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.

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.

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.

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.

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.

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.

Clearance of cardiovascular medications during hemodialysis.

PURPOSE OF REVIEW: To review the current understanding of hemodialysis-mediated clearance of commonly used cardiovascular medications. RECENT FINDINGS: Although cardiovascular drug dialyzability is poorly understood, many drug classes appear to include agents with substantially different degrees of dialyzability. Recent data suggest that more readily dialyzable beta-blockers associate with higher short-term mortality in patients initiating these drugs when on hemodialysis. Although this relationship was not observed in a later study with angiotensin-converting enzyme inhibitors of varying dialyzability, studies of this kind are currently limited by pharmacokinetic data that are either incomplete or no longer applicable to modern hemodialysis procedures. SUMMARY: There are substantial deficits in our understanding of cardiovascular medication dialyzability, which relates in large part to advances in the process of hemodialysis that have rendered older studies of dialyzability irrelevant. The importance of cardiovascular disease in patients receiving hemodialysis demands a better understanding of the effect hemodialysis exerts on cardiovascular drug pharmacokinetics.

Effect of renal impairment on atherosclerosis: only partially mediated by homocysteine.

BACKGROUND: Cardiovascular risk and plasma total homocysteine (tHcy) are high in patients with renal failure. High tHcy may account for a substantial part of the increased risk. We assessed mediation by tHcy of the association of estimated glomerular filtration rate (eGFR CKD/EPI) with carotid total plaque area (TPA) and carotid stenosis. METHODS: TPA and carotid stenosis were measured by ultrasound. Multiple linear regression was used to assess the effects of eGFR and/or tHcy after adjustment for age, sex, systolic blood pressure (SBP), smoking, LDL, HDL and weight. RESULTS: Complete data were available for 1967 patients. eGFR decreased, and TPA and total stenosis increased linearly with age. After adjustment [age, sex, SBP, smoking (in pack years), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and weight], eGFR and tHcy were independently associated with TPA (P < 0.01), but when both were added to the model, their significance was attenuated (P = 0.06 for eGFR, 0.03 for tHcy). Mediation analysis showed that tHcy seems to contribute to a significant mediation of the association of eGFR with TPA but not stenosis; after adjustment for the set of risk factors listed above, tHcy still demonstrated significant mediation on TPA (P = 0.03), but not on stenosis (P = 0.16). CONCLUSIONS: tHcy accounts for a significant part, but not all of the effect of renal impairment on atherosclerosis. Other uremic toxins including metabolic products of the intestinal microbiome may explain residual effects of renal failure on atherosclerosis. Therapeutic approaches arising from that hypothesis are discussed.

Effect of CKD and dialysis modality on exposure to drugs cleared by nonrenal mechanisms.

BACKGROUND: Patients with kidney disease frequently experience adverse effects from medication exposure, even when drugs are cleared by nonrenal pathways. Although many studies suggest that nonrenal drug clearance is decreased in chronic kidney disease (CKD), there remains a paucity of in vivo studies in patients with varying degrees of decreased kidney function and those comparing the impact of dialysis modality (eg, hemodialysis [HD] and peritoneal dialysis [PD]). STUDY DESIGN: We performed in vivo clinical pharmacokinetic studies of midazolam, a nonrenally cleared specific probe for CYP3A4, and fexofenadine, a nonspecific probe for hepatic and intestinal transporters. SETTING & PARTICIPANTS: Healthy controls (n=8), patients with non-dialysis-dependent (NDD)-CKD (n=8), and patients receiving HD (n=10) or PD (n=8). OUTCOMES: Exposure to midazolam and fexofenadine were quantified using area under the curve (AUC). Comprehensive pharmacokinetic parameters also were calculated for both probes. RESULTS: Midazolam AUC was significantly higher in the HD group (382.8 h·ng/mL) than in the healthy-control (63.0 h·ng/mL; P<0.001), NDD-CKD (84.5 h·ng/mL; P=0.002), and PD (47.4 h·ng/mL; P<0.001) groups. Fexofenadine AUC was significantly higher in each of the NDD-CKD (2,950 h·ng/mL; P=0.003), HD (2,327 h·ng/mL; P=0.01), and PD (2,095 h·ng/mL; P=0.04) groups compared with healthy controls (1,008 h·ng/mL). LIMITATIONS: Small study groups had different proportions of diabetic patients, early stages of CKD not available. CONCLUSIONS: Our data suggest that selection of dialysis modality is a major determinant of exposure to the CYP3A4 probe midazolam. Exposure to the intestinal and hepatic transporter probe fexofenadine is altered in patients with NDD-CKD and PD and HD patients. Thus, drug development and licensing of nonrenally cleared drugs should include evaluation in these 3 patient groups, with these results included in approved product information labeling. This reinforces the critical need for more in vivo studies of humans that evaluate the exposure to drugs cleared by these pathways.

