alpha 1- and beta 2-adrenergic receptor expression in the Madin-Darby canine kidney epithelial cell line

The Madin-Darby canine kidney (MDCK) cell line, derived from distal tubule/collecting duct, expresses differentiated properties of renal tubule epithelium in culture. We studied the expression of adrenergic receptors in MDCK to examine the role of catecholamines in the regulation of renal function. Radioligand-binding studies demonstrated, on the basis of receptor affinities of subtype-selective adrenergic agonists and antagonists, that MDCK cells have both alpha 1- and beta 2- adrenergic receptors. To determine whether these receptor types were expressed by the same cell, we developed a number of clonal MDCK cell lines. The clonal lines had stable but unique morphologies reflecting heterogeneity in the parent cell line. Some clones expressed only beta 2-adrenergic receptors and were nonmotile, whereas others expressed both alpha 1- and beta 2-receptors and demonstrated motility on the culture substrate at low cell densities. In one clone, alpha- and beta- receptor expression was stable for more than 50 passages. Catecholamine agonists increased phosphatidylinositol turnover by activating alpha- adrenergic receptors and cellular cyclic adenosine monophosphate accumulation by activating beta-adrenergic receptors. Guanine nucleotide decreased the affinity of isoproterenol for the beta 2- receptor but did not alter the affinity of epinephrine for the alpha 1- receptor. These results show that alpha 1- and beta 2-receptors can be expressed by a single renal tubular cell and that the two receptors behave as distinct entities in terms of cellular response and receptor regulation. Heterogeneity of adrenergic receptor expression in MDCK clones may reflect properties of different types of renal tubule cells.     

The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule

Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues.     

Characterization of Zn(2+) transport in Madin-Darby canine kidney cells

The aim of this study was to characterize the mechanism implicated in Zn(2+) transport in MDCK cells. Trace elements such as Zn(2+), Cd(2+) or Cu(2+) induced MDCK cell depolarization at the micromolar level as demonstrated by bis-oxonol fluorescence and whole-cell patch experiments. This depolarization was inhibited by La(3+) and Gd(3+) and was not related to the activation of Na(+) or Cl(-) channels. Uptake of 65Zn was assessed under initial rate conditions. The kinetic parameters obtained at 37 degrees C were a K(m) of 18.9 microM and a V(max) of 0.48 nmol min(-1) (mg protein(-1)). Intracellular pH measurements using BCECF probe demonstrated that Zn(2+) transport induced a cytoplasmic acidification. The cytoplasmic acidification resulting from Zn(2+) uptake activated Na(+)/H(+) antiporter, which allowed for the recycling of protons. These data suggest that Zn(2+) enters MDCK cells through a proton-coupled metal-ion transporter, the characteristics of which are slightly different from those described for the metal transporter DCT1. This mechanism could be in part responsible of the metal transport evidenced in the distal parts of the renal tubule.     

Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney.

The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule. The level of 3':5'-cyclic AMP in JTC-12.P3 (monkey kidney) cells increased in 2 min as much as 2.5-5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1-34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin E1 (14 micronM) and isopropylnorepinephrine (10 micronM) were also found to increase the concentration of cyclic AMP by more than 30- and 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell. In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E1 and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E1 was observed. The cell lines JTC-12, MDCK and MDBK, when incubated with H235SO4, incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate. The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.     

Development of an AT<Subscript>2</Subscript>-deficient proximal tubule cell line for transport studies

