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Influence of heredity on response of inbred rats to diet
Influence of Heredity on Response of Inbred Rats to Diet I. Differences in Body Size, Food Intakes, Incidence of Spontaneous Kidney Defects, Kidney Weights, Urine pH and Protein By MARY W. MARSHALL ANDRICHARDP. LEHMANN Inbreeding our laboratory strain of rats (BHE) resulted in 2 lines significantly different with respect to body size, food consumption, occurrence and degree of gross nephrosis, renal stones and urine protein excretion although average kidney size, occurrence of hydronephrosis and urine pH were not significantly different. Inbred BHE female rats had less nephrosis in both lines than male rats. Body size explained the sex difference in this defect in the line with less nephrosis (line 1) but not in the other (line 2). Response of the individual lines to 2 kinds of protein in semipurified diets indicated that heredity is a more potent factor than dietary protein in occurrence of nepbrosis and excretion of
urine protein. Within each line, stock diet provoked the largest excretion of urine protein and 24 per cent of either defatted egg or lactalbumin, the lowest. However, line 2 rats excreted twice as much protein with all diets as line 1 rats. Stock diet produced the highest urine pH and stone incidence in both lines. Females had more kidney stones than males; stones occurred in virgin female rats as well as in breeders. Results of this study point out the need for caution in interpretation of nutrition studies; individual differences may be related to “hidden” genetic defects. (Metabolism 16: No. 8, August, 763774, 1967)
REPORTS most but not all male rats of our laboratory strain (BHE), when compared with a strain of Wistar rats, were shown to have many unique and inherent differences in metabolic response, particularly with respect to lipid metabolism, and length of life, if fed under similar conditions a semipurified diet containing 25 per cent cooked whole egg.la2 It has been demonstrated with the egg-containing diet that BHE rats develop larger livers with higher levels of fat and cholesterol, and have higher urinary protein excretion, serum cholesterol and carcass fat than do Wistar rats. In addition, a higher incidence of nephrosis, hydronephrosis and renal stones occurs spontaneously in apparently healthy BHE than in Wistar rats of the same age. In contrast, BHE rats fed a semipurified diet with less fat and without egg do not develop severe hyperlipemia or nephrosis by 330 days of age but their carcass lipid is higher with this diet than with the egg-containing diet.l
I
N PREVIOUS
From the Human Nutrition Research Research Service, United States Department
Division and Biometrical Services, of Agricultuw, Beltsville, Maryland.
Agricultural
Received for publication Dec. 1, 1966. MARY W. MARSHALL, M.S.: Research Nutritionist, Experimental Nutrition Laboratory, Human Nutrition Research Division. RICHARDP. LEHMANN, PH.D.: Biometrician in charge of Livestock Research Staff, Biometrical Services. Both Research Service of the U. S. Department of Agriculture. 763
authors
are
from
Agricultural
764
hfARSHALL
AND
LEHMANN
It is well known that pathological consequences of a gene may manifest themselves only in certain environmental conditions and that the effects may be altered considerably without causing changes in the gene itself. The rat is currently used as the test animal in a wide variety of investigations, pharmacological as well as physiological and knowledge of specific genetic traits is important particularly in nutrition studies. Certain kidney defects and associated symptoms have been shown to occur spontaneously in certain strains of rats3-* and some defects can be experimentally produced and/or manipulated by dietary means .9-12 Therefore, it is of considerable significance in nutrition investigations that rats with specific genetic traits be produced and studied with the view of understanding some of the reasons for individual variations and perhaps of shedding some light on the solution of some of the practical problems of nutrition of human populations where genetic history of individuals is not always known. To this end, eighth generation inbred BHE rats were fed a stock diet or diets containing 1 of 2 levels of defatted egg or lactalbumin until they were killed at 300 days of age. The results for growth, food intake, kidney defects, kidney weights, urine pH, and protein are reported herein. Findings for liver composition, thyroid, adrenal and heart weights, and serum cholesterol will b e presented in a subsequent report. Evidence is presented that individual animals with unknown defects may provide the explanation for many of the individual differences found in response to diet. EXPERIMENTAL
Animals. were
inbred
animals
Pairs of BHE
rats with known family histories,
by continuing
full-sib
were fed the same pelleted
matings stock diet’
as the BHE
eighth generation were kept until weaned at 21-26 in the litters. Weights of the animals when weaned 487 animals of the eighth generation Diets.
Composition
of the diets
quality were fed, lactalbumin was fed at the same level when
the lower
were included is shown
selected
of offspring
from the stock on hand,
in successive colony.
generations.
days old depending upon the numbers are referred to as initial weight. In all, in the study.
in Table
1. Two
kinds
of protein
and defatted whole egg containing 0.5 per cent fat.7 in all the semipurified diets. Cornstarch replaced
level was fed.
Littermates
The
All animals born in the
of first litters
of 2 family
lines
of high Sucrose protein
of rats,
both
males and females, were housed individually and fed by litter and sex 1 of 3 diets: pelleted stock (diet 1 ), and 1 of 2 levels of defatted egg (diets 3 and 4) or of lactalbumin (diets 5 and 8). Rats designated as line 1 received only defatted egg as the source of protein. Rats Line 2 rats which received defatted egg designated as line 2 were fed only lactalbumin. but no stock diet were designated as line 2a. Each litter of each line received one kind of protein. Male and female littermates received the same kind of protein. For almost every litter fed one combination of 3 diets, a litter from the same family was group-grown (3 to 5 rats each) in the stock colony for the same period of time and was fed only the pelleted stock diet. The stock diet was fed in pelleted
form except
during
collection
of excreta
when it was
“Animal Foundation Laboratory diet, Standard Brands, Inc. (diet 1). Purina Laboratory Chow, Ralston Purina Company (diet 2), to be referred to in a later report. )Defatted whole egg was prepared in our laboratory by cold extraction with a 50:50 mixture of skellysolve “B” and 95 per cent ethyl alcohol.
HEREDITARY
INFLUENCES
ON
RAT
Table
765
DIET
l.-Composition
of Diets Diet No.
Ingredients* Gm./lOO Gm.
Defatted
3
4
23.72
47.43
43.19 23.71
43.19
30.00 0.08 2.6 1.6
18.81 0.01 0.6 0.5
37.44 0.02 0.7
3.93
4.15
4.38
It
whole egg
Lactalbumin Sucrose Cornstarch (edible Proximate
grade)
5
6
23.72
47.43
43.19
43.19
23.71
Composition
Protein, X$ Cholesterol, X$ Calcium. % Phosphorus, % Calories, kcal/Gm. Da
38.56 0.08 0.7
19.31 0.04 0.7 0.5
0.6
0.6 4.68
4.24
“The following vitamins were added per Kg. to each semipurified diet: niacin, pyridoxine*HCl and thiamine*HCl, 5 mg. each; riboflavin, 10 mg.; Ca-pantothenate (d), 25 mg.; u-tocopherol acetate, 25 mg.; folic acid and 2-methyl-l, 4-naphthoquinone, 2 mg. each; vitamin II,,, 30 pg.; biotin, 100 pg.; and later 400 pg. (see text). The following ingredients
were
fed
at the
(Mazola), 5.00; Jones p-amino benzoic acid, vitamins A and D. The fAnima1 Foundation
same percentage level in all semipurified diets: corn oil and Foster13 salt mix, 4.00; choline chloride, 0.20; inositol, 0.10; 0.03; and Percomorph Oil (Mead Johnson Co.), 0.05; to supply oil contained 8,500 units D and 60,000 units A/Gm. Laboratory Diet, Standard Brands, Inc.
SKjeldahl nitrogen X 6.25. Average cent; average N content of lactalbumin $Calculated respectively.
from
‘ODefatted
ground
analysis
whole
to facilitate
fed the pelleted
N content of defatted was 12.64 per cent.
of defatted
egg, 4.70
kcal/Gm.;
the collection
whole
egg and lactalbumin, lactalbumin,
of scattered
food.
