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Xinyi (Cindy) Chen- Sigma Xi Presentation 2015
- 1. VITAMIN D MITIGATES DIABETIC AND OXIDATIVE STRESSES ON KIDNEY CELLS Xinyi (Cindy) Chen Research in Biology North Carolina School of Science and Mathematics
- 2. Background: Diabetes and P-glycoprotein • Diabetes is now the seventh leading cause of death in the world. • The elevated glucose levels due to diabetes yield a down regulation of P-glycoprotein, a membrane transporter that eliminates toxins from cells. • P-glycoprotein plays a critical role in the absorption and elimination of drugs and toxins, so increasing the expression level of P-glycoprotein can delay or even prevent the onset of diabetes.
- 3. A proposed mechanism by which P-glycoprotein secretes substrates 2 P-gp substrate 1 P-gp Cell membrane Intracellular space Extracellular space Secretion 1. Passive uptake of substrate across cellular membrane. 2. Formation of P- glycoprotein channel through which the substrate is secreted out into the extracellular space.
- 4. Background: Vitamin D • The active form of vitamin D, calcitriol, has been shown to up regulate P-glycoprotein levels in the cell membrane. • The effects of vitamin D on diabetes have not yet been tested on the kidney, an organ that can be damaged by high glucose levels. • One of my goals was to determine if vitamin D can beneficially elevate P-glycoprotein expression levels to prevent diabetes by an in vitro study of canine kidney cells.
- 5. Background: Vitamin D and Hydrogen Peroxide • Research has shown that reactive oxygen species (ROS) including hydrogen peroxide (H2O2) significantly reduce P- glycoprotein expression, but this reduction was later prevented by co-treatment with various antioxidants. • Vitamin D can act as a membrane antioxidant, and this ability may be useful in counteracting the down regulation of P-glycoprotein expression induced by H2O2. • Investigating this link between vitamin D and H2O2 can shed light on the mechanism that vitamin D takes to up regulate P-glycoprotein levels.
- 6. Digoxin as an indicator for P-glycoprotein activity level • Digoxin is a toxin that’s pumped out of cells via the P-glycoprotein transporter in the cell membrane. • Lowered activity of P-glycoprotein causes a higher accumulation of digoxin in the cells, thus leading to greater cell death. 2 P-gp substrate (such as digoxin) 1 P-gp Cell membrane Intracellular space Extracellular space • Therefore, digoxin can be used to quantitatively measure P-glycoprotein activity levels via cell viability.
- 7. Objectives 1. Viability assay: Determine the duration of exposure to digoxin necessary to induce MDCK-II (canine kidney) cell death as well as the optimal concentration of digoxin that must be used. 2. Vitamin D and Glucose experiment: Determine if vitamin D has a beneficial effect on MDCK-II cells in different glucose concentrations by up regulating P-glycoprotein activity. 3. Vitamin D and H2O2 experiment: Determine if vitamin D can act as an antioxidant and reverse the down regulation of P- glycoprotein activity induced by H2O2, a reactive oxygen species.
- 8. General Methods • An established cell line of canine kidney cells (MDCK-II) was continuously cultured in Minimum Essential Media (MEM). • For the glucose and H2O2 experiments, all cells were kept in their respective treatments for 24 hours before they were removed, stained with trypan blue, and counted under a light microscope using a hemocytometer. • The number of live cells per unit volume (mL) was counted for each well, and the ratio of the number of live cells per mL in each well to the number of live cells per mL in the control well was calculated as the response variable.
- 9. Cell Viability Assays: Methods • In my first viability assay, the duration of exposure to digoxin was varied (6 time points) and the concentration of digoxin was kept constant at 40 nM (from previous research). Cell death was measured after each time point to determine which, if any, time point would induce significant cell death. • However, since 40 nM of digoxin wasn’t enough to induce cell death at any time point, a second test for viability was conducted using higher concentrations of digoxin, as shown in the next slide, and measuring cell death after a set time of 24 hours. • In both tests for cell viability, normal glucose (5 mM) and diabetic glucose (25 mM) concentrations were tested. • Photos were taken and analyzed using Fiji software to indicate live cell density.
