Endogenous estrogen may prevent bone loss in postmenopausal hemodialysis patients throughout life.

SUMMARY: Postmenopausal hemodialysis patients are at risk of complications related to renal mineral and bone disorder, and postmenopausal osteoporosis. In 112 postmenopausal hemodialysis patients, free estrogen index was positively correlated with bone mineral density (BMD) Z-score and the annual percent change of BMD in multiple regression analysis. Endogenous estrogen may prevent bone loss in postmenopausal hemodialysis patients throughout life. INTRODUCTION: Women on dialysis are not only at risk of developing mineral and bone disorder, but also suffer from postmenopausal osteoporosis. We assessed the effect of sex hormones on bone metabolism in postmenopausal hemodialysis patients. METHODS: We enrolled 112 postmenopausal hemodialysis patients with a mean age of 68.4 ± 10.4 years. We measured the serum levels of estradiol, testosterone, sex hormone-binding globulin (SHBG), and intact parathyroid hormone (intact-PTH), as well as bone metabolism parameters and radial bone mineral density (BMD). The free estrogen index (FEI) was calculated from the estradiol and SHBG values. After conventional dialysis was performed for 12 months, BMD was measured again and the annual percent change was calculated. Estradiol and SHBG were also measured in 25 postmenopausal women without chronic kidney disease. RESULTS: Estradiol levels were higher in the hemodialysis patients than in the postmenopausal women without chronic kidney disease. In patients with relatively normal bone turnover (intact-PTH: from 150 to 300 pg/ml), the FEI showed a positive correlation with the BMD Z-score. The annual percent change of BMD showed a positive correlation with the FEI according to multiple regression analysis. CONCLUSIONS: Endogenous estrogen may prevent bone loss in postmenopausal hemodialysis patients throughout life.

Plasma osteoprotegerin, arterial stiffness, and mortality in normoalbuminemic Japanese hemodialysis patients.

UNLABELLED: A high circulating osteoprotegerin (OPG) level may be a risk factor for vascular calcification and mortality in hemodialysis patients. OPG and pulse wave velocity (PWV) were measured at baseline in 151 normoalbuminemic, long-term (>3 years) Japanese hemodialysis patients who were prospectively followed for 6 years. In long-term normoalbuminemic Japanese hemodialysis patients, OPG levels were strongly linked with both arterial stiffness and worse outcome. INTRODUCTION: A high circulating OPG level is reported to be a risk factor for vascular calcification and mortality in Western chronic kidney disease (CKD) patients but it is not known if this is true for Japanese CKD patients, where a different risk profile may operate. METHODS: OPG and PWV were measured at baseline in 151 normoalbuminemic, long-term (>3 years) Japanese hemodialysis patients (median age 62 years) who were prospectively followed for 6 years. RESULTS: OPG levels were associated in multivariate analysis with age, dialysis vintage, history of cardiovascular disease (CVD) and parathyroid hormone levels. C-reactive protein levels did not correlate with OPG. Patients with clinical history of CVD had significantly higher OPG levels and OPG levels were positively correlated to PWV, an index of arterial stiffness. These associations were independent of age, sex, dialysis vintage, and diabetes. During the follow-up period, 40 deaths, including 25 cardiovascular deaths, were recorded. In crude analysis, each unit of increase in OPG was associated with increased all-cause (hazard ratios 1.14, 95% confidence interval 1.08-1.20) and CVD mortality (1.14 [1.07-1.21]), which persisted after adjustment for age, sex, dialysis vintage, diabetes, and baseline CVD (1.12 [1.05-1.19] and 1.11 [1.02-1.19], all-cause and CVD mortality, respectively). CONCLUSIONS: In long-term normoalbuminemic Japanese hemodialysis patients, with low prevalence of inflammation, OPG levels were strongly linked with both arterial stiffness and worse outcome.

Correlation between mammary tumor and blood glucose, serum insulin, and free fatty acids in mice.

