Expression Of Relevant Transporters Research Articles

Development of a Novel HEK293 Cell Model Lacking SLC29A1 to Study the Pharmacology of Endogenous SLC29A2-Encoded Equilibrative Nucleoside Transporter Subtype 2.

Equilibrative nucleoside transporters (ENTs) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogs used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of coexpressed ENT1. We created a cell model where ENT1 was removed from human embryonic kidney (HEK293) cells using CRISPR/cas9 [ENT1 knockout (KO) cells]; this cell line has ENT2 as the only functional purine transporter. Transporter function was assessed through measurement of [3H]2-chloroadenosine uptake. ENT1 protein was quantified based on the binding of [3H]nitrobenzylthioinosine, and ENT1/ENT2 protein was detected by immunoblotting. Changes in expression of relevant transporters and enzymes involved in purine metabolism were examined by quantitative polymerase chain reaction. Wild-type HEK293 cells and ENT1KO cells had a similar expression of SLC29A2/ENT2 transcript/protein and ENT2-mediated [3H]2-chloroadenosine transport activity (Vmax values of 1.02 ± 0.06 and 1.50 ± 0.22 pmol/μl/s, respectively). Of the endogenous nucleosides/nucleobases tested, adenosine had the highest affinity (Ki) for ENT2 (2.6 μM), while hypoxanthine was the only nucleobase with a submillimolar affinity (320 μM). A range of nucleoside/nucleobase analogs were also tested for their affinity for ENT2 in this model, with affinities (Ki) ranging from 8.6 μM for ticagrelor to 2,300 μM for 6-mercaptopurine. Our data suggest that the removal of endogenous ENT1 from these cells does not change the expression or function of ENT2. This cell line should prove useful for the analysis of novel drugs acting via ENT2 and to study ENT2 regulation. SIGNIFICANCE STATEMENT: We have created a cell line whereby endogenous ENT2 can be studied in detail in the absence of the confounding influence of ENT1. Loss of ENT1 has no impact on the expression and function of ENT2. This novel cell line will provide an ideal model for studying drug interactions with ENT2 as well as the cellular regulation of ENT2 expression and function.

Kidney and Gut Microbiome Crosstalk in Response to High Dietary Potassium

In this study, we tested the hypothesis that the gut microbiome influences the homeostatic response to a dietary K+ load. Male Specific Pathogen Free (SPF) and Germ‐Free (GF) mice (n = 5 per group) received 5% potassium citrate or K+alimentary matched control diets for 7 days. At the time of sacrifice, blood and urine parameters were compared, and the expression of relevant transporters and signaling molecules was assessed in the kidney by western blotting. No significant differences in whole blood K+ were observed between SPF and GF mice on control or high K+ diets. However, differences in other parameters were observed. Compared to control diet treated SPF mice, K+ loaded SPF mice exhibited a significant decrease in hematocrit (37 vs 39; p ≤ 0.0152) and hemoglobin (12.58 vs 13.26; p ≤ 0.0150), whereas GF mice exhibited no differences in these parameters regardless of dietary maneuver. Blood urea nitrogen (BUN) levels were significantly decreased in SPF mice (23.4mg/dL vs 27; p ≤ 0.0180) on High K+compared to SPF controls, while this was not the case for GF mice. These findings suggest that K+ loaded GF mice were less volume expanded than K+ loaded SPF mice. Consistent with this, plasma aldosterone measurements were higher in K+ loaded GF mice compared to K+ loaded SPF mice (966pg/mL vs 614; p ≤ 0.0155). In whole kidney homogenates, dietary K+ loading markedly increased the cleaved (active) form of the epithelial sodium channel gamma subunit (γENaC) relative to control diet treated mice in both the SPF and GF groups. However, cleaved γENaC was one‐fold higher in the K+ loaded GF mice compared to K+ loaded SPF mice. K+ citrate loading significantly upregulated pendrin in both GF and SPF groups (p ≤ 5.7146E‐05 and p ≤ 5.6654E‐06), likely an effect of the alkaline content of the diet. Interestingly, we also observed a significant 91% increase in Aquaporin 2 (AQP2) levels in the GF high K+ group (p ≤ 0.0168) relative to GF mice treated with control diet, an effect that was not observed in SPF mice (p= 0.165). These findings indicate that male germ‐free mice maintain plasma [K+] in response to a dietary alkaline potassium load, but develop reduced volume status compared to K+ loaded SPF controls. To compensate, germ free mice exhibit plasma elevated aldosterone concentrations, and upregulate cleaved γENaC and AQP2 abundance in the kidney. This suggests that an intact gut microbiome helps to defend against excessive volume depletion during the homeostatic response to alkaline K+ loading.

Expression of basolateral organic anion and cation transporters in experimental cadmium nephrotoxicity in rat kidney.