- 10. 5 mM (Normal) 25 mM (Diabetic) Glucose concentration 128 nM Digoxinconcentration 400 nM 700 nM 1000 nM 1280 nM N=3 Cell Viability Assays: Experimental Design *Control = Ethanol
- 11. Cell Viability Assays: Results EtOH 700 nM 1000 nM 1280 nM • Each photo represents cells treated with a different digoxin concentration (shown in upper right of photos). • As digoxin concentration increased, cell density decreased. • Both 700 and 1000 nM of digoxin gave reasonable percentages of dead cells, so 850 nM of digoxin was used for the following two experiments investigating glucose and H2O2. • In the figure, dark gray areas represent areas with live cells and light gray areas represent areas with no cells. Photo credit: Xinyi Chen. Magnification: 100x
- 12. Vitamin D and Glucose: Methods • This experiment tested the effects of 5 concentrations of vitamin D on MDCK-II cells in 3 glucose concentrations. • Vitamin D concentrations: • 0 ng/mL = control • 12 ng/mL = concentration associated with a vitamin D deficiency • 20 ng/mL = lower bound for an adequate amount of vitamin D for health • 35 ng/mL = adequate concentration for health • 50 ng/mL = concentration above which vitamin D may yield adverse effects • Glucose concentrations: • 5 nM = normal glucose concentration in healthy individuals • 15 nM = concentration associated with prediabetes • 25 nM = concentration associated with diabetes
- 13. Vitamin D and Glucose: Experimental Design N = 3 5 25 12 20 50 35 15 VitaminDconcentration(ng/mL) Glucose concentration (mM) 0
- 14. Vitamin D and Glucose: Results 0 1 2 3 4 0 12 20 35 50 proportionoflivecells, relativetocontrol vitamin D concentration (ng/mL) 5 mM glucose 15 mM glucose 25 mM glucose *Control = cells treated with 5 mM of glucose and 0 ng/mL of vitamin D
- 15. Vitamin D and Glucose: Results • There is a clear upward trend in the proportion of live cells as vitamin D concentration increases, with a significant increase from 0 to 35 ng/mL of vitamin D, indicating that vitamin D is beneficial to MDCK-II cells. • A slight dip follows as vitamin D increases from 35 to 50 ng/mL, indicating that 50 ng/mL may be nearing the range where vitamin D becomes harmful. • For the cells treated with 15 mM glucose, all vitamin D concentrations brought the proportion of live cells up to or above the control, indicating that vitamin D can help cells survive in prediabetic conditions. • There is no upward trend among the cells treated with 25 mM glucose, suggesting that vitamin D is probably not an effective diabetes treatment when glucose levels are high but more so as a prevention strategy when glucose levels are lower.
- 16. Vitamin D and H2O2: Methods • This experiment tested the beneficial effects of vitamin D as an antioxidant by varying vitamin D and H2O2 concentrations. • Vitamin D concentrations: • 0 ng/mL = control • 12 ng/mL = concentration associated with a vitamin D deficiency • 20 ng/mL = lower bound for an adequate amount of vitamin D for health • 35 ng/mL = adequate concentration for health • 50 ng/mL = concentration above which vitamin D may yield adverse effects • H2O2 concentrations: • 0 μM = control • 200 μM = concentration used in previous experiment done on MDCK-II cells • 400 μM = double 200 μM to investigate the effects of increasing H2O2 concentrations on MDCK-II cells
- 17. Vitamin D and H2O2: Experimental Design N = 3 0 12 20 50 35 VitaminDconcentration(ng/mL) Hydrogen Peroxide concentration (μM) 200 400 0
- 18. Vitamin D and H2O2: Results 0 1 2 3 4 5 0 12 20 35 50 proportionoflivecells. relativetocontrol vitamin D concentration (ng/mL) 0 μM H2O2 200 μM H2O2 400 μM H2O2 *Control = cells treated with 0 ng/mL of vitamin D and 0 μM of H2O2 * * *
- 19. Vitamin D and H2O2: Results • Once again, increasing vitamin D concentrations yielded greater proportions of live cells, with a clear upward trend in cell viability as vitamin D concentration increases from 0 to 50 ng/mL. • Higher concentrations of H2O2 yielded lower proportions of live cells, with the harmful consequences becoming more pronounced with each increase in vitamin D concentration. • Increasing vitamin D concentrations counteracted the negative effects of increasing H2O2 concentrations, as shown by the 3 asterisked bars in the graph that have approximately the same proportion of live cells. • According to the dotted reference line, as little as 12 ng/mL of vitamin D is enough to offset the detrimental effects of the highest level of H2O2, further indicating the beneficial effects of vitamin D.
