review article molecular mechanisms of renal cellular ... · e opacity of iodine to x-rays renders...

Review ArticleMolecular Mechanisms of Renal Cellular Nephrotoxicity due toRadiocontrast Media

Ashour Michael1 Teresa Faga1 Antonio Pisani2 Eleonora Riccio2 Placido Bramanti3

Massimo Sabbatini2 Michele Navarra4 and Michele Andreucci1

1 Department of Health Sciences Nephrology Unit ldquoMagna Graeciardquo University I-88100 Catanzaro Italy2 Department of Nephrology ldquoFederico IIrdquo University I-80131 Naples Italy3 IRCCS Centro Neurolesi ldquoBonino Pulejordquo I-98124 Messina Italy4Department of Drug Sciences and Health Products University of Messina I-98168 Messina Italy

Correspondence should be addressed to Michele Andreucci andreucciuniczit

Received 18 October 2013 Revised 9 December 2013 Accepted 24 December 2013 Published 18 March 2014

Academic Editor Elizabeth N Pearce

Copyright copy 2014 Ashour Michael et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Modern iodinated radiocontrast media are all based on the triiodinated benzene ring with various chemical modifications havingbeen made over the last few decades in order to reduce their toxicity However CIN remains a problem especially in patients withpre-existing renal failure In vitro studies have demonstrated that all RCM are cytotoxic RCM administration in vivomay lead to adecrease in renal medullary oxygenation leading to the generation of reactive oxygen species that may cause harmful effects to renaltissue In addition endothelin and adenosine release and decreased nitric oxide levels may worsen the hypoxic milieu In vitro cellculture studies together with sparse in vivo rat model data have shown that important cell signalling pathways are affected by RCMIn particular the prosurvival and proproliferative kinases Akt and ERK12 have been shown to be dephosphorylated (deactivated)whilst proinflammatorycell death molecules such as the p38 and JNK kinases and the transcription factor NF-120581Bmay be activatedby RCM accompanied by activation of apoptotic mediators such as caspases Increasing our knowledge of themechanisms of RCMaction may help to develop future therapies for CIN

1 Introduction

Radiocontrast media (RCM) are commonly used in med-ical practice but their use may lead to contrast-inducednephropathy (CIN) The continued growth in radiographicexaminations means that increasing numbers of patients areexposed to RCM which in turn has resulted in increasingincidence of CIN CIN is the third most common causeof hospital-acquired kidney failure accounting for 12 ofall cases [1 2] Whilst the toxicity of RCM is not fullyunderstood it is believed to be due to many factors the twopossible principal mechanisms being their effects on renalhemodynamics and direct toxicity on renal cells [3 4]

The opacity of iodine to X-rays renders it a suitablecompound as a contrastmediumHowever due to its toxicityiodine is not suitable to be used in itsmolecular or ionic formWhilst many iodinated organic compounds (with covalently

bound iodine within the molecule) had been proposed thesearch was for a compound that was less toxic more solubleand having more opacity (ie containing more iodine atomsper molecule) It was in the 1950s that certain derivativesof iodinated benzoic acid were suggested as possible safecontrast media [5] and since then all modern iodinatedcontrast media are based on the triiodinated benzene ringThe ratio of iodine atoms to dissolved particles is importantsince a greater number of iodine atoms would give betteropacification and fewer particles in solution would resultin a lower osmotic effect and bearing these characteristicsin mind the evolution of the RCM have involved succes-sive chemical modifications Firstly hydrogen atoms on thebenzene ring were substituted with acetamido groups toreduce protein binding (protein bindingwas believed to causeanaphylactoid reactions) giving rise to the acetrizoates anddiatrizoates These compounds are ionic and dissociate in

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 249810 10 pageshttpdxdoiorg1011552014249810

2 BioMed Research International

solution and are termed as high-osmolar contrast media(HOCM) Then the carboxyl groups were replaced by non-polar groups giving nonionic soluble molecules with lowerosmolality These were termed as the low-osmolar contrastmedia (LOCM) and were further improved by the additionof more hydroxyl groups for increased hydrophilicity andthen a more even distribution of the hydroxyl groups onthe molecule Finally the dimerization of two molecules viaside chains on the benzene ring gave rise to the nonioniciso-osmolar contrast media (IOCM) with increased iodineatoms per molecule [6] Of interest is the RCM ioxaglatethat whilst it is ionic it is classified as an LOCM as it is adimer containing more iodine atoms per particle in solutionA summary of the most commonly used RCM are presentedin Table 1The improvements in contrast media developmenthave resulted in the acknowledgement that in clinical usethe newer LOCM and ICOM are less toxic than the originalHOCM [7] but in vitro cell culture studies have suggestedthat all types of RCM have a direct toxic effect in manydifferent types of cells (see Table 2) It has been suggested thatmolecular iodine may be present in solutions of RCM due todegradation [8] but this may be negligible in solutions thathave been properly stored Moreover a study has shown thatincubating isolated proximal tubule segments with varyingconcentrations of NaI had no adverse effects on cell viability[9] and a further study showed that sodium iodide alone didnot cause significant cell death in cultured renal cells [10]

2 Toxicity of Radiocontrast Media inIn Vitro Cell Culture Studies

Many in vitro studies have investigated the toxicity of RCMusing different types of cultured cells including renal epithe-lial cells mesangial cells endothelial cells smooth musclecells hepatic cells human fibroblasts pulmonary mast cellshuman embryonic kidney cells and human neutrophils

The most common in vitro studies addressing the patho-physiology of RCM-induced apoptosis have been criticizedbecause of their limitations which include (1) the assessmentof only one potential mechanism of the RCM-induced renalcell damage in the absence of several conflicting variablesthat can be found in vivo (2) the exposure to a constantconcentration of RCM to all cell lines whereas in vivo themore distal epithelial tubular cells are exposed to a muchhigher concentration than the proximal tubular cells (3) thepotentially high dose of RCM commonly used for the cellculture experiments (4) the fact that effects of the RCM areusually investigated as cellular ldquoshort termrdquo-induced effects(ie just after their exposure to cells) not along several hours(as ldquolong termrdquo effects) of exposure to them Finally researchgroups rarely compare different types of RCM (especiallythose with different osmolarities) in the same study

The most commonly used types of renal tubular cells forin vitro studies include the canine-derived MDCK cells (amodel of distal tubular cells) the porcine cell line LLC-PK1(a model of proximal tubular cells) and the humanHK-2 cellline The last one is a commonly used immortalized humanproximal tubular cell line which retains the phenotypic

expression and functional characteristic of human proximaltubular cells as described by others [29 30]

Different measures of cellular functionalstructuralchanges have been used to indicate cell toxicity due to RCMas outlined in the Table 2

