Effects of progesterone and selective oestrogen receptor modulators on chronic allograft nephropathy in rats

Balazs Antus1,2, Shanying Liu1, Yousheng Yao1, Hequn Zou1, Erwei Song1, Jens Lutz1 and Uwe Heemann1

1 Department of Nephrology, Klinikum rechts der Isar, D-81576 Munich, Germany and 2 Semmelweis University, Department of Pathophysiology, H-1089 Budapest, Hungary

Correspondence and offprint requests to: Balazs Antus MD, PhD, Semmelweis University, Department of Pathophysiology, Nagyvarad ter 4, H-1089 Budapest, Hungary. Email: antbal{at}net.sote.hu



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. We recently demonstrated that oestrogens ameliorate the progression of chronic allograft nephropathy (CAN). In our present study, we investigated the role of progesterone and selective oestrogen receptor modulators (SERMs) in this process.

Methods. Female Fisher (F344) kidneys were orthotopically transplanted into intact or ovariectomized female Lewis recipients. Ovariectomized recipients were divided into four groups and were treated with either progesterone alone or in combination with oestradiol, oestradiol alone or vehicle. Intact recipients were divided into three groups and were treated with SERMs such as tamoxifen and one of its new derivatives, droloxifene or vehicle. Animals were harvested 24 weeks after transplantation for histological and immunohistological studies as well as for molecular analysis.

Results. Administration of progesterone resulted in increased urinary protein excretion as well as profound glomerulosclerosis and mononuclear cell infiltration. The combined treatment had similar detrimental effects on the development of CAN. In contrast, oestradiol treatment alone improved graft function, reduced glomerulosclerosis and diminished cellular infiltration. SERMs again impaired allograft function and promoted the development of CAN. Renal allograft damage paralleled intragraft mRNA expression of transforming growth factor-ß1 in all groups.

Conclusions. Our results suggest that addition of progesterone diminishes the beneficial effects of oestrogens on the development of CAN in rats. Similarly to progesterone, SERMs worsened long-term renal allograft outcome.

Keywords: chronic allograft nephropathy; growth factor; oestradiol; progesterone



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Despite improved immunosuppression, chronic allograft nephropathy (CAN) still remains the major obstacle for long-term success after kidney transplantation. It is characterized by a progressive deterioration of renal function associated with glomerulosclerosis, tubular atrophy, interstitial fibrosis and intimal thickening of graft arteries (graft vasculopathy) [1].

The aetiology of CAN is clearly multifactorial and has been related to both alloantigen-dependent and -independent factors. Among alloantigen-independent factors, sex hormones may be of importance. In favour of this concept, we previously demonstrated that oestrogens ameliorate allograft injury both in male [2] and female recipients [3].

Whether progesterone, the other major female sex steroid, influences the development of CAN is unclear. Similarly, little information is available as to whether the addition of progesterone to oestrogens influences the effects of oestrogens on allograft injury.

Several lines of evidence indicate that progesterone is an effective immunosuppressive agent [4]. In vitro assays have established that progesterone inhibits lymphocyte proliferation [5], and suppresses the production of various pro-inflammatory cytokines and chemokines in monocytes [6]. The immunosuppressive effects of progesterone were also demonstrated in animal studies. For example, it has been shown that progesterone delays the rejection of xenogenic cells transplanted into the uterus of sheep [7], and prolongs survival of xenografts near silastic implants containing high concentrations of progesterone in rats [4]. Based on these data, we hypothesized that progesterone may suppress rejection of solid organ allografts such as kidney transplants.

