a, Abcb1b and Abcg2 that mediate the excretion of apixaban as well as that of Cyp1a2 and Cyp3a11, which are the main enzymes that metabolize it (especially the latter). We grouped animals treated with control or IS to analyze the effect of sex on the expression of genes involved in drug metabolism (Ungrouped data is displayed in Fig. 2S). The first barrier that apixaban faces before entering the bloodstream is the intestinal epithelium of the ileum: Higher expression of Abcb1a, Abcb1b and Abcg2 was observed in males compared to females (Fig. 2). We hypothesized that this would potentially limit apixaban absorption in males and could be the first step to explaining the lower levels of apixaban observed in males compared to females. Due to apixaban being mainly metabolized in the liver, higher Cyp3a11 mRNA expression in males (Fig. 2) could help reduce circulating apixaban levels. Furthermore, higher Abcg2 levels in males would promote apixaban excretion into the bile, contributing to reducing its accumulation. Abcb1a showed higher expression rates in females, but due to overall lower expression in the liver, this is expected to minimally affect apixaban clearance.

Although only 27% of Apixaban is metabolized by the kidneys[16], it should not be overlooked. As with the precedent organs, Abcg2 expression in the kidneys was higher in males (Fig. 2), contributing to its elimination. Concerning, Abcb1a and Abcb1b, despite being higher expressed in females, their contribution to total kidney clearance is minor (lower expression rates).

These results suggest that higher accumulation of apixaban in females could be due to a sex-dependent expression of genes involved in apixaban metabolization: Cyp3a11 and excretion: Abcg2, both less expressed in females.

Apixaban and IS accumulation are correlated in male mice but not in females

Despite lower accumulation in plasma, apixaban and IS showed a positive correlation in males (Fig. 3A), while in females no association was found (Fig. 3C). IS seemed to have a modulatory effect on liver Cyp3a11 expression in the presence of apixaban in males (Fig. 3B). Indeed, while apixaban promotes its own metabolization by increasing Cyp3a11 mRNA levels in control (KCl) males, in combination with IS, impeded its own metabolization by lowering Cyp3a11, suggesting a crosstalk between these two molecules. In females (Fig. 3D), Cyp3a11 remained unchanged regardless of the treatment. This alteration in Cyp3a11 expression could explain the positive correlation observed between these two molecules in male mice.

Organic anion transporters (OATs) in the kidneys have major mRNA expression differences regarding sex that could contribute to IS accumulation in female mice

Organic anionic transporters Scl22a6 (OAT1) and Scl22a8 (OAT3) are the main efflux transporters that regulate the passage of IS from the circulating blood to the proximal renal tubule towards excretion. Scl22a6 mRNA expression was more abundant in males whereas Scl22a8 was scarcer when compared to female levels, which could impact IS renal clearance. Sex dimorphic expression of these transporters remains unchanged independently of treatment or genotype and could explain IS accumulation in female plasma (Fig. 4, Fig. 3S).

Sexual dimorphism in mRNA expression of efflux transporters and genes related to IS production influences IS accumulation

To understand the differences in the accumulation of IS between male and female mice, we studied the expression of the genes implicated in its excretion and production. No sex-related differences in mRNA expression were found at the ileum level (Fig. 5A). In the liver, the site of transformation of indole to indoxyl sulfate, we observed higher Sult1a1 in females (Fig. 5B), which could facilitate endogenous IS production. Concerning efflux transporters in the kidney, changes in Abcb1a and Abcb1b were less relevant, due to Abcg2 showing higher expression rates overall (Fig. 5C). Abcg2 mRNA remained either higher in males (liver), where higher quantities of IS could be excreted via this efflux transporter to the bile, or unchanged (kidney) compared to females (Fig. 5B and 5C). Results regarding Control (KCl) vs. IS-treated mice and sex-dependent differences regarding non-pooled mice, that justify the pooling realized can be found in Figs. 2S, 4S and 5S.

IS effect in mRNA expression of genes modulated by activation AhR in the liver

To further understand, the influence of IS in liver metabolism, we analyzed several cytochromes and efflux transporters related to IS production or with genes whose expression is modulated by AhR. In this case, males and females were pooled when no sex-related differences were observed to provide a better insight into treatment effects, non-pooled data can be found in Fig. 5S.

