Comparative impact of cephaloridine on glutathione and related enzymes in LLC-PK1, LLC-RK1, and primary cultures of rat and rabbit proximal tubule cells

Among kidney tubular epithelial cell types, proximal tubule cells are one of the major renal targets for xenobiotics. Several in vitro culture models have been proposed for use of proximal tubule cells for in vitro pharmacotoxicology studies. This paper reports a comparative study of the response to cephaloridine exposure of two established cell lines from pig (LLC-PK1) and rabbit (LLC-RK1) kidneys and primary cultures of rat and rabbit proximal tubule cells. These cultured cells were first compared for their levels of activity of -methylglucopyranoside transport, alkaline phosphatase, succinate dehydrogenase, and NADPH cytochrome c reductase, their glutathione-dependent activity levels, and their adenylate cyclase response pattern to stimulation by PTH and AVP. The results presented show major phenotypic differences between these four cellular models. The differences observed in glutathione-dependent mechanism activities and regulation may in part be responsible for the variability of the responses of these four cellular models when exposed to cephaloridine.Abbreviations AVP arginine vasopressin - GGT -glutamyl transpeptidase - GRED glutathione reductase - GSH glutathione - GST glutathione S-transferase - PTC proximal tubule cells - PTH parathyroid hormone - SDH succinate dehydrogenase     

Heterogeneity of the Na(+)-H+ antiport systems in renal cells.

This study analyzes the differential characteristics of the Na(+)-H+ antiport systems observed in several epithelial and non-epithelial renal cell lines. Confluent monolayers of LLC-PK1A cells have a Na(+)-H+ antiport system located in the apical membrane of the cell. This system, however, is not expressed during cell proliferation or after incubation in the presence of different mitogenic agents. In contrast, confluent monolayers of MDCK4 express minimal Na(+)-H+ antiport activity in the confluent monolayer state but reach maximal antiport activity during cell proliferation or after activation of the cells by different mitogenic agents. Similar results were obtained with the renal fibroblastic cell line BHK. The system present in MDCK4 cells is localized in the basolateral membrane of the epithelial cell. In LLC-PK1A cells, an increase in the extracellular Na+ concentration produces a hyperbolic increase in the activity of the Na(+)-H+ antiporter. In MDCK4 and BHK cells, however, an increase in external Na+ produces a sigmoid activation of the system. Maximal activation of the system occur at a pHo 7.5 in LLC-PK1A cells and pHo 7.0 in MDCK4 cells. The Na(+)-H+ antiporter of LLC-PK1A cells is more sensitive to the inhibitory effect of amiloride (Ki 1.8 x 10(-7) M) than is the antiporter of MDCK4 cells (Ki 7.0 x 10(-6) M). Moreover, 5-(N-methyl-N-isobutyl)amiloride is the most effective inhibitor of Na(+)-H+ exchange in LLC-PK1A cells, but the least effective inhibitor in MDCK4 cells. Conversely, the analog, 5-(N,N-dimethyl)amiloride, is the most effective inhibitor of Na(+)-H+ exchange in MDCK4 cells, but is the least effective inhibitor in LLC-PK1A cells. These results support the hypothesis that Na(+)-H+ exchange observed in LLC-PK1A and other cell lines may represent the activity of different Na(+)-H+ antiporters.     

Polarized nature of taurine transport in LLC-PK1 and MDCK cells: Further characterization of divergent transport models

Summary Taurine transport was measured in cultured epithelial cells-LLC-PK1 and MDCK-grown on permeable membrane supports. Taurine transport by LLC-PK1 cells was greater on the apical surface compared to the basolateral surface. MDCK cells exhibited greater taurine uptake from the basolateral side. Transepithelial taurine flux was in the direction of apical to basolateral in the LLC-PK1 monolayers. There was no net transepithelial movement of taurine in the MDCK monolayers. Efflux of taurine from the apical and the basolateral membrane surfaces of LLC-PK1 cell monolayers was stimulated by external-alanine but not L-alanine. Efflux of taurine from MDCK cell monolayers was stimulated by-alanine on the basolateral surface. While the competitive inhibitor guainidinoeithane sulfonate (GES) competitively inhibited taurine uptake to a similar degree on the apical and basolateral surface of LLC-PK1 cell monolayers, GES had a more potent inhibitory effect on the basolateral taurine uptake in MDCK cells when compared to its inhibition of apical taurine transport. We conclude that there are characteristic differences in transport of taurine by apical and basolateral surfaces of LLC-PK1 and MDCK cells which may be the consequence of asymmetric distribution or unique structural properties of the taurine transporter.Supported by a grant from the National Institutes of Health (DK 37223), the American Heart Association (92-004470).     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

