Estrogen accelerates the development of renal disease in female obese Zucker rats

Kidney International, Vol. 53 (1998), pp. 130 –135

Estrogen accelerates the development of renal disease in female obese Zucker rats MATTHEW D. GADES, JUDITH S. STERN, HARRY VAN GOOR, DUNG NGUYEN, PATRICIA R. JOHNSON, and GEORGE A. KAYSEN Department of Nutrition, Division of Clinical Nutrition and Metabolism, and Division of Nephrology, Department of Internal Medicine University of California Davis School of Medicine, Davis California, USA; Department of Pathology University of Groningen, Groningen, The Netherlands; and Department of Veterans Affairs, Northern California Health Care System, Pleasant Hill, California, USA

Estrogen accelerates the development of renal disease in female obese Zucker rats. Renal failure is the primary cause of death in obese Zucker rats (OZR). We previously found that renal injury occurred earlier and with greater severity in female OZR; also, prevention of hyperphagia decreased renal damage in females more than males. To examine the relationship between estrogen (E), hyperphagia, hyperlipidemia, and renal injury in female OZR, we studied four groups from 5 to 10 or 21 weeks of age: Sham-operated (Sham), ovariectomized (Ovx), Ovx with estrogen treatment (Ovx1E), and, since Ovx increases food intake, Ovx pair-fed to sham (Ovx-PF). By only six weeks of age, albumin excretion (UAE) increased significantly in Ovx1E (9.9 6 4.1 mg/day). Ovx1E also ate least and gained the least weight, but had the highest plasma lipid levels. In contrast, UAE in Ovx did not increase by 10 weeks of age, despite a significantly greater food consumption. The hyperlipidemia of Ovx1E was due primarily to triglycerides. Both plasma triglycerides and renal injury, judged from either histology or UAE, were greatest in the Ovx1E group. Fasting plasma glucose was lower and insulin was higher in Ovx1E compared to Ovx rats at 15 weeks of age. Estrogen may promote renal injury in female OZR by increasing the plasma concentration of triglyceride-rich lipoproteins.

The obese Zucker rat (OZR), a widely used model of obesity, is hyperinsulinemic but not hyperglycemic up to one year of age [1]. The OZR spontaneously develops renal failure characterized by histopathologic changes similar to those found in patients with NIDDM including mesangial matrix expansion and thickening of the glomerular basement membrane [2]. At 9 to 13 weeks of age, transglomerular capillary pressures in obese and lean male Zucker rats do not differ [3]. Lipids are increased though, and treatment with the lipid-lowering drugs clofibrate and mevolinin markedly decrease glomerular damage [4]. These observations led Keane and coworkers to propose that hyperlipidemia was important in initiating renal injury in the OZR, but hemodynamics was not involved [2, 3]. Additionally, macrophage infiltration and hyperlipidemia occurs as early as four weeks of age in OZR [5]. Key words: albuminuria, diabetes, estrogen, nephrotic syndrome, obese rat. Received for publication April 7, 1997 and in revised form August 20, 1997 Accepted for publication August 21, 1997

© 1998 by the International Society of Nephrology

Cholesterol has been the primary focus of the possible contribution of lipids to the development of renal disease. However, in female OZR, proteinuria has been observed as early as seven weeks of age, a period when only very-low density lipoprotein (VLDL) is elevated [6]. Usually lipid levels are lower in female than in male rats [7]. Exceptions to this include the Nagase analbuminemic rat (NAR) and rats with experimentally-induced nephrotic syndrome. Females in both have higher lipid levels [8, 9]. The hyperlipidemia in these rats, due to hepatic overproduction, is corrected by ovariectomy and exacerbated by estrogen administration [10]. Similarly, some women given unopposed estrogens respond with severe hyperlipidemia [11, 12], though on average, there is a small but significant increase in plasma triglycerides [13]. In the OZR, we reported that renal failure occurs earlier in females [14]. Lean Zucker rats also have a lower incidence of end-stage renal disease at spontaneous death than do OZR. When hyperphagia in OZR is prevented by pair-feeding to lean littermates, renal disease is diminished in both sexes, but to a greater degree in females than males [13, 15]. We conducted this study in part to establish whether the gender differences in renal disease seen in OZR are due to estrogen. Since estrogen has been shown to increase triglycerides in female OZR [16], and lipids have been implicated in the development of renal pathology in OZR, we were interested in the role of lipids. These studies were designed both to investigate the temporal relationship between blood lipid levels and renal injury and to determine estrogen’s influences on both. METHODS Experimental animals Obese (fa/fa) female Zucker rats were obtained at four weeks of age from the UC Davis NIH-funded Clinical Nutrition Research Unit Animal Model Core. In study one, 31 rats were assigned to one of four experimental groups while keeping initial body wts equal: Sham (N 5 9), Ovx (N 5 7), Ovx1E (N 5 7) Ovx-PF (N 5 8). The Ovx-PF group was fed daily only the average amount eaten by the sham group. This group was necessary because Ovx causes increased food intake [17]. In study two, 18 obese female Zucker rats were assigned to one of three groups: Sham (N 5 3), Ovx (N 5 7), Ovx1E (N 5 8). For both groups, bilateral