5.15
Food
0.42
12.58
and
mg./Gm.,
1.86
intake
was measured
stock diet during the 5 days prior to the lOOth, 20Oth, and 300th
When
the rats reached
300
j,
per
kcal/Gm.
of the rats. Food intakes of the semipurified diets were measured experimental period, with intakes corrected for scattered food. Procedures.
egg was
weekly
for rats birthdays
during
the entire
5 days of age, urine was collected
quantita-
tively from representative groups of rats for 3 or 4 days during the week of autopsy. On the day of autopsy, pH of freshly voided urine was determined* for most of the rats. Then the rats were anesthetized, without previous fasting, with sodium amytal solution. The body cavity was opened and 3 ml. blood removed by heart puncture. Livers were excised, blotted to remove excess blood, weighed and frozen prior to analysis. Kidneys were removed,
trimmed
weighed
(without
and graded
removing
the
capsule),
sagittally,
examined
The
heart,
base, was blotted free of blood in its chambers and weighed. (including the parathyroid) were removed, trimmed and weighed.
Left
adrenals
of
fresh
urine
for gross occurrence
cut
of nephrosis.3
Aliquots
O-4+
were
precipitated
with
washed and after recentrifuging for the second time, was calculated by multiplying by the factor, 6.25. Fat
was
determined
in liver
homogenates
after
trichloroacetic analyzed acid
acid,
for nitrogen.
hydrolysis
and
for
separated
stones, at its
and thyroids
the
precipitate
Urine extraction
protein with
ethyl and petroleum ethers. In composites of defatted egg, diets and lactalbumin, measurements were made of fat by a modified AOAC method using acid hydrolysis, cholesterol,14 calories in the Parr Bomb Adiabatic Calorimeter, nitrogen by the Kjeldahl-GunningArnold method, calcium,rs~1s and phosphorus.17 *Combistix
reagent
strips, Ames Company.
7%
in the inbred BHE rats: upper left: hydronephrosis with stone; upper right: stones and early nephrosis; lower left: cysts in cortex; lower right: congestion at the corticomedullary border. Later stages of nephrosis are similar to illustrations by Durnnd et al.”
St~tistic~EAnalysis. Data from eighth generation rats were analyzed by the method of least squares employing models which evaluated the effects due to (1) line, litters in lines, sex, and line x sex interaction, and (2) sex, diet and sex x diet interaction. Other models were then used which adjusted all variables to average initial, maximum, or final weights of rats. Body weight was taken into account to determine whether the differences observed between the 2 lines were primarily the result of differences in body size. Since significant litter differences were observed each analysis was conducted on a within-litter basis. For some variables, only littermates were included in the analysis and for others, all rats fed the stock diet (“family mates”) were included (line comparison). Differences within the 2 lines of eighth generation rats were analyzed separately, i.e., for line 1, diets 1, 3 and 4 were compared, and for line 2, diets 1, 5 and 6 were compared. Unadjusted means are used in the text when discussing results. For the kidney defects statistical procedures that follow closely those of general least squares were applied according to the methods outlined by Gabriel. 1s Because of differences in feeding plans, rats designated as line 2a were not included in the analysis of variance.
RESULTS Gene&
By the fourth
families
in lines
accrued
by the eighth
a distinct
generation,
1 and 2. With generation
full-sib
matings
was calculated
separation the
degree
occurred
between
of homozygosity
by methods
presented
by
85 per cent. Some of the more frequent defects are shown in Figure 1. One of the most frequent findings in line 1 was severe congestion at the corticomedullary border of the kidneys. Another finding was that of kidney shaped transparent cysts; often, solitary cysts were found in one or both kidneys. The ureter was often greatly dilated.
Wright rg to
be
approximately
HEREDITARY
Typical
INFLUENCES
ON
of the structural
R.4T
767
DIET
characteristics
of line 1 rats was
an
increased
collateral circulation in the region of the left kidney and frequently the left adrenal was embedded on the upper left corner of the left kidney. A small area of necrotic tissue was often attached to the mesentery at the esophageal opening to the stomach and simultaneously to the smallest lobe of liver. Nephrosis was more severe in line 2 rats than in line 1, and was generally Rats of line 2 greatly resembled the rats accompanied by hyperlipemia. described by Zucker and Zucker,4 with the “fafa” genotype except for the unusually large amount of carcass fat which their rats contained. One incident that occurred early during the feeding trials of eighth generation line 1 rats should be noted. After receiving the diets containing defatted egg for 4 to 6 weeks, typical symptoms of biotin deficiency were evident.20 Eighteen of 19 males and 10 of 17 females receiving diet 4 developed the symptoms which disappeared when biotin was given orally, 4 pg./rat/day for 2 weeks. Thereafter, 400 pg. biotin instead of 100 pg./Kg. diet were incorporated in all semipurified diets. However, none of the rats of line 2a fed the defatted egg-containing diets developed the symptoms. Differences in biotin requirements of rats have been reported”* but were not evaluated in the present study. Body Weights and Food Intakes. In Table 2 are average body weights and food intakes (semipurified diets) of eighth generation male and female rats.* Rats of both sexes of line 1 fed the stock diet had on the average significantly larger initial, maximum (16 per cent higher), and final body weights than rats of line 2. When littermates were compared within lines, rats fed defatted egg or lactalbumin grew as well as those fed stock diet and there were no significant diet differences in initial, maximum or final weights of these rats. The tendency was, however, for maximum and final weights of rats within lines to be smaller when fed 47 per cent protein than when fed 24 per cent protein. Rats within line 1 ate more of all diets than rats within line 2. Rats within each line ate less of the diets containing 47 per cent of either protein than of those containing 24 per cent protein. As is usually the case, all differences in food intakes were removed when adjustment for maximum body weight was made and a significant regression coefficient of maximum body weight on food intakes was observed for each line. Kidney Size. Kidney weights are presented in Table 3. Rats of lines 1 and 2 fed stock diet had almost identical average kidney weights, 3.44 vs. 3.-19 (means derived by averaging kidney weights of both sexes within lines) but this weight was a significantly larger percentage of the final body weight of ____ _ ‘Detailed tables of data are submitted for inspection with this report. Data are included in these tables for 141, 300-day-old, sixth and seventh generation rats to support evidence of prior separation of the lines with respect to some of the traits. For the stock diet, average daily food intakes for 21 line 1 males at 100, 200. and 300 days of age were 93.7, 24.9, and 23.8 Gm., respectively; for 18 line 1 females, 16.3. 18.8 (17) and 17.3 Gm. Corresponding figures for 20 line 2 males were 20.8. 27.1. and 22.0 Gm.: and for 15 line 2 femalrs, 15.3, 16.3, and 17.8 Gm.
Weights,
29 5
Line 2a, 24% DFE Line 2a, 47% DFE
*Number in group is same unless indicated iStandard error. $Littermates. gLittermates.
Lact.9 Line 2,47% Lact.$
Line 2,24%
1 2 2 2
5 7 6 11
+ -t f +
7 13 16 15
+ t + +
4 7 7 10
ZlI 5 -+ 7 rtr: 12 z!I 11
263 f 7 250 * 9
272 290 326 278
299 338 321 298
Females
412 ?z 5 414 * 8
444 Z!I 6 464+6 505 + 8 474 ?z 13
522 554 549 492
Males
Final
in parentheses.
274 i- 7 253 -e 8
by other number
47 * 2 58 I!= 2
* + * f
2 & + A
Line 2, stock, in colony
Line 2, stock9
278 298 329 291
32 15 12 10
1, stock, in colony Line 1, stockt Line 1, 24% DFEj Line 1, 47% DFEd 47 42 41 42
54 18 12 14
Line
1 2 2 3 5 7 12 11
303 F 347 -t323 -t 300+
55 51 54 50
F * t &
419 + 14 430 AZ8
55 I!I 5 53 -t- 1
5 22
” 6 z!z 6 3- 9 -+ 9
458 476 518 487
Line 2a, 24% DFE Line 2a, 47% DFE
1 1 1 1
t -c + rt
50 46 46 46
2, stock, in colony 2, stock$ 2,24% Lact.4 2,47% La&Q
41 20 19 14
6 13 16 15
t + + 4
538 563 555 497
Maximum
Line Line Line Line
2f 2 2 2
Initial
Averane Body Weights. Gm.
3501 + 64 3260 f 119
3288 z!z 54 3032 I!I 101
3567 z!z 77 3495 I!I 84
3973 -c 157 4252 +- 84
4384 Z!I 79 4137 * 78
4862 I!I 107 4493 + 110
Total Food Intake, Gm.
and Zncidence of Kidney Defects of Male and Female Either Stock or 1 of 4 Semipurified Diets
* + _’ -t
Rat.9
NO.
