Pharmacometabolomics in Drug Disposition, Toxicity and Precision Medicine.

The precision medicine initiative has driven a substantial change in the way scientists and health care practitioners think about diagnosing and treating disease. While it has long been recognized that drug response is determined by the intersection of genetic, environmental and disease factors, improvements in technology have afforded precision medicine guided dosing of drugs to improve efficacy and reduce toxicity. Pharmacometabolomics aims to evaluate small molecule metabolites in plasma and/or urine to help evaluate mechanisms that predict and/or reflect drug efficacy and toxicity. In this mini review, we provide an overview of pharmacometabolomic approaches and methodologies. Relevant examples where metabolomic techniques have been used to better understand drug efficacy and toxicity in major depressive disorder and cancer chemotherapy are discussed. In addition, the utility of metabolomics in drug development and understanding drug metabolism, transport and pharmacokinetics is reviewed. Pharmacometabolomic approaches can help understand factors mediating drug disposition, efficacy and toxicity. While important advancements in this area have been made, their remain several challenges that must be overcome before this approach can be fully implemented into clinical drug therapy. Significance Statement Pharmacometabolomics has emerged as an approach to identify metabolites that allow for implementation of precision medicine approaches to pharmacotherapy. This review article provides an overview pharmacometabolomics including highlights of important examples.

Investigation of N,N,N-Trimethyl-L-alanyl-L-proline Betaine (TMAP) as a Biomarker of Kidney Function.

Glomerular filtration rate (GFR) is the most widely used tool for the measurement of kidney function, but endogenous biomarkers such as cystatin C and creatinine have limitations. A previous metabolomic study revealed N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP) to be reflective of kidney function. In this study, we developed a quantitative LCMS assay for the measurement of TMAP and evaluated TMAP as a biomarker of GFR. An assay to measure TMAP was developed using liquid chromatography-mass spectrometry. After validation of the method, we applied it to plasma samples from three distinct kidney disease patient cohorts: nondialysis chronic kidney disease (CKD) patients, patients receiving peritoneal and hemodialysis, and living kidney donors. We investigated whether TMAP was conserved in other mammalian and nonmammalian species, by analyzing plasma samples from Wistar rats with diet-induced CKD and searching for putative matches to the m/z for TMAP and its known fragments in the raw sample data repository "Metabolomics Workbench". The assay can measure plasma TMAP at a lower limit of quantitation (100 ng/mL) with an interday precision and accuracy of 12.8 and 12.1%, respectively. In all three patient cohorts, TMAP concentrations are significantly higher in patients with CKD than in controls with a normal GFR. Further, TMAP concentrations are also elevated in rats with CKD and TMAP is present in the sap produced from Acer saccharum trees. TMAP concentration is inversely related to GFR suggesting that it is a marker of kidney function. TMAP is present in nonmammalian species suggesting that it is part of a biologically conserved process.

Metabolomics for the identification of early biomarkers of nephrotoxicity in a mouse model of cisplatin-induced acute kidney injury.

BACKGROUND AND PURPOSE: Cisplatin-induced nephrotoxicity manifests as acute kidney injury (AKI) in approximately one third of patients receiving cisplatin therapy. Current measures of AKI are inadequate in detecting AKI prior to significant renal injury, and better biomarkers are needed for early diagnosis of cisplatin-induced AKI. EXPERIMENTAL APPROACH: C57BL/6 and FVB/N mice were treated with a single intraperitoneal injection of cisplatin (15 mg kg-1) or saline. Plasma, urine, and kidney samples were collected prior to cisplatin injection and 24-, 48-, 72-, and 96-hours following cisplatin injection. Untargeted metabolomics was employed using liquid chromatography-mass spectrometry to identify early diagnostic biomarkers for cisplatin nephrotoxicity. PRINCIPAL RESULTS: There was clear metabolic discrimination between saline and cisplatin-treated mice at all timepoints (day 1 to day 4). In total, 26 plasma, urine, and kidney metabolites were identified as exhibiting early alterations following cisplatin treatment. Several of the metabolites showing early alterations were associated with mitochondrial function and energetics, including intermediates of the tricarboxylic acid cycle, regulators of mitochondrial function and indicators of fatty acid ß-oxidation dysfunction. Furthermore, several metabolites were derived from the gut microbiome. MAJOR CONCLUSIONS: Our results highlight the detrimental effects of cisplatin on mitochondrial function and demonstrate potential involvement of the gut microbiome in the pathophysiology of cisplatin-induced AKI. We provide a panel of metabolites to guide future clinical studies of cisplatin-induced AKI and provide insight into potential mechanisms behind cisplatin nephrotoxicity.

