Renal Drug Elimination Research Articles

Venlafaxine induces P‐glycoprotein in human Caco‐2 cells

The objective of this study was to evaluate the effect of a treatment with venlafaxine on the expression of multidrug resistance-associated protein (MRP) gene and multidrug resistance-related proteins (MDR) in human colon carcinoma cells (Caco-2) compared to a known P-glycoprotein (PGY1) inducer, rifampine. Caco-2 cells were treated with venlafaxine (50 microM, 100 microM, 250 microM, and 500 microM) and rifampin (25 microM and 50 microM) to test the possible induction of MRP and MDR expression. The treatment times used were 1.5, 3, 6, 12, 24, 48, and 72 h. RNA was isolated from the cells, and MDR and MRP genes were amplified using PCR. Both venlafaxine and rifampine had the most dramatic effect at the 50 microM concentration. There was an increase in MDR and MRP expression in Caco-2 cells after the acute treatment (1.5, 3, and 6 h) with venlafaxine. These results were similar to those with rifampine. PGY1 contributes to renal and biliary elimination of drugs by transporting the drug out of the cell and back into the intestinal lumen, where drugs may be further metabolized by intestinal enzymes such as Cytochrome P (CYP)-450 3A4. Its function is to limit the bioavailability of orally administered compounds. Due to the increase in MDR and MRP gene expression seen after the acute treatment with venlafaxine, there could be a potential drug-drug interaction with other medications that are metabolized via CYP450-3A4 when coadministered with venlafaxine.

Cysteine Accessibility in the Hydrophilic Cleft of Human Organic Cation Transporter 2

Organic cation transporters (OCTs) are involved in the renal elimination of many cationic drugs and toxins. A hypothetical three-dimensional structure of OCT2 based on a homology model that used the Escherichia coli glycerol 3-phosphate transporter as a template has been described (Zhang, X., Shirahatti, N. V., Mahadevan, D., and Wright, S. H. (2005) J. Biol. Chem. 280, 34813-34822). To further define OCT structure, the accessibility to hydrophilic thiol-reactive reagents of the 13 cysteine residues contained in the human ortholog of OCT2 was examined. Maleimide-PEO2-biotin precipitated (surface biotinylation followed by Western blotting) and reduced tetraethylammonium transport by OCT2 expressed in Chinese hamster ovary cells, effects that were largely reversed by co-exposure to substrates and transport inhibitors, suggesting interaction with cysteines that are near to or part of a substrate-binding surface. Cysteines at amino acid position 437, 451, 470, and 474 were identified from the model as being located in transmembrane helices that participate in forming the hydrophilic cleft, the proposed region of substrate-protein interaction. To determine which residues are exposed to the solvent, a mutant with all four of these cysteines converted to alanine, along with four variants of this mutant each with an individual cysteine restored, were created. Maleimide-PEO2-biotin was only effective at precipitating and reducing transport by wild-type OCT2 and the mutant with cysteine 474 restored. Additionally, the smaller thiol-reactive reagent, methanethiosulfonate ethylsulfonate, reduced transport by wild-type OCT2 and the mutant with cysteine 474 restored. These data demonstrate that cysteine 474 of OCT2 is exposed to the aqueous milieu of the cleft and contributes to forming a pathway for organic cation transport.

The human organic anion transporter 3 (OAT3;SLC22A8): genetic variation and functional genomics

The human organic anion transporter, OAT3 (SLC22A8), plays a critical role in renal drug elimination, by mediating the entry of a wide variety of organic anions, including a number of commonly used pharmaceuticals, into the renal proximal tubular cells. To understand the nature and extent of genetic variation in OAT3, and to determine whether such variation affects its function, we identified OAT3 variants in a large, ethnically diverse sample population and studied their transport activities in cellular assays. We identified a total of 10 distinct coding-region variants, which altered the encoded amino acid sequence, in DNA samples from 270 individuals (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). The overall prevalence of these OAT3 variants was relatively low among the screened population, with only three variants having allele frequencies of >1% in a particular ethnic group. Clones of each variant were created by site-directed mutagenesis, expressed in HEK-293 cells, and tested for function using the model substrates, estrone sulfate (ES) and cimetidine (CIM). The results revealed a high degree of functional heterogeneity among OAT3 variants, with three variants (p. Arg149Ser, p. Gln239Stop, and p. Ile260Arg) that resulted in complete loss of function, and several others with significantly reduced function. One of the more common variants (p. Ile305Phe), found in 3.5% of Asian-Americans, appeared to have altered substrate specificity. This variant exhibited a reduced ability to transport ES, but a preserved ability to transport CIM. These data suggest that genetic variation in OAT3 may contribute to variation in the disposition of drugs.

