Compensatory kidney growth in estrogen receptor- null mice
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《美国生理学杂志》
Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska
ABSTRACT
Females are relatively protected in many progressive kidney diseases. Processes of kidney scarring and growth are intricately linked, and female kidneys are smaller than male kidneys. To better understand links between sex, growth, and the kidney, we examined compensatory kidney growth (CKG) after uninephrectomy (Unx) in wild-type and estrogen receptor- null mice (ERKO). Mice (10 wk old) underwent Unx or sham procedure, with removal of all remaining kidney(s) 48 h later. Studies included kidney weight, renal content of protein, DNA, and insulin-like growth factor-I (IGF-I), serum IGF-I, mean glomerular area, and immunostaining for proliferating cell nuclear antigen (PCNA). Sham Unx produced no differences between left and right kidneys. Unx altered kidney weight, glomerular area, DNA content, IGF-I content, and PCNA regardless of sex or genotype. Females showed greater increases in kidney weight (26 vs. 19%) and glomerular area (73 vs. 51%) than males. Differences in kidney weight were restricted to wild-type females (32% increase); ERKO females showed an increase in kidney weight similar to males (19%). Genotype did not influence glomerular growth in this model. Both male and female mice exhibit hyperplastic growth 48 h after Unx, with more pronounced enlargement in females. Lack of estrogen receptor- is associated with reduced CKG in females, probably via suppression of proliferation. ERKO mice did not demonstrate any alterations in compensatory glomerular enlargement. Kidney IGF-I content doubled after Unx, regardless of sex or genotype, implicating other mechanisms with regard to these findings.
hypertrophy; hyperplasia; uninephrectomy; insulin-like growth factor I; sex
THE PROCESSES OF RENAL HYPERTROPHY and progression of kidney disorders are intimately linked (8, 9, 15). Virtually all progressive nephropathies are associated with enlargement of the kidney and the glomeruli. Numerous factors that control normal renal growth have been implicated in nephrosclerosis, and all therapies that ameliorate sclerosis also prevent early enlargement in kidney disease (8).
Clinical and animal model studies suggest that females are relatively protected from a number of progressive nephropathies (10, 30, 31, 41, 42). Females also have smaller kidneys than males, so there may be a direct link between kidney growth and risk of kidney failure. Secondary sex differences are produced by gonadal steroids, and it is likely that these hormones influence the kidney as well. It has been suggested that androgens, known to promote kidney enlargement, are detrimental in a number of clinical and experimental conditions (2, 22, 35–38). There is also evidence that estrogens may play a beneficial role in kidney and vascular disease (4, 13, 35, 40).
Transgenic and knockout mice provide powerful tools to delineate the role of various genes in physiology. We have recently reported that female mice lacking estrogen receptor- (ERKO) are protected from diabetic glomerular hypertrophy, although not from overall diabetic kidney growth (19). Differences in kidney weight were noted in nondiabetic mice, suggesting that this genotype also altered normal kidney development. The following study examined the effects of this genotype in another model of kidney growth, compensatory hypertrophy after uninephrectomy (Unx).
Sex differences in compensatory kidney growth (CKG) have received direct attention in the rat but not in the mouse (23–28). Female rats show initial hyperplastic growth, whereas males show hypertrophy (27, 28). Upregulation of the intrarenal insulin-like growth factor-I (IGF-I) system was implicated in females, but no role was apparent for growth hormone (GH; see Refs. 23–26). In contrast, CKG in adult males was blocked by castration or GH antagonists, but no differences in the IGF-I system were identified (28). Testosterone has thus been implicated as a driving force in CKG, but a role for estrogen has been less clear. Sex differences in this model in mice have not been directly examined.
CKD was examined in male and female wild-type and -ERKO mice at 48 h. We show that there are significant differences in CKD in mice compared with the existing literature on rats (23–28) and that female -ERKO mice show suppressed CKD, similar to that seen in males of either genotype.
METHODS
Animals. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center. Homozygous wild-type and -ERKO mice of both sexes began these experiments at 10 wk of age. Four groups of mice of each genotype and sex underwent Unx at time 0. Similar groups of mice underwent sham Unx. For both procedures, all mice were anesthetized with ketamine and xylazine. For the sham procedure, the left kidney was manipulated out of its fossa and then returned, and the incision was closed. Unx animals had the kidney manipulated out of its fossa and then dissected free of its capsule. The renal vessels were tied off, and the left kidney was removed. After the initial procedure (48 h), all animals underwent a terminal procedure under anesthesia with collection of blood and removal of all remaining kidneys.
