Renal Transport of Drugs: An Overview of Methodology with Application to Cimetidine

The development of new methods to study transport processes in renal epithelia has greatly enhanced our knowledge of the mechanisms involved in the transport of a number of endogenous compounds. More recently, these methods have been applied to study mechanisms of specific drug transport. This article is intended to provide an overview of the various methods used to study renal elimination of compounds. References to more detailed reviews of the individual methods are provided. Studies of the renal transport of cimetidine, a histamine H₂-receptor antagonist, are presented to illustrate the application of these methods to the study of specific drugs. Methods such as clearance techniques and the Sperber chicken preparation used to study renal elimination of compounds in whole animals are briefly described. Techniques to identify the site of renal transport including stop flow, isolated perfused tubules, and micropuncture methods are discussed and references to more technical reviews are cited. The more recently developed methods of isolated membrane vesicles for studying transport across the individual polar membranes of the proximal tubule are discussed along with the relevant studies of the use of these membranes in elucidating the mechanisms involved in the renal transport of cimetidine. Finally, the use of cultured renal epithelial cell lines in studying renal transport is described. Knowledge of drug transport mechanisms in the kidney is important both in drug targeting to the kidney and in understanding the pharmacokinetics of renally eliminated drugs. As exemplified by the studies with cimetidine, only by combining the data from experiments using diverse methodology can the mechanisms involved in the renal excretion of compounds be delineated. With the use of existing methods and the development of new technologies, many of the questions related to drug transport mechanisms can be addressed.     

The effect of naringenin on the intestinal and renal transport of organic solutes

Summary Naringenin (4,5,7-trihydroxy-flavanone) inhibits the accumulation of glycine, -methyl-glucoside, p-amino-hippurate and N¹-methyl-nicotinamide in dog renal cortex slices. It also inhibits oxygen consumption by this tissue. Since the sensitivity of amino-acid uptake to the drug is less than the sensitivity of oxygen consumption, the inhibition of this transport might be secondary to an effect on intermediary metabolism. The inhibition of PAH uptake occurs at a lower concentration of the drug, and so naringenin may affect this process at the membrane level.Naringenin inhibits the transport of sugars and amino-acids by dog, guinea-pig and rat small intestine. Both steady-state accumulation and initial rates of entry are affected. Amino-acid uptake is depressed both in the presence or absence of sodium ions. The inhibition is reversible provided short contact times are employed. According to kinetic analysis, naringenin appeared to be a fully non-competitive inhibitor of phenylalanine influx. Examination of the unidirectional transmural fluxes of phenylalanine across guinea-pig intestine revealed that only the mucosal-serosal flux was affected, and then only if the flavanone was added to the solution bathing the mucosal face of the tissue. Naringenin does not inhibit mucosal Na⁺-K⁺-ATPase, but it does alter the intracellular ion concentrations.Although some of the results can be explained in terms of an effect of naringenin on metabolism, others can not. It is argued that naringenin has a direct action on cell membranes.Some of these results have been presented to the Physiological Society and a short report has appeared in their proceedings (Robinson, 1979)     

基层医院小儿危重症的急救转运128例报道

目的探讨基层医院急救转运模式及方法,提高本地区危重患儿的抢救成功率,降低危重患儿死亡率及致残率。方法对丰顺县人民医院近3年来转运的危重患儿128例进行回顾性分析。结果全部危重病患儿均安全转运至上级医院相应专科病房,在转运患儿的原发病以新生儿疾病、重症肺炎和各种感染为主,无1例在途中发生意外或死亡。结论加强转运前、转运途中质量控制,充分估计病情变化,密切监测病情是安全转运的前提条件,快捷安全的转运,提高了本地区危重患儿的抢救成功率,降低危重患儿死亡率及致残率是非常有效的。     

Inner Blood-Retinal Barrier Transporters: Role of Retinal Drug Delivery

The inner blood-retinal barrier (inner BRB) forms complex tight junctions of retinal capillary endothelial cells to prevent the free diffusion of substances between the circulating blood and the neural retina. Thus, understanding of the inner BRB transport mechanisms could provide a basis for the development of strategies for drug delivery to the retina. Recent progress in inner BRB research has revealed that retinal endothelial cells express a variety of unique transporters which play a role in the influx transport of essential molecules and the efflux transport of xenobiotics. In this review we focus on the transport mechanism at the inner BRB in relation to its importance in influencing the inner BRB permeability of drugs.     