Interaction of Furosemide with the Human Sodium-Dependent Dicarboxylate Transporter (hNaDC-3)

Renal elimination of drugs bound to plasma proteins is mediated mainly by tubular secretion. Furosemide, a frequently used diuretic, is tightly bound to plasma proteins and is believed to be secreted. It contains a carboxyl group and a sulfamoyl moiety and may therefore be a substrate for the sodium-dependent dicarboxylate cotransporter from human kidney (hNaDC-3). Furosemide, besides inhibiting [14C]succinate uptake, reduced succinate-associated currents in a dose-dependent manner with an IC50 of 2.2 mM. Furosemide showed sodium-dependent inward currents as evidence for its translocation by hNaDC-3. The concentrations necessary to affect hNaDC-3, however, are far higher than the therapeutically relevant plasma concentrations of furosemide. This implies that dicarboxylate uptake necessary for drug excretion via organic anion/dicarboxylate exchange will not be altered by therapeutical doses of furosemide. Keywords: Furosemide, human kidney, organic anion transporters 1 and 3, sodium-dependent dicarboxylate transporter

Regulating Renal Drug Elimination?

Among the clinically highly relevant functions of the kidney is the elimination of xenobiotics, including drugs. In the past years a wide variety of transport systems have been identified serving renal transport of organic cations and anions ([1][1]–[4][2]). The operation of several of those

Functional analysis of polymorphisms in the organic anion transporter, SLC22A6 (OAT1)

The organic anion transporter, OAT1 (SLC22A6), plays a role in the renal elimination of many drugs and environmental toxins. The goal of this study was to identify and functionally characterize OAT1 variants as a first step towards understanding whether genetic variation in OAT1 may contribute to interindividual differences in renal elimination of xenobiotics. As part of a larger study, 276 DNA samples from an ethnically diverse population were screened and 12 coding region variants of OAT1 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. A small family-based clinical study was conducted to determine the renal elimination of a model OAT1 substrate, adefovir (an antiviral agent) in human subjects who possessed a non-functional variant, OAT1-R454Q. Six non-synonymous variants were identified; two (OAT1-R50 H and OAT1-R293W) were present at > or = 1% in at least one ethnic population. These two variants exhibited normal uptake of p-aminohippurate, ochratoxin A and methotrexate assayed in X. laevis oocytes. One variant, OAT1-R454Q, was non-functional with respect to the above substrates. In the clinical study, there was no significant decrease in the renal secretory clearance of adefovir in family members heterozygous for OAT1-454Q in comparison to those with the reference transporter, OAT1-454R. These data indicate that the coding region of OAT1 has low genetic and functional diversity and suggest that coding region variants of OAT1 may not contribute substantially to interindividual differences in renal elimination of xenobiotics.

Heritability of metformin renal clearance

Background Twin studies have been used to determine the genetic contribution to variation in hepatic drug metabolism. In contrast, little is known about the contribution of genetic factors to variation in renal drug elimination. In this study, we used monozygotic twin pairs to assess the heritability of the clearance of the anti-diabetic agent, metformin, a drug that is eliminated exclusively in the kidneys. Methods We administered 850 mg metformin to six monozygotic twin pairs and collected plasma and urine samples over 24 hours. Concentrations of metformin were determined by LC-MS. A modified calculation was used for assessing the heritability (H) of renal clearance: H= (Vbetween pairs− Vwithin pairs)/Vbetween pairs, where V is variance. Results In this interim analysis, we observed that 95% of the variation in renal clearance of metformin could be attributed to genetic factors (H=0.95), with an overall correlation coefficient of 0.97. Conclusions While the formal heritability calculation will require the enrollment of dizygotic twin pairs, this initial finding indicates that genetic factors contribute substantially to the renal clearance of metformin. The degree of heritability in renal clearance is similar to that observed in classical twin studies of drugs that are eliminated by hepatic metabolism. Clinical Pharmacology & Therapeutics (2005) 77, P61–P61; doi: 10.1016/j.clpt.2004.12.123

