Involvement of de novo ceramide synthesis in radiocontrast-induced renal tubular cell injury

We reported previously that various radiocontrast media cause apoptosis in porcine proximal tubular (LLC-PK1) cells, in which reduction in B-cell lymphoma (Bcl)-2 expression and caspase-3 activation are implicated. In the present study, we investigated a role for ceramide in radiocontrast media-induced apoptosis in renal tubular cells. LLC-PK1 cells were exposed to radiocontrast media for 30 min, followed by incubation for 24 h in normal medium. Cell viability was assessed by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, while apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling stain. Immunofluorescent stains were performed using antibodies against phosphorylated Akt (pAkt) and cAMP response element binding protein (CREB) (pCREB), and ceramide. The mRNA expression and protein content of Bcl-2 were determined by reverse transcriptase-polymerase chain reaction and enzyme immunoassay, respectively. In vivo model of contrast-induced renal injury was induced in mice with unilateral renal occlusion. The cell injury induced by the nonionic radiocontrast medium ioversol was reversed by inhibiting de novo ceramide synthesis with fumonisin B1 (FB1) and L-cycloserine, but not by suppressing sphingomyelin breakdown with D609. FB1 reversed ioversol-induced decrease in the immunoreactivities of pAkt and pCREB, reduction in Bcl-2 expression and caspase-3 activation. Like ioversol, C2 ceramide and the Akt inhibitor Src homology-6 induced apoptosis by reducing pAkt and pCREB-like immunoreactivities, lowering Bcl-2 expression and enhancing caspase-3 activity. Indeed, various radiocontrast media, excluding iodixanol which showed the least nephrotoxicity, enhanced ceramide-like immunoreactivity. The role for de novo ceramide synthesis was also shown in the in vivo model of radiocontrast nephropathy. We demonstrated here for the first time that the enhancement of de novo ceramide synthesis contributes to radiocontrast nephropathy. We reported previously that various radiocontrast media cause apoptosis in porcine proximal tubular (LLC-PK1) cells, in which reduction in B-cell lymphoma (Bcl)-2 expression and caspase-3 activation are implicated. In the present study, we investigated a role for ceramide in radiocontrast media-induced apoptosis in renal tubular cells. LLC-PK1 cells were exposed to radiocontrast media for 30 min, followed by incubation for 24 h in normal medium. Cell viability was assessed by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, while apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling stain. Immunofluorescent stains were performed using antibodies against phosphorylated Akt (pAkt) and cAMP response element binding protein (CREB) (pCREB), and ceramide. The mRNA expression and protein content of Bcl-2 were determined by reverse transcriptase-polymerase chain reaction and enzyme immunoassay, respectively. In vivo model of contrast-induced renal injury was induced in mice with unilateral renal occlusion. The cell injury induced by the nonionic radiocontrast medium ioversol was reversed by inhibiting de novo ceramide synthesis with fumonisin B1 (FB1) and L-cycloserine, but not by suppressing sphingomyelin breakdown with D609. FB1 reversed ioversol-induced decrease in the immunoreactivities of pAkt and pCREB, reduction in Bcl-2 expression and caspase-3 activation. Like ioversol, C2 ceramide and the Akt inhibitor Src homology-6 induced apoptosis by reducing pAkt and pCREB-like immunoreactivities, lowering Bcl-2 expression and enhancing caspase-3 activity. Indeed, various radiocontrast media, excluding iodixanol which showed the least nephrotoxicity, enhanced ceramide-like immunoreactivity. The role for de novo ceramide synthesis was also shown in the in vivo model of radiocontrast nephropathy. We demonstrated here for the first time that the enhancement of de novo ceramide synthesis contributes to radiocontrast nephropathy. Acute renal failure associated with iodinated radiocontrast media is still the major complication after radiographic examination, since it is associated with increased morbidity and mortality.1.Gruberg L. Mintz G.S. Mehran R. et al.Prognostic implications of further renal function deterioration within 48 h of interventional coronary procedures in patients with pre-existent chronic renal insufficiency.J Am Coll Cardiol. 