Nancy Solís

Farnesoid X Receptor Critically Determines the Fibrotic Response in Mice but Is Expressed to a Low Extent in Human Hepatic Stellate Cells and Periductal Myofibroblasts

The nuclear bile acid receptor, farnesoid X receptor (FXR), may play a pivotal role in liver fibrosis. We tested the impact of genetic FXR ablation in four different mouse models. Hepatic fibrosis was induced in wild-type and FXR knock-out mice (FXR(-/-)) by CCl(4) intoxication, 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding, common bile duct ligation, or Schistosoma mansoni (S.m.)-infection. In addition, we determined nuclear receptor expression levels (FXR, pregnane X receptor (PXR), vitamin D receptor, constitutive androstane receptor (CAR), small heterodimer partner (SHP)) in mouse hepatic stellate cells (HSCs), portal myofibroblasts (MFBs), and human HSCs. Cell type-specific FXR protein expression was determined by immunohistochemistry in five mouse models and prototypic human fibrotic liver diseases. Expression of nuclear receptors was much lower in mouse and human HSCs/MFBs compared with total liver expression with the exception of vitamin D receptor. FXR protein was undetectable in mouse and human HSCs and MFBs. FXR loss had no effect in CCl(4)-intoxicated and S.m.-infected mice, but significantly decreased liver fibrosis of the biliary type (common bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine). These data suggest that FXR loss significantly reduces fibrosis of the biliary type, but has no impact on non-cholestatic liver fibrosis. Since there is no FXR expression in HSCs and MFBs in liver fibrosis, our data indicate that these cells may not represent direct therapeutic targets for FXR ligands.

Effect of losartan on early liver fibrosis development in a rat model of nonalcoholic steatohepatitis.

Background and Aim:  Nonalcoholic steatohepatitis (NASH) is a metabolic disorder of the liver that may evolve into fibrosis or cirrhosis. Recent studies have shown reduction of experimental liver fibrosis with the use of angiotensin‐converting‐enzyme inhibitors or angiotensin‐receptor antagonists. The aim of this study was to determine whether losartan can influence the early phase of fibrogenesis in an animal model of NASH.

Down-regulation of the Na+/taurocholate cotransporting polypeptide during pregnancy in the rat

BACKGROUND Experimental studies have shown decreased bile acid (BA) uptake and reduced excretion of cholephilic compounds in pregnant rodents. AIM To assess the expression and function of the main BA importer, the Na(+)/taurocholate cotransporting polypeptide (Ntcp) in pregnant rats. METHODS BA uptake and Ntcp expression were studied in control and timed-pregnant rats in late gestation. Ntcp protein, messenger RNA (mRNA) expression, and Ntcp tissue localization were determined by Northern blotting, Western analysis, and tissue immunofluorescence. The activity of three transactivators of the Ntcp promoter: hepatocyte nuclear factor 1-alpha (HNF1-alpha), nuclear receptor heterodimer retinoid X receptor:retinoid acid receptor (RXR:RAR) and signal transducer and activator of transcription 5 (Stat5) was assessed using gel electrophoretic mobility shift assays. RESULTS A significantly reduced BA uptake and decreased Ntcp mRNA levels (-40%) and protein mass (-60%) was observed in pregnant rats. Nuclear extracts from pregnant rats showed a marked decrease of HNF1-alpha and RXR:RAR binding activities by -80 and -40% of basal activity, respectively. In contrast, binding activity of Stat-5 was increased by 50% in nuclear extracts from pregnant rats. CONCLUSIONS Pregnancy is associated with reduced Ntcp expression and function in the rat. Our findings suggest that Ntcp down-regulation during pregnancy occurs primarily at the transcriptional level.

