Studies on the renal pigment of estrogen-treated rats Histochemistry and electron microscopy

ESPERIhIENTAL

AND

Studies

nmxcuLm

on the Renal Histochemistry ALBERTO

Centro

PAT~IOLOGY

29,

211-227

( 1978)

Pigment of Estrogen-Treated and Electron Microscopy

J. MONSERRAT,~

Rats

AND EDTJARDO A. PORTA

de Patologia Experimental, II C&e&a de Patologia, Facultad de Medicina, Uniuersidad de Buenos Aires, Argentina, and Department of Pathology, University of Hawaii School of Medicine, Honolulu, Hawaii Received

June

23, 1977,

and

form March

in revised

6, 1978

Although it has been shown that the administration of estrogen to rats is associated with renal pigmentation, neither the nature nor the precise ultrastructural location of the pigment have been yet established. Sequential histochemical and electron microscopic studies carried out in kidneys of Wistar female rats injected intramuscularly with estradiol at various intervals during 7 months, indicated that the pigment accumulating in the epithelium of proximal convoluted tubules fundamentally consisted of hemosiderin, although some other constituents appear to be also present. The pigment deposits were located within lysosomes that with time became post-lysosomes. These deposits differ from those of lipofuscin or ceroid pigments. The possible functional implications of this estrogenassociated hemosiderin pigment remain unknown.

INTRODUCTION In 1966, Harris reported the consistent presence of a golden brown pigment in the epithelium of proximal convoluted tubules of rats treated for prolonged periods with large amounts of estrogen (17 p-estradiol). Dietary vitamin E did not prevent its formation and the histochemical tests performed by this author, although inconclusive, led to the suggestion that the pigment could be pseudomelanin or a variant of lipofuscin. No other investigations on this pigment were reported. It is evident that the nature of this pigment remained uncertain and, since no electron microscopic or systematic cytoenzymatic studies have been performed, its precise subcellular location is unknown. These studies were conducted, therefore, to explore these, as yet indefinite, aspects of the pigment in order to gain an insight on its possible pathogenesis and functional significance. MATERIALS

AND METHODS

Thirty five Wistar female rats ( 116 to 140 g initial body weight) were allotted to three groups (I, II, and III). Rats of group I ( 15 animals) were fed a stock 1 Career tina.”

award

of the “Consejo

National

de Investigaciones

Cientificas

y Tecnicas

of Argen-

211 0014-4800/78/0292-0211$02.00/O All

Copyright @ 1978 rights of reproduction

by Academic Press, Inc. in any form reserved.