Angiotensin II is a major regulatory peptide for proximal tubule Na(+) reabsorption acting through two distinct receptor subtypes: AT(1) and AT(2). Physiological or pathological roles of AT(2) have been difficult to unravel because angiotensin II can affect Na(+) transport either directly via AT(2) on luminal or peritubular plasma membranes of proximal tubule cells or indirectly via the renal vasculature. Furthermore, separate systemic and intratubular renin-angiotensin systems impart considerable complexity to angiotensin's regulation. A transport-competent, proximal tubule cell model that lacks AT(2) is a potentially useful tool to assess cellular angiotensin II regulation. To this end, AT(2)-receptor-deficient mice were bred with an Immortomouse, which harbors the thermolabile immortalization gene SV40 large-T antigen (Tag), and AT(2)-receptor-deficient [AT(2) (-/-)], Tag heterozygous [Tag (+/-)] F(2) offspring were selected for cell line generation. S1 proximal tubule segments were microdissected, and epithelial cell outgrowth was expanded in culture. Cells that formed confluent, electrically resistive monolayers were selected for cryopreservation, and one isolate was extensively characterized for conductance (2 mS/cm(2)), short-circuit current (Isc; 0.2 microA/cm(2)), and proximal tubule-specific Na3(+) - succinate (DeltaIsc = 0.8 microA/cm(2) at 2 mM succinate) and Na3(+) - phosphate cotransport (DeltaIsc = 3 microA/cm(2) at 1 mM phosphate). Light microscopy showed a uniform, cobblestone-shaped monolayer with prominent cilia and brush borders. AT(2) receptor functionality, as demonstrated by angiotensin II inhibition of ANF-stimulated cGMP synthesis, was absent in AT(2)-deficient cells but prominent in wild-type cells. This transport competent cell line in conjunction with corresponding wild type and AT(1)-deficient lines should help explain angiotensin II signaling relevant to Na(+) transport.     

Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney

The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule.The level of 3′: 5′-cyclic AMP in JTC-12.P3 (monkey kidney) cells increased in 2 min as much as 2.5–5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1–34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin E₁ (14 μM) and isopropylnorepinephrine (10 μM) were also found to increase the concentration of cyclic AMP by more than 30- and 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell.In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E₁ and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E₁ was observed.The cell lines JTC-12, MDCK and MDBK, when incubated with H₂³⁵SO₄, incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate.The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.     

Characterization of glucose transport by cultured rabbit kidney proximal convoluted and proximal straight tubule cells

Rabbit kidney proximal convoluted tubule (RPCT) and proximal straight tubule (RPST) cells were independently isolated and cultured. The kinetics of the sodium-dependent glucose transport was characterized by determining the uptake of the glucose analog alpha-methylglucopyranoside. Cell culture and assay conditions used in these experiments were based on previous experiments conducted on the renal cell line derived from the whole kidney of the Yorkshire pig (LLC-PK1). Results indicated the presence of two distinct sodium-dependent glucose transporters in rabbit renal cells: a relatively high-capacity, low-affinity transporter (V(max) = 2.28 +/- 0.099 nmoles/mg protein min, Km = 4.1 +/- 0.27 mM) in RPCT cells and a low-capacity, high-affinity transporter (V(max) = 0.45 +/- 0.076 nmoles/mg protein min, K(m) = 1.7 +/- 0.43 mM) in RPST cells. A relatively high-capacity, low-affinity transporter (V(max) = 1.68 +/- 0.215 nmoles/mg protein min, Km = 4.9 +/- 0.23 mM) was characterized in LLC-PK1 cells. Phlorizin inhibited the uptake of alpha-methylglucopyranoside in proximal convoluted, proximal straight, and LLC-PK1 cells by 90, 50, and 90%, respectively. Sodium-dependent glucose transport in all three cell types was specific for hexoses. These data are consistent with the kinetic heterogeneity of sodium-dependent glucose transport in the S1-S2 and S3 segments of the mammalian renal proximal tubule. The RPCT-RPST cultured cell model is novel, and this is the first report of sodium-dependent glucose transport characterization in primary cultures of proximal straight tubule cells. Our results support the use of cultured monolayers of RPCT and RPST cells as a model system to evaluate segment-specific differences in these renal cell types.     

