Frog Kidney Research Articles

Tissue-Specific Distribution and Maternal Transfer of Persistent Organic Halogenated Pollutants in Frogs.

Persistent organic pollutants pose a great threat to amphibian populations, but information on the bioaccumulation of contaminants in amphibians remains scarce. To examine the tissue distribution and maternal transfer of organic halogenated pollutants (OHPs) in frogs, seven types of tissues from black-spotted frog (muscle, liver, kidney, stomach, intestine, heart, and egg) were collected from an e-waste-polluted area in South China. Among the seven frog tissues, median total OHP concentrations of 2.3 to 9.7 μg/g lipid weight were found (in 31 polychlorinated biphenyl [PCB] individuals and 15 polybrominated diphenyl ether [PBDE], dechlorane plus [syn-DP and anti-DP], bexabromobenzene [HBB], polybrominated biphenyl] PBB153 and -209], and decabromodiphenyl ethane [DBDPE] individuals). Sex-specific differences in contaminant concentration and compound compositions were observed among the frog tissues, and eggs had a significantly higher contaminant burden on the whole body of female frogs. In addition, a significant sex difference in the concentration ratios of other tissues to the liver was observed in most tissues except for muscle. These results suggest that egg production may involve the mobilization of other maternal tissues besides muscle, which resulted in the sex-specific distribution. Different parental tissues had similar maternal transfer mechanisms; factors other than lipophilicity (e.g., molecular size and proteinophilic characteristics) could influence the maternal transfer of OHPs in frogs. Environ Toxicol Chem 2024;43:1557-1568. © 2024 SETAC.

Ontogenetic Expression of Aquaporins in the Kidney and Urinary Bladder of the Japanese Tree Frog, Dryophytes japonicus.

For adult anuran amphibians, the kidney and urinary bladder play important osmoregulatory roles through water reabsorption. In the present study, we have examined ontogenetic expression of aquaporins, i.e., AQP2, AQPamU (AQP6ub, AQPa2U), and AQP3, in these organs using the Japanese tree frog, Dryophytes japonicus. Immunohistochemistry using the metamorphosing larvae at stages 40-43 localized AQP2 protein to the collecting ducts in the dorsal zone of the mesonephric kidney. At prometamorphic stages 40 and 41, labelling of AQP2 protein was observed in the apical/ subapical regions of the collecting duct cells. At climax stages 42 and 43, labels for AQP2 and AQP3 became observed in the apical/subapical regions and basolateral membrane of the collecting duct cells, respectively, as seen in the adults. As for the urinary bladder, immuno-positive labels for AQPamU were localized to the apical/subapical regions of granular cells in the mucosal epithelium at stages 40-43. On the other hand, AQP3 immunoreactivity was hardly observed in the urinary bladder at stage 40, and weakly appeared in many granular cells at stage 41. Thereafter, labels for AQP3 became evident along the basolateral membrane of granular cells at stages 42 and 43, together with AQPamU in the apical/subapical regions. These results suggest that the kidney and urinary bladder might be capable of water reabsorption, via AQP2, AQPamU, and AQP3, at stage 42, contributing to the acclimation of the tree frogs to terrestrial environments.

The Mechanism of Elizabethkingia miricola Infection of the Black Spotted Frog as Revealed by Multi-Omics Analysis

