Estrogen Alters Ischemia-Reperfusion Injury-Induced Glomerular Barrier Failure In Vivo and In Vitro
Michael P Hutchens, M.D.1, Yasuharu Kosaka, M.D.1, Paco Herson, Ph.D.1, Patricia D. Hurn Ph.D.1, Sharon Anderson,M.D.2
1Department of Anesthesiology & Perioperative Medicine, Oregon Health & Science University
2Division of Nephrology & Hypertension, Oregon Health & Science University
ABSTRACT
RESULTS
Introduction: Estrogen is renoprotective after cardiac arrest and is known to be active at endothelial barriers. Investigation into renal
ischemia has focused on tubular epithelial cells, but the protective effect of estrogen suggests endothelium may be importantly
involved. The glomerular endothelium forms an important part of the filtration barrier in the kidney, and is damaged by ischemiareperfusion. We hypothesized cardiac arrest damages the glomerular barrier, and that estrogen might protect glomerular endothelial
cells.
Methods: In vivo: Ovariectomized female C57BL/6 mice were treated with vehicle (VEH) and subjected to sham procedure or treated
with VEH or estradiol (EST) and subjected to 10 min apneic cardiac arrest, induced by potassium chloride, followed by cardiopulmonary
resuscitation (CA/CPR) as previously described. 24h after CA/CPR, fluorescent conjugated Ficoll-70, a 50kD polysaccharide (FITCFicoll), was administered intravenously. 1h later, mice were killed, urine and blood samples taken, and BUN, creatinine, and urine
fluorescence measured. In vitro: Immortomouse-derived glomerular endothelial cells were raised to 80% confluence on transwells
(Corning, Lowell, MA). Medium containing 0.1 uM EST or VEH was applied and cells subjected to 3h oxygen-glucose deprivation.
Medium was then replaced with color/drug-free media with FITC-Ficoll (some concentration or other) in the upper chamber only. After
4h, fluorescence of lower chamber media was measured with a VICTOR fluorescence spectrometer (Perkin Elmer, Waltham, MA).
Analysis: Data analysis was performed with Prism 5.0 software. All data are reported as mean±SEM. Three group comparisons were
performed with 1-way ANOVA. Two group matched pair comparisons were performed using Wilcoxon’s analysis. Statistical
significance was set at p < 0.05.
Results: CA/CPR massively increased glomerular macromolecular flux (sham 0.061±0.010, CA/VEH 0.510±0.033, CA/EST
0.390±0.072, mcg/hr, p<0.05 v sham, n=3-4/group) in vivo. There was a trend toward reduced flux in EST treated animals compared
with VEH. In vitro, 3h OGD massively increased macromolecular flux. EST treatment during OGD significantly reduced flux compared
with VEH (EST 0.013±0.0005, VEH 0.015±0.0009 mcg/hr/cm2,*=p<0.05, figure 1).
Discussion: Endothelial injury is a common sequel of ischemia reperfusion injury. In the kidney, glomerular endothelium is a component
of the filtration barrier but is also uniquely positioned sero- and uro-luminally upstream of tubular epithelial cells, the loss of which
produces the clinical findings of acute kidney injury. We have shown that function of glomerular endothelium is altered after ischemia
reperfusion in vivo and in vitro. In addition we found that estrogen protects barrier function in vitro, and perhaps in vitro.
BACKGROUND
Estrogen protects against ischemia-reperfusion (IRI) induced AKI in a mouse model of
cardiac arrest.(1)
IRI damages the glomerular filtration barrier, of which a part is glomerular endothelium.(2)
Estrogen is active on renal endothelium in IRI.(3)
Short (3h) estrogen exposure (only during 3h OGD) reduced
the OGD-induced increase in macromolecular flux as
measured after 4h reoxygenation. Means±SEM are shown.
Serum-to-urine macromolecular flux was massively increased 4h
after surgery in animals undergoing CA/CPR procedure as
compared with sham. Numbers in bars are group sizes. Shown
are means ±SEM.
HYPOTHESIS
We hypothesized that ischemia-reperfusion damages
glomerular endothelial barrier function and estrogen might
protect against this damage.
METHODS
In Vivo Cardiac Arrest (CA):
•Ovariectomized female C57BL/6 mice were treated with implanted silastic pellets
containing either vehicle (VEH) or 17β-estradiol (EST, 6.3 mcg) for 7 days.
•CA Induced with KCL (0.5M ).
•During CA, Rectal temperature was controlled 36.5-37.5°C.
•Resuscitation with 0.3 -0.6 mg/kg epinephrine (8-16 mcg) and chest compressions
(300 beats/min)
•24h after CA/CPR TRITC-Ficoll and bumetanide 0.25 mg given IV
•1h later, mice were killed, and BUN, serum creatinine, and urine TRITC fluorescence
measured
Treatment
24 hours
7 days
10
min
(EST or VEH
Cardiac arrest
Pellet
Implantation)
Normal Rectal temperature
Administer IV TRITC/Ficoll
& bumetanide
1 hour
Measure Urine
TRITC/Ficoll
In Vitro Gomerular endothelial cell (GENDO) oxygen/glucose deprivation (OGD):
•GENDOs (a gift of Dr. Michael Madaio) grown on membrane supports.
•Transendothelial resistance (TEER) measured daily, experiments performed when
TEER>20Ω
•Cells treated with 17β-estradiol (100nM) or vehicle, only during OGD
•3 or 8h OGD followed by 4h reoxygenation
•At removal from OGD, FITC-Ficoll (50kD) to upper chambers after thorough rinsing
•Fluorescence quantification at 2,4h from lower chambers
•TEER at 4h
Upper Chamber with FITC-Ficoll added
GENDO cell layer
Lower chamber
REFERENCES:
1. Hutchens M, et al, Anesthesiology 2010; 112.
2. Andersson M, et al, Am J Renal Physiol 2007; 292(6)
3. Takaoka M, et al, Clin Sci 2002; 103 Suppl 48
Longer OGD increased the damage to barrier function and
estrogen ameliorated this effect. 8h OGD followed by 4h
reoxygenation, mean±SEM shown.
A nonsignificant trend toward reduced serum-to-urine
macromolecular flux was observed in estrogen-treated animals.
Power analysis suggests group sizes of 15 may be significantly
different. These experiments are ongoing. Numbers in bars are
group sizes. Shown are means ±SEM.
After 8h OGD, TEER was lower in vehicle-treated groups
than in estrogen-treated groups. Measurement at 4h
reoxygenation, mean ±SEM shown.
CONCLUSION
In vitro, estrogen treatment protects against OGD-induced glomerular endothelial barrier failure. In vivo, CA/CPR massively
increases serum-to-urine macromolecular flux, and estrogen may protect against this insult. Further investigation is ongoing.