Mary Taub

Glomerulonephritis induced in the rabbit by antiendothelial antibodies.

The effects of interaction between endothelial angiotensin converting enzyme (ACE) and goat anti-rabbit ACE (GtARbACE) antibodies were studied in rabbit glomeruli. By immunofluorescence ACE was not detectable in normal glomeruli. However, when kidneys were perfused with GtARbACE antibodies glomerular bound IgG was seven times higher than that of non-immune IgG and granular deposits of goat IgG were found on the endothelium of glomeruli and arteries. Rabbits injected intravenous for 4 d with GtARbACE antibodies showed on day 1 granular deposits of goat IgG on the glomerular endothelium; from day 3 to 24 there was gradual development of subepithelial deposits of goat IgG, rabbit IgG and C3. When GtARbACE antibodies were similarly injected into proteinuric rabbits there was formation of subepithelial granular deposits of goat IgG and ACE. The results document that a glomerular endothelial antigen is redistributed in vivo by a specific ligand, an event associated with formation of immune deposits. Furthermore, if the glomerular permeability is artificially increased, immune complexes shed from nonglomerular endothelia into the circulation can contribute to form subepithelial immune deposits.

Tissue culture of epithelial cells

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TOXICITY OF IFOSFAMIDE AND ITS METABOLITE CHLOROACETALDEHYDE IN CULTURED RENAL TUBULE CELLS

SummaryRenal injury is a common side effect of the chemotherapeutic agent ifosamide. Current evidence suggests that the ifosfamide metabolite chloroacetaldehyde may contribute to this nephrotoxicity. The present study examined the effects of ifosfamide and chloroacetaldehyde on rabbit proximal renal tubule cells in primary culture. The ability of the uroprotectant medication sodium 2-mercaptoethanesulfonate (mesna) to prevent chloroacetaldehyde-induced renal cell injury was also assessed. Chloroacetaldehyde (12.5–150 µM) produced dose-dependent declines in neutral red dye uptake, ATP levels, glutathione content, and cell growth. Coadministration of mesna prevented chloroacetaldehyde toxicity while pretreatment of cells with the glutathione-depleting agent buthionine sulfoximine enhanced the toxicity of chloroacetaldehyde. Ifosfamide (1000–10 000 µM) toxicity was detected only at concentrations of 4000 µM or greater. Analysis of media collected from ifosfamide-treated cell cultures revealed the presence of several ifosfamide metabolites, demonstrating that renal proximal tubule cells are capable of biotransforming this chemotherapeutic agent. This primary renal cell culture system should prove useful in studying the cause and prevention of ifosfamide nephrotoxicity.

COMPARATIVE TOXICITY OF IFOSFAMIDE METABOLITES AND PROTECTIVE EFFECT OF MESNA AND AMIFOSTINE IN CULTURED RENAL TUBULE CELLS

Renal injury is a common side effect of the chemotherapeutic agent ifosfamide. Current evidence suggests that the ifosfamide metabolite chloroacetaldehyde contributes to this nephrotoxicity. The present study examined the effects of chloroacetaldehyde and acrolein, another ifosfamide metabolite, on rabbit proximal renal tubule cells in primary culture. The ability of the uroprotectant medications sodium 2-mercaptoethanesulfonate (mesna) and amifostine to prevent chloroacetaldehyde- and acrolein-induced renal cell injury was also assessed. Chloroacetaldehyde and acrolein (25-200 M) produced dose-dependent declines in neutral red dye uptake, glucose transport and glutathione content. Chloroacetaldehyde was a more potent toxin than acrolein. Pretreatment of cells with the glutathione-depleting agent buthionine sulfoximine enhanced the toxicity of both chloroacetaldehyde and acrolein while co-administration of mesna or amifostine prevented metabolite toxicity. These results support the hypothesis that chloroacetaldehyde is responsible for ifosfamide-induced nephrotoxicity. The protective effect of mesna and amifostine in vitro contrasts with clinical experience showing that these medications do not eliminate ifosfamide nephrotoxicity.

Primary cultures of rabbit renal proximal tubule cells: I. Growth and biochemical characteristics

SummayBefore the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures within 7 d after plating. Multicellular done formation, an indicator of transepithelial solute transport, was expressed after confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme,N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture. respectively. In contrast, the activity of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers into the culture medium, which are routinely used to measure cytoxicity, stabilized after 8 d in culture. The ratio of cellular protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/μg DNA in preconfluent cultures to 0.059 mg protein/μg DNA in confluent cultures. These results document the characteristics of a primary proximal tubule cell culture system for future studies in in vitro toxicology.