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ovariectomy or sham surgery by dorsal approach and estrogen or placebo treatment was performed at five weeks of age. Presence or absence of ovaries was confirmed on termination. Rats in studies one and two were terminated at 21 and 10 weeks of age, respectively. An additional group of seven rats was terminated at five weeks of age to provide baseline plasma lipid values. Estrogen treatment Rats in the Ovx1E groups were implanted with time release pellets of 17b-estradiol, which release 0.0153 mg/day (Innovative Research of America, Sarasota, FL, USA). The pellets used in studies one and two were 90- and 60-day release, respectively. Those in Sham groups were implanted with a placebo pellet. All pellets were inserted subcutaneously via a small incision on the nape of the neck. In study two, following kidney perfusion at termination, uteri were removed, blotted dry, and weighed. Uterine weights were used as an indicator of estrogen levels. Diet, food intake and body weight Rats were housed individually in stainless steel cages with wire mesh floors and had free access to water and, except for Ovx-PF, food. The light cycle was 0700 to 1900. All rats received a soy protein-based (20%) refined diet that was previously found to minimize renal injury in rats [18]. Body weights were measured twice weekly for all rats. Food cups were replaced between 0800 and 1000 hours daily for the Ovx-PF group and twice weekly for the other groups. Food intakes were measured at those times and adjusted for spillage. Urinary albumin excretion Urine was taken over a 24-hour period by placing rats in stainless steel metabolic cages and collecting the urine in flasks containing three drops of 10% sodium azide solution to prevent bacterial growth. Water was freely available, as was food moistened with water to minimize spillage, except for the Ovx-PF group which was given only their prescribed amount of food. Urinary albumin excretion was determined using electroimmunodiffusion using rabbit anti-rat albumin and purified rat albumin as previously described [19]. In study one, urine was collected at 21 weeks for all groups, as well as at 8 and 10 weeks for Ovx, Sham, and Ovx1E, and at six weeks for Ovx1E. In study two, urine was collected weekly for all groups. Plasma triglycerides and cholesterol Prior to blood collection, all rats were fasted overnight. For study one, in Ovx and Sham groups at 10 weeks of age, blood was obtained by nicking the tail tip with a sterile scalpel and collected in EDTA-coated capillary tubes. In all studies, including the baseline rats, blood was taken by exsanguination at termination. Rats were anesthetized with sodium pentobarbital and blood was collected from the abdominal aorta in an EDTA-coated syringe. Plasma was obtained by centrifugation, removed, and stored at 270°C until assayed. Measurements of plasma triglyceride and cholesterol were made using enzymatic, colorimetric assays (Sigma Chemical Co., St. Louis, MO, USA). Insulin and glucose In study one, fasted tail blood was collected again at 15 weeks. Plasma glucose concentrations were determined using a YSI 2300 STAT plus glucose analyzer (Yellow Springs Instruments, Colum-

