Kishor Devalaraja-Narashimha* and
Babu J. Padanilam*,
* Department of Cellular and Integrative Physiology and Department of Internal Medicine, Section of Nephrology, University of Nebraska Medical Center, Omaha, Nebraska
Received for publication March 26, 2008.
Accepted for publication August 21, 2008.
After ischemic renal injury (IRI), selective damage occurs inthe S3 segments of the proximal tubules as a result of inhibitionof glycolysis, but the mechanism of this inhibition is unknown.We previously reported that inhibition of poly(ADP-ribose) polymerase-1(PARP-1) activity protects against ischemia-induced necrosisin proximal tubules by preserving ATP levels. Here, we testedwhether PARP-1 activation in proximal tubules after IRI leadsto poly(ADP-ribosyl)ation of the key glycolytic enzyme glyceraldehyde-3-phosphatedehydrogenase (GAPDH), a modification that inhibits its activity.Using in vitro and in vivo models, under hypoxic conditions,we detected poly(ADP-ribosyl)ation and reduced activity of GAPDH;inhibition of PARP-1 activity restored GAPDH activity and ATPlevels. Inhibition of GAPDH with iodoacetate exacerbated ATPdepletion, cytotoxicity, and necrotic cell death of LLCPK1 cellssubjected to hypoxic conditions, whereas inhibition of PARP-1activity was cytoprotective. In conclusion, these data indicatethat poly(ADP-ribosyl)ation of GAPDH and the subsequent inhibitionof anaerobic respiration exacerbate ATP depletion selectivelyin the proximal tubule after IRI.
Compromised perfusion of renal tissues leading to ischemic renalinjury (IRI), generally accepted as the major cause of acutekidney injury (AKI), usually results from hypoxia-induced renalvascular and tubular dysfunction. The outer medullary regionof the kidney receives <10% of the blood delivered to thekidney via the renal artery. After IRI, a persistent perfusiondeficit exists even at 24 h after reperfusion, and the outermedullary partial pressure of oxygen is restored to only 10%of its normal levels, rendering this region susceptible to injuryat both the tubular and vascular levels.1–3 Thus, theprolonged perfusion deficit shuts down oxidative phosphorylationin the cells of the outer medullary segments of the nephronand reverts to anaerobic metabolism for ATP synthesis. Nevertheless,ischemia causes selective injury to the outer medullary proximalstraight tubules (PST), causing the PST cells to undergo celldeath and/or sublethal injury to instigate renal dysfunction.4,5The medullary thick ascending limb, although situated in thesame region, does not undergo injury to the same level.6–8Despite ongoing debate for more than two decades, the molecularmechanisms by which PST cells undergo selective injury are notknown.
Hypoxia resulting from decreased blood flow leads to a varietyof secondary effects, including a breakdown in cellular energymetabolism and generation of reactive oxygen species (ROS) andreactive nitrogen species.9,10 The superoxides induce DNA strandbreaks in ischemic kidneys as early as 1 h after IRI.11 Thesevere DNA damage that ensues results in excessive activationof the DNA repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1),which exacerbates ATP depletion and triggers signaling cascades,leading to cellular suicide.12 Recent data from our laboratoryshowed selective upregulation of PARP-1 expression and its activityin PST cells after renal ischemia.13,14 Gene ablation or pharmacologicinhibition of PARP-1 activity offers both functional and histopathologicprotection from IRI.13,15 The ATP levels are significantly preservedin both in vivo and in vitro models of IRI after PARP gene ablationor inhibition, respectively13,15,16; however, the exact mechanismsby which PARP activation leads to ATP depletion and whetherthese mechanisms are linked to selective damage to PST afterrenal ischemia are not defined.
According to the "cell suicide" hypothesis, PARP-1 activationinduces energy failure by depleting NAD+, and the cell consumesATP to replete the NAD+ level, ultimately leading to energyfailure and cell death12; however, the role of NAD+ in energydepletion is controversial, and its depletion alone may notbe lethal to cells.17,18 Moreover, PST cells can carry out anaerobicrespiration under hypoxic conditions and be protected from injury.Nevertheless, under anoxic conditions, significant amounts ofATP are generated by anaerobic glycolysis by thick ascendinglimbs but not by the proximal tubular cells.19 These findingsprompted us to investigate whether PARP activation interfereswith glycolytic ATP synthesis and thus exacerbates ATP depletionand PST cell injury.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzymein the glycolytic pathway and is susceptible to several modificationsthat alter its activity, including oxidative modification ofthiols and mono-ADP-ribosylation.20–22 Recently, PARP-1was reported to inhibit GAPDH activity by poly(ADP-ribosyl)ationafter hyperglycemia-induced aortic endothelial cell injury.23The temporal and spatial expression pattern of PARP-1 in PSTduring the time of ischemic injury prompted us to hypothesizethat poly(ADP-ribosyl)ation and inhibition of the GAPDH leadsto inhibition of glycolysis, reducing ATP synthesis and exacerbatingenergy depletion and cell injury.
