Hepatic Transporter Alterations by Nuclear Receptor Agonist T0901317 in Sandwich-Cultured Human Hepatocytes: Proteomic Analysis and PBPK Modeling to Evaluate Drug-Drug Interaction Risk.

In vitro approaches for predicting drug-drug interactions (DDIs) caused by alterations in transporter protein regulation are not well established. However, reports of transporter regulation via nuclear receptor (NR) modulation by drugs are increasing. This study examined alterations in transporter protein levels in sandwich-cultured human hepatocytes (SCHH; n = 3 donors) measured by liquid chromatography-tandem mass spectrometry-based proteomic analysis after treatment with N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide (T0901317), the first described synthetic liver X receptor agonist. T0901317 treatment (10 µM, 48 hours) decreased the levels of organic cation transporter (OCT) 1 (0.22-, 0.43-, and 0.71-fold of control) and organic anion transporter (OAT) 2 (0.38-, 0.38-, and 0.53-fold of control) and increased multidrug resistance protein (MDR) 1 (1.37-, 1.48-, and 1.59-fold of control). The induction of NR downstream gene expression supports the hypothesis that T0901317 off-target effects on farnesoid X receptor and pregnane X receptor activation are responsible for the unexpected changes in OCT1, OAT2, and MDR1. Uptake of the OCT1 substrate metformin in SCHH was decreased by T0901317 treatment. Effects of decreased OCT1 levels on metformin were simulated using a physiologically-based pharmacokinetic (PBPK) model. Simulations showed a clear decrease in metformin hepatic exposure resulting in a decreased pharmacodynamic effect. This DDI would not be predicted by the modest changes in simulated metformin plasma concentrations. Altogether, the current study demonstrated that an approach combining SCHH, proteomic analysis, and PBPK modeling is useful for revealing tissue concentration-based DDIs caused by unexpected regulation of hepatic transporters by NR modulators. SIGNIFICANCE STATEMENT: This study utilized an approach combining sandwich-cultured human hepatocytes, proteomic analysis, and physiologically based pharmacokinetic modeling to evaluate alterations in pharmacokinetics (PK) and pharmacodynamics (PD) caused by transporter regulation by nuclear receptor modulators. The importance of this approach from a mechanistic and clinically relevant perspective is that it can reveal drug-drug interactions (DDIs) caused by unexpected regulation of hepatic transporters and enable prediction of altered PK and PD changes, especially for tissue concentration-based DDIs.

Mechanistic Modeling of the Hepatic Disposition of Estradiol-17ß-Glucuronide in Sandwich-Cultured Human Hepatocytes.

Estradiol-17ß-glucuronide (E217G) is an estrogen metabolite that has cholestatic properties. In humans, circulating E217G is transported into hepatocytes by organic anion transporting polypeptides (OATPs) and is excreted into bile by multidrug-resistance associated protein 2 (MRP2). E217G is also a substrate of the basolateral efflux transporters MRP3 and MRP4, which translocate E217G from hepatocytes to blood. However, the contribution of basolateral efflux to hepatocyte disposition of E217G has not been evaluated previously. To address this question, E217G disposition was studied in sandwich-cultured human hepatocytes and mechanistic modeling was applied to calculate clearance values (mean ± S.D.) for uptake, intrinsic biliary excretion (CLint,bile) and intrinsic basolateral efflux (CLint,BL). The biliary excretion index of E217G was 45% ± 6%. The CLint,BL of E217G [0.18 ± 0.03 (ml/min)/g liver) was 1.6-fold higher than CLint,bile [0.11 ± 0.06 (ml/min)/g liver]. Simulations were performed to study the effects of increased CLint,BL and a concomitant decrease in CLint,bile on hepatic E217G exposure. Results demonstrated that increased CLint,BL can effectively reduce hepatocellular and biliary exposure to this potent cholestatic agent. Simulations also revealed that basolateral efflux can compensate for impaired biliary excretion and, vice versa, to avoid accumulation of E217G in hepatocytes. However, when both clearance processes are impaired by 90%, hepatocyte E217G exposure increases up to 10-fold. These data highlight the contribution of basolateral efflux transport, in addition to MRP2-mediated biliary excretion, to E217G disposition in human hepatocytes. This elimination route could be important, especially in cases where basolateral efflux is induced, such as cholestasis. SIGNIFICANCE STATEMENT: The disposition of the cholestatic estrogen metabolite estradiol-17ß-glucuronide (E217G) was characterized in sandwich-cultured human hepatocytes. The intrinsic basolateral efflux clearance was estimated to be 1.6-fold higher than the intrinsic biliary excretion clearance, emphasizing the contribution of basolateral elimination in addition to biliary excretion. Simulations highlight how hepatocytes can effectively cope with increased E217G through the regulation of both basolateral and biliary transporters.