Primary cultures of rabbit renal proximal tubule cells: III. Comparative cytotoxicity of inorganic and organic mercury

The present study further developed primary cultures of rabbit renal proximal tubule cells (RPTC) as an in vitro model to study chemical-induced toxicity by investigating the comparative cytotoxicity of mercuric chloride (HgCl2) and methyl mercury chloride (CH3HgCl) to RPTC. Confluent monolayer cultures of RPTC exposed to HgCl2 and CH3HgCl for 24 hr exhibited a concentration-dependent loss in cell viability at culture medium concentrations greater than 25 and 2.5 microM, respectively. Vital dye exclusion was a more sensitive indicator of cytotoxicity than the amount of lactate dehydrogenase activity, alkaline phosphatase activity, N-acetylglucosaminidase activity, and protein content remaining on the culture dish. On the basis of vital dye exclusion, HgCl2 was less toxic to proximal tubule cells in culture than CH3HgCl after 24 hr of exposure, whether cytotoxicity was based on LC50 values (34.2 microM HgCl2 vs 6.1 microM CH3HgCl) or total cellular mercury uptake (4.6 nmol Hg2+/10(5) cells vs 1.25 nmol CH3Hg+/10(5) cells). Differences in the extent and rate of metal uptake were also evident. Maximum cellular uptake of Hg2+ occurred within 6-24 hr after exposure and was not concentration-dependent, whereas maximum uptake of CH3Hg+ occurred within 3 hr of exposure and was concentration-dependent. The intracellular distribution of both mercurials between acid-soluble and acid-insoluble binding sites also differed. At noncytotoxic concentrations of HgCl2 (0.04-5 microM), intracellular Hg2+ bound increasingly to acid-soluble binding sites as a function of time, from 15-30% after 6 hr of exposure to 40-60% after 72 hr of exposure. However, at subcytotoxic (25 microM) and cytotoxic (34.2 microM) concentrations, Hg2+ binding to acid-soluble binding sites remained constant at approximately 30-40% for 6, 12, 24, and 72 hr after exposure. In contrast, only 20% of total cellular CH3Hg+ was bound to acid-soluble binding sites after exposure to 0.039 to 6.1 microM CH3HgCl for 6, 12, and 24 hr. Total cellular glutathione content was unaffected after exposure to 0.04-5 microM HgCl2 and 0.039-6.1 microM CH3HgCl, but was depleted 6 hr after exposure to 25 and 34.2 microM HgCl2. These results indicate that CH3HgCl was a more potent cytotoxicant to RPTC in primary culture than HgCl2. Furthermore, compared to Hg2+, the low binding of CH3Hg+ to acid-soluble binding sites and the absence of a redistribution of CH3Hg+ from acid-insoluble to acid-soluble binding sites appeared to contribute to its more potent toxicity to cultured cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Both mitogen activated protein kinase and the mammalian target of rapamycin modulate the development of functional renal proximal tubules in matrigel

Tubules may arise during branching morphogenesis through several mechanisms including wrapping, budding, cavitation and cord hollowing. In this report we present evidence that is consistent with renal proximal tubule formation through a process of cord hollowing (a process that requires the concomitant establishment of apicobasal polarity and lumen formation). Pockets of lumen filled with Lucifer Yellow were observed within developing cords of rabbit renal proximal tubule cells in matrigel. The observation of Lucifer Yellow accumulation suggests functional polarization. In the renal proximal tubule Lucifer Yellow is initially transported intracellularly by means of a basolaterally oriented p-aminohippurate transport system, followed by apical secretion into the lumen of the nephron. Consistent with such polarization in developing tubules, Triticum vulgare was observed to bind to the lumenal membranes within pockets of Lucifer Yellow-filled lumens. As this lectin binds apically in the rabbit renal proximal tubule, T. vulgare binding is indicative of the emergence of an apical domain before the formation of a contiguous lumen. Both epidermal growth factor and hepatocyte growth factor stimulated the formation of transporting tubules. The stimulatory effect of both epidermal growth factor and hepatocyte growth factor on tubulogenesis was inhibited by PD98059, a mitogen activated protein kinase kinase inhibitor, rather than by wortmannin, an inhibitor of phosphoinositide 3-kinase. Nevertheless, Lucifer Yellow-filled lumens were observed in tubules that formed in the presence of PD98059 as well as with wortmannin, indicating that these drugs did not prevent the process of cavitation. By contrast, rapamycin, an inhibitor of the mammalian target of rapamycin, prevented the process of cavitation without affecting the frequency of formation of developing cords. Multicellular cysts were observed to form in 8-bromocyclic AMP-treated cultures. As these cysts did not similarly accumulate Lucifer Yellow lumenally, it is very likely that processes other than organic anion accumulation are involved in the process of cystogenesis, including the Na,K-ATPase.