3 Radiocontrast Agents Cause RenalHypoxia-Role of Reactive Oxygen Species

Many studies have reported that administration of radiocon-trast agents causes a decrease in renal medullary oxygena-tion [31] This may be due to mechanical factors such asincreased blood viscosity (in part related to red blood cellaggregation) and urine viscosity as well as changes in thelevels of vasoactivemediators such as endothelins natriureticpeptides nitric oxide adenosine and prostaglandins [31] Ithas also been proposed that the medullary hypoperfusion iscaused by constriction of the descending vasa recta (DVR)due to cytotoxic damage of the endothelial cells of the DVRcaused by RCM [32] Using isolated perfused human andrat DVR [32] it was observed that the IOCM iodixanol atphysiologically relevant concentrations caused constrictionof DVR and caused structural damage of endothelial cellsfrom rat renal interlobular arteries Thus it is possible thatsuch RCM-induced effects lead to reduced medullary bloodflow in the kidney A decrease in blood flow and hence inoxygen supply may lead to perturbations in the mitochon-drial electron transport chain leading to the production ofreactive oxygen species (ROS) that may have a detrimentaleffect within the cell by oxidizing membrane lipids inacti-vating proteins oxidizing DNA and activating cell signallingpathways leading to inflammation and cell death [33 34]In vitro studies have suggested that RCM may also leadto ROS production Sendeski et al using isolated singlespecimens of rat descending vasa recta (DVR) demonstratedthat iodixanol caused vasoconstriction of the DVR and theuse of the superoxide dismutase (SOD) mimetic Tempolreduced this iodixanol-induced vasoconstriction [35] Inaddition to demonstrating that iodixanol causes structuraldamage to endothelial cells from isolated arteries the samegroup has demonstrated that iodixanol caused an increasedpermeability of HUVEC (human umbilical vein endothelialcell)monolayers and an increased phosphorylation ofmyosinlight chain an indicator of endothelial cell retraction andincreased permeability [32] Hence it is feasible that RCMmay penetrate through the cell membrane and once inthe cytosol may also inflict similar damage to intracellularorganelles Indeed plasma membrane damage (measured asloss of the membrane proteins caveolin and NaK-ATPase)andmitochondrial damage (cytochrome c release) by ioversolhas been reported [9] As mentioned earlier disruption ofmitochondria may lead to the production of ROS and thismay be how RCM can induce the formation of ROS invitro without the need for hypoxia [34 36] Zager et al[9] questioned the role of oxidative stress in RCM renaltubular and cell injury They found that plasma membranedamage to proximal tubule segments isolated from mice andsubjected to the LOCM ioversol was not due to oxidant

BioMed Research International 3

Table 1 Iodinated contrast media commonly used in clinical practice

Name Type Iodine content Osmolality Osmolality type(mgmL) (mOsmkg)

IonicDiatrizoate (Hypaque 76) Monomer 370 2016 HOCMMetrizoate (Isopaque 370) Monomer 370 2100 HOCMIothalamate (Conray 400) Monomer 400 2300 HOCMIoxaglate (Hexabrix) Dimer 320 580 LOCM

NonionicIopamidol (Isovue 370) Monomer 370 796 LOCMIohexol (Omnipaque 350) Monomer 350 884 LOCMIopromide (Ultravist 370) Monomer 370 774 LOCMIoversol (Optiray 350) Monomer 350 792 LOCMIomeprol (Iomeron 400) Monomer 400 720 LOCMIobitridol (Xenetix 350) Monomer 350 915 LOCMIodixanol (Visipaque 320) Dimer 320 290 IOCMIotrolan (Isovist 300) Dimer 300 320 IOCM

Ionic and nonionic contrast media may be monomeric or dimeric 3 iodine atoms are present on each benzene ring of the contrast medium if a contrastmolecule contains only 1 benzene ring it is called a monomer if it contains 2 benzene rings it is called a dimer In solution ionic contrast media break up intotheir anion and cation components thereby increasing osmolality while nonionic contrast media do not break up in solution Nonionic dimers are the idealcontrast media as they deliver the most iodine with the least effect on osmolalityThe osmolality of contrast media is compared with the osmolality of plasma HOCM (high-osmolar contrast media) have the highest osmolality that is 5ndash8times the osmolality of plasma LOCM (low-osmolar contrast media) have an osmolality still higher than plasma which is 2-3 times the osmolality of plasmaIOCM (iso-osmolar contrast media) have the same osmolality as plasma

stress since no lipid peroxidation of the tubules was observedFurthermore using HK-2 cells Zager et al [9] observedthat the antioxidant N-acetylcysteine (NAC) failed to protectagainst RCM toxicity as assessed by lactate dehydrogenaserelease and MTT reduction

However more recent work using a recombinant man-ganese superoxide dismutase (SOD) administered in vivo torats undergoing diatrizoate treatment caused an improve-ment in the glomerular filtration rate and a reduction inrenal histologic damage [37] But the use of antioxidantsas therapeutic agents for the alleviation of CIN has yieldedconflicting data The use of the antioxidant NAC had beensuggested as a means of reducing CIN in patients [38] butsuccessive trials have been contradictory [39] and recentlythe Acetylcysteine for Contrast-induced nephropathy Trial(ACT) has concluded that NAC does not reduce the riskof CIN [40] Other antioxidants have been reported to beeffective In a clinical trial administration of ascorbic acid(vitamin C) protected against CIN whilst in an animalmodel rats fed with doses of alpha-tocopherol (vitaminE) before iopromide injection showed decreased tubularinjury due to the RCM increased SOD levels and reducedmalondialdehyde levels [41] Furthermore rats fed with thegrape seed proanthocyanidin extract and treated with theHOCM diatrizoate showed decreases in biochemical mark-ers of oxidative stress apoptosis and renal tissue damagecaused by the RCM Hizoh and Haller [20] observed aprotective effect by taurine on DNA fragmentation inducedby RCM in MDCK cells but not by NAC Nonetheless itshould be mentioned that intracellular peroxide levels incultured glomerularmesangial cells increased upon exposure

to diatrizoate and iohexol and those levels were attenuatedby alpha-tocopherol in diatrizoate-treated but not in iohexol-treated cells [42] Nonetheless it has been argued that theoxidative stress observed with the use of RCM may be aconsequence of the toxicity of the RCM rather than the cause[8]

4 RCM Effects on Endothelin Release

RCM have been reported to induce the release of the potentvasoconstrictor peptide endothelin (ET) both in vivo andin vitro as well as upregulating ET mRNA transcription[43ndash45] and also mediating the upregulation of the renalmedullary endothelin converting enzyme-1 expression andsynthesis [46] It is believed that the ET-A receptor is involvedin vasoconstriction whilst stimulation of the ET-B receptorhas the opposite effect A study in humans receiving RCMin which both ET receptors were blocked resulted in ahigher incidence of CIN in patients receiving the blockerthan those receiving a placebo [47] However the use of aspecific ET-A antagonist gave a more positive outcome in anin vivo rat study but this was explained by inhibitory effectsof the antagonist on tubular transport mechanisms therebydecreasing the oxygen demand and reducing hypoxia [48]

5 Role of Adenosine in CIN

Adenosine is a product of ATP degradation and may ariseunder the hypoxic conditions arising from RCM adminis-tration Whilst it may cause vasodilatation in most vesselsadenosine causes vasoconstriction in the renal vasculature


Table 2 Summary of some in vitro cell culture studies using different types of RCM

Authorsyear of publication Radiocontrast media used cell type used Cell functionalstructural changes observed

Laerum 1983 [11] HOCM and LOCM human endothelialcells

Chromium-51 release as measure of cell toxicityHOCMmore toxic than LOCM

Andersen et al 1994 [12] Ionic monomericdimeric nonionicLOCM MDCK and LLC-PK1 cells

RCM caused formation of large cytoplasmic vacuolesincrease in brush border and lysosomal marker enzymeactivity

Dascalu and Peer 1994 [13] Ionicnonionic RCM endothelial andrenal cells Acidification of internal pH decrease in cell viability

Andersen et al 1995 [14] Nonionic LOCM and IOCM MDCK andLLC-PK1 cells

RCM caused concentration-dependent formation oflarge cytoplasmic vacuoles cell deathdecrease in cellviability increase in brush border and lysosomalmarker enzyme activity These effects were morepronounced with LOCM than with IOCM