All kidneys were weighed at the time they were removed. A 2- to 3-mm transverse slice from the midportion of each kidney was immersed in formalin for histological studies, including measurement of glomerular area and proliferating cell nuclear antigen (PCNA)-positive cells. The remaining portions of the kidneys were snap-frozen for later biochemical studies.
Protein, DNA, and IGF-I studies. Protein was extracted from 20 mg of both kidneys from every mouse using a homogenization buffer containing Tris-buffered saline (pH 8.0), 1% Nonidet P-40, 10% glycerol, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 10 mM phenylmethylsulfonyl fluoride, and 0.5 mM sodium vanadate. Total protein in the extracts was assessed by the Coomassie method (Pierce Biotechnology, Rockford, IL). Kidney IGF-I levels were measured using an ELISA (DuoSet; R&D Systems, Minneapolis, MN). Renal total DNA was isolated with a DNeasy Tissue Kit (QIAGEN, Valencia, CA) from 15 mg tissue from each kidney from every mouse. Final values of total protein and total DNA were expressed as micrograms per milligram tissue; the ratio of protein to DNA (μg/μg) was also analyzed as an index of hypertrophy. Kidney IGF-I was expressed as picogram per milligram tissue. Serum IGF-I levels were measured by using the OCTEIA Rat/Mouse IGF1 kit (IDS, Fountain Hills, AZ). Serum IGF-I was expressed as nanograms per milliliter.
Histology. Formalin-fixed tissue was embedded in paraffin and sectioned at 5 μm. Tissue was stained with periodic acid-Schiff. Digital images were captured at a final magnification of x100, as determined by stage micrometry, and glomerular profile areas were measured using ScionImage Software (Scion, Frederick, MD). At least 15 profiles from each animal were averaged as an index of glomerular size. We have previously shown that glomerular area determined by this method is a reasonable proxy measurement of glomerular volume determined by strict stereological techniques (16, 17).
Immunohistochemistry studies. Kidney sections were mounted on coated slides. Paraffin was removed with Hemo-D, and then the slides were rehydrated with ethanol to tap water. All subsequent steps were separated by PBS rinses of 5 min. Slides were treated with 0.1% trypsin in PBS for 30 min and 10% H2O2 for 5 min and then blocked with 10% normal goat serum. Monoclonal antibody to PCNA (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:100, was used for an overnight incubation. Goat anti-mouse IgG-horseradish peroxidase, diluted 1:100, was applied for 1 h, followed by localization with 3-amino-9-ethylcarbazole (AEC; Chemicon International, Temecula, CA) for 10 min and Mayer's hematoxylin stain for 5 min. The slides were mounted using an aqueous-based medium. PCNA-positive nuclei stained red-brown. Slides were scored by a single individual masked to the identity of the tissue. Values are expressed as the total number of PCNA-positive cells for the 30 fields of cortex examined per kidney.
Statistics. Raw data from left kidneys not exposed to CKG were examined by two-factor ANOVA for the influence of the major factors sex (male or female) and genotype (wild-type or ERKO) on normal kidney growth. Raw data from right and left kidneys of sham animals were compared by paired Student's t-test to determine any effect(s) of the sham procedure. Data were then expressed as right (remnant) kidney value as a percentage of that of the left (Unx) kidney value. Because the sham procedure did not alter any parameter in the study between the two kidneys, two-factor ANOVA was then performed for the major factors in only the Unx mice. Significant results by ANOVA were followed by post hoc Tukey's tests. If data were not normally distributed, similar nonparametric tests were performed. All analyses were performed with SigmaStat 3.0 (Systat Software, Point Richmond, CA) A P value <0.05 was considered significant for all comparisons.
RESULTS
The sham procedure resulted in no differences in any parameter when the left and right kidneys were compared (Table 1). To examine differences in baseline status of the kidneys, raw values for these parameters were then considered for all left kidneys, since none would have been subjected to compensatory growth (Table 2). Kidney weight (156 ± 24 vs. 125 ± 32 mg), glomerular area (1,518 ± 279 vs. 1,421 ± 487 μm2), and total protein content (128 ± 35 vs. 115 ± 38 μg/mg) were influenced by sex (all greater in males), and kidney weight (150 ± 25 vs. 130 ± 28 mg), total protein content (132 ± 35 vs. 112 ± 38 μg/mg), and DNA content (0.42 ± 0.24 vs. 0.32 ± 0.32 μg/mg) were all greater in ERKO mice, whereas the protein-to-DNA ratios (349 ± 91 vs. 679 ± 103 μg/μg) were lower with the ERKO genotype. PCNA staining showed no variation with either sex or genotype alone but varied with genotype in a sex-dependent manner (Table 2). Wild-type males and ERKO females had similar low levels of PCNA staining, with statistical significance when ERKO females were compared with ERKO males or wild-type females.