The effect of micro-particle curcumin on chronic kidney disease progression: the MPAC-CKD randomized clinical trial.

BACKGROUND: Curcumin is a commonly used herbal supplement with anti-inflammatory and anti-fibrotic properties. Animal studies and small human trials suggest that curcumin reduces albuminuria in patients with chronic kidney disease (CKD). Micro-particle curcumin is a new, more bioavailable formulation of curcumin. METHODS: To determine whether micro-particle curcumin versus placebo slows the progression of albuminuric CKD we conducted a randomized, double-blind, placebo-controlled trial with 6-month follow-up. We included adults with albuminuria [a random urine albumin-to-creatinine ratio >30 mg/mmol (265 mg/g) or a 24-h urine collection with more than 300 mg of protein] and an estimated glomerular filtration rate (eGFR) between 15 and 60 mL/min/1.73 m2 within the 3 months before randomization. We randomly allocated participants 1:1 to receive micro-particle curcumin capsules (90 mg/day) or matching placebo for 6 months. After randomization, the co-primary outcomes were the changes in albuminuria and the eGFR. RESULTS: We enrolled 533 participants, but 4/265 participants in the curcumin group and 15/268 in the placebo group withdrew consent or became ineligible. The 6-month change in albuminuria did not differ significantly between the curcumin and placebo groups [geometric mean ratio 0.94, 97.5% confidence interval (CI) 0.82 to 1.08, P = .32]. Similarly, the 6-month change in eGFR did not differ between groups (mean between-group difference -0.22 mL/min/1.73 m2, 97.5% CI -1.38 to 0.95, P = .68). CONCLUSIONS: Ninety milligrams of micro-particle curcumin daily did not slow the progression of albuminuric CKD over 6 months. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02369549.

Metabolomic identification of predictive and early biomarkers of cisplatin-induced acute kidney injury in adult head and neck cancer patients.

AIM: Cisplatin causes acute kidney injury (AKI) in approximately one third of patients. Serum creatinine and urinary output are poor markers of cisplatin-induced AKI. Metabolomics was utilized to identify predictive or early diagnostic biomarkers of cisplatin-induced AKI. METHODS: Thirty-one adult head and neck cancer patients receiving cisplatin (dose ≥70 mg/m2 ) were recruited for metabolomics analysis. Urine and serum samples were collected prior to cisplatin (pre), 24-48 h after cisplatin (24-48 h) and 5-14 days (post) after cisplatin. Based on serum creatinine concentrations measured at the post timepoint, 11/31 patients were classified with clinical AKI. Untargeted metabolomics was performed using liquid chromatography-mass spectrometry (LC-MS). RESULTS: Metabolic discrimination was observed between "AKI" patients and "no AKI" patients at all timepoints. Urinary glycine, hippuric acid sulfate, 3-hydroxydecanedioc acid and suberate were significantly different between AKI patients and no AKI patients prior to cisplatin infusion. Urinary glycine and hippuric acid sulfate were lower (-2.22-fold and -8.85-fold), whereas 3-hydroxydecanedioc acid and suberate were higher (3.62-fold and 1.91-fold) in AKI patients relative to no AKI patients. Several urine and serum metabolites were found to be altered 24-48 h following cisplatin infusion, particularly metabolites involved with mitochondrial energetics. CONCLUSIONS: We propose glycine, hippuric acid sulfate, 3-hydroxydecanedioc acid and suberate as predictive biomarkers of predisposition to cisplatin-induced AKI. Metabolites indicative of mitochondrial dysfunction may serve as early markers of subclinical AKI.

Pharmacokinetics of recombinant human annexin A5 (SY-005) in patients with severe COVID-19.