Elizabethkingia miricola (E. miricola) is a significant pathogen that causes the crooked head disease in black spotted frogs. This disease has plagued numerous frog farms in China and has resulted in substantial losses to the frog farming industry. Nonetheless, the exact mechanism that causes the disease in frogs remains unknown. In this study, transcriptomic and microbiomic analyses were conducted to analyze frog samples infected with E. miricola to reveal the infection mechanism of the pathogen. Liver transcriptomic analysis indicated that the livers of infected frogs had 1469 differentially expressed genes when compared with an uninfected group. These DEGs are mainly involved in immunity and metabolism, including neutrophil extracellular trap formation, the NOD-like receptor signaling pathway, leukocyte transendothelial migration, chemokine signaling pathway, Fc gamma R-mediated phagocytosis, and “metabolism”-related pathways such as the pentose phosphate pathway, carbon metabolism, glycerophospholipid metabolism, and glycerolipid metabolism. Similarly, 4737 DEGs were found in the kidney of infected frogs. These DEGs are mainly involved in immunity, including neutrophil extracellular trap formation, the NOD-like receptor signaling pathway, B cell receptor signaling pathway, C-type lectin receptor signaling pathway, complement and coagulation cascade, and Toll-like receptor signaling pathway. Ten immune-associated DEGs were screened in liver and kidney DEGs, respectively. And it was hypothesized that E. miricola infection could influence the host immune response. Microbiome analysis results showed that some opportunistic pathogens such as Citrobacter, Shigella, and Providencia were significantly elevated (p < 0.05) in infected frogs. Additionally, functional prediction confirmed that most of the microbiota in infected frogs were linked to metabolism-related KEGG pathways. In this study, the screened genes linked to immunity showed an association with the gut microbiome. The majority of these genes were found to be linked with the abundance of opportunistic pathogens. The results showed that E. miricola infection led to the downregulation of immune and metabolic-related genes, which led to the inhibition of immune function and metabolic disorder, and then increased the abundance of opportunistic pathogens in the gut microbiota. The findings of this study offer a preliminary foundation for comprehending the pathogenic processes of E. miricola infection in black spotted frogs.

Characteristics of the interferon regulatory factor IRF5 and its stimulation expression by Elizabethkingia miricola Infection in black-spotted frog (Pelophylax nigromaculatus)

Interferon regulatory factors (IRFs) have been confirmed as vital transcription factors for the immune system, which play a certain role in anti-infection defense, immune regulation, hematopoietic cell development, and maturation of the immune system. In this study, the cDNA of Interferon regulatory factor 5 (IRF5) was cloned from black-spotted frog, Pelophylax nigromaculatus, and termed PnIRF5. The results indicated, the full-length cDNA of PnIRF5 was 2090 bp, and a putative protein of 504 amino acids was encoded. The results of protein domain prediction suggested that PnIRF5 exhibited a DNA-binding domain (DBD), a middle region (MR), an IRF association domain (IAD), a virus activated domain (VAD), as well as two nuclear localization signals (NLSs). Phylogenetic analysis clustered PnIRF5 into the amphibian IRF5 subgroup in vertebrate IRF5 group. The results of qPCR show that PnIRF5 mRNA was expressed in all examined tissues, with higher levels were identified in the kidney, intestine, and lung. The gene expression of PnIRF5 was analyzed in kidney, spleen, and liver of black-spotted frog after challenged with Elizabethkingia miricola, and the maximum expression level of PnIRF5 in liver, spleen, and kidney were 1.80, 2.33 and 2.88 times that at 0 days post challenged, respectively. As indicated by the results, the PnIRF5 protein may take on critical significance in protecting black-spotted frog from E. miricola infection, and they may play a certain role in the immune response of black-spotted frog against bacteria.

Renal protein reabsorption impairment related to a myxosporean infection in the grass frog (Rana temporaria L.).

A morphophysiological study of tubular reabsorption and mechanisms of protein endocytosis in the kidney of frogs (Rana temporaria L.) during parasitic infection was carried out. Pseudoplasmodia and spores of myxosporidia, beforehand assigned to the genus Sphaerospora, were detected in Bowman's capsules and in the lumen of individual renal tubules by light and electron microscopy. Remarkable morphological alteration and any signs of pathology in kidney tissue related to this myxosporean infection have not been noted. At the same time, significant changes in protein reabsorption and distribution of molecular markers of endocytosis in the proximal tubule (PT) cells in infected animals were detected by immunofluorescence confocal microscopy. In lysozyme injection experiments, the endocytosed protein and megalin expression in the infected PTs were not revealed. Tubular expression of cubilin and clathrin decreased, but endosomal recycling marker Rab11 increased or remained unchanged. Thus, myxosporean infection resulted in the alterations in lysozyme uptake and expression of the main molecular determinants of endocytosis. The inhibition of receptor-mediated clathrin-dependent protein endocytosis in amphibian kidneys due to myxosporidiosis was shown for the first time. Established impairment of the endocytic process is a clear marker of tubular cell dysfunction that can be used to assess the functioning of amphibian kidneys during adaptation to adverse environmental factors.