Translocation of MARCKS and reorganization of the cytoskeleton by PMA correlates with the ion selectivity,the confluence,and transformation state of kidney epithelial cell lines

The role of protein kinase C (PKC) in the regulation of the cytoskeleton of epithelial cells with tightly sealed contacts, poor contacts, and without contacts were investigated by incubating them with a protein kinase C activator phorbol myristoyl acetate (PMA). The morphology and organization of the membrane skeleton and stress fibers as well as the localization of an actin-bundling PKC substrate MARCKS in confluent MDCK cells originating from the distal tubulus of dog kidney, LLC-PK1 cells originating from the proximal tubulus of pig kidney, src-transformed MDCK cells, epidermoid carcinoma A431 cells, and MDCK cells grown in low calcium medium (LC medium) in low density were visualized with phase contrast and immunofluorescence microscopy. Four different responses to the PMA-treatment in actin-based structures of cultured epithelial cells were observed: 1) disintegration of the membrane skeleton in confluent MDCK cells; 2) depolymerization of the stress fibers in confluent MDCK and LLC-PK1 cells; 3) formation of the membrane skeleton in A431 cells, and 4) formation of the stress fibers and membrane skeleton in LC-MDCK cells. Thus, it seems that in fully confluent tightly sealed epithelium, activation of PKC has a deleterious effect on actin-based structures, whereas in cells without contacts or loose contacts, activation of PKC by PMA results in improvement of actin-based cytoskeletal structures. The main difference between the two kidney cell lines used is their selectivity to ion transport: the monolayer of LLC-PK1 cells is anion selective and MDCK cells cation selective. We propose a model where alterations in the ionic milieu within the MDCK cells by means of cation channels affect the disintegration of the membrane skeleton. The distribution of MARCKS followed the distribution of fodrin in both cell lines upon PMA-treatment, suggesting that phosphorylation of MARCKS by PKC may contribute in the regulation of the integrity of the membrane skeleton. J. Cell. Physiol. 181:83–95, 1999. © 1999 Wiley-Liss, Inc.     

Enzyme histochemical studies on rat lungs 2. Normal enzyme distribution in the alveolar tissue of the mature lung

Synopsis The activity and distribution of the following eighteen oxidative and hydrolytic enzyme systems have been investigated in the lung of the adult rat: reduced NAD dehydrogenase, reduced NADP dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase, glutamate dehydrogenase, -hydroxybutyrate dehydrogenase, acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, adenosine triphosphatase, 5-nucleotidase, non-specific esterase, cytochrome oxidase and -glucuronidase.The low concentration of cells in sections of inflated lung may have made histochemical demonstration of some enzymes impossible because the enzyme concentration was below that detectable by the method employed.The carboxylic acid cycle and the hexose monophosphate shunt were potentially active but fatty acid metabolism was not indicated.The granular reaction sometimes encountered in alveolar cell cytoplasm may be useful for differentiating alveolar cell types, but further cytochemical studies are required to resolve the possible metabolic differences of alveolar cells.     

The effect of cryptorchidism on the quantitative histology, histochemistry and hydrolytic enzyme activity of the rat testis.

Cryptorchidism of the mature rat testis led to degeneration of the seminiferous tubules and changes in enzyme patterns and activities. Spermatogenic stages 1-4, containing pachytene primary spermatocytes in late meiotic prophase, and stage 5, containing recently formed round spermatids, were damaged by 48 h. Within 96 h stages showed a loss of germinal cells into the lumen and this was almost complete by 192 h. Acid phosphatase showed increased histochemical activity in the basal area of the seminiferous tubule up to 96 h of cryptorchidism, and at 192 h much of the activity was located in large lipidcontaining bodies within the remaining seminiferous epithelium. Total and free biochemical acid phosphatase decreased during cryptorchidism in parallel with cell loss; there were no significant changes in total cathepsin D activity but free enzyme activity was increased throughout the experimental period indicating increased lability of lysosomes in the Sertoli cell. Lactate dehydrogenase activity was mainly tubular but succinate dehydrogenase also showed interstitial activity. Lipoamide dehydrogenase (NADH) was found mainly in the interstitium. During cryptorchidism both lactate and succinate dehydrogenase activity decreased in the tubules parallel to the loss of germinal cells, whereas lipoamide dehydrogenase (NADH) activity increased in both interstitial and tubular areas. It is suggested that the initial lesion in the seminiferous epithelium, produced by cryptorchidism is in the Sertoli cell and that germ cell damage may result from reduced function of the Sertoli cell.     

