Antispasmodic Effect of Asperidine B, a Pyrrolidine Derivative, through Inhibition of L-Type Ca2+ Channel in Rat Ileal Smooth Muscle.

Antispasmodic agents are used for modulating gastrointestinal motility. Several compounds isolated from terrestrial plants have antispasmodic properties. This study aimed to explore the inhibitory effect of the pyrrolidine derivative, asperidine B, isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, on spasmodic activity. Isolated rat ileum was set up in an organ bath. The contractile responses of asperidine B (0.3 to 30 µM) on potassium chloride and acetylcholine-induced contractions were recorded. To investigate its antispasmodic mechanism, CaCl2, acetylcholine, Nω-nitro-l-arginine methyl ester (l-NAME), nifedipine, methylene blue and tetraethylammonium chloride (TEA) were tested in the absence or in the presence of asperidine B. Cumulative concentrations of asperidine B reduced the ileal contraction by ~37%. The calcium chloride and acetylcholine-induced ileal contraction was suppressed by asperidine B. The effects of asperidine B combined with nifedipine, atropine or TEA were similar to those treated with nifedipine, atropine or TEA, respectively. In contrast, in the presence of l-NAME and methylene blue, the antispasmodic effect of asperidine B was unaltered. These results suggest that the antispasmodic property of asperidine B is probably due to the blockage of the L-type Ca2+ channel and is associated with K+ channels and muscarinic receptor, possibly by affecting non-selective cation channels and/or releasing intracellular calcium.

Tiliacora triandra (Colebr.) Diels Leaf Aqueous Extract Inhibits Hepatic Glucose Production in HepG2 Cells and Type 2 Diabetic Rats.

This study investigated the effects of Tiliacora triandra (Colebr.) Diels aqueous extract (TTE) on hepatic glucose production in hepatocellular carcinoma (HepG2) cells and type 2 diabetic (T2DM) conditions. HepG2 cells were pretreated with TTE and its major constituents found in TTE, epicatechin (EC) and quercetin (QC). The hepatic glucose production was determined. The in vitro data were confirmed in T2DM rats, which were supplemented daily with 1000 mg/kg body weight (BW) TTE, 30 mg/kg BW metformin or TTE combined with metformin for 12 weeks. Results demonstrate that TTE induced copper-zinc superoxide dismutase, glutathione peroxidase and catalase genes, similarly to EC and QC. TTE decreased hepatic glucose production by downregulating phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and increasing protein kinase B and AMP-activated protein kinase phosphorylation in HepG2 cells. These results correlated with the antihyperglycemic, antitriglyceridemic, anti-insulin resistance, and antioxidant activities of TTE in T2DM rats, similar to the metformin and combination treatments. Consistently, impairment of hepatic gluconeogenesis in T2DM rats was restored after single and combined treatments by reducing PEPCK and G6Pase genes. Collectively, TTE could potentially be developed as a nutraceutical product to prevent glucose overproduction in patients with obesity, insulin resistance, and diabetes who are being treated with antidiabetic drugs.

Antidiabetic and Renoprotective Effects of Coffea arabica Pulp Aqueous Extract through Preserving Organic Cation Transport System Mediated Oxidative Stress Pathway in Experimental Type 2 Diabetic Rats.

