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Regular Article |
a Biology Department, Facultad de Química, Universidad Nacional Autónoma de México, México D.F., Mexico
b Reproductive Biology Department, Hospital Juárez de México, Mexico City, Mexico
ABSTRACT
Chronic aminonucleoside nephrosis in rats is an experimental analogue of human focal segmental glomerulosclerosis. This study was undertaken to define the effects of chronic nephrosis on the pituitary-ovarian axis and on fertility. Chronic nephrosis was induced by puromycin aminonucleoside and followed for 112 days. The estrous cycle was evaluated daily in all rats, whereas biochemical parameters, hormonal concentrations, and fertility were measured on Days 7, 14, 28, 56, 84, and 112 (n = 8). Animals were divided in four experimental groups: A, B, C, and D. Group A was used to determine LH, FSH, progesterone, and estradiol concentrations. Group B was used to evaluate fertility, and groups C and D were added to clarify the role of male rats in the fertility of nephrotic female rats. The results showed a persistent proteinuria in nephrotic rats; the estrous cycle of nephrotic animals was disrupted. The LH and estradiol concentrations were significantly low at all time points evaluated, whereas no significant changes were noted in FSH or progesterone values. In addition, fertility and litter size were diminished in nephrotic female rats. Interestingly, the presence of a male rat or its urine resulted in a positive influence on serum estradiol concentrations of nephrotic female rats. These data indicate that experimental chronic nephrosis results in a pituitary-ovarian dysfunction that is characterized by low LH concentration, hypoestrogenism, failure of the hormonal feedback control, and diminution of fertility. In addition, they show the positive effect of a male rat on the fertility of a nephrotic female, which strongly suggests the participation of pheromones.
fertilization, hormone action, LH, ovulatory cycle, pheromones
INTRODUCTION
Chronic nephrotic syndrome as a focal and segmental glomerular sclerosis is the histologic description for a form of glomerular injury that is associated with proteinuria and progressive loss of renal function [1]. This disease is found in approximately 15% to 20% of adults with idiopathic nephrotic syndrome, which predominantly affects males [2, 3]. The focal glomerular sclerosing lesions follow a clinical course that is characterized by exacerbations and remissions that progress to renal failure [2]. Although the pathogenesis of this disease remains unclear [4], immunologic, hemodynamic, genetic, racial, viral, and metabolic factors may be involved in its development [5].
Renal disorders are commonly associated with endocrine dysfunction [6, 7]; however, little is known about the endocrine status of the nephrotic syndrome. To our knowledge, the effect of heavy proteinuria on the female reproductive process in particular has not yet been evaluated. We have previously studied and reported the existence of endocrine alterations in the experimental acute stage of nephrotic syndrome, which was specifically characterized by low circulating values of LH, undetectable levels of estradiol, loss of estrous cycle, and presence of follicular atresia and infertility [8, 9]. Accordingly, in the present study, we focus our attention on evaluation of the reproductive function during the chronic stage of the illness. Knowledge of the complete spectrum of endocrine disorders in addition to that of the metabolic alterations led by the nephrotic syndrome will support the evaluation and establishment of new therapeutic treatments of patients with nephrosis.
MATERIALS AND METHODS
Reagents
Puromycin aminonucleoside (PAN) was obtained from Sigma Chemical Co. (St. Louis, MO). Reagents for RIA of LH and FSH were kindly provided by the National Hormone and Pituitary Program (National Institutes of Health [NIH], Baltimore, MD). The RIA kits for progesterone and estradiol were purchased from Diagnostic Products Corporation (Los Angeles, CA). All other chemicals were of reagent grade and available commercially.
Animals
Adult female Wistar rats (age = 2 mo) weighing 220 to 250 g at the start of the experiments were used. Only those animals showing 4-day estrous cycles were included. Animals were fed with Lab Diet brand animal food containing not less than 23% crude protein from PMI Feeds (St. Louis, MO). Water and food pellets were available ad libitum. Intact fertile male rats were also included to evaluate the fertility of nephrotic female rats.
Monitoring the Estrous Cycle
The ovarian cycle was monitored throughout the study based on vaginal smear patterns, considering that ovulation occurs approximately every 4 days with proestrus, estrus, diestrus 1, and diestrus 2 phases and classifying the stages accordingly to the criteria described by Fox [10] and Freeman [11].
