Severe Diabetes and Leptin Resistance Cause Differential Hepatic and Renal Transporter Expression in Mice

Background: Type-2 Diabetes is a major health concern in the United States and other Westernized countries, with prevalence increasing yearly. There is a need to better model and predict adverse drug reactions, drug-induced liver injury, and drug efficacy in this population. Because transporters significantly contribute to drug clearance and disposition, it is highly significant to determine whether a severe diabetes phenotype alters drug transporter expression, and whether diabetic mouse models have altered disposition of acetaminophen (APAP) metabolites. Results: Transporter mRNA and protein expression were quantified in livers and kidneys of adult C57BKS and db/db mice, which have a severe diabetes phenotype due to a lack of a functional leptin receptor. The urinary excretion of acetaminophen-glucuronide, a substrate for multidrug resistance-associated proteins transporters was also determined. The mRNA expression of major uptake transporters, such as organic anion transporting polypeptide Slco1a1 in liver and kidney, 1a4 in liver, and Slc22a7 in kidney was decreased in db/db mice. In contrast, Abcc3 and 4 mRNA and protein expression was more than 2 fold higher in db/db male mouse livers as compared to C57BKS controls. Urine levels of APAP-glucuronide, -sulfate, and N-acetyl cysteine metabolites were higher in db/db mice. Conclusion: A severe diabetes phenotype/presentation significantly altered drug transporter expression in liver and kidney, which corresponded with urinary APAP metabolite levels

Constitutive activation of nuclear factor‐E2‐related factor 2 induces biotransformation enzyme and transporter expression in livers of mice with hepatocyte‐specific deletion of Kelch‐like ECH‐associated protein 1

Chemicals that activate nuclear factor‐E2‐related factor 2 (Nrf2) often increase multidrug‐resistance‐associated protein (Mrp) expression in liver. Hepatocyte‐specific deletion of Kelch‐like ECH‐associated protein 1 (Keap1) activates Nrf2. Use of hepatocyte‐specific Keap1 deletion represents a nonpharmacological method to determine whether constitutive Nrf2 activation upregulates liver transporter expression in vivo. The mRNA, protein expression, and localization of several biotransformation and transporters were determined in livers of wild‐type and hepatocyte‐specific Keap1‐null mice. Sulfotransferase 2a1/2, NADP(H):quinone oxidoreductase 1, cytochrome P450 2b10, 3a11, and glutamate–cysteine ligase catalytic subunit expression were increased in livers of Keap1‐null mice. Organic anion‐transporting polypeptide 1a1 expression was nearly abolished, as compared to that detected in livers of wild‐type mice. By contrast, Mrp 1–5 mRNA and protein levels were increased in Keap1‐null mouse livers, with Mrp4 expression being more than 15‐fold higher than wild types. In summary, Nrf2 has a significant role in affecting Oatp and Mrp expressions

Differential \u3cem\u3eFmo3\u3c/em\u3e gene expression in various liver injury models involving hepatic oxidative stress in mice

Flavin-containing monooxygenase-3 (FMO3) catalyzes metabolic reactions similar to cytochrome P450 monooxygenase, however, most metabolites of FMO3 are considered non-toxic. Recent findings in our laboratory demonstrated Fmo3 gene induction following toxic acetaminophen (APAP) treatment in mice. The goal of this study was to evaluate Fmo3 gene expression in other diverse mouse models of hepatic oxidative stress and injury. Fmo3 gene regulation by Nrf2 was also investigated using Nrf2 knockout (Nrf2 KO) mice. In our studies, male C57BL/6J mice were treated with toxic doses of hepatotoxicants or underwent bile duct ligation (BDL, 10 days). Hepatotoxicants included APAP (400 mg/kg, 24–72 h), alpha-naphthyl isothiocyanate (ANIT; 50 mg/kg, 2–48 h), carbon tetrachloride (CCl4; 10 or 30 μL/kg, 24 and 48 h) and allyl alcohol (AlOH; 30 or 60 mg/kg, 6 and 24 h). Because oxidative stress activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2), additional studies investigated Fmo3 gene regulation by Nrf2 using Nrf2 knockout (Nrf2 KO) mice. At appropriate time-points, blood and liver samples were collected for assessment of plasma alanine aminotransferase (ALT) activity, plasma and hepatic bile acid levels, as well as liver Fmo3 mRNA and protein expression. Fmo3 mRNA expression increased significantly by 43-fold at 12 h after ANIT treatment, and this increase translates to a 4-fold change in protein levels. BDL also increased Fmo3 mRNA expression by 1899-fold, but with no change in protein levels. Treatment of mice with CCl4 decreased liver Fmo3 gene expression, while no change in expression was detected with AlOH treatment. Nrf2 KO mice are more susceptible to APAP (400 mg/kg, 72 h) treatment compared to their wild-type (WT) counterparts, which is evidenced by greater plasma ALT activity. The Fmo3 mRNA and protein expression increased in Nrf2 KO mice after APAP treatment. Collectively, not all hepatotoxicants that produce oxidative stress alter Fmo3 gene expression. Along with APAP, toxic ANIT treatment in mice markedly increased Fmo3 gene expression. While BDL increased the Fmo3 mRNA expression, the protein level did not change. The discrepancy with Fmo3 induction in cholestatic models, ANIT and BDL, is not entirely clear. Results from Nrf2 KO mice with APAP suggest that the transcriptional regulation of Fmo3 during liver injury may not involve Nrf2