- 20. Conclusions 2 P-gp substrate (such as digoxin) 1 P-gp Cell membrane Intracellular space Extracellular space Secretion In the presence of vitamin D: • The expression of P-glycoprotein is up-regulated, allowing more digoxin molecules that enter the kidney cells to be secreted out. • Cell mortality is lowered because more cells are able to effectively secrete out the toxic molecules.
- 21. Conclusions • Higher vitamin D concentrations generally yielded less cell death, thus implying increased expression of P-glycoprotein; however, there is an optimal concentration of approximately 35 ng/mL of vitamin D. • Vitamin D had a more beneficial effect on cells treated with 5 mM and 15 mM glucose than with 25 mM glucose. • Vitamin D, as an antioxidant, neutralizes the down regulating effects of H2O2, with increasing levels of vitamin D counteracting the negative effects of increasing H2O2 concentrations. • Vitamin D can potentially be used as a supplement to delay or prevent the onset of diabetes by up-regulating P-glycoprotein expression through the same mechanism that H2O2, a ROS, takes to down regulate P-glycoprotein activity levels.
- 22. Future Work • Measure the percentage of live or dead cells in each well rather than just counting the number of live cells in each well and dividing it by the number of live cells in the control well. • Focus on a narrower range of vitamin D concentrations around 35 ng/mL in smaller gradations, since 35 ng/mL appears to be approximately the optimal concentration. • Study the mechanisms of vitamin D at the expression and transcription levels using molecular biology techniques. • Use human kidney cells, instead of canine kidney cells, to gain a more accurate picture of how vitamin D affects P-glycoprotein levels in humans.
- 23. Acknowledgments • Dr. Amy Sheck, Dean of Science at NCSSM • Ms. Korah Wiley, Instructor of Biology at NCSSM • Dr. Floyd Bullard, Instructor of Mathematics at NCSSM • Research in Biology Class of 2014 • Research in Biology Class of 2015 • Glaxo endowment at NCSSM
- 24. Works Cited 1. American Diabetes Association. Statistics About Diabetes. 2014. http://www.diabetes.org/diabetes-basics/statistics/. Accessed 7/15/14. 2. Chow, E., M. Durk, C. Cummins, and K. Pang. 2011. 1α, 25-Dihydroxyvitamin D3 up- regulates P-glycoprotein via the vitamin D receptor and not farnesoid X receptor in both fxr(-/-) and fxr(+/+) mice and increased renal and brain efflux of digoxin in mice in vivo. The Journal of Pharmacology and Experimental Therapeutics 337: 846-859. 3. Matheny, C., M. Lamb, K. Brouwer, and G. Pollack. 2001. Pharmacokinetic and pharmacodynamic implications of P-glycoprotein modulation. Pharmacotherapy 21: 778-796. 4. Qiu, J., H. Gao, Y. Liang, H. Yu, and R. Zhou. 2008. Comparative proteomics analysis reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial cells. Biochimica et Biophysica Acta 1784: 1857-1864. 5. Yeh, S., H. Pan, C. Lin, Y. Kao, Y. Chen, and C. Lin. 2012. Hyperglycemia induced down regulation of renal P-glycoprotein expression. European Journal of Pharmacology 690: 42-50.