Aminoglycosides inhibit hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells

The clinical use of aminoglycosides often leads to renal magnesium wasting and hypomagnesemia. Of the nephron segments, both the thick ascending limb of Henle's loop and the distal tubule play significant roles in renal magnesium conservation but the distal convoluted tubule exerts the final control of urinary excretion. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. Peptide hormones, such as parathyroid hormone (PTH), glucagon, calcitonin, and arginine vasopressin (AVP) stimulate Mg2+ uptake in MDCT cells that is modulated by extracellular polyvalent cations, Ca2+ and Mg2+. The present studies determined the effect of aminoglycosides on parathyroid hormone (PTH)-mediated cAMP formation and Mg2+ uptake in MDCT cells. Gentamicin, a prototypic aminoglycoside, elicited transient increases in intracellular Ca2+ from basal levels of 102 +/- 13 nM to 713 +/- 125 nM, suggesting a receptor-mediated response. In order to determine Mg2+ transport, MDCT cells were Mg(2+)-depleted by culturing in Mg(2+)-free media for 16 h and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.0 mM MgCl2. The mean rate of Mg2+ uptake, d([Mg2+]i)/dt, was 138 +/- 24 nM/s in control MDCT cells. Gentamicin (50 microM) did not affect basal Mg2+ uptake (105 +/- 29 nM/s), but inhibited PTH stimulated Mg2+ entry, decreasing it from 257 +/- 36 nM/s to 108 +/- 42 nM/s. This was associated with diminished PTH-stimulated cAMP formation, from 80 +/- 2.5 to 23 +/- 1 pmol/mg protein x 5 min. Other aminoglycosides such as tobramycin, streptomycin, and neomycin also inhibited PTH-stimulated Mg2+ entry and cAMP formation. As these antibiotics are positively charged, the data suggest that aminoglycosides act through an extracellular polyvalent cation-sensing receptor present in distal convoluted tubule cells. We infer from these studies that aminoglycosides inhibit hormone-stimulated Mg2+ absorption in the distal convoluted tubule that may contribute to the renal magnesium wasting frequently observed with the clinical use of these antibiotics.     

1H NMR spectroscopic studies on the characterization of renal cell lines and identification of novel potential markers of in vitro nephrotoxicity

Cell cultures are increasingly used in the evaluation of chemically-induced nephrotoxicity. The utili of renal cell culture systems in toxicology would be improved, however, if better characterized and more specific markers of toxicity were available. High resolution proton nuclear magnetic resonance (¹H NMR) spectroscopy is well suited to the study of toxicological events and has identified many novel markers of nephrotoxicity in vivo. In this study, ¹H NMR spectroscopy has been used to characterize the biochemical composition of two renal cell lines of different nephronal origin, LLC-PK₁ (pig proximal tubule) and Madin-Darby canine kidney (MDCK, distal tubule). The early biochemical responses of these cell lines to the model proximal tubular toxin S-(1,2dichlorovinyl)i-L-cysteine (DCVC) and the renal medullary toxin 2-chloroethanamine (CEA) have also been investigated. For each line, 500 MHz ¹H NMR spectra of protein-free acetone extracts of cells and culture medium gave characteristic and reproducible profiles of low MW constituents, including amino and organic acids, glucose and soluble membrane precursors, such as choline and myo-inositol. Treatment-related changes in several low MW compounds not routinely measured in toxicological studies were revealed by NMR specboscopy before marked cytotoxicity was observed by phase contrast microscopy. For example, LLC-PK₁ cells treated with 60 μM DCVC showed a marked decrease in intracellular choline levels within 3 h which suggests an effect on the balance of choline synthesis and utilization. Wrthin 9 h of treatment with DCVC there were decreases in intracellular acetate and alanine concentrations which may be indicative of a decrease in fatty acid oxidation and biglyceride metabolism accompanied by an increase in gluconeogenesis. In MDCK cells, 1 h post treatment with 5 mM CEA, intracellular glycine was decreased. This study indicates the potential power and applicability of ¹H NMR spectroscopy for evaluating the biochemical and metabolic effects of toxins in cell culture systems and provides a novel approach to identifying new markers of tissue damage.     

