Beneficial Effects of Myo-Inositol Oxygenase Deficiency in ...

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Beneficial Effects of Myo-Inositol OxygenaseDeficiency in Cisplatin-Induced AKI

Rajesh K. Dutta,* Vinay K. Kondeti,* Isha Sharma,* Navdeep S. Chandel,†

Susan E. Quaggin,† and Yashpal S. Kanwar*†

Departments of *Pathology and †Medicine, Northwestern University, Chicago, Illinois

ABSTRACTOverexpression of the proximal tubular enzymemyo-inositol oxygenase (MIOX) induces oxidant stress invitro. However, the relevance of MIOX to tubular pathobiology remains enigmatic. To investigate the roleof MIOX in cisplatin-induced tubular AKI, we generated conditional MIOX-overexpressing transgenic(MIOX-TG)mice andMIOX-knockout (MIOX2/2) mice with tubule-specificMIOX overexpression or knock-out, respectively. Compared with cisplatin-treated wild-type (WT) mice, cisplatin-treated MIOX-TG micehad even greater increases in urea, creatinine, and KIM-1 levels andmore tubular injury and apoptosis, butthese effectswere attenuated in cisplatin-treatedMIOX2/2mice. Similarly,MIOX-TGmice had the highestand MIOX2/2 mice had the lowest renal levels of Bax, cleaved caspase-3, and NADPH oxidase-4 expres-sion and reactive oxygen species (ROS) generation after cisplatin treatment. In vitro, cisplatin dose-dependently increased ROS generation in LLC-PK1 cells. Furthermore, MIOX overexpression in thesecells accentuated cisplatin-induced ROS generation and perturbations in the ratio of GSH to oxidizedGSH, whereas MIOX-siRNA or N-acetyl cysteine treatment attenuated these effects. Additionally, thecisplatin-induced enhancement of p53 activation, NF-kB binding to DNA, andNF-kB nuclear translocationin WTmice was exacerbated in MIOX-TGmice but absent in MIOX2/2mice. In vitro, MIOX-siRNA or NACtreatment reduced the dose-dependent increase in p53 expression induced by cisplatin. We alsoobserved a remarkable influx of inflammatory cells and upregulation of cytokines in kidneys of cisplatin-treated MIOX-TG mice. Finally, analysis of genomic DNA in WT mice revealed cisplatin-induced hypome-thylation of the MIOX promoter. These data suggest that MIOX overexpression exacerbates, whereasMIOX gene disruption protects against, cisplatin-induced AKI.

J Am Soc Nephrol 28: 1421–1436, 2017. doi: https://doi.org/10.1681/ASN.2016070744

AKI is encountered in up to 30% of critically illhospitalized patients, and it is quite often associatedwith acute tubular necrosis (ATN) leading to rapiddeterioration of renal functions.1,2 There are a widerange of clinico-pathologic states that may be asso-ciated with ATN, and the latter can be classifiedunder two broad categories, i.e., ischemic versustubulo-toxic injury.3 The pathophysiologic mecha-nisms leading to AKI between these two categoriesmay differ to a certain extent, and conceivably thismay be one of the factorswhydifferent segments of thenephron are differentially affected, possibly involvingdifferent cellular and molecular mechanisms.4 For in-stance, although all of the three segments of proxi-mal tubules are vulnerable to ischemic injury, the S3segment is highly susceptible to tubulo-toxins by

involving pathogeneticmechanisms that are restrictiveto molecules expressed in this segment of the neph-ron.5 With respect to cellular specificity, past experi-ments have demonstrated that mice with selectiveablation of p53 in proximal tubules are resistant to

Received July 12, 2016. Accepted October 17, 2016.

R.K.D. and V.K.K. contributed equally to this work.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Yashpal S. Kanwar, Department of Pathol-ogy, Northwestern University Medical School, 303 E. Chicago Ave-nue, Chicago, Illinois 60611. Email: [email protected]

Copyright © 2017 by the American Society of Nephrology

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tubulo-toxins such as cisplatin, whereas p53 deletion in othersegments of the nephron provided no protection against toxicinjury.6 Intriguingly, repetitive tubulo-toxic assault can lead toboth adaptive as well as maladaptive changes.7 Early adaptivechanges could be beneficial in the form of cytoresistance, whereaslate maladaptive changes may be detrimental; these include upre-gulation of inflammatory and profibrogenic cytokines along withoxidant stress, which ultimately could lead to tubulo-interstitialfibrosis, CKD, and renal failure.7–9

Myo-inositol oxygenase (MIOX), a proximal tubular enzyme,has been implicated in the pathogenesis of diabetic tubulopathy.10

It catabolizesmyo-inositol, that is synthesizedby isomerization anddephosphorylation of glucose 6-phospate, to D-glucuronic acidvia the Glucuronate-Xylulose pathway (G-X pathway).11,12 It hasosmotic-, carbohydrate-, and sterol- response elements in its pro-moter, and its expression is modulated by organic osmolytes, fattyacids, and obesity besides high glucose ambience or diabetes.13,14

In addition, the MIOX promoter region includes antioxidant andoxidant response elements and its transcription is regulated by aredox-sensitive response transcription factor, Nrf2.13 Moreover,MIOX overexpression in vitro accentuates the generation of reac-tive oxygen species (ROS), apparently derived both from mito-chondrial and NADPH oxidase systems.15 The kidney is one ofthemajor organs vulnerable to oxidant damage by ROS because ofits high concentration of long-chain polyunsaturated fatty acids.16

Conceivably, the free radicals or ROS attack long-chain highly-unsaturated fatty acids and promote lipid peroxidation causingoxidative damage. In general, the ROS adversely affect homeostasisof the kidney in a variety of disease states, including diabetes mel-litus, hypertension, obesity, ischemia, and ARF secondary to theadministration of analgesics, antibiotics, and chemotherapeuticagents.16 Some of these states, e.g., secondary to the administrationof therapeutic agents, are associated with AKI, which necessitatesthe development of various animal model systems to delineate themechanisms that are relevant to thepathobiologyof renal tubules.17

Cisplatin-induced nephropathy is a prototype toxic injurymodel which has been extensively utilized to investigate themechanisms relevant to the pathogenesis of AKI.5 Cisplatin istaken up by proximal tubules, and inhibition of organic cationtransporters with cimetidine decreases its cellular uptake.In 1view of the fact that cellular damage is confined to prox-imal tubules in toxin-induced nephropathy, and MIOX, ametabolic enzyme, is expressed in proximal tubules and itsoverexpression in vitro leads to the generation of ROS,studies were initiated to assess the effect of up- or downregu-lation of MIOX on the outcomes of cisplatin-inducednephropathy and associated signaling events in transgenicand knockout mice.

RESULTS

Characterization of MIOX Transgenic and Null MiceWestern blot analyses revealed an increased expression (2–3-fold) of MIOX in kidneys of transgenic mice compared with

the flox/flox or wild-type (WT) and PEPCK-Cre mice (Figure1, C and D). No significant renal expression of MIOX wasobserved in MIOX-knockout (MIOX2/2) mice (Figure 1, Eand F). By immuno-histochemistry a marked increase inMIOX expression was observed in renal proximal tubules inMIOX transgenic (MIOX-TG)mice (Figure 1, H versusG). Noimmuno-histochemical signal of MIOX was observed inMIOX2/2 mice (Figure 1I). Both the mutant mice generatedwere proximal tubule–specific. Moreover, MIOX is a proximaltubule–specific enzyme. We did not observe any MIOX stain-ing in any other renal compartment.

