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.

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.

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.

The anthraquinone drug rhein potently interferes with organic anion transporter-mediated renal elimination.

Rhein, a major metabolite of the prodrug diacerein and a major component of the medicinal herb Rheum sp., is used for its beneficial effects in a variety of clinical applications including the treatment of osteoarthritis and diabetic nephropathy. The physicochemical properties of rhein are consistent with those of known organic anion transporter (OAT) substrates and inhibitors. Therefore, the inhibitory effect of rhein on human (h) OAT1, hOAT3, hOAT4, and murine (m) Oat1 and mOat3 was examined in heterologous cell lines stably expressing each transporter in isolation. Rhein was shown to potently inhibit hOAT1 and hOAT3, with IC50 estimates in the low nanomolar range (IC50=77.1±5.5 nM and 8.4±2.5 nM, respectively), while poor affinity was observed for hOAT4 (IC50>100 µM). Marked species differences were observed with hOAT1 and hOAT3 exhibiting 3- and 28-fold higher affinity for rhein as compared to their murine orthologs. The estimated drug-drug interaction (DDI) indices (>>0.1) indicated a very strong potential for clinically relevant, rhein perpetrated DDIs mediated by inhibition of hOAT1 (DDI index=5.0; 83% inhibition) and/or hOAT3 (DDI index=46; 98% inhibition) transport activity. These results suggested that rhein, from herbal medicines and/or prodrug conversion, may significantly impact the dosing, efficacy and toxicity (i.e., pharmacokinetics and pharmacodynamics) of co-administered hOAT1 and/or hOAT3 drug substrates.

Simultaneous determination of renal plasma flow and glomerular filtration rate in conscious mice using dual bolus injection.

INTRODUCTION: The present report describes and evaluates a simple protocol for serial measurements of glomerular filtration rate (GFR) and renal plasma flow (RPF) in conscious mice. METHODS: In conscious mice, a bolus of [(3)H]methoxy-inulin and [(14)C]para-amino-hippuric (PAH) was injected in the tail vein whereupon eight blood samples were taken during the following 75min. Plasma concentrations were determined by liquid scintillation and clearances of the injected markers were calculated by non-compartmental pharmacokinetic data analysis of the plasma disappearance curves. In anaesthetized mice, the renal extraction ratio of PAH was determined by infusion of PAH and subsequent analysis of blood taken from the carotid artery and the renal vein. The acquired value (0.70±0.02) was used for all subsequent calculations of RPF. To evaluate the protocol, a crossover study was performed where either the vehicle or the angiotensin II AT1 receptor antagonist candesartan was given prior to the clearance measurements. RESULTS: Baseline values of GFR and RPF were in line with those earlier reported in mice. Administration of candesartan increased RPF and reduced the filtration fraction, whereas GFR was unaltered. These changes are supported by earlier findings and demonstrate that GFR and RPF can be determined independently. Furthermore, modelling experiments demonstrated that acceptable results are obtained even if the number of blood samples is reduced to four which is a way to further simplify the procedure. DISCUSSION: The method provides an effective way for repeated measurements of GFR and RPF in mice without potentially confounding effects of anaesthesia.

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.

Determination of p-aminohippuric acid in rat plasma by liquid chromatography-tandem mass spectrometry.

A rapid, simple and sensitive method was developed for the determination of para-aminohippuric acid (PAH) in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS-MS). Acetaminophen was used as the internal standard. Chromatographic separation was performed using a Symmetry C18 column and the mobile phase was composed of A: 2 mM ammonium formate and 0.1% formic acid in water and B: 2 mM ammonium formate and 0.1% formic acid in acetonitrile (ACN) (A:B, 30:70, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 195.2-->120.2 and 152.1-->110.1 for PAH and acetaminophen, respectively. Good linearity is observed over the concentration range of 0.1-500 microg/ml. The method was proved to be accurate and reliable and was applied to a pharmacokinetic study in rat.

Determination of para-aminohippuric acid (PAH) in human plasma and urine by liquid chromatography-tandem mass spectrometry.

