The step-by-step development of a mammalian kidney, from its early beginnings in the embryo to its adult role as a vital filtration system, has been described by UCSD School of Medicine researchers using DNA gene-chip technology and novel software.
In research reported in the May 1, 2001 issue of Proceedings of the National Academy of Sciences (PNAS), researchers in the lab of Sanjay Nigam, M.D., professor of pediatrics and medicine, studied rat kidneys to find the specific genes that become active, then turn off, during kidney development. Nigam holds the Nancy Kaehr Endowed Chair in Pediatric Research.
According to Nigam, "roughly a third of all chronic kidney disease in children is related to a disorder of kidney development. This study is hopefully the first of a series in which we aim to identify specific subsets of genes necessary for different processes, which, together, lead to formation of the kidney."
"In essence," he adds, "the questions of how to engineer a kidney, how it regenerates and how it develops in the embryo are variations of the same broader question: how do you make a kidney?"
The study’s lead author, nephrologist and assistant professor of medicine Robert Stuart, M.D., agrees, noting that "the ultimate goal of kidney research is to one day grow replacement kidneys in the lab. Until then, we’re trying to understand all that is going on in the kidney as it develops."
"Although scientists have investigated specific target genes and diseases, this is the first really broad description of gene expression during organ development," Stuart says.
"Although it is commonly said that the huge volume of data makes gene expression analysis difficult, it really is the amazing complexity of tracking perhaps 30,000 variables which is the hard part."
That data comes from the high-tech use of gene chips, also called DNA microarrays, which permit scientists to track the expression – the turning on and off – of thousands of genes in a single, high-speed test.
In this relatively new technology, DNA fragments representing known sequences of genes are synthesized on silicon chips using technology related to that used to produce computer chips. Samples of RNA (the expressed genes) – in this case, RNA from the rat kidney – are labeled with fluorescent dye and applied to the gene chip. The RNA binds to just the DNA fragments on the chip with the correct, or complimentary sequence, thus indicating which rat kidney genes are activated at specific points in time. As the precise position of the highlighted DNA fragments on the chip is known, a computer can deconstruct a chip image into thousands of numbers.
However, the challenge has been the interpretation of the complex data provided by the chips. The field is relatively young and the few commercial software applications designed for gene expression analysis don’t meet all needs. Stuart wrote customized programs – called Equalizer and eBlot – to track gene expression and organize the findings into meaningful groups. The goal was to separate activated genes from the unchanging "housekeeping" genes.
"The thousands of housekeeping genes represent the haystack in which the needles – or genes that turn on or off during development – are buried," Stuart says. "Our goal was to whittle away all the chaff in the haystack by placing each gene expression measurement in the context of all the others."
First, the investigators isolated RNA from embryonic rats at gestational days 13, 15, 17 and 19, at birth, one week of age, and as non-pregnant adults. The researchers used DNA chips and their custom tools to identify 873 kidney genes out of 8,740 genes present on the DNA chips that significantly changed expression during development. These genes clustered into five distinct groups, based on their peak expression during development:
· The first stage of development focused on proliferation with many genes involved in DNA, RNA and protein production. This group also included several master regulatory genes.
· Peaking at the mid-embryonic stage, this group included continued expression of master regulatory genes and a marked production of extracellular matrix (ECM) genes, fibrous proteins outside cells that interact with each other and play a key role in determining the shape and activities of the cell.
· In newborn rats, the researchers witnessed a number of retrotransposon RNA genes, which are retroviruses that long ago inserted themselves in DNA and were then passed on to future generations as a footprint of a past event. Stuart notes that the function of these genes is unknown, but they may be involved in the stress of birth.
· Following birth, the developing kidney genes focused on energy production and the transport mechanisms that move substances such as toxins, water and urea from one compartment to another.
· Active genes in the adult rat included a mix of transporter genes not previously seen, detoxification enzymes, and additional genes involved in immune recognition and defense against oxidative stress.
An additional author on the PNAS paper is Kevin T. Bush, Ph.D. The work was conducted at the UCSD Departments of Medicine and Pediatrics and the UCSD Cancer Center. The research was funded by grants from the National Institutes of Health, the American Heart Association, and the Medicine Education and Research Foundation.
University of California – San Diego. April 2001.