Microvascular changes in rat glomeruli as a consequence of small differences in selenium exposure

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

57, 222-234 (1992)

Microvascular Changes in Rat Glomeruli as a Consequence Small Differences in Selenium Exposure

of

C. D. ECKHERT,’ M. K. LOCKWOOD, M. H. Hsu, J. Ho, AND R. KANG Department

Received

of Environmental Health Sciences, University Los Angeles, California 90024-1772 February

6, 1992, and in revised

form

November

of California,

9, 1992

Thispaperevaluatesthe effect of small differences in selenium exposure, within the safe range, on the glomerular vascular tufts of rats fed high-sucrose diets. In the first experiment male Wistar rats were housed in galvanized cages and were provided sucrose-based diets to induce a mild chronic insult to the microcirculation. One group of rats received the diet prepared to contain 0.10 mg Se/kg and another group 0.21 mg Se/kg. To assure that the galvanized metal cages were not influencing the results of the experiment this protocol was repeated in a second experiment wherein rats were housed in stainless steel cages. The levels of Se used supported normal activity of the long-term indicator of Se sufftciency, erythrocyte glutathione peroxidase. In both experiments rats fed diets containing 0.21 mg Se/kg had larger Bowman’s capsules (P < 0.01) and vascular tufts (P < 0.01). Vascular tufts from these rats also contained a higher proportion of open capillary lumen (P < O.Ol), contained less cytoplasmic and extracellular material (P < O.OOl), and had larger nuclei (P < 0.001) than those fed 0.10 mg Se/kg. A third study was designed to determine if the selenium-dependent differences in nuclear size were indicative of this being a site of incorporation. Year-old rats subjected to the same protocol as those in the second experiment were given ‘%e, by injection into the femoral vein, to label the sites of incorporation. Glomeruli were purified and subjected to subcellular fractionation. Ninety percent of the radioactivity was associated with the crude nuclear fraction. Purification of the crude nuclear fraction demonstrated that the radioactivity was associated with the nuclei. 0 1992 Academic

Press, Inc.

INTRODUCTION Selenium (Se) is required for maintaining capillary integrity but when the level of exposure is either too low or high the microvasculature suffers damage. Animals foraging on plants grown in soils of low Se concentration and those exposed to environments with high levels of the element develop a wide array of pathological lesions due to the breakdown in capillary integrity (National Research Council, 1976). Attempts to determine the basis for the importance of Se in the maintenance of capillary integrity have not been successful. The use of Se deficiency and toxicity states to study this problem is too severe since both of these conditions produce widespread tissue damage that masks the primary effect of Se on microvessels. We have previously shown that rat retinal capillaries are sensitive to Se status when they are subjected to mild injury resulting from chronic exposure to elevated blood lipids, glucose, and insulin (Lockwood and Eckhert, 1992). Under these conditions supplementation with Se within the safe range (between deficiency and toxicity) protected the retinal capillaries from mild injury. The conditions of the experiment provided a means to study the effect of Se directly on the microcirculation free from the confounding variables induced by secondary pathologically ’ To whom correspondence should be addressed at Department of Environmental University of California, Los Angeles, CA 90024-1772. Fax: (310) 794-2106. 222 0014-4800/92 $5.00 Copyright All rights

8 1992 by Academic Press, Inc. of reproduction in any form reserved.

Health

Sciences,

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223

alterations encountered in experiments involving Se deficiency and toxicity. The retinal circulation of the rat is rather limited in size and it would be useful to identify a more abundant source of Se-sensitive microvessels for characterizing the molecular basis of the Se effect. Kidney is a good candidate for evaluation. Selenium is present in relatively high concentrations in this highly vascular tissue. In a comparative study Lindberg (1968) reported the concentration of Se (mg/g dry wt) in the organs of the adult pig to be: 11.47, kidney; 1.82, liver; 1.42, pancreas; 1.26, spleen; 1.13, lung; 1.05, heart; and 0.52, skeletal muscle. Like the retinal circulation the glomerular vascular tuft is also susceptible to mild injury induced by the long-term consumption of diets containing sucrose as the sole carbohydrate (see Poulsom, 1986 for review). In the present report, high-sucrose diets have been used to provide evidence that glomerular capillaries of normal rats are sensitive to small differences in the level of selenium exposure and that their nuclei represent the major subcellular site of Se incorporation. MATERIALS Animals

