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Parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) control serum phosphate levels by downregulating the renal Na-phosphate transporter NPT2A, thereby decreasing phosphate absorption and augmenting urinary excretion. This mechanism requires NHERF1, a PDZ scaffold protein, and is governed by the regulator of G protein signaling-14 (RGS14), which harbors a carboxy-terminal PDZ ligand that binds NHERF1. RGS14 is part of a triad of structurally related RGS proteins that includes RGS12 and RGS10. Like RGS14, RGS12 contains a class 1 PDZ ligand. However, unlike RGS14, the larger RGS12 contains an upstream PDZ-binding domain. The studies outlined here examined and characterized the binding of RGS12 with NHERF1 and NPT2A and its function on hormone-regulated phosphate transport. Immunoblotting experiments revealed RGS12 C-terminal PDZ ligand binding to NHERF1. Further structural analysis disclosed that NPT2A engaged full-length RGS12 and the upstream fragment containing the PDZ domain. Neither the downstream RGS12 portion nor RGS14 interacted with NPT2A. PTH and FGF23 profoundly inhibited phosphate uptake in opossum kidney proximal tubule cells. Transfection with human RGS14, or human RGS12, abolished hormone-sensitive phosphate transport as reported for human proximal tubule cells. RGS12 inhibitory activity resides in the downstream region and is comparable to RGS14. The carboxy-terminal RGS12(667-1447) splice variant is prominently expressed in the kidney and may contribute to regulating hormone-sensitive phosphate transport., Competing Interests: Declaration of competing interest The authors avow that the described work has not been published previously in any form. The manuscript and results are not under consideration for publication elsewhere. All authors approved by and tacitly or explicitly by the authorities where the work was performed. If accepted, the article will not be published elsewhere in any form, in English or in any other language, including electronically without the written consent of the copyright-holder., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Trypanosoma cruzi is the etiologic agent of American trypanosomiasis and can infect humans and different species of domestic and wild animals. The marsupials are important wild reservoirs of T. cruzi, aiding in the maintenance of this agent in sylvatic and peri-domestic environments. The objective of this study was to report the parasitological and clinicopathological findings of a natural infection by T. cruzi in one specimen of Philander opossum that originated from the Brazilian Amazon. The animal was captured in a forest fragment near a rural community with reports of human Chagas disease. T. cruzi infection was diagnosed by blood smear examinations, blood culture, scent glands secretion culture, histopathological examination, and nested-PCR. Positive samples were subjected to PCR to characterize the discrete typing units (DTUs) of T. cruzi. Characteristic trypomastigotes of T. cruzi were observed in the blood smear, and spheromastigotes, epimastigotes, and trypomastigotes were visualized in the cultures. Non-suppurative myocarditis associated with amastigote clusters was the principal histopathological finding. DNA from T. cruzi was detected in samples of blood, blood cultures, scent glands secretion cultures, cardiac muscles, and the spleen. The TcI and the TcII/V/VI group DTUs were detected in blood culture and scent glands secretion cultures. Infection by T. cruzi can cause myocarditis in P. opossum and DTUs TcI and TcII/V/VI group mixed infection can be detected in the acute phase. P. opossum can be a source of infection for triatomine vectors and has the potential source for direct transmission of T. cruzi by secretions from the scent glands. These data are important to improve the understanding of the complex enzootic transmission cycle of T. cruzi in the Brazilian Amazon., (Copyright © 2020. Published by Elsevier B.V.)

Worldwide, Bartonella species are known to infect a wide range of mammalian and arthropod hosts, including humans. The current study aimed to investigate the prevalence of Bartonella spp. in synanthropic mammals captured in peri-urban areas from Central-Western and Southern Brazil and their ectoparasites. For this aim, 160 mammals belonging to four species, and 218 associated arthropods were sampled. DNA was extracted and subjected to different Bartonella screening assays. Additionally, blood samples from 48 small rodents were submitted to liquid BAPGM culture followed by qPCR assay and solid culture. Two out of 55 Rattus captured in Santa Catarina state were PCR-positive for Bartonella when targeting the nuoG, 16S, and ITS loci. Sequences showed high homology with Bartonella coopersplainsensis. Conversely, all 48 small rodents, 14 capybaras and 43 opossum DNA samples from animals trapped in Mato Grosso do Sul were Bartonella negative in the HRM real time PCR assays targeting the ITS locus and gltA gene. Additionally, all mammal-associated ectoparasites showed negativity results based on HRM real time PCR assays. The present study showed, for the first time, the occurrence of B. coopersplainsensis in Brazil, shedding some light on the distribution of rats-related Bartonella in South America. In addition, the majority of rodents and marsupials were negative for Bartonella spp. Since B. coopersplainsensis reservoirs - Rattus spp. - are widely dispersed around the globe, their zoonotic potential should be further investigated., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Equine protozoal myeloencephalitis (EPM) is an important equine neurologic disorder, and treatments for the disease are often unrewarding. Prevention of the disease is the most important aspect for EPM, and a killed vaccine was previously developed for just that purpose. Evaluation of the vaccine had been hampered by lack of post vaccination challenge. The purpose of this study was to determine if the vaccine could prevent development of clinical signs after challenge with Sarcocystis neurona sporocysts in an equine challenge model. Seventy horses that were negative for antibodies to S. neurona and were neurologically normal were randomly assigned to vaccine or placebo groups and divided into short-term duration of immunity (study #1) and long-term duration of immunity (study #2) studies. S. neurona sporocysts used for the challenge were generated in the opossum/raccoon cycle isolate SN 37-R. Study #1 horses received an initial vaccination and a booster, and were challenged 34days post second vaccination. Study #2 horses received a vaccination and two boosters and were challenged 139days post third vaccination. All horses in study #1 developed neurologic signs (n=30) and there was no difference between the vaccinates and controls (P=0.7683). All but four horses in study #2 developed detectable neurologic deficits. The neurologic signs, although not statistically significant, were worse in the vaccinated horses (P=0.1559). In these two studies, vaccination with the S. neurona vaccine failed to prevent development of clinical neurologic deficits., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The main transport mechanism of reabsorption of sodium bicarbonate and fluid in the renal proximal tubules involves Na(+)/H(+) exchanger 3 (NHE3), which is acutely and chronically downregulated by parathyroid hormone (PTH). Although PTH is known to exert an inhibitory effect on NHE3 expression and transcription, the molecular mechanisms involved remain unclear. Here, we demonstrated that, in opossum kidney proximal tubule (OKP) cells, PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter that controls expression of the reporter gene. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways transformed PTH from an inhibitor of promoter activity into an activator of that same activity, as did point mutations in the EGR1, Sp1, and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1 mRNA and of some Sp3 isoforms. Electrophoretic mobility shift assay showed a supershift of the -61 to -42-bp probe with an anti-EGR1 antibody in PTH-treated cells, suggesting that EGR1 binding is relevant for the inhibitory activity of PTH. We conclude that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression; to EGR1 binding to the proximal and core promoters; and to PKA and JAK/STAT pathway activation. This mechanism might be responsible, at least in part, for lower NHE3 expression and sodium reabsorption in renal proximal tubules in the presence of high PTH levels., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Our goal was to understand rickettsial spotted fevers' circulation in areas of previous outbreaks reported from 2006 to 2008 in Colombia. We herein present molecular identification and isolation of Rickettsia sp. Atlantic rainforest strain from Amblyomma ovale ticks, a strain shown to be pathogenic to humans. Infected ticks were found on dogs and a rodent in Antioquia and Córdoba Provinces. This is the first report of this rickettsia outside Brazil, which expands its known range considerably., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Hepcidin, the key regulatory hormone of iron homeostasis, and iron carriers such as transferrin receptor1 (TFR1), divalent metal transporter1 (DMT1), and ferroportin (FPN) are expressed in kidney. Whether hepcidin plays an intrinsic role in the regulation of renal iron transport is unknown. Here, we analyzed the renal handling of iron in hemochromatosis Hepc(-/-) and Hjv(-/-) mouse models, as well as in phenylhydrazine (PHZ)-treated mice. We found a marked medullary iron deposition in the kidneys of Hepc(-/-) mice, and iron leak in the urine. The kidneys of Hepc(-/-) mice exhibited a concomitant decrease in TFR1 and increase in ferritin and FPN expression. Increased FPN abundance was restricted to the thick ascending limb (TAL). DMT1 protein remained unaffected despite a significant decrease of its mRNA level, suggesting that DMT1 protein is stabilized in the absence of hepcidin. Treatment of kidney sections from Hepc(-/-) mice with hepcidin decreased DMT1 protein, an effect confirmed in renal cell lines where hepcidin markedly decreased (55)Fe transport. In the kidneys of Hjv(-/-) mice exhibiting low hepcidin expression, the iron overload was similar to that in the kidneys of Hepc(-/-) mice. However, in PHZ mice, iron accumulation resulting from hemoglobin leak was detected in the proximal tubule. Thus, kidneys exhibit a tissue-specific handling of iron that depends on the extra iron source. Hepcidin may control the expression of iron transporters to prevent renal iron overload.