Experimental Design
Chronic nephrosis was induced by the administration of three s.c. doses of PAN at 7.5, 5, and 5 mg per 100 g body weight on Days 0, 21, and 35, respectively. Control rats received a sham injection of saline [9]. Four experiments in duplicate were conducted as follows: In experiment A, 12 groups of animals, divided into six nephrotic and six control groups, were used. The estrous cycle was evaluated daily for each rat, and on Days 7, 14, 28, 56, 84, and 112, one nephrotic and one control group of rats (n = 8) were killed by decapitation at 0800 h to collect the blood and 24-h urine samples. In experiment B, as in experiment A, another set of 12 groups of female rats were recruited, and the estrous cycle was followed for 112 days of study. On the days as specified previously, fertility was evaluated in each nephrotic or control rat. In experiment C, eight groups of female rats were used to evaluate the effect of male rats on the circulating estradiol concentration and on the fertility of female nephrotic rats on Days 7 and 14. Thus, on Day 7 or 14, four groups of female rats were divided into two nephrotic and two control groups. All rats were kept in individual cages, and one of each group was selected to be with or without a fertile male during a 4-day period. Serum samples were collected daily at 0800 h from the caudal vein until female rats were mated by the fertile male rat. In experiment D, in the same way, another eight groups of female rats were used to evaluate the effect of urine from a fertile male rat on the circulating estradiol concentration and on the fertility of female nephrotic rats. On Days 7 and 14, the daily serum estradiol concentration was measured during a 4-day period, in which each nephrotic or control female rat was separated in individual cages and exposed to the fresh 24-h urine samples, which were obtained daily from a fertile male rat. Blood samples were collected at 0800 h from the caudal vein.
Fertility Evaluation
Fertility as evaluated in experiment B was based on the rate of pregnancies on Days 7, 14, 28, 56, 84, and 112. Different groups of female rats were recruited on each testing day. All nephrotic or control animals were kept in individual cages for mating with one fertile male rat for a 4-day period, as described previously [9, 12]. If mating and fertilization occurred, the number of pregnant rats as well as the weight, sex, and litter size were registered. The same procedure was used in experiment C to evaluate fertility on Days 7 and 14.
Hormonal Assay
Serum LH and FSH levels were measured with specific RIA by using the double-antibody technique with reagents and protocol supplied by the NIH. The intra- and interassay coefficients of variations were 5.1% and 6.5% for LH and 4% and 7.9% for FSH, respectively. The results are expressed as nanograms per milliliter of international reference standards NIH LH-RP-3 and FSH-RP-2. Steroid hormones, progesterone, and estradiol were also measured by specific RIA. The intra- and interassay coefficients of variation were 2% and 5.4% for progesterone and 6.1% and 7.4% for estradiol, respectively.
Analytical Determinations
Total protein in serum and urine as well as cholesterol and creatinine in serum were measured by spectrophotometric methods according to the method described by Pedraza-Chaverrí et al. [13].
Statistical Analysis
Results of biochemical determinations and fertility evaluations are expressed as the mean ± SD (Tables 1 and 2), and two-way ANOVA was used to evaluate significance. Hormonal results are expressed as the mean ± SEM. Data in Figure 2 were analyzed by one-way ANOVA with the Dunnett post test, and data in Figures 3 and 4 were evaluated by two-way ANOVA. A mean of all samples for each female rat was used in the statistical evaluation; P < 0.05 was considered to be significant. Statistical analysis was performed using Analyzing Data with Graph Pad Prism (version 3; Graph Pad Software Inc., San Diego, CA).
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RESULTS
Table 1 shows the laboratory evaluation of the nephrotic syndrome. Values for 24-h urine protein (P < 0.0001) and serum cholesterol (P < 0.0001) were markedly elevated in nephrotic rats on all days studied, whereas values for serum proteins were significantly depressed (P < 0.002), even though they tended to reach the intact female values. Creatinine concentrations were no greater than 88.4 µmol/L for each nephrotic rat and not different from that of its respective control group (P = not significant).
Evaluation of the estrous cycle, based on the daily picture of vaginal smears, revealed rapid loss of the normal pattern in nephrotic female rats. Figure 1 shows the results as percentages of diestrous, proestrous, and estrous stages and represents the mean of three different experiments. As shown, the estrous cycle was disrupted in the nephrotic groups from Days 3 to 53, when a pattern of abundant leukocytes indicated predominance of the diestrous stage. On the remaining days, the estrous cycle tended to normalize in nephrotic female rats, but the exfoliate cytology of the vaginal lumen showed an abnormal cell composition. Thus, during the proestrus period, a mixture of leukocytes and cornified cells were observed in addition to nucleated epithelial cells; likewise, during the estrous period, not only cornified cells were registered but also many leukocytes and nucleated epithelial cells.