As shown in Fig. 6A, IS-treated AhR^−/− mice were the only experimental group with similar IS accumulation levels in males and females. This could be because Abcg2 levels in AhR^−/− male mice, although still higher than female levels, were highly diminished, which could influence IS accumulation in AhR^−/− males (Fig. 6B). Foreseeably, Cyp1a1 and Cyp1a2 were greatly diminished in the AhR^−/− mice validating the correct functionality of the model (Fig. 6C). Concerning genes related to IS production, Cyp2e1 showed lower mRNA expression in the AhR^−/− mice and Sult1a1 was increased under IS treatment in all females which could lead to induced IS production, an effect that we don’t retrieve in males treated under the same conditions.

Whole proteome analysis revealed that the main agent driving sample separation is sex

It has been extensively described in the literature that the liver shows high sexual dimorphism. Not surprisingly, from a total of 60 liver samples analyzed, constituted by 4 experimental groups per sex (WT-KCl, WT-IS, AhR^−/− KCl, AhR^−/− IS), sample separation is mainly driven by mice sex, as showed on the principal component analysis (Fig. 7A). Therefore, regardless of treatment or genotype, sex is still the most powerful differentiating agent.

Differentially expressed proteins amongst sexes lead to remarkable differences in coagulation and xenobiotic metabolization pathways

Differences among all the sex groups compared are shown in Fig. 7B-F. Most interestingly, pathway enrichment analysis reveals that at the liver level, pathways such as coagulation and bile acid metabolism were downregulated while xenobiotic metabolism and fatty acid metabolism were upregulated in females with regards to males (Fig. 7G). Corroborating the need to study both sexes in preclinical models involving any of the aforementioned pathways. Proteins contributing to each pathway can be found in Table 1S.

Most of the sex-dependent differences that we have previously mentioned at the mRNA level, have been confirmed by whole proteome analysis. Important differences at the Abcg2, upregulated in males, and Sult1a1, upregulated in females, remained significant amongst all the comparisons, and could reinforce the idea that the sexual dimorphism that governs its expression could influence increased accumulation in the bloodstream of different compounds, in this case, apixaban and IS (Fig. 8).

STUDY LIMITATIONS

Both our apixaban and IS treated models, are overdose models that allow us to assess changes in healthy mice. For reference, an average mice of 25g was receiving an apixaban dose of 32 µg per gavage, which equals to giving a 60 Kg human being a dose of 76.8 mg per serving (between 15 and 30 times the real dose used in patients) [43]. Nonetheless, those are the doses widely used for pre-clinical models [44] [45].

Pre-clinical trials tend to be executed in males only alleging that high variability due to the estrous cyclicity (which has been showed to be not relevant for most of the cases [46]) would mean to increase sample size and costs, leading to lack of female data. This study has stated the importance of sex segregation in order to get a closer look at the real drug metabolism panorama. Furthermore, we have identified a significant impact of sex in the expression of metabolism enzymes and efflux transporters implicated in the metabolization of IS and apixaban in the ileum, liver and kidneys of both treated and control mice, as basal expression levels already differ in a sex-dependent manner.

Our results in a pre-clinical model highlight the need to document the impact of sex in clinical trials with well-balanced sex ratios more precisely, particularly in the context of cardio-kidney metabolic syndrome and CKD where women are still underrepresented [47] [48]. For instance, in the DAPA-CKD trial, females comprised only 33% of the cohort [49]. A recent review study concerning sodium glucose cotransporter-2 inhibitors, a drug that reduces the risk of cardiovascular events and/or chronic kidney disease progression in CKD patients, [50] have showed a potential bias linked to sex in efficacy and adverse side effects. When more women were enrolled in the trials the reduction in cardiovascular mortality was smaller and drug-related complications were detected in a higher proportion in women. In the ARISTOTLE trial, females represented only 35% of the clinical trial population, limiting the power to observe specific outcomes related to the drug [51] and in another prospective study it was observed that during heart failure treatment, the optimal dose of RAS inhibitors to prevent the latter outcome is not the full dose but the half dose in women compared to men [52].

