Frog Kidney Research Articles

Amphibia-Related Viruses

The chapter discusses the present knowledge of both the suspected and established amphibia-related viruses. The former category includes viruses associated with a lipoma of Wisconsin frogs ( Rana pipiens ), an epithelioma of the newt, Triturus alpestris , and a lymphosarcoma of the toad, Xenopus laevis. The basis of the suspected viral etiology in these cases rests' usually on cell-free filtrate transmission experiments. Experiments of this kind illustrates this point for it has recently been convincingly demonstrated to be caused by a bacterium, and will be considered in more detail at the end of this section on suspected amphibian viruses. Physical data concerning the purification and morphology of postulated viruses in the other cases have yet to be presented. The first two genera, involving an anuran (Rana pipiens) liposarcoma and a urodele (Triton alpestris) epithelioma, are less convincing than the last genus, that of a toad (Xenopus Zuevis) lymphosarcoma. Established amphibian viruses include those isolated by tissue culture techniques and by physical methods. Particular emphasis will be placed on the virus typically associated with the Luck´e frog renal adenocarcinoma. The term “Luck´e” as used in the chapter refers to characteristic spheroid virus particles repeatedly observed in frog kidney tumors and in intraocular tumor implants. Such particles have not been seen in extensive examinations of healthy frog kidneys. One of the most important immediate problems in amphibian virus research is to define the relationship of the various frog virus isolates both to each other and to the more typical Luck´e tumor-associated viruses. Although filtration experiments and morphological studies strongly indicate a viral etiology for the frog renal adenocarcinoma, intrarenal injection of purified (mature) virus preparations into adult frogs has thus far failed to induce tumor formation.

Viruses and renal carcinoma of Rana pipiens: II. Ultrastructural studies and sequential development of virus isolated from normal and tumor tissue

The sequential development of a virus (FV 1) isolated from cultured frog kidney cells has been studied by electron microscopy in fathead minnow (FHM) and baby hamster kidney (BHK 21/13) cells. Regions of virus synthesis were first seen in the cell cytoplasm at 7 and 10 hours after infection of FHM and BHK 21/13 cells, respectively. These sites contained virus particles in all stages of development from partial capsids to complete capsids with nucleoids. The virus was hexagonal and measured approximately 120 by 130 mμ. At 12 hours after infection, virus was seen budding from the cytoplasmic membrane, thus acquiring an envelope. This process continued throughout the course of the infection. Late in the infection (24–48 hours) one or more virus crystals were regularly seen in the cytoplasm of FHM cells and occasionally in BHK 21/13 cells. FV 1 replication in adult and embryonic frog cell monolayers, cultured Lucké tumor cells, and chick embryo monolayers was identical to that seen in FHM and BHK 21/13 cells. Eleven additional isolates from both normal and tumor (Lucké) frog tissue were examined in FHM cells. The morphology and site of synthesis of these isolates were identical to FV 1 and contrasted with the nuclear site of synthesis of virus seen in the Lucké tumor.

Viruses and renal carcinoma of Rana pipiens: I. The isolation and properties of virus from normal and tumor tissue

Properties of a virus, frog virus 1 (FV 1), isolated from plaques appearing in uninoculated frog kidney cell monolayers were studied. The virus multiplied at 24 ° in frog, fish, chick embryo, and hamster cell monolayers with cytopathology resulting in cell death. Although each cell system could be used for plaque assay the efficiency of plating was higher (10- to 100-fold) in chick embryo and hamster cells. Growth curves in cultured cells from both poikilothermic and homothermic animals were similar with a latent period between 6 and 10 hours. Most of the virus (50–99%) remained cell-associated. Virus multiplication was temperature dependent. At 33 ° plaques did not develop and little or no infectious virus was produced although adsorption and penetration occurred. Virus multiplication ceased when infected cultures were removed from 24 ° to 33 ° at any time during the growth cycle, a phenomenon suggesting that virus maturation was thermosensitive. The site of virus synthesis appeared to be the cell cytoplasm where DNA, identified by cytochemical stains and isotope incorporation, accumulated during infection. Infectivity was lost by exposure of virus to lipid solvents or to pH 3.0. It was stable to storage at low temperature (−20 ° to −60 °) but was rapidly inactivated at 50 °. Additional virus isolations were made from spontaneously degenerating frog kidney cell cultures and from homogenates of frog livers and frog renal tumors (Lucké adenocarcinoma). There was no correlation between the presence of intranuclear inclusions or virus particles (as observed by light and electron microscopy) and the ability to isolate virus from tumor homogenates. Comparative tests showed no distinguishing differences between properties of an isolate from a Lucké tumor, FV 3, and FV 1. The results are discussed as they relate to a viral etiology of the Lucké tumor.

