United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-84-007 June 1984
Project Summary
The Effects of Cadmium on
Renal Aging -- A Chronic
Cadmium Feeding Study in Rats
Susan Ann Perlin, Kazuyoshi Kawata, and John M. Frazier
Cadmium (Cd), a nephrotoxin, is
known to accumulate in the kidney
cortex, preferentially in the renal
proximal tubules. Animal and human
autopsy studies have shown that
damage to the renal proximal tubular
cells is associated with toxicity from
chronic Cd exposure. Several
investigations have shown an age-
associated change in the levels of renal
cortical cadmium resulting from
chronic exposure in laboratory animals
and long-term environmental exposure
in humans.
This study was undertaken to
determine if Cd exposure influences
the natural aging process in the kidney
and the accumulation patterns of renal
copper (Cu) and zinc (Zn). The concen-
trations of Cd, Cu, and Zn in the cortical
tubules were quantitated to determine
if the tubules preferentially accumulate
these three metals relative to the whole
cortex.
Two hundred seventy-two male
Wistar rats were treated for 24 months
with 0.0, 0.5, 5.0, and 50 mg/l CdCI2
in the drinking water. Every three
months, data were collected pertaining
to renal structure, function and concen-
trations of Cd, Zn, and Cu.
The levels of all three metals studied
were higher in the tubules than in the
intact cortex, on a dry weight basis,
indicating that these three metals were
concentrated by the tubular cells
relative to the other cortical cell types.
The concentration of Cd in both the
cortex and tubules increased with dose
level and duration of exposure. This
increase was linear up to 12 months in
the two low dose groups and up to 15
months in the highest dose group. After
15 months, the levels of Cd decreased
in both the tubules and the whole
cortex. Zn and Cu concentrations in the
cortex and tubules were significantly
elevated by Cd exposure, with the
greater effect on Cu. Zinc and Cu levels
in the tubules also decreased signifi-
cantly after 15 months.
The toxic effects from Cd exposure
were mild and limited primarily to the
highest dosed group. The combination
of age and Cd dosing had the greatest
effect on the accumulation patterns of
renal Cd, Zn and Cu.
Age alone had the greatest effect on
changes in the tissue structure of the
kidneys and may have accounted for the
decrease in the concentration of renal
metals seen in older individuals. Age
alone also had the greatest effect on the
physiological functioning of the kidney
as measured by proteinuria, decreased
urine and renal leucine aminopeptidase
activity, changing diurnal urine
volumes and decreased urine pH and
specific gravity. The combination of old
age and high Cd dosing appeared to
affect renal functioning as indicated by
increased urine volume, increased urine
glucose levels and decreased specific
gravity.
Increased Cd dosing appeared to be
asociated with certain structural
changes in the kidney. Although not
significant, the highest dosed animals
had a greater prevalence of moderate to
severe tubular hyperplasia and tubular
dilation/cast formation. The highest
dosed rats also had significantly larger
kidneys as measured by their percent of
the total body mass.
This Project Summary was developed
by EPA's Health Effects Research Lab-
oratory. Research Triangle Park, NC, to
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announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Cadmium, a non-essential trace
element used in many industrial
processes and consumer products, is
now widely distributed throughout the
environment. Since cadmium is a
nephrotoxin, there is increased concern
over the potential health effects from
low-level, chronic exposures.
Previous animal and human autopsy
studies have shown that chronic
cadmium exposure is associated with
characteristic renal morphological
changes. Such studies have also
demonstrated that the pattern of renal
cadmium accumulation varies as a
function of age
Because of the documented age-
associated change m renal cadmium
levels, a study was designed to examine
the effects of both natural aging and
cadmium exposure on the tissue
structure and physiological functioning of
the kidney The primary hypothesis tested
was whether cadmium exposure
influences the physiological, morpholog-
ical, and biochemical aging of the kidney
Since cadmium is thought to accumu-
late preferentially in renal proximal
tubules, it was desirable to separate out
the tubules and relate the physiological
changes in the kidney to the cadmium
levels m the tubules. Traditionally, the
renal cadmium burden has been
evaluated as the concentration of
cadmium m the intact cortex; therefore, a
comparison of the levels of metals in the
tubules with that of the cortex was
needed to determine if the cortex levels
were good indicators of tubular levels It
was hoped that by separating out the
tubules, it would be possible to gam a
more accurate picture of the relationship
between renal cadmium accumulation
and renal toxicity A secondary
hypothesis tested was whether cadmium
concentrations in the renal cortex are
sufficient to predict the cadmium dose to
the renal tubules
Since tissue accumulation of cadmium
is closely associated with that of zinc and
copper, additional hypotheses tested
were whether the renal tubules preferen-
tially concentrate zinc and copper and if
cadmium affects the accumulation of
these two metals.
Every three months, eight rats from
each dosage group (i.e., 0 0, 0 5, 5.0, and
50.0 mg/l cadmium) were sacrificed to
obtain kidneys from which enriched
cortical tubule concentrations were
determined for both the intact cortex and
the tubule preparation from each rat.
Changes in renal function were assessed
by performing urinalysis on a regular
basis. Changes in renal tissue structure
were assessed by morphometric analysis
of fixed kidney sections.
Results and Discussion
The effect of cadmium exposure on the
general health status of the rats was
assessed by several parameters,
including body and organ weight gain,
hematocrit, and food and water
consumption.
Increased cadmium exposure was
associated with decreased food and
water consumption, body weight and
hematocrit. Reduction in the values of
these variables indicated that cadmium
exposure adversely affected the general
health of the individuals; however, the
effect was not severe at the levels of
cadmium given to the rats.
The concentration of cadmium in the
intact renal cortex(Figure 1 )andthe renal
cortical tubules (Figure 2) versus time
have shown that in all exposure groups,
cortical and cortical tubular cadmium
concentrations increase with time of
exposure up to 15 months and then begin
to decrease. The 15-month time point
appears to be a critical time for cadmium
kinetics independent of renal cadmium
burden, suggesting that the effect is
related more to aging than to cadmium
toxicity.
Comparison with control rats showed
that cadmium exposure elevated renal
copper and zinc levels in a dose
dependent manner. The greater effect
was on copper concentrations (Figure 3).
As with cadmium accumulation,
tubular and cortical copper and tubular
zinc levels peaked around 15 months and
declined thereafter. These results
support the idea that an age component
affects the renal accumulation of these
three metals.
The data support the hypothesis that
the renal cortical tubules preferentially
accumulate cadmium, zinc and copper.
The relationship between the
concentration of cadmium in the intact
cortex and the concentration of cadmium
in the cortical tubules for the high dose
group is indicated in Figure 4. For all
dosage groups up to 1 8 months, with few
exceptions, the tubules consistently had
significantly higher concentrations of
cadmium than did the intact cortices
(p ^0.05). The same effect was observec
for both zinc and copper. Dramatic
decreases of cortical tubular metals seer
at 21 and 24 months may have been more
artifactual than real. This was due to £
significant loss of physical integrity of the
tubule preparations recovered during this
late phase of the study.
Further, the relative rate of
accumulation of cadmium in the cortical
tubules versus the intact cortex was
calculated for each group for the linear
portion of the cadmium accumulation
curve (i.e., 0-12 months for the low dose
groups and 0-15 months for the high dose
group). The ratios of the rates of cortical
tubule-cortical accumulation were found
to be 1.2, 1.3, and 1.4 for the high,
midrange and low groups, respectively.
Although the differences in these ratios
are very small, they do show a dose-related
trend. This would suggest that the rate of
accumulation of cadmium in the intact
cortex may not be a good indicator of the
rate of accumulation of the metal in the
cortical tubules at all doses. It does
suggest, however, that at higher doses,
the rate of cadmium accumulation in the
intact cortex would be a good estimator of
the rate of accumulation in the cortical
tubules. Since the preparation of tubule
pellets is extremely time-consuming, it is
reasonable to use the cortical data as an
approximation for the cortical tubule data.
By separately analyzing the cortical
tubules and the whole cortex, it was hoped
that the cause of decreased cortical
cadmium levels with old age could be
explained. It was theorized that if the
cortical levels declined with age but the
cortical tubular levels plateaued or
continued to increase, then the decreased
cortical levels could be attributed to the
age-related infiltration of renal fibrous
tissue, which concentrates minimal
amounts of cadmium. The data partially
support this theory. First, the
morphometrics showed that there was a
significant decrease with age of the
numerical density of glomeruli and,
therefore, the number of nephrons.
Second, there was a significant age-
associated decrease in the volume
density of intact proximal tubular walls in
the outer cortex. Third, there was a
significant age-associated increase in the
amount of interstitial fibrotic tissue. All
three effects were seen equally among
the controls and the dosed animals.
These data suggest that the decrease of
renal cadmium with age is due to at least
two factors. First, with age, there is an,
actual physical loss of intact proximal'
tubules, the component of the renal
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500-
450 •-
400- -
350' -
300'-
250 —
200 -
150"
100
50 ••
Figure 1.
Time of Exposure (months)
Concentration of cadmium in the intact renal cortex (fjg Cd/g dried cortex) as a
function of exposure to cadmium.
cortex that sequesters the highest
proportion of the cadmium organ burden.
Second, with increasing age, there is a
change in the proportion of tissue types in
the kidney. Presumably, each tissue type
has a characteristic metal burden so that
alterations in the tissue composition
could lead to alterations in the renal
metal levels. Given the present data,
these factors seem to apply to the
accumulation patterns of zinc and copper
as well as cadmium.
All study groups showed many age-
related changes in kidney structure.
Consistent with the findings of others,
age correlated significantly (p<0.05)
with increased inflammation, interstitial
fibrosis, glomerular and tubular
basement membrane thickening, tubular
dilation/cast formation, and hyperplasia.
The glomeruli became significantly larger
(p < 0.05) with age, but their density in the
outer cortex became significantly
smaller. Although there were no statistic-
ally significant differences between the
groups, the high dose group, in compari-
son to controls, tended to have a higher
percentage of individuals with moderate
to severe hyperplasia and tubular dilation/
cast formation.
The data suggest that cadmium
exposure may have had a toxic effect on
the kidney that was expressed by an
increase in renal mass. Although there
was no statistically significant difference
between the groups in the total wet
weight of the kidneys, the highest dose
group consistently had heavier kidneys
than all other groups throughout most of
the experiment. Also, when the kidney
weight was recalculated as the percent of
body weight, kidney values for the
highest dose group were significantly
higher (p<0.05) than those of the
controls.
Since the renal wet/dry weight ratios
were not significantly different between
the groups, increased hyperplasia in the
high dosed group seemed the likely cause
of the larger kidneys. Morphometric
analysis supporte this idea.
At the levels tested, renal
morphological changes typical of
cadmium toxicity were not detected.
In general, cadmium at the doses
tested had little adverse effect on the
functioning of the kidney. Significant
dose-response relationships were not
anticipated since the maximum cortical
concentrations of cadmium were approx-
imately 90 mg/g wet weight (high dose
group) and it has been suggested that 200
mg/g wet weight is the critical
concentration associated with detectable
renal dysfunction.
Age alone caused more significant
changes in renal function than did
cadmium exposure. For certain
parameters, including diurnal urine
volume, urine glucose and specific
gravity, the effect of old age in combina-
tion with high cadmium exposure
appeared to have a greater effect on renal
function than did age alone. The data
were suggestive of a mild toxic effect of
cadmium expressed only in late life.
The rat presented a picture of gradual
renal change with time that included
decreased levels of renal and urine
leucine aminopeptidase proteinuria,
changing diurnal urine volumes,
decreasing pH, and decreasing specific
gravity.
Conclusions and
Recommendations
Data from this study suggest many age-
related renal changes which alter the
kidney's physiological handling of the
toxic metal, cadmium, and the essential
metals, copper and zinc.
The hypothesis that chronic cadmium
exposure influences the physiological,
morphological and biochemical aging of
the kidney has been supported partially
by the findings. This hypothesis was not
fully supported because of the levels of
cadmium exposure tested. In general,
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a
'9 '72 "75 '78
Time of Exposure (months)
21
24
Figure 2.
Concentration of cadmium in the renal cortical tubules as a function of exposure to
cadmium.
* 3
91 VI
tj
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600
550
500
450
400
350
300
250
200
750
700
50
0
* Significant difference between tubules and cortex, p < 0.05.
*(x) Significant difference at p = x.
o- — -o Tubules
• • Cortex
(0 084)
*
"9 \12 \15
Time of Exposure {months)
18
21
<24
Figure 4. Concentration of cadmium in the intact renal cortex and cortical tubules as a
function of exposure in Group 4 (50 ng Cd/ml).
hyperplasia and tubular dilation in the
high dose group as compared to the
controls. There appeared to be a small,
but definite shift toward larger glomeruli
with high cadmium exposure.
There was a significant (p < 0.05) corre-
lation between renal cortical cadmium
levels and inflammation of the renal
tissue. However, this relationship is
confounded by the finding that both
inflammation and renal cadmium
concentrations also correlate positively
with age.
Of all the parameters studied, age and
cadmium exposure had the most
significant influence on the renal
handling of cadmium, copper and, to a
lesser extent, zinc.
At the levels tested, cadmium exposure
had a mild effect on renal aging in terms
of disturbing the tubular reabsorption of
proteins, and altering both the physical
integrity of the renal cortical tubules and
the size of the glomeruli. Age alone did
not appear to have a significant effect on
diurnal urine volumes or excretion of
urine glucose. However, old age, in
combination with high cadmium
exposure, showed a significant, positive
correlation with increased urine volumes
and glucose excretion. These observa-
tions suggest that cadmium exposure in
the older animals compromised renal
tubular functioning to a certain extent.
Cadmium exposure did not appear to
significantly affect age-associated
changes in the kidney structure such as
increased inflammation, glomerular and
tubular basement membrane thickening
or interstitial fibrosis. On the other hand,
cadmium exposure did appear to have a
mild effect on renal structure as
evidenced by a dose-response relation-
ship with the degree of tubular hyper-
plasia.
Fifteen months of exposure appeared
to be the critical time point for renal metal
kinetics as evidenced by the peak in the
metals accumulation curves. It also may
have been a point at which the renal
physiology was changing in other ways,
as evidenced by changes in certain renal
function tests.
In examining the relationship between
the level of cadmium exposure and the
concentration of cadmium in the kidney,
it was found that the rate of accumulation
of cadmium in the whole cortex did not
increase linearly with the actual dose to
the rats. These results implied that the
actual dose to the rat could not be used to
predict the renal cadmium concentration
at a given time.
The results of this study indicate the
need for further research in order to more
fully answer the question of whether or
not cadmium exposure influences normal
renal aging.
Data from this study have demonstra-
ted that renal concentrations of 90 pg
Cd/g wet weight of cortex are associated
with mild functional and structural
changes in the kidney in association with
old age. Since these data are suggestive
of a possible effect of cadmium on renal
aging, further research is needed using
higher exposures. Exposures which
would result in attaining the critical con-
centration of 200 //g Cd/g wet weight in
the cortex should be used in conjunction
with morphometric techniques for
analyzing renal tissue pathology. Also,
appropriate measurements of the total
amount of cortical tissue in the fresh and
fixed kidney should be made so that an
estimate of the total number of nephrons
and total mass of proximal tubular tissue
per kidney can be calculated. Estimates of
the total amount of a specific tissue type
rather than the proportion of that tissue
type in the kidney may result in a more
sensitive measure of the effects of
cadmium exposure on renal structure
and aging.
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Susan Ann Perlin, Kazuyoshi Kawata, and John M. Frazier are with The Johns
Hopkins University, School of Hygiene and Public Health. Baltimore, MD21205.
Norman E. Kowal is the EPA Project Officer (see below).
The complete report, entitled "The Effects of Cadmium on Renal Aging: A Chronic
Cadmium Feeding Study in Rats," (Order No. PB 84-191 022; Cost: $22.00.
subject to change} will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
D utrt«rtU*i
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*USGPO: 1984-759-102-106C
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