Coffea arabica pulp (CP) is a by-product of coffee processing. CP contains polyphenols that have exhibited beneficial effects, including antioxidant and lipid-lowering effects, as well as enhanced insulin sensitivity, in in vitro and in vivo models. How polyphenols, as found in CP aqueous extract (CPE), affect type 2 diabetes (T2D) has not been investigated. Thus, the present study examined the potential antidiabetic, antioxidant, and renoprotective effects of CPE-rich polyphenols, using an experimental model of T2D in rats induced by a high-fat diet and a single low dose of streptozotocin. The T2D rats received either 1000 mg/kg body weight (BW) of CPE, 30 mg/kg BW of metformin (Met), or a combination treatment (CPE + Met) for 3 months. Plasma parameters, kidney morphology and function, and renal organic transport were determined. Significant hyperglycemia, hypertriglyceridemia, insulin resistance, increased renal lipid content and lipid peroxidation, and morphological kidney changes related to T2D were restored by both CPE and CPE + Met treatments. Additionally, the renal uptake of organic cation, 3H-1-methyl-4-phenylpyridinium (MPP+), was reduced in T2D, while transport was restored by CPE and CPE + Met, through an up-regulation of antioxidant genes and protein kinase Cα deactivation. Thus, CPE has antidiabetic and antioxidant effects that potentially ameliorate kidney function in T2D by preserving renal organic cation transport through an oxidative stress pathway.

Spirogyra neglecta Aqueous Extract Attenuates LPS-Induced Renal Inflammation.

Spirogyra neglecta (SN), commonly named "Tao" in Thai, is a genus of filamentous green macroalgae. SN contains polyphenols such as isoquercetin, catechin, hydroquinone and kaempferol. These constituents exhibit beneficial effects including anti-oxidant, anti-gastric ulcer, anti-hyperglycaemia and anti-hyperlipidaemia in both in vitro and in vivo models. Whether SN extract (SNE) has an anti-inflammatory effect in vivo remains unclear. This study examined the effect of SNE on renal function and renal organic transport in lipopolysaccharide (LPS)-induced renal inflammation in rats. Rats were randomised and divided into normal saline (NS), NS supplemented with 1000 mg/kg body weight (BW) of SNE (NS + SNE), intraperitoneally injected with 12 mg/kg BW of LPS and LPS treated with 1000 mg/kg BW of SNE (LPS + SNE). Biochemical parameters in serum and urine, lipid peroxidation concentration, kidney function and renal organic anion and cation transports were determined. LPS-injected rats developed renal injury and inflammation by increasing urine microalbumin, total malondialdehyde (MDA) and inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß protein expression, respectively. In addition, uptake of renal organic anion, [3H]-oestrone sulphate (ES), was reduced in LPS-injected rats together with increased expression of organic anion transporter 3 (Oat3). However, the renal injury and inflammation, as well as impaired Oat3 function and protein expression, were restored in LPS + SNE rats. Accordingly, SNE could be developed as nutraceutical product to prevent inflammation-induced nephrotoxicity.

Asperidines A-C, pyrrolidine and piperidine derivatives from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178.

One new pyrrolidine derivative, asperidine A (1), and two new piperidine derivatives, asperidines B (2) and C (3), were isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178 together with two known alkaloids. Compound 3 possessed an unprecedented 7-oxa-1-azabicyclo[3.2.1]octane skeleton with four chiral centers. Their structures were determined by spectroscopic evidence. The absolute configurations of compounds 2 and 3 were established using Mosher's method and further confirmed for compound 3 by X-ray crystallographic data. Compound 2 dose-dependently inhibited the CFTR-mediated chloride secretion in T84 cells with an IC50 value of 0.96 µM whereas 3 displayed the same activity with the IC50 value of 58.62 µM. Compounds 2 and 3 also significantly reduced intracellular ROS under both normal and H2O2-treated conditions compared with their respective controls in a dose-dependent manner without cytotoxic effect on Caco-2 cells. In addition, compound 3 was inactive against noncancerous Vero cells whereas compound 2 was considered to be inactive with the IC50 value of >10 µM.

Lovastatin Analogues from the Soil-Derived Fungus Aspergillus sclerotiorum PSU-RSPG178.

Three new lovastatin analogues (1, 4, and 5) together with four known lovastatin derivatives, namely, lovastatin (2), α,ß-dehydrolovastatin (3), α,ß-dehydrodihydromonacolin K (6), and α,ß-dehydro-4a,5-dihydromonacolin L (7), were isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178. Their structures were established using spectroscopic evidence. Compound 5 exhibited the most potent activity against HMG-CoA reductase, with an IC50 value of 387 µM. In addition, the present study indicated the direct interaction of compound 5 with HMG-CoA reductase. Compound 5 was considered to be noncytotoxic against noncancerous Vero cells, with an IC50 value of 40.0 µM, whereas compound 2 displayed much stronger activity, with an IC50 value of 2.2 µM.

