Placental transfer of the HIV integrase inhibitor dolutegravir in an ex vivo human cotyledon perfusion model.

OBJECTIVES: Data on fetal exposure to antiretroviral agents during pregnancy are important to estimate their potential for prevention of mother-to-child transmission (PMTCT) and possible toxicity. For the recently developed HIV integrase inhibitor dolutegravir, clinical data on fetal disposition are not yet available. Dual perfusion of a single placental lobule (cotyledon) provides a useful ex vivo model to predict the in vivo maternal-to-fetal transfer of this drug. The aim of this study was to estimate the transfer of dolutegravir across the human term placenta, using a dual-perfusion cotyledon model. METHODS: After cannulation of the cotyledons (n = 6), a fetal circulation of 6 mL/min and maternal circulation of 12 mL/min were initiated. The perfusion medium consisted of Krebs-Henseleit buffer (pH = 7.2-7.4) supplemented with 10.1 mM glucose, 30 g/L human serum albumin and 0.5 mL/L heparin 5000IE. Dolutegravir was administered to the maternal circulation (∼ 4.2 mg/L) and analysed by UPLC-MS/MS. RESULTS: After 3 h of perfusion, the mean ± SD fetal-to-maternal (FTM) concentration ratio of dolutegravir was 0.6 ± 0.2 and the mean ± SD concentrations in the maternal and fetal compartments were 2.3 ± 0.4 and 1.3 ± 0.3 mg/L, respectively. CONCLUSIONS: Dolutegravir crosses the blood-placental barrier with a mean FTM concentration ratio of 0.6. Compared with other antiretroviral agents, placental transfer of dolutegravir is moderate to high. These data suggest that dolutegravir holds clinical potential for pre-exposure prophylaxis and consequently PMTCT, but also risk of fetal toxicity.

Sunitinib does not attenuate contractile force following a period of ischemia in isolated human cardiac muscle.

Concerns have been raised about the development of heart failure in patients treated for cancer with angiogenesis inhibitors, such as the tyrosine kinase inhibitor sunitinib. Patients with previous coronary artery disease and hypertension have an increased risk of developing heart failure. Therefore, we studied the effect of sunitinib on the contractility of isolated human atrial trabeculae and the effect on recovery after ischemic stimulation. After informed consent, the atrial appendage of patients undergoing cardiac surgery was harvested and isolated trabeculae were placed in an organ bath with a force transducer. During electrical stimulation, contractile force was measured during normal pacing or after simulated ischemia. Of each patient, one trabecula was perfused with control and one with sunitinib. Contractile force (expressed as percentage of baseline force) declined over time to 57 ± 8 and 73 ± 20% after 150 min of stimulation for solvent- and sunitinib-treated trabeculae, respectively (mean ± SE; n = 8; p > 0.1). After simulated ischemia and reperfusion, contractile force was 40 ± 6% in the control compared to 39 ± 6% in the sunitinib-treated trabeculae during the last final 5 min of reperfusion (n = 12; p > 0.1). Sunitinib at low, but clinically relevant, concentrations does not have a direct effect on function of human atrial cardiomyocytes nor does it attenuate the recovery in contractile force of atrial cardiomyocytes after a period of ischemia. A direct and acute toxic effect on cardiomyocytes does not explain the development of heart failure in patients treated with sunitinib.

Renal glucuronidation and multidrug resistance protein 2-/ multidrug resistance protein 4-mediated efflux of mycophenolic acid: interaction with cyclosporine and tacrolimus.

