A Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid Receptors: GPR40 and GPR84.

Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases.

PBI-4050 via GPR40 activation improves adenine-induced kidney injury in mice.

PBI-4050 (3-pentylbenzenacetic acid sodium salt), a novel first-in-class orally active compound that has completed clinical Phases Ib and II in subjects with chronic kidney disease (CKD) and metabolic syndrome respectively, exerts antifibrotic effects in several organs via a novel mechanism of action, partly through activation of the G protein receptor 40 (GPR40) receptor. Here we evaluate the effects of PBI-4050 in both WT and Gpr40-/- mice on adenine-induced tubulointerstitial injury, anemia and activation of the unfolded protein response (UPR) pathway. Adenine-induced CKD was achieved in 8-week-old C57BL/6 mice fed a diet supplemented with 0.25% adenine. After 1 week, PBI-4050 or vehicle was administered daily by oral-gavage for 3 weeks. Gpr40-/- mice were also subjected to adenine-feeding, with or without PBI-4050 treatment. PBI-4050 improved renal function and urine concentrating ability. Anemia was present in adenine-fed mice, while PBI-4050 blunted these effects and led to significantly higher plasma erythropoietin (EPO) levels. Adenine-induced renal fibrosis, endoplasmic reticulum (ER) stress and apoptosis were significantly decreased by PBI-4050. In parallel, Gpr40-/- mice were more susceptible to adenine-induced fibrosis, renal function impairment, anemia and ER stress compared with WT mice. Importantly, PBI-4050 treatment in Gpr40-/- mice failed to reduce renal injury in this model. Taken together, PBI-4050 prevented adenine-induced renal injury while these beneficial effects were lost upon Gpr40 deletion. These data reinforce PBI-4050's use as a renoprotective therapy and identify GPR40 as a crucial mediator of its beneficial effects.

PBI-4050 Reduces Stellate Cell Activation and Liver Fibrosis through Modulation of Intracellular ATP Levels and the Liver Kinase B1/AMP-Activated Protein Kinase/Mammalian Target of Rapamycin Pathway.

Hepatic fibrosis is a major cause of morbidity and mortality for which there is currently no effective therapy. We previously showed that 2-(3-pentylphenyl)acetic acid (PBI-4050) is a dual G protein-coupled receptor GPR40 agonist/GPR84 antagonist that exerts antifibrotic, anti-inflammatory, and antiproliferative action. We evaluated PBI-4050 for the treatment of liver fibrosis in vivo and elucidated its mechanism of action on human hepatic stellate cells (HSCs). The antifibrotic effect of PBI-4050 was evaluated in carbon tetrachloride (CCl4)- and bile duct ligation-induced liver fibrosis rodent models. Treatment with PBI-4050 suppressed CCl4-induced serum aspartate aminotransferase levels, inflammatory marker nitric oxide synthase, epithelial to mesenchymal transition transcription factor Snail, and multiple profibrotic factors. PBI-4050 also decreased GPR84 mRNA expression in CCl4-induced injury, while restoring peroxisome proliferator-activated receptor γ (PPARγ) to the control level. Collagen deposition and α-smooth muscle actin (α-SMA) protein levels were also attenuated by PBI-4050 treatment in the bile duct ligation rat model. Transforming growth factor-ß-activated primary HSCs were used to examine the effect of PBI-4050 and its mechanism of action in vitro. PBI-4050 inhibited HSC proliferation by arresting cells in the G0/G1 cycle phase. Subsequent analysis demonstrated that PBI-4050 signals through a reduction of intracellular ATP levels, activation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK), and blockade of mammalian target of rapamycin (mTOR), resulting in reduced protein and mRNA levels of α-SMA and connective tissue growth factor and restored PPARγ mRNA expression. Our findings suggest that PBI-4050 may exert antifibrotic activity in the liver through a novel mechanism of action involving modulation of intracellular ATP levels and the LKB1/AMPK/mTOR pathway in stellate cells, and PBI-4050 may be a promising agent for treating liver fibrosis.

Comparison of open and closed abdomen techniques for the delivery of intraperitoneal pemetrexed using a murine model.