Thus, in this study we investigated the role of progesterone in the development of CAN in the established Fisher-to-Lewis rat model [1]. Additionally, we tested whether co-administration of progesterone with oestrogens modulates the protective effects of oestrogens on this process. Finally, we were interested in the role of selective oestrogen receptor modulators (SERMs) such as tamoxifen and one of its new derivatives, droloxifene in the development of CAN. These compounds are known to bind to oestrogen receptors and elicit agonist or antagonist responses depending on the target tissue and hormonal milieu. Because transforming growth factor (TGF)-ß is generally believed to be one of the most important growth factors related to the pathogenesis of CAN, we also analysed levels of TGF-ß mRNA in the allograft tissues.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Animals and renal transplantation
Kidneys of female Fisher rats (F344, RT1v1) (180–220 g) (Charles River, Sulzfeld, Germany) were orthotopically transplanted into intact or ovariectomized female Lewis (Lew, RT1) (180–220 g) rats in all experimental groups, as previously described [2,3]. Animals were kept under standard conditions and were fed rat chow and water ad libitum. All animals were treated with low-dose cyclosporine A (1.5 mg/kg/day s.c.; Novartis GmbH, Nürnberg, Germany) from day 0 to day 10 [2,3], at which time the right recipient kidney was removed. All experiments were approved by a governmental committee on animal welfare.

Experimental design
Progesterone and oestradiol were administered into ovariectomized female recipients in order to exclude the interference with regularly secreted endogenous sex hormones. SERMs, on the other hand, were applied in intact female rats. Accordingly, animals were divided into the following seven experimental groups (n = 6/group): intact recipients were treated either with tamoxifen (T); droloxifene (D) or vehicle (IV), while ovariectomized recipients were treated either with progesterone (OV + P); progesterone + 17ß-oestradiol (OV + P + E); 17ß-oestradiol alone (OV + E) or vehicle (OV). Tamoxifen (3 mg/kg, Klinge Pharma, Munich, Germany), droloxifene (3 mg/kg, Klinge Pharma), progesterone (10 mg/kg, Sigma, Deisenhofen, Germany) and 17ß-oestradiol (20 mg/kg, Sigma) were dissolved in sesame oil and were administered subcutaneously following transplantation every second day (0.1 ml) until harvesting. Doses of progesterone and oestradiol were similar to our previous studies [2,8], while doses of SERMs were chosen on the basis of the literature [9,10]. Intact (IV) and ovariectomized (OV) control animals were given sesame oil alone.

To test the possible toxic effect of progesterone to the transplanted kidney, a pilot study was performed. A group of six ovariectomized female Lewis rats was transplanted with syngenic (Lewis) kidney grafts, and treated with cyclosporine A for 10 days and with the same dose of progesterone until harvesting for histology. In the pilot experiment, we observed that progesterone-treated rats did not become proteinuric (<10 mg/24 h) and the kidney grafts remained intact (glomerulosclerosis index <5%). Thus, progesterone has no direct toxic effect on the transplanted kidney and does not enhance the nephrotoxicity of cyclosporine A either.

After 24 weeks, rats were anaesthetized with ketamine and immediately thereafter the intra-aortic blood pressure was measured (Sirecust 404, Siemens, Germany). Animals were then bled, and the transplanted kidneys were removed. Samples were snap frozen in liquid nitrogen for immunohistology and ribonuclease protection assay or fixed in buffered formalin (4%) for light microscopical evaluation.

Functional measurements
Twenty-four hour urinary protein excretion was measured every 4 weeks, while serum and urinary creatinine levels as well as serum hormone concentrations were determined at the end of the study as previously described [2,3].

Histology
For histology, fixed kidney tissues were embedded in paraffin, and stained with haematoxylin/eosin or periodic acid-Schiff (PAS). Glomerulosclerosis was defined as the accumulation of extracellular matrix in the mesangium. At least 200 glomeruli were counted per kidney section, and the proportion of sclerosed to total glomeruli was expressed as a percentage (glomerulosclerosis index). CAN was graded according to the parameters adapted from the Banff 1997 classification: grade 0, no signs of CAN; grade 1, mild CAN with mild interstitial fibrosis and tubular atrophy (affecting 5–15% of the section); grade 2, moderate CAN with moderate interstitial fibrosis and tubular atrophy (affecting 15–50% of the section); and grade 3, severe CAN with severe interstitial fibrosis and tubular atrophy (affecting >50% of the section). Grading also included vasculopathy, with mild vasculopathy as defined by intimal proliferation with luminal obstruction <25%; moderate vasculopathy with luminal obstruction between 25 and 50%; and severe vasculopathy with luminal obstruction >50%. Two independent observers examined the slides by light microscopy in a blinded fashion.

