Organic Cation/carnitine Transporters Research Articles

Meldonium Supplementation in Professional Athletes: Career Destroyer or Lifesaver?

Meldonium is a substance with known anti-anginal effects demonstrated by numerous studies and human clinical trials; however, it does not possess marketing authorization within the European Union, only in ex-Soviet republics. Since 2016, meldonium has been included by the World Anti-doping Agency (WADA) on the S4 list of metabolic modulators. In performance athletes, meldonium is now considered a doping agent due to its capacity to decrease lactate production during and after exercise, its capability to enhance the storage and utilization of glycogen, and its protective action against oxidative stress. Together, these attributes can significantly improve aerobic endurance, cardiac function, and capacityas well as shorten recovery times (allowing higher intensity training), thereby enhancing performance. The purpose of this review is to highlight the most important mechanisms underlying the protective effect of meldonium against mitochondrial dysfunction (MD), which is responsible for oxidative stress, inflammation, and the cardiac changes known as "athletic heart syndrome."Meldonium acts as an inhibitor of γ-butyrobetaine hydroxylase (BBOX), preventing the de novo synthesis of carnitine and its absorption at the intestinal level via the organic cation/carnitine transporter 2 (OCTN2) and directing the oxidation of fatty acids to the peroxisomes. The decrease in mitochondrial β-oxidation of fatty acids leads to a reduction in lipid peroxidation products that cause oxidative stress and prevent the formation of acyl/acetyl-carnitines involved in numerous pathological disorders. Given the recent findings of the potentially detrimental effects of prolonged high-intensity exercise on cardiovascular health and coronary atherosclerosis, there may be legitimate arguments for the justification of the use of substances likemeldonium as protective supplements for athletes.

OCTN2- and ATB0,+-targeted nanoemulsions for improving ocular drug delivery

Traditional eye drops are administered via topical instillation. However, frequent dosing is needed due to their relatively rapid precorneal removal and low ocular bioavailability. To address these issues, stearoyl L-carnitine-modified nanoemulsions (SC-NEs) were fabricated. The physicochemical properties of SC-NEs in terms of size, morphology, zeta potential, encapsulation efficiency, and in vitro drug release behavior were characterized. The cellular uptake and mechanisms of SC-NEs were comprehensively studied in human corneal epithelial cells and the stearoyl L-carnitine ratio in SC-NEs was optimized. The optimized SC-NEs could target the novel organic cation/carnitine transporter 2 (OCTN2) and amino acid transporter B (0 +) (ATB0,+) on the corneal epithelium, which led to superior corneal permeation, ocular surface retention ability, ocular bioavailability. Furthermore, SC-NEs showed excellent in vivo anti-inflammatory efficacy in a rabbit model of endotoxin-induced uveitis. The ocular safety test indicated that the SC-NEs were biocompatible. In general, the current study demonstrated that OCTN2 and ATB0,+-targeted nanoemulsions were promising ophthalmologic drug delivery systems that can improve ocular drug bioavailability and boost the therapeutic effects of drugs for eye diseases.Graphical

OCTN2 enhances PGC-1α-mediated fatty acid oxidation and OXPHOS to support stemness in hepatocellular carcinoma.

The Metabolic reprogramming of tumor cells plays a vital role in the progression of hepatocellular carcinoma. Organic cation/carnitine transporter 2 (OCTN2), a sodium-ion dependent carnitine transporter and a sodium-ion independent tetraethylammonium (TEA) transporter, has been reported to contribute tumor malignancies and metabolic dysregulation in renal and esophageal carcinoma. However, the role of lipid metabolism deregulation mediated by OCTN2 in HCC cells has not been clarified. Bioinformatics analyses and immunohistochemistry assay were employed to identify OCTN2 expression in HCC tissues. The correlation between OCTN2 expression and prognosis was elucidated through K-M survival analysis. The expression and function of OCTN2 were examined via the assays of western blotting, sphere formation, cell proliferation, migration and invasion. The mechanism of OCTN2-mediated HCC malignancies was investigated through RNA-seq and metabolomic analyses. Furthermore, xenograft tumor models based on HCC cells with different OCTN2 expression levels were conducted to analyze the tumorigenic and targetable role of OCTN2 in vivo. We found that gradually focused OCTN2 was significantly upregulated in HCC and tightly associated with poor prognosis. Additionally, OCTN2 upregulation promoted HCC cells proliferation and migration in vitro and augmented the growth and metastasis of HCC. Moreover, OCTN2 promoted the cancer stem-like properties of HCC by increasing fatty acid oxidation and oxidative phosphorylation. Mechanistically, PGC-1α signaling participated in the HCC cancer stem-like properties mediated by OCTN2 overexpression, which is confirmed by in vitro and in vivo analyses. Furthermore, OCTN2 upregulation may be transcriptionally activated by YY1 in HCC. Particularly, treatment with mildronate, an inhibitor of OCTN2, showed a therapeutic influence on HCC in vitro and in vivo. Our findings demonstrate that OCTN2 plays a critical metabolic role in HCC cancer stemness maintenance and HCC progression, providing evidence for OCTN2 as a promising target for HCC therapy.