The interaction of positively-charged antibiotic gentamicin with cell membranes was studied to determine if any changes in membrane organization were induced by the drug. Opossum kidney epithelia (OK) cells were used as models of eukaryotic cells. Two methods were used: laurdan fluorescence spectroscopy and fluorescence anisotropy recordings on 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) labeled cell suspensions. Both methods showed an altered membrane hydration and fluidity of gentamicin treated cells. Liposomes prepared from dimyristoyl-phosphatidylcholine (DMPC) mixed with cardiolipin, which mimics the heterogeneous charge composition of the natural cell membrane, were used to determine the effect of gentamicin on artificial bilayers. The membrane lipid packing as revealed by generalized polarization (GP) and fluorescence anizotropy variation with increasing temperature was studied. It was found that the generalized polarization of liposomal membranes containing a negatively charged lipid (cardiolipin) is higher in the presence of gentamicin; in the membrane of living cell (OK), gentamicin induces, on the contrary, a decrease of general polarization. Considering the role of membrane organization in the function of transmembrane channels and receptors, our findings suggest hypotheses that may explain the permeation of gentamicin through the living cell membrane by using these channels., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ischemic renal injury is a formidable clinical problem, the pathophysiology of which is incompletely understood. As the Na/H exchanger-3 (NHE3) mediates the bulk of apical sodium transport and a significant fraction of oxygen consumption in the proximal tubule, we examined mechanisms by which ischemia-reperfusion affects the expression of NHE3. Ischemia-reperfusion dramatically decreased NHE3 protein and mRNA (immunohistochemistry, immunoblot, and RNA blot) in rat kidney cortex and medulla. The decrease in NHE3 protein was uniform throughout all tubules, including those appearing morphologically intact. In the kidney cortex, a decrease in NHE3 surface protein preceded that of NHE3 total protein and mRNA. Kidney homogenates from rats exposed to mild renal ischemia-reduced cell surface NHE3 protein expression in opossum kidney cells in vitro, whereas homogenates from animals with moderate-to-severe ischemia reduced both total NHE3 protein and mRNA. The decrease in total NHE3 protein was dependent on the proteasomal degradation associated with NHE3 ubiquitylation measured by coimmunoprecipitation. The transferable factor(s) from the ischemic homogenate that reduce NHE3 expression were found to be heat sensitive and to be associated with a lipid-enriched fraction, and did not include regulatory RNAs. Thus, transferable factor(s) mediate the ischemia-reperfusion injury-induced decrease in NHE3 of the kidney.

Hexokinase (HK), the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. Since HK isoforms I and II have mitochondrial targeting sequences, we attempted to separate the protective effects of HK on cell metabolism from those on apoptosis. We exposed renal epithelial cells to metabolic stress causing ATP depletion in the absence of glucose and found that this activated glycogen synthase kinase 3β (GSK3β) and Bax caused mitochondrial membrane injury and apoptosis. ATP depletion led to a progressive HK II dissociation from mitochondria, released mitochondrial apoptosis inducing factor and cytochrome c into the cytosol, activated caspase-3, and reduced cell survival. Compared with control, adenoviral-mediated HK I or II overexpression improved cell survival following stress, but did not prevent GSK3β or Bax activation, improve ATP content, or reduce mitochondrial fragmentation. HK I or HK II overexpression increased mitochondria-associated isoform-specific HK content, and decreased mitochondrial membrane injury and apoptosis after stress. In vivo, HK II localized exclusively to the proximal tubule. Ischemia reduced total renal HK II content and dissociated HK II from proximal tubule mitochondria. In cells overexpressing HK II, Bax and HK II did not interact before or after stress. While the mechanism by which HK antagonizes Bax-mediated apoptosis is unresolved by these studies, one possible scenario is that the two proteins compete for a common binding site on the outer mitochondrial membrane.

In this paper, we characterize the uptake mechanism of fluorescein isothiocyanate-labeled human immunoglobulin G (FITC-hIgG) in opossum kidney (OK) epithelial cells, which have been shown to express megalin and cubilin. Confocal immunofluorescence microscopy showed the punctate expression of the neonatal Fc receptor FcRn in the cytoplasm, but not on the cell surface membrane. Temperature- and energy-dependent uptake of FITC-hIgG was observed at pH 7.4 but not at pH 6.0, indicating that the internalization of FITC-hIgG might not be due to FcRn, which has a binding affinity for IgG under acidic conditions. Under physiological pH conditions, human and bovine serum γ-globulin decreased FITC-hIgG uptake in a concentration-dependent manner. In addition, FITC-hIgG uptake was inhibited by various megalin and/or cubilin ligands including albumin, cytochrome c, transferrin and gentamicin. Endosomal acidification inhibitors (bafilomycin A(1) and chloroquine) significantly decreased the uptake of FITC-hIgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) decreased FITC-hIgG uptake. Potassium depletion and hypertonicity, conditions known to inhibit clathrin-dependent endocytosis, also decreased FITC-hIgG uptake. In contrast, caveolin-dependent endocytosis inhibitors (nystatin and methyl-β-cyclodextrin) did not decrease, but rather increased the uptake of FITC-hIgG. These observations suggest that the internalization of FITC-hIgG in OK cells might be, at least in part, due to megalin/cubilin-mediated, clathrin-dependent endocytosis.

Metabolically generated acid is the major physiological stimulus for increasing proximal tubule citrate reabsorption, which leads to a decrease in citrate excretion. The activity of the Na-citrate cotransporter, NaDC-1, is increased in vivo by acid ingestion and in vitro by an acidic pH medium. In opossum kidney cells the acid stimulatory effect and the ability of endothelin-1 (ET-1) to stimulate NaDC-1 activity are both blocked by the endothelin B (ET(B)) receptor antagonist, BQ788. Acid feeding had no effect on brush border membrane NaDC-1 activity in mice in which ET(B) receptor expression was knocked out, whereas a stimulatory effect was found in wild-type mice. Using ET(A)/ET(B) chimeric and ET(B) C-terminal tail truncated constructs, ET-1 stimulation of NaDC-1 required a receptor C-terminal tail from either ET(A) or ET(B). The ET-1 effect was greatest when either the ET(B) transmembrane domain and C-terminal tail were present or the ET(B) C-terminal tail was linked to the ET(A) transmembrane domain. This effect was smaller when the ET(B) transmembrane domain was linked to the ET(A) C-terminal tail. Thus, the acid-activated pathway mediating stimulation of NaDC-1 activity requires a functional ET(B) receptor in vivo and in vitro, as does acid stimulation of NHE3 activity. Since increased NaDC-1 and NHE3 activities constitute part of the proximal tubule adaptation to an acid load, these studies indicate that there are similarities in the signaling pathway mediating these responses.