Interaction of green tea catechins with renal organic cation transporter 2.

1. Green tea extract (GTE) and EGCG have previously shown to increase the uptake of MPP+ into Caco-2 cells. However, whether GTE and its derivatives interact with renal basolateral organic cation transporter 2 (Oct2) which plays a crucial role for cationic clearance remains unknown. Thus, this study assessed the potential of drug-green tea (GT) catechins and its derivatives interactions with rat Oct2 using renal cortical slices and S2 stably expressing rat Oct2 (S2rOct2). 2. Both GTE and ECG inhibited MPP+ uptake in renal slices in a concentration-dependent manner (IC50 = 2.71 ± 0.360 mg/ml and 0.87 ± 0.151 mM), and this inhibitory effect was reversible. Inhibition of [3H]MPP+ transport in S2rOct2 by either GTE or ECG (IC50 = 1.90 ± 0.087 mg/ml and 1.67 ± 0.088 mM) was also observed. 3. The weak and reversible interactions of GTE and ECG with rOct2 indicate that consumption of GT beverages could not interfere with cationic drugs secreted via renal OCT2 in humans. However, the rise of therapeutic use of GTE and ECG might have to take into account the significant possibility of adverse drug-green tea catechins interactions which could alter renal organic cation drug clearance.

Regulation of renal organic anion transporter 3 (SLC22A8) expression and function by the integrity of lipid raft domains and their associated cytoskeleton.

BACKGROUND/AIMS: In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function. METHODS: LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices. RESULTS: Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), ß-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol depletion by methyl-ß-cyclodextrin (MßCD) also led to a dose dependent reduction Oat3 expression and ES transport by rat renal cortical slices. Moreover, the up-regulation of rOat3-mediated transport seen following insulin stimulation was completely prevented by MßCD. CONCLUSION: We have demonstrated that renal Oat3 resides in LRD-rich membranes in proximity to cytoskeletal and signaling proteins. Disruption of LRD-rich membranes by cholesterol-binding agents or protein trafficking inhibitors altered Oat3 expression and regulation. These findings indicate that the integrity of LRD-rich membranes and their associated proteins are essential for Oat3 expression and function.

Coffea arabica bean extract inhibits glucose transport and disaccharidase activity in Caco-2 cells.

The major constituents of Coffea arabica (coffee), including caffeine, chlorogenic acid and caffeic acid, exhibit antihyperglycemic properties in in vitro and in vivo models. However, whether Coffea arabica bean extract (CBE) regulates glucose uptake activity and the underlying mechanisms involved remain unclear. The aim of the present study was to examine the effects of CBE on glucose absorption and identify the mechanisms involved using an in vitro model. The uptake of a fluorescent glucose analog into Caco-2 colorectal adenocarcinoma cells was determined. The expression levels of sodium glucose co-transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) were evaluated. In addition, glycoside hydrolase enzyme activity was investigated. It was observed that CBE inhibited disaccharidase enzyme activity. Furthermore, CBE exerted an inhibitory effect on intestinal glucose absorption by downregulating SGLT1- and GLUT2-mediated 5' AMP-activated protein kinase phosphorylation and suppressing hepatocyte nuclear factor 1α expression. These data suggest that CBE may attenuate glucose absorption and may have potentially beneficial antihyperglycemic effects in the body; however, the mechanisms underlying the effects of CBE must be elucidated through further investigation.

High-Efficacy α,ß-Dehydromonacolin S Improves Hepatic Steatosis and Suppresses Gluconeogenesis Pathway in High-Fat Diet-Induced Obese Rats.