Decreased nuclear receptor activity and epigenetic modulation associates with down-regulation of hepatic drug-metabolizing enzymes in chronic kidney disease.

Patients with chronic kidney disease (CKD) require many medications. CYP2C and CYP3A drug-metabolizing enzymes play a critical role in determining the pharmacokinetics of the majority of prescribed medications. These enzymes are transcriptionally regulated by the nuclear receptors pregnane X receptor (PXR) and hepatic nuclear factor 4α (HNF-4α). Expression of CYP2C and CYP3A is decreased in CKD; however, the mechanisms by which this occurs is unknown. We induced CKD in rats by 5/6 nephrectomy and used chromatin immunoprecipitation (ChIP) to determine nuclear receptor- and epigenetic alteration-mediated differences in the promoter region of the CYP2C and CYP3A genes. RNA polymerase II and HNF-4α binding was decreased 76 and 57% in the CYP2C11 promotor and 71 and 77% in the CYP3A2 promoter, respectively (P<0.05). ChIP also revealed a 57% decrease in PXR binding to the CYP3A2 promoter in CKD rats (P<0.05). The decrease in PXR and HNF-4α binding was accompanied by diminished histone 4 acetylation in the CYP3A2 promoter (48%) and histone 3 acetylation in the CYP2C11 (77%) and CYP3A2 (77%) promoter loci for nuclear receptor activation (P<0.05). This study suggests that decreased nuclear receptor binding and histone acetylation may contribute to the mechanism of drug-metabolizing enzyme down-regulation and altered pharmacokinetics in CKD.

Enzymatic and non-enzymatic mechanisms of dimesna metabolism.

The chemical reduction of the disulfide homodimer dimesna to its constituent mesna moieties is essential for its mitigation of nephrotoxicity associated with cisplatin and ifosfamide anticancer therapies and enhancement of dialytic clearance of the cardiovascular risk factor homocysteine. The objective of this study was to investigate potential enzymatic and non-enzymatic mechanisms of intracellular dimesna reduction. Similar to endogenous intracellular disulfides, dimesna undergoes thiol-disulfide exchange with thiolate anion-forming sulfhydryl groups via the two-step SN2 reaction. Determination of equilibrium constants of dimesna reduction when mixed with cysteine or glutathione provided a mechanistic explanation for dramatic cysteine and homocysteine depletion, but sparing of the endogenous antioxidant glutathione, previously observed during mesna therapy. Dimesna was reduced by recombinant enzymes of the thioredoxin system; however, oxidation of NADPH by the glutaredoxin system was only observed in the presence of combined dimesna and reduced glutathione, suggesting formation of oxidized glutathione following an initial non-enzymatic reduction of dimesna. Production of mesna by enzymatic and non-enzymatic mechanisms in HeLa cell lysate following dimesna incubation was demonstrated by a loss in mesna production following protein denaturation and prediction of residual non-enzymatic mesna production by mathematical modeling of thiol-disulfide exchange reactions. Reaction modeling also revealed that mixed disulfides make up a significant proportion of intracellular thiols, supporting their role in providing additional nephroprotection, independent of direct platinum conjugation.

Protein restoration in low-birth-weight rat offspring derived from maternal low-protein diet leads to elevated hepatic CYP3A and CYP2C11 activity in adulthood.

The World Health Organization has identified hypercholesterolemia to be one of the major symptoms encompassing the metabolic syndrome. Moreover, epidemiologic evidence indicates that low-birth-weight offspring are at greater risk of developing the metabolic syndrome. Previous work in our laboratory demonstrated that maternal protein restriction (MPR) results in impaired fetal growth and hypercholesterolemia in adulthood. This was attributed to repression of hepatic CYP7A1, a rate-limiting enzyme that catabolizes cholesterol to bile acids. Another important function of hepatic cytochrome P450 enzymes is the phase I oxidative metabolism of drugs (i.e., statins for hypercholesterolemia), which can significantly impact pharmacokinetics. We hypothesized that MPR offspring may have altered ability to metabolize drugs in adulthood. To address this hypothesis, we maintained Wistar rats on a 20% protein diet (control) or a low 8% protein diet throughout prenatal and postnatal life (LP1) or exclusively during prenatal life and weaning (LP2). Intriguingly CYP3A and CYP2C11 intrinsic clearance (Vmax/Km) was significantly increased exclusively in LP2 offspring at postnatal day 130 compared with control or LP1 offspring, as evaluated by testosterone enzyme kinetics in liver microsomes. The increase in activity was secondary to an increase in CYP3A23 and CYP2C11 mRNA. Collectively, these findings suggest that a low-birth-weight offspring with postnatal catch-up growth may have a diminished response to xenobiotics metabolized by CYP3A and CYP2C11 enzymes.