PS-B08-7: L-CARNITINE AMELIORATES DIABETIC KIDNEY DISEASE BY ALLEVIATING MITOCHONDRIAL ABNORMALITY IN SDT-FATTY RATS

Background and Aims: Abnormal fatty acid metabolism is associated with the progression of diabetic kidney disease (DKD). Carnitine plays a central role in β;-oxidation and energy production by transporting long-chain fatty acids from the cytoplasm to the mitochondria. Recently, we found that significant ectopic fat was accumulated in the kidney tissue in DKD patients compared with those in minimal change kidney disease patients. Juvenile visceral steatosis (jvs) mice, described as an animal model for systemic carnitine deficiency, introduce a marked ectopic fat accumulation in the kidney. These findings suggest that abnormal carnitine dynamics in DKD cause ectopic fat accumulation due to abnormality of the fatty acid metabolism. However, the association between abnormalities of carnitine metabolism and the progression of DKD is unknown. Method: Spontaneously Diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model was used. Unilateral nephrectomy was performed and 0.3% salt water was administered at 6 weeks of age, and created a renal dysfunction model in SDT fatty rat (DKD model). Age-matched male Sprague-Dawley (SD) rat was used as a control. Experiment 1: We compared 3 groups; SD rats (n = 8), SDT fatty rats (n = 8), DKD rats (n = 8). Experiment 2: We compared 3 groups; SD rats with sham operation (n = 8), DKD rats (n = 7), DKD rats treated with L-carnitine administration (0.75% L-carnitine diet) for 10 weeks (n = 6). All rats were sacrificed at 17 weeks old. Carnitine was measured by the enzyme cycling method. Acylcarnitine was analyzed by the tandem-mass spectrometry. Results: Plasma and renal free carnitine levels and short/middle-long chain acylcarnitine ratio were significantly decreased in DKD rats compared with those in SD and SDT fatty rats. The renal expression levels of organic cation / carnitine transporter 2 (OCTN2), carnitine palmitoyl transferase 1a (CPT1a), CPT2 and carnitine acetyltransferase (CrAT) were significantly decreased in DKD rats compared with those in SD and SDT fatty rats. Similarly, peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α) activity was decreased in DKD rats. L-carnitine administration significantly improved renal function, urinary albumin excretion, interstitial fibrosis and renal ectopic fat accumulation in DKD model rats. L-carnitine administration ameliorated the decrease of renal OCTN2, CPT1a, CPT2 and CrAT protein expression, and significantly improved PGC1α activity in DKD model rats. Furthermore, L-carnitine administration mitigated lipid peroxidation and mitochondrial dysfunction in DKD rats. Conclusion: L-carnitine administration might be a promising therapeutic strategy for improving renal function by alleviating mitochondrial dysfunction in DKD rat model.

Transcriptional Regulation of Solute Carrier (SLC) Drug Transporters.