BACKGROUND AND OBJECTIVES: Pemetrexed is an appealing agent to use for cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (HIPEC). However, the optimal method of pemetrexed delivery still remains undefined. Using a murine model, we compared the use of open and closed abdomen techniques on the absorption of intraperitoneal (IP) pemetrexed in different compartments. METHODS: Eleven Sprague-Dawley rats were submitted to a fixed dose of IP pemetrexed (1000 mg/m2 ) at a perfusion temperature of 40°C during 25 min according to two techniques: open and closed. At the end of perfusion, samples in different compartments were harvested and the concentrations of pemetrexed were measured by high performance liquid chromatography. RESULTS: Absorption of IP pemetrexed in portal and systemic blood was significantly higher using the open compared to the closed abdomen technique (93.17 vs 52.50 µg/mL, P < 0.001) and (76.26 vs 51.65 µg/mL, P < 0.001), respectively. No difference was found between the two techniques on the peritoneal tissue concentration of pemetrexed (18.07 vs 19.17 µg/g, P = 0.51). CONCLUSION: Peritoneal absorption of pemetrexed is not modified by the use of either technique. However, systemic concentrations of pemetrexed increased using the open technique, suggesting it could increase systemic toxicity.

Effects of Chronic Renal Failure on Brain Cytochrome P450 in Rats.

Chronic renal failure (CRF) impedes renal excretion of drugs and their metabolism by reducing the expression of liver cytochrome P450 (P450). Uremic serum contains factors, such as parathyroid hormone (PTH), that decrease liver P450s. The P450s are also involved in the metabolism of xenobiotics in the brain. This study investigates: 1) the effects of CRF on rat brain P450, 2) the role of PTH in the downregulation of brain P450s in CRF rats, and 3) the effects of PTH on P450s in astrocytes. Protein and mRNA expression of P450s were assessed in the brain of CRF and control (CTL) rats, as well as from CTL or CRF rats that underwent parathyroidectomy (PTX) 1 week before nephrectomy. CYP3A activity was measured using 3-[(3, 4-difluorobenzyl) oxy]-5, 5-dimethyl-4-[4-methylsulfonyl) phenyl] furan-2(5H)-1 metabolism in brain microsomal preparation. CYP3A protein expression was assessed in primary cultured astrocytes incubated with serum obtained from CRF or CTL rats or with PTH. Significant downregulations (≥40%) of CYP1A, CYP2C11, and CYP3A proteins were observed in microsomes from CRF rat brains. CYP3A activity reduction was also observed. CYP3A expression and activity were unaffected in PTX-pretreated CRF rats. Serum of PTX-treated CRF rats had no impact on CYP3A levels in astrocytes compared with that of untreated CRF rats. Finally, PTH addition to normal calf serum induced a reduction in CYP3A protein similar to CRF serum, suggesting that CRF-induced hyperparathyroidism is associated with a significant decrease in P450 drug-metabolizing enzymes in the brain, which may have implications in drug response.

Electrocautery effect on intestinal vascularisation in a murine model.

BACKGROUND: The use of electrocautery devices is associated with complications such as perforation or fistulisation when used near intestinal structures. This is likely due to its effect on vascularisation of the bowel wall. To test this hypothesis we established a murine model to quantify the effect of electrocautery injury on the intestinal microvascularisation. METHODS: Sprague-Dawley rats were subjected to five electrocautery injuries on the small bowel in coagulation mode (30 W intensity) and in cut mode (40 W, 80 W and 200 W intensities) for durations of 1, 2 and 5 s. 5 mg/kg of fluorescein was injected intravenously, the injured bowel segments harvested and the rat sacrificed. The segments were analysed to measure the fluorescence of injured bowel compared to adjacent unharmed tissue. RESULTS: A significant decrease in bowel wall microvascularisation occurred with increasing intensity (coag 30 W/cut 40 W versus cut 200 W 1 s: p < 0.05) and duration of electrocautery injury (cut 40 W 1/2 s versus 5 s: p < 0.05). There was a 40% perforation rate when decreased bowel wall microvascularisation was 25% or more. Despite similar electrocautery injury, a significantly greater microvascularisation decrease was observed in jejunum compared to ileum (p < 0.05). CONCLUSION: We successfully established a murine model to quantify the decrease of bowel wall microvascularisation associated with electrocautery use. Unsurprisingly, the decrease in microvascularisation is greater with higher intensity and duration of electrocautery and is associated with more perforations in the experimental model. The jejunum seems more vulnerable to electrocautery injury than the ileum. These observations support caution when using electrocautery devices near intestinal structures.