Modulation of hepatic content and biliary excretion of P-glycoproteins in hepatocellular and obstructive cholestasis in the rat

BACKGROUND/AIMS Release into bile of canalicular membrane enzymes, such as alkaline phosphatase and gamma-glutamyl transpeptidase, is significantly increased in rats subjected to experimental models of hepatocellular or obstructive cholestasis. This effect appears to be related to a greater susceptibility of these membrane intrinsic proteins to the solubilizing effects of secreted bile acids. It is not known whether canalicular membrane transport proteins, such as P-glycoprotein isoforms, involved in ATP-dependent xenobiotic biliary excretion and phospholipid secretion, are excreted into bile and whether this process is modified in cholestasis. The aims of this work have been to investigate in the rat: a) whether P-glycoproteins are normally excreted into bile, b) whether their excretion is modified in two experimental models of cholestasis, i.e., hepatocellular cholestasis induced by ethynylestradiol and obstructive cholestasis, and c) whether observed changes correlate with bile acid and phospholipid secretion and enzyme release into bile and with relative P-glycoprotein content in hepatic tissue and isolated and purified canalicular membranes. METHODS P-glycoproteins in bile and hepatic tissue were identified and quantitated by Western-blotting and immunohistochemistry using the C219 MAb. Changes in total mdr mRNA were analyzed by Northern-blotting. RESULTS Like canalicular membrane enzymes, P-glycoproteins are normally excreted into bile. Ethynylestradiol-induced cholestasis was associated with a 4.9-fold increase in P-glycoprotein excretion compared with controls while, in contrast, the excretion of the carrier decreased markedly in obstructive cholestasis to 2% of control values. P-glycoprotein excretion per nmol of secreted bile acids increased 4.4-fold in ethynylestradiol-induced cholestasis but decreased to 2% of control values in obstructive cholestasis. Total mdr mRNA levels in hepatic tissue were markedly increased (3.4-fold) in rats subjected to obstructive cholestasis and moderately increased (1.6-fold) in the ethynylestradiol group, compared with controls. P-glycoprotein content in isolated canalicular membranes was slightly decreased by 15% in ethynylestradiol-induced cholestasis, while it increased 4.7-fold in obstructive cholestasis. Immunohistochemistry of rat livers showed that P-glycoprotein reaction at the canalicular domain of hepatocytes at acinar zone 1 was decreased in ethynylestradiol-treated rats and markedly increased in obstructive cholestasis. CONCLUSIONS Ethynylestradiol-induced cholestasis is associated with increased P-glycoprotein biliary excretion and decreased hepatic content. In contrast, obstructive cholestasis results in decreased P-glycoprotein biliary excretion and increased hepatic content. These results suggest that biliary P-glycoprotein excretion might be a modulating factor in canalicular membrane P-glycoprotein content. Increased P-glycoprotein release into bile in ethynylestradiol-treated rats is apparently not a consequence of cholestasis, but it might be a primary event and play a pathogenetic role in ethynylestradiol-induced cholestasis.

Overexpression of 11β-hydroxysteroid dehydrogenase type 1 in visceral adipose tissue and portal hypercortisolism in non-alcoholic fatty liver disease.

The enzyme 11β‐hydroxysteroid‐dehydrogenase type 1 (11β‐HSD1) catalyses the reactivation of intracellular cortisol. We explored the potential role of 11β‐HSD1 overexpression in visceral adipose tissue (VAT) in non‐alcoholic fatty liver disease (NAFLD) assessing sequential changes of enzyme expression, in hepatic and adipose tissue, and the occurrence of portal hypercortisolism in obese mice. 11β‐HSD1 expression was also assessed in tissues from obese patients undergoing bariatric surgery.

Overexpression of hepatic 5α-reductase and 11β-hydroxysteroid dehydrogenase type 1 in visceral adipose tissue is associated with hyperinsulinemia in morbidly obese patients