The effects of Unx were then examined by expressing values of the right (remnant) kidney as a percentage of the left (Unx) kidney. When all mice were studied, regardless of sex or genotype, Unx altered kidney weight, glomerular area, DNA content, IGF-I content, and PCNA staining (Table 3).
Females showed a greater increase in kidney weight with Unx than males (126 ± 2 vs. 119 ± 2%), especially in wild-type mice (Fig. 1). Genotype did not influence the increase in kidney weight in males. Mean glomerular area increased more in females than in males after Unx (173 ± 7 vs. 151 ± 6%), but no differences could be demonstrated by genotype (Fig. 2). Total protein content of the kidney was not affected by sex (males 97 ± 4%, females 100 ± 4%; P = 0.52) or genotype (wild type 103 ± 4%, ERKO 94 ± 4%; P = 0.15) after Unx. No interactions of sex and genotype could be demonstrated for total protein content (P = 0.14). Kidney DNA content was greater in females than in males (98 ± 7 vs. 78 ± 6%) but was unaffected by genotype (Fig. 3). The ratio protein/DNA did not vary with sex (males 131 ± 10%, females 112 ± 12%; P = 0.22) or genotype (wild type 123 ± 11%, ERKO 120 ± 11%; P = 0.86). No interactions of sex and genotype were identified for this ratio (P = 0.18). Cortical PCNA-positive cells were greater in males than females (12,444 ± 1,682 vs. 5,796 ± 1,885%) but were also not influenced by genotype (Fig. 4).
Kidney IGF-I content was increased in all groups after Unx, but results were not influenced by sex (males 229 ± 24%, females 188 ± 29%; P = 0.28) or genotype (wild type 192 ± 26%, ERKO 225 ± 27%; P = 0.38). Terminal serum IGF-I levels did not differ between sham and Unx mice (428 ± 147 vs. 450 ± 138 ng/ml; P = 0.54). Sham animals also showed no differences in these levels with sex or genotype. After Unx, serum IGF-I showed significant interaction between sex and genotype, with wild-type males greater than wild-type females (546 ± 36 vs. 404 ± 40 ng/ml, P = 0.01) and ERKO males (380 ± 40 ng/ml, P = 0.002). ERKO females (466 ± 36 ng/ml) did not differ from wild-type females or ERKO males.
DISCUSSION
ERKO mice showed sex-specific differences in compensatory increases in kidney weight but not glomerular area after Unx. Female wild-type mice showed greater CKG than ERKO females. Levels in ERKO females were similar to males of either genotype, suggesting that a lack of estrogen receptor (ER)--mediated processes suppresses CKG. Unx increased DNA content and PCNA staining in the remnant kidney without altering protein content or the protein-to-DNA ratio, suggesting that hyperplasia is the major component of CKG at 48 h in all of these mice. Genotype was otherwise not associated with differences in these parameters in Unx mice.
ERKO mice also showed alterations in normal kidney growth as demonstrated in the analysis of left kidneys. Lack of ER- was associated with greater kidney weight, protein content, and DNA content, but lower protein-to-DNA ratios. ERKO mice also showed a decrease in PCNA staining in female left kidneys, suggesting less baseline proliferation of tubular cells in this sex and genotype.
Proliferation was histologically assessed by using immunostaining to PCNA. PCNA is a 36-kDa auxiliary protein to DNA polymerase-, which is found in various concentrations within cells throughout the cell cycle, reaching a maximum during the S phase, making it a marker of active cell proliferation (20). We report it only in the tubular compartment of the cortex; staining was virtually absent in glomeruli, suggesting that if proliferation occurred after Unx it was completed before 48 h in the glomerular compartment. This index of proliferation was elevated in the tubules in all groups after Unx, with greater numbers in males than in females. DNA content, an index of cell number, was greater in females than in males, contrary to these PCNA results. This could be explained if hyperplasia occurred earlier in females so that DNA was increased, but active proliferation was abating by 48 h. Further study at earlier time points after Unx would be necessary to confirm this supposition.
The GH-IGF-I system has been linked to sex differences in CKG in other studies, so its role was examined in the present experiments (6, 27, 28). Kidney IGF-I content increased approximately twofold after Unx and was not influenced by sex or genotype. Serum IGF-I did not differ significantly between sham and Unx. The kidney produces IGF-I for paracrine and autocrine functions (5). Increased IGF-I was likely the result of this intrarenal system rather than systemic effects; however, other alterations of GH-IGF-I cannot be ruled out from our data (26–28).