Altered Expression and Function of Hepatic Transporters in a Rodent Model of Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common form of inherited polycystic kidney disease (PKD) and is a leading cause of kidney failure. Fluid-filled cysts develop in the kidneys of patients with ADPKD, and cysts often form in their liver and other organs. Previous data have shown that bile acids are increased in the liver of polycystic kidney (PCK) rats, a rodent model of PKD; these changes may be associated with alterations in liver transporter expression and function. However, the impact of PKD on hepatic transporters has not been characterized. Therefore, this preclinical study was designed to investigate hepatic transporter expression and function in PCK compared with wild-type (WT) Sprague-Dawley rats. Transporter gene expression was measured by quantitative polymerase chain reaction, and protein levels were quantified by Western blot and liquid chromatography-tandem mass spectroscopy (LC-MS/MS)-based proteomic analysis in rat livers. Transporter function was assessed in isolated perfused livers (IPLs), and biliary and hepatic total glutathione content was measured. Protein expression of Mrp2 and Oatp1a4 was decreased 3.0-fold and 2.9-fold, respectively, in PCK rat livers based on Western blot analysis. Proteomic analysis confirmed a decrease in Mrp2 and a decrease in Oatp1a1 expression (PCK/WT ratios, 0.368 ± 0.098 and 0.563 ± 0.038, respectively; mean ± S.D.). The biliary excretion of 5(6)-carboxy-2',7'-dichlorofluorescein, a substrate of Oatp1a1, Mrp2, and Mrp3, was decreased 28-fold in PCK compared with WT rat IPLs. Total glutathione was significantly reduced in the bile of PCK rats. Differences in hepatic transporter expression and function may contribute to altered disposition of Mrp2 and Oatp substrates in PKD.

Protein expression and function of organic anion transporters in short-term and long-term cultures of Huh7 human hepatoma cells.

Human-derived hepatic cell lines are a valuable alternative to primary hepatocytes for drug metabolism, transport and toxicity studies. However, their relevance for investigations of drug-drug and drug-organic anion (e.g., bile acid, steroid hormone) interactions at the transporter level remains to be established. The aim of the present study was to determine the suitability of the Huh7 cell line for transporter-dependent experiments. Huh7 cells were cultured for 1 to 4 weeks and subsequently were analyzed for protein expression, localization and activity of solute carrier (SLC) and ATP-binding cassette (ABC) transporters involved in organic anion transport using liquid chromatography-tandem mass spectroscopy, immunocytochemistry, and model substrates [3H]taurocholate (TCA), [3H]dehydroepiandrosterone sulfate (DHEAS) and 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) diacetate. The extended 4-week culture resulted in a phenotype resembling primary hepatocytes and differentiated HepaRG cells: cuboidal hepatocyte-like cells with elongated bile canaliculi-like structures were surrounded by epithelium-like cells. Protein expression of OSTα, OSTß and OATP1B3 increased over time. Moreover, the uptake of the SLC probe substrate DHEAS was higher in 4-week than in 1-week Huh7 cultures. NTCP, OATP1B1, BSEP and MRP3 were barely or not detectable in Huh7 cells. OATP2B1, MRP2 and MRP4 protein expression remained at similar levels over the four weeks of culture. The activity of MRP2 and the formation of bile canaliculi-like structures were confirmed by accumulation of CDF in the intercellular compartments. Results indicate that along with morphological maturation, transporters responsible for alternative bile acid secretion pathways are expressed and active in long-term cultures of Huh7 cells, suggesting that differentiated Huh7 cells may be suitable for studying the function and regulation of these organic anion transporters.