The Roles of Amphibian (Xenopus laevis) Macrophages during Chronic Frog Virus 3 Infections.

Infections by Frog Virus 3 (FV3) and other ranavirus genus members are significantly contributing to global amphibian decline. The Xenopus laevis frog is an ideal research platform upon which to study the roles of distinct frog leukocyte populations during FV3 infections. Frog macrophages (MΦs) are integrally involved during FV3 infection, as they facilitate viral dissemination and persistence but also participate in immune defense against this pathogen. In turn, MΦ differentiation and functionality depend on the colony-stimulating factor-1 receptor (CSF-1R), which is ligated by CSF-1 and iterleukin-34 (IL-34) cytokines. Our past work indicated that X. laevis CSF-1 and IL-34 give rise to morphologically and functionally distinct frog MΦ subsets, and that these CSF-1- and IL-34-MΦs respectively confer susceptibility and antiviral resistance to FV3. Because FV3 targets the frog kidneys and establishes chronic infections therein, presently we examined the roles of the frog CSF-1- and IL-34-MΦs in seeding and maintaining these chronic kidney infections. Our findings indicate that the frog CSF-1-MΦs result in more prominent kidney FV3 infections, which develop into greater reservoirs of lingering FV3 marked by infiltrating leukocytes, fibrosis, and overall immunosuppressive states. Moreover, the antiviral effects of IL-34-MΦs are short-lived and are lost as FV3 infections progress.

MO082THE EFFECTS OF DIFFERENT MICROMOLAR CONCENTRATIONS OF CADMIUM IONS ON THE PERITUBULAR MEMBRANE POTENTIAL OF PROXIMAL TUBULAR CELLS IN PERFUSED FROG KIDNEYS

Abstract Background and Aims Cadmium (Cd2+) is toxic metal and environmental pollutant. Accumulation of cadmium in the kidney results initially in proximal tubule dysfunction. Although Cd2+ toxicity is well documented, all mechanisms that are involved in the early stages of nephrotoxicity, especially considering low micromolar concentrations of Cd2+ ions are still unknown. The Aim of this study was to investigate the effects acute exposure to different peritubular micromolar concentrations of cadmium (0.25, 0.50, 1.0, 2.0, 3.0, 5.0 μmol/L) on the peritubular cell membrane potential in proximal tubular cells of frog kidney. Method The experiments were performed on isolated, doubly perfused kidneys of Rana esculenta of both sexes. Aortic and portal vein were cannulated in order to perfusate luminal and peritubular cell membraine. In controled conditions, Ringer solution was simultaneously used to perfusate both cell membraines. Cadmium chloride (different concentrations: 0.25, 0.50, 1.0, 2.0, 3.0, 5.0 μmol/L) were added to the peritubular perfusate separately, by switching the peritubular perfusate from the control Ringer solution to Ringer solution with addition of cadmium ions. Peritubular cell membrane potentials (PD) were measured with conventional 3 mol/L KCl microelectrodes. Results The peritubular application of different micromolar Cd2+ concentrations led to a rapid, sustained, reversible hyperpolarization of the peritubular cell membrane: 0.25 µmol/L, by −3.3±0.4 mV (n=8, p<0.001); 0.50 µmol/L, by −3.0±0.5 mV (n=11, p<0.001); 1.0 µmol/L, by −2.9±0.6 mV (n=8, p<0.01); 2.0 µmol/L, by −4.2±0.4 mV (n=13, p<0.01); 3.0 µmol/L, by −3.4±0.3 mV (n=14, p<0.001); 5.0 µmol/L, by −3.0±0.4 mV (n=10, p<0.001). After switching the perfusion from Ringer solution with addition of cadmium ions to control Ringer, the peritubular membraine potential returned to the average values that were maintained before the peritubular Cd2+ application (p>0.05). Comparing the effect of different Cd2+ concentrations, there was no difference in the hyperpolarization of the peritubular cell membrane (p>0.05).Each cell served as its own control. Conclusion Different low micromolar concentrations of Cd2+ provoked rapid and sustained hyperpolarization of peritubular membrane potential that did not show concentration-dependent response.

