Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK1 pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced 'permanent' stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK1 cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK1 cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.     

In vitro growth and differentiation of human kidney tubular cells on a basement membrane substrate

Summary Kidney cortical tubular cells, mainly proximal tubular cells, isolated from human kidney and grown either on a basement membrane substrate in chemically defined medium or on plastic in serum-supplemented medium, had substantial proliferative potential and could be propagated for more than 10 generations or 8 passages before senescence. Basement membrane produced on a plastic substrate by the HR-9 endodermal cell line could replace serum supplementation in promoting tubular cell growth. Tubular cells grown on an HR-9 basement membrane substrate exhibited stable epithelial morphology over an extended period of time; in the presence of 5% serum they differentiated into organized structures such as hemicysts and cell cords. Cells grown on plastic failed to differentiate and gradually degenerated. Tubular cells on HR-9 basement membrane were characterized by densely packed microvilli, abundant rough endoplasmic reticulum and free polysomes, basal cell membrane interdigitations, a well-developed endocytotic apparatus, and conspicuous junctional complexes—all features of the proximal tubular cell. Compared with cells on plastic substrate, there were higher levels of the brush border enzymes γ-glutamyl transpeptidase,l-leucine aminopeptidase, and alkaline phosphatase in cells maintained on an HR-9 basement membrane substrate, further supporting the conclusion that a basement membrane substrate promoted differentiation of tubular cells. These data and morphological observations indicate that a basement membrane substrate can promote growth and both functional and morphologic differentiation of human kidney tubular cells. This work was supported by the Veterans Administration.     

Nucleoside transport in cultured LLC-PK1 epithelia.

The transport of nucleosides by LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent Km approximately 34 microM at 22 degrees C) and inhibited by greater than 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK1 cells exhibited both a high affinity for cytidine (apparent Ki approximately 65 microM for influx) and differential 'mobility' of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 microM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the N1 sodium-dependent nucleoside carrier in bovine renal outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27-33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na(+)-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.     

Nephrotoxic cell death by diclofenac and meloxicam

The nephrotoxicity of diclofenac, a non-steroidal anti-inflammatory drug that inhibits both isoforms of cyclooxygenase (COX) has been reported to be fatal to vultures but this was not so with meloxicam which is COX-2 selective. Our study showed that diclofenac was more toxic than meloxicam to both the proximal tubular LLC-PK1 cells and the distal tubular Madin-Darby canine kidney type II (MDCKII) cells, and that LLC-PK1 cells were more susceptible. Exposure of MDCKII cells to meloxicam caused activation of caspase-9/-3 and release of cytochrome c. These observations together with a positive annexin V-FITC staining implicate an intrinsic mitochondrial cell death pathway by apoptosis. Diclofenac-treated MDCKII cells on the other hand showed extensive propidium iodide staining, suggestive of cell death by necrosis. The mode of cell death in LLC-PK1 cells was however less well-defined with positive annexin V-FITC staining but minimal increase in caspase-3 activity alluding to a caspase-independent pathway.     

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.     

Growth, enzyme activity, sugar transport, and hormone supplement responses in cells cloned from a pig kidney cell line LLC-PK1

Three clones of the pig kidney cell line LLC-PK1 were isolated and characterized with regard to morphology, growth, proximal tubule enzyme activity, sugar uptake capacity, and hormone and drug responsiveness in a defined medium. Clone N4 was similar in morphology to the wild type (WT), whereas clone F8 showed loose attachment to the substrate, formed large, sweeping domes, and had an elongated desmosome junction between cells. The third clone, F2, did not form domes and showed a marked reduction in growth rate. Cultures of WT, N4, and F8 had higher specific activities of the enzyme alkaline phosphatase and gamma-glutamyl transpeptidase at confluence relative to growing cells; however, there was no evidence of an increase in activity of either enzyme at confluence in F2. Phlorizin-sensitive alpha-methyl-D-glucoside uptake and cytochalasin B-sensitive 2-deoxy-D-glucose uptake were measured in confluent cultures grown on porous filter supports. None of the clones lacked either of the hexose transport systems, although quantitative differences were evident. N4 cells grown in a defined medium in 96-well culture plates were tested in situ for their enzyme responses to differentiation inducers, tumor promoters, and hormones. Alkaline phosphatase activity was significantly increased at confluence by serum, parathyroid hormone (PTH), and vasopressin (AVP), and was decreased by tetradecanoylphorbol acetate (TPA) and epinephrine (EPI). Glutamyl transpeptidase activity was decreased at confluence by serum, TPA, and EPI. Similar tests on alpha-methyl-D-glucoside uptake showed that serum, TPA, PTH, and AVP had no significant effect on phlorizin-sensitive uptake; however, calcitonin increased uptake by 84% (n = 18). It was concluded that LLC-PK1 clones maintained in a defined medium are useful models for studying renal cell function.     