In this manuscript, we present a simple and reliable method for the quantitation of para-aminohippuric acid (PAH; 2-(4-aminobenzamido)acetic acid) in human plasma and urine using liquid chromatography coupled to electrospray ionisation low-energy collision-induced dissociation tandem mass spectrometry (HPLC-ESI-CID-MS/MS) analysis (negative ion mode) via multiple reaction monitoring (MRM). Sample preparation involved protein precipitation of plasma samples with acetonitrile and subsequent dilution of urine samples with the mobile phase. The internal standard (IS) adopted was para-aminosalicylic acid (PAS; 4-amino-2-hydroxybenzoic acid). Chromatographic separation was achieved on a Cosmosil HILIC column using an isocratic mobile phase consisting of ammonium acetate buffer (20mM) and acetonitrile (45:55, v/v) at a flow rate of 200microl/min. The transactions monitored were m/z 192.9-->149.1 for PAH and m/z 152.1-->108.1 for IS. Linear calibration curves were generated over the PAH concentration range of 0.2-100mg/L in human plasma and urine. The method was validated for selectivity, accuracy, precision, recovery and stability according to USFDA criteria, and has been successfully applied to a pharmacokinetic study in healthy volunteers administered an intravenous dose of 440mg PAH.

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.

Regulation of human organic anion transporter 1 by ANG II: involvement of protein kinase Calpha.

Human organic anion transporter 1 (hOAT1) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT1 is abundantly expressed in the kidney. In the current study, we examined the regulation of hOAT1 by ANG II in kidney COS-7 cells. ANG II induced a concentration- and time-dependent inhibition of hOAT1 transport activity. Such inhibition mainly resulted from a decreased cell surface expression without a change in total cell expression of the transporter, kinetically revealed as a decreased maximal velocity without significant change in Michaelis constant. ANG II-induced inhibition of hOAT1 activity could be prevented by treating hOAT1-expressing cells with staurosporine, a general protein kinase C (PKC) inhibitor. To obtain further information on which PKC isoform mediates ANG II regulation of hOAT1 activity, cellular distribution of various PKC isoforms was examined in cells treated with or without ANG II. We showed that ANG II treatment resulted in a significant translocation of PKCalpha from cytosol to membrane, and such translocation was blocked by treating hOAT1-expressing cells with Gö-6976, a PKCalpha-specific inhibitor. We further showed that ANG II-induced inhibition of hOAT1 activity and retrieval of hOAT1 from the cell surface could also be prevented by treating hOAT1-expressing cells with Gö-6976. We concluded that ANG II inhibited hOAT1 activity through activation of PKCalpha, which led to the redistribution of the transporter from the cell surface to the intracellular compartments.

The effect of oral contraceptives on the nitric oxide system and renal function.

We have demonstrated that oral contraceptive (OC) users exhibit elevated angiotensin II levels and angiotensin II type 1 receptor expression, indicative of renin-angiotensin system (RAS) activation, yet the renal and systemic consequences are minimal, suggesting that there is increased vasodilatory activity, counteracting the effect of RAS activation. We hypothesized that the nitric oxide (NO) system would be upregulated in OC users and that this would be reflected by a blunted hemodynamic response to l-arginine infusion. All subjects were studied after a 7-day controlled sodium and protein diet. Inulin and para-aminohippurate clearance techniques were used to assess renal function. l-Arginine was infused at 100, 250, and 500 mg/kg, each over 30 min. Skin endothelial NO synthase mRNA expression was assessed by real-time PCR. While OC nonusers exhibited significant increases in effective renal plasma flow (670.8 +/- 35.6 to 816.2 +/- 59.7 ml.min(-1).1.73 m(-2)) and glomerular filtration rate (133.4 +/- 4.3 to 151.0 +/- 5.7 ml.min(-1).1.73 m(-2), P = 0.04) and declines in renal vascular resistance (81.1 +/- 6.1 to 63.5 +/- 6.2 mmHg.ml(-1).min, P = 0.001) at the lower l-arginine infusion rates, the responses in OC users were blunted. While l-arginine reduced mean arterial pressure at the 250 and 500 mg/kg doses in OC nonusers, OC users only exhibited a decrease in mean arterial pressure at the highest infusion rate. In contrast, tissue endothelial NO synthase mRNA levels were higher in the OC users (P = 0.04). In summary, these findings suggest that the NO system is upregulated by OC use in young, healthy women. Increased activity of the NO pathway may modulate the hemodynamic effects of RAS activation in OC users.

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.

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.

Decreased renal transplant function after parathyroidectomy.

