Abstract

Over 1 out of 10 people are expected to experience kidney stones in their lifetime. The pain has been compared to childbirth. That is why we aim to ease the pain with the use of enzymes. The problem with current treatments is their invasive and sometimes painful nature and the need for stones of specific sizes, making doctor visits a must.

Moreover, fifty percent of people get kidney stones again after having experienced them once. To avoid people having to endure having kidney stones multiple times. We propose using GMO Lactic acid bacteria, engineered to express the enzyme oxalate oxidase when oxalate is present. The bacteria will be administered through yogurt since it is a widely consumed beverage in arid parts of the world where low water quality and high temperatures lead to increased kidney stone formation.

Furthermore we successfully transformed our bacteria with the enzyme and proved its ability to break down oxalate. More so we designed four new programmes to ease the process of conducting docking simulations for future iGEM’ers.

The Issue - Kidney Stones

Kidney stones can affect anyone, with over 1 in 10 people expected to experience them at some point in their lives. The pain is often compared to the intensity of childbirth. Moreover, individuals who have had kidney stones are 30% more likely to develop another. This issue is especially pronounced in arid regions, which led us to visit Morocco. However, kidney stones are still commonplace in northern countries; in Denmark, for instance, 10% of men and 5% of women will endure the pain of kidney stones during their lifetime [1]. Kidney stones form when the concentration of certain waste products, most commonly oxalate, increases in the bladder, normally due to insufficient hydration [2]. These crystals develop in the kidneys and cause significant discomfort as they must pass through the ureter, a narrow tube with an average diameter of 3 to 4 mm [3].Regarding the typical size of kidney stones is the data unreliable since most kidney stones are passed at home and not in hospitals.

The Problems with Current Treatments

There are already ways to treat kidney stones, nonetheless, they all have some vital flaws, from being extremely painful to invasive.

• The painkillers and pray: The most common way to treat kidney stones is to simply drink a large amount of water and take over-the-counter painkillers. This can however be extremely painful. This is the primary treatment for stones from less than 2mm to 7mm. Which means that the other treatments (that try to make the stones smaller) usually also ends with the “painkillers and pray”-method, since their goal is to simply make this step less painful.

• Ureteroscopy: Works by inserting a small ureteroscope through the urethra, the bladder and the ureter up to where the stone is located. If it's small it can be snared with a basket device and removed. If it's bigger it will first need to be fragmented with a laser, before being removed [4]. This approach is normally done on stones from 7-10 mm

• Shock-wave lithotripsy: Works by blasting stones into fragments with the use of high-energy sound waves, to make them easier to pass. This means that this still has the same end as the “natural” treatment, however, the crystals may be smaller after the shock-wave lithotripsy making it less painful. This approach is normally done with stones around 10-30mm.

• Percutaneous nephrolithotomy: Works by surgery removing the stones, this does however have the same problems as normal surgery with the risk of infections and so on. That is also the reason why this approach is normally only done on stones bigger than 30mm [5][6].

Our Solution

Our initial idea was to develop a product aimed at breaking down the most common type of kidney stone: calcium oxalate. The goal was to create a less painful treatment option for kidney stone sufferers.

With this in mind, we began researching enzymes capable of breaking down calcium oxalate. During this process, we discovered an enzyme from barley (Hordeum Vulgare), which can degrade oxalate through the following reaction [7]:

Oxalate + O₂ + 2H⁺ ⇌ 2CO₂ + H₂O₂

There are two main problems with this reaction; the first is that it breaks down oxalate and not necessarily calcium oxalate. The second is that it creates hydrogen peroxide. Furthermore, if we had to create a therapeutic that worked directly on the kidney stones, we needed to be able to deliver it to the kidneys. While methods for drug delivery in the kidneys are still researched, targeting a medicine for the kidney is an extensive procedure, and something we did not attend the main focus of our project to be [8].This meant we had to go back to the drawing board and revise our ideas.

We went through revisions for our final product and landed on the final concept being genetically modified lactic acid bacteria expressing the enzyme oxalate oxidase. Bacteria naturally have pathways that would be able to break down the hydrogen peroxide, making it much safer [9].

Articles showed that lowering the concentration of oxalate in the gut largely reduces the risk of getting kidney stones [10]. So therefore we should be able to create a bacteria that breaks down the oxalate in the gut and prevents kidney stones in this way. Here we also solve the problem of delivery by making our product into a probiotic. While avoiding having to research whether oxalate oxidase can break down oxalate in the form calcium oxalate.

Further evidence that this product may work is the fact that species of bacteria with the capability to break down oxalate in the gut, are often lacking in individuals suffering from kidney stones. For example Oxalobacter Formigenes [11]. This is a specialist oxalotroph bacteria living in the gut. Meaning that the bacterias only source of energy is oxalate. One might think, oh lets just introduce this bacterium as a probiotic. but the fact that they only depend on oxalate, and are very sensitive to antibiotics [12] means that they may very easily be wiped out from the microbiome. in the practicals they seem to be hard to culture, and oral administration seems improbable cause the bacteria are sensitive to pH and most likely will be killed by the gastric acid in the stomach. (though some strains may be more pH tolerant) [13]. Therefore purely introducing Oxalobacter Formigenes will not suffice. and our project still has merit.