易化运输机制下的膜运载动力学的基本方程

各种膜的性质是一个进行广泛研究的题目。有许多分子,特别是各种代谢物质,如糖类和氨基酸,并不遵循Firk定律。本文将提出物质通过细胞膜进入细胞进行营养运输的不遵循Firk定律方式的易化运输过程,给出它的动力学基本方程和某些结论,并指出膜运载动力学的发展前景。     

Transport of N-acetyl-S-pentachloro-1,3-butadienylcysteine by rat renal cortex

N-acetyl-S-pentachloro-1,3-butadienyl-L-cysteine (PCBD-NAC) is a postulated metabolite derived from glutathione conjugation of hexachloro-1,3-butadiene and is nephrotoxic in the rat. Because PCBD-NAC causes selective necrosis to the pars recta of the proximal tubule, and is an organic anion it might be expected to be transported by the renal organic anion transport system. Rat renal cortical slices were used to characterise the transport. ¹⁴C-PCBD-NAC uptake was temperature dependent and reduced by the metabolic inhibitors cyanide and iodoacetate. Probenecid and sulphinpyrazone, specific competitive inhibitors of the anion transport system, and dinitrophenol, a metabolic inhibitor as well as a competitive inhibitor of anion transport, reduced PCBD-NAC transport. Organic cations or uric acid transport inhibitors did not alter PCBD-NAC accumulation by the slices. These data are consistent with the transport of PCBD-NAC by the renal organic anion secretory system.     

Response of rat small intestinal active aldohexose transport to elevation of mucosal cyclic AMP by forskolin and 3-isobutyl-1-methylxanthine in vitro

Summary The phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX) was able to elevate rat small intestinal cyclic AMP levels to 300% of basal values. Active jejunal d-glucose transport was enhanced parallel to the rise of intracellular cyclic AMP levels to 140% of control values at 100 mol/l IBMX. Transport parameters, as determined in a three compartment model in vitro using a dual label method, indicate increased uphill glucose transport at the site of the brush border membrane, higher intracellular accumulation of the sugar, with unchanged passive permeabilities. Phlorizin-inhibited d-glucose transport and l-glucose transfer in the rat were not affected by the persisting cyclic AMP elevation produced by IBMX. Stimulating effects could also be demonstrated with d-galactose as a substrate. IBMX 100 mol/l also increased active d-glucose as well as 3-O-methylglucose transport in mouse jejunum.Stimulatory effects on intestinal hexose transport and mucosal cyclic AMP levels were also found with the adenylate-cyclase activator forskolin. In the present study, forskolin effects on jejunal mucosal cyclic AMP levels were enhanced in the presence of 100 mol/l IBMX, resulting in a 20-fold increase compared to controls at 20 mol/l forskolin. The concentration response for the effect of forskolin in the presence of 100 mol/l IBMX on d-glucose transport did not produce a significant increase compared to transport stimulation with IBMX alone. At higher concentrations of forskolin however, glucose transport decreased to levels well below the IBMX controls.The elevation of cellular cyclic AMP levels had no effects on passive permeability.Both IBMX 100 mol/l as well as forskolin 20 mol/l inhibited rat jejunal net fluid transport by 40%, combination of both agents resulted in a 55% reduction of net fluid absorption in everted sacs of rat jejunum.These results indicate a functional relationship between jejunal mucosal cyclic AMP levels and active hexose absorption different from the inhibitory role of cyclic AMP in intestinal fluid transport.A preliminary account of this work has been given at the 26th Spring Meeting (March 1985) of the Deutsche Pharmakologische Gesellschaft in Mainz, FRG     

