Grace Swaim is a senior majoring in Biology. She was awarded a Spring 2016 Independent Grant which she used to conduct research on the effect of lithium on kidney cells under Dr. Mitsi Blount.
This semester has had a drastic change in research for me. For the past year and a half I have been studying the effect of lithium on kidney cells-specifically, how long term lithium treatment causes irreversible kidney fibrosis. Through my research, I investigated MMP-9, a protein that is responsible for remodeling the extracellular matrix and is implicated in fibrosis in a number of systems.
In order to study the interactions between lithium and MMP-9 last year, I performed a series of Western Blots to test for expression of MMP-9 and other components of the above pathway in a cell model that had been treated with increasing concentrations of lithium chloride. I found that:
This representative western blot shows an increase in MMP-9 expression in mTEC (mouse tubular epithelial) cells treated with lithium chloride up to 50mM LiCl. This seemed to support our original hypothesis that lithium is causing fibrosis through MMP-9.
Last fall, I continued my research by turning to a new method in order to see whether or not all the extra MMP-9 that was being produced was also being activated. Inactive MMP-9, after all, was not going to be able to contribute to fibrosis. To do so, I had to use a technique that was new to the rest of my lab, called gelatin zymography. Gelatin zymography is a technique that is similar to a Western Blot, with two exceptions. It is much faster than a western blot, and it specifically detects MMP-9 activity. If MMP-9 is active, it will look like a huge white blotch on the gel. We found.
This representative gelatin zymograph shows almost no MMP-9 activity from these treated cells (here I was using a similar cell type called mouse inter-medullary collecting duct 3). What could this mean for our hypothesis? At this point, I had actually already submitted my application for the grant. Now I was going to have to completely change direction! I repeated this experiment several times, but eventually my mentor and I were forced to start thinking of a new direction to move in. This time, lithium would be the good guy.
Acute kidney injury (AKI) is a potentially life-threatening condition where kidney function declines suddenly. If the kidney can repair the renal tubular epithelia damaged by AKI, the risk of developing future chronic kidney disease is decreased. Cellular recovery from AKI is regulated by multiple signaling pathways that are not fully explored and lithium, a common mood stabilizer, activates many of those same pathways. Remember the first figure? This is the first figure.
My mentor found a few recent papers that show short-term administration of lithium following AKI to have a protective effect. However, these papers did not fully analyze the signaling pathways involved. We decided to turn our attention to AKI and try and understand some of the signaling that was going on behind the scenes of the kidney recovery.
During last semester, I actually began mentoring a new student to the lab, and we set him up in cell culture at the beginning of this semester to get our finicky new HK-2 cells (human kidney 2) to start growing. They kept dying on us almost for an entire month, but Ashwin (former mentee, now super awesome) managed to get them to stay alive. I quickly relearned how to do RNA extraction, and now I’m busy extracting RNA from 18 different plates of these cells, which will keep me busy for a while. The plan is to study expression of all the proteins in that first image using both Western Blots and qPCR. Sadly, I don’t actually have any data yet, but hopefully we’ll find some interesting stuff in the data yet to come!
Visit the Undergraduate Research Programs website to learn more about applying for Independent Research Grants.
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