The blood glucose level and serum levels of insulin, glucagon, and free fatty acids were examined in 7- to 8-mo-old female SHN mice with or without spontaneous mammary tumors (MT). Blood glucose levels in the females with MT were significantly higher than in those without MT, rising in proportion to the increase in size of MT up to 30 mm in diameter. In 4-mo-old male SHN and 11-mo-old female C57BL mice bearing mammary tumor grafts (MTg), the blood glucose level was significantly higher than in mice without MTg. Serum insulin and free fatty acids in female SHN mice with MT rose to higher levels than in mice without MT, whereas serum glucagon levels were unaltered. In 50% of mice with MT, pancreatic islets contained a large number of pyknotic cells. Livers of mice with MT or MTg were significantly heavier than those of mice without MT or MTg. In both female SHN mice with spontaneous MT and male SHN and female C57BL mice with MTg, the total number of hepatocytes and the total amount of liver DNA increased significantly compared with values from corresponding controls without MT or MTg. These findings suggest that MT or MTg induce a hyperglycemic state and an enhanced production of free fatty acids and insulin, which may in turn stimulate the growth of mammary tumors and the liver.

C terminus of presenilin is required for overproduction of amyloidogenic Abeta42 through stabilization and endoproteolysis of presenilin.

Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). To identify a PS subdomain responsible for overproduction of Abeta42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in Abeta42 secretion, and Abeta42 overproduction was dependent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of Abeta42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.

Postnatal vaginal nodules induced by prenatal diethylstilbestrol treatment correlate with later development of ovary-independent vaginal and uterine changes in mice.

Pregnant ICR/JCL mice were given 4 daily subcutaneous injections of 0.2-2000 micrograms diethylstilbestrol (DES) starting on day 15 of gestation. Offspring of mothers given DES were killed at 1-10 days of age and examined for nodules of enlarged polygonal cells under the epithelium of the Müllerian (upper) vagina. Some offspring were ovariectomised at 30 days and killed at 120 days. The nodules which appeared in the prenatally DES-exposed mice (2-2000 micrograms/day) at 3-7 days were not connected with the epithelium of the sinus vagina and reacted positively to an antibody to epidermal growth factor. Nodule formation may prove to be prodromic of later ovary-independent vaginal changes in the DES-exposed mice. Epithelial stratification (2-2000 micrograms/day) and downgrowths and/or pegs (20-2000 micrograms/day) occurred in vaginae of ovariectomized mice exposed prenatally to DES; however, adenosis-like lesions occurred only in the offspring of mothers given the highest prenatal injections of 2000 micrograms DES. Wolffian remnants and hypospadias (2-2000 micrograms/day) were also encountered in the DES-exposed mice. Ovary-independent stratification of the uterine epithelium (20-2000 micrograms/day) and disorganization of the circular musculature (2-2000 micrograms/day) were also observed in the DES-exposed mice. None of these changes was found in ovariectomised 0.2 micrograms DES-exposed and control mice.

Quantitative analysis of the development of genital organs from the urogenital sinus of the fetal male mouse treated prenatally with a 5 alpha-reductase inhibitor.

The role of 5 alpha-dihydrotestosterone (DHT) in the development of the genital organs and in the differentiation of the genital tract into prostate, coagulating gland (CG), bulbo-urethral gland (BUG) and seminal vesicle (SV) in male mice exposed prenatally to the 5 alpha-reductase inhibitor 6-methylene-4-pregnene-3,20-dione (6-MP) has been examined quantitatively. Female ICR mice were given 7 daily s.c. injections of the inhibitor (400 mg/day) starting on day 12 of gestation and the experiment was terminated on day 19 when the fetuses were removed by Caesarean section. In the prenatally 6-MP-exposed male mice the anogenital distance was significantly shorter than in the controls. Feminization of the nipples and hypospadias of the phallic urethra were noted. Development of prostate, CG and BUG was significantly suppressed. SV and testis development were not affected. These results lend further support to the conclusion that DHT is necessary for the development of the urogenital sinus (prostate, CG and BUG) and penis, and for the regression of the nipples in male mice. Reproductive abnormalities were not found in 90-day-old mice of both sexes exposed to 6-MP in utero. The 6-MP-exposed male and female mice had a normal reproductive capacity when mated with normal mice. These results show that 6-MP-induced growth retardation of reproductive organs is evident on day 19 of gestation, but that such retardation is no longer apparent in the adult.