Differential regulation of basolateral and canalicular transporter expression in rat liver in chronic renal failure

Background Renal failure (RF) decreases the hepatic elimination of substrates of basolateral Organic Anion Transporting Polypeptides (Oatp) and canalicular Multidrug Resistance Protein 2 (Mrp2), e.g. bromosulfophthalein, cerivastatin, and pravastatin. Therefore we investigated, the expression of these hepatocellular transporters in RF. Methods Male SD rats (120–140g) underwent 2-step 5/6-nephrectomy (6, N) or sham operation (8, C), were pair fed and killed after 6 weeks. Hepatic transporter expression was evaluated in a total liver membrane fraction (TLMF) by semiquantitative Western Blot analysis. Results N and C exhibited similar body weights, liver weights and transaminases. N had uremia, i. e. 314% increased serum creatinine, 698% increased serum urea and 73% decreased creatinine clearance as compared to C (100%). TLMF showed similar recovery and enrichment of canalicular marker enzyme activities in N and C. In N liver, transporter expression showed the following changes as compared to C (100%): Ntcp +20±13%, Oatp1a1 −61±6%, Oatp1a4 −43±13%, Oatp1b2 −72±10%, Mrp2 +25±41%, Mdr1 +7±18%, Mdr2 +96±34%. Thus chronic RF decreases the expression of basolateral Oatps in rat liver, whereas expression of canalicular transporters remains stable or increases. Conclusion Downregulation of hepatic basolateral Oatps might explain the decreased hepatic drug elimination, while upregulation of some canalicular transporters may compensate for reduced renal drug elimination in chronic RF. Clinical Pharmacology & Therapeutics (2005) 77, P34–P34; doi: 10.1016/j.clpt.2004.12.023

Cystatin C as a New Covariate to Predict Renal Elimination of Drugs

The individual dosing of drugs that are mainly eliminated unchanged in the urine is made possible by assessing renal function. Most of the methods used are based on serum creatinine (SCr) levels. Cystatin C (CysC) has been proposed as an alternative endogenous marker of the glomerular filtration rate (GFR). Carboplatin is one of the drugs for which elimination is most dependent on the GFR. A prospective clinical trial including 45 patients was conducted to assess the value of serum CysC as a predictor of carboplatin clearance (CL). The patients were receiving carboplatin as part of established protocols. Carboplatin was administered as a daily 60-minute infusion at doses ranging from 290 to 1700mg. A population pharmacokinetic analysis was performed using the nonlinear mixed effect modelling NONMEM program according to a two-compartment pharmacokinetic model. Data from 30 patients were used to test the relationships between carboplatin CL and morphological, biological and demographic covariates previously proposed for prediction of the GFR. The interindividual variability of carboplatin CL decreased from 31% (no covariate) to 14% by taking into account five covariates (SCr, CysC, bodyweight [BW], age and sex). Prospective evaluation of these relationships using the data from the other 15 patients confirmed that the best equation to predict carboplatin CL was based on these five covariates, with a mean absolute percentage error of 13% as an assessment of precision. NONMEM analysis of the whole dataset (n = 45 patients) was performed. The best covariate equation corresponding to the overall analysis was: CL (mL/min) = 110 x (SCr/75)-0.512 x (CysC/1.0)-0.327 x (BW/65)0.474 x (age/56)-0.387 x 0.854sex, with SCr in micromol/L, CysC in mg/L, BW in kilograms, age in years and sex = 0 if male and 1 if female. To put the value of CysC as an endogenous marker of the GFR into perspective, covariate equations without SCr were also evaluated; a better prediction was obtained by considering CysC together with age and BW (interindividual variability of 16.6% vs 23.3% for CysC alone). CysC is a marker of drug elimination that is at least as good as SCr for predicting carboplatin CL. The model based on five covariates was superior to those based on only four covariates (with BW, age and sex combined with either SCr or CysC), indicating that CysC and SCr are not completely redundant to each other. Further pharmacokinetic evaluation is needed to determine whether SCr or CysC is the better marker of renal elimination of other drugs.