High-responding opossums are susceptible to developing hypercholesterolemia on a high-cholesterol diet, but low-responding opossums are resistant. The observation of low biliary cholesterol and low biliary phospholipids in high responders suggested that the ABCB4 gene affects response to dietary cholesterol. Two missense mutations (Arg29Gly and Ile235Leu) were found in the ABCB4 gene of high responders. High responders (ATHH strain) were bred with low responders (ATHE or ATHL strain) to produce F1 and F2 progeny in two different genetic crosses (KUSH6 and JCX) to determine the effect of ABCB4 allelic variants on plasma cholesterol concentrations after a dietary challenge. Pedigree-based genetic association analyses consistently implicated a variant in ABCB4 or a closely linked locus as a major, but not the sole, genetic contributor to variation in the plasma cholesterol response to dietary cholesterol. High responders, but not low responders, developed liver injury as indicated by elevated plasma biomarkers of liver function, probably reflecting damage to the canalicular membrane by bile salts because of impaired phospholipid secretion. Our results implicate ABCB4 as a major determinant of diet-induced hypercholesterolemia in high-responding opossums and suggest that other genes interact with ABCB4 to regulate lipemic response to dietary cholesterol.

DNA delivery to cells can be improved by using particle carriers made from biodegradable polymers such as poly(lactic-co-glycolic)acid (PLGA). It is speculated that addition of targeting moieties to the particle surface to facilitate uptake can further enhance gene expression in specific cells or tissues. Taking advantage of well-known receptor/ligand interactions in intestinal and renal epithelial cells, we formulated PLGA particles with high density of surface-bound bovine serum albumin (BSA; approximately 768 molecules/particle). BSA-coated particles exhibited significantly higher uptake by cells expressing the albumin receptor, megalin, and resisted degradation in low pH. However, gene expression from BSA-coated particles was 3- to 10-fold lower than that from unmodified particles; this reduction in transfection efficiency was probably due to the slower DNA release rate from modified particles. In this setting, addition of a targeting feature to particles reduced their effectiveness. Our study highlights the importance of the interplay between cell uptake and payload release in the design of polymer drug carriers., From the Clinical Editor: DNA delivery to cells can be improved by using particle carriers such as PLGA. Taking advantage of known receptor/ligand interactions in intestinal and renal epithelial cells, PLGA particles with high density surface-bound BSA were formulated. BSA-coated particles exhibited significantly higher uptake; however, gene expression was 3 to 10-fold lower. Unexpectedly, the addition of a targeting feature to these particles reduced their overall effectiveness., (Copyright 2010 Elsevier Inc. All rights reserved.)

We have previously reported the cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) induces renal proximal epithelial cell death through NF-kappaB inhibition. However, the upstream and down-stream signaling pathways that NF-kappaB inhibition mediates 15d-PGJ(2)-induced apoptosis remain to be defined. In the present study, we determined whether NF-kappaB inhibition induces cell death through the mitochondrial apoptotic pathway and whether protein kinase A (PKA) functions upstream of NF-kappaB inhibition by 15d-PGJ(2). The role of NF-kappaB inhibition in this apoptotic pathway was evaluated using NF-kappaB p65 transfected cells. 15d-PGJ(2) induced cell death by a PPARgamma-independent mechanism and the cell death was prevented by NF-kappaB p65 transfection. 15d-PGJ(2) treatment caused disruption of mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, suggesting that 15d-PGJ(2) induces cell death through a mitochondria-dependent apoptotic mechanism. These changes by 15d-PGJ(2) were attenuated by NF-kappaB p65 transfection. 15d-PGJ(2) treatment resulted in an increase in Bax expression, which were blocked by NF-kappaB p65 transfection. 15d-PGJ(2) treatment caused PKA inhibition and 15d-PGJ(2)-induced cell death was enhanced by the PKA specific inhibitor H89. Inhibition of NF-kappaB by 15d-PGJ(2) was prevented by addition of forskolin, a PKA activator. Taken together, these results suggest that PKA-dependent NF-kappaB inhibition stimulates 15d-PGJ(2)-mediated mitochondrial apoptotic pathway through alterations in expression of the NF-kappaB target genes Bax.

BLAT (BLAST-Like Alignment Tool) analyses and interrogations of the recently published opossum genome were undertaken using previously reported rat ADH amino acid sequences. Evidence is presented for six opossum ADH genes localized on chromosome 5 and organized in a comparable ADH gene cluster to that reported for human and rat ADH genes. The predicted amino acid sequences and secondary structures for the opossum ADH subunits and the intron-exon boundaries for opossum ADH genes showed a high degree of similarity with other mammalian ADHs, and four opossum ADH classes were identified, namely ADH1, ADH3, ADH6 and ADH4 (for which three genes were observed: ADH4A, ADH4B and ADH4C). Previous biochemical analyses of opossum ADHs have reported the tissue distribution and properties for these enzymes: ADH1, the major liver enzyme; ADH3, widely distributed in opossum tissues with similar kinetic properties to mammalian class 3 ADHs; and ADH4, for which several forms were localized in extrahepatic tissues, especially in the digestive system and in the eye. These ADHs are likely to perform similar functions to those reported for other mammalian ADHs in the metabolism of ingested and endogenous alcohols and aldehydes. Phylogenetic analyses examined opossum, human, rat, chicken and cod ADHs, and supported the proposed designation of opossum ADHs as class I (ADH1), class III (ADH3), class IV (ADH4A, ADH4B and ADH4C) and class VI (ADH6). Percentage substitution rates were examined for ADHs during vertebrate evolution which indicated that ADH3 is evolving at a much slower rate to that of the other ADH classes.

We have previously demonstrated that the synthetic peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist ciglitazone induces apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) and nuclear translocation of apoptosis inducing factor (AIF) in opossum kidney (OK) renal epithelial cells. However, the precise mechanism by which ciglitazone induces activation of p38 MAPK and the role of AIF in the induction of the apoptosis are not defined. This study was therefore undertaken to determine whether the roles of reactive oxygen species (ROS) generation and intracellular Ca(2+) in the ciglitazone-induced activation of p38 MAPK and whether AIF nuclear translocation is responsible for the ciglitazone-induced apoptosis in OK renal epithelial cells. Ciglitazone caused generation of ROS and an increase in intracellular Ca(2+). Ciglitazone-induced cell death was reduced by the antioxidant Trolox, the Ca(2+) chelator EGTA, and the store-operated Ca(2+) channels (SOCC) blocker lanthanum chloride (La(3+)), indicating involvement of ROS and Ca(2+) in the ciglitazone-induced cell death. Ciglitazone-induced intracellular Ca(2+) increase was decreased by Trolox, while ROS generation was not affected by EGTA and La(3+), suggesting that ROS generation promote the increase of intracellular Ca(2+). Transfection of small interfering RNA (siRNA) of p38 MAPK or vector expressing microRNA (miRNA) of AIF prevented the ciglitazone-induced cell death. Activation of p38 MAPK, mitochondrial membrane depolarization, and AIF nuclear translocation induced by ciglitazone were inhibited by Trolox, EGTA and La(3+). Taken together, these results suggest that ROS-dependent intracellular Ca(2+) increase is responsible for activation of p38 MAPK and nuclear translocation of AIF by ciglitazone.

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

Secondary hyperparathyroidism and human cytomegalovirus (hCMV) seropositivity are highly prevalent in patients undergoing renal transplantation, and both are linked to the development of chronic allograft nephropathy (CAN). We investigated the hypothesis that parathyroid hormone (PTH) 1-84 regulates hCMV immediate-early gene (IEG) promoter activation in proximal renal tubular cells. PTH 1-84 enhanced hCMV IEG promoter (-548 to +92) activity in opossum kidney cells. Deletion analysis from the 5' end of the promoter localized the PTH 1-84 associated activity to the DNA sequence between -123 and -45. Mutation of an imperfect ATF/AP-1 DNA element within this region abrogated the PTH 1-84 effect and also strongly attenuated basal gene expression. Mobility shift analyses using this DNA element revealed that a member of the ATF-1 family was in the binding complex. In summary, we present evidence for a novel pathogenic role of PTH 1-84 in promoting hCMV immediate-early gene transcription.

