UMaine Honors College: Molecular Mechanisms of Human Disease

An eight-day laboratory short course offering broad training in physiology, molecular biology, and microscopy research techniques for undergraduate students from The University of Maine.

Overview

This eight day course in functional genomics is sponsored by an INBRE grant from the National Center for Research Resources at the National Institutes of Health. 

Molecular Mechanisms Human Disease is an intensive and focused research short course for undergraduate students from the Honors College at The University of Maine. The goal of the course is to deepen students’ understanding of fundamental physiological mechanisms of human disease, and to teach hands-on research techniques. This course will give students an insight into fundamental elements underlying the molecular mechanisms of disease, and strengthen their ability to apply this knowledge to future research and clinical applications.

Undergraduate students will use a number of techniques and animal models (zebrafish, killifish) to study water balance, kidney function, and development. Students will learn and utilize molecular biology to understand the scientifc processes behind genetic screens for human disease.

Students will be assessed on the basis of their participation in lectures and laboratory research, as well as on group research presentations to be given on the final day of the course. After the course each student team will be required to put together a research poster that will be presented at the Maine Biological and Medical Sciences Symposium at MDIBL in April, 2010.

1. Human Diseases of Water Balance

Jennifer Bomberger, Ph.D.

Water balance is essential for human life, and the kidneys play a major role in regulating water balance. Water absorption is regulated in the collecting duct of the kidney by the antidiuretic hormone (ADH, a.k.a., vasopressin). Water is absorbed across the collecting duct when AVP binds to the vasopressin V2 receptor and promotes insertion of aquaporin-2 (AQP2) water channels into the apical membrane of collecting duct cells. Mutations in several genes affect the ability of the kidney to regulate water balance in humans. The genetic disease nephrogenic diabetes insipidus is caused by mutations in the AQP2 gene, and is characterized by an inability of the kidneys to concentrate the urine in response to ADH.

In this module, we will perform three experiments to examine water balance and kidney function in humans. In the first experiment, we will examine osmoregulation using an osmometer to measure urine osmolalities. We will assign group members to ingest different osmotic loads for the day and record the effects of these osmotic loads on the urine osmolality and volume during the day. The following day, we will discuss the results in the context of the renal physiology presented on day one. Second, the students will perform voluntary urinalysis using ChemiStrip 10 with SG urine testing system. Each Chemstrip contains several reagent pads that measure urine: specific gravity, pH, leukocytes, nitrites, protein, glucose, ketones, urobilogen, bilirubin, blood and hemoglobin. We will discuss what the Chemstrip measures, normal values, what abnormal values mean and how these values relate to renal function on day two. Finally, the third experiment will examine the molecular regulation of AQP2 channel function in collecting duct cells of the kidney using confocal microscopy (based on Nunes, et al 2008). After a brief introduction to confocal microscopy, students will investigate the effects of ADH on insertion of AQP2 water channels into the apical cell membrane in collecting duct cells. Pharmacological inhibitors of this process will also be examined. The goal of this module is to introduce basic principles of renal physiology using our own “in vivo” experiments and in vitro experiments utilizing cutting-edge technologies.

References

1. Robben JH, Knoers NV, Deen PM. Cell biological aspects of the vasopressin type-2 receptor and aquaporin 2 water channel in nephrogenic diabetes insipidus. Am J Physiol Renal Physiol. 2006. 291 (2): F257-70.
2. Nunes P, Hasler U, McKee M, Lu H, Bouley R, Brown D. A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK1 cells expressing aquaporin-2. Am J Physiol Cell Physiol. 2008. 295: C1476-1487.

2. Genetic Screening of Human Diseases

Bruce A. Stanton, Ph.D. and J. Denry Sato, D.Phil.

Many human diseases have a genetic cause or an underlying genetic predisposition. In many cases it is likely that diseases are caused by genetic variations in multiple genes, while it is clear that in some cases the disease phenotype is caused by mutation in a single gene. With the cloning and sequencing of the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan, et al., 1989), it became apparent that cystic fibrosis, the most common genetic disease in Caucasians, was caused by recessive mutations in a single gene. The most common of these mutations is delta-F508 (Kerem, et al., 1990), which is a deletion of the three-nucleotide codon encoding amino acid residue 508 in the CFTR protein. This mutation causes CFTR protein instability leading to degradation before it can be inserted into the plasma membrane where it functions as a channel for chloride ions moving into and out of the cell. Because the CFTR gene product is a chloride channel that mediates the hydration of the airways, mutations in CFTR results in a dehydation of the airway, and a reduced ability of cilia to clear mucus and entrapped pathogens and particulate material. This results in a persistent bacterial infection that is dominated by Pseudomonas aeruginosa, which elicits a profound inflammatory response. The bacterial infection and prolonged inflammatory response, and its sequella, lead to the degradation of lung function, which is the proximate cause of death n >95% of CF patients (Boucher, 2007).