Properties of isolated frog liver and kidney nuclei.

AbstractIn order for a nuclear preparation to be used for analytical purposes, the method of isolation and composition of the suspension medium must be carefully examined. Accordingly, satisfactory techniques for the isolation of frog liver and kidney nuclei were developed. The medium for frog liver nuclei consisted of: 55% glycerol, 0.001 M magnesium chloride, 0.033 M sodium β‐glycerophosphate and/or 0.002 M KH2PO4, K2HPO4 (pH 6.8), however, the addition of 0.15 M sucrose was essential for satisfactory isolation of kidney nuclei. Inclusion of sucrose (0.15 M) in the isolation medium promoted nucleolar swelling and a decrease in nuclear volume in liver cell nuclei. Nucleolar migration and extrusion were noted in solutions with high cationic content.The morphological appearance of isolated nuclei was found to be extremely sensitive to the ionic strength of the isolation medium, as was the isolation procedure in toto. Effects were considered to be the result of precipitation and swelling of nucleoprotein. Dissociation of nucleoprotein was considered to be associated with temperature change. The uptake of supra‐vital dyes aided in recognition of the morphological alterations and was also an indicator of nuclear viability.Trypsin readily altered the nuclear membrane and a rapid decrease in nuclear density occurred, but the nucleolus remained intact. The diverse response of liver and kidney nuclei as compared with the nucleated red blood cells (a contaminant) to treatment with trypsin was noted and its implications discussed.

Leptospires isolated from frog kidneys.

IN the summer of 1964, cases of leptospirosis developed in man as a result of swimming in a farm creek. During epidemiological studies, six leopard frogs (Rana pipiens) were collected from these waters. Blood samples were obtained from the frogs. Two pools of kidneys (A and B) were made from three frogs each. Leptospires were not observed on dark-field microscopy of the kidney suspensions. These were diluted with Stuart's liquid media (1 : 10–1: 100,000) and 2–3 drops from the dilutions inoculated1 into semi-solid media: bovine-albumin ‘Tween 80’ (ref. 2) containing 5-fluorouracil3 (96 µg/ml.) (Hoffman-LaRoche, Inc., Nutley, New Jersey) and Fletcher's semi-solid media1. The inoculated media were incubated at 28°–30° C for 90 days and examined periodically by dark-field microscopy. Leptospires were isolated from pool B in albumin ‘Tween 80’ medium on the twentieth day after inoculation. Observations of the frog isolate by electron microscopy indicated that the organisms were morphologically characteristic of the genus Leptospira4.

Implant-induced accessory limbs in urodeles: fresh, frozen, and boiled tissues.

AbstractImplants were made into forelimbs of Triturus viridescens using fresh, frozen and boiled kidney and liver of T. viridescens and R. pipiens. Limbs were recovered at intervals up to 70 days post‐implantation.Kidney implants from Wisconsin R. pipiens gave twice as many extensive accessory structures as did Vermont frog kidney. Total induction percentages, however, were similar.Quantitative and qualitative parameters for implant‐induction of accessory structures were investigated. The decrease in antigenicity and increased rate of cytolysis of frozen implants resulted in increased similarity between frog and newt kidney in rate and pattern of breakdown and in rates of induction. Modification of rate and duration of the release of the stimulating factor from the implant did not result in induction by liver implants.No evidence was found for any increase in innervation prior to or coincident with blastema formation. Implantation and implant cytolysis may cause hypersensitivity of limb tissues to the normal innervation pattern or trophic stimuli from the implant may act with those from the injured limb tissues to produce growth.The general pattern of host reaction to the implanted material was studied and described.