Elizabethkingia miricola infection in Chinese spiny frog (Quasipaa spinosa).

Elizabethkingia miricola is a Gram-negative rod which has been incriminated in severe infections in humans. Recently, a serious infectious disease was identified in Chinese spiny frogs (Quasipaa spinosa), in the Sichuan Province of China; the disease was characterized by corneal opacity, the presence of ascites and neurological symptoms. A Gram-negative bacillus was isolated from the liver, spleen and kidney of the diseased frogs. Experimental infection test revealed that the bacillus could infect the frogs Q.spinosa and the LD50 value was 1.19×106 cfu per frog. The isolated Gram-negative bacillus was identified as E.miricola according to phenotypic characteristics, 16S rRNA and gyrB gene sequence analysis. The isolated strain was only susceptible to florfenicol among all investigated chemotherapeutic agents. Histological examination revealed that E.miricola infection caused pathological lesions to multiple organs and tissues, especially in the liver, brain, kidney. These results confirmed that E.miricola is an emerging pathogen of Chinese spiny frogs.

Modulation of luminal L-alanine transport in proximal tubular cells of frog kidney induced by low micromolar Cd2+ concentration

The kidneys are recognized as a major target of cadmium-induced toxicity. However, all mechanisms that are involved in the early stages of cadmium nephrotoxicity, particularly considering low micromolar concentrations of cadmium ions (Cd2+) are still unknown. Therefore, the aim of this study was to investigate the effects of peritubular acute exposure to micromolar Cd2+ concentration (2.3 μmol/L) on the rapid depolarization and the rate of slow repolarization of peritubular membrane potential difference (PD), induced by luminal application of L-alanine in proximal tubular cells of frog kidney. The results showed that the luminal application of L-alanine rapidly depolarized the peritubular membrane PD of −42.00 ± 11.68 mV by 23.89 ± 4.15 mV with an average rate of slow repolarization of 5.64 ± 0.81 mV/min. Additionally, peritubular acute exposure to Cd2+ induced change in rapid depolarization of peritubular membrane PD of −53.33 ± 13.01 mV by 18.78 ± 3.31 mV (P < 0.01) after luminal application of L-alanine. Also, peritubular acute exposure to Cd2+ led to statistically significant decrease in the rate of slow repolarization of peritubular membrane PD (3.53 ± 0.35 mV/min; P < 0.05). In conclusion, these results suggest that peritubular acute exposure to low micromolar Cd2+ concentration decreased the rapid depolarization and the rate of slow repolarization of peritubular membrane PD induced by luminal application of L-alanine. This is followed by reduced luminal sodium-coupled transport of L-alanine and this change may be one of the possible mechanisms involved in the early stages of Cd2+-induced nephrotoxicity.

Amphibian (Xenopus laevis) Tadpoles and Adult Frogs Differ in Their Use of Expanded Repertoires of Type I and Type III Interferon Cytokines.

While amphibians around the globe are facing catastrophic declines, in part because of infections with pathogens such as the Frog Virus 3 (FV3) ranavirus; the mechanisms governing amphibian susceptibility and resistance to such pathogens remain poorly understood. The type I and type III interferon (IFN) cytokines represent a cornerstone of vertebrate antiviral immunity, while our recent work indicates that tadpoles and adult frogs of the amphibian Xenopus laevis may differ in their IFN responses to FV3. In this respect, it is notable that anuran (frogs and toads) tadpoles are significantly more susceptible to FV3 than adult frogs, and thus, gaining greater insight into the differences in the tadpole and adult frog antiviral immunity would be invaluable. Accordingly, we examined the FV3-elicited expression of a panel of type I and type III IFN genes in the skin (site of FV3 infection) and kidney (principal FV3 target) tissues and isolated cells of X. laevis tadpoles and adult frogs. We also examined the consequence of tadpole and adult frog skin and kidney cell stimulation with hallmark pathogen-associated molecular patterns (PAMPs) on the IFN responses of these cells. Together, our findings indicate that tadpoles and adult frogs mount drastically distinct IFN responses to FV3 as well as to viral and non-viral PAMPs, while these expression differences do not appear to be the result of a distinct pattern recognition receptor expression by tadpoles and adults.