Facilitated transport is necessitated for large size, charged, and/or hydrophilic drugs to move across the membrane. The drug transporters in the solute carrier (SLC) superfamily, mainly including organic anion-transporting polypeptides (OATPs), organic anion transporters (OATs), organic cation transporters (OCTs), organic cation/carnitine transporters (OCTNs), peptide transporters (PEPTs), and multidrug and toxin extrusion proteins (MATEs), are critical facilitators of drug transport and distribution in human body. The expression of these SLC drug transporters is found in tissues throughout the body, with high abundance in the epithelial cells of major organs for drug disposition, such as intestine, liver, and kidney. These SLC drug transporters are clinically important in drug absorption, metabolism, distribution, and excretion. The mechanisms underlying their regulation have been revealing in recent years. Epigenetic and nuclear receptor-mediated transcriptional regulation of SLC drug transporters have particularly attracted much attention. This review focuses on the transcriptional regulation of major SLC drug transporter genes. Revealing the mechanisms underlying the transcription of those critical drug transporters will help us understand pharmacokinetics and pharmacodynamics, ultimately improving drug therapeutic effectiveness while minimizing drug toxicity. Significance Statement It has become increasingly recognized that solute carrier (SLC) drug transporters play a crucial, and sometimes determinative, role in drug disposition and response, which is reflected in decision-making during not only clinical drug therapy but also drug development. Understanding the mechanisms accounting for the transcription of these transporters is critical to interpret their abundance in various tissues under different conditions, which is necessary to clarify the pharmacological response, adverse effects, and drug-drug interactions for clinically used drugs.

Characterization of Aripiprazole Uptake Transporter in the Blood-Brain Barrier Model hCMEC/D3 Cells by Targeted siRNA Screening.

Identification of blood-brain barrier (BBB) uptake transporters is a major challenge in the research and development of central nervous system (CNS) drugs. However, conventional methods that consider known drug uptake characteristics have failed at identifying the responsible transporter molecule. The present study aimed at identifying aripiprazole uptake transporters in BBB model hCMEC/D3 cells using a knockdown screening study targeting various transporters, including uncharacterized ones. We evaluated the effect of 214 types of siRNA targeting transporters on the uptake of aripiprazole, an atypical antipsychotic drug, in hCMEC/D3 cells. Aripiprazole uptake was determined using Xenopus oocytes expressing the candidate genes extracted from the siRNA screening assay. The estimated unbound brain to plasma concentration ratio (Kp,uu,brain) of aripiprazole was estimated as 0.67 in wild-type mice and 1.94 in abcb1a/1b/abcg2 knockout mice, suggesting the involvement of both uptake and efflux transporters in BBB permeation. According to siRNA knockdown screening studies, organic cation/carnitine transporter 2 (OCTN2) and long-chain fatty acid transporter 1 (FATP1) were identified as candidate genes. The uptake of aripiprazole by hCMEC/D3 cells was decreased by OCTN2 inhibitors, but not by FATP1 inhibitors. A partially increased uptake of aripiprazole was observed in OCTN2-expressing Xenopus oocytes. Finally, to evaluate transporter-mediated BBB permeation of drugs, the reported and estimated Kp,uu,brain values were summarized. A knockdown screening study in combination with Kp,uu,brain values showed that aripiprazole was a potential substrate of OCTN2. The technique described in this study can be applied to identifying novel BBB transporters for CNS drugs.

POS-331 L-carnitine ameliorates diabetic kidney disease by alleviating mitochondrial abnormality and fatty acid accumulation in SDT-fatty rats