Intakes
57 54 54 52
Food
36 21 19 17
Group and Diet
2.-Body
1, stock, in colony Line 1, stock$ Line 1,24% DFE $ Line 1,47% DFE 1
Line
Table
Rats,
90 100
84 93 83 90
100 75 64
91
100 86
100 I.00 100 100
100 100 84 82
All
300 Days
Old
41 60
37 47 33 70
2 17 0 14
80 77
100 100 95 100
86 86 10 41
65 60
16 0 25 40
17 17 8 29
40 54
63 55 42 57
31 38 16 59
0 20
41 80 17 0
63 83 I.7 0
0 0
I.5 50 0 0
44 71 0 0
Hydronephmsis Stones
37 40 67 40
63 67 7s 57
20 41
19 20 21 50
36 33 79 29
Other
Incidence of Kidney Defects
Fed until
Nephrosis
Percentage
Inbred
$ % g
29 5
*Number in group is same unless indicated +Standard error. SLittermates. $Littermates.
Line 2a, 24% DFE Line 2a, 47% DFE
2, stock, in colony 2, stock$ 2, 24% la&.$ 2,47% lact.$
Line Line Line Line
32 15 12 10
54 18 12 14
1, stock, in colony 1, stockt 1, 24% DFE$ 1, 47% DFE j
Line Line Line Line
41 20 19 14
36 21 19 17
NO. Rats’
0.12f 0.22 0.09 0.19
t 0.09 F 0.24 A 0.41 X!I0.55
+rt ++
+ 0.05 & 0.12 z!z 0.11 * 0.40
Z!I 0.05 I+ 0.11 I 0.10 t 0.09
by other number
Weight
* i t *
.02 .05 .08 .15
t .03 -t .04 x!z .Ol -t .03
* t t +-
r+ & -t ?z .02 .04 .03 .18
.Ol .03 .02 .03
1.09 x!z .03 1.59 I!z .18
0.94 0.95 0.83 1.41
0.81 0.84 0.79 0.91
Fetnales
0.78 A .lO 1.15 =!I .13
0.96 0.99 0.96 1.51
0.82 0.82 0.59 0.76
Alalcs
Body Weight
in parentheses.
2.88 * 0.12 3.93 AI 0.39
2.55 2.76 2.71 3.84
2.41 2.83 2.51 2.68
3.26 + 0.49 4.83 -+ 0.60
4.27 4.58 4.83 7.02
4.27 4.52 3.20 3.73
Weight
Kidney
.l (35) .l 0 0
.l .3 0 0
6.2 + .l (25) 6.0 2 0
0.9 2.5
& .l (30) * .2 zlz .l ” .l (9)
+ i it I+ 6.3 6.6 5.9 5.9
7.1 6.5 6.0 6.0 0.6 0.8 0.5 2.2
0.1 0.2 0 0.1
6.7 & .l 6.1 f .l
1.3 1.9
_c .l (39) & .2 ? .l -+- .l (13)
i* ?I +
PH
Urine Protein
300 Days
478 + 53 829 rt 242
645 & 99 (10) 202?52(8) 431f lOO(8)
334*78(13) 163*41(10) 211+26(H)
434 t 133 801 AZ67
930 -+ 56 (9) 721 * 150 (8) 773*99(g)
595 f 61 (13) 200 r+ 46 (12) 410 + 63 (13)
Rats, Fed until
7.2 7.1 6.1 5.9
7.5 7.2 6.0 6.0
-_
Inbred
2.0 2.0 2.0 3.4
1.4 1.6 0.2 0.9
Degree of Nephrosis
of Nephrosis, Urine pH and Protein of Male and Female Either Stock or 1 of 4 Semipurified Diets
5 22
Degree
Line 2a, 24% DFE Line 2a, 47% DFE
2, stock, in colon) 2, stock$ 2,24% 1act.g 2, 47% lact.$
Line Line Line Line
and Diet
~______
Weights,
1, stock, in colony 1, stockt 1, 24% DFE$ 1, 47% DFE]
Group
3.-Kidney
Line Line Line Line
Table
old
770
MARSHALL
AND
LEH.IfASS
the smaller rats of line 2 than of larger rats of line 1. When adjusted to average maximum body weight, significant line differences were observed but differences due to sex were removed. Littermates within lines 1 and 2 exhibited similar response to diet with respect to kidney size. Sex differences but not diet differences in kidney size occurred and these were removed when maximum body weight was considered. However, results for individual diets should not be ignored. Both males and females of line 2 fed diet 6 had larger kidneys than rats fed either diet 1 or diet 5 but kidneys of male rats of line 1 fed diet 4 were smaller than those of rats fed diet 1. Line 2(a) inbred male rats fed diet 4 had kidneys of identical size as line 2 rats fed diet 5. Thus it appears that heredity as well as kind and quantity of protein are factors in determining kidney size. Gross Kidney Defects. Similar incidence, 88 vs. 90 per cent (Table 2) of gross kidney damage occurred in both lines of rats but there was a considerable difference in kind and degree of damage. More kidneys (70 per cent) of line 2 rats had some degree of nephrosis than kidneys of line 1 rats (41 per cent) fed the stock diet and this difference between lines was not explained by body size. Males had a significantly higher incidence than females fed diet 1 or any diet, within each line. Within line 2, average occurrence of nephrosis was higher for each diet than for line 1 rats fed stock diet or corresponding levels of protein. Within each line, rats fed either 24 per cent defatted egg or lactalbumin had the lowest incidence per line. Kind of protein was less important than genetic differences because line 2(a) rats fed defatted egg had more nephrosis than line 1 rats fed this protein. Within line 1, a significant sex by diet interaction occurred but not within line 2; body weight was a significant factor in the development of nephrosis in line 1 rats but not in line 2 rats. Differences in severity of nephrosis were calculated (Table 3) by averaging grades (O-4+) assigned to each pair of kidneys. By this technic within line 1, male rats fed stock diet had the highest score, avg. 1.5, and females fed diet 3, the lowest, zero. Within line 2, male rats fed diet 6 had the highest score, 3.4, and females fed diet 5, the lowest, 0.5. Within each line, rats fed 47 per cent protein had a higher degree of nephrosis in the kidneys than those fed 24 per cent protein. The detrimental role of the stock diet used in this study is indicated by increased kidney size and occurrence of nephrosis in male rats. However, female rats of line 1 showed considerable resistance to nephrosis regardless of diet. Hydronephrosis occurred in both lines, significantly more in males than in females when the stock diet was fed although line differences were not significant. Right kidneys were affected more often in male rats while left kidneys were affected more often in female rats. Overall, rats of line 2 had a higher incidence of hydronephrosis, 36 vs. 22 per cent, than rats of line 1. Within each line, rats fed 4i per cent of either protein had the highest incidence of hydro-
HEREDITARY
nephrosis.