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The hormonal status of nephrotic rats is summarized in Figure 2. Results for each hormone were divided into the three stages of the estrous cycle (diestrous, proestrous, and estrous), and data for nephrotic female rats from each stage at all testing days were compared with those obtained from all sham-injected rats (controls) at the diestrous, proestrous, or estrous stage respectively. No important changes were noted for FSH and progesterone (P > 0.05). The main disturbance of LH concentration was observed during the proestrous stage, because at all testing days, nephrotic female rats showed a significantly reduced LH concentration compared with the control proestrous value (P < 0.05 for proestrous). Regarding the estradiol concentration, more profound damage was perceived at all stages. In fact, estradiol concentrations were significantly decreased compared with the mean of control values at the diestrous or proestrous stage (P < 0.01 for diestrous and < 0.05 for proestrous).
Results of the fertility evaluation are shown in Table 2. Compared with the saline control group, nephrotic female rats had a sustained diminution of reproductive capacity in terms of the percentage of fertility. At all time points, only 60% of nephrotic rats were fertile, compared with 100% of control rats (P < 0.0021). Regarding the characteristics of the litters, the number of offspring per litter decreased in nephrotic female rats compared with control female rats (P < 0.0074). The remaining parameters did not differ in either nephrotic or control rats.
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Figure 3 shows the effect of a fertile male rat on the female circulating estradiol concentration. Starting on Day 7, when 100% of nephrotic rats had presented a persistent diestrous stage (>3 determinations), data indicate that during the following 4-day period, nephrotic females increased significantly their estradiol concentration only when they were accompanied by a fertile male rat. Statistical analysis showed a significant effect of group (nephrotics or controls; P < 0.0165) and a significant effect of a male rat (P < 0.0195) on the estradiol concentration of nephrotic female rats. Estradiol concentrations from nephrotic animals alone were undetectable on Day 7 as well as after 4 days. Similar data were obtained on Day 14 (estradiol levels of nephrotic rats + male, Day 14 = 1 ± 1 pg/ml vs. + 4-day estrous cycle = 21 ± 2 pg/ml, P < 0.05). Data from control animals with and without a male rat during a 4-day period did not significantly differ, either from each other or from control values on Days 7 and 14. Results of the fertility evaluation of these animals were equivalent to those showed in Table 2: Fertility decreased significantly in nephrotic female rats, and the fertility rate was 60% and 67% from nephrotic animals and 92% and 92% from controls on Days 7 and 14, respectively (P < 0.05).
The effect of a 24-h urine sample from a fertile male rat on the circulating estradiol concentration is shown in Figure 4. Initially, on Day 7, 100% of female nephrotic rats presented the diestrous stage at more than three consecutive determinations. After a 4-day estrous cycle period, control animals maintained their estradiol concentration, whereas nephrotic animals increased their estradiol values significantly. Statistical analysis revealed a significant effect of both groups (P < 0.0001) and male urine (P < 0.00151) on the estradiol concentration in nephrotic female rats.
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DISCUSSION
The results of this study reveal an endocrine dysfunction during the chronic stage of the nephrotic syndrome. The main abnormalities observed were an alteration of the estrous cycle, low LH concentrations and hypoestrogenism, and diminution of fertility. In addition, results of this study show the positive role of fertile male rats on the female reproductive capacity in chronic nephrosis.
In this study, proteinuria, which is the hallmark of nephrotic stage, and hypercholesterolemia were the characteristics that mimicked the clinical condition of the nephrotic syndrome [14–16]. Transient hypoproteinemia, observed in the female rat with chronic nephrosis, has been previously described [12, 17] and explained as an hepatic response to compensate for the metabolic imbalance produced by the protein losses. In addition, uremia was excluded based on the serum creatinine concentration.
Data from the hormone measurements reflect the existence of pituitary-ovarian abnormalities associated with the chronic nephrosis. The main characteristics of the hormonal pattern at all time points were permanently decreased LH and estradiol concentrations. These results suggest both pituitary damage with a more harmful effect on LH secretion and an ovarian impairment, because the estradiol concentration was greatly reduced. The association between chronic renal failure and alterations on the hypothalamo-pituitary-gonadal axis in humans has already been established by several authors [18–20], who have shown that circulating LH, FSH, and estradiol levels are elevated in patients with uremia [21]. On the other hand, our data show the coexistence of reduced gonadotropin concentrations with low gonadal steroids, particularly extremely low estradiol values. These observations show the hypothalamic-pituitary-gonadal axis dysfunction to be a common feature associated with both renal diseases but with a typical expression in the nephrotic syndrome, in which, in addition to the hormonal withdrawal, a total failure of the feedback-axis control mechanism was also recognized. The reduced estradiol concentration is quite interesting, because as in the acute stage of experimental nephrotic syndrome of male and female rats [9] and in the chronic stage of nephrosis in male rats [11] as well as during the recently evaluated nephrosis in male patients [22], estradiol was undetectable or rather low in female rats with chronic nephrosis. Thus, hypoestrogenism might be an additional hallmark of the nephrotic stage. If we consider that a usually nephrotic condition leads the patient to chronic renal failure, which is characterized by high estradiol concentrations [23], the data presented here could be interpreted as showing that the hypoestrogenism is a transitory or still recovery stage before renal failure. This hypothesis is also supported by the results of Gades et al. [24] and Joles et al. [25], who showed that in the hypertriglyceridemic condition, as in obese Zucker rats, estrogens hasten the development of renal disease.