11-β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts cortisone to cortisol, mainly in the liver and visceral adipose tissue (VAT), and has been implicated in several metabolic disorders. The absence of systemic hypercortisolism in central obesity could be due to increased inactivation of cortisol to its tetrahydrometabolites by the hepatic enzymes 5α- and 5β-reductases. Our aim was to assess the expression of the reductases in the liver and of 11β-HSD1 in the liver and VAT in morbidly obese patients and to analyze their association with clinical, anthropometric, and biochemical parameters. Hepatic and VAT samples were obtained during bariatric surgery. 5α- and 5β-reductases, 11β-HSD1, and 18S expression was measured using real-time polymerase chain reaction. Anthropometric and biochemical variables were analyzed. Forty-one patients were recruited (age, 41.8 ± 10.6 years; body mass index, 42.1 ± 6.6 kg/m(2); 71% women). The expression of hepatic 5α- and 5β-reductases was positively correlated (r = +0.53, P = .004), and their expression levels were correlated with hepatic 11β-HSD1 expression (r = +0.61, P < .001 for 5α-reductase and r = +0.50, P < .001 for 5β-reductase). Hepatic 5α-reductase was associated with insulin (r = +0.34, P = .015). Visceral adipose tissue 11β-HSD1 expression was associated with glucose (r = +0.37, P = .025) and insulin (r = +0.54, P = .002). Our results showed that 5α-reductase and VAT 11β-HSD1 expressions were associated with insulinemia. These findings suggest that overexpression of 5α-reductase, through a higher inactivation of cortisol in the liver, could have a protective role in preserving hepatic sensitivity to insulin. The overexpression of liver reductases in obesity could be an adaptive response to an increase in cortisol production by the liver and visceral 11β-HSD1 to avoid systemic hypercortisolism.

Enhanced biliary excretion of canalicular membrane enzymes in estrogen-induced and obstructive cholestasis, and effects of different bile acids in the isolated perfused rat liver

BACKGROUNDS/AIMS Canalicular membrane enzymes are normally released into bile by partially known processes. This study was undertaken to investigate whether hepatocellular cholestatis induced in rats by ethynylestradiol or obstructive cholestasis produced by complete biliary obstruction for 24 h is associated with an increased release of alkaline phosphatase and gamma-glutamyl transpeptidase into bile, and to clarify how this process is affected by different bile acids. METHODS The studies were performed in the isolated perfused liver during infusion of sodium taurocholate, taurochenodeoxycholate and tauroursodeoxycholate at increasing rates. RESULTS Maximum sodium taurocholate, taurochenodeoxycholate and tauroursodeoxycholate secretory rates were decreased in both cholestatic groups (complete biliary obstruction > ethynylestradiol) compared with controls. Maximum biliary outputs of alkaline phosphatase and gamma-glutamyl transpeptidase were significantly increased in the ethynylestradiol group during infusion of sodium taurocholate and taurochenodeoxycholate, but not of tauroursodeoxycholate, and were increased in the complete biliary obstruction group during the infusion of sodium taurocholate and tauroursodeoxycholate but not of taurochenodeoxycholate. The biliary outputs of alkaline phosphatase and gamma-glutamyl transpeptidase showed a significant and direct linear relationship with sodium taurocholate and taurochenodeoxycholate secretory rates in both cholestatic groups. However, only in the complete biliary obstruction group did alkaline phosphatase and gamma-glutamyl transpeptidase excretion show a significant correlation with tauroursodeoxycholate secretory rates. The slope of the line, which indicated the mU of enzyme activity secreted per nmol of sodium taurocholate or taurochenodeoxycholate, was greater for gamma-glutamyl transpeptidase and alkaline phosphatase in both cholestatic groups (ethynylestradiol > complete biliary obstruction) than in the control group. Alkaline phosphatase activity in purified isolated canalicular and sinusoidal membranes was significantly increased in both cholestatic groups (complete biliary obstruction > ethynylestradiol), while gamma-glutamyl transpeptidase activity was unchanged compared with controls. CONCLUSION The marked increase in sodium taurocholate and taurochenodeoxycholate-mediated release of alkaline phosphatase and gamma-glutamyl transpeptidase into bile in cholestatic rats suggests an increased lability of these intrinsic membrane proteins to the detergent effects of secreted bile acids. It remains to be elucidated whether this phenomenon, which was particularly intense in ethynylestradiol induced cholestasis, is important in the pathogenesis and perpetuation of bile secretory failure. In contrast, tauroursodeoxycholate administration did not result in enhanced biliary excretion of these membrane enzymes, in either the control group or the ethynylestradiol group, supporting the concept that this bile salt lacks the membrane toxicity of common bile acids.

