Kidney Involvement

"Research Approaches"

by Carol Feghali-Bostwick, Ph.D., Assistant Professor of Medicine, University of Pittsburgh, (originally published in "Scleroderma Voice," 2004 #2)

If we view scleroderma as a puzzle, then research is the process by which the disease cause, treatment, and eventual cure will be pieced together. A clear understanding of the ongoing disease process in scleroderma is only possible as additional parts of the puzzle are identified, piece by piece. Such pieces include a better understanding of the cause of the vascular abnormalities in scleroderma, the role of the immune system in the disease process, and the role of various cells in the development of fibrosis.

Carol Feghali-Bostwick, Ph.D.

New Techniques Are Generating New Data

New advanced techniques and novel methodologies used in research worldwide now make it feasible to generate data that promotes increased understanding of the ongoing disease process in scleroderma.

These techniques are being applied in research laboratories and include approaches such as DNA microarray analysis, tissue arrays, proteomics, and laser capture microdissection among others.

Such techniques are becoming pervasive in academic and non-academic research settings. They allow the identification of genes and their protein products that are part of the disease process. Results generated using these methods provide the basic information needed to identify drugs that can block the effects of these proteins and thus stop the disease process.

Figure 7. DNA serves as a template for the transcription of RNA. RNA is then translated into protein. The protein is made of a chain of amino acids which folds in a specific manner before the protein becomes functional.

DNA Microarrays

DNA (Deoxyribonucleic acid, a chemical structure that forms chromosomes) microarrays are a powerful tool for assessing which genes are turned on or off in a group of cells or tissues.

The first microarray experiment was described in 1995. Since then, hundreds of investigators have published research data using microarray analysis.

The popularity of this technique has mushroomed over the past few years. Its usefulness lies in the fact that the technique allows the comparison of gene expression levels of thousands of genes simultaneously.

The technique is relatively simple: DNA sequences representing thousands of known genes are printed on small slides or chips. Each DNA is printed at a precise location on every chip.

RNA (Ribonucleic acid/RNA carries the genetic information from DNA to those parts of the cell where proteins are made) is then extracted from cells grown in the laboratory or from tissues.

Figure 8. DNA Microarray Analysis. DNA representing thousands of genes is placed in specific spots on a slide. An DNA copy of RNA from cells of patient A and patient B is prepared and allow to find its match on the slide. Slides are scanned and data analyzed on a computer. Each column of the final result represents a patient's sample, and each row represents a gene. Red indicates genes turned on in one sample compared to the other. Green indicates genes turned down or off in one sample compared to the other.

A DNA copy of the RNA is generated in the laboratory, and each DNA copy is allowed to find its match on the chip. The chip can then be scanned, and the “profile” or list of genes turned on (activated or expressed) or off (inactivated or not expressed) can be compared in different samples from the same individual or from different individuals.

Microarray analysis has thus made the study of gene expression faster and less arduous.

One of the challenges of DNA microarrays includes the analysis and interpretation of the data necessary for the generation of useful and accurate results. One emerging application of this technique is the development of new drugs that target the identified genes.

Tissue Arrays

Tissue arrays differ from DNA microarrays in that pieces of tissue are used instead of DNA. Briefly, pieces of tissue from different samples or individuals are transferred by a core needle ‘biopsy’ from pre-existing tissue and placed on a microscope slide. Tissue spots are circular and each spot is less than 10 mm in diameter with spots spaced approximately 1 mm apart.

In contrast to the traditional approach of analyzing tissues by placing one section of tissue on an individual slide in specific spots, tissue arrays allow the simultaneous analysis of hundreds of tissue spots from multiple patients on one slide.

Slides are then analyzed for protein content and the amount of protein in tissues from different individuals is compared. Comparisons can be made from tissues, such as skin, of healthy individuals and patients with scleroderma.

Proteomics

Proteomics is derived from “protein” and ‘“genomics.” Proteomics is the study of proteins that are the gene products of DNA. It is based on the analysis of global differences in thousands of proteins in different samples.

Figure 9. Proteomics. Proteins are prepared from cells or tissues of patients A and B. The proteins are separated on a two-dimensional gel. Each protein is represented by a spot. Spots circled in red indicate proteins found in cells of patient A but not B. Spots circled in blue indicate proteins found in cells of patient B but not A.

Proteins, like cells, do not work in isolation. They interact with other proteins in a complex network. The protein content of cells is not static and can differ depending on the cell’s environment.

In addition, there are roughly 10–30 times as much proteins as genes. Proteomics allows researchers to characterize a large set of such interacting proteins by separating them in a two-dimensional gel based on their electrical charge and molecular weight.

After separation, each individual protein is represented by a spot on the gel. Proteins from different samples separated in such a manner can be compared. Spots representing individual proteins can be cut out and the proteins in them identified.

Thus, researchers can identify proteins that are present at different concentrations in different samples.

For example, one can determine whether a protein which serves as a “messenger” in the interactions of different cell types is present at different levels in scleroderma patients compared to healthy individuals.

This protein analysis can reveal information about the specific proteins present or absent in a sample, their levels, their potential function, and their interactions with other proteins.

Laser Capture Microdissection

Laser capture microdissection, also referred to as Laser Capture Microscopy (LCM), is rapidly becoming a very useful tool in the understanding of human disease. It allows the isolation of individual cells from thin sections of skin, lung, or other tissues.

Figure 10. A Representative 2-D Gel.

Cells or tissue sections are placed on a microscope slide on the microscope stage, and visualized using a microscope and a camera. Cells of interest or areas of tissue are identified and visualized on a monitor. The area around them is marked using standard computer graphics. A UV laser then cuts around the marked area. Cells in that area are then “captured” and transferred to a small cap. Captured cells can then be used for further analysis using a variety of assays such as polymerase chain reaction (PCR), DNA microarray, and proteomics.

Comparing which genes are activated (turned on) or inactivated (turned off), and which proteins are present in abnormally low or high levels in cells isolated directly from tissues eliminates the effect of artificial (in vitro) conditions used in the laboratory when growing cells outside the body, and thus allows us to understand real-life disease mechanisms.

Summary

The methods described above will enable us to design more effective drugs for the general treatment of scleroderma as well as individ-ualized therapies.

Not all patients respond to the same treatments. Thus, the ability to predict patient response to therapy and drug toxicity will allow us to use new therapeutic approaches, so that a drug will be used only in individuals who respond favorably to it.

For example, patients can be screened to allow medical professionals to select the most effective treatments for each individual patient.

Ongoing research will also provide us with tools for improved and faster diagnosis of scleroderma variants. In addition, data generated will yield information about the cause(s)/trigger(s) of scleroderma and a better understanding of the disease process.

Research analysis methods such as microarray and proteomics will also lead to increased interdisciplinary interactions between researchers and new research avenues. As these recent research tools identify new pieces of the puzzle and how they fit in the overall picture of scleroderma, new discoveries in scleroderma by researchers worldwide will be greatly accelerated.