Objective: Annexin A5 is a phosphatidylserine binding protein with anti-inflammatory, anticoagulant and anti-apoptotic properties. Preclinical studies have shown that annexin A5 inhibits pro-inflammatory responses and improves organ function and survival in rodent models of sepsis. This clinical trial aimed to evaluate the pharmacokinetic (PK) properties of the recombinant human annexin A5 (SY-005) in severe COVID-19. Methods: This was a pilot randomized, double-blind, placebo-controlled trial. Severe COVID-19 patients were randomly assigned to receive intravenous 50 µg/kg (low dose, n = 3), 100 µg/kg (high dose, n = 5) of SY-005 or placebo (n = 5) every 12 h for 7 days. Plasma SY-005 levels were assessed using enzyme-linked immunosorbent assay (ELISA) and the PK parameters were determined using non-compartmental analysis. Results: All patients treated with SY-005 had a normal baseline estimated glomerular filtration rate (eGFR, 104-125 mL/min/1.73 m2). Both low and high doses of SY-005 were cleared within 6 h after intravenous administration. Plasma maximum concentrations (Cmax), half-life, clearance and volume distribution of low and high doses of SY-005 were 402.4 and 848.9 ng/mL, 0.92 and 0.96 h, 7.52 and 15.19 L/h, and 9.98 and 20.79 L, respectively. Daily pre-dose circulating annexin A5 levels were not significantly different when SY-005 was administered at the low or the high dose 12-h intervals. There was no significant effect on activated partial thromboplastin time (aPTT) or INR (international normalized ratio of prothrombin time) during 7 days of SY-005 treatment. Conclusion: SY-005 doses of 50 and 100 µg/kg were detectable and subsequently cleared from the plasma in severe COVID-19 patients with normal baseline renal function. There was no significant plasma SY-005 accumulation 6 h after drug administration and coagulation was not altered during 7 days of treatment. Clinical trials Registration: This study was registered with ClinicalTrials.gov (NCT04748757, first posted on 10 February 2021).

Cerebrovascular Disease, Cardiovascular Disease, and Chronic Kidney Disease: Interplays and Influences.

PURPOSE OF REVIEW: We reviewed reasons for the high cardiovascular risk (CVD) of patients with chronic kidney disease (CKD), and explored alternatives to treatment of traditional risk factors to reduce CVD in CKD. RECENT FINDINGS: Besides traditional risk factors, patients with CKD are exposed to uremic toxins of two kinds: systemically derived toxins include asymmetric dimethylarginine (ADMA), total homocysteine (tHcy), thiocyanate, tumor necrosis factor alpha, and interleukin 6. Gut-derived uremic toxins (GDUT), products of the intestinal microbiome, include hippuric acid, indoxyl sulfate, p-cresyl sulfate, p-cresyl glucuronide, phenylacetylglutamine, and trimethylamine N-oxide (TMAO). Cyanocobalamin is toxic in patients with CKD. Approaches to reducing plasma levels of these uremic toxins would include diet to reduce GDUT, kidney transplantation, more intensive dialysis, and vitamin therapy to lower tHcy with methylcobalamin rather than cyanocobalamin. The high CVD risk in CKD requires consideration of therapies beyond treatment of traditional risk factors.

A Canadian Study of Cisplatin Metabolomics and Nephrotoxicity (ACCENT): A Clinical Research Protocol.