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists have been reported to induce apoptosis in a variety of cell types including renal proximal epithelial cells. However, the underlying mechanism of cell death induced by PPARgamma agonists has not been clearly defined in renal proximal tubular cells. This study was therefore undertaken to determine the mechanism by which ciglitazone, a synthetic PPARgamma agonist, induces apoptosis in opossum kidney (OK) cells, an established renal epithelial cell line. Ciglitazone treatment induced apoptotic cell death in a dose- and time-dependent manner. Ciglitazone caused a transient activation of ERK and sustained activation of p38 MAP kinase. Ciglitazone-mediated cell death was attenuated by the p38 inhibitor SB203580 and transfection of dominant-negative form of p38, but not by the MEK inhibitor U0126, indicating that p38 MAP kinase activation is involved in the ciglitazone-induced cell death. Although ciglitazone-induced caspase-3 activation, the ciglitazone-mediated cell death was not affected by the caspase-3 inhibitor DEVD-CHO. Ciglitazone-induced mitochondrial membrane depolarization and apoptosis-inducing factor (AIF) nuclear translocation and these effects were prevented by the p38 inhibitor. These results suggest that ciglitazone induces caspase-independent apoptosis through p38 MAP kinase-dependent AIF nuclear translocation in OK renal epithelial cells.

Introduction: In obstructive liver disease bile salts are known to accumulate in and damage specific kidney cells. High Performance Thin-Layer Chromatography (HPTLC) was used to determine the membrane lipid composition of a range of kidney cells., Methods: Kidney cells were exposed to three hydrophobic bile salts (lithocholic, deoxycholic and chenodeoxycholic acids) and cytotoxicity was determined. In addition membrane lipids from the cells were extracted in a chloroform:methanol (2:1, v/v) solution and quantified by HPTLC., Results: The results reveal a differential toxicity to the bile acids with IC(50) values ranging from 79+/-5 microM to 394+/-13 microM. When the lipid composition of the most and least susceptible cells were assayed, the least susceptible cells had a much higher lipid composition (46.6+/-3.7 microg/mg protein compared to 28.1+/-5.2 microg/mg protein for the least susceptible cells)., Discussion: These results suggest that HPTLC may be a useful technique when determining the mechanisms of toxicity of compounds which cause the disruption of the cell membrane.

NaSi-1 encodes a Na(+)-sulfate cotransporter expressed on the apical membrane of renal proximal tubular cells, which is responsible for body sulfate homeostasis. Limited information is available on NaSi-1 protein structure and the mechanisms controlling its apical membrane sorting. The aims of this study were to biochemically determine the quaternary structure of the rat NaSi-1 protein and to characterize its expression in renal epithelial cell lines. Hexahistidyl-tagged NaSi-1 (NaSi-1-His) proteins expressed in Xenopus oocytes, appeared as two bands of about 60 and 75 kDa. PNGase F treatment shifted both bands to 57 kDa while endoglycosidase H treatment led to a downward shift of the lower molecular mass band only. Mutagenesis of a putative N-glycosylation site (N591S) produced a single band that was not shifted by endoglycosidase H or PNGase F, confirming a single glycosylation site at residue 591. Blue native-PAGE and cross-linking experiments revealed dimeric complexes, suggesting the native form of NaSi-1 to be a dimer. Transient transfection of EGFP/NaSi-1 in renal epithelial cells (OK, LLC-PK1 and MDCK) demonstrated apical membrane sorting, which was insensitive to tunicamycin. Transfection of the EGFP/NaSi-1 N591S glycosylation mutant also showed apical expression, suggesting N591 is not essential for apical sorting. Treatment with cholesterol depleting compounds did not disrupt apical sorting, but brefeldin A led to misrouting to the basolateral membrane, suggesting that NaSi-1 sorting is through the ER to Golgi pathway. Our data demonstrates that NaSi-1 forms a dimeric protein which is glycosylated at N591, whose sorting to the apical membrane in renal epithelial cells is brefeldin A-sensitive and independent of lipid rafts or glycosylation.

The extracellular signal-regulated kinase (ERK) and Akt have been reported to be activated by ischemia/reperfusion in vivo. However, the signaling pathways involved in activation of these kinases and their potential roles were not fully understood in the postischemic kidney. In the present study, we observed that these kinases are activated by hypoxia/reoxygenation (H/R), an in vitro model of ischemia/reperfusion, in opossum kidney (OK) cells and elucidated the signaling pathways of these kinases. ERK and Akt were transiently activated during the early phase of reoxygenation following 4-12h of hypoxia. The ERK activation was inhibited by U0126, a specific inhibitor of ERK upstream MAPK/ERK kinase (MEK), but not by LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K), whereas Akt activation was blocked by LY294002, but not by U0126. Inhibitors of epidermal growth factor receptor (EGFR) (AG 1478), Ras and Raf, as well as antioxidants inhibited activation of ERK and Akt, while the Src inhibitor PP2 had no effect. PI3K/Akt activation was shown to be associated with up-regulation of X chromosome-linked inhibitor of apoptosis (XIAP), but not survivin. Reoxygenation following 4-h hypoxia-stimulated cell proliferation, which was dependent on ERK and Akt activation and was also inhibited by antioxidants and AG 1478. Taken together, these results suggest that H/R induces activation of MEK/ERK and PI3K/Akt/XIAP survival signaling pathways through the reactive oxygen species-dependent EGFR/Ras/Raf cascade. Activation of these kinases may be involved in the repair process during ischemia/reperfusion.

Renal Fanconi syndrome occurs in about 1-5% of all children treated with Ifosfamide (Ifo) and impairment of renal phosphate reabsorption in about 20-30% of them. Pathophysiological mechanisms of Ifo-induced nephropathy are ill defined. The aim has been to investigate whether Ifo metabolites affect the type IIa sodium-dependent phosphate transporter (NaPi-IIa) in viable opossum kidney cells. Ifo did not influence viability of cells or NaPi-IIa-mediated transport up to 1 mM/24 h. Incubation of confluent cells with chloroacetaldehyde (CAA) and 4-hydroperoxyIfosfamide (4-OH-Ifo) led to cell death by necrosis in a concentration-dependent manner. At low concentrations (50-100 microM/24 h), cell viability was normal but apical phosphate transport, NaPi-IIa protein, and -mRNA expression were significantly reduced. Coincubation with sodium-2-mercaptoethanesulfonate (MESNA) prevented the inhibitory action of CAA but not of 4-OH-Ifo; DiMESNA had no effect. Incubation with Ifosfamide-mustard (Ifo-mustard) did alter cell viability at concentrations above 500 microM/24 h. At lower concentrations (50-100 microM/24 h), it led to significant reduction in phosphate transport, NaPi-IIa protein, and mRNA expression. MESNA did not block these effects. The effect of Ifo-mustard was due to internalization of NaPi-IIa. Cyclophosphamide-mustard (CyP-mustard) did not have any influence on cell survival up to 1000 microM, but the inhibitory effect on phosphate transport and on NaPi-IIa protein was the same as found after Ifo-mustard. In conclusion, CAA, 4-OH-Ifo, and Ifo- and CyP-mustard are able to inhibit sodium-dependent phosphate cotransport in viable opossum kidney cells. The Ifo-mustard effect took place via internalization and reduction of de novo synthesis of NaPi-IIa. Therefore, it is possible that Ifo-mustard plays an important role in pathogenesis of Ifo-induced nephropathy.