CD73-TNAP crosstalk regulates the hypertrophic response and cardiomyocyte calcification due to α1 adrenoceptor activation.

Cluster of differentiation 73 (CD73) is an ecto-5' nucleotidase which catalyzes the conversion of AMP to adenosine. One of the many functions of adenosine is to suppress the activity of tissue nonspecific alkaline phosphatase (TNAP), an enzyme important in regulating intracellular calcification. Since myocardial calcification is associated with various cardiac disease states, we studied the individual roles and crosstalk between CD73 and TNAP in regulating myocyte responses to the α1 adrenoceptor agonist phenylephrine in terms of calcification and hypertrophy. Cultured neonatal rat cardiomyocytes were treated with 10 µM phenylephrine for 24 h in the absence or presence of the stable adenosine analog 2-chloro-adenosine, the TNAP inhibitor tetramisole or the CD73 inhibitor α,ß-methylene ADP. Phenylephrine produced marked hypertrophy as evidenced by significant increases in myocyte surface area and ANP gene expression, as well as calcification determined by Alizarin Red S staining. These responses were associated with reduced CD73 gene and protein expression and CD73 activity. Conversely, TNAP expression and activity were significantly increased although both were suppressed by 2-chloro-adenosine. CD73 inhibition alone significantly reduced myocyte-derived adenosine levels by >50 %, and directly induced hypertrophy and calcification in the absence of phenylephrine. These responses and those to phenylephrine were abrogated by TNAP inhibition. We conclude that TNAP contributes to the hypertrophic effect of phenylephrine, as well as its ability to produce cardiomyocyte calcification. These responses are minimized by CD73-dependent endogenously produced adenosine.

Design it yourself (DIY): in-house instructional design for online pharmacology.

Demand for e-learning courses has risen dramatically placing pressure on institutions to offer more online courses. Third party vendors now offer courses that can be embedded directly into learning management systems. When transitioning from in-class to e-learning formats, instructors must decide whether to use commercially available courses or design in-house. The objective of this study was to evaluate our transition from delivering introductory pharmacology via a purchased e-pack to an in-house designed course. A team that included an instructional designer, an education specialist and a content expert created an online course in pharmacology. Merrill's first principles of instruction were used as a guide for the design of our online course. Where appropriate, multiple forms of media were introduced to reinforce concepts. We compared grades and design strategy from a previous iteration that was delivered using a commercially available e-pack. A cost analysis was conducted to determine the institutional setup and maintenance costs of in-house course design. The mean final grade from the in-house designed course was 81.9 (0.5) % compared to 76.4 (0.5) % for the e-pack course (P < 0.001). Course evaluations were significantly improved for the in-house course compared to the e-pack. Cost-analysis demonstrated that designing a course in-house has a high initial cost ($111,180.57) but can be maintained with minimal institutional cost ($500) in future offerings. Our results demonstrate that effective courses can be designed in-house and this should be a viable option for institutions that have appropriate resources to support instructional design.

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.

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.

Down-regulation of hepatic CYP3A and CYP2C mediated metabolism in rats with moderate chronic kidney disease.

Expression and activity of drug-metabolizing enzymes are decreased in severe kidney disease; however, only a small percentage of patients with chronic kidney disease (CKD) are at the final stage of the disease. This study aimed to determine the changes in drug-metabolizing enzyme function and expression in rats with varying degrees of kidney disease. Sprague-Dawley rats were subjected to surgical procedures that allowed the generation of three distinct models of kidney function: normal kidney function, moderate kidney function, and severe kidney disease. Forty-two days after surgery, rats were sacrificed and hepatic CYP3A and CYP2C expression was determined. In addition, enzymatic activity was determined in liver microsomes by evaluating midazolam (CYP3A), testosterone (CYP3A and CYP2C), and tolbutamide (CYP2C) enzyme kinetics. Both moderate and severe kidney disease were associated with a reduction in CYP3A2 and CYP2C11 expression (p < 0.05). Likewise, moderate kidney disease resulted in more than a 60% decrease in enzyme activity (V(max)) for CYP2C11 and CYP3A, compared with controls (p < 0.05). When the degree of kidney disease was correlated with metabolic activity, an exponential decline in CYP2C- and CYP3A-mediated metabolism was observed. Our results demonstrate that CYP3A and CYP2C expression and activity are decreased in both moderate and severe CKD. Our data suggest that drug metabolism is significantly decreased in the earlier stages of CKD and imply that patients with moderate CKD may be subject to unpredictable pharmacokinetics.

Human skeletal muscle drug transporters determine local exposure and toxicity of statins.

RATIONALE: The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity. OBJECTIVE: We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro. METHODS AND RESULTS: We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance-associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1. CONCLUSIONS: These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.

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.