INFLUENCES
ON RAT
DIET
771
Male rats in both lines and with all diets had a greater incidence
than female rats. When found, renal stones were usually imbedded
in the pelvis beneath
the papillae of the kidney. They were of different shapes and sizes, were unilateral or bilateral and occurred with or without hydronephrosis. Line 1 rats had a significantly higher incidence of renal stones (Table 2) than line 2 rats fed stock diet, avg. 61 vs. 39 per cent. Female rats of both lines had more stones than male rats, Within each line, the significant diet difference observed is due almost entirely to stone formation in rats fed the stock diet. None of the 96 eighth generation male rats of either line and only 5 of 82 of the female rats fed any of the semipurified diets had kidney stones. It may appear that the higher average urine pH (Table 3) of rats fed the stock diet is associated with the high stone incidence. However, when fed the stock diet, rats with urine pH of 6.0 or 6.5, showed average incidence of 41 per cent for males and 57 per cent for females; in rats with urine pH of 7.0, 7.5, or 8.0, 40 per cent of the males and 70 per cent of females had stones. Thus, urine pH did not seem to affect stone production in males but a higher urine pH was associated with a higher incidence of stones in females. Within lines stone incidence was not influenced by body size, neither was the significant sex by diet interaction for urine pH influenced by body size. Kidney stones occurred in 17 of 28 female rats that had litters and in 50 virgin female rats. With respect to the production of stones in virgin female rats, BHE inbred rats differ from the Sprague-Dawley rats of Wexler.‘” Rats of line 1 fed the stock diet had nearly twice the number of miscella?leozls defects 49 vs. 29 per cent, as rats of line 2 (Table 2). These included various degrees of congestion at the corticomedullary border and the occurrence of cysts of various sizes in one or both kidneys. Female rats of both lines had more of these defects than males. There was no consistent diet trend. Line 2 rats with the highest incidence of nephrosis excreted on the average a significantly larger amount of urinary protein than did rats of line 1 fed the stock diet, 787 vs. 464 mg./day, although a large variation occurred in protein excretion of some individual rats (Table 3). The stock diet provoked the largest excretion of protein in both sexes of the two lines. The significant sex difference was eliminated when values were adjusted for maximum body weight. The differences in urine protein may be related to the larger food intakes of the line 1 rats. Within lines, line 1 rats fed diet 4 excreted more protein than those eating diet 3 though because of the large variation differences due to protein level were not significant. Line 2(a) rats fed defatted egg, showed similar differences due to protein level but the amounts were more than double those excreted by line 1 rats fed these diets. Line 2 rats fed stock diet and lactalbumin showed the same trend of response to protein level as line 1 rats but excreted twice as much protein with all diets. A significant regression of urine protein on maximum body weight found for line 1 rats may also be related to food intake of these rats. A small positive relationship was found between kidney
772
MARSHALL AND LEHMANS
weight and urine protein excretion for both sexes in line 1 but no relationship between kidney size and protein excretion in male or female rats of line 2. Thus kidney weight is not the major factor. Relationship between degree of nephrosis and urine protein excretion was almost identical with that for kidney weight and protein excretion for the 2 lines of rats. DISCUSSION
Segregation of genetic factors by inbreeding rats with a history of kidney damage resulted in 2 lines of rats with distinct characteristics with respect to body size and occurrence of kidney defects. Rats with some characteristics similar to these have been produced previously but under different conditions. Kleiber and Cole23 produced 2 strains of rats significantly different in body size after I3 generations of inbreeding grey or white rats with the same ancestry. Astarabadi and Bell7 observed cysts in the kidneys of rats in a colony whose origin was not known but believed to be of the Wistar strain. Zucker and Zucker4 observed nephrosis in 25 per cent of a strain of rats resulting from a mutant gene which also produced unusually large amounts of body fat. We are not aware of any studies in rats where response to kind and level of protein has been tested in rats prone to develop nephrosis spontaneously. The influence of quantity of protein consumed on kidney size has been previously reported 24-26but genetic factors were not considered. Previously when BHE male rats were fed a diet with 25 per cent cooked whole egg their kidneys were enlarged. 1-3 Inbred BHE male rats of line 1, in the present study, consumed large amounts of defatted whole egg which did not result in enlarged kidneys. Thus egg protein per se was not responsible for the enlarged kidneys of BHE rats. In addition, results reported here indicate that semipurified diets with an intermediate level of protein may have a more protective effect with respect to kidney size and development of nephrosis in rats prone to nephrosis (line 2) than diets with high levels of protein. It is well known that kidney stone production can be influenced by diet, particularly by mineral composition and balance,10~27~28and by the interaction between minerals and vitamin B 6.20 The large production of kidney stones in the inbred BHE rats primarily with the stock diet may be related to its high grain content and/or to its high ash content” and the added limestone. In his early studies in India, McCarrison30 reported that gram diets deficient in vitamin A and containing high calcium to phosphorus ratios increased stone production in rats. Kinard31 associated stone production in mice with heredity and with alkalinity of urine. In the present study, it appears that both lines of rats have the genetic potential for stone production which is influenced by diet. Urine pH is not the major factor here since females had lower urine pH values than males with all diets but were the largest stone producers. Proteinuria is known to increase in nephrosis. Increased proteinuria with increased protein intake of nephrotic rats has been reported in Long-Evans ‘Ash content 4.1 per cent.
of the stock diet was 9.9 per cent. Semipurified
diets ranged
from 3.0 to
HEREDITARY
INFLUENCES
ON RAT
773
DIET
and Wistar rats,32 in an inbred hooded strain of rats33 and in SpragueDawley rats.34 Similar sex differences as those reported herein were found by Sellers et a1.35 Genetic differences in spontaneous proteinuria of BHE rats vs. Wistar rats were reported from this laboratory.lv2 However, spontaneous excretion of the inbred BHE rats of line 2 reported herein far exceeds that previously reported for 300-day-old rats; these values demonstrate that differences in protein excretion of apparently healthy rats are related more to heredity than to diet. ACKNOWLEDGMENTS We
to express our appreciation to Dr. H. Menge,
wish
Division
for assistance
with the method
of preparation
Knox, Mrs. Elsie Crump, Miss Rose Harrison, and Mrs. Ethel feeding of animals; and to Mrs. Barbara Smith, Mrs. Vestine technical
assistance.
In addition,
we wish to thank
Poultry
Husbandry
of the defatted
Dr.
Officer, Human Nutrition Research Division, for valuable abnormalities in the animals. The statistical assistance
Research
egg; to Mrs. Vestine
Hall for assistance in care and Knox. and Mrs. Ethel Hall for
Anna
M. Allen
Durand,
advice in the evaluation of Dr. Frank Dickinson
Medical of gross is also
appreciated.
REFERENCES 1. Marshall,
M. W., and Hildebrand,
Differences
in rat
three
of
diets
strain
H. E.:
response
different
to
composition.
J. Nutrition 79:227, 1963. 2. Adams, M.: Diet as a factor
in length
8. Fiegelson, Recant,
E. L.:
B.,
Drake,
J.
W.,
and
Experimental aminonucleoside nephrosis in rats. J. Lab. Clin. Med. 50:437, 1957.
9. March,
J. B., and Drabkin,
D. L.:
Ex-
of life and in structure and composition of tissues of the rat with aging.
perimental reconstruction of metabolic pattern of lipid nephrosis: Key role
USDA
of hepatic protein synthesis in hyperlipemia. Metabolism 9:946, 1960.
port
Home Economics No.
24.
Agriculture, 3. Durand, A. Adams.
M.:
U.
S.
Research
Re-
Department
of
Washington, D. C., 1964. M. A., Fisher, M., and Histology
in rats
as in-
fluenced by age and diet. I. Renal an d cardiovascular systems. Arch. Path. 77~268, 1964. 4. Zucker, T. F., and Zucker, L. M.: Hereditary
obesity
in the
ated with high serum terol.
Proc.
Sot.
rat
associ-
fat and choles-
Exp.
Biol.
Med.
110:165, 1962. 5. Berg, B. N.: Spontaneous nephrosis. with proteinuria, hyperglohulinemia and hypercholesterolemia in the rat. Proc. Sot. Exp. Biol. Med. 119:417, 1965. 6. Sellers, A. L., Rosenfeld,
S., and Fried-
man, N. B.: Spontaneous hydronephrosis in the rat. Proc. Sot. Exp. Biol. Med. l-04:512, 1960. 7. Astnrahadi, T., and Bell, E. T.: Spontaneous hydronephrosis in albino rats. Nature 195:392, 1962.
10. Rohhins, J. D., Oltjen, C. A., and Dolnick, of varying
levels
R. R., Cabbell, E.: Influence
of dietary
minerals
on the development of urolithiasis, hair growth and weight gains in rats. J. Nutrition 11. Hinman, mental
85:355,
1965.
F., and Belt, A. E.: hydronephrosis. The
Experifailure
of diuresis to affect its rate of development. J. Urol. 9:397,
1923.
12. Bras, G., and Ross, M. H.: Kidney disease and nutrition in the rat. Tox. Appl. Pharm. 6:247, 1964. 13. Jones, J. H., and Foster, C.: A salt mixture for use with basal diets either low or high in phosphorus. J. Nutrition 24:245,
1942.