Involvement of Claudin-11 in Disruption of Blood-Brain, -Spinal Cord, and -Arachnoid Barriers in Multiple Sclerosis.

It is important to understand the molecular mechanisms of barrier disruption in the central nervous system (CNS) of patients with multiple sclerosis (MS). The purpose of the present study was to clarify whether claudin-11 is involved in the disruption of two endothelial barriers (blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB)) and two epithelial barriers (blood-arachnoid barrier (BAB) and blood-CSF barrier (BCSFB)) in the CNS in MS. Immunohistochemical analysis revealed that, in both normal human and mouse, claudin-11 is co-localized with claudin-5 in the brain and spinal cord capillaries. The absolute protein expression level of claudin-11 was nearly equal to that of claudin-5 in rat brain capillaries, but was 2.81-fold greater in human brain capillaries. The protein expressions of claudin-11 were significantly downregulated in the brain and spinal cord capillaries of an MS patient and experimental autoimmune encephalomyelitis (EAE) mice. Specific downregulation of claudin-11 with siRNA significantly increased the transfer of membrane-impermeable FITC-dextran across human brain capillary endothelial cell (hCMEC/D3) monolayer. As for the epithelial barrier, claudin-11 protein expression was not decreased in choroid plexus epithelial cells forming the BCSFB in EAE mice, whereas it was decreased in brain and spinal cord meninges that form the BAB. Specific downregulation of claudin-11 with siRNA in a rat choroid plexus epithelial cell (TR-CSFB) monolayer significantly increased the permeability of FITC-dextran. In conclusion, our present findings indicate that claudin-11 expression at the BBB, BSCB, and BAB, but not the BCSFB, is downregulated in multiple sclerosis, impairing the functional integrity of these barriers.

Effects of high-sodium intake on systemic blood pressure and vascular responses in spontaneously diabetic WBN/Kob-Leprfa/fa rats.

The prevalence of type 2 diabetes mellitus (T2DM) and hypertension has markedly increased worldwide. The purpose of the present study was to examine the effects of a high-salt intake on the systolic blood pressure (SBP) and vascular responses in WBN/Kob-Leprfa/fa (WBKDF) rats, a new spontaneous animal model of T2DM. Male WBKDF rats and age-matched Wistar rats at 6 weeks of age were each divided into two groups and fed either a normal-sodium (NS, 0.26%) diet or high-sodium (HS, 8%) diet for 14 weeks: (i) Wistar rats on NS diet (Wistar-NS); (ii) Wistar rats on HS diet (Wistar-HS); (iii) WBKDF rats on NS diet (WBKDF-NS); (iv) WBKDF rats on HS diets (WBKDF-HS). Neither WBKDF-NS nor Wistar-NS rats showed significant changes in SBP throughout the experiment, but both WBKDF-HS and Wistar-HS exhibited significant elevation of SBP, which was more prominent (P<.01) in WBKDF-HS than in Wistar-HS. Phenylephrine-induced contractions of isolated thoracic aortic rings were significantly (P<.01) enhanced in WBKDF-HS and Wistar-HS compared with the respective strain of rats on the NS diet. In contrast, acetylcholine- and nitroprusside-induced relaxation were significantly (P<.01) diminished in both WBKDF-HS and Wistar-HS, and these HS diet-induced changes were more profound (P<.01) in WBKDF rats than in Wistar rats. Significantly (P<.05) higher plasma concentrations of 8-iso-prostaglandin F2α and sodium ions were observed in WBKDF-HS than in Wistar-HS. The current study demonstrated that WBKDF-HS rats developed salt-sensitive hypertension associated with vascular dysfunction. The WBKDF rat may be a useful model for investigating the etiology of hypertension with T2DM.

Major involvement of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) in uptake of biotin and pantothenic acid by human brain capillary endothelial cells.