Pharmacogenomic Variants May Influence the Urinary Excretion of Novel Kidney Injury Biomarkers in Patients Receiving Cisplatin

Nephrotoxicity is a dose limiting side effect associated with the use of cisplatin in the treatment of solid tumors. The degree of nephrotoxicity is dictated by the selective accumulation of cisplatin in renal tubule cells due to: (1) uptake by organic cation transporter 2 (OCT2) and copper transporter 1 (CTR1); (2) metabolism by glutathione S-transferases (GSTs) and γ-glutamyltransferase 1 (GGT1); and (3) efflux by multidrug resistance-associated protein 2 (MRP2) and multidrug and toxin extrusion protein 1 (MATE1). The purpose of this study was to determine the significance of single nucleotide polymorphisms that regulate the expression and function of transporters and metabolism genes implicated in development of acute kidney injury (AKI) in cisplatin treated patients. Changes in the kidney function were assessed using novel urinary protein biomarkers and traditional markers. Genotyping was conducted by the QuantStudio 12K Flex Real-Time PCR System using a custom open array chip with metabolism, transport, and transcription factor polymorphisms of interest to cisplatin disposition and toxicity. Traditional and novel biomarker assays for kidney toxicity were assessed for differences according to genotype by ANOVA. Allele and genotype frequencies were determined based on Caucasian population frequencies. The polymorphisms rs596881 (SLC22A2/OCT2), and rs12686377 and rs7851395 (SLC31A1/CTR1) were associated with renoprotection and maintenance of estimated glomerular filtration rate (eGFR). Polymorphisms in SLC22A2/OCT2, SLC31A1/CTRI, SLC47A1/MATE1, ABCC2/MRP2, and GSTP1 were significantly associated with increases in the urinary excretion of novel AKI biomarkers: KIM-1, TFF3, MCP1, NGAL, clusterin, cystatin C, and calbindin. Knowledge concerning which genotypes in drug transporters are associated with cisplatin-induced nephrotoxicity may help to identify at-risk patients and initiate strategies, such as using lower or fractionated cisplatin doses or avoiding cisplatin altogether, in order to prevent AKI

Regulation of hepatobiliary transporters during drug-induced liver injury

Drug-induced liver injury impairs hepatobiliary function and results in altered disposition of xenobiotics. Previous studies have demonstrated that gene and protein expression of hepatobiliary transporters is altered in response to chemical-mediated hepatotoxicity. The work presented in this dissertation includes a comprehensive temporal characterization of mRNA and protein expression for mouse uptake and efflux transporters in response to acetaminophen and carbon tetrachloride exposure. In general, a hepatotoxic dose of acetaminophen or carbon tetrachloride decreased mRNA and protein levels of uptake transporters (organic anion transport proteins, Oatps, and sodium-taurocholate co-transporting polypeptide, Ntcp) and increased levels of efflux transporters (multidrug resistance-associated proteins, Mrps) coordinately with detoxification, stress and antioxidant enzymes. Induction of Mrp3 and Mrp4 occurred in centrilobular hepatocytes, and Mrp4 staining localized primarily to proliferating cells. Administration of a second higher dose of acetaminophen to mice when Mrp3 and Mrp4 levels and hepatocellular regeneration were maximal resulted in lower hepatotoxicity or autoprotection. Livers from protected mice demonstrated marked increases not only in hepatocyte proliferation but also Mrp4 protein far beyond those seen after a single dose of acetaminophen. Inhibition of hepatocyte proliferation blocked acetaminophen autoprotection and also diminished induction of Mrp4. It is hypothesized that upregulation of Mrp4 is a protective response to minimize accumulation of potentially toxic chemicals in hepatocytes and may also be a mechanism for paracrine signaling within the liver during recovery from injury. ^ Work in this dissertation also investigates regulatory factors underlying transporter regulation during liver injury. Previous data demonstrate that increased expression of detoxification and antioxidant genes in mice exposed to acetaminophen is mediated by the transcription factor, NFE2-related factor 2, Nrf2. Experiments were designed to investigate whether up-regulation of Mrp mRNA and protein after acetaminophen was dependent on Nrf2 expression. The results showed that induction of Mrp3 and Mrp4 mRNA and protein is absent in Nrf2 knockout mice suggesting that Nrf2 signaling is critical for regulation of these two transporters during APAP toxicity. These findings provide insightful information on the regulation of hepatobiliary transporter expression in mice during drug-induced liver injury. Further studies are necessary to establish the functional consequences of altered transporter levels during hepatotoxicity.