14. Koval, G. J.: Cholesterol measurement in normal and lipemic sera: Elimination of extraneous chromogen. J. Lipid Res. 2:419, 1961. 15. Gilbert, D. L.. and McGann, J.: Titrimetric analysis of calcium and mag-
774
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
MARSHALL
nesium in muscle. Proc. Sot. Exp. Biol. Med. 97:791, 1958. Harrison, H. E., and Harrison, H. C.: A micromethod for determination of serum calcium. J. Lab. Clin. Med. 46:662, 1955. Chen, P. S., Torikara, T. Y.. and Warner, H.: Microdetermination of phosphorus. Anal. Chem. 28:1756, 1956. Gabriel, K. R.: Analysis of variance of proportions with unequal frequencies. J. Amer. Stat. Ass. 58:1133, 1963. Wright, S.: Systems of mating. II. The effects of inbreeding on the genetic composition of a population. Genetics 6:124, 1921. Okey, R., Pencharz, R., and Lepkovsky, S.: Sex hormone effects in incipient biotin deficiency. Amer. J. Physiol. 161:1, 1950. Nielsen, E., and Elvehjem, C. A.: Cure of paralysis in rats with biotin concentrates and crystalline biotin. J. Biol. Chem. I44:405, 1942. Wexler, B. C.: Spontaneous development of renal calculi in repeatedly bred male and female rats. J. Urol. 89:332, 1963. Kleiber, M., and Cole, H. H.: Body size, growth rate and metabolic rate in two inbred strains of rats. Amer. J. Physiol. 161:294, 1950. Mackay, E. M., Mackay, L. L., and Addis, T.: Factors which determine renal weight. VI. Influence of age on the relation of renal weight to the protein intake and the degree of renal hypertroph, produced by high protein diets. Amer. J. Physiol. 86:466, 1928. Hamilton, T. S.: The growth, activity, and composition of rats fed diets
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
AND
LEHMANN
balanced and unbalanced with respect to protein. J. Nutrition 17:565, 1939. Moise, T. S., and Smith, A. H.: The effect of high protein diet on the kidneys. Arch. Path. 4:530, 1927. Forbes, R. M.: Mineral utilization in the rat. I. Effects of varying dietary ratios of calcium, magnesium, and phosphorus. J. Nutrition 80:321, 1963. Coburn, S. P., and Packett, L. V., Jr.: Calcium, phosphorus and citrate interactions in oxalate urolithiasis produced with a low-phosphorus diet in rats. J. Nutrition 76:385, 1962. Faragella, F. F., and Gershoff, S. N.: Interrelations among magnesium, vitamin B,, sulfur, and phosphorus in the formation of kidney stones in the rat. J. Nutrition 81:60, 1963. McCarrison, R.: The causation of stone in India. Brit. Med. J. No. 3675: 1009.1931. Kinard, R.: Occurrence of bladder stones in inbred and hybrid mice. J. Urol. 88:223, 1962. Marsh, J. B., and Drabkin, D. L.: Metabolic channeling in experimental nephrosis. III. Influence of diet and of adrenalectomy; liver hypertrophy. J. Biol. Chem. 230:1063, 1958. Farr, L. E., and Smadel, J. E.: The effect of dietary protein on the course of nephrotoxic nephritis in rats. J. Exp. Med. 70:615, 1939. Rumsfeld, H. W. J.: Role of dietary protein in normal rat proteinuria. Amer. J. Physiol. 184:473, 1956. Sellers, A. L., Goodman, H. C., Marmorston, J., and Smith, M.: Sex difference in proteinuria in the rat. Amer. J. Physiol. 163:662, 1950.
urine protein. Within each line, stock diet provoked the largest excretion of urine protein and 24 per cent of either defatted egg or lactalbumin, the lowest. However, line 2 rats excreted twice as much protein with all diets as line 1 rats. Stock diet produced the highest urine pH and stone incidence in both lines. Females had more kidney stones than males; stones occurred in virgin female rats as well as in breeders. Results of this study point out the need for caution in interpretation of nutrition studies; individual differences may be related to “hidden” genetic defects. (Metabolism 16: No. 8, August, 763774, 1967)
REPORTS most but not all male rats of our laboratory strain (BHE), when compared with a strain of Wistar rats, were shown to have many unique and inherent differences in metabolic response, particularly with respect to lipid metabolism, and length of life, if fed under similar conditions a semipurified diet containing 25 per cent cooked whole egg.la2 It has been demonstrated with the egg-containing diet that BHE rats develop larger livers with higher levels of fat and cholesterol, and have higher urinary protein excretion, serum cholesterol and carcass fat than do Wistar rats. In addition, a higher incidence of nephrosis, hydronephrosis and renal stones occurs spontaneously in apparently healthy BHE than in Wistar rats of the same age. In contrast, BHE rats fed a semipurified diet with less fat and without egg do not develop severe hyperlipemia or nephrosis by 330 days of age but their carcass lipid is higher with this diet than with the egg-containing diet.l
I
N PREVIOUS
From the Human Nutrition Research Research Service, United States Department
Division and Biometrical Services, of Agricultuw, Beltsville, Maryland.
Agricultural
Received for publication Dec. 1, 1966. MARY W. MARSHALL, M.S.: Research Nutritionist, Experimental Nutrition Laboratory, Human Nutrition Research Division. RICHARDP. LEHMANN, PH.D.: Biometrician in charge of Livestock Research Staff, Biometrical Services. Both Research Service of the U. S. Department of Agriculture. 763
authors
are
from
Agricultural
764
hfARSHALL
AND
LEHMANN
It is well known that pathological consequences of a gene may manifest themselves only in certain environmental conditions and that the effects may be altered considerably without causing changes in the gene itself. The rat is currently used as the test animal in a wide variety of investigations, pharmacological as well as physiological and knowledge of specific genetic traits is important particularly in nutrition studies. Certain kidney defects and associated symptoms have been shown to occur spontaneously in certain strains of rats3-* and some defects can be experimentally produced and/or manipulated by dietary means .9-12 Therefore, it is of considerable significance in nutrition investigations that rats with specific genetic traits be produced and studied with the view of understanding some of the reasons for individual variations and perhaps of shedding some light on the solution of some of the practical problems of nutrition of human populations where genetic history of individuals is not always known. To this end, eighth generation inbred BHE rats were fed a stock diet or diets containing 1 of 2 levels of defatted egg or lactalbumin until they were killed at 300 days of age. The results for growth, food intake, kidney defects, kidney weights, urine pH, and protein are reported herein. Findings for liver composition, thyroid, adrenal and heart weights, and serum cholesterol will b e presented in a subsequent report. Evidence is presented that individual animals with unknown defects may provide the explanation for many of the individual differences found in response to diet. EXPERIMENTAL
Animals. were
inbred
animals
Pairs of BHE
rats with known family histories,
by continuing
full-sib
were fed the same pelleted
matings stock diet’
as the BHE
eighth generation were kept until weaned at 21-26 in the litters. Weights of the animals when weaned 487 animals of the eighth generation Diets.
Composition
of the diets
quality were fed, lactalbumin was fed at the same level when
the lower
were included is shown
selected
of offspring
from the stock on hand,
in successive colony.
generations.
days old depending upon the numbers are referred to as initial weight. In all, in the study.
in Table
1. Two
kinds
of protein
and defatted whole egg containing 0.5 per cent fat.7 in all the semipurified diets. Cornstarch replaced
level was fed.
Littermates
The
All animals born in the
of first litters
of 2 family
lines
of high Sucrose protein
of rats,
both
males and females, were housed individually and fed by litter and sex 1 of 3 diets: pelleted stock (diet 1 ), and 1 of 2 levels of defatted egg (diets 3 and 4) or of lactalbumin (diets 5 and 8). Rats designated as line 1 received only defatted egg as the source of protein. Rats Line 2 rats which received defatted egg designated as line 2 were fed only lactalbumin. but no stock diet were designated as line 2a. Each litter of each line received one kind of protein. Male and female littermates received the same kind of protein. For almost every litter fed one combination of 3 diets, a litter from the same family was group-grown (3 to 5 rats each) in the stock colony for the same period of time and was fed only the pelleted stock diet. The stock diet was fed in pelleted
form except
during
collection
of excreta
when it was
“Animal Foundation Laboratory diet, Standard Brands, Inc. (diet 1). Purina Laboratory Chow, Ralston Purina Company (diet 2), to be referred to in a later report. )Defatted whole egg was prepared in our laboratory by cold extraction with a 50:50 mixture of skellysolve “B” and 95 per cent ethyl alcohol.
