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Originally published In Press as doi:10.1074/jbc.M407629200 on September 8, 2004

J. Biol. Chem., Vol. 279, Issue 50, 52282-52292, December 10, 2004
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Testosterone Is Responsible for Enhanced Susceptibility of Males to Ischemic Renal Injury*

Kwon Moo Park{ddagger}§, Jee In Kim{ddagger}, Youngkeun Ahn¶||, Andrew J. Bonventre{ddagger}, and Joseph V. Bonventre{ddagger}**{ddagger}{ddagger}

From the {ddagger}Renal Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, the §Department of Anatomy, School of Medicine, Kyungpook National University, Daegu, 700-422, Korea, the Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, and the **Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Boston, Massachusetts 02115

Received for publication, July 7, 2004 , and in revised form, September 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Female mice are much more resistant to ischemia/reperfusion (I/R)-induced kidney injury when compared with males. Although estrogen administration can partially reduce kidney injury associated with I/R, we demonstrated that the presence of testosterone, more than the absence of estrogen, plays a critical role in gender differences in susceptibility of the kidney to ischemic injury. Testosterone administration to females increases kidney susceptibility to ischemia. Dihydrotestosterone, which can not be aromatized to estrogen, has effects equal to those of testosterone. Castration reduces the I/R-induced kidney injury. In contrast, ovariectomy does not affect kidney injury induced by ischemia in females. Testosterone reduces ischemia-induced activation of nitric oxide synthases (NOSs) and Akt and the ratio of extracellular signal related kinase (ERK) to c-jun N-terminal kinase (JNK) phosphorylation. Pharmacological (N{omega}-nitro-L-arginine) or genetic (endothelial NOS or inducible NOS) inhibition of NOSs in females enhances kidney susceptibility to ischemia. Nitric oxide increases Akt phosphorylation and protects Madin-Darby canine kidney epithelial cells from oxidant stress. Antagonists of androgen or estrogen receptors do not affect the gender differences. In conclusion, testosterone inhibits the post-ischemic activation of NOSs and Akt and the ratio of ERK to JNK phosphorylation through non-androgen receptor-medicated mechanisms, leading to increased inflammation and increased functional injury to the kidney. These findings provide a new paradigm for the design of therapies for ischemia/reperfusion injury and may be important to our understanding of the pathophysiology of acute renal failure in pregnancy where plasma androgen levels are elevated.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gender differences characterize the susceptibility or expression of many diseases. In general, the gender disparity has been interpreted primarily as reflecting estrogen-mediated protection against pathological conditions (1–4). Recent studies, however, suggest that male hormones may also play important roles in gender differences in disease susceptibility (5–7).

Steroid hormones, including sex hormones, regulate inflammation, an important contributor to the pathophysiology of ischemia/reperfusion (I/R)1-induced tissue injury (8–11). Postischemic tissues generate inflammatory mediators and up-regulate leukocyte-endothelial adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), which can attract and/or activate leukocytes and potentiate small vessel occlusion (8, 9). Our laboratory and others have reported that the reduction of inflammation secondary to the inhibition of ICAM-1 protects the kidney from I/R (8, 12).

An important endogenous modulator of I/R-induced tissue injury is nitric oxide (NO) (13, 14). NO, the product of NO synthases (NOSs), possesses anti-inflammatory and vasodilative activity and is anti-apoptotic through Akt signaling pathways (13–17). Sex hormones are known to regulate NO synthesis (18, 19).

We have characterized the differences in susceptibility of male and female mice to kidney I/R injury and have studied the relationship of these differences to estrogen or testosterone. Our findings reveal that the kidneys of males are much more susceptible to I/R than those of females. There is increased post-ischemic proximal tubule injury, apoptosis, and inflammation in males. This higher susceptibility in males is due more to the presence of testosterone than the absence of estrogen. Testosterone acts to inhibit NOS activation, Akt phosphorylation, and the post-ischemic increase in the ratio of extracellular signal related kinase (ERK) to c-jun N-terminal kinase (JNK) activation, leading to greater inflammatory responses. The differences in ischemia susceptibility between male and female kidneys can be eliminated by castration of males or treatment of females with testosterone or dihydrotestosterone, which can not be aromatized to estrogen. The effects of testosterone and dihydrotestosterone are not inhibited by androgen receptor antagonists.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Preparation—Experiments were performed in age-matched (12–14 weeks) BALB/c (Charles River Laboratory) mice unless other-wise described. In some studies C57BL/6, SV 129, C57BL/6 + SV129, iNOS +/+, and iNOS –/– mice (The Jackson Laboratory) were used. In all cases studies were done according to animal experimental procedures approved by the Institutional Animal Care and Use Committee. Each animal group consisted of more than four mice. Blood was collected from mice to determine plasma creatinine (Pcr), urea nitrogen (BUN), estrogen, or testosterone concentration. To determine creatinine clearance (Ccr) and fractional excretion of sodium (FENa) urinary creatinine and sodium concentrations were measured in a sample that was collected for 24 h.