2000; 36: 1542-1548Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar, 2.Dangas G. Iakovou I. Nikolsky E. et al.Contrast-induced nephropathy after percutaneous coronary interventions in relation to chronic kidney disease and hemodynamic variables.Am J Cardiol. 2005; 95: 13-19Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar Unfortunately, there have been few effective medications for prevention of radiocontrast nephropathy. Although little is known about the etiology of radiocontrast nephropathy, the decrease in renal blood flow3.Liss P. Nygren A. Erikson U. Ulfendahl H.R. Injection of low and iso-osmolar contrast medium decreases oxygen tension in the renal medulla.Kidney Int. 1998; 53: 698-702Abstract Full Text PDF PubMed Scopus (119) Google Scholar and/or direct toxic action on renal tubular cells4.Zager R.A. Johnson A.C. Hanson S.Y. Radiographic contrast media-induced tubular injury: evaluation of oxidant stress and plasma membrane integrity.Kidney Int. 2003; 64: 128-139Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar are postulated to be implicated in the pathogenesis of radiocontrast nephropathy. Thus, a number of agents that improve renal vascular circulation, including endothelin antagonists, adenosine antagonists such as theophylline, atrial natriuretic peptide, dopamine agonists and calcium channel blockers, have been clinically investigated for the prevention of radiocontrast nephropathy, but most of them failed to succeed in preventing radiocontrast nephropathy in high-risk patients (for a review, see Cox and Tsikouris5.Cox C.D. Tsikouris J.P. Preventing contrast nephropathy: what is the best strategy? A review of the literature.J Clin Pharmacol. 2004; 44: 327-337Crossref PubMed Scopus (51) Google Scholar). On the other hand, it has recently been reported that the antioxidant compound N-acetylcysteine6.Tepel M. van der Giet M. Schwarzfeld C. et al.Prevention of radiographic contrast medium-induced reductions in renal function by acetylcysteine.N Engl J Med. 2000; 343: 180-184Crossref PubMed Scopus (1376) Google Scholar, 7.Pannu N. Manns B. Lee H. Tonelli M. Systematic review of the impact of N-acetylcysteine on contrast nephropathy.Kidney Int. 2004; 65: 1366-1374Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar has a slight and not complete protective action against radiocontrast nephropathy, thereby suggesting that the radiocontrast nephropathy is associated with renal tubular injury due to the oxidative stress. We have recently found that a variety of radiocontrast media cause cell injury in porcine renal tubular (LLC-PK1) cell line cells, as characterized by changes in the expression of B-cell lymphoma (Bcl)-2 family proteins such as Bcl-2 and Bax, activation of caspase-3 and nuclear fragmentation.8.Yano T. Itoh Y. Sendo T. et al.Cyclic AMP reverses radiocontrast media-induced apoptosis in LLC-PK1 cells by activating A kinase/PI3 kinase.Kidney Int. 2003; 64: 2052-2063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar We also found that the radiocontrast medium inhibits the phosphorylation of Akt.9.Yano T. Itoh Y. Kubota T. et al.A prostacyclin analog beraprost sodium attenuates radiocontrast media-induced LLC-PK1 cells injury.Kidney Int. 2004; 65: 1654-1663Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Since Akt is a serine/threonine kinase involved in cell survival and cell growth, the radiocontrast medium-induced apoptosis in renal tubular cells may be due to the inhibition of Akt phosphorylation. Indeed, the nonhydrolyzable cyclic AMP (cAMP) analog dibutyryl cAMP (DBcAMP) was found to attenuate renal tubular cell injury induced by the radiocontrast medium through activation of Akt, followed by phosphorylation of cAMP response element binding protein (CREB).10.Yano T. Itoh Y. Kubota T. et al.A prostacyclin analog prevents radiocontrast nephropathy via phosphorylation of cyclic AMP response element binding protein.Am J Pathol. 