The present study evaluated the hypothesis of whether increases in vectorial Na+ transport translate into facilitation of Na+-dependent L-DOPA uptake in cultured renal epithelial tubular cells. Increases in vectorial Na+ transport were obtained in opossum kidney (OK) cells engineered to overexpress Na+-K+-ATPase after transfection of wild type OK cells with the rodent Na+-K+-ATPase alpha1 subunit. The most impressive differences between wild type and transfected OK cells are that the latter overexpressed Na+-K+-ATPase accompanied by an increased activity of the transporter. Non-linear analysis of the saturation curve for l-DOPA uptake revealed a Vmax value (in nmol mg protein/6 min) of 62 and 80 in wild type and transfected cells, respectively. The uptake of a non-saturating concentration (0.25 microM) of [14C]-L-DOPA in OK-WT cells was not affected by Na+ removal, whereas in OK-alpha1 cells accumulation of [14C]-L-DOPA was clearly dependent on the presence of extracellular Na+. When Na+ was replaced by choline, the inhibitory profile of neutral l-amino acids, but not of basic and acidic amino acids, upon [14C]-L-DOPA uptake in both cell types, was significantly greater than that observed in the presence of extracellular Na+. It is concluded that enhanced ability of OK cells overexpressing Na+-K+-ATPase to translocate Na+ from the apical to the basal cell side correlates positively with their ability to accumulate L-DOPA, which is in agreement with the role of Na+ in taking up the precursor of renal dopamine.

Angiogenesis is critical to the growth and regeneration of tissue but is also a key component of tumor growth and chronic inflammatory disorders. Endoglin plays a key role in angiogenesis by modulating cellular responses to transforming growth factor-beta (TGF-beta) signaling and is upregulated in proliferating endothelial cells. To gain insights into the transcriptional hierarchies that govern endoglin expression, we used a combination of comparative genomic, biochemical, and transgenic approaches. Both the promoter and a region 8 kb upstream of exon 1 were active in transfection assays in endothelial cells. In transgenic mice, the promoter directed low-level expression to a subset of endothelial cells. By contrast, inclusion of the -8 enhancer resulted in robust endothelial activity with additional staining in developing ear mesenchyme. Subsequent molecular analysis demonstrated that both the -8 enhancer and the promoter depend on conserved Ets sites, which were bound in endothelial cells in vivo by Fli-1, Erg, and Elf-1. This study therefore establishes the transcriptional framework within which endoglin functions during angiogenesis.

A liquid chromatographic-electrospray ionization mass spectrometric technique was developed for direct quantitation of iophenoxic acid (IA) in serum. IA was spiked into canine, feline, bovine, equine, and porcine sera, extracted, and quantified using negative ion monitoring following chromatographic separation on a Luna C18(2) 3 microm (100 mm x 2.1mm) reversed-phase column. The limit of detection was 25 ng/mL and the limit of quantification was 50 ng/mL. Inter- and intra-assay accuracy (86-113% and 87-115%, respectively) and precision (1.8-7.7%) were calculated. Analysis of serum collected from feral pigs, raccoons, and opossums following ingestion of IA-marked baits confirmed the appropriateness of this method for bait acceptance studies.

An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the final destination.

Background: Parathyroid hormone (PTH) rapidly down-regulates type IIa sodium-dependent phosphate transporter (NaPi-IIa) via an endocytic pathway. Since the relationship between PTH signaling and NaPi-IIa endocytosis has not been explored, we investigated the role of membrane microdomains in this process., Methods: We examined the submembrane localization of NaPi-IIa in opossum kidney (OK-N2) cells that stably expressed human NaPi-IIa, and searched for a PTH-induced specific phosphorylating substrate on their membrane microdomains by immunoblotting with specific antibody against phospho substrates of protein kinases., Results: We found that NaPi-IIa was primarily localized in low-density membrane (LDM) domains of the plasma membrane; PTH reduced the levels of immunoreactive NaPi-IIa in these domains. Furthermore, PTH activated both protein kinase A (PKA) and protein kinase Calpha (PKCa) and increased the phosphorylation of 250 kD and 80 kD substrates; this latter substrate was identified as ezrin, which a member of the ezrin-radixin-moesin (ERM) protein family. In response to PTH, ezrin was phosphorylated by both PKA and PKC. Dominant negative ezrin blocked the reduction in NaPi-IIa expression in the LDM domains that was induced by PTH., Conclusion: These data suggest that NaPi-IIa and PTH-induced phosphorylated proteins that include ezrin are compartmentalized in LDM microdomains. This compartmentalization may play an important role in the down-regulation of NaPi-IIa via endocytosis.

Mercury produces acute renal failure in experimental animal models, but the mechanism of tubular injury has not completely been clarified. There is an increased interest in the role of apoptosis in the pathogenesis of renal diseases that result primarily from injury to renal tubular epithelial cells. However, detailed studies of morpho-functional alterations induced by mercuric chloride in kidney cell lines are scarce. This work characterizes these alterations in OK cell cultures. Morphological alterations were profiled using light microscopy, transmission electron microscopy, and confocal microscopy, as well as mitochondrial functional assays in the cells exposed to low concentrations of HgCl2. At concentrations of 1 and 10 microM of HgCl2 there were no morphological or ultrastructural alterations, but the mitochondrial function (MTT assay) and intracellular ATP content was increased, especially at longer incubation times (6 and 9 h). At 15 microM HgCl2, both the mitochondrial activity and the endogenous ATP decreased significantly. At this concentration the OK cells rounded up, had increased number of cytoplasmic vacuoles, and detached from the cell monolayer. At 15 microM HgCl2 ultrastructural changes were characterized by dispersion of the ribosomes, dilatation of the cisterns of the rough endoplasmic reticulum, increase of number of cytoplasmic vacuoles, chromatin condensation, invaginations of the nuclear envelope, presence of cytoplasmic inclusion bodies, and alterations in the size and morphology of mitochondria. At 15 microM HgCl2 apoptotic signs included membrane blebbing, chromatin condensation, mitochondrial alterations, apoptotic bodies, and nuclear envelope rupture. Using confocal microscopy and the mitochondrial specific dye MitoTracker Red, it was possible to establish qualitative changes induced by mercury on the mitochondrial membrane potential after incubation of the cells for 6 and 9h with 15 microM HgCl2. This effect was not observed at short times (1 and 3h) with this same concentration, neither with 1 and 10 microM HgCl2 in all the studied times. Taken together, these findings indicate that low concentrations of HgCl2 induce apoptosis by inhibiting mitochondrial function, and the OK cell line may be considered a useful tool for the study of programmed cell death involving mercurial species and other heavy metals.

Background: Daily rhythms in mammalian physiology are generated by a transcription/translation feedback loop orchestrated by a set of clock genes. However, little is known about the molecular cascade from the clock gene oscillators to cellular function., Methods: The mRNA expression profiles of NHE3 and clock genes were examined in mice and rat kidneys. First, luciferase assays followed by a site directed mutagenesis of an E-box sequence were used to assess the CLOCK:BMAL1-transactivated NHE3 promoter activity. A direct binding of CLOCK:BMAL1 heterodimers to an E-box sequences of NHE3 promoter was confirmed by electrophoretic mobility shift assay (EMSA)., Results: We present evidence that renal tubular NHE3, the Na(+)/H(+) exchanger critical for systemic electrolyte and acid-base homeostasis, is a clock-controlled gene regulated directly by CLOCK:BMAL1 heterodimers in kidneys. NHE3 mRNA level in rat kidney displayed circadian kinetics, and this circadian expression was severely blunted in homozygous CRY1/2 double-deficient mice, suggesting that the transcriptional machinery of peripheral clocks in renal tubular cells directly regulates the circadian expression of NHE3. By analyzing the 5' upstream region of the NHE3 gene, we found an E box critical for the transcription of NHE3 via the CLOCK:BMAL1-driven circadian oscillator. The circadian expression of NHE3 mRNA was reflected by oscillating protein levels in the proximal tubules of the rat kidney., Conclusion: NHE3 should represent an output gene of the peripheral oscillators in kidney, which is regulated directly by CLOCK:BMAL1 heterodimers.

