Preliminary Flow Modeling by Hybrid Automata Alternating Continuous Reaction and Discrete Transit for Pharmacokinetics.

In traditional pharmacokinetic models, blood flow or liquid transit is often expressed as first-order kinetics. When the flow expression by first-order kinetics is used for dynamic simulation, the flow velocity illogically depends on the step size of a solver of ordinary differential equations. In this study, we propose flow modeling using hybrid automata that combine ordinary differential equations and recursive equations, and we have preliminarily applied the constructed models to several examples. The blood concentration-time profiles of p-aminohippurate and propranolol after intravenous administration were successfully reproduced by simple hybrid automata. The simulation results of one-dimensional tube flow have demonstrated that the fluid velocity in the hybrid automata was independent of the step size of the ordinary differential equation solver. A body fluid model coordinated various flows in a human body with scheduled daily activities and could be used as a drug container to describe formulation-dependent disposition of 5-aminosalicylic acid and enterohepatic circulation of a virtual drug. These findings suggested that flow modeling using hybrid automata could avoid the logical inconsistency in the traditional pharmacokinetic modeling and that the hybrid automata have high versatility and a wide range of applicability to pharmacokinetic analysis. Because our method can define various intervals for multiple recursive equations, the resolution of a specific part of a model can be adjusted relatively freely while the whole body is being roughly modeled, which would be beneficial to refine a coarse model into a fine model in future. SIGNIFICANCE STATEMENT: There is a logical inconsistency in flow expression by first-order kinetics in ordinary differential equations used in traditional pharmacokinetic modeling. It is difficult to model a whole human body using flow models in partial differential equations because of the excessive calculation costs. Our simulations on tube flow and body fluids have demonstrated that the flow modeling using hybrid automata could avoid the problems. The preliminary applications of hybrid automata to several examples highlighted its high versatility in pharmacokinetic analysis.

Net release and uptake of xenometabolites across intestinal, hepatic, muscle, and renal tissue beds in healthy conscious pigs.

Xenometabolites from microbial and plant sources are thought to confer beneficial as well as deleterious effects on host physiology. Studies determining absorption and tissue uptake of xenometabolites are limited. We utilized a conscious catheterized pig model to evaluate interorgan flux of annotated known and suspected xenometabolites, derivatives, and bile acids. Female pigs (n = 12, 2-3 mo old, 25.6 ± 2.2 kg) had surgically implanted catheters across portal-drained viscera (PDV), splanchnic compartment (SPL), liver, kidney, and hindquarter muscle. Overnight-fasted arterial and venous plasma was collected simultaneously in a conscious state and stored at -80°C. Thawed samples were analyzed by liquid chromatography-mass spectrometry. Plasma flow was determined with para-aminohippuric acid dilution technology and used to calculate net organ balance for each metabolite. Significant organ uptake or release was determined if net balance differed from zero. A total of 48 metabolites were identified in plasma, and 31 of these had at least one tissue with a significant net release or uptake. All bile acids, indole-3-acetic acid, indole-3-arylic acid, and hydrocinnamic acid were released from the intestine and taken up by the liver. Indole-3-carboxaldehyde, p-cresol glucuronide, 4-hydroxyphenyllactic acid, dodecanendioic acid, and phenylacetylglycine were also released from the intestines. Liver or kidney uptake was noted for indole-3-acetylglycine, p-cresol glucuronide, atrolactic acid, and dodecanedioic acid. Indole-3-carboxaldehyde, atrolactic acid, and dodecanedioic acids showed net release from skeletal muscle. The results confirm gastrointestinal origins for several known xenometabolites in an in vivo overnight-fasted conscious pig model, whereas nongut net release of other putative xenometabolites suggests a more complex metabolism.NEW & NOTEWORTHY Xenometabolites from microbe origins influence host health and disease, but absorption and tissue uptake of these metabolites remain speculative. Results herein are the first to demonstrate in vivo organ uptake and release of these metabolites. We used a conscious catheterized pig model to confirm gastrointestinal origins for several xenometabolites (e.g., indolic compounds, 4-hydroxyphenyllactic acid, dodecanendioic acid, and phenylacetylgycine). Liver and kidney were major sites for xenometabolite uptake, likely highlighting liver conjugation metabolism and renal excretion.