The purpose of this study was to clarify the expression of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) and its contribution to the supply of biotin and pantothenic acid to the human brain via the blood-brain barrier. DNA microarray and immunohistochemical analyses confirmed that SLC5A6 is expressed in microvessels of human brain. The absolute expression levels of SLC5A6 protein in isolated human and monkey brain microvessels were 1.19 and 0.597 fmol/µg protein, respectively, as determined by a quantitative targeted absolute proteomics technique. Using an antibody-free method established by Kubo et al. (2015), we found that SLC5A6 was preferentially localized at the luminal membrane of brain capillary endothelium. Knock-down analysis using SLC5A6 siRNA showed that SLC5A6 accounts for 88.7% and 98.6% of total [(3) H]biotin and [(3) H]pantothenic acid uptakes, respectively, by human cerebral microvascular endothelial cell line hCMEC/D3. SLC5A6-mediated transport in hCMEC/D3 was markedly inhibited not only by biotin and pantothenic acid, but also by prostaglandin E2, lipoic acid, docosahexaenoic acid, indomethacin, ketoprofen, diclofenac, ibuprofen, phenylbutazone, and flurbiprofen. This study is the first to confirm expression of SLC5A6 in human brain microvessels and to provide evidence that SLC5A6 is a major contributor to luminal uptake of biotin and pantothenic acid at the human blood-brain barrier. In humans, it was unclear (not concluded) about what transport system at the blood-brain barrier (BBB) is responsible for the brain uptakes of two vitamins, biotin and pantothenic acid, which are necessary for brain proper function. This study clarified for the first time that the solute carrier 5A6/Na(+) -dependent multivitamin transporter SLC5A6/SMVT is responsible for the supplies of biotin and pantothenic acid into brain across the BBB in humans. DHA, docosahexaenoic acid; NSAID, non-steroidal anti-inflammatory drug; PGE2, prostaglandin E2.

Comparison of the blood coagulation profiles of ferrets and rats.

The aim of this study was to examine the blood coagulation profiles of ferrets and compare them with those of rats. The ferret activated partial thromboplastin time (aPTT) was slightly longer than the rat aPTT. In contrast, the ferret prothrombin time and thrombin time were profoundly shorter than the corresponding rat values. The fibrinogen level in ferret plasma was 2 times higher than that in rats. Heparin prolonged all blood coagulation times in a concentration-dependent manner in both ferret and rat plasma. A significantly (P<0.01) higher concentration of heparin was required to double the aPTT in ferrets than rats. These blood coagulation data for ferrets will be useful in experimental animal studies.

High-efficiency NO(x) absorption in water using equipment packed with a glass fiber filter.

NO(X) absorption in water is quite difficult by comparison with other exhausted gas, such as SO(2), CO(2), and NH(3) because of low solubility of NO(X) in water. We have been developed a NO(X) absorption equipment with a glass fiber filter having high porosity and surface area. When feed NO(X) gas concentration was high, high NO(X) removal efficiency was obtained. This was because the surface area per glass fiber filter volume was about 40 to 600 times higher than for common packing materials. For verification test and industrial application, a high concentration of NO(X) gas (206,000 ppm) produced by a metal dissolution process was treated with a series of two absorption experiments. We can attain 97.6% of NO(X) removal efficiency, and HNO(3) concentration in water was concentrated up to 56.3 wt %. Furthermore, ozone addition to gas and usage of ozone saturated water as an absorbent resulted in complete removal of NO(X) in the gas (up to 120 ppm). This result indicated the importance of aqueous phase oxidation of HNO(2), which produces NO in the gas phase.

Quantitative membrane protein expression at the blood-brain barrier of adult and younger cynomolgus monkeys.

Cynomolgus monkey has been used as a model for the prediction of drug disposition in human brain. The purpose of this study was to clarify protein expression levels of membrane proteins affecting drug distribution to brain, such as transporters, receptors, and junctional proteins, in cynomolgus monkey brain microvessels by using liquid chromatography tandem mass spectrometry. In adult monkeys, three ATP-binding cassette transporters (multidrug resistance 1 (MDR1), breast cancer resistance protein (BCRP), and multidrug resistance protein 4 (MRP4)), six solute carrier transporters (glucose transporter 1 (GLUT1), GLUT3/14, monocarboxylate transporter 1 (MCT1), MCT8, organic anion transporting polypeptide 1A2, and equilibrative nucleoside transporter 1), two junctional proteins (claudin-5 and vascular endothelial cadherin), and two receptors (insulin receptor and low-density lipoprotein receptor-related protein 1) were detected. Comparison of the expression levels with those in mouse, which we reported previously, revealed a pronounced species difference. BCRP expression in monkey was greater by 3.52-fold than that in mouse, whereas MDR1 and MRP4 expression levels in monkey were lower by 0.304- and 0.180-fold, respectively, than that in mouse. This study also investigated the developmental changes in expression of membrane proteins in neonate and child monkeys. Expression of MDR1 was similar in neonate and adult monkeys, whereas in rat, P-glycoprotein expression was reported to be significantly lower in brain microvessels of neonate as compared with adult rat. These results will be helpful to understand and predict brain concentrations of drugs in different species and at different ages of primates.