Isolated α,ß-dehydromonacolin S (C5) from soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178 was recently shown to exhibit an inhibitory effect against 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity in vitro. In this study, we investigated the effects of C5 on lipid-lowering, hepatic steatosis, and hepatic gluconeogenesis in vivo. The control rats received a daily dose of either vehicle or C5 at 10 mg/kg, while the high-fat diet-induced obese (HFD) rats were administered vehicle; 1, 3, or 10 mg/kg C5; or 10 mg/kg lovastatin (LO) for 6 weeks. C5 significantly improved dyslipidemia and diminished liver enzymes, HMGR activity, insulin resistance, and hepatic steatosis, comparable to LO without any hepatotoxicity and nephrotoxicity in HFD rats. A higher efficacy of C5 in lipid-lowering activity and anti-hepatic steatosis was associated with a significant decrease in genes involved in lipid metabolism including sterol regulatory element binding protein (SREBP) 1c, SREBP2, liver X receptor alpha (LXRα), and peroxisome proliferator-activated receptor (PPAR) gamma (PPARγ) together with an increase in the PPAR alpha (PPARα). Correspondingly, C5 was able to down-regulate the lipid transporters cluster of differentiation 36 (CD36) and Niemann-Pick C1 Like 1 (NPC1L1), increase the antioxidant superoxide dismutase gene expression, and decrease the proinflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1ß). Impairment of hepatic gluconeogenesis and insulin resistance in HFD rats was restored by C5 through down-regulation of the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), and the activation of AMP-dependent kinase serine (AMPK) and serine/threonine protein kinase B (Akt). Collectively, this novel C5 may be a therapeutic option for treating dyslipidemia, hepatic steatosis, and reducing potential risk for diabetes mellitus.

High affinity of 4-(4-(dimethylamino)styryl)-N-methylpyridinium transport for assessing organic cation drugs in hepatocellular carcinoma cells.

Human organic cation transporter 1 (hOCT1) and human organic cation transporter 3 (hOCT3) are highly expressed in hepatocytes and play important roles in cationic drug absorption, distribution, and elimination. A previous study demonstrated that downregulation of hOCT1 and hOCT3 mRNA was related to hepatocellular carcinoma (HepG2) prognosis and severity. Whether these transporters expressed in HepG2 cells serve for cationic drug delivery has not been investigated. Besides radioactive transport, options for assessing hOCTs in hepatocytes are limited. This study clarified the significant roles of hOCTs in HepG2 by comparing cationic fluorescent 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+ ) with traditional [3 H]-1-methyl-4-phenylpyridinium (MPP+ ). The results showed ASP+ was preferably transported into HepG2 compared to [3 H]-MPP+ with high affinity and a high maximal transport rate. Selective transport of ASP+ mediated by hOCTs was influenced by extracellular pH, temperature, and membrane depolarization, corresponding to hOCT1 and hOCT3 expressions. Furthermore, transport of cationic drugs, metformin, and paclitaxel in HepG2 cells was blunted by OCT inhibitors, suggesting that hOCT1 and hOCT3 expressed in HepG2 cells exhibit notable impacts on cationic drug actions. The fluorescent ASP+ -based in vitro model may also provide a rapid and powerful analytical tool for further screening of cationic drug actions and interactions with hOCTs, particularly hOCT1 and hOCT3 in hepatocellular carcinoma.

Role of estrogen in renal handling of organic cation, tetraethylammonium: in vivo and in vitro studies.