Andrographolide is neither a human organic anion transporter 1 (hOAT1) substrate nor inhibitor.

Andrographolide, a major bioactive compound isolated from Andrographis paniculata (Burm. F.) Nees, was evaluated for its effects on the hOAT1 membrane transporter. Substrate determination and inhibition of hOAT1-mediated uptake transport assay was carried out using recombinant CHO-hOAT1 cells. The results showed that the uptake ratio of andrographolide was less than 2.0 at all concentrations tested, indicating that andrographolide is not a hOAT1 substrate. Andrographolide has no significant effects on the p-aminohippuric acid uptake and on the mRNA and protein expression of hOAT1. In conclusion, andrographolide may not pose a drug-herb interaction risk related to hOAT1.

Impact of the induced organic anion transporter 1 (Oat1) renal expression by furosemide on the pharmacokinetics of organic anions.

AIM: Furosemide is a loop diuretic. Different authors demonstrated that continuous administration of furosemide modulates the expression of organic anion transporters. This study was undertaken to simultaneously evaluate the effects of furosemide pretreatment on organic anion transporter 1 (Oat1) and multidrug resistance protein 2 (Mrp2) renal expressions, on p-aminohippurate (PAH) pharmacokinetics and on renal and urinary PAH levels in rats. METHODS: Male Wistar rats were treated with furosemide (6 mg/100 g body weight per day, subcutaneously, 4 days) (treated group) or saline (control group). On the fifth day, PAH was administered as a bolus infusion in the femoral vein, and plasma samples were obtained from femoral artery at different time points. PAH levels in renal tissue and urine were also assessed. Renal Oat1 and Mrp2 expressions were evaluated by western blotting. RESULTS: Furosemide pretreatment increased both the expression of Oat1 and Mrp2. PAH plasma concentrations decreased following a biexponential function. The furosemide-treated group showed higher PAH plasma levels, a lower systemic clearance and elimination rate constant from the peripheral compartment, indicating that PAH renal elimination was decreased. PAH levels in renal tissue were significantly elevated and in urine appeared to be significantly lower as compared with control animals. CONCLUSIONS: Furosemide pretreatment caused a significant decrease of PAH renal elimination, despite Oat1 and Mrp2 augmented renal expression. The goal of the present study is the addition of important information in the wide gap of knowledge that exists about drug-drug interactions. Because of furosemide worldwide use, the data obtained are interesting and useful in terms of translation to clinical practice.

A phase 1 study to evaluate the effect of dolutegravir on renal function via measurement of iohexol and para-aminohippurate clearance in healthy subjects.

AIM: Dolutegravir (DTG; S/GSK1349572) is under clinical development as a once daily, unboosted integrase inhibitor for the treatment of HIV infection. The effect of DTG on glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and creatinine clearance (CLcr ) was evaluated in 34 healthy volunteers. METHODS: Subjects received DTG 50 mg (once daily or twice daily) or placebo for 14 days. GFR was measured by iohexol plasma clearance, ERPF was assessed by para-aminohippurate plasma clearance and CLcr was measured by 24 h urine collection. RESULTS: All treatments were generally well tolerated. A modest decrease (10-14%) in CLcr was observed, consistent with clinical study observations. DTG 50 mg once daily and twice daily had no significant effect on GFR or ERPF compared with placebo over 14 days in healthy subjects. CONCLUSIONS: These findings support in vitro data that DTG increases serum creatinine by the benign inhibition of the organic cation transporter 2, which is responsible for tubular secretion of creatinine.