Mycophenolic acid (MPA) is an immunosuppressant used in transplant rejection, often in combination with cyclosporine (CsA) and tacrolimus (Tac). The drug is cleared predominantly via the kidneys, and 95% of the administered dose appears in urine as 7-hydroxy mycophenolic acid glucuronide (MPAG). The current study was designed to unravel the renal excretory pathway of MPA and MPAG, and their potential drug-drug interactions. The role of multidrug resistance protein (MRP) 2 and MRP4 in MPA disposition was studied using human embryonic kidney 293 (HEK293) cells overexpressing the human transporters, and in isolated, perfused kidneys of Mrp2-deficient rats and Mrp4-deficient mice. Using these models, we identified MPA as substrate of MRP2 and MRP4, whereas its MPAG appeared to be a substrate of MRP2 only. CsA inhibited MPAG transport via MRP2 for 50% at 8 µM (P < 0.05), whereas Tac had no effect. This was confirmed by cell survival assays, showing a 10-fold increase in MPA cytotoxicity (50% reduction in cell survival changed from 12.2 ± 0.3 µM to 1.33 ± 0.01 µM by MPA + CsA; P < 0.001) and in perfused kidneys, showing a 50% reduction in MPAG excretion (P < 0.05). The latter effect was observed in Mrp2-deficient animals as well, supporting the importance of Mrp2 in MPAG excretion. CsA, but not Tac, inhibited MPA glucuronidation by rat kidney homogenate and human uridine 5'-diphospho-glucuronosyltransferase-glucuronosyltransferase 1A9 (P < 0.05 and P < 0.01, respectively). We conclude that MPA is a substrate of both MRP2 and MRP4, but MRP2 is the main transporter involved in renal MPAG excretion. In conclusion, CsA, but not Tac, influences MPA clearance by inhibiting renal MPA glucuronidation and MRP2-mediated MPAG secretion.

Nitric oxide down-regulates the expression of organic cation transporters (OCT) 1 and 2 in rat kidney during endotoxemia.

In the kidney, P-glycoprotein (Abcb1), an ATP-driven drug efflux pump, plays an important role in the detoxification of proximal tubule cells through the excretion of cationic and amphipathic organic compounds. We recently found that NO, produced by renal inducible NO synthase (iNOS), is involved in an up-regulation of P-glycoprotein during endotoxemia in rats. In the present study, we investigated the functional consequences of endotoxemia on the renal handling of rhodamine 123 by using isolated perfused rat kidneys. Wistar Hannover rats were injected intraperitoneally with 5 mg/kg body weight lipopolysaccharide (LPS) or with both LPS and the iNOS inhibitor, aminoguanidine. Despite an increased P-glycoprotein expression, we found a diminished urinary rhodamine 123 clearance 12 h after LPS (P<0.001). In addition, we found a diminished perfusate clearance (P<0.05) for rhodamine 123 after LPS treatment, suggesting a predominant role of influx carriers in urinary rhodamine 123 excretion. We examined the expression levels of organic cation transporter 1 (Slc22a1/Oct1) and Slc22a2/Oct2. Both appeared to be down-regulated at the mRNA and protein level, 12 h after LPS. Co-administration of aminoguanidine attenuated the down-regulation of both Oct1 and Oct2 protein expression and reversed the decrease in rhodamine 123 clearance (P<0.001). These findings indicate that NO, produced by iNOS, is responsible for a down-regulation of the influx carriers, Oct1 and Oct2.

Nitric oxide differentially regulates renal ATP-binding cassette transporters during endotoxemia.

Nitric oxide (NO) is an important regulator of renal transport processes. In the present study, we investigated the role of NO, produced by inducible NO synthase (iNOS), in the regulation of renal ATP-binding cassette (ABC) transporters in vivo during endotoxemia. Wistar-Hannover rats were injected with lipopolysaccharide (LPS(+)) alone or in combination with the iNOS inhibitor, aminoguanidine. Controls received detoxified LPS (LPS(-)). After LPS(+), proximal tubular damage and a reduction in renal function were observed. Furthermore, iNOS mRNA and protein, and the amount of NO metabolites in plasma and urine, increased compared to the LPS(-) group. Coadministration with aminoguanidine resulted in an attenuation of iNOS induction and reduction of renal damage. Gene expression of 20 ABC transporters was determined. After LPS(+), a clear up-regulation in Abca1, Abcb1/P-glycoprotein (P-gp), Abcb11/bile salt export pump (Bsep), and Abcc2/multidrug resistance protein (Mrp2) was found, whereas Abcc8 was down-regulated. Up-regulation of Abcc2/Mrp2 was accompanied by enhanced calcein excretion. Aminoguanidine attenuated the effects on transporter expression. Our data indicate that NO, produced locally by renal iNOS, regulates the expression of ABC transporters in vivo. Furthermore, we showed, for the first time, expression and subcellular localization of Abcb11/Bsep in rat kidney.

