Modulation of glycolysis induction in primary cultures of rabbit kidney proximal tubule cells: the role of shaking,glucose and insulin.

We have assessed the impact of increasing oxygen availability on cellular phenotype expression of rabbit proximal tubule cells in primary culture developed with variable glucose and/or insulin contents. To mitigate hypoxia at the cell/medium interface, cells were shaken for the whole culture duration and their expressed phenotype was compared with those expressed by static cultures. O₂ and CO₂ tensions were kept constant in the incubator atmosphere. Glycolysis and gluconeogenesis pathways, detoxication system, and mitochondrial, apical and basolateral membrane marker enzyme activities were assessed. This study showed that the induction of glycolysis which appear in primary cultures of proximal tubule cells may be partially prevented by continuously shaking the cultures. This effect was more marked in the presence of glucose, suggesting better substrate oxidation in shaken cultures.     

Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.     

Response of primary rabbit kidney proximal tubule cells to estrogens

The effects of estrogens on the growth and function of primary rabbit kidney proximal tubule (RPT) cells have been examined in hormonally defined phenol red–free medium. 17β-estradiol was observed to stimulate growth at dosages as low as 10⁻¹⁰ M. The growth stimulatory effects of 17β-estradiol were mitigated in the presence of hydrocortisone, suggesting that these two steroid hormones acted at least in part by common mechanisms. The effects of other steroids known to interact with the estrogen receptor were examined. Alpha estradiol was found to be growth stimulatory over a concentration range of 10⁻⁹ to 10⁻⁸ M, albeit to a lower extent than beta estradiol. In addition, the anti-estrogen tamoxifen was also growth stimulatory (unlike the case with the human mammary tumor cell line MCF-7). The effects of several metabolic precursors of 17β-estradiol were examined, including testosterone, which was growth stimulatory, and progesterone, which was growth inhibitory. The growth stimulatory effects of 17β-estradiol, alpha estradiol, and tamoxifen could possibly be explained by their interaction with an estrogen receptor. Indeed, metabolic labelling and immunoprecipitation studies indicated the presence of such an estrogen receptor in the primary cultures. The rate of biosynthesis of the estrogen receptor was found to be affected by the presence of exogenously added 17β-estradiol. 17β-estradiol was also observed to increase the activity of two brush border enzymes, alkaline phosphatase and gamma glutamyl transpeptidase, during the growth phase of the primary cultures. J Cell Physiol 178:35–43, 1999. © 1999 Wiley-Liss, Inc.     

Polarized membrane expression of brush-border hydrolases in primary cultures of kidney proximal tubular cells depends on cell differentiation and is induced by dexamethasone

To analyze the influence of cell differentiation and the effects of hormones on the subcellular distribution of apical antigens in polarized epithelial cells, we have compared the localization of three brush border (BB) hydrolases [neutral endopeptidase (ENDO), aminopeptidase N (APN), and dipeptidylpeptidase IV (DPPIV)] in primary cultures of renal proximal tubule cells grown in various culture media. The degree of cell differentiation modulated by medium composition was estimated by measuring proximal functions, including glucose transport, specific enzymatic activities, and PTH responsiveness. In the dedifferentiated state observed in cells grown in 1% fetal calf serum (FCS)-supplemented medium, the three hydrolases are abnormally concentrated in a cytoplasmic vesicle compartment with weak expression on both membrane domains. By contrast, in serum-free hormonally defined medium (DM: insulin, 5 microgram/ml; dexamethasone, 5 x 10(-8) M), which markedly enhances morphological and functional cell differentiation, the distribution of hydrolases parallels that observed in the normal tubule. When added to the DM devoid of hormones, insulin has little polarizing effect, whereas dexamethasone dramatically increases the apical expression of the hydrolases, which then almost disappear from the basolateral membrane and cytoplasmic vesicular compartments. This glucocorticoid hormone augments the amount of immunoreactive antigen detectable on the apical domain in paraformaldehyde-fixed cells but does not change the total enzymatic activity. This suggests the presence in tubular cells of a dexamethasone-dependent polarizing machinery that requires de novo RNA and protein synthesis, and probably acts mainly by targeting a storage cytoplasmic pool of enzyme to the apical domain.     