Editor's Notes
- Change template? Change high glucose to stress? Add graphics (VISUAL :D)
- Over 29 million people in the USA currently have diabetes, which is now the seventh leading cause of death. The elevated glucose levels due to diabetes yield a down regulation of P-glycoprotein, a membrane transporter that eliminates toxins from the cells. P-glycoprotein plays a critical role in the absorption, distribution, and elimination of various drugs and toxins, so increasing the expression level of P-glycoprotein can potentially delay or prevent the onset of diabetes. Delete: which is an ATP-powered pump that transports hazardous substances out of cells. This protein is highly expressed in tumorous tissues as well as many normal organs and tissues throughout the human body, including the blood-brain barrier, intestines, and kidneys.
- Passive drug uptake across the cellular membrane. (2a) Formation of a hydrophobic channel (P-gp) between the intracellular and extracellular space through which the substrate is secreted into the extracellular space. Put this diagram 3 times: one now, one on digoxin slide, one at the end with vitamin D hydrogen peroxide
- The active form of vitamin D, calcitriol, has been shown to up regulate P-glycoprotein levels in the cell membrane. Also, research has shown that reactive oxygen species (ROS) including hydrogen peroxide significantly reduces P-glycoprotein expression and this reduction was later prevented by co-treatment with various antioxidants. Vitamin D acts as a membrane antioxidant, so this ability may be useful in counteracting the down regulation of P-glycoprotein expression induced by hydrogen peroxide. However, the effects of vitamin D on diabetes have not yet been tested on the kidney, an organ that can be damaged by high glucose levels. (delete: For example, one research experiment focused on the cell line Caco-2, which is a continuous line of heterogeneous human epithelial colorectal cells, and found that the expression levels of P-glycoprotein increased in the presence of calcitriol, with an increased level of transport of digoxin across the cell monolayer when vitamin D was added.) (Delete: Thus, high glucose more ROS lowered P-glycoprotein expression, and the pathway that reactive oxygen species take to down regulate P-glycoprotein expression can possibly be reversed by the action of vitamin D as an antioxidant. ) Make the connection that vitamin D’s mechanism is antioxidant more clear!!
- Therefore, digoxin can be used to quantitatively measure P-glycoprotein activity levels via cell viability. Digoxin is a toxin, and it is pumped out of cells via the P-glycoprotein transporter in the cell membrane. Previous research has shown that exposure to digoxin reduces cell viability in various cell types including neurons and endothelial cells. Not only does digoxin affect cell viability, but it also helps to indicate the activity level of P-glycoprotein in a variety of cells; low P-glycoprotein expression yields a high cellular accumulation of digoxin, thus leading to greater cell death while a high level of P-glycoprotein expression yields a lower cellular accumulation of digoxin and a lower percentage of cell death. (copied, but no need: In my experiment, digoxin performs 2 functions: firstly, exposure to digoxin reduces cell viability, and secondly, high cellular digoxin levels are linked to low P-glycoprotein expression. digoxin performs two functions: firstly, exposure to digoxin reduces cell viability. secondly, high cellular digoxin levels are linked to low P-glycoprotein expression in kidney cells (Yeh et al., 2012), allowing digoxin to serve as an indicator for P-glycoprotein activity level. In my experiment, these findings were effectively used to link cell viability to the activity level of P-glycoprotein in the presence of digoxin.) Move this slide to methods part of first objective