A dileucine motif targets the sulfate anion transporter sat-1 to the basolateral membrane in renal cell lines

The sat-1 transporter mediates sulfate/bicarbonate/oxalate anion exchange in vivo at the basolateral membrane of the kidney proximal tubule. In the present study, we show two renal cell lines [Madin-Darby canine kidney (MDCK) and porcine proximal tubular kidney (LLC-PK1) cells] that similarly target sat-1 exclusively to the basolateral membrane. To identify possible sorting determinants, we generated truncations of the sat-1 cytoplasmic COOH terminus, fused to enhanced green fluorescence protein (EGFP) or the human IL-2 receptor -chain (Tac) protein, and both fusion constructs were transiently transfected into MDCK cells. Confocal microscopy revealed that removal of the last three residues on the sat-1 COOH terminus, a putative PDZ domain, had no effect on basolateral sorting in MDCK cells or on sulfate transport in Xenopus oocytes. Removal of the last 30 residues led to an intracellular expression for the GFP fusion protein and an apical expression for the Tac fusion protein, suggesting that a possible sorting motif lies between the last 3 and 30 residues of the sat-1 COOH terminus. Elimination of a dileucine motif at position 677/678 resulted in the loss of basolateral sorting, suggesting that this motif is required for sat-1 targeting to the basolateral membrane. This posttranslational mechanism may be important for the regulation of sulfate reabsorption and oxalate secretion by sat-1 in the kidney proximal tubule. enhanced green fluorescence protein; Tac; polarized cells; sorting; transport     

Cytotoxicity,apoptosis, DNA damage and methylation in mammary and kidney epithelial cell lines exposed to ochratoxin A

This study aimed to investigate the in vitro damage induced by ochratoxin A (OTA) in BME-UV1 and MDCK epithelial cells. Both cells lines were treated with OTA (0 up to 10 μg/mL), and cell viability (MTT assay), membrane stability (lactate dehydrogenase (LDH) release assay) and apoptotic cell rate (Tunel assay) were investigated. Further, the effect of the incubation with OTA has been evaluated at DNA level by the determination of DNA integrity, by the quantification of DNA adduct formation (8-hydroxy-2′-deoxyguanosine (8-OHdG)) and by the assessment of the global DNA methylation status (5-methyl-cytosine (5-mC)). The obtained results showed that after 24 h of OTA treatment, BME-UV1 cell viability was reduced in a dose-dependent way. OTA significantly (P?<?0.05) increased LDH release in BME-UV1 cells at all concentrations tested. OTA (1.25 μg/mL) induced 35 % LDH release in MDCK cells (P?<?0.05). A significant (P?<?0.05) change in percentages of apoptotic BME-UV1 (10?±?0.86) and MDCK (25?±?0.88) cells was calculated when the cells were co-incubated with OTA. The level of 8-OHdG adduct formation was significantly (P?<?0.05) increased in BME-UV1 cells treated with 1.25 μg/mL of OTA. The results of the present study suggest that a different mechanism of action may occur in these cell lines.

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Graphical abstract Study results overview

     

Assessment of unscheduled DNA synthesis in a cultured line of renal epithelial cells exposed to cysteine S-conjugates of haloalkenes and haloalkanes

The ability of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFEC) and S-(2-chloroethyl)-L-cysteine (CEC) to induce DNA repair was investigated in LLC-PK1, a cultured line of porcine kidney tubular epithelial cells. DNA repair due to exposure of the cells to the S-conjugates was determined as unscheduled DNA synthesis (UDS) after inhibition of replicative DNA synthesis in confluent LLC-PK1 monolayers. DCVC, TCVC and PCBC induced dose-dependent UDS in LLC-PK1 at concentrations which did not impair the viability of the cells compared to untreated controls; higher concentrations were cytotoxic, resulting in lactate dehydrogenase leakage into the medium. Cell death was also induced by CTFEC, which failed to exert genotoxicity. CEC induced the highest response among these cysteine conjugates without impairing cell viability. Inhibition of cysteine conjugate beta-lyase with aminooxyacetic acid abolished the effects of DCVC, TCVC, PCBC and CTFEC but did not influence the genotoxicity of CEC.     

