Hormonal Replacement Therapy in an Animal Model with Chronic Renal Failure and Gonadectomy: Biochemical and Hematological Study

Abstract

The aim of the present study was to investigate the effects of subcutaneous administration of estradiol propionate (450 μg/kg female rat/day) or testosterone propionate (2 mg/kg male rat/day) for 4 weeks on some biochemical and hematological variables in intact and gonadectomized male and female rats with chronic renal failure (CRF) induced by 7/8 nephrectomy (remnant kidney model). Twenty-four hours after the last injection, rats were decapitated and blood samples were collected for complete hemogram and for measuring the concentrations of creatinine, urea, and indoxyl sulphate in plasma. Body weights of all rats were taken every week during the experimental period. The hematological and biochemical parameters measured in the sham–operated and gonadectomized rats were not significantly different from those in intact rats. Induction of CRF significantly increased the concentrations of creatinine, urea, and indoxyl sulphate by about 90–300% (P < 0.05), and caused signs indicative of anemia. These effects were significantly exacerbated in gonadectomized rats with CRF, and were partially and significantly reversed by exogenous administration of testosterone/estradiol. The changes induced by CRF and gonadectomy on the hematocrit (HTC) and hemoglobin concentration (HGB) were more pronounced in females than in males. The HTC and HGB in gonadectomized male rats with CRF were not significantly different from the controls. In the rest of the groups, there were no significant gender effects in the measured variables. It is suggested that, in the used rat model of CRF, there is depressed growth; significant increases in the plasma concentrations of creatinine, urea, and indoxyl sulphate; and anemia. All these signs were significantly and partially reversed by estradiol and testosterone therapy equally in female and male rats, respectively.

Keywords:

• rats
• chronic renal failure
• hemogram
• testosterone
• estradiol
• gonadectomy

INTRODUCTION

It is evident that chronic renal failure (CRF) produces several metabolic consequences, including many endocrine aberrations.[1–4] Uremia may alter endocrine function through its effect on the hypothalamo-pituitary axis, the individual end organs, and the peripheral metabolism of various hormones.[4–6] Deficiency of some hormones and excess of others coexist in experimental animals and patients with renal failure.[7–9] Reproductive hormonal replacement therapy has been extensively used in clinical and experimental studies without renal impairment. However, there are only a few studies concerning estrogen and androgen replacement in chronic renal failure.[[10]]

The purpose of this work was to ascertain the effect of subtotal nephrectomy on the concentrations of creatinine, urea, and indoxyl sulphate in plasma, and on the hematological profile in rats. Further, the aim was to find out if replacement therapy in intact and surgically-gonadectomized male and female rats would influence these parameters.

MATERIALS AND METHODS

Animals

A total of 96 young male and female locally bred Wistar rats weighing initially 150–200 g were obtained from the Animal House of King Saud University and housed at a temperature of 22 ± 2 °C, relative humidity of 50–60% and under 12–12 h dark–light cycle (lights on at 0700). The rats were maintained on normal rat pellets and tap water ad libitum. An acclimatization period of at least 3 days was allowed to the animals before any experimental work was undertaken. All experiments were conducted according to the NIH Guide for the Care and Use of Laboratory Animals, NIH publication no. 85–23, 1985. For each gender (n = 48), rats were randomly divided into eight equal groups as follows:

Group 1: intact control rats; Group 2: sham-operated rats; Group 3: CRF rats; Group 4: gonadectomized rats; Group 5: gonadectomized rats with CRF, subcutaneously (s/c) injected with corn oil (0.2 mL/rat) for 4 weeks; Group 6: CRF rats, given estradiol propionate (450 μg/kg/day) or testosterone propionate (2 mg/kg male rats/day) in corn oil (0.2 ml) for 4 weeks, s/c; Group 7: gonadectomized rats treated, as in group 6; and Group 8: gonadectomized rats with CRF and treated, as in group 6.