BACKGROUND: Cisplatin, a chemotherapy used to treat solid tumors, causes acute kidney injury (AKI), a known risk factor for chronic kidney disease and mortality. AKI diagnosis relies on biomarkers which are only measurable after kidney damage has occurred and functional impairment is apparent; this prevents timely AKI diagnosis and treatment. Metabolomics seeks to identify metabolite patterns involved in cell tissue metabolism related to disease or patient factors. The A Canadian study of Cisplatin mEtabolomics and NephroToxicity (ACCENT) team was established to harness the power of metabolomics to identify novel biomarkers that predict risk and discriminate for presence of cisplatin nephrotoxicity, so that early intervention strategies to mitigate onset and severity of AKI can be implemented. OBJECTIVE: Describe the design and methods of the ACCENT study which aims to identify and validate metabolomic profiles in urine and serum associated with risk for cisplatin-mediated nephrotoxicity in children and adults. DESIGN: Observational prospective cohort study. SETTING: Six Canadian oncology centers (3 pediatric, 1 adult and 2 both). PATIENTS: Three hundred adults and 300 children planned to receive cisplatin therapy. MEASUREMENTS: During two cisplatin infusion cycles, serum and urine will be measured for creatinine and electrolytes to ascertain AKI. Many patient and disease variables will be collected prospectively at baseline and throughout therapy. Metabolomic analyses of serum and urine will be done using mass spectrometry. An untargeted metabolomics approach will be used to analyze serum and urine samples before and after cisplatin infusions to identify candidate biomarkers of cisplatin AKI. Candidate metabolites will be validated using an independent cohort. METHODS: Patients will be recruited before their first cycle of cisplatin. Blood and urine will be collected at specified time points before and after cisplatin during the first infusion and an infusion later during cancer treatment. The primary outcome is AKI, defined using a traditional serum creatinine-based definition and an electrolyte abnormality-based definition. Chart review 3 months after cisplatin therapy end will be conducted to document kidney health and survival. LIMITATIONS: It may not be possible to adjust for all measured and unmeasured confounders when evaluating prediction of AKI using metabolite profiles. Collection of data across multiple sites will be a challenge. CONCLUSIONS: ACCENT is the largest study of children and adults treated with cisplatin and aims to reimagine the current model for AKI diagnoses using metabolomics. The identification of biomarkers predicting and detecting AKI in children and adults treated with cisplatin can greatly inform future clinical investigations and practices.

CONTEXTE: Le cisplatine, un agent utilisé en chimiothérapie pour traiter les tumeurs solides, entraîne de l'insuffisance rénale aiguë (IRA); un facteur de risque connu de néphropathie chronique et de mortalité. Le diagnostic de l'IRA repose sur des biomarqueurs qui ne sont mesurables qu'après l'apparition d'une lésion rénale et d'une déficience fonctionnelle; ce qui empêche le diagnostic et le traitement précoce de la maladie. La métabolomique s'efforce d'établir le profil des métabolites impliqués dans le métabolisme des tissus cellulaires en relation avec des facteurs liés à la maladie ou au patient. Une étude canadienne portant sur la métabolomique et la néphrotoxicité du cisplatine (ACCENT) s'est amorcée, elle explore la puissance de la métabolomique dans l'identification de nouveaux biomarqueurs permettant de prédire le risque de néphrotoxicité du cisplatine et d'en distinguer la présence. L'objectif étant de mettre en œuvre des stratégies d'intervention précoce, dès l'apparition de l'IRA, et de limiter la gravité de la maladie. OBJECTIFS: Décrire la conception et la méthodologie de l'étude ACCENT. Cette étude vise à établir et à valider des profils métabolomiques, dans l'urine et le sérum, associés au risque de néphrotoxicité médiée par le cisplatine chez les enfants et les adultes. TYPE D'ÉTUDE: Étude de cohorte prospective. CADRE: Six centres canadiens d'oncologie (trois centres pédiatriques, un centre pour adultes et deux centres mixtes). SUJETS: L'étude porte sur 300 adultes et 300 enfants pour qui un traitement par cisplatine est prévu. MESURES: L'IRA sera confirmée par mesure de la créatinine et des électrolytes dans le sérum et l'urine au cours de deux cycles de perfusion de cisplatine. De nombreuses variables relatives au patient et à la maladie seront recueillies prospectivement avant et pendant le traitement. Les analyses métabolomiques des échantillons de sérum et d'urine seront effectuées par spectrométrie de masse. Une approche métabolomique non ciblée sera utilisée pour analyser les échantillons avant et après les perfusions de cisplatine pour identifier les biomarqueurs candidats d'une IRA découlant du traitement par cisplatine. Les métabolites candidats seront validés dans une cohorte indépendante. MÉTHODOLOGIE: Les patients seront recrutés avant le premier cycle de cisplatine. Le sang et l'urine seront recueillis à des moments précis, soit avant et pendant le traitement; plus précisément lors de la première perfusion, puis d'une perfusion subséquente au cours du traitement contre le cancer. Le principal critère d'évaluation est la présence d'IRA, laquelle sera établie selon la définition classique fondée sur la mesure de la créatinine sérique et d'une autre définition fondée sur les anomalies électrolytiques. Un examen des dossiers trois mois après la fin du traitement par cisplatine sera effectué afin de documenter la santé rénale et la survie des patients. LIMITES: Il pourrait être impossible de corriger tous les facteurs confusionnels mesurés et non mesurés lors de l'évaluation de la prédiction de l'IRA à l'aide de profils de métabolites. La collecte de données sur plusieurs sites sera un défi. CONCLUSION: ACCENT est la plus vaste étude portant sur des enfants et des adultes traités avec le cisplatine; cette étude tente de revoir le modèle actuel en utilisant la métabolomique pour diagnostiquer l'IRA. L'identification de biomarqueurs permettant de prédire et de détecter l'IRA chez les enfants et les adultes traités par cisplatine pourrait grandement éclairer les futures études et pratiques cliniques. RENSEIGNEMENTS SUR L'ENREGISTREMENT DE L'ESSAI CLINIQUE: ClinicalTrials.gov, insuffisance rénale induite par le cisplatine, NCT04442516.

