Estrogen-Mediated Renoprotection following Cardiac Arrest and Cardiopulmonary Resuscitation Is Robust to GPR30 Gene Deletion

<div><p>Introduction</p><p>Acute kidney injury is a serious,sexually dimorphic perioperative complication, primarily attributed to hypoperfusion. We previously found that estradiol is renoprotective after cardiac arrest and cardiopulmonary resuscitation in ovariectomized female mice. Additionally, we found that neither estrogen receptor alpha nor beta mediated this effect. We hypothesized that the G protein estrogen receptor (GPR30) mediates the renoprotective effect of estrogen.</p><p>Methods</p><p>Ovariectomized female and gonadally intact male wild-type and GPR30 gene-deleted mice were treated with either vehicle or 17β-estradiol for 7 days, then subjected to cardiac arrest and cardiopulmonary resuscitation. Twenty four hours later, serum creatinine and urea nitrogen were measured, and histologic renal injury was evaluated by unbiased stereology.</p><p>Results</p><p>In both males and females, GPR30 gene deletion was associated with reduced serum creatinine regardless of treatment. Estrogen treatment of GPR30 gene-deleted males and females was associated with increased preprocedural weight. In ovariectomized female mice, estrogen treatment did not alter resuscitation, but was renoprotective regardless of GPR30 gene deletion. In males, estrogen reduced the time-to-resuscitate and epinephrine required. In wild-type male mice, serum creatinine was reduced, but neither serum urea nitrogen nor histologic outcomes were affected by estrogen treatment. In GPR30 gene-deleted males, estrogen did not alter renal outcomes. Similarly, renal injury was not affected by G1 therapy of ovariectomized female wild-type mice.</p><p>Conclusion</p><p>Treatment with 17β-estradiol is renoprotective after whole-body ischemia-reperfusion in ovariectomized female mice irrespective of GPR30 gene deletion. Treatment with the GPR30 agonist G1 did not alter renal outcome in females. We conclude GPR30 does not mediate the renoprotective effect of estrogen in ovariectomized female mice. In males, estrogen therapy was not renoprotective. Estrogen treatment of GPR30 gene-deleted mice was associated with increased preprocedural weight in both sexes. Of significance to further investigation, GPR30 gene deletion was associated with reduced serum creatinine, regardless of treatment.</p></div

Baseline and Resuscitation Data.

<p>*<i>p</i> values shown for 2-way ANOVA. Post-ANOVA individual comparisons are within the text. Abbreviations: WT: Wild Type; GPR30KO: GPR30 gene-deleted mice; VEH: vehicle-treated mice; EST: 17β-estradiol-treated mice; sUN: serum urea nitrogen; sCr: serum creatinine; VNC: volume of necrotic tubules; CPR: cardiopulmonary resuscitation; CA/CPR:cardiac arrest and cardiopulmonary resuscitation.</p

Representative Photomicrographs: Mouse kidneys were perfusion-fixed, removed, and stained with Flourojade-B, which stains early-necrotic cells bright green.

<p>Images taken of the cortico-medullary junction at 100× with no postprocessing other than pseudocolor simulating 532 nM flourescence. A)Female, wild-type vehicle control. B)Female, wild-type, estradiol-treated. C)Female, GPR30KO vehicle control. D)Female, GPR30KO, estradiol-treated. E)Male, wild-type vehicle control. F)Male, wild-type, estradiol-treated. G)Male, GPR30KO vehicle control. H)Male, GPR30KO, estradiol-treated. Second panel, wild-type vehicle vs. control. I)Wild-type, female vehicle control. J)Wild-type, female G1-treated.</p

Summary of Quantitative Outcomes in Ovariectomized Female Mice.

<p>*<i>p</i><0.05, values shown for 2-way ANOVA, with respect to control (vehicle-treated) in same strain. Abbreviations: WT: Wild Type; GPR30KO: GPR30 gene-deleted mice; VEH: vehicle-treated mice; EST: 17β-estradiol-treated mice; sUN: serum urea nitrogen; sCr: serum creatinine; VNC: volume of necrotic tubules.</p

Serum urea nitrogen, serum creatinine, and histologic outcomes 24/CPR in male mice.

<p>Estradiol treatment significantly reduced serum creatinine, but not serum urea nitrogen, or tubular cell death in wild-type (WT) males. GPR30 gene deletion (GPR30KO) was associated with reduced serum creatine overall, but estrogen treatment did not reduce renal injury injury in GPR30 gene-deleted male mice. (Mean±SEM, n = 6,8,9,10 respectively, for WT-VEH, WT-EST, GPR30KO-VEH, and GPR30KO-EST, *p<0.05).</p

Summary of Quantitative Outcomes in Male Mice.

<p>*<i>p</i><0.05, values shown for 2-way ANOVA, with respect to control (vehicle-treated) in same strain. Abbreviations: WT: Wild Type; GPR30KO: GPR30 gene-deleted mice; VEH: vehicle-treated mice; EST: 17β-estradiol-treated mice; sUN: serum urea nitrogen; sCr: serum creatinine; VNC: volume of necrotic tubules.</p

Serum urea nitrogen, serum creatinine, and histologic outcomes 24/CPR in ovariectomized female mice.

<p>Estradiol (EST) reduced renal injury in both wild-type (WT) and GPR30 gene-deleted (GPR30KO) mice, as evidenced by reduced serum urea nitrogen, serum creatinine, and volume fraction of necrotic tubules (% tubular cell death). (Mean±SEM, n = 8,13,7,6 respectively, for WT-VEH, WT-EST, GPR30KO-VEH, and GPR30KO-EST, *p<0.05).</p