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.     

Generation of anti-idiotypic monoclonal antibodies recognizing vasopressin receptors in cultured cells and kidney sections.

To produce anti-idiotypic antibodies against receptors for the neurohypophyseal hormone vasopressin, an anti-vasopressin monoclonal antibody with a ligand specificity similar to that of vasopressin receptors was employed for immunization. Three anti-idiotypic monoclonal antibodies were obtained which induced, like vasopressin, plasminogen activator production in the renal epithelial cell line LLC-PK1 (expressing V2-receptors). Induction of plasminogen activator synthesis by the anti-idiotypic antibodies could be inhibited by coincubation with a vasopressin antagonist. In a fashion similar to that of vasopressin itself, the anti-idiotypic antibodies induced receptor down-regulation. The anti-idiotypic antibodies were employed to visualize vasopressin receptors on LLC-PK1 and A7r5 (V1-receptor-expressing) smooth muscle cells by immunofluorescence. Antibody-mediated fluorescence was not observed in receptor-deficient mutant cell lines or vasopressin-receptor-down-regulated cells. Furthermore, these antibodies were used for immunohistochemical localization of vasopressin receptors in rat and bovine kidney preparations. In accordance with earlier physiological and biochemical observations, vasopressin receptors were detected predominantly in collecting ducts in cortex and medulla. On the cellular level, a differential staining pattern was observed.     

Mitochondrial cytochrome c oxidase as a target site for cephalosporin antibiotics in renal epithelial cells (LLC-PK(1)) and renal cortex

We reported previously that treatment of the pig kidney proximal tubular epithelial cell line LLC-PK(1) with cephaloridine (CLD) decreased the activity of cytochrome c oxidase in the mitochondria of the cells followed by increases in lipid peroxidation and cell necrosis. In this study, we investigated the effects of CLD on the activity of cytochrome c oxidase in mitochondria isolated from LLC-PK(1) cells and purified the enzyme from mitochondria of the rat renal cortex. The activity of cytochrome c oxidase in the isolated mitochondria from LLC-PK(1) cells was significantly decreased from 1 h after addition of 1 mM CLD. Other cephalosporin antibiotics, cefazolin and cefalotin, also decreased the activity of cytochrome c oxidase in the isolated mitochondria. The activity of cytochrome c oxidase purified from the mitochondria of the rat renal cortex was also decreased from 2 h after addition of 1 mM CLD in a non-competitive manner. These results suggest that the direct inhibition of cytochrome c oxidase activity in the mitochondrial electron transport chain by cephlosporins may result from the observed nephrotoxicity.     

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     

Protective Role of AMP-Activated Protein Kinase-Evoked Autophagy on an In Vitro Model of Ischemia/Reperfusion-Induced Renal Tubular Cell Injury

Ischemia/reperfusion (I/R) injury is a common cause of injury to target organs such as brain, heart, and kidneys. Renal injury from I/R, which may occur in renal transplantation, surgery, trauma, or sepsis, is known to be an important cause of acute kidney injury. The detailed molecular mechanism of renal I/R injury is still not fully clear. Here, we investigate the role of AMP-activated protein kinase (AMPK)-evoked autophagy in the renal proximal tubular cell death in an in vitro I/R injury model. To mimic in vivo renal I/R injury, LLC-PK1 cells, a renal tubular cell line derived from pig kidney, were treated with antimycin A and 2-deoxyglucose to mimic ischemia injury followed by reperfusion with growth medium. This I/R injury model markedly induced apoptosis and autophagy in LLC-PK1 cells in a time-dependent manner. Autophagy inhibitor 3-methyladenine (3MA) significantly enhanced I/R injury-induced apoptosis. I/R could also up-regulate the phosphorylation of AMPK and down-regulate the phosphorylation of mammalian target of rapamycin (mTOR). Cells transfected with small hairpin RNA (shRNA) for AMPK significantly increased the phosphorylation of mTOR as well as decreased the induction of autophagy followed by enhancing cell apoptosis during I/R. Moreover, the mTOR inhibitor RAD001 significantly enhanced autophagy and attenuated cell apoptosis during I/R. Taken together, these findings suggest that autophagy induction protects renal tubular cell injury via an AMPK-regulated mTOR pathway in an in vitro I/R injury model. AMPK-evoked autophagy may be as a potential target for therapeutic intervention in I/R renal injury.     

Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.     

Conditioned medium from renal tubular epithelial cells initiates differentiation of human mesenchymal stem cells

Objectives: Mesenchymal–epithelial interactions play a pivotal role in tubular morphogenesis and in maintaining the integrity of the kidney. During renal repair, similar mechanisms may regulate cellular reorganization and differentiation. We have hypothesized that soluble factors from proximal tubular epithelial cells (PTC) induce differentiation of adipose-derived adult mesenchymal stem cells (ASC). This hypothesis has been tested using cultured ASC and PTC.
Material and methods: Conditioned medium was prepared from injured PTC and transferred to ASC cultures. ASC proliferation was analysed by a fluorometric and photometric assay. Signal transduction was analysed by phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2). Grade of ASC differentiation was assessed by morphological analysis and cell expression of characteristic markers.
Results: Conditioned medium significantly induced proliferation and phosphorylation of ERK1/ERK2 of ASC. After 12 days of incubation, cell morphology changed to an epithelial-like monolayer. Expression of cytokeratin 18 was induced by conditioned medium, while α-smooth muscle actin, CD49a and CD90 expression decreased. These alterations strongly indicate onset of the differentiation process to the epithelial lineage. In summary, soluble factors from PTC induce signal transduction and differentiation of ASC.
Conclusions: Our study shows that conditioned medium from renal tubular epithelial cells provides a convenient source of inductive signals to initiate differentiation of ASC towards epithelial lineage. We deduce that these interactions may play an important role during renal repair mechanisms.     

Regulation by adenosine of the vasopressin-sensitive adenylate cyclase in pig-kidney cells (LLC-PK1L) grown in defined media

LLC-PK1L cells, a kidney-derived cell line, had sustained growth in a defined medium. When compared to the parent cell line growing with 10% fetal bovine serum, LLC-PK1L cells had about 100-times fewer vasopressin receptors. Upon modifications of the cell culture medium, the vasopressin response of the adenylate cyclase could be increased by more than 10-fold with a parallel increase in vasopressin receptor number. Using cells with high or low receptor densities, the stimulatory and inhibitory effects of N6-L-2-phenylisopropyl-adenosine on the modulation of the adenylate cyclase responsiveness to vasopressin were investigated. When high concentrations of GTP were added, low concentrations of phenylisopropyladenosine inhibited the enzyme, while higher concentrations were found to be stimulatory. The adenylate cyclase activity stimulated by vasopressin could only be inhibited by phenylisopropyladenosine under these conditions in membranes with high receptor density; only the increase in enzyme activity due to high GTP concentration was inhibitable. The analysis of the dependency of the adenylate cyclase activity as a function of the vasopressin concentration showed that, besides reducing the maximum velocity of the system for vasopressin, the addition of phenylisopropyladenosine generated an heterogeneity in the adenylate cyclase response to vasopressin (as judged by a curvilinear Eadie plot). A high-affinity component in the adenylate cyclase response appeared when phenylisopropyladenosine was added. The growth of the cells in a medium containing adenosine deaminase gave results identical to those obtained for control cells. However, growing the cells with both phenylisopropyladenosine and adenosine deaminase abolished the inhibitory effects of the former on the adenylate cyclase and greatly reduced its stimulatory action. Under these conditions, the vasopressin response of the adenylate cyclase was not further regulated by phenylisopropyladenosine. These results indicate a role of adenosine on vasopressin response, especially at low physiological concentrations of the hormone where a high-affinity component of the hormonal response could be demonstrated.     

Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of γ-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, γ-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by a grant from The Polycystic Kidney Foundation. We gratefully acknowledge the support of the Children’s Medical Research Institute and Children’s Miracle Network Foundation.     

Effects of confluence on phosphate transport capacity in cultured renal cell lines

The LLC-PK1 cell line transports phosphate (Pi), glucose, and amino acids using carriers similar to those in proximal tubular cells. Others have reported that when monolayers reach confluence, hexose transport increases and activity of the A-amino acid transporter falls. The present study evaluates Pi uptake by two continuous cell lines derived from renal proximal tubule, and demonstrates that phosphate uptake falls sharply upon reaching confluence in LLC-PK1 cells but not in cultured opossum kidney (OK) cells. The fall in Pi uptake in LLC-PK1 cells at confluence represents a halving in Vmax for Na-dependent phosphate uptake (2.33 vs. 5.00 nmol/mg protein/5 min) without a change in Km (82 vs. 94 microM). Suppression of phosphate transport in confluent monolayers of LLC-PK1 cells is completely reversed by bringing the cells into suspension. As has been shown for the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), exposure of monolayers to serum stimulates phosphate uptake, but unlike phorbol ester, serum does so without stimulating alanine uptake. OK cells differ from LLC-PK1 in that no change occurs in Pi uptake at confluence, although they resemble LLC-PK1 cells in that sugar uptake rises and alanine uptake falls at confluence. The different temporal patterns for Pi uptake in the two cell lines indicates that developmental change in the uptake of Pi is not linked to that of glucose or alanine.     

Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK)

Madin-Darby canine kidney (MDCK) cells grown in tissue culture have the morphological properties of distal tubular epithelial cells, form tight junctions, and lack several proximal tubular enzyme markers. Adenylate cyclase in these cells was stimulated by vasopressin, oxytocin, prostaglandins E1 and E2, glucagon, and cholera toxin. Hormone-stimulated adenylate cyclase activity in isolated membrane preparations was dependent on low concentrations of GTP and had the MgCl2 and pH optima expected for the kidney enzyme. The results, as well as the demonstration of enhanced hemicyst formation induced by cyclic AMP, suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cell of origin. When MDCK cells were injected into baby nude mice, continuous nodule growth was observed until adulthood was attained. Histological studies revealed the presence of two cell types: normal mouse fibroblasts which comprise 80--90% of the solid nodule mass, and MDCK cells, which formed epithelial sheets lining internal fluid-filled glands. Electron microscope analysis showed that the mucosal surfaces of the cells were characterized by microvilli which faced the lumen of the glands, that adjacent MDCK cells were joined by tight junctions, and that the serosal surfaces of the epithelial sheets were characterized by smooth plasma membranes which were lined by a continuous basement membrane. These observations lead to the conclusion that the MDCK cells retain regional differentiation of their plasma membranes and the ability to regenerate kidney tubule-like structures in vivo.     

Specific binding activities and cyclic GMP responses by atrial natriuretic polypeptide in kidney epithelial cell line (LLC-PK1)

Receptor binding activities and cyclic GMP responses by alpha-human atrial natriuretic polypeptide (alpha-hANP) and its fragments were studied in a kidney epithelial cell line (LLC-PK1). Binding of 125I-alpha-hANP to the cells at 0 degrees C was saturable, time-dependent and reversible, indicating the presence of a single class of binding sites. alpha-hANP (7-23)NH2 fragment inhibited most effectively the specific binding of 125I-alpha-hANP to the LLC-PK1 cells, followed by alpha-hANP (17-28) and alpha-hANP (8-22), while alpha-hANP (1-6) and alpha-hANP (24-28) did not. alpha-hANP stimulated the formation of cyclic GMP in the LLC-PK1 cells dose-dependently. Although no fragments of alpha-hANP used were effective for cyclic GMP formation in the LLC-PK1 cells, alpha-hANP (7-23) NH2 antagonized the action of alpha-hANP on cyclic GMP formation. These data suggest that the LLC-PK1 cells retain specific receptors for atrial natriuretic polypeptide (ANP) and respond to ANP by stimulating cyclic GMP formation, and therefore this cell line may be useful for studying the mechanism of action for ANP in renal tubular cells.