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Background/aims: Estrogen-mediated cholestasis is an important clinical entity, but its molecular pathophysiology is still not fully understood. Impaired sodium-dependent uptake of bile acids has been associated with diminished expression of a basolateral Na(+)/bile acid cotransporter (Ntcp), whereas sodium-independent uptake is maintained despite a down-regulation of the organic anion transporter Oatp1. Thus, expression of the two other rat Oatps (Oatps2 and -4) was determined in estrogen-induced cholestasis. In addition, known transactivators of Oatp2 and Ntcp were studied to further characterize transcriptional regulation of these transporter genes., Methods: Hepatic protein and mRNA expression of various Oatps (1, 2, 4) in comparison to Ntcp were analyzed after 0.5, 1, 3 and 5 days of ethinylestradiol (EE) treatment (5 mg/kg) in rats. Binding activities of Oatp2 and Ntcp transactivators were assessed by electrophoretic mobility shift assays., Results: All basolateral Oatps (1, 2 and 4) were specifically down-regulated at the protein level by 30-40% of controls, but less pronounced than Ntcp (minus 70-80%). In contrast to unaltered Oatp4 mRNA levels, Oatp1 and Oatp2 mRNAs were reduced to various extents (minus 40-90% of controls). Binding activity of known transactivators of Ntcp and Oatp2 such as hepatocyte nuclear factor 1 (HNF1), CAAT enhancer binding protein alpha (C/EBPalpha) and pregnane X receptor (PXR) were also diminished during the time of cholestasis., Conclusions: Estrogen-induced cholestasis results in a down-regulation of all basolateral organic anion transporters. The moderate decline in expression of Oatp1, -2 and -4 may explain the unchanged sodium-independent transport of bile acids due to overlapping substrate specificity. Reduction in transporter gene expression seems to be mediated by a diminished nuclear binding activity of transactivators such as HNF1, C/EBP and PXR by estrogens.

Earlier studies indicated that chemically crosslinking glomerular basement membrane (GBM) rendered it more permeable to water and to macromolecules. Here possible mechanisms for the introduction of crosslinks into GBM under pathological conditions were explored. Glycation with glucose and with fructose over periods of 2 wk (fructose) and 6 weeks (glucose) rendered the GBM more permeable to water and myoglobin as judged from in vitro ultrafiltration behaviour. The membranes were also made more permeable to serum following glycation. The permeation changes were shown to be dependent on glycoxidative reactions judging by their inhibition by EDTA and DTPA. Aminoguanidine also prevented glycation from altering the permeability of GBM. Fluorescence studies indicated the formation of bityrosine in glycated GBM. Studies with oxidants showed that while hydrogen peroxide superoxide and peroxynitrite had little effect on GBM, hypochlorite anion was capable of increasing GBM permeability to water, myoglobin, albumin and serum. Changes in permeation were induced by very low quantities of hypochlorite, well within the range of the amounts of hypochlorite formed by activated neutrophils. Thus glycoxidation, or oxidation by hypochlorite, are chemical mechanisms by which GBM permeability can be increased.

It has been hypothesized that Maillard reaction products form in basement membranes during aging and may affect protein turnover. The purpose of this study was to localize Maillard reaction products in intact lens capsules and Descemet's membranes by immunoelectron microscopy to determine whether Maillard products accumulated with age and whether basement membrane thickness increased to a similar degree. The monoclonal antibodies antiglucitollysine and antipyrraline were employed to detect the products in native and glucose-treated bovine basement membranes. The content of basic amino acids, furosine, and fluorophores (370/440), as well as resistance to trypsin digestion showed that the basement membranes formed significant quantities of Maillard products when incubated with 200 mM glucose in vitro (P < 0.05). Likewise, incubation in 200 mM glucose resulted in at least a 4-fold increase in immunoreactivity (P < 0.001). Native basement membranes increased in thickness more than 2-fold with age (P < 0.001). Immunoreactivity varied similarly in that bound antiglucitollysine increased approx. 2-fold and antipyrraline approx. 3-fold in old vs. young basement membranes, but these differences were significant only in pyrraline immunoreactivity in the lens capsule (P < 0.01). Advanced products other than pyrraline may accumulate in Descemet's membrane since significant increases in fluorescence and resistance to trypsin were noted. These data suggest that the Maillard reaction may, to a small degree, contribute to basement membrane thickening.

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine epsilon-NH2 groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.

In renal basal-lateral membranous vesicles, the probenecid-sensitive p-aminohippurate uptake was stimulated by alloxan. This stimulation of uptake was observed only after a lag period of 15 seconds, and it reached a maximal value after one minute. Stimulation was increased by 1 mM to 5 mM alloxan in a linear fashion. The effect was maximal and constant between 5 mM and 20 mM alloxan. Alloxan affected neither the glucose space of the vesicle nor the rate of transport or diffusion of glutamate, another organic anion. The mechanism of stimulation by alloxan was not clear. Its effect was blocked by the sulfhydryl reagent N-ethylmaleimide and weakly mimicked by H2O2, an oxidizing reagent. However, ninhydrin, a structural analogue of alloxan which reacts with sulfhydryl groups, and glucose, a neutral structural analogue of alloxan, failed to stimulate probenecid-sensitive uptake.

Effect of inorganic anions on p-amino[3H]hippurate transport in renal basolateral membranes has been studied using the vesicles preloaded with unlabeled p-aminohippurate (countertransport condition). The uptake of p-amino[3H]hippurate was stimulated by the outward gradient of unlabeled p-aminohippurate and the labeled substrate was accumulated into the vesicles against its concentration gradient in the presence of Cl-. The substitution of SCN- and SO4(2-) for Cl- in both sides of the vesicles depressed the initial rate and the overshoot magnitude of p-amino[3H]hippurate uptake. These results suggest that Cl- may play an important role for the carrier-mediated transport system of organic anion in renal basolateral membranes.