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• W2007656576 abstract "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 (Nω-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. 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 (Nω-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. 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 (1Fukuda K. Yao H. Ibayashi S. Nakahara T. Uchimura H. Fujishima M. Hall E.D. Stroke. 2000; 31: 155-160Crossref PubMed Scopus (102) Google Scholar, 2Camper-Kirby D. Welch S. Walker A. Shiraishi I. Setchell K.D. Schaefer E. Kajstura J. Anversa P. Sussman M.A. Circ. Res. 2001; 88: 1020-1027Crossref PubMed Scopus (246) Google Scholar, 3Squadrito F. Altavilla D. Squadrito G. Campo G.M. Arlotta M. Arcoraci V. Minutoli L. Serrano M. Saitta A. Caputi A.P. Eur. J. Pharmacol. 1997; 335: 185-192Crossref PubMed Scopus (104) Google Scholar, 4Sugden P.H. Clerk A. Circ. Res. 2001; 88: 975-977Crossref PubMed Scopus (51) Google Scholar). Recent studies, however, suggest that male hormones may also play important roles in gender differences in disease susceptibility (5Ling S. Dai A. Williams M.R. Myles K. Dilley R.J. Komesaroff P.A. Sudhir K. Endocrinology. 2002; 143: 1119-1125Crossref PubMed Scopus (94) Google Scholar, 6Muller V. Losonczy G. Heemann U. Vannay A. Fekete A. Reusz G. Tulassay T. Szabo A.J. Kidney Int. 2002; 62: 1364-1371Abstract Full Text Full Text PDF PubMed Google Scholar, 7Yang S.H. Perez E. Cutright J. Liu R. He Z. Day A.L. Simpkins J.W. J. Appl. Physiol. 2002; 92: 195-201Crossref PubMed Scopus (4) Google Scholar).Steroid hormones, including sex hormones, regulate inflammation, an important contributor to the pathophysiology of ischemia/reperfusion (I/R) 1The 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, Nω-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.1The 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, Nω-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.-induced tissue injury (8Kelly K.J. Williams W.J. Colvin R.B. Meehan S.M. Springer T.A. Gutierrez R.J. Bonventre J.V. J. Clin. Investig. 1996; 97: 1056-1063Crossref PubMed Scopus (672) Google Scholar, 9Sheridan A.M. Bonventre J.V. Curr. Opin. Nephrol. Hypertens. 2001; 9: 427-434Crossref Scopus (250) Google Scholar, 10Park K.M. Kramers C. Vayssier-Taussat M. Chen A. Bonventre J.V. J. Biol. Chem. 2002; 277: 2040-2049Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Park K.M. Chen A. Bonventre J.V. J. Biol. Chem. 2001; 276: 11870-11876Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). 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 (8Kelly K.J. Williams W.J. Colvin R.B. Meehan S.M. Springer T.A. Gutierrez R.J. Bonventre J.V. J. Clin. Investig. 1996; 97: 1056-1063Crossref PubMed Scopus (672) Google Scholar, 9Sheridan A.M. Bonventre J.V. Curr. Opin. Nephrol. Hypertens. 2001; 9: 427-434Crossref Scopus (250) Google Scholar). Our laboratory and others have reported that the reduction of inflammation secondary to the inhibition of ICAM-1 protects the kidney from I/R (8Kelly K.J. Williams W.J. Colvin R.B. Meehan S.M. Springer T.A. Gutierrez R.J. Bonventre J.V. J. Clin. Investig. 1996; 97: 1056-1063Crossref PubMed Scopus (672) Google Scholar, 12Rabb H. Mendiola C.C. Saba S.R. Dietz J.R. Smith C.W. Bonventre J.V. Ramirez G. Biochem. Biophys. Res. Commun. 1995; 211: 67-73Crossref PubMed Scopus (137) Google Scholar).An important endogenous modulator of I/R-induced tissue injury is nitric oxide (NO) (13Park K.M. Byun J.Y. Kramers C. Kim J.I. Huang P.L. Bonventre J.V. J. Biol. Chem. 2003; 278: 27256-27266Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 14Simoncini T. Hafezi M.A. Brazil D.P. Ley K. Chin W.W. Liao J.K. Nature. 2000; 407: 538-541Crossref PubMed Scopus (1218) Google Scholar). NO, the product of NO synthases (NOSs), possesses anti-inflammatory and vasodilative activity and is anti-apoptotic through Akt signaling pathways (13Park K.M. Byun J.Y. Kramers C. Kim J.I. Huang P.L. Bonventre J.V. J. Biol. Chem. 2003; 278: 27256-27266Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 14Simoncini T. Hafezi M.A. Brazil D.P. Ley K. Chin W.W. Liao J.K. Nature. 2000; 407: 538-541Crossref PubMed Scopus (1218) Google Scholar, 15Schramm L. La M. Heidbreder E. Hecker M. Beckman J.S. Lopau K. Zimmermann J. Rendl J. Reiners C. Winderl S. Wanner C. Schmidt H.H. Kidney Int. 2002; 61: 1423-1432Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 16Alexander B.T. Cockrell K. Cline F.D. Granger J.P. Hypertension. 2002; 39: 586-590Crossref PubMed Scopus (14) Google Scholar, 17Hickey M.J. Clin. Sci. (Lond.). 2001; 100: 1-12Crossref PubMed Scopus (78) Google Scholar). Sex hormones are known to regulate NO synthesis (18Friedl R. Brunner M. Moeslinger T. Spieckermann P.G. Life Sci. 2000; 68: 417-429Crossref PubMed Scopus (75) Google Scholar, 19Neugarten J. Ding Q. Friedman A. Lei J. Silbiger S. J. Am. Soc. Nephrol. 