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3
*
Department of Nutrition and Food Sciences and
Center for Research on Women’s Health, Texas Woman’s University, Denton, TX 76204;
Department of Surgical Pathology, University of Texas Southwestern Medical School, Dallas, TX 75235
3To whom correspondence should be addressed at: Department of Foods and Nutrition, H506 Duff Roblin, 190 Dysart Road, University of Manitoba, Winnipeg, MB, Canada R3T 2N2.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • high dietary fat • rats • kidney disease • testosterone • estrogen
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Ogborn et al. 1998
and 1999
, Tomobe et al. 1994
).
Although one study has demonstrated that dietary fish oil enriched in
(n-3) polyunsaturated fatty acids reduces life span in male, but not
female pcy mice (Aukema et al. 1992
), the
effect of total fat level in the diet on progression of PKD has not
been examined. High levels of fat, common in the Western diet, are
associated with increased plasma concentrations of sex hormones
(Adlercreutz 1990
, Wu et al. 1999
).
Studies in humans (Hamalainen et al. 1984
, Reed et al. 1987
) and in rats (Clinton et al. 1997
)
also show that high levels of dietary fat can increase serum sex
hormone concentrations.
Several studies suggest that sex hormones play an important role
in the progression of disease in animal models of PKD. In the
Han:SPRD-cy rat model of PKD, castration of males slows
disease progression, and administering testosterone to females or
castrated males increases cystic development (Cowley et al. 1997
, Zeier et al. 1994
). In this rat model,
dietary flaxseed or soy protein containing estrogen-like compounds
also ameliorates disease progression (Ogborn et al. 1998
and 1999
). In the CD1-pcy/pcy mouse model of
PKD, the effects of dietary soy protein are more pronounced in female
mice (Aukema et al. 1999
).
Although the endogenous serum concentrations of sex hormones in these
animals have not been reported, several studies indicate that serum sex
hormone concentrations may be altered in renal diseases
(Carlstrom et al. 1990
, Joven et al. 1985
). At the cellular level, testosterone stimulates fluid
secretion and ion transport in kidney cells (Sandhu et al. 1997
), suggesting that testosterone may contribute to the
hypersecretion of fluids, resulting in increased cyst expansion in PKD.
Prolonged administration of androgens to ovariectomized rats also
reduces renal function (Blantz et al. 1988
), whereas
castration preserves renal function in other animal models of renal
disease (Gafter et al. 1990
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Animals were
obtained from our breeding colony, which was derived from animals
provided by Dr. B. D. Cowley (University of Kansas Medical Center,
Kansas City, KS). The experimental protocol was in accordance with the
NIH guidelines (NRC 1996
) and was approved by
the University Animal Care and Use Committee.
The experimental diets (Table 1
) were based on the AIN-93G purified rodent diet (Reeves et al. 1993
), with the low fat (LF) and high fat (HF) diets providing
soybean oil as the sole lipid source at levels of 5 and 20 g/100 g of
diet, respectively. Lipid was substituted for carbohydrate, and the
levels of the other nutrients were adjusted to maintain a constant
nutrient-to-energy ratio. All diet ingredients were obtained from Dyets
(Bethlehem, PA). Han:SPRD-cy offspring from matings of
heterozygous (cy/+) rats were assigned randomly to either
the LF or HF diet group. Homozygous (cy/cy) rats
do not survive to weaning, and the surviving rats are either normal
(+/+) or diseased (cy/+). Random assignment of 15 males and
15 females to each dietary group resulted in the distribution of 9 and
12 diseased rats in the LF and HF male groups, and 12 and 8 diseased
rats in the LF and HF female groups, respectively, with the remainder
in each group being normal.
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and 1997
). Food
intakes (24 h) were determined during wk 2 and 5 of the experiment. During the last 3 wk of the study, vaginal smears were performed daily between 0900 and 1000 h in all female rats to determine the phase of the reproductive cycle. At the end of the 6-wk feeding period, rats were weighed, briefly anesthetized with CO2, decapitated and trunk blood samples were collected. Females were killed during the luteal phase of the estrous cycle, which is characterized by low estradiol levels. Serum was collected and kidneys and livers were removed, weighed and frozen immediately in liquid nitrogen. Rats across treatments were killed at the same time of day to account for diurnal variations in hormone levels.
The right kidney was frozen at -80°C and lyophilized to determine
water content. The left kidney was fixed in alcoholic Bouin’s reagent
and embedded in paraffin blocks. Sections (4 µm) for
measurement of cyst area were stained with hematoxylin and eosin.
Morphometric analysis of randomly selected sections was performed as
described (Aukema et al. 1999
). At an object-to-screen
magnification of 470X, cyst area was determined from nonoverlapping
fields until the whole kidney section was covered. Cyst score was
calculated by multiplying the percentage of cyst area by kidney weight,
standardized for body weight (Aukema et al. 1999
).
Urea nitrogen in the serum and creatinine in the serum and urine were
analyzed using reagents from Sigma kits 640 and 555, respectively
(Sigma, St. Louis, MO). Urinary protein was determined using the
Bio-Rad Coomassie dye binding assay (Hercules, CA) with bovine
serum albumin as the standard (Bradford 1976
).
Testosterone and estradiol were determined by RIA, using kits DSL-4000
and -4800, respectively (Diagnostic Systems Laboratories, Webster, TX).
Data for many variables did not display homogeneity of variance between
normal and diseased rats; because the purpose of these analyses (except
sex hormones) was not to examine genotype effects, data from normal and
diseased rats for all variables except sex hormones were analyzed
separately by two-way (gender x diet) ANOVA. Because only one
gender was examined for each sex hormone and these data passed tests of
normality and homogeneity of variance, serum sex hormone data from
normal and polycystic rats were analyzed together by two-way (diet
x genotype) ANOVA. If interactions were present, simple effect
differences were determined using Duncan’s Multiple RangeTest.
Differences and interactions were considered significant at
P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The effects appeared to be more pronounced in male rats, which is not
surprising because the disease progresses at a more rapid rate in males
than in females (Cowley et al. 1993
and 1997
). However,
a significant interaction was detected for only one variable measured
(serum creatinine, see Table 2
and below), and significance is reported
for all others on the basis of main effects of dietary fat level (Table 2)
. Kidneys of diseased rats were 19% larger in males and 12% larger
in females fed the HF diets compared with those fed the LF diets.
Congruent with increased cyst fluid accumulation during disease
progression, renal fluid content (g) was higher in diseased rats fed
the HF diet, by 21 and 15% in males and females, respectively. For
rats fed the HF diets, cyst scores calculated from morphometric
analysis were 37 and 8% higher in males and females, respectively. The
liver does not become cystic until much later in the disease process
(if at all), and the level of dietary fat had no effect on the liver
weights of either male or female rats (data not shown). Body weights
also did not differ in rats fed the two levels of dietary fat. Overall,
food intakes were significantly lower in rats fed the HF compared with
the LF diets, as would be expected with a more energy-dense diet
(15.1 ± 0.4 vs. 16.8 ± 0.4 g/d, respectively, P
< 0.01). Energy intakes, therefore, were not different between
rats fed the HF and LF diets (286 ± 7 vs. 272 ± 6 kJ/d,
respectively); because all ingredients were present at a constant
energy-to-nutrient ratio, intakes of nutrients also were similar (Table 2)
.
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. Although males had a higher concentration of urinary protein
than females (0.0040 ± 0.0003 vs. 0.0018 ± 0.0003 g/L,
P < 0.0001), neither disease state nor diet altered
the normal level of proteinuria in rats (data not shown).
Serum testosterone concentrations were lower in diseased male rats
compared with normal males. In normal males fed the HF diet, serum
testosterone concentrations were twice those in rats consuming the LF
diet. In contrast, testosterone concentrations were unaltered by
dietary fat levels in diseased rats (Table 3
). Serum estrogen concentrations were not different in diseased compared
with normal rats, and dietary fat level did not have an effect on the
estrogen concentrations in females (Table 3)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several markers of renal function were altered adversely in diseased
male rats fed the HF diet compared with the LF diet. This may have been
observed predominantly in males because disease progression is more
aggressive in male Han:SPRD-cy rats, and renal function is
less perturbed in females compared with males at this age. Prognosis
for renal patients with different etiologies is typically worse in men
compared with women. In PKD, the average age of renal failure is 
5 y
later in women than in men (Choukroun et al. 1995
,
Gabow et al. 1992
). In addition to the
gender-associated dimorphism in PKD, experimental evidence from
animal models suggests that the level or balance of sex hormones plays
an important role in the etiology of this disease (Cowley et al. 1997
, Zeier et al. 1994
).
A high fat diet, commonly consumed in Western cultures, is associated
with higher concentrations of serum sex hormones, and reducing dietary
intake of fat can lower these concentrations (Hamalainen et al. 1984
, Ingram et al. 1987
, Wu et al. 1999
). In a study of normal rats, a high fat diet resulted in
increased serum testosterone concentrations (Clinton et al. 1997
). In Han:SPRD-cy rats with kidney disease,
castration slows disease progression, whereas administration of
testosterone to castrated males or intact females accelerates disease
progression (Cowley et al. 1997
, Zeier et al. 1994
). The concentration of serum sex hormones, however, was
not reported in those studies. The hypothesis of this study was that a
high level of dietary fat would increase serum sex hormone
concentrations, resulting in a worsening of disease progression.
Consistent with the previous study in normal rats (Clinton et al. 1997
), a high fat diet resulted in higher serum
testosterone concentrations in normal rats in the current study. In
Han:SPRD-cy rats with kidney disease, however, the level of
dietary fat had no effect on serum concentrations of total
testosterone, suggesting that elevated serum sex hormone concentrations
is not the mechanism by which high dietary fat exacerbates disease
progression in Han:SPRD-cy rats. It should be noted that the
serum total testosterone concentration was determined in this study,
and it is possible that the serum concentration of free (unbound) sex
hormones could be altered in rats fed different levels of dietary fat.
The level of dietary fat can alter the level of serum sex
hormone–binding globulin (SHBG) (Reed et al. 1987
),
thus altering the balance between bound and free testosterone. The
commercial availability of an antibody that recognizes the rat binding
protein would aid in assessing whether the level of SHBG is altered in
normal and diseased rats fed varying levels of dietary fat.
Similarly, diet may also affect the free levels of serum estrogen by altering SHBG levels and not affecting total levels. Another caveat of the estrogen data is that samples were obtained from rats in the luteal phase. It is possible that a dietary effect may have been observed during other phases of the reproductive cycle, although the inherent variability in estrogen levels in the other stages would make it very difficult to distinguish a dietary effect.
Serum total testosterone concentrations were significantly higher in
normal rats fed both diets compared with the diseased rats. This may be
surprising, given the studies that demonstrate that castration
ameliorates and exogenous testosterone exacerbates disease progression
in Han:SPRD-cy rats (Cowley et al. 1997
,
Zeier et al. 1994
). These studies, however, do not
report endogenous sex hormone levels in these rats. In contrast,
several human studies have reported lower serum testosterone
concentrations in men with renal disease (Carlstrom et al. 1990
, Joven et al. 1985
).
Alternative mechanisms by which dietary fat can influence PKD
progression in Han:SPRD-cy rats remain to be examined.
Amelioration of PKD in Han:SPRD-cy rats with dietary
flaxseed (Ogborn et al. 1999
), which (in addition to
containing estrogenic compounds) alters the eicosanoid profile in
kidneys, suggests indirectly that eicosanoids may be important in this
renal disorder. We recently found evidence that steady-state levels
of the rate-limiting enzyme in eicosanoid synthesis, phospholipase
A2, are altered in the pcy mouse with
PKD (Mishra et al. 1999
). Functional changes in
glomerular filtration rate and renal plasma flow in animals fed high
protein diets also are accompanied by increased kidney synthesis and
excretion of eicosanoids in normal rats and experimental models of
renal disease (Breyer and Badr 1996
, Hostetter 1995
, Klahr and Harris 1989
). In the diseased
kidney, eicosanoids appear to play a role in maintaining glomerular
filtration rate, as well as being involved in inflammatory processes in
response to renal injury (Breyer and Badr 1996
,
Klahr and Harris 1989
). In some types of renal disease,
a reduction in eicosanoid formation is associated with amelioration of
the disease process, whereas in others, it appears to have a protective
effect (Breyer and Badr 1996
, Hostetter 1995
, Klahr and Harris 1989
). Increased levels
of dietary fat increase eicosanoid synthesis and may be involved in the
mechanism by which the high fat diet increased disease progression in
the Han:SPRD-cy rats in this study.
Currently no treatment for PKD exists, aside from treating secondary disorders associated with the disease progression. Animal studies, however, indicate that this disease is very sensitive to dietary constituents. The current study demonstrates that a low level of dietary fat compared with a high level slows disease progression in Han:SPRD-cy rats with kidney disease, without compromising body growth. This study also demonstrates that serum testosterone concentrations are reduced in these rats. The degree of fat restriction that is effective in retarding disease progression, the effect of diet on sex hormones in this disorder and the potential long-term effects remain to be elucidated.
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FOOTNOTES |
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2 Supported by Texas State Human Nutrition and by a Texas Woman’s University Research Enhancement Award.
4 Abbreviations used: HF, high fat; LF, low fat; PKD, polycystic kidney disease; SHBG, sex hormone–binding globulin.
Manuscript received November 12, 1999. Initial review completed March 1, 2000. Revision accepted May 19, 2000.
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