MIOX Gene Disruption Ameliorates Cisplatin-InducedAKICisplatin (20mg/kg) induced significantweight loss inMIOX-TGand WT mice, whereas MIOX2/2 mice had minimal bodyweight loss (Figure 2A). Maximal renal functional changeswere seen in cisplatin-treated MIOX-TG mice. Both serumcreatinine and urea were markedly elevated in MIOX-TGmice (Figure 2, B and C). A minimal increase in creatinineor urea levels was seen in MIOX2/2 mice. A significant in-crease in expression of Kim-1 mRNA, a marker of AKI,18 wasobserved in kidneys of cisplatin-treated MIOX-TGmice (Fig-ure 2D).WTmice showed cytolysis of tubular cells with loss ofbrush border (Figure 2, H versus E). These changes were ac-centuated in MIOX-TG mice (Figure 2, I versus F). Interest-ingly, the kidneys of cisplatin-treated MIOX2/2 mice had mildcytolytic changes compared with WTor TG strains (Figure 2, Jversus G).

MIOX Gene Disruption Inhibits, Whereas ItsOverexpression Accentuates, Renal Tubular CellApoptosis in Cisplatin-Induced AKIBy immuno-histochemistry and immunoblot analyses an in-creased Bax expression was seen in tubules of cisplatin-treatedWT mice (Figures 3, D versus A, and M). It was markedlyincreased in MIOX-TG mice (Figure 3, E versus B, and M).No significant increase was observed in MIOX2/2 mice(Figure 3, F versus C, and M). On the other hand, Bcl-2 expres-sion remarkably decreased in both WT and MIOX-TG mice,whereas it remained unchanged in MIOX2/2 mice (Figure3M). The status of another executioner of the apoptosis path-way, known as caspase-3, was investigated. It is activated aftercleavage and catalyzes specific cleavage of multiple cellularproteins and thus activates apoptosis.19 An increased expres-sion of cleaved caspase-3 was observed in kidneys of cisplatin-treated WTmice (Figure 3M). This increase was accentuatedin MIOX-TG mice. No expression of cleaved caspase-3 was ob-served in MIOX2/2mice (Figure 3M). An increase in caspase-3activity in kidneys of cisplatin-treated WT mice was observed(Figure 3N), but it was accentuated in MIOX-TG mice. A mildincrease in its activitywas observed in cisplatin-treatedMIOX2/2

mice (Figure 3N).Using the terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling(TUNEL) procedure, a notable degree of apoptosis was observed

1422 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1421–1436, 2017


in tubules of cisplatin-treated WTmice (Figure 3, J versus G).Whereas a fulminant apoptosis was observed in cisplatin-treatedMIOX-TG mice (Figure 3, K versus H), no TUNEL-positivenuclei were seen in MIOX2/2 mice (Figure 3, I and L).

MIOX Accentuates Cisplatin-Induced ROS GenerationGeneration of ROS is one of the major mechanism(s) respon-sible for cisplatin-induced AKI.20 The status of ROS was in-vestigated because it is known that MIOX overexpressionaccentuates ROS generation.15 Flow cytometric analysesrevealed a dose-dependent increase in ROS levels in cells trans-fected with empty vector after cisplatin (20 mM) treatment(Figure 4, A–C, and J), whereas MIOX-overexpressing cellsexhibited a remarkable further increase in ROS levels aftercisplatin treatment (Figure 4, F–H, and J). AfterMIOX-siRNAor anti-oxidant NAC treatment the cisplatin-induced increasein ROS was reduced (Figure 4, D and E, I and J). Fluorescentmicroscopy of cells subjected to various treatments confirmedthe observations of flow cytometry (Figure 4, L–O). A notableincrease in intensity of staining was observed in cisplatin-treated cells (Figure 4M), and MIOX siRNA and NAC treat-ments reduced the intensity of fluorescence, suggesting thatcisplatin-induced ROS generation is mediated by MIOX(Figure 4, N and O). Likewise, a decrease in the ratio of re-duced GSH/oxidized glutathione (GSSG) was observed aftercisplatin treatment (Figure 4K). A further decrease in theGSH/GSSG ratio was observed inMIOX-overexpressing cells.Treatment of MIOX siRNA normalized the cisplatin-induceddecrease in the GSH/GSSG ratio (Figure 4K).

The ROS generation was also assessed in vivo by stainingrenal tissues with CM-H2DCFDA. An accentuated ROS gen-eration in cisplatin-treated MIOX-TG mice, compared withcontrol WTmice, was observed (Figures 5, E versus D). How-ever, MIOX2/2 mice showed no induction of ROS generation(Figure 5, F versus C). A relatively higher signal of ROS gen-eration, compared with WT mice, was also detected in un-treatedMIOX-TGmice (Figures 5, B versus A). During a seriesof reactions in the G-X pathway there were perturbations inNADPH/NADP+ and NAD+/NADH ratios, and as a resultthere was increased generation of ROS and NOX-4 expressionin diabetic and obesity states.13–15 Similarly, a mild increase inNOX-4 expression was observed in cisplatin-treated WTmice

Figure 1. Characterization of MIOX transgenic and Null mice. (A)Schematic drawing of the construct used for generation of MIOXflox/flox transgenic mice. MIOX gene was inserted after thesecond LoxP site. Its expression would be under the control ofchicken–b-actin hybrid promoter. The flox/flox mice were gen-erated and cross-bred with PEPCK-Cre mice to direct the

expression primarily into proximal tubules. (B) Schematic drawingof the construct used to generate flox/Nullmice. The strategy wasto delete exons 2–10 of the MIOX gene in Null mice or flox/floxmice cross-bred with PEPCK-Cre mice. Western blots revealed anincreased expression (2–3-fold) of MIOX in kidneys of transgenicmice (TG) compared with the flox/flox or WT and PEPCK-Cre mice(C andD). No significant renal expression of MIOXwas observed inMIOX2/2 (KO) mice (E and F). Immuno-histochemistry revealed anincreased MIOX expression in renal proximal tubules of TG miceextending into the deeper cortex (H versus G). No expression ofMIOX was observed in cortical tubules of MIOX2/2 mice (I).*, P,0.01 compared with the flox/flox and PEPCK-Cre groups.

J Am Soc Nephrol 28: 1421–1436, 2017 Myo-Inositol Oxygenase in Renal Injury 1423

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(Figures 5, J versus G, and M), and it was accentuated inMIOX-TG mice (Figure 5, K versus H, and M). No significantincrease in NOX-4 expressionwas observed inMIOX2/2mice(Figure 5, L versus I, and M).

Modulation of ROS-Dependent p53 Induction inCisplatin-Induced AKICisplatin induces p53 expression in tubular cells via ROS gen-eration resulting in apoptosis.5,20,21 Because MIOX facilitatesROS generation, which in turn could modulate the inductionof p53, we evaluated the expression of p53 in WT, MIOX-TG,and MIOX2/2 mice and cultured cells. In cisplatin-treatedWTmice a notable increase in the nuclear expression of p53in renal tubules was observed (Figure 6, D versus A, arrow-heads andG), and it was highly accentuated inMIOX-TGmice(Figure 6, E versus B, arrowheads and G). A very mild increasein nuclear p53 expression was observed in cisplatin-treatedMIOX2/2 mice (Figure 6, F versus C, and G). Likewise, cisplatin-treated HK-2 cells revealed a dose-dependent increase in theexpression of p53 and it was notably reduced by NAC orMIOX-siRNA treatment (Figure 6G, right panel), suggestingthat p53 activation is related to ROS generation partlymediatedvia upregulation of MIOX.