Absence of gap junctional complexes in two established renal epithelial cell lines (LLC-PK1 and MDCK)

The type of junctions present in the membranes of the two renal epithelial cell lines, LLC-PK1 and MDCK, and of subcultured porcine aortic endothelial (PAE) cells have been studied by freeze-fracture. No gap junctions were observed in the two renal cell lines, while they were numerous in the endothelial cells. Tight junctions were abundant in LLC-PK1 and MDCK cells and varied in numbers of ridges from one to ten. ONly a few simple tight junctions unconnected with gap junctions were observed in PAE cells. The occurrence of gap junctions in these cells correlates with their ability to form intercellular communicating channels.     

Calyculin A-induced actin phosphorylation and depolymerization in renal epithelial cells

This study reports actin phosphorylation and coincident actin cytoskeleton alterations in renal epithelial cell line, LLC-PK1. Serine phosphorylation of actin was first observed in vitro after the cell lysate was incubated with phosphatase inhibitors and ATP. Both the phosphorylated actin and actin kinase activities were found in the cytoskeletal fraction. Actin phosphorylation was later detected in living LLC-PK1 cells after incubation with the phosphatase inhibitor calyculin A. Calyculin A-induced actin phosphorylation was associated with reorganization of the actin cytoskeleton, including net actin depolymerization, loss of cell-cell junction and stress fiber F-actin filaments, and redistribution of F-actin filaments in the periphery of the rounded cells. Actin phosphorylation was abolished by 3-h ATP depletion but not by the non-specific kinase inhibitor staurosporine. These results demonstrate that renal epithelial cells contain kinase/phosphatase activities and actin can be phosphorylated in LLC-PK1 cells. Actin phosphorylation may play an important role in regulating the organization of the actin cytoskeleton in renal epithelium.     

Experimentally induced colon cancer metastases in rat liver increase the proliferation rate and capacity for purine catabolism in liver cells

Metastases in rat liver were generated experimentally by intraportal injection of colon cancer cells to investigate the effects of cancerous growth on the metabolism of surrounding liver tissue. Maximum activities (capacity) of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, lactate dehydrogenase, succinate dehydrogenase, alkaline phosphatase, 5-nucleotidase, xanthine oxidoreductase, purine nucleoside phosphorylase and adenosine triphosphatase have been determined. Two types of metastases were found, a small type surrounded by stroma and a larger type in direct contact with hepatocytes. Both types affected the adjacent tissue in a similar way suggesting that the interactions were not mediated by stroma. High capacity of the degradation pathway of extracellular purines released from dead cells of either tumours or host tissue was found in stroma and sinusoidal cells. Metastases induced both an increase in the number of Kupffer cells and proliferation of hepatocytes. The distribution pattern in the liver lobulus of most enzymes investigated did not change distinctly. However, activity of alkaline phosphatase, succinate dehydrogenase and phosphogluconate dehydrogenase was increased in hepatocytes directly surrounding metastases. These data imply that the overall metabolic zonation in liver lobuli is not dramatically disturbed by the presence of cancer cells despite the fact that various metabolic processes in liver cells are affected.In honour of Prof. Dr. Z. Lojda for his 65th birthday     

Organization of the endoplasmic reticulum in renal cell lines MDCK and LLC-PK1

The spatial organization of the endoplasmic reticulum has been studied in two renal cell lines, MDCK and LLC-PK1, which originate from the distal and proximal portions of the mammalian nephron, respectively, and which form a polarized epithelium when they reach confluence in tissue culture. The two renal cell lines, grown to confluence on either solid or permeable supports, were investigated by fluorescence microscopy, confocal microscopy, and transmission electron microscopy. Fluorescence labeling of the endoplasmic reticulum was achieved using the cationic fluorescent dye DIOC₆ (3). In order to differentiate fluorescent labeling of the endoplasmic reticulum from that of the mitochondria, cells were also labeled with rhodamine 123. For electron microscopy, the spatial organization of the endoplasmic reticulum was examined in thick sections using the long-duration osmium impregnation technique or the ferrocyanide/osmium technique. In both cell lines, the endoplasmic reticulum formed an abundant tubular network of canaliculi that frequently abutted the basolateral domain of the plasma membrane and occasionally the apical membrane. Elements of the endoplasmic reticulum were also found in close proximity to mitochondria that, as in the nephron, formed branched structures. Canaliculi appeared circular or flattened and had an inner diameter of 10–70 nm for MDCK cells and 20–90 nm for LLC-PK1 cells. Such a three-dimensional organization might facilitate the translocation of defined lipid species between the endoplasmic reticulum and the plasma membrane, and between the endoplasmic reticulum and mitochondria.     

