Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling.

The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysis of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.

Human proximal tubular epithelium actively secretes but does not retain rosuvastatin.

Rosuvastatin is a potent HMG-CoA reductase inhibitor that has proven to be effective in the treatment of dyslipidemia. Rosuvastatin is cleared from the body by both biliary and renal clearance, the latter believed to be due to active tubular secretion. Whereas the mechanisms of hepatic clearance of rosuvastatin are well documented, those of renal clearance are not. Because rosuvastatin (and other statins) may alter proximal tubular function, this study aimed to characterize the mechanisms of tubular rosuvastatin secretion to define the factors that could influence the presence/concentration of rosuvastatin in proximal tubular cells. Hereto, polarized monolayers of primary human tubular cells were used. We found rosuvastatin net secretion across proximal tubule cells, which was saturable (K50=20.4+/-4.1 microM). The basolateral uptake step was rate-limiting and mediated by OAT3. Rosuvastatin efflux at the apical membrane was mediated by MRP2/4 and ABCG2 together with a small contribution from MDR1 or P-glycoprotein. These data, obtained in an intact human tubule cell model, provide a detailed insight into rosuvastatin's renal handling and the possible factors influencing it.

The effect of stress on the expression of the amyloid precursor protein in rat brain.

The abnormal processing of the amyloid precursor protein (APP) is a pivotal event in the development of the unique pathology that defines Alzheimer's disease (AD). Stress, and the associated increase in corticosteroids, appear to accelerate brain ageing and may increase vulnerability to Alzheimer's disease via altered APP processing. In this study, rats were repeatedly exposed to an unavoidable stressor, an open elevated platform. Previous studies in this laboratory have shown that a single exposure produces a marked increase in plasma corticosterone levels but animals develop tolerance to this effect between 10 and 20 daily sessions. Twenty-four hours after stress, there was an increase in the ratio of the deglycosylated form of APP in the particulate fraction of the brain, which subsequently habituated after 20 days. The levels of soluble APP (APPs) tended to be lower in the stress groups compared to controls except for a significant increase in the hippocampus after 20 days of platform exposure. Since APPs is reported to have neurotrophic properties, this increased release may represent a neuroprotective response to repeated stress. It is possible that the ability to mount this response decreases with age thus increasing the vulnerability to stress-induced AD-related pathology.

Association of a salivary acetylcholinesterase with Alzheimer's disease and response to cholinesterase inhibitors.

OBJECTIVES: A decrease in cholinergic activity is a key event in the biochemistry of Alzheimer's disease (AD). The aim of the study was to investigate the expression levels of markers of cholinergic function in saliva, which is a readily accessible body fluid that can be obtained from subjects with minimal distress. DESIGN AND METHODS: Salivary samples were obtained from people with NINCDS-ARDRA "probable" Alzheimer's disease and age- and sex-matched controls. Salivary acetylcholinesterase enzyme (AChE) activity was determined colorometrically. RESULTS: Robust AChE catalytic activity was detected in the saliva samples that was stable for up to 6 h at room temperature following the provision of the salivary sample. The activity of the enzyme was significantly lower in people with AD than in age-matched controls. In addition, there were significant differences in activity between those who responded to acetylcholinesterase inhibitor (AChE-I) therapy and those who did not. CONCLUSIONS: Salivary enzyme activity may therefore prove to be a useful marker of central cholinergic activity.