- I used MDCK-II cells, which are canine kidney cells, in my experiment. My first objective was to determine the duration of exposure to digoxin necessary to induce cell death as well as the optimal concentration of digoxin that must be used. My second objective entails my first experiment regarding vitamin D, and I planned to study if vitamin D has a beneficial effect on MDCK-II cells in different glucose concentrations by up regulating P-glycoprotein activity. My third objective was to determine if vitamin D can act as an antioxidant and reverse the down regulation of P-glycoprotein activity induced by hydrogen peroxide, which is a reactive oxygen species. (Delete: If vitamin D does have a beneficial effect by up regulating P-glycoprotein activity, the cells treated with higher concentrations of vitamin D should have lower percentages of cell death after treatment with digoxin, and supplementing vitamin D may be helpful in delaying or preventing the onset of diabetes.) (Delete: If greater concentrations of vitamin D should lower the percentage of dead cells due to increased P-glycoprotein activity and vice versa, then I can conclude that vitamin D up regulates P-glycoprotein activity by the same pathway that hydrogen peroxide down regulates P-glycoprotein activity by, thus providing information about the mechanisms of vitamin D.) Name objectives: viability assay ,glucose, peroxide
- According to previous research, 40 nM of digoxin was added to MDCK-II cells and incubated for 1.5 hours before cellular accumulation of digoxin was measured using western blot. Therefore, my first cell viability assay was conducted at 40 nM digoxin to determine which, if any, time point shown on the slide will induce significant cell death. Since 40 nM of digoxin was not enough to induce cell death at any time point, a second cell viability assay was performed by testing 5 concentrations of digoxin at one time point, as indicated by the figure on the right. In both tests for cell viability, both normal and high glucose conditions were tested so I know at which time point and what concentration digoxin can be used to test for cell viability in both treatments of glucose.
- According to previous research, 40 nM of digoxin was added to MDCK-II cells and incubated for 1.5 hours before cellular accumulation of digoxin was measured using western blot. Therefore, my first cell viability assay was conducted at 40 nM digoxin to determine which, if any, time point shown on the slide will induce significant cell death. Since 40 nM of digoxin was not enough to induce cell death at any time point, a second cell viability assay was performed by testing 5 concentrations of digoxin at one time point, as indicated by the figure on the right. In both tests for cell viability, both normal and high glucose conditions were tested so I know at which time point and what concentration digoxin can be used to test for cell viability in both treatments of glucose. Make blocks narrower Tell us what is normal and what is diabetes Color code control? (5 mM) Don’t put important words in dead areas Possibly take out left block because no significant results (just talk about it in words) Focus on right diagram
- Both qualitative and quantitative data was obtained from the second cell viability assay where I varied the digoxin concentration and kept the duration of exposure constant. MENTION THAT THE DEAD CELLS WERE WASHED OUT! These 4 photos have been altered with Fiji software to better show the cell density, with the dark gray areas representing areas where cells reside and the light gray areas representing areas with no cells. Each photo represents cells treated with 25 mM glucose and a different digoxin concentration: a) 128 nM, b) 400 nM, c) 700 nM, d) 1000 nM, e) 1280 nM, and f) ethanol control. Clearly, as digoxin concentration increased, cell density decreased, with a marked contrast between photo 1c to photo 1d (indicating that there may be some sort of threshold for the concentration of digoxin necessary to induce cell death. Both 700 and 1000 nM seem to give a reasonable percentage of dead cells, so 850 nM of digoxin was used for my two actual experiments investigating glucose and hydrogen peroxide. Change the color of f Just c, d, and f? No a or b? Don’t put in a-f, use the actual concentrations
- 0 = control 12 = concentration associated with vitamin D deficiency 20 = lower bound of an adequate amount of vitamin D for bone and overall health 35 = adequate concentration for health 50 = anything greater than this concentration can yield adverse effects 5 mM = normal glucose concentration 15 mM = concentration associated with prediabetes 25 mM = high glucose concentration common in diabetics
- The first experiment tested the effects of 5 concentrations of vitamin D on the MDCK-II cells in 3 glucose concentrations. 0 = control 12 = concentration associated with vitamin D deficiency 20 = lower bound of an adequate amount of vitamin D for bone and overall health 35 = adequate concentration for health 50 = anything greater than this concentration can yield adverse effects 5 mM = normal glucose concentration 15 mM = concentration associated with prediabetes 25 mM = high glucose concentration common in diabetics Put units in title and just have numbers on the sides Put the glucose higher