Cyclic-AMP deficient MDCK cells form tubules

It has known for many years that MDCK cells blister structures, termed domes. During an examination of the morphbology of a large number of MDCK clones, we found that two stable morphotypes exist in an MDCK cell population namely, dome-forming and tubule-forming clones. When maintained at high cell density, tubule-forming clones displayed large numbers of anastomosing tubules which contained lumens. The frequency of obseration of the tubule forming clones in an MDCK population was 0.7% Tubule-forming MDCK clones should be useful in studying tubule morphogenesis. While agents that affect protein kinase A actiity increased dome formation, the same agents abolished the formation of tubules in all tubule-forming clones. In contrast, drugs that stimulate protein kinase C actity (phorbol esters and staurosporine) decreased dome formation and increased tubule morphogenesis in all MDCK morphotyes. Tubules-forming clones were found to have lower resting levels of cyclic-AMP and to respond to forskolin stimulation of adenylate cyclase readily. Hence, sigals transmitted by the protein kinase C pathway appear to lead to tubule formation MDCK cells, while signals transmitted through the protein A pathway lead to dome formation. © 1995 Wiley-Liss, Inc.     

Stimulation by parathyroid hormone of calcium absorption in confluent Madin-Darby canine kidney cells

Parathyroid hormone (PTH) increases renal calcium absorption exclusively in cortical thick limbs and distal tubules. Lack of sufficient tissue has precluded detailed biochemical study of the mechanisms responsible for the hypercalcemic effect of PTH. Therefore, we assessed PTH action on calcium transport in Madin-Darby canine kidney (MDCK) cells, a cell line expressing distal characteristics, to determine its suitability as a model for analyzing PTH action. Calcium transport across MDCK cells grown to confluence on porous filters was measured at 37 degrees C in Ussing chambers. Mucosal-to-serosal calcium fluxes (JCa, mol/min cm-2 x 10(-9)) were measured with 45Ca at -3, -1, 5, 10, and 20 min; agonist was added at 0 min. Basal JCa averaged 0.98. PTH at 0.2 microM increased JCa by 12% (P less than 0.05) and 1 microM PTH by 70% (P less than 0.01). Calcitonin (1 microM) had no effect on JCa. The fact that high concentrations of dibutyryl cAMP (1 mM) and forskolin (10 microM) increased JCa by only 37% and 22%, respectively, suggested that cAMP-independent mechanisms may participate in PTH-stimulated JCa. Therefore we examined the effect of other putative second messengers. In the presence of 2 mM external [Ca], 10 nM A23187 increased JCa by 88%, and 10 microM A23187 increased JCa by 121%. Addition of 10 microM phorbol 12-myristate 13-acetate (PMA) increased JCa by 60%. We conclude that: 1) PTH specifically stimulates unidirectional calcium absorption in MDCK cells; 2) both adenylate cyclase-coupled and calcium-coupled receptors may participate in signaling the response to PTH; and 3) confluent MDCK cells represent a useful experimental model for elucidating the biochemical mechanisms involved in the renal hypercalcemic action of PTH.     

