Distribution of the organic anion transporters Oat1 and Oat3 between renal membrane microdomains in obstructive jaundice.

Relation between the renal function and the membrane environment where the organic anion transporters Oat1 and Oat3 are localized is scarce. The aim of this study was to examine the Oat1 and Oat3 distribution in different cellular fractions under physiological conditions as well as the effects of extrahepatic cholestasis on membrane distribution of both proteins. Besides, the potential role of jaundice serum on the Oat1 and Oat3 expression in suspensions of renal tubular cells was evaluated. Cellular and membrane fractions of renal cortex were obtained from control rats to evaluate Oat1 and Oat3 protein expressions. Other rats were subjected to bile duct ligation (BDL) or Sham operation to determine the membrane distribution of Oat1 and Oat3 between lipid raft domains (LRD) and non-LRD. Incubation of renal cortical cells with serum from Sham and BDL were also performed to study Oat1 and Oat3 protein expressions. In physiological conditions, Oat1 and Oat3 were concentrated in LRD. The pathology induced a shift of Oat1 from LRD to non-LRD, while Oat3 showed no changes in its distribution. In cells exposed to BDL serum, Oat1 protein expression in membranes significantly increased. For Oat3, no difference between groups was observed. The Oat1 redistribution to non-LRD in BDL could be favoring the increase in renal transport of organic anions previously observed. This change was specific to Oat1. The in vitro experiment allows to conclude that some component present in BDL serum is responsible for the alterations observed in Oat1 expression in cortical membranes.

Expression of renal Oat5 and NaDC1 transporters in rats with acute biliary obstruction.

AIM: To examine renal expression of organic anion transporter 5 (Oat5) and sodium-dicarboxylate cotransporter 1 (NaDC1), and excretion of citrate in rats with acute extrahepatic cholestasis. METHODS: Obstructive jaundice was induced in rats by double ligation and division of the common bile duct (BDL group). Controls underwent sham operation that consisted of exposure, but not ligation, of the common bile duct (Sham group). Studies were performed 21 h after surgery. During this period, animals were maintained in metabolic cages in order to collect urine. The urinary volume was determined by gravimetry. The day of the experiment, blood samples were withdrawn and used to measure total and direct bilirubin as indicative parameters of hepatic function. Serum and urine samples were used for biochemical determinations. Immunoblotting for Oat5 and NaDC1 were performed in renal homogenates and brush border membranes from Sham and BDL rats. Immunohistochemistry studies were performed in kidneys from both experimental groups. Total RNA was extracted from rat renal tissue in order to perform reverse transcription polymerase chain reaction. Another set of experimental animals were used to evaluate medullar renal blood flow (mRBF) using fluorescent microspheres. RESULTS: Total and direct bilirubin levels were significantly higher in BDL animals, attesting to the adequacy of biliary obstruction. An important increase in mRBF was determined in BDL group (Sham: 0.53 ± 0.12 mL/min per 100 g body weight vs BDL: 1.58 ± 0.24 mL/min per 100 g body weight, P < 0.05). An increase in the urinary volume was observed in BDL animals. An important decrease in urinary levels of citrate was seen in BDL group. Besides, a decrease in urinary citrate excretion (Sham: 0.53 ± 0.11 g/g creatinine vs BDL: 0.07 ± 0.02 g/g creatinine, P < 0.05) and an increase in urinary excretion of H(+) (Sham: 0.082 ± 0.03 µmol/g creatinine vs BDL: 0.21 ± 0.04 µmol/g creatinine, P < 0.05) were observed in BDL animals. We found upregulations of both proteins Oat5 and NaDC1 in brush border membranes where they are functional. Immunohistochemistry technique corroborated these results for both proteins. No modifications were observed in Oat5 mRNA and in NaDC1 mRNA levels in kidney from BDL group as compared with Sham ones. CONCLUSION: Citrate excretion is decreased in BDL rats, at least in part, because of the higher NaDC1 expression. Using the outward gradient of citrate generated by NaDC1, Oat5 can reabsorb/eliminate different organic anions of pathophysiological importance.

Expression and function of renal and hepatic organic anion transporters in extrahepatic cholestasis.

Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct. The absorption, distribution and elimination of drugs are impaired during this pathology. Prolonged cholestasis may alter both liver and kidney function. Lactam antibiotics, diuretics, non-steroidal anti-inflammatory drugs, several antiviral drugs as well as endogenous compounds are classified as organic anions. The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds. It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions. The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis, such as multidrug resistance-associated protein 2, organic anion transporting polypeptide 1, organic anion transporter 3, bilitranslocase, bromosulfophthalein/bilirubin binding protein, organic anion transporter 1 and sodium dependent bile salt transporter. The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.

Characterization of the mechanisms involved in the increased renal elimination of bromosulfophthalein during cholestasis: involvement of Oatp1.