Both estrogen receptors, ER- and ER-, may affect cellular function in three ways (29). The classic genomic signaling mechanism is via binding to a receptor that then translocates to the cell nucleus, binds to the DNA, and alters cellular transcription. ERs may also be found in the membrane of cells; ligand binding produces rapid, nongenomic effects via these receptors. Estrogen may also directly bind to ion channels and other membrane proteins to rapidly alter cellular functions. Both ERs can modulate these genomic and nongenomic effects. ER- produces classical feminizing effects, including proliferation of the uterus and breasts, and is proproliferative in a number of tumors (14). It has also been shown to mediate virtually all feedback to the neuroendocrine system (3). ER- is often a negative regulator of ER-, producing opposite effects (21, 34). ER- agonism inhibits proliferation in a number of tissues and tumors (14).
Suppression of compensatory renal growth in ERKO females may be the result of loss of ER--mediated events, increased ER--mediated events, and/or elevated androgens or gonadotropins. The hypothalamic-pituitary-gonadal axis has recently been characterized in ERKO mice (3). ER- is essential for negative feedback on production of luteinizing hormone, and females lacking this receptor show elevated plasma levels of this gonadotropin. Because they lack negative feedback on the hypothalamus and pituitary, circulating estradiol levels are elevated approximately eightfold over wild-type females. The ovaries of these mice produce 17-hydroxysteroid dehydrogenase type III, an enzyme normally found only in the testes that converts androstenedione to testosterone. Intact ER- null females have elevated plasma levels of testosterone, similar to those seen in wild-type males (3). Both androgens and luteinizing hormone may promote renal growth, perhaps explaining the larger baseline kidney weights in the ERKO mice (1, 32, 33, 39, 43).
Because our female mice are known to have elevated levels of estradiol and still express ER- (3), this receptor pathway may mediate the antiproliferative effect seen in the present study. The kidney has also been demonstrated to be the most ER--regulated organ in the mouse outside of the reproductive neuroendocrine axis (11), so a lack of proproliferative ER- effects could also explain our results (14).
Sex differences in CKG have been examined in rats. Both males and females show a similar increase in remnant kidney weight of 20% at 24–48 h after Unx (18, 27, 28). After 24 h, both sexes show similar increases in protein content, whereas DNA increases only in females (27, 28). Bromodeoxyuridine uptake and staining were also increased in adult females, confirming a hyperplastic component of early CKG not seen in male rats. After Unx (2 mo), males showed a doubling of remnant kidney weight, whereas female kidneys weighed 50–60% more than the Unx specimen (28). At the 2-mo observation point, glomerular volume had increased 25% in females, whereas males were up 125%.
Before the present experiments, sex differences in mice had not been examined directly in a single study. Male Swiss mice, 6–8 mo of age, were shown to increase remnant kidney dry weight by 10–15% at 48 h, similar to the 17% increase seen in wild-type males in the present study (12). With the use of radioactive uptake studies, both hyperplasia and hypertrophy were demonstrated in these experiments. Within 1 h of nephrectomy, synthesis of protein and RNA doubled in the remnant kidney and remained stable through 48 h. DNA synthesis began to rise after 10 h and peaked at 48 h at levels five to six times those of sham animals. Levels were approaching baseline 120 h after Unx. This predominance of hyperplasia at 48 h is also consistent with our observations in male mice. In another study, C57Bl6 male mice, the same background strain as our mice, showed 20% increases in both remnant kidney weight and the protein-to-DNA ratio at 4 days after Unx when hypertrophy would be expected to predominate (18).
Other studies have used only female mice. Adult Balb/C females showed an 20% increase in kidney weight 48 h after Unx, with an increase in glomerular volume of 50% over 7 days (6). Adult female NMRI mice developed a 26% increase in kidney weight on day 2, whereas glomerular volume increased by 20% after 7 days in this strain (7). In the present experiment, wild-type females showed a 30% increase in kidney weight and a 70% increase in glomerular area after 2 days, values not inconsistent with those previously reported. All of these results in female mice appear to be greater than those reported in males, although only the present study examines males and females of the same strain simultaneously.
Mice thus appear to respond to Unx differently than rats. In both male and female mice, an initial period of hyperplastic growth occurs during the first 48 h after Unx. Enlargement of both kidney weight and glomerular size is more pronounced in female mice at this time point. This growth is associated with a doubling of IGF-I content in the kidney but not levels of this growth factor in the serum. Lack of ER- is associated with reduced CKG in females, probably via a suppression of tubular proliferation. -ERKO mice did not demonstrate any alterations in compensatory glomerular enlargement. Further study will be necessary to determine the mechanism of these phenomena.
GRANTS
Funding was provided by the Edna Ittner Pediatric Research Fund of the University of Nebraska Medical Center.