Uremic toxin buildup during CKD could exert an influence not only in the elimination of drugs excreted by the kidneys but also on the metabolism of drugs subjected to non-renal clearance, mainly the liver and the gut [53]. How modifications in these complex processes translate to patient pathology is poorly understood due to the large number of enzymes, transporters and metabolites implicated. Nonetheless, their understanding is essential as CKD patients are often prescribed several medications simultaneously. This poly-pharmacology results in frequently occurring drug-related adverse events, that could, in many cases be avoided if physicians were aware of the changes in drug disposition. [54]. Therefore, future clinical trials should also consider how pre-existent conditions, in this case CKD patients: uremic toxin accumulation, could alter the metabolism of a given drug and whether the dose should be sex-adjusted to minimize side effects and treatment rejection.

A correct management of haemostasis is essential to avoid bleeding and thrombosis in patients with CKD. Excess IS due to poor renal clearance can tip the scale towards a pro-coagulant state and over-activate AhR target genes which could interfere with xeno- and endobiotic metabolism. Understanding how IS interacts with commonly used anticoagulants, such as apixaban, and how it might affect its metabolization is required to better deal with haemostasis disbalance in patients with CKD.

To date, no human trials have revealed a link between IS accumulation and gender. Concerning apixaban, it is unclear whether its levels differ with gender in humans or not, and the implications it might have [16], [55]. According to the review work of Byon et al [16], despite apixaban accumulation in female subjects being up to 18% higher, no clinically relevant effects were observed. This could be due to the adherence not being closely monitored in some of the studies included and to a poorer collection of bleeding events. In our study, female mice showed a higher accumulation of IS (except from the AhR -/- mice treated with IS) and apixaban in plasma. Uneven efflux transporters expression, especially in the liver, could be behind this phenomenon. U.S Food and Drug Administration (FDA) recommends avoiding strong inhibitors and inducers of CYP3A and P-gp during treatment with apixaban (ELIQUIS (apixaban) tablets for oral use Initial U.S. Approval: 2012), nonetheless no dose adjustment is considered for patients at a higher apixaban accumulation/metabolization risks. Yet, several studies show that inhibition or genetic variation in the drug transporter gene ABCG2 affects the pharmacokinetics of apixaban[56] [57], which in some cases leads to increased bleeding risk [58].

The gut, a major drug absorption site, can be affected by sex-dependent differences in efflux transporter expression and metabolism disturbances caused by exogenous molecules like apixaban or IS, as seen in our study. When treating patients on multiple medications, addressing these factors is crucial to prevent treatment failure.

In the liver, females showed higher Abcb1a levels than males, however, this difference could not compensate for the fact that Abcg2 showed greater expression rates in all males, which is over 10 times greater than Abcb1a, and which would contribute to the excretion of apixaban and IS in the bile quicker. Furthermore, apixaban-treated males showed higher Cyp3a11 than females, which implies that apixaban metabolization rates could be enhanced. Moreover, apixaban induced Cyp3a11 expression in male liver, which would lead to even higher metabolization rates that could explain the lower accumulation, but could also lead to treatment ineffectiveness. Nonetheless, both of these effects disappear when apixaban is given in combination with IS, showing how these two compounds interact with each other metabolism.

In the liver of Ahr ^−/− mice, IS decreases Abcg2 expression (main efflux transporter) in both males and females. We hypothesize that when a drug inhibits AhR at the hepatic level, if IS concentration is high enough, a lower clearing rate could occur through the bile duct of such drug or others that are cleared through the affected transporters. Cyp1a2 is lowered under basal levels when AhR is knocked out, occasioning lower metabolization rates of other substances that are broken down by this cytochrome.

The whole liver proteomic analysis performed supported our previous findings at the mRNA level, and remarked the need to study both sexes in pre-clinical models.

On a different note, organic anion transporters OAT1 and OAT3, that import IS and apixaban from the bloodstream onto the kidney, show sex-dependent differences, a fact widely described in the literature both at the mRNA and the protein level, that seems to be due to testosterone production [59] [60]. If Scl22a6 (OAT1) and Scl22a8 (OAT3) were to have dissimilar transportation rates for apixaban and/or IS, they could play a very significant role in the plasma accumulation of the aforementioned substances in females. In humans, OAT1 and OAT3 are the second and first transporters of the Scl22a family with the highest mRNA expression, and this would be the equivalent to our female mice situation. hOAT1 mRNA was significantly lower in the kidney of patients with renal disease compared to normal controls [61], which could be one of the factors that could explain increasing IS accumulation as CKD progresses. Furthermore, several studies demonstrate that Oat1 knockout mice show an increase in IS plasma levels, that has not been observed in Oat3 knockout mice and that IS accumulation has been seen to inhibit OAT1 in vitro [62]. The kinetic parameters for rat OATs have been previously described: IS had a 10-fold greater K_m value for rOat3 than rOat1 (K_m = 174 µmol/L and K_m = 18 µmol/L respectively). Moreover, kinetic analysis of the IS uptake by human kidney slices revealed two saturable components with K_m1 =24 µmol/L and K_m2 = 196 µmol/L similar to those of rOat1 and rOat3 [63]. Therefore, higher affinity of IS for OAT1 could be playing a major role in its excretion.