Comparison of morphometric and gravimetric measurements of Common Skittering Frog (Euphlyctis cyanophlyctis) from paddy fields and urban wetlands

Frogs are common in aquatic habitats such as marshes, edges of water bodies and paddy fields. High prevalence of deformed frogs as well as reduction in body length, head length, limb size, growth rate and increase in liver weight are known from various frog species occurring in pesticide contaminated sites. We hypothesized that snout-vent length, liver, kidney, body weight and growth of Common Skittering Frog inhabiting areas contaminated by pesticides and fertilizers such as paddy fields of District Gujranwala, Punjab, Pakistan, do not differ than those from areas with less pesticide and fertilizer use such as urban wetlands in Rawalpindi-Islamabad Districts. Our results revealed statistically significant differences in measurements such as in snout-vent length, inter-orbital width, distance from front of eyes to the tip of snout, distance between the nostrils, inter-nasal space, distance from front of eyes to the nostrils, distance from nostrils to the tip of snout, distance from tympanum to the back of eyes, forelimb length and body weight of frogs from paddy fields and urban wetlands. We found a strong significant relationship between snout-vent length and body weight of female frogs collected from paddy fields and urban wetlands and of male frogs collected from paddy fields only. We found that Common Skittering Frog had a higher growth in paddy fields, presumably due to abundance of food. Our study documented first record of a deformity (micromelia) and an abnormality (gas bubble disease) in Common Skittering Frog from Pakistan. We suggest detailed studies on food availability and consumption by Common Skittering Frog in the paddy fields and urban wetlands to understand factors influencing the growth pattern.

Osmolyte regulation by TonEBP/NFAT5 during anoxia-recovery and dehydration-rehydration stresses in the freeze-tolerant wood frog (Rana sylvatica).

BackgroundThe wood frog, Rana sylvatica, tolerates freezing as a means of winter survival. Freezing is considered to be an ischemic/anoxic event in which oxygen delivery is significantly impaired. In addition, cellular dehydration occurs during freezing because water is lost to extracellular compartments in order to promote freezing. In order to prevent severe cell shrinkage and cell death, it is important for the wood frog to have adaptive mechanisms for osmoregulation. One important mechanism of cellular osmoregulation occurs through the cellular uptake/production of organic osmolytes like sorbitol, betaine, and myo-inositol. Betaine and myo-inositol are transported by the proteins BGT-1 and SMIT, respectively. Sorbitol on the other hand, is synthesized inside the cell by the enzyme aldose reductase. These three proteins are regulated at the transcriptional level by the transcription factor, NFAT5/TonEBP. Therefore, the objective of this study was to elucidate the role of NFAT5/TonEBP in regulating BGT-1, SMIT, and aldose reductase, during dehydration and anoxia in the wood frog muscle, liver, and kidney tissues.MethodsWood frogs were subjected to 24 h anoxia-4 h recovery and 40% dehydration-full rehydration experiments. Protein levels of NFAT5, BGT-1, SMIT, and aldose reductase were studied using immunoblotting in muscle, liver, and kidney tissues.ResultsImmunoblotting results demonstrated downregulations in NFAT5 protein levels in both liver and kidney tissues during anoxia (decreases by 41% and 44% relative to control for liver and kidney, respectively). Aldose reductase protein levels also decreased in both muscle and kidney tissues during anoxia (by 37% and 30% for muscle and kidney, respectively). On the other hand, BGT-1 levels increased during anoxia in muscle (0.9-fold compared to control) and kidney (1.1-fold). Under 40% dehydration, NFAT5 levels decreased in liver by 53%. Aldose reductase levels also decreased by 42% in dehydrated muscle, and by 35% in dehydrated liver. In contrast, BGT-1 levels increased by 1.4-fold in dehydrated liver. SMIT levels also increased in both dehydrated muscle and liver (both by 0.8-fold).DiscussionOverall, we observed that osmoregulation through an NFAT5-mediated pathway is both tissue- and stress-specific. In both anoxia and dehydration, there appears to be a general reduction in NFAT5 levels resulting in decreased aldose reductase levels, however BGT-1 and SMIT levels still increase in certain tissues. Therefore, the regulation of osmoregulatory genes during dehydration and anoxia occurs beyond the transcriptional level, and it possibly involves RNA processing as well. These novel findings on the osmoregulatory mechanisms utilized by the wood frog advances our knowledge of osmoregulation during anoxia and dehydration. In addition, these findings highlight the importance of using this model to study molecular adaptations during stress.