Antibodies and immunohistology
For immunohistology, cryostat sections (4 mm) were fixed in acetone, air dried and stained individually with primary, monoclonal, mouse derived antibodies against monocytes/macrophages (ED1) and CD5+ T lymphocytes (OX19) (Serotec Labor-Service GmbH, Wiesbaden, Germany). After incubation with primary antibody, sections were incubated with rabbit anti-mouse IgG and thereafter with the alkaline phosphatase anti-alkaline phosphatase (APAAP) complex (DAKO A/S, Hamburg, Germany). Cells staining positive were counted and expressed as cells per field of view (cells/fv). At least 20 fields of view per section and per specimen were evaluated at 400x magnification.

Ribonuclease protection assay
Total RNA was extracted as previously described [2,3]. Intragraft mRNA expression specific for TGF-ß1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was determined by a ribonuclease protection assay (Riboquant, Pharmingen Becton Dickinson GmbH, Hamburg, Germany) according to the protocol provided by the manufacturer. Briefly, antisense RNA probes were prepared with the use of T7 RNA polymerase transcription in the presence of [{alpha}-32P]UTP (template set was provided by the manufacturer). Radiolabelled RNA probes were then extracted with Tris-saturated phenol and chloroform:isoamyl alcohol and precipitated with ethanol. The dried pellets were dissolved in 50 ml of hybridization buffer and the activity measured in a scintillation counter. Thereafter, 15 mg of total RNA from each samples was hybridized together with a specific radiolabelled antisense probe (activity >3 x 105 c.p.m.) at 56°C overnight. After purification and precipitation, protected fragments were separated by electrophoresis on a 5% polyacrylamide gel. Intensities of the specific bands were quantified by a PhosphorImaging analyser (Fuji-BAS 2000, Düsseldorf, Germany), and the ratios of the density of the investigated genes to GAPDH (internal control) were calculated.

Statistical analysis
Data are presented as mean±SEM. Parametric data were compared using one-way analysis of variance, followed by multiple pair-wise comparisons according to the Newman-Keuls test. Non-parametric data were tested using the Kruskal-Wallis one-way analysis of ranks. A P-value of <0.05 was considered significant.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Animals
At the beginning of the study, body weights were comparable between the groups (Table 1). At week 24, body weight was significantly higher in vehicle-treated ovariectomized animals (OV) as compared to vehicle-treated intact (IV) and sex hormone-treated ovariectomized animals (OV + P, OV + P + E, OV + E). In addition, we noted a decreased body weight gain in tamoxifen- (T) and droloxifene-treated (D) animals as compared to vehicle-treated intact rats.


View this table:
[in this window]
[in a new window]
 
Table 1. Body weights at the time of the transplantation and harvesting

 
Histology
Glomerulosclerosis. Administration of progesterone alone (OV + P) or in combination with oestradiol (OV + P + E) resulted in extensive glomerular sclerosis as compared to vehicle treatment (OV) in ovariectomized rats (Table 2). Similarly, glomerulosclerosis was significantly increased in tamoxifen- (T) and droloxifene-treated (D) animals as compared to their vehicle-treated counterparts (IV). In contrast, glomerulosclerosis was reduced in animals treated with oestradiol alone (OV + E). Ovariectomy did not significantly influence glomerulosclerosis; no differences in the degree of glomerulosclerosis were noted between OV- and IV-treated animals.


View this table:
[in this window]
[in a new window]
 
Table 2. Histological and immunohistological analysis of the transplanted kidneys

 
CAN. The grade of CAN was significantly increased in progesterone- and progesterone plus oestradiol-treated rats as compared to vehicle-treated ovariectomized animals (Table 2). Similarly, tamoxifen- and droloxifene-treated animals developed a higher degree of CAN than vehicle-treated intact rats (IV). In contrast, the grade of CAN was significantly ameliorated in oestradiol-treated animals as compared to controls (OV).