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bus, OH, USA). Immunoreactive insulin was determined by radioimmunoassay as previously described [20]. Renal histology Histology was performed in study one. Following exsanguination under anesthesia, rats were infused via the abdominal aorta with phosphate buffered saline (0.1 M PBS, pH 7.4) containing 6% sucrose and 500 U heparin/liter for a sufficient time to blanch the kidneys. A 0.05% glutaraldehyde/4% paraformaldehyde in PBS fixative was then perfused for one minute or until the kidneys became rigid. The fixative was removed by re-perfusion with the PBS/sucrose/heparin solution. Tissue was then processed in glycol methacrylate for light microscopy and immunohistochemistry as described [21]. Sections were then stained by the periodic acidSchiff technique. Glomeruli were scored by the method of Raij, Azar and Keane as previously reported [22]. Thirty to 40 glomeruli were scored from each kidney for three parameters: focal and segmental glomerulosclerosis (FGS), mesangial matrix expansion (MME), and visceral epithelial cell adhesion formation to Bowman’s capsule (Adh). Each glomerulus was scored semiquantitatively on a scale of 0 to 4 for each parameter depending on the percentage of the glomerulus involved. A final score was obtained by multiplying the degree of change by the percent of glomeruli with the degree of injury and adding scores. The maximum score obtainable for each rat was thus 400. Focal and segmental glomerulosclerosis were scored positive when MME, adhesion formation and capillary obliteration were present in the same segment of the glomerulus. Statistical analysis Data were analyzed using Sigma Stat 2.0, a commercial statistics program (Jandel, San Rafael, CA, USA). Data were first analyzed for distribution. If the data were normally distributed, they were analyzed for differences using one-way ANOVA. If differences between the groups were detected, then a post hoc analysis was performed using the Tukey test for multiple comparisons. If data were not normally distributed, the significant differences between median values were determined using Kruskal-Wallis one-way ANOVA on ranks. If differences were detected, then multiple comparisons were performed using Dunn’s method. Differences were considered statistically significant at P # 0.05. Data are presented as mean 6 standard error or median and range. RESULTS Food intake and body weight Food intake and body wt in Ovx-PF and Sham groups did not differ significantly (Table 1). Ovx rats ate significantly more and were heavier than the other groups. In contrast, Ovx1E rats weighed the least and had the lowest food intake throughout the study. Food intake for Ovx1E was similar to that of the genetically lean (Fa/Fa) female Zucker rat [13]. Uterine mass per 100 g body wt at 10 weeks was significantly lower in Ovx (Table 1). Uterine weight in Ovx1E was slightly higher but not significantly different than the sham animals, suggesting that the estrogen dose was at least as much or slightly more than replacement levels.

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Gades et al: Estrogen accelerates renal disease in OZR Table 1. Body weight, total food intake, uterine mass, and fasted plasma insulin and glucose in female obese Zucker rats

Age

Body Wt grams 20 weeks

Uterine mass mg/100 g body weight 10 weeks

Total Food Intake grams 5 to 20 weeks

Insulin pm/liter 15 weeks

Glucose mg/dl 15 weeks

Ovx-PF Ovx Ovx1E Sham

450 6 5c 537 6 15a 266 6 7b 434 6 3c

— 3.5 6 1.1a 10.7 6 0.8b 8.4 6 0.6b

1800 6 10c 2090 6 10a 1390 6 30b 1790 6 40c

1170 6 170b 1720 6 550b 6180 6 1310a 1300 6 170b

154 6 4a,b 165 6 9a 119 6 14b 130 6 5b

Abbreviations are: Ovx-PF, ovariectomized and pair-fed to Sham; Ovx, ovariectomized; Ovx1E, ovariectomized and estrogen treated; Sham, sham operated. Values with different superscripts differ significantly (P , 0.05).

Fig. 1. Twenty-four-hour urinary albumin excretion (UAE) of female obese Zucker rats at 21 weeks of age. Values are group average 6 standard error. Groups were compared to each other (ANOVA). Bars with different letters differ significantly (P , 0.05). Abbreviations are: Sham, Sham-operated control (N 5 9); Ovx1E, ovariectomized and estrogen treated (N 5 7); Ovx, ovariectomized (N 5 7); Ovx-PF, ovariectomized and pair-fed to Sham (N 5 8).

