Overexpression of SSAT in Kidney Cells Recapitulates Various Phenotypic Aspects of Kidney Ischemia-reperfusion Injury

Abstract

Abstract. To ascertain the role of spermidine/spermine N-1-acetyl-transferase (SSAT; the rate-limiting enzyme in polyamine catabolism) in cell injury, cultured kidney (HEK 293) cells conditionally overexpressing SSAT were generated. The SSAT expression was induced and its enzymatic activity increased 24 h after addition of tetracycline and remained elevated over the length of the experiments. Induction of SSAT upregulated the expression of polyamine oxidase and resulted in the reduction of cellular concentration of spermidine and spermine, increased concentration of putrescine, and inhibited cell growth. SSAT overexpression increased the expression of heme oxygenase-1 (HO-1) by 350% 24 h after addition of tetracycline, indicating the induction of oxidative stress. The presence of catalase significantly prevented the upregulation of HO-1 in SSAT overexpressing cells, indicating that generation of H 2 O 2 is partially responsible for the induction of oxidative stress. Overexpression of SSAT caused rounding and loss of cell anchorage and significantly altered the morphology of actin-containing filopodia, suggesting an adhesion defect. SSAT upregulation may mediate majority of the oxidative stress in kidney ischemia-reperfusion injury (IRI) as manifested by decreased cell growth, generation of toxic metabolites (H 2 O 2 and putrescine), upregulation of HO-1, disruption of cell anchorage, and defect in cell adhesion. These data point to the inhibition of polyamine catabolism as a therapeutic approach for the prevention of tissue injury in kidney IRI.Ischemia-reperfusion injury (IRI) is the major cause of morbidity and mortality in diseases such as stroke, myocardial infarction, and acute renal tubular necrosis. Ischemic conditions result in ATP depletion and accumulation of toxic metabolites. Reperfusion results in the production of reactive oxygen intermediates (1,2). The resulting alteration in cellular metabolism and generation of toxic molecules contribute to tissue damage in IRI (1,2), which is characterized by the presence of necrotic and apoptotic areas in the affected organs (1,3). Despite important developments in our understanding of the pathophysiology of IRI in kidney, heart, and other organs, there is no specific therapy for patients except for supportive care.Polyamines (spermidine, spermine, and putrescine) are aliphatic cations derived from ornithine (4,5). They play a fundamental role in the stabilization of DNA structure; they modulate gene expression and regulate protein synthesis, signal transduction, and cell growth and differentiation (4,6 -8). Spermidine/spermine N-1-acetyl-transferase (SSAT; the rate-limiting enzyme in polyamine catabolism) acetylates both spermidine and spermine. As a result, the cellular contents of spermidine and spermine are decreased and the concentrations of N-acetyl spermidine and N-acetyl spermine are increased (9). The subsequent activity of polyamine oxidase (PAO) on acetylated polyamines results in the production of spermidine or putrescine (depending on the startin...

Hepatocyte-specific ablation of spermine/spermidine-N1-acetyltransferase gene reduces the severity of CCl4-induced acute liver injury

“…Previous studies indicate that the induction of SSAT expression in cultured cells is associated with coincidental increases in the expression of ODC and SMO mRNAs (32). The results presented here indicate that the hepatic expression of SMO transcript is lower in CCl 4 -treated Hep-SSAT-Cko than similarly treated WT mice (Fig.…”

“…The maladaptive role of SSAT in the induction of cellular damage and tissue injury is supported by in vitro and in vivo observations (1,4,5,11,25,32,(35)(36)(37). In vitro studies indicate that expression of SSAT in cultured cells leads to induction of oxidative stress, DNA damage, cell cycle arrest, and onset of the intrinsic/ caspase-dependent apoptosis (7,36).…”

“…RNA was extracted and subjected to Northern blot analysis as previously described (32,36). Equal loading of RNA samples was confirmed by assessment of the 28 s or 18 s rRNA band intensities.…”

Roles of the Exogenous H2S-Mediated SR-A Signaling Pathway in Renal Ischemia/ Reperfusion Injury in Regulating Endoplasmic Reticulum Stress-Induced Autophagy in a Rat Model

“…Renal ischemic-reperfusion injury (IRI) is a typical complication of organ transplantation, leading to adenosine triphosphate (ATP) depletion, accumulation of toxic metabolites, and consequent tissue damage [1]. During the process of renal transplantation, transient cessation in renal blood flow leads to acute ischemic insult, and the reperfusion further deepens the functional and structural damage to the human kidney [2].…”

Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cells

“…The effects of overexpression of SAT1 were previously reported by others using cell lines overexpressing SAT1 stably or in an inducible manner (22)(23)(24)33), SAT1 transgenic mice (34)(35)(36), and a SAT1 adenovirus (37,38). Although retardation of growth and decreases in spermidine and spermine were consistently associated with SAT1 overexpression, the degree of growth inhibition and polyamine depletion were much lower than those reported in this study.…”