Enhanced biliary excretion of canalicular membrane enzymes in ethynylestradiol-induced cholestasis. Effects of ursodeoxycholic acid administration

Cholestasis is associated with a marked increase in the release of canalicular membrane enzymes into bile. This phenomenon has been related to an increased lability of these canalicular membrane integral proteins to the solubilizing effects of secreted bile salts. To further characterize the effects of oral ursodeoxycholic acid (UDCA) administration on ethynylestradiol (EE)-induced cholestasis, the influence of this bile acid on changes in biliary excretion of membrane-bound enzymes was investigated. Bile flow, basal bile salt and biliary lipid secretory rates, the maximum secretory rate of taurocholate (TC SRm), and the biliary excretion of the canalicular membrane-bound ectoenzymes alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGT) were measured in rats after EE and/or UDCA administration. The activities of ALP, GGT and Na+,K(+)-ATPase in purified isolated canalicular and sinusoidal membrane fractions and the ultrastructure of hepatic acinus, including histochemical studies of ALP distribution, were also examined. EE significantly reduced bile flow, bile salt and biliary lipid secretory rates, and TC SRm, and caused dilatation and loss of microvilli at the canalicular pole of hepatocytes. Biliary excretion of ALP increased 2-fold, whereas biliary excretion of GGT was unchanged. The relationship between biliary excretion of ALP or GGT and bile salt secretion (units of enzyme activity secreted per nanomole of bile salt) was greater in EE-treated rats compared with controls (2.1- and 1.5-fold greater for ALP and GGT, respectively), indicating that in EE-induced cholestasis more enzyme was released into bile per nanomole of bile salt. Na+,K(+)-ATPase activity in sinusoidal membrane fraction was reduced significantly, whereas ALP activity increased in both membrane fractions in EE-treated rats. The histochemical distribution of ALP in the acinus showed a strong reaction in acinar zone 3 and at both the canalicular and sinusoidal membranes. Oral administration of UDCA prevented EE-induced bile secretory failure by normalizing bile flow, bile salt and biliary phospholipid secretory rates, and TC SRm. UDCA also prevented the EE-induced changes in the biliary excretion of enzymes. On the contrary, UDCA did not modify either the enzyme activity in isolated membrane fractions or the morphological or ALP histochemical changes associated with EE administration. These data indicate that in EE-induced cholestasis changes occur at the canalicular membrane, enabling this portion of the plasma membrane to be more susceptible to the solubilizing effect of bile salt, and that oral administration of UDCA prevents bile secretory failure and changes in the biliary excretion of ALP and GGT in EE-treated rats.

Beneficial effects of mineralocorticoid receptor blockade in experimental non‐alcoholic steatohepatitis

Therapeutic options to treat Non‐alcoholic steatohepatitis (NASH) are limited. Mineralocorticoid receptor (MR) activation could play a role in hepatic fibrogenesis and its modulation could be beneficial for NASH.

Development of ACLEEM questionnaire, an instrument measuring residents' educational environment in postgraduate ambulatory setting

Background: Students’ perceptions of their educational environment (EE) have been studied in undergraduate and postgraduate curricula. Postgraduate EE has been measured in hospital settings. However, there are no instruments available to measure the EE in postgraduate ambulatory settings. Aim: The aim of this study was to develop the “Ambulatory Care Learning Education Environment Measure” (ACLEEM). Methods: A mixed methodology was used including three stages: (1) Grounded theory (focus groups); (2) Delphi technique to identify consensus; and (3) Pilot study. Results: Three quota samples of approximately 60 stakeholders were formed, one as Focus Groups and two as Delphi panels. Eight focus groups were carried out including 58 residents (Latin-American Spanish speakers). The results were analysed and 173 items were offered to a National Delphi panel (61 residents and teachers). They reduced in two rounds the number of important items to 54. The 54-item questionnaire was then piloted with 63 residents and refined to the final version of the ACLEEM with 50 items and three domains. Conclusions: The 50-item inventory is a valid instrument to measure the EE in postgraduate ambulatory setting in Chile. Large-scale administration of the ACLEEM questionnaire to evaluate its construct validity and reliability are the next steps to test the psychometric properties of the instrument.