Occurrence of genital tract abnormalities and bladder hernia in female mice exposed neonatally to tamoxifen.

Three groups of female C57BL/Tw mice given 5 daily injections of 2, 20 or 100 micrograms tamoxifen (Tx) starting on the day of birth were killed at 35 and 150 days of age. About a half of the mice killed at 150 days had been ovariectomized at 90 days. Uterine hypoplasia, myometrial involution and suppression of the uterine-gland genesis were found in the 2 age-groups of Tx-treated mice. Vaginal hypoplasia and hypospadia were common abnormalities in 150-day-old Tx-treated mice. Vaginal adenosis was encountered in 35-day-old mice treated neonatally with 20 or 100 micrograms Tx, but not in 150-day-old group. Permanent proliferation of vaginal epithelium was not induced by Tx. More than 80% of oocytes in small follicles were degenerated in Tx-exposed mice at 150 days, but not so in those at 35 days. Ovaries of neonatally Tx-exposed mice lacking corpora lutea made no luteinizing response to human chorionic gonadotropin injected prepubertally. Urinary-bladder hernia with or without caecum hernia frequently occurred in 150-day-old mice given 20 or 100 micrograms Tx. The present study revealed that neonatally administered Tx causes various abnormalities in gonad and genito-urinary tract of female C57BL/Tw mice.

Blockade by vitamin A of the occurrence of permanent vaginal changes in mice treated neonatally with 5alpha-dihydrotestosterone.

Estrogen-independent, persistent proliferation and cornification of the vaginal epithelium occurred in ovariectomized adult mice which had received neonatal injections of 50 microgram 5alpha-dihydrotestosterone (DHT). The occurrence of the permanent vaginal changes was prevented by injections of 200 IU vitamin A acetate (VA) given simultaneously with DHT. In 45% of the DHT plus VA-treated, ovariectomized mice, clear cells with pale cytoplasm appeared in the basal layer of the degenerating epithelium, while they came out in the epithelium of 13% of the ovariectomized mice receiving neonatal injections of DHT alone. For suppressing the permanent vaginal changes, including high mitotic rate, more than 1 month was required after the combined treatment. However, the occurrence of the permanent changes was not blocked by VA given after the DHT treatment. Permanent proliferation and squamous metaplasia took place in the uterine epithelium of the DHT-treated mice. Neonatal injections of VA also prevented the occurrence of the permanent uterine changes when given simultaneously with DHT but not when given after the androgen treatment.

Polyovular follicles in the ovary of immature mice exposed prenatally to diethylstilbestrol.

Polyovular follicles (PF) occur in the ovary of 30-day-old offspring of ICR/JCL mice given 4 daily subcutaneous injections of 20-2,000 micrograms diethylstilbestrol (DES)/day from days 15 to 18 of gestation. PF containing 2-9 oocytes per follicle in the prenatally DES-exposed mice are increased 33- to 112-fold as compared to controls. In 5- to 25-day-old offspring of mothers given injections of 2,000 micrograms DES/day, PF are observed 17-65 times more frequent than in controls.

Deciduoma formation in rats treated neonatally with the anti-estrogens, tamoxifen and MER-25.

Female rats of the T strain were given single daily injections of 100 micrograms and 200 micrograms of tamoxifen (Tx) and MER-25 (MER) for 5 days beginning on the day of birth (DAY 1). When sacrificed on Day 60, the Tx-treated rats (Tx rats) exhibited continued vaginal diestrus, whereas the females given MER or the vehicle alone showed regular estrous cycles. Ovaries from Tx rats were polyfollicular without corpora lutea, while those from MER rats, as well as from the controls given the vehicle alone, invariably contained both follicles and corpora lutea. In Tx rats, the uteri underwent atrophy, containing few uterine glands in an endometrium largely occupied by fibroblasts. Decidual response of the uterus to intraluminal oil instillation was markedly reduced in Tx rats given an appropriate regimen of progesterone and estradiol injections following ovariectomy on Day 60. By contrast, MER given neonatally had little effects on decidualization. Since ovariectomy on Day 10 brought about no amelioration of the decidualization in Tx rats, it is suggested that the lowered deciduogenic responsiveness to the instillation was caused by a direct action of Tx on the uterus of neonatal rats rather than by a Tx-induced alteration of hypothalamo-hypophyseal-ovarian system. Differences in effect on the female reproductive system between Tx and MER were discussed.

