<p>OBJECTIVETo study the effect of Zhusha Anshen pill, cinnabar, HgS, HgCl2 and MeHg on the gene expression of renal transporters in mice.p><p>METHODHealthy male mice were given equivalent physiological saline, Zhusha Anshen pill (1.8 g · kg(-1), containing 0.17 g · kg(-1) of mercury), cinnabar (0.2 g · kg(-1), containing 1.7 g · kg(-1) of mercury), high dose cinnabar (2 g · kg(-1), containing 1.7 g · kg(-1) of mercury), HgS (0.2 g · kg(-1), containing 0.17 g · kg(-1) of mercury), HgCl2 (0.032 g · kg(-1), containing 0. 024 g · kg(-1) of mercury), MeHg (0.026 g · kg(-1), containing 0.024 g · kg(-1) of mercury), once daily, for 30 d, measuring body mass gain. 30 days later, the mice were sacrificed. The mercury accumulation in kidneys was detected with atomic fluorescence spectrometer. Expressions of Oat1, Oat2, Oat3, Mrp2, Mrp4, Urat1 were detected with RT-PCR.p><p>RESULTCompared with the normal control group, a significant accumulation of Hg in kidney in HgCl2 and MeHg groups was observed (P <0.05), but these changes were not found in other groups. Compared with normal control group, mRNA expressions of Oat1 and Oat2 were evidently lower in HgCl2 and MeHg groups, but mRNA expressions of Mrp2 were apparently higher in HgCl2 group (P <0.05), mRNA expression of Mrp4 was significant higher in HgCl2 and MeHg groups, and mRNA expression of Urat1 was apparently lower in MeHg group.p><p>CONCLUSIONHgCl2 and MeHg groups show significant difference from the normal group in mercury accumulation in kidneys and gene expression of kidney transporters, but with no difference between other groups and the normal group. Compared with HgCl2 and MeHg, cinnabar and its compounds could cause lower renal toxicity to mice.p>
Animals ; Carrier Proteins ; genetics ; Drugs, Chinese Herbal ; toxicity ; Gene Expression ; drug effects ; Kidney ; drug effects ; metabolism ; Male ; Mercuric Chloride ; toxicity ; Mercury Compounds ; toxicity ; Methylmercury Compounds ; toxicity ; Mice ; Multidrug Resistance-Associated Proteins ; genetics ; Organic Anion Transport Protein 1 ; genetics ; Organic Anion Transporters, Sodium-Independent ; genetics

AIM: The present study was undertaken to characterize the effects of Wuling San on urate excretion and renal function, and explore its possible mechanisms of action in hyperuricemic mice. METHODS: Mice were administered with 250 mg·kg(-1) potassium oxonate by gavage once daily (10 animals/group) for seven consecutive days to develop a hyperuricemia model. Different doses of Wuling powder were orally initiated on the day 1 h after oxonate was given, separately. Allopurinol was used as a positive control. Serum and urine levels of uric acid and creatinine, and fractional excretion of uric acid (FEUA) were measured in hyperuricemic mice treated with Wuling San and allopurinol. Simultaneously, renal mRNA and protein levels of urate transporter 1 (mURAT1), glucose transporter 9 (mGLUT9), organic anion transporter 1 (mOAT1), as well as organic cation/carnitine transporters mOCT1, mOCT2 and mOCTN2, were assayed by semi-quantitative RT-PCR and Western blot methods, respectively. RESULTS AND CONCLUSION Compared to the hyperuricemia control group, Wuling San significantly reduced serum uric acid and creatinine levels, increased 24 h urate and creatinine excretion, and FEUA in hyperuricemic mice, exhibiting its ability to enhance urate excretion and improve kidney function. Wuling San was found to down-regulate mRNA and protein levels of mURAT1 and mGLUT9, as well as up-regulate mOAT1 in the kidney of hyperuricemic mice. Moreover, Wuling San up-regulated renal mRNA and protein levels of mOCT1, mOCT2 and mOCTN2, leading to kidney protection in this model.
Animals ; Drugs, Chinese Herbal ; administration & dosage ; Glucose Transport Proteins, Facilitative ; genetics ; metabolism ; Humans ; Hyperuricemia ; drug therapy ; genetics ; metabolism ; Kidney ; drug effects ; metabolism ; Male ; Mice ; Organic Anion Transport Protein 1 ; genetics ; metabolism ; Organic Anion Transporters ; genetics ; metabolism ; Up-Regulation ; drug effects ; Uric Acid ; metabolism

Liver is regarded as one of the most important organs for drug clearance in the body, which mediates both the metabolism and biliary excretion of drugs. Transporters are a class of functional membrane proteins and control the movement of substances into or out of cells. Transporters, which are extensively expressed in the liver, play important roles in the drug hepatic disposition by regulating the uptake of drugs from blood into hepatocytes or the efflux of drugs and their metabolites into bile. In this review, the localization, functions and substrate selectivity of the major transporters in the liver will be summarized, and the impacts of these transporters on drug hepatic disposition, the potential drug-drug interactions as well as their genetic polymorphisms will also be reviewed.
ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Bile ; metabolism ; Biological Transport ; Drug Interactions ; Humans ; Liver ; metabolism ; Membrane Transport Proteins ; genetics ; metabolism ; Metabolic Clearance Rate ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Organic Anion Transporters ; genetics ; metabolism ; Organic Anion Transporters, Sodium-Dependent ; metabolism ; Organic Anion Transporters, Sodium-Independent ; genetics ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Pharmacokinetics ; Polymorphism, Genetic ; Symporters ; metabolism

