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. 2010 Jul;299(1):C164-74.
doi: 10.1152/ajpcell.00512.2009. Epub 2010 Apr 14.

The role of spermidine/spermine N1-acetyltransferase in endotoxin-induced acute kidney injury

Kamyar Zahedi  1 , Sharon Barone, Debora L Kramer, Hassane Amlal, Leena Alhonen, Juhani Jänne, Carl W Porter, Manoocher Soleimani
Affiliations

The role of spermidine/spermine N1-acetyltransferase in endotoxin-induced acute kidney injury

Kamyar Zahedi et al. Am J Physiol Cell Physiol. 2010 Jul.

Abstract

The expression of catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO) increases after ischemic reperfusion injury. We hypothesized that polyamine catabolism is upregulated and that this increase in catabolic response contributes to tissue damage in endotoxin-induced acute kidney injury (AKI). SSAT mRNA expression peaked at threefold 24 h following LPS injection and returned to background levels by 48 h. The activity of SSAT correlated with its mRNA levels. The expression of SMO also increased in the kidney after LPS administration. Serum creatinine levels increased significantly at approximately 15 h, peaking by 24 h, and returned to background levels by 72 h. To test the role of SSAT in endotoxin-induced AKI, we injected wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice with LPS. Compared with SSAT-wt mice, the SSAT-ko mice subjected to endotoxic-AKI had less severe kidney damage as indicated by better preservation of kidney function. The role of polyamine oxidation in the mediation of kidney injury was examined by comparing the severity of renal damage in SSAT-wt mice treated with MDL72527, an inhibitor of both polyamine oxidase and SMO. Animals treated with MDL72527 showed significant protection against endotoxin-induced AKI. We conclude that increased polyamine catabolism through generation of by-products of polyamine oxidation contributes to kidney damage and that modulation of polyamine catabolism may be a viable approach for the treatment of endotoxin-induced AKI.