The doses of the two hormones were based on our previous work.[[11]] Rats were sacrificed 24 h after the last treatment by decapitation after light ether anesthesia. Blood was collected in heparinized tubes, and some of it was immediately used for hematological determinations, and the rest centrifuged at 900 X g at 5°C for 15 min to separate plasma. The plasma obtained was stored at −20°C to await biochemical analysis.

Induction of CRF

After anesthetizing the rats by intraperitoneal injection of ketamine (75 mg/kg) and xylazine (5 mg/kg), CRF was surgically induced by two-stage partial resection of the renal tissue (equivalent to 7/8 nephrectomy using ligation method) as previously reported.[[12]] Gonadectomy was performed surgically as previously described.[[13]] Both procedures were performed in the same surgical intervention. Prior to experimentation, animals were allowed to recover from the surgery for about 10 days.

Hematological and Biochemical Analysis

A complete hemogram was automatically conducted using a Cell-Dyne Hematology Analyzer (Abbot, USA). The concentrations of urea and creatinine were measured using commercial kits (bioMerieux, Marcy I'Etoile, France), and the concentration of indoxyl sulphate was measured as described previously.[[14]]

Statistical Analysis

Values reported are the mean ± SEM (number of observations). Differences between the mean of the groups were estimated by one-way analysis of variance followed by Tukey-Kramer multiple comparison tests. P < 0.05 was considered significant.

RESULTS

Weekly body weight measurements of the eight groups (for each sex) indicated that all the groups, except those subjected to partial nephrectomy, had a progressive increase in body weight amounting to about 19.6–23.7% at the end of the 4 weeks of treatments. The body weights of the rats subjected to partial nephrectomy grew much less than the other groups (0.7–1.8%). Rats with CRF and treated with testosterone or estradiol grew by about 5.6–8.5%. (Data not shown.)

The biochemical results of this work are shown in Tables 1 and 2. The parameters measured in the sham–operated and the gonadectomized rats were not significantly different from those in intact rats (P > 0.1). Induction of CRF induced a significant increase in the concentrations of creatinine, urea, and indoxyl sulphate amounting to about 90–300% (P < 0.05). These effects were exacerbated in gonadectomized rats with CRF (P < 0.05). There were no significant gender effects in the measured variables (data in Tables 1 and 2). Replacement therapy with testosterone (males) or estradiol (females) was effective in partially reversing the deleterious effects of CRF and gonadectomy on the studied indices of renal function.

Table 1 Effect of chronic renal failure (CRF), gonadectomy, and testosterone treatment on plasma creatinine, urea, and indoxyl sulphate concentrations in male rats

Group	Treatment	Creatinine (μmol/L)	Urea (mmol/L)	Indoxyl sulphate (mg/L)
1	Intact, control	36.7 ± 3.4^a	5.56 ± 0.46^a	0.21 ± 0.01^a
2	Sham-operated	36.3 ± 3.2^a	5.47 ± 0.49^a	0.25 ± 0.02^a
3	CRF	96.9 ± 8.8^b	22.45 ± 3.33^b	0.40 ± 0.03^b
4	Gonadectomy	38.3 ± 3.6^a	5.71 ± 0.56^a	0.22 ± 0.02^a
5	CRF + gonadectomy	119.6 ± 10.8^c	35.85 ± 3.92^c	0.49 ± 0.04^c
6	CRF + testosterone	84.5 ± 9.8^b	25.75 ± 3.08^b	0.31 ± 0.03^d
7	Gonadectomy + testosterone	37.3 ± 3.5	5.70 ± 0.48^a	0.23 ± 0.01^a
8	Gonadectomy + CRF + testosterone	89.8 ± 9.08^b	23.45 ± 3.6^b	0.33 ± 0.03^d

The values in the table are means ± SEM (n = 6 rats).

For each variable, values with different superscripts are significantly different (P < 0.05).

*Testosterone propionate was given at a dose of 2 mg/kg male rats/day in corn oil (0.2 mL) for 4 weeks, s/c. Measurements were taken 24 h after the last injection.

