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Background and Objectives:
This project is part of an
ongoing program whose overall goal is composed of 2 parts:
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To identify diet and food
products (including supplements, nutraceuticals, functional foods)
that influence health and disease progression, specifically renal
health and disease.
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To understand how these diet
and food products influence renal health and disease, so that more
rational approaches to diet intervention can be made.
The specific project outlined in
this proposal is to examine mechanisms underlying the beneficial effect
of flaxseed oil on renal disease, using a genetic mouse model of chronic
renal injury. We have demonstrated that flax has a beneficial effect,
when compared to other oils containing polyunsaturated fatty acids.
Histological analyses show that dietary flaxseed oil diets rich in
18:3n-3 retard early kidney fibrosis progression compared to diets rich
in 18:2n-6 or in 22:6n-3. ARDI funding was sought to probe the
mechanisms by which flaxseed oil appears to mediate its beneficial
effects.
We have shown that flaxseed can
alter the fatty acid composition of the kidneys, and we have some
evidence from previous studies which show that eicosanoid production is
altered when flax is fed to animals with renal diseases. Our studies
with conjugated linoleic acid (CLA) indicate that the enzymes regulating
kidney eicosanoid production are altered when animals with renal disease
are fed CLA. We therefore proposed to examine these enzymes by
immunoblotting to determine whether the steady state levels of these
enzymes, which we have shown to be altered in renal disease, can be
modified by flax feeding. This type of study will help us understand
how flax mediates it beneficial effects in kidney disease (re: part 2 of
the overall goals of the research program) and give greater rationale
for the use of this prairie product in promoting health.
Procedure
and Project Activities:
Male and female pcy mice, at 4 weeks of age,
were fed three types of dietary fat, namely corn oil, flaxseed oil and
an algal oil containing 40% 22:6n-3. These mice have a genetic defect
which causes the development of kidney disease. The oils were fed at 2
different levels - high (20%) and low (4%) levels. The 3 oils were fed
at both high and low levels to the weanling polycystic mice, to
determine the effect of early dietary fat intervention in these animals.
At the end of the 8 week feeding
period, the animals were killed. Trunk blood, kidneys and the livers
were collected. Analysis of select serum and histological markers of
renal function and disease progression (serum blood urea nitrogen
creatinine, serum lipids, renal fibrosis and inflammation) showed that
flaxseed oil reduces disease progression the most of these 3 types of
oils. Therefore, we analyzed the expression of the key enzymes in the
20-carbon fatty acid metabolism, cyclooxygenases 1 and 2 (COX-1 and
COX-2) and cytosolic phospholipase A2 (cPLA2), in
order to determine whether these beneficial effects of dietary flaxseed
oil were related to modulating the 20-carbon fatty acid metabolism.
The levels of theses enzymes
were determined by immunoblotting. To do this, kidneys were
freeze-dried and a representative sample was homogenized in 100 volumes
of ice-cold homogenization buffer containing protease inhibitors to
prevent degradation of proteins. Homogenates were centrifuged at
100,000 x g for 30 min at 4°C and the supernatant, which represents the
cytosolic fraction, was collected. The remaining pellet was resuspended
in 15 volumes of the homogenization buffer containing a detergent (1%
Triton X-100) to solubilize the cell membrane fractions. This was
incubated on ice for 10 minutes and centrifuged at 100,000 x g for 30
min at 4°C. The resulting supernatant was collected as the particulate
extract and represents the Triton-soluble fractions of the plasma and
intracellular membranes. Protein concentrations of all fractions were
determined by protein assay using the Bradford assay, with bovine serum
albumin as the standard.
Denaturing gel electorphoresis (SDS-PAGE)
was then used to separate out the proteins to be measured. In this
procedure proteins are separated by loading them on a gel and applying
an electrical field to cause them to move through the gel. This
separates the proteins according to size and then the proteins are
transferred to a membrane so that they can be probed using antibodies
specific to the protein of interest. By linking a reporter to the
specific antibodies, the relative amounts of the protein can be
determined. Antibodies to COX-1 and COX-2 were purchased from Cayman
Chemical Co., and to cPLA2 from Santa Cruz, Inc. After
incubation of the membranes with the specific antibodies, a peroxidase
conjugated secondary antibody was incubated for 1 h at room temperature
at a dilution of 1/20000 - 1/50000. Immunoblots were incubated with
Chemi GlowTM, a commercial product that is converted by the
peroxidase in the secondary antibody into chemiluminescence. To
measure the amount of chemiluminescence produced due to each specific
protein, the Fluorchem Imager was used. A range of protein amounts was
loaded onto gels for each antibody to ensure that quantitative
comparisons could be made for results for each protein. The amount of
protein that was in the middle of the linear response and used for
analyses of these enzymes was 14ug of protein.
Data were
analyzed by ANOVA to determine whether there were main effects of
the type and level of fat. Differences between the types of fat were
determined by Least Significant Difference tests. A p value <0.05 was
accepted as statistically significant. Data were expressed as means ±
standard errors.
 Results and Discussion:
In summary, the analyses of the
proteins showed that when compared to the mice fed the corn oil
(control) diet, the level of kidney COX-2 was elevated in the mice that
were fed the diet containing the 22:6n-3, but not in the mice that were
fed the flaxseed oil (see figures below). This parallels the protective
effect of the flaxseed oil diet on the progression of early kidney
disease, when compared to the diet high in 22:6n-3. Of note, there was
no effect of these diets on either the COX-1 or cPLA2 levels,
other enzymes involved in eicosanoid synthesis. This shows that the
effect of these diets is specific for COX-2.
Flaxseed contains an n-3 fatty
acid, 18:3n-3. This study demonstrates that the length of the fatty
acid may be important in the dietary effects of n-3 fatty acids on the
kidney in the early stages of kidney disease. In the currently used
model, flaxseed oil containing 18:n-3 appears to be more beneficial in
slowing disease progression than the oil containing a longer chain n-3
fatty acid, namely 22:6n-3. COX-2 is generally associated with
increased inflammation, which is a significant contributor to the
progression of kidney disease. Although this study does not demonstrate
cause and effect, it does show that there is a positive correlation
between flaxseed oil, a slowing of early kidney disease progression and
preventing the rise in a protein associated with increased inflammation,
i.e. COX-2. With the current interest in aspirin-like anti-inflammatory
drugs that specifically target the COX-2 protein, this may have
important implications for the use of flaxseed oil in relation to in the
treatment of patients with kidney disease for inflammatory disorders
(including kidney disease itself).
Conclusions:
Understanding the mechanisms by
which flax promotes renal health will give scientific validity and
understanding to the use of flax as a dietary supplement and as a
functional food product. Renal disease as a result of diabetes is one
of the major causes of morbidity in those individuals afflicted with
diabetes. This is a very significant health issue and cost in
Manitoba.
Understanding how flax is
helpful in promoting renal health will aid in designing future studies
that examine flax effects in not only renal disease, but also many other
disorders. The research on flax in all diseases is not done in
isolation, and any molecular understanding of how flax benefits the
kidney will have an impact on research in other tissues and diseases as
well, just as findings with other oils and flax oil in other research
studies on other diseases influences research on the kidney. The
combination of all of this work will add credibility to any claims that
flax is a desirable component in our diets.
A significant issue that relates
to the credibility of health claims for a particular food product,
nutraceutical or supplement is the issue of safety in health. Not only
is it important to demonstrate added health benefits for a specific food
product, there must also be demonstrated an absence of any negative
health benefits. Therefore, research in a number of areas of health is
imperative. In addition, understanding the mechanisms by which a
product has its effects, will allow a more rational approach to its
use. Not only will this understanding be useful in applying this
product to our diets, but it also will help target that product to uses
that are most efficacious. Just as important is steering the use of a
specific product away from applications that are at not useful, and
possibly counterproductive. In the case of the current study, we have
demonstrated that the potentially detrimental effect of a dietary fat
source high in 22:6n-3 on elevating COX-2 levels is not observed in the
kidneys of the mice given the flaxseed oil, adding to the data which
shows that dietary flaxseed is not only effective in promoting health in
many conditions, but is also safe.
With respect specifically to
kidney disease, current technology of dialysis and transplantation
allows prolonged survival even with total kidney failure. However, the
costs of dialysis in Canada is close to $100,000 per year, and with over
10,000 patients on dialysis alone, the costs of this treatment
approaches $1 billion per year. The number of individuals requiring
dialysis continues to increase, due to the aging of the population and
the increased incidence of diabetes in the Western world. The benefits
to the health care system to even delaying the onset of renal failure
therefore is tremendous, in terms of not only economic costs, but also
social and personal costs.
The extent to which an increased
awareness and understanding of the benefits of flax in renal health is
achieved will determine what the economic benefits will be. It is not
possible to estimate with any certainty how great the economic impact
will be, but clearly the potential is large. The soybean industry is a
good example of how a large volume of research has resulted in the
understanding of North Americans of the beneficial effects of soy
products for heart disease. The legitimacy of the health claims due to
the large volume of data in relation to heart disease has also spilled
over into other areas of health that soy may impact, even though the
data for those specific areas may not be as persuasive.
Therefore, this type of research
will add to the bulk of evidence that points to the benefits of flax in
overall health and disease. While this one study may not have
measurable effects, as part of a growing volume of research, its effects
are potentially vast. Also, this project will generate data which will
be used as background information and supporting evidence for future
grant proposals – i.e. a small investment here may result in a much
larger amount of work being done on flax in the future in our program.
Acknowledgements:
The overall project was
supported by funding from the Natural Sciences and Engineering Research
Council. That funding was used to perform the study and to determine
the effects of these diets on the progression of early kidney disease
and on the composition of these kidneys. This data was published in
Lipids 39 (3): 207-214, 2004. The portion of the work supported by
ARDI, namely the analyses of the COX and cPLA2 proteins, as
described herein, was matched by a scholarship to a student in the PhD
program in Food and Nutritional Sciences at the University of Manitoba.
This student obtained a fellowship from the Manitoba Health Research
Council. Hence ARDI support allowed the extension of the work so that
the mechanisms underlying the demonstrated beneficial effect of the
flaxseed oil could begin to be probed.
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