Effect of experimental kidney disease on the functional expression of hepatic reductases.

Chronic kidney disease (CKD) affects the nonrenal clearance of drugs by modulating the functional expression of hepatic drug-metabolizing enzymes and transporters. The impact of CKD on oxidative and conjugative metabolism has been extensively studied. However, its effect on hepatic drug reduction, an important phase I drug-metabolism pathway, has not been investigated. We aimed to assess the effect of experimental CKD on hepatic reduction using warfarin as a pharmacological probe substrate. Cytosolic and microsomal cellular fractions were isolated from liver tissue harvested from five-sixths-nephrectomized and control rats (n = 10 per group). The enzyme kinetics for warfarin reduction were evaluated in both fractions, and formation of warfarin alcohols was used as an indicator of hepatic reductase activity. Selective inhibitors were employed to identify reductases involved in warfarin reduction. Gene and protein expression of reductases were determined using quantitative real-time polymerase chain reaction and Western blotting, respectively. Formation of RS/SR-warfarin alcohol was decreased by 39% (P < 0.001) and 43% (P < 0.01) in cytosol and microsomes, respectively, in CKD rats versus controls. However, RR/SS-warfarin alcohol formation was unchanged in the cytosol, and a trend toward its decreased production was observed in microsomes. Gene and protein expression of cytosolic carbonyl reductase 1 and aldo-keto reductase 1C3/18, and microsomal 11ß-hydroxysteroid dehydrogenase type 1 were significantly reduced by >30% (P < 0.05) in CKD rats compared with controls. Collectively, these results suggest that the functional expression of hepatic reductases is selectively decreased in kidney disease. Our findings may explain one mechanism for altered nonrenal clearance, exposure, and response of drugs in CKD patients.

In Vitro and In Vivo Mechanistic Studies toward Understanding the Role of 1-Aminobenzotriazole in Rat Drug-Drug Interactions.

1-Aminobenzotriazole (ABT) is regularly used in vivo as a nonspecific and irreversible cytochrome P450 inhibitor to elucidate the role of metabolism on the pharmacokinetic profile of xenobiotics. However, few reports have considered the recent findings that ABT can alter drug absorption or have investigated the possible differential inhibition of ABT on intestinal and hepatic metabolism. To address these uncertainties, pharmacokinetic studies under well controlled and defined ABT pretreatment conditions (50 mg/kg, 1 hour ABT i.v. and 16 hours ABT p.o.) were conducted prior to the oral administration of metoprolol, a permeable P450 probe that undergoes extensive intestinal and hepatic metabolism. The pharmacokinetic profile of metoprolol was affected differently by the two ABT pretreatments. An increase in area under the curve of 16-fold with ABT p.o. and 6.5-fold with ABT i.v. was observed compared with control. Based on in vitro studies, this difference could not be attributed to a differential inhibition of intestinal and hepatic metabolism. In the ABT i.v. pretreatment group, the increase in area under the curve was also associated with a prolonged time at maximal concentration (24-fold versus control), suggesting a delay in absorption. This was further confirmed by the administration of a charcoal meal, which resulted in a 7-fold increase in stomach weights in the 1-hour ABT pretreated groups compared with the untreated or 16-hour ABT pretreated rats. Based on these results, we recommend pretreating rats with ABT p.o. 16 hours before the administration of a test compound to preserve the inhibitory effect on intestinal and hepatic metabolism and avoid the confounding effect on drug absorption.

Transplantation of mesenchymal stem cells promotes tissue regeneration in a glaucoma model through laser-induced paracrine factor secretion and progenitor cell recruitment.