Immunohistology. Immunohistological analysis of graft tissues revealed mononuclear cell infiltration in all groups, localizing preferentially in perivascular and periglomerular areas (Table 2). In vehicle-treated ovariectomized animals (OV) slightly elevated numbers of CD5+ T lymphocytes as well as ED1-positive macrophages were observed as compared to vehicle-treated intact animals (IV). Administration of progesterone alone or in combination with oestradiol enhanced the number of infiltrating CD5+ T lymphocytes as well as ED1-positive macrophages, particularly around the vessels. Nevertheless, differences reached statistical significance only with progesterone treatment alone. SERMs increased mononuclear cell infiltration, most pronounced in droloxifene-treated rats. In contrast again, the number of T lymphocytes and macrophages was markedly reduced in animals treated with oestradiol alone.

Urinary protein excretion. Proteinuria was significantly increased in animals treated with progesterone alone (OV + P) or in combination with oestradiol (OV + P + E) (Figure 1a). Similarly, administration of tamoxifen (T) and droloxifene (D) enhanced proteinuria (Figure 1b). One animal with extremely high proteinuria even died due to renal failure among tamoxifen-treated rats. The level of protein excretion in oestradiol-treated animals (OV + E) remained almost at the same level as compared to vehicle-treated ovariectomized animals during the whole follow-up period. Ovariectomy did not significantly influence urinary protein excretion between vehicle-treated animals (IV, OV).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. (a) Changes in 24-h urinary protein excretion during the course of the experiment in ovariectomized female recipients. *P<0.05 group OV + P + E vs group OV; §P<0.05 group OV + P vs group OV. (b) Changes in 24-h urinary protein excretion during the course of the experiment in intact female recipients. *P<0.05 group T vs group IV; §P<0.05 group D vs group IV.

 
Ribonuclease protection assay. The intragraft mRNA expression of TGF-ß1 paralleled the development of CAN in all groups (Figure 2). Accordingly, TGF-ß1 mRNA levels were most pronounced following the administration of tamoxifen, droloxifene and progesterone. Similarly, the combined treatment increased TGF-ß1 levels. In contrast, TGF-ß1 mRNA was significantly reduced in oestradiol-treated rats.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2. TGF-ß1 mRNA expression in renal allografts 24 weeks after transplantation. *P<0.05 vs group IV; §P<0.05 vs group OV.

 
Functional measurements
Animals treated with anti-oestrogens, progesterone or progesterone plus oestradiol tended to have higher serum creatinine concentrations and a lower creatinine clearance as compared to vehicle-treated rats (Table 3). Serum creatinine and creatinine clearance did not differ between vehicle-treated intact and ovariectomized rats.


View this table:
[in this window]
[in a new window]
 
Table 3. Mean arterial blood pressure and serum constituents at the time of harvesting

 
With respect to sex hormones, oestradiol levels in animals treated with oestradiol alone (OV + E) or in combination with progesterone (OV + P + E) were higher than those in vehicle-treated intact animals (IV). In progesterone- (OV + P), tamoxifen- (T), droloxifene- (D) and vehicle-treated ovariectomized animals (OV) oestradiol levels remained below the limits of detection.

With respect to serum progesterone, we observed a trend towards decreased progesterone levels in vehicle-treated ovariectomized rats as compared to vehicle-treated intact ones. Animals treated with progesterone alone or in combination with oestradiol developed increased progesterone levels. Both tamoxifen and droloxifene decreased progesterone levels. However, these differences did not reach statistical significance.

Mean arterial blood pressure did not significantly differ between the groups, although there was a trend towards an increased blood pressure in animals treated with progesterone alone or in combination with oestradiol as compared to vehicle-treated animals (Table 3).



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Our results demonstrated that (i) progesterone treatment promoted the development of CAN, (ii) addition of progesterone to oestradiol treatment abolished the oestradiol-mediated protection of allograft injury, and (iii) treatment with SERMs such as tamoxifen and droloxifene impaired allograft function and exacerbated CAN in the established rat model.