AND METHODS

and Diets

The diets used in the experiments were designed to evaluate Se-dependent alterations in microvascular morphology within a range of exposure above deliciency (as measured by erythrocyte glutathione peroxidase activity) and below toxicity levels. Milk casein was used as the protein source since, unlike torula yeast, the protein usually used to induce deficiency status (0.05 mg Se/kg diet), the Se concentration of casein is high enough to maintain growth (Witting and Horwitt, 1964). Sucrose was used as the sole carbohydrate as it served the dual purpose of providing a mild injurious challenge to the microvascular system and served as a Se-free energy source (Thornber and Eckhert, 1984). Forty weanling 50 to 60-g male Wistar rats (Charles Rivers, Wilmington, MA) were assigned to four diet groups. The purified diets were prepared in our laboratory using New Zealand casein, Hawaiian cane sugar, cellulose (solka floe), and corn oil. Two different vitamin and mineral mixes without Se were used. Diet A was that used by Papachristodoulou et al. (1976) and Thornber and Eckert (1984). This diet was used in the first experiment because of its proven ability to induce a mild microvascular injury (Papachristodoulou et al., 1976). It is an old diet formulation used for rats housed in galvanized cages where the metal frame provides animals with an adequate supply of some of the essential elements. However, diet A’s micronutrient content falls short of recent standards for rat diets when they are housed in stainless steel cages. Asecond experiment was therefore used to replicate the experiment to be sure the results were not influenced by something in the galvanized metal. In the replicate experiment rats were housed in stainless steel cages and fed diet B. Diet B contained all the vitamins and elements in amounts currently recommended for the rat (American Institute of Nutrition, 1977) except for Se, which was varied as described, and vitamin E. RRR-ol-tocopherol was provided at double the recommended level to assure the diet contained adequate amounts of this membrane-associated antioxidant (Eckhert, 1982, 1987). It did not contain vitamin C, as the rat can synthesize this vitamin and it is no longer recommended as preservative in rat diets (American Institute of Nutrition, 1977; National Research Council, 1978). Selenium as sodium selenite (Na,SeO, * 5 H,O) was prepared as a separate mix with sucrose for

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ET AL.

supplementation. Diets were prepared fresh every three weeks and stored at 4°C. The diet compositions are given in Table I. The selenium concentrations were 0.10 and 0.21 mg Se/kg diet, respectively, for the basal and supplemented diets. A group of four Wistar rats was also fed a commercial closed-formula cereal-based diet (Wayne Rodent Blox chow) to provide an external comparison group. Rats were housed individually and provided free access to their food and water during the year-long feeding trial. The cages of both groups were in the same room and maintained under a 12-hr light (060&18OO)/dark cycle. Rats were handled once each week for weighing. Food consumption records were maintained for each rat in each experiment. Se Analysis

Diets were analyzed for Se content by the fluorometric procedure described previously (Hoffman et al., 1968; Olson et al., 1975). Briefly, diets were digested with nitric and sulfuric acid to remove organic material. The Se was then complexed with 2,3-diaminonaphthalene, extracted with cyclohexane, and measured fluorometrically. The coefficient of variation of the assay was 10%. The commerTABLE I Composition of Diets Ingredient

Diet A (%)

Diet B (%)

Casein Corn oil Cane sugar Vitamin mix A (93% sucrose) Vitamin mix B (%% sucrose) Mineral mix A (12% sucrose) Mineral mix B (0.03% sucrose) Cellulose DL methionine Choline chloride