Requirement of focal adhesion kinase in branching tubulogenesis

We previously demonstrated that α3β1 integrins are essential to hepatocyte growth factor (HGF)-independent branching tubulogenesis in Mardin-Darby Canine Kidney (MDCK) cells. However, the involvement of integrin downstream signaling molecules remains unclear. In the present study, we successfully isolated cell lines possessing different tubulogenic potentials from the MDCK cells; cyst clones (CA4, CA6) forming cystic structures when cultured in 0.3% type I collagen gel and mass clones (M610, M611, M612) forming aggregated masses. Cyst clones maintained cystic structure in 0.1% collagen gel, whereas mass clones spontaneously developed into tubules. Both clones exhibited various morphologies when cultured on a dish: cyst clones formed aggregated islands, while mass clones were more scattered and exhibited higher migration capacity. Among several focal adhesion machinery proteins examined, only the expression and phosphorylation level of focal adhesion kinase (FAK) in mass clones was higher than in cyst clones, while other proteins showed no obvious differences. However, overexpression of wild type FAK in CA6 cells did not facilitate branching tubule formation in 0.1% collagen gel. Targeted decrease in the expression level of FAK in M610 cells with the application of antisense cDNA resulted in a marked reduction of branching tubule formation in 0.1% collagen gel and showed a down-regulation of fibronectin assembly, which is known to promote tubulogenesis. In contrast, overexpression of wild type FAK in CA6 cells had no effect on fibronectin assembly. Taken together, our data demonstrates that FAK is required, but not sufficient for HGF-independent branching tubulogenesis in MDCK cells.     

Requirement of regulated activation of Ras for response of MDCK cells to hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering, migration, and branching tubule formation of MDCK cells. To examine the role of the Ras protein in the HGF/SF-induced responses, we constructed MDCK cell clones expressing either inducible dominant-negative Ras or constitutively activated Ras and analyzed their effects on responses of cells to HGF/SF. Induced expression of dominant-negative Ras prevented cell dissociation required for cell scattering, migration, and cystic formation as well as branching morphology required for branching tubule formation. Constitutively activated Ras induced cell dissociation, but not a scattered fibroblastic morphology even in the presence of HGF/SF. MDCK cells expressing constitutively activated Ras migrated at a level similar to that of wild-type MDCK cells stimulated by HGF/SF. MDCK cells expressing constitutively activated Ras showed disorganized growth in three-dimensional culture and did not form the branching tubule structures. These results indicate that activation of the Ras protein is essential for the cell scattering, migration, and branching tubule formation of MDCK cells induced by HGF/SF, and a properly regulated activation is required for some stages of the HGF/SF-induced responses of MDCK cells.     

Cyclosporine A-induced toxicity in two renal cell culture models (LLC-PK1 and MDCK)

Renal damage caused by therapeutic treatment with cyclosporine A has been well documented. Clinical experiences have shown that cyclosporine A nephrotoxicity is determined by interstitial fibrosis with tubular atrophy. However, the exact mechanism by which this drug causes nephrotoxicity has not yet been clarified. This study used an in vitro model in an attempt to identify the cellular mechanisms underlying kidney cyclosporine A damage. We used two cell lines with the characteristics of proximal and distal tubule cells (pig kidney proximal tubular epithelial cell line [LLC-PK1] and Madin–Darby canine kidney cell line [MDCK]. The cell lines were treated with cyclosporine A for 24h. After the treatment, the cells were stained with Trypan Blue to estimate cell viability and processed by histochemical reactions to evaluate their cellular metabolism. Four enzymes (acid phosphatase, alkaline phosphatase, lactate dehydrogenase and succinate dehydrogenase) were considered. The cell viability assay showed that the LLC-PK1 cell line was more sensitive to cyclosporine A than MDCK. Remarkably, the LLC-PK1 cells disappeared with cyclosporine A treatment. As for the hydrolytic enzymes, only acid phosphatases showed an increased positivity in the treated LLC-PK1 cells. Similarly, lactate dehydrogenase showed a different activity histochemically. No statistically significant alterations were observed in the succinate dehydrogenase reaction.The cyclosporine A-treated MDCK cell lines did not show any difference in either their hydrolytic or succinate dehydrogenase enzyme positivity with respect to the control line. In contrast, there was a significant increase in lactate dehydrogenase activity. This study allowed the possible mechanism of cyclosporine A-induced damage in renal tubular cells to be evaluated. The enzymatic changes happened rapidly (during the 24h of treatment), suggesting that this alteration was one of the steps by which cyclosporine A induced toxicity. Moreover, since acid phosphatase is a marker of protein catabolism, the variation in the activity of this enzyme, in the LLC-PK1 line only, showed that cyclosporine can induce alterations leading to cellular toxicity. The modifications in lactate dehydrogenase activity, in both lines, suggested that this drug caused cell stress, inducing the production of lactic acid from glucose in the presence of oxygen. In conclusion, cyclosporine A treatment may force LLC-PK1 and MDCK cells to use anaerobic glycolysis preferentially. Further, these enzyme alterations may represent an epiphenomenon or a consequence of cyclosporine A toxicity.     