Normothermic Ex-vivo Kidney Perfusion in a Porcine Auto-Transplantation Model Preserves the Expression of Key Mitochondrial Proteins: An Unbiased Proteomics Analysis.

Normothermic ex-vivo kidney perfusion (NEVKP) results in significantly improved graft function in porcine auto-transplant models of donation after circulatory death injury compared with static cold storage (SCS); however, the molecular mechanisms underlying these beneficial effects remain unclear. We performed an unbiased proteomics analysis of 28 kidney biopsies obtained at three time points from pig kidneys subjected to 30 min of warm ischemia, followed by 8 h of NEVKP or SCS, and auto-transplantation. 70/6593 proteins quantified were differentially expressed between NEVKP and SCS groups (false discovery rate < 0.05). Proteins increased in NEVKP mediated key metabolic processes including fatty acid ß-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation. Comparison of our findings with external datasets of ischemia-reperfusion and other models of kidney injury confirmed that 47 of our proteins represent a common signature of kidney injury reversed or attenuated by NEVKP. We validated key metabolic proteins (electron transfer flavoprotein subunit beta and carnitine O-palmitoyltransferase 2, mitochondrial) by immunoblotting. Transcription factor databases identified members of the peroxisome proliferator-activated receptors (PPAR) family of transcription factors as the upstream regulators of our dataset, and we confirmed increased expression of PPARA, PPARD, and RXRA in NEVKP with reverse transcription polymerase chain reaction. The proteome-level changes observed in NEVKP mediate critical metabolic pathways. These effects may be coordinated by PPAR-family transcription factors and may represent novel therapeutic targets in ischemia-reperfusion injury.

Uremic Toxins in the Progression of Chronic Kidney Disease and Cardiovascular Disease: Mechanisms and Therapeutic Targets.

Chronic kidney disease (CKD) is a progressive loss of renal function. The gradual decline in kidney function leads to an accumulation of toxins normally cleared by the kidneys, resulting in uremia. Uremic toxins are classified into three categories: free water-soluble low-molecular-weight solutes, protein-bound solutes, and middle molecules. CKD patients have increased risk of developing cardiovascular disease (CVD), due to an assortment of CKD-specific risk factors. The accumulation of uremic toxins in the circulation and in tissues is associated with the progression of CKD and its co-morbidities, including CVD. Although numerous uremic toxins have been identified to date and many of them are believed to play a role in the progression of CKD and CVD, very few toxins have been extensively studied. The pathophysiological mechanisms of uremic toxins must be investigated further for a better understanding of their roles in disease progression and to develop therapeutic interventions against uremic toxicity. This review discusses the renal and cardiovascular toxicity of uremic toxins indoxyl sulfate, p-cresyl sulfate, hippuric acid, TMAO, ADMA, TNF-α, and IL-6. A focus is also placed on potential therapeutic targets against uremic toxicity.

Association of Urine Platinum With Acute Kidney Injury in Children Treated With Cisplatin for Cancer.