22.6 5.0 63.2 2.0

20.0 5.0 65.0

Total

1.0 4.0

0.2

3.5 5.0 0.3 0.2

100.0

100.0

3.0

Nore. Chemical analysis showed that these diets contained 0.1 mg Se/kg. Identical diets were provided with an additional 0.1 mg Se/kg diet by the addition of 5 g/kg diet of a mix containing 70 mg Na,SeO, . 5 H,O/kg sucrose. These were shown by chemical analysis to contain 0.2 mg Seikg. Diet A Vitamin mix per kg; ascorbic acid, 3.75 g; thiamin . HCl, 500 mg; riboflavin, 500 mg; pyridoxine * HCI, 500 mg; nicotinic acid, 3 g; calcium D-pantothenate, 2 g; folic acid, 250 mg; d-biotin, 50 mg; vitamin BL2 as 0.1% in mannitol, 2500 mg; RRR-a-tocopherol, 5000 IU; vitamin A pahnitate, 650,000 IU; cholecalciferol, 400,000 IU; and menadione, 50 mg. The salt mix contained per kg mix: dibasic calcium phosphate, 325 g; calcium carbonate, 205 g; potassium chloride, 205 g; dibasic sodium phosphate, 185 g; magnesium sulfate * H,O, 70 g; manganese sulfate * H,O, 4.5 g; ferric citrate * 5 H,O, 4.4 g; zinc carbonate, 0.75 g; potassium iodate, 25 mg; cupric sulfate .5 H,O, 380 mg. Each mix was made up to 1 kg with sucrose. Diet B vitamin mix contained per kg: thiamin * HCl, 600 mg; riboflavin, 600 mg; pyridoxine * HCl, 700 mg; nicotinic acid, 3 g; DL-calcium pantothenate, 1.6 g; folic acid, 200 mg; d-biotin, 20 mg; vitamin B,,, 1 mg; RRR-a-tocopherol acetate, 10,000 IU; vitamin A palmitate, 400,000 IU; cholecalciferol, 100,000 IU; and menadione, 5 mg. The salt mix contained per kg mix: dibasic calcium phosphate, 500 g; sodium chloride, 74 g; potassium citrate, 220 g; potassium sulfate, 52 g; magnesium oxide, 24 g; manganese carbonate, 3.5 g; ferric citrate, 6 g; zinc carbonate, 1.6 g; cupric carbonate, 0.3 g; potassium iodate, 10 mg; and chromium potassium sulfate, 0.55 g. Each mix was made up to 1 kg with sucrose. The total sucrose concentration of diet A was 65.5% and of diet B, 66.0%.

MICROVASCULAR

cial chow ration contained supplier.

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0.2 mg Se/kg based on mean analysis of lots by the

Tissue Preparation Pairs of rats from each group were anesthetized with ketamine (0.5 ml ketamine hydrochloride containing 20% acepromazine maleate), and kidneys were prepared for light microscopy using the procedure of Hirose et al. (1982). Kidneys were removed, bisected longitudinally, and fixed by immersion in 10% neutral-buffered formalin, pH 7.4. Blocks of cortical tissue were excised from kidneys from each diet group and concurrently dehydrated through a graded series of alcohols and embedded in paraffin. Serial sections (6 pm) were mounted on glass slides and dried at 60°C. Slides were deparaffinized with xylene, rehydrated using a graded series of alcohols, and stained with hematoxylin and eosin. Sampling of 300 glomeruli from the superficial and midcortex was done by the systematic lattice point count method of Weibel (1979). Glomerular measurements were determined by the stereological methods of Elias and Hennig (1967) and Elias and Hyde (1983). The renal corpuscle was defined for convenience as that portion of the nephron composed of Bowman’s capsule lined with parietal epithelial cells and the glomerular tuft which included the outer visceral layer of podocytes, the capillary endothelial cells, and the mesangial cells (Tisher and Madsen, 1986). The frequency of distribution of nuclear size was based on cross-sectional area measurements of 30% individual nuclei in 30 glomeruli from seven rats provided 0.21 mg Se/kg diet and 2892 individual nuclei from 30 glomeruli from eight rats provided 0.10 mg Se/kg diet. Control for Bias Slides were coded in the following manner to ensure impartial evaluation. Slides from each rat in each dietary group were selected by one of us (M.K.L.) and coded with numbers from a random number table by another (C.D.E.). Quantification of coded sections was accomplished using a Zeiss ZIDAS image-analysis system by another (J.H.). Morphological evaluation included measuring the size of Bowman’s capsules and their inclusive glomerular vascular tuft, nuclei, and capillary lumina. The contribution of the cytoplasmic and extracellular material was estimated by subtracting from the area of the tuft the combined total areas of nuclei and capillary lumina with inclusive erythrocytes. Evaluation of Morphological Quantification Procedure The accuracy and precision of the quantitative morphological procedure was evaluated for both linear and area measurement. A microscope slide containing a reference linear scale was used to standardize image-analysis arbitrary units into the SI units of micrometers. Each day prior to making measurements on histological sections, the evaluator made 20 replicate measurements on a Graticules microscope linear reference scale. The coefficient of variation of the 20 replications was used as a measure of the precision of the quantitative procedure. The average precision was 0.5%. Glycated Hemoglobin Glycated hemoglobin was measured to ensure that the high-sucrose diets were not inducing chronic hyperglycemia. This condition has been observed in some