212

MONSERRAT

AND

PORTA

laboratory ration 2 ad libitum and injected intramuscularly with estradiol dipropionate 3 according to the following schedule: 0 time, 10 mg; 20 days, 10 mg; 40 days, 5 mg; 60 days, 5 mg; 180 days, 5 mg; and 210 days, 5 mg (total = 40 mg). Rats of group II (10 animals) were fed the same diet ad Zil~itum and injected at the same periods with equivalent amounts of the vehicle alone (sesame oil); and rats of group III (10 animals) were fed the same diet ad libitum and no substance was administered. All animals were housed in wire bottomed individual cages in airconditioned rooms and had free access to water. A number of rats from each group was killed, following light ether anesthesia, from the 2nd and up to the 8th months of the experiment and sections of the kidneys were fixed for light microscopic studies in cold 4% formaldehydesaturated calcium carbonate-5% sucrose (pH 7.2) for 12 to 16 hr or in 3% glutaraldehyde in cacodylate buffer (Sabatini et al., 1963). For electron microscopy, pieces of the renal cortex were fixed in osmium tetroxide (Dalton, 1955) or in 3% glutaraldehyde for about 4 hr, and then washed in buffer-sucrose, refixed in osmium (Dalton, 1955) or fixed in 3% glutaraldehyde alone. Paraffin sections were stained with hematoxilin-eosin ( McManus and Mowry, 1960a), PAS (McManus and Mowry, 1960b) ( wl ‘t’n or without previous diastase digestion), alcian blue ( McManus and Mowry, 196Oc), Muller-Mowry colloidal iron (McManus and Mowry, 1960d), Feulgen (Lillie, 1965a), Mallory’s method for hemofuscin (Lillie, 1965b), Gomori-Burtner’s methenamine silver method for argentaffin cells (Lillie, 1965c), ferrocyanide reaction of Perls (Lillie, 1965d), Schmorl’s method (Pearse, 1960a), Ziehl-Nielsen’s method for acid fastness (Pearse, 1960b), bromophenol blue (Pearse, 196Oc; Barka and Anderson, 1963a), chromotrope 2R/light green (Roque, 1953), and oil red 0 (Wilson, 1950). Unstained sections were studied by conventional light microscopy, uv light and polarizing light microscopy. In addition, after removal of the iron from tissues (Lillie et al., 1963; Goldfischer et al., 1966) sections were studied unstained, after Perls’ reaction or after PAS stain. Frozen sections were stained with oil red 0 (Wilson, 1950) or the unstained ones were studied by uv and polarizing microscopy. For enzymatic histochemical studies the material was kept in acacia gumsucrose (Burstone, 1962) when formalin was used as fixative, or in buffer-sucrose when glutaraldehyde was used instead. Frozen sections were incubated freefloating in appropriate media for the detection of activities of thiaminepyrophos1961) , inosine-di-phosphatase phatase ( TPP-ase) (Novikoff and Goldfischer, and Goldfischer, 1961), dihydronicotinamide-adeninedi( IDP-ase) (Novikoff nucleotide-tetrazolium reductase (NADH-TR) (Novikoff et al., 1961), esterases (Delellis and Fischman, 1965), aryl sulphatase (Goldfischer, 1965) and acid phosphatase using either Gomori’s (Gomori, 1952; Novikoff, 1963; Goldfischer et al., 1964) or Barka and Anderson’s method ( 1962). Control sections incubated in media without any substrate or with suitable inhibitors were also examined. For electron microscopic studies the blocks were embedded in Epon 812 (Luft, 1961) and “thick” sections (0.5 to 1 p) were stained with toluidine blue (Trump et al., 1961) and thin adjacent sections were stained with phosphotungstic acid 2 Purina Laboratory Chow. 3 DI, Ovocyline, Ciba.

RENAL

PIGMENT

IN

ESTROGEN-TREATED

BODY

213

RATS

WEIGHT

180

0

1

2

3

4

5

6

7

8

Months 1. Growth

FIG.

curves

in the

different

groups.

(Watson, 1958), lead citrate (Reynolds, 1963) or a combination of uranyl acetate (Watson, 1958) and lead citrate. At the 6th month of the experiment blood from the three groups was analyzed for hemoglobin, red and white cell counts, mean corpuscular hemoglobin index, reticulocytes, and urea nitrogen. RESULTS Changes in body weight in the different groups are presented in Fig. 1. The estrogen-treated rats grew substantially less than the controls injected or not with the vehicle alone. The levels of hemoglobin, mean corpuscular hemoglobin index and the number of red blood cells in the estrogen-treated rats at the 6th month were significantly lower than those in control rats (Table I). No significant differences between groups were observed in the other parameters studied, After the 5th to 6th month the renal cortex of estrogen-treated rats displayed on gross examination a dark discoloration. This was not observed in the control groups. TABLE Blood Group

I II III 0 b c d

Hb (9%)

RBC (X 109

9.7 f 0.9 (S)b 12.8 zt 0.7 (8) 13.4 f 0.3 (4)

4.1 f0.5 (8)~ 5.0 f 0.3 (8) 5.6 f 0.1 (4)

Mean VdU~28 f SEM. Significantly different, Significantly different Significantly different

from from from

group group group

Analysis

at the 6th MCH (Pg)

23.5 25.8 23.8

I

f 0.3 (t3)d zt 0.5 (8) f 0.6 (4)

Montha WBC (XlO’l 6.9 zt 1.5 (8) 8.3 f 1.1 (8) 7.6 f 1.1 (4)

II (P < 0.02) and III (I’ < 0.05). III (Z’ < 0.02). II (P < 0.001). Number of determinations

Retie. (%) 1.2 f0.3 (8) 1.9 f 0.2 (8) 1.9 f 0.4 (4)

in brackets.