HEREDITARY
INFLUENCES
ON
RAT
Table
765
DIET
l.-Composition
of Diets Diet No.
Ingredients* Gm./lOO Gm.
Defatted
3
4
23.72
47.43
43.19 23.71
43.19
30.00 0.08 2.6 1.6
18.81 0.01 0.6 0.5
37.44 0.02 0.7
3.93
4.15
4.38
It
whole egg
Lactalbumin Sucrose Cornstarch (edible Proximate
grade)
5
6
23.72
47.43
43.19
43.19
23.71
Composition
Protein, X$ Cholesterol, X$ Calcium. % Phosphorus, % Calories, kcal/Gm. Da
38.56 0.08 0.7
19.31 0.04 0.7 0.5
0.6
0.6 4.68
4.24
“The following vitamins were added per Kg. to each semipurified diet: niacin, pyridoxine*HCl and thiamine*HCl, 5 mg. each; riboflavin, 10 mg.; Ca-pantothenate (d), 25 mg.; u-tocopherol acetate, 25 mg.; folic acid and 2-methyl-l, 4-naphthoquinone, 2 mg. each; vitamin II,,, 30 pg.; biotin, 100 pg.; and later 400 pg. (see text). The following ingredients
were
fed
at the
(Mazola), 5.00; Jones p-amino benzoic acid, vitamins A and D. The fAnima1 Foundation
same percentage level in all semipurified diets: corn oil and Foster13 salt mix, 4.00; choline chloride, 0.20; inositol, 0.10; 0.03; and Percomorph Oil (Mead Johnson Co.), 0.05; to supply oil contained 8,500 units D and 60,000 units A/Gm. Laboratory Diet, Standard Brands, Inc.
SKjeldahl nitrogen X 6.25. Average cent; average N content of lactalbumin $Calculated respectively.
from
‘ODefatted
ground
analysis
whole
to facilitate
fed the pelleted
N content of defatted was 12.64 per cent.
of defatted
egg, 4.70
kcal/Gm.;
the collection
whole
egg and lactalbumin, lactalbumin,
of scattered
food.
5.15
Food
0.42
12.58
and
mg./Gm.,
1.86
intake
was measured
stock diet during the 5 days prior to the lOOth, 20Oth, and 300th
When
the rats reached
300
j,
per
kcal/Gm.
of the rats. Food intakes of the semipurified diets were measured experimental period, with intakes corrected for scattered food. Procedures.
egg was
weekly
for rats birthdays
during
the entire
5 days of age, urine was collected
quantita-
tively from representative groups of rats for 3 or 4 days during the week of autopsy. On the day of autopsy, pH of freshly voided urine was determined* for most of the rats. Then the rats were anesthetized, without previous fasting, with sodium amytal solution. The body cavity was opened and 3 ml. blood removed by heart puncture. Livers were excised, blotted to remove excess blood, weighed and frozen prior to analysis. Kidneys were removed,
trimmed
weighed
(without
and graded
removing
the
capsule),
sagittally,
examined
The
heart,
base, was blotted free of blood in its chambers and weighed. (including the parathyroid) were removed, trimmed and weighed.
Left
adrenals
of
fresh
urine
for gross occurrence
cut
of nephrosis.3
Aliquots
O-4+
were
precipitated
with
washed and after recentrifuging for the second time, was calculated by multiplying by the factor, 6.25. Fat
was
determined
in liver
homogenates
after
trichloroacetic analyzed acid
acid,
for nitrogen.
hydrolysis
and
for
separated
stones, at its
and thyroids
the
precipitate
Urine extraction
protein with
ethyl and petroleum ethers. In composites of defatted egg, diets and lactalbumin, measurements were made of fat by a modified AOAC method using acid hydrolysis, cholesterol,14 calories in the Parr Bomb Adiabatic Calorimeter, nitrogen by the Kjeldahl-GunningArnold method, calcium,rs~1s and phosphorus.17 *Combistix
reagent
strips, Ames Company.
7%
in the inbred BHE rats: upper left: hydronephrosis with stone; upper right: stones and early nephrosis; lower left: cysts in cortex; lower right: congestion at the corticomedullary border. Later stages of nephrosis are similar to illustrations by Durnnd et al.”
St~tistic~EAnalysis. Data from eighth generation rats were analyzed by the method of least squares employing models which evaluated the effects due to (1) line, litters in lines, sex, and line x sex interaction, and (2) sex, diet and sex x diet interaction. Other models were then used which adjusted all variables to average initial, maximum, or final weights of rats. Body weight was taken into account to determine whether the differences observed between the 2 lines were primarily the result of differences in body size. Since significant litter differences were observed each analysis was conducted on a within-litter basis. For some variables, only littermates were included in the analysis and for others, all rats fed the stock diet (“family mates”) were included (line comparison). Differences within the 2 lines of eighth generation rats were analyzed separately, i.e., for line 1, diets 1, 3 and 4 were compared, and for line 2, diets 1, 5 and 6 were compared. Unadjusted means are used in the text when discussing results. For the kidney defects statistical procedures that follow closely those of general least squares were applied according to the methods outlined by Gabriel. 1s Because of differences in feeding plans, rats designated as line 2a were not included in the analysis of variance.
RESULTS Gene&
By the fourth
families
in lines
accrued
by the eighth
a distinct
generation,
1 and 2. With generation
full-sib
matings
was calculated
separation the
degree
occurred
between
of homozygosity
by methods
presented
by
85 per cent. Some of the more frequent defects are shown in Figure 1. One of the most frequent findings in line 1 was severe congestion at the corticomedullary border of the kidneys. Another finding was that of kidney shaped transparent cysts; often, solitary cysts were found in one or both kidneys. The ureter was often greatly dilated.
Wright rg to
be
approximately
HEREDITARY
Typical
INFLUENCES
ON
of the structural
R.4T
767
DIET
characteristics
of line 1 rats was
an
increased
collateral circulation in the region of the left kidney and frequently the left adrenal was embedded on the upper left corner of the left kidney. A small area of necrotic tissue was often attached to the mesentery at the esophageal opening to the stomach and simultaneously to the smallest lobe of liver. Nephrosis was more severe in line 2 rats than in line 1, and was generally Rats of line 2 greatly resembled the rats accompanied by hyperlipemia. described by Zucker and Zucker,4 with the “fafa” genotype except for the unusually large amount of carcass fat which their rats contained. One incident that occurred early during the feeding trials of eighth generation line 1 rats should be noted. After receiving the diets containing defatted egg for 4 to 6 weeks, typical symptoms of biotin deficiency were evident.20 Eighteen of 19 males and 10 of 17 females receiving diet 4 developed the symptoms which disappeared when biotin was given orally, 4 pg./rat/day for 2 weeks. Thereafter, 400 pg. biotin instead of 100 pg./Kg. diet were incorporated in all semipurified diets. However, none of the rats of line 2a fed the defatted egg-containing diets developed the symptoms. Differences in biotin requirements of rats have been reported”* but were not evaluated in the present study. Body Weights and Food Intakes. In Table 2 are average body weights and food intakes (semipurified diets) of eighth generation male and female rats.* Rats of both sexes of line 1 fed the stock diet had on the average significantly larger initial, maximum (16 per cent higher), and final body weights than rats of line 2. When littermates were compared within lines, rats fed defatted egg or lactalbumin grew as well as those fed stock diet and there were no significant diet differences in initial, maximum or final weights of these rats. The tendency was, however, for maximum and final weights of rats within lines to be smaller when fed 47 per cent protein than when fed 24 per cent protein. Rats within line 1 ate more of all diets than rats within line 2. Rats within each line ate less of the diets containing 47 per cent of either protein than of those containing 24 per cent protein. As is usually the case, all differences in food intakes were removed when adjustment for maximum body weight was made and a significant regression coefficient of maximum body weight on food intakes was observed for each line. Kidney Size. Kidney weights are presented in Table 3. Rats of lines 1 and 2 fed stock diet had almost identical average kidney weights, 3.44 vs. 3.-19 (means derived by averaging kidney weights of both sexes within lines) but this weight was a significantly larger percentage of the final body weight of ____ _ ‘Detailed tables of data are submitted for inspection with this report. Data are included in these tables for 141, 300-day-old, sixth and seventh generation rats to support evidence of prior separation of the lines with respect to some of the traits. For the stock diet, average daily food intakes for 21 line 1 males at 100, 200. and 300 days of age were 93.7, 24.9, and 23.8 Gm., respectively; for 18 line 1 females, 16.3. 18.8 (17) and 17.3 Gm. Corresponding figures for 20 line 2 males were 20.8. 27.1. and 22.0 Gm.: and for 15 line 2 femalrs, 15.3, 16.3, and 17.8 Gm.