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.     

Serum renotropic factor stimulates prostaglandin synthesis in primary cultures of rabbit kidney cells

Compensatory growth of the kidney occurs in response to a partial reduction in renal mass. This compensatory renal growth may be regulated by a circulating renotropic factor. Prostaglandin synthesis has been shown to be increased in kidneys undergoing compensatory renal growth in vivo. In the present study we observed that the addition of rabbit sera obtained after uninephrectomy enhanced DNA synthesis in primary cultures of rabbit kidney cells compared to sera obtained prenephrectomy. The stimulated kidney cells produced more prostaglandin E2 than control cells. Furthermore, the addition of prostaglandin E2 to rabbit kidney cells in the presence of control sera also stimulated DNA synthesis. These results provide further evidence that prostaglandins may participate in the biological events which regulate renal growth in response to a circulating renotropic factor.     

Growth and function of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium.

The properties of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose-free and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium.     

Morphological and biochemical characterization of the opossum kidney cell line and primary cultures of rabbit proximal tubule cells in serum-free defined medium.

Proliferation, morphology and time course patterns of marker enzyme activities of primary cultures of renal rabbit proximal tubule cells (RPT cells) and Opossum kidney cells (OK cells) in antibiotic-free and serum-free defined medium were investigated. Both RPT and OK cells grew to confluency within 6-8 days. RPT cells were thicker and displayed higher density of both microvilli and mitochondria when compared with OK cells. RPT cells exhibited higher activity of glutathione-S-transferase when compared with OK cells, whereas in the latter, higher glutathione content could be detected. Apical and basolateral membrane enzymes were higher in RPT cells than in OK cells. Stable high glycolytic activity and low gluconeogenesis activity in OK cells pointed out a strict dependence on glycolysis, whereas RPT cells exhibited glucose metabolism shift towards the glycolysis pathway.     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

Stimulation by calcium and carbamoylcholine of the ouabain-sensitive uptake of 86Rb+ in isolated rat pancreatic acinar cells

The uptake of 86Rb+ was assayed in isolated rat pancreatic acinar cells to determine the effect of calcium and carbamoylcholine on the ouabain-sensitive and ouabain-insensitive components. The presence of calcium in the medium bathing the cells during the preincubation and the main incubation periods was needed to preserve in optimum conditions the uptake of 86Rb+, the stimulation by carbamoylcholine and the sensitivity to ouabain. In the presence of calcium, the ouabain-sensitive component of 86Rb+ uptake was higher than the ouabain-insensitive. The ouabain-sensitive component was 3-times lower in cells incubated in a medium lacking calcium and containing 1 mM EGTA, as compared to cells incubated in the presence of calcium. Carbamoylcholine, at 5 X 10(-4) M, stimulated the uptake of 86Rb+ and this effect depended on the presence of calcium in the bathing medium. Maximal stimulation by carbamoylcholine was reached at 0.2 mM calcium. The nett stimulation by carbamoylcholine was inhibited up to 85% by 1 mM ouabain. As judged by digitonin-disruption of plasma membrane, the above-indicated effects were limited to a cytoplasmic pool of 86Rb+ and a leaky plasma membrane could be ruled out. The results suggest that in rat pancreatic acinar cells, carbamoylcholine stimulated the ouabain-sensitive uptake of 86Rb+ and required the presence of calcium in the bathing medium.     

Antigenic expression of aminopeptidase M,dipeptidyl-peptidase IV and endopeptidase by primary cultures from rabbit kidney proximal tubule

Summary Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured, to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occured during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occured only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.     