2005; 166: 1333-1342Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar However, it is still uncertain how the radiocontrast medium inhibits phosphorylation of Akt in renal tubular cells. It has been demonstrated that ceramide, a sphingolipid metabolite, decreases the level of phosphorylated Akt (pAkt)11.Schubert K.M. Scheid M.P. Duronio V. Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine473.J Biol Chem. 2000; 275: 13330-13335Crossref PubMed Scopus (207) Google Scholar, 12.Stoica B.A. Movsesyan V.A. Lea IV, P.M. Faden A.I. Ceramide-induced neuronal apoptosis is associated with dephosphorylation of Akt, BAD, FKHR, GSK-3β, and induction of the mitochondrial-dependent intrinsic caspase pathway.Mol Cell Neurosci. 2003; 22: 365-382Crossref PubMed Scopus (130) Google Scholar and leads to apoptosis in a variety of cells.13.Iwata M. Herrington J. Zager R.A. Sphingosine: a mediator of acute renal tubular injury and subsequent cytoresistance.Proc Natl Acad Sci USA. 1995; 92: 8970-8974Crossref PubMed Scopus (51) Google Scholar, 14.Ueda N. Kaushal G.P. Hong X. Shah S.V. Role of enhanced ceramide generation in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells.Kidney Int. 1998; 54: 399-406Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Therefore, it seems likely that the contrast medium causes acute renal failure by elevating ceramide concentration in renal tubular cells. To ascertain this idea, we investigated the effects of several compounds that interfere with sphingolipid metabolism on renal tubular cell injury induced in vitro as well as in vivo by the radiocontrast medium. The effects of various nonionic radiocontrast media on the ceramide-like immunoreactivity were also examined in cultured renal tubular cells. As shown in Figure 1, ioversol caused a significant decrease in cell viability, which was reversed by fumonisin B1 (FB1), a specific inhibitor of ceramide synthase,15.Wang E. Norred W.P. Bacon C.W. et al.Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme.J Biol Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar and L-cycloserine, an inhibitor of serine palmitoyltransferase,16.Holleran W.M. Williams M.L. Gao W.N. Elias P.M. Serine-palmitoyl transferase activity in cultured human keratinocytes.J Lipid Res. 1990; 31: 1655-1661Abstract Full Text PDF PubMed Google Scholar but not by either D609, a xanthogenate compound that inhibits acidic sphingomyelinase activity through the inhibition of phosphatidylcholine-specific phospholipase C,17.Luberto C. Hannun Y.A. Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?.J Biol Chem. 1998; 273: 14550-14559Crossref PubMed Scopus (262) Google Scholar or the tricyclic antidepressant imipramine that inhibits sphingomyelinase activity.18.Albouz S. Vanier M.T. Hauw J.J. et al.Effect of tricyclic antidepressants on sphingomyelinase and other sphingolipid hydrolases in C6 cultured glioma cells.Neurosci Lett. 1983; 36: 311-315Crossref PubMed Scopus (38) Google Scholar As shown in Figure 2a, the exposure of LLC-PK1 cells to insulin-like growth factor-1 (IGF-1) markedly enhanced the immunoreactivity for pAkt (Ser473), which was completely inhibited by Src homology (SH)-6, a specific Akt inhibitor that binds selectively to the pleckstrin homology domain of Akt to prevent the association of Akt with plasma membrane phosphatidylinositol-3-phosphate,19.Meuillet E.J. Mahadevan D. Vankayalapati H. et al.Specific inhibition of the Akt1 pleckstrin homology domain by D-3-deoxy-phosphatidyl-myo-inositol analogues.Mol Cancer Ther. 2003; 2: 389-399PubMed Google Scholar thereby suggesting that the immunoreactivity is derived from pAkt. Ioversol markedly inhibited the IGF-1-stimulated phosphorylation of Akt in a manner dependent on de novo ceramide synthesis. C2 ceramide also markedly inhibited the phosphorylation of Akt. On the other hand, protein level of pAkt as determined by the enzyme immunoassay was also reduced by ioversol, C2 ceramide and SH-6, in which the effect of ioversol was reversed by FB1 (Figure 2b). Similar changes were observed for the phosphorylation of CREB and the level of phosphorylated CREB (pCREB). As shown in Figure 3a, fluorescent images for 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were changed in color from red (J-aggregates) to green (monomers) after exposure to ioversol, suggesting that ioversol causes depolarization of mitochondrial membranes. FB1 abolished, while C2 ceramide and SH-6 mimicked, the action of ioversol. Since mitochondrial membrane potential is reported to be regulated by Bcl-2 family proteins such as Bcl-2 and Bax,20.Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis.Nat Med. 1997; 3: 614-620Crossref PubMed Scopus (1673) Google Scholar we investigated the changes in mRNA expression of and protein levels of Bcl-2 and Bax after exposure to ioversol in the absence or presence of FB1. As shown in Figure 3b and c, ioversol inhibited mRNA expression of Bcl-2 and decreased the level of Bcl-2 protein, while it enhanced mRNA expression of Bax and increased the level of Bax protein. These actions of ioversol were almost completely reversed by FB1. Similar changes in mRNA expressions and protein contents of these Bcl-2 family proteins were observed after exposure to C2 ceramide and SH-6. As shown in Figure 4a, z-Asp(O-Me)-Glu(O-Me)-Val-Asp(O-Me) fluoromethyl ketone a specific caspase-3 inhibitor, reversed the appearance of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells induced by ioversol, C2 ceramide and SH-6. Moreover, the loss of cell viability induced by these agents was also reversed by z-Asp(O-Me)-Glu(O-Me)-Val-Asp(O-Me) fluoromethyl ketone (Figure 4b). As shown in Figure 4c, the caspase-3 activity determined by the enzymatic degradation of caspase-3-specific substrate peptide was markedly enhanced by ioversol, C2 ceramide and SH-6. The exposure of LLC-PK1 cells to ioversol for 30 min increased the ceramide-like immunoreactivity, as determined by immunofluorescent stain with anticeramide antibody, followed by flow-cytometric analysis (Figure 5a). The ioversol-induced increase in ceramide-like immunoreactivity was reversed by FB1 (300 nM), but not by D609 (1 μM). Like ioversol, several nonionic radiocontrast media, including iohexol, iomeprol, iopamidol and iotrolan, enhanced the ceramide-like immunoreactivity (Figure 5b). Moreover, these contrast media reduced the cell viability determined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) assay, although the reduction in cell viability was slight (decrease by 10.5%) and not significant in iomeprol-exposed cells (Figure 5c). By contrast, iodixanol had no significant influence on ceramide-like immunoreactivity. The cell viability also did not change (decrease by 2.4%) after exposure to iodixanol. As we reported previously,9.Yano T. Itoh Y. Kubota T. et al.A prostacyclin analog beraprost sodium attenuates radiocontrast media-induced LLC-PK1 cells injury.Kidney Int. 2004; 65: 1654-1663Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar a single intravenous injection of ioversol-induced apoptosis in renal tubular cells (Figure 6a) and significant elevation of urinary activity of N-acetyl-β-D-glucosaminidase (NAG), a specific marker of proximal renal tubular cell injury,21.Bourbouze R. Baumann F.C. Bonvalet J.P. Farman N. Distribution of N-acetyl-β-D-glucosaminidase isoenzymes along the rabbit nephron.Kidney Int. 1984; 25: 636-642Abstract Full Text PDF PubMed Scopus (61) Google Scholar in mice with unilateral renal occlusion (Figure 6b). FB1 (0.075 and 0.25 mg/kg) inhibited the apoptosis and enhancement of urinary NAG excretion induced by ioversol in a dose-dependent manner. Ioversol also lowered protein levels of pAkt (Figure 6c) and pCREB (Figure 6d) and enhanced caspase-3 activity (Figure 6e) in renal tissues. Interestingly, these cellular events induced by ioversol were all remarkably reversed by 0.25 mg/kg FB1. However, a high dose of FB1 (2 mg/kg) was not effective in attenuating the ioversol-induced increase in urinary NAG excretion (NAG in U/g Cr: 242.8±10.4, mean±s.e.m., N=4, for ioversol alone vs 235.7±14.5, N=4 for ioversol+FB1 at 2 mg/kg). We have previously shown that ioversol causes concentration- and time-dependent decreases