Potier et al 1997 [15] Ionicnonionic LOCM and HOCMmesangial cells

Dye uptake as measure of cell viabilityHOCMmore toxic than LOCM

Haller et al 1997 [16] HOCM and LOCM MDCK andLLC-PK1 cells

HOCMmore toxic LLC-PK1 cells more susceptible toRCM cytotoxicity

Hizoh et al 1998 [17] MDCK HOCM DNA fragmentation caused by RCM

Hardiek et al 2001 [18] LLC-PK1 cells and human renal proximaltubule cells HOCM LOCM and IOCM

Cell viability was affected by all RCM with HOCMhaving a greater affect than IOCM which in turn had agreater effect than LOCM

Fanning et al 2002 [19] Human neutrophils HOCM LOCM andIOCM

All types of RCM induced neutrophil apoptosis withHOCM having greater effect

Hizoh and Haller 2002 [20] MDCK cells HOCM HOCM induced DNA fragmentationYano et al 2003 [21] LLC-PK1 cells HOCM and LOCM HOCM caused a greater decrease in cell viability

Ribeiro et al 2004 [22] Renal artery smooth muscle cells HOCMand LOCM HOCM caused a greater decrease in cell viability

Heinrich et al 2005 [23] LLC-PK1 cells HOCM LOCM andIOCM

All types of RCM cause a decrease in cell viability withHOCM showing greatest effect

Andreucci et al 2006 2011 2014[24ndash26] HK-2 cells HOCM LOCM and IOCM All RCM caused a decrease in cell viability in the order

HOCM gt LOCM gt IOCM

Heinrich et al 2007 [27] LLC-PK1 cells LOCM and IOCM Cell viability measured no difference in toxicitybetween the 2 types of RCM

Yang et al 2013 [28] LOCM (ioversol) rat renal proximaltubular cell line (NRK-52E)

Decrease in cell viability and increase in intracellularCa2+ ion concentration

[49] thereby worsening the hypoxic conditions in the kidneyparenchyma Hence unselective (theophylline) and adeno-sine A1-receptor selective antagonists have been used toprevent CIN with some reported positive outcomes [50 51]In vitro mitochondrial damage by RCM may lead to disrup-tion of the electron transport chain which as in hypoxicconditions will lead to ATP degradation and adenosineproduction The adenosine produced may act as a substratefor xanthine oxidase leading to production of the ROShydrogen peroxide which would be harmful [52] Howeverwhilst it has been observed that RCM can increase adenosinelevels threefold in cultured HK-2 cells inhibition of xanthineoxidase with oxypurinol did not confer protection [9]

6 RCM May Impair Nitric Oxide (NO)Production

In a study by Ribeiro et al [22] using cultured smoothmusclecells obtained from rat renal artery HOCM and LOCM but

not IOCM were found to lower NO levels Thus in thiscase RCM may act to block a vasodilatory pathway It hasbeen suggested that one mechanism by which NO levels arelowered is by reactionwith superoxide ions that are generatedin the kidney by the RCM [53] which would lead to theformation of the even more potent oxidant peroxynitriteanion [54]

7 Cellular Signalling PathwaysAffected by RCM

Over the course of the last two decades our knowledgeof signal transduction pathways by which cells respond tochanges in their environment has increased immenselyTheseintracellular signalling pathways may determine cell fatefor example death survival proliferation and release ofhormones and may be triggered by mechanical chemicallight and thermal stimuli


71 Pathways Involved in Cell Survival and ProliferationSaito et al [55] reported a reduction in cyclic adenosinemonophosphate (cAMP) levels inmast cellsThis was quicklyfollowed by reports of the LOCM ioversol inducing apoptosisin LLC-PK1 cells [21] Incubation of LLC-PK1 cells withioversol for 30 minutes followed by a further incubationfor 24 h in the absence of the RCM caused an increase inthe activities of caspases-3 and -9 (proteases involved inapoptosis) and an increase in the mRNA for the proapoptoticprotein Bax whilst the mRNA levels of the antiapoptoticBcl-2 decreased It was also found that use of the cAMPanalogue dibutyl(DB)cAMP reversed the changes in a waythat was dependent on the signalingmolecules protein kinaseA (PKA) and phosphatidylinositol 3-kinase (PI 3-K) Thesame group also found that the prostaglandin I2 analogueberaprost sodium could also reverse the effects of ioversolon caspases and BaxBcl-2 [56] by phosphorylation of thecAMP-responsive element binding protein (CREB) via aPKA-dependent mechanism Further studies again usingLLC-PK1 cells suggested that the prosurvival kinase Akt mayalso be involved in the effects of beraprost sodium [57]Workfrom our laboratory has shown that incubation of HK-2 cellswith an HOCM (sodium diatrizoate) LOCM (iopromideand iomeprol) and IOCM (iodixanol) causes the dephos-phorylation of Akt at the Serine473 and Threonine308 sites[24 26] This was accompanied by effects on downstreamtargets of Akt such as p70S6 kinase (inactivated) which isinvolved in protein synthesis and the FoxO (Forkhead-box)family of transcription factors (dephosphorylated and henceactivated) [24] Both diatrizoate and iopromide caused adecrease in HK-2 cell viability which was partially alleviatedby transfection with plasmids encoding constitutively activeAkt [24] Diatrizoate also caused a greater dephosphorylationof mTOR (mammalian target of rapamycin) and ERK12(extracellular signal regulated kinases 12) with respect toiopromide iomerol or iodixanol [24 26] It should alsobe noted that the dephosphorylation of Akt p70S6 kinaseand FoxO proteins was observed in primary cultures ofhuman proximal tubule cells [24] It should be noted thatperturbations in the activity of kinases such as the ERK12 andAktmay affect the synthesis of vasodilatory and vasoconstric-tory molecules For example endothelin-1 gene transcriptionis negatively regulated by Akt and positively regulated byFoxO1 [58] whilst COX-2 expression and prostaglandinF2120572 synthesis may be dependent on ERK12 activity [59]Akt has also been implicated in vasorelaxation [60] West-ern blot analysis of whole lysates prepared from kidneysremoved from rats treated with the LOCM iomeprol andthe IOCM iodixanol also showed lowered levels of phospho-Akt (pAkt) and phospho-ERK12 (pERK12) compared withlysates obtained from control nontreated rats [26] An earlierstudy using the LOCM ioversol also indicated that RCMmay cause a decrease in the basal levels of pAkt in mousekidneys [61] These authors suggested that RCM enhance denovo ceramide synthesis and cause the activation of proteinphosphatase 2A (PP2A) which in turn can dephosphorylateAkt However PP2A may also act as an ERK- and JNK-phosphatase [62] but our in vitro results show discrepancybetween the effects of RCM on the phosphorylation status of

Akt ERK12 and the JNK (c-jun N-terminal kinase) familyof mitogen activated protein kinases (MAPKs) NonethelessItoh et al also found that use of ceramide synthase inhibitorsattenuated renal tubular cell injury induced by ioversol inLLC-PK1 cells and reduced the decreased pAkt levels invivo Our observations of the decrease in pAkt and pERK12by RCM was surprising since RCM have been associatedwith ROS production and our experience with HK-2 cellsis that these kinases are phosphorylated upon stimulationwith hydrogen peroxide [63 64] A possible explanationfor the decrease in phosphorylation of Akt may be due todisruption of cell membrane rafts that organize membrane-associated molecules such as receptors and kinases and reg-ulate cellular signal transduction [65] It has been proposedthat these membrane rafts allow for the close proximity andinteraction between Akt and kinases that phosphorylate itfor example PDK-1 (phosphoinositide-dependent kinase-1)and any perturbation of the raft structure may interfere withthe phosphorylation of Akt Given the already mentionedphysical impairment of the cell membrane by RCM [9 32]it is possible that RCM could be affecting signalling pathwaysin this way