药物跨膜转运细胞模型影响因素及应用

本文详细介绍了Caco-2细胞系和MDCK细胞系的特点、跨膜转运细胞模型的建立及其影响因素,包括细胞模型的选择、细胞接种密度、细胞单层的紧密性等细胞因素和Transwell多微孔膜的性质等环境因素。概述了国内外关于利用Caco-2和MDCK细胞系作为模型进行药物筛选、药物相互作用和研究药物吸收转运机制等方面的内容及MDCK细胞模型作为肠道模型、肾脏模型及血脑屏障模型的应用。比较了Caco-2细胞和MDCK细胞在肠道模型方面的差别,MDCK细胞主要用于选择性研究药物在小肠吸收及转运机制,特别用于细胞旁被动转运药物的研究,而Caco-2细胞用于双向转运或能量依赖主动转运研究。MDCK细胞模型可在体外培养条件下平稳转染人类MDR1基因,因此可高表达P-gp基因,可作为可用于评估肾脏药物相互作用、快速进行候选药物筛选及研究药物转运机制的理想模型。     

An inter-laboratory study to evaluate the effects of medium composition on the differentiation and barrier function of Caco-2 cell lines

Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, trans-epithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.     

Developmental origin of segmental differences in the leech ectoderm: survival and differentiation of the distal tubule cell is determined by the host segment

The body plan of the adult leech is metameric, with each hemisegmental complement of ectodermal and mesodermal tissues being produced from a set of seven serially repeated embryonic blast cells. Previous studies have shown that homologous o blast cells give rise to an almost identical complement of descendant cells in each of the 21 abdominal segments, but that one o blast cell derivative--the distalmost cell of the nephridial tubule--is only present in 15 abdominal segments in the mature leech. Here we show that all o blast cells generate a presumptive distal tubule cell and that this cell migrates to its normal position in all abdominal segments. However, in segments which normally do not contain the mesodermal portion of the nephridium, the distal tubule cell dies before undergoing its terminal morphological differentiation. To ascertain whether the fate of the distal tubule cell is determined by its lineage history or by the segmental environment into which it is born, we utilized a previously described procedure for altering the segmental register between different embryonic cell lines. This procedure allowed us to effectively transplant o blast cells into more posterior segments prior to the cell divisions which generate their descendant clones. The results indicate that the survival or death of the distal tubule cell is determined by the identity of the host segment and that a given distal tubule cell could be effectively murdered or rescued by slipping its blast cell precursor into an appropriate segment. These findings suggest that the segment-specific pattern of distal tubule cell survival is not inherent to the O cell line, but arises from interactions with surrounding tissues.     

Tissue and cell distribution of the multidrug resistance-associated protein (MRP) in mouse intestine and kidney.

The multidrug resistance-associated protein (MRP) that is involved in drug resistance and the export of glutathione-conjugated substrates may not have the same epithelial cell membrane distribution as the P-glycoprotein encoded by the MDR gene. Because intestinal and kidney epithelial cells are polarized cells endowed distinct secreting and absorptive ion and protein transport capacities, we investigated the tissue and cell distribution of MRP in adult mouse small intestine, colon, and kidney by immunohistochemistry. Western blot analyses revealed the 190-kD MRP protein in these tissues. MRP was found in the basolateral membranes of intestinal crypt cells, mainly Paneth cells, but not in differentiated enterocytes. All the cells lining the crypt-villous axis of the colon wall contained MRP. MRP was found in the glomeruli, ascending limb cells, and basolateral membranes of the distal and collecting tubule cells of the kidney but not in proximal tubule cells. Cultured mouse intestinal m-ICcl2 cells and renal distal mpkDCT cells that have retained the features typical of intestinal crypt and renal distal epithelial cells, respectively, also possess MRP in their basolateral membranes. The patterns of subcellular and cellular distribution indicate that MRP may have a specific role in the basolateral transport of endogenous compounds in Paneth, renal distal, and collecting tubule cells.     

