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
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Abstract |
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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
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Introduction |
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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.
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Subjects and methods |
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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 515% of the section); grade 2, moderate CAN with moderate interstitial fibrosis and tubular atrophy (affecting 1550% 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 [-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.
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Results |
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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).
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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).
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Discussion |
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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- 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-
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.
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Acknowledgments |
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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.
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References |
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