In this module we will screen student volunteers for the presence of the delta-F508 mutation in one of the two copies of their CFTR gene. In Caucasians 1/30 individuals have one allele of a mutant CFTR. The polymerase chain reaction technique (Saiki, et al., 1985) will be used to amplify a region of the CFTR gene from cells obtained in buccal swabs. The CFTR amplicons will be generated from cDNA templates synthesized on total cellular RNA or from genomic DNA templates. The amplicons will be subjected to enzymatic DNA sequencing, and the region containing codon 508 will be examined. Control template DNA will be obtained from cells expressing human delta-F508 CFTR.

References

1. Boucher RC. Evidence for airway surface dehydration as the initiating event in CF airway disease. Journal of Internal Medicine 261: 5-16, 2007.
2. Kerem E, Corey M, Kerem B, Rommens J, Markiewicz D, Levison H, Tsui L-C, Durie P. (1990) The relation between genotype and phenotype in cystic fibrosis--analysis of the most common mutation (delta-F508). New Eng. J. Med. 323: 1517-1522.
3. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066-1073.
4. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354.

3. Acute Kidney Injury: Zebrafish Kidney Function and Disease

Sharon L. Ashworth, Ph.D.

Acute kidney injury (AKI) occurs at a high frequency in the American adult population with the nation wide cost for patient care estimated at over twelve billion dollars a year (1). Although the occurrence of AKI is high and leads to significant mortality rates in intensive care units, very little progress has been made in the last thirty years to overcome these statistics. In addition, very expensive medical procedures such as long term dialysis or renal transplantation often follow an occurrence of AKI. Ischemic AKI is not a simple disease. It is characterized by altered blood flow to the kidney that results in cell injury and inflammation. This leads to decreased urine production and increased toxic end products in the circulating blood. Normal and abnormal kidney development have been extensively studied in the many organisms, but several recent studies have focused on the zebrafish model system (2, 3). The zebrafish is a good model system to use for these studies because the zebrafish is small, transparent, has a short generation time, has large numbers of offspring, and has a simple kidney or pronephros.

For the AKI module, we will examine pronephros development in zebrafish embryos exposed to a nephrotoxin or hypoxia and compare their development to pronephros development in control embryos. Forty-eight hour post fertilization control and treated zebrafish embryos will be dechorinated and euthanized. These embryos will be fixed with paraformaldehyde for microscopic examination. The fixed specimens will be blocked and probed with pronephros specific antibodies followed by fluorescently labeled secondary antibodies. The embryos will be mounted in agarose and the fluorescently labeled pronephros will be examined by confocal laser scanning microscopy. Kidney function will be evaluated by clearance studies (4). Fluorescently labeled 70 kDa dextrans will be microinjected into the blood stream of forty-eight hour post fertilization zebrafish embryos. Two hours later the labeled dextran will be visualized in the zebrafish pupil and the fluorescent intensity will be recorded. The fluorescent intensity readings will be repeated every 24 hours to determine if the fluorescently labeled dextran has been cleared from the blood stream of the zebrafish. In a functional pronephros, the labeled dextrans will slowly clear from the blood. If the kidney is obstructed, clearance of the dextran will be delayed or not cleared.

References

1. Needham E. Management of acute renal failure. American Family Physician 72(9):1739-1746
2. Drummond IA. The zebrafish pronephros: a genetic system for studies of kidney development. Pediatr Nephrol 14: 428-435, 2000.
3. Drummond IA, Majumdar A, Hentschel H, Elger M, Solnica-Krezel L, Schier AF, Neuhauss SCF, Stemple DL, Zwarkruis F, Rangini Z, Driever W and Fishman MC. Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. Development 125:4655-4667, 1998.
4. Hentschel DM, Park KM, Cilenti L, Zervso AS, Drummond I and Bonventre JV. Acute renal failure in zebrafish: a novel system to study a complex disease. Am J Physiol Renal Physiol 288: F923-F929, 2005.

Faculty/Affiliations

Sharon Ashworth, Ph.D.
Assistant Professor of Biology
The University of Maine

Jennifer Bomberger, Ph.D.
Dartmouth Medical School

Angela Parton
MDI Biological Laboratory

Denry Sato, Ph.D.
MDI Biological Laboratory

Bruce Stanton, Ph.D.
Professor of Physiology
Dartmouth Medical School

Seminar Speakers/Affiliations

Keith Hutchison, Ph.D.
The University of Maine

Randy Dahn, Ph.D.
MDI Biological Laboratory

Keith DiPetrillo, Ph.D.
Novartis Research Institute

Carol Kim, Ph.D.
University of Maine at Orono

Carolyn Mattingly, Ph.D.
MDI Biological Laboratory

Tony Planchart, Ph.D.
MDI Biological Laboratory

Housing: Undergraduate participants will be assigned to a room in either Spruce or Birch Hall.

Travel: Students are encouraged to carpool to the MDI Biological Laboratory. On campus parking is available adjacent to the student dormitories.

Driving directions and a campus map are available on the MDIBL Travel Information page.

The Bar Harbor Chamber of Commerce has information regarding local attractions, including Acadia National Park.

This course is organized and supported by the Maine IDeA Network of Biomedical Research Excellence (ME-INBRE), with support from the National Center for Research Resources (NCRR) and the National Institutes of Health (NIH). (2P20-RR016463)