ACKNOWLEDGMENTS
These studies were presented at the International Union of Physiological Sciences, April 2, 2005, San Diego, CA.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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ABSTRACT
Females are relatively protected in many progressive kidney diseases. Processes of kidney scarring and growth are intricately linked, and female kidneys are smaller than male kidneys. To better understand links between sex, growth, and the kidney, we examined compensatory kidney growth (CKG) after uninephrectomy (Unx) in wild-type and estrogen receptor- null mice (ERKO). Mice (10 wk old) underwent Unx or sham procedure, with removal of all remaining kidney(s) 48 h later. Studies included kidney weight, renal content of protein, DNA, and insulin-like growth factor-I (IGF-I), serum IGF-I, mean glomerular area, and immunostaining for proliferating cell nuclear antigen (PCNA). Sham Unx produced no differences between left and right kidneys. Unx altered kidney weight, glomerular area, DNA content, IGF-I content, and PCNA regardless of sex or genotype. Females showed greater increases in kidney weight (26 vs. 19%) and glomerular area (73 vs. 51%) than males. Differences in kidney weight were restricted to wild-type females (32% increase); ERKO females showed an increase in kidney weight similar to males (19%). Genotype did not influence glomerular growth in this model. Both male and female mice exhibit hyperplastic growth 48 h after Unx, with more pronounced enlargement in females. Lack of estrogen receptor- is associated with reduced CKG in females, probably via suppression of proliferation. ERKO mice did not demonstrate any alterations in compensatory glomerular enlargement. Kidney IGF-I content doubled after Unx, regardless of sex or genotype, implicating other mechanisms with regard to these findings.
hypertrophy; hyperplasia; uninephrectomy; insulin-like growth factor I; sex
THE PROCESSES OF RENAL HYPERTROPHY and progression of kidney disorders are intimately linked (8, 9, 15). Virtually all progressive nephropathies are associated with enlargement of the kidney and the glomeruli. Numerous factors that control normal renal growth have been implicated in nephrosclerosis, and all therapies that ameliorate sclerosis also prevent early enlargement in kidney disease (8).
Clinical and animal model studies suggest that females are relatively protected from a number of progressive nephropathies (10, 30, 31, 41, 42). Females also have smaller kidneys than males, so there may be a direct link between kidney growth and risk of kidney failure. Secondary sex differences are produced by gonadal steroids, and it is likely that these hormones influence the kidney as well. It has been suggested that androgens, known to promote kidney enlargement, are detrimental in a number of clinical and experimental conditions (2, 22, 35–38). There is also evidence that estrogens may play a beneficial role in kidney and vascular disease (4, 13, 35, 40).
Transgenic and knockout mice provide powerful tools to delineate the role of various genes in physiology. We have recently reported that female mice lacking estrogen receptor- (ERKO) are protected from diabetic glomerular hypertrophy, although not from overall diabetic kidney growth (19). Differences in kidney weight were noted in nondiabetic mice, suggesting that this genotype also altered normal kidney development. The following study examined the effects of this genotype in another model of kidney growth, compensatory hypertrophy after uninephrectomy (Unx).
Sex differences in compensatory kidney growth (CKG) have received direct attention in the rat but not in the mouse (23–28). Female rats show initial hyperplastic growth, whereas males show hypertrophy (27, 28). Upregulation of the intrarenal insulin-like growth factor-I (IGF-I) system was implicated in females, but no role was apparent for growth hormone (GH; see Refs. 23–26). In contrast, CKG in adult males was blocked by castration or GH antagonists, but no differences in the IGF-I system were identified (28). Testosterone has thus been implicated as a driving force in CKG, but a role for estrogen has been less clear. Sex differences in this model in mice have not been directly examined.
CKD was examined in male and female wild-type and -ERKO mice at 48 h. We show that there are significant differences in CKD in mice compared with the existing literature on rats (23–28) and that female -ERKO mice show suppressed CKD, similar to that seen in males of either genotype.
METHODS
Animals. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center. Homozygous wild-type and -ERKO mice of both sexes began these experiments at 10 wk of age. Four groups of mice of each genotype and sex underwent Unx at time 0. Similar groups of mice underwent sham Unx. For both procedures, all mice were anesthetized with ketamine and xylazine. For the sham procedure, the left kidney was manipulated out of its fossa and then returned, and the incision was closed. Unx animals had the kidney manipulated out of its fossa and then dissected free of its capsule. The renal vessels were tied off, and the left kidney was removed. After the initial procedure (48 h), all animals underwent a terminal procedure under anesthesia with collection of blood and removal of all remaining kidneys.
All kidneys were weighed at the time they were removed. A 2- to 3-mm transverse slice from the midportion of each kidney was immersed in formalin for histological studies, including measurement of glomerular area and proliferating cell nuclear antigen (PCNA)-positive cells. The remaining portions of the kidneys were snap-frozen for later biochemical studies.