in the viability of LLC-PK1 cells, in which significant injury is observed in as low as 25 mg iodine/ml of the contrast medium.8.Yano T. Itoh Y. Sendo T. et al.Cyclic AMP reverses radiocontrast media-induced apoptosis in LLC-PK1 cells by activating A kinase/PI3 kinase.Kidney Int. 2003; 64: 2052-2063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Moreover, the toxic effect of ioversol reaches maximum at 100 mg iodine/ml and the half-maximal reduction in the cell viability is observed at 24 h after exposure to 100 mg iodine/ml of the contrast medium. Thus, in the present study, we examined the effects of compounds that interfere with sphingolipid metabolisms on the cell injury induced at 24 h by exposure to 100 mg iodine/ml of ioversol. In the present study, the ioversol-induced injury of LLC-PK1 cells was reversed concentration-dependently by the ceramide synthase inhibitor FB1 and the serine palmitoyltransferase inhibitor L-cycloserine, but not by inhibitors of sphingomyelin breakdown, such as D609 and imipramine. Therefore, it is suggested that ioversol causes renal tubular cell damage by enhancing de novo ceramide synthesis. In our previous report, ioversol-induced injury of renal tubular cells is characterized by nuclear fragmentation, Annexin V-positive stain and the activation of caspase-9 and caspase-3, suggesting apoptosis.8.Yano T. Itoh Y. Sendo T. et al.Cyclic AMP reverses radiocontrast media-induced apoptosis in LLC-PK1 cells by activating A kinase/PI3 kinase.Kidney Int. 2003; 64: 2052-2063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 22.Itoh Y. Yano T. Sendo T. Oishi R. Critical review: clinical and experimental evidence for prevention of acute renal failure induced by radiographic contrast media.J Pharmacol Sci. 2005; 97: 473-488Crossref PubMed Scopus (76) Google Scholar Moreover, the apoptotic cell death is attributable to the mitochondrial stress due to changes in Bcl-2 family proteins such as Bcl-2 and Bax.8.Yano T. Itoh Y. Sendo T. et al.Cyclic AMP reverses radiocontrast media-induced apoptosis in LLC-PK1 cells by activating A kinase/PI3 kinase.Kidney Int. 2003; 64: 2052-2063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar It has been demonstrated that the relative increase in the expression of Bax over Bcl-2 leads to the depolarization of mitochondrial membranes and stimulates the release of cytochrome c, which, in turn, activates caspase-9 through the action of the adaptor molecule apoptotic protease-activating factor-1.23.Robertson J.D. Orrenius S. Role of mitochondria in toxic cell death.Toxicology. 2002; 181–182: 491-496Crossref PubMed Scopus (76) Google Scholar Consistent with our previous data, in the present study, ioversol caused the depolarization of mitochondrial membranes as assessed by JC-1 stain, activation of caspase-3 and ultimately led to caspase-3-dependent apoptosis determined by TUNEL stain. We also found previously that DBcAMP protects renal tubular cells against ioversol-induced apoptosis through phosphorylation of Akt and CREB and subsequent enhancement of Bcl-2 expression.8.Yano T. Itoh Y. Sendo T. et al.Cyclic AMP reverses radiocontrast media-induced apoptosis in LLC-PK1 cells by activating A kinase/PI3 kinase.Kidney Int. 2003; 64: 2052-2063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 9.Yano T. Itoh Y. Kubota T. et al.A prostacyclin analog beraprost sodium attenuates radiocontrast media-induced LLC-PK1 cells injury.Kidney Int. 2004; 65: 1654-1663Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10.Yano T. Itoh Y. Kubota T. et al.A prostacyclin analog prevents radiocontrast nephropathy via phosphorylation of cyclic AMP response element binding protein.Am J Pathol. 2005; 166: 1333-1342Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 22.Itoh Y. Yano T. Sendo T. Oishi R. Critical review: clinical and experimental evidence for prevention of acute renal failure induced by radiographic contrast media.J Pharmacol Sci. 2005; 97: 473-488Crossref PubMed Scopus (76) Google Scholar Interestingly, ioversol was found to inhibit phosphorylation of Akt at Ser473 and phosphorylation of CREB at Ser133.10.Yano T. Itoh Y. Kubota T. et al.A prostacyclin analog prevents radiocontrast nephropathy via phosphorylation of cyclic AMP response element binding protein.Am J Pathol. 