Renal Tumors in a Western Population of Rana pipiens

A new locality for a species specific frog kidney tumor is reported. While the cytological characteristics appear identical to the original tumors from Vermont, the new tumors differ in population incidence, sex of hosts and in tumor inductive capability. In the leopard frog from Vermont, Rana pipiens pipiens, there occurs a tumor of the kidney, first described as an adenocarcinoma, (Lucke, 1934) which is used widely in experimental tumor transfer. New tumors, specific to kidney, arise after implantation of the living tissue into the eyes of the homologous species (Lucke and Schlumberger, 1939; Rose and Rose, 1952; Tweedell, 1955). In addition to organ and tissue specificity, induction of tumors ordinarily occurs onlv when host and donor animals are from the same population in Vermont and environs. Heterologous species of frogs and other alien species are refractory to ordinary tumor passage (Lucke, 1938, 1952; Lucke and Schlumberger, 1940; Tweedell, 1955). Populations of Rana pipiens, as close as New Jersey (Lucke, 1952) are resistant to kidney tumor induction. Similarly, Rose and Rose (1952) and Tweedell (1955) report that Lucke eye tumor grafts do not induce renal tumors in R. pipiens from Wisconsin, Illinois or Kentucky, unless special adaptive measures are used (Tweedell, 1953, 1955). The range of natural tumor occurrence was believed to be rather limited since it was obtained only in populations of R. pipiens from the Lake Champlain Valley in northern Vermont and adjacent Quebec (Lucke, 1938; Lucke and Schlumberger, 1949; Rose and Rose, 1952; Auclair, 1961). Lucke (1952) did report that tumor bearing frogs were obtained from the Mississippi Valley, North Dakota and Indiana but this has never been confirmed. Several hundred R. pipiens collected from west of the Rocky Mountain barriers in northwestern Minnesota failed to yield any Lucke adenocarcinomas (Meteyko, 1957). Recently, Rafferty (1964) has indicated that renal tumors are reportedly found also in R. pipiens from Wisconsin. During the last 7 years, similar renal tumors have been detected periodically in R. pipiens obtained from Wisconsin. It thus appeared that either the range of histologically similar if not identical tumors might be broader than once suspected or that a different biological strain of the renal tumor existed in a more western population of R. pipiens. The present observations summarize the characteristics of the new renal tumors and compare them briefly with tumors from Vermont R. pipiens. METHODS When the animals were first received into the laboratory, they were palpated for renal tumors and each injected with 0.5 mg of chloromycetin in 0.5 ml of distilled water to counteract redleg. Animals

METABOLIC PATHWAYS FOR UREA PRODUCTION BY THE AMPHIBIAN KIDNEY.

Homogenized kidneys, pooled from bullfrogs ( Rana catesbiana), produce urea in amounts of 1.4–6.4 µmole/g of wet tissue after 1 hr of incubation in substrate-free media. The maximal rate of endogenous urea production under present conditions, 16 µmoles/g kidney per hr, was found during the first 10 min of incubation. In standard tests with added substrates, activity of frog kidney arginase was greater than that of frog liver arginase. However, argininosuccinase, easily detected in frog liver, dog liver, and dog kidney cortex, could not be detected in frog kidney. Additional tests showed that frog kidney homogenates contain uricase, allantoicase, and allantoicase in concentrations similar to those of frog liver tissue. This means that urea forming in frog kidney tissue comes from at least two pathways: one terminating in hydrolysis of arginine, and one terminating in hydrolysis of allantoic acid. In the absence of argininosuccinase, arginine could be derived from degradation of protein or peptides. In view of the oxygen dependency of uric acid-loaded homogenates, allantoic acid is derived from hydrolysis of allantoin which, in turn, derives from the oxidation of uric acid.

The isolation and morphology of the Lucké frog kidney tumor virus

Viruses have been isolated from “spontaneous” Lucké frog renal adenocarcinomas by means of equilibrium centrifugation in a Ficoll gradient. Visualization was accomplished by negative contrast staining and thin section electron microscopy. Particles in negative contrast consist of either “empty” or “full” capsids approximately 100 mμ in diameter, occasionally surrounded by a loose-fitting envelope 160–240 mμ in diameter. In thin section the capsid can be seen to enclose a dense core or nucleoid 55–80 mμ in diameter. The capsid contains 162 hollow, elongated capsomeres arranged in an ordered architecture suggestive of icosahedral symmetry. The close morphological relationship between the Lucké virus and those of the herpes group is discussed.

ARGENTAFFIN GRANULES IN TOAD KIDNEY TUBULES.

A considerable amount of argentaffin granules was localised in the proximal tubules of the toad kidney. While they were found to give argentaffin, metal chelating and alkaline azo coupling reactions in keeping with the general characteristics of the chromaffin cells, the chromaffin and Gibbs reactions could not be obtained. It is, however, peculiar that the granules gave some acid azo coupling reaction and benzylidene reaction even after formol fixation. Positive fluorescence in ultraviolet light and some other reactions were also obtained to indicate that the compound studied was a tryptamine derivative with a tertiary amine at the end of the side chain.

NUCLEOLAR ABERRATIONS IN FROG KIDNEY TUMOUR CELLS.

ACCORDING to Gates1, the nucleolus was apparently the first intranuclear organelle described; yet its function in the normal cell has never been fully clarified2, nor has its possible malfunction in malignant cells3,4. But Caspersson5 has held that chromatin associated with the nucleolus and the genes which govern its function must play a part in carcinogenesis.

Thin-section election microscopy of the mature Lucke frog kidney tumor virus

Thin-section election microscopy of the mature Lucke frog kidney tumor virus

HANDLING OF UREA AND RELATED COMPOUNDS BY THE RENAL TUBULES OF THE FROG.