Protein, DNA, and IGF-I studies. Protein was extracted from 20 mg of both kidneys from every mouse using a homogenization buffer containing Tris-buffered saline (pH 8.0), 1% Nonidet P-40, 10% glycerol, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 10 mM phenylmethylsulfonyl fluoride, and 0.5 mM sodium vanadate. Total protein in the extracts was assessed by the Coomassie method (Pierce Biotechnology, Rockford, IL). Kidney IGF-I levels were measured using an ELISA (DuoSet; R&D Systems, Minneapolis, MN). Renal total DNA was isolated with a DNeasy Tissue Kit (QIAGEN, Valencia, CA) from 15 mg tissue from each kidney from every mouse. Final values of total protein and total DNA were expressed as micrograms per milligram tissue; the ratio of protein to DNA (μg/μg) was also analyzed as an index of hypertrophy. Kidney IGF-I was expressed as picogram per milligram tissue. Serum IGF-I levels were measured by using the OCTEIA Rat/Mouse IGF1 kit (IDS, Fountain Hills, AZ). Serum IGF-I was expressed as nanograms per milliliter.
Histology. Formalin-fixed tissue was embedded in paraffin and sectioned at 5 μm. Tissue was stained with periodic acid-Schiff. Digital images were captured at a final magnification of x100, as determined by stage micrometry, and glomerular profile areas were measured using ScionImage Software (Scion, Frederick, MD). At least 15 profiles from each animal were averaged as an index of glomerular size. We have previously shown that glomerular area determined by this method is a reasonable proxy measurement of glomerular volume determined by strict stereological techniques (16, 17).
Immunohistochemistry studies. Kidney sections were mounted on coated slides. Paraffin was removed with Hemo-D, and then the slides were rehydrated with ethanol to tap water. All subsequent steps were separated by PBS rinses of 5 min. Slides were treated with 0.1% trypsin in PBS for 30 min and 10% H2O2 for 5 min and then blocked with 10% normal goat serum. Monoclonal antibody to PCNA (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:100, was used for an overnight incubation. Goat anti-mouse IgG-horseradish peroxidase, diluted 1:100, was applied for 1 h, followed by localization with 3-amino-9-ethylcarbazole (AEC; Chemicon International, Temecula, CA) for 10 min and Mayer's hematoxylin stain for 5 min. The slides were mounted using an aqueous-based medium. PCNA-positive nuclei stained red-brown. Slides were scored by a single individual masked to the identity of the tissue. Values are expressed as the total number of PCNA-positive cells for the 30 fields of cortex examined per kidney.
Statistics. Raw data from left kidneys not exposed to CKG were examined by two-factor ANOVA for the influence of the major factors sex (male or female) and genotype (wild-type or ERKO) on normal kidney growth. Raw data from right and left kidneys of sham animals were compared by paired Student's t-test to determine any effect(s) of the sham procedure. Data were then expressed as right (remnant) kidney value as a percentage of that of the left (Unx) kidney value. Because the sham procedure did not alter any parameter in the study between the two kidneys, two-factor ANOVA was then performed for the major factors in only the Unx mice. Significant results by ANOVA were followed by post hoc Tukey's tests. If data were not normally distributed, similar nonparametric tests were performed. All analyses were performed with SigmaStat 3.0 (Systat Software, Point Richmond, CA) A P value <0.05 was considered significant for all comparisons.
RESULTS
The sham procedure resulted in no differences in any parameter when the left and right kidneys were compared (Table 1). To examine differences in baseline status of the kidneys, raw values for these parameters were then considered for all left kidneys, since none would have been subjected to compensatory growth (Table 2). Kidney weight (156 ± 24 vs. 125 ± 32 mg), glomerular area (1,518 ± 279 vs. 1,421 ± 487 μm2), and total protein content (128 ± 35 vs. 115 ± 38 μg/mg) were influenced by sex (all greater in males), and kidney weight (150 ± 25 vs. 130 ± 28 mg), total protein content (132 ± 35 vs. 112 ± 38 μg/mg), and DNA content (0.42 ± 0.24 vs. 0.32 ± 0.32 μg/mg) were all greater in ERKO mice, whereas the protein-to-DNA ratios (349 ± 91 vs. 679 ± 103 μg/μg) were lower with the ERKO genotype. PCNA staining showed no variation with either sex or genotype alone but varied with genotype in a sex-dependent manner (Table 2). Wild-type males and ERKO females had similar low levels of PCNA staining, with statistical significance when ERKO females were compared with ERKO males or wild-type females.