Effects of Bone Morphogenetic Protein-2 and Transforming Growth Factor-β1 on Gene Expression of Decorin and Biglycan by Cultured Osteoblastic Cells

The influence of bone morphogenetic protein-2 (BMP-2) and transforming growth factor (TGF-) on the expression of small proteoglycans, decorin and biglycan was investigated in a clonal rat osteoblastic cell line, ROS-C26 (C26) cells, which is a potential osteoblast precursor cell line and capable of differentiating into mature osteoblasts after treatment with recombinant BMP-2 (rhBMP-2). Following the culture of C26 cells for 3, 6, and 9 days in the presence or absence of rhBMP-2, alkaline phosphatase activity increased in the rhBMP-2 treated cells in direct proportion to their differentiation into more mature osteoblastic cells, whereas decorin mRNA decreased in the cells, when compared to control cells without rhBMP-2 treatment. These results were evident 6 days after treatment. However, rhBMP-2 treatment had no effect on biglycan mRNA expression in the cells. Subsequently, after removal of rhBMP-2 from the culture media, the cells were further cultured for 24h with graded concentrations of TGF-1 (0, 0.1, 1.0, 5.0, and 10ng/ml). TGF-1 decreased decorin mRNA expression in the cells dose dependently, but did not affect their biglycan mRNA expression. Furthermore, either removal of rhBMP-2 from the culture media or addition of TGF-1 significantly decreased alkaline phosphatase activity of rhBMP-2-induced cells. These results indicate that osteoblastic differentiation is accompanied by increased alkaline phosphatase activity and decreased expression of decorin mRNA, but continuous expression of biglycan mRNA. Both rhBMP-2 and TGF-1 inhibit decorin mRNA expression in osteoblasts at varying stages of differentiation, but their effects on biglycan mRNA expression and alkaline phosphatase are different.     

Comparative cytotoxicity of 5-aminosalicylic acid (mesalazine) and related compounds in different cell lines

Nonsteroidal anti-inflammatory drugs can cause serious side-effects such as tubulo-interstitial nephritis. Mesalazine (5-ASA, 5-aminosalicylic acid) is used for the treatment of colitis ulcerosa, Crohn disease, and other diseases; it has been found to induce necrosis of both proximal convoluted tubules and renal papillaries. The comparative cytotoxicity of 3-, 4-, and 5- aminosalicylic acid, acetylsalicylic acid (AcSA), and the parent compound salicylic acid (SA) was investigated for the free acids and for their sodium salts. The interaction with endogenous glutathione (GSH) was also investigated. Four established cell lines were used: MDCK, LLC-PK1, NRK as renal cells, and HepG2 as hepatic cells. The free acid compounds were less toxic than their corresponding salts. Acidic 5-ASA was the most toxic of the three isomers in MDCK and LLC-PK1 cells, while NRK and HepG2 were more susceptible to acidic 3-ASA. Addition of NaOH modified the relative toxicity of 3-ASA and 5-ASA. The LLC-PK1 and HepG2 cells were more sensitive to the test chemicals as their salts than were the NRK and MDCK cells. SA and 5-ASA decreased the GSH content in renal cells and increased it in HepG2. GSH depletion with l-buthionine-(S,R)-sulfoximine enhanced the toxicity only for SA in NRK and for 5-ASA and AcSA in HepG2. No correlation between endogenous GSH and the susceptibility of MDCK and LLC-PK1 to the test compounds was observed. The results suggest that no typical nephrotoxic effect occurred. No explanation could be found for the tubulo-interstitial nephritis caused by 5-ASA therapy.     

Enzymatic coordination indices of leukocytes in the prognosis of mouse resistance to staphylococcal toxin

The activity of succinate, lactate and alpha-glycerophosphate dehydrogenases, as well as acid phosphatase, in the lymphocytes and neutrophils of the blood was studied in noninbred white mice prior to the intraperitoneal injection of 1 LD50 of staphylococcal toxin. As the result of intoxication, a half of the animals died and the other half survived. The two groups of the animals differed in the levels of the activity of succinate dehydrogenase and hyaloplasmatic alpha-glycerophosphate dehydrogenase in the neutrophils and lymphocytes, and also differed in the stability of correlations between the activity of succinate and lactate dehydrogenases in the lymphocytes and neutrophils and, besides, between the activity of lactate dehydrogenase in these types of cells.     