- Talk about the response variable!! Graph of proportion of live cells relative to control vs. vitamin D concentration, with each color representing a different glucose concentration and a reference line placed at y = 1, the control proportion, which was measured using the cells treated with 0 ng/mL of vitamin D and 5 mM of glucose. There is a clear upward trend in the proportion of live cells as vitamin D concentration increases, with a significant increase from 0 ng/mL to 35 ng/mL of vitamin D, indicating that vitamin D is beneficial to MDCK-II cells. A slight dip follows from the 35 ng/mL concentration as vitamin D levels increase to 50 ng/mL, indicating that 50 ng/mL may be nearing the range where vitamin D becomes harmful, which is consistent with previous literature. At every vitamin D concentration, 5 mM glucose yielded a greater proportion of live cells than 15 mM glucose and 25 mM glucose, with no suggestion of an upward trend among the cells treated with 25 mM glucose. This suggests that vitamin D is not strong enough to have a beneficial effect in diabetics, so it is probably not adequate as a diabetes treatment but more so as a preventative strategy against diabetes. For 15 mM glucose, all vitamin D concentrations brought the proportion of live cells up to or above the control. Scatterplot and fit a line? Threshold (not a direct linear relationship) Mention the y=1 line more (more explicit) make 2 slides: one with just first 3 bars and line and then another with the entire graph
- There is a clear upward trend in the proportion of live cells as vitamin D concentration increases, with a significant increase from 0 ng/mL to 35 ng/mL of vitamin D, indicating that vitamin D is beneficial to MDCK-II cells. A slight dip follows from the 35 ng/mL concentration as vitamin D levels increase to 50 ng/mL, indicating that 50 ng/mL may be nearing the range where vitamin D becomes harmful, which is consistent with previous literature. At every vitamin D concentration, 5 mM glucose yielded a greater proportion of live cells than 15 mM glucose and 25 mM glucose, with no suggestion of an upward trend among the cells treated with 25 mM glucose. This suggests that vitamin D is not strong enough to have a beneficial effect in diabetics, so it is probably not adequate as a diabetes treatment but more so as a preventative strategy against diabetes. For 15 mM glucose, all vitamin D concentrations brought the proportion of live cells up to or above the control.
- The second experiment tested the beneficial effects of vitamin D as an antioxidant by varying the vitamin D and hydrogen peroxide concentrations. 0 μM = control 200 μM = amount of hydrogen peroxide used in a previous experiment done on the same type of cells, which showed that treatment with various antioxidants reversed the down regulation induced by hydrogen peroxide 400 μM = double the previous amount
- The second experiment tested the beneficial effects of vitamin D as an antioxidant by varying the vitamin D and hydrogen peroxide concentrations. ((0 = control 12 = concentration associated with vitamin D deficiency 20 = lower bound of an adequate amount of vitamin D for bone and overall health 35 = adequate concentration for health 50 = anything greater than this concentration can yield adverse effects)) **don’t need to say this again I don’t think?? 0 μM = control 200 μM = amount of hydrogen peroxide used in a previous experiment done on the same type of cells, which showed that treatment with various antioxidants reversed the down regulation induced by hydrogen peroxide 400 μM = double the previous amount Put units in the title again
- Graph of proportion of live cells relative to control vs. vitamin D concentration, with each color representing a different hydrogen peroxide concentration and a reference line placed at y = 1, the control proportion, which was measured using the cells treated with 0 ng/mL of vitamin D and 0 μM of hydrogen peroxide. Once again, increasing vitamin D concentrations yielded greater proportions of live cells relative to the control and there is a clear upward trend in cell viability as vitamin D concentration increases from 0 ng/mL to 50 ng/mL, thus confirming the results of the first experiment. Also, higher concentrations of hydrogen peroxide yielded lower proportions of live cells, with the harmful consequences becoming more pronounced with each increase in vitamin D concentration (the proportion of live cells in