肿瘤相关蛋白分子核转运机制研究进展

信号分子的核转运是多种信号传导通路的基础。肿瘤的发生和发展常与多种信号蛋白异常的亚细胞定位相关,因此阐明肿瘤相关蛋白的核转运机制对于研究肿瘤的发病机制和开发新的疗法至关重要。本文介绍了核转运系统的基本组成,并对多种重要的肿瘤相关蛋白的核转运机制进行综述。     

安石榴苷在MDCK细胞单层模型上的转运机制研究

摘 要 目的：采用MDCK细胞单层模型考察安石榴苷跨膜转运特性。方法: CCK8法筛选安石榴苷对MDCK细胞作用的安全浓度,Millicell ERS测量细胞单层的TEER值确定细胞单层的完整性及致密性,考察给药浓度、方向、时间、温度、维拉帕米和EDTA Na2不同条件下安石榴苷的转运情况,采用HPLC法测定安石榴苷浓度,并计算表观渗透系数( Papp ) 和外排率（ER）。结果:安石榴苷在MDCK模型上累积转运量具有时间和浓度依赖性,在100~300 μg·mL^-1浓度范围内测得的顶侧（AP）到基底侧（BL）的Papp值为（6.13±0.12）×10-7cm·s-1、（6.96±0.26）×10-7cm·s-1、（5.94±0.10）×10-7cm·s-1,未随浓度升高而增大,4℃时转运量降低,P gp 抑制药维拉帕米可以促进AP BL方向的转运,加入EDTA Na2后Papp(AP BL)显著增大。结论:安石榴苷以被动转运为主兼有主动转运参与,是P 糖蛋白（P gp）的底物受到P gp的外排作用,同时有细胞旁路转运途径。     

Percutaneous absorption of steroids: Relative contributions of epidermal penetration and dermal clearance

The permeation of triamcinolone, hydrocortisone, prednisolone, corticosterone, triamdnolone acetonide, testosterone, and betamethasone-17-valerate through excised human stratum corneum was quantified. The time course of permeation could be adequately described by a simple diffusion model suggesting that shunt transport may not be important. The disappearance of these steroids from aqueous solutions applied to human and rat dermis was also monitored. The concentrations of unbound steroid in the viable epidermis appeared to be mainly related to the blood perfusion rate in the dermis and, more importantly, to the lipophilicity of the steroid. The most lipophilic steroids penetrated the human epidermis at the fastest rates but are cleared from the viable epidermis at rates comparable to those found for more polar steroids.     

Transport of citrinin by rat renal cortex

Citrinin, a secondary product of fungal metabolism, is nephrotoxic in the rat. Because citrinin is an organic anion, it might be expected to be transported by the renal organic anion transport system. Rat renal cortical slices were used to characterize the transport. ¹⁴C-Citrinin uptake was enhanced by lactate and reduced by probenecid, a specific inhibitor of anion transport. Dinitrophenol is a metabolic inhibitor as well as competitive inhibitor of anion transport, and it also reduced citrinin transport. Organic cations did not alter citrinin accumulation by the slices. These data are consistent with the transport of citrinin by the renal organic anion secretory system.     

Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories

Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1 > GLUT3 > GLUT5 > GST1A > OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.     

Saturable Absorptive Transport of the Hydrophilic Organic Cation Ranitidine in Caco-2 Cells: Role of pH-Dependent Organic Cation Uptake System and P-Glycoprotein