Cisplatin is a chemotherapeutic agent highly excreted in urine and known to cause acute kidney injury (AKI). As AKI diagnosis by serum creatinine (SCr) is usually delayed, endeavors for finding early AKI biomarkers continue. This study aims to determine if urine platinum (UP) concentration 24 hours after cisplatin infusion is associated with AKI, and to evaluate the association between urine platinum and tubular damage biomarkers: neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1). Children treated with cisplatin in 12 Canadian centers (April 2013 to December 2017) were included. Urine from the morning after the first cisplatin infusion of the first or second cisplatin cycle was measured for urine platinum, NGAL, and KIM-1. SCr and serum electrolytes were used to detect AKI by either SCr elevation or urinary electrolyte wasting (potassium, magnesium, phosphate). The associations of urine platinum with AKI, NGAL, and KIM-1 were assessed. A total of 115 participants (54% boys, median age, 8.5 years; interquartile range, 4.0-13.4) were included, of which 29 (25%) and 105 (91%) developed AKI defined by SCr and electrolyte criteria, respectively. Higher urine platinum was associated with higher cisplatin dose (Spearman rho, 0.21) and with younger age (Spearman rho, -0.33). Urine platinum was not associated with postinfusion AKIor KIM-1, but was weakly associated with NGAL, particularly in participants without SCr AKI (Pearson's r, 0.22). Urine platinum may be a marker of mild tubular injury but is not likely to be a useful biomarker of clinically evident AKI.

Protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc tissues.

The comprehensiveness of data collected by "omics" modalities has demonstrated the ability to drastically transform our understanding of the molecular mechanisms of chronic, complex diseases such as musculoskeletal pathologies, how biomarkers are identified, and how therapeutic targets are developed. Standardization of protocols will enable comparisons between findings reported by multiple research groups and move the application of these technologies forward. Herein, we describe a protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc (IVD) tissues, building from the combined expertise of our collaborative team. This protocol covers dissection of murine IVD tissues, sample isolation, and data analysis for both proteomics and metabolomics applications. The protocol presented below was optimized to maximize the utility of a mouse model for "omics" applications, accounting for the challenges associated with the small starting quantity of sample due to small tissue size as well as the extracellular matrix-rich nature of the tissue.

Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing.

The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.

Crohn's Disease Is Associated with Decreased CYP3A4 and P-Glycoprotein Protein Expression.

Cytochrome P450 (CYP) 3A4 and P-glycoprotein (P-gp) have broad substrate overlap and are involved in the metabolism and transport of nearly 50% of currently prescribed medications. In the intestine, CYP3A4 and P-gp are coexpressed in the enterocytes at the intestinal villous tip and act in a coordinated manner to limit drug and xenobiotic oral bioavailability prior to further metabolism and disposition in the liver. Crohn's disease (CD), a form of inflammatory bowel disease, introduces a transmural intestinal insult that disrupts the intestinal barrier function; it therefore has the potential to affect intestinal drug metabolism and transport. We hypothesized that individuals with CD have reduced intestinal expression of CYP3A4 and P-gp. We obtained intestinal biopsy samples from individuals with and without CD and quantified the expression of CYP3A4 and P-gp. When we carried out Western analysis for protein expression, we observed a significant reduction in ileal (45% decrease) and colonic (78% decrease) CYP3A4 protein expression in subjects with CD compared with those without. Similarly, an 85% reduction in colonic P-gp protein expression was seen in the CD patients. Our data highlight important and novel findings pertaining to CD-associated changes to the intestinal expression of CYP3A4 and P-gp that are of relevance to better predict substrate drug dosing for patients with CD.

Untargeted metabolomics reveals N, N, N-trimethyl-L-alanyl-L-proline betaine (TMAP) as a novel biomarker of kidney function.

The diagnosis and prognosis of chronic kidney disease (CKD) currently relies on very few circulating small molecules, which can vary by factors unrelated to kidney function. In end-stage renal disease (ESRD), these same small molecules are used to determine dialysis dose and dialytic clearance. Therefore, we aimed to identify novel plasma biomarkers to estimate kidney function in CKD and dialytic clearance in ESRD. Untargeted metabolomics was performed on plasma samples from patients with a single kidney, non-dialysis CKD, ESRD and healthy controls. For ESRD patients, pre- and post-dialysis plasma samples were obtained from several dialysis modalities. Metabolomics analysis revealed over 400 significantly different features in non-dialysis CKD and ESRD plasma compared to controls while less than 35 features were significantly altered in patients with a single kidney. N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP, AUROC = 0.815) and pyrocatechol sulfate (AUROC = 0.888) outperformed creatinine (AUROC = 0.745) in accurately identifying patients with a single kidney. Several metabolites accurately predicted ESRD; however, when comparing pre-and post-hemodialysis, TMAP was the most robust biomarker of dialytic clearance for all modalities (AUROC = 0.993). This study describes TMAP as a novel potential biomarker of kidney function and dialytic clearance across several hemodialysis modalities.

