Estrogen protects renal endothelial barrier function from ischemia-reperfusion in vitro and in vivo

Abstract

Emerging evidence suggests that renal endothelial function may be altered in ischemia-reperfusion injury. Acute kidney injury is sexually dimorphic, and estrogen protects renal tubular function after experimental ischemic injury. This study tested the hypothesis that during ischemia-reperfusion, estrogen alters glomerular endothelial function to prevent hyperpermeability. Glomerular endothelial cells were exposed to 8-h oxygen-glucose deprivation (OGD) followed by 4- and 8-h reoxygenation-glucose repletion. After 4-h reoxygenation-glucose repletion, transendothelial permeability to Ficoll-70 was reduced, and transendothelial resistance increased, by 17β-estradiol vs. vehicle treatment during OGD (OGD-vehicle: 91.0 ± 11.8%, OGD-estrogen: 102.6 ± 10.8%, P < 0.05). This effect was reversed by coadministration of G protein-coupled receptor 30 (GPR30) antagonist G15 with 17β-estradiol (OGD-estrogen-G15: 89.5 ± 6.9, P < 0.05 compared with 17β-estradiol). To provide preliminary confirmation of this result in vivo, Ficoll-70 was administered to mice 24 h after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Blood urea nitrogen (BUN) and serum creatinine (SCr) in these mice were elevated within 12 h following CA/CPR and reduced at 24 h by pretreatment with 17β-estradiol (BUN/SCr 17β-estradiol: 34 ± 19/0.2 ± 0.1 vehicle: 92 ± 49/0.5 ± 0.3, n = 8-12, P < 0.05). Glomerular sieving of Ficoll 70 was increased by CA/CPR within 2 h of injury and 17β-estradiol treatment (θ; 17β-estradiol: 0.74 ± 0.26 vs. vehicle: 1.05 ± 0.53, n = 14-15, P < 0.05). These results suggest that estrogen reduces postischemic glomerular endothelial hyperpermeability at least in part through GPR30 and that estrogen may regulate post CA/CPR glomerular permeability in a similar fashion in vivo.

Fig. 1.

RT-PCR for cell markers on glomerular endothelial cells (gENCs). gENCs express endothelial cell markers CD102, CD31, and CD34. They do not express epithelial cell markers S100a and EPCAM -1. All 3 known estrogen receptors (ER), ERα, ERβ, and G protein-coupled receptor 30 (GPR30), are expressed on gENCs.

Fig. 2.

Transendothelial resistance after 8-h oxygen-glucose deprivation (OGD) and 4-h reoxygenation-glucose repletion (RGR). Cells were treated with vehicle (VEH), 100 nM 17β-estradiol (EST), or 100 nM 17β-estradiol (EST) and 1,000 nM G15 (a selective GPR30 antagonist). Control cells were exposed to normoxic conditions in vehicle (Normox). Drug treatments were removed following OGD. Estrogen-treated cell monolayers exhibited significantly more electrical resistance than those treated with vehicle and were not significantly different from non-OGD exposed cells. This protective effect was reversed by the addition of G15. NS, not significant. Values are means ± SD.

Fig. 3.

Transendothelial resistance after 8-h OGD and 8-h RGR. Cells were treated with vehicle, 100 nM 17β-estradiol, or 100 nM 17β-estradiol and 1,000 nM G15 (a selective GPR30 antagonist). Control cells were exposed to normoxic conditions in vehicle (Normox). Drug treatments were removed following OGD. At the 8-h time point, the protective effect of estrogen was lost. Drug-treated cells were not different from vehicle-treated cells, and all OGD-exposed cell monolayers exhibited reduced electrical resistance compared with control (Normox) cell monolayers. Values are means ± SD.

Fig. 4.

Transendothelial flux of FITC-labeled Ficoll-70 after 8-h OGD and 4-h RGR. Cells were treated with vehicle, 100 nM 17β-estradiol, or 100 nM 17β-estradiol and 1,000 nM G15 (a selective GPR30 antagonist). Control cells were exposed to normoxic conditions in vehicle. Drug treatments were removed following OGD. Estrogen-treated cell monolayers transited significantly less FITC-Ficoll-70 than those treated with vehicle and were not significantly different from non-OGD exposed cells. This protective effect was reversed by the addition of G15. Values are means ± SD.

Fig. 5.

Transendothelial flux of FITC-Ficoll-70 after 8-h OGD and 8-h RGR. Cells were treated with vehicle, 100 nM 17β-estradiol, or 100 nM 17β-estradiol and 1,000 nM G15 (a selective GPR30 antagonist). Control cells were exposed to normoxic conditions in vehicle. Drug treatments were removed following OGD. At the 8-h time point, the protective effect of estrogen was lost. Drug-treated cells were not different from vehicle-treated cells, and all OGD-exposed cell monolayers transited more FITC-Ficoll-70 compared with control (Normox) cell monolayers. Values are means ± SD.

Fig. 6.

Serum creatinine and blood urea nitrogen (BUN) following sham or cardiac arrest/cardiopulmonary resuscitation (CA/CPR) surgery. After sham surgery (time 0) or 2, 6, 12, 18, 24, or 48 h after CA/CPR, separate cohorts of mice were assessed for serum content of creatinine and BUN. Values peaked and were significantly elevated relative to sham (P < 0.05, n = 5–7/group) at 12 h post-CA/CPR.

Fig. 7.

Glomerular sieving coefficient for Ficoll-70 (θ_Ficoll-70) in gonadally intact CA/CPR-naive mice (Naive-INT), ovariectomized CA/CPR-naive mice (Naive-OVX), and CA/CPR-treated mice at 2, 6, 12, and 24 h following arrest and resuscitation. At the specified time point, mice were reanesthetized and administered fluorescent-labeled Ficoll-70, bumetanide, and intravenous 0.9% sodium chloride solution. Eighty minutes later, the mice were euthanized and blood and serum fluorescence were evaluated to determine Ficoll-70 concentration. Removal of physiological estrogen by ovariectomy did not alter θ_Ficoll-70. Twenty-four hours following CA/CPR, θ_Ficoll-70 was significantly elevated, but then rapidly declined to values similar to those found in CA/CPR-naive animals. Numbers in bars are n.

Fig. 8.

Serum BUN (A) and creatinine (B) were measured 24 h following CA/CPR and were significantly lower in estrogen-treated animals than in controls (P < 0.05, n = 8–12/group).

Fig. 9.

θ_Ficoll-70 in vehicle- and estrogen-treated animals 2 h (A) and 24 h (B) following CA/CPR. At the specified time point, mice were reanesthetized and administered fluorescent-labeled Ficoll-70, bumetanide, and intravenous 0.9% sodium chloride solution. Eighty minutes later, the mice were euthanized and blood and serum fluorescence were evaluated to determine Ficoll-70 concentration. Administration of 17β-estradiol (estrogen) significantly reduced glomerular sieving of Ficoll-70 at the 2-h, but not the 24-h post-CA/CPR time point. Numbers in bars are n.