Hyperoxia Increases Kidney Injury During Renal Ischemia and Reperfusion in Mice

Abstract

Background: Renal ischemia and reperfusion (IR) contribute to perioperative acute kidney injury, and oxygen is a key regulator of this process. We hypothesized that oxygen administration during surgery and renal IR would impact postoperative kidney function and injury in mice.

Methods: Mice were anesthetized, intubated, and mechanically ventilated with a fraction of inspired oxygen (F io2 ) 0.10 (hypoxia), 0.21 (normoxia), 0.60 (moderate hyperoxia), or 1.00 (severe hyperoxia) during 67 minutes of renal IR or sham IR surgery. Additional mice were treated before IR or sham IR surgery with 50 mg/kg tempol, a superoxide scavenger. At 24 hours, mice were sacrificed, and blood and kidney collected. We assessed and compared kidney function and injury across groups by measuring blood urea nitrogen (BUN, primary end point), renal histological injury, renal expression of neutrophil gelatinase-associated lipocalin (NGAL), and renal heme oxygenase 1 ( Ho-1 ), peroxisome proliferator-activated receptor gamma coactivator 1-α ( Pgc1-α ), and glutathione peroxidase 4 ( Gpx-4 ) transcripts, to explore potential mechanisms of any effect of oxygen.

Results: Hyperoxia and hypoxia during renal IR surgery decreased renal function and increased kidney injury compared to normoxia. Baseline median (interquartile range) BUN was 22.2 mg/dL (18.4-26.0), and 24 hours after IR surgery, BUN was 17.5 mg/dL (95% confidence interval [CI], 1.3-38.4; P = .034) higher in moderate hyperoxia-treated animals, 51.8 mg/dL (95% CI, 24.9-74.8; P < .001) higher in severe hyperoxia-treated animals, and 64.9 mg/dL (95% CI, 41.2-80.3; P < .001) higher in hypoxia-treated animals compared to animals treated with normoxia ( P < .001, overall effect of hyperoxia). Hyperoxia-induced injury, but not hypoxia-induced injury, was attenuated by pretreatment with tempol. Histological injury scores, renal NGAL staining, and renal transcription of Ho-1 and suppression of Pgc1- α followed the same pattern as BUN, in relation to the effects of oxygen treatment.

Conclusions: In this controlled preclinical study of oxygen treatment during renal IR surgery, hyperoxia and hypoxia impaired renal function, increased renal injury, and impacted expression of genes that affect mitochondrial biogenesis and antioxidant response. These results might have implications for patients during surgery when high concentrations of oxygen are frequently administered, especially in cases involving renal IR.

Figure 1:

Experimental design matrix. One hundred and sixty-four mice were assigned to one of four oxygen treatment groups and underwent either arterial blood gas analysis, renal ischemia and reperfusion (IR) surgery, or sham IR surgery. Some mice were pre-treated with the superoxide scavenger tempol and others with vehicle.

Figure 2:

Oxygenation. Partial pressure of arterial oxygen (PaO₂) in mice ventilated with hypoxia (FiO₂ 0.10), normoxia (FiO₂ 0.21), moderate hyperoxia (FiO₂ 0.60), and severe hyperoxia (FiO₂ 1.00). P value represents the effect of oxygen treatment on PaO₂ (n=5/group).

Figure 3:

Renal Function. Blood urea nitrogen (BUN) at 24-hours in mice exposed to hypoxia, normoxia, moderate hyperoxia, and severe hyperoxia during renal IR surgery (A) and sham IR surgery (B). Effect of hyperoxia treatment determined by Jonckheere-Terpstra test for trend across normoxia, moderate hyperoxia, and severe hyperoxia groups. * indicates p <0.05 for comparison between animals administered hypoxia vs. normoxia and # indicates p<0.05 for comparison of tempol vs. vehicle within each oxygen treatment, determined by Mann-Whitney U-Test.

Figure 4:

Histological injury at 24-hours in mice exposed to hypoxia, normoxia, moderate hyperoxia, and severe hyperoxia during renal IR surgery. A) Representative images of PAS-stained corticomedullary kidney sections at 400x magnification. B) Tissue injury score. Effect of hyperoxia treatment determined by Jonckheere-Terpstra test for trend across normoxia, moderate hyperoxia, and severe hyperoxia groups. * indicates p <0.05 for comparison between animals administered hypoxia vs. normoxia, and # indicates p<0.05 for comparison of tempol vs. vehicle within each oxygen treatment, determined by Mann-Whitney U-Test.

Figure 5:

Neutrophil gelatinase associated lipocalin (NGAL) staining at 24 hours in mice exposed to hypoxia, normoxia, moderate hyperoxia, and severe hyperoxia during renal IR surgery. A) Representative images of immunohistochemical staining for NGAL. B) Threshold quantification of NGAL expression. Effect of hyperoxia treatment determined by Jonckheere-Terpstra test for trend across normoxia, moderate hyperoxia, and severe hyperoxia groups. * indicates p <0.05 for comparison between animals administered hypoxia vs. normoxia, and # indicates p<0.05 for comparison of tempol vs. vehicle within each oxygen treatment, determined by Mann-Whitney U-Test.

Figure 6:

Renal Ho-1 (A), Pgc1-α (B), and Gpx-4 (C) expression at 24-hours in mice exposed to hypoxia, normoxia, moderate hyperoxia, and severe hyperoxia during renal IR surgery. Effect of hyperoxia treatment determined by Jonckheere-Terpstra test for trend across normoxia, moderate hyperoxia, and severe hyperoxia groups. * indicates p <0.05 for comparison between animals administered hypoxia vs. normoxia, and # indicates p<0.05 for comparison of tempol vs. vehicle within each oxygen treatment, determined by Mann-Whitney U-Test.