The effects of Unx were then examined by expressing values of the right (remnant) kidney as a percentage of the left (Unx) kidney. When all mice were studied, regardless of sex or genotype, Unx altered kidney weight, glomerular area, DNA content, IGF-I content, and PCNA staining (Table 3).
Females showed a greater increase in kidney weight with Unx than males (126 ± 2 vs. 119 ± 2%), especially in wild-type mice (Fig. 1). Genotype did not influence the increase in kidney weight in males. Mean glomerular area increased more in females than in males after Unx (173 ± 7 vs. 151 ± 6%), but no differences could be demonstrated by genotype (Fig. 2). Total protein content of the kidney was not affected by sex (males 97 ± 4%, females 100 ± 4%; P = 0.52) or genotype (wild type 103 ± 4%, ERKO 94 ± 4%; P = 0.15) after Unx. No interactions of sex and genotype could be demonstrated for total protein content (P = 0.14). Kidney DNA content was greater in females than in males (98 ± 7 vs. 78 ± 6%) but was unaffected by genotype (Fig. 3). The ratio protein/DNA did not vary with sex (males 131 ± 10%, females 112 ± 12%; P = 0.22) or genotype (wild type 123 ± 11%, ERKO 120 ± 11%; P = 0.86). No interactions of sex and genotype were identified for this ratio (P = 0.18). Cortical PCNA-positive cells were greater in males than females (12,444 ± 1,682 vs. 5,796 ± 1,885%) but were also not influenced by genotype (Fig. 4).
Kidney IGF-I content was increased in all groups after Unx, but results were not influenced by sex (males 229 ± 24%, females 188 ± 29%; P = 0.28) or genotype (wild type 192 ± 26%, ERKO 225 ± 27%; P = 0.38). Terminal serum IGF-I levels did not differ between sham and Unx mice (428 ± 147 vs. 450 ± 138 ng/ml; P = 0.54). Sham animals also showed no differences in these levels with sex or genotype. After Unx, serum IGF-I showed significant interaction between sex and genotype, with wild-type males greater than wild-type females (546 ± 36 vs. 404 ± 40 ng/ml, P = 0.01) and ERKO males (380 ± 40 ng/ml, P = 0.002). ERKO females (466 ± 36 ng/ml) did not differ from wild-type females or ERKO males.
DISCUSSION
ERKO mice showed sex-specific differences in compensatory increases in kidney weight but not glomerular area after Unx. Female wild-type mice showed greater CKG than ERKO females. Levels in ERKO females were similar to males of either genotype, suggesting that a lack of estrogen receptor (ER)--mediated processes suppresses CKG. Unx increased DNA content and PCNA staining in the remnant kidney without altering protein content or the protein-to-DNA ratio, suggesting that hyperplasia is the major component of CKG at 48 h in all of these mice. Genotype was otherwise not associated with differences in these parameters in Unx mice.
ERKO mice also showed alterations in normal kidney growth as demonstrated in the analysis of left kidneys. Lack of ER- was associated with greater kidney weight, protein content, and DNA content, but lower protein-to-DNA ratios. ERKO mice also showed a decrease in PCNA staining in female left kidneys, suggesting less baseline proliferation of tubular cells in this sex and genotype.
Proliferation was histologically assessed by using immunostaining to PCNA. PCNA is a 36-kDa auxiliary protein to DNA polymerase-, which is found in various concentrations within cells throughout the cell cycle, reaching a maximum during the S phase, making it a marker of active cell proliferation (20). We report it only in the tubular compartment of the cortex; staining was virtually absent in glomeruli, suggesting that if proliferation occurred after Unx it was completed before 48 h in the glomerular compartment. This index of proliferation was elevated in the tubules in all groups after Unx, with greater numbers in males than in females. DNA content, an index of cell number, was greater in females than in males, contrary to these PCNA results. This could be explained if hyperplasia occurred earlier in females so that DNA was increased, but active proliferation was abating by 48 h. Further study at earlier time points after Unx would be necessary to confirm this supposition.
The GH-IGF-I system has been linked to sex differences in CKG in other studies, so its role was examined in the present experiments (6, 27, 28). Kidney IGF-I content increased approximately twofold after Unx and was not influenced by sex or genotype. Serum IGF-I did not differ significantly between sham and Unx. The kidney produces IGF-I for paracrine and autocrine functions (5). Increased IGF-I was likely the result of this intrarenal system rather than systemic effects; however, other alterations of GH-IGF-I cannot be ruled out from our data (26–28).