Abnormal fatty acid metabolism is associated with the progression of diabetic kidney disease (DKD). Carnitine plays a central role in fatty acid β-oxidation and energy production by transporting long-chain fatty acids from the cytoplasm to the mitochondria. Recently, we found that significant ectopic fat was accumulated in the kidney tissue in DKD patients compared with those in minimal change kidney disease patients independent of proteinuria levels, suggesting the abnormal carnitine dynamics in DKD. Juvenile visceral steatosis (jvs) mice, described as an animal model for systemic carnitine deficiency caused by a mutation in slc22a5 gene, introduce a marked ectopic fat accumulation in the kidney. These findings suggest that abnormal carnitine dynamics in DKD cause ectopic fat accumulation due to abnormality of the fatty acid metabolism. However, the association between abnormalities of carnitine metabolism and the progression of DKD is unknown. Therefore, we investigated the kinetics of carnitine and the effect of L-carnitine therapy in type 2 DKD model rats. Spontaneously Diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model was used. Unilateral nephrectomy was performed and 0.3% salt water was administered at 6 weeks of age, and created a renal dysfunction model in SDT fatty rat (DKD model). Age-matched male Sprague-Dawley (SD) rat was used as a control. Experiment 1: We compared 3 groups; ①SD rats (n=8) ②SDT fatty rats (n=8) ③DKD rats (n=8). Experiment 2: We compared 3 groups; ①SD rats with sham operation (n=8) ②DKD rats (n=7) ③DKD rats treated with L-carnitine administration (0.75% L-carnitine diet) for 10 weeks (n=6). All rats were sacrificed at 17 weeks old. Carnitine was measured by the enzyme cycling method. Acylcarnitine was analyzed by the tandem-mass spectrometry. Plasma and renal free carnitine levels were significantly decreased and long-chain acylcarnitine levels were significantly increased in DKD rats compared with those in SD and SDT fatty rats. The renal expression levels of organic cation / carnitine transporter 2 (OCTN2), carnitine palmitoyl transferase 1a (CPT1a), CPT2 and carnitine acetyltransferase (CrAT) were significantly decreased in DKD rats compared with those in SD and SDT fatty rats. Similarly, acetyl-CoA carboxylase (ACC) activity was significantly increased and peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α) and peroxisome proliferator-activated receptor α (PPARα) activity were decreased in DKD rats. Further, marked ectopic fat accumulation in the renal tubules and glomeruli in DKD model rats was observed. L-carnitine administration significantly improved renal function, urinary albumin excretion, interstitial fibrosis and renal ectopic fat accumulation in DKD model rats. L-carnitine administration ameliorated the decrease of renal OCTN2, CPT1a, CPT2 and CrAT protein expression, and significantly improved ACC, PGC1α, and PPARα activity in DKD model rats. Furthermore, L-carnitine administration mitigated lipid peroxidation and mitochondrial dysfunction in DKD rats Abnormal carnitine kinetics and dynamics might play an important role in the progression of DKD. L-carnitine administration might be a promising therapeutic strategy for improving renal function by alleviating mitochondrial dysfunction in DKD rat model.

The Role of the Carnitine/Organic Cation Transporter Novel 2 in the Clinical Outcome of Patients With Locally Advanced Esophageal Carcinoma Treated With Oxaliplatin.

Esophageal cancer is the ninth most common malignancy worldwide, ranking sixth in mortality. Platinum-based chemotherapy is commonly used for treating locally advanced esophageal cancer, yet it is ineffective in a large portion of patients. There is a need for reliable molecular markers with direct clinical application for a prospective selection of patients who can benefit from chemotherapy and patients in whom toxicity is likely to outweigh the benefit. The cytotoxic activity of platinum derivatives largely depends on the uptake and accumulation into cells, primarily by organic cation transporters (OCTs). The aim of the study was to investigate the impact of OCT expression on the clinical outcome of patients with esophageal cancer treated with oxaliplatin. Twenty patients with esophageal squamous cell carcinoma (SCC) were prospectively enrolled and surgical specimens used for screening OCT expression level by western blotting and/or immunostaining, and for culture of cancer cells. Sixty-seven patients with SCC who received oxaliplatin and for whom follow-up was available were retrospectively assessed for organic cation/carnitine transporter 2 (OCTN2) expression by real time RT-PCR and immunostaining. OCTN2 staining was also performed in 22 esophageal adenocarcinomas. OCTN2 function in patient-derived cancer cells was evaluated by assessing L-carnitine uptake and sensitivity to oxaliplatin. The impact of OCTN2 on oxaliplatin activity was also assessed in HEK293 cells overexpressing OCTN2. OCTN2 expression was higher in tumor than in normal tissues. In patient-derived cancer cells and HEK293 cells, the expression of OCTN2 sensitized to oxaliplatin. Patients treated with oxaliplatin who had high OCTN2 level in the tumor tissue had a reduced risk of recurrence and a longer survival time than those with low expression of OCTN2 in tumor tissue. In conclusion, OCTN2 is expressed in esophageal cancer and it is likely to contribute to the accumulation and cytotoxic activity of oxaliplatin in patients with esophageal carcinoma treated with oxaliplatin.