Immunolocalization of the TASK2 Potassium Channel in Frog Kidney.

TASK2 (K2P5. 1, KCNK5) is a two-pore domain K⁺ channel belonging to the TALK subgroup of the K2P family of proteins. TASK2 expression has been reported in a variety of cells and tissues ranging from kidney to immune cells and including specific neurons, its proposed functions spanning from involvement in the regulation of cell volume to control of excitability. The purpose of this study was to determine the tubule location ofthe TASK2 K⁺ channel protein in frog kidney applying polyclonal antibody against the carboxyl terminus of human TASK2 (KCNK5) protein. Immunohistochemical analysis revealed that TASK2 is expressed on distal tubules and proximal epithelial cells. TASK2 is strongly expressed predominantly on the luminal part ofthe proximal epithelial cells and slightly cytoplasmatic staining is expressed. Distal tubules showed diffuse cytoplasmatic staining as well as slight staining on the apical parts ofthe cells. These findings suggest that the TASK2 K⁺ channel has cell-specific roles in renal potassium ion transport.

Effect of different proteins on reabsorption of yellow fluorescent protein in the kidney of the brown frog Rana temporaria

Protein reabsorption in the proximal tubules (PT) of the frog kidney was studied by the methods of immunohistochemistry, fluorescent and confocal microscopy. Yellow fluorescent protein (YFP) was introduced in combination with other proteins. Reabsorption of YFP introduced simultaneously with ly- sozyme or green fluorescent protein (GFP) did not differ from the result of YFP injection only. Previous lysozyme injection did not change YFP absorption in contrast to YFP uptake reduced after GFP pretreat- ment. Lysozyme loading for 4 days resulted in a significant reduction in YFP absorption. The results show that receptor-mediated endocytosis in the frog kidney depends on the molecular nature of absorbable ligands, conditions of their competitive absorption and lysosomal accumulation in epithelial PT cells.

Biospectroscopy reveals the effect of varying water quality on tadpole tissues of the common frog (Rana temporaria)

Amphibians are undergoing large population declines in many regions around the world. As environmental pollution from both agricultural and urban sources has been implicated in such declines, there is a need for a biomonitoring approach to study potential impacts on this vulnerable class of organism. This study assessed the use of infrared (IR) spectroscopy as a tool to detect changes in several tissues (liver, muscle, kidney, heart and skin) of late-stage common frog (Rana temporaria) tadpoles collected from ponds with differing water quality. Small differences in spectral signatures were revealed between a rural agricultural pond and an urban pond receiving wastewater and landfill run-off; these were limited to the liver and heart, although large differences in body size were apparent, surprisingly with tadpoles from the urban site larger than those from the rural site. Large differences in liver spectra were found between tadpoles from the pesticide and nutrient impacted pond compared to the rural agricultural pond, particularly in regions associated with lipids. Liver mass and hepatosomatic indices were found to be significantly increased in tadpoles from the site impacted by pesticides and trace organic chemicals, suggestive of exposure to environmental contamination. Significant alterations were also found in muscle tissue between tadpoles from these two ponds in regions associated with glycogen, potentially indicative of a stress response. This study highlights the use of IR spectroscopy, a low-cost, rapid and reagent-free technique in the biomonitoring of a class of organisms susceptible to environmental degradation.