Purpose The purpose of this work was to investigate the involvement of carrier-mediated apical (AP) uptake and efflux mechanisms in the absorptive intestinal transport of the hydrophilic cationic drug ranitidine in Caco-2 cells. Methods Absorptive transport and AP uptake of ranitidine were determined in Caco-2 cells as a function of concentration. Permeability of ranitidine in the absorptive and secretory directions was assessed in the absence or presence of the P-glycoprotein (P-gp) inhibitor, GW918. Characterization of the uptake mechanism was performed with respect to inhibitor specificity, pH, energy, membrane potential, and Na⁺ dependence. Efflux from preloaded monolayers was evaluated over a range of concentrations and in the absence or presence of high extracellular ranitidine concentrations. Results Saturable absorptive transport and AP uptake of ranitidine were observed with K_m values of 0.27 and 0.45 mM, respectively. The ranitidine absorptive permeability increased and secretory permeability decreased upon inhibition of P-gp. AP ranitidine uptake was inhibited in a concentration-dependent fashion by a diverse set of organic cations including tetraethylammonium, 1-methyl-4-phenylpyridinium, famotidine, and quinidine. AP ranitidine uptake was pH and membrane potential dependent and reduced under conditions that deplete metabolic energy. Efflux of [³H]ranitidine across the basolateral membrane was neither saturable as a function of concentration nor trans stimulated by unlabeled ranitidine. Conclusions Saturable absorptive transport of ranitidine in Caco-2 cells is partially mediated via a pH-dependent uptake transporter for organic cations and is subject to attenuation by P-gp. Inhibition and driving force studies suggest the uptake carrier exhibits similar properties to cloned human organic cation transporters. The results also imply ranitidine transport is not solely restricted to the paracellular space.     

Utilization of endothelial cell monolayers of low tightness for estimation of transcellular transport characteristics of hydrophilic compounds

The determination of transcellular transport characteristics using filter-grown brain capillary endothelial cells in conventional permeation studies requires extremely tight cell layers, exhibiting electrical resistance values of more than 500 ohm · cm², for avoiding interferences with the paracellular flux. The present investigations demonstrate that rate and asymmetry of transcellular transport processes can be evaluated sufficiently reliably for orientation and comparison purposes also by use of more easily obtainable leaky cell layers exhibiting electrical resistance values of about 20 ohm · cm² only. The studies, relying on measurements of rates for influx and efflux into and out of the cells, respectively, of the cell barrier were performed with aortic endothelial cell monolayers grown on permeable supports. Alanine, leucine, 3-(O-methyl)-D-glucose served as representative hydrophilic substrates being transported actively or by facilitated diffusion. HPLC, combined with a simple precolumn benzoylation appropriate for a simultaneous analysis of both substance groups, was used for the quantitation.     

The group of Professor Qiang Zhang and Bing He has made important progress in nanodrug transport mechanism

The group of Professor Qiang Zhang and Bing He has made important progress in nanodrug transport mechanism.     

Determination of Transport Rates for Arginine and Acetaminophen in Rabbit Intestinal Tissues in Vitro

The in vitro Ussing technique was employed to examine transport rates for acetaminophen and arginine across rabbit intestinal tissues. Mannitol and transepithelial conductance were used to monitor the integrity of rabbit intestinal tissues and the basal and stimulated short-circuit current were measured to assess functional viability. Transepithelial transport of acetaminophen, arginine, and mannitol was determined in rabbit jejunum, ileum, and distal colon. Transepithelial transport of arginine in the ileum and jejunum was composed of both passive (nonsaturable) (P _m = 0.06) and saturable components (K _T = 0.6-0.7 mM; J _max = 0.3-0.4 µmol/hr · cm²). The saturable component of arginine fluxes was abolished by pretreatment of the tissue with serosal ouabain (0.1 mM). In the distal colon, both unidirectional arginine fluxes were nonsaturable. In the segments examined, both unidirectional fluxes of acetaminophen were nonsaturable over the concentration range from 0.1 to 30 mM. These results provide values for maximal permeabilities attained by molecules traversing both the cellular and the paracellular pathways and, by comparison to their in vivo bioavailabilities, provide selection criteria for evaluating drug candidates for oral activity.     

