Hepatic and renal expression of organic anion transporters during jaundice, and improvement of transcellular organic anion transport after transfection with Multidrug Resistance-associated Protein 2(MRP2) gene.

多种胆红素udp -葡萄糖醛基转移酶基因（UGT1A1）突变已报道在家族性非偶联高胆红素血症患者。为了阐明该基因突变在日本人群中的发生率，我们调查了一组患有Crigler - Najjar综合征（CNS） 2型和Gilbert综合征（GS）的日本患者以及健康无黄疸受试者中UGT1A1突变的存在。UGT1A1编码区多态性在日本GS患者和健康受试者中普遍存在。高胆红素血症的遗传基础似乎在日本人和白种人之间有所不同。在这项研究中，我们还发现了一种新的突变，杂合P364L。P364L UGT1A1酶活性比野生型酶活性低64.4%。Gunn大鼠是CNSⅰ型的动物模型，有报道称UGT1A1在CNSⅰ型中存在缺陷。我们检测了有机阴离子转运多肽（oatp） 1、oatp2、多耐药相关蛋白（mrp） 2和mrp3在Gunn大鼠肝脏和肾脏中的表达。Gunn大鼠肝、肾组织中oatpl表达降低，mrp3表达升高。卫材高胆红素尿鼠（EHBR）是由于胆红素葡萄糖醛酸苷胆排泄受损而导致黄疸的Dubin-Johnson综合征动物模型。在EHBR中，mrp2的缺乏导致大量有机阴离子的胆道排泄缺陷。EHBR中mrp3在肝脏和肾脏的表达明显高于SD大鼠。EHBR和SD大鼠肝脏和肾脏mrp1和mrp6 mRNA的表达无显著差异。Mrp1和mrp6蛋白在EHBR和SD大鼠的肝脏和肾脏中几乎不表达。与mrp3相比，EHBR大鼠肝脏中oatp1和oatp2 mRNA的表达低于SD大鼠。免疫组化显示肝脏和肾脏mrp3蛋白定位于基底外侧膜。我们构建了包含人MRP2 cDNA全长的蛋白表达载体（pDEST_<26>），该载体被日本血液凝集病毒（HJV）的包膜蛋白包裹。用pDEST_<26>转染EHBR。我们发现，MRP2转染EHBR后，结合胆红素的跨细胞转运在肝脏中恢复。转染EHBR后，血清结合胆红素水平降至正常水平（35.7 ~ 6.4 μmol/L）。转染EHBR后，肝脏中Mrp3表达降低，Oatp1和Oatp2表达升高，但与对照SD大鼠无显著差异。少

Various mutations of bilirubin UDP-glucronosyltransferase gene (UGT1A1) have been reported in patients with familial unconjugated hyperbilirubinemia. To clarify the incidence of this gene mutation in the Japanese population, we investigated the presence of UGT1A1 mutation in a group of Japanese patients with Crigler Najjar syndrome(CNS) type 2 and Gilbert's syndrome(GS), as well as in healthy anicteric subjects. Polymorphisms in the coding region of UGT1A1 were commonly observed in Japanese patients with GS and in healthy subjects. The genetic basis of hyperbilirubinemia appears to be different between the Japanese population and Caucasians. In this study, we additionally identified a novel mutation, heterozygous P364L. The P364L UGT1A1 enzyme activity was 64.4% lower than the wild type enzyme activity.Gunn rat is an animal model of CNS type I. UGT1A1 has been reported to be deficient in CNS type 1. We evaluated expressions of organic anion transporting polypeptide(oatp) 1, oatp2, mult … More idrug resistance-associated protein(mrp) 2, and mrp3 in the liver and kidney of Gunn rats. Decreased expression of oatpl and increased expression of mrp3 were observed in the liver and kidney of Gunn rats.Eisai hyperbilirubinuria rat(EHBR) is an animal model of Dubin-Johnson syndrome that suffers from jaundice due to impaired biliary excretion of bilirubin glucuronides. In EHBR, deficiency of mrp2 causes defective biliary excretion of numerous organic anions. Hepatic and renal expression of mrp3 was significantly higher in EHBR than in SD rats. Hepatic and renal expression of mrp1 and mrp6 mRNA was not significantly different between EHBR and SD rats. Mrp1 and mrp6 proteins were hardly expressed in liver and kidney of both EHBR and SD rats. In contrast to mrp3, hepatic expression of oatp1 and oatp2 mRNA were lower in EHBR than in SD rats. Immunohistochemistry disclosed that hepatic and renal mrp3 protein was localized at the basolateral membrane.We developed a protein expression vector (pDEST_<26>) which includes the full length of human MRP2 cDNA, which is encapsulated by the envelope protein of the hemoagglutinating virus of Japan (HJV). Gene transfection with pDEST_<26> was performed on EHBR. We disclosed that transcellular transport of conjugated bilirubin was recovered in the liver from EHBR after transfection with MRP2. Serum conjugated bilirubin level decreased to normal levels in EHBR (35.7 μmol/L to 6.4 μmol/L) after transfection. Mrp3 expression decreased while Oatp1 or Oatp2 expression increased in the liver from transfected EHBR but these expressions were not significantly different from control SD rats. Less