Background: Nephrotoxicity of cancer therapy using radiolabeled somatostatin analogues such as octreotide is due to ultrafiltration and reuptake by proximal tubular cells (PTCs). The mechanism of uptake is unknown. It could occur either by receptor-mediated endocytosis via a somatostatin receptor or, alternatively, the multiligand megalin/cubilin tandem receptor, or by fluid-phase endocytosis. To define the mechanisms of internalization and to identify potential receptors, we have studied the uptake and processing of octreotide by the PTC-derived opossum kidney (OK) cell line., Methods: We compared the kinetics of uptake and fate of (111)In-diethylenetriamine pentaacetic acid (DTPA)-D-Phe(1)-octreotide and (125)I-human serum albumin ((125)I-HSA). To determine the contribution of receptor-mediated endocytosis, we tested competition for uptake by octreotide and somatostatin and by various megalin/cubilin ligands [receptor-associated protein (RAP), albumin, transferrin, insulin, polymixin B] or basic amino acids. The subcellular localization of fluorescein isothiocyanate (FITC)-D-Phe(1)-octreotide was studied by confocal microscopy., Results: Kinetics of uptake of (111)In-DTPA-D-Phe(1)-octreotide and (125)I-HSA by OK cells were comparable, but only the somatostatin analogue was significantly retained intact. All megalin/cubilin ligands and basic amino acids strongly inhibited (125)I-HSA uptake, but these could not compete for >50% of (111)In-DTPA-D-Phe(1)-octreotide uptake. The same was found for somatostatin and octreotide. The noncompetable uptake of (111)In-DTPA-D-Phe(1)-octreotide was comparable to the clearance of Lucifer Yellow, a marker of fluid-phase endocytosis. By confocal microscopy, FITC-D-Phe(1)-octreotide colocalized with transferrin in endosomes, then accumulated in lysosomes., Conclusion: Receptor-mediated endocytosis via megalin/cubilin and fluid-phase endocytosis contribute about equally to the uptake of radiolabeled somatostatin analogues by OK cells.

Unlabelled: Background. Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors with multiple effects on target cell function. PPAR gamma activity is regulated by extracellular signal-regulated protein kinase (ERK), mitogen-activated protein (MAP) kinase, and PPAR gamma ligands have varying effects on activity of ERK. Different PPAR gamma ligands have been shown to have both protective and detrimental effects in the kidney. Since transcriptional activation by different PPAR agonists is ligand- and depot-specific PPAR gamma, we have examined the effects of different agonists on PPAR activity in the proximal tubule., Methods: Opossum kidney cells were used in all experiments, transiently transfected with a PPAR response element luciferase reporter and subject to stimulation with various PPAR ligands. The role of ERK and phosphorylation in PPAR gamma activation were studied, as were the effects of PPAR agonists on ERK activation and cell proliferation., Results: Transcriptional activity of PPAR was not stimulated by PPAR alpha agonists, and only very modestly stimulated by a PPAR beta agonist. The PPAR gamma agonists 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and troglitazone stimulated significant transcriptional activation and phosphorylation of PPAR gamma. These effects were more marked with 15d-PGJ(2). Thiazolidinediones attenuated 15d-PGJ(2) evoked PPAR gamma activation and phosphorylation. ERK activity positively regulated PPAR activation. Only 15d-PGJ(2) stimulated ERK activity and cell proliferation, and these effects were also inhibited by thiazolidinediones., Conclusion: PPAR gamma agonists exert differential effects in proximal tubule cells with thiazolidinediones behaving as partial agonists.

Background: Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, which are known to be critical factors in lipid metabolism, have also been reported to reduce proteinuria. The mechanism and its relevance to progressive nephropathy have not been determined. The aims of this study were to assess the direct effects of a PPARgamma agonist on tubular cell albumin uptake, proinflammatory and profibrotic markers of renal pathology, using an opossum kidney model of proximal tubular cells., Methods: Cells were exposed to pioglitazone (10 micromol/L) in the presence and absence of low-density lipoprotein (LDL) 100 microg/mL +/- exposure to albumin 1 mg/mL. Results were expressed relative to control (5 mmol/L glucose) conditions., Results: Pioglitazone caused a dose-dependent increase in tubular cell albumin uptake (P < 0.0001). Despite the increase in albumin reabsorption, no concurrent increase in inflammatory or profibrotic markers were observed. Exposure to LDL increased monocyte chemoattractant protein-1 (MCP-1) (P < 0.05) and transforming growth factor-beta1 (TGF-beta1) (P < 0.05) production, which were reversed in the presence of pioglitazone. LDL induced increases in MCP-1 and TGF-beta1 were independent of nuclear factor-kappaB (NF-kappaB) transcriptional activity. In contrast, tubular exposure to albumin increased tubular protein uptake, in parallel with an increase in MCP-1 (P= 0.05), TGF-beta1 (P < 0.02) and NF-kappaB transcriptional activity (P < 0.05), which were unaffected by concurrent exposure to pioglitazone., Conclusion: These findings suggest that dyslipidemia potentiates renal pathology through mechanisms that may be modified by PPARgamma activation independent of NF-kappaB transcriptional activity. In contrast, tubular exposure to protein induces renal damage through NF-kappaB-dependent mechanisms that are unaffected by PPARgamma activation.

Background: Chronic renal failure has been associated with oxidative stress. Serum sulfite, sulfate, cysteine, homocysteine, cysteine sulfinic acid, and gamma-glutamylcysteine are elevated in patients on hemodialysis, suggesting an accelerated catabolism of sulfur-containing amino acids or a reduced elimination of sulfite/sulfate, or both. Administration of metabisulfite has also been shown to damage kidney cells., Methods: Measurement of reactive oxygen species (ROS) was performed with the fluorescence of dichlorofluorescein (DCF), and that of intracellular ATP was by the luciferin-luciferase reaction. Oxidation of sulfite and succinate by isolated mitochondria from rat kidney was monitored polarographically. The fluorescent probe, 5, 5', 6, 6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was employed to assess any loss in membrane potential in energized respiring mitochondria. Activities of glutamate and malate dehydrogenases (GDH, MDH, respectively) were assayed by the spectrophotometric measurement of NADH. Sulfite was determined by HPLC-fluorimetric measurement of monochlorobimane-sulfite and cell viability was by the MTT procedure., Results: An immediate increase in ROS followed exposure of Madin-Darby canine kidney (MDCK), type II, and opossum kidney (OK) cells to 5-500 micromol/L sulfite. Depletion of intracellular ATP was also observed. A low rate of oxidation of 100 micromol/L sulfite was observed polarographically in isolated kidney mitochondria, but ADP-stimulated State 3 respiration was not apparent. ATP biosynthesis from the oxidation of glutamate in rat kidney mitochondria was significantly inhibited by coincubation with 100 micromol/L sulfite; this was not the case with malate, succinate, and TMPD/ascorbate. However, activities of both GDH and MDH in kidney mitochondrial extract were inhibited. The mitochondrial membrane potential and cell viability were not compromised., Conclusion: Micromolar sulfite elicited an immediate increase in ROS in MDCK, type II, and OK cells. This was accompanied by a depletion of intracellular ATP, which could be explained by its inhibitory effect on mitochondrial GDH. Although MDH was similarly inhibited, the impact was buffered by the high level of this enzyme in kidney mitochondria.