Proton-Cotransport of Pravastatin Across Intestinal Brush-Border Membrane

Purpose. The purpose of the present study is to clarify the intestinal brush-border transport mechanism of a weak organic acid, pravastatin, an HMG-CoA reductase inhibitor. Methods. The transport of pravastatin was studied by using intestinal brush-border membrane vesicles prepared from rabbit jejunum, and uptake by the membrane vesicles was measured using rapid filtration technique. Results. The initial uptake of [¹⁴C]pravastatin was markedly increased with decreases in extravesicular pH and showed a clear overshoot phenomenon in the presence of a proton gradient (pH_in/out = 7.5/5.5). A protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, significantly reduced the uptake of [¹⁴C]pravastatin. In addition, an ionophore for sodium, potassium and proton, nigericin, stimulated the uptake of [¹⁴C]pravastatin in the presence of a potassium gradient ([K ⁺ ]_in/[K⁺ ]_out = 0/145 mM). On the other hand, neither the imposition of an inwardly directed sodium gradient nor an outwardly directed bicarbonate gradient stimulated the uptake of [¹⁴C]pravastatin. In the presence of a proton gradient (pH_in/out = 7.5/5.5), the initial uptake of pravastatin was saturable with the apparent K_t of 15.2 ± 3.2 mM and J_max of 10.6 ± 1.21 nmol/mg protein/10 sec. The uptake of pravastatin was significantly inhibited by monocarboxylic acid compounds such as acetic acid and nicotinic acid in a competitive manner but not by di- or tri-carboxylic acids, or acidic amino acid. Conclusions. It was concluded that a pH-dependent transport of pravastatin across the brush-border membrane occurs by a proton-gradient dependent carrier-mediated mechanism rather than by simple diffusion of its unionized form.     

Forskolin-induced elevation of rat jejunal cyclic AMP levels and stimulation of active glucose transport in vitro

Summary The adenylate cyclase stimulator forskolin enhanced both mucosal cyclic AMP levels and d-aldohexose transport in rat jejunum in vitro in a concentration-dependent manner. With mucosal cyclic AMP elevated to 400% of basal values, active d-glucose transport was at 200% of control values. Transport parameters, as determined in a three compartment model using a dual label method, indicate a) increased uphill glucose transport at the brush border membrane with higher intracellular accumulation, b) unchanged serosal to mucosal glucose fluxes and passive permeabilities.Transport stimulating effects of forskolin were also present with d-galactose in the rat. In mouse jejunum active transport of d-glucose and 3-O-methylglucose was increased by forskolin 10 mol/l.Phlorizin inhibited d-glucose transport as well as l-glucose transport in the rat were not changed by the persisting cyclic AMP elevation induced by forskolin.The results show a positive correlation of active intestinal hexose transport with a modest elevation of mucosal cyclic AMP.In accordance to current models of cyclic AMP-induced changes in intestinal fluid absorption, rat jejunal net fluid transport was reduced by 40% with 20 mol/l forskolin.Part of this work was presented at the 25th Spring Meeting (April 1984) of the Deutsche Pharmakologische Gesellschaft in Mainz, FRG     

成骨细胞跨膜转运锌特性及其机制的初步研究

目的　研究锌在成骨细胞跨膜转运的动力学特点及影响因素 ,为阐明锌在骨发育中的作用提供科学依据。方法　采用65Zn同位素示踪法研究锌在成骨细胞跨膜转运的动力学特点 ;分别研究组氨酸、Na+、K+泵抑制剂、Ca2 +离子通道阻滞剂对成骨细胞锌转运的影响。结果　细胞外锌浓度增加可以促进锌转运入胞内 ,但锌缺乏使锌胞内转运减少 ;组氨酸、Na+、K+泵抑制剂对锌转运无影响 ;Ca2 +离子通道阻滞剂促进锌内流。结论　成骨细胞外环境锌水平可以影响成骨细胞锌转运及胞内锌水平 ,Ca2 +离子通道与Zn2 +通道可能相互影响     

Endocrine regulation of calcium transport in epithelia

1. Calcium (re)absorption occurs in epithelia, including the intestine, kidney, mammary glands, placenta and gills (in the case of fish). 2. Calcium is transported across epithelia by two transport mechanisms, paracellular and transcellular, and the movement is regulated by a complex array of transport processes that are mediated by hormonal, developmental and physiological factors involving the gastrointestinal tract, bone, kidney and the parathyroids. 3. Clear understanding of the calcium transport pathways and their endocrine regulation is critical for minimizing various metabolic and health disorders at different physiological stages. Here, we first briefly review the calcium transport mechanisms before discussing in detail the endocrine factors that regulate calcium transport in the epithelia.