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Figures

Fig. 1.
Fig. 1.
Schematic presentation of the polyamine metabolic pathway. Spermidine (Spd) and spermine (Spm) are synthesized by the sequential addition of aminopropyl groups derived from decarboxylated S-adenosylmethionine onto putrescine (Put) and Spd to form Spd and Spm, respectively. Catabolism of polyamines proceeds via 2 distinct pathways: 1) the back conversion pathway [spermidine/spermine N1-acetyltransferase/polyamine oxidase (SSAT/PAO)] or 2) the oxidation of Spm by spermine oxidase (SMO). In the polyamine back conversion pathway, Spd and Spm are acetylated by SSAT and then oxidized by PAO, leading to generation of Put and catabolic by-products [i.e., hydrogen peroxide (H2O2), acetyl-3-aminopropionaldehyde]. The SMO oxidizes Spm directly and generates Spd, H2O2, and 3-aminopropanal.
Fig. 2.
Fig. 2.
Effect of LPS administration on kidney function. The effect of LPS treatment (10 mg/kg ip) on renal function was examined by measuring the serum creatinine levels at timed intervals up to 72 h after LPS administration (n = 6/group). Data were analyzed for statistical significance using ANOVA and P values <0.05. *P ≤ 0.05.
Fig. 3.
Fig. 3.
Expression and activity of SSAT increase in SSAT-treated wild-type (SSAT-wt) mice subjected to endotoxin-induced acute kidney injury (AKI). A: total RNA (30 μg/well) was extracted from kidneys at timed intervals following LPS or saline treatment (n = 3/treatment time point and n = 2 for control animals), size-fractionated by gel electrophoresis, and subjected to Northern blot analysis using radiolabeled SSAT cDNA probe. Equal loading was confirmed by the examination of 28s rRNA bands (bottom). A single band of ∼1.3 kb was recognized by the SSAT probe (top). B: SSAT activity was measured in kidney extracts 24 h after saline or LPS injections (n = 3/group). Data are expressed as pmol·min−1·mg protein−1. Statistical significance was determined using P values <0.05. *P ≤ 0.05.
Fig. 4.
Fig. 4.
Expression of SMO increases in SSAT-wt mice subjected to endotoxin-induced AKI. Total RNA (30 μg/well) from kidneys of LPS- or saline-treated animals (n = 3) was size-fractionated and then subjected to Northern blot analysis using radiolabeled SMO cDNA probe. A single band was recognized by the SMO probe (top). Equal loading was confirmed by the examination of 28s rRNA bands (bottom).
Fig. 5.
Fig. 5.
Measurement of polyamine levels in the kidneys of SSAT-wt mice subjected to endotoxin-induced AKI. Polyamine levels were measured in the kidneys of animals given intraperitoneal injection of saline or 10 mg/kg LPS (n = 3). Kidney polyamine contents were determined by HPLC. A: kidney Put levels were determined at timed intervals after treatment with LPS and compared with polyamine levels in the kidneys of saline-treated animals. *P ≤ 0.05; **P ≤ 0.01. B: Spd and Spm levels in the kidneys of saline- and LPS-treated animals. Polyamine levels are expressed as pmol/mg protein.
Fig. 6.
Fig. 6.
Comparison of Put levels in SSAT-wt and SSAT-deficient (SSAT-ko) mice in endotoxin-induced AKI. Kidney Put levels were measured in SSAT-wt and SSAT-ko animals (n = 3) 24 h after saline and LPS treatment. The Put contents in the kidneys of saline-treated (Cont) SSAT-wt and SSAT-ko animals were similar. Subsequent to LPS administration, Put levels increased by ∼4-fold in the kidneys of SSAT-wt animals compared with saline-treated animals of the same genotype. The kidney Put contents of the LPS-treated SSAT-ko animals were not significantly different from those of the saline-treated SSAT-ko and SSAT-wt mice. **P ≤ 0.01.
Fig. 7.
Fig. 7.
Comparison of renal function in SSAT-wt and SSAT ko mice after endotoxin-induced AKI. Serum creatinine levels were measured to assess the effect of SSAT deficiency on the severity of endotoxin-induced AKI. A colorimetric assay was utilized to obtain creatinine levels in the sera of vehicle- and LPS-treated SSAT-wt and SSAT-ko animals (n = 8/group) at 24 h posttreatment. Whereas serum creatinine levels of saline-treated SSAT-ko and SSAT-wt mice were similar, those of SSAT-ko animals subjected to LPS treatment were significantly (*P ≤ 0.05) lower than those of similarly treated SSAT-wt animals. Serum creatinine levels of LPS-treated SSAT-wt mice were significantly (+P ≤ 0.05) higher than those of saline-treated animals.
Fig. 8.
Fig. 8.
Comparison of renal histology of SSAT-wt and SSAT-ko mice after endotoxin-induced AKI. Renal histology of SSAT-wt and SSAT-ko mice were compared to assess the effect of SSAT deficiency on the severity of endotoxin-induced AKI. No histological differences were detected when kidneys of saline-treated SSAT-wt (A) and SSAT-ko (B) animals were compared. The tissue damage caused by endotoxin-induced AKI was not very severe; however, SSAT-wt (C) mice showed increased vacuolization in the affected tubular epithelium compared with SSAT-ko (D) littermates (arrows).
Fig. 9.
Fig. 9.
Comparison of SMO expression in the kidneys of SSAT-wt and SSAT-ko mice after endotoxin-induced AKI. Total RNA (10 μg/well) from kidneys of saline (n = 2/genotype)- or LPS-treated (n = 3/genotype) SSAT-wt and SSAT-ko animals was size-fractionated and then subjected to Northern blot analysis using radiolabeled SMO cDNA probe. Equal loading was confirmed by the examination of 18s rRNA bands (bottom). The intensity of SMO signals (top) was determined by densitometry and normalized against the intensity of the 18s rRNA band. The semiquantitative results are shown in the graph and indicate that SMO expression levels were not significantly affected by SSAT deletion in endotoxin-induced AKI. C, control (saline treatment).
Fig. 10.
Fig. 10.
Comparison of the inflammatory response in the kidneys of SSAT-wt and SSAT-ko mice in endotoxin-induced AKI. A: comparison of ICAM-1 expression in the kidneys of SSAT-wt and SSAT-ko mice. Total RNA (10 μg/well) from kidneys of saline (n = 2/genotype)- or LPS-treated (n = 3/genotype) SSAT-wt and SSAT-ko animals was subjected to Northern blot analysis using radiolabeled ICAM-1 cDNA probe. Equal loading was confirmed by the examination of 18s rRNA bands (bottom). ICAM-1 signal strength was assessed by densitometry and normalized against the strength of the 18s rRNA band signal. The semiquantitative value of induction profiles of ICAM-1 are depicted in the graph. B: infiltration of neutrophils in kidneys of SSAT-wt and SSAT-ko mice was assessed at 24 h after saline or LPS injection by immunofluorescent microscopy. Infiltrating neutrophils were detected in the kidney (×20 magnification) using polyclonal rat anti-mouse neutrophil antibody. Quantitative comparison of the average number of infiltrating neutrophils per field (×20 magnification) in the kidney is shown in the graph. *P ≤ 0.05; **P ≤ 0.01, LPS- vs. saline-treated animals of the same genotype. +P ≤ 0.05, LPS-treated SSAT-wt vs. SSAT-ko mice.
Fig. 11.
Fig. 11.
Measurement of polyamine levels in the kidneys of saline- and MDL72527-treated control mice and mice subjected to endotoxin-induced AKI. Kidney polyamine contents were determined by HPLC and expressed as pmol/mg protein. A: comparison of Spd and Spm levels in the kidneys of mice given intraperitoneal injection of saline or LPS and then treated with vehicle or MDL72527. B: comparison of kidney Put levels in the same animals. C: comparison of kidney acetylated polyamine levels in the same animals. *P ≤ 0.05; **P ≤ 0.01, animals subjected to a particular treatment vs. saline-treated control animals.
Fig. 12.
Fig. 12.
Effect of MDL72527 on renal function after endotoxin-induced AKI. The effect of PAO inhibition on the renal function of animals was determined 24 h post-LPS administration by measuring their serum creatinine levels. Animals given intraperitoneal injection of LPS or saline were treated with 100 mg/kg MDL72527 or vehicle (n = 8/group). Serum creatinine levels of saline-injected animals treated with MDL72527 or vehicle were not significantly different (P ≥ 0.05). Animals given intraperitoneal LPS injection and then treated with MDL72527 had significantly (*P ≤ 0.05) lower serum creatinine levels than LPS-treated animals receiving vehicle. Serum creatinine levels of the LPS-treated mice were significantly (+P ≤ 0.01) higher than those of saline-treated animals.
Fig. 13.
Fig. 13.
Comparison of renal histology in vehicle- or MDL72527-treated SSAT-wt mice after endotoxin-induced AKI. Renal histology of SSAT-wt mice treated with vehicle or 100 mg/kg MDL72527 were compared to assess the effect of inhibition of polyamine oxidation on the severity of endotoxin-induced AKI. No histological differences were detected when the kidneys of vehicle- (A) or MDL72527-treated (B) animals receiving saline injection (control) were compared. The tissue damage caused by endotoxin-induced AKI was not severe; however, vehicle-treated SSAT-wt (C) animals showed increased vacuolization in the affected tubular epithelium compared with MDL72527-treated SSAT-wt (D) animals (arrows).
Fig. 14.
Fig. 14.
Effect of treatment with MDL72527 on neutrophil infiltration in LPS-induced AKI. Infiltration of neutrophils in kidneys of vehicle- and MDL72527-treated SSAT-wt mice was assessed at 24 h after saline or LPS injection by performing immunofluorescent microscopy. Top: infiltrating neutrophils were detected in the kidney (×20 magnification) using polyclonal rat anti-mouse neutrophil antibody. Bottom: quantitative comparison of the average number of infiltrating neutrophils per field (×20 magnification) in the kidney is presented in the graph. *P ≤ 0.05; **P ≤ 0.01, LPS- vs. saline-treated animals receiving vehicle or MDL72527 injections. +P ≤ 0.05 compared with LPS-treated mice receiving vehicle or MDL72527 injections.
Fig. 15.
Fig. 15.
Comparison of GSSG-to-GSH ratio in the kidneys of SSAT-wt, SSAT-ko, and MDL72527-treated SSAT-wt (MDL/SSAT-wt) animals after endotoxin-induced AKI. The onset of oxidative stress in the kidneys of SSAT-wt, SSAT-ko, and MDL/SSAT-wt mice 24 h after administration of LPS or saline was examined by determining the ratio of GSSG to GSH (GSSG/GSH). GSSG/GSH increased in LPS-treated animals in all 3 groups; however, after LPS treatment, the deficiency of SSAT or inhibition of PAOs significantly (*P ≤ 0.05) dampened the increase in GSSG/GSH compared with LPS-treated SSAT-wt animals.
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References