The most surprising finding of the present study was that progesterone adversely influenced allograft injury. On one hand, it is generally believed that progesterone is an immunosuppressive hormone, and that it plays a major role in the regulation of immune homeostasis during pregnancy [4]. However, depending on the actual hormone concentrations, progesterone can have both suppressive and stimulatory effects on immune cells. In general, high doses inhibit lymphocyte activation and proliferation [5], suppress the production of pro-inflammatory cytokines such as IL-1 and TNF-{alpha} in monocytes [6] and favour the development of T-helper cells producing Th2-type cytokines including IL-4 [11]. In contrast, low physiological concentrations are stimulatory, for example, by increasing IL-1 and TNF-{alpha} mRNA expression or super oxide production in rat and human macrophages [12].

Doses of progesterone administered in our experiment are known to induce decidual reaction in ovariectomized female rats [13]. With these doses, however, we achieved much lower tissue concentrations of the hormone as compared to those during pregnancy or to those applied in cell culture experiments demonstrating immunosuppressive effects of progesterone. This may explain, at least in part, the stimulatory rather than the suppressive effect of progesterone on the development of CAN in this study.

Furthermore, a number of other mechanisms may also be involved in progesterone-mediated allograft injury. For example, progestins are known to exert partial androgenic effects weakly bindings to androgen receptors in various tissues including the kidney [14]. As androgens promote renal fibrosis [2,3], it is possible that activation of androgen receptors was responsible for the effects of progesterone. Furthermore, interactions between progesterone and oestrogen or progesterone and angiotensin type I receptor expression [15] could have been involved in the process. Finally, progesterone-treated animals tended to have a slightly elevated blood pressure that may have contributed to the more pronounced allograft injury in these animals. Nevertheless, allograft function was also impaired in tamoxifen- and droloxifene-treated rats despite normal blood pressure. Thus, factors other than hypertension seem to be more important in determining allograft function.

Oestradiol treatment attenuated renal allograft injury in this experiment, which confirms the results of our previously published study [2]. These data are in accordance with the general hypothesis that oestrogens are nephroprotective, and that they ameliorate transplant vasculopathy in a manner analogous to their protective effects on atherosclerosis [16].

Whether addition of progesterone to oestrogens antagonizes the nephroprotective effects of oestrogens in animal models of renal injury is a matter of debate. In the rat remnant kidney model we recently demonstrated that progesterone tended to reduce the beneficial effects of oestradiol [8]. In this study co-administration of progesterone completely abolished the protective effect of oestradiol on CAN.

Of note is the fact, that ovariectomy itself did not significantly influence the development of CAN in our experiment. The reason for this finding is not clear. However, both oestradiol and progesterone levels were decreased in ovariectomized rats. Thus, we speculate that the potential beneficial effects of lower progesterone levels on allograft injury were counteracted by the adverse effects of oestrogen deficiency on this process, and therefore no major impact on long-term allograft outcome could be observed.

In recent years, several synthetic compounds have been developed that display mixed oestrogen receptor agonist/antagonist profiles depending on the target tissues [17]. The non-steroid tamoxifen, for example, exerts profound anti-oestrogenic effects in reproductive tissues, and became therefore a widely accepted therapeutic approach in the treatment of women with advanced breast cancer. In some other tissues, however, tamoxifen exerts intrinsic activity on oestrogen receptors. Therefore, it protects against atherosclerosis and inhibits bone loss in postmenopausal women [18]. More recently, a new tamoxifen derivative, droloxifene, has been reported to possess higher anti-oestrogenic effects on reproductive tissues than tamoxifen [19]. Nevertheless, the bone-protective properties of droloxifene are probably similar to those of oestrogens, indicating again the intrinsic activity of the drug on some non-reproductive tissues [9].

More recently, it has also been demonstrated that tamoxifen suppresses collagen type IV synthesis in glomerular mesangial cells [20]. Therefore, it has been postulated that some SERMs might be useful in the treatment of chronic renal diseases. Our results, however, do not support this hypothesis, as both tamoxifen and droloxifene impaired allograft function and promoted the development of CAN.

