This summer, I had the opportunity to investigate the effect of environmental acidity on the mitochondrial shape of human kidney cells.
African Americans have a much higher chance of getting kidney disease than European Americans because they frequently carry risk mutations in a gene called APOL1. Two mutations, termed as G1 and G2, have been discovered only in African American populations. Those who carry two copies of G1 or G2 variants are more likely to develop chronic kidney disease (CKD). Thirteen percent of African Americans carry two copies of APOL1 variants, and these two variants contribute to at least 70% of CKD in this population (Science, 2010). However, only 20% of African Americans with two copies of APOL1 gene variants eventually develop CKD. Therefore, we believe there must be a “second stressor” working together with the APOL1 variants to cause CKD. Prior studies at Wake Forest School of Medicine have concluded that APOL1 G1 and G2 variants induced malfunction of the mitochondrion, a critical organelle providing energy for normal cell activity (Ma, JASN 2017). An acidic environment may have additional negative effects on cells carrying defective APOL1 G1 or G2 variants, for two reasons. The first reason is that the protein encoded by the APOL1 gene variants is sensitive to acid and an acidic environment affects its function (Thomson and Finkelstein, PNAS 2015). The second reason is that kidney tissue accumulates more acid than other tissues in the body and presents an acidic environment that may affect the function of APOL1 protein or potentially be that “second stressor”.
Therefore, we investigated how the difference in environmental acidity level, defined by pH value, affects the mitochondrial shape of cultured human kidney cells — that is whether the mitochondrial pattern is normal or fragmented in the cells exposed to environments of different acidity (=different pH). We performed confocal microscopy to scan serial images of mitochondria from human kidney cells expressing normal APOL1 G0 and G1/G2 variants, and used Fiji software to measure the relative mitochondrial length. These experiments were done when APOL1 expression levels were comparable among cells of different APOL1 genotypes, and the cell viability was intact when APOL1 was moderately expressed. We found that an acidic environment enhanced the negative effect of APOL1 risk variants on the mitochondrial pattern of kidney cells. According to these preliminary experiments, an acidic environment appears to elicit more mitochondrial fragmentation, which typically suggests that mitochondria may not be working properly.
Our preliminary studies suggest that the effect of environmental acidity in the kidney may be important for understanding how APOL1 variants pose a high risk for CKD in the African American community. Understanding why products of APOL1 gene variants, which are expressed around body, damage only the kidney and how and why kidney disease develops in those individuals who carry G1 and G2 variants of this gene will have a huge impact on the African American community and help in the fight against kidney disease. This study is also a part of the larger project to identify other possible “second stressors” to APOL1 gene associated kidney disease, and relatively high environmental acidity of the kidney may be one of those.
Realities of Research
When I first came into the lab, my mind was filled with awe and admiration because I saw how dedicated the lab members were every day. I have found that scientific research is such a complex process. In order for a research project to be successful, every step of the experiment should be planned ahead of time and in minute detail. My mentor and lab instructor have been so considerate. They discussed every aspect of the experiment, such as when to seed cells, when to add doxycycline, when to purify and extract RNA from cells, etc. This allowed me to perform the experiments efficiently and saved me a lot of time so that I would not make too many mistakes and have to start all over again. Without their instruction and guidance, I cannot imagine how I could have done the cell culture (including doxycycline controlled gene overexpression), taken images on the confocal microscope, isolated RNA from cultured cells, and run RT-PCR all in a short period of ten weeks. The procedures were overwhelming to someone like me who had no previous experience with cell experiments. After the first two weeks of “playing” with cells, I realized it is delicate work requiring patience and fine motor skills. For example, I struggled opening and closing the cap of the falcon tube only with one hand or pipetting minute amounts of fluid for PCR experiments. After several rounds of practice, I felt comfortable performing the task. Now, I have a true understanding of “practice makes perfect”. To analyze the mitochondrial patterns, we used a new technique called Fiji/MiNA software to measure the length of the mitochondria of kidney cells. Based on literature, Fiji/MiNA software was used to measure the length of mitochondria in neuron cells. We adopted this software and successfully adjusted the settings to apply to mitochondrial lengths in kidney cells and accurately captured the small fragments of mitochondria, which made the measurement more precise. Thanks to my mentor and lab instructor, their dedication and precision greatly influenced me. From time to time, my mentor praised me for my contributions to the study. As a team, we have been able to complete the study and obtain our results as we expected.
Life of a Scientist
Doing scientific research has always been my passion. Since my high school years, I longed that someday I could make new discoveries, which would eventually change people’s lives. Working in the lab, I have been so excited to learn new techniques needed to complete my project. I never get tired of tedious repeats of 200 scans of images. The best part of the fellowship is that I have a tremendous amount of guidance from my mentor to successfully complete the project in the time limit and obtain the results we are expecting. I have realized that scientific research not only requires patience and proper time management, but also requires thorough knowledge from a variety of disciplines such as physiology, anatomy, molecular/cell biology, etc. This is quite challenging for me as an undergraduate. I shared the frustrations of other fellows, whose experiments did not go as planned. They had to rely on trial and error, and were even unfortunate to find out that there was not enough knowledge to do the study in their lab and started moving in another direction. The worst part of my research project was that I had to spend days measuring and recording the mitochondrial lengths of human kidney cells after treatment with different pH solutions using software and manually enter the data first in Word, then into an Excel spread sheet. But overall, being a scientist especially working as a team member of this lab filled me with joy and pride as we were rewarded with a successful project.