Relationships between intermediate filaments and cell-specific functions in renal cell lines derived from transgenic mice harboring the temperature-sensitive T antigen

Four renal cell lines were derived from glomeruli, proximal, distal, and cortical collecting tubules microdissected from the kidneys of transgenic mice carrying the temperature-sensitive mutant of the simian virus 40 large T antigen under the control of the vimentin promoter. All four cell lines contained large T antigen in their nuclei, grew rapidly, and contained vimentin filaments when grown in serum-enriched medium at the permissive temperature of 33°C. The glomerular cell line formed multiple layers of cells and contained smooth muscle actin and desmin filaments, features of mesangial cells. The three tubule cell lines formed monolayers of polarized cuboid cells separated by tight junctions and having a patchy distribution of cytokeratins K8-K18. A shift from 33°C to the restrictive temperature (39.5°C) stopped cell growth in all cell lines and caused profound changes in the content of intermediate filaments. Vimentin was still present in mesangial-like cells, but the proximal, distal, and collecting tubule cells contained uniform networks of cytokeratins K8-K18 and desmoplakin I and II around the cell peripheries. Potassium transport, mediated by NA⁺-K⁺ ATPase pumps and specific cAMP hormonal sensitivities, significantly increased in proximal, distal, and collecting tubule cells when shifted from 33°C to 39.5°C. Thus, the temperature-dependent inactivation of large T antigen, responsible for the arrest of cell growth, did not affect the phenotype of mesangial-like glomerular cells but induced some changes in the expression of intermediate filaments and restored, at least partially, the main parental cell-specific functions in proximal, distal, and collecting tubule cultured cells. © 1996     

Polarized monolayers formed by epithelial cells on a permeable and translucent support

An epithelial cell line (MDCK) was used to prepare monolayers which, in vitro, develop properties of transporting epithelia. Monolayers were formed by plating cells at high densities (10(6) cells/cm2) on collagen- coated nylon cloth disks to saturate the area available for attachment, thus avoiding the need for cell division. An electrical resistance developed within 4-6 h after plating and achieved a steady-state value of 104 +/- 1.8 omega-cm2 after 24 h. Mature monolayers were morphologically and functionally polarized. They contained junctional complexes composed of desmosomes and tight junctions with properties similar to those of "leaky" epithelia. Monolayers were capable of maintaining a spontaneous electrical potential sensitive to amiloride, produced a net water flux from the apical to basal side, and discriminated between Na+ and Cl- ions. The MDCK permeability barrier behaves as a "thin" membrane with negatively charged sites. It has: (a) a linear conductance/concentration relationship; (b) an asymmetric instantaneous current/voltage relationship; (c) a reduced ability to discriminate between Na+ and Cl- caused by lowering the pH; and (d) a characteristic pattern of ionic selectivity which suggests that the negatively charged sites are highly hydrates and of medium field strength. Measurements of Na+ permeability of electrical and tracer methods ruled out exchange diffusion as a mechanism for ion permeation and the lack of current saturation in the I/deltapsi curves does not support the involvement of carriers. The discrimination between Na+ and Cl- was severely but reversibly decreased at low pH, suggesting that Na+-specific channels which exclude Cl- contain acidic groups dissociated at neutral pH. Bound Ca++ ions are involved in maintaining the integrity of the junctions in MDCK monolayers as was shown by a reversible drop of resistance and opening of the junctions in Ca++-free medium containing EGTA. Several other epithelial cell lines are capable of developing a significant resistance under the conditions used to obtain MDCK monolayers.