BUN (%) 26.2 22.0 24.5

f f f

1.4 (7) 1.9 (4) 0.9 (4)

214

MONSERRAT

FIG. 2. Pigmented these granules show (gray in this figure). stain, X1125).

Light

AND

PORTA

granules located in proximal convoluted tubules of the cortex. Most of a dark zone and clear one; the latter sometimes stained metachromatically Group I 250 days. (Osmium fixation, Epon embedded. Toluidine blue

Microscopy

In estrogen-treated rats a granular, golden or golden-brown pigment exclusively located in the epithelium of proximal convoluted tubules was first detected at the 3rd month of the experiment. The number and size of pigmented granules as well as the number of cells and tubules affected increased with time. In the control rats only few dispersed pigmented granules were observed in these tubules after the 5th month. In paraffin embedded sections the granules were composed of a dense zone and a clear one located either on one side or surrounding the dense zone. In frozen sections the clear zone was less conspicuous. In epon embedded sections stained with toluidine blue the granules also displayed two different zones; one of these, usually located on one side was deeply blue and of high optical density while the other was either clear or more often stained metachromatically in different shades of purple-red (Fig. 2). In Dalton fixed tissue the purple metachromasia was quite deep, but in sections fixed in glutaraldehyde alone the metachomatic zone was pale red. The tinctorial affinities, histochemical reactions and physical characteristics of the pigment found in estrogen treated rats are summarized in Table II. In control rats the enzymatic histochemical reactions showed the well-known characteristics previously described in normal rats (Novikoff, 1963, Delellis and Fischman, 1965; Monserrat et al., 1969),

RENAL

PIGMENT

IN

ESTROGEN-TREATED

TABLE Characteristics (A)

II

of the pigmented-granules.

Solubility Water Alcohol Xylol

Insoluble Insoluble Insoluble

Stains Hemotoxylin Eosin Light green Chromotrope 2 R Feulgen Silver-Methenamine OR0 (frozen sections) OR0 (paraffin sections) Bromphenol blue PAS” PAS (after diastase) Hemofuscin Schmorl Ziehl-Nielsen Ferric pigment 6 Colloidal-iron Alcian blue Toluidine blue (epoxy sections)

Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive in part” Positive in part Positive in part Positive in part Positive in part Positive in part Positive in part Positive in part Blue, red-purple,d

(C)

Ultraviolet

Negative

(D)

Polarizing

(E)

Histoenzymatic activities NADH-TR IDPase TPPase Esterases Acid Pase (Barka and Anderson) Acid Pase (Gomori) Aryl sulphatase

(B)

215

RATS

light light

11The PAS positivity after treatment with sodium dithionite. 6 Negative after treatment with sodium dithionite. c Dense zone of the granules. d Deep red-purple in osmium fixed sections, pale red-purple c Positive in early stages and negative later.

white

Negative

Negative Negative Positive Positive Positive Positive Positive

or or or or or

negativec negative negative negative negative

in gluturaldehyde

fixed

sections.

In the initial stages of pigment formation in treated rats, due to the small number and size of the granules, it was difficult to ascertain whether the tinctorial affinities and particularly the histochemical reactions were positive or negative. The positivity for PAS, hemofuscin, alcian blue, Schmorl and acid-fastness was detected in the dense part of the granules while the clear zone was negative. The dense zone usually gave a positive reaction for ferric iron although occasionally some small areas of this zone were negative (Fig. 3). When sections were kept for 15 min in sodium dithionite, the reaction for ferric iron became

216

MONSERRAT

FIG. 3. ‘The pigmented zone of most pigment. Group I 145 days (Formaldehyde

AND

PORTA

of the granules display a positive reaction for ferric fixation, paraffin cml~edded. Perls’ reaction, x450).