The kidneys and liver are the major routes for organic anion elimination. We have recently shown that acute obstructive jaundice is associated with increased systemic and renal elimination of two organic anions, p-aminohippurate and furosemide, principally excreted through urine. This study examined probable adaptive mechanisms involved in renal elimination of bromosulfophthalein (BSP), a prototypical organic anion principally excreted in bile, in rats with acute obstructive jaundice. Male Wistar rats underwent bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BSP renal clearance was performed by conventional techniques. Renal organic anion-transporting polypeptide 1 (Oatp1) expression was evaluated by immunoblotting and IHC. Excreted, filtered, and secreted loads of BSP were all higher in BDL rats compared with Sham rats. The higher BSP filtered load resulted from the increase in plasma BSP concentration in BDL rats, because glomerular filtration rate showed no difference with the Sham group. The increase in the secreted load might be explained by the higher expression of Oatp1 observed in apical membranes from kidneys of BDL animals. This likely adaptation to hepatic injury, specifically in biliary components elimination, might explain, at least in part, the huge increase in BSP renal excretion observed in this experimental model.

Oxidized low-density-lipoprotein accumulation is associated with liver fibrosis in experimental cholestasis.

OBJECTIVE: The aim of the present study was to examine the probable relationship between the accumulation of oxLDL and hepatic fibrogenesis in cholestatic rats. INTRODUCTION: There is growing evidence to support the current theories on how oxidative stress that results in lipid peroxidation is involved in the pathogenesis of cholestatic liver injury and fibrogenesis. One of the major and early lipid peroxidation products, OxLDL, is thought to play complex roles in various immuno-inflammatory mechanisms. METHODS: A prolonged (21-day) experimental bile duct ligation was performed on Wistar-albino rats. Biochemical analysis of blood, histopathologic evaluation of liver, measurement of the concentration of malondialdehyde (MDA) and superoxide-dismutase (SOD) in liver tissue homogenates, and immunofluorescent staining for oxLDL in liver tissue was conducted in bile-duct ligated (n=8) and sham-operated rats (n=8). RESULTS: Significantly higher levels of MDA and lower concentrations of SOD were detected in jaundiced rats than in the sham-operated rats. Positive oxLDL staining was also observed in liver tissue sections of jaundiced rats. Histopathological examination demonstrated that neither fibrosis nor other indications of hepatocellular injury were found in the sham-operated group, while features of severe hepatocellular injury, particularly fibrosis, were found in jaundiced rats. CONCLUSION: Our results support the finding that either oxLDLs are produced as an intermediate agent during exacerbated oxidative stress or they otherwise contribute to the various pathomechanisms underlying the process of liver fibrosis. Whatever the mechanism, it is clear that an association exists between elevated oxLDL levels and hepatocellular injury, particularly with fibrosis. Further studies are needed to evaluate the potential effects of oxLDLs on the progression of secondary biliary cirrhosis.

Oxidized low-density-lipoprotein accumulation is associated with liver fibrosis in experimental cholestasis

OBJECTIVE: The aim of the present study was to examine the probable relationship between the accumulation of oxLDL and hepatic fibrogenesis in cholestatic rats. INTRODUCTION: There is growing evidence to support the current theories on how oxidative stress that results in lipid peroxidation is involved in the pathogenesis of cholestatic liver injury and fibrogenesis. One of the major and early lipid peroxidation products, OxLDL, is thought to play complex roles in various immuno-inflammatory mechanisms. METHODS: A prolonged (21-day) experimental bile duct ligation was performed on Wistar-albino rats. Biochemical analysis of blood, histopathologic evaluation of liver, measurement of the concentration of malondialdehyde (MDA) and superoxide-dismutase (SOD) in liver tissue homogenates, and immunofluorescent staining for oxLDL in liver tissue was conducted in bile-duct ligated (n = 8) and sham-operated rats (n = 8). RESULTS: Significantly higher levels of MDA and lower concentrations of SOD were detected in jaundiced rats than in the sham-operated rats. Positive oxLDL staining was also observed in liver tissue sections of jaundiced rats. Histopathological examination demonstrated that neither fibrosis nor other indications of hepatocellular injury were found in the sham-operated group, while features of severe hepatocellular injury, particularly fibrosis, were found in jaundiced rats. CONCLUSION: Our results support the finding that either oxLDLs are produced as an intermediate agent during exacerbated oxidative stress or they otherwise contribute to the various pathomechanisms underlying the process of liver fibrosis. Whatever the mechanism, it is clear that an association exists between elevated oxLDL levels and hepatocellular injury, particularly with fibrosis. Further studies are needed to evaluate the potential effects of oxLDLs on the progression of secondary biliary cirrhosis.

