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The kidney is the target for a wide variety of chemical agents, including heavy metals, haloalkenes, analgesics and antibiotics. The functional and metabolic characteristics of the proximal tubule (PT) predispose it as the primary site for xenobiotic damage. The aim of this study was to isolate and characterise human and rat PT cells in suspension and primary culture for use as defined models to investigate drug-induced PT cell damage in vitro . A second aim was to compare the response of human and rat systems to known nephrotoxins. Human and rat PT cells (90&'37 viable) were isolated from kidney cortex by collagenase digestion followed by isopycnic Percoll density centrifugation. This resulted in the formation of two distinct bands of cell at densities 1.040g/ml (A) and 1.060g/ml (B) for both preparations. Characterisation of human cells in terms of morphology, marker enzymes, retention of active transport systems and responsiveness to parathyroid hormone indicated that &'62 95&'37 of the cells in band B were proximal tubular. Each transport system demonstrated Michaelis-Menten kinetics; kinetic parameters suggested that a higher proportion of PT cells from the S1-S2 segment of the nephron were present in human isolates. Human isolated cells also contained levels of glutathione and cytochrome P450, in particular ethoxyresorufin-O-deethylase activity, a marker for the P4501A family, similar to the intact kidney. Both human and rat cells were successfully cultured in serum-supplemented medium (10&'37 v/v) with human cells reaching confluence by 3-4 days and rat by 5-6. Maximal attachment was seen when cells from both preparations were inoculated onto collagen coated plates with an additional layer of fibronectin. Only human cells were able to reach confluence on porous membranes and demonstated an enhanced morphology when compared to normal cultured cells. Cultured cells from both preparations retained an epithelial morphology and showed minimal secondary cell contamination as shown by light microscopy and in the case of human cultures additionally through immunohistochemical staining. Immunohistochemical staining also demonstrated that human cells in culture were depositing components of the extracellular matrix. The maintenance of PT cell function, throughout the time in culture, was shown following maintenance of active transport systems, in particular the glucose carrier system and on porous membranes the organic anion system. Only rat cells maintained the organic cation system in primary culture. In addition human cells maintained the preferential response to parathyroid hormone. Except for the transport of organic cations, the other carrier systems and responsiveness to hormones were evident at both sub-confluent and confluent stages of cell culture.

Class of 2010 Abstract / OBJECTIVES: To determine the clinical significance of a population of scorpions, C. sculpturatus, found in New Mexico. This includes the toxicity of the venom as well as the interactions of venom and antivenom. METHODS: This project will include a descriptive retrospective study of clinical and laboratory data obtained through a patient chart and analytical laboratory procedures to positively identify the species of scorpion responsible for the envenomation. Scorpion Collection Scorpions from the location of the reported sting will be collected for venom analysis. Specific details and directions will be obtained from the grandparent of the victim regarding the campsite at Caballo Lake State Park. Human Subject – N/A Sample Size The sample size of specimens needed should be a minimum of 50 scorpions. This is due to factors which include; extremely small volumes of venom produced by each individual, the possibility of no venom production, damaged telson during collection (anatomical feature used in the delivery of venom), size variation in specimens and short window of opportunity to collect. Since these scorpions are more active during summer months, and travel time must also be accounted for, only a few months a year are acceptable. Instrumentation and Variables This is primarily laboratory assays rather than clinical. The clinical aspect, a case study involving the victim of a scorpion envenomation, was the reason behind needing to identify this Centruroides species. The analytical laboratory findings will be what will determine the exact species of Centruroides. A number of laboratory instrumentations and tests will be used or performed. These include; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), SDS-PAGE with hyaluronic acid, turbidimetric absorbances of hyaluronidase, reverse phase high pressure liquid chromatography (RPHPLC), enzyme-linked immunosorbent assay (ELISA), lethal dose 50 (LD%) and effective dose 50 (ED50). Variables regarding these findings will include two main factors; the human factor and the instrumentation factor. First the human factor, samples collected must remain free of contaminants up to the point of analysis. The instruments RESULTS: 104 specimens were collected at Caballo Lake State Park. Four of these specimens were sent to a taxonomist and identified as C. sculpturatus. SDS-PAGE, turbidimetric HA, and RPHPLC showed no significant difference in venom between the New Mexico and Arizona Centruroides, but did show a significant difference between these two groups and the Mexico Centruroides. SDS-PAGE/HA and ELISA assays showed no significant differences between groups. LD50 and ED50 data were similar between New Mexico and Mexico Centruroides, both being more potent and more readily reversed by antivenom than the Arizona Centruroides. CONCLUSIONS: The assays which show possible differences between species, SDS-PAGE, SDS-PAGE-HA, turbidimetric HAase, RPHPLC, and ELISA were all identical between the New Mexico Centruroides species and the Arizona Centruroides sculpturatus. These findings were opposite when comparing New Mexico Centruroides species and the Mexico Centruroides limpidus limpidus. Three of the five assays showed a significant difference. Since the Mexico Centruroides limpidus limpidus is a known different species, this was expected. With this data the scorpion specimens collected in New Mexico have been identified as Centruroides sculpturatus. An interesting difference between the New Mexico and Arizona Centruroides sculpturatus was toxicity of the venom. The New Mexico groups had close to a two fold increase in toxicity. In fact, the toxicity of the New Mexico groups was equivalent to the Mexico Centruroides limpidus limpidus which is well documented as having an increased toxicity.

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The responses of freshwater (Daphnia magna. Pimephales promelas) and saltwater (Mysidopsis bahia. Cyprinodon variegatus) toxicity test species to elevated calcium concentrations and changing salinity conditions were investigated. The use of salinity as a criterion for selection between saltwater and freshwater test species was investigated by conducting both calcium and salinity toxicity tests. Salinity was determined to be an inappropriate criterion under conditions encountered in this study.