the three hydrogen peroxide levels differ most when the cells were treated with 50 ng/mL of vitamin D). In addition to this, the (point out 2nd blue bar, 4th pink bar, and 5th yellow bar) all yielded approximately the same proportion of live cells, showing that increasing vitamin D concentrations counteract increasing H2O2 concentrations. Finally, according to the dotted reference line, as little as 12 ng/mL of vitamin D was enough to offset the effects of the highest level of hydrogen peroxide, further indicating the beneficial effects of vitamin D. Positive linear relationship No threshold response Make 2 slides just like before: one with just first 3 bars and the reference line and another with the entire graph
- Once again, increasing vitamin D concentrations yielded greater proportions of live cells relative to the control and there is a clear upward trend in cell viability as vitamin D concentration increases from 0 ng/mL to 50 ng/mL, thus confirming the results of the first experiment. Also, higher concentrations of hydrogen peroxide yielded lower proportions of live cells, with the harmful consequences becoming more pronounced with each increase in vitamin D concentration (the proportion of live cells in the three hydrogen peroxide levels differ most when the cells were treated with 50 ng/mL of vitamin D). In addition to this, the (point out 2nd blue bar, 4th pink bar, and 5th yellow bar) all yielded approximately the same proportion of live cells, showing that increasing vitamin D concentrations counteract increasing H2O2 concentrations. Finally, according to the dotted reference line, as little as 12 ng/mL of vitamin D was enough to offset the effects of the highest level of hydrogen peroxide, further indicating the beneficial effects of vitamin D.
- To conclude: increasing vitamin D concentrations increased cell viability, thus implicitly increasing P-glycoprotein activity levels, and this effect was most pronounced in the control glucose concentration of 5 mM. In my second experiment, I confirmed this result and also showed that vitamin D, as an antioxidant, neutralizes the down regulating effects of hydrogen peroxide, with increasing levels of vitamin D counteracting increasing concentrations of hydrogen peroxide. Vitamin D can effectively be used as a supplement to prevent or delay the onset of diabetes when blood glucose levels are still normal, and vitamin D possibly up regulates P-glycoprotein activity through the same mechanism that hydrogen peroxide, an ROS, down regulates P-glycoprotein activity by. (Delete: With diabetes becoming a prevalent health problem in our world today, it is imperative that scientists and researchers search for both treatments and prevention strategies for diabetes. My research looked into using vitamin D as a possible chemical to prevent or delay the onset of diabetes by up regulating P-glycoprotein expression, thus neutralizing the down regulating effects of high glucose levels on P-glycoprotein expression levels. ) (Delete: There is a continual upward trend in cell viability as vitamin D concentration increases, whether the stressor is glucose or hydrogen peroxide. In the first experiment, I was able to show that ) (Delete: In the second experiment, I was able to confirm my results from the previous experiment while also showing that ) Say what 35 ng/mL of vitamin D translates to in human terms First line is to general (because cell viability decreased from 35 to 50) Second bullet: vitamin D is more useful in 5 mM and 15 mM glucose than in 25 mM glucose
- Even though my method of standardization was effective in measuring the response variable, it would have been better to measure the percentage of live or dead cells for each well to give me a more accurate measurement of the effects of vitamin D, because each well probably had slightly different cell densities. Also, since I now know that 35 ng/mL of vitamin D appears to be the optimal concentration, I can focus on a narrower range of vitamin D concentrations around 35 ng/mL (ex: every 5 ng/mL from 25 ng/mL to 45 ng/mL). In the future, western blot and RT-PCR can be implemented in my experiment to study the mechanisms at the expression and the transcription levels and to look at whether or not vitamin D actually up-regulates P-glycoprotein activity and how much P-glycoprotein expression is up-regulated by. Finally, rather than using canine kidney cells, human kidney cells could be used as a more accurate description that pertains to us in general. Broaden the future work more (not TOO specific)