This study examined the effect of estrogen (17beta-estradiol) on renal handling of organic cation, tetraethylammonium (TEA), both in vivo and in vitro. Clearance of TEA in ovariectomized (OVX) mice was increased by 38% above intact animals, which was able to be returned to control level by estrogen supplementation. The mechanism of this effect was examined in isolated mouse renal proximal tubules (mRPT), showing that [(3)H]-TEA uptake was higher in OVX mice than control, and estrogen supplementation returned uptake to normal level. Kinetics analysis of [(3)H]-TEA uptake indicated an increase in numbers of organic cation transporters in OVX mice but no change in substrate affinity. However, mRNA levels determined by quantitative real time polymerase chain reaction of the relevant transporters at basolateral (organic cation transporter (OCT)1, OCT2 and OCT3) and apical (organic cation/carnitine transporter (OCTN)1, OCTN2 and multidrug and toxin extrusion (MATE)1) membranes of OVX mice were not significantly changed, with only MATE2 mRNA of OVX mice being significantly decreased. The realization that estrogen status affects renal clearance of organic cations will be of importance when assessing the susceptibility of an individual to drug-induced toxicity.

Siamese neem flower extract suppresses cholesterol absorption by interfering NPC1L1 and micellar property in vitro and in intestinal Caco-2 cells.

Siamese neem (Azadirachta indica A. Juss var. siamensis Valeton) (A. indica) leaf extract, a traditional ayurvedic medicine, has been reported to exhibit antipyretic, antibacterial, antidyslipidemic, and antihyperglycemia effects. This study investigated the mechanism of hypocholesterolemic effect of methanolic extract of Siamese neem flowers in in vitro studies and in Caco-2 cells. Pancreatic cholesterol esterase and 3-hydroxy 3-methylglutaryl-CoA (HMG-CoA) reductase activities were assessed. Cholesterol micelle formation was prepared for in vitro cholesterol physicochemical property analyses, micelle size and solubility, and transport of cholesterol into the Caco-2 cells. The expression of niemann-pick C1 like 1 (NPC1L1), and its major regulator, peroxisome proliferator-activated receptor δ (PPARδ), were determined by western blot and real time polymerase chain reaction, respectively. A. indica flower extract inhibited pancreatic cholesterol esterase activity and increased cholesterol micelles size. Uptake of cholesterol into Caco-2 cells was inhibited by A. indica flower extract in a dose-dependent manner. In addition, A. indica extract inhibited HMG-CoA reductase activity, resulting in low level of intracellular cholesterol accumulation, together with increased cytosolic NPC1L1 protein expression and decreased PPARδ gene expression. In conclusion, A. indica flower extract has cholesterol-lowering effects by inhibiting intestinal cholesterol absorption, interfering micellar cholesterol formation, and attenuating cholesterol synthesis. As such, A. indica flower extract has potential for developing into nutraceutical product for prevention of hypocholesterolemia.

Lipid-lowering effects of Coffea arabica pulp aqueous extract in Caco-2 cells and hypercholesterolemic rats.

BACKGROUND: Coffea arabica pulp (CP) is the first by-product obtained from coffee berries during coffee processing. The major constituents of CP, including chlorogenic acid, caffeine, and epicatechin exhibit anti-hyperlipidemic effects in in vitro and in vivo models. Whether Coffea arabica pulp aqueous extract (CPE) has a lipid-lowering effect remains unknown. PURPOSE: This study examined the effect of CPE on cholesterol absorption, and identified the mechanisms involved in lowered cholesterol in in vitro and in vivo models. METHODS: Uptake of [3H]-cholesterol micelles and the mode of CPE inhibition were determined using human intestinal Caco-2 cells, and subsequently, confirmed using isolated rat jejunal loops. In addition, the 12-week high-fat diet-induced hypercholesterolemic rats (HF) received either CPE (1000  mg/kg BW), a sole and high dose which was selected because it contained approximately 12  mg of CGA that was previously shown to have lipid-lowering effects, or ezetimibe (10  mg/kg BW), a cholesterol inhibitor. The rats were divided into HF, HF  ++ CPE, and HF  ++ ezetimibe groups for the next 12 weeks. Normal rats received a normal diet (ND) and CPE (ND  +  CPE). Body weights and lipid profiles were evaluated. Cholesterol transporter, Niemann-Pick C1-Like 1 (NPC1L1), protein expression and liver X receptor alpha (LXRα) mRNA expression were determined. In vitro micellar complex properties were also investigated. RESULTS: CPE inhibited [3H]-cholesterol micelle transport in Caco-2 cells and rat jejunal loops in a dose-dependent, non-competitive manner partly by decreasing membrane NPC1L1 expression. Congruently, CPE and its major constituents activated LXRα which, in turn, down-regulated NPC1L1. Furthermore, CPE interfered with physicochemical characteristics of cholesterol mixed micelles. These data were consistent with decreased body weight and slowed body weight gain and improved lipid profiles by CPE in hypercholesterolemic rats while no change occurred in these parameters in normal rats. Down-regulated intestinal NPC1L1 expression mediated by increased LXRα mRNA were also observed in HF  ++ CPE and ND  +  CPE rats. CONCLUSION: CPE has a cholesterol-lowering effect in in vitro and in vivo via inhibition of intestinal cholesterol absorption by down-regulating NPC1L1 mediated LXRα activation and interfering with micellar complex formation. Accordingly, CPE could be developed as nutraceutical product to prevent dyslipidemia-induced obesity and insulin resistance.