The effects of mangiferin on uric acid excretion, kidney function and related renal transporters were investigated in hyperuricemic mice induced by potassium oxonate. Mice were divided into normal control group, and 5 hyperuricemic groups with model control, 50, 100, and 200 mg x kg(-1) mangiferin, and 5 mg x kg(-1) allopurinol. Mice were administered by gavage once daily with 250 mg x kg(-1) potassium oxonate for seven consecutive days to create the model. And 3 doses of mangiferin were orally initiated on the day 1 h after potassium oxonate was given, separately. Serum uric acid, creatinine and urea nitrogon levels, as well as urinary uric acid creatinine levels were measured. Mouse uromodulin (mUMOD) levels in serum, urine and kidney were determined by ELISA method. The mRNA and protein levels of related renal transporters were assayed by RT-PCR and Western blotting methods, respectively. Compared to model group, mangiferin significantly reduced serum uric acid, creatinine and urea nitrogon levels, increased 24 h uric acid and creatinine excretion, and fractional excretion of uric acid in hyperuricemic mice, exhibiting uric acid excretion enhancement and kidney function improvement. Mangiferin was found to down-regulate mRNA and protein levels of urate transporter 1 (mURAT1) and glucose transporter 9 (mGLUT9), as well as up-regulate organic anion transporter 1 (mOAT1) in the kidney of hyperuricemic mice. These findings suggested that mangiferin might enhance uric acid excretion and in turn reduce serum uric acid level through the decrease of uric acid reabsorption and the increase of uric acid secretion in hyperuricemic mice. Moreover, mangiferin remarkably up-regulated expression levels of renal organic cation and carnitine transporters (mOCT1, mOCT2, mOCTN1 and mOCTN2), increased urine mUMOD levels, as well as decreased serum and kidney mUMOD levels in hyperuricemic mice, which might be involved in mangiferin-mediated renal protective action.
Animals ; Blood Urea Nitrogen ; Carrier Proteins ; genetics ; metabolism ; Creatinine ; blood ; Glucose Transport Proteins, Facilitative ; genetics ; metabolism ; Hyperuricemia ; blood ; chemically induced ; physiopathology ; urine ; Kidney ; metabolism ; physiopathology ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Octamer Transcription Factor-1 ; genetics ; metabolism ; Organic Anion Transport Protein 1 ; genetics ; metabolism ; Organic Anion Transporters ; genetics ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Organic Cation Transporter 2 ; Oxonic Acid ; Protective Agents ; pharmacology ; RNA, Messenger ; metabolism ; Random Allocation ; Solute Carrier Family 22 Member 5 ; Uric Acid ; blood ; urine ; Uromodulin ; blood ; urine ; Xanthones ; pharmacology

Drug metabolism will change significantly during inflammation, including the reduction of expression and activity of many drug metabolizing enzymes and transporters. Body would release a series of inflammatory cytokines which can regulate drug metabolizing enzymes. Recent studies have revealed that drug transporters are also regulated by the cytokines with obvious species difference. Mechanism studies show that several transcription factors play important roles during the signal pathways of regulation. This review focuses on the progress in the regulation of drug transporters during inflammation.
ATP Binding Cassette Subfamily B Member 11 ; ATP Binding Cassette Transporter, Sub-Family B ; metabolism ; ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; metabolism ; Animals ; Biological Transport ; Humans ; Inflammation ; metabolism ; Membrane Transport Proteins ; metabolism ; Multidrug Resistance-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Organic Anion Transporters ; metabolism ; Organic Cation Transport Proteins ; metabolism ; Signal Transduction

ATP Binding Cassette Subfamily B Member 11 ; ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; physiology ; ATP-Binding Cassette, Sub-Family B, Member 1 ; physiology ; Animals ; Bile ; metabolism ; Biliary Tract ; physiology ; Drug Interactions ; Drug Resistance, Multiple ; Humans ; Liver ; physiology ; Multidrug Resistance-Associated Proteins ; physiology ; Neoplasm Proteins ; physiology ; Organic Anion Transporters ; physiology ; Organic Anion Transporters, Sodium-Dependent ; physiology ; Organic Cation Transport Proteins ; physiology ; Symporters ; physiology

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.
Humans ; Cell Membrane ; Mutation, Missense ; Phosphates/metabolism* ; Sodium-Phosphate Cotransporter Proteins, Type III/genetics*