Permanent chondrification in the pelvis and occurrence of hernias in mice treated neonatally with tamoxifen.

Male and female C57BL/Tw mice were given 5 daily subcutaneous injections of 100 micrograms tamoxifen (Tx), starting on the day of birth (Tx mice). In untreated fetal mice on day 18 of gestation, the greater part of the pubic and ischial bones were cartilaginous. At more than 30 days of age, however, untreated mice showed completely calcified pelvic bone, whereas in age-matched Tx mice the greater part of the junctional regions in the pelvis remained cartilaginous. Treatment with Tx starting within 5 days of age caused bladder hernia with or without cecum hernia. The pubic ligament in Tx mice at ages of 30-540 days was markedly expanded as compared with that in age-matched controls. The permanent chondrification in the pelvis was found in all mice given Tx starting within 10 days of age. By contrast, neonatal treatments of mice with other antiestrogens, clomiphene and nafoxidine (100 micrograms/day), induced neither permanent chondrification in the pelvis nor expansion of the pubic ligament nor hernia. These findings suggest that Tx has a specific effect on the symphysis pubis and some junctional regions of the developing pelvis in mice when given neonatally.

Induction of estrogen receptor and cell division in genital tracts of male mice by neonatal exposure to diethylstilbestrol.

Estrogen receptor (ER) and ER mRNA expression and cell division induced by neonatal diethylstilbestrol (DES) exposure in male C57BL/Tw mouse genital organs were studied using immunohistochemistry, in situ hybridization, and cell counting at metaphase. A single injection of 3 micrograms DES on the day of birth induced ER in both epithelial (E) and stromal (S) cells of the epididymis and the seminal vesicles, in E cells of the ductus deferens, and in S cells of the ductus efferens and the anterior prostate 24 h after the injection. After 5 daily injections of DES, ER was induced in S cells of the ventral prostrate at 5 days of age. The staining intensity of ER increased in E cells of the epididymis and the ductus efferens at 5 days. A single injection of DES induced ER mRNA in both E and S cells of the ductus efferens, the caput epididymis, and the ductus deferens 4 to 12 h after injection of the newborn mice. A single injection of DES stimulated cell division of both E and S cells in the ductus deferens and the epididymis; cell division of S cells of the ductus efferens, the anterior prostate, and the urethra was stimulated 24 h after the injection. After 5 daily injections of DES, cell division was stimulated significantly in E and S cells of the epididymis, the ductus deferens, and the ventral prostate, and in S cells of the seminal vesicle at 5 days. At 30 days of age, mitotic rates were significantly higher in E cells of the seminal vesicles and the epididymides of mice given 5 daily injections of DES.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of postpubertal treatment with diethylstilbestrol and tamoxifen on protein expression in the vagina and uterus of neonatally diethylstilbestrol-exposed mice.

Postpubertal injections of a synthetic estrogen, diethylstilbestrol (-DES), and an anti-estrogen, tamoxifen (-Tx), stimulate proliferation of vaginal and uterine epithelial cells of ovariectomized (OVX) adult mice. In vaginae of two groups of OVX mice treated neonatally with DES (DES-) and oil vehicle alone (Oil-), postpubertal injections of DES altered all and 25 out of 37 protein expressions examined, except for keratin polypeptides, respectively. Twenty-one of the DES- and Tx-altered protein expressions showed the same behavior, suggesting that Tx acts as an estrogen agonist on these proteins. Both neonatally Oil- and DES-treated OVX mice given postpubertal injections of DES (Oil-DES and DES-DES) and Tx (Oil-Tx and DES-Tx) showed similar histological changes in the vagina. Epithelial proliferation and superficial cornification were observed in vaginae of five groups of Oil-DES, Oil-Tx, DES-DES, DES-Tx and DES-Oil (given oil postpubertally) OVX mice. In these groups, 13 proteins showed the same behavior, implying that these proteins are related to proliferation and cornification of mouse vagina. The histology of the uterus in DES-Oil OVX mice closely resembled that in Oil-Oil OVX mice; in protein expressions of the uterus, 5 proteins in DES-DES and 7 proteins in DES-Tx OVX mice were different from those in Oil-DES and Oil-Tx OVX mice, respectively. These findings indicate that neonatal DES treatment alters the behavior of the vagina and uterus in response to postpubertally administered DES and Tx.