MIOX Overexpression Promotes NF-kB ActivationWhile Dampened by Its Gene Disruption in Cisplatin-Induced AKIActivation of p53 by NF-kB is one of the major mechanism(s)in the pathogenesis of folate-induced AKI.22 Conceivably,NF-kBbinds to the p53 promoter andmodulates various patho-biologic processes; on the other hand p53 is regarded as theregulator of NF-kB repression.22–25 This suggests an intricatecrosstalk between p53 and NF-kB.22 In view of the intercon-nectivity of these various signaling events, we investigated thepathobiology of NF-kB among three strains of MIOX mice.Electrophoretic mobility shift assays (EMSA) demonstrated anincreased binding of NF-kB–specific probe in cisplatin-treated WTmice (Figure 7A, lane 3, arrowhead). The bindingwas accentuated in cisplatin-treated MIOX-TG mice (Figure7A, lane 5). No significant increase in NF-kB binding wasobserved inMIOX2/2mice (Figure 7A, lane 7). The specificity

Figure 2. MIOX overexpression exacerbates cisplatin-inducedAKI, whereas its gene disruption ameliorates AKI. Cisplatintreatment (20 mg/kg) led to a significant weight loss in MIOXtransgenic (TG) and WT mice, whereas MIOX2/2 (KO) mice had alesser degree of body weight loss (A). Cisplatin administrationalso led to a marked elevation of both serum creatinine and urealevels in TG mice compared with WT and KO mice (B and C). The

KO mice had minimal increase in creatinine and urea levels. A re-markable increase in Kim-1 mRNA expression was observed inkidneys of MIOX-TG mice compared with WT or KO mice (D).Kidney sectionsofWTmice that hadundergone cisplatin treatmentshowed cytolysis of tubular cells with detachment from the un-derlying basal lamina and loss of brush border (H versus E). Thesechanges were accentuated in MIOX-TG mice receiving cisplatin,andamarkedvacuolizationof tubular cellswasobserved (I versusF).The glomeruli had collapse of the capillary loops. The kidneys ofMIOX2/2 KO mice had a relatively lesser degree of cellular dam-age comparedwith theWTorMIOX-TG strains ofmice (J versusG).



Figure 3. MIOX gene disruption inhibits, whereas its overexpression accentuates, renal tubular cell apoptosis. Expression of proa-poptogenic Bax was increased after cisplatin treatment in kidney tubules of WT mice (D versus A, and M). It was highly accentuated inMIOX-TG mice (E versus B, and M). No increase was observed in MIOX-KO mice (F versus C, and M). Intriguingly, at times a milddecrease in BAX expression was noted in KO mice (M), whereas antiapoptogenic Bcl-2 expression drastically decreased in both the WTandMIOX-TGmice, and it was unchanged in KOmice (M). An increased expression of cleaved caspase-3 was observed in kidneys of WTmice after cisplatin treatment (M), which was accentuated inMIOX-TGmice. No expression of cleaved caspase-3 was observed in treatedor untreated MIOX-KO mice (M). After cisplatin treatment, a marked increase in caspase-3 activity was observed in MIOX-TG mice,whereas a moderate increase was also observed inWTmice (N). No significant increase in caspase-3 was observed in kidneys of KOmice.Along these lines, a fulminant degree of apoptosis was observed in kidneys of MIOX-TG mice (K versus H), although a mild-to-moderatedegree of apoptosis was also observed in WT mice (J versus G), and no apoptosis was detected in MIOX-KO mice (I and L).



Figure 4. MIOX accentuates cisplatin-induced ROS generation in vitro. LLC-PK1 cells transfected with empty vector and MIOX pcDNA(overexpressing proximal tubular cell line) were treated with cisplatin (20 mM), and ROS generation was assessed by CM-H2DCF-DAstaining and flow cytometric analyses. The analyses revealed a dose-dependent increase in ROS levels in cells transfected with emptyvector (A–C, and J). MIOX-overexpressing cells had higher ROS levels after cisplatin treatment (F–H, and J). After MIOX-siRNA orantioxidant NAC treatment the cisplatin-induced increase in ROS was notably reduced (D, E, I, and J). Fluorescent microscopy of cells



of the NF-kB probe binding was confirmed by incubatingDNA-protein complexes of renal extracts from WT cisplatin-treated mice with antibody against the p65 subunit of NF-kB.Another high molecular band indicating supershift was ob-served after incubation with the antibody (Figure 7B, lane 3,

arrow). Western blot analyses revealed a translocation of p65into the nucleus from cytoplasm in cisplatin-treated WTmice,and it was accentuated inMIOX-TGmice (Figure 7C, lanes 2 and4). No significant p65 translocation was observed in MIOX2/2

mice (Figure 7C, lanes 5 and 6). Cisplatin-treated HK-2 cells

confirmed the observations made by flow cytometric analyses (L–O). Cellular redox was quantified by assessing the levels of GSSG andreduced GSH (K). A decrease in the ratio of GSH/GSSG was seen after cisplatin treatment, which was further reduced in MIOX-over-expressing cells. MIOX siRNA treatment largely normalized the GSH/GSSG ratio (K). *, P,0.01 compared with the control group;#, P,0.05 compared with the control group.

Figure 5. MIOX accentuates cisplatin-induced ROS generation andNOX-4 expression in vivo. The ROS generation was assessed by stainingrenal tissues of mice with CM-H2DCFDA (DCFA) after cisplatin treatment. A mild increase in DCFA staining was observed in WT type mice (Dversus A), which was accentuated in MIOX-TG mice (E versus B). The MIOX-KO mice showed no induction of ROS generation after cisplatintreatment (F versus C). Compared with WT mice, a relatively higher degree of signal of DCFA staining was observed in TG mice that did notreceive the cisplatin treatment (B versus A). Likewise, NOX-4 expression was markedly increased in MIOX-TG mice compared with WT mice(J versus G, K versus H, and M). No significant increase in the expression was observed in MIOX-KO mice (L versus I, and M).



also showed cytoplasm-to-nuclear translocation of the p65, thusestablishing the role of NF-kB p65 in AKI (Figure 7D).

MIOX Modulates the Expression of InflammatoryCytokines in Cisplatin-Induced AKINF-kB plays a role in inflammation via induction of proin-flammatory cytokines.26,27 Because they are implicated in thepathogenesis of AKI, we investigated the expression of variouscytokines, e.g., IL-1b, IL-6, TNF-a, and MCP-1, in differentstrains of mice (Figures 8, A –D). Overall, the changes in theirexpression were similar. A remarkable increase in expression ofall of the cytokines was observed in cisplatin-treated MIOX-TGmice, although a moderate increase in TNF-a expression wasobserved in cisplatin-treated WT mice (Figure 8C). The sourceof these cytokines in AKI could be from the tubular or resident

dendritic cells or cells influxed into the tubulo-interstitium.5,28,29

Keeping in view these possibilities we assessed if there was adifferential increase in infiltrating cells among various strainsof mice. Normally, very few resident inflammatory cells wereseen in the interstitium (Figure 8E, arrowhead). After cisplatintreatment, a mild-to-moderate increase in the influxed inflam-matory cells was observed (Figure 8, F–H, arrowheads). A re-markable increase was seen in cisplatin-treated MIOX-TGmice (Figure 8G, arrowheads). The inset shows the morphol-ogy of infiltrating inflammatory cells (green arrowheads). A fewtubular cells undergoing apoptosis were also seen in MIOX-TGmice (Figure 8G, arrow).

Cisplatin Increases MIOX Expression via Demethylationof Its PromoterHere, we addressed the question of whether cisplatin can in-crease the expression of MIOX and if as a result there is anexcessive ROS generation, which would further accentuate re-nal injury. The answer seems to be affirmative because cisplatinadministration caused an increased MIOX expression (Figure9, A–C). The increase was remarkable and was confined totubules (Figure 9B). Next, mechanism(s) that induced the in-creased MIOX expression were explored. Genomic DNA sam-ples were isolated from kidneys of WT and cisplatin-treatedmice. They were subjected to bisulfite treatment, followed byPCR and nucleotide sequencing to assess the efficiency of con-version (cytosine to thymine) of CpG dinucleotides. Theirnucleotide analyses revealed that the MIOX promoter isdevoid of CpG islands but it has eight CpG dinucleotidesat 219, 222, 242, 248, 273, 2424, 2436, and 2456 bp sites.InWTcontrolmiceCpGat222 and2436bpwereunmethylated,whereas six others were methylated (Figure 9, D and E). Thesamples from cisplatin-treated mice had all of the CpG dinu-cleotides unmethylated, except for 2424 and 2456. Overall,the efficiency of conversion of C to T nucleotide was .95%.The analyses of percentage of methylation of individual dinu-cleotide residues between control and cisplatin-treated micewere plotted as bar graphs, which suggested that cisplatin-induced hypomethylation is most likely responsible for theMIOX upregulation in AKI (Figures 9F).

DISCUSSION

Several years ago, MIOX was identified in proximal tubules ofnewborn mice.30 Since then its relevance in the pathogenesisof diabetic nephropathy has been described in several publi-cations.10,13–15 These studies highlight its biochemical char-acteristics pertaining to transcriptional, translational, andpost-translational events that modulate MIOX expression.However, its relevance to the pathobiology of tubules remainsenigmatic, and that warranted generation of mice with itsgenetic overexpression or deletion. We employed Cre/loxPtechnology to direct its overexpression in renal tubules bycross-breeding floxed mice with PEPCK-Cre mice. These

Figure 6. MIOX accentuates cisplatin-induced ROS-dependentp53 induction in vivo and in vitro. A moderate increase in the nu-clear expression of p53 was observed in WT mice after cisplatintreatment (D versus A, arrowheads, and G, left). The p53 nuclearexpression was markedly accentuated in MIOX-TG mice (E versusB, arrowheads, and G, left). A very mild increase in the p53 nuclearexpression was observed in MIOX-KO mice (F versus C, and G,left). After cisplatin treatment a dose-dependent increase in p53expression was observed in HK-2 cells, and it was reduced afterMIOX-siRNA or antioxidant NAC treatment (G, right).



mice express Cre recombinase gene, and the latter is under thecontrol of mutated PEPCK promoter and thus would expressMIOXmainly in proximal tubules.31 Incidentally, the PEPCKCretransgene is expressed on the X chromosome, and thereforemalemice exhibit greater penetrance than females. In view of this, weselected male mice for our studies. Nevertheless, both male andfemale mice exhibited identical phenotypes, i.e., MIOX overex-pression in proximal tubules of MIOX-TG mice and no expres-sion in MIOX2/2 mice (Figure 1).

Using these mutant mice we exploredadditional functionalities of MIOX in thecontext of pathobiology of proximal tu-bules. The latter are adversely affected invarious disease states that result in AKI. AprototypeofAKI is cisplatin-inducedneph-rotoxicity, where proximal tubules bear thebrunt of injury, possibly due to their uptakeof cisplatin.32 In this scenario, in whichproximal tubules are targeted and whereMIOX is expressed, it would indicate thatthe cisplatin-induced AKI is an appropriatemodel to delineate the pathogenetic role ofMIOX in renal injury. Numerous reportshave documented the cellular toxicity inproximal tubules accompanied with com-promise in renal functions.4,5,32 Likewise,we observed vacuolization and necrosis oftubular cells accompanied with a rise in se-rum urea and creatinine, and loss of bodyweight in C57BL/6J mice (Figure 2). Thesechanges were highly accentuated inMIOX-TGmice. Interestingly, the MIOX2/2 micehad amuch lesser degree of cisplatin-inducedtubular toxicity and minimal elevation ofserum creatinine. An affirmation of theseresults was derived by assessing the statusof KIM-1, a marker of tubular injury.18 Aremarkable elevation of KIM-1 levels wasobserved in MIOX-TG mice, whereas therewas a modest increase in control mice anda minimal rise in MIOX2/2 mice. Besidesnecrosis, cellular apoptosis has been welldescribed in cisplatin nephrotoxicity, whichapparently relates to the activation of proa-poptotic protein Bax and caspases whilediminishing the expression/activity of an-tiapoptotic Bcl-2.20,21,33,34 Interestingly,apoptosis, Bax activation, and expressionof cleaved caspase-3 and its activity weremarkedly accentuated in MIOX-TG micecompared with control animals (Figure 3).Notably, the MIOX2 /2 mice hadno apoptosis or Bax activation or expres-sion of cleaved caspase-3, and only a mar-ginal increase in the caspase-3 activity,

whereas a marked downregulation of antiapoptotic proteinBcl-2 was observed in MIOX-TG after cisplatin treatment.Taken together, these data indicate that MIOX overexpressionaccentuates AKI, whereas its gene disruption is protective forkidney homeostasis.

The next question that was addressed was how MIOX ac-centuates AKI. Besides necrosis/apoptosis, oxidant stress hasbeen regarded as one of the factors for induction of nephro-toxicity.16 Previous studies indicate that MIOX promoter

Figure 7. MIOX overexpression promotes NF-kB activation while dampened by itsgene disruption in AKI. EMSA revealed an increased binding of the NF-kB–specificprobe with the nuclear extracts isolated from kidneys of WT mice treated with cisplatin(A, lane 3, arrowhead). The binding was further accentuated in MIOX-TG mice, asgauged by the intensity of the band (A, lane 5). Minimal binding of NF-kB probe wasobserved in MIOX-KO mice receiving cisplatin (A, lane 7). The specificity of the NF-kBprobe binding was confirmed by incubating renal nuclear extracts of WT mice treatedwith anti-p65 antibody before EMSA. An additional high molecular band indicatingsupershift was observed after incubation with antibody against the p65 subunit ofNF-kB (B, lane 3, arrow). Subcellular compartmentalization of the transcriptionalsubunit of NF-kB p65 after cisplatin treatment demonstrated the activation of NF-kB.Western blot analyses revealed a translocation of p65 from the cytoplasm into thenucleus after cisplatin administration in WT mice (C, lane 2), and the translocation wasfurther accentuated in MIOX-TG mice, whereas no significant translocation was ob-served in MIOX-KO mice after cisplatin treatment (C, lanes 4 and 6). The b-actin andlamin–b-1 served as controls. The cisplatin-induced p65 translocation was confirmedby in vitro experiments in HK-2 cells treated with cisplatin (D).



includes oxidant response elements, and its overexpression invitro accentuates generation of ROS.10,14 The current data re-garding accentuated expression, as well as activity, of caspase-3points towardmitochondria as the source of excessive generationof ROS in MIOX-TGmice. In this regard, MIOX overexpressionin LLC-PK1 cells has been reported to cause exaggerated mito-chondrial dysfunctions, such as cytochrome C release, DNA

fragmentation, and mitochondrial fission, under high glucoseambience.14 However, in cisplatin-induced nephropathy the in-creased ROS generation in MIOX-overexpressing cells could bealso related to the pathobiology of the NADPH oxidase system.In this regard, perturbations in NAD+/NADH ratios have beendescribed in the MIOX-initiated G-X pathway.14 In support ofthe latter contention our in vitro data indicate a perturbed GSH(oxidized)/GSSG (reduced) ratio after cisplatin treatment,which was largely normalized by the concomitant administra-tion of MIOX-siRNA (Figure 4). Likewise, MIOX-siRNA treat-ment of LLC-PK1 cells markedly reduced the generation ofROS after cisplatin exposure, as assessed by FACS analysesand immunofluorescence microscopy after DCFDA staining(Figure 4). The production of ROS was markedly accentuatedin the renal tubular compartment of MIOX-TG mice, whereasMIOX2/2 mice had minimal generation of ROS, even aftercisplatin treatment (Figure 5). Further support for the involve-ment of NADPH oxidase in the current model comes fromexpression studies on the induction of tubular–NOX-4. Aprominent expression of NOX-4 was observed after cisplatintreatment in MIOX-TG mice, whereas it was barely detectablein WT and MIOX2/2 mice (Figure 5). Taking these data to-gether, one can conclude that MIOX accentuates cisplatin-induced AKI, most likely via boosting ROS generation.

Besides ROS and apoptosis, various interlinked p53-associatedpathways and molecules are also involved in the pathogen-esis of cisplatin toxicity. They may include p53 upregulatedmodulator of apoptosis (PUMA), histone deacetylase inhibitors,taurine transporter (TauT) gene, and Sirt1.20,21,35–37 The induc-tion of PUMA is dependent on the activation of p53 becauseinhibition of p53 with pifithrin or its genetic ablation reducesapoptosis.21 Interestingly,N-acetyl-cyteine (NAC) or dimethylth-iourea administration reduces hydroxyl radical generation, i.e.,oxidant stress, p53 activation, and suppression of PUMA-a andapoptosis.20 Along these lines the TauT transgenic mice exhibitattenuation of cisplatin-induced apoptosis by dampening the ac-tivation of p53.36 Similarly, Sirt1, anNAD-dependent deacetylase,has been reported to reduce ROS-induced apoptosis most likelyvia deacetylation of p53, thereby reducing its transcriptional ac-tivation.38 On the other hand, histone deacetylase inhibitor tri-chostatin Awas administered alongwith cisplatin, and although itenhances tumor cell death it limits cisplatin-induced toxicity orapoptosis in the kidney, probably by increasing phosphorylationof cAMP-response element binding protein and reversing cAMP-response element binding protein–mediated gene repression.39 Inline with these literature data we observed increased p53 expres-sion after cisplatin administration, especially in MIOX-TG micehaving accentuated ROS generation (Figure 6). The question ofwhether MIOX-generated ROS induces p53 expression wasassessed by in vitro experiments. The cisplatin-treated cells hadincreased expression of p53, which was abrogated with MIOX-siRNAorNAC treatment, suggesting thatMIOXaccentuates p53-mediated injury via excessive ROS generation (Figure 6).

Another pathobiologic process that plays a noteworthy rolegermane to cisplatin-induced AKI relates to inflammation.28

Figure 8. MIOX modulates the expression of inflammatory cy-tokines in cisplatin-induced AKI. A remarkable increase in theexpression of IL-1b, IL-6, TNF-a, and MCP-1 was observed inMIOX-TG mice receiving cisplatin compared with WT or MIOX-KOmice or untreated control mice (A–D), although a moderateincrease of TNF-a was observed in WT mice after cisplatintreatment (C). Examination of kidneys revealed a remarkableincrease of interstitial inflammatory cells in the MIOX-TG micereceiving cisplatin (G, arrowheads). The inset shows the mor-phologic nature of the infiltrating inflammatory cells (greenarrowheads). A mild increase was also observed in cisplatin-treated WT mice (F, arrowheads). The MIOX-KO mice had nosignificant increase of inflammatory cells compared with the un-treated WT control (H versus E, arrowheads). A few foci of apo-ptosis were also seen in MIOX-TG mice (G, arrow). *, P,0.01compared with the control group; #, P,0.05 compared with thecontrol group.



In this realm there may be activation of transcription factors,generation of cytokines, and influx of inflammatory cells. Oneof the important transcription factors that is induced afteroxidant stress includes NF-kB.40 It modulates diverse biologicprocesses, including inflammatory responses, while at thesame time modulating p53 expression.22 Interestingly, recentdata also suggest that p53 itself can regulate NF-kB repres-sion.25 This would suggest an intricate reciprocal crosstalkbetween these two molecules during an inflammatory re-sponse in AKI.22 The role of NF-kB has been ascribed to di-verse diseases associated with renal inflammation.27 Also, itsrole in the folic acid–induced toxic injury model is wellknown, where folic acid administration was shown to con-comitantly upregulate expression of p53, NF-kB, and its ac-tive transcriptional subunit, relA/p65.22 Administration ofpyrrolidine dithio-carbamate ammonium, an NF-kB inhibi-tor, reduced the upregulated expression of all of these mole-cules along with improvement in renal functions. Along theselines, we investigated the role of NF-kB in cisplatin-inducedinjury in MIOX-TG and MIOX2/2 mice. By EMSA, an in-creasedNF-kB binding was seen in cisplatin-treatedWTmice,and it was accentuated in MIOX-TG, although no significantbinding was seen in MIOX2/2 mice (Figure 7). The role ofNF-kB in cisplatin-induced nephropathy was further suppor-ted by accentuated translocation of p65 into the nuclear com-partment in renal cells ofMIOX-TGmice. The downstream ofNF-kB activation would include upregulation of variousinflammatory cytokines, including TNF-a.41 In fact, TNF-ageneration after the activation of NF-kB in cisplatin-inducednephropathy iswell described.42 In addition, there is an increasedexpression of other cytokines, including IL-1b, MCP-1, andRANTES. We also observed an upregulation of inflammatorycytokines after cisplatin administration, especially in MIOX-TGmice (Figure 8). The concomitant rise of different cytokines maysuggest that they synergistically stimulate one another’s produc-tion in MIOX-TG mice that are endowed with increased gener-ation of ROS in their kidneys. Some of these cytokines, e.g.,MCP-1, are chemotactic for a variety of inflammatory cells, in-cluding neutrophils, monocytes, and lymphocytes.42 In concur-rence with the production of cytokines an accentuated influx ofinflammatory cells into renal parenchyma was observed in

Figure 9. Cisplatin treatment per se upregulates MIOX via de-methylation of its promoter. WT mice treated with cisplatin hadan increased renal expression of MIOX, and it was confined tothe tubules (A–C). No significant MIOX expression was seen inglomeruli. Genomic DNA was isolated from kidneys of WT andcisplatin-treated mice and subjected to bisulfite treatment. Pro-moter fragments were isolated by PCR, cloned into pGEM-Tplasmid, and transformed in DH5a cells. Ten clones from each

variable were selected and subjected to nucleotide sequencing toassess the efficiency of conversion (cytosine to thymine) of CpGdinucleotides. Nucleotide analyses revealed eight CpG dinucle-otides at 219, 222, 242, 248, 273, 2424, 2436 and 2456 bpsites. In WT, untreated control mice samples, CpG at 222 and 2436 bp were found to be unmethylated, whereas six others weremethylated (D and E). The samples from cisplatin-treated micewere found to have all of the CpG dinucleotides unmethylated,except for2424 and2456. Overall, the efficiency of conversion ofC to T nucleotide was .95%. The percentages of methylation ofindividual dinucleotide residues are presented as bar graphs,which indicate that cisplatin induces hypomethylation that is likelyresponsible for the MIOX upregulation (F).



MIOX-TGmice (Figure 8). No significant influx was observed inMIOX2/2mice, suggesting that increasedMIOXexpression leadsto an activation of various inflammatory responses.

Several publications indicate that in a variety of pathobiologicstates the increasedexpressionofMIOXseems tobe the resultof itstranscriptional modulation, where conceivably various transcrip-tion factors bind to specific consensus sequences localized withinits promoter.10,13–15,43 Therefore, the question that warrants ananswer is, whether there is an increased expression of MIOX aftercisplatin treatment, and if so, what are the mechanism(s) respon-sible for such an upregulation? Indeed, there was increased ex-pression of MIOX after cisplatin treatment (Figure 9). Becausethere are no known transcription factors which could bind to theMIOX promoter and induce its upregulation in cisplatin toxicity,we therefore proceeded to search for an alternative mechanism.One such mechanism that we explored pertains to the epigeneticregulation of MIOX, i.e., methylation/demethylation of itspromoter. Epigenetic modification of various genes has been im-plicated in various renal diseases with consequential altered ex-pression.44 In this regard, hypomethylation/demethylation hasbeen reported in AKI induced by ischemia reperfusion or contactfreezing of the kidney surface.45–47Althoughamultitude ofmech-anisms have been described in cisplatin-induced AKI, the studiesrelating to epigenetic modifications are sparsely reported.5,32,48 Inlinewith the observationsmade in the ischemia reperfusion injurymodel, we observed a marked demethylation of the CpG dinu-cleotides in the MIOX promoter (Figure 9), which could explainits increased expression and ultimately exacerbation of cisplatin-induced injury in MIOX-TG mice.

In summary, this investigation highlights the worsening ofAKI in cisplatin-induced injury via a multitude of mechanismsin states of overexpression of MIOX, although its gene disrup-tion seems to ameliorate toxic renal proximal tubular injury.

CONCISE METHODS

ReagentsReagents were purchased from the following vendors. Sigma-Aldrich:

cisplatin (# P4394), anti–b-actin antibody (# A5441), Caspase-3 assay

kit, Colorimetric (# CASP-3C), poly-(deoxyinosinic-deoxycytidylic)

acid sodium salt (# P4929), NAC (# A7250); BioAssay Systems: Quanti-

Chrom creatinine assay kit (# DICT-500) and QuantiChrom urea

assay kit (# DIUR-500); Life Technologies: Power SYBR Green PCR

Master Mix (# 4367659), Chloromethyl derivative of 29, 79-dichloro-

fluorescein diacetate (CM-H2DCF-DA, # C6827), 49-6-diamidino-2-

phenylindole (DAPI, # D1306), and TRIzol Reagent (# 15596026);

Cell Signaling Technology: anti–cleaved caspase-3 antibody

(# D175), anti-p53 (1C12) antibody (# 2524), and anti-p65 antibody

(# D14E12); Abcam: anti–lamin B1(ab16048); Enzo Life Sciences:

DNA methylation gold kit (# D5005); Santa Cruz Biotechnology:

anti-Bax antibody (# SC-526), anti–NOX-4 antibody (# SC-21860),

anti-p53 antibody (# SC-6243); Roche Diagnostic: In Situ Cell Death

Detection Kit, Fluorescein (# 11684795910); Cayman Chemicals:

GSH assay kit (# 703002); Promega Corporation: NF-kB EMSA oligo

(# E329A) and pGEM-T plasmid vector; Perkin Elmer: ATP g-32p

(# BLU002A); and Thermo Scientific: polynucleotide kinase

(EK0032). OriGene Technologies: MIOX siRNA (# SR310776).

Anti-MIOX polyclonal antibody was prepared in our laboratory using

recombinant mouse MIOX as the immunogen.10

Generation of MIOX Transgenic and Null MiceThe transgenic CBA-flox mice were generated using the pCMV flox

vector (a generous gift fromDr.Holzman).49 TheCMVpromoter was

replaced with the cytomegalovirus–chicken b-actin hybrid promoter

which contains the cytomegalovirus immediate early enhancer linked

to chicken b-actin promoter and a chimeric intron of b-globin and

immunoglobin genes.50,51 This hybrid promoter demonstrates en-

hanced activity in vivo and was used to drive the expression of a

flanked b-gal-stop-codon cassette.52 The MIOX cDNA was inserted

downstream of the b-gal-stop-codon cassette at the NheI and PmeI

sites (Figure 1A). This construct enables the expression of the b-gal

gene (as verified by X-gal staining in LLC-PK1 cells), but it does not

express the MIOX gene. After recombination of the Loxp sites in the

presence of Cre recombinase, the b-gal gene is deleted and MIOX is

expressed under the regulation of the b-actin hybrid promoter. The

CBA-flox construct was released from the plasmid vector by diges-

tion with PacI and microinjected into the pronucleus of fertilized

ova from C57BL/6 F1 mice. The litter was screened for the presence

of transgene using b-gal–specific primers as previously described.52

The founders were cross-bred with PEPCK-Cre C57BL/6J mice

(a generous gift from Dr. Volker Hasse)31 to direct expression pri-

marily into the proximal tubules. The mice were then screened for

the MIOX transgene using primers encompassing the NheI and

PmeI restriction sites. Their kidneys were tested for the extent of

MIOX expression by Western blot analyses and immuno-histochemical

techniques.

MIOXnullmicewere generated atCellMolecularTechnology Inc.,

Phillipsburg, New Jersey. The design of the targeting vector for gen-

eration of MIOX knockout mice is included in Figure 1B. Briefly, the

targeting vector was designed to generate both Null and Flox mice

lines. A 26 kb BAC subclone was cloned by applying Gene Bridges

technology.53 A 59 LoxP site was placed in intron 1 at the AfIII site

and a FRT-Neo-FRT-39 LoxP cassette at the AatII site after exon 10. In

doing so, Cre-recombinase would excise exons 2–10 along with the

Neo cassette leaving behind a single LoxP site to generate Null mice

lines; whereas FLP recombinase would excise only the Neo cassette

leaving exons 2–10 and Loxp sites intact to generate the conditional

(Flox) line in embryonic stem cell (ES) recombinants. The targeting

vector was electroporated into 129/SVEV ES cells. The ES recombi-

nant clones were picked and tested by PCR tomake sure the excisions

had taken place. After injection of ES recombinants into blastocysts

and subsequent implantation, chimeras were generated and mouse

lines expanded. The Flox mouse lines were also cross-bred with

PEPCK-Cre mice to selectively delete the MIOX gene from the prox-

imal tubules. Both the mouse lines were backcrossed with C57BL/6J

for seven generations to generate mouse lines with a homogenous

background. Their kidneys were harvested to confirm deletion of

MIOX genes by Western blot analyses and immuno-histochemical

techniques.14,15



Design of In Vivo ExperimentsFor AKI experiments, 8-week-old male WTmice and sex- and age-

matched MIOX2/2 (KO) and MIOX-overexpressing transgenic

(MIOX-TG) mice were utilized. Initially, various dosages (5–25

mg/kg body wt) of cisplatin (single intraperitoneal injection) were

tested for tolerance among these three strains of mice. A dose of 20

mg/kg (1 mg/ml solution in sterile normal saline) was tolerated by all

three of the strains without having significant mortality, and this dose

was chosen for all of the subsequent experiments (n=6). The control

mice were injected with normal saline only. Mice were euthanized 3

days after the cisplatin treatment. Blood samples were collected for

measuring levels of serum creatinine and urea using QuantiChrom

Creatinine and QuantiChrom Urea assay kits (BioAssay Systems).

Their kidneys were utilized for various morphologic, biochemical,

and molecular biology studies. All animal procedures used in this

study were approved by the Animal Care and Use committee of

Northwestern University.

Cell Culture StudiesHK-2 cells and LLC-PK1 cells were purchased fromATCC (Manassas,

VA) and used in this study. The HK-2 cells were grown in low glucose

DMEM medium supplemented with 5% heat-inactivated FBS,

100 U/ml penicillin, and 100 mg/ml streptomycin. LLC-PK1 cells were

grown in low glucose M199 medium supplemented with 5% heat-

inactivated FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin.

The cells in quadruplicate plates were maintained in a humidified at-

mosphere of 5% CO2, 95% air, at 37°C. In addition, MIOX-overexpressing

LLC-PK1 cells were also used in some of the experiments. The gener-

ation of overexpressing cell lines has been detailed in previous publi-

cations.15 Approximately 13 106 cells were seeded per well in six-well

collagen-coated plates and allowed to adhere to the culture plates

overnight. The cells were then treated with various concentrations

of cisplatin (5–20 mM) for 12–24 hours, and then processed for var-

ious morphologic and biochemical studies. In addition, MIOX siRNA

(50 nM) and ROS scavenger (NAC, 10 mM) studies were also carried

out. The LLCPK1 cells were transfected with siRNA against MIOX14

or scramble (serve as a control) using Lipofectamine 2000 reagent

(Invitrogen) in the presence of 20 mM of cisplatin. Forty-eight hours

later the ROS generation, GSH/GSGG ratios, and p53 expression were

assessed in the cells.

Renal Tissues’ Histologic and Immuno-HistochemicalStudiesKidneys from control (WT) and experimental (TG/KO) mice were

harvested, and 3–4 mm thick slices were prepared. They were fixed in

10% buffered formalin, dehydrated by graded concentrations (50%–

100%) of ethanols, and then transferred into xylene and embedded in

paraffin. For immuno-histochemistry, 4 mM thick sections were pre-

pared and mounted on glass slides. The tissue sections were air-dried

overnight. They were then deparaffinized, hydrated, and then pro-

cessed for antigen retrieval. After antigen retrieval, endogenous per-

oxidase activity in the tissues was quenched by immersing glass slides

with tissue sections in 3% H2O2 for 3 minutes. Tissue sections were

immersed in 3% BSA for 1 hour and incubated in a primary antibody

overnight at 4°C. After washing with PBS the sections were incubated

with horse radish peroxidase (HRP)–conjugated anti-rabbit IgG,

immPRESS (Vector Laboratories), for 1 hour. The sections were sub-

jected to another PBS wash and then incubated with HRP substrate

diaminobenzadine for color development. Sections were rewashed

and counterstained with hematoxylin to delineate the cellular details.

The sections were then dehydrated in a graded series of ethanols,

treated with xylene, and coverslip-mounted after immersion in a

drop of mounting media (Vector Labs). Slides were then viewed

and photographed.

Immunoblotting Analyses of Relevant ProteinsExpressed in Kidney TissuesKidney tissues from control and cisplatin-treatedWT,MIOX-TG, and

MIOX2/2 mice were harvested and lysed in RIPA buffer. Superna-

tants were collected and protein concentration was measured using

Bradford reagent. Equal amounts of protein for each sample were

mixed with 53 SDS sample buffer and boiled for 5 minutes. The

proteins in the lysates were fractionated by SDS-PAGE and electro-

blotted onto PVDF membranes. The blots were then probed with

various primary antibodies, followed by incubation with secondary

antibodies conjugated withHRP. Autoradiograms were developed for

detection of the bands using an Enhanced ChemiLuminence kit

(Thermoscientific). Equal loading of the samples was confirmed by

probing the immunoblots with mouse b-actin or lamin-b1 anti-

bodies.15

TUNEL Staining Studies in Kidney TissuesThe extent of apoptosis in kidney tissues was determined by estab-

lished TUNEL procedures.14 Deparaffinized kidney sections were hy-

drated and then digested with Proteinase K (240 unit/ml; Promega,

Madison) for 30 minutes at 22°C. After washing tissue sections with

PBS, they were incubated with TUNEL reagent for enzymatic labeling

of the nicked DNA with a fluorescent nucleotide probe (Roche Ap-

plied Science).

Assessment of Caspase-3 Activity in Kidney TissuesThe assay was performed using a Colorimetric caspase-3 assay kit

(Sigma), as previously described.15 Kidney tissues from WT,

MIOX-TG, and MIOX2/2 mice were harvested. Their cortices were

dissected and homogenized in a lysis solution provided in the kit. The

tissue lysates were then centrifuged at 16,0003 g for 10 minutes at 4°

C. Supernatants were transferred into a new tube and protein con-

centration measured by using the Biorad Bradford Reagent. After

adjusting the protein concentration to 5 mg/ml the caspase-3 activity

was determined. Each sample was assayed in duplicate in a 96-well

plate in 200 ml reaction volume. The reaction mixture included 10 ml

of lysate containing 50 mg of protein per sample, 170 ml of 13 assay

buffer, and 20ml of caspase-3 substrate. Wells were then covered with

adhesive film and incubated at 37°C for 3 hours. The sample absor-

bance was read at 405 nm.

Assessment of ROS in Tissues and CellsFor determination of ROS in kidneys, tissues from both control and cis-

platin-treatedWT,MIOX-TG,andMIOX2/2micewereembeddedinOCT

compound and processed for frozen sectioning. Eight-micrometer-thick



frozen sections of kidney were prepared, transferred onto glass slides, and

air dried for 5minutes. The slideswere then rinsedwith PBS to remove the

OCT compound from tissue sections. Tissue sections were then stained

with CM-H2DCFDA for 15 minutes at 37°C in the dark and visualized

using a fluorescent microscope equipped with UV illumination.

ROS levels were determined in cisplatin-treated cells by CM-

H2DCFDA staining using flow cytometry and fluorescent micros-

copy.14,15 For flow cytometry, cells transfected with empty vector

and MIOX-overexpressing LLCPK1 cells were seeded onto six-well

plates and treated overnight with different concentrations of cis-

platin, as indicated above. Cells were then washed in PBS and de-

tached from the plate using BD Accutase cell detachment solution.

Cells were then stained with of CM-H2DCFDA (5 mM) for 15 min-

utes at 37°C in dark. After washing twice with PBS the cells were

resuspended in 300 ml of PBS and processed for acquisition of fluo-

rescence with a flow cytometer. After which, mean fluorescent inten-

sity of the CM-H2DCFDA was measured using standard operational

procedures of flow cytometry and employing FACSDiva software

(Becton Dickinson). For fluorescence microscopy, HK-2 cells were

seeded onto coverslips and treated with different concentrations of

cisplatin (10 and 20 mM) for 12 hours. Cells were then washed and

stained with 5 mM of CM-H2DCFDA for 15 minutes at 37°C in the

dark. After a brief rewash with PBS the coverslips with attached cells

were inverted and mounted on the glass slides after placing a drop of

DAKO mounting medium. The cells were then examined with a UV

microscope.

Determination of GSH/GSSG Ratio in Cultured CellsThiswasmeasuredusingGSHassay kit (CaymenChemicals) using the

manufacturer’s instructions, as previously described.15 Both empty

vector control LLCPK1 cells andMIOX-overexpressing LLCPK1 cells

were used in this study. Cells that had undergone various treatments

were collected and sonicated in 500ml of MES buffer. Lysed cells were

then centrifuged to collect supernatant. Protein concentration in su-

pernatant (#1) wasmeasured using the Biorad Bradford Reagent. The

supernatants were then deproteinated by using equal volumes of

freshly prepared 10% metaphosphoric acid. The precipitates were

allowed to settle and supernatants (#2) were utilized for GSH mea-

surement. Fifty microliters of triethanolamine reagent was added to

each 1 ml sample of the supernatants (#2) and mixed by immediate

vortexing. Fifty microliters of mixture was used for determination of

GSH. For GSSG determination, 10 ml of 1 M 2-vinylpyridine was

added to 1 ml of triethanolamine reagent mixture, as prepared above.

The mixture was vortexed and incubated at 22°C for 1 hour. Fifty

microliters of this mixture was added to the 150 ml of assay solution

provided in the kit and gently shaken in the dark on an orbital shaker

for 45minutes to achieve full color development. Spectrophotometer

readings were made from four experiments at 415 nm, and concen-

trations of GSH and GSSG were measured using a standard curve.

Preparation of Cytosolic and Nuclear Extracts fromKidney Tissues for EMSAKidney tissues harvested from control and cisplatin-treated WT,

MIOX-TG, and MIOX2/2 mice were first homogenized in cytoplas-

mic extraction buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA,

and 0.1 mM EGTA) and incubated for 15 minutes on ice. Then, 12.5

ml of 10% NP-40 per 400 ml of lysis buffer was added and vortexed

vigorously for 10 seconds to lyse the cells. The lysate was then centri-

fuged for 1 minute, and the supernatant was collected and designated

as the cytosolic extract. Pellets were then processed for nuclear extract

preparation. They were incubated in nuclear extraction buffer

(20 mM HEPES, 400 mM NaCl, 1 mM EDTA, and 1 mM EGTA)

for 30 minutes on ice with intermittent vigorous vortexing to lyse

nuclei. Nuclear extract was collected as supernatant after 5minutes of

centrifugation, and protein concentration in various samples was

measured. The nuclear extracts prepared above from various kidney

tissues were utilized for EMSA to assess the NF-kB activation and its

nuclear translocation. Ten micrograms of nuclear extract from each

sample was incubated with 32P-labeled double stranded NF-kB

probe (Promega) for 15 minutes at 37°C. The DNA-protein complex

formed was subjected to 6.6% native PAGE. Gels were dried and

exposed to x-ray film and autoradiograms prepared. Specificity of

binding of NF-kB oligo probe with NF-kB protein was assessed by

performing a supershift assay. Nuclear extract was prepared from

cisplatin-treated WT mice kidneys. It was incubated with antibody

against the p65 subunit of NF-kB for 15 minutes at 22°C before incuba-

tion with labeled NF-kB probe. The shift in the DNA-protein-antibody

complex migration in the gel was visualized after autoradiography.

Cellular Translocation of NF-kBTo assess the translocation of NF-kB from cytoplasm to the nucleus in

HK-2 cells, immunofluorescence studies were performed. Briefly, 5

3 105 cells were seeded onto coverslips in a six-well plate and allowed

to attach overnight. Cells were then treated with 20 mM of cisplatin

for 12 hours. After treatment, the cells were fixed with 4% parafor-

maldehyde for 5 minutes in PBS and washed three times with PBS.

They were then permeabilized with ice cold methanol for 10 minutes

at 220°C. Cells were rewashed twice with PBS and incubated in a

blocking solution (13 PBS/1% BSA/0.3% Triton X-100) for 1 hour.

After blocking, fixed cells were incubated with anti-p65 antibody di-

luted in blocking solution for 2 hours at 22°C. Cells were thenwashed

three times with PBS and incubated with FITC-conjugated secondary

antibody for 2 hours in the dark. Cells were then rewashed with PBS,

and nuclei were stained with 0.5 mg/ml of DAPI for 5 minutes. After

another PBS wash, a drop of DAKO fluorescent mounting media was

placed on the coverslips, and they were then inverted and mounted

onto the glass slides. The images were captured using a Nikon A1R

Spectral Confocal microscope.

Assessment of Tissue Expression of InflammatoryCytokines and Kidney Injury Biomarkers by Real-TimePCRTotal RNAwas isolated from kidneys using TRIzol Reagent (Invitro-

gen) and cDNAwas synthesized using Go Script reverse transcription

system (Promega). ThemRNA levels of various genes were quantified

using Step One Plus System Real Time PCR (Applied Biosystems). A

20 ml total reaction mix included 100 ng of cDNA, 50 nmol/L for-

ward/reverse primers, and 13 Fast SYBR Green Master Mix (Life

Techologies). GAPDHwas used as an internal control and the amount of

mRNA was calculated by comparative CTmethod. PCR reactions were



run in quadruplicate. Primers used were, Kim 1: Forward, 59-GGAAG-

TAAAGGGGGTAGTGGG-39, Reverse, 59-AAGCAGAAGATGGGCATTGC-

39; IL-1b: Forward, 59-ACCTGTCCTGTGTAATGAAAGACG-39, Reverse,

59-TGGGTATTGCTTGGG ATCC-39; MCP-1: Forward, 59-CCCAATGAG-

TAGGCTGGAGAG-39, Reverse, 59-TGGTTG AAAAGGTAGTGGATG-39;

TNF-a: Forward, 59-AGAAGAGGCACTCCCCCAAAAG-39, Reverse,

59-TTCAGTAGACAGAAGAGCGTGGTG-39; GAPDH: Forward,

59-GAATACGGCTACAGCAACAGG 39, Reverse, 59-GGTCTGGGATG-

GAAATTGTG-39; and IL-6: Forward, 59-CCGGAGAGGAGACTTCA-

CAG-39, Reverse, 59-CAGAATTGCCATTGCACAAC-39.

Analysis of MIOX Promoter Methylation/Demethylation in Kidney Tissues after CisplatinTreatmentKidneys of mice treated with cisplatin were harvested for isolation of

DNA. Their cortices were dissected, and an aliquot of cortex (20 mg)

was homogenized in 550 ml of lysis buffer (50 mM Tris-HCI, 10 mM

EDTA, 100 Mm NaCl, 1% SDS) containing RNAase1 (1 mg/ml). The

homogenate was kept at 55°C for 2 hours to achieve complete solu-

bilization of cellular proteins. After a phenol-choloroform extraction

the DNA was precipitated with the addition of isopropanol at 4°C.

The precipitated DNAwas pelleted by a centrifugation at 10,0003 g

for 5 minutes. The DNA pellet was washed with 70% ethanol, air-

dried, and dissolved in nuclease-free water. The isolated DNA (2 mg)

was subjected to bisulphite modification, using EZ DNAmethylation

Gold Kit (Zymo Res. Irvine, CA), and by following the methodology

originally described by Frommer et al.54 In doing so, the sodium

bisulphite in an acidic environment will convert all cytosine residues

in single stranded DNA to uracil, excluding the methylated cytosine.

The bisulfite-treated DNAwas loaded into spin columns provided in

the kit and eluted with 20 ml of elution buffer. This purified and

bisulfite-treated DNAwas used to determine the frequency of meth-

ylation of CpG islands within the MIOX promoter, using bisulfite-

specific PCR primers. These primers were devoid of CpG sites. For

amplification of the mouseMIOX promoter, 2ml of treated DNAwas

amplified in a reaction mixture containing: 4 mM primers (Forward-

59-GTTTTTATAGTAAAAGGTGGG-39 Reverse- 59-CCTAAAAAAA-

CAAACACC-39), 13 PCR buffer, 0.4 mM dNTP mix, 1 U Taq

polymerase (1 U/ml), and the final volume of the reaction mixture

was adjusted to 25ml with nuclease-free water. Amplification conditions

were as follows: 94°C for 5minutes, 39 cycles at 94°C for 30 seconds, 55°

C for 45 seconds, 70°C 2 minutes, and final extension at 70°C for 7

minutes. Amplified PCR products were subjected to 1.5% agarose gel

electrophoresis, and the relevant band was eluted using QIA quick gel

extraction kit (Qiagen). The PCRproduct was then cloned into pGEM-T

Vector System I. After transformation in the DH5a strain of E.coli, the

transfectants were plated on ampicillin agar plates. Ten positive colonies

of each sample were amplified for preparation and purification of plas-

mid DNA containing the MIOX promoter. For sequencing of plasmid

DNA, T7 promoter and SP6 promoter primers were used.

Statistical AnalysesResults were expressed asmean6SD after statistical analyses. t test was

used to compare the data between groups. A P value of ,0.05 was

considered statistically significant.

ACKNOWLEDGMENTS

We are thankful to Dr. Anupam Agarwal, Dr. Subhashini Bolisetty,

Dr. Volker Hasse, and Dr. Larry Holzman for providing the plasmid

constructs and PEPCK mice.

Supported by National Institutes of Health grants DK60635,

DK78314, and T32 DK007139.

DISCLOSURESNone.

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