Among bone marrow cells, hematopoietic and mesenchymal components can contribute to repair damaged organs. Such cells are usually used in acute diseases but few options are available for the treatment of chronic disorders. In this study, we have used a laser-induced model of open angle glaucoma (OAG) to evaluate the potential of bone marrow cell populations and the mechanisms involved in tissue repair. In addition, we investigated laser-induced tissue remodeling as a method of targeting effector cells into damaged tissues. We demonstrate that among bone marrow cells, mesenchymal stem cells (MSC) induce trabecular meshwork regeneration. MSC injection into the ocular anterior chamber leads to far more efficient decrease in intraocular pressure (IOP) (p < .001) and healing than hematopoietic cells. This robust effect was attributable to paracrine factors from stressed MSC, as injection of conditioned medium from MSC exposed to low but not to normal oxygen levels resulted in an immediate decrease in IOP. Moreover, MSC and their secreted factors induced reactivation of a progenitor cell pool found in the ciliary body and increased cellular proliferation. Proliferating cells were observed within the chamber angle for at least 1 month. Laser-induced remodeling was able to target MSC to damaged areas with ensuing specific increases in ocular progenitor cells. Thus, our results identify MSC and their secretum as crucial mediators of tissue repair in OAG through reactivation of local neural progenitors. In addition, laser treatment could represent an appealing strategy to promote MSC-mediated progenitor cell recruitment and tissue repair in chronic diseases.

Postnatal exposure to voluntary exercise but not the antioxidant catechin protects the vasculature after a switch to an atherogenic environment in middle-age mice.

We aimed to evaluate the lasting functional imprinting of exercise (EX) and catechin (CAT) on the vascular function of middle-age mice switched to a proatherogenic environment. C57BL/6J mice (n = 10-15 in each group) fed a regular diet (RD) were exposed from the age of 1 to 9 months either to EX (voluntary running; 2.7 ± 0.2 km/day), to the polyphenol CAT (30 mg/kg/day in drinking water), or to physical inactivity (PI). At 9 months of age, EX and CAT were stopped and mice either remained on the RD or were fed a Western diet (WD) for an additional 3 months. At 12 months of age, mice from all groups fed a WD had similar body mass, systolic blood pressure, and plasma total cholesterol, glucose, insulin, and isoprostane. Compared to the RD, the WD induced an indomethacin-sensitive aortic endothelium-dependent and independent dysfunction in PI mice (p < 0.05) that was prevented by both EX and CAT; this benefit was associated with a higher (p < 0.05) non-nitric oxide/non-prostacyclin endothelium-dependent relaxation. While EX, but not PI or CAT, prevented vascular dysfunction induced by the WD in cerebral arteries, it had no effect in femoral arteries. The profiles of activity of antioxidant enzymes and of proinflammatory gene expression in the aorta suggest a better adaptation of EX > CAT > PI mice to stress. In conclusion, our data suggest that a postnatal exposure to EX, but not to CAT, imprints an adaptive defense capacity in the vasculature against a deleterious change in lifestyle.

Longitudinal Control of Lipid Levels in Patients With Premature Coronary Artery Disease.

Background: Lipid-lowering therapy (LLT) is a central aspect of the treatment of patients with coronary artery disease (CAD), and the benefits of LLT accrue over time. However, there are limited real-world data on longitudinal lipid control in patients with premature CAD. Objectives: The purpose of this study was to assess longitudinal attainment of guideline-recommended lipid goals and outcomes in a contemporary cohort of patients with premature CAD. Methods: We enrolled males younger than 50 years and females younger than 55 years with coronary stenosis of >50% and examined achievement of lipid goals, LLT characteristics, and cardiovascular outcomes (major adverse cardiovascular event [MACE]). Results: Of 476 patients who presented with acute coronary syndrome (ST-elevation myocardial infarction, non-ST-segment elevation myocardial infarction, unstable angina) (68%), stable angina (28%), or other symptoms, 73.2% achieved low-density lipoprotein cholesterol (LDL-C) <1.8 mmol/L on at least 1 occasion, but only 27.3% consistently stayed in the target range for 3 years after diagnosis. Although 73.9% of patients received high-intensity LLT at the time of diagnosis, only 43.5% had good adherence over the following 3 years. In multivariable analysis, 1 mmol/L increase in time-weighted average exposure to LDL-C, but not the lowest achieved LDL-C, was associated with a higher risk of MACE, hazard ratio 2.02 (95% CI: 1.48-2.76), when adjusted for sex, age, hypertension, diabetes, and smoking. Conclusions: We found low rates of longitudinal lipid target achievement in patients with premature CAD. Cumulative LDL-C exposure, but not lowest achieved LDL-C, was associated with risk of MACE. This highlights the critical importance of longitudinal control of lipids levels and identifies opportunities to improve LLT and maximize the time-dependent benefits of lipid-lowering.