The Product - A Yogurt Drink

We wanted the product to be accessible and cheap, so a large number of people would benefit from the solution. That's why we landed on a yogurt drink (more specifically Ayran since it's common in the drier parts of the world). This would make our idea a drink that everyone could enjoy while giving it the possibility of easing the pain involved with kidney stones by making them smaller. This would work by choosing a promoter that would react when oxalate was present, and therefore not activating, (or activating in smaller amounts) if no kidney stones would be present.

Our solution is better than the current treatments, even though it still ends with the “painkillers and pray” method since it can be introduced and potentially help with stones of all sizes. Furthermore, it could have a longer-lasting effect if the bacteria we introduce can become a part of the microbiome and thereby continue to produce the enzyme whenever oxalate is available. This is however not a burden and the product would most likely need to have been consumed over a large amount of time before, to make sure the bacteria have a chance to become a part of the microbiome. The bacteria could however at one point lose the plasmid due to factors such as horizontal gene transfer which would make it “lighter” and thereby overpower the bacteria that still have the plasmid taking over their part of the microbiome. This is why it would not be a permanent solution respectfully.

The Wetlab Experiments

To make our proof of concept we attempted to transform E. Coli. Even though our final concept was GMO of lactic acid bacteria since E.coli is a standard in biotechnology, which meant that all our members that were in the GMO lab, had already worked with E.coli before, making it safer and a more secure choice for our project. The transformation was successful and the enzymes were isolated for testing which showed that they were able to break down oxalate 7, 9, 13,

Figure 1: Expression of Oxalate Oxidase in E. coli Using an Inducible Plasmid

This schematic illustrates the plasmid design used to express the oxalate oxidase enzyme in E. coli. The expression is driven by the araBAD promoter, which is inducible by the addition of arabinose. The B0034 ribosome binding site (RBS) facilitates efficient translation of the oxalate oxidase coding sequence. The oxalate oxidase enzyme catalyzes the breakdown of oxalate into hydrogen peroxide (H₂O₂) and carbon dioxide (CO₂). Transcription is terminated by the B0015 terminator, ensuring proper regulation of gene expression.

The Drylab Simulations

In dry lab, we assessed the possibilities of molecular interactions with naturally present compounds in the body inhibiting the activity of our main enzyme, oxalate oxidase. To run the docking we used AutoDock4 which enables the docking of metalloenzymes such as main enzyme. To complete the screening process of ligand-enzyme interactions a total of four individual python programs were developed to streamline the individual steps of the docking process. The docking simulations showed that histidine could bind in the active site leading to problems. This realization led us to use GST-tag instead of 6xHis-tag. Additionally, we employed AlphaFold from Google DeepMind to predict the final protein structure of the chain of GST-tag, TEV-site and oxalate oxidase. This provided us with theoretical proof that the enzyme’s functionality remained intact with the purification tag.

References

1. Admin, & Admin. (2024, August 8). 10 Surprising Facts about Kidney Stones. Keck Medicine of USC. https://www.keckmedicine.org/blog/10-surprising-facts-about-kidney-stones/
2. Kidney stones. (n.d.). National Kidney Foundation. https://www.kidney.org/kidney-health/kidney-stones
3. Rossiaky, D. (2022, October 13). What are the different types of kidney stones? Healthline. https://www.healthline.com/health/types-of-kidney-stones-chart#treatment
4. Ureteroscopy. (2021, August 8). Johns Hopkins Medicine. https://www.hopkinsmedicine.org/health/treatment-tests-and-therapies/ureteroscopy
5. Kidney Stones. (n.d.). National Kidney Foundation. https://www.kidney.org/kidney-topics/kidney-stones
6. Mathis, A., PhD. (2023, June 28). Kidney stone size chart for determining treatment in 2024. Healthcanal.com. https://www.healthcanal.com/health/kidney-stones/kidney-stone-size-chart
7. Bank, R. P. D. (n.d.). RCSB PDB - 1FI2: CRYSTAL STRUCTURE OF GERMIN (OXALATE OXIDASE). https://www.rcsb.org/structure/1FI2
8. Huang, L., Ye, Q., Chen, X., Huang, X., Zhang, Q., Wu, C., Liu, H., & Yang, C. (2024). Research progress of drug delivery systems targeting the kidneys. Pharmaceuticals, 17(5), 625. https://doi.org/10.3390/ph17050625
9. Sen, A., & Imlay, J. A. (2021). How microbes defend themselves from incoming hydrogen peroxide. Frontiers in Immunology, 12. https://doi.org/10.3389/fimmu.2021.667343
10. Chen, T., Qian, B., Zou, J., Luo, P., Zou, J., Li, W., Chen, Q., & Zheng, L. (2023). Oxalate as a potent promoter of kidney stone formation. Frontiers in Medicine, 10. https://doi.org/10.3389/fmed.2023.1159616
11. Siener, R., Bangen, U., Sidhu, H., Hönow, R., Von Unruh, G., & Hesse, A. (2013). The role of Oxalobacter formigenes colonization in calcium oxalate stone disease. Kidney International, 83(6), 1144–1149. https://doi.org/10.1038/ki.2013.104
12. Stewart, C. S., Duncan, S. H., & Cave, D. R. (2003). Oxalobacter formigenes and its role in oxalate metabolism in the human gut. FEMS Microbiology Letters, 230(1), 1–7. https://doi.org/10.1016/s0378-1097(03)00864-4
13. Ellis, M. L., Dowell, A. E., Li, X., & Knight, J. (2016). Probiotic properties of Oxalobacter formigenes: an in vitro examination. Archives of Microbiology, 198(10), 1019–1026. https://doi.org/10.1007/s00203-016-1272-y