Interferon gamma-knockout mice were challenged with 5000 Sarcocystis neurona sporocysts acquired from a naturally infected opossum. Ponazuril was administered once, by gavage, at day 1, 3, 7, 10, or 14 post-infection (pi). Ponazuril was given at either 20 or 200mg/kg. Mice that survived to day 30 pi were euthanized. Severity of CNS infection was quantified as schizont density in the cerebellum. Unchallenged mice in treatment and non-treatment groups remained free of disease and gained weight throughout the experiment. All challenged mice, regardless of treatment, developed histologic evidence of CNS infection even though clinical signs were prevented in some groups. The greatest treatment benefits were seen in mice given 200mg/kg ponazuril between days 4 and 14 pi. Weight gain over the course of the experiment occurred only in mice that were given 200mg/kg ponazuril on day 7 or 10 pi. With the exception of groups given 200mg/kg ponazuril on day 7 or 14 pi, mice in groups that got sporocysts developed abnormal neurologic signs. No deaths before day 30 pi occurred in mice given ponazuril at 20mg/kg on day 7 pi or 200mg/kg on day 1, 7, 10, or 14 pi. This effect was not significant. Mice given 200mg/kg on day 7 pi had significantly fewer cerebellar schizonts than did those of the control group that was not given ponazuril. These results indicate that single-dose administration of ponazuril for prevention of CNS infection is partially protective when given between days 4 and 14 pi.

OK cells are a transformed cell line derived from opossum kidney proximal tubule cells. Prior studies have utilized this cell line to study both positive and negative transcriptional responses to Vitamin D. However, there was a noticeable decrease in sensitivity on the part of these cells to respond to Vitamin D treatment in transfection assays, particularly when assessing transcriptional activity from a heterologous promoter construct that used the chicken parathyroid hormone (cPTH) repressor Vitamin D response element (VDRE). Western blotting revealed the apparently diminished expression of both the Vitamin D receptor (VDR) together with its heterodimeric DNA-binding partner, the retinoid X receptor (RXR), in these cellular extracts. Co-transfection of either a VDR or RXR expression vector alone had little effect on hormone-dependent enhancer transcriptional activity from the human osteocalcin (hOC) reporter construct, or the degree of repression from the cPTH construct. Indeed, significant effects on repressor or enhancer activity were only observed in these cells when expression vectors for both the VDR and RXR were simultaneously introduced into the cells via transfection experiments. Analogous results were obtained irrespective of the identity of RXR isoform; co-transfection of either RXRalpha or RXRbeta expression vectors together with the VDR-produced similar improvements in repressor activity. Titration of Vitamin D hormone under conditions of co-expression of the two receptors indicated that half-maximal responses were comparable for both VDREs and occurred at <1nM concentration. In summary, these results are consistent with prior in vitro studies indicating interaction of the VDR with these VDREs occurs as a heterodimer complex with RXR. The decreased expression of both heterodimer partners observed in these cells could explain the requirement for additional VDR/RXR expression, in particular in order to compensate for the reportedly lower binding affinity of the heterodimer with the repressor cPTH VDRE. The extent of expression of both heterodimer partners, therefore, may act to modulate the available responses to Vitamin D in target cells.

In the kidney, 25-hydroxyvitamin D(3) (25(OH)D) is converted to 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D) by the 25(OH)D(3)-1alpha-hydroxylase enzyme, which contains a terminal cytochrome P450 (CYP1alpha) (systematic name: CYP27B1). Likewise, the kidney also produces 24,25-dihydroxyvitamin D(3) and 1,24,25-trihydroxyvitamin D(3) via a 24-hydroxylase whose terminal cytochrome P450 is CYP24. The purpose of this study was to characterize the transcriptional regulation of the CYP1alpha and CYP24 genes by parathyroid hormone (PTH) and 1,25(OH)(2)D in the kidney. Promoter-reporter gene constructs were transfected into opossum kidney (OK) cells, a renal proximal tubular cell line with endogenous PTH and 1,25(OH)(2)D receptors. PTH and forskolin stimulated CYP1alpha promoter activity via a cAMP-dependent pathway acting through the phosphorylation of CREB (cAMP-dependent response element-binding protein). This stimulation did not require new protein synthesis but may be modulated by short-lived proteins. 1,25(OH)(2)D modestly inhibited basal and forskolin-stimulated CYP1alpha promoter activity. The stimulation of CYP1alpha promoter activity by PTH and forskolin can account for the effect of these hormones on renal CYP1alpha mRNA levels. CYP24 promoter activity in transfected cells was increased by both 1,25(OH)(2)D and PTH, but there was no interaction between the two. The modest effects of 1,25(OH)(2)D and PTH on promoter activity and their lack of interaction do not account for the effects of these hormones on renal CYP24 mRNA levels. This suggests that there may be important posttranscriptional regulation of CYP24 mRNA in the kidney.

Sodium monofluoroacetate (1080) is a vertebrate pesticide widely used for possum control in New Zealand. Fluoroacetate is also a toxic component of poisonous plants found in Australia, South Africa, South America, and India. Because of its importance and effectiveness in pest control and the highly toxic nature of this compound, its acute sub-lethal and target organ toxicity have been extensively studied. In relation to its use as a pesticide its environmental fate, persistence, non-target impacts and general toxicology have been and continue to be extensively studied. Toxic baits must be prepared and used with extreme care, otherwise humans, livestock, and non-target wildlife will be put at risk. The high risk of secondary poisoning of dogs is a cause for concern. 1080 acts by interfering with cellular energy production. Possums die from heart failure, usually within 6-18 h of eating baits. Long-term exposure to sub-lethal doses can have harmful effects and strict safety precautions are enforced to protect contractors and workers in the bait manufacturing industry. Considerable care is taken when using 1080 to ensure that the risks of using it are outweighed by the ecological benefits achieved from its use. When its use is controversial, risk communicators must take care not to trivialise the toxicity of the compound. The benefits of 1080 use in conservation, pest control, and disease control should be weighed up alongside the risks of using 1080 and other techniques for pest control.

A simple HPLC method to separate human luteinizing hormone releasing hormone (LHRH) from its metabolites using an isocratic elution is described. Intact LHRH and five metabolites were separated in 11.4 min. The calibration curve (peak area versus concentration) was linear over the concentration range 1.25-35 microg/ml (r(2)=0.99) with the intercept not significantly different from zero (P>0.05). Intra-day and inter-day variability of the assay was less than 5% for repeat injections of 5, 14.5 and 29 microg/ml. The method was applied to evaluate the susceptibility of LHRH to enzymes present in the lumen and mucosal extracts of the gastrointestinal tract of possums. The major degradation products of LHRH were identified by HPLC separation, amino acid analysis and mass spectrometry as LHRH (1-5), LHRH (1-4), LHRH (1-3) and LHRH (3-4).

Background: Administration of cisplatin, cis-diamminedichloroplatinum (II) (CDDP), causes a severe impairment of renal function, including increases in urinary excretion of proteins. We recently found that CDDP inhibits vacuolar H+-ATPase, which plays an important role in receptor-mediated endocytosis in the renal proximal tubules. Therefore, CDDP-induced proteinuria may be due to an inhibition of the receptor-mediated endocytosis in the renal proximal tubules following a decrease in vacuolar H+-ATPase activity by the drug., Methods: Effects of CDDP on receptor-mediated endocytosis of albumin in opossum kidney (OK) epithelial cells, and on urinary excretion of albumin and vitamin D binding protein, which are reabsorbed in the renal proximal tubules by endocytosis, in rats were examined., Results: CDDP inhibited uptake of fluorescein-isothiocyanate (FITC)-albumin, a receptor-mediated endocytosis marker, by OK cells in a time- and concentration-dependent fashion. In contrast, CDDP treatment did not affect the uptake of FITC-inulin, a fluid-phase endocytosis marker. CDDP caused a decrease in the affinity and in the maximal velocity of FITC-albumin uptake. The adenosine 5'-triphosphate (ATP) content in OK cells was not changed by CDDP at concentrations that inhibited FITC-albumin uptake. The endosomal pH in OK cells was increased by CDDP treatment. Administration of CDDP to rats increased the urinary excretion of albumin and vitamin D binding protein., Conclusions: These results suggest that CDDP decreases the receptor-mediated endocytosis of protein following the inhibition of vacuolar H+-ATPase in the renal proximal tubules, and the inhibition of receptor-mediated endocytosis would be the mechanisms underlying the proteinuria induced by CDDP.