L-tetrahydropalmatine reduces oxaliplatin accumulation in the dorsal root ganglion and mitochondria through selectively inhibiting the transporter-mediated uptake thereby attenuates peripheral neurotoxicity

Oxaliplatin (OXA) is a third-generation platinum drug; however, its application is greatly limited due to the severe peripheral neurotoxicity. This study aims to confirm the transport mechanism of OXA and to explore whether L-tetrahydropalmatine (L-THP) would alleviate OXA-induced peripheral neurotoxicity by selectively inhibiting these uptake transporters in vitro and in vivo. Our results revealed that organic cation transporter 2 (OCT2), organic cation/carnitine transporter 1 (OCTN1) and organic cation/carnitine transporter 2 (OCTN2) were involved in the uptake of OXA in dorsal root ganglion (DRG) neurons and mitochondria, respectively. L-THP (1−100 μM) reduced OXA (40 μM) induced cytotoxicity in MDCK-hOCT2 (Madin–Darby canine kidney, MDCK), MDCK-hOCTN1, MDCK-hOCTN2, and rat primary DRG cells, and decreased the accumulation of OXA in above cells and rat DRG mitochondria, but did not affect its efflux from MDCK-hMRP2 cells. Furthermore, Co-administration of L-THP (5–20 mg/kg for mice, 10–40 mg/kg for rats; twice a week, iv or ig) attenuated OXA (8 mg/kg for mice, 4 mg/kg for rats; twice a week, iv) induced peripheral neurotoxicity and reduced the platinum concentration in the DRG. Whereas, L-THP (1−100 μM for cells; 10–20 mg/kg for mice) did not impair the antitumour efficacy of OXA (40 μM for cells; 8 mg/kg for mice) in HT29 tumour-bearing nude mice nor in tumour cells (HT29 and SW620 cells). In conclusion, OCT2, OCTN1 and OCTN2 contribute to OXA uptake in the DRG and mitochondria. L-THP attenuates OXA-induced peripheral neurotoxicity via inhibiting OXA uptake but without impairing the antitumour efficacy of OXA. L-THP is a potential candidate drug to attenuate OXA-induced peripheral neurotoxicity.

Intestinal Absorption of Alogliptin Is Mediated by a Fruit-Juice-Sensitive Transporter.

Alogliptin (ALG), an inhibitor of dipeptidylpeptidase-4, is used in the management of type 2 diabetes mellitus, and has a high absorption rate (>60-71%), despite its low lipophilicity (logP=-1.4). Here, we aimed to clarify the mechanism of its intestinal absorption. ALG uptake into Caco-2 cells was time-, temperature-, and concentration-dependent, but was not saturated at concentrations up to 10 mmol/L. The uptake was significantly inhibited by the organic anion transporting polypeptide (OATP) substrate fexofenadine and by the OATP inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), but was not inhibited by organic cation transporter (OCT)/organic cation/carnitine transporter (OCTN) or peptide transporter 1 (PEPT1) substrates. Grapefruit, orange, and apple juices and their constituents, which are known to strongly inhibit intestinal OATPs, significantly inhibited ALG uptake into Caco-2 cells. The pH dependence was bell-shaped, indicating the involvement of a pH-sensitive transporter. However, ALG uptake by HEK293 cells overexpressing OATP2B1, a key intestinal OATP transporter of amphiphilic drugs, was not different from that of mock cells. In a rat in vivo study, apple juice reduced systemic exposure to orally administered ALG without changing the terminal half-life. These observations suggest that intestinal absorption of ALG is carrier-mediated, and involves a fruit-juice-sensitive transporter other than OATP2B1.

Pharmacokinetics and Toxicokinetics Roles of Membrane Transporters at Kidney Level.

Transporters are large membrane proteins, which control the passage of various compounds through biological membranes. These proteins are divided into uptake and efflux transporters and play an important role in the toxicokinetics of many endobiotics and xenobiotics. The uptake transporters facilitate the absorption of these compounds from the blood into the proximal tubular cells, while the efflux transporters eliminate these compounds into tubular fluid (urine). Overall, the uptake is performed by the superfamily solute carrier (SLC) transporters, which are, mostly, located in the basolateral membrane. The organic anion transporters (OATs; SLC22), the organic cation transporters (OCTs; SLC22), the organic cation/carnitine transporters (OCTNs), and the organic anion transporting polypeptides (OATP; SLC21/SLCO) are some examples of uptake transporters of the SLC superfamily. On the other hand, the superfamily ATP-binding cassette (ABC) transporters carry out the elimination of the substances through the apical membrane of the proximal tubular cells. The multidrug resistance proteins 1 (MDR; ABCB), the multi resistance protein (MRP2; ABCC) and the breast cancer resistance protein (BCRP, ABCG) along with the multidrug and toxin extrusion (MATE), which is an SLC transporter, carry out the substance efflux of the cell, However, uptake transporters seem to be more efficient than efflux transporters, leading to an accumulation of compounds in proximal tubular cells and, consequently, to renal damage. The accumulation of compounds can also occur due to variations in the number of transporters that exist due to differences in sex, age, genetic polymorphisms and epigenetics. Furthermore, some substances can inhibit, induce or, eventually, activate these transporters, with consequent drug-drug interactions (DDIs) as a result of alterations on the toxicokinetics of xenobiotics, leading to an increase of their accumulation and, consequently, to renal damage. These compounds may be exogenous, such as antibiotics, antivirals, cisplatin, metals, herbicides, mycotoxins and drugs; or endogenous, like uric acid, bile acids, bilirubin conjugates and conjugated steroids. Thus, in this review, we will focus on the accumulation of exogenous compounds due to variations on renal transporters and the consequent biological effects caused by them.