Effects of chronic renal failure on brain drug transporters in rats.

Studies demonstrated that chronic renal failure (CRF) affects the expression and activity of intestinal, hepatic, and renal drug transporters. Such drug transporters are expressed in brain cells and at the blood-brain barrier (BBB), where they limit the entry and distribution of drugs in the brain. Perturbations in brain drug transporter equilibrium by CRF could lead to central drug toxicity. This study evaluates how CRF affects BBB drug transporters using a 5/6 nephrectomized rat model. Protein and mRNA expression of influx transporters [organic anion-transporting polypeptide (Oatp), organic anion transporter (Oat)] and efflux transporters [P-glycoprotein (P-gp), multidrug resistance-related protein (Mrp), breast cancer resistance protein (Bcrp)] were measured in CRF and control rat brain. Intracerebral accumulation of radiolabeled benzylpenicillin, digoxin, doxorubicin, and verapamil was used to evaluate BBB drug permeability. Protein expression of the transporters was evaluated in rat brain endothelial cells (RBECs) and astrocytes incubated with control and CRF rat serum. We demonstrated significant decreases (30-50%) in protein and mRNA levels of Bcrp, Mrp2 to -4, Oat3, Oatp2 and -3, and P-gp in CRF rat brain biopsies, as well as in astrocytes and RBECs incubated with CRF serum. These decreases did not correlate with in vivo changes because BBB permeability of benzylpenicillin was decreased by 30% in CRF rats, whereas digoxin, doxorubicin, and verapamil permeabilities were unchanged. It thus seems that even with decreased drug transporters, BBB integrity and function is conserved in CRF.

Rationale for heating oxaliplatin for the intraperitoneal treatment of peritoneal carcinomatosis: a study of the effect of heat on intraperitoneal oxaliplatin using a murine model.

OBJECTIVE: To study the effect of heat on the absorption of intraperitoneal (IP) oxaliplatin using a murine model. BACKGROUND: Because of its efficiency in the systemic treatment of colorectal cancer, oxaliplatin is currently used in hyperthermic intraperitoneal chemotherapy (HIPEC) for peritoneal carcinomatosis. However, its properties when administered by the IP route have not been well characterized by preclinical studies. METHODS: Under general anesthesia, 35 Sprague-Dawley rats were submitted to 3 different doses of IP oxaliplatin (460, 920, and 1840 mg/m(2)) at 3 different perfusion temperatures (37, 40, and 43°C) during 25 minutes. At the end of perfusion, samples in different compartments (peritoneum, portal blood, and systemic blood) were harvested and the concentrations of oxaliplatin were measured by high performance liquid chromatography. RESULTS: As the dose of IP oxaliplatin was increased, higher concentrations were observed in every compartment. When the temperature of IP oxaliplatin was increased, it resulted in an increase of its peritoneal concentration (linear regression 0.38; 95% CI: 0.28-0.47) and in a decrease of its systemic blood (linear regression -1, 02; 95% CI: -1.45 to -0.60) and portal blood (linear regression -1.08; 95% CI: -1.70 to -0.47) concentrations. CONCLUSION: Proportionally to the dose administered, IP oxaliplatin leads to high concentration of drug in peritoneal tissues. Furthermore, heat enhances peritoneal tissue concentration of Oxaliplatin while reducing its systemic absorption. This last effect may possibly lead to decreased systemic toxicity. These observations support the use of oxaliplatin for HIPEC.

Effects of chronic renal failure on kidney drug transporters and cytochrome P450 in rats.