Sarcocystis neurona is the most important cause of a neurologic disease of horses, equine protozoal myeloencephalitis (EPM). Cats and other carnivores can act as its intermediate hosts and horses are aberrant hosts. Little is known of the sero-epidemiology of S. neurona infections in cats. In the present study, antibodies to S. neurona were evaluated by the S. neurona agglutination test (SAT). Cats fed sporocysts from the feces of naturally infected opossums or inoculated intramuscularly with S. neurona merozoites developed high levels (> or =1:4000) of SAT antibodies. Antibodies to S. neurona were not found in a cat inoculated with merozoites of the closely related parasite, Sarcocystis falcatula. These results should be useful in studying sero-epidemiology of S. neurona infections in cats.

Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT1 antagonist losartan. AT2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1 x 10(-6) M) but were partially attenuated by the PKC inhibitor H-7 (1 x 10(-6) M), Ca2+ channel blocker nicardipine (1 x 10(-5) M), Rho kinase inhibitor HA-1077 (1 x 10(-7) M) or p(44/42) MAP kinase inhibitor PD 98059 (5 x 10(-5) M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT1 and AT2 receptors. We conclude that Ang lI-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca2+, and PKC, Rho kinase and p(44/42) MAP kinase.

Two primary tumours, squamous cell carcinoma of the tongue and multiple bronchioloalveolar carcinomas, were diagnosed in a Virginia opossum (Didelphis virginiana). Two oral masses were located in the right ventrolateral surface of the tongue, near the frenulum, and the lungs contained multiple, widely distributed, nodular masses. Microscopically, the oral masses were composed of invasive cords of pleomorphic, polyhedral cells, typical of squamous cells. The multiple pulmonary masses consisted of non-ciliated, cuboidal, columnar, or occasionally polyhedral cells arranged in an alveolar pattern with multifocal areas of necrosis. This is the first report of spontaneous oropharyngeal squamous cell carcinoma in the Virginia opossum. However, multiple pulmonary adenomas have been reported previously in this species, the lesions being similar to those in sheep pulmonary adenomatosis (jaagsiekte). In the present study, immunohistochemical examination of the pulmonary tumours with a rabbit polyclonal antiserum to jaagsiekte retroviral capsid protein proved negative., (Copyright Harcourt Publishers Ltd.)

The aim of our study was to determine the effect of lipopolysaccharide (LPS) on sphincter of Oddi (SO) motility. Opossums received saline, Escherichia coli LPS (1.0 mg/kg), or E. coli LPS (1.0 mg/kg) and aminoguanidine (50 mg/kg), and the SO was removed 6-24 h later. At 12 h LPS decreased electrical field stimulation (EFS)-induced relaxation and increased baseline tone. These changes were reversed when the animals were pretreated with aminoguanidine. The dose-dependent decrease in EFS-induced relaxation by N(omega)-nitro-l-arginine was impaired after LPS, but not in animals that received LPS and aminoguanidine. The impaired EFS-induced relaxation after LPS was reversed when l-arginine was added to the tissue bath. Serum levels of NO(-)(2)/NO(-)(3) were increased with LPS as compared to saline or both LPS and aminoguanidine. Inducible nitric oxide synthase mRNA was readily seen in SO segments after LPS. LPS impairs EFS-induced relaxation and increases baseline tone of the SO. The effects of LPS on SO motility appear to be mediated by nitric oxide.

Background: Aristolochic acid (AA), present in Aristolochia plants, appears to be the toxin responsible for Chinese herbs nephropathy (CHN), a rapidly progressive tubulointerstitial nephritis. One of the earliest sign of CHN is the urinary excretion of low-molecular-weight proteins (LMWP), suggesting that AA is toxic to proximal tubules (PT)., Methods: The effects of AA on PT functions including reabsorption of LMWP were investigated on the well-established opossum kidney (OK) cell line, a model for PT, and compared with those of the classical PT toxin cadmium chloride (CdCl2)., Results: OK cell monolayers internalized albumin and beta2-microglobulin by receptor-mediated endocytosis, both proteins apparently competing for the same receptor, a complex of megalin and cubulin. The process was significantly impaired by 24-hour preincubation with AA (10 or 20 micromol/L) or CdCl2 (15 micromol/L). Furthermore, 24-hour exposure to AA followed by its removal during one to six days led to a persistent inhibition of the uptake of albumin, in contrast to the substantial recovery observed after CdCl2 removal. Neither AA nor CdCl2 affected cell viability, Na+-glucose cotransport or total rate of protein synthesis. AA significantly decreased megalin expression and formed specific DNA adducts in OK cells, similar to those found in kidneys from CHN patients., Conclusions: The present data support the involvement of AA in the early PT dysfunction found in CHN; furthermore, they suggest a causal relationship between DNA adduct formation, decreased megalin expression, and inhibition of receptor-mediated endocytosis of LMWP.

Background: Patients undergoing successful kidney transplantation often manifest overt hypophosphatemia associated with exaggerated phosphaturia during the early post-transplant period (2 weeks to 3 months). The mechanism for this phenomenon has not been fully elucidated. We tested the hypothesis that a circulating serum factor [non-parathyroid hormone (non-PTH)], which operates during chronic renal failure (CRF) to maintain phosphate (Pi) homeostasis, can increase fractional excretion of Pi (FE(PO4)) in normal functioning kidney grafts during the early post-transplant period, thereby causing phosphaturia and hypophosphatemia., Methods: Five groups of patients were studied: control subjects (group 1, N = 16), "early" (2 weeks to 1 month) post-transplant patients (group 2, N = 22), "late" (9 to 12 months) post-transplant patients (group 3, N = 14), patients with advanced CRF (glomerular filtration rate = 30 to 40 mL/min; group 4, N = 8), and patients who suffered from end-stage renal failure and were treated by chronic hemodialysis (group 5, N = 14). Group 2 manifested significant hypophosphatemia and phosphaturia when compared with groups 1 and 3 (Pi = 0.9 +/- 0.003 mg/dL, FE(PO4) = 68+/- 5%, P < 0.0005 vs. groups 1 and 3). Sera were taken from each of the five subject groups and applied to the proximal tubular opossum kidney (OK) cells. The activity of Na/Pi-type 4 (that is, OK-specific type II transporter) was evaluated by measuring Na(+)-dependent (32)Pi flux. The expression of Na/Pi type II mRNA and the abundance of Na/Pi protein were determined by Northern and Western blot assays, respectively., Results: When compared with sera from groups 1 and 3, 10% sera taken from groups 2, 4, and 5 (incubated overnight with OK cells) inhibited (32)Pi flux by 25 to 30% (P < 0.0003). Both Na/Pi mRNA and the expression of Na/Pi protein were markedly augmented under the same conditions (P < 0.05 groups 2, 4, and 5 vs. groups 1 and 3). Time-course analysis revealed that the up-regulation of Na/Pi protein by sera from groups 2, 4, and 5 was observed as early as four hours of incubation, whereas augmented abundance of Na/Pi mRNA was only seen after eight hours of incubation. The addition of PTH (1-34) to sera from groups 2, 4, and 5 abolished the augmented expression of NaPi protein. We labeled OK cell surface membrane proteins with N-hydroxysuccinimide bound to biotin (NHS-SS-biotin). Biotinylated transporters incubated with the different sera were precipitated by strepavidin and identified by Western blot analysis. Cells incubated in sera from group 2 showed increased membrane bound transporter when compared with control sera, whereas the intracellular pool of the transporter was comparable between the two groups., Conclusion: A non-PTH circulating serum factor (possibly phosphatonin) that increases FE(PO4) during CRF is also responsible for phosphaturia and hypophosphatemia in the early period following successful kidney transplantation. The putative factor inactivates Na/Pi activity along with inhibition of the transporter trafficking from the cell membrane into the cytosol.