Browsing by Subject "Kidney Tubules, Proximal"

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Isoflurane Preserves Viability of Highly Metabolic Renal Epithelial Cells Exposed to Anoxia
(2016-04-01) Mantravadi, Vasudha; Kojima, Koji; Ambardekar, Aditee; Lin, Xihui; Gingrich, Kevin
BACKGROUND: Cells subjected to ischemia, whether in the context of hypoxia, hypovolemia, or circulatory collapse, undergo damage and death as a result of oxygen deprivation. Previous studies have shown that general anesthetics can protect cells from ischemic injury by lowering their aerobic metabolism and decreasing production of toxic metabolites, among other mechanisms (1, 2, 3). This very preliminary study investigated the potential protective effect of isoflurane on the survival of cells that have a fairly high baseline metabolic rate, human renal proximal tubular epithelial cells (HK-2) and human microvascular endothelial cells (HMEC), in an anoxic environment. METHODS: Cultured HK-2 and HMEC cells were incubated in a Forma Scientific Anaerobic System at 37C either in the absence (control) or presence (experimental) of 5% isoflurane for 0, 24, 48, 72, and 96 hours. Cell viability and metabolic activity were then assessed using live/dead fluorescence imaging and an MTT cell metabolism assay, respectively. RESULTS: In vitro exposure of cells to anoxia without isoflurane over a period of 96 hours, resulted in a reduction of viability of HK-2 cells from a baseline of 98%, to approximately 8-9%. Over the same period of time, viability of cells exposed to isoflurane and anoxia decreased to 35%. This represented a fourfold increase in survival of HK-2 cells exposed to isoflurane at 96 hours. At earlier time points, both cell death in anoxia, and the protective effect of isoflurane were less dramatic. HMECs did not undergo significant cell death upon exposure to either anoxia or anoxia with isoflurane, with 98% of the cells surviving the exposure to anoxia in both cases. The net metabolic activity, as assessed by absorbance using the MTT assay, decreased in HK-2 cells over increasing periods of anoxia, a trend that did not change with the addition of isoflurane. Metabolic activity of HMECs remained intact and relatively stable throughout the course of anoxic exposure. CONCLUSION: In this preliminary study, continuous exposure of HK-2 cells to 5% isoflurane during anoxic incubation had a protective effect on cell viability over a period of 96 hours. Whether this effect was also present in the less metabolically active HMECs, was not determined, as anoxia over the time period of the study had little effect on cell viability in either the experimental group or in the control group. The protective effect observed for HK-2 cells will likely vary with differences in metabolic requirements of different cell types, types and concentrations of anesthetic agents, and duration of anesthetic exposure. Anesthetic treatment may need to be tailored specifically to a cell type to confer the protective effects desired.
Mitochondrial Dynamics in Renal Cell with Burn Serum Stimulation: An Observational Study
(2017-01-17) Maxwell, Christian T. W.; Huebinger, Ryan M.; Song, Juquan; Sehat, Alvand J.; Wolf, Steven E.
BACKGROUND: Myoblasts and myocardiocytes have increased cell death with burn serum stimulation, which is associated with mitochondrial fission and function impairment. Acute kidney injury is a significant issue in burn patients; however, the role of the mitochondrial response in renal cells (RCs) has not been explored. The purpose of this summer project was to establish a method that observes mitochondrial dynamics in renal epithelial cells upon exposure to burn serum. HYPOTHESIS: The mitochondrial fission/fusion cycle in human RCs is disrupted following exposure to burn serum. METHODS: Human primary renal proximal tubule epithelial cells were cultured with Renal Epithelial Cell Growth Media in an incubator at 37°C with 5% CO2. Upon reaching 65-70% confluency, RCs were treated with growth media containing 10% rat serum (RS) either from control rats or 40% total body surface area scald burn rats. 3nM of MitoTracker Green FM dye was added to all cell treatments to stain the mitochondria (MT). Live cell images were taken under a Nikon Ti Eclipse Confocal microscope at 6, 24, 48, 72, and 96 hour time points; subsequently, images were analyzed with Nikon software to identify fluorescent intensity, mitochondrial volume and elongation. T-tests were then applied to analyze the significance with Bonferroni correction. Significance after correction was determined if p < .01 . OUTCOMES: RC morphology was cuboidal and refractile in the culture media. During serum stimulation experiments, the MT of RCs gradually increased in fluorescent intensity and maintained a rod-like shape in both groups. Specifically, the MT exposed to normal RS had significantly higher intensity at the 72 and 96 hour marks than those in burn RS. Despite these results, cell viability was determined to be 48 hours, since 60% or more of cells had detached at the 72 and 96 hour marks, indicating cell death. There were no significant differences in elongation and volume between burn and normal serum stimulation at each time point. CONCLUSIONS: The cell culture protocol for human primary renal epithelial cells was established this summer. Although rats and humans share evolutionarily conserved traits, the RS did not significantly influence the more evolved human RC.