Chronic renal failure (CRF) leads to decreased drug renal clearance due to a reduction in the glomerular filtration rate. However, little is known about how renal failure affects renal metabolism and elimination of drugs. Because both depend on the activity of uptake and efflux by renal transporters as well as enzymes in tubular cells, the purpose of this study was to investigate the effects of CRF on the expression and activity of select renal drug transporters and cytochrome P450. Two groups of rats were studied: control and CRF (induced by 5/6 nephrectomy). Compared with control rats, we observed reductions in the expression of both protein and mRNA of Cyp1a, sodium-dependent phosphate transport protein 1, organic anion transporter (Oat)1, 2, and 3, OatK1/K2, organic anion-transporting polypeptide (Oatp)1 and 4c1, P-glycoprotein, and urate transporter 1, whereas an induction in the protein and mRNA expression of Mrp2, 3, and 4 and Oatp2 and 3 was observed. Cyp3a expression remained unchanged. Similar results were obtained by incubating a human proximal tubule cell line (human kidney-2) with sera from CRF rats, suggesting the presence of uremic modulators. Finally, the renal elimination of [(3)H]digoxin and [(14)C]benzylpenicillin was decreased in CRF rats, compared with controls, as shown by a 4- and 9-fold accumulation, respectively, of these drugs in kidneys of rats in CRF. Our results demonstrate that CRF affects the expression and activity of several kidney drug transporters leading to the intrarenal accumulation of drugs and reduced renal clearance that could, at least partially, explain the tubular toxicity of many drugs.

Reduced hepatic synthesis of calcidiol in uremia.

Calcidiol insufficiency is highly prevalent in chronic kidney disease (CKD), but the reasons for this are incompletely understood. CKD associates with a decrease in liver cytochrome P450 (CYP450) enzymes, and specific CYP450 isoforms mediate vitamin D(3) C-25-hydroxylation, which forms calcidiol. Abnormal levels of parathyroid hormone (PTH), which also modulates liver CYP450, could also contribute to the decrease in liver CYP450 associated with CKD. Here, we evaluated the effects of PTH and uremia on liver CYP450 isoforms involved in calcidiol synthesis in rats. Uremic rats had 52% lower concentrations of serum calcidiol than control rats (P < 0.002). Compared with controls, uremic rats produced 71% less calcidiol and 48% less calcitriol after the administration of vitamin D(3) or 1alpha-hydroxyvitamin D(3), respectively, suggesting impaired C-25-hydroxylation of vitamin D(3). Furthermore, uremia associated with a reduction of liver CYP2C11, 2J3, 3A2, and 27A1. Parathyroidectomy prevented the uremia-associated decreases in calcidiol and liver CYP450 isoforms. In conclusion, these data suggest that uremia decreases calcidiol synthesis secondary to a PTH-mediated reduction in liver CYP450 isoforms.

Chemokine expression patterns in the systemic and genital tract compartments are associated with HIV-1 infection in women from Benin.

INTRODUCTION: Understanding the genital mucosal immunity and the factors involved in linking innate to adaptive immunity is crucial for the design of efficient preventive strategies against human immunodeficiency virus (HIV)-1. METHODS: Levels of both genital mucosal and blood chemokines were compared between 58 HIV-1-uninfected and 50 HIV-1-infected female commercial sex workers (CSWs) as well as 53 HIV-1-uninfected non-CSW control women at low risk for exposure, recruited in Cotonou, Benin. RESULTS: HIV-1-infected CSWs had significantly higher blood and genital levels of monocyte chemotactic protein (MCP-3/CCL7) and monokine induced by gamma interferon (MIG/CXCL9) compared with those in both the HIV-1-uninfected CSW and non-CSW groups. In the HIV-1-infected group, levels of MCP-3 and MIG were significantly higher in the genital mucosa than in the blood. However, the blood levels of macrophage inflammatory protein (MIP-1a/CCL3) and MIP-1b/CCL4 were higher in HIV-1-uninfected CSWs compared with those in the other groups. CONCLUSION: Increased production of chemokines in the genital tract may favour the recruitment of HIV-1 target cells causing a mucosal environment that promotes viral replication and dissemination, whereas higher expression of beta-chemokines at the systemic level is associated with protection from HIV-1 infection.

Down-regulation of liver drug-metabolizing enzymes in a murine model of chronic renal failure.