<p>BACKGROUNDHuman urate anion exchanger (hURAT1) as a major urate transporter expressed on renal tubular epithelial cells regulates blood urate level by reabsorbing uric acid. Antibody is an important tool to study hURAT1. This study aimed, by genetic immunization, to produce mouse anti-hURAT1 polyclonal antibody with high throughput and high specificity and to detect the location of hURAT1 in human kidney.p><p>METHODSHuman renal total RNA was isolated and the entire cDNA of hURAT1 was amplified by RT-PCR. The sequence of intracellular high antigenicity fragment (A280 to R349) was chosen by prediction software of protein antigenicity, and its cDNA was amplified from cDNA of hURAT1, and then cloned into pBQAP-TT vector to construct recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization. Mice were inoculated with this recombinant plasmid and two other adjuvant plasmids, pCMVi-GMCSF and pCMVi-Flt3L, which helped to enhance the antibody's generation. After four weeks, the mice were sacrificed to obtain the anti-hURAT1 antibody from serum. The antibody was identified by western blot analysis and immunohistochemistry. At the same time, rabbit anti-hURAT1 antibody was produced by protein immunization. The specificity and efficiency between the rabbit and mouse anti-hURAT1 antibody were compared by western blot analysis and immunohistochemistry.p><p>RESULTSThe entire cDNA of hURAT1 and cDNA of its intracellular high immunogenic fragment were amplified successfully. Recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization was confirmed by restriction digestion and sequencing. Both the mouse anti-hURAT1 antibody and rabbit anti-hURAT1 antibody recognized 58 kD hURAT1 and 64 kD glycosylated hURAT1 protein bands in western blot. Immunohistochemically, hURAT1 was located at the brush border membrane of renal proximal tubular cells. In addition, the throughput and specificity of the mouse anti-hURAT1 antibody were higher than those of the rabbit anti-hURAT1 antibody.p><p>CONCLUSIONGenetic immunization can generate anti-hURAT1 polyclonal antibody of high throughput and specificity.p>
Animals ; Antibodies ; analysis ; Blotting, Western ; Carrier Proteins ; analysis ; immunology ; Female ; Humans ; Immunization ; Immunohistochemistry ; Kidney ; chemistry ; Male ; Mice ; Organic Anion Transporters ; analysis ; immunology ; Organic Cation Transport Proteins ; Plasmids ; Rabbits

Citrin is a liver-type mitochondrial aspartate-glutamate carrier encoded by the SLC25A13 gene, and its deficiency causes adult-onset type II citrullinemia and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). Here, the authors investigated clinical findings in Korean infants with NICCD and performed mutation analysis on the SLC25A13 gene. Of 47 patients with neonatal cholestasis, three infants had multiple aminoacidemia (involving citrulline, methionine, and arginine) and galactosemia, and thus were diagnosed as having NICCD. Two of these three showed failure to thrive. The laboratory findings showed hypoproteinemia and hyperammonemia, and liver biopsies revealed micro-macrovesicular fatty liver and cholestasis. The three patients each harbored compound heterozygous 1,638-1,660 dup/ S225X mutation, compound heterozygous 851del4/S225X mutation, and heterozygous 1,638-1,660 dup mutation, respectively. With nutritional manipulation, liver functions were normalized and catch-up growth was achieved. NICCD should be considered in the differential diagnosis of cholestatic jaundice in Korean infants.
Amino Acids/blood ; Calcium-Binding Proteins/*deficiency ; Cholestasis, Intrahepatic/*etiology/genetics ; Citrullinemia/genetics ; Humans ; Infant ; Membrane Transport Proteins/genetics ; Mitochondrial Proteins/genetics ; Mutation ; Organic Anion Transporters/*deficiency

<p>OBJECTIVETo assess the feasibility of high-resolution melting (HRM) analysis for screening patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).p><p>METHODSBased on previous studies on SLC25A13 gene in Chinese patients with NICCD, four hotspot mutations (851del4, 1638ins23, IVS6+5G>A and IVS16ins3kb) were selected. Results of the HRM analysis was validated using 50 negative controls and 20 patients with NICCD whose genotypes were confirmed previously by direct sequencing. With the established protocol, 171 suspected patients were enrolled. Samples with abnormal melting curves were further validated by DNA sequencing.p><p>RESULTSHRM analysis can accurately determine the genotypes of all negative controls and patients. The sensitivity and specificity of the technique reached 100% (70/70). The melting curves of samples with the same genotype were highly reproducible. In 171 suspected patients, seven NICCD patients were detected by HRM. Identified mutations have included one case of 851del4 homozygote, one case of IVS6+5G>A heterozygote, 3 cases of 851del4 heterozygotes, one case of [IVS6+5G>A]+[ 851del4] and one case of [1638ins23+IVS16ins3kb]+[1638ins23]. All mutations were subsequently confirmed by DNA sequencing.p><p>CONCLUSIONHRM analysis is a convenient, high-throughput and rapid technique for the screening of NICCD patients.p>
Anion Transport Proteins ; genetics ; Base Sequence ; Calcium-Binding Proteins ; deficiency ; China ; Citrullinemia ; diagnosis ; genetics ; metabolism ; DNA ; chemistry ; genetics ; Genetic Predisposition to Disease ; Genotype ; Humans ; Mitochondrial Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Nucleic Acid Denaturation ; Organic Anion Transporters ; deficiency ; Sensitivity and Specificity