FIG. 4. Some pigmented granules show a positive days (Glutaraldehyde fixation, Aryl sulfatase, 45 min,

reaction x500).

for

aryl

sulfatase.

Group

I 145

RENAL

PIGMENT

IN ESTROGEN-TREATED

RATS

217

FIG. 5. Irregularity in size and distribution of the aryl sulfatase positive droplets. Large pigmented granules are negative. Group I 250 days. (Formaldehyde fixation. Aryl sulfatase, GO min, X500).

FIG. 6. Irregularity in size of the acid phosphatase-positive droplets. granules display a positive reaction. Group I 145 days. (Fonnnltlcl~ytle Gomori’s technique, 30 min, x500).

Numerous pigmented fixation. Acid Pnse;

MONSERRAT

218

FIG. 7. Irregularity in size and distribution as large positive aggregates. Large pigmented tive rim. Group I 250 days. (Formaldehyde x500).

AND

PORTA

of the acid phosphatase-positive granules are negative, but fixation. Acid Pase, Gomori’s

droplets some display Technique,

as well a posi30 min,

negative, but the PAS positiveness and the natural color of the pigment as seen in unstained sections persisted. In sections stained with oil red 0 or with chromotrope 2R, faint staining was only occasionally detected. The pigmented granules did not give positive reactixr for NADH-TR or for IDP-ase. In those tubules with heavy pigmentation the overall NADH-TR activity was in fact diminished. Some of the granules were positive for TPP-ase but others, particularly those of large size, were negative. Similarly, the reactions for aryl sulphatase and acid phosphatase (Barka and Anderson’s technic) were positive in small granules and negative in the larger ones. In tubules with heavy deposits there was a marked irregularity in size distribution of granules positive for aryl sulphatase (Figs. 4 and 5). In sections incubated for the demonstration of acid phosphatase following Gomori’s technic, it was seen again that the small gramdes were positive while the larger ones were generally negative although some of these had a positive rim and tiny positive sites of activity within the granular matrix. In tubules with heavy deposition of large granules there were few acid phosphatase positive droplets (Figs. 6 and 7). Electron

Microscopy

The ultrastructural features observed in control rats were practically similar to those described in normal rats (Latta ef LIZ.,1967; Trump and Bulger, 1968). In

RENAL

ing

PIGMENT

IN

ESTROGEN-TREATED

219

RATS

FIG. 8. Granules in proximal tubules of the cortex. There is diminution cytosomes and cytosegresomes. Group I 145 days. (Osmium fixation,

of normally lead stain,

appearX8580).

the control rats killed at the latest periods of the experiment, small number of granules ultrastructurally similar to those observed at earlier stages in the experimental group were also found.

FIG. rounded

9. The two zones of the granules are clearly clelimited; most by a single membrane. Group I 145 days. (Osmium fixation,

of the granules are surlead stain, X 14,080).

In the estrogen-treated rats granular bodies, 1 to 14 p in diameter, were observed in proximal convoluted tubules (Fig. 8). The number and size of these granules increased with time. Most granules were surrounded by a single membrane and consisted of a clear and a dark zone (Fig. 9). The clear zone generally had a faint electron density due to the presence of a fine and sparse granularity somewhat resembling ferritin. The internal configuration of the dark zones was in many instances rather polymorphic due to the presence of granular conglomerates, tortuous membranes and myelin-like figures (Fig. 10). A clear “halo” (Daems et al., 1972) was occasionally found. Images interpreted as confluence of these bodies were frequently encountered. Large bodies or granular conglomerates usually lacked a limiting membrane. Huge laminated bodies (myelin bodies) were occasionally observed in the epithelial cells of proximal convoluted tubules (Fig. 11). The substructure of these bodies consisted of closely packed concentric fine lamellae and were sometimes partly surrounded by granular or more compact deposits. Along with the appearance of the above mentioned bodies there was a conspicuous decrease in the number of normally appearing cytosegresomes and particularly cytosomes. Other cytoplasmic organelles did not show any significant change.