Table 2 Effect of chronic renal failure (CRF), gonadectomy, and estradiol treatment on plasma creatinine, urea, and indoxyl sulphate concentrations in female rats

Group	Treatment	Creatinine (μmol/L)	Urea (mmol/L)	Indoxyl sulphate (mg/L)
1	Intact, control	35.3 ± 2.6	5.86 ± 0.56	0.23 ± 0.02^a
2	Sham-operated	34.0 ± 2.5	5.78 ± 0.46	0.20 ± 0.02^a
3	CRF	97.8 ± 8.6^b	22.45 ± 3.66^b	0.42 ± 0.04^b
4	Gonadectomy	36.9 ± 3.1	5.71 ± 0.48	0.21 ± 0.01^a
5	CRF + gonadectomy	116.7 ± 11.1^c	36.65 ± 3.79^c	0.47 ± 0.04^c
6	CRF + estradiol	83.4 ± 8.5^b	25.75 ± 3.08^b	0.31 ± 0.03^d
7	Gonadectomy + estradiol	38.1 ± 3.7	5.81 ± 0.46^a	0.24 ± 0.01^a
8	Gonadectomy + CR + estradiol	85.8 ± 8.0^b	26.23 ± 2.9^b	0.34 ± 0.03^d

The values in the table are means ± SEM (n = 6 rats).

For each variable, values with different superscripts are significantly different (P < 0.05).

*Estradiol was given at a dose of 450 μg/kg/day in corn oil (0.2 mL) for 4 weeks, s/c. Measurements were taken 24 h after the last injection.

As shown in Tables 3 and 4, CRF induced significant decreases in the Ht, Hg, and RBCs in both species. The changes induced by CRF and gonadectomy on the hematocrit (Ht) and hemoglobin concentration (Hg) were more pronounced in females than in males. The Ht and Hg in gonadectomized male rats with CRF were not significantly different from the controls. In the rest of the groups, there were no significant gender effects in the measured variables. Replacement therapy with testosterone (males) or estradiol (females) was partially effective in reversing the deleterious effects of CRF and gonadectomy on the hematological indices studied.

Table 3 Effect of chronic renal failure (CRF), gonadectomy, and testosterone treatment on the hemogram of male rats

Group	Treatment	HGB (g/dL)	RBCs (×10^6μL)	HCT (%)
1	Intact, control	14.89 ± 1.9^a	7.15 ± 0.37^a	41.69 ± 4.51^a
2	Sham-operated	14.36 ± 1.4^a	7.02 ± 0.49^a	40.20 ± 4.01^a
3	CRF	10.32 ± 1.9^b	5.18 ± 0.60^b	29.79 ± 4.13^b
4	Gonadectomy	14.89 ± 1.8^a	7.15 ± 0.37^a	41.22 ± 5.01^a
5	CRF + Gonadectomy	08.99 ± 1.9^c	5.04 ± 0.57^c	23.20 ± 2.11^c
6	CRF + testosterone	12.56 ± 1.79^b	5.98 ± 0.48^c	29.29 ± 3.30^c
7	Gonadectomy + testosterone	14.89 ± 1.9^a	7.15 ± 0.37^a	42.31 ± 4.57^a
8	Gonadectomy + CRF + testosterone	14.00 ± 2.4^a	7.02 ± 0.57^a	40.24 ± 5.81^a

The values in the table are means ± SEM (n = 6 rats).

HGB, hemoglobin concentration; RBCs, erythrocyte count; HCT, hematocrit.

For each variable, values with different superscripts are significantly different (P < 0.05).

*Testosterone propionate was given at a dose of 2 mg/kg male rats/day in corn oil (0.2 mL) for 4 weeks, s/c. Measurements were taken 24 h after the last injection.

Table 4 Effect of chronic renal failure (CRF), gonadectomy, and estradiol treatment on the hemogram of female rats

Group	Treatment	HGB (g/dL)	RBCs (×10^6μL)	HCT (%)
1	Intact, control	12.89 ± 1.4^a	6.04 ± 0.49^a	37.69 ± 4.51^a
2	Sham-operated	12.02 ± 1.6^a	6.32 ± 0.51^a	36.27 ± 4.01^a
3	CRF	09.62 ± 1.9^b	4.78 ± 0.50^b	24.71 ± 3.813^b
4	Gonadectomy	12.69 ± 1.7^a	6.19 ± 0.41^a	35.77 ± 3.91^a
5	CRF + gonadectomy	07.82 ± 1.4^b	4.79 ± 0.52^b	20.20 ± 2.11^b
6	CRF + estradiol	09.46 ± 1. 7^b	5.83 ± 0.45^c	29.32 ± 3.30^c
7	Gonadectomy + estradiol	12.04 ± 1.7^a	7.15 ± 0.37^a	35.71 ± 4.21^a
8	Gonadectomy + CR + estradiol	09.04 ± 1.4^b	7.02 ± 0.57^a	34.26 ± 4.31^a

The values in the table are means ± SEM (n = 6 rats).

HGB, hemoglobin concentration; RBCs, erythrocyte count; HCT, hematocrit.

For each variable, values with different superscripts are significantly different (P < 0.05).

*Estradiol was given at a dose of 450 μg/kg/day in corn oil (0.2 mL) for 4 weeks, s/c. Measurements were taken 24 h after the last injection.

DISCUSSION

In the present work we measured the concentrations of creatinine and urea, which are established “classical” variables for renal function. Moreover, we also measured indoxyl sulphate, a harmful uremic toxin that accumulates during chronic renal failure, and is a useful indicator of the progression of CRF in humans and animals.[[15]] The present work showed that gonadectomy (which causes production defects in male and female sex hormones) exacerbated the biochemical and hematological adverse effects of experimental CRF on renal function in rats, emphasizing the involvement of the hypothalamo-hypophyseal-gonadal axis in CRF. These effects were partially and significantly reversed by exogenous administration of testosterone/estradiol for 4 consecutive weeks.

In this work, the effects of the partial nephrectomy were similar in the two sexes, and the effects of estradiol and testosterone in reversing these effects were also not markedly different, although the changes induced by CRF and gonadectomy on the HTC and HGB were more pronounced in females than in males. The HTC and HGB in gonadectomized male, but not female rats, with CRF were not significantly different from the controls. This is different from the reports that suggest female sex hormones do not protect against renal disease progression, and that male sex hormones lead to acceleration of CRF.[[16]] Progression of renal disease has been reported by some authors to be faster in males, and others have suggested that estrogens are responsible for slowing renal disease progression in females.[[16]], [[17]] The reasons behind these differences are not certain.

Clinically, it is observed that women with end-stage renal failure frequently exhibit disturbances of ovarian function,[[1]] an effect that can be reversed by dialysis or renal transplant. In addition to hormonal replacement therapy, the sexual impairments in uremic women have been treated by non-hormonal interventions that include dialysis and raising hemoglobin concentration by erythropoietin.[[1]] In males, CRF is known to impair spermatogenesis, steroidogenesis, fertility, and sexual function through effects at all levels of the hypothalamic–pituitary-testicular axis.[[18]] Disturbances in several hormones (e.g., testosterone and prolactin) are common in uremic men. Management of uremic men with impaired renal function includes transplantation or dialysis, and treatment with erythropoietin, zinc, and vitamin D. Although there are currently no proven indications of androgen therapy in CRF, this type of therapy can be useful in renal anemia.

The present finding suggests that in the used rat model of CRF there is depressed growth; significant increases in the plasma concentrations of creatinine, urea, and indoxyl sulphate; and anemia. All these signs were partially and significantly equally reversed by estradiol and testosterone therapy in gonadectomized female and male rats, respectively. The lack of a clear beneficial effect of hormonal treatment in intact uremic rats may be explained by the early stage of renal failure (4 weeks after surgical nephrectomy), at a time when hormone levels are not yet significantly altered. The use of rats at end-stage CRF might have been more appropriate, and will be considered in future work.

ACKNOWLEDGMENT

We thank King Saud University, Al Gaseem branch, for providing the animals and facilities to conduct this research.