2005; 166: 1333-1342Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar It has been demonstrated that Akt plays an important role in the cell survival since inhibition of propidium iodide 3-kinase/Akt stimulates caspase activity and leads to apoptosis in a variety of cells.24.Kandel E.S. Hay N. The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB.Exp Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (771) Google Scholar, 25.Goswami R. Dawson S.A. Dawson G. Multiple polyphosphoinositide pathways regulate apoptotic signalling in a dorsal root ganglion derived cell line.J Neurosci Res. 2000; 59: 136-144Crossref PubMed Scopus (17) Google Scholar, 26.Datta S.R. Brunet A. Greenberg M.E. Cellular survival: a play in three Akts.Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3647) Google Scholar Moreover, phosphorylation of Akt is reported to upregulate the expression of Bcl-2 through phosphorylation of CREB.27.Pugazhenthi S. Nesterova A. Sable C. et al.Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein.J Biol Chem. 2000; 275: 10761-10766Crossref PubMed Scopus (670) Google Scholar, 28.Pugazhenthi S. Miller E. Sable C. et al.Insulin-like growth factor-I induces bcl-2 promoter through the transcription factor cAMP-response element-binding protein.J Biol Chem. 1999; 274: 27529-27535Crossref PubMed Scopus (178) Google Scholar In the present study, IGF-1 enhanced not only the pAkt-like immunoreactivity but also the pCREB-like immunoreactivity in LLC-PK1 cells. In addition, IGF-1-mediated phosphorylation of CREB was diminished by SH-6, thereby suggesting that the phosphorylation of CREB is mediated through the activation of Akt in these cells. It has been demonstrated that the membrane sphingolipid ceramide decreases the level of phosphorylated form of Akt by facilitating the dephosphorylation of Akt at Ser473 through the activation of protein phosphatase 2A11.Schubert K.M. Scheid M.P. Duronio V. Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine473.J Biol Chem. 2000; 275: 13330-13335Crossref PubMed Scopus (207) Google Scholar and/or by inhibiting the association of pleckstrin homology domain of Akt with membrane proximal intraglomerular pressure 3 through the activation of protein kinase Cζ.29.Powell D.J. Hajduch E. Kular G. Hundal H.S. Ceramide disables 3-phosphoinositide binding to the pleckstrin homology domain of protein kinase B (PKB)/Akt by a PKCzeta-dependent mechanism.Mol Cell Biol. 2003; 23: 7794-7808Crossref PubMed Scopus (247) Google Scholar In the present study, it was noteworthy that the inhibition by ioversol of IGF-1-induced phosphorylation of Akt was abolished by FB1. Moreover, C2 ceramide showed a marked inhibition on IGF-1-induced phosphorylation of Akt. Therefore, it is highly probable that the inhibition of the phosphorylation of Akt induced by ioversol is attributable to the enhancement of de novo ceramide synthesis. FB1 was also effective in suppressing ioversol-induced inhibition of IGF-1-stimulated phosphorylation of CREB, while C2 ceramide and SH-6 markedly reduced the IGF-1-stimulated phosphorylation of CREB. Moreover, downregulation of Bcl-2 and upregulation of Bax induced by ioversol were reversed by FB1, while C2 ceramide and SH-6 caused similar changes in the expression of these Bcl-2 family proteins to ioversol. Taken together, it is suggested that ioversol stimulates de novo ceramide synthesis, which leads to apoptosis by inhibiting the phosphorylation of Akt and CREB and subsequent reduction of Bcl-2 expression and activation of caspase-3. It has been demonstrated that a number of apoptotic stimuli such as reactive oxygen species30.Martin D. Salinas M. Fujita N. et al.Ceramide and reactive oxygen species generated by H2O2 induce caspase-3-independent degradation of Akt/protein kinase B.J Biol Chem. 2002; 277: 42943-44952Crossref PubMed Scopus (155) Google Scholar and tumor necrosis factor (TNF)-α31.Liu B. Andrieu-Abadie N. Levade T. et al.Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death.J Biol Chem. 1998; 273: 11313-11320Crossref PubMed Scopus (318) Google Scholar increase the ceramide content by facilitating sphingomyelin breakdown, but not through the activation of the de novo synthesis pathway. In the present study, both the enhancement of ceramide-like immunoreactivity and cell injury induced by ioversol were not affected by inhibiting sphingomyelin breakdown with D609 or imipramine. Therefore, it is unlikely that sphingomyelin breakdown plays a role in renal tubular injury induced by the radiocontrast medium. On the other hand, a role for reactive oxygen species in the pathogenesis of radiocontrast nephropathy has been shown by several clinical findings indicating that the antioxidant N-acetylcysteine is effective for the prevention of radiocontrast nephropathy in patients with pre-existing renal insufficiency.6.Tepel M. van der Giet M. Schwarzfeld C. et al.Prevention of radiographic contrast medium-induced reductions in renal function by acetylcysteine.N Engl J Med. 2000; 343: 180-184Crossref PubMed Scopus (1376) Google Scholar, 7.Pannu N. Manns B. Lee H. Tonelli M. Systematic review of the impact of N-acetylcysteine on contrast nephropathy.Kidney Int. 2004; 65: 1366-1374Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar We do not know whether N-acetylcysteine is effective for prevention of apoptosis of LLC-PK1 cells induced by radiocontrast media. However, Hizoh and Haller32.Hizoh I. Haller C. Radiocontrast-induced renal tubular cell apoptosis: hypertonic versus oxidative stress.Invest Radiol. 2002; 37: 428-434Crossref PubMed Scopus (130) Google Scholar have shown in cultured Madin Darby Canine Kidney cells that N-acetylcysteine has no protective action against the nuclear damage induced by the ionic contrast medium amidotrizoate. At present, it is uncertain whether reactive oxygen species contribute to the increase in ceramide content or apoptosis induced by radiocontrast media. In the present study, like ioversol, a variety of iodinated radiocontrast media enhanced ceramide-like immunoreactivity in renal tubular cells. However, iomeprol caused only a slight increase in the ceramide-like immunoreactivity, while iodixanol had no effect on the immunoreactivity. It was noteworthy that the increase in the ceramide-like immunoreactivity was closely associated with the loss of cell viability. It has been demonstrated by the clinical studies that the risk of radiocontrast nephropathy is much lower in iodixanol than in other low osmolar media.33.Aspelin P. Aubry P. Fransson S.G. et al.Nephrotoxicity in high-risk patients study of iso-osmolar and low-osmolar non-ionic contrast media study investigators. Nephrotoxic effects in high-risk patients undergoing angiography.N Engl J Med. 2003; 348: 491-499Crossref PubMed Scopus (953) Google Scholar It is generally considered that high osmolality is the major causative factor of radiocontrast nephropathy.34.Barrett B.J. Carlisle E.J. Metaanalysis of the relative nephrotoxicity of high- and low-osmolality iodinated contrast media.Radiology. 1993; 188: 171-178Crossref PubMed Scopus (627) Google Scholar However, in the present study, another iso-osmolar contrast medium iotrolan, whose osmolality is equivalent to that of iodixanol, produced a marked decrease in cell viability as well as an enhancement of ceramide-like immunoreactivity. Taken together, it is suggested that the enhancement of de novo ceramide synthesis is the common cellular mechanism underlying renal tubular injury induced by a variety of iodinated radiocontrast media. Finally, we investigated the effect of FB1 on the in vivo renal injury induced by ioversol in mice with unilateral renal occlusion. Deray et al.35.Deray G. Dubois M. Martinez F. et al.Renal effects of radiocontrast agents in rats: a new model of acute renal failure.Am J Nephrol. 1990; 10: 507-513Crossref PubMed Scopus (73) Google Scholar reported that the radiocontrast medium enhances urinary excretion of NAG in rats with renal artery occlusion, but not in intact animals. Generally consistent with their data, in the present study, ioversol enhanced urinary NAG excretion in mice with unilate