226

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ET

AL.

diabetes-prone rat strains fed high-sucrose diets (Michaelis et al., 1984). Glycated hemoglobin was separated from lysed red blood cell hemoglobin and analyzed spectrophotometrically by the method of Abraham et al. (1983). Glutathione

Peroxidase

Activity

Glutathione peroxidase activity was determined method of Paglia and Valentine (1967). Subcellular

Location

of 75Se in Isolated

in lysed red blood cells by the

Glomeruli

A third year-long study was employed to determine if the Se-dependent differences in nuclear size were indicative of nuclei representing a major site of Se incorporation. For this study groups of rats were provided diet B at both 0.10 and 0.21 mg Se/kg diet for 1 year to establish the same conditions as the previous two experiments for the subcellular 75Se incorporation study. After 12.5 months rats were anesthetized with 0.5 ml ketamine * HCl (100 mg/ml) containing 20% acepromazine maleate and 50 t.&i 75Se as sodium selenite was injected into the femoral vein. The rats were allowed to recover for 72 hr and then killed by CO,. The time interval of 72 hr was chosen since this is the time period required for Se incorporation to come to equilibration (Evenson and Sunde, 1988). The kidneys were perfused with ice-cold saline and their glomeruli isolated by the method of Blau and Michael (1971) using macroporous filters of 101 and 53 pm*. Evaluation of the glomerular preparations, performed by counting debris and glomeruli in a hemocytometer, showed they were greater than 95% pure. Glomeruli from each rat were homogenized in 0.24 M sucrose containing 0.1 mM EDTA, 1 m&I phenylmethylsulfonyl fluoride, and 0.01 M Tris-HCl buffer, pH 7.4, at 4°C and centrifuged using a Beckman L8-70M ultracentrifuge to separate subcellular fractions (Hogeboom, 1955). Crude nuclear pellets were purified using the procedure of Kaufmann et al. (1981) in order to obtain pure nuclei. Nuclear pellets were washed in buffer containing 0.25 M sucrose, 0.5 mM Mg SO,, 1 mM PMSF, and 50 mM Tris buffer, pH 7.4, at 4°C. Statistical

Evaluation

Comparison of data between groups was made using analysis of variance and Duncan’s new multiple range test (1955). Precision of the morphological procedure was evaluated as described. Differences in the number of animals per group in the tables were a result of rats dying due to anesthesia. Data are expressed as means + SEM. RESULTS Kidney Size Kidney weights were similar in all groups and the relative weight of the kidney compared to total body weight was not affected by Se supplementation. The weights (g) for groups A + Se, A, B + Se, B, and chow-fed rats were 4.3 f 0.1, 4.8 + 0.3, 4.5 + 0.2, 3.9 + 0.3, and 4.5 + 0.1, respectively. The ratio of kidney weight (g) per body weight (kg) for groups A + Se, A, B + Se, B, and chow-fed rats were 6.1 + 0.2, 7.0 + 0.4, 5.4 + 0.3, 5.1 + 0.4, and 5.5 + 0.1, respectively. The relationship between the Se-dependent differences in the Bowman’s capsule and tuft size to body weight were not significant. The correlation coefficient

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between body weight and the size of Bowman’s capsule was r = -0.1. The correlation coefficient between body weight and the size of the tuft was r = 0.2. Glycated

Hemoglobin

Glycated hemoglobin was within the normal age glycated hemoglobin in rats provided diet ever, higher (*P < 0.05) than values obtained groups A + Se, A, B + Se, and B were 4.5 ? +- 0.1, respectively. Glutathione

Peroxidase

range for all groups. The percentA (0.10 mg Se/kg diet) was, howfrom other groups. The values for 0.4, 7.1 + l.O,* 4.0 + 0.5, and 4.0