Primary rabbit kidney proximal tubule cell cultures maintain differentiated functions when cultured in a hormonally defined serum-free medium

SummaryA primary rabbit kidney epithelial cell culture system has been developed which retains differentiated functions of the renal proximal tubule. In addition, the cells have a distinctive metabolism and spectrum of hormone responses. The primary cell were observed to retain in vitro a Na+-dependent sugar transport system (distinctive of the proximal segment of the nephron) and a Na+-dependent phosphate transport system. Both of these transport processes are localized on the apical membrane of proximal tubule cells in vivo. In addition, probenicid-sensitivep-aminohippurate (PAH) uptake was observed in basolateral membranes of the primary tubule cells, and the PAH uptake by these vesicles occurred at a rate that was very similar to that observed with membranes derived from the original tissue. Several other characteristics of the primary cells were examined, including hormone-sensitive cyclic AMP production and phosphoenolpyruvate carboxykinase (PEPCK) activity. Like the cells in vivo, the primary proximal tubule cells were observed to produce significant cyclic AMP in response to parathyroid hormone, but not in response to arginine vasopressin or salmon calcitonin. Significant PEPCK acivity was observed in the particulate fraction derived from a homogenate of primary rabbit kidney proximal tubule cells.

THE DEVELOPMENT OF SERUM‐FREE HORMONE‐SUPPLEMENTED MEDIA FOR PRIMARY KIDNEY CULTURES AND THEIR USE IN EXAMINING RENAL FUNCTIONS

We have demonstrated the primary kidney cultures and defined medium can be used for several purposes. First, primary kidney cultures can be examined in defined medium to evaluate the physiological significance of studies made with established kidney cell lines. Secondly, new kidney epithelial cell culture systems can be developed, using primary cultures and defined medium. The studies presented here have demonstrated several possible approaches towards these ends. First, a hormone-supplemented serum-free medium (Medium K-1) developed for an established dog kidney epithelial cell line (MDCK) was used to grow primary cultures of baby mouse kidney epithelial cells without fibroblast overgrowth. The primary cultures of baby mouse cells not only grew in response to each of the five supplements in Medium K-1, but also possessed several of the differentiated kidney functions observed in the MDCK cell line. Secondly, defined media were developed for primary cultures of rabbit kidney epithelial cells directly. The rabbit cells were shown to require a different set of supplements than MDCK cells to attain optimal growth. Furthermore primary cultures derived from purified rabbit proximal tubules required a different set of supplements than primary cultures derived from unpurified rabbit kidney tissue. These new primary culture systems should prove invaluable in examining renal transport functions in vitro.

Phospholipids regulate growth and function of MDCK cells in hormonally defined serum free medium

SummaryThe effects of the simple phospholipids phosphatidic acid (PA) and lysophosphatidic acid (LPA) on the growth and function of Madin Darby Canine Kidney (MDCK) cells has been studied. We observed that PA and LPA not only stimulated the growth of MDCK cells (at 20µM), but also stimulated the growth of normal rabbit kidney cells in serum free medium (albeit at a lower dosage of 5µM). Evidence was obtained that PA interacts synergistically with insulin so as to elicit a growth stimulatory effect. Recently, extracellular PA and LPA were proposed to stimulate mitogenesis in several types of animal cells by binding to particular sites on the plasma membrane which are coupled to signaling mechanisms such as adenylate cyclase via a pertussis toxin sensitive, inhibitory guanosine triphosphate binding protein (Gi protein) (15). However, even when the pertussis toxin dosage was increased to 50 ng/ml, LPA still had a dramatic growth stimulatory effect on MDCK cells. In the absence of LPA pertussis toxin was slightly growth stimulatory to MDCK cells. Phospholipids such as PA and LPA have been observed to prevent prostaglandin-induced increases in adenylate cyclase activity in other cell types via their effects on such a pertussis toxin sensitive Gi protein. If PA and LPA act on MDCK cells in this manner, then these phospholipids may possibly prevent the effect of PGE1 on the growth of normal MDCK cells. However PGE1 was still growth stimulatory to normal MDCK cells. The effects of PA on PGE1 independent variants of MDCK cells, which have elevated intracellular cyclic AMP levels (22), were also examined. In the presence of PA, PGE1 remained growth inhibitory, rather than growth stimulatory to the PGE1 independent cells. However, the PA dosage required to elicit an optimal growth response (5µM) was dramatically reduced, as compared with normal MDCK cells (20µM). This altered dosage requirement could be explained by the elevated intracellular cyclic AMP levels in the PGE1 independent variants. Like PGE1 and 8-bromocyclic AMP, PA and LPA also significantly increased the initial rate of Rb+ uptake by confluent monolayers of MDCK cells. The increase in the initial rate of Rb+ uptake could be explained by an increase in the ouabain-sensitive component of Rb+ uptake. An increase in the initial rate of ouabain-insensitive Rb+ uptake was also observed in LPA treated MDCK cell cultures.