Diet is not enough: The prevalence of calcium oxalate stones when combining an altered diet with genetic factors

Introduction •Kidneys are a vital set of organs which filter 200 quarts of blood daily •Filtration allows the blood to be cleaned and balanced chemically •These organs are capable of removing 2 quarts of waste daily •Filtration process occurs in the cortex •Filtration has 3 steps:

1. Filtration 2. Re-absorption3. Secretion

•Calcium oxalate stones: most common and formed by the binding of a waste product (Oxalate) and an excess of calcium in the kidney forming a precipitate•OPN and THP-proteins known to inhibit calcium oxalate stone formation•OPN is a phosphor-glyco protein which is an inhibitor of crystal nucleation in kidneys•THP is a glycoprotein which is an inhibitor of crystal aggregation and is proposed to form a gel-like webThe development of kidney stones is a common occurrence. Understanding how the kidney functions helps to determine how the formation of calcium oxalate stones occurs. The excess calcium and oxalate can bind in the medulla of the kidney to form a precipitate. Much research has been conducted which has found that OPN and THP are proteins which inhibit stone formation. These findings have lead doctors to look at genetics as a cause for calcium oxalate stone formation. This experiment combines both the genetic predisposition and dietary intake of mice in order to look at the combined effects of calcium oxalate stone formation.

By: Brandi VanFleet York College of Pennsylvania

(Marieb and Hoehn 2010)

(Marieb and Hoehn 2010)

HypothesisThe lack of OPN and THP (the double knockout) in combination with a change in diet will increase the number of renal stones produced in the kidney compared to double knock out with a normal diet

Research Design & Methods

Double Knockout OPN and THP

Wild Type Diet 1: Unlimited H2O

1.3% CaCO3 added to chow

Diet 2: 15ml H20/mouse/day

1.3%CaCO3 added to chow

Diet 3: Unlimited H2O0.4% CaCO3 added to

chow

Diet 4: 15ml H20/mouse/day 0.4% CaCO3 added to chow

Diet 5: 20mL H20/day/ 100g mouse

Normal chow

The above groups will receive their selected diet for a 3 month period. During this time, all 100 mice will be collected on a monthly basis for urinalysis. After the completion of the 3 month diet the mice will be euthanized for kidney dissection. After dissection, kidneys will be placed in paraffin and sectioned into 4µm portions. Theses sections will then be stained using Von Kossa staining procedures, which will show the presence of Ca2+ deposits. The rest of the urinary tract will also be removed from the mouse and examined to look for calcium oxalate stones which had begun to pass through the urinary tract.

Acknowledgements:I would like to thank Dr. Boehmler for all of her time in helping me to develop my research design, knowledge in all subject matter, and her help throughout the last year. I would like to thank Dr. Kleiner and Dr. Landau for their help with statistical analysis of my theoretical data. I would like to thank Prof. Hodgson for her vast knowledge of the kidneys and taking the time to teach me everything she knows. Finally, I would like to thank the entire Biology Department for their time over the last several years. Without their dedication we would not receive the help, knowledge, and skills that we will need to know for the rest of our lives.

Diet 1

Euthanized

Urine Samples: Once per month Measure pH and

volume

Kidney Histology: Von Kossa staining of kidney Ca+2

deposits Dissection of urinary tract for presence

of stones

Diet 2

Diet 1 Diet 2

Diet 3

Diet 3

Diet 4 Diet 5

Diet 4 Diet 5

Project Summary For years the main preventative treatment for kidney stones has been diet alteration. It is known that eliminating specific foods from a diet will help to lower the risks of future stone formation. In the last few years much research has been conducted on genes found inthe kidney which are involved with stone formation. The scientific world has yet to study the effects combining both the genetics and diet on kidney stone formation. This experiment would test the relationship between the genetics and diet for stone formation. It will include mice both with and without the genes believed to be associated with kidney stone formationas well as using several altered diets to determine which combination shows the most stone formation.The results of this study will give valuable insight intothe role genes and diet play in kidney stone formation which could lead to novel treatments in the future.

Review of Literature Kidneys &Kidney Stones (Marieb and Hoehn 2010)The kidneys have two main sections the medulla where the waste product is collected and the cortex. The cortex uses small cells known as nephrons to conduct the filtration process. With in each of the estimated 1 million nephrons per kidney is a golmerulus. This is where most of the filtration occurs. This is where the blood is filtered and a waste filtrate is produced. Kidney stones are created during the filtration process. After the final step of filtration, the filtrate sits in the medulla where certain wastes will bind to others causing them to precipitate out of solution and form kidney stones. Most commonly, excess calcium binds with the waste oxalate and forms calcium oxalate stones.

Osteopontin (OPN) While doctors are aware of effects of dietary intake increase the risk of stone formation, they are now looking into genetics as a key factor. OPN is one protein shown to be involved in the inhibition of stone formation . While involved with many body systems, it is known to be an inhibitor of crystal nucleation and Calcium oxalate stone formation (Worcester 1994). Tamm-horsfall protein (THP)THP is a glycoprotein synthesized only in the kidneys (Raffi 2009). THP is an inhibitor of crystal aggregation (Bleyer 2007). THP’s function is unknown, but they have proposed it is capable of folding into a gel like web, which then prevents small particles from binding to form a precipitate and produced calcium oxalate stones (Schaeffer 2009).

Previous findings of OPN, THP, and stone formation (Mo et.al. 2007)•Wild type mice did not spontaneously produce stones •OPN knock out mice showed about 10% spontaneously produce stones •THP knock out mice showed about 14% spontaneously produce stones •OPN and THP knock out mice (double knockout) showed about 40% spontaneously produced stones

Expected Results

• Increasing the amount of calcium in the diet will increase the number of calcium oxalate stones• Decreasing the amount of water in the diet will also increase the number of calcium oxalate stones • There is a genetic component responsible in spontaneously forming calcium oxalate stones•Mice which are lacking the genetic components as well as receiving a diet rich in calcium and low in water will produce the greatest amount of calcium oxalate stones

SignificanceThere is very little understood about the circumstances in which kidney stones form. With this experiment we would be able to develop further knowledge of this mechanism. It is an important step in determining how diet and genetics combine to produce kidney stones. It will also be important in determining if one of these components plays a larger role in kidney stone formation than another.

Literature CitedBleyer, A.J., Gupta, K. 2007. UMDO-Related Kidney Disease. Gene

Reviews [Internet]. Available from: National Library of Medicine and National Institutes of Health.

Marieb, E.N., and Hoehn,K. 2010. Human Anatomy and Physiology. Eight Edition. Pearson Education Inc., SanFransisco, CA.

Raffi,H.S., Bates, J.M. Jr., Laszik, Z., and Kumar, S. 2009. Tamm-Horsfall Protein Protects Against Urinary Tract Infections by Proteus mirabilis. The Journal of Urology [Serial Online] 181(5): 2332-2338.

Available from: PubMed CentralSchaeffer, C., Santambrogio, S., Perucca, S., Casari, G, and Rampoldi,

L. 2009. Analysis of Uromodulin Polymerization Provides New Insights into the Mechanisms Regulating ZP Domain-mediated

Protein Assembly. Molecular Biology of the Cell [Serial Online] 20(2): 589-599. Available from PubMed Central

Worcester, E.M.1994. Urinary Calcium oxalate crystal growth inhibitors. Journal of the American Society of Nephrology [Serial

Online] 5(5): S46-S 53. Available from: JASN

Staghorn Kidney Stone

Struvite Kidney Stone

Uric Acid Kidney Stone

Calcium Oxalate Monohydrate partially encrusted w/ Dihydrate

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