Urinary albumin excretion At 21 weeks of age Ovx1E UAE was four- to sixfold higher than the other groups (Fig. 1). Ovx1E UAE was significantly higher than Ovx and Ovx-PF, but not Sham. In study two, Ovx1E UAE was significantly higher than Ovx at all post-treatment time points (Fig. 2). With the pooled data from studies one and two, differences between Ovx1E and Ovx were significant by six weeks. Compared to baseline UAE at five weeks (0.38 6 0.05 mg/day), Ovx1E and Sham increased significantly by 10 weeks (114 6 28 and 20 6 10 mg/day), respectively, but Ovx did not (1.6 6 0.9 mg/day). Remarkably, Ovx1E UAE was elevated only one week after treatment (9.9 6 4.1 mg/day). Plasma lipids As with UAE, triglyceride levels at 21 weeks of age were highest in Ovx1E (Fig. 3). Average triglycerides in Ovx1E were more than twice as high as sham and 6 to 7 times higher than Ovx and Ovx-PF. Despite the high variability in triglyceride values, differences between Ovx1E and both Ovx and Ovx-PF were highly significant (P , 0.01). Cholesterol levels at 21 weeks in Ovx1E were twice as high as the other groups, and this is the only group

Fig. 2. Weekly 24-hour urinary albumin excretion (UAE) of female obese Zucker rats from 5 to 10 weeks of age. Values are group average 6 standard error. Groups were compared to each other at the same time point as well as the pooled five week average (ANOVA). Values with different letters differ significantly (P , 0.05). Bars with an asterisk differ significantly from the pooled five week average. Number per group is indicated below each time point in the table. Symbols and abbreviations are: (F) Sham, sham-operated control; (f) Ovx1E, ovariectomized and estrogen treated; (L) Ovx, ovariectomized.

that differed from the others (Fig. 3). The situation at 10 weeks of age was much the same. Ovx1E triglycerides were much higher than Ovx and Sham (Fig. 4), while Ovx1E had the highest cholesterol levels, though differing significantly from Sham only (Fig. 4). Insulin and glucose Insulin levels at 15 weeks were highest in Ovx1E, while glucose was not elevated (Table 1). Ovariectomy led to increased blood glucose, as Ovx was significantly higher than Sham and Ovx1E. Histology Histologic evaluation revealed significantly more FGS and MME in Ovx1E than in any of the other three groups (Table 2 and Fig. 5), and significantly greater glomerular adhesion in Ovx1E than in Ovx and Ovx-PF. The interstitium from Ovx-PF animals was normal, while in the interstitium from the other three groups, areas of tubular dilation were found.

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Table 2. Renal histopathology from obese female Zucker rats at 21 weeks of age MME N Sham Ovx1E Ovx Ovx-PF

5 7 4 6

Median a

10 125b 23a,b 9a

Range 3–33 38 –235 10 –33 0 –13

FGS Median a

0 3b 0a 0a

Adhesions

Range 0 –10 3–168 0–0 0–0

Median a,b

0 6b 0a,b 0a

Range 0 –15 3–168 0–8 0–0

Abbreviations are: Sham, sham operated; Ovx1E, ovariectomized and estrogen treated; Ovx, ovariectomized; Ovx-PF, ovariectomized and pairfed to Sham: MME, mesangial matrix expansion; FGS, focal and segmental glomerulosclerosis; Adhesions, visceral epithelial cell adhesions to Bowman’s capsule. Values with different superscripts differ significantly (P , 0.05).

Fig. 3. Plasma triglycerides (M) and cholesterol (u) of female obese Zucker rats at 21 weeks of age. Values are group average 6 standard error. Groups were compared to each other by ANOVA. Bars with different letters differ significantly (P , 0.05). Abbreviations are: Sham, sham-operated control (N 5 9); Ovx1E. ovariectomized and estrogen treated (N 5 7); Ovx, ovariectomized (N 5 7); Ovx-PF, ovariectomized and pair-fed to Sham (N 5 8).

Fig. 4. Plasma triglycerides (M) and cholesterol (u) of female obese Zucker rats at 10 weeks of age. Values are group average 6 standard error. Groups were compared to each other as well as values from a five weeks old control group (ANOVA). Bars with different letters differ significantly (P , 0.05). Bars with an asterisk differ significantly (P , 0.05 from baseline) from the five week value of the control group (N 5 7). Abbreviations are: Sham, sham-operated control (N 5 12); Ovx1E, ovariectomized and estrogen treated (N 5 8); Ovx, ovariectomized (N 5 13); dotted line, baseline triglycerides; dashed line, cholesterol.