Cadmium induces apoptotic program imbalance and cell cycle inhibitor expression in cultured human astrocytes.

Cadmium is a highly neurotoxic heavy metal impairing neurogenesis and induces neurodegenerative disorders. Toxic concentrations of cadmium induce astrocytic apoptosis by depleting intracellular glutathione levels, elevating intracellular calcium levels, altering mitochondria membrane potentials, and activating JNK and PI3K/Akt signaling pathways. Cadmium suppresses cell proliferation in kidney epithelial cells, lung fibroblasts, and primary myelocytes; however, cadmium's effects on proteins regulating oxidative stress, apoptosis, and cell proliferation in astrocytes are less known. The present study hypothesized that cadmium alters levels of antioxidant enzymes, apoptotic regulator proteins, and cell cycle inhibitor proteins, resulting in apoptosis and cell cycle arrest. Concentrations ≥20 µM cadmium induced apoptosis and led to intracellular changes including DNA fragmentation, reduced mRNA expression of antioxidant enzymes (i.e., catalase and glutathione S transferase-A4), downregulation of B-cell lymphoma 2 (Bcl-2), and upregulation of Bcl-2-associated X protein (Bax). Moreover, cadmium suppressed astrocytic proliferation by inducing S and G2/M phase cell cycle arrest and promoting p53, p21, and p27 expression. In conclusion, this study provides mechanistic insight into cadmium-induced cytotoxicity of astrocytes and highlights potential targets for prevention of cadmium-induced apoptosis and cell cycle arrest.

Impaired renal organic anion transport 1 (SLC22A6) and its regulation following acute myocardial infarction and reperfusion injury in rats.

Acute kidney injury (AKI) is a high frequent and common complication following acute myocardial infarction (AMI). This study examined and identified the effect of AMI-induced AKI on organic anion transporter 1 (Oat1) and Oat3 transport using clinical setting of pre-renal AKI in vivo. Cardiac ischaemia (CI) and cardiac ischaemia and reperfusion (CIR) were induced in rats by 30-min left anterior descending coronary artery occlusion and 30-min occlusion followed by 120-min reperfusion, respectively. Renal hemodynamic parameters, mitochondrial function and Oat1/Oat3 expression and function were determined along with biochemical markers. Results showed that CI markedly reduced renal blood flow and pressure by approximately 40%, while these parameters were recovered during reperfusion. CI and CIR progressively attenuated renal function and induced oxidative stress by increasing plasma BUN, creatinine and malondialdehyde levels. Correspondingly, SOD, GPx, CAT mRNAs were decreased, while TNFα, IL1ß, COX2, iNOS, NOX2, NOX4, and xanthine oxidase were increased. Mitochondrial dysfunction as indicated by increasing ROS, membrane depolarisation, swelling and caspase3 activation were shown. Early significant detection of AKI; KIM1, IL18, was found. All of which deteriorated para-aminohippurate transport by down-regulating Oat1 during sudden ischaemia. This consequent blunted the trafficking rate of Oat1/Oat3 transport via down-regulating PKCζ/Akt and up-regulating PKCα/NFκB during CI and CIR. Thus, this promising study indicates that CI and CIR abruptly impaired renal Oat1 and regulatory proteins of Oat1/Oat3, which supports dysregulation of remote sensing and signalling and inter-organ/organismal communication. Oat1, therefore, could potentially worsen AKI and might be a potential therapeutic target for early reversal of such injury.