Metal concentrations in common skittering frog (Euphlyctis cyanophlyctis) inhabiting Korang River, Islamabad, Pakistan

The current study investigated metal concentrations (Zn, Cd, Pb, Mn and Fe) in the habitat and body tissues (blood, liver and kidney) of common skittering frog Euphlyctis cyanophlyctis along the Korang River, Islamabad, Pakistan. Samples were collected from three selected contaminated sites and a reference site at four different occasions from October 2010 to March 2012. All studied metals showed elevated levels in river water, as well as in blood, liver and kidneys of frogs from all contaminated sites. Specifically, Mn showed significantly higher levels in all analysed matrices from contaminated sites compared to the reference site. Histological sections of frog livers exposed to elevated metals showed abnormal hepatocytes, while their kidney sections showed discontinuous glomeruli with dead or ruptured cells exhibiting improper shapes with cytoplasmic depositions. This study indicates that frogs inhabiting Korang River are at potential risk of metal toxicity from the environment.

Comparative analysis of fluorescent protein reabsorption in rat and frog kidney

GFP signal intensity revealed similar patterns of GFP absorption in rat and frog proximal tubular cells depending on the amount of the administered protein [1, 3]. At the same time, in frog kidney there were demonstrated some peculiarities of GFP fluorescence distribution associated with a smaller number of nephrons, their intermittent activity and a reduced glomerular filtration under the effect of antidiuretic hormone [2, 3]. Efficiency of the model fluorescent proteins in physiological experiments and a paucity of evidence concerning functional differences between the mammalian metanephros and amphibian mesonephros urged a detailed comparative analysis of protein filtration and absorption in kidneys of rats and frogs. The goal of this investigation was to compare functional parameters of the kidney activity in the above species including the glomerular filtration rate (GFR) and dynamics of GFP and YFP uptake and accumulation by proximal tubular cells during endocytosis. Experiments were carried out on Wistar rat females (Rattus norvegicus) and hibernating brown frog males (Rana temporaria). Highly purified recombinant GFP and YFP were obtained from the Laboratory of protein synthesis mechanisms (Institute of Protein Research RAS, Pushchino, SHORT COMMUNICATIONS

Reabsorption of yellow fluorescent protein in the Rana temporaria kidney by receptor-mediated endocytosis

The absorption of yellow fluorescent protein (YFP) and the expression of the endocytic receptors, megalin and cubilin, were investigated in the renal proximal tubules (PT) in frogs Rana temporaria after parenteral YFP injections. The methods of confocal microscopy and immunohistochemistry were used. The dynamics of YFP absorption was analyzed 2 h after injection. The logarithmic time dependence of the accumulation of YFP-containing endocytic vesicles in PT cells and the completion of absorption process 90-120 min after injection were shown. Unlike substantial megalin and cubilin expression 15-30 min after YFP introduction, immunolabeled endocytic receptors were not detected in PT cells after 2 h. The re-injection of YFP led to the appearance of apical endocytic vesicles containing megalin or cubilin colocalized with YFP. At the same time, the decrease of YFP uptake associated with reduction in the number of receptor-containing vesicles was demonstrated, suggesting a failure of megalin and cubilin expression. The decrease of absorption capacity of PT cells after YFP re-injection was similar to that found previously under conditions of the competitive absorption of green fluorescent protein (GFP) and YFP injected in different sequences. The data are the further demonstration of the proposed mechanism limiting the tubular protein absorption in the frog kidney and suggest the involvement of megalin and cubilin in uptake and vesicular transport of YFP.