Cadmium (Cd)-intoxicated experimental animals exhibit impaired renal secretion of organic anions (OA) and cations (OC), indicating their transporters (Oats and Octs) in the proximal tubule (PT) basolateral membrane as possible targets of Cd. To correlate transport data from the literature with the expression of relevant transporters, we performed immunochemical and RT-PCR studies of renal Oats and Octs in the subchronic (treatment with CdCl2; 2 mg Cd/kg b.m./day, for 2 weeks) and acute (treatment with Cd-metallothionein (CdMT); 0.4 mg Cd/kg b.m., 6 or 12 h before killing) models of Cd nephrotoxicity. In the subchronic model, PT exhibited a minor loss of basolateral invaginations and overall unchanged expression of Na(+)/K(+)-ATPase and GAPDH proteins and mRNAs, while the expression of Oat and Oct proteins and their mRNAs was strongly downregulated. In the acute model, a time-related redistribution of basolateral transporters to the intracellular vesicular compartment was a major finding. However, 6 h following CdMT treatment, the total abundance of Oat and Oct proteins in the renal tissue remained unchanged, the expression of mRNAs decreased only for Oats, while a limited Oat1 and Na(+)/K(+)-ATPase immunoreactivity in the PT apical membrane indicated loss of cell polarity. As tested in rats treated with colchicine, the observed loss/redistribution of basolateral transporters in both models may be independent on microtubules. Therefore, the diminished renal secretion of OA and OC via PT in Cd nephrotoxicity may result from (a) limited loss of secretory surface (basolateral invaginations), (b) selective loss of Oats and Octs, and

Regulatory effect of leonurus extracts on hyperuricemia in rats

In this study, SD rats were orally administrated with oteracil potassium (300 mg . kg-1 . d-1 ) to prepare the hyperuricemia model, and divided into normal, model, Allopurinol, LE high dosage, middle dosage and low dose (200, 100, 50 mg . kg-1 . d-1) groups. The rats were orally administrated with test drugs 1 hour later after being orally administrated with Oteracil potassium. After 7 days, serum uric acid, serum creatinine, uric acid and expression of relevant transporters in kidney were tested to study the regulatory effect of leonurus extracts on serum uric acid, renal function and relevant transporters in kidney of rats with hyperuricemia. Compared with the model group, the leonurus extract group could significantly down-regulate serum uric acid and creatinine levels of rats with hyperuricemia, and increase the urine uric acid level. Meanwhile, leonurus extracts could notably down-regulate the mRNA expressions of urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9), up-regulate the mRNA expressions of organic cation transportanter (OCT) and Carnitine transporter (OCTN) and promote the excretion of uric acid of kidney.

Mechanisms of Reduced Maternal and Fetal Lopinavir Exposure in a Rat Model of Gestational Diabetes

Lopinavir (LPV) is the preferred HIV protease inhibitor in pregnancy, but it is unknown if gestational diabetes mellitus (GDM) affects its disposition. Hepatic protein expression and plasma protein binding are altered in rodent models of GDM. Because LPV is influenced by hepatic transporters and metabolic enzymes and is highly protein bound, it was hypothesized that streptozotocin-induced GDM would alter its disposition. Maternal and fetal tissues were collected from GDM rats and controls 45 min after LPV injection. In another cohort, fetuses were serially extracted 5 to 60 min after injection. LPV was quantified using liquid chromatography tandem mass spectrometry. Expression of relevant transporters, such as Multidrug resistance protein 1 (Mdr1), and cytochrome P450 3a2 (Cyp3a2), which metabolizes LPV in rodents, was measured in maternal liver via quantitative reverse transcriptase polymerase chain reaction and Western blot analysis. Expression of relevant transporters also was measured in placenta via quantitative reverse transcriptase polymerase chain reaction. Protein binding was determined by ultrafiltration. Relative to controls, we observed dramatically reduced maternal and fetal LPV exposure in GDM. Compared with controls, maternal hepatic Mdr1 and Cyp3a2 were up-regulated, and protein binding was reduced in the GDM group. Increased Mdr1- and Cyp3a2-mediated hepatobiliary clearance, coupled with a larger unbound LPV fraction, is likely to have facilitated hepatic elimination, thereby decreasing maternal and fetal exposure. Not surprisingly, up-regulation of Mdr1 and Cyp3a2's transcriptional regulator, pregnane X receptor, was demonstrated in maternal liver via Western blot analysis. Up-regulation of Mdr1 in placentas isolated from the GDM group likely also contributed to decreased fetal exposure to LPV. This study provides preclinical support for an as yet unreported drug-disease (LPV-GDM) interaction.

Cyclosporin a and enterohepatic circulation of bile salts in rats: decreased cholate synthesis but increased intestinal reabsorption.

Cyclosporin A (CsA) has been shown to inhibit synthesis and hepatobiliary transport of bile salts. However, effects of CsA on the enterohepatic circulation of bile salts in vivo are largely unknown. We characterized the effects of CsA on the enterohepatic circulation of cholate, with respect to synthesis rate, pool size, cycling time, intestinal absorption, and the expression of relevant transporters in liver and intestine in rats. CsA (1 mg. 100 g(-1). day(-1) s.c.) or its solvent was administered daily to male rats for 10 days. Cholate synthesis rate and pool size were determined by a 2H4-cholate dilution technique. Bile and feces were collected for determination of cholate and total bile salts, respectively. Cycling time and intestinal absorption of cholate were calculated. The mRNA levels and corresponding transporter protein levels in liver and intestine were assessed by real-time polymerase chain reaction and Western analysis, respectively. CsA treatment decreased cholate synthesis rate by 71%, but did not affect pool size or cycling time. CsA reduced the amount of cholate lost per enterohepatic cycle by approximately 70%. Protein levels of the apical sodium-dependent bile salt transporter (Asbt) were 2-fold increased in distal ileum of CsA-treated rats, due to post-transcriptional events. In conclusion, chronic CsA treatment markedly reduces cholate synthesis rate in rats, but does not affect cholate pool size or cycling time. Our results strongly suggest that CsA enhances efficacy of intestinal cholate reabsorption through increased Asbt protein expression in the distal ileum, which contributes to maintenance of cholate pool size in CsA-treated rats.