Increased apical insertion of the multidrug resistance protein 2 (MRP2/ABCC2) in renal proximal tubules following gentamicin exposure.

Multidrug resistance protein (MRP) 2 (MRP2; ABCC2), an organic anion transporter apically expressed in liver, kidney, and intestine, plays an important protective role through facilitating the efflux of potentially toxic compounds. We hypothesized that upon a toxic insult, MRP2 is up-regulated in mammalian kidney, thereby protecting the tissue from damage. We studied the effects of the nephrotoxicant gentamicin on the functional expression of MRP2 in transfected Madin-Darby canine kidney type II (MDCKII) cells and rat kidney. Transport of glutathionemethyl fluorescein by cells or calcein by isolated perfused rat kidney was measured to monitor MRP2 activity. MDCKII cells were exposed to gentamicin (0-1000 microM) for either 1 h, 24 h, or for 1 h followed by 24-h recovery. No effect was observed on MRP2 after 1-h exposure. After 24-h gentamicin exposure or after a 24-h recovery period following 1-h exposure, an increase in MRP2-mediated transport was seen. This up-regulation was accompanied by a 2-fold increase in MRP2 protein expression in the apical membrane, whereas the expression in total cell lysates remained unchanged. In perfused kidneys of rats exposed to gentamicin (100 mg/kg) for seven consecutive days, an increase in Mrp2 function and expression was found, which was prevented by addition of a dual endothelin-receptor antagonist, bosentan. We conclude that an increased shuttling of the transporter to the apical membrane takes place in response to gentamicin exposure, which is triggered by endothelin. Up-regulation of MRP2 in the kidney may be interpreted as part of a protective mechanism.

Intravenously administered short interfering RNA accumulates in the kidney and selectively suppresses gene function in renal proximal tubules.

Different gene-silencing methods, like antisense and short interfering RNA (siRNA), are widely used as experimental tools to inhibit gene expression. In the present study, the in vivo behavior of siRNA in rats and siRNA-mediated silencing of genes in the renal proximal tubule were investigated. To study the biodistribution of siRNA, rats were injected i.v. with radiolabeled siRNA or radiolabel alone (control), and scintigraphic images were acquired at different time intervals postinjection. The siRNA preferentially accumulated in the kidneys and was excreted in the urine. One hour after injection, the amount of siRNA present in both kidneys (1.7 +/- 0.3% of injected dose/g tissue) was on average 40 times higher than in other tissues (liver, brain, intestine, muscle, lung, spleen, and blood). Besides the biodistribution, the effect of siRNA on multidrug resistance protein isoform 2 (Mrp2/Abcc2, siRNAMrp2) in renal proximal tubules was investigated. Mrp2 function was assessed by measuring the excretion of its fluorescent substrate calcein in the isolated perfused rat kidney. Four days after administration, siRNAMrp2 reduced the urinary calcein excretion rate significantly (35% inhibition over the period 80-150 min of perfusion). This down-regulation was specific because another siRNA sequence directed against a different transporter in the proximal tubule, Mrp4 (Abcc4, siRNAMrp4), did not alter the Mrp2-mediated excretion of calcein. In conclusion, siRNA accumulates spontaneously in the kidney after i.v. injection, where it selectively suppresses gene function in the proximal tubules. Therefore, i.v. administered siRNA provides a novel experimental and potential therapeutic tool for gene silencing in the kidney.

Iron chelators do not reduce cold-induced cell injury in the isolated perfused rat kidney model.