Moderate Renal Impairment and Toxic Metabolites Produced by the Intestinal Microbiome: Dietary Implications.

OBJECTIVE: Toxic metabolites produced by the intestinal microbiome from animal proteins, carnitine (mainly from red meat), or phosphatidylcholine (mainly from egg yolk), have important adverse effects on cardiovascular disease. These are renally eliminated and may be termed gut-derived uremic toxins (GDUT). We hypothesized that even moderate renal impairment and intake of nutrient precursors would raise plasma levels of GDUT. DESIGN: A cohort study. SETTING: Academic medical center. SUBJECTS: Patients attending stroke prevention clinics at a university medical center were recruited. MAIN OUTCOME MEASURE: Nutrient intake was assessed by the 131-item Harvard Food Frequency Questionnaire; estimated glomerular filtration rate (eGFR) was caculated using the Chronic Kidney Disease-Epidemiology (EPI) equations. Plasma levels of trimethylamine n-oxide, p-cresyl sulfate, hippuric acid, p-cresyl glucuronide, pheny acetyl glutamine, and phenyl sulfate were measured by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. RESULTS: Among 316 patients recruited, the mean (standard deviation [SD]) age was 66.74 (10.42) years; 59.7% were men. Mean eGFR was 76.03 ± 20.01; 57 (18%) had eGFR<60 mL/min/1.73 m2. Plasma levels of all GDUT were significantly higher even with moderate reduction of eGFR. Nutrient intake affected plasma levels of some GDUT; the effects differed by eGFR above and below 60 mL/min/1.73 m2. Plasma levels were obtained fasting, so we probably underestimated the effect of nutrient intake. CONCLUSIONS: Even moderate impairment of renal function was associated with higher plasma levels of GDUT. This has dietary implications for patients at risk of atherosclerosis, particularly in those with impaired renal function (including the elderly): they should limit intake of animal protein, red meat, and egg yolk. It also points the way to novel approaches to vascular prevention, including more intensive dialysis, renal transplantation, and modification of the intestinal microbiome with probiotics or fecal transplantation.

Mediterranean Diet Score: Associations with Metabolic Products of the Intestinal Microbiome, Carotid Plaque Burden, and Renal Function.

Metabolic products of the intestinal microbiome such as trimethylamine N-oxide (TMAO) that accumulate in renal failure (gut-derived uremic toxins, GDUTs) affect atherosclerosis and increase cardiovascular risk. We hypothesized that patients on a Mediterranean diet and those consuming lower amounts of dietary precursors would have lower levels of GDUTs. Patients attending vascular prevention clinics completed a Harvard Food Frequency Questionnaire (FFQ) and had plasma levels of TMAO, p-cresylsulfate, hippuric acid, indoxyl sulfate, p-cresyl glucuronide, phenyl acetyl glutamine, and phenyl sulfate measured by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Carotid plaque burden was measured by ultrasound; CKD-Epi equations were used to estimate the glomerular filtration rate. In total, 276 patients completed the study. Even moderate renal function significantly increased plasma GDUTs, which were significantly associated with higher carotid plaque burden. There was no significant difference in plasma levels of any GDUT associated with a Mediterranean diet score or with intake of dietary precursors. In omnivorous patients with vascular disease, the intake of dietary precursors of intestinal metabolites or adherence to a Mediterranean diet did not change plasma GDUT. Approaches other than diet, such as probiotics and repopulation of the intestinal microbiome, may be required to mitigate the adverse effects of GDUTs.

Metabolic products of the intestinal microbiome and extremes of atherosclerosis.