Effects of postpubertal treatment with progesterone on protein expression in the vagina and uterus of mice exposed neonatally to diethylstilbestrol.

Postpubertal progesterone injections slightly inhibited the proliferation and cornification of the vaginal epithelium induced by neonatal injections of diethylstilbestrol (DES). In vaginae of neonatally DES-exposed mice (DES mice), 9 protein expressions (PEX) appeared newly; 13 PEX decreased; 5 PEX increased compared to those in the controls. In vaginae of DES mice, PEX were altered by postpubertal injections of progesterone (DES-P); 3 PEX disappeared; 11 PEX were reversed to those in the controls. Progesterone injections impaired the proliferation of the vaginal epithelium, reversing 11 PEX compared to those in DES mice. In uteri of DES-P mice, 3 PEX increased, 2 PEX decreased, 2 PEX appeared and 1 PEX disappeared compared to those in DES mice. In conclusion, it was shown that postpubertal injections of progesterone alter the protein expression in the vagina and uterus of DES mice.

Effects of neonatal exposure to diethylstilbestrol on protein expression by vagina and uterus in mice.

Neonatal treatment of female mice with diethylstilbestrol (DES) results in genital tract abnormalities including ovary-independent vaginal proliferation and cornification. Protein profiles were examined in vagina and uterus from 45-day-old, ovariectomized C57BL/Tw mice which had been given 5 daily injections of 2 micrograms DES or oil vehicle alone from the day of birth, and in those from 45-day-old, ovariectomized mice given 3 daily injections of 0.1 microgram DES from 42 days of age. Proteins extracted were analyzed by two-dimensional polyacrylamide gel electrophoresis. In the vagina, expression of 37 non-keratin proteins was altered by postpubertal injections of DES as compared with the controls. In the neonatally DES-exposed vagina, expression of 26 of 37 proteins was altered as compared with controls. In the uterus, expression of 22 proteins was altered in the postpubertally DES-exposed group as compared with that in the control; however, the protein expression pattern of the neonatally DES-exposed group closely resembled that of the control except for one protein (no. 23, pI = 6.1, MW = 39.5 kDa) which was specifically increased in the neonatally DES-exposed group. By immunoblot analysis, 6 keratin polypeptides (49.5, 50, 52, 53, 57 and 58 kDa) were identified in vaginae of ovariectomized mice exposed neonatally and postpubertally to DES and of the controls. These results indicate that neonatal DES exposure induces organ specific alterations in the synthesis of proteins in mouse vagina and uterus.

In vitro fertilization of oocytes from polyovular follicles in mouse ovaries exposed neonatally to diethylstilbestrol.

In 35-day-old female ICR/JCL mice given 5 daily injections of 1 microgram diethylstilbestrol (DES) from the day of birth, a significantly higher incidence of polyovular follicles was found in the ovaries than in those of age-matched control mice. Gap junctions of granulosa cells of mature follicles in neonatally DES-exposed mice were larger than those of the controls. When stimulated by gonadotropins (PMSG and hCG) and caged with males, the number of tubal embryos in DES-exposed mice was less, but the rate of fertilization and development was not different compared to the controls. Division of oocytes collected from the ovaries of 40-day-old DES-exposed and control mice after stimulation of gonadotropins was examined 24 to 72 h after in vitro insemination to ascertain whether fertilization had occurred in oocytes from polyovular follicles. Seventy-seven % of oocytes from uniovular follicles of control mice developed up to 8-cell stage embryos following in vitro insemination; 66% of those from similar follicles of DES-exposed mice developed into the same stage. By contrast, only 47% of oocytes from polyovular follicles of DES-exposed mice showed the division up to 8-cell stage 72 h after insemination, indicating a significantly lower fertilization rate compared to the oocytes from uniovular follicles of control and DES-exposed mice. Without insemination, oocytes taken from the same pools in these experiments never divided during the period of manipulation and incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistent changes in protein synthesis by vagina of ovariectomized mice exposed neonatally to diethylstilbestrol.