Multiple efflux pumps are involved in the transepithelial transport of colchicine: combined effect of p-glycoprotein and multidrug resistance-associated protein 2 leads to decreased intestinal absorption throughout the entire small intestine.

The purpose of this study was to thoroughly characterize the efflux transporters involved in the intestinal permeability of the oral microtubule polymerization inhibitor colchicine and to evaluate the role of these transporters in limiting its oral absorption. The effects of P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on colchicine bidirectional permeability were studied across Caco-2 cell monolayers, inhibiting one versus multiple transporters simultaneously. Colchicine permeability was then investigated in different regions of the rat small intestine by in situ single-pass perfusion. Correlation with the P-gp/MRP2 expression level throughout different intestinal segments was investigated by immunoblotting. P-gp inhibitors [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), verapamil, and quinidine], and MRP2 inhibitors [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), indomethacin, and p-aminohippuric acid (p-AH)] significantly increased apical (AP)-basolateral (BL) and decreased BL-AP Caco-2 transport in a concentration-dependent manner. No effect was obtained by the BCRP inhibitors fumitremorgin C (FTC) and pantoprazole. P-gp/MRP2 inhibitors combinations greatly reduced colchicine mucosal secretion, including complete abolishment of efflux (GF120918/MK571). Colchicine displayed low (versus metoprolol) and constant permeability along the rat small-intestine. GF120918 significantly increased colchicine permeability in the ileum with no effect in the jejunum, whereas MK571 augmented jejunal permeability without changing the ileal transport. The GF120918/MK571 combination caused an effect similar to that of MK571 alone in the jejunum and to that of GF120918 alone in the ileum. P-gp expression followed a gradient increasing from proximal to distal segments, whereas MRP2 decreased from proximal to distal small intestinal regions. Overall, it was revealed that the combined effect of P-gp and MRP2, but not BCRP, dominates colchicine transepithelial transport, leading to complete coverage of the entire small intestine, and makes the efflux transport dominate the intestinal permeability process.

Development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry method for the simultaneous determination of iohexol, p-aminohippuric acid and creatinine in porcine and broiler chicken plasma.

In order to study the renal function, in terms of glomerular filtration and effective renal plasma flow, in broiler chickens and pigs, an ultra-high performance liquid chromatography-tandem mass spectrometry method for the simultaneous determination of iohexol, p-aminohippuric acid (PAH) and exogenously administered creatinine in plasma was developed and validated. Sample preparation consisted of a deproteinization step using methanol for porcine plasma and an Ostro™ Protein Precipitation & Phospholipid Removal Plate was used for broiler chicken plasma. Chromatographic separation was achieved on a Hypersil Gold aQ column using 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phases. The total run time was limited to 10 min. Matrix-matched calibration curves for iohexol and PAH were prepared and good linearity (r ≥ 0.9973; gof ≤ 6.17%) was achieved over the concentration range tested (0.25-90 µg/mL). Limits of quantification were 0.25 µg/mL for iohexol and PAH. Water was used as surrogate matrix for analysis of creatinine in plasma. This surrogate calibration curve showed good linearity over the concentration range tested (0.25-90 µg/mL) (r ≥ 0.9979; gof ≤ 5.66%). For creatinine, the relative lower limit of quantification was 201.03 ±â€¯49.20% and 60.14 ±â€¯7.64% for chicken and porcine plasma, respectively. The results for within-day and between-day precision and accuracy fell within the specified ranges. This straightforward, cost-effective and rapid method, determining iohexol, PAH and creatinine within one single chromatographic run, has been successfully used for the analysis in porcine and broiler chicken plasma samples in order to determine the renal function of these species.

Renal elimination of p-aminohippurate (PAH) in response to three days of biliary obstruction in the rat. The role of OAT1 and OAT3.