Regulation of epithelial sodium channel activity in A6 cells via the peptide hormone, prolactin (1181.9)

Prolactin (PRL) is a peptide hormone well‐known for its role in lactation. PRL also has osmoregulatory actions via alteration of ion and water transport, actions characterized mostly in aquatic life. In 2003, researchers reported that PRL stimulates epithelial sodium channel (ENaC) activity in adult frog skin. Since that time, PRL‐mediated regulation of ENaC has been largely ignored. Given that ENaC present in the kidney plays an essential part in blood pressure control, we hypothesized that PRL may control blood volume and blood pressure via regulation of ENaC activity. To investigate this hypothesis, we used a well‐known frog kidney cell model (A6‐2F3) possessing amiloride‐sensitive transepithelial current and highly selective sodium channels with a conductance of ~5pS. Amiloride‐sensitive (ENaC‐mediated) current dramatically increased within 30 minutes after application of 1µg/mL PRL to the basal side of the cells ‐ an effect sustained even at 24 hours after treatment. We confirmed this finding using cell attached patch clamp. We found an almost 6 fold increase in ENaC NPo in PRL‐treated versus vehicle‐treated cells. The mechanism, although still not clear, at least partially involves JAK2 signaling since AG‐490, a JAK2 inhibitor, reduced the effect of PRL on amiloride‐sensitive current in A6 cells. Inhibitors of PI3K and SRC had no effect on PRL’s induction of amiloride‐sensitive current. In conclusion, we observed a significant induction of ENaC activity in frog kidney cells exposed to PRL ‐ an effect partially mediated through JAK signaling pathways. Future studies will finalize the signaling mechanisms involved in PRL’s activation of ENaC and the role of these pathways in blood pressure control.Grant Funding Source: Supported by NIH K12GM000680 and T32HL076118

The Amphibian (Xenopus laevis) Type I Interferon Response to Frog Virus 3: New Insight into Ranavirus Pathogenicity

The increasing prevalence of ranavirus (RV; Iridoviridae) infections of wild and commercially maintained aquatic species is raising considerable concerns. While Xenopus laevis is the leading model for studies of immunity to RV, amphibian antiviral interferon (IFN) responses remain largely uncharacterized. Accordingly, an X. laevis type I interferon was identified, the expression of the gene for this IFN was examined in RV (frog virus 3 [FV3])-infected tadpoles and adult frogs by quantitative PCR, and a recombinant form of this molecule (recombinant X. laevis interferon [rXlIFN]) was produced for the purpose of functional studies. This rXlIFN protected the kidney-derived A6 cell line and tadpoles against FV3 infection, decreasing the infectious viral burdens in both cases. Adult frogs are naturally resistant to FV3 and clear the infection within a few weeks, whereas tadpoles typically succumb to this virus. Hence, as predicted, virus-infected adult X. laevis frogs exhibited significantly more robust FV3-elicited IFN gene expression than tadpoles; nevertheless, they also tolerated substantially greater viral burdens following infection. Although tadpole stimulation with rXlIFN prior to FV3 challenge markedly impaired viral replication and viral burdens, it only transiently extended tadpole survival and did not prevent the eventual mortality of these animals. Furthermore, histological analysis revealed that despite rXlIFN treatment, infected tadpoles had considerable organ damage, including disrupted tissue architecture and extensive necrosis and apoptosis. Conjointly, these findings indicate a critical protective role for the amphibian type I IFN response during ranaviral infections and suggest that these viruses are more pathogenic to tadpole hosts than was previously believed, causing extensive and fatal damage to multiple organs, even at very low titers. Ranavirus infections are threatening wild and commercially maintained aquatic species. The amphibian Xenopus laevis is extensively utilized as an infection model for studying ranavirus-host immune interactions. However, little is known about amphibian antiviral immunity and, specifically, type I interferons (IFNs), which are central to the antiviral defenses of other vertebrates. Accordingly, we identified and characterized an X. laevis type I interferon in the context of infection with the ranavirus frog virus 3 (FV3). FV3-infected adult frogs displayed more robust IFN gene expression than tadpoles, possibly explaining why they typically clear FV3 infections, whereas tadpoles succumb to them. Pretreatment with a recombinant X. laevis IFN (rXlIFN) substantially reduced viral replication and infectious viral burdens in a frog kidney cell line and in tadpoles. Despite reducing FV3 loads and extending the mean survival time, rXlIFN treatments failed to prevent tadpole tissue damage and mortality. Thus, FV3 is more pathogenic than was previously believed, even at very low titers.