72 Pathways Involved in Cell Death and Inflammation Ourgroup also demonstrated the phosphorylation of the p38 andJNK MAPKs and of the transcription factor NF-120581B (Ser276) by sodium diatrizoate and iomeprol in HK-2 cells [25]Sodium diatrizoate had a greater effect than iomeprol (at thesame concentration of iodine) on phosphorylation of thesemolecules which have been implicated in inflammation andupregulation of the proinflammatory cytokine IL-8 [66 67]Incubation of HK-2 cells with HOCM LOCM and IOCMat concentrations of 75 and 100mgImL for up to 3 h didnot result in caspase-3 cleavage [26] However in HK-2 cellsthat had been previously exposed to diatrizoate for 2-3 hand then incubated for a further 22 h (after removal of theRCM) caspase-3 cleavage was observed [25 26] It shouldbe noted that the presence of phosphorylated JNKs wasdetected in renal tubular epithelial cells collected from urinesamples of patients 24 h and 48 h after RCM administration[68] A possible role for the transcription factor NF-120581B wasalso suggested by Xu et al [69] who noted that the DNA-binding activity of NF-120581B in rat increased after diatrizoateadministration

In contrast Romano et al have demonstrated that bothLOCM and IOCM caused a marked increase in caspases-3and -9 activities and poly(ADP-ribose) polymerase fragmen-tation in HEK293 cells no effect was observed by them oncaspase-8 and -10 thus indicating that the RCM activatedapoptosis mainly through the intrinsic pathway [10] BothRCM induced an increase in protein expression levels ofproapoptotic members of the Bcl2 family Bim and BadTheyalso demonstrated that pretreatment with NAC and ascorbicacid but notwith sodiumbicarbonate could prevent apoptosisin a dose-dependent fashion

Using HEK293T cells Lee et al [70] analyzed the effectsof four different contrast media ionic high-osmolar diatri-zoate ionic low-osmolar iothalamate nonionic low-osmolar


iohexol and nonionic iso-osmolar iodixanol They showedthat diatrizoate iodixanol and iothalamate but interestinglynot iohexol induced the expression of ATF-2 (activatingtranscription factor-2) mRNA and phosphorylation of ATF-2 in HEK293T cells in a time-dependent manner Moreapoptotic cells were detected in diatrizoate-treated kidneycells than in the saline injection group Cell death wassignificantly increased by knockdown ATF-2 expression inthe presence of diatrizoate indicating a protective role ofATF-2 in contrast media-induced apoptosis Despite thestudyrsquos limitations such as the absence of any controlconditions (eg the use of hyperosmolar solutions sincediatrizoate is a high-osmolar RCM) in assessing the roleof hypertonicity in CIN pathogenesis and their choice ofcells that did not include an adult human proximal tubulecell line (such as HK-2 cells) the work provided newevidence that iodinated contrast media with the exceptionof iohexol (a nonionic LOCM) could activate the JNKATF-2 signaling pathways So this provided a new insight intothe mechanism and a potential way of prevention of CINshowing a differential activation of ATF-2 by different RCM[70]

Gong et al studied the apoptotic signaling mechanism inCIN and testedwhether the new antioxidantN-acetylcysteineamide (NACA) could prevent it using the RCM iohexolIn this study [71] LLC-PK1 cells were exposed to iohexolin order to observe their cell death with apoptotic featuresin a dose- and time-dependent manner they showed thatthe initiation of iohexol-induced apoptosis was mediated byupregulation of Bax and downregulation of Bcl-2 and Mcl-1(another anti-apoptotic protein) which was preceded by p38MAPK activation and iNOS (inducible nitric oxide synthase)inductionThe use of inhibitors of p38MAPK and iNOS par-tially abolished iohexol-induced apoptosis They also foundthat pretreatment with NACA partially protected cells fromiohexol-induced death by reverting the expression of Bcl-2Mc1-1 and Bax expression through inhibition of p38 MAPKand iNOS pathway NACA partially protecting LLC-PK1cells from iohexol-induced apoptosis by suppression of p38MAPK activation and iNOS protein expression was moreeffective than NAC a widely used antioxidant compoundThe authors claimed that a possible explanation is that NACAhas better membrane permeation and could therefore beexpected to be even more effective than NAC in vivo [71]However our study with p38 MAPK inhibition only yieldeda small increase in cell viability after exposure of HK-2 cellsto diatrizoate [26]

Recently Gong et al implicated the role of p38 MAPKas well as FoxO1 pathways in RCM toxicity [72] Using anexperimental model of CIN in rats they showed that tetram-ethylpyrazine (TMP) could significantly attenuate the result-ing renal dysfunction and renal tubular cellular apoptosisThese functional changes were accompanied by the decreasedlevels of phospho-p38 MAPK protein and attenuation ofthe increased FoxO1 mRNA and nuclear protein expression[72] Figure 1 shows a summary of the signaling moleculesthat play roles in cell death survival inflammation and invasoconstrictionvasodilation as discussed above

73 Cell Survival Pathways Induced by RCM The cellularstress evoked by RCM has also been shown to prompt anunfolded protein response [UPR] (which is believed to bea prosurvival response) in a rat renal proximal tubular cellline NRK-52E [73] Wu et al found that treatment of thecells with diatrizoate caused the expression of the chaperonesGRP (Glucose regulated protein) 78 and GRP94 which actto protect the cell under conditions of stress FurthermoreGRP78 dissociates from the endoplasmic reticulum trans-membrane receptor PERK [PKR (double-stranded RNA-activated protein kinase) like ER kinase] and in so doingallowing PERK to be activatedWu et al suggested that activePERK phosphorylates and activates the eukaryotic initiationfactor (eIF)2120572 which in turn reduces RCM-induced cellapoptosis [73]

Moon et al [74] suggested that angiopoietin-1 (Ang1)mayprotect vascular endothelial cells from iopromide-inducedapoptosis through PI 3-K and mTORS6 kinase postulatingthat the pretreatment with Ang1 could help in maintainingnormal vascular endothelial cell integrity before and duringsystemic RCM administration In that work Ang1 reducediopromide-induced apoptosis in a dose-dependent mannerTwo PI 3-K inhibitors wortmannin and LY294002 decreasedthe Ang1-induced anti-apoptotic effect Since Ang1 mediatesthe activation of mTORribosomal protein p70 S6 kinasethrough PI 3-K wortmannin and rapamycin an inhibitor ofmTOR suppressedAng1-induced p70S6 kinase phosphoryla-tion and partially inhibited the Ang1-induced anti-apoptoticeffect [74]