Animals were anesthetized with pentobarbital sodium (50 mg/kg of body weight (BW); intraperitoneally) prior to surgery. Kidney (10, 11) ischemia was carried out as described previously. In some animals ovariectomy or castration was carried out 15 days before bilateral renal ischemia or sham surgery. Different groups of animals were administrated 17{beta}-estradiol benzoate (40, 100, or 500 µg/kg of BW; estrogen), testosterone propionate (40, 100, or 500 µg/kg of BW; testosterone), dihydrotestosterone (500 µg/kg of BW), cyproterone (5 mg/kg of BW), flutamide (5 mg/kg of BW), tamoxifen (5 mg/kg of BW), or vehicle (sesame oil) (all from Sigma) by subcutaneous injection every day for 14 days prior to ischemia. In other experiments BALB/c mice were administrated L-arginine (30 mg/kg; Sigma), N{omega}-nitro-l-arginine (L-NNA, 12 mg/kg; Sigma), or 0.9% NaCl intraperitoneally 30 min before and after either ischemia or sham operation. Kidneys were harvested for Western blot analysis, myeloperoxidase (MPO) activity, NOS activity, and histological studies as described previously (10, 11).

Histological Examination—Kidney sections were stained with hematoxylin and eosin. The percentage of damaged tubules (identified by the presence of at least one of the following: dilatation, atrophy, or casts) was determined by scoring 200 renal outer medulla tubules/kidney in randomly selected microscopic fields.

Monocyte-Macrophage Homing to Kidney—RAW 264.7 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were washed with serum-free Dulbecco's modified Eagle's medium three times and incubated with Dulbecco's modified Eagle's medium containing microspheres (FluoSpheres® carboxylate-modified microsphere, 0.2 µm, yellow-green fluorescent (505/515); Molecular Probes, Eugene, Oregon), which were phagocytized for 75 min. Cells were then harvested with 0.25% trypsin-EDTA and injected into the tail vein 30 min after initiation of reperfusion in ischemic or sham-operated mice. The kidneys were harvested 24 h after ischemia and fixed. Five-micron sections were prepared. The numbers of infiltrated RAW 264.7 cells were counted using fluorescence microscopy. This technique is similar to that used by Pasceri et al. (20) to detect vascular inflammation.

Nitrite Measurement—For measurement of nitrite production, RAW264.7 cells were grown in the phenol red-free Dulbecco's modified Eagle's medium containing 10% charcoal-deprived fetal bovine serum, incubated with either testosterone or 17{beta}-estradiol for 24 h and then stimulated with lipopolysaccharide (Sigma) for 14 h. The nitrite levels in medium were measured using a commercial nitrite assay kit (Cayman Chemical).

NOS Catalytic Assay—Calcium-dependent or calcium-independent NOS activity was measured using a commercial NOS activity assay kit, and the values were normalized to the protein amount and presented as the -fold increase over control.

MPO Activity—Kidney MPO activity was measured as described previously (10).

LDH Assay—MDCK cells were grown in the phenol red-free Dulbecco's modified Eagle's medium containing 10% charcoal-deprived fetal bovine serum. Cells were pretreated in serum-free medium for 14 h with DETA NONOate and then treated with 1 mM H2O2 or vehicle for 4 h. LDH release was measured as previously described (10)

Immunoblot Analysis—Immunoblot analyses were performed as described previously (10, 11) with antibodies against phospho-Akt (Ser-473), phospho-JNK1/2, phospho-ERK1/2, or cleaved poly(ADP-ribose) polymerase obtained from Cell Signaling Technology (Beverly, Massachusetts). JNK1 and ERK1/2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, California), and ICAM-1 antibody was obtained from M. A. Arnaout (Massachusetts General Hospital).

Statistics—The results are expressed as the means ± S.E. Statistical differences among groups were calculated using an analysis of variance. Differences between groups were evaluated with Student's t test. Differences were considered statistically significant at a p value of <0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gender Differences on Renal Function and Morphology after Ischemia/Reperfusion—There were no significant changes of body weight after gonadectomy or treatment of BALB/c mice with hormones. Thirty minutes of kidney bilateral ischemia in male mice markedly reduced renal function as reflected by creatinine clearance (Fig. 1a, Ccr), fractional excretion of sodium (Fig. 1b, FENa), and plasma creatinine (Fig. 1c, Pcr), whereas this period of ischemia in females results in no measurable change in renal function (Fig. 1, a–c). Even when the ischemic time is extended to 45 min in females, there is only a small increase of Pcr (Fig. 1c). Consistent with the functional differences in Ccr and FENa, post-ischemic mortality is greater in males than in females exposed to 60 min of ischemia (Fig. 1d). Tubule damage is most severe in the outer medullary S3 segment of the proximal tubule (Fig. 1e) and much more severe in males than in females (Fig. 1, e and f). The gender differences in kidney ischemia are observed in a variety of mouse strains including C57BL/6, SV 129, and C57BL/6 + SV129 in addition to BALB/c (Fig. 2).



