Several lines of evidence suggest that both humoral and cell-mediated immunity are more active in females than in males 1 . Sex steroid hormones could play a pivotal role 2 3 in this process, and act as regulators of inflammatory processes. Indeed, numerous inflammatory cell functions, such as neutrophil chemoattractant generation, phagocytic responses of neutrophils 4 , and nitric oxide production by alveolar macrophages 5 show gender differences.

Ischemia-reperfusion injury (IRI) is known to exacerbate a pro-inflammatory milieu. The influence of gender on IRI has been mainly studied in cardiac ischemia and it is now established that female steroid hormones play a protective role 6 7 8 . In liver, it was reported that female mice were protected from liver ischemia-reperfusion compared to males, linked to an estrogen-dependent mechanism 9 10 . In a renal ischemia-reperfusion model, the mortality rate was significantly greater in male rodents 11 12 . Orchidectomy significantly improved male survival 11 and testosterone may increase kidney ischemic injury in mice 13 . These results suggest a protective effect of female hormones, and a detrimental effect of male hormones. However, although sex hormones could play an important role, the direct gender influence has not been fully demonstrated in renal ischemia-reperfusion models and the mechanisms involved remain to be elucidated.

We have previously demonstrated, in a rodent kidney ischemia-reperfusion model, that extensive renal tissue injury such as proximal tubule necrosis and outer medulla congestion occurred during the reperfusion phase following 60 min of ischemia 14 . In these conditions, tissue remodelling is determined by the severity of ischemia as we have previously shown in a pig model of renal ischemia reperfusion 15 . Tissular regeneration could be a pivotal key of organ outcome, limiting or counteracting injury development. Different markers of tissue remodelling or regeneration have been used in renal tissue such as vimentin, alpha-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA) or translocator protein (TSPO) expressions. TSPO, formerly known as the peripheral-type benzodiazepine receptor 16 , is a widely distributed transmembrane protein that is localized mainly in the outer mitochondrial membrane. Many functions are associated directly or indirectly with TSPO, including the regulation of cholesterol transport and steroid hormones synthesis, porphyrin transport and heme synthesis, apoptosis, cell proliferation, anion transport, regulation of mitochondrial functions and immunomodulation 16 17 . TSPO expression has been shown to be modulated by renal ischemia-reperfusion injury, in particular during the repair process 18 19 . Under these conditions, IRI could affect TSPO expression and sex hormone production respectively, modulating cellular response.

The aim of this study was to investigate sex hormone production in male rats subjected to renal ischemia-reperfusion, and analyze the influence of gender differences on tissue remodelling markers, particularly on cell proliferation processes and TSPO expression in the early phase of reperfusion.

In uninephrectomized mature rats, renal ischemia-reperfusion resulted in a severe acute renal failure, which affected more predominantly male than female animals as previously reported 11 12 13 22 23 . In our study, we showed a higher mortality rate for males in comparison to females following 60-min of renal ischemia with contralateral nephrectomy, associated with further deterioration of kidney function. The modulation of systemic male sex hormone levels by renal ischemia-reperfusion could explain male sensitivity to renal injury. Experiments with orchidectomy or ovariectomy as well as with various hormone treatments in male and female animals helped demonstrate the crucial role of sex hormones. Indeed, administration of estradiol in myocardial ischemia-reperfusion induced a rapid recovery of hemodynamic parameters 6 8 . Moreover, exogenous estradiol attenuates the hepatocellular injury induced by ischemia reperfusion in a reduced-size liver mouse model 9 . These results support a protective effect of female sex hormone in several organs subjected to IRI. In male rats, renal IRI was improved by orchidectomy and by estradiol administration, suggesting that either lowering androgen concentrations, or increasing the estradiol-androgen ratio, may have a protective role 11 13 . In contrast, in females, ovariectomy and or testosterone administration did not have any impact on renal function after IRI 11 13 . In addition, it was reported that male sex steroids cause immune suppression, while female sex hormones maintain immune response following trauma-hemorrhage 24 . In the present study, we showed a decrease in testosterone plasma levels after 1 day of reperfusion and a concomitant large increase in estradiol plasma levels. In accordance with our findings, this modulation has also been reported in male rats during experimental septic shock 25 and trauma-hemorrhage shock 26 . The activation of extra-testis aromatase transforming testosterone into estradiol has been found to be involved in experimental septic and hemorrhagic animal models and potentially due to upregulation of aromatase expression likely induced by acute increase in adrenal cortisol production 27 . However, the influence of decreased plasma testosterone and increased estradiol levels on tissue remodelling remains to be elucidated. Following the hypothesis of a detrimental effect of testosterone, and a protective effect of estradiol, we can speculate that our results may be due to compensatory regulatory effects. In addition, the trend towards a decrease in progesterone levels could explain, at least in part, the sensitivity of males to renal IRI.

After ischemia, kidneys from uninephrectomized male and female rats develop, at early reperfusion time points, a similar pattern of histopathological damages, according to previous reports 14 . Importantly, we showed that prolonged reperfusion (5 days) induced more severe tubular damage in kidneys from male compared to female rats. In the past, only few studies, including histological data, have been performed both in male and female rodents 11 12 13 22 23 . Hu et al. showed that ischemia time necessary to obtain IRI in mice was 35 to 45 min in male and 75 min in female, suggesting increased sensitivity of males to IRI 12 . According to these results, in a limited number of mice, Park et al. showed higher severe proximal tubular damage in males in comparison to females after 30 min of ischemia combined with 24 h of reperfusion 13 . In contrast, using a similar model, Muller et al. failed to show any histological differences between sexes after reperfusion 11 . This discrepancy with our results is likely due to the different ischemia-reperfusion times.

Inflammation is a major component of renal IRI. In our study, kidney from female exhibited a less degree of inflammation as shown by CD3 positive cells and neutrophils recruitment. Previous studies have already demonstrated the role of T lymphocytes as modulators of renal IRI and neutrophils as critical mediators of renal parenchymal injury 15 suggesting in our condition a protection against cellular infiltration related to gender.

Renal tubular epithelial cell dedifferentiation is thought to be a prerequisite for regenerative proliferation after IRI 28 . During the recovery process, surviving kidney cells dedifferentiated to mesenchymal phenotype and then proliferated and migrated to replace lost cells, commonly accepted as an epithelial mesenchymal transition process 29 . Under these conditions, new expression of mesenchymal markers like vimentin occurred in deteriorating and atrophic tubules. Vimentin is an intermediate filament protein expressed only in the mesenchymal cells and has been used as a marker for epithelial mesenchymal transition 30 . In a rat renal vascular occlusion model, Witzgall et al. previously reported that vimentin was detected 1 day post-clamping and prominently expressed 5 days after reperfusion 28 . Concomitantly with vimentin expression, authors described a maximal expression of PCNA 2 days post-ischemia in the proximal tubule 28 . PCNA is a marker for the G1-S transition phase cell cycle and hence mitogenesis. In our study, PCNA and vimentin expression analysis indicated a nephrogenic repair process more pronounced in male rats than in the female rats, related to a more severe injury after reperfusion. These results support a predominant tissue remodelling in male rats related to an extensive renal injury. However, at Day 5, in the female group a decrease in PCNA index and vimentin expression was associated with attenuation of tubular disorders, whereas in males these markers remained elevated in relation to a persistence of major tubular disorders. This underscores gender differences in renal tubular alterations and tissue remodelling after IRI.

There is increasing evidence that mitochondria play a central role in IRI mechanisms 31 32 . Our results supported a previous study in a porcine renal ischemia-reperfusion model, which showed that mitochondrial TSPO protein expression was increased 24h after reperfusion, particularly in the proximal tubule, which does not express this protein constitutively 33 . In addition, we have recently suggested that TSPO integrity is required for renal proximal tubular cell survival 18 and previous studies demonstrated its implication in cell cycle regulation 16 34 . In the present study, TSPO expression was more pronounced in proximal tubules of male rats after 24h of reperfusion, in association with the severity of tubular injury and PCNA staining. The renal distal tubular epithelial cells which express constitutively TSPO, are well adapted to the hypoxic environment of the renal medulla and appear to be more able to withstand hypoxic stress than the epithelium of the proximal tubule 35 36 . The transient expression of TSPO in proximal tubular cells paralleled with PCNA staining results at D1, suggested a transient putative role of TSPO in the mitochondria biogenesis and cell functions at the early time of reperfusion. However, TSPO modulation was not clearly correlated with steroid synthesis. Collectively, these data support the different roles of TSPO depending on its location. In steroid-producing tissues (adrenal glands and gonads), TSPO has been implicated in the regulation of cholesterol transport into the mitochondria, where it is metabolized by CYP11A1 to pregnenolone, the precursor of all steroids. In tissues or organs that are not directly involved in steroidogenesis, TSPO could be involved in a protective effect against lesion-promoting mechanisms like apoptosis and maintain mitochondrial integrity 18 .

In conclusion, we showed that male rats were more sensitive to renal IRI than female rats. These gender differences were found in tubular injury development, as well as in the nephrogenic repair process indicated by PCNA and vimentin expression. This sensitivity was associated with modulation of male sex hormone production in response to renal IRI. This adaptive response could be related to an extension of organ failure or a compensatory mechanism to limit renal injury. In addition, TSPO expression was transiently increased in proximal tubules in male rats during IRI process, supporting a putative role of this protein, which will be defined with further studies.

The authors declare that they have no competing interests.

DAH participated in the design of the study and performed experiments. FF participated in the design of the study and performed experiments and the statistical analysis. JMG performed histopathological analysis, including immunostaining. RR and TH conceived of the study, and participated in its design and coordination. RR, TH and GM helped to draft the manuscript. All authors read and approved the final manuscript.