As oestradiol and SERMs exerted opposite effects on allograft injury, it is reasonable to assume that SERMs elicited oestrogen receptor antagonist properties in the renal tissue and this was responsible for their effects. Alternatively, one could assume that SERMs plus oestrogen yielded too much oestrogenic action in the single kidney, which was already toxic. However, oestrogens are nephroprotective even in rats with reduced nephron mass (5/6 nephrectomized), as observed previously [8]. Thus, this theory is not likely.

Our finding that oestradiol levels are lower in SERM-treated animals than in controls is in line with the observations of several other studies [10,21]. This effect is thought to be a consequence of decreased FSH and LH levels caused by the agonistic effects of these drugs on the pituitary gland.

It is well established that dietary protein intake modifies the progression of renal disease. In our study, oestradiol-treated rats gained less body weight as compared to their vehicle-treated counterparts. Thus, one may argue that lower protein consumption was responsible for the beneficial effects of oestradiol on allograft injury. However, in our previous study, in which we used a pair-feeding protocol, we clearly showed that oestradiol-mediated renal protection is independent of food intake [8]. Therefore, differences in allograft injury between the groups in this study are also not likely to be due to various protein intakes.

There are several mechanisms that may contribute to oestradiol-mediated allograft protection: oestrogens act directly on glomerular mesangial cells, inhibit cellular proliferation and synthese types I and IV collagen [22].

Furthermore, oestrogens may modulate the synthesis of vasoactive agents, cytokines and other growth factors, which in turn are capable of altering allograft injury. In CAN, TGF-ß1 is recognized as one of the most important mediators of tissue fibrosis [1]. It promotes matrix synthesis, inhibits its degradation by several mechanisms, stimulates mesangial cell proliferation and regulates various inflammatory processes. In our experiment, oestradiol suppressed intragraft TGF-ß1 mRNA levels, and this may be responsible, at least in part, for the better outcome in this group. In contrast, TGF-ß1 expression was enhanced in progesterone-, tamoxifen- and droloxifene-treated animals that may have contributed to the more pronounced allograft injury in these rats.

In conclusion, we demonstrated that progesterone promoted the development of CAN, and that its addition to oestradiol treatment abolished the protective effects of oestradiol on allograft injury. Similarly to progesterone, both tamoxifen and droloxifene worsened long-term renal allograft outcome in this model.



   Acknowledgments
 
This work was supported by a grant from BMBF/DRL (UNG/056/96) and OTKA (F046526) and ETT 94/2003. Tamoxifen and Droloxifene were a kind gift from Klinge Pharma Germany, made possible by Dr Tinhof. Dr Antus is a recipient of a scholarship from the German Academic Exchange Service (DAAD).

