Male C57BL/6 mice aged 8 to 10 weeks were purchased from Orient Bio (Seongnam, Korea). All mouse experiments were performed in accordance with the animal protocols approved by the Institutional Animal Care and Use Committee of Jeju National University. Mice were subjected to 30 minutes of bilateral kidney ischemia followed by 6 hours or 24 hours of reperfusion or sham operation under anesthesia through an intraperitoneal injection of a cocktail containing ketamine (200 mg/kg body weight) and xylazine (16 mg/kg body weight), as previously described [14151617]. Sham-operated mice underwent the same surgical procedure except for the induction of ischemia. Spermidine (10 mg/kg body weight; Sigma, St. Louis, MO, USA) was dissolved in distilled water (vehicle) and administered orally at 24 hours and 1 hour before ischemia or sham operation. PARP1 inhibitor PJ34 (10 mg/kg body weight, R&D Systems, Minneapolis, MN, USA) or saline (vehicle) was administered intraperitoneally at 24 hours and 1 hour before ischemia or sham operation [1819]. To use a small interfering RNA (siRNA) targeting ODC in vivo, ODC siSTABLE siRNA (50 µg in 1 ml phosphate buffered saline [PBS]) modified from siGENOME (Dharmacon, Lafayette, CO, USA) or siSTABLE non-targeting siRNA (control siRNA; 50 µg in 1 ml PBS) was injected within 10 seconds into the tail vein at 48 hours and 24 hours before ischemia, as previously described [1620].

Histological damage due to tubular injury in the outer medulla of periodic acid-Schiff (PAS)-stained kidney sections was scored by counting the percentage of tubules that displayed tubular necrosis, cast formation, and tubular dilation as follows: 0, normal; 1, 1% to 10%; 2, 11% to 25%; 3, 26% to 50%; 4, 51% to 75%; and 5, 76% to 100%. Five randomly chosen high-power (×200 magnification) fields per kidney were used for the scoring [141516181921].

Bood samples were taken from the retro-ocular venous plexus at 6 hours or 24 hours after reperfusion. Renal function was then assessed by determining the creatinine concentration in plasma using a QuantiChrom Creatinine kit (BioAssay Systems, Hayward, CA, USA) [1415161819].

PARP1 activity, 8-nitroguanine, and 8-hydroxy-2′-deoxyguanosine assays were performed in whole kidneys using a universal PARP colorimetric assay kit (Trevigen, Gaithersburg, MD, USA), a 8-nitroguanine DNA/RNA damage ELISA kit (Cell Biolabs, San Diego, CA, USA) and a DNA/RNA oxidative damage ELISA kit (Cayman, Ann Arbor, MI, USA), respectively, according to the manufacturer's instructions [181922232425].

Analysis of variance was used to compare data among groups using Systat SigmaPlot (Systat Software Inc., San Jose, CA, USA). Differences between two groups were assessed by two-tailed unpaired Student's t tests. P-values <0.05 were considered statistically significant. Each experimental group consisted of nine animals (n=9).

Here, it has been confirmed that IRI time-dependently and markedly increased tubular damage scored in PAS-stained kidney sections and kidney dysfunction represented by high concentrations of plasma creatinine at 6 hours and 24 hours after reperfusion in vehicle-treated mice (Fig. 1A–C). Although it has been reported that loss of spermine/spermidine-N(1)-acetyltransferase, an enzyme in spermidine degradation protects against IRI [26], a direct effect of spermidine supplement on IRI has not yet been investigated. In the present study, exogenous spermidine significantly reduced tubular damage during IRI (Fig. 1A, B). Consistent with the histological data, the increases in plasma creatinine concentrations were also significantly lessened by exogenous spermidine (Fig. 1C). These data indicate that exogenous spermidine attenuates histological and functional damage induced by IRI.

As excessive activation of PARP1, which catalyzes poly (ADP-ribosyl)ation of various proteins as well as PARP1 itself, contributes to tubular damage and kidney dysfunction during AKI induced by IRI or cisplatin nephrotoxicity [1418192223], PARP1 activity during IRI was evaluated in the kidneys treated with spermidine or vehicle. Vehicle-treated mouse kidneys showed marked increases in PARP1 activity at 6 hours and 24 hours after IRI, whereas treatment with spermidine significantly diminished the increases in PARP1 activity at both time-points (Fig. 2A). Since PARP1 activation is considered a downstream effector of DNA oxidative and nitrative stress [2728], nitrative and oxidative levels of DNA in these kidneys were assessed using measurements of 8-nitroguanine and 8-hydroxy-2′-deoxyguanosine concentrations, respectively. DNA nitration was time-dependently increased in kidneys of mice treated with vehicle at 6 hours and 24 hours after IRI, but spermidine-treated mouse kidneys showed a significant reduction in DNA nitration at both time-points (Fig. 2B). Intriguingly, DNA oxidation was significantly attenuated by treatment with spermidine at 24 hours after IRI, but not at 6 hours after IRI (Fig. 2C). According to the results of nitration and oxidation of DNA, it is possible to suggest that inhibition of PARP1 activation is caused by the reduction in DNA nitration in IRI-subjected kidneys of spermidine-treated mice, and that the inhibition of PARP1 activation may lead to the reduction in DNA oxidation.

Spermidine is generated from ODC, which catalyzes the decarboxylation of ornithine to form putrescine, and the latter is metabolized to spermidine. Thus, to determine whether spermidine depletion exaggerates IRI in vivo, genetic inhibition of ODC was induced via siRNA transfection into mice. DNA nitration in ODC siRNA (siODC)-transfected mouse kidneys was significantly more enhanced at 6 hours and 24 hours after IRI, compared with that in control siRNA (siControl)-transfected mouse kidneys (Fig. 3A). PARP1 activity in siODC-transfected mouse kidneys was also consistently more increased at both time-points (Fig. 3B). Next, it has been investigated whether ODC inhibition exacerbates histological and functional damage induced by IRI. The data shown in Fig. 3C and D indicate that ODC inhibition significantly increased tubular damage at 6 hours after IRI, but not at 24 hours after IRI, because the score at the time-point in siControl-transfected kidneys almost reached the highest level (5). Levels of plasma creatinine in siODC-transfected mouse kidneys at 6 hours and 24 hours after IRI were significantly higher than those in siControl-transfected mouse kidneys (Fig. 3E). These data indicate that genetic inhibition of ODC exaggerates the consequences of IRI.

To determine whether there is a hierarchy in spermidine depletion, DNA nitration, and PARP1 activation involved in IRI, pharmacological inhibition of PARP1 and supplementation of spermidine were carried out in ODC-knockdown IRI kidneys, respectively. As shown in Fig. 4A and B, spermidine supplement prevented IRI-induced DNA nitration and PARP1 activation in siODC-transfected mouse kidneys. Meanwhile, pharmacological PARP1 inhibition attenuated PARP1 activation in siODC-transfected mouse kidneys, but not DNA nitration. These results suggest that DNA nitration is upstream of PARP1 activation, and spermidine prevents PARP1 activation through DNA nitration in mouse kidneys after IRI. Finally, PARP1 inhibition and spermidine supplement attenuated functional and histological damage induced by IRI in siODC-transfected mouse kidneys (Fig. 4C–E).