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.