L-Carnitine conjugated chitosan-stearic acid polymeric micelles for improving the oral bioavailability of paclitaxel

A delivery system based on l-carnitine (LC) conjugated chitosan (CS)-stearic acid polymeric micelles has been developed for improving the oral bioavailability of paclitaxel (PTX) through targeting intestinal organic cation/carnitine transporter 2 (OCTN2). Stearic acid grafted chitosan (CS-SA), as micelle skeleton material, was synthesized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction. The PTX-loaded micelles were prepared by solvent evaporation-hydration method, and the ligand LC was conjugated onto the micelle surface by anchoring its derivative stearoyl group to the lipophilic core of micelle. The modified polymeric micelles showed regular spherical shapes with small particle size of 157.1 ± 5.2 nm and high drug loading capacity of 15.96 ± 0.20 wt%, and the micelle stability in water was supported by low critical micelle concentration of 14.31 ± 0.21 μg/ml. The drug-loaded micelles presented a slow and incomplete in vitro release, and the pharmacokinetic studies indicated the micelle carriers increased the relative bioavailability of PTX to 165.8% against the commercial formulation. The enhancement effect on intestinal absorption was also confirmed by the intracellular uptake of Caco-2 cells. The proposed micelle carrier system manifested a prospective tool for oral drug delivery.

Glucocorticoids attenuate the sensitivity of glucocorticoid-resistant lymphoid cells to doxorubicin via reduction in OCTN2.

Glucocorticoid (GC) resistance is associated with poor response to the following chemotherapy in lymphoid malignancies, such as lymphoma and leukemia. However, it remains unclear whether GCs interfere with the cytotoxic effects of anti-cancer drugs on GC-resistant cells. In this study, we examined whether GCs affected the sensitivities to vincristine (VCR)/doxorubicin (DOX) and the expression of drug transporters in GC-resistant cells. The dexamethasone (DEX)/prednisolone (PSL)-resistant lymphoid and non-lymphoid cell lines Raji and HL60 were cultured with DEX for 7days and then treated with VCR or DOX for 3days. Seven days of DEX treatment increased the IC50s of both VCR and DOX in Raji cells but not in HL60 cells. The mRNA and protein expression levels of organic cation/carnitine transporter (OCTN) 2, one of the drug uptake transporters expressed in both cell lines, were decreased only in Raji cells. When Raji cells were cultured with PSL, the IC50 of DOX but not VCR increased as the expression of OCTN2 decreased. No significant increases in efflux transporter expression were induced by DEX or PSL. When siRNA against OCTN2 was introduced into Raji cells, the IC50 of DOX but not VCR increased significantly. These data suggested that both DEX and PSL decreased the sensitivity of the DEX/PSL-resistant Raji cells to DOX, a change that was at least partially due to reductions in OCTN2. Thus, the continuous usage of GCs may interfere with the effects of chemotherapy on GC-resistant lymphoid cells.

Effect of Acetyl-L-carnitine Used for Protection of Neonatal Hypoxic-Ischemic Brain Injury on Acute Kidney Changes in Male and Female Rats.