Activity

Erythrocyte glutathione peroxidase activity (enzyme unit = nmoles NADPH oxidized/min/mg hemoglobin) was normal in all groups, and no differences were observed between groups. The enzyme units/mg hemoglobin for groups A + Se, A, B + Se, and B were 918 + 145, 917 * 158, 585 + 123, and 760 + 140, respectively. Glomerular

Evaluation

At 1 year, kidneys from rats provided 0.21 mg Se/kg diet had larger Bowman’s capsule diameters (P < 0.01) than those provided 0.10 mg Se/kg diet (Table II). The diameters of the capillary tufts were also larger (P < 0.01) in the rats provided 0.21 mg Se/kg diet (Table II). The proportion of the tuft occupied by extracellular and cytoplasmic material was smaller (P < 0.001) and they had more open capillary lumen than rats receiving the 0.10 mg Se/kg diets A and B (P < 0.01) (Table II) (Fig. 2). The diameters of cell nuclei were larger (P < 0.001) in the vascular tuft of rats provided supplemental Se, and they occupied more of the total cross-sectional area of the tuft (P < 0.05) (Table II). A plot of the relative frequency distribution of nuclear areas in the tuft revealed that nuclei from rats provided 0.21 mg Se/kg diet fell into three different population sizes, whereas those from rats provided 0.10 mg Se/kg diet fell into two sizes (Fig. 1). Subcellular

Location

of 15Se

A 75Se tracer study was carried out to identify the subcellular site of glomerular Se. 75Se was injected into the femoral vein and its location determined within purified glomeruli. The subcellular distribution of ‘?Se within the glomeruli is given in Table III. Almost all the radioactivity (90%) was associated with the crude nuclear fraction which contained nuclei from all glomerular cells. The remaining 10% was distributed between the cytosol and mitochondria, with a small amount going to the heavy and light microsome fractions. In order to assure that the radioactivity was associated with nuclei, and not a contaminant of cellular membranes, nuclei were purified using the procedure of Kaufmann et al. (1981) (see Materials and Methods). Sixty-eight percent of the radioactivity was associated with the purified nuclei (Fig. 3) and 32% with the broken nuclei, glomerular membrane, and matrix contamination that were removed with the supernatant in the purification procedure.

0.2 0.1 0.2 0.1

mg Se/kg

4

10 7 7 8

N+

f 2 f 5

3* 4 3* 3

116 2 3

138 114 137 118

f 2 2 *

3* 3 3* 2

105 -+ 3

128 108 126 110

Tuft diameter (v) f 2 + 2

0.70** 1.02 0.53** 0.78

” 2 2 2

O.lO* 0.06 0.11* 0.09

2.84 e 0.05

2.34 1.41 3.53 1.84

Capillary lumen area (1 X 10m3 mm*)

+ 3.6 + 2.5 -+ 3.2 2 4.2 89.6 + 3.6

96.1 82.7 91.9 96.0

Mean number of nuclei/tuft

+- 0.05* + 0.05 + 0.06* f 0.06 4.40 f 0.10

4.40 3.30 3.90 3.20

Mean diameter of nuclei (i.4

evaluated. Values expressed as means 2 SEM. Values for rats provided values from chow rats was deemed inappropriate because of the multithe ingredients used to prepare chow vary seasonally. mg Se/kg (p < 0.01). mg Se/kg (P < 0.001).

65.70 f 2.00

71.55 75.82 77.18 80.65

Proportion of tuft present as cytoplasmic and extracellular matrix (%)

Note. +N represents the number of rats; see Methods for number of glomeruli cereal-based chow diets are included for comparison. Statistical evaluation of component differences of closed-formula commercial animal feed and the fact * Significantly different from group within same experiment containing 0.1 ** Significantly different from group within same experiment containing 0.1

Chow

II

I

Experiment

Bowman’s capsule diameter km)

TABLE II Effect of Exposure to Selenium on Bowman’s Capsule Parameters

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20

10

0

0

10 Cro6s~alareaofnlJek!l

20

30

(1x104m2,

FIG. 1. The cross-sectional areas of the cell nuclei in the glomerular vascular tufts were plotted as a relative frequency distribution. Cell nuclei of rats provided 0.21 mg Se/kg diet were larger and fell into three major group sizes. In contrast, rats provided 0.10 mg Se/kg diet had smaller nuclei and completely lacked the population of large nuclei. The relative frequency distribution was based on cross-sectional area measurements of 30% individual nuclei in 30 glomeruli from rats provided 0.21 mg Se/kg diet and 2892 individual nuclei from 30 glomeruli from rats provided 0.10 mg Se/kg diet.