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  		FIG. 1.
Effect of gender on ischemia-induced kidney injury. Except where indicated (c and d) BALB/c mice were subjected to 30 min of bilateral ischemia at 36–38 °C. Some mice were subjected to castration (e and f) 15 days before the bilateral ischemia. Creatinine clearance (a), fractional excretion of sodium (FENa) (b), plasma creatinine concentrations (c), and morphological damage (e and f) were evaluated 24 h after bilateral ischemia. In d mice were exposed to 60 min of ischemia, and survival was observed for 7 days. Results are the means ± S.E. of (n) experiments. * and +, p < 0.05 versus control, respectively. #, p < 0.05 versus ischemia of M. C, castration; F, female; M, male.

 



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  		FIG. 2.
Effect of mouse strain differences on ischemia-induced kidney injury. Various strains of mice were subjected to 30 min of bilateral ischemia at 36–38 °C. Plasma creatinine concentration was evaluated 24 h after bilateral ischemia. Results are the means ± S.E. *, p < 0.05 versus the respective control.

 
Castration reduces plasma testosterone levels and daily testosterone administration (500 µg/kg of body weight) for 14 days to females significantly increases plasma testosterone levels to levels measured in intact male mice pretreated with vehicle (IMV) (Fig. 3a). Estrogen administration to males significantly increases the plasma estrogen levels to within the range measured in intact female mice pretreated with vehicle (IFV) plasma concentrations, and ovariectomy decreases the levels in females treated with vehicle (OFV) (Fig. 3b). There are no significant changes in plasma testosterone and estrogen after ischemia (Fig. 3, a and b).



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  		FIG. 3.
Effect of gender and hormonal modification on plasma testosterone (a) and estradiol (b) concentrations. Some BALB/c mice were subjected to gonadectomy on day 0. Mice were administered testosterone (500 µg/kg of BW), 17{beta}-estradiol (40 µg/kg of BW), or vehicle (sesame oil) by subcutaneous injection daily for 14 days. Ischemia or sham surgery was performed on day 15. Plasma testosterone (a) and 17{beta}-estradiol (b) concentrations were evaluated on day 16, 24 h after the last dose of estrogen, testosterone, or vehicle. Results are the means ± S.E. * and +, p < 0.05 versus respective controls of intact males (IMV) or intact females (IFV). C, castration; E, 17{beta}-estradiol-treated; F, female; I, intact; M, male; O, ovariectomy; T, testosterone-treated; V, vehicle-treated.

 
In intact (IFT) or ovariectomized females treated with testosterone, Pcr, and BUN levels 24 h after ischemia increase to levels indistinguishable from those reached post-ischemia in intact males pretreated with vehicle (IMV) (Figs. 4 and 5). By contrast in ovariectomized females treated with vehicle (OFV), ischemia does not increase BUN and Pcr (Fig. 4, c and d). Thus increased plasma testosterone levels in females increases kidney susceptibility to ischemic injury whereas reduced estrogen levels in females does not enhance susceptibility to ischemia injury. Dihydrotestosterone (DHT), a non-aromatizable analogue of testosterone, administration to intact females results in an increase in Pcr 24 h after a 30-min period of ischemia (Fig. 4a). When tamoxifen, an antagonist of the estrogen receptor, is administrated to females, there is no increase in Pcr 24 h after 30 min of ischemia (Fig. 4b). Cyproterone, an antagonist of the androgen receptor, does not prevent the increased susceptibility to ischemia of testosterone-treated females (Fig. 4b) indicating that the effect of testosterone is not androgen receptor-mediated.



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  		FIG. 4.
Effect of hormonal modification on ischemia-induced kidney injury in female mice. Some BALB/c female mice were ovariectomized on day 0. Mice were administered testosterone (500 µg/kg of BW with the exception of the dose response in a), dihydrotestosterone (DHT, 500 µg/kg of BW), 17{beta}-estradiol (40 µg/kg of BW), tamoxifen (5 mg/kg of BW), testosterone (500 µg/kg of BW) plus cytproterone (5 mg/kg of BW), or vehicle by subcutaneous injection daily for 14 days. On day 15 mice were subjected to 30 min of bilateral ischemia at 36–38 °C. Plasma creatinine (a–c) and BUN concentrations (d) were evaluated 24 h after 30 min of bilateral ischemia. Results are the means ± S.E. *, p < 0.05 versus control of intact females treated with vehicle (IFV). E, 17{beta}-estradiol-treated; I, intact; T, testosterone-treated; V, vehicle-treated.

 



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  		FIG. 5.
Effect of hormonal modification on ischemia-induced kidney injury in male mice. Some BALB/c male mice were castrated on day 0. Mice were administrated testosterone (500 µg/kg of BW), 17-{beta} estradiol (40 µg/kg of BW with the exception of the dose response in c), flutamide (5 mg/kg of BW), cyproterone (5 mg/kg of BW), testosterone (500 µg/kg of BW) plus 17{beta}-estradiol (40 µg/kg of BW), 17{beta}-estradiol (40 µg/kg BW) plus tamoxifen (5 mg/kg of BW), or vehicle by subcutaneous injection daily for 14 days. On day 15 mice were subjected to 30 min of bilateral ischemia at 36–38 °C. Plasma creatinine concentrations (a, c, and d) and BUN concentration (b) were evaluated 24 h after ischemia. Results are the means ± S.E. * and #, p < 0.05 versus control and ischemia of intact males pretreated with vehicle, respectively. E, 17{beta}-estradioltreated; I, intact; T, testosterone-treated; V, vehicle-treated.

 
In males, when testes are removed 15 days prior to ischemia, there is protection against ischemia with no increase observed in Pcr and BUN 24 h after I/R (Fig. 5, a and b). Testosterone treatment of castrated males restores susceptibility to I/R injury (Fig. 5, a and b). Estrogen treatment of intact males reduces the post-ischemic increase of Pcr and BUN compared with intact male mice pretreated with vehicle (Fig. 5, a–c) but not as much as what occurs with castration (Fig. 5, a and b). Neither treatment of males with cyproterone or flutamide, antagonists of the androgen receptor, affect the degree to which Pcr rises after ischemia (Fig. 5d). Tamoxifen, an antagonist of the estrogen receptor, does not prevent a beneficial effect of estrogen in males (Fig. 5d). Thus the presence of testosterone, rather than the absence of estrogen, appears to be more relevant to the increased susceptibility of males to I/R injury through non-genomic mechanisms, although pharmacological doses of estrogen have a protective effect in males.

Tissue MPO Activity, RAW 264.7 Cell Trapping, and Expression of ICAM-1 as a Result of Ischemia/Reperfusion—Tissue MPO activity, an index of tissue leukocyte infiltration, is much greater in intact males after ischemia (IMV) than in females (IFV) (Fig. 6a). Gonadectomy reduces the post-ischemic MPO activity in males but not in females (Fig. 6a). Testosterone treatment of females results in increased post-ischemic MPO activity (Fig. 6a). When RAW 264.7 cells, a mouse monocyte/macrophage cell line, are injected intravenously into animals 30 min after ischemia, trapping is greater in males (IMV) than females (IFV) (Fig. 6b). Castration (CMV) decreases RAW cell trapping in males (Fig. 6b). Ischemia increases the expression of ICAM-1 to a greater extent in males than in females (Fig. 6c), and gonadectomy results in a diminished ICAM-1 response to ischemia in males and females (Fig. 6c). Thus increased postischemic MPO activity, RAW 264.7 cell trapping, and ICAM-1 expression are mitigated by castration in males (Fig. 6, a–c). Testosterone treatment of females potentiates ICAM-1 expression post-ischemia (Fig. 6c).



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  		FIG. 6.
Effect of gender and hormonal modification on ischemia-induced inflammatory responses. Some BALB/c mice were gonadectomized on day 0. Mice were administered testosterone (500 µg/kg of BW), 17{beta}-estradiol (40 µg/kg of BW), or vehicle by subcutaneous injection daily for 14 days. On day 15, mice were subjected to 30 min of bilateral ischemia at 36–38 °C. a, tissue myeloperoxidase (MPO) activity was measured 4 h after 30 min of bilateral renal ischemia (n = 4–8). b, RAW 264.7 cells labeled with fluorescence microspheres were injected into the tail vein 30 min after 30 min of bilateral ischemia. The number of RAW 264.7 cells counted in an area of 10 mm2 in a 5-µm section of mouse kidney is presented. c, Western blot analysis of ICAM-1 expression 24 h after ischemia. The blot is representative of 3–5 independent experiments. The densities of the Western blot bands were quantified by the NIH Image program. Values presented are expressed as mean ± S.E. *, p < 0.05 versus their respective control. # and +, p < 0.05 versus intact males (IMV) and females (IFV) after ischemia, respectively. Control, sham-operation; C, castration; E, 17{beta}-estradiol-treated; F, female; I, intact; M, male; O, ovariectomy; T, testosterone-treated; V, vehicle-treated.

 
Tissue NOSs Activity and Effect of Pharmacological or Genetic Modulation of NOSs on Ischemia Injury—Base-line levels of both calcium-dependent NOS (cNOS) and calcium-independent NOS activity (ciNOS) are significantly higher in females than in males (Fig. 7, a and b). Castrated males (CMV) have no significant increase in base-line kidney cNOS activity but have a significant increase in post-ischemic cNOS activity when compared with post-ischemic activity in intact male kidneys (IMV) (Fig. 7a). By contrast, testosterone administration to females (IFT) inhibits the post-ischemic activation of kidney cNOS (Fig. 7a). Except for the IMV and IFT groups, ischemia resulted in increased kidney cNOS activity when compared with cNOS activity in their respective controls prior to ischemia (Fig. 7a). Castration significantly increases the basal activity of ciNOS, which is further increased with ischemia (Fig. 7b). Testosterone treatment of females inhibits the post-ischemic increase of ciNOS activity (Fig. 7b).




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  		FIG. 7.
Calcium-dependent (a) and calcium-independent (b) NOS activity in the mouse kidney. Effect of genetic or pharmacological modification of NO/NOS on plasma creatinine levels 24 h after ischemia (c and d) and tissue MPO activity (e). f and g, effect of hormones on nitrite production in RAW 264.7 cells. With the exception of c, BALB/c mice (a, b, d, e) were used for the experiments. Some BALB/c mice were gonadectomized on day 0 (a and b). Mice were administered testosterone (500 µg/kg of BW), 17{beta}-estradiol (40 µg/kg of BW), or vehicle by subcutaneous injection daily for 14 days. On day 14 mice were subjected to 30 min of bilateral ischemia at 36–38 °C (a and b). Some mice (n = 4–12) are treated with L-arginine (Arg), L-NNA (NNA), or 0.9% NaCl (Veh, vehicle) 30 min before and after ischemia (d and e). Kidneys were harvested 30 min (a), 4 h (b), or 24 h (e) after ischemia to measure calcium-dependent (a) or calcium-independent (b) NOS activity and MPO (e) activity. Plasma creatinine levels were measured 24 h after ischemia (c and d). g, RAW 264.7 cells were pretreated for 24 hr with testosterone or estrogen and then treated with lipopolysaccharide (LPS) (2 mg/ml) or vehicle for 14 h after which nitrite levels in the medium were measured (n = 9). Values presented are expressed as mean ± S.E. * and #, p < 0.05 versus control and ischemia of intact males (IMV), respectively. +, p < 0.05 versus ischemia of intact female (IFV); !, p < 0.05 versus their respective control; ^, p < 0.05 versus lipopolysaccharide alone. Control, sham-operation; C, castration; E, 17{beta}-estradiol-treated; F, female; I, intact; M, male; T, testosterone-treated; V, vehicle-treated.

 
We further evaluated the contribution of NO to gender differences in susceptibility to ischemia by using genetic or pharmacological modification of NO/NOSs. Either iNOS or eNOS gene deletion in females partially increases kidney susceptibility to I/R (Fig. 7c). This increase is modest compared with the increase in ischemia susceptibility seen with testosterone pretreatment perhaps because of the fact that in either knock-out animal other NOSs are intact. L-Arginine, a donor of NO, reduces the post-ischemic increase of Pcr in males (Fig. 7d). L-NNA, an inhibitor of NOSs, results in increased post-ischemic Pcr in males and females (Fig. 7d). Three days after ischemia the mortality of six L-NNA-treated males is 100%, whereas all five vehicle (0.9% NaCl)-treated and six L-arginine-treated males survived. L-NNA treatment of females dramatically increases the levels of post-ischemic Pcr, but the increase in Pcr does not reach the levels measured in males (Fig. 7d). The increases of post-ischemic Pcr with L-NNA pretreatment is greater than increases in post-ischemic Pcr seen in iNOS–/– or eNOS–/– females (Fig. 7, c and d) as could be expected, because L-NNA will inhibit both iNOS and eNOS, and in the knock-out animals only one form is eliminated. L-NNA pretreatment of females results in enhanced post-ischemic tissue MPO activity (Fig. 7e). Thus decreased NOS activity may serve as a downstream effector mechanism for enhanced kidney susceptibility to the ischemic injury seen with testosterone.

Testosterone may also exert an effect on the inflammatory cell itself. At a concentration of 10 nM, which is within the physiological range (21), testosterone significantly inhibits the production of nitrite, a metabolite of NO, from RAW 264.7 cells activated by exposure to lipopolysaccharide. By contrast, high doses of estrogen (22) increase nitrite production from these cells (Fig. 7, f and g).

Basal and Post-ischemic Activation of Akt—Because Akt has been implicated in the protection against cell death and is an upstream regulator and downstream target of NO and NOS (14, 23), we evaluated whether activation of this kinase might explain the differences in kidney ischemic injury between males and females. Fifteen days after castration of males or testosterone treatment of females and prior to ischemia, the base-line levels of activated Akt are higher in vehicle-treated females (IFV) and castrated males (CMV) than in vehicle-treated males (IMV) or testosterone-treated females (IFT) (Fig. 8, a–c). Estrogen treatment of normal and castrated males increases the base-line but not to levels seen in IFV (Fig. 8, a–c). Ischemia dramatically increases the levels of phosphorylated Akt in all experimental groups (Fig. 8, a–c), but this increase is much more transient in non-protected groups of animals. Twenty-four hours after ischemia the phosphorylated Akt levels in intact females are higher than in intact males (Fig. 8a). Testosterone treatment of females reduces the 24 h post-ischemic levels of activated Akt (Fig. 8, a and c). Testosterone treatment of ovariectomized or castrated mice also results in reduced 24-h post-ischemic levels of activated Akt when compared with OFV or CMV, respectively (Fig. 8, a–c). Neither cyproterone in males or tamoxifen in females changes the 24-h post-ischemic activation of Akt (Fig. 8d).



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  		FIG. 8.
Effect of gender and hormonal modification on ischemia-induced Akt phosphorylation (a–d) or poly(ADP-ribose) polymerase activation (g) in kidney or effects of DETA NONOate, a donor of nitric oxide, on phosphorylation of Akt (e) and LDH release (f) in MDCK cells. Some BALB/c mice were gonadectomized on day 0. Mice were administered testosterone (500 µg/kg of BW), 17{beta}-estradiol (40 µg/kg of BW), tamoxifen (5 mg/kg of BW), cyproterone (5 mg/kg of BW), or vehicle by subcutaneous injection daily for 14 days (a–d, and g). On day 15 mice were subjected to 30 min of bilateral ischemia at 36–38 °C, and kidneys were harvested at 30 min (a, c, and g), 90 min (g) or 24 h (a–d, and g) after reperfusion. c, the density of phospho-Akt (Ser-473) Western blot bands was quantified by the NIH Image program. Each blot is representative of 3–5 independent experiments. e, phosphorylation of Akt in MDCK cells incubated with vehicle, DETA NONOate, L-NNA, an inhibitor of nitric oxide synthase, or L-NNA plus DETA NONOate for 14 h. f, LDH release from MDCK cells preincubated with various concentrations of DETA NONOate or vehicle for 14 h and then treated with 1 mM of H2O2 for 4 h (n = 9). *, p < 0.05 versus their respective base line. # and +, p < 0.05 versus 30 min and 24 h after ischemia of intact males (IMV), respectively. !, p < 0.05 versus baseline of intact males (IMV). ^, p < 0.05 versus 1 mM H2O2 alone. B, baseline; C, castration; Cypro, cyproterone-treated; DETA, DETA NONOate-treated; E, 17{beta}-estradioltreated; F, female; I, intact; M, male; O, ovariectomy; T, testosterone-treated; Tam, Tamoxifen-treated; V, vehicle-treated.

 
DETA NONOate (3 µM), a donor of NO, activates Akt (Fig. 8e) and protects MDCK cells from H2O2-induced injury as measured by LDH release (Fig. 8f). The protective effects are lost at higher concentrations, a biphasic effect with precedence in the literature in other models of injury (24). Akt activation has been associated with an anti-apoptotic effect (25). The cleavage of poly(ADP-ribose) polymerase to form an 89-kDa signature fragment cleavage is a well characterized event in the apoptotic pathway of animal cells (26). After ischemia/reperfusion, poly(ADP-ribose) polymerase cleavage is much greater in males than females at all times tested (Fig. 8g).

Effect of Ischemia on JNK and ERK Activation—We have recently shown that the ratio of ERK to JNK phosphorylation correlates with protection against ischemic injury (11, 12) and H2O2-induced toxicity in renal epithelial cells (27). JNK is much more highly activated in intact males (IMV) when compared with intact females (IFV) at 30 min (Fig. 9a) and 90 min (Fig. 9b) after ischemia. Interestingly, testosterone removal by castration reduces post-ischemic activation of JNK, whereas testosterone treatment of females increases post-ischemic phosphorylation of JNK (Fig. 9, a and b). Ischemia dramatically increases ERK1/2 activation in the both males and females (Fig. 9c). Although there is no significant post-ischemic functional and histological injury in females, the phosphorylated levels of ERK1/2 in females is even higher than those in males (Fig. 9c) suggesting that the increase of ERK 1/2 may be involved with the resistance of females to ischemia (Fig. 9c). Ovariectomy does not alter the ERK 1/2 activation post-ischemia, whereas testosterone treatment of females reduced the activation of ERK 1/2 (Fig. 9c). Thus functional protection is highly correlated with an increase in the ratio of ERK phosphorylation to JNK phosphorylation post-ischemia.



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  		FIG. 9.
Effect of gender and hormonal modification on phosphorylation of JNK (a and b) and ERK (c). Some BALB/c mice were gonadectomized on day 0. Mice were administered testosterone (500 µg/kg of BW), 17{beta}-estradiol (40 µg/kg of BW), or vehicle by subcutaneous injection daily for 14 days. On day 15 mice were subjected to 30 min of bilateral ischemia at 36–38 °C, and kidneys were harvested 30 min (a and c), 90 min (c), or 4 h (b) after either sham-operation (control) or 30 min of bilateral ischemia. JNK and ERK phosphorylation was determined with Western blot analysis using phospho-JNK or phospho-ERK1/2 specific antibodies. a and c, the density of Western blot bands was quantified by the NIH Image program. Each blot is representative of 3–5 independent experiments. *, p < 0.05 versus their respective baseline control. # and +, p < 0.05 versus ischemia of intact male (IMV) and intact female (IFV), respectively. C, castration; E, 17{beta}-estradiol-treated; F, female; I, intact; M, male; O, ovariectomy; T, testosterone-treated; V, vehicle-treated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our findings indicated that the presence of testosterone, rather than the absence of estrogen, appears to be more relevant to the gender difference in susceptibility to kidney I/R injury. Although estrogen treatment of males reduced ischemic kidney injury, consistent with other studies in the heart (3) and brain (1), a marked reduction in serum estrogen secondary to ovariectomy does not enhance ischemic injury in females. Although many studies have emphasized the beneficial effects of estrogen in gender differences in I/R injury (1, 4), some studies have implicated male sexual hormones (5–7). After our paper was published in abstract form (28), Muller et al. (6) reported that male rats are more susceptible to kidney ischemia than females and that castration and estrogen treatment in the male improve post-ischemic survival. In this study, however, they did not observe increased post-ischemic mortality in females pretreated with testosterone for 7 days (6). The discrepancy with our results is probably due to the length of time of administration, perhaps the levels of plasma testosterone, and possibly species differences. In our studies the degree of sensitivity of female mice to I/R depends on the plasma concentration of testosterone (Fig. 4a). We found that acute injection (intravenous, 30 min before ischemia) of testosterone (500 µg/kg of body weight) in females does not change the kidney susceptibility to ischemia.

A reduction in the inflammatory response protects the kidney from functional ischemic injury (8, 10–12). We observed that ICAM-1 expression, post-ischemic MPO activity, and RAW 264.7 cell trapping after I/R are greater in males than in females. Some investigators have reported that estrogen inhibits inflammatory actions (29–31). Although estrogen treatment in males reduces ICAM-1 expression and MPO activity, these levels remain well above those found in protected females or castrated males. Ovariectomy does not increase ICAM-1 expression or MPO activity in females, consistent with the absence of an effect of an ovariectomy on renal functional impairment post-ischemia. Interestingly, ovariectomy actually reduced post-ischemic ICAM-1 expression suggesting that estrogens may contribute to the post-ischemic inflammatory response in females. This also suggested that although an effect of an ovariectomy on post-ischemic renal function was not seen, using plasma creatinine and BUN as indicators, ovariectomy may induce differences on the post-ischemic inflammatory response in intact females. By contrast, with testosterone treatment of females there is increased ICAM-1 expression and MPO activity with ischemia, whereas the castration of males results in reduced post-ischemic ICAM-1 expression and MPO activity. Thus our results suggested that functionally significant post-ischemic kidney inflammatory responses likely depend more on testosterone than estrogen. Testosterone has been shown to accentuate vascular responses to vasopressor agents (32), increase the thromboxane/prostaglandin ratio (33), increase platelet aggregation (34), and increase monocyte adhesion to endothelial cells (35), properties that can contribute to vasoconstriction, small vessel occlusion, and acute renal failure (9).

NO/NOSs have been implicated in kidney I/R injury (13, 36–38). Estrogen up-regulates eNOS expression in kidney, but testosterone does not (39, 19). Neugarten et al. (19) found that the expression of eNOS and iNOS in the medulla is greater in female rats then in males. We found that both calcium-dependent and calcium-independent NOS activities are greater in females than in males. Testosterone treatment of females results in a mitigated increase of post-ischemic cNOS and ciNOS activity, whereas castration in males enhances cNOS and ciNOS activation. This effect of testosterone in vivo is mimicked in vitro in RAW 264.7 cells where we found that testosterone mitigated NO synthesis when cells were activated by lipopolysaccharide, whereas estrogen enhanced NO synthesis. L-Arginine ameliorates I/R injury in heart (41) and in the kidney (15) correlating with a decreased inflammatory reaction and an anti-apoptotic effect. In our studies, L-arginine modestly reduces renal ischemic injury in males, whereas an eNOS or iNOS gene deletion or L-NNA modestly increases I/R-induced kidney injury in females. The effects of L-NNA, an inhibitor of multiple forms of NOS, are much greater when compared with either iNOS or eNOS gene deletion in females. We propose that the inhibition of NO/NOSs by testosterone plays an important role in the gender differences in functional consequences of ischemia, although, as reflected by the effects of L-NNA in females and L-arginine in males, NO/NOS is not the only contributor to gender differences in susceptibility to ischemic injury of the kidney.

It has been suggested that activation of the phosphatidylinositol 3-kinase/Akt pathway protects organ or cells against I/R injury and hypoxia through suppression of the cell death machinery. NO has been reported to protect against apoptotic cell death through the phosphatidylinositol 3-kinase/Akt pathway (42). Camper-Kirby et al. (2) reported that young women possess higher levels of activated Akt than comparably aged men or postmenopausal woman, and sexually mature female mice have higher levels of activated Akt than male mice. These authors suggest that the higher resistance of females to cardiovascular diseases might be related to these differences in Akt activation. Recently Simoncini et al. (14) reported that estrogen prevents vascular leukocyte accumulation in muscle via the phosphatidylinositol 3-kinase/Akt/eNOS signal pathway. Recently Chao et al. (43) demonstrated that local insulin-like growth factor-I gene transfer, which constitutively activates Akt but does not increase serum insulin-like growth factor-I, thus avoiding increased inflammatory reactions, protects cardiomyocytes from ischemia/reperfusion. Some have demonstrated that insulin-like growth factor-I treatment ameliorates ischemic acute renal failure (44, 45), whereas others have reported that insulin-like growth factor-1 worsens kidney I/R injury because of greater inflammatory responses (46, 47).