Neonatal hypoxia-ischemia (HI) is a common cause of brain injury in infants. Acute kidney injury frequently occurs after birth asphyxia and is associated with adverse outcome. Treatment with acetyl-L-carnitine (ALCAR) after HI protects brain and improves outcome. Rat pups underwent carotid ligation and 75min hypoxia on postnatal day 7 to determine effects of HI on kidney which is understudied in this model. HI + ALCAR pups were treated at 0, 4 and 24h after HI. The organic cation/carnitine transporter 2 (OCTN2), transports ALCAR and functions to reabsorb carnitine and acylcarnitines from urine. At 24h after injury OCTN2 levels were significantly decreased in kidney from HI pups, 0.80 ± 0.04 (mean± SEM,p < 0.01), compared to sham controls 1.03 ± 0.04, and HI + ALCAR pups 1.11 ± 0.06. The effect of HI on the level of pyruvate dehydrogenase (PDH) was determined since kidney has high energy requirements. At 24h after HI, kidney PDH/β-actin ratios were significantly lower in HI pups, 0.98 ± 0.05 (mean ± SEM,p < 0.05), compared to sham controls 1.16 ± 0.06, and HI + ALCAR pups 1.24 ± 0.03, p < 0.01. Treatment of pups with ALCAR after HI prevented the decrease in renal OCTN2 and PDH levels at 24h after injury. Protection of PDH and OCTN2 after HI would improve energy metabolism in kidney, maintain tissue carnitine levels and overall carnitine homeostasis which is essential for neonatal health.

The anti-hyperuricemic effect of four astilbin stereoisomers in Smilax glabra on hyperuricemic mice

Ethnopharmacological relevanceSmilax glabra Roxb. (SG), a Chinese medicinal herb which called “tufuling”, is believed to be effective in treating hyperuricemia and gout symptoms. But the active substance and pharmacological mechanism of reducing uric acid remain unknown. This study aimed to obtain the total flavonoids including four astilbin stereoisomers and to examine their effects on reducing uric acid content in hyperuricemic mice. Materials and methodsThe total flavonoids of S. glabra (TFSG) were purified and then analysed by HPLC-PDA-MS. The effect of TFSG on the content of serum uric acid (SUA), Serum creatinine (SCr), blood urea nitrogen (BUN) and the activities of xanthine oxidase (XOD) in hyperuricemic mouse model induced by potassium oxonate were examined. Western blot and PCR method were also used to investigate whether TFSG have effect on renal transport protein organic anion transporter 1 (OAT1), organic cation/carnitine transporter 2 (OCTN2) and their mRNA in hypeuricemic mice. ResultTotal flavonoids were obtained from EtOAc soluble portion of S. glabra. Four compounds were identified as neoastilbin, astilbin, neoisoastilbin and isoastilbin, which accounted for 55.6% of total flavonoids. TFSG could significantly reduce the serum uric acid content in hyperuricemic mouse (p < 0.01 or p < 0.05). The activities of hepatic XOD have been reduced in hyperuricemic mouse administered with 125 mg/kg TFSG (p < 0.05). The expressions of renal OAT1, OCTN2 and their mRNA have been up-regulated in hyperuricemic mice administered with TFSG (250, 125 mg/kg) (p < 0.01or p < 0.05). TFSG (62.5 mg/kg) could also elevated the expression of renal OCTN2 (p < 0.05). ConclusionA novel and simple method for preparative separation of astilbin stereoisomers from S. glabra was developed. It was the first time to obtain total flavonoids (including four marker compounds) of S. glabra, and the total content was up to 55.6%. The results suggested TFSG has significant effect on reducing uric acid in hyperuricemic mice by inhibiting the XOD activities and up-regulating the expression of OAT1, OCTN2 and their mRNA in kidney tissue.

Transporter-Mediated Drug-Drug Interactions and Their Significance.

Drug transporters are considered to be determinants of drug disposition and effects/toxicities by affecting the absorption, distribution, and excretion of drugs. Drug transporters are generally divided into solute carrier (SLC) family and ATP binding cassette (ABC) family. Widely studied ABC family transporters include P-glycoprotein (P-GP), breast cancer resistance protein (BCRP), and multidrug resistance proteins (MRPs). SLC family transporters related to drug transport mainly include organic anion-transporting polypeptides (OATPs), organic anion transporters (OATs), organic cation transporters (OCTs), organic cation/carnitine transporters (OCTNs), peptide transporters (PEPTs), and multidrug/toxin extrusions (MATEs). These transporters are often expressed in tissues related to drug disposition, such as the small intestine, liver, and kidney, implicating intestinal absorption of drugs, uptake of drugs into hepatocytes, and renal/bile excretion of drugs. Most of therapeutic drugs are their substrates or inhibitors. When they are comedicated, serious drug-drug interactions (DDIs) may occur due to alterations in intestinal absorption, hepatic uptake, or renal/bile secretion of drugs, leading to enhancement of their activities or toxicities or therapeutic failure. This chapter will illustrate transporter-mediated DDIs (including food drug interaction) in human and their clinical significances.

Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in cardiac hypertrophy and failure.

Heart failure is characterized by structural and metabolic cardiac remodelling. The aim of the present study is to expand our understanding of the complex metabolic alterations in the transition from pathological hypertrophy to heart failure and exploit the results from a translational perspective. Mice were subjected to transverse aortic constriction (TAC) or sham surgery and sacrificed 2 weeks, 4 weeks, or 6 weeks after the procedure. Samples from plasma, liver, skeletal muscle, and heart were collected and analysed using metabolomics. Cardiac samples were also analysed by transcriptional profiling. Progressive alterations of key cardiac metabolic pathways and gene expression patterns indicated impaired mitochondrial function and a metabolic switch during transition to heart failure. Similar to the heart, liver, and skeletal muscle revealed significant metabolic alterations such as depletion of essential fatty acids and glycerolipids in late stages of heart failure. Circulating metabolites, particularly fatty acids, reflected cardiac metabolic defects, and deteriorating heart function. For example, inverse correlation was found between plasma and the heart levels of triacylglycerol (C18:1, C18:2, C18:3), and sphingomyelin (d18:1, C23:0) already at an early stage of heart failure. Interestingly, combining metabolic and transcriptional data from cardiac tissue revealed that decreased carnitine shuttling and transportation preceded mitochondrial dysfunction. We, thus, studied the therapeutic potential of OCTN2 (Organic Cation/Carnitine Transporter 2), an important factor for carnitine transportation. Cardiac overexpression of OCTN2 using an adeno-associated viral vector significantly improved ejection fraction and reduced interstitial fibrosis in mice subjected to TAC. Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in heart failure, which can be used for identification of novel biomarkers and potential therapeutic targets.

P4776Targeting metabolic remodeling in a murine heart failure model by AAV-mediated over-expression of organic cation/carnitine transporter 2 (OCTN2)

P4776Targeting metabolic remodeling in a murine heart failure model by AAV-mediated over-expression of organic cation/carnitine transporter 2 (OCTN2)

The SLC22 Transporter Family: A Paradigm for the Impact of Drug Transporters on Metabolic Pathways, Signaling, and Disease.

The SLC22 transporter family consists of more than two dozen members, which are expressed in the kidney, the liver, and other tissues. Evolutionary analysis indicates that SLC22 transporters fall into at least six subfamilies: OAT (organic anion transporter), OAT-like, OAT-related, OCT (organic cation transporter), OCTN (organic cation/carnitine transporter), and OCT/OCTN-related. Some-including OAT1 [SLC22A6 or NKT (novel kidney transporter)] and OAT3 (SLC22A8), as well as OCT1 (SLC22A1) and OCT2 (SLC22A2)-are widely studied drug transporters. Nevertheless, analyses of knockout mice and other data indicate that SLC22 transporters regulate key metabolic pathways and levels of signaling molecules (e.g., gut microbiome products, bile acids, tricarboxylic acid cycle intermediates, dietary flavonoids and other nutrients, prostaglandins, vitamins, short-chain fatty acids, urate, and ergothioneine), as well as uremic toxins associated with chronic kidney disease. Certain SLC22 transporters-such as URAT1 (SLC22A12) and OCTN2 (SLC22A5)-are mutated in inherited metabolic diseases. A new systems biology view of transporters is emerging. As proposed in the remote sensing and signaling hypothesis, SLC22 transporters, together with other SLC and ABC transporters, have key roles in interorgan and interorganism small-molecule communication and, together with the neuroendocrine, growth factor-cytokine, and other homeostatic systems, regulate local and whole-body homeostasis.

Urinary Excretion of Tetrodotoxin Modeled in a Porcine Renal Proximal Tubule Epithelial Cell Line, LLC-PK₁.

This study examined the urinary excretion of tetrodotoxin (TTX) modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK1. Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA), l-carnitine, and cimetidine, slightly reduced by p-aminohippuric acid (PAH), and unaffected by 1-methyl-4-phenylpyridinium (MPP+), oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs) and organic cation/carnitine transporters (OCTNs), partially transported by organic anion transporters (OATs) and multidrug resistance-associated proteins (MRPs), and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs).