The present study is the first to evaluate the effect of E2 and of synthetic progestins with different pharmacological profiles on renal injury in rats with an excess activation of the mineralocorticoid receptor. We demonstrate that a progestin, which is devoid of partial AR and GR activities and that acts as a potent MR antagonist (DRO) confers protective functions, whereas MPA caused adverse effects on renal structure and function and on gene expression in female AST rats.
Mineralocorticoids and sex hormones affect gene expression and kidney morphology and function via specific nuclear hormone receptors for estrogens, progestins, glucocorticoids, and androgens. In models of hypertension employing male rats, activation of the mineralocorticoid receptor by MR agonists contributes to kidney damage, and administration of MR antagonists or adrenalectomy attenuates renal injury (Rocha et al. 1999
). In addition, AST treatment in male rats is characterized by vascular and glomerular sclerosis, fibrinoid necrosis, tubular damage, and increased inflammation markers in addition to increased cellular proliferation, which is detectable by PCNA staining (Blasi et al. 2003
; Peng et al. 2001
Although it is conceivable that sex differences in renal disease might be explained in part by different sex hormone levels, chronic AST, which causes kidney hypertrophy and injury in male rodents, has so far not been studied in female rats. Here, we describe the renal effects of AST treatment on female rats. In contrast to male animals, aldosterone did not cause the degree of extensive renal damage that has been reported for male rats. Although kidney mass is lower in female versus male rats, AST treatment caused a similar extent of kidney hypertrophy as that observed previously in male AST rats. Kidney weight: 1404 mg female AST vs. 711 mg female control; 3618 mg male AST vs. 2192 mg male control as reported by Peng (2001). Sex-associated differences in aggravation of renal hemodynamics in response to angiotensin II have been analyzed in single nephrons from male and female rats, showing that male nephrons present lower preglomerular resistance (Baylis 1994
). Similar to what has been reported in male rats (Peng et al. 2001
), the increase of kidney weight in female AST rats was accompanied by augmented PCNA staining, suggesting that aldosterone induced increased cellular proliferation. However, the onset of inflammatory processes and renal fibrosis appears to be delayed in female compared to male rats as, except for MPA-treated animals, we were not able to detect a significant inflammation and renal fibrosis in the kidneys of female AST rats. In line with this hypothesis, Caron et al. (2006)
reported on a sexual dimorphism with a delayed onset of inflammation in female adrenomedullin one-copy:Ren transgenic mice. Furthermore, testosterone is known to increase plasma IL-1B concentrations (Machowska et al. 2004
). Interestingly, kidney hypertrophy and hypertension were differentially affected by estradiol substitution, because blood pressure, but not kidney mass, responded favorably to estrogen treatment. These observations are in agreement with previous studies on blood pressure regulation in estrogen-treated AST rats and match with persistent kidney hypertrophy in estrogen-treated stroke-prone SHR rats (Arias-Loza et al. 2006
; Gross et al. 2004
). Esqueda, Craig, and Hinojosa-Laborde (2007)
recently reported that estrogen status affects renal ERα and ERβ expression in salt-sensitive and salt-resistant Dahl rats. Therefore, it is important to note that global ERα and ERβ content did not differ among kidneys samples from all treatment groups.
The current findings therefore substantiate the hypothesis that sex differences also play an important role in the development of aldosterone-salt–induced renal injury similar to what is already known in cardiovascular disease (Kim and Levin 2006
; Meyer, Haas, and Barton 2006
; Reckelhoff 2001
). But it appears unlikely that estrogens play the dominant role in this process, since estrogen depletion did not aggravate the renal phenotype of AST rats. Instead, varying androgen levels or genetic factors may act as more important mediators of sex-specific differences in renal injury owing to chronic MR activation. Aldosterone plays an important role in both cardiac and renal disease, and patients with chronic renal failure face an increased incidence of concomitant heart disease (Herzog, Ma, and Collins 1998
; Silberg et al. 1989
). Of note, the current effects of E2, DRO, and MPA on renal disease in AST rats are generally in good agreement with our recent observations on cardiac and vascular injury in the very same animal model (Arias-Loza et al. 2006
). However, increased kidney mass persisted in E2-treated rats despite a significant reduction in blood pressure.
A second and novel finding of this study is the observation that MPA aggravated several key features of renal injury that have been previously attributed to excess mineralocorticoid receptor activity such as increased renal mass, excessive fluid uptake, increased renal sodium absorption, and potassium loss. These observations were accompanied by extensive structural damage that included the vascular, glomerular, tubular, and interstitial compartments of the remnant kidney and decreased serum albumin levels (please see online supplement). In particular, co-treatment of AST rats with MPA plus E2 caused myointimal proliferation of the small cortical arterioles with occasional perivascular fibrosis, including occasional complete occlusion of the vessels. There was generalized glomerular sclerosis necrosis and mesangiolysis, tubular degeneration and necrosis, and interstitial collagen and lymphocyte accumulations. No such alterations were observed in intact or in ovx AST rats receiving placebo, E2, DRO, or SPIRO. Therefore it is likely that MPA treatment also promotes renal injury by mechanisms that are blood pressure independent.
Because chronic aldosterone infusion in the absence of a high-salt diet causes only a very mild degree of hypertension and cardiovascular damage, enhanced sodium uptake might provide another rationale to explain excessive renal damage in MPA-treated rats (Brilla and Weber 1992
; Silberg et al. 1989
; Yoshida et al. 2005
). In support of this concept, sodium uptake reached maximum levels in MPA-treated animals. Renal sodium absorption in the collecting duct is facilitated via the amiloride-sensitive Na+
channel (ENaC), which consists of three subunits, α, β, and γ. Moreover, the mineralocorticoid receptor plays a critical role in regulating ENaC activity because aldosterone up-regulates α-ENaC expression via the MR and because MR knockout mice exhibit severely impaired ENaC activity that is accompanied by excessive renal sodium wasting (Berger et al. 2000
; Masilamani et al. 1999
). However, renal MR expression was not different among all rats, which is in line with previous observations in male AST rats (Silvestre et al. 2000
). Interestingly, Thomas, Liu, and Vats (2006)
have recently reported that MPA up-regulates α-ENaC promoter activity in renal collecting duct epithelia via binding and trans-activating mineralocorticoid and glucocorticoid receptors. Although not formally tested here, it appears conceivable that a very similar mechanism might have also been operational in MPA-treated AST rats, which would provide a mechanistic concept to explain increased sodium uptake and thus renal injury in female rats exposed to chronic AST.
Excess MR activity is associated not only with altered fluid, potassium, and sodium homeostasis, but also with increased oxidative stress (Nishiyama et al. 2004
). Medroxyprogesterone acetate treatment of AST rats enhanced renal nuclear DHE fluorescence and glomerular protein tyrosin-nitrosylation, and elevated the expression levels of the NADPH-oxidase subunit p67phox (Touyz 2004
). Collectively, these observations suggest that oxidative stress may have aggravated structural injury in the kidneys of MPA-treated rats. In support of this concept, enhanced oxidative stress has recently been identified as a mechanism for early podocyte damage in male rats receiving AST treatment (Shibata et al. 2007
). However, future and more specific studies will be required to prove this hypothesis, the clarification of which was beyond the scope of this article.
In contrast to MPA, DRO not only acts as a potent progestogen, but it also possesses a strong MR-antagonist activity (Muhn et al. 1995
; White et al. 2006
). Therefore, it is interesting to note that the medium and high dosages of DRO were as potent as SPIRO in attenuating kidney hypertrophy in E2-treated AST rats. The fact that compared to the medium dose of DRO, the high dose did not cause a further reduction of kidney mass and blood pressure is most likely explained by a maximum effect of DRO that is achieved already with the medium dose of the drug. In addition, increasing dosages of DRO gradually relieved the suppression of systemic angiotensin II serum concentrations that occurred in response to aldosterone-salt treatment. An identical effect was observed in AST rats receiving SPIRO treatment. Perhaps more important, renal pathology was completely normal among all rats receiving DRO or SPIRO. Together, these observations provide the first evidence for a possible renal-protective function of DRO in female AST rats that most likely relates to the very different pharmacological profiles and partial agonist activities of DRO and MPA.
Estrogens, progestins, and aldosterone interact with the renin angiotensin aldosterone system (RAAS). The very different effects of MPA and DRO on renal morphology and function might thus also relate to differential interactions with the RAAS. Serum angiotensin II concentrations are decreased in response to chronic AST treatment and MR blockade with SPIRO or DRO relieved this suppression, which by itself does not explain the reduction of blood pressure in DRO- or SPIRO-treated AST rats (Arias-Loza et al. 2006
). Instead, the expression pattern and the divergent function of both angiotensin II receptor subtypes, AT1R and AT2R, may provide an additional explanation for the blood pressure–lowering effect of DRO and SPIRO. Renal AT1R activation by angiotensin II causes vasoconstriction and increased blood pressure (Blume Kaschina, and Unger 2001
; Matsubara 1998
). But although female sex hormones down-regulate AT1R expression in vascular cells, AT1R expression levels were not different among all groups, which is unlikely to explain the protective function of DRO and SPIRO (Nickenig et al. 1998
). The function of the AT2R largely opposes those of the AT1R, since pharmacological activation or genetic manipulation of AT2R expression revealed important natriuretic and blood pressure–lowering effects. Activation of AT2R by Ang II thus counteracts and balances AT1R activity (Hein et al. 1995
; Inagami et al. 1997
; Lo et al. 1995
). Up-regulation of renal AT2R expression in E2-substituted rats may indicate a protective function, but the constant suppression of serum AII levels may have limited the extent of AT2R activation. However, co-treatment with DRO or SPIRO not only caused an additional increase of renal AT2R expression in E2-treated rats, but it also alleviated the suppression of serum AII levels. Although we have not formally tested the hypothesis that enhanced renal AT2R activation promoted increased renal sodium excretion and lower blood pressure in AST rats receiving combined treatment with E2 plus DRO (or SPIRO), such a concept is supported by recent studies by Gross et al. (2000)
, who reported that deletion of the AT2R results in a threefold reduction of renal sodium and water excretion. Together, these data provide novel and mechanistic hypotheses that might explain the protective function of DRO and SPIRO on kidney hypertrophy and renal sodium excretion.
In conclusion, the choice of specific synthetic progestins has profound implications on the development of hypertension, kidney injury, and renal gene expression under conditions of elevated aldosterone serum levels and salt intake. Clinical and experimental data indicate that mineralocorticoid and estrogen receptors do functionally interact in the development of vascular and renal injury. The current report shows that different synthetic progestins that are currently employed for HRT confer either adverse (MPA) or protective (DRO) effects on aldosterone-induced renal injury. The present information could be useful to enhance the pharmacological safety of HRT in female patients who are at risk to develop or may already be diagnosed with hypertension and coexisting renal disease.