Identification of basolateral membrane targeting signal of human sodium-dependent dicarboxylate transporter 3

Sodium-dependent dicarboxylate transporters (NaDC) include low-affinity NaDC1 and high-affinity NaDC3. Despite high similarities structurally and functionally, both are localized to opposite surfaces of renal tubular cells. The molecular mechanisms and localization signals leading to this polarized distribution remain unknown. In this study, distribution of NaDC3 in human kidney tissue was firstly observed by immunohistochemistry and immunofluorescence. Then, EGFP-fused wild-type, NH2- and COOH-terminal deletion and point mutants of NaDC3, and chimera between NaDC3 and NaDC1, were generated and transfected into polarized renal cells lines, LLC-PK1 and MDCK. Their subcellular localizations were analyzed by laser confocal microscopy. Immunolocalization results revealed that NaDC3 was expressed at basolateral membrane of human renal proximal tubular epithelia. Confocal examinations showed that wild-type NaDC3 was targeted to the basolateral membrane of MDCK and LLC-PK1. Deletion mutations indicated that the basolateral targeting signal of NaDC3 located within a short sequence AKKVWSARR of its amino-terminal cytoplasmic domain. Addition of this sequence could redirect apical NaDC1 to the basolateral membrane of LLC-PK1. Point mutagenesis revealed that mutation of either of two hydrophobic amino acids V and W in this short sequence largely redirected NaDC3 to both apical and basolateral surfaces of LLC-PK, indicating that the two hydrophobic amino acids are critical for the basolateral targeting of NaDC3. Our studies provide direct evidence of the localization of NaDC3 at the basolateral membrane of human renal proximal tubule cells and identify a di-hydrophobic amino acid motif VW as basolateral localization signal in the N-terminal cytoplasmic domain of NaDC3.     

Renal epithelial cell lines (BSC-1, MDCK, LLC-PK1) express 11 beta-hydroxysteroid dehydrogenase activity

Renal tissue of several species has been shown to express considerable 11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) activity. However, it is uncertain as to which renal cell types exhibit 11-HSD activity. In the present study, we investigated corticosterone metabolism in BSC-1 cells, a continuous renal epithelial cell line derived from the African green monkey (Cercopithecus aethiops). In incubation experiments using 3H-labelled corticosterone and HPLC, we have demonstrated oxidative 11-HSD activity in intact monolayers of BSC-1 cells as well as in BSC-1 cell homogenates. 11-HSD activity in cell homogenates could be stimulated 7-9-fold by the addition of exogenous NADP+ (1 mM). In contrast, no reductive 11-HSD could be detected either in intact cells or in cell homogenates under various experimental conditions which were designed to favor reductive 11-HSD activity. Pilot experiments were performed in cell homogenates from two other renal epithelial cell lines derived from canine (MDCK) and porcine (LLC-PK1) kidney. They also revealed oxidative but no reductive 11-HSD activity. The data provide evidence for an epithelial localization of renal oxidative 11-HSD activity.     

The cell culture medium — a functional extracellular compartment of suspension-cultured cells

The spent medium of suspension-cultured cells of Lupinus polyphyllus was analyzed by capillary GC and GC-MS and shown to contain ethanol (up to 160 mmol l^-1), organic acids (lactate, benzoate, succinate, fumarate, malate), amino acids (main components: alanine, glycine, serine, aspartate, ornithine, glutamate), and quinolizidine alkaloids (lupanine and an uncharacterized malonylderivative). In addition, cells obviously secrete polysaccharides and enzymes (acid phosphatase, phosphodiesterase, DNAse, esterase, -mannosidase, -galactosidase, -glucosidase, lipase, protease and peroxidase) into the medium. Typically these enzymes are localized in the vacuole of intact cells. Cytosolic enzymes, such as glutamate dehydrogenase and malate dehydrogenase were retained by the cells. Peroxidase is overexpressed in suspension-cultured lupin cells but only one basic isoenzyme is secreted, whereas the others are retained in the vacuole. In lupin leaves this isoenzyme is sequestered in the vacuole, implying that secretion is selective and needs a change in the sorting signals of the peroxidase protein. The cell culture medium shares many features of the vacuole. We assume therefore that the medium functions as a lytic compartment. In addition it provides a sink-source system for nutrients and metabolites.Abbreviations ADH alcohol dehydrogenase - FW fresh weight - GC gas chromatography - GC-MS gas chromatography — mass spectrometry - POD peroxidase - QA quinolizidine alkaloids