DISCUSSION The essentiality of Se as a nutrient is based on its role as a component of selenocysteine, the 21st amino acid recognized to have its own t-RNA and be encoded into protein (Lee et al., 1990). In addition to being essential, Se is also one of the most toxic elements in the biosphere (Toxicological profile for selenium, 1989). The pathology of Se deficiency and excess are similar and separated by a narrow safe range of exposure. In the rat, the safe range is between 0.05 to 4.0 mg/kg diet. The pathological lesions for both Se deficiency and toxicity cover a wide spectrum of alterations that include myocardial hemorrhages, liver and kidney necrosis, pulmonary edema, and skeletal degeneration. An evaluation of the pathology of Se deficiency and toxicity has concluded that the basis for both is rooted in a localized consequence of Se on vascular tissue (National Research Council, 1976). How selenium is involved in maintaining and disrupting capillary integrity is not understood. The induction of microvascular lesions by the chronic consumption of diets containing sucrose as the sole carbohydrate has been used by many laboratories over the past 20 years (for review, see Poulsom, 1986). We have shown that the sucrose-fed rat model provides a means of providing rigorous control over the level of Se exposure at the same time it induces a mild microangiopathy in the rat (Thornber and Eckhert, 1984; Lockwood and Eckhert, 1992). Using this model we have been able to show that Se supplementation within the safe range, from 0.1 to 0.2 mg/kg diet, protects the microcirculation of the

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FIG. 2. Glomeruli of rats provided 0.21 mg Se/kg diet B (a, c, e) and 0.10 mg Se/kg diet B (b, d, f). Representative glomeruli were photographed from histological sections of mean capsular size under different optical conditions. Bright field, a and b; fluorescence, c and d, showing bright eosin fluorescing red blood cells; confocal imaging with Nomarski, e and f. Numerous red blood cells can be seen in the tuft of 0.21 mg Se/kg rats (c) but not in d. Nuclei within the tuft in rats provided 0.21 mg Sekg (a and e) are larger than those within the tufts in 0.10 mg Se/kg rats (b and f). Magnification, 390x.

retina from sucrose induced damage. The use of this model provides the opportunity to probe for the site of Se action in a setting free from the secondary pathological complications encountered in models of Se deficiency and toxicity, conditions which have obscured the identification of the primary site of Se. The

MICROVASCULAR Subcellular Incorporation Subcellular fraction of puritled glomerular microvessels Crude nuclei Cytosol Mitochondria Light microsomes Heavy microsomes

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231

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TABLE III of “Se in Glomeruli of Year-old Rats Distribution Exposure level: 0.2 mg Se/kg (%) 89.04 5.36 3.64 1.20 0.79

2 1.11 -t- 0.74 2 0.27 1 0.01 ? 0.08

of radioactivity Exposure level: 0.1 mg Se/kg (%) 90.18 4.50 3.62 0.93 0.75

f 1.18 -+ 0.45 + 0.55 + 0.13 f 0.10

Note. Following the observation that Se exposure resulted in a difference in the size of nuclei in the microvascular tuft, a new group of weaning rats was assigned to diet B at levels of 0.1 and 0.2 mg Se/kg for a period of 1 year. At 12.5 months 75Se was injected into the femoral vein and purified fractions of glomerular microvessels were isolated 72 hr later. Values are expressed as mean % ?SEM and represent five replications using two rats per replication for each level of Se exposure. The sum of the variation from the individual steps in the fractionation procedure would obscure differences in “Se incorporation between the two levels of Se exposure.

sucrose model was applied in the’ present study to determine if Se dependent microvascular changes codd also be observed in the glomenrlus. The levels of Se used in the experiment were all sufficient for maintaining normal activity of the selenoprotein, erythrocyte glutathione peroxidase, a good indicator of long-term Se status (Deagen et al., 1987). Diet A was used in the first experiment because of its proven ability to induce mild microvascular injury even though it was an old diet formulation designed for rats housed in galvanized cages. Once the impact of Se on the renal corpuscle was documented using diet A, the experiment was repeated using diet B. This diet was formulated to contain all vitamins and elements at levels currently recommended for rats housed in modern stainless steel cages. The only exceptions were the levels of Se, which were varied at 0.10 and 0.21 mg Se/kg diet, and vitamin E. RRR-cw-tocopherol acetate was provided at 100 mg/kg diet (100 W/kg diet), a level double the requirement, to assure the diet contained adequate amounts of this membrane-associated antioxidant (Eckhert,