Weights,
29 5
Line 2a, 24% DFE Line 2a, 47% DFE
*Number in group is same unless indicated iStandard error. $Littermates. gLittermates.
Lact.9 Line 2,47% Lact.$
Line 2,24%
1 2 2 2
5 7 6 11
+ -t f +
7 13 16 15
+ t + +
4 7 7 10
ZlI 5 -+ 7 rtr: 12 z!I 11
263 f 7 250 * 9
272 290 326 278
299 338 321 298
Females
412 ?z 5 414 * 8
444 Z!I 6 464+6 505 + 8 474 ?z 13
522 554 549 492
Males
Final
in parentheses.
274 i- 7 253 -e 8
by other number
47 * 2 58 I!= 2
* + * f
2 & + A
Line 2, stock, in colony
Line 2, stock9
278 298 329 291
32 15 12 10
1, stock, in colony Line 1, stockt Line 1, 24% DFEj Line 1, 47% DFEd 47 42 41 42
54 18 12 14
Line
1 2 2 3 5 7 12 11
303 F 347 -t323 -t 300+
55 51 54 50
F * t &
419 + 14 430 AZ8
55 I!I 5 53 -t- 1
5 22
” 6 z!z 6 3- 9 -+ 9
458 476 518 487
Line 2a, 24% DFE Line 2a, 47% DFE
1 1 1 1
t -c + rt
50 46 46 46
2, stock, in colony 2, stock$ 2,24% Lact.4 2,47% La&Q
41 20 19 14
6 13 16 15
t + + 4
538 563 555 497
Maximum
Line Line Line Line
2f 2 2 2
Initial
Averane Body Weights. Gm.
3501 + 64 3260 f 119
3288 z!z 54 3032 I!I 101
3567 z!z 77 3495 I!I 84
3973 -c 157 4252 +- 84
4384 Z!I 79 4137 * 78
4862 I!I 107 4493 + 110
Total Food Intake, Gm.
and Zncidence of Kidney Defects of Male and Female Either Stock or 1 of 4 Semipurified Diets
* + _’ -t
Rat.9
NO.
Intakes
57 54 54 52
Food
36 21 19 17
Group and Diet
2.-Body
1, stock, in colony Line 1, stock$ Line 1,24% DFE $ Line 1,47% DFE 1
Line
Table
Rats,
90 100
84 93 83 90
100 75 64
91
100 86
100 I.00 100 100
100 100 84 82
All
300 Days
Old
41 60
37 47 33 70
2 17 0 14
80 77
100 100 95 100
86 86 10 41
65 60
16 0 25 40
17 17 8 29
40 54
63 55 42 57
31 38 16 59
0 20
41 80 17 0
63 83 I.7 0
0 0
I.5 50 0 0
44 71 0 0
Hydronephmsis Stones
37 40 67 40
63 67 7s 57
20 41
19 20 21 50
36 33 79 29
Other
Incidence of Kidney Defects
Fed until
Nephrosis
Percentage
Inbred
$ % g
29 5
*Number in group is same unless indicated +Standard error. SLittermates. $Littermates.
Line 2a, 24% DFE Line 2a, 47% DFE
2, stock, in colony 2, stock$ 2, 24% la&.$ 2,47% lact.$
Line Line Line Line
32 15 12 10
54 18 12 14
1, stock, in colony 1, stockt 1, 24% DFE$ 1, 47% DFE j
Line Line Line Line
41 20 19 14
36 21 19 17
NO. Rats’
0.12f 0.22 0.09 0.19
t 0.09 F 0.24 A 0.41 X!I0.55
+rt ++
+ 0.05 & 0.12 z!z 0.11 * 0.40
Z!I 0.05 I+ 0.11 I 0.10 t 0.09
by other number
Weight
* i t *
.02 .05 .08 .15
t .03 -t .04 x!z .Ol -t .03
* t t +-
r+ & -t ?z .02 .04 .03 .18
.Ol .03 .02 .03
1.09 x!z .03 1.59 I!z .18
0.94 0.95 0.83 1.41
0.81 0.84 0.79 0.91
Fetnales
0.78 A .lO 1.15 =!I .13
0.96 0.99 0.96 1.51
0.82 0.82 0.59 0.76
Alalcs
Body Weight
in parentheses.
2.88 * 0.12 3.93 AI 0.39
2.55 2.76 2.71 3.84
2.41 2.83 2.51 2.68
3.26 + 0.49 4.83 -+ 0.60
4.27 4.58 4.83 7.02
4.27 4.52 3.20 3.73
Weight
Kidney
.l (35) .l 0 0
.l .3 0 0
6.2 + .l (25) 6.0 2 0
0.9 2.5
& .l (30) * .2 zlz .l ” .l (9)
+ i it I+ 6.3 6.6 5.9 5.9
7.1 6.5 6.0 6.0 0.6 0.8 0.5 2.2
0.1 0.2 0 0.1
6.7 & .l 6.1 f .l
1.3 1.9
_c .l (39) & .2 ? .l -+- .l (13)
i* ?I +
PH
Urine Protein
300 Days
478 + 53 829 rt 242
645 & 99 (10) 202?52(8) 431f lOO(8)
334*78(13) 163*41(10) 211+26(H)
434 t 133 801 AZ67
930 -+ 56 (9) 721 * 150 (8) 773*99(g)
595 f 61 (13) 200 r+ 46 (12) 410 + 63 (13)
Rats, Fed until
7.2 7.1 6.1 5.9
7.5 7.2 6.0 6.0
-_
Inbred
2.0 2.0 2.0 3.4
1.4 1.6 0.2 0.9
Degree of Nephrosis
of Nephrosis, Urine pH and Protein of Male and Female Either Stock or 1 of 4 Semipurified Diets
5 22
Degree
Line 2a, 24% DFE Line 2a, 47% DFE
2, stock, in colon) 2, stock$ 2,24% 1act.g 2, 47% lact.$
Line Line Line Line
and Diet
~______
Weights,
1, stock, in colony 1, stockt 1, 24% DFE$ 1, 47% DFE]
Group
3.-Kidney
Line Line Line Line
Table
old
770
MARSHALL
AND
LEH.IfASS
the smaller rats of line 2 than of larger rats of line 1. When adjusted to average maximum body weight, significant line differences were observed but differences due to sex were removed. Littermates within lines 1 and 2 exhibited similar response to diet with respect to kidney size. Sex differences but not diet differences in kidney size occurred and these were removed when maximum body weight was considered. However, results for individual diets should not be ignored. Both males and females of line 2 fed diet 6 had larger kidneys than rats fed either diet 1 or diet 5 but kidneys of male rats of line 1 fed diet 4 were smaller than those of rats fed diet 1. Line 2(a) inbred male rats fed diet 4 had kidneys of identical size as line 2 rats fed diet 5. Thus it appears that heredity as well as kind and quantity of protein are factors in determining kidney size. Gross Kidney Defects. Similar incidence, 88 vs. 90 per cent (Table 2) of gross kidney damage occurred in both lines of rats but there was a considerable difference in kind and degree of damage. More kidneys (70 per cent) of line 2 rats had some degree of nephrosis than kidneys of line 1 rats (41 per cent) fed the stock diet and this difference between lines was not explained by body size. Males had a significantly higher incidence than females fed diet 1 or any diet, within each line. Within line 2, average occurrence of nephrosis was higher for each diet than for line 1 rats fed stock diet or corresponding levels of protein. Within each line, rats fed either 24 per cent defatted egg or lactalbumin had the lowest incidence per line. Kind of protein was less important than genetic differences because line 2(a) rats fed defatted egg had more nephrosis than line 1 rats fed this protein. Within line 1, a significant sex by diet interaction occurred but not within line 2; body weight was a significant factor in the development of nephrosis in line 1 rats but not in line 2 rats. Differences in severity of nephrosis were calculated (Table 3) by averaging grades (O-4+) assigned to each pair of kidneys. By this technic within line 1, male rats fed stock diet had the highest score, avg. 1.5, and females fed diet 3, the lowest, zero. Within line 2, male rats fed diet 6 had the highest score, 3.4, and females fed diet 5, the lowest, 0.5. Within each line, rats fed 47 per cent protein had a higher degree of nephrosis in the kidneys than those fed 24 per cent protein. The detrimental role of the stock diet used in this study is indicated by increased kidney size and occurrence of nephrosis in male rats. However, female rats of line 1 showed considerable resistance to nephrosis regardless of diet. Hydronephrosis occurred in both lines, significantly more in males than in females when the stock diet was fed although line differences were not significant. Right kidneys were affected more often in male rats while left kidneys were affected more often in female rats. Overall, rats of line 2 had a higher incidence of hydronephrosis, 36 vs. 22 per cent, than rats of line 1. Within each line, rats fed 4i per cent of either protein had the highest incidence of hydro-
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nephrosis.