BACKGROUND AND AIMS: There is increasing awareness that the intestinal microbiome plays an important role in human health. We investigated its role in the burden of carotid atherosclerosis, measured by ultrasound as total plaque area. METHODS: Multiple regression with traditional risk factors was used to identify three phenotypes among 316/3056 patients attending vascular prevention clinics. Residual score (RES; i.e. the distance off the regression line, similar to standard deviation) was used to identify the 5% of patients with much less plaque than predicted by their risk factors (Protected, RES <-2), the 90% with about as much plaque as predicted (Explained, RES -2 to 2), and the 5% with much more plaque than predicted (Unexplained RES >2). Metabolic products of the intestinal microbiome that accumulate in renal failure - gut-derived uremic toxins (GDUT) - were assayed in plasma by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. RESULTS: Plasma levels of trimethylamine n-oxide (TMAO), p-cresyl sulfate, p-cresyl glucuronide, and phenylacetylglutamine were significantly lower among patients with the Protected phenotype, and higher in those with the Unexplained phenotype, despite no significant differences in renal function or in dietary intake of nutrient precursors of GDUT. In linear multiple regression with a broad panel of risk factors, TMAO (p = 0.011) and p-cresyl sulfate (p = 0.011) were significant independent predictors of carotid plaque burden. CONCLUSIONS: The intestinal microbiome appears to play an important role in atherosclerosis. These findings raise the possibility of novel approaches to treatment of atherosclerosis such as fecal transplantation and probiotics.

ß-Blocker Dialyzability in Maintenance Hemodialysis Patients: A Randomized Clinical Trial.

BACKGROUND AND OBJECTIVES: There is a paucity of data available to describe drug dialyzability. Of the available information, most was obtained before implementation of modern hemodialysis membranes. Our study characterized dialyzability of the most commonly prescribed ß-blockers in patients undergoing high-flux hemodialysis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Patients on hemodialysis (n=8) were recruited to an open label, pharmacokinetic, four-way crossover trial. Single doses of atenolol, metoprolol, bisoprolol, and carvedilol were administered on separate days in random order to each patient. Plasma and dialysate drug concentrations were measured, and dialyzability was determined by the recovery clearance and arterial venous difference methods. RESULTS: Using the recovery clearance method, the dialytic clearance values for atenolol, metoprolol, bisoprolol, and carvedilol were 72, 87, 44, and 0.2 ml/min, respectively (P<0.001). Applying the arterial venous difference method, the dialytic clearance values of atenolol, metoprolol, bisoprolol, and carvedilol were 167, 114, 96, and 24 ml/min, respectively (P<0.001). CONCLUSIONS: Atenolol and metoprolol are extensively cleared by hemodialysis compared with the negligible dialytic clearance of carvedilol. Contrary to estimates of dialyzability on the basis of previous literature, our data indicate that bisoprolol is also dialyzable. This finding highlights the importance of conducting dialyzability studies to definitively characterize drug dialytic clearance.

Untargeted plasma and tissue metabolomics in rats with chronic kidney disease given AST-120.

Chronic kidney disease (CKD) results in the accumulation of metabolic waste products that are normally cleared by the kidney, known as uremia. Many of these waste products are from bacteria metabolites in the gut. Accumulation of uremic toxins in plasma and tissue, as well as the gut-plasma-tissue metabolic axis are important for understanding pathophysiological mechanisms of comorbidities in CKD. In this study, an untargeted metabolomics approach was used to determine uremic toxin accumulation in plasma, liver, heart and kidney tissue in rats with adenine-induced CKD. Rats with CKD were also given AST-120, a spherical carbon adsorbent, to assess metabolic changes in plasma and tissues with the removal of gut-derived uremic toxins. AST-120 decreased >55% of metabolites that were increased in plasma, liver and heart tissue of rats with CKD. CKD was primarily defined by 8 gut-derived uremic toxins, which were significantly increased in plasma and all tissues. These metabolites were derived from aromatic amino acids and soy protein including: indoxyl sulfate, p-cresyl sulfate, hippuric acid, phenyl sulfate, pyrocatechol sulfate, 4-ethylphenyl sulfate, p-cresol glucuronide and equol 7-glucuronide. Our results highlight the importance of diet and gut-derived metabolites in the accumulation of uremic toxins and define the gut-plasma-tissue metabolic axis in CKD.