A high arginase activity is found in the kidney of the adult frog Rana catesbeiana which actively secretes urea. Urea accumulates in the kidney in concentrations around seven to eight times the blood concentration. To test whether the kidney arginase causes significant formation of urea in tubular cells at normal plasma arginine levels, C14 urea, chemical urea, and creatinine clearances were measured. C14 urea and chemical urea clearances were identical, both being about seven times the GFR. 2,4-Dinitrophenol (DNP) inhibited the active secretion of urea, but C14 and chemical urea clearances remained identical to each other. When arginine was administered, urea was formed in the kidney and excreted in the urine at the rate of 35 µm/g kidney hr. The chemical urea clearance was then about twice the C14 urea clearance. DNP administered with arginine caused the C14 urea clearance to fall below the GFR but did not inhibit the formation of urea as shown by the chemical urea clearance being three times the GFR. Compounds chemically related to urea were handled in different ways by the frog kidney. Acetamide and methylurea were not secreted and did not accumulate in the kidney. DNP had little or no affect on their excretion. Thiourea was found to be actively secreted and to accumulate in the kidney. DNP inhibited thiourea secretion. It is concluded that urea is normally actively transported across the tubular wall and that formation of urea in the tubular cells is insignificant.

ACTIVATION OF PHOSPHORYLASE IN TOAD BLADDER AND MAMMALIAN KIDNEY BY ANTIDIURETIC HORMONE.

An increase in phosphorylase activity has been demonstrated in the intact urinary bladder of the toad and in slices of rabbit renal medulla and dog renal cortex and medulla incubated in vitro with vasopressin. A similar response occurred in the toad bladder incubated with adenosine-3',5'-monophosphate (cyclic-3',5'-AMP). Phosphorylase activation in the bladder was also produced by incubation with oxytocin or protopituitrin, but not by incubation with ACTH or epinephrine. The metabolic significance of hormone-induced phosphorylase activation is unclear. However, since activation of phosphorylase is a characteristic tissue response to hormones that act through cyclic-3',5'-AMP, the present observations support the suggestion that vasopressin alters the permeability to water of the urinary bladder of the toad and mammalian kidney through a pathway involving stimulation of the production of cyclic-3',5'-AMP.

An improved technique of preparing primary cultures of isolated cells from adult frog kidney

Eine verbesserte Technik zur Gewinnung der ersten Einzelschichtbildungen von Froschnierenzellen wurde entwickelt. Die Zellen werden in neuem auflosendem Nahrboden isoliert und wachsen auf modifiziertem Eagle-Nahrboden mit 13% Kalbsserum. Nach 10 Tagen zeigt sich stark aktives mitotisches Zellwachstum in der Kultur.

Cultivation of monolayer cultures of frog renal cells.

CONSIDERABLE evidence has been accumulated by Lucke1, Duryee2, Rose3, Tweedell4, and others for an agent which induces an organ-specific adenocar-cinoma in the kidney of the leopard frog, Rana pipiens. Lucke1 has postulated that this agent is probably of a viral nature. Although a percentage of the animals develop palpable renal tumours after a long and variable period of time following injection of tumour material, it is still desirable to devise other methods to propagate and possibly isolate the infective agent. At the same time, the large dimensions of the frog cells make them an ideal system with which to study the initiation of any subcellular modifications following incubation of cells with the tumour-inducing agent.

Retention of urea by frog and mammalian kidney slices in vitro

Retention of urea by frog and mammalian kidney slices <i>in vitro</i>

An electron microscope study of the uptake, transport, and storage of colloidal materials by the cells of the vertebrate nephron

Trypan blue and colloidal thorium dioxide were used to study the uptake, transport, and storage of colloidal particles by the tubular epithelium of frog and rat kidneys. Colloidal particles apparently enter the proximal tubular cells by passing between the microvilli of the brush border, first appearing within small, apical vesicles. The absorbed materials appear to be conveyed to the mid-region of the cells by larger, smooth-walled vesicles, and are segregated or stored within membrane-limited bodies or cytosomes. The cytosomes are limited by single membranes, have a characteristic internal structure and are considered to represent lysosomes. These observations suggest a mechanism by which native colloidal materials, such as proteins, could be taken up and stored by the tubular cells in vivo ; a similar mechanism may be responsible for the formation of so-called “hyaline droplets.” Evidence is also provided for the concept that lysosomes play an important role in the cellular storage of reabsorbed materials.

Histochemical localization of L-gulonolactone oxidase activity in tissues of several species.

SummaryA histochemical method was described which localized the activity of L-gulonolactone oxidase, the final oxidative step in synthesis of ascorbic acid from glucose. The enzyme was found to be active in the centrilobular cells of the rat liver and in selected portions of the mesonephric tubules of the frog kidney. It was absent from guinea pig kidney and liver, and from frog liver.

A histochemical study of the kidney of a Japanese frog.

A histochemical study of the kidney of a Japanese frog.