Conflict of interest statement. All authors of this manuscript declare that they have no involvements that might raise the question of bias in the work reported or in the conclusions, implications, or opinions stated.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Joosten SA, van Kooten C, Sijpkens YW, de Fijter JW, Paul LC. The pathobiology of chronic allograft nephropathy: immune-mediated damage and accelerated aging. Kidney Int 2004; 65: 1556–1559[CrossRef][ISI][Medline]
  2. Antus B, Yao Y, Song E, Liu S, Lutz J, Heemann U. Opposite effects of testosterone and estrogens on chronic allograft nephropathy. Transpl Int 2002; 15: 494–501[ISI][Medline]
  3. Müller V, Szabo A, Viklicky O et al. Sex hormones and gender related differences: their influence on chronic renal allograft rejection. Kidney Int 1999; 55: 2011–2020[CrossRef][ISI][Medline]
  4. Siiteri PK, Febres F, Clemens LE, Chang RJ, Gondos B, Stites D. Progesterone and maintenance of pregnancy: is progesterone nature's immunosuppressant? Ann N Y Acad Sci 1977; 286: 384–397[ISI][Medline]
  5. Schust DJ, Anderson DJ, Hill JA. Progesterone-induced immunosuppression is not mediated through the progesterone receptor. Hum Reprod 1997; 11: 980–985[CrossRef][ISI]
  6. Miller L, Hunt JS. Regulation of TNF-{alpha} production in activated mouse macrophages by progesterone. J Immunol 1998; 160: 5098–5104[Abstract/Free Full Text]
  7. Majewski AC, Hansen PJ. Progesterone inhibits rejection of xenogeneic transplants in the sheep uterus. Horm Res 2002; 58: 128–135[CrossRef][ISI][Medline]
  8. Antus B, Hamar P, Kokeny G et al. Estradiol is nephroprotective in the rat remnant kidney. Nephrol Dial Transplant 2003; 18: 54–61[Abstract/Free Full Text]
  9. Ke HZ, Simmons HA, Pirie CM, Crawford TD, Thompson DD. Droloxifene, a new estrogen antagonist/agonist, prevents bone loss in ovariectomized rats. Endocrinology 1995; 136: 2435–2441[Abstract]
  10. Donath J, Nishino Y. Effects of partial versus pure antiestrogens on ovulation and the pituitary-ovarian axis in the rat. J Steroid Biochem Mol Biol 1998; 66: 247–254[CrossRef][ISI][Medline]
  11. Piccinni MP, Giudizi MG, Biagiotti R et al. Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established Th1 cell clones. J Immunol 1995; 155: 128–133[Abstract]
  12. Miller L, Hunt JS. Sex steroid hormones and macrophage function. Life Sci 1996; 59: 1–14[CrossRef][ISI][Medline]
  13. Neumann F, Elger W. Critical considerations on the biological basis of toxicity studies with sexual steroid hormones. In: Plotz EJ, Haller J, eds, Methodik der Steroidtoxikologie. Georg Thieme Verlag: Stuttgart, 1971; 6–24
  14. Bullock LP, Bardin CW, Sherman MR. Androgenic, antiandrogenic and synandrogenic actions of progestins: role of steric and allosteric interactions with androgen receptors. Endocrinology 1978; 103: 1768–1782[ISI][Medline]
  15. Nickenig G, Strehlow K, Wassmann S et al. Differential effects of estrogen and progesterone on AT1 receptor gene expression in vascular smooth muscle cells. Circulation 2000; 102: 1828–1833[Abstract/Free Full Text]
  16. Lou H, Kodama T, Zhao YJ et al. Inhibition of transplant arteriosclerosis in rabbits by chronic estradiol treatment is associated with abolition of MHC class II antigen expression. Circulation 1996; 94: 3355–3361[Abstract/Free Full Text]
  17. Mitlak BH, Cohen FJ. Selective estrogen receptor modulators. A look ahead. Drugs 1999; 57: 653–666[ISI][Medline]
  18. Grainger DJ, Metcalfe JC. Tamoxifen: teaching an old drug new tricks? Nat Med 1996; 2: 381–385[ISI][Medline]
  19. Eppenberger U, Wosikowski K, Kung W. Pharmacologic and biologic properties of droloxifene, a new antiestrogen. Am J Clin Oncol 1991; 14: S5–S14[ISI][Medline]
  20. Neugarten J, Acharya A, Lei J, Silbiger S. Selective estrogen receptor modulators suppress mesangial cell collagen synthesis. Am J Physiol 2000; 279: 309–318
  21. Geisler J, Ekse D, Hosch S, Lonning PE. Influence of droloxifene (3-hydroxytamoxifen), 40mg daily, on plasma gonadotrophins, sex hormone binding globulin and estrogen levels in postmenopausal breast cancer patients. J Steroid Biochem Mol Biol 1995; 55: 193–195[CrossRef][ISI][Medline]
  22. Kwan G, Neugarten J, Sherman M et al. Effects of sex hormones on mesangial cell proliferation and collagen synthesis. Kidney Int 1996; 50: 1173–1179[ISI][Medline]
Received for publication: 13. 7.04
Accepted in revised form: 20.10.04





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
20/2/329    most recent
gfh602v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Disclaimer
Request Permissions
Google Scholar
Articles by Antus, B.
Articles by Heemann, U.
PubMed
PubMed Citation
Articles by Antus, B.
Articles by Heemann, U.