Clinical trial: modulation of human placental multidrug resistance proteins in cholestasis of pregnancy by ursodeoxycholic acid

Dipartimento di Medicina Interna e Gastroenterologia, Università degli Studi di Bologna, Bologna, Italy/
Alimentary Pharmacology & Therapeutics (Impact Factor: 5.73). 10/2007; 26(8):1139-46. DOI: 10.1111/j.1365-2036.2007.03462.x
Source: PubMed


The effects of ursodeoxycholic acid on human placental bile acids and bilirubin transporters in intrahepatic cholestasis of pregnancy are still undefined.
To evaluate whether ursodeoxycholic acid affects MRP2, MRP3 and MRP4 expression in the placenta.
Forty-three pregnant women were enrolled; fourteen subjects had physiological pregnancies. Intrahepatic cholestasis of pregnancy patients were divided into two groups: (i) 13 received ursodeoxycholic acid (20 mg/kg/day) and (ii) 16 untreated. Total bile acid and bilirubin in serum and cord blood were determined in each subject. Multidrug resistance proteins expression (immunoblot, quantitative real-time PCR) was evaluated in placentas collected at delivery. anova test was used for statistical analysis of data.
Ursodeoxycholic acid administration significantly improved maternal serum bile acid and cord blood bilirubin and bile acid levels. MRP2 protein and RNA expression was significantly increased in placentas from treated patients compared to controls (P < 0.001 and P < 0.01, respectively). MRP3 protein expression was not significantly different between the groups while RNA expression was significantly decreased in treated patients (P < 0.01). MRP4 did not show significant differences between the groups.
Ursodeoxycholic acid administration induces placental MRP2 expression, and reduces bilirubin and bile acid levels in cord blood.