Drug metabolism could be altered in patients with chronic renal failure (CRF). In rats, this phenomenon is related to a decrease in liver cytochrome P450 (P450) and phase II enzymes, particularly N-acetyltransferase 2 (NAT2). This study attempted to determine the effects of CRF on liver P450 isoforms and NAT2 expressions by using a CRF mouse model. Two groups of mice were studied: CRF induced by 3/4 nephrectomy and control. Liver protein expression and mRNA levels of the major P450 isoforms involved in drug metabolism (CYP1A2, 2C29, 2D, 2E1, and 3A11) and NAT2 were measured by Western blot and real-time polymerase chain reaction (PCR), respectively. CYP3A activity was also assessed by the N-demethylation of erythromycin. Results showed a significant reduction in the protein expression of CYP1A2 (56%), 2C29 (31%), and 3A11 (37%) in CRF mice compared with control animals. Real-time PCR revealed a similar reduction in mRNA levels of CYP1A2, 2C29, and 3A11 (59, 56, and 37%, respectively), in CRF mice. There was no significant modification in protein expression and mRNA of CYP2D and 2E1. Compared with control animals, CRF mice displayed a 25% reduction in N-demethylation of erythromycin. For NAT2, protein expression decreased by 33% and mRNA levels decreased by 23%. In conclusion, this study demonstrates that protein expression of liver CYP1A2, CYP2C29, and CYP3A11 is down-regulated in CRF mice, secondary to reduced gene expression. Phase II enzymes are similarly affected by CRF. Our results will allow the use of knockout mice to determine the mechanism underlying CRF-induced down-regulation of liver drug-metabolizing enzymes.

ESRD impairs nonrenal clearance of fexofenadine but not midazolam.

ESRD can affect the pharmacokinetic disposition of drugs subject to nonrenal clearance. Cytochrome P450 (CYP) enzymes, including CYP3A, and multiple intestinal and hepatic drug transporters are thought to mediate this process, but the extent to which kidney disease alters the function of these proteins in humans is unknown. We used midazolam and fexofenadine to assess CYP3A (intestinal and hepatic) and drug transport, respectively, in patients with ESRD and healthy control subjects. We evaluated the effect of uremia on CYP3A and transporter expression in vitro by incubating normal rat hepatocytes and enterocytes with serum drawn from study participants. ESRD dramatically reduced nonrenal transporter function, evidenced by a 63% decrease in clearance (P < 0.001) and a 2.8-fold increase in area under the plasma concentration-time curve for fexofenadine (P = 0.002), compared with control subjects. We did not observe significant differences in midazolam or 1'-hydroxymidazolam clearance or area under the curve after oral administration, suggesting that CYP3A function is not changed by ESRD. Changes in hepatocyte and enterocyte protein expression in the presence of uremic serum were consistent with in vivo results. These findings demonstrate a mechanism for altered drug disposition in kidney disease, which may partially account for the high rates of drug toxicity in this population.

Alk1 haploinsufficiency causes glomerular dysfunction and microalbuminuria in diabetic mice.

Endothelial dysfunction has been shown to play an important role in the pathogenesis of glomerular damage during diabetic kidney disease (DKD). As such, a better understanding of the molecular mechanisms involved in glomerular endothelial dysfunctions could provide novel therapeutic strategies for the prevention of DKD. We have previously shown that Alk1/BMP9 signaling plays an important function to maintain vascular integrity in diabetic animals. As such, we evaluated the effects of Alk1 suppression on glomerular endothelial function in diabetic mice. In the present study, we used mice with conditional heterozygote deletion of Alk1 in the endothelium (Alk1ΔEC) to evaluate the role of Alk1 on kidney function during STZ-induced diabetes. DKD was investigated in diabetic control and Alk1ΔEC mice euthanized eight weeks after the onset of diabetes. We showed that Alk1 expression is reduced in the glomeruli of human DKD patients. While renal function was not altered in Alk1ΔEC non-diabetic mice, we showed that Alk1 haploinsufficiency in the glomerular endothelium leads to microalbuminuria, thickening of the glomerular basement membrane, glomerular apoptosis and podocyte loss in diabetic mice. These data suggest that Alk1 is important for the proper function of glomerular endothelial cells and that decreased Alk1 combined with chronic hyperglycemia can impair renal function.