One unexpected finding of this study, however, was the transient disruption of the ovarian cycle. Between Days 3 and 53, nephrotic animals presented anovulatory cycles, recovering later on their cyclical behavior. This suggests damage at the pituitary-ovarian level, because control of the ovarian cycle results from a complex feedback mechanism that mainly involves gonadotropin hormones from the pituitary gland and steroids and proteins secreted by the ovaries [26]. In addition, considering the strict correlation between the estrous cycle as an ovulatory process and fertility, the data presented here are paradoxical, because from group A, nephrotic animals lost, in transition, the estrous cycle. In group B, even though the reproductive capacity was reduced, the fertility rate of nephrotic female rats was almost the same on all days (
60%). As the only difference between these two groups was the presence of an intact, fertile male rat with each female nephrotic rat in group B, we traced the fertility maintenance caused by the male pheromones. The existence of pheromones as chemical signals that are released by an individual into the environment and that affect the physiology or behavior of other members of the same species is already accepted [27]. Above all, the clear effect of pheromones from male and female rats on the hypothalamic-pituitary-gonadal axis has indicated the existence of a functional vomeronasal organ-hypothalamic-pituitary-gonadal axis [28]. To support the hypothesis of pheromonal action on the fertility of nephrotic female rats, our results (Fig. 3) show that the fertile male rat could reinstall the cyclicity of the estrous cycle in the nephrotic female rats, which consequently presented the proestrous stage, increased the estradiol concentrations, and became pregnant. In addition, results of another experiment (Fig. 4) revealed that the factor eliciting ovulation in nephrotic animals was probably pheromonal in nature, because female nephrotic animals exposed to urine obtained from an adult male rat started the regular cyclicity and increased their estradiol concentrations. These data are in agreement with the information showing that pheromones, which are present in urine samples, potentially affect behavior and sexual response [29, 30]. The role and efficacy of pheromones to restore the reproductive function in patients with renal diseases represent an interesting possibility in clinical treatment and deserve more profound study.
Contrary to what occurs in male rats during chronic nephrosis, in which reproductive capacity is initially greatly affected but totally restored later [11], the fertility of nephrotic female rats was equally depressed at all time points. This can be explained by gender influence. In this respect, deterioration of renal function in patients with chronic renal disease is more rapid in men than in women [2, 31].
Results from several recent studies have shown the harmful effect of nephrotic syndrome on the reproductive process, but not on the litter characteristics [8, 9, 12]. Thus, the significant decrease of litter size observed in female rats with chronic nephrosis was unexpected, and it suggests ovarian damage that could be more profound than that found during the acute stage of experimental nephrotic syndrome [8]. Finally, the results from this investigation show that chronic nephrosis has an adverse effect on the hypothalamic-pituitary-ovarian axis, along with a marked drop in female rat reproductive capacity.
ACKNOWLEDGMENTS
The authors thank the National Hormone and Pituitary Program for providing the rat LH and FSH biological materials for assay performance.
FOOTNOTES
First decision: 17 April 2000.
1 Supported by a grant from CONACYT N°25126M, Mexico City, Mexico. 
2 Correspondence: Marta Menjívar, Facultad de Química, Depto. Biología, Edif. "B", 2°piso, Lab. 209, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, México D.F., Mexico. FAX: 52 56 22 35 15; menjivar{at}servidor.unam.mx
Accepted: July 6, 2000.
Received: March 23, 2000.
REFERENCES
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  N. P.Y. Lee and C. Y. Cheng Adaptors, Junction Dynamics, and Spermatogenesis Biol Reprod, August 1, 2004; 71(2): 392 - 404. [Abstract] [Full Text] [PDF]
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 P. Dan, J. C. Y. Cheung, D. R. L. Scriven, and E. D. W. Moore Epitope-dependent localization of estrogen receptoralpha , but not -beta , in en face arterial endothelium Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1295 - H1306. [Abstract] [Full Text] [PDF]
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