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Available from: Antonio Colecchia, Sep 23, 2014
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    • "In the reproductive system MRP4 is expressed in testes (Morgan et al., 2012), placenta (Azzaroli et al., 2007), endometrium (Lacroix-Pépin et al., 2011) and ovary (Maher et al., 2005). It has been shown that in human and rodent testicular Leydig cells MRP4 plays a relevant role in testosterone production (Morgan et al., 2012). "
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    ABSTRACT: Sperm capacitation has been largely associated with an increase in cAMP, although its relevance in the underlying mechanisms of this maturation process remains elusive. Increasing evidence shows that the extrusion of cAMP through multidrug resistance associated protein 4 (MRP4), regulates cell homeostasis not only in physiological but also in pathophysiological situations and studies from our laboratory strongly support this assumption. In the present work we sought to establish the role of cAMP efflux in the regulation of sperm capacitation. Sperm capacitation was performed in vitro by exposing bovine spermatozoa to bicarbonate 40 and 70 mM; cAMP; probenecid (a MRPs general inhibitor) and an adenosine type 1 receptor (A1 adenosine receptor) selective antagonist (DPCPX). Capacitation was assessed by chlortetracycline assay and lysophosphatidylcholine-induced acrosome reaction assessed by PSA-FITC staining. Intracellular and extracellular cAMP was measured by radiobinding the regulatory subunit of PKA under the same experimental conditions. MRP4 was detected by Western blot and immunohistochemistry assays. Results showed that the inhibition of soluble adenylyl cyclase significantly inhibited bicarbonate-induced sperm capacitation. Furthermore, in the presence of 40 and 70 mM bicarbonate bovine spermatozoa synthesized and extruded cAMP. Interestingly, in the absence of IBMX (a PDEs inhibitor) cAMP efflux still operated in sperm cells, suggesting that cAMP extrusion would be a physiological process in the spermatozoa complementary to the action of PDE. Blockade of MRPs by probenecid abolished the efflux of the cyclic nucleotide resulting not only in the accumulation of intracellular cAMP but also in the inhibition of bicarbonate-induced sperm capacitation. The effect of probenecid was abolished by exposing sperm cells to cAMP. The high affinity efflux pump for cAMP, MRP4 was expressed in bovine spermatozoa and localized to the mid-piece of the tail as previously reported for soluble adenylyl cyclase and A1 adenosine receptor. Additionally, blockade of A1 adenosine receptor abolished not only bicarbonate-induced sperm capacitation but also that stimulated by cAMP. Present findings strongly support that cAMP efflux, presumably through MRP4, and the activation of A1 adenosine receptor regulate some events associated to bicarbonate-induced sperm capacitation, and further suggest a paracrine and/or autocrine role for cAMP.
    Molecular Human Reproduction 08/2013; 20(1). DOI:10.1093/molehr/gat053 · 3.75 Impact Factor
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    • "Reports of the expression of human MRP2 range from very low or undetectable in the human placenta (Pascolo et al., 2003; Evseenko et al., 2006) to comparable to hepatic expression in term placentae (Meyer Zu Schwabedissen et al., 2005), while other reports do not provide quite enough information to make this comparison (St-Pierre et al., 2000; Azzaroli et al., 2007). Some reports demonstrate high interpatient variability in MRP2 expression (Meyer Zu Schwabedissen et al., 2005; Vaidya et al., 2009). "
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    ABSTRACT: Quantitation of the expression levels of proteins involved in drug transport and disposition is needed to overcome limitations of film-based detection of chemiluminescent immunoblots. The purpose was to describe and validate a quantitative immunofluorescent blotting method for detection of ATP-binding cassette transporter isoform C2/multidrug resistance-associated protein 2 (ABCC2/MRP2). Western blotting was performed by electrophoresis of membrane vesicle protein isolated from Sf9 cells overexpressing MRP2 subsequently blotted with infrared labeled secondary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities, followed by linear regression of the intensity data. The limits of quantitation for the time-insensitive technique described here were from 0.001 μg to 0.5 μg of total membrane protein, the coefficient of variation of the slope was 8.9%; r2 values were 0.986 ± 0.012. The utility and sensitivity of this technique were demonstrated in quantitating expression of MRP2 in human placental tissue samples, in which MRP2 was present in low abundance. The immunofluorescent blotting technique described provides sensitive, reproducible, and quantitative determinations of large, integral membrane proteins such as MRP2, all with potential long-term cost savings.
    Journal of pharmacological and toxicological methods 05/2011; 63(3):279-82. DOI:10.1016/j.vascn.2011.01.003 · 2.39 Impact Factor
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    • "In addition to the obvious effects on renal urate excretion, MRP4 presumably has a function in regulating urate levels in various tissues where it is expressed, including the liver (Gradhand et al., 2007), vascular smooth muscle (Mitani et al., 2003), brain (Hirrlinger et al., 2005), intestine (Zimmermann et al., 2005), blood cells (Kock et al., 2007) and placenta (Azzaroli et al., 2007). The function of MRP4 may have different implications depending on the tissue and Table 1 IC 50 values of various drugs tested on MRP4-mediated [ 14 C]urate transport Drugs MRP4-mediated urate transport (mM) "
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    ABSTRACT: The xanthine oxidase inhibitors allopurinol and oxypurinol are used to treat hyperuricaemia, whereas loop and thiazide diuretics can cause iatrogenic hyperuricaemia. Some uricosuric drugs and salicylate have a bimodal action on urate renal excretion. The mechanisms of action of these hypo- and hyperuricaemic drugs on the handling of urate in renal tubules have not been fully elucidated. Recently, we identified the multidrug resistance protein (MRP) 4 as a luminal efflux transporter for urate in the proximal tubule. Here, we studied the effect of these drugs on [(14)C]urate transport using human embryonic kidney 293 cells overexpressing human MRP4 and in membrane vesicles isolated from these cells. Allopurinol stimulated MRP4-mediated cellular urate efflux and allopurinol and oxypurinol both markedly stimulated urate transport by MRP4 in membrane vesicles. Bumetanide and torasemide had no effect, whereas furosemide, chlorothiazide, hydrochlorothiazide, salicylate, benzbromarone and sulfinpyrazone inhibited urate transport, at concentrations ranging from nanomolar up to millimolar. Probenecid stimulated urate transport at 0.1 microM and inhibited transport at higher concentrations. These data suggest that inhibition of MRP4-mediated urate efflux by furosemide and thiazide diuretics could have an important function in their hyperuricaemic mechanisms. Furthermore, stimulation of MRP4-mediated renal urate efflux could be a new mechanism in the hypouricaemic action of allopurinol and oxypurinol. In conclusion, MRP4 may provide a potential target for drugs affecting urate homoeostasis, which needs to be further evaluated in vivo.
    British Journal of Pharmacology 09/2008; 155(7):1066-75. DOI:10.1038/bjp.2008.343 · 4.84 Impact Factor
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