Neonatal treatment of female mice with a synthetic estrogen, diethylstilbestrol (DES), results in adenosis, ovary-independent epithelial proliferation and cornification, and downgrowths in the vagina. Protein synthesis was examined in the vagina of 45-day-old, ovariectomized C57BL/Tw mice which had been given 5 daily injections of 2 micrograms DES or the oil vehicle alone from the day of birth, and in those of age-matched, ovariectomized mice given 3 daily injections of 0.1 microgram DES from 42 days of age. [35S]Methionine-labeled proteins of the vagina were analyzed by two-dimensional polyacrylamide gel electrophoresis. Major changes were observed in 11 proteins of the vagina in both neonatally and postnatally DES-exposed groups. Six of the 11 proteins were increased in expression, but one of the remaining proteins was decreased. In the group of neonatally DES-exposed mice alone, expressions of 4 proteins (MW. 128, 90, 46 and 44 kDa) were markedly increased. These results indicate that neonatal exposure of mice to DES induced ovary-independent, persistent alteration in the protein synthesis of the vagina.

Estrogen receptor expression in the genital tract of female mice treated neonatally with diethylstilbestrol.

The distribution of the estrogen receptor (ER) was investigated immunohistochemically in the genital tract of 0 (day of birth)--to 50-day-old C57BL/Tw female mice given 5 daily injections of 2 micrograms diethylstilbestrol (DES) from the day of birth using an anti-ER monoclonal antibody. In the oviduct, cervix and vagina of control mice, both epithelial and stromal cell nuclei showed positive ER immunoreaction at 0 day. Nuclei of uterine stromal cells also showed strong ER immunostaining even at 0 day. In contrast, the ER in uterine epithelial cells began to appear by day 5. The number of positive epithelial cells and staining intensity gradually increased until 10 days. In neonatally DES-exposed mice, ER appeared in uterine epithelial cells 24 h after the injection. Polygonal cells appearing in the vaginal epithelium of DES-exposed 3-day-old mice showed weak ER staining. Neonatal DES exposure reduced staining intensity of columnar vaginal epithelial cells and uterine stromal cells at 3 days. Vaginal epithelial cells undergoing ovary-independent stratification and cornification in neonatally DES-exposed mice and normal estrous mice showed ER immunoreaction only in the basal cells.

Incidence of neuroblastoma in Sapporo city.

The incidence of neuroblastoma in Sapporo City for the period before the enforcement of the mass screening of neuroblastoma (1974 to 1980) was compared with the period after it (1981 to 1987). No large difference was found in incidence between them. In 1981 to 1987 the occurrence cluster at 2 to 4 years of age found in the prescreening period disappeared and an accumulation of patients at 0 year of age was noted. The sensitivity of the mass screening was almost stable (76.9% to 80.0%) throughout the 7 years. By the mass screening system of Sapporo City, early detection will be expected in 60% to 70% of all the neuroblastoma patients, the deficiency of which is due to the patients who neglected the mass screening.

Improvement in prognosis of neuroblastoma through mass population screening.

Since April 1981, the city of Sapporo, Japan, has conducted a mass screening program to measure urinary vanillylmandelic acid and homovanillylic acid using high-performance liquid chromatography. This program was expanded to the entire island of Hokkaido in October 1987. Mass screening proved beneficial, resulting in an increase in patients diagnosed with neuroblastoma under 1 year of age from 17% to 66%, an increase in stage I cases from 9% to 26%, an increase in stage III cases from 9% to 32%, and an increase in the tumor resectability rate from 15.1% to 58%. These improvements raised the 5-year survival rate for neuroblastoma from 23% to 66.7%. An additional study of these mass screening cases using Shimada's classification showed a wide range of histopathological distribution, and it demonstrated the usefulness of such a program in identifying the tumors most in need of early treatment.