DISCUSSION The female OZR is infertile. It has delayed puberty, which does not occur until eight or nine weeks of age [23, 24]. Infertility is thought to be linked to abnormal estrous cycles, since estrogen and gonadotropin levels are normal [25, 26]. Ovariectomy of OZR has been previously reported to reduce plasma triglycerides, an effect reversed by estrogen replacement [27]. The reduced estrogen level both decreased triglyceride production and increased

clearance [26]. Female OZR are not unique in this respect. Estrogen massively increases triglyceride levels in the female NAR and in female rats rendered nephrotic with adriamycin [28, 29]. The resultant hypertriglyceridemia initiates renal injury in the normal remaining kidney of the uninephrectomized NAR and exacerbates renal injury in the adriamycin model. In our model, hyperlipidemia itself, principally hypertriglyceridemia, may play a role in the initiation of renal injury since no alternative initiation events have been identified. While Ovx1E exhibited markedly increased hyperinsulinemia, fasting insulin levels were the same in Ovx and Sham groups at 15 weeks. Thus, we find that in adult OZR, within which ovariectomy does cause a decrease in estrogen [25], there is no evidence for the direct dependence of insulin levels on estrogen. This increased severity of NIDDM in Ovx1E may instead be a result of hypertriglyceridemia. For example, while the fractional catabolic rate of glucose is positively correlated with insulin in normal humans, the same is not true of hypertriglyceridemics [29]. These same individuals become more sensitive to insulin when their hyperlipidemia is treated pharmacologically. Thus, estrogen’s influence on insulin resistance here is likely indirect only, by means of the amplified hypertriglyceridemia. Hypercholesterolemia is an independent risk factor for the development of renal disease in patients with diabetes [30, 31]. While this association has been made with respect to low density lipoprotein (LDL) cholesterol, non-insulin-dependent diabetes mellitus (NIDDM) is also characterized by increased lipoprotein remnant particles [32, 33]. Clinical studies on patients with renal disease, including NIDDM patients, and previous studies on the OZR indicate that the hypertriglyceridemia observed is primarily due to VLDL [6, 34, 35]. While VLDL may be metabolized to either VLDL remnant particles or LDL, the low levels of LDL that are seen in the rat [36] suggest that the primary pathway for VLDL metabolism is through the generation of remnants, which in turn are taken up by the liver. Very-low density lipoprotein has not been directly shown to be either cytotoxic or to be taken up directly by macrophages or mesangial cells. However, remnants are atherogenic [37]. They are taken up by macrophages and are toxic to them [38]. It is therefore possible that the hypertriglyceridemia induced by estrogen in the female OZR elevates circulating remnant particles, leading to glomerulosclerosis. An interesting result is that the effects of estrogen outweigh the effects of reduced food intake. Reduction of total caloric and/or protein intake consistently protects against renal damage in rat

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Fig. 5. Photomicrograph of representative female obese Zucker rat glomeruli at 21 weeks of age. (A) Ovariectomized and estrogen treated; (B) sham-operated; (C) ovariectomized; (D) ovariectomized and pair-fed to sham.

models, including the OZR [12, 13, 16]. Here, however, ovariectomy was protective despite a 17% increase in food intake compared to sham, while the 22% decrease seen in Ovx1E resulted in greater damage. A likely explanation for this seeming contradiction is that while reduced food intake has otherwise salutary effects on plasma lipids, these effects can be overpowered by the actions of estrogen [39]. We have now demonstrated that estrogen accelerates the development of glomerular damage in the obese female Zucker rat. Our observations that plasma lipids were significantly elevated in Ovx1E compared to other groups, and that they had the most severe level of histopathology and UAE support the hypothesis that lipids play a role in initiation and/or progression of glomerular injury [40]. Specifically, estrogen-induced VLDL elevations may contribute to glomerular injury, causing proteinuria and ultimately renal failure in the OZR. These findings have important clinical implications for the large numbers of postmenopausal women using hormone replacement therapy. We suggest that the subset of these women who respond to estrogen with severely elevated plasma triglycerides may be at risk for nephropathy as well as atherosclerosis. ACKNOWLEDGMENTS This study was supported by the research service of the Department of Veterans Affairs and NIH grants (NIA/DK 09945, T32 DK7355, DK

35747), the Nora Eccles Treadwell Foundation, and a Nestle Fellowship to M. Gades. We acknowledge the assistance of Ms. Susan Hansen and Ms. Susan Bennett.

Reprint requests to Judith S. Stern, ScD., Department of Nutrition, University of California, Davis, 3150B Meyer Hall, One Shields Avenue, Davis, California 95616, USA.

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