Obstructive jaundice induces early depression of mitochondrial respiration in rat hepatocytes

INTRODUCTION: Oxidative phosphorylation dysfunction of hepatocyte mitochondria is involved in the pathophysiology of organ dysfunction following obstructive jaundice (OJ). However the time period from biliary occlusion to the occurrence of the dysfunction has not been determined decisively. PURPOSE: To evaluate the early effects (1 d and 7 d) of OJ on liver mitochondria respiratory function in rats. METHODS: Male Wistar rats (200-250 g) were randomly divided into the following 3 groups: laparotomy plus OJ for 24 h (1d group) (n = 10); laparotomy plus OJ for 7 d (7d group) (n = 10); sham control procedure (CTR group) (n = 12). At the end of OJ periods, total serum bilirubin level, hepatic enzyme activity levels (GOT, GTP, Gama-GT, ALP), mitochondrial respiration phases S3 and S4, as well as the respiratory control ratio (RC = S3/S4), and ADP consumption/oxygen consumption (ADP/O) ratio, were determined. RESULTS: Total serum bilirubin, activity of most hepatic enzymes, and O2 consumption during basal (S4) respiration were increased in the 1d and 7d groups (ANOVA, p = 0.05 vs. CTR). After ADP addition, the O2 consumption rate (S3) in the 1d group remained similar to the CTR rate (ANOVA p > .05), while the RC rate was reduced (ANOVA, p = 0.001) vs. CTR. The effects observed on mitochondrial respiration in the 1d group were exacerbated in the 7d group. CONCLUSION: These results indicate that OJ induces early (24 h) depression of liver mitochondria respiration, and thus may lead to early reduction in the production of high energy bonds.

INTRODUÇÃO: A disfunção da fosforilação oxidativa das mitocôndrias do hepatócito está envolvida na fisiopatologia da disfunção orgânica subseqüente à icterícia obstrutiva (IO). Entretanto, a precocidade da ocorrência desta disfunção permanece obscura. OBJETIVO: Avaliar o efeito precoce da IO na função respiratória mitocondrial em ratos. MÉTODOS: Ratos Wistar machos (200 a 250g) foram randomizados em 3 grupos que foram submetidos a laparotomia mais: IO por 24hs (grupo 1d)(n=10); IO por 7 dias (grupo 7d)(n=10; procedimento simulado (grupo CTR)(n=12). Ao final dos períodos de IO, foram determinados: bilirrubina sérica total, atividade de enzimas hepáticas (TGO, TGP, Gama-GT, FA), e as fases S3 e S4 da respiração mitocondrial, bem como o razão do controle respiratório (RC = S3/S4), e a razão entre consumo de ADP/consumo de oxigênio (ADP/O). RESULTADOS: Observou-se significativo aumento de bilirrubina sérica total, enzimas hepáticas, e consumo de O2 durante a respiração basal (S4) no grupo de IO por 24hs (ANOVA, p=0.009). Após adição de ADP, a taxa de consumo de O2 (S3) não diminuiu significativamente no grupo de IO, comparado com o CTR (ANOVA, p>0.05); entretanto, a razão do controle respiratório (RC) foi significativamente mais baixa comparada com o CTR (ANOVA, p=0.001). Os efeitos observados na respiração mitocondrial no grupo do dia 1d estavam exacerbados no grupo 7d. CONCLUSÃO: Estes resultados indicam que a icterícia obstrutiva induz depressão precoce (24hs) da respiração mitocondrial, e pode assim levar à redução da produção de ligações de alta energia.

Obstructive jaundice induces early depression of mitochondrial respiration in rat hepatocytes.

INTRODUCTION: Oxidative phosphorylation dysfunction of hepatocyte mitochondria is involved in the pathophysiology of organ dysfunction following obstructive jaundice (OJ). However the time period from biliary occlusion to the occurrence of the dysfunction has not been determined decisively. PURPOSE: To evaluate the early effects (1 d and 7 d) of OJ on liver mitochondria respiratory function in rats. METHODS: Male Wistar rats (200-250 g) were randomly divided into the following 3 groups: laparotomy plus OJ for 24 h (1d group) (n = 10); laparotomy plus OJ for 7 d (7d group) (n = 10); sham control procedure (CTR group) (n = 12). At the end of OJ periods, total serum bilirubin level, hepatic enzyme activity levels (GOT, GTP, Gama-GT, ALP), mitochondrial respiration phases S3 and S4, as well as the respiratory control ratio (RC = S3/S4), and ADP consumption/oxygen consumption (ADP/O) ratio, were determined. RESULTS: Total serum bilirubin, activity of most hepatic enzymes, and O2 consumption during basal (S4) respiration were increased in the 1d and 7d groups (ANOVA, p = 0.05 vs. CTR). After ADP addition, the O2 consumption rate (S3) in the 1d group remained similar to the CTR rate (ANOVA p > .05), while the RC rate was reduced (ANOVA, p = 0.001) vs. CTR. The effects observed on mitochondrial respiration in the 1d group were exacerbated in the 7d group. CONCLUSION: These results indicate that OJ induces early (24 h) depression of liver mitochondria respiration, and thus may lead to early reduction in the production of high energy bonds.