BACKGROUND: Persistent secondary hyperparathyroidism after renal transplantation may require parathyroidectomy (PTX). Clinical experience suggests that these patients commonly develop decreased renal function thereafter. METHODS: To test this notion, we evaluated 76 transplant patients who underwent pararhyroidectomy between 1997 and 2003. RESULTS: In half the patients (47%), creatinine clearance decreased >20% (before vs after PTX, 57 +/- 21 vs 38 +/- 17 ml/min, P = 0.001). The patients with decreased creatinine clearance had higher parathyroid hormone (PTH) concentrations before and lower values after PTX compared with those who did not (594 +/- 392 vs 447 +/- 234 pg/ml before PTX, P = 0.03; 35 vs 123 pg/ml thereafter, P = 0.002). They also had lower serum calcium concentrations after PTX (2.0 vs 2.2 mmol/l, P = 0.005) and they required more calcium and vitamin D analogues. These patients also more commonly underwent total PTX with autotransplantation, compared with subtotal (75 vs 50%, P = 0.03). However, in multivariate analysis, only the delta PTH decline (%) after PTX was a significant predictor of deteriorating renal function (P = 0.005) and was correlated with the creatinine clearance decrease (R = 0.369, P = 0.001). Prospectively measured inulin and para-amino-hippuric acid (PAH) clearance decreased significantly after PTX in a subgroup of 19 patients (inulin before vs after PTX 67 vs 55 ml/min/1.73 m(2), P = 0.001; PAH 360 vs 289 ml/min/1.73 m(2), P = 0.001). Transplant biopsies revealed calcification in 70% of biopsied cases. CONCLUSION: Since PTH has a known positive regulatory effect on renal perfusion and glomerular filtration rate, we conclude that relative hypoparathyroidism after PTX is the main mechanism contributing to decreased renal function in these patients. There was no difference in 10-year-graft survival between the deteriorating and the non-deteriorating group.

Ifosfamide toxicity in cultured proximal renal tubule cells.

Renal injury is a common side effect of the chemotherapeutic agent ifosfamide. Current evidence suggests that ifosfamide metabolites, particularly chloroacetaldehyde, produced within the kidney contribute to nephrotoxicity. The present study examined the effects of ifosfamide and its metabolites, chloroacetaldehyde and acrolein, on rabbit proximal renal tubule cells in primary culture, using a transwell culture system that allows separate access to apical and basolateral cell surfaces. The ability of the uroprotectant medications sodium 2-mercaptoethanesulfonate (mesna) and amifostine to prevent chloroacetaldehyde-and acrolein-induced renal cell injury was also assessed. Ifosfamide (2,000-4,000 microM) did not affect transcellular inulin diffusion but caused a modest but significant impairment in organic ion transport; this impairment was greater when ifosfamide was added to the basolateral compartment of the transwell. Chloroacetaldehyde and acrolein (6.25-100 microM) produced dose-dependent impairments in transcellular inulin diffusion and organic ion transport. Chloroacetaldehyde was a more potent toxin than acrolein. Co-administration of mesna or amifostine prevented metabolite toxicity. Amifostine was only protective when added to the apical compartment of transwells. These results show that ifosfamide is taken up by renal tubule cells preferentially through their basolateral surfaces, and supports the hypothesis that chloroacetaldehyde is primarily responsible for ifosfamide-induced nephrotoxicity. The protective effect of mesna and amifostine in vitro contrasts with clinical experience showing that these medications do not eliminate ifosfamide nephrotoxicity in vivo.

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.

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.

Differences in decline in GFR with age between males and females. Reference data on clearances of inulin and PAH in potential kidney donors.

BACKGROUND: To ensure that potential kidney donors have no renal impairment, it is extremely important to have accurate methods for evaluating the glomerular filtration rate (GFR). The golden standard, clearance of inulin, has been used in the present study. The aim was to evaluate the effects of age and sex on renal function and present reference data. METHODS: A total of 122 potential kidney donors, 62 females, aged 21-67 years, were investigated with the GFR and effective renal plasma flow (ERPF) determined by clearances of inulin and para-amino hippurate. RESULTS: The mean +/- SD GFR and ERPF were 105 +/- 13 and 545 +/- 108 ml/min/1.73 m(2), respectively, and we found no difference between the males and females. When relating GFR and ERPF to age, however, a significant decline was found in GFR and ERPF in males, but not in females in the age range of 20-50 years. GFR fell by a mean of 8.7 ml/min/1.73 m(2) and ERPF by 90 ml/min/1.73 m(2) per decade in male donors. CONCLUSION: With adequate methods for determining GFR and ERPF, a clear difference in the effect of age was seen between the sexes. Males showed a significant decrease between 20 and 50 years of age, which was not seen in females. Females seem to be protected in the pre-menopausal period probably by oestrogens. These results confirm clinically those found in rats.