BACKGROUND: In vitro, cold-induced injury is an important contributor to renal tubular cell damage. It is mediated by iron-dependent formation of reactive oxygen species and can be prevented by iron chelation. We studied whether iron chelators can prevent cold-induced damage in the isolated perfused rat kidney (IPK) model both after cold perfusion (CP) and after cold storage (CS). We hypothesized that in the CP model iron-dependent cold-induced injury is more pronounced, since oxygen is constantly provided. METHODS: The IPK was either flushed with University of Wisconsin (UW) solution and stored for 4, 18 or 24 h at 4 degrees C or perfused during 4 h at 4 degrees C with UW for machine perfusion. The iron chelators 2,2'-dipyridyl or desferal, or the negative control 4,4'-dipyridyl were added during the cold perfusion. Kidney function was measured during 2 h reperfusion at 37.5 degrees C and compared to a control group (without cold preservation). RESULTS: Compared to control perfusion, kidney function was decreased in all experimental protocols. glomerular filtration rate and FR(H2O) were significantly decreased, while FE(gluc) and FE(Na) were higher after 4 h CS and CP. After 4 h CP, also renal vascular resistance was increased. Addition of 2,2'-dipyridyl did not improve kidney function after either CS or CP. Prolonged periods of CS worsened kidney function. The addition of 2,2'-dipyridyl or desferal did not improve kidney function after longer periods of CS. CONCLUSIONS: Addition of an iron chelator to the preservation solution UW did not improve kidney function after both CS and CP. Iron chelation is not able to prevent cold-induced damage in the isolated perfused rat kidney.

Contribution of multidrug resistance protein 2 (MRP2/ABCC2) to the renal excretion of p-aminohippurate (PAH) and identification of MRP4 (ABCC4) as a novel PAH transporter.

p-Aminohippurate (PAH) is the classical substrate used in the characterization of organic anion transport in renal proximal tubular cells. Although basolateral transporters for PAH uptake from blood into the cell have been well characterized, there is still little knowledge on the apical urinary efflux transporters. The multidrug resistance protein 2 (MRP2/ABCC2) is localized to the apical membrane and mediates ATP-dependent PAH transport, but its contribution to urinary PAH excretion is not known. In this report, we show that renal excretion of PAH in isolated perfused kidneys from wild-type and Mrp2-deficient (TR(-)) rats is not significantly different. Uptake of [(14)C]PAH in membrane vesicles expressing two different MRP2 clones isolated from Sf9 and MDCKII cells exhibited a low affinity for PAH (Sf9, 5 +/- 2 mM; MDCKII, 2.1 +/- 0.6 mM). Human MRP4 (ABCC4), which has recently been localized to the apical membrane, expressed in Sf9 cells had a much higher affinity for PAH (K(m) = 160 +/- 50 microM). Various inhibitors of MRP2-mediated PAH transport also inhibited MRP4. Probenecid stimulated MRP2 at low concentrations but had no effect on MRP4; but at high probenecid concentrations, both MRP2 and MRP4 were inhibited. Sulfinpyrazone only stimulated MRP2, but inhibited MRP4. Real-time PCR and Western blot analysis showed that renal cortical expression of MRP4 is approximately fivefold higher as compared with MRP2. MRP4 is a novel PAH transporter that has higher affinity for PAH and is expressed more highly in kidney than MRP2, and may therefore be more important in renal PAH excretion.

Impaired renal secretion of substrates for the multidrug resistance protein 2 in mutant transport-deficient (TR-) rats.

Previous studies with mutant transport-deficient rats (TR(-)), in which the multidrug resistance protein 2 (Mrp2) is lacking, have emphasized the importance of this transport protein in the biliary excretion of a wide variety of glutathione conjugates, glucuronides, and other organic anions. Mrp2 is also present in the luminal membrane of proximal tubule cells of the kidney, but little information is available on its role in the renal excretion of xenobiotics. The authors compared renal transport of the fluorescent Mrp2 substrates calcein, fluo-3, and lucifer yellow (LY) between perfused kidneys isolated from Wistar Hannover (WH) and TR(-) rats. Isolated rat kidneys were perfused with 100 nM of the nonfluorescent calcein-AM or 500 nM fluo3-AM, which enter the tubular cells by diffusion and are hydrolyzed intracellularly into the fluorescent anion. The urinary excretion rates of calcein and fluo-3 were 3 to 4 times lower in perfused kidneys from TR(-) rats compared with WH rats. In contrast, the renal excretion of LY (10 micro M, free anion) was somewhat delayed but appeared unimpaired in TR(-) rats. Membrane vesicles from Sf9 cells expressing human MRP2 or human MRP4 indicated that MRP2 exhibits a preferential affinity for calcein and fluo-3, whereas LY is a better substrate for MRP4. We conclude that the renal clearance of the Mrp2 substrates calcein and fluo-3 is significantly reduced in TR(-) rat; for LY, the absence of the transporter may be compensated for by (an)other organic anion transporter(s).