Organic anion transporter 3 (oat3/slc22a8) interacts with carboxyfluoroquinolones, and deletion increases systemic exposure to ciprofloxacin.

Carboxyfluoroquinolones, such as ciprofloxacin, are used for the treatment of numerous infectious diseases. Renal secretion is a major determinant of their systemic and urinary concentration, but the specific transporters involved are virtually unknown. In vivo studies implicate the organic anion transporter (OAT) family as a pivotal component of carboxyfluoroquinolone renal secretion. Therefore, this study identified the specific renal basolateral OAT(s) involved, thereby highlighting potential sources of carboxyfluoroquinolone-drug interactions and variable efficacy. Two heterologous expression systems, Xenopus laevis oocytes and cell monolayers, were used to determine the roles of murine and human renal basolateral mOat1/hOAT1 and mOat3/hOAT3. Ciprofloxacin was transported by mOat3 in both systems (K(m) value, 70 +/- 6 microM) and demonstrated no interaction with mOat1 or hOAT1. Furthermore, ciprofloxacin, norfloxacin, ofloxacin, and gatifloxacin exhibited concentration-dependent inhibition of transport on mOat3 in cells with inhibition constants of 198 +/- 39, 558 +/- 75, 745 +/- 165, and 941 +/- 232 microM, respectively. Ciprofloxacin and gatifloxacin also inhibited hOAT3. Thereafter, in vivo elimination of ciprofloxacin was assessed in wild-type and Oat3 null mice [Oat3-/-]. Oat3-/- mice exhibited significantly elevated plasma levels of ciprofloxacin at clinically relevant concentrations (P < 0.05, male mice; P < 0.01, female mice). Oat3-/- mice also demonstrated a reduced volume of distribution (27%, P < 0.01, male mice; 14%, P < 0.01, female mice) and increased area under the concentration-time curve (25%, P < 0.05, male mice; 33%, P < 0.01, female mice). Female Oat3-/- mice had a 35% (P < 0.01) reduction in total clearance of ciprofloxacin relative to wild type. In addition, putative ciprofloxacin metabolites were significantly elevated in Oat3-/- mice. The present findings indicate that polymorphisms of and drug interactions on hOAT3 may influence carboxyfluoroquinolone efficacy, especially in urinary tract infections.

Effect of Exercise Training on Signaling of Interleukin-6 in Skeletal Muscles of Type 2 Diabetic Rats.

OBJECTIVES: Diabetes and exercise training have been shown to involve interleukin 6 (IL-6) signaling in muscle. However, the relationship between the actions of these two stimuli on muscle IL-6 and their downstream components is still unknown. Thus, the effect of endurance training on the key components of muscle IL-6 signaling transduction was investigated in a rat model of type 2 diabetes. METHODS: Diabetes was induced by streptozotocin (STZ) in male Wistar rats fed a high-fat diet, with normal rats acting as controls. The animals were left to conduct their normal activities or assigned to endurance training in a treadmill. At the end of 8 weeks, blood biochemical profiles, exercise performance, muscle oxidative capacity, glucose transporter 4 (GLUT4) protein distribution, and expressions of IL-6 and its downstream proteins were determined. RESULTS: Blood biochemical profiles of the diabetic rats were altered compared to normal rats, whereas endurance training improved blood chemistry and exercise performance. It also increased muscle oxidative capacity, and promoted GLUT4 subcellular localization to the membrane in muscles. Furthermore, protein expression of IL-6 receptor (IL-6Rα) was increased in both normal and diabetic rats after endurance training, but no significant changes in IL-6, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), or suppressor of cytokine signaling 3 (SOC3) were observed in muscles of normal and diabetic rats. CONCLUSIONS: IL-6 signaling pathway mediating muscle response to endurance training was conserved in type 2 diabetes. There was no link between training-induced IL-6 downstream targets in skeletal muscles and IL-6-induced type 2 diabetes.