INFLUENCES
ON RAT
DIET
771
Male rats in both lines and with all diets had a greater incidence
than female rats. When found, renal stones were usually imbedded
in the pelvis beneath
the papillae of the kidney. They were of different shapes and sizes, were unilateral or bilateral and occurred with or without hydronephrosis. Line 1 rats had a significantly higher incidence of renal stones (Table 2) than line 2 rats fed stock diet, avg. 61 vs. 39 per cent. Female rats of both lines had more stones than male rats, Within each line, the significant diet difference observed is due almost entirely to stone formation in rats fed the stock diet. None of the 96 eighth generation male rats of either line and only 5 of 82 of the female rats fed any of the semipurified diets had kidney stones. It may appear that the higher average urine pH (Table 3) of rats fed the stock diet is associated with the high stone incidence. However, when fed the stock diet, rats with urine pH of 6.0 or 6.5, showed average incidence of 41 per cent for males and 57 per cent for females; in rats with urine pH of 7.0, 7.5, or 8.0, 40 per cent of the males and 70 per cent of females had stones. Thus, urine pH did not seem to affect stone production in males but a higher urine pH was associated with a higher incidence of stones in females. Within lines stone incidence was not influenced by body size, neither was the significant sex by diet interaction for urine pH influenced by body size. Kidney stones occurred in 17 of 28 female rats that had litters and in 50 virgin female rats. With respect to the production of stones in virgin female rats, BHE inbred rats differ from the Sprague-Dawley rats of Wexler.‘” Rats of line 1 fed the stock diet had nearly twice the number of miscella?leozls defects 49 vs. 29 per cent, as rats of line 2 (Table 2). These included various degrees of congestion at the corticomedullary border and the occurrence of cysts of various sizes in one or both kidneys. Female rats of both lines had more of these defects than males. There was no consistent diet trend. Line 2 rats with the highest incidence of nephrosis excreted on the average a significantly larger amount of urinary protein than did rats of line 1 fed the stock diet, 787 vs. 464 mg./day, although a large variation occurred in protein excretion of some individual rats (Table 3). The stock diet provoked the largest excretion of protein in both sexes of the two lines. The significant sex difference was eliminated when values were adjusted for maximum body weight. The differences in urine protein may be related to the larger food intakes of the line 1 rats. Within lines, line 1 rats fed diet 4 excreted more protein than those eating diet 3 though because of the large variation differences due to protein level were not significant. Line 2(a) rats fed defatted egg, showed similar differences due to protein level but the amounts were more than double those excreted by line 1 rats fed these diets. Line 2 rats fed stock diet and lactalbumin showed the same trend of response to protein level as line 1 rats but excreted twice as much protein with all diets. A significant regression of urine protein on maximum body weight found for line 1 rats may also be related to food intake of these rats. A small positive relationship was found between kidney
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MARSHALL AND LEHMANS
weight and urine protein excretion for both sexes in line 1 but no relationship between kidney size and protein excretion in male or female rats of line 2. Thus kidney weight is not the major factor. Relationship between degree of nephrosis and urine protein excretion was almost identical with that for kidney weight and protein excretion for the 2 lines of rats. DISCUSSION
Segregation of genetic factors by inbreeding rats with a history of kidney damage resulted in 2 lines of rats with distinct characteristics with respect to body size and occurrence of kidney defects. Rats with some characteristics similar to these have been produced previously but under different conditions. Kleiber and Cole23 produced 2 strains of rats significantly different in body size after I3 generations of inbreeding grey or white rats with the same ancestry. Astarabadi and Bell7 observed cysts in the kidneys of rats in a colony whose origin was not known but believed to be of the Wistar strain. Zucker and Zucker4 observed nephrosis in 25 per cent of a strain of rats resulting from a mutant gene which also produced unusually large amounts of body fat. We are not aware of any studies in rats where response to kind and level of protein has been tested in rats prone to develop nephrosis spontaneously. The influence of quantity of protein consumed on kidney size has been previously reported 24-26but genetic factors were not considered. Previously when BHE male rats were fed a diet with 25 per cent cooked whole egg their kidneys were enlarged. 1-3 Inbred BHE male rats of line 1, in the present study, consumed large amounts of defatted whole egg which did not result in enlarged kidneys. Thus egg protein per se was not responsible for the enlarged kidneys of BHE rats. In addition, results reported here indicate that semipurified diets with an intermediate level of protein may have a more protective effect with respect to kidney size and development of nephrosis in rats prone to nephrosis (line 2) than diets with high levels of protein. It is well known that kidney stone production can be influenced by diet, particularly by mineral composition and balance,10~27~28and by the interaction between minerals and vitamin B 6.20 The large production of kidney stones in the inbred BHE rats primarily with the stock diet may be related to its high grain content and/or to its high ash content” and the added limestone. In his early studies in India, McCarrison30 reported that gram diets deficient in vitamin A and containing high calcium to phosphorus ratios increased stone production in rats. Kinard31 associated stone production in mice with heredity and with alkalinity of urine. In the present study, it appears that both lines of rats have the genetic potential for stone production which is influenced by diet. Urine pH is not the major factor here since females had lower urine pH values than males with all diets but were the largest stone producers. Proteinuria is known to increase in nephrosis. Increased proteinuria with increased protein intake of nephrotic rats has been reported in Long-Evans ‘Ash content 4.1 per cent.
of the stock diet was 9.9 per cent. Semipurified
diets ranged
from 3.0 to
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INFLUENCES
ON RAT
773
DIET
and Wistar rats,32 in an inbred hooded strain of rats33 and in SpragueDawley rats.34 Similar sex differences as those reported herein were found by Sellers et a1.35 Genetic differences in spontaneous proteinuria of BHE rats vs. Wistar rats were reported from this laboratory.lv2 However, spontaneous excretion of the inbred BHE rats of line 2 reported herein far exceeds that previously reported for 300-day-old rats; these values demonstrate that differences in protein excretion of apparently healthy rats are related more to heredity than to diet. ACKNOWLEDGMENTS We
to express our appreciation to Dr. H. Menge,
wish
Division
for assistance
with the method
of preparation
Knox, Mrs. Elsie Crump, Miss Rose Harrison, and Mrs. Ethel feeding of animals; and to Mrs. Barbara Smith, Mrs. Vestine technical
assistance.
In addition,
we wish to thank
Poultry
Husbandry
of the defatted
Dr.
Officer, Human Nutrition Research Division, for valuable abnormalities in the animals. The statistical assistance
Research
egg; to Mrs. Vestine
Hall for assistance in care and Knox. and Mrs. Ethel Hall for
Anna
M. Allen
Durand,
advice in the evaluation of Dr. Frank Dickinson
Medical of gross is also
appreciated.
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man, N. B.: Spontaneous hydronephrosis in the rat. Proc. Sot. Exp. Biol. Med. l-04:512, 1960. 7. Astnrahadi, T., and Bell, E. T.: Spontaneous hydronephrosis in albino rats. Nature 195:392, 1962.
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