Pharmacokinetic studies of the drugs administered to subjects with mechanical cholestasis are scarce. The purpose of the present study was to examine the effects of bile duct ligation of 3 days (peak of elevation of serum bile acids and bilirubin) on the systemic and renal PAH clearance and on the expression of cortical renal OAT1 and OAT3 in a rat model. PAH is the prototypical substrate of the renal organic anion transport system. Male Wistar rats underwent a bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BDL rats displayed a significantly lower systemic PAH clearance. Renal studies revealed a reduction in the renal clearance and in the excreted and secreted load of PAH in BDL rats. The OAT1 protein expression in kidney homogenates was not modified, but it decreased in the basolateral membranes from BDL rats. In contrast, OAT3 abundance in both kidney cortex homogenates and in basolateral membranes increased by 3 days after the ligation. Immunocytochemical studies (light microscopic and confocal immunofluorescence microscopic analyses) confirmed the changes in the renal expression of these transport proteins. The present study demonstrates the key role of OAT1 expression in the impaired elimination of PAH after 3 days of obstructive cholestasis.

Alteration in renal organic anion transporter 1 after ischemia/reperfusion in cadaveric renal allografts.

We have previously shown that postischemic injury to renal allografts results in profound impairment of p-aminohippuric acid (PAH) extraction. To elucidate the cellular integrity of the human organic anion transporter 1 (hOAT1) in postischemic acute renal failure (ARF), immunohistochemical analysis of hOAT1 was performed in cadaveric renal allografts using confocal microscopy for three-dimensional reconstruction of serial optical images. Biopsy samples were obtained from 10 cadaveric renal allografts 1 hr after reperfusion during transplant operation. Control tissues were obtained from four living donors of healthy kidneys immediately before an arterial clamp was applied to the renal artery. Control tissues demonstrated hOAT1 distributed to basolateral membrane of proximal tubule cells. In contrast, maldistribution of hOAT1 to cytoplasm and/or diminution of the protein was noted in cadaveric allografts. Characteristics of maldistribution were variable: disappearance of lateral distribution, diffuse cytoplasmic aggregates, apical cytoplasmic aggregates, and disappearance of the staining. In addition, iothalamate and PAH clearances were performed on posttransplant days 3-7 in 18 recipients of a cadaveric renal allograft. PAH clearance was depressed <250 ml/min in all but three subjects. We conclude that reperfused, transplanted kidneys exhibit maldistribution of hOAT1 in proximal tubule cells, resulting in impairment of PAH clearance. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Difference between pharmacokinetics of mycophenolic acid (MPA) in rats and that in humans is caused by different affinities of MRP2 to a glucuronized form.

PURPOSE: Mycophenolic acid (MPA), an immunosuppressant, is excreted as its glucuronized form, MPAG. In humans, MPAG is mostly excreted into urine, whereas more than 80% of the dose is excreted into bile in rats. The aim of this study was to clarify the cause of the species difference. We investigated whether MPAG is a substrate of human organic anion transporters (hOATs), and we compared the affinities of multi-drug resistance-associated protein 2 (MRP2) for MPAG in rats and humans. METHODS: The inhibitory effects of MPAG on the uptake of typical substrates via hOAT1 and hOAT3 were determined using HeLa cells heterologously expressing hOAT1 and Xenopus laevis oocytes heterologously expressing hOAT3. MPAG transport activity via hOAT1 and hOAT3 was determined by the two-microelectrode voltage-clamp technique using Xenopus laevis oocytes expressing hOAT1 and hOAT3. The affinities of MPAG for hMRP2 and rMrp2 were determined by the inhibitory effects of MPAG on p-aminohippuric acid (a typical substrate) uptake using membrane vesicles expressing hMRP2 or rMrp2. RESULTS: MPAG inhibited the uptake of PAH via hOAT1 and hOAT3, and calculated IC50 values were 222.6+/-26.6 microM and 41.5+/-11.5 microM, respectively. However, MPAG was not transported by hOAT1 and hOAT3. MPAG strongly inhibited the uptake of PAH via both rMrp2 and hMRP2. However, the magnitudes of inhibitory effects were different. The calculated IC50 values were 286.2+/-157.3 microM and 1036.8+/-330.5 microM, respectively. CONCLUSION: MPAG is not a substrate but is an inhibitor of hOAT1 and hOAT3. The affinity of rMRP2 to MPAG was about 3.6 times as high as that of hMRP2. Therefore, the difference of affinity between hMRP2 and rMrp2 is a possible mechanism of the difference of excretion ratio of MPAG between rats and human.