Antidiabetic and renoprotective effects of Cladophora glomerata Kützing extract in experimental type 2 diabetic rats: a potential nutraceutical product for diabetic nephropathy.

Cladophora glomerata extract (CGE) has been shown to exhibit antigastric ulcer, anti-inflammatory, analgesic, hypotensive, and antioxidant activities. The present study investigated antidiabetic and renoprotective effects of CGE in type 2 diabetes mellitus (T2DM) rats. The rats were induced by high-fat diet and streptozotocin and supplemented daily with 1 g/kg BW of CGE for 12 weeks. The renal transport function was assessed by the uptake of para-aminohippurate mediated organic anion transporters 1 (Oat1) and 3 (Oat3), using renal cortical slices. These two transporters were known to be upregulated by insulin and PKCζ while they were downregulated by PKCα activation. Compared to T2DM, CGE supplemented rats had significantly improved hyperglycaemia, hypertriglyceridemia, insulin resistance, and renal morphology. The baseline uptake of para-aminohippurate was not different among experimental groups and was correlated with Oat1 and 3 mRNA expressions. Nevertheless, while insulin-stimulated Oat1 and 3 functions in renal slices were blunted in T2DM rats, they were improved by CGE supplementation. The mechanism of CGE-restored insulin-stimulated Oat1 and 3 functions was clearly shown to be associated with upregulated PKCζ and downregulated PKCα expressions and activations. These findings indicate that CGE has antidiabetic effect and suggest it may prevent diabetic nephropathy through PKCs in a T2DM rat model.

Impaired insulin signaling affects renal organic anion transporter 3 (Oat3) function in streptozotocin-induced diabetic rats.

Organic anion transporter 3 (Oat3) is a major renal Oats expressed in the basolateral membrane of renal proximal tubule cells. We have recently reported decreases in renal Oat3 function and expression in diabetic rats and these changes were recovered after insulin treatment for four weeks. However, the mechanisms by which insulin restored these changes have not been elucidated. In this study, we hypothesized that insulin signaling mediators might play a crucial role in the regulation of renal Oat3 function. Experimental diabetic rats were induced by a single intraperitoneal injection of streptozotocin (65 mg/kg). One week after injection, animals showing blood glucose above 250 mg/dL were considered to be diabetic and used for the experiment in which insulin-treated diabetic rats were subcutaneously injected daily with insulin for four weeks. Estrone sulfate (ES) uptake into renal cortical slices was examined to reflect the renal Oat3 function. The results showed that pre-incubation with insulin for 30 min (short term) stimulated [3H]ES uptake into the renal cortical slices of normal control rats. In the untreated diabetic rats, pre-incubation with insulin for 30 min failed to stimulate renal Oat3 activity. The unresponsiveness of renal Oat3 activity to insulin in the untreated diabetic rats suggests the impairment of insulin signaling. Indeed, pre-incubation with phosphoinositide 3-kinase (PI3K) and protein kinase C zeta (PKCζ) inhibitors inhibited insulin-stimulated renal Oat3 activity. In addition, the expressions of PI3K, Akt and PKCζ in the renal cortex of diabetic rats were markedly decreased. Prolonged insulin treatment in diabetic rats restored these alterations toward normal levels. Our data suggest that the decreases in both function and expression of renal Oat3 in diabetes are associated with an impairment of renal insulin-induced Akt/PKB activation through PI3K/PKCζ/Akt/PKB signaling pathway.