Yokomaku et al have suggested that asialoerythropoi-etin may have potential as a new therapeutic approach toprevent CIN given its ability to preserve renal functionand directly protect renal tissue as demonstrated in ratsin which nephropathy was induced by injection with theRCM ioversol in addition to inhibition of prostaglandinand nitric oxide synthesis [75] The administration of asingle dose of asialoerythropoietin before the induction ofnephropathy could significantly attenuate the resulting renaldysfunction and the histologic renal tubular injury RCM-induced apoptosis of renal tubular cells was inhibited byasialoerythropoietin both in vivo and in vitro and this effectwas blocked by a Janus kinase 2 (JAK2) inhibitor in vitro Fur-thermore phospho-JAK2signal transducer and activator oftranscription 5 (STAT5) andheat-shock protein 70 expressionincreased after injection of asialoerythropoietin suggestingthat the effects of asialoerythropoietin might be mediated bythe activation of the JAK2STAT5 pathway

Another cellular mechanism that may be protectiveagainst RCM injury was reported by Goodman et al [76]They demonstrated that heme oxygenase-1 induction bycobalt protoporphyrin could prevent the increase in plasmacreatinine and in superoxide ion formation in both the cortexand medulla in uninephrectomized salt depleted male Sabrarats treated with the RCM sodium iothalamate comparedwith untreated ratsThis protective effect of heme oxygenase-1 was associated with increased anti-apoptotic proteins Bcl-2and Bcl-xl and with a decrease of proapoptotic caspase-3 andcaspase-9 together with increased expression of inactive Bax


RCMAkt ERK12

cAMP

ET-1 mRNA

Vasoconstriction

PKA

CREB

Bcl-2 mRNA

COX-2

Prostaglandins

p70S6 kinase

Proteinsynthesis

FoxOtranscription

factors

Celldeath

Hypoxia mitochondrial

damage

Vasodilation

ROS

Adenosineproduction Vasoconstriction

Cell death

Cell death

inflammation

Bax Bim Bad

Cell death inflammation

JNKs p38 and NF-120581B

(minus)

(minus)(minus)

(minus)

(+) (+)(+)

Xanthine oxidase

Caspases-3 and -9

Figure 1 Scheme showing the effects of RCM on signaling molecules The scheme relates to effects on signaling molecules that mayunderlie the toxic effects of RCM RCM cause inactivation of the kinase Akt (as shown by the minus symbol) leading to activation of FoxOtranscription factors which may lead to cell death upregulation of ET-1 transcription and hence vasoconstriction deactivation of the p70S6kinase and hence downregulation of protein synthesis ERK12 are also inactivated which may lead to a decrease in activity of COX-2 andprostaglandin production and hence inhibition of vasodilation RCM also downregulate cAMP and hence the cAMP dependent kinase PKAThe antiapoptotic protein Bcl-2 is downregulated whilst the proapoptotic proteins Bax Bim Bad and caspases-3 and -9 are upregulated(as shown by the plus symbol) by RCM The JNK and p38 MAP kinases are activated as also is the transcription factor NF-120581B all three ofwhich may play a role in cell death and inflammation Hypoxia and mitochondrial damage caused by RCM may lead to the formation ofROS (reactive oxygen species) that can cause cell death and inflammation and to the formation of adenosine that can cause vasoconstrictionwhilst its metabolism by xanthine oxidase can lead to further formation of ROS

8 Conclusions

It is clear that RCM induce a drastic effect both in vitroand in vivo Whilst in vivo they cause changes in the tissueenvironment causing hypoxic conditions and changes inlevels of vasoconstrictory and vasodilatory factors that mayexacerbate the hypoxic milieu in vitro cell culture studieshave demonstrated that RCM cause changes in a variety ofcell signaling molecules that play important roles in cellularhomeostasis These include the deactivation of moleculessuch as Akt and ERK12 that enable cells to survive stress andto proliferate as well as to regulate the synthesis of vasoactivemolecules At the same time other molecular species areincreased or activated that may be detrimental such as ROSand signalling molecules such as the p38 and JNK kinasesand transcription factors such asNF-120581B thatmaymediate celldeath and inflammation [77] Delineation of the molecularmechanisms of RCM may help future strategies to reducetheir detrimental effects

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Ashour Michael is recipient of a Research Check fromldquoMagna Graeciardquo University of Catanzaro Italy

References

[1] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39 no5 pp 930ndash936 2002

[2] T G Gleeson and S Bulugahapitiya ldquoContrast-inducednephropathyrdquo American Journal of Roentgenology vol 183 no6 pp 1673ndash1689 2004

[3] S N Heyman M Brezis F H Epstein K Spokes P Silvaand S Rosen ldquoEarly renal medullary hypoxic injury fromradiocontrast and indomethacinrdquo Kidney International vol 40no 4 pp 632ndash642 1991

[4] H D Humes D A Hunt and M D White ldquoDirect toxiceffect of the radiocontrast agent diatrizoate on renal proximaltubule cellsrdquo American Journal of PhysiologymdashRenal Fluid andElectrolyte Physiology vol 252 no 2 pp F246ndashF255 1987

[5] V H Wallingford H G Decker and M Kruty ldquoX-ray contrastmedia I Iodinated acylaminobenzoic acidsrdquo Journal of theAmerican Chemical Society vol 74 no 17 pp 4365ndash4372 1952


[6] R W Katzberg ldquoUrography into the 21st century new contrastmedia renal handling imaging characteristics and nephrotox-icityrdquo Radiology vol 204 no 2 pp 297ndash312 1997

[7] S K Morcos ldquoContrast-induced nephropathy are there differ-ences between low osmolar and iso-osmolar iodinated contrastmediardquo Clinical Radiology vol 64 no 5 pp 468ndash472 2009

[8] M M Sendeski ldquoPathophysiology of renal tissue damage byiodinated contrast mediardquo Clinical and Experimental Pharma-cology and Physiology vol 38 no 5 pp 292ndash299 2011

[9] R A Zager A C M Johnson and S Y Hanson ldquoRadiographiccontrast media-induced tubular injury evaluation of oxidantstress and plasma membrane integrityrdquo Kidney Internationalvol 64 no 1 pp 128ndash139 2003

[10] G Romano C Briguori C Quintavalle et al ldquoContrast agentsand renal cell apoptosisrdquo EuropeanHeart Journal vol 29 no 20pp 2569ndash2576 2008

[11] F Laerum ldquoAcute damage to human endothelial cells by briefexposure to contrast media in vitrordquo Radiology vol 147 no 3pp 681ndash684 1983

[12] K-J Andersen E I Christensen and H Vik ldquoEffects of iodi-nated x-ray contrast media on renal epithelial cells in culturerdquoInvestigative Radiology vol 29 no 11 pp 955ndash962 1994

[13] A Dascalu and A Peer ldquoEffects of radiologic contrast media onhuman endothelial and kidney cell lines intracellular pH andcytotoxicityrdquoAcademic Radiology vol 1 no 2 pp 145ndash150 1994

[14] K J Andersen H Vik H P Eikesdal and E I ChristensenldquoEffects of contrast media on renal epithelial cells in culturerdquoActa Radiologica Supplementum vol 399 pp 213ndash218 1995

[15] M Potier I Lagroye B Lakhdar J Cambar and J IdeeldquoComparative cytotoxicity of low- and high-osmolar contrastmedia to human fibroblasts and rat mesangial cells in culturerdquoInvestigative Radiology vol 32 no 10 pp 621ndash626 1997

[16] C Haller C S Schick M Zorn andW Kubier ldquoCytotoxicity ofradiocontrast agents on polarized renal epithelial cell monolay-ersrdquo Cardiovascular Research vol 33 no 3 pp 655ndash665 1997

[17] I Hizoh J Strater C S Schick W Kubier and C HallerldquoRadiocontrast-induced DNA fragmentation of renal tubularcells in vitro role of hypertonicityrdquo Nephrology Dialysis Trans-plantation vol 13 no 4 pp 911ndash918 1998