Altered renal elimination of organic anions in rats with chronic renal failure.

The progress of chronic renal failure (CRF) is characterized by the development of glomerular and tubular lesions. However, little is known about the expression of organic anions renal transporters. The objective of this work was to study, in rats with experimental CRF (5/6 nephrectomy), the expression of the organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) and their contribution to the pharmacokinetics and renal excretion of p-aminohippurate (PAH). Two groups of animals were used: Sham and CRF. Six months after surgery, systolic blood pressure and plasma creatinine concentrations were significantly higher in CRF groups. CRF rats showed a diminution in: the filtered, secreted and excreted load of PAH; the systemic clearance of PAH; the renal OAT1 expression; and the renal Na-K-ATPase activity. No remarkable modifications were observed in the OAT3 expression from CRF kidneys. The diminution in the systemic depuration and renal excretion of PAH may be explained by the decrease in its filtered and secreted load. The lower OAT1 expression in remnant renal mass of CRF rats or/and the lower activity of Na-K-ATPase might justify, at least in part, the diminished secreted load of this organic anion.

Characterization of prostaglandin E1 transport in rat renal brush-border membrane.

Transport of prostaglandin E(1) (PGE(1)) was investigated in rat renal brush-border membrane vesicles. The uptake of [(3)H]PGE(1) was sensitive to osmosis and temperature. This uptake was saturable and mediated by high-affinity (K(m)=2.1 microM)/low-capacity (V(max)=17.4 pmol/mg protein/30 sec) and low-affinity (K(m)=526.5 microM)/high-capacity (V(max)=1,032.5 pmol/mg protein/30 sec) transport systems. [(3)H]PGE(1) uptake was Na(+)-independent and inhibited by various eicosanoids including PGE(2) and PGF(2alpha). Bromcresol green and sulfobromophthalein, potent inhibitors of prostaglandin transporter (PGT), significantly decreased [(3)H]PGE(1) uptake. Uptake was also inhibited by indomethacin and probenecid, which reportedly have little effect on PGT. Benzylpenicillin and taurocholate decreased the uptake of [(3)H]PGE(1). Like p-[(14)C]aminohippurate (PAH) uptake by vesicles, the uptake of [(3)H]PGE(1) was stimulated by an inside-positive membrane potential, created by applying an inward K(+) gradient and valinomycin. However, the uptake of [(3)H]PGE(1) was not inhibited by PAH, suggesting that PAH and PGE(1) are transported by separate transport systems. [(3)H]PGE(1) uptake was not stimulated by outwardly directed gradients of Cl(-) nor unlabeled PGE(1), indicating that an anion exchanger may not be involved in PGE(1) transport. These findings suggest that the transport of PGE(1) in rat renal brush-border membrane is mediated by specific transport system(s), at least in part, by a potential-sensitive transport system.

Anion transport through the contraluminal cell membrane of renal proximal tubule. The influence of hydrophobicity and molecular charge distribution on the inhibitory activity of organic anions.