Regulation of hepatobiliary transporters during drug-induced liver injury

Drug-induced liver injury impairs hepatobiliary function and results in altered disposition of xenobiotics. Previous studies have demonstrated that gene and protein expression of hepatobiliary transporters is altered in response to chemical-mediated hepatotoxicity. The work presented in this dissertation includes a comprehensive temporal characterization of mRNA and protein expression for mouse uptake and efflux transporters in response to acetaminophen and carbon tetrachloride exposure. In general, a hepatotoxic dose of acetaminophen or carbon tetrachloride decreased mRNA and protein levels of uptake transporters (organic anion transport proteins, Oatps, and sodium-taurocholate co-transporting polypeptide, Ntcp) and increased levels of efflux transporters (multidrug resistance-associated proteins, Mrps) coordinately with detoxification, stress and antioxidant enzymes. Induction of Mrp3 and Mrp4 occurred in centrilobular hepatocytes, and Mrp4 staining localized primarily to proliferating cells. Administration of a second higher dose of acetaminophen to mice when Mrp3 and Mrp4 levels and hepatocellular regeneration were maximal resulted in lower hepatotoxicity or autoprotection. Livers from protected mice demonstrated marked increases not only in hepatocyte proliferation but also Mrp4 protein far beyond those seen after a single dose of acetaminophen. Inhibition of hepatocyte proliferation blocked acetaminophen autoprotection and also diminished induction of Mrp4. It is hypothesized that upregulation of Mrp4 is a protective response to minimize accumulation of potentially toxic chemicals in hepatocytes and may also be a mechanism for paracrine signaling within the liver during recovery from injury. ^ Work in this dissertation also investigates regulatory factors underlying transporter regulation during liver injury. Previous data demonstrate that increased expression of detoxification and antioxidant genes in mice exposed to acetaminophen is mediated by the transcription factor, NFE2-related factor 2, Nrf2. Experiments were designed to investigate whether up-regulation of Mrp mRNA and protein after acetaminophen was dependent on Nrf2 expression. The results showed that induction of Mrp3 and Mrp4 mRNA and protein is absent in Nrf2 knockout mice suggesting that Nrf2 signaling is critical for regulation of these two transporters during APAP toxicity. These findings provide insightful information on the regulation of hepatobiliary transporter expression in mice during drug-induced liver injury. Further studies are necessary to establish the functional consequences of altered transporter levels during hepatotoxicity.

Establishment of Metabolism and Transport Pathways in the Rodent and Human Fetal Liver

The ultimate fate of drugs and chemicals in the body is largely regulated by hepatic uptake, metabolism, and excretion. The liver acquires the functional ability to metabolize and transport chemicals during the perinatal period of development. Research using livers from fetal and juvenile rodents and humans has begun to reveal the timing, key enzymes and transporters, and regulatory factors that are responsible for the establishment of hepatic phase I and II metabolism as well as transport. The majority of this research has been limited to relative mRNA and protein quantification. However, the recent utilization of novel technology, such as RNA-Sequencing, and the improved availability and refinement of functional activity assays, has begun to provide more definitive information regarding the extent of hepatic drug disposition in the developing fetus. The goals of this review are to provide an overview of the early regulation of the major phase I and II enzymes and transporters in rodent and human livers and to highlight potential mechanisms that control the ontogeny of chemical metabolism and excretion pathways