Bleeding time is higher in females treated with KCl + Apixaban than in males which could be due to the plasma accumulation of apixaban. Females treated with IS + Apixaban showed lower bleeding time than those only treated with IS or apixaban alone. An IS-rich milieu, could lead to endothelial damage and increased tissue factor and activation of coagulation [29], which could increase platelet activation [64] making them more reactive at the time of an injury, and therefore, lowering the bleeding time. These results suggest that during a procoagulant state, like CKD, the hemorrhagic effect of the accumulation of apixaban could be mitigated by the activation of coagulation. Therefore, since the monitoring of the thrombotic and bleeding risk during CKD is a real challenge, better identification of specific risk patients could reduce the chances of under- or over-treatment.

Further studies need to be carried out to assess the efficiency of the different efflux transporters and metabolization enzymes to better determine the cause of the apixaban and IS accumulation in female mice. Specifically, since our study is focused on short term effects, it would be interesting to see if this accumulation has any deleterious long-term effects. Moreover, recognizing the impact of sex differences in drug management is crucial for improving the care provided to women. It is essential to be correctly represented in clinical trials, so that possible underlying biological divergences related to sex can be regarded in order to better understand renal pathophysiology, disease progression and drug metabolization in both men and women.

ACKNOWLEDGMENTS

This work was supported by the European Union’s Horizon 2020 research and innovation program

(860329 Marie-Curie ITN “STRATEGY-CKD”) (SB, JSR) and the EU Horizon 2020 program Epic-XS

(project 0000432) (BPB, SP, SB).

AUTHOR CONTRIBUTIONS

Conceptualization: BPB, SP, SB. Methodology: BPB, DD, JPI ,SP. Formal Analysis: BPB, DD. Funding acquisition: BPB, JPI, JSR, SP, SB. Investigation: BPB, MG, SP, VP, DP. Visualization: BPB. Resources: JSR, SB. Supervision: SP, SB. Writing—original draft: BPB. Writing—review & editing: BPB, DD, MG, VP, DP, ZZ, JPI, SP, SB

COMPETING INTERESTS

Authors declare that they have no competing interests

DATA AVAILABILITY

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [65] partner repository with the dataset identifier PXD053404.

Reviewer access details:

Project accession: PXD053404

Token: DXFU5QG3BsoV

Alternatively, reviewer can access the dataset by logging in to the PRIDE website using the following account details:

Username: [email protected]

Password: 3DfiWu1rY1vs

CODE AVAILABILITY

All code used for analysis and the processed data is available at https://github.com/bpina-beltran/AhR_IndoxylSulfate_liverproteomics

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Unraveling the influence of Indoxyl Sulfate and apixaban in drug metabolism and elimination: Is sex a major player?

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Mice trial

Indoxyl Sulfate (IS) administration

Apixaban administration

Tail Bleeding Time, blood and tissue collection

Obtention of platelet-poor plasma (PPP)

Indoxyl Sulfate plasma concentrations

Apixaban plasma concentrations

Organ dissection, RNA Extraction and RT-qPCR Analysis of mRNA Expression

Protein Extraction and Whole proteome analysis of liver samples

Statistical Analysis

RESULTS

IS and apixaban accumulate in WT female mice

Apixaban and IS accumulation are correlated in male mice but not in females

IS effect in mRNA expression of genes modulated by activation AhR in the liver

Whole proteome analysis revealed that the main agent driving sample separation is sex

Differentially expressed proteins amongst sexes lead to remarkable differences in coagulation and xenobiotic metabolization pathways

STUDY LIMITATIONS

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

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