Three different mechanisms of anion transport have been identified for the contraluminal membrane in the proximal tubule of the rat kidney. These mechanisms are specific for the transport of sulfate, dicarboxylate and p-aminohippurate anions. Sulfate transport is inhibited by bivalent organic anions with a distance between the charges of less than 7 A. The sulfate system acts in two modes: in a planar mode for anions with flat charged residues such as COO- and a charge separation of 3-4 A or in a bulky mode for groups such as SO3H- and a charge separation of 4-7 A. Monovalent anions can be accepted if there is a hydrophobic core next to the negative charges. Dicarboxylate transport is inhibited exclusively by anions with two charge centers located within 5 to 9 A, one of those possibly being a partial charge of -0.5 elementary charges. p-Aminohippurate transport is inhibited by monovalent anions, if these have a hydrophobic domain with a minimal length of about 4 A. Bivalent anions inhibit, if they have a charge distance of 6-10 A; both charges can be partial charges of about -0.5 elementary charges. Longer bivalent anions can be effective provided they have a sufficiently large hydrophobic domain. For the sulfate and p-aminohippurate systems it is found that anions with high acidity yield good inhibition. The overlapping specificities of the three systems with respect to charge distance and hydrophobicity allow them to accept a large variety of organic anions.

The influence of the lipid bilayer phase state on the p-aminohippurate (PAH) transport and the activity of the alkaline phosphatase in brush-border membrane vesicles from normal and mutant rats.

The kinetic parameters of p-aminohippurate transport and activity of the alkaline phosphatase were studied using brush-border membrane vesicles isolated from the kidney cortex of normal and mutant (strain of Campbell) rats. p-Aminohippurate (PAH) transport of both normal and mutant animals was carried out by the mechanism of facilitated diffusion. The apparent Michaelis constant at 36 degrees C was equal to 7 mM, the maximal rate of PAH transport was 15 nmol/min per mg protein and the constant of inhibition by probenecid was 0.5 mM for normal rats and, respectively, 29 mM, 62 nmol/min per mg protein and 1.4 mM for mutant rats. The Arrhenius plot for the PAH transport and activity of the alkaline phosphatase showed the breakpoints at 28-30 degrees C for normal rats and at 36-38 degrees C for the Campbell strain rats. The thermotropic phase transitions detected by the EPR method with 5-doxylstearate as a probe were recorded at 21-30 degrees C and 30-35 degrees C for normal and mutant rats, respectively. Therefore, characteristic features of the PAH carrier and alkaline phosphatase activity in normal and Campbell strain rats are determined by the difference in the phase state of their membrane lipid bilayers. We suppose that mutation in the Campbell strain gives rise to a membrane pleiotropic effect which enables us to understand the mechanism of genetic control of the lipid structure and membrane fluidity.

Renal elimination of organic anions in rats with bilateral ureteral obstruction.

Urinary tract obstruction is an important cause of acute renal failure. Several abnormalities in renal tubular function may occur in obstructive nephropathy. The tubular secretion of organic anions is an important function of the kidney that eliminates potentially toxic organic anions from the body, however, the mechanisms involved in organic anions renal elimination in rats with bilateral ureteral obstruction (BUO) have not been elucidated. In this study, it was evaluated the renal handling of p-aminohippurate (PAH) in adult male Wistar rats with BUO. A diminished renal clearance of PAH was observed in BUO rats as consequence of a diminution in the secreted load of this organic anion. The increase in the abundance of organic anions transporter 1 (OAT1) and the absence of modification in cortical renal blood flow, measured with fluorescence microspheres, do not explain the altered secretion of PAH. The diminished Na,K-ATPase activity in cortex from obstructed kidneys might condition OAT1 function. Additionally, it is also possible to conclude that in the presence of BUO, PAH clearance is not a good estimate of renal plasma flow.

Impairment of cellular redox status and membrane protein activities in kidneys from rats with ischemic acute renal failure.