The NPC motif of aquaporin-11, unlike the NPA motif of known aquaporins, is essential for full expression of molecular function.

The recently identified molecule aquaporin-11 (AQP11) has a unique amino acid sequence pattern that includes an Asn-Pro-Cys (NPC) motif, corresponding to the N-terminal Asn-Pro-Ala (NPA) signature motif of conventional AQPs. In this study, we examined the effect of the mutation of the NPC motif on the subcellular localization, oligomerization, and water permeability of AQP11 in transfected mammalian cells. Furthermore, the effect was also assessed using zebrafish. Site-directed mutation at the NPC motif did not affect the subcellular localization of AQP11 but reduced its oligomerization. A cell swelling assay revealed that cells expressing AQP11 with a mutated NPC motif had significantly lower osmotic water permeability than cells expressing wild-type AQP11. Zebrafish deficient in endogenous AQP11 showed a deformity in the tail region at an early stage of development. This phenotype was dramatically rescued by injection of human wild-type AQP11 mRNA, whereas the effect of mRNA for AQP11 with a mutated NPC motif was less marked. Although the NPA motif is known to be important for formation of water-permeable pores by conventional AQPs, our observations suggest that the corresponding NPC motif of AQP11 is essential for full expression of molecular function.

The protective effect of radicicol against renal ischemia--reperfusion injury in mice.

Overexpression of heat shock protein 70 kDa (HSP70) is known to confer cellular protection against ischemia-reperfusion (I/R) injury. Radicicol, a HSP90 inhibitor, has been reported to induce the expression of HSP70 protein. Here we studied whether radicicol attenuated renal I/R injury in vivo. Treatment of mice with radicicol ameliorated renal I/R injury and increased renal HSP70 mRNA and protein. Administration of radicicol with quercetin, an inhibitor of HSP70 induction, eliminated the renoprotective effect of radicicol. Our results suggest that the up-regulation of renal HSP70 protein by radicicol leads to a novel drug therapy against renal I/R injury.

Decreased abundance of urinary exosomal aquaporin-1 in renal ischemia-reperfusion injury.

Urinary exosomes, secreted into urine from renal epithelial cells, are known to contain many types of renal functional membrane proteins. Here, we studied whether renal ischemia-reperfusion (I/R) affects urinary exosomal aquaporin-1 (AQP1) excretion in rats subjected to renal I/R and patients who underwent renal transplantation. Immunoblotting studies demonstrated reduction of the urinary exosomal AQP1 level even at 6 h after renal I/R, and the level continued to be low over 96 h after I/R. Renal AQP1 mRNA and protein analyses revealed that the decreased excretion of urinary exosomal AQP1 is associated with renal AQP1 protein retention in the early phase and with a decreased expression level of renal AQP1 in the later phase of renal I/R injury. Decreased abundance of urinary exosomal AQP1 in a recipient patient was also observed at 48 h after renal allograft transplantation. No significant decrease in urinary exosomal AQP1 was observed in a rat model of nephropathy or in patients with proteinuria. Our studies suggest that the renal AQP1 expression level is possibly controlled by its urinary exosomal excretion and indicate that urinary exosomal AQP1 is a novel urinary biomarker for renal I/R injury.

Amelioration of renal ischemia-reperfusion injury by inhibition of IL-6 production in the poloxamer 407-induced mouse model of hyperlipidemia.

It is largely unknown whether hyperlipidemia is involved in the pathobiology of renal ischemia-reperfusion (I/R) injury that is an important cause of acute kidney injury. Here we studied the effect of experimental dyslipidemia on renal I/R injury. Renal I/R injury was less severe in hyperlipidemic mice treated with poloxamer 407 than in the control mice. Cytokine analyses revealed decreased levels of renal and serum IL-6 in the hyperlipidemic mice after renal I/R. Protection from renal I/R injury in the hyperlipidemic mice was diminished by administration of recombinant IL-6. Concanavalin A-induced IL-6 release from cultured splenocytes derived from the hyperlipidemic mice was lower than that from splenocytes of normal mice. In hypercholesterolemic apolipoprotein E-knockout mice, in which renal I/R injury is less severe than in control mice, renal I/R-induced IL-6 production was also less than that in controls. In angiopoietin-like 3-deficient mice, which were hypolipidemic, renal dysfunction and renal IL-6 level after I/R were similar to those of control mice. Our data indicate that the presence of experimental hyperlipidemia may be associated with a decreased risk of renal I/R injury, possibly mediated by reduced renal IL-6 production after the insult and extend the notion that an anti-IL6 agent would be useful for the treatment of acute kidney injury.

Involvement of multidrug resistance-associated protein 1 in intestinal toxicity of methotrexate.

PURPOSE: Methotrexate (MTX) causes dose-limiting gastrointestinal toxicity due to exposure of intestinal tissues, and is a substrate of the multidrug resistance-associated protein (MRP) 1. Here we examine the involvement of MRP1, which is reported to be highly expressed in the proliferative crypt compartment of the small intestine, in the gastrointestinal toxicity of MTX. METHODS: MTX was intraperitoneally administered to mrp1 gene knockout (mrp1 ((-/-))) and wild-type (mrp1 ((+/+))) mice. Body weight, food and water intake were monitored, intestinal histological studies and pharmacokinetics of MTX were examined. RESULTS: mrp1 ((-/-)) mice more severely decreased body weight, food and water intake than mrp1 ((+/+)) mice. Almost complete loss of villi throughout the small intestine in mrp1 ((-/-)) mice was observed, whereas the damage was only partial in mrp1 ((+/+)) mice. Plasma concentration and biliary excretion profiles of MTX were similar in mrp1 ((-/-)) and mrp1 ((+/+)) mice, though accumulation of MTX in immature proliferative cells isolated from mrp1 ((-/-)) mice was much higher compared to mrp1 ((+/+)) mice. Immunostaining revealed localization of Mrp1 in plasma membrane of the intestinal crypt compartment in mrp1 ((+/+)) mice, but not in mrp1 ((-/-)) mice. CONCLUSION: Mrp1 determines the exposure of proliferative cells in the small intestine to MTX, followed by gastrointestinal toxicity.

The protective effect of a newly developed molecular chaperone-inducer against mouse ischemic acute kidney injury.

Activation of the unfolded protein response (UPR) has been suggested to attenuate renal ischemia-reperfusion (I/R) injury. We recently found a compound, namely BIX, that activated the UPR selectively through the activating transcription factor 6 pathway. This study examined the effect of BIX on renal I/R injury in mice. BIX selectively up-regulated renal BiP mRNA and protein. Pretreatment with BIX significantly ameliorated renal I/R injury. Co-administration of BIX and tunicamycin, a non-selective UPR inducer, provided no additional protection. Our results suggest that the UPR activation by BIX leads to a novel drug therapy against renal I/R injury.

P-glycoprotein (Abcb1) is involved in absorptive drug transport in skin.

The purpose of the present study was to investigate the role of P-glycoprotein (P-gp) in drug disposition in skin. The distribution of P-gp substrates (rhodamine 123 and itraconazole) to the skin after administration from the epidermal side was lower in P-gp gene knockout (mdr1a/1b(-/-)) mice than that in wild-type mice. Coadministration of propranolol, a P-gp inhibitor, decreased the distribution of itraconazole to the skin in wild-type mice, but not in mdr1a/1b(-/-) mice. These results suggest that P-gp contributes to the influx (from the epidermal side) of its substrates into skin, although P-gp is generally involved in efflux of drugs from various tissues. This finding was supported by the lower vectorial transport of rhodamine 123 from the epidermal to the hypodermal side in mdr1a/1b(-/-) mice in Ussing-type chamber experiments and by the immunohistochemical localization of P-gp throughout the dermal layer. Distribution of itraconazole after intravenous administration, on the other hand, was higher in mdr1a/1b(-/-) mice than that in wild-type mice, suggesting that P-gp transports this drug from the skin to the circulation. The present findings are the first to demonstrate involvement of P-gp in dermal drug disposition.

A protective role of unfolded protein response in mouse ischemic acute kidney injury.

Although renal ischemia-reperfusion is known to activate the unfolded protein response, the renal site and role of activation of this response following the insult in vivo remains largely unknown. Here we studied the renal spatio-temporal expression pattern of glucose-regulated protein (GRP) 78, a central regulator of the unfolded protein response network, following renal ischemia-reperfusion and the effects of the specific chemical unfolded protein response inducers, tunicamycin and thapsigargin, on renal ischemia-reperfusion injury in mice. Renal ischemia-reperfusion resulted in expression of the spliced form of the X-box binding protein-1 (XBP-1s) transcript, an unfolded protein response target, at 1 and 2 h after the insult. This response was followed by an increase in the GRP78 transcript and protein. The increased amount of GRP78 protein after ischemia-reperfusion was largely localized in proximal tubule cells. Pretreatment with tunicamycin or thapsigargin significantly ameliorated renal dysfunction and injury after ischemia-reperfusion. Taken together with these results, the unfolded protein response was activated following renal ischemia-reperfusion at sites that are susceptible to ischemia-reperfusion injury, and this activation had a protective effect against renal ischemia-reperfusion injury in vivo. Molecules involved in the unfolded protein response may offer new opportunities for pharmacological intervention against renal ischemia-reperfusion injury, which is an important cause of acute kidney injury.

Pravastatin improves renal ischemia-reperfusion injury by inhibiting the mevalonate pathway.

Statins are known to lessen the severity of renal ischemia-reperfusion injury. The present study was undertaken to define the mechanism of renoprotective actions of statins using a mouse kidney injury model. Treatment of mice with pravastatin, a widely used statin, improved renal function after renal ischemia-reperfusion without lowering the plasma cholesterol level. Administration of pravastatin with mevalonate, a product of HMG-CoA reductase, eliminated renal protection suggesting an effect of pravastatin on mevalonate or its metabolism. In hypercholestrolemic apolipoprotein E knockout mice with reduced HMG-CoA reductase activity; the degree of injury was less severe than in control mice, however, there was no protective action of pravastatin on renal injury in the knockout mice. Treatment with a farnesyltransferase inhibitor (L-744832) mimicked pravastatin's protective effect but co-administration with the statin provided no additional protection. Both pravastatin and L-744832 inhibited the injury-induced increase in plasma IL-6 concentration to a similar extent. Our results suggest the protective effect of pravastatin on renal ischemia-reperfusion injury is mediated by inhibition of the mevalonate-isoprenoid pathway independent of its lipid lowering action.

Evaluation of olmesartan medoxomil in the rat monocrotaline model of pulmonary hypertension.

It is suggested that angiotensin II is involved in the pathogenesis of pulmonary hypertension and subsequent right ventricular hypertrophy; therefore, an angiotensin AT1 receptor antagonist could be beneficial for the treatment of this disease. We tested the effect of the new AT1 receptor antagonist olmesartan medoxomil on monocrotaline-induced pulmonary hypertension in rats. At 3 weeks after a single subcutaneous injection of monocrotaline (50 mg/kg), the lung/body weight ratio, the right ventricle/(left ventricle plus septum) weight ratio [RV/(LV+S)], and right ventricular systolic pressure were increased, indicating establishment of pulmonary hypertension and right ventricular hypertrophy. Oral administration of olmesartan medoxomil (2 or 5 mg/kg/day for 3 weeks) restored RV/(LV+S) and right ventricular systolic pressure, and a higher dose (5 mg/kg/day) improved the lung/body weight ratio. Pulmonary arteries isolated from monocrotaline-treated rats exhibited an increase in basal tone in the resting state, indicating that they had intrinsic tone. Three weeks of treatment with olmesartan decreased this intrinsic tone. These data suggest that long-term treatment with olmesartan has beneficial effects on monocrotaline-induced pulmonary hypertension and subsequent right ventricular hypertrophy.