[18] K Hardiek R E Katholi V Ramkumar and C DeitrickldquoProximal tubule cell response to radiographic contrast mediardquoAmerican Journal of PhysiologymdashRenal Physiology vol 280 no1 pp F61ndashF70 2001

[19] N F Fanning B J Manning J Buckley and H P RedmondldquoIodinated contrast media induce neutrophil apoptosis throughamitochondrial and caspase mediated pathwayrdquo British Journalof Radiology vol 75 no 899 pp 861ndash873 2002

[20] I Hizoh and C Haller ldquoRadiocontrast-induced renal tubularcell apoptosis hypertonic versus oxidative stressrdquo InvestigativeRadiology vol 37 no 8 pp 428ndash434 2002

[21] T Yano Y Itoh T Sendo T Kubota and R Oishi ldquoCyclic AMPreverses radiocontrast media-induced apoptosis in LLC-PK1cells by activating a kinasePI3 kinaserdquo Kidney Internationalvol 64 no 6 pp 2052ndash2063 2003

[22] L Ribeiro F de Assuncao e Silva R S Kurihara N Schorand E M S Higa ldquoEvaluation of the nitric oxide productionin rat renal artery smooth muscle cells culture exposed toradiocontrast agentsrdquo Kidney International vol 65 no 2 pp589ndash596 2004

[23] M C Heinrich M K Kuhlmann A Grgic M HeckmannB Kramann and M Uder ldquoCytotoxic effects of ionic high-osmolar nonionic monomeric and nonionic iso-osmolar

dimeric iodinated contrastmedia on renal tubular cells in vitrordquoRadiology vol 235 no 3 pp 843ndash849 2005

[24] M Andreucci G Fuiano P Presta et al ldquoRadiocontrast mediacause dephosphorylation of Akt and downstream signalingtargets in human renal proximal tubular cellsrdquo BiochemicalPharmacology vol 72 no 10 pp 1334ndash1342 2006

[25] MAndreucci G Lucisano T Faga et al ldquoDifferential activationof signaling pathways involved in cell death survival andinflammation by radiocontrast media in human renal proximaltubular cellsrdquo Toxicological Sciences vol 119 no 2 pp 408ndash4162011

[26] M Andreucci T Faga D Russo et al ldquoDifferential activationof signaling pathways by low-osmolar and iso-osmolar radio-contrast agents in human renal tubular cellsrdquo Journal of CellularBiochemistry vol 115 no 2 pp 281ndash289 2014

[27] M Heinrich M Scheer M Heckmann W Bautz andM Uder ldquoReversibility and time-dependency of contrastmedium induced inhibition of 3-(45-dimethylthiazol-2-yl)-25-diphenyl-tetrazolium bromide (MTT) conversion in renalproximal tubular cells in vitro comparison of amonomeric anda dimeric nonionic iodinated contrast mediumrdquo InvestigativeRadiology vol 42 no 11 pp 732ndash738 2007

[28] D Yang D Yang R Jia andGDing ldquoSelective inhibition of thereverse mode of Na(+)Ca(2+) exchanger attenuates contrast-induced cell injuryrdquoAmerican Journal of Nephrology vol 37 pp264ndash273 2013

[29] L C Racusen C Monteil A Sgrignoli et al ldquoCell lines withextended in vitro growth potential from human renal proximaltubule characterization response to inducers and comparisonwith established cell linesrdquo Journal of Laboratory and ClinicalMedicine vol 129 no 3 pp 318ndash329 1997

[30] M J Ryan G Johnson J Kirk S M Fuerstenberg R A Zagerand B Torok-Storb ldquoHK-2 an immortalized proximal tubuleepithelial cell line from normal adult human kidneyrdquo KidneyInternational vol 45 no 1 pp 48ndash57 1994

[31] S N Heyman S Rosen and C Rosenberger ldquoRenal parenchy-mal hypoxia hypoxia adaptation and the pathogenesis ofradiocontrast nephropathyrdquo Clinical Journal of the AmericanSociety of Nephrology vol 3 no 1 pp 288ndash296 2008

[32] M M Sendeski A B Persson Z Z Liu et al ldquoIodinatedcontrast media cause endothelial damage leading to vasocon-striction of human and rat vasa rectardquo American Journal ofPhysiologymdashRenal Physiology vol 303 pp F1592ndashF1598 2012

[33] C Li and R M Jackson ldquoReactive species mechanisms ofcellular hypoxia-reoxygenation injuryrdquo American Journal ofPhysiologymdashCell Physiology vol 282 no 2 pp C227ndashC2412002

[34] M P Murphy ldquoHow mitochondria produce reactive oxygenspeciesrdquo Biochemical Journal vol 417 no 1 pp 1ndash13 2009

[35] M Sendeski A Patzak T L Pallone C Cao A E Persson andP B Persson ldquoIodixanol constriction of medullary descendingvasa recta and risk for contrastmedium-induced nephropathyrdquoRadiology vol 251 no 3 pp 697ndash704 2009

[36] G Ozkan S Ulusoy A Orem et al ldquoProtective effect of thegrape seed proanthocyanidin extract in a rat model of contrast-induced nephropathyrdquo Kidney and Blood Pressure Research vol35 pp 445ndash453 2012

[37] A Pisani M Sabbatini E Riccio et al ldquoEffect of a recom-binant manganese superoxide dismutase on prevention ofcontrast-induced acute kidney injuryrdquo Clinical and Experimen-tal Nephrology 2013


[38] M Tepel M van der Giet C Schwarzfeld U Laufer DLiermann andW Zidek ldquoPrevention of radiographic-contrast-agent-induced reductions in renal function by acetylcysteinerdquoThe New England Journal of Medicine vol 343 no 3 pp 180ndash184 2000

[39] S Fishbane ldquoN-acetylcysteine in the prevention of contrast-induced nephropathyrdquo Clinical Journal of the American Societyof Nephrology vol 3 no 1 pp 281ndash287 2008

[40] ACT Investigators ldquoAcetylcysteine for prevention of renal out-comes in patients undergoing coronary and peripheral vascularangiography main results from the randomized acetylcysteinefor contrast-induced nephropathy trial (ACT)rdquoCirculation vol124 no 11 pp 1250ndash1259 2011

[41] S Kongkham S Sriwong and A Tasanarong ldquoProtective effectof alpha tocopherol on contrast-induced nephropathy in ratsrdquoNefrologia vol 33 pp 116ndash123 2013

[42] M Wasaki J Sugimoto and K Shirota ldquoGlucose alters thesusceptibility of mesangial cells to contrast mediardquo InvestigativeRadiology vol 36 no 7 pp 355ndash362 2001

[43] S N Heyman B A Clark N Kaiser et al ldquoRadiocontrastagents induce endothelin release in vivo and in vitrordquo Journalof the American Society of Nephrology vol 3 no 1 pp 58ndash651992

[44] S N Heyman B A Clark L Cantley et al ldquoEffects of ioversolversus iothalamate on endothelin release and radiocontrastnephropathyrdquo Investigative Radiology vol 28 no 4 pp 313ndash3181993

[45] J-M Sung G H F Shu J-C Tsai and J-J Huang ldquoRadio-contrast media induced endothelin-1 mRNA expression andpeptide release in porcine aortic endothelial cellsrdquo Journal of theFormosan Medical Association vol 94 no 3 pp 77ndash86 1995