1997; 8: 1240-1246PubMed Google Scholar).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 METHODSAnimal Preparation—Experiments were performed in age-matched (12Rabb H. Mendiola C.C. Saba S.R. Dietz J.R. Smith C.W. Bonventre J.V. Ramirez G. Biochem. Biophys. Res. Commun. 1995; 211: 67-73Crossref PubMed Scopus (137) Google Scholar, 13Park K.M. Byun J.Y. Kramers C. Kim J.I. Huang P.L. Bonventre J.V. J. Biol. Chem. 2003; 278: 27256-27266Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 14Simoncini T. Hafezi M.A. Brazil D.P. Ley K. Chin W.W. Liao J.K. Nature. 2000; 407: 538-541Crossref PubMed Scopus (1218) Google Scholar 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 (10Park K.M. Kramers C. Vayssier-Taussat M. Chen A. Bonventre J.V. J. Biol. Chem. 2002; 277: 2040-2049Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Park K.M. Chen A. Bonventre J.V. J. Biol. Chem. 2001; 276: 11870-11876Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) 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 17β-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), Nω-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 (10Park K.M. Kramers C. Vayssier-Taussat M. Chen A. Bonventre J.V. J. Biol. Chem. 2002; 277: 2040-2049Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Park K.M. Chen A. Bonventre J.V. J. Biol. Chem. 2001; 276: 11870-11876Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar).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. (20Pasceri V. Wu H.D. Willerson J.T. Yeh E.T. Circulation. 2000; 101: 235-238Crossref PubMed Scopus (499) Google Scholar) 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 17β-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 (10Park K.M. Kramers C. Vayssier-Taussat M. Chen A. Bonventre J.V. J. Biol. Chem. 2002; 277: 2040-2049Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar).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 (10Park K.M. Kramers C. Vayssier-Taussat M. Chen A. Bonventre J.V. J. Biol. Chem. 2002; 277: 2040-2049Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar)Immunoblot Analysis—Immunoblot analyses were performed as described previously (10Park K.M. Kramers C. Vayssier-Taussat M. Chen A. Bonventre J.V. J. Biol. Chem. 2002; 277: 2040-2049Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Park K.M. Chen A. Bonventre J.V. J. Biol. Chem. 2001; 276: 11870-11876Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) 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.RESULTSGender 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).Fig. 2Effect 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.View Large Image Figure ViewerDownload (PPT)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).Fig. 3Effect 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), 17β-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 17β-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, 17β-estradiol-treated; F, female; I, intact; M, male; O, ovariectomy; T, testosterone-treated; V, vehicle-treated.View Large Image Figure ViewerDownload (PPT)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.Fig. 4Effect 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), 17β-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, 17β-estradiol-treated; I, intact; T, testosterone-treated; V, vehicle-treated.View Large Image Figure ViewerDownload (PPT)Fig. 5Effect 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-β 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 17β-estradiol (40 μg/kg of BW), 17β-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, 17β-estradioltreated; I, intact; T, testosterone-treated; V, vehicle-treated.View Large Image Figure ViewerDownload (PPT)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).Fig. 6Effect 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), 17β-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, 17β-estradiol-treated; F, female; I, intact; M, male; O, ovariectomy; T, testosterone-treated; V, vehicle-treated.View Large Image Figure ViewerDownload (PPT)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).Fig. 7Calcium-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), 17β-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" @default.
• W2007656576 created "2016-06-24" @default.
• W2007656576 creator A5030182387 @default.
• W2007656576 creator A5034147174 @default.
• W2007656576 creator A5046481926 @default.
• W2007656576 creator A5086651935 @default.
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• W2007656576 date "2004-12-01" @default.
• W2007656576 modified "2023-10-16" @default.
• W2007656576 title "Testosterone Is Responsible for Enhanced Susceptibility of Males to Ischemic Renal Injury" @default.
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