Epoxyeicosatrienoic acid analogue mitigates kidney injury in a rat model of radiation nephropathy

Abstract

Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) by CYP epoxygenases, and EETs are kidney protective in multiple pathologies. We determined the ability of an EET analogue, EET-A, to mitigate experimental radiation nephropathy. The kidney expression of the EET producing enzyme CYP2C11 was lower in rats that received total body irradiation (TBI rat) compared with non-irradiated control. At 12 weeks after TBI, the rats had higher systolic blood pressure and impaired renal afferent arteriolar function compared with control, and EET-A or captopril mitigated these abnormalities. The TBI rats had 3-fold higher blood urea nitrogen (BUN) compared with control, and EET-A or captopril decreased BUN by 40-60%. The urine albumin/creatinine ratio was increased 94-fold in TBI rats, and EET-A or captopril attenuated that increase by 60-90%. In TBI rats, nephrinuria was elevated 30-fold and EET-A or captopril decreased it by 50-90%. Renal interstitial fibrosis, tubular and glomerular injury were present in the TBI rats, and each was decreased by EET-A or captopril. We further demonstrated elevated renal parenchymal apoptosis in TBI rats, which was mitigated by EET-A or captopril. Additional studies revealed that captopril or EET-A mitigated renal apoptosis by acting on the p53/Fas/FasL (Fas ligand) apoptotic pathway. The present study demonstrates a novel EET analogue-based strategy for mitigation of experimental radiation nephropathy by improving renal afferent arteriolar function and by decreasing renal apoptosis.

Keywords: Fas; afferent arteriole; apoptosis; novel small lipid molecule; radiotherapy.

Figure 1

Renal mRNA and protein expressions of CYP2C11 (A) and CYP2C23 (B) at 12-week after total-body radiation (TBI) compared to control (non-irradiated, vehicle-treated and age-matched). In immunoblotting experiments, protein expressions of CYP2C23 and CYP2C11 are normalized to the expression of β-actin. *P<0.05 vs. Control, data expressed as mean ± SEM, and n=5.

Figure 2

Systolic blood pressure (A) and renal afferent arteriolar responses to acetylcholine (B) in the experimental groups at the end of 12-week protocol. In determining renal afferent arteriolar response to acetylcholine, the baseline afferent arteriolar diameters in Vehicle, 11Gy+Vehicle, 11Gy+EET-A and 11Gy+Captopril were 26.2±2.1 (n=7), 26.7±0.9 (n=8), 25.8±0.6 (n=5) and 22.4±0.6 µm (n=8), respectively. *P<0.05 vs. Vehicle; #P<0.05 vs. 11Gy+Vehicle. All data expressed as mean ± SEM, and in systolic blood pressure measurement n=8 for each group.

Figure 3

a: (A) Blood urea nitrogen (BUN), (B) albuminuria, (C) nephrinuria, and (D) histopathological injury score in the experimental groups at the end of 12-week protocol. (E) Representative photomicrographs of Periodic Acid-Schiff staining (200×) depicting renal tubular casts and damaged glomeruli (yellow arrows) in the kidney sections of different experimental groups. P<0.05 vs. Vehicle, and #P<0.05 vs. 11Gy+Vehicle. Data expressed as mean ± SEM, and n=8 for each group. b: (A) Representative photomicrographs of Picrosirius Red staining (200×) depicting collagen formation (black arrows) in the kidney sections of the experimental groups. (B) Percentage of collagen positive area relative to total area of a kidney section analyzed (200×). Staining was done at the end of 12-week protocol. P<0.05 vs. Vehicle, and #P<0.05 vs. 11Gy+Vehicle. Data expressed as mean ± SEM, and n=8 for each group.

Figure 3

a: (A) Blood urea nitrogen (BUN), (B) albuminuria, (C) nephrinuria, and (D) histopathological injury score in the experimental groups at the end of 12-week protocol. (E) Representative photomicrographs of Periodic Acid-Schiff staining (200×) depicting renal tubular casts and damaged glomeruli (yellow arrows) in the kidney sections of different experimental groups. P<0.05 vs. Vehicle, and #P<0.05 vs. 11Gy+Vehicle. Data expressed as mean ± SEM, and n=8 for each group. b: (A) Representative photomicrographs of Picrosirius Red staining (200×) depicting collagen formation (black arrows) in the kidney sections of the experimental groups. (B) Percentage of collagen positive area relative to total area of a kidney section analyzed (200×). Staining was done at the end of 12-week protocol. P<0.05 vs. Vehicle, and #P<0.05 vs. 11Gy+Vehicle. Data expressed as mean ± SEM, and n=8 for each group.

Figure 4

Renal mRNA expression ratio of pro-apoptotic Bax (A) and Bak (B) relative to the mRNA expression of anti-apoptotic Bcl2 in the experimental groups. All measurements were done at the end of 12-week protocol. P<0.05 vs. Vehicle, and #P<0.05 vs. 11Gy+Vehicle. Data expressed as mean ± SEM, and n=6–8 for each group.

Figure 5

Renal mRNA expression (A) and the activity (B) of caspase 3, and (C) percentage (%) of TUNEL positive renal apoptotic cells in the kidney of different experimental groups. (D) Representative photomicrographs of TUNEL staining in the kidney of the experimental groups. All measurements were done at the end of 12-week protocol. P<0.05 vs. Vehicle, and #P<0.05 vs. 11Gy+Vehicle. Data expressed as mean ± SEM, and n=8 for each group.

Figure 6

Renal mRNA expressions of (A) FasL, (B) FasR, (C) caspase 8, (D) p53, (E) TNF-α, and (F) TNFR1 in the kidney of the experimental groups. All measurements were done at the end of 12-week protocol. P<0.05 vs. Vehicle, and #P<0.05 vs. 11Gy+Vehicle. Data expressed as mean ± SEM, and n=8 for each group.

Figure 7

Renal mRNA expression (A) and the activity (B) of caspase 9 in the experimental groups. All measurements were done at the end of 12-week protocol. P<0.05 vs. Vehicle, and #P<0.05 vs. 11Gy+Vehicle. Data expressed as mean ± SEM, and n=8 for each group.