In our studies, the levels of activated Akt in kidney prior to and post-ischemia are much greater in females than in males. Castration increases the levels of baseline and post-ischemic-activated Akt in males, whereas testosterone treatment of females or castrated males decreases the levels of activated Akt. The Akt activation in the protected animals is greater and sustained longer than in non-protected animals. Thus, the greater and prolonged activation of Akt after ischemia may contribute to gender-related kidney resistance to ischemia, and the sustained activation of Akt may be down-regulated by testosterone. Our data indicate that the exposure of renal MDCK epithelial cells to DETA NONOate, a NO donor, activates Akt and reduces oxidant-induced cell injury. The cleavage of poly(ADP-ribose) polymerase is higher in males than in females. We propose that testosterone regulation of the Akt/NOS pathway may explain the significant gender differences in susceptibility to ischemic injury. The higher susceptibility of males to ischemia is mediated by reduced production and anti-inflammatory effects of NO and reduced levels of sustained Akt phosphorylation leading to enhanced necrosis and apoptosis.

We have reported previously that post-ischemic activation of JNK is positively correlated with the extent of renal injury, whereas ERK activation is correlated with protection both in vivo and in vitro (10, 11, 27). These prior observations are consistent with our findings that post-ischemic activation of JNK is much less, and post-ischemic activation of ERK is much greater, in females than in males. Testosterone treatment of females increases post-ischemic JNK phosphorylation and decreases ERK and castration increases the ratio of ERK to JNK phosphorylation. di Mari et al. (48) have proposed that ERK and JNK have counteracting influences on kidney survival during stress. JNK accentuates inflammatory reactions and apoptotic cell death. Akt activation down-regulates JNK activation in human melanoma cells (49). Ischemia-induced JNK activation is negatively correlated with Akt activation. Thus our overall hypothesis for the mechanism of testosterone-induced potentiation of post-ischemic injury involves the down-regulation of NOS enzymes resulting in less NO and hence less Akt phosphorylation, which results in reduced cellular stress and reduced activation of the JNK kinases. Whether the protective effect involves up-regulation of ER stress proteins, which act through up-regulation of the ERK and down-regulation of the JNK kinase (27), awaits additional evaluation. It is possible that the forkhead family of transcription factors, which we have recently found to be up-regulated with ischemia in males (50), are involved in this effect of Akt.

Our data may have important implications for understanding the pathophysiology of toxemia of pregnancy. Total and free testosterone levels are elevated in preeclamptic women in the third trimester of pregnancy compared with normotensive controls (40). These elevated testosterone levels could be responsible for enhanced renal inflammation and endothelial and tubular cell injury that might lead to toxemia.

In summary, our data indicated that the presence of testosterone, rather than the absence of estrogen, via non-androgen receptor-mediated effects, plays a critical role in gender differences in susceptibility to kidney ischemic injury. The vulnerability of males is associated with much less activation of NOSs, less activation of Akt and ERK, greater activation of JNK, greater expression of ICAM-1, greater infiltration of leukocytes, and more apoptosis. Our findings may have important implications for understanding the pathophysiology of toxemia of pregnancy and should lead to consideration of androgens as targets for protective strategies to avoid acute renal failure in acutely ill patients.


   FOOTNOTES
 
* This work was supported by the Young Investigator Grant of the National Kidney Foundation, the Korea Research Foundation KRF-2004-003-E00245, and Kyungpook National University Research Fund 2004 (to K. M. P.) and by Grants DK 39773, DK 38452, DK46267, and NS 10828 from the National Institutes of Health (to J. V. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Present address: Dept. of Cardiology, School of Medicine, Chonnam National University, Kwangju, 501–190, Korea. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-525-5960; Fax: 617-525-5965; E-mail: joseph_bonventre{at}hms.harvard.edu.

1 The abbreviations used are: I/R, ischemia/reperfusion; ICAM-1, intracellular adhesion molecule-1; NO, nitric oxide; NOS, NO synthase; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; BUN, urea nitrogen; Pcr, plasma creatinine; Ccr, creatinine clearance; BW, body weight; L-NNA, N{omega}-nitro-l-arginine; MPO, myeloperoxidase; MDCK, Madin-Darby canine kidney; DHT, dihydrotestosterone; cNOS, calcium-dependent NOS; ciNOS, calcium-independent NOS; eNOS, endothelial NOS; IMV, intact males treated with vehicle; IFV, intact females treated with vehicle; CMV, castrated males treated with vehicle; OFV, ovarectomized females treated with vehicle; DETA NONOate, (Z)-1[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; LDH, lactate dehydrogenase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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