Study of renal function matching between banna minipig inbred line and human

Pigs have been thought to be ideal candidates for xenotransplant donors. However besides the immunological barrier, physiological and pharmacokinetic comparabilities of kidney function between donor animals and humans are important factors for successful xenotransplantation. As a unique large inbred animal, Banna Minipig Inbred (BMI) has been reported to be a potential large animal suitable for xenotransplantation. However, its physical and pharmacokinetic compatibilities with humans have not been documented. The purpose of this investigation was to measure renal routine function, glomerular filtration rate (GFR), renal blood flow (ERPF), and drug metabolism to evaluate comparability to humans. The results suggested that the renal function of BMI was similar to that of humans to eliminate nonprotein nitrogenous end products of metabolism. Pharmacokinetics of p-aminohippurate (PAH) and inulin—the most widely used methods to assess ERPF and GFR—showed that BMI showed lower values than humans for GFR, but similar function to humans in ERPF. The pharmacokinetics of cefazolin; a widely used model drug to study kidney drug metabolizing capacity, showed greater overall renal drug elimination of BMI than of humans. These results suggested that BMI did show comparable data to human renal function.

Hypertension, renal disease, and drug considerations.

The incidence of chronic kidney disease is steadily increasing in the United States. The magnitude of this problem is such that virtually all health care providers are being called upon to manage these patients. The interplay between chronic kidney disease and drug therapy is complex in that the kidney is both a target for drug effect as well as a moderator of drug elimination. Renal drug elimination occurs by filtration, secretion, and/or metabolism. For renally-cleared compounds, drug clearance typically falls in tandem with the loss of renal function. This process is noteworthy for drug accumulation when the glomerular filtration rate approaches the 30-cc/min range. The kidney is a target for drug effect in relationship to blood pressure and protein excretion. Angiotensin-converting enzyme inhibitor and angiotensin receptor blocker therapy (usually given along with a diuretic) are the drug classes that have been shown to be effective for reduction in both blood pressure and protein excretion in the chronic kidney disease patient. A number of questions still remain unanswered in the pharmacotherapy of chronic kidney disease, including the optimal dose for these drugs as well as what represents the most favorable achieved blood pressure.

Transporters and renal drug elimination.

Carrier-mediated processes, often referred to as transporters, play key roles in the reabsorption and secretion of many endogenous and xenobiotic compounds by the kidney. The renal proximal tubule is the primary site of active transport for a wide variety of substrates, including organic anions/cations, peptides, and nucleosides. During the past decade, significant advances in molecular identification and characterization of transporter proteins have been made. Although it is generally noted that these transporters significantly contribute to renal drug handling and variability in drug disposition, the extent of our knowledge regarding the specific roles of such transporters in drug disposition and drug-drug interactions remains, for the most part, limited. In this review, we summarize recent progress in terms of molecular and functional characterization of renal transporters and their clinical relevance to drug therapy.

Identification and functional characterization of variants of the human organic anion transporter, OAT3 (SLC22A8)

The human organic anion transporter, OAT3 (SLC22A8), mediates renal excretion of various endogenous substances and xenobiotics. Genetic variants in OAT3 may alter its activity, and thus affect the elimination of certain pharmaceuticals. We identified ten non-synonymous OAT3 variants by screening the exonic regions of DNA samples from an ethnically diverse population of 256 subjects. One variant, OAT3–V>A, had an allele frequency of 6% in African Americans; all others were less common. We then screened all ten variants for function. Samples of the variants were produced by site-directed mutagenesis of reference OAT3 cDNA. Reference and variant sequences were transiently expressed in a vaccinia-infected HeLa cell system, and function of their encoded transporters assessed by measuring cellular uptake of 3H-estrone sulfate. Compared with reference, five of the OAT3 variants, OAT3-F>L, -R>S, -Q>X, -I>R, and -R>W, had significantly reduced substrate transport (range = 18-36% of control). Four variants, OAT3-V>A, -A>V, -A>S, and -V>I, displayed substrate transport similar to, or exceeding, reference (range = 127-178% of control). The common variant, OAT3-I>F, showed an intermediate uptake (53% of control) of 3H-estrone sulfate, but an uptake of cimetidine similar to reference, suggesting that this variant may exhibit altered specificity. The data suggest that genetic variation in OAT3 may contribute to interindividual variation in the renal elimination of various drugs and toxins. Clinical Pharmacology & Therapeutics (2004) 75, P93–P93; doi: 10.1016/j.clpt.2003.11.356

Pharmacokinetics of Tocolytic Agents

Tocolytic agents are drugs designed to inhibit contractions of myometrial smooth muscle cells. Such an effect has been demonstrated in vitro or in vivo for several pharmacological agents, including beta-adrenergic agonists, calcium channel antagonists, oxytocin antagonists, NSAIDs and magnesium sulfate. However, the aim of tocolysis is not only to stop uterine contractions or to prevent preterm delivery, but to prevent perinatal morbidity and mortality associated with preterm birth. The achievement of this goal has not yet been clearly demonstrated for any of the drugs available, and the use of tocolytic agents may appear controversial. Therefore, it is important to avoid maternal and fetal toxicity when tocolytic agents are used. During pregnancy, all steps of drug pharmacokinetics are altered. Absorption of drugs administered orally is limited because of delayed stomach emptying and reduced intestinal motility. The volume of distribution of drugs is increased. The metabolic activity of the liver is increased, accelerating the metabolism of lipophilic drugs. Renal filtration is increased, leading to enhanced renal elimination of water-soluble drugs. These modifications are generally responsible for reduced plasma concentration and reduced half-life of most drugs. These specific modifications have to be taken into account when using a drug in pregnant women. The aim of this review is to provide the reader with pharmacological data about drugs currently used to treat preterm labour. Such data in pregnant women may affect the choice of optimal drug dosage and route of administration.

A review of the effects of chronic exercise and physical fitness level on resting pharmacokinetics.

Pharmacological intervention in cooperation with physical activity is currently being used in the prevention and treatment of diseases like cardiovascular disease and obesity. Physical activity, both acute and chronic, can cause changes in physiology that can alter the observed pharmacokinetics of drugs. The purpose of this paper is to focus on how chronic exercise can change pharmacokinetics. Chronic exercise can affect drug absorption by the increase in collateral blood flow and absorption may also be affected by changes in gastrointestinal transit times. Chronic exercise may affect volume of distribution of drugs by the increases in lean body mass, decreased fat mass, increased plasma protein and increased plasma volume that occurs with physical conditioning. Changes in hepatic clearance of drugs may explain the differences in systemic clearance seen when comparing physically trained subjects to sedentary ones. Some studies have shown that hepatic enzymes are increased with training but other studies have found no change or lower activities. Finally, renal elimination of drugs may be altered by changes in protein binding but there is little evidence that renal elimination of drugs changes with long-term exercise. Therefore, changes in pharmacokinetics associated with chronic exercise can differ from those during acute exercise and in sedentary subjects. The differences between the physically active and sedentary individuals may require individualization of dosing regimens. It should be noted that there are no standardized protocols to evaluate the influence of exercise on drug disposition.

Molecular Diversity of Organic Cation Transporter (OCT) Mediating Renal Excretion of Drugs

AbstractFor Abstract see ChemInform Abstract in Full Text.

Impaired tubular excretory function as a late renal side effect of chemotherapy in children.

Renal drug excretion is variously influenced by nephrotoxic drugs. This study was designed to evaluate renal function as a late renal side effects in children receiving combination chemotherapy for malignancy. Follow-up studies of 30 newly diagnosed patients were performed a median of 12 months after completion of chemotherapy. The glomerular filtration rate (GFR) was measured using sodium thiosulfate. The following were also assessed: urinary high-molecular-weight fraction (urinary albumin/urinary creatinine ratio); para-aminohippurate (PAH) clearance; urinary low-molecular-weight fraction (urinary beta2-microglobulin/urinary creatinine ratio); and routine serum and urinary parameters. Serum and urinary electrolytes were normal in most patients. GFR was low in four patients (13%). Urinary high-molecular-weight fraction was elevated in two patients. Urinary low-molecular-weight fraction was elevated in one patient. PAH clearance was below the referenced normal value in 73% of the patients. This report demonstrates decreased PAH clearance as a late renal side effect of chemotherapy and suggests disturbed function of the organic anion transport system. The unexpected high serum concentration of drugs excreted through the organic anion transport system may induce severe side effects. Elucidation of the mechanism and clinical relevance of decreased PAH clearance is warranted.

Structure, Function, and Regulation of Renal Organic Anion Transporters

AbstractFor Abstract see ChemInform Abstract in Full Text.

Transport of organic anions across the basolateral membrane of proximal tubule cells.

Renal proximal tubules secrete diverse organic anions (OA) including widely prescribed anionic drugs. Here, we review the molecular properties of cloned transporters involved in uptake of OA from blood into proximal tubule cells and provide extensive lists of substrates handled by these transport systems. Where tested, transporters have been immunolocalized to the basolateral cell membrane. The sulfate anion transporter 1 (sat-1) cloned from human, rat and mouse, transported oxalate and sulfate. Drugs found earlier to interact with sulfate transport in vivo have not yet been tested with sat-1. The Na(+)-dicarboxylate cotransporter 3 (NaDC-3) was cloned from human, rat, mouse and flounder, and transported three Na(+) with one divalent di- or tricarboxylate, such as citric acid cycle intermediates and the heavy metal chelator 2,3-dimercaptosuccinate (succimer). The organic anion transporter 1 (OAT1) cloned from several species was shown to exchange extracellular OA against intracellular alpha-ketoglutarate. OAT1 translocated, e.g., anti-inflammatory drugs, antiviral drugs, beta-lactam antibiotics, loop diuretics, ochratoxin A, and p-aminohippurate. Several OA, including probenecid, inhibited OAT1. Human, rat and mouse OAT2 transported selected anti-inflammatory and antiviral drugs, methotrexate, ochratoxin A, and, with high affinities, prostaglandins E(2) and F(2alpha). OAT3 cloned from human, rat and mouse showed a substrate specificity overlapping with that of OAT1. In addition, OAT3 interacted with sulfated steroid hormones such as estrone-3-sulfate. The driving forces for OAT2 and OAT3, the relative contributions of all OA transporters to, and the impact of transporter regulation by protein kinases on renal drug excretion in vivo must be determined in future experiments.

Principles and clinical application of assessing alterations in renal elimination pathways.

Drugs and metabolites are eliminated from the body by metabolism and excretion. The kidney makes the major contribution to excretion of unchanged drug and also to excretion of metabolites. Net renal excretion is a combination of three processes - glomerular filtration, tubular secretion and tubular reabsorption. Renal function has traditionally been determined by measuring plasma creatinine and estimating creatinine clearance. However, estimated creatinine clearance measures only glomerular filtration with a small contribution from active secretion. There is accumulating evidence of poor correlation between estimated creatinine clearance and renal drug clearance in different clinical settings, challenging the 'intact nephron hypothesis' and suggesting that renal drug handling pathways may not decline in parallel. Furthermore, it is evident that renal drug handling is altered to a clinically significant extent in a number of disease states, necessitating dosage adjustment not just based on filtration. These observations suggest that a re-evaluation of markers of renal function is required. Methods that measure all renal handling pathways would allow informed dosage individualisation using an understanding of renal excretion pathways and patient characteristics. Methodologies have been described to determine individually each of the renal elimination pathways. However, their simultaneous assessment has only recently been investigated. A cocktail of markers to measure simultaneously the individual renal handling pathways have now been developed, and evaluated in healthy volunteers. This review outlines the different renal elimination pathways and the possible markers that can be used for their measurement. Diseases and other physiological conditions causing altered renal drug elimination are presented, and the potential application of a cocktail of markers for the simultaneous measurement of drug handling is evaluated. Further investigation of the effects of disease processes on renal drug handling should include people with HIV infection, transplant recipients (renal and liver) and people with rheumatoid arthritis. Furthermore, changes in renal function in the elderly, the effect of sex on renal function, assessment of living kidney donors prior to transplantation and the investigation of renal drug interactions would also be potential applications. Once renal drug handling pathways are characterised in a patient population, the implications for accurate dosage individualisation can be assessed. The simultaneous measurement of renal function elimination pathways of drugs and metabolites has the potential to assist in understanding how renal function changes with different disease states or physiological conditions. In addition, it will further our understanding of fundamental aspects of the renal elimination of drugs.