In this study, we evaluated the role of GAPDH-poly(ADP-ribosyl)ationas a mechanism to inhibit GAPDH activity in PST after IRI usingin vitro and in vivo models. We explored the role of GAPDH-poly(ADP-ribosyl)ationin inhibiting glycolysis, exacerbating ATP depletion, and inducingcell death. Our findings suggest that PARP-1–mediatedanaerobic glycolytic inhibition is a key mechanism of selectivePST injury after IRI.
GAPDH Activity Assay in Wild-Type and PARP–/– Mouse Kidneys after IRI
To test our hypothesis that GAPDH activity is inhibited by PARP,we first evaluated GAPDH activity in wild-type (WT) and PARP–/–renal outer medullary proteins after IRI. As shown in Figure 1,GAPDH activity was significantly inhibited at 3 and 6 h afterIRI in WT mouse kidneys compared with that of sham-operatedkidneys, whereas its activity was significantly improved atboth 3 and 6 h after IRI in PARP–/– mouse kidneyscompared with their WT counterparts. These data suggest a rolefor PARP-1 in the inhibition of GAPDH activity after IRI. Nosignificant change in GAPDH activity was observed between WTand PARP–/– sham-operated kidneys.
Figure 1. Effect of IRI on GAPDH activity in WT and PARP–/– renal tissues. Each bar represents mean ± SEM. *P < 0.0005 versus sham-operated WT mice; P < 0.005 versus WT renal ischemic mice.
GAPDH-Poly(ADP-ribosyl)ation in Ischemic Renal Tissues
Because activated PARP-1 ADP ribosylates several different proteinsand modulates their activities,14 we next examined whether poly(ADP-ribosyl)ationof GAPDH could be a possible underlying mechanism for its reducedactivity after IRI. Proteins isolated from the outer medullaryregion of WT or PARP–/– animals that underwent eithersham surgery or IRI were subjected to immunoblot analysis. Severalpoly(ADP-ribosyl)ated protein bands, including a band at approximately37 kD, were observed at 3 and 6 h after IRI in WT kidney lysates(Figure 2A, lanes 1 and 2, respectively) with anti-PAR antibody,suggesting possible GAPDH-poly(ADP-ribosyl)ation after IRI.No PAR bands were observed in either sham-operated or PARP–/–-ischemicrenal tissues (data not shown).
Figure 2. (A) Poly(ADP-ribosyl)ation of GAPDH in renal ischemic tissues. Lanes 1 and 2 represent Western blotting analysis showing poly(ADP-ribosyl)ated proteins in whole-cell lysates of 3 and 6 h postischemic WT outer medullary renal tissues, respectively (100 µg of protein was loaded). Lanes 3, 4, and 5 represent Western blotting analysis using GAPDH antibody on PAR-immunoprecipitated proteins from sham and 3- and 6-h postischemic WT kidney lysates, respectively. (B) Western blotting analysis showing the expression of GAPDH in whole-cell lysates of outer medullary region. Lane 1, WT sham; lane 2, PARP–/– sham; lane 3, WT 3 h after IRI; lane 4, PARP–/– 3 h after IRI; lane 5, WT 6 h after IRI; and lane 6, PARP–/– 6 h after IRI (50 µg of protein was loaded).
To confirm the possibility that GAPDH is poly(ADP-ribosyl)ated,we performed immunoprecipitation with anti-PAR antibody followedby immunoblotting with anti-GAPDH antibody. The results showeda prominent band at 37 kD at both 3 and 6 h after IRI in WTkidney lysates (Figure 2A, lanes 4 and 5, respectively) butnot in sham-operated kidneys (Figure 2A, lane 3) clearly demonstratingGAPDH-poly(ADP-ribosyl)ation after ischemia. No change in themolecular weight of GAPDH was observed as a result of poly(ADP-ribosyl)ationas other PARP-1 targets in the previous reports.24,25 GAPDHexpression per se did not change between WT and PARP–/–sham-operated or ischemic renal tissues (Figure 2B).
Lactate and ATP levels in WT and PARP–/– Mouse Kidneys after IRI
To show that the inhibition of GAPDH activity by PARP-1 doesindeed inhibit anaerobic glycolysis, we measured lactate productionand ATP levels in the outer medulla of WT and PARP–/–mouse kidneys after IRI. As shown in Figure 3A, lactate productionwas significantly higher in the outer medulla of PARP–/–mice compared with WT mice at 12 h after IRI. As shown in Figure 3B,the ATP levels in the outer medulla of WT mice at 12 h afterIRI were decreased to 58.560 ± 8.018% of the sham-operatedmice (P < 0.03; n = 4), whereas the absence of PARP-1 preservedATP levels to 91.570 ± 9.228% of sham-operated mice.The ATP levels in sham-operated kidneys were measured to be11.146 nmol/mg protein.
Figure 3. (A) Effect of IRI on lactate production in WT and PARP–/– mice. Each bar represents the mean ± SEM of four separate experiments. *P < 0.05 versus WT mice. (B) Effect of IRI on ATP levels in WT and PARP–/– mice. Each bar represents the mean ± SEM of four separate experiments. *P < 0.05 versus WT mice.
Effect of PARP Inhibition on GAPDH Activity in Hypoxia-Injured LLCPK1 Cells
To confirm our in vivo finding that PARP-1 mediates the inhibitionof GAPDH activity and to demonstrate that this mechanism occursin PST cells, we used the porcine-derived proximal tubular cellline LLCPK1. LLCPK1 cells expressed proximal tubular markeraquaporin 1 (AQP1) and the PST marker AQP7 (see SupplementaryFigure S1A). Furthermore, 6 h of hypoxia induced poly(ADP-ribose)(Figure S1B) and several poly(ADP-ribosyl)ated protein bands,including a band at approximately 37 kD, (Supplemental FigureS1C, lanes 3 and 4), suggesting potential PARP activation andGAPDH-poly(ADP-ribosyl)ation in LLCPK1 cells after hypoxia.We subjected LLCPK1 cells to hypoxia with or without the PARPinhibitor GPI-15427 and assayed GAPDH activity. GAPDH activitywas significantly inhibited in LLCPK1 cells after 6 or 12 hof hypoxic treatment (Figure 4, bars 3 and 5, respectively)compared with untreated control cells (Figure 4, bar 1). Subjectingthe cells to hypoxia in the presence of GPI 15427 resulted insignificant improvement of GAPDH activity at 6 h (Figure 4,bar 4) but not at 12 h (Figure 4, bar 6), compared with hypoxiatreatment only (Figure 4, bars 3 and 5). Inhibition of PARPin untreated control cells (Figure 4, bar 2) did not cause significantchanges in the GAPDH activity compared with controls (Figure 4,bar 1).
Figure 4. Effect of hypoxia with or without GPI-15427 on GAPDH activity in LLCPK1 cells. Each bar represents the mean ± SEM. P < 0.02 versus control; *P < 0.05 versus 6 h of hypoxia only.
Inhibition of GAPDH Activity by PARP-1 Is Due to its Poly(ADP-ribosyl)ation
To confirm further the direct role of PARP-1 in the inhibitionof GAPDH activity, we performed an in vitro cell-free assayinvolving purified PARP-1 and GAPDH proteins. GAPDH activitywas significantly inhibited by activated PARP-1 (PARP-1 + activatedDNA) in the presence of NAD+ compared with GAPDH alone (Figure 5,bar 3 versus bar 1). Presence of the PARP-1 inhibitor GPI-15427reversed the effect of activated PARP-1 on GAPDH activity (Figure 5,bars 4 and 5 versus bar 3), indicating a direct role of PARP-1activity in the inhibition of GAPDH activity. Inclusion of NAD+alone in the incubation medium, without activated PARP-1, didnot change GAPDH activity significantly (Figure 5, bar 2 versusbar 1). The proteins from the in vitro cell-free assay, whensubjected to immunoblotting with anti-PAR antibody, demonstratedPAR modification of PARP and GAPDH in a PARP-1–dependentmanner (see Supplementary Figure S2, B through E).
Figure 5. Effect of PARP-1 activity on GAPDH activity in in vitro cell-free system. Each bar represents the mean ± SEM. **P < 0.0002 versus GAPDH enzyme only; *P < 0.0005 versus GAPDH enzyme in the presence of NAD+ and activated PARP-1 enzyme.
Effect of Hypoxia on ATP Levels in Cultured LLCPK1 Cells
To examine whether energy levels are compromised after hypoxicinjury in LLCPK1 and whether PARP inhibitor preserves them,we measured ATP levels in LLCPK1 cells immediately after hypoxictreatment so as to isolate ATP production by anaerobic respirationfrom that of aerobic respiration. As a positive control to indicatethe significance of GAPDH activity in anaerobic ATP production,we used a GAPDH inhibitor, sodium iodoacetate (IA), as describedpreviously.26,27 A 45-min incubation of LLCPK1 cells with IAresulted in significant inhibition of GAPDH activity comparedwith control. The inhibitory effect of IA was dosage-dependentlyenhanced with the increased concentration of the inhibitor (seeSupplementary Figure S3). The concentration of iodoacetamidethat was used in the study is comparable or lesser than theconcentrations used in previous reports.26,28,29 ATP levelswere significantly decreased in LLCPK1 cells after 6 h of hypoxictreatment when compared with control treatment (Figure 6, bar3 versus bar 1). The PARP inhibitor GPI-15427 significantlypreserved ATP levels in 6-h hypoxic-LLCPK1 cells compared withhypoxia only (Figure 6, bar 4 versus bar 3). Interestingly,inhibiting GAPDH enzyme activity by IA along with 6 h of hypoxictreatment further decreased ATP levels significantly comparedwith hypoxia only, and this decrease was enhanced with the increasingconcentration of GAPDH inhibitor (Figure 6, bars 6 through 8versus bar 3), suggesting the significance of preserving GAPDHactivity and ATP production from anaerobic glycolytic pathway.A concentration of 1 µM IA did not significantly decreaseATP levels compared with hypoxia only (Figure 6, bar 5 versusbar 3). Inhibition of PARP activity in control LLCPK1 cellsdid not cause significant change in ATP levels (Figure 6, bar2 versus bar 1).
Figure 6. Effect of hypoxia on ATP levels in LLCPK1 cells with or without PARP/GAPDH inhibitors. Each bar represents the mean ± SEM of four separate experiments except 6 h of hypoxia (n = 5). **P < 0.0002 versus control; *P < 0.05 versus 6 h of hypoxia only; *P < 0.0005 versus 6 h of hypoxia.
Effect of GAPDH/PARP Inhibition on Cytotoxicity in Hypoxic LLCPK1 Cells
We then examined the percentage of cytotoxicity in hypoxic LLCPK1cells by measuring the concentration of lactate dehydrogenase(LDH) that was released from injured cells. We subjected theLLCPK1 cells to hypoxia with and without reoxygenation and inthe presence/absence of GAPDH or PARP inhibitors and measuredthe percentage of cytotoxicity. The cytotoxicity percentagewas significantly increased when GAPDH inhibitor was used alongwith 6 h of hypoxia compared with hypoxia only treatment (Figure 7A,bar 2 versus bar 1), whereas PARP inhibition significantly protectedcells from injury after 6 h of hypoxia (Figure 7A, bar 3 versusbar 1). Similar results were found when LLCPK1 cells were subjectedto 6 h of hypoxia followed by 6 h of reoxygenation; GAPDH inhibitionsignificantly increased cytotoxicity, whereas PARP inhibitionprotected cells (Figure 7B).
Figure 7. LDH cytotoxicity assay. (A) Effect of hypoxia on cytotoxicity in LLCPK1 cells in the presence/absence of GAPDH or PARP inhibitors. Each bar represents the mean ± SEM. *P < 0.05 versus 6 h of hypoxia only; *P < 0.0005 versus 6 h of hypoxia only. (B) Effect of hypoxia and reoxygenation on cytotoxicity in LLCPK1 cells in the presence/absence of GAPDH or PARP inhibitors. Each bar represents the mean ± SEM. *P < 0.05 versus 6 h of hypoxia with 6 h of reoxygenation; *P < 0.0005 versus 6 h of hypoxia with 6 h of reoxygenation.
Our results from LDH assay experiments were further bolsteredby Trypan blue spectrophotometric assay for necrotic cell death.We subjected LLCPK1 cells to 6 h of hypoxia followed by 6 hof reoxygenation and measured Trypan blue absorbance, whichwas directly proportional to necrotic cell death. As in thecase of LDH assay experiments, GAPDH inhibition together withhypoxia/reoxygenation significantly increased cell death comparedwith hypoxia/reoxygenation only (Figure 8, bar 2 versus bar1), whereas PARP inhibition significantly decreased cell death(Figure 8, bar 3 versus bar 1). The images in Figure 8 representTrypan blue–stained cells corresponding to the respectivetreatments. Trypan blue staining seemed to be enhanced whenGAPDH inhibitor was used along with hypoxia/reoxygenation comparedwith hypoxia/reoxygenation only. Only trace levels of Trypanblue staining were visible in the hypoxic cells treated withthe PARP inhibitor GPI-15427. Collectively, these data clearlyindicate that PARP-mediated GAPDH inhibition after simulatedischemia led to exacerbation of cell injury in LLCPK1 cells.
Figure 8. Trypan blue absorbance cytotoxicity assay. Effect of hypoxia and reoxygenation on cytotoxicity in LLCPK1 cells in the presence/absence of GAPDH or PARP inhibitors. Each bar represents the mean ± SEM of four separate experiments. *P < 0.0005 versus 6 h of hypoxia with 6 h of reoxygenation. (Top) Trypan blue staining images of the respective treatments.
The S3 segment of the proximal tubule or PST is extremely susceptibleto IRI compared with other segments of the nephron4,5; however,the S3 segment is less vulnerable to injury compared with S1and S2 segments after selective inhibition of glycolytic processor mitochondrial respiration as opposed to IRI.30 Although alteredhemodynamic factors and differential glycolytic enzyme concentrationsor activities were proposed to be the underlying causes,1,31–33the exact mechanisms leading to selective PST injury are largelyunknown.
Our data clearly demonstrated that GAPDH activity is improvedin the absence of PARP-1 compared with its presence after renalischemia in the outer medullary nephron segments. We showedthat GAPDH is poly(ADP-ribosyl)ated after IRI, suggesting thereduced GAPDH activity is due to its modification by poly(ADP-ribosyl)ation.Our previous finding that PARP-1 expression and activity areselectively upregulated in the S3 segments13,14 but not in othertubular segments after renal ischemia suggests that GAPDH-poly(ADP-ribosyl)ationand consequent inhibition of its activity exclusively occurin the S3 segment. Thus, although significant, the relativelysmall absolute difference that is observed in GAPDH activity(Figure 1) may be due to the relative changes in its activityin the whole outer medullary nephron segments used in the assay.GAPDH expression level was not altered among sham, WT, or PARP–/–ischemic renal tissues, thereby excluding the possibility ofreduced expression as a mechanism of altered activity. Althoughwe did not examine the role of ROS in GAPDH activity inhibitionin this study, our previous studies did not observe any changesin the generation of ROS between WT and PARP–/–ischemic renal tissues.13 Moreover, data from other laboratoriessuggested that oxidative modification is not responsible forthe reduced GAPDH activity.23,34
To define further the mechanism and the significance of PARP-1–mediatedinhibition of anaerobic ATP synthesis in exacerbation of hypoxiccell injury, we used LLCPK1 cells as an in vitro cell culturemodel of IRI. It has been shown that, under in vitro cell cultureconditions, LLCPK1 cells tend to depend mainly on glycolysis.35Similarly, under control conditions, the freshly isolated proximalstraight tubular cells have higher glycolytic rates comparedwith proximal convoluted tubules.36 Hence, proximal straighttubular cells can revert to anaerobic glycolysis and be protectedunder hypoxia. Our data also show that lactate and ATP levelswere significantly increased in the outer medulla of PARP–/–mouse kidneys compared with WT. This suggests that, under ischemia/hypoxicconditions, the glycolytic capacity of proximal straight tubularcells can be enhanced in the absence of PARP-1. We acknowledgethat, although we cannot compare the glycolytic capacities ofPST cells and LLCPK1 cells, our data suggest that the glycolyticcapacity is increased in PST cells under hypoxia and thus theuse of LLCPK1 cells as a model system can be justified. We conductedmost of our in vitro experiments immediately after subjectingLLCPK1 cells to hypoxia to isolate anaerobic respiration fromaerobic metabolism as the sole source of ATP production. Ourdata clearly demonstrated that GAPDH activity was significantlyblunted in LLCPK1 cells subjected to 6 or 12 h of hypoxia, whereasPARP inhibition restored its activity after 6 h of hypoxic treatment.PARP inhibition could not restore GAPDH activity after 12 hof hypoxia, suggesting that either PARP-independent inhibitionof GAPDH activity occurred under severe hypoxia or the injuryat 12 h was too severe for recovery of cellular functions. AlthoughGAPDH-poly(ADP-ribosyl)ation has been suggested to be the underlyingmechanism for its inhibited activity in hyperglycemic aorticendothelial cells23 and in both our in vivo and in vitro modelsof ischemia, direct evidence is lacking toward this end. Toconfirm this proposed mechanism, we performed in vitro cell-freeGAPDH assay using purified GAPDH and PARP-1 enzymes. We showedthat GAPDH activity was significantly inhibited in the presenceof NAD+ and activated PARP-1. That GAPDH activity was restoredto control levels in the presence of PARP inhibitor clearlyindicates that GAPDH modification by poly(ADP-ribosyl)ationleads to inhibition of its activity. Furthermore, it rules outthe possibility of direct binding of PARP-1 to GAPDH as an alternatemechanism of inhibition. Western blot analysis of these proteinsat the end of the in vitro cell-free GAPDH assay experimentrevealed that both PARP-1 and GAPDH were poly(ADP-ribosyl)ated.To rule out the possibility that the reduction in GAPDH activityis due to nonspecific leakage of GAPDH out of the cytosol, possiblyas a result of membrane damage, we assessed the levels of GAPDHprotein and its activity after 6 h of hypoxia with or withoutPARP inhibitor in the cultured medium. Unlike the changes inthe level of LDH, we observed no changes in the protein levelsor activity of GAPDH (data not shown).
Under hypoxic/ischemic environment, cells cannot carry out oxidativephosphorylation because of unavailability of oxygen and hencerevert to anaerobic metabolism. Measurement of ATP levels immediatelyafter hypoxia shows the level of activation of anaerobic respiration.Our data demonstrate that ATP levels were depleted in hypoxic-LLCPK1cells in concordance with reduced GAPDH activity. PARP inhibitionpreserved ATP levels, suggesting restoration of anaerobic glycolysisby improved GAPDH activity; however, PARP inhibition could notreplete ATP levels completely, suggesting that GAPDH may notbe the "rate-limiting" enzyme and modulation of other key glycolyticenzymes such as phosphofructokinase or its product, fructose1,6-biphosphate,37 may also be involved in the metabolic controlof anaerobic ATP synthesis under hypoxic conditions.34,38 Nevertheless,the significant preservation of energy levels may be sufficientto reduce the necrotic cell death that was revealed by our cytotoxicityexperiments.
To confirm that reduced GAPDH activity in fact leads to decreasedATP production by anaerobic glycolysis, we inhibited GAPDH activityusing iodoacetamide, a GAPDH inhibitor, which inhibits the activityof GAPDH by reacting with the cysteines in its active site.26,27We demonstrated that IA dosage-dependently (1 to 100 µM)inhibited GAPDH activity. Subjecting LLCPK1 cells to hypoxiain the presence of IA resulted in ATP depletion in a dosage-dependentmanner, suggesting that inhibiting GAPDH activity prevents anaerobicglycolysis and consequent ATP production.
We and others previously showed that PARP-1 mediates ATP depletionand subsequent necrotic cell death after simulated renal ischemia13,16;however, the mechanisms by which PARP-1 elicits ATP depletionand exacerbation of PST injury after simulated ischemia wasnot elucidated. In this study, we showed that necrotic celldeath was significantly enhanced in LLCPK1 cells subjected tohypoxia with or without reoxygenation in a PARP-1–dependentmanner; however, we did not observe any apoptotic cells immediatelyafter hypoxia (data not shown). PARP inhibition offered significantprotection whereas inhibition of GAPDH enhanced necrotic celldeath in hypoxic LLCPK1 in concordance with ATP depletion, suggestingGAPDH is a limiting factor in mediating anaerobic ATP synthesisand subsequent cell survival.
In summary, the study provides novel insights into the mechanismsby which PARP-1 mediates targeted acute PST cell injury underischemia/hypoxia. We showed that GAPDH was poly(ADP-ribosyl)atedand its activity was inhibited in both in vivo and in vitromodels of simulated renal ischemia. Inhibition of GAPDH activityby PARP-1 led to decreased ATP production by anaerobic glycolysis.PARP-1–mediated inhibition of anaerobic ATP synthesisresulted in exacerbation of proximal tubular cell injury andconsequent necrosis. Taken together, our data indicate thatGAPDH-poly(ADP-ribosyl)ation and subsequent inhibition of anaerobicrespiration lead to exacerbation of ATP depletion in PST toinduce selective injury after IRI.
Materials
All chemicals were purchased from Sigma-Aldrich Corp. (St. Louis,MO) unless otherwise noted.
Animal Models, Surgical Procedures, and Tissue Preparation
All animal procedures were performed after previous approvalby the Institutional Animal Care and Use Committee at the Universityof Nebraska Medical Center. A total of 129 SV (WT) or PARP-1–/–mice (approximately 20 g) were purchased from Jackson Laboratories(Bar Harbor, ME). IRI was induced by bilateral renal pedicleclamping, as described previously.13 Briefly, we clamped bothrenal pedicles for 37 min by using microaneurysm clamps andobserved reflow visually after the clamps were removed. Themice were allowed to recover for variable duration before beingkilled. Sham-operated mice underwent the same surgical procedureexcept the renal pedicles were not occluded. All mice were givenfree access to food and water. At the end of the each experiment,we obtained tissue from the outer medullary region (rich inPST/S3 segments) from slices made using a Stadie-Riggs microtome(Thomas Scientific, Swedesboro, NJ),36 snap-frozen with liquidnitrogen, and stored at –80°C for future experiments.
Cell Culture Conditions
The porcine-derived proximal tubular cell line LLCPK1 (ATCC,Rockville, MD) was cultured to 80 to 90% confluent monolayercultures as described previously.39 We used hypoxia to simulateischemic injury in in vitro experiments using a pouch systemproviding a CO2-enriched anaerobic environment (BBL GasPak PouchSystem; Becton Dickinson, Sparks, MD) as described previously.40Hypoxic injury was induced by incubating 80 to 90% confluentmonolayer of LLCPK1 cells for 6 or 12 h in Krebs-Ringer bicarbonatebuffer (in mM: 115 NaCl, 1 KH2PO4, 3.5 KCl, 1 MgSO4, 1.25 CaCl2,and 25 NaHCO3) at 37°C.41,42 In some experiments, cellswere allowed to recover after hypoxia for 6 h by reoxygenationin normal growth medium. GAPDH inhibition was achieved by exposureto 1 to 100 µM (10 µM in most of the experiments)sodium IA (a specific GAPDH inhibitor).26,27 A novel highlypotent PARP inhibitor, GPI-15427 (MGI Pharmaceuticals, Andover,MA), was used in some studies.43,44,45 The final concentrationof GPI-15427 used in the experiments was 20 µM unlessotherwise stated.
GAPDH Activity Assay
We centrifuged protein lysates from frozen kidney tissue orcultured LLCPK1 cells at 4°C for 10 min at 13,000 rpm toremove cellular debris. We prepared the cytosolic fraction bycentrifuging the tissue or cell lysate at 100,000 x g at 4°Cfor 30 min. We measured protein concentration with Bio-Rad Dcprotein assay reagents using the manufacturer's protocol. Wedetermined the GAPDH enzyme activity by the reduction of NAD+with GAPDH as a substrate in arsenate buffer, as described previously.23,45Briefly, we added 1 to 5 µg of cytosolic protein to 1ml of assay buffer at 25°C and measured the rate of absorbanceby continuous monitoring for 30 min at 340 nm using a spectrophotometer(Biomate 3; Thermo Scientific, Waltham, MA). The enzyme activitywas expressed in nmol/min per mg of protein.
Immunoprecipitation
We immunoprecipitated total protein (500 µg) from tissuelysate with 4 µg of indicated antibody and 20 µlof protein A/G plus (Santa Cruz Biotechnology, Santa Cruz, CA)as per the manufacturer's protocol. We resuspended the immunoprecipitatedprotein in SDS loading dye and performed Western blotting analysisas described previously,13 using antibodies against PAR (BDBiosciences, San Jose, CA), GAPDH (Novus, Littleton, CO), orPARP (Trevigen, Gaithersburg, MD). The signal was detected withECL-plus system according to the manufacturer's instructions(Amersham Biosciences, Piscataway, NJ). For biotinylated proteins,we performed blocking with 1% BSA in PBST, followed by biotindetection with 1:50,000 dilutions of streptavidin–horseradishperoxidase.
Immunofluorescence Microscopy
We performed immunofluorescence staining of LLCPK1 cells withprimary antibodies against PAR (Trevigen), AQP1 (Alpha Diagnostic,San Antonio, TX), or AQP7 (Santa Cruz Biotechnology) as describedpreviously.39 Samples were viewed using a Leica DMR fluorescencemicroscope and the images were captured with an Optronics digitalcamera.
In Vitro Biotinylation Assay to Detect PARP Activity
We performed in vitro biotinylation to detect PARP activityin hypoxia-injured LLCPK1 cells as described previously.21,46Briefly, 50 µg of protein was incubated with 60 µMbiotinylated NAD in a 50-µl final volume of PARP reactionbuffer (50 mM Tris-HCl [pH 8.0] and 25 mM MgCl2) at 37°Cfor 1 h. We terminated the reaction with SDS loading dye andsubjected the samples to Western blot analysis to detect biotinexpression, an indirect method to detect PAR expression.
ATP Assay
We performed ATP assay on tissue/cellular extracts using theEnlighten ATP assay system (Promega, Madison, WI) as previouslydescribed.13 Cellular ATP levels were expressed as nmol/µgprotein.
Lactate Assay
We used the same tissue extracts used to measure ATP levelsto measure lactate levels. We measured the tissue lactate levelsusing Lactate Assay Kit, EnzyChrom (BioAssay Systems, Hayward,CA) according to the manufacturer's protocol. The lactate levelswere expressed in µM/µg protein.
We performed in vitro cell-free GAPDH assay as described previously,47,48using unlabeled NAD+ or 6-biotin-17-NAD+ (biotin-NAD+), purifiedrabbit muscle GAPDH enzyme, activated DNA, and recombinant humanPARP-1 enzyme (Trevigen, MD). The final reaction was performedin a reaction buffer (50 mM Tris-HCl [pH 8.0] and 25 mM MgCl2)containing 5 µg of GAPDH, 5 µg of activated DNA,10 µM biotin-NAD+ or 1 mM of NAD+, 20 or 200 µMGPI-15427, and 1 µl of PARP-1 enzyme (20 U). The samplewas mixed well and incubated at 37°C for 1 h. Immediatelyafter the reaction, we used 1 µl of the sample to measureGAPDH activity and mixed the rest of the sample with SDS loadingdye. We performed Western blot analysis to detect biotin-labeledproteins, PAR, GAPDH, or PARP-1 with the indicated antibodies.
Cell Death Determination by LDH Release Assay
We performed LDH release assay using the cytotoxicity detectionkit (Roche, Indianapolis, IN). We performed cell death determinationby Trypan blue absorbance assay as described previously,49 withslight modifications. Briefly, we added 0.05% Trypan blue toeach culture well at the end of each treatment and placed theplate in the incubator at 37°C for 15 min. We removed dye-containingmedium by three washes with ice-cold PBS and lysed the cellswith 1 ml of 1% SDS followed by absorbance measurements at 590nm. The absorbance value from 2% Triton-X 100–treatedcells was considered as a high control, whereas absorbance valuefrom cells growing in normal medium without any treatment wasconsidered as low control. We measured the cytotoxicity percentageby the formula
Statistical Analysis
All data are expressed as means ± SEM. We used one-wayANOVA to compare the mean values of all groups. We used unpairedt test to compare the means of two different groups. P <0.05 was considered statistically significant.
This work was supported by a grant-in-aid from the AmericanHeart Association to B.J.P., a research grant from NebraskaKidney Association, and a graduate assistantship/fellowshipfrom the University of Nebraska Medical Center to K.D.N.
We thank Dr. Pamela K. Carmines for critical reading of themanuscript and Dr. Jie Zhang, MGI Pharmaceuticals, for kindlyproviding the PARP inhibitor GPI-15427.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Molitoris BA, Sandoval R, Sutton TA: Endothelial injury and dysfunction in ischemic acute renal failure.
Crit Care Med 30
: S235
–S240, 2002[CrossRef][Medline]
Bonventre JV: Mechanisms of ischemic acute renal failure.
Kidney Int 43
: 1160
–1178, 1993[Medline]
Yamamoto K, Wilson DR, Baumal R: Outer medullary circulatory defect in ischemic acute renal failure.
Am J Pathol 116
: 253
–261, 1984[Abstract]
Venkatachalam MA, Bernard DB, Donohoe JF, Levinsky NG: Ischemic damage and repair in the rat proximal tubule: Differences among the S1, S2, and S3 segments.
Kidney Int 14
: 31
–49, 1978[Medline]
Shanley PF, Rosen MD, Brezis M, Silva P, Epstein FH, Rosen S: Topography of focal proximal tubular necrosis after ischemia with reflow in the rat kidney.
Am J Pathol 122
: 462
–468, 1986[Abstract]
Lieberthal W, Nigam SK: Acute renal failure