[46] M Khamaisi I Raz V Shilo et al ldquoDiabetes and radiocontrastmedia increase endothelin converting enzyme-1 in the kidneyrdquoKidney International vol 74 no 1 pp 91ndash100 2008

[47] A Wang T Holcslaw T M Bashore et al ldquoExacerbation ofradiocontrast nephrotoxicity by endothelin receptor antago-nismrdquo Kidney International vol 57 no 4 pp 1675ndash1680 2000

[48] P Liss P-O Carlsson A Nygren F Palm and P Hansell ldquoET-A receptor antagonist BQ123 prevents radiocontrast media-induced renal medullary hypoxiardquoActa Radiologica vol 44 no1 pp 111ndash117 2003

[49] P BHansen and J Schnermann ldquoVasoconstrictor and vasodila-tor effects of adenosine in the kidneyrdquo American Journal ofPhysiologymdashRenal Physiology vol 285 no 4 pp F590ndashF5992003

[50] CM Erley SHDuda S Schlepckow et al ldquoAdenosine antago-nist theophylline prevents the reduction of glomerular filtrationrate after contrast media applicationrdquo Kidney International vol45 no 5 pp 1425ndash1431 1994

[51] R E Katholi G J Taylor W P McCann et al ldquoNephrotoxicityfrom contrast media attenuation with theophyllinerdquo Radiologyvol 195 no 1 pp 17ndash22 1995

[52] P C Y Wong Z Li J Guo and A Zhang ldquoPathophysiology ofcontrast-induced nephropathyrdquo International Journal of Cardi-ology vol 158 pp 186ndash192 2012

[53] C Cao A Edwards M Sendeski et al ldquoIntrinsic nitric oxideand superoxide production regulates descending vasa rectacontractionrdquoAmerican Journal of PhysiologymdashRenal Physiologyvol 299 no 5 pp F1056ndashF1064 2010

[54] P Pacher J S Beckman and L Liaudet ldquoNitric oxide andperoxynitrite in health and diseaserdquo Physiological Reviews vol87 no 1 pp 315ndash424 2007

[55] M Saito Y Itoh T Yano et al ldquoRoles of intracellular Ca(2+)and cyclic AMP in mast cell histamine release induced byradiographic contrast mediardquo Naunyn-Schmiedebergrsquos Archivesof Pharmacology vol 367 no 4 pp 364ndash371 2003

[56] T Yano Y Itoh T Kubota T Sendo and R Oishi ldquoA prostacy-clin analog beraprost sodium attenuates radiocontrast media-induced LLC-PK1 cells injuryrdquoKidney International vol 65 no5 pp 1654ndash1663 2004

[57] T Yano Y Itoh T Kubota et al ldquoA prostacyclin analog preventsradiocontrast nephropathy via phosphorylation of cyclic AMPresponse element binding proteinrdquo The American Journal ofPathology vol 166 no 5 pp 1333ndash1342 2005

[58] L R Stow M E Jacobs C S Wingo and B D CainldquoEndothelin-1 gene regulationrdquoThe FASEB Journal vol 25 no1 pp 16ndash28 2011

[59] H N Jabbour K J Sales S C Boddy R A Anderson andA R W Williams ldquoA positive feedback loop that regulatescyclooxygenase-2 expression and prostaglandin F2120572 synthesisvia the F-series-prostanoid receptor and extracellular signal-regulated kinase 12 signaling pathwayrdquo Endocrinology vol 146no 11 pp 4657ndash4664 2005

[60] T Kobayashi T Matsumoto and K Kamata ldquoThe PI3-KAktpathway roles related to alterations in vasomotor responses indiabetic modelsrdquo Journal of SmoothMuscle Research vol 41 no6 pp 283ndash302 2005

[61] Y Itoh T Yano T Sendo et al ldquoInvolvement of de novoceramide synthesis in radiocontrast-induced renal tubular cellinjuryrdquo Kidney International vol 69 no 2 pp 288ndash297 2006

[62] Q Liu and P A Hofmann ldquoProtein phosphatase 2A-mediatedcross-talk between p38 MAPK and ERK in apoptosis of cardiacmyocytesrdquo American Journal of PhysiologymdashHeart and Circula-tory Physiology vol 286 no 6 pp H2204ndashH2212 2004

[63] M Andreucci T Faga G Lucisano et al ldquoMycophenolic acidinhibits the phosphorylation of NF-120581B and JNKs and causes adecrease in IL-8 release in H

2O2-treated human renal proximal

tubular cellsrdquoChemico-Biological Interactions vol 185 no 3 pp253ndash262 2010

[64] M Andreucci G Fuiano P Presta et al ldquoDownregulation ofcell survival signalling pathways and increased cell damage inhydrogen peroxide-treated human renal proximal tubular cellsby alpha-erythropoietinrdquo Cell Proliferation vol 42 no 4 pp554ndash561 2009

[65] D Calay D Vind-Kezunovic A Frankart S Lambert YPoumay and R Gniadecki ldquoInhibition of akt signaling byexclusion from lipid rafts in normal and transformed epidermalkeratinocytesrdquo Journal of Investigative Dermatology vol 130 no4 pp 1136ndash1145 2010

[66] J M Kyriakis and J Avruch ldquoMammalian MAPK signaltransduction pathways activated by stress and inflammation a10-year updaterdquo Physiological Reviews vol 92 no 2 pp 689ndash737 2012

[67] D E Nowak B Tian M Jamaluddin et al ldquoRelA Ser276phosphorylation is required for activation of a subset of NF-120581B-dependent genes by recruiting cyclin-dependent kinase 9cyclint1 complexesrdquoMolecular and Cellular Biology vol 28 no 11 pp3623ndash3638 2008

[68] C Quintavalle M Brenca F de Micco et al ldquoIn vivo andin vitro assessment of pathways involved in contrast media-induced renal cells apoptosisrdquo Cell Death and Disease vol 2no 5 article e155 2011

[69] X Xu T Wu X Ding J Zhu J Zou and J He ldquoThe roleof nuclear factor-120581B in rats of radiocontrast-media-induced


nephropathyrdquo Journal of Biochemical and Molecular Toxicologyvol 22 no 6 pp 416ndash421 2008

[70] H Lee S Sheu H Yen W Lai and J Chang ldquoJNKATF2 path-way is involved in iodinated contrastmedia-induced apoptosisrdquoAmerican Journal of Nephrology vol 31 no 2 pp 125ndash133 2010

[71] X Gong G Celsi K Carlsson S Norgren and M Chen ldquoN-acetylcysteine amide protects renal proximal tubular epithelialcells against iohexol-induced apoptosis by blocking p38 MAPKand iNOS signalingrdquo American Journal of Nephrology vol 31no 2 pp 178ndash188 2010

[72] X Gong Q Wang X Tang et al ldquoTetramethylpyrazine pre-vents contrast-induced nephropathy by inhibiting p38 MAPKand FoxO1 signaling pathwaysrdquo American Journal of Nephrol-ogy vol 37 pp 199ndash207 2013

[73] C T Wu M L Sheu K S Tsai T I Weng C K Chiangand S H Liu ldquoThe role of endoplasmic reticulum stress-related unfolded protein response in the radiocontrastmedium-induced renal tubular cell injuryrdquoToxicological Sciences vol 114no 2 pp 295ndash301 2010

[74] S O Moon W Kim D H Kim et al ldquoAngiopoietin-1 reducesiopromide-induced endothelial cell apoptosis through activa-tion of phosphatidylinositol 31015840-kinaseP70 S6 kinaserdquo Interna-tional Journal of Tissue Reactions vol 27 no 3 pp 115ndash124 2005