    1. Alhonen L, Karppinen A, Uusi-Oukari M, Vujcic S, Korhonen VP, Halmekyto M, Kramer DL, Hines R, Janne J, Porter CW. Correlation of polyamine and growth responses to N1,N11-diethylnorspermine in primary fetal fibroblasts derived from transgenic mice overexpressing spermidine/spermine N1-acetyltransferase. J Biol Chem 273: 1964–1969, 1998 - PubMed
    1. Alhonen L, Parkkinen JJ, Keinanen T, Sinervirta R, Herzig KH, Janne J. Activation of polyamine catabolism in transgenic rats induces acute pancreatitis. Proc Natl Acad Sci USA 97: 8290–8295, 2000 - PMC - PubMed
    1. Alhonen L, Rasanen TL, Sinervirta R, Parkkinen JJ, Korhonen VP, Pietila M, Janne J. Polyamines are required for the initiation of rat liver regeneration. Biochem J 362: 149–153, 2002 - PMC - PubMed
    1. Barone S, Okaya T, Rudich S, Petrovic S, Tenrani K, Wang Z, Zahedi K, Casero RA, Lentsch AB, Soleimani M. Distinct and sequential upregulation of genes regulating cell growth and cell cycle progression during hepatic ischemia-reperfusion injury. Am J Physiol Cell Physiol 289: C826–C835, 2005 - PubMed
    1. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987 - PubMed

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