Both estrogen receptors, ER- and ER-, may affect cellular function in three ways (29). The classic genomic signaling mechanism is via binding to a receptor that then translocates to the cell nucleus, binds to the DNA, and alters cellular transcription. ERs may also be found in the membrane of cells; ligand binding produces rapid, nongenomic effects via these receptors. Estrogen may also directly bind to ion channels and other membrane proteins to rapidly alter cellular functions. Both ERs can modulate these genomic and nongenomic effects. ER- produces classical feminizing effects, including proliferation of the uterus and breasts, and is proproliferative in a number of tumors (14). It has also been shown to mediate virtually all feedback to the neuroendocrine system (3). ER- is often a negative regulator of ER-, producing opposite effects (21, 34). ER- agonism inhibits proliferation in a number of tissues and tumors (14).
Suppression of compensatory renal growth in ERKO females may be the result of loss of ER--mediated events, increased ER--mediated events, and/or elevated androgens or gonadotropins. The hypothalamic-pituitary-gonadal axis has recently been characterized in ERKO mice (3). ER- is essential for negative feedback on production of luteinizing hormone, and females lacking this receptor show elevated plasma levels of this gonadotropin. Because they lack negative feedback on the hypothalamus and pituitary, circulating estradiol levels are elevated approximately eightfold over wild-type females. The ovaries of these mice produce 17-hydroxysteroid dehydrogenase type III, an enzyme normally found only in the testes that converts androstenedione to testosterone. Intact ER- null females have elevated plasma levels of testosterone, similar to those seen in wild-type males (3). Both androgens and luteinizing hormone may promote renal growth, perhaps explaining the larger baseline kidney weights in the ERKO mice (1, 32, 33, 39, 43).
Because our female mice are known to have elevated levels of estradiol and still express ER- (3), this receptor pathway may mediate the antiproliferative effect seen in the present study. The kidney has also been demonstrated to be the most ER--regulated organ in the mouse outside of the reproductive neuroendocrine axis (11), so a lack of proproliferative ER- effects could also explain our results (14).
Sex differences in CKG have been examined in rats. Both males and females show a similar increase in remnant kidney weight of 20% at 24–48 h after Unx (18, 27, 28). After 24 h, both sexes show similar increases in protein content, whereas DNA increases only in females (27, 28). Bromodeoxyuridine uptake and staining were also increased in adult females, confirming a hyperplastic component of early CKG not seen in male rats. After Unx (2 mo), males showed a doubling of remnant kidney weight, whereas female kidneys weighed 50–60% more than the Unx specimen (28). At the 2-mo observation point, glomerular volume had increased 25% in females, whereas males were up 125%.
Before the present experiments, sex differences in mice had not been examined directly in a single study. Male Swiss mice, 6–8 mo of age, were shown to increase remnant kidney dry weight by 10–15% at 48 h, similar to the 17% increase seen in wild-type males in the present study (12). With the use of radioactive uptake studies, both hyperplasia and hypertrophy were demonstrated in these experiments. Within 1 h of nephrectomy, synthesis of protein and RNA doubled in the remnant kidney and remained stable through 48 h. DNA synthesis began to rise after 10 h and peaked at 48 h at levels five to six times those of sham animals. Levels were approaching baseline 120 h after Unx. This predominance of hyperplasia at 48 h is also consistent with our observations in male mice. In another study, C57Bl6 male mice, the same background strain as our mice, showed 20% increases in both remnant kidney weight and the protein-to-DNA ratio at 4 days after Unx when hypertrophy would be expected to predominate (18).
Other studies have used only female mice. Adult Balb/C females showed an 20% increase in kidney weight 48 h after Unx, with an increase in glomerular volume of 50% over 7 days (6). Adult female NMRI mice developed a 26% increase in kidney weight on day 2, whereas glomerular volume increased by 20% after 7 days in this strain (7). In the present experiment, wild-type females showed a 30% increase in kidney weight and a 70% increase in glomerular area after 2 days, values not inconsistent with those previously reported. All of these results in female mice appear to be greater than those reported in males, although only the present study examines males and females of the same strain simultaneously.
Mice thus appear to respond to Unx differently than rats. In both male and female mice, an initial period of hyperplastic growth occurs during the first 48 h after Unx. Enlargement of both kidney weight and glomerular size is more pronounced in female mice at this time point. This growth is associated with a doubling of IGF-I content in the kidney but not levels of this growth factor in the serum. Lack of ER- is associated with reduced CKG in females, probably via a suppression of tubular proliferation. -ERKO mice did not demonstrate any alterations in compensatory glomerular enlargement. Further study will be necessary to determine the mechanism of these phenomena.
GRANTS
Funding was provided by the Edna Ittner Pediatric Research Fund of the University of Nebraska Medical Center.
ACKNOWLEDGMENTS
These studies were presented at the International Union of Physiological Sciences, April 2, 2005, San Diego, CA.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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