Antigenic expression of aminopeptidase M, dipeptidyl-peptidase IV and endopeptidase by primary cultures from rabbit kidney proximal tubule

Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occurred during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occurred only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.     

Changes in expression and subcellular localization of annexin IV in rabbit kidney proximal tubule cells during primary culture

In the present study, we investigated the polarized expression of annexin IV at various stages in the growth of rabbit kidney proximal tubule cells (PTC) in primary cultures. The results of immunoblotting analysis and indirect immunofluorescence studies using a specific anti-annexin IV monoclonal antibody, indicated that annexin IV is expressed in proximal tubule cultured cells, although it was not detected in the proximal tubules present in frozen sections of kidney cortex and freshly isolated proximal tubule cells. In either non-confluent or confluent cells which remained attached to the collagen-coated support, annexin IV was mainly concentrated around the nucleus, whereas in PTC forming the monolayer of domes, it was restricted to the basolateral membrane domain. This basolateral localization was identical to that observed in other polarized epithelial cell types such as enterocytes. When the domes burst, the cells returned to the collagen-coated support and the annexin IV was again localized around the nuclei. The fact that the change of localization was very rapid suggested the existence of a considerable difference between the differentiation states of dome forming and adherent confluent cells. Moreover, a transient association of annexin IV with the basal body of apically located cilia also seemed to be correlated with a particular polarization state and/or differentiation states of adherent cultured cells, corresponding to the beginning of the polarized expression of aminopeptidase N, a hydrolase located in the apical brush border membrane, and to the falling of cells onto the support, subsequent to the bursting of the domes. In conclusion, these results provide evidence that annexin IV may constitute a new marker of the basolateral membrane domain of polarized epithelial renal cells in primary cultures. © 1995 Wiley-Liss, Inc.     

Regulation of 86Rb influx during accumulation of Rb+ or K+ in yeast

The influx rate of ⁸⁶Rb decreases during accumulation of K⁺ or Rb⁺ into metabolizing yeast cells under anaerobic conditions with glucose as substrate. Possible causes for the decrease in the influx rate are examined. It is ruled out that the decrease in the influx rate is caused by an increased turgor pressure of the cells or to an impairment of their energization. During the accumulation of K⁺ or Rb⁺, no decrease but an increase in the protonmotive force of the cells is found. The concomitant increase in cell pH accounts only in small part for the decrease in the influx rates. Acidification of the cells on adding butyrate to the suspension causes a transient increase in the influx rates, whereas the cellular monovalent cation content is still increased. Consequently, no direct relationship is found between the influx rate and the cellular content of K⁺ and Rb⁺.     

Effect of aldosterone on 86Rb fluxes in cultured kidney cells (A6)

This study was designed to evaluate the relative contributions of hormone induced changes in active and passive K+ transport in an epithelial cell line in continuous culture derived from toad kidney (A6) using 86Rb as a tracer for measuring unidirectional K+ fluxes. The effects of 24 h exposure to aldosterone (A) and aldosterone plus insulin (A+I) on unidirectional K+ fluxes were evaluated under short-circuited conditions and under open circuit conditions. In epithelia exposed to A, a small but significant amount of active K+ secretion was found, although it was not significantly greater than in control epithelia. The bidirectional fluxes in both A and A+I treated epithelia, under short-circuited conditions, increased by a similar amount over control values indicating an increase in apparent permeability of passive transepithelial K+ transport. Under open circuit conditions, A stimulated net K+ transport by about 5-fold over controls. The increase in K+ secretion produced by A under open circuit conditions could be explained by the combined effects of an increase in transepithelial K+ permeability and an increase in the transepithelial electrical potential difference (PD). The presence of I produced no additional effects to that of A on K+ transport under the conditions used in this study. It is concluded that the substantial increase in K+ secretion induced in A6 cells by 24 h exposure to A is primarily passive in nature. It is possible that the changes in both PD and transepithelial K+ permeability, which can account for the observed increase in K+ secretion, are secondary to the stimulation of active Na+ transport.