FIG.3. In the subcellular fractionation evaluation 90% of the 75Se radioactivity was associated with nuclei in the crude nuclear fraction (Table III). Crude nuclei were purified (see Materials and Methods) and evaluated to determine if the radioactivity was associated with the nuclei or with contaminants of the crude nuclear fraction. The distribution of radioactivity from the crude nuclear fraction was 68% purified nuclei and 32% with the broken nuclei, glomerular membrane, and matrix contamination that were removed with the supernatant in the puritication procedure. This dgure shows a preparation of purified nuclei. Magnification, 1742 X .

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1987). The results of both year-long experiments using diets A and B demonstrated that supplementation to a level of 0.21 mg Se/kg prevented the mild sucrose induced expansion of the cytoplasmic and extracellular matrix and reduction in open capillary lumen. The chow-fed rats had a similar amount of open capillary lumen as rats provided 0.21 mg Se/kg diet, but since they had a smaller proportion of their tufts present as cytoplasmic and extracellular matrix, their tuft sizes were smaller. Replication of the results using both diets A and B demonstrated that the observed effect of Se was unique to the element and not secondary to an antioxidant deficiency correctable by vitamin E (Witting and Horwitt, 1964) or an unknown variable associated with the cage material. These results were in agreement with the studies investigating the relationship between Se status and the integrity of retinal capillaries which showed that supplementation to a level of 0.2 mg Se/kg prevented the collapse of capillaries and degeneration of capillary cells in the retinal circulation (Thornber and Eckhert, 1984; Lockwood and Eckhert, 1992). These and the present results are important as they show that the effect of Se status on the microcirculation is not limited to the extremes of deficiency and toxicity, but also occurs within the safe range. The basis for the microangiopathy observed in extreme Se imbalance may be based on an interference with a requisite role for the element in microvascular cells that is manifested under the conditions induced by the chronic sucrose challenge. Cellular glutathione peroxidase has been shown to reside in the cytoplasm and mitochondria of liver from growing animals (Behne et al., 1990; Burk et al., 1982; Flohe and Schlegel, 1971; Stults ef al., 1977) and mammary epithelial cells in the log phase of growth (Morrison et al., 1988). Incorporation studies using radiolabeled [75Se]selenite have also shown that these are the major subcellular sites of Se incorporation in growing organisms (Behne et al., 1990; Flohe and Schlegel, 1971; Stults et al., 1977) and cells (Morrison et al., 1988). The Se-dependent differences in nuclear size observed in the present study were therefore unexpected. This effect on nuclear size occurred in rats fed both diets A and B. These observations led us to undertake the third year-long study to examine the incorporation of radiolabeled Se into subcellular fractions of purified glomeruli. Tracer studies using 75Se indicated that the majority of the glomerular 75Se incorporated into the crude nuclear fraction of the glomerular cells and the radioactivity remained associated with the nuclei after they were purified. Recent observations suggest that renal dysfunction in populations living in Se depleted soils in the Balkan peninsula and Italy and can be partially corrected by Se supplementation (Mihailovic ef al., 1992; Guidi et al., 1992). The results of the present study suggest that the protective effect of Se could be due to the direct utilization of the element by renal microvessels. Using the procedures developed in this report, the molecular target of Se within microvascular nuclei can be radiolabeled. This should facilitate the isolation and characterization of the molecular basis for the role of Se in microangiopathy. REFERENCES ABRAHAM, E. C., PERRY, R. E., and STALLINGS, M. (1983). Applications of affinity chromatography for separation and quantification of glycosylated hemoglobins. J. Lab. Clin. Med. 102, 187-197. AMERICAN INSTITUTE OF NUTRITION (1977). Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. J. Nutr. 107, 1340-1348. BEHNE, D., SCHEID, S., KYRIKOPOULOS, A., and HILMERT, H. (1990). Subcellular distribution of selenoproteins in the liver of the rat. Biochim. Biophys. Acta 1033, 219-22.5.

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