[75] Y Yokomaku T Sugimoto S Kume et al ldquoAsialoerythropoietinprevents contrast-induced nephropathyrdquo Journal of the Ameri-can Society of Nephrology vol 19 no 2 pp 321ndash328 2008

[76] A I Goodman R Olszanecki L M Yang et al ldquoHemeoxygenase-1 protects against radiocontrast-induced acute kid-ney injury by regulating anti-apoptotic proteinsrdquo Kidney Inter-national vol 72 no 8 pp 945ndash953 2007

[77] M Andreucci ldquoContrast media and nephrotoxicity a molecu-lar conundrumrdquo Giornale Italiano di Nefrologia vol 28 no 4p 355 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


solution and are termed as high-osmolar contrast media(HOCM) Then the carboxyl groups were replaced by non-polar groups giving nonionic soluble molecules with lowerosmolality These were termed as the low-osmolar contrastmedia (LOCM) and were further improved by the additionof more hydroxyl groups for increased hydrophilicity andthen a more even distribution of the hydroxyl groups onthe molecule Finally the dimerization of two molecules viaside chains on the benzene ring gave rise to the nonioniciso-osmolar contrast media (IOCM) with increased iodineatoms per molecule [6] Of interest is the RCM ioxaglatethat whilst it is ionic it is classified as an LOCM as it is adimer containing more iodine atoms per particle in solutionA summary of the most commonly used RCM are presentedin Table 1The improvements in contrast media developmenthave resulted in the acknowledgement that in clinical usethe newer LOCM and ICOM are less toxic than the originalHOCM [7] but in vitro cell culture studies have suggestedthat all types of RCM have a direct toxic effect in manydifferent types of cells (see Table 2) It has been suggested thatmolecular iodine may be present in solutions of RCM due todegradation [8] but this may be negligible in solutions thathave been properly stored Moreover a study has shown thatincubating isolated proximal tubule segments with varyingconcentrations of NaI had no adverse effects on cell viability[9] and a further study showed that sodium iodide alone didnot cause significant cell death in cultured renal cells [10]

2 Toxicity of Radiocontrast Media inIn Vitro Cell Culture Studies

Many in vitro studies have investigated the toxicity of RCMusing different types of cultured cells including renal epithe-lial cells mesangial cells endothelial cells smooth musclecells hepatic cells human fibroblasts pulmonary mast cellshuman embryonic kidney cells and human neutrophils

The most common in vitro studies addressing the patho-physiology of RCM-induced apoptosis have been criticizedbecause of their limitations which include (1) the assessmentof only one potential mechanism of the RCM-induced renalcell damage in the absence of several conflicting variablesthat can be found in vivo (2) the exposure to a constantconcentration of RCM to all cell lines whereas in vivo themore distal epithelial tubular cells are exposed to a muchhigher concentration than the proximal tubular cells (3) thepotentially high dose of RCM commonly used for the cellculture experiments (4) the fact that effects of the RCM areusually investigated as cellular ldquoshort termrdquo-induced effects(ie just after their exposure to cells) not along several hours(as ldquolong termrdquo effects) of exposure to them Finally researchgroups rarely compare different types of RCM (especiallythose with different osmolarities) in the same study

The most commonly used types of renal tubular cells forin vitro studies include the canine-derived MDCK cells (amodel of distal tubular cells) the porcine cell line LLC-PK1(a model of proximal tubular cells) and the humanHK-2 cellline The last one is a commonly used immortalized humanproximal tubular cell line which retains the phenotypic

expression and functional characteristic of human proximaltubular cells as described by others [29 30]

Different measures of cellular functionalstructuralchanges have been used to indicate cell toxicity due to RCMas outlined in the Table 2

3 Radiocontrast Agents Cause RenalHypoxia-Role of Reactive Oxygen Species

Many studies have reported that administration of radiocon-trast agents causes a decrease in renal medullary oxygena-tion [31] This may be due to mechanical factors such asincreased blood viscosity (in part related to red blood cellaggregation) and urine viscosity as well as changes in thelevels of vasoactivemediators such as endothelins natriureticpeptides nitric oxide adenosine and prostaglandins [31] Ithas also been proposed that the medullary hypoperfusion iscaused by constriction of the descending vasa recta (DVR)due to cytotoxic damage of the endothelial cells of the DVRcaused by RCM [32] Using isolated perfused human andrat DVR [32] it was observed that the IOCM iodixanol atphysiologically relevant concentrations caused constrictionof DVR and caused structural damage of endothelial cellsfrom rat renal interlobular arteries Thus it is possible thatsuch RCM-induced effects lead to reduced medullary bloodflow in the kidney A decrease in blood flow and hence inoxygen supply may lead to perturbations in the mitochon-drial electron transport chain leading to the production ofreactive oxygen species (ROS) that may have a detrimentaleffect within the cell by oxidizing membrane lipids inacti-vating proteins oxidizing DNA and activating cell signallingpathways leading to inflammation and cell death [33 34]In vitro studies have suggested that RCM may also leadto ROS production Sendeski et al using isolated singlespecimens of rat descending vasa recta (DVR) demonstratedthat iodixanol caused vasoconstriction of the DVR and theuse of the superoxide dismutase (SOD) mimetic Tempolreduced this iodixanol-induced vasoconstriction [35] Inaddition to demonstrating that iodixanol causes structuraldamage to endothelial cells from isolated arteries the samegroup has demonstrated that iodixanol caused an increasedpermeability of HUVEC (human umbilical vein endothelialcell)monolayers and an increased phosphorylation ofmyosinlight chain an indicator of endothelial cell retraction andincreased permeability [32] Hence it is feasible that RCMmay penetrate through the cell membrane and once inthe cytosol may also inflict similar damage to intracellularorganelles Indeed plasma membrane damage (measured asloss of the membrane proteins caveolin and NaK-ATPase)andmitochondrial damage (cytochrome c release) by ioversolhas been reported [9] As mentioned earlier disruption ofmitochondria may lead to the production of ROS and thismay be how RCM can induce the formation of ROS invitro without the need for hypoxia [34 36] Zager et al[9] questioned the role of oxidative stress in RCM renaltubular and cell injury They found that plasma membranedamage to proximal tubule segments isolated from mice andsubjected to the LOCM ioversol was not due to oxidant































































RCMAkt ERK12

cAMP

ET-1 mRNA

Vasoconstriction

PKA

CREB

Bcl-2 mRNA

COX-2

Prostaglandins

p70S6 kinase

Proteinsynthesis

FoxOtranscription

factors

Celldeath


damage

Vasodilation

ROS


Cell death

Cell death

inflammation

Bax Bim Bad



(minus)

(minus)(minus)

(minus)

(+) (+)(+)

Xanthine oxidase

Caspases-3 and -9


8 Conclusions




Acknowledgment


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































































RCMAkt ERK12

cAMP

ET-1 mRNA

Vasoconstriction

PKA

CREB

Bcl-2 mRNA

COX-2

Prostaglandins

p70S6 kinase

Proteinsynthesis

FoxOtranscription

factors

Celldeath


damage

Vasodilation

ROS


Cell death

Cell death

inflammation

Bax Bim Bad



(minus)

(minus)(minus)

(minus)

(+) (+)(+)

Xanthine oxidase

Caspases-3 and -9


8 Conclusions




Acknowledgment


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article molecular mechanisms of renal cellular ... · e opacity of iodine to x-rays renders...

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