Granules of various dimensions and showing myelin-like internal configuration were also occasonally found between the microvilli of the bursh border (Fig. 12). They were not snrrountlctl 1)~ a 1~w1~~1nxn~. In the interstitial cells of the i-end cortex few hcterogcnolIs granules were seen.

RENAL

FIG. 10. within the x 13,500). They

were

Two huge latter

zone,

granules Group

ultrastructurally

and Hartroft, 1973).

MS),

PIGMENT

IN ESTROCXN-TREATED

show distinctly I 250 days.

delimited (Osmium

clear fixation,

RATS

and dark zones, uranyl acetate,

221

with membranes and lead stain,

similar to those described as ceroid pigment (Porta as well as iron pigment (Daems et al., 1969; Trump et al.,

222

FIG. 11. Numerous myeh is a decrease in the number days. ( Glutaraldehyde and

MOI’SERRAT

AND

PORTA

figures, some of them in relation with pigmented granules. of normally appearing cytosomes and cytosegresomes. Group osmium fixation, uranyl acetate and lead stain, X 14,800).

There I 250

DISCUSSION Pigmentation of the renal ‘cortex has been observed in various animal species under normal and pathological conditions. Ceroid deposits have been found in

RENAL

FIG. 12. Residual 145 days. (Osmium

bodies fixation,

PIGMENT

IN

ESTROGEN-TREATED

among the lead stain,

microvilli X 14,800).

of a proximal

223

RATS

tubule

of the

cortex.

Group

I

the proximal convoluted tubules of vitamin E-deficient rats (Mason and Emmel, 1945), monkeys (Mason and Telford, 1947), and minks (Mason and Hartsough, 1951); particularly when the deficient diets contained large amounts of cod liver oil (Mason et al., 1946). Ceroid pigment, predominantly located in proximal convoluted tubules has been also found in human albinos with hemorrhagic diathesis ( Bednar et al., 1964). Another lipid pigment, lipofuscin (age pigment), has been described in the proximal convoluted tubules of different animal species including non-human primates and in man (Colcolough et al., 1970). Cain and Kraus ( 1972), have reported the presence of “lipofuscin-like” pigment in lysosomes of proximal convoluted tubules of old rats. The ultrastructural configuration of many of the granules found by these authors had a resemblance to those found in our estrogen-treated rats. However, while the pigment described by Cain and Kraus was sudanophilic and strongly autofluorescent, the pigment in our rats was not. Although lipid pigments are characteristically heterogeneous, the ultrastructural features generally described for lipofuscin and ceroid pigments (Porta and Hartroft, 1969) differ from those in our animals. In addition, unlike lipofuscin granules, the reaction for NADH-TR (Goldfischer et al., 1966) was negative in our studies. In consequence, the estrogen-associated pigment of rats cannot be considered a lipid pigment. Hemosiderin pigment has been observed in cytosomes of epithelial cells of proximal convoluted tubules in golden hamsters with malarial nephropathy, (Sesta et al., 1968), as well as in the human tubular epithelium in a great variety of conditions (Remmele and Hinrichs, 1970). This pigment may also occur

“spontaneously” in the renal cortex of goats (“cloisonne kidney”) ( Light, 1960). Ultrastructural studies of these caprine kidneys revealed thickening of the basement membrane of proximal convoluted tubules with ferritin aggregates on its capillary side, while the epithelial cells of these tubules contained membranebound aggregates of hemosiderin (Grossman et al., 1969). Our present results are clearly at variant with those of Harris (1966), since he found that the pigment of estrogen-treated rats was not anatomically associated with ferric iron or with sites of acid phosphatase activity. It seems unlikely that the differences between experimental procedures could be the cause of the conflictual results. Based on the histochemical and histoenzymatic reactions as well as the ultrastructural features displayed by the pigment in the present study, we conclude that although it apparently has a rather complex nature, it is fundamentally a hemosiderin type of pigment (Pearse, 1960d; Bnrka and Anderson, 196313; Engberg et al., 1969; Trump et al., 1973), initially located in lysosomes and later on in post-lysosomes (De Duve and Wattiaux, 1966; Ericsson and Trump, 1969). Lysosomes with clear and dark zones have been also reported in other circumstances (Koenig, 1969; Daems et al., 1972). Although lysosomes are generally autofluorescent (Ericsson and Trump, 1964; Maunsback, 1966; Koenig, 1969; Monserrat et al., 1969), they can lose this property in pathologic conditions ( Monserrat et al., 1969). The possible functional implications, if any, of the renal accumulation of this estrogen-associated pigment remain unknown. While the main purpose of this experiment was to study the nature of the pigment, the presence of anemia in the estrogen-treated rats merits some comments on its possible origin. Although the limited hematologic data do not allow adequate determination of the type of anemia, the normal mean corpuscular hemoglobin index indicates, at least, that the anemia is of a saturated type. The norma (or relatively reduced) number of reticuIocytes found in the estrogentreated rats does not preclude the eventual presence of a hemolytic mechanism. Anemi‘as with reduced erythrocytic survival, reduced reticulocytes and hemosiderosis have been described in certain nutritional deficiencies such as protein, vitamins E, Bs, B1z and folate (Wintrobe et al., 1974a). On the other hand, it is well known that ineffective erythropoiesis can be associated with tissue deposition of hemosiderin pigment, as seen in either megaloblastic (Chanarin, 1969) or sideroblastic anemias (Mollin, 1965). Although in our experiment no attempts have been made to evaluate the food intake of the animals, the reduced growth curve (Fig. 1) of the estrogen-treated rats would suggest a diminished food intake with the possibility of resulting nutritional deficiencies. It has been shown that the administration of large amounts of estrogen to rats is associated with reduced erythropoietic response and reduced production of erythropoietin ( Wintrobe et al., 1974b). It has been also postulated that prolonged estrogen treatment may induce hepatic damage (Schaffner, 1966), and therefore, possible metabolic alterations of the erythrocytic maturation factors. Furthermore, in women treated with estrogen-containing oral contraceptives, the levels of serum folate are generally reduced ( Shojania and Hornady, 1968). Although it has been recently suggested that this reduction might be due to a primary nutritional deficit (Ross et al., 1976), a more direct effect of estrogen

RENAL

PIGMENT

IN

ESTROGEN-TREATED

225

RATS

on folate metabolism cannot be presently excluded. Since at the moment of this writing there are no reports on the possible association between the use of oral contraceptives or prolonged estrogen therapy and the presence of renal changes in man, such as the pigment deposits in our rats, it would be of obvious interest to explore this aspect in human renal biopsies. All the speculative possibilities mentioned above, clearly indicate the need of further hematologic and nutritional studies to determine more precisely the origin of this pigment in estrogen-treated rats. ACKNOWLEDGMENT The

authors

wish

to express

their

appreciation

to Dr.

Arturo

Mario

Musso

for

his advice.

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435492. DELELLIS, R., and FISHMAN, W. H. (1965). The variable of pH in the Bromoindoxyl acetate method for the demonstration of esterase. J. Histochem. Cytochem. 13, 297. ENGBERG, A., ERICSSON, J. L. E., and ORREMUS, S. (1969). Electron microscopic observations on iron labelled secondary lysosomes in renal tubules in mice. 2. Zellforsch. 98, 378-398. ERICSSON, J. L. E., and TRUMP, B. F. (1964). Ebctron microscopic studies of the epithelium of the proximal tubule of the rat kidney. I. The intracellular localization of acid phosphatase. Lab. Inuest. 13, 1427-1456. ERICSSON, J. L. E., and TRUMP, B. F. (1969). Electron microscopy of the uriniferous tubules. In “The kidney. Morphology, Biochemistry, Physiology” (Ch. Rouiller and A. F. Muller, eds.), pp. 351447. Academic Press, New York. GOLDFISCHER, S. ( 1965). The cytochemical demonstration of lysosomal aryl sulfatase activity by light and electron microscopy. J. Histochem. Cytochem. 13,, 520-523. GOLDFISCHER, S., ESSNER, E., and NOVIKOFF, A. B. (1964). The localization of phosphatase activities at the level of ultrastructure. J. H&o&em, Cytochem. 12, 72-95. GOLDFISCHER, S., VILLAVERDE, H. and FORSCHIRM, R. (1966). The demonstration of acid hydrolase thermostable reduced diphosphopyridine nucleotide tetrazolium reductase and

MONSERRAT

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granules.

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G. (1952). Microscopic histochemistry. Principles and practice, pp. 189-194. University of Chicago Press, Chicago. GROSSMAN, I. W., ALTMAN, N. H., and ERSENAL, E. ( 1969). Caprine cloisonne renal lesions. ( Ultrastructure of the thickened proximal convoluted tubular basement membrane). Arch. GOMORI,

Path. 88, 609-612. HARRIS, CH. ( 1966). Pathol. 82, 353-355.

A lipofuscin-like

pigment

in the kidneys of estrogen-treated

rats. Arch.

KOENINC, H. ( 1969). Lysosomes in the nervous system. In “Lysosomes in Biology and Pathology” (J. T. Dingle and H. B. Fells, eds. ), Vol. 2, pp. 111-162. North Holland Pub. Co., Amsterdam. LATTA, H., MAUNSUACH, A. B., and OSVALDO, L. (1967). The fine structure of renal tubules in cortex and medulla. In “Ultrastructure of the kidney” (A. J. Dalton and F. Haguenau, eds.), pp. l-56. Academic Press, New York. LIGHT, F. W. ( 1960). Pigmented thickening of the basement membranes of the renal tubules of the goat (“cloisonne kidney”). Lab. Invest. 9, 228-238. LILLIE, Ft. D. (1965a). Histopathologic technic and practical histochemistry, pp. 149-150, 3rd ed. McGraw-Hill Book Co., Toronto. LILIJE, R. D. (1965b). Histopathologic technic and practical histochemistry, p. 417, 3rd ed. McGraw-Hill Book Co., Toronto. LILLIE, R. D. (1965~). Histopathologic technic and practical histochemistry, pp. 240-241, 3rd ed. McGraw-Hill Book Co., Toronto. LILLIE, R. D. (1965d). Histopathologic technic and practical histochemistry, pp. 405407, 3rd ed. McGraw-Hill Book Co., Toronto. LILLIE, R. D., GEER, J. C., and GUTIERREZ, A. ( 1963). Th e removal of histochemically demonstrable iron from tissue sections by brief exposure to sodium dithionite solution. J. Histochem. Cytochem. 11, 662-664. LUFT, J. H. (1961). Improvements in Epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414. MASON, K. E., DAM, H., and GRANADOS, H. ( 1946). Histological changes in adipose tissue of rats fed a vitamin E deficient diet high in cod liver oil. Anat. Rec. 94, 265-287. MASON, K. E., and TELFORD, I. R. ( 1947). Some manifestations of vitamin E deficiency in tht monkey. Arch. Pathol. 43, 363-373. MASON, K. E., and EhfhfEL, A. F. ( 1945). Vitamin E and muscle pigment in the rat. Anat. Rec. 92, 33-59.

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