Cellular redox status and membrane protein activities were analyzed in kidneys from rats with ischemic acute renal failure (ARF). ARF was induced by clamping the left renal artery for 50 min. A parallel group of control animals was processed. In the ischemic group urea plasma levels were statistically increased as compared with the control group. Studies employing whole kidney homogenates revealed that ischemia produces an increment in lipid peroxidation levels and a reduction in glutathione concentration and in superoxide dismutase and glutathione peroxidase activities. Since lipid peroxidation may alter the function of membrane proteins we determined succinate cytochrome c reductase (SuccR), sodium-potassium ATPase (Na-K-ATPase), glucose-6-phosphatase (G-6-Pase) and alkaline phosphatase (ALP) activities in whole renal homogenates. Only G-6-Pase and ALP activities were modified by ischemia. Since ALP is a brush border membrane (BBM) enzyme and BBM is one of the main target structures in ARF, we assessed some parameters of BBM functionality. ALP, gamma-glutamyl transferase (gamma-GT) and 5'-nucleotidase (5'-NT) showed diminished activities in BBM from ischemic kidneys. Ischemia also modified the Vmax of paraaminohippuric acid (PAH) uptake without altering Km. An increment of lipid peroxidation and membrane fluidity in BBM was observed after the treatment. Total membrane proteins and protein recoveries in BBM were similar in both experimental groups. Sialic acid and sulfhydryl levels were similar in BBM from ischemic kidney and control ones. In summary, ARF induced by renal artery clamping for 50 min takes place with a significant increase in urea plasma levels. A decrease in the antioxidant defense system is detected. This induces lipid peroxidation in whole renal tissue, which may justify the diminished activities of some membrane enzymes such as G-6-Pase and ALP. A specific analysis of BBM function reveals a significant increment of lipid peroxidation which may be the cause of an increased membrane fluidity. This latter parameter might be, at least in part, responsible for the damaged function of apical ALP, 5'-NT, gamma-GT and PAH carrier.

Functional expression of pig renal organic anion transporter 3 (pOAT3).

With the cloning of pig renal organic anion transporter 1 (pOAT1) (Biochimie 84 (2002) 1219) we set up a model system for comparative studies of cloned and natively isolated membrane located transport proteins. Meanwhile, another transport protein involved in p-aminohippurate (PAH) uptake on the basolateral side of the proximal tubule cells was identified, designated organic anion transporter 3 (OAT3). To explore the contribution of pOAT1 to the PAH clearance in comparison to OAT3, it was the aim of this study to extend our model by cloning of the pig ortholog of OAT3. Sequence comparisons of human organic anion transporter 3 (hOAT3) with the expressed sequence tag (EST) database revealed a clone and partial sequence of the pig renal organic anion transporter 3 (pOAT3) ortholog. Sequencing of the entire open reading frame resulted in a protein of 543 amino acid residues encoded by 1632 base pairs (EMBL Acc. No. AJ587003). It showed high homologies of 81%, 80%, 76%, and 77% to the human, rabbit, rat, and mouse OAT3, respectively. A functional characterization of pOAT3 in Xenopus laevis oocytes yielded an apparent Km (Kt) for [3H]estrone sulfate of 7.8 +/- 1.3 microM. Moreover, pOAT3 mediated [3H]estrone sulfate uptake was almost abolished by 0.5 mM of glutarate, dehydroepiandosterone sulfate, or probenecid consistent with the hallmarks of OAT3 function.

Caffeine attenuates the renal vascular response to angiotensin II infusion.

Non-modulation has been proposed as an intermediate phenotype in human essential hypertension. The trait is characterized by blunted aldosterone and renal plasma flow responses to short-term angiotensin II (Ang II) infusion. Elevated tissue Ang II levels or decreased tissue adenosine levels could account for this decreased sensitivity to Ang II. In support of the latter possibility, endogenous adenosine has been shown to contribute to the renal vasoconstrictive response to Ang II in animals. We therefore tested the hypothesis that endogenous adenosine contributes to modulation of renal plasma flow in sodium-replete humans. We examined the effect of long-term administration of the adenosine receptor antagonist caffeine on baseline renal plasma flow and on the renal plasma flow response to short-term Ang II infusion in six salt-replete normotensive subjects in a single-blind, placebo-controlled study. para-Aminohippurate clearance was used to assess renal plasma flow. Ang II was infused in graded doses (0.3 to 3 ng/kg per minute) in the presence and absence of caffeine (250 mg PO TID for 7 days). Blood pressure, plasma renin activity, Ang II, electrolytes, and para-aminohippurate clearance were measured before and after each dose of Ang II. Caffeine did not alter either baseline blood pressure or the blood pressure response to Ang II but did increase baseline plasma renin activity from 0.72 +/- 0.09 to 1.42 +/- 0.26 ng angiotensin I/mL per hour (P = .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperglycemia and angiotensin-mediated control of the renal circulation in healthy humans.

Type 1 and type 2 diabetics have an enhanced renal vasodilator response to angiotensin-converting enzyme (ACE) inhibition despite suppressed plasma renin activity (PRA), indicating possible activation of the intrarenal renin angiotensin system. To investigate the role of hyperglycemia, we evaluated the renal hemodynamic response to ACE inhibition in 9 healthy subjects in high-salt balance after steady-state hyperglycemia (8.4+/-1 mmol/L) was achieved via intravenous glucose administration. Renal plasma flow (RPF) and glomerular filtration rate (GFR) responses to captopril and to angiotensin II (Ang II) were measured as paraminohippuric acid and inulin clearances. Hyperglycemia produced a significant increase in RPF of 117 mL. min-1. 1.73 m-2 after 90 minutes but not GFR. Administration of captopril at a dose of 25 mg during glucose infusion led to an increase in RPF of 173+/-24 mL. min-1. 1.73 m-2 (P<0.01) but did not significantly change RPF in the absence of hyperglycemia (7+/-21 mL. min-1. 1.73 m-2). Captopril did not alter GFR in the presence or absence of hyperglycemia. Ang II infusion during hyperglycemia decreased RPF by 45+/-16 mL. min-1. 1. 73 m-2, and this was significantly enhanced by captopril (-98+/-26 mL. min-1. 1.73 m-2, P<0.05). In contrast, there was no enhancement of the vasoconstrictor response to Ang II in the absence of hyperglycemia. PRA did not change with hyperglycemia. Enhancement of renal vasodilation during hyperglycemia by captopril without alteration of PRA suggests activation of the intrarenal renin angiotensin system.

Vancomycin pharmacokinetics, renal handling, and nonrenal clearances in normal human subjects.

The renal handling of vancomycin is unknown. Previously reported studies have not achieved steady-state conditions with constant vancomycin concentrations. We measured systemic vancomycin clearance simultaneously with the renal clearances of vancomycin, creatinine, inulin, and para-aminohippurate in nine healthy subjects at steady-state serum vancomycin concentrations of 7 and 14 mg/L. For all steady-state observations the renal clearance of vancomycin was 89 +/- 11 ml/min (mean +/- SE), the clearance of inulin 105 +/- 9 ml/min, the clearance of creatinine 117 +/- 9 ml/min, and the clearance of para-aminohippuric acid 496 +/- 41 ml/min. The systemic clearance of vancomycin was 131 +/- 7 ml/min. The clearances of creatinine, inulin, and para-aminohippuric acid and the renal clearance of vancomycin were not statistically different at both steady-state vancomycin concentrations. The ratio of the renal clearance of vancomycin to the clearance of inulin was 0.89 +/- 0.06 and to creatinine clearance 0.79 +/- 0.05. Both ratios were independent of vancomycin concentration, urine flow rate, and filtration fraction. The systemic clearance of vancomycin was 10% greater at serum vancomycin concentrations of 14 mg/L than at 7 mg/L (p less than 0.05) because of an increase in the nonrenal clearance. Therefore in healthy subjects, 30% of the systemic vancomycin clearance is by nonrenal mechanisms and this nonrenal clearance is concentration dependent. Assuming protein binding to be between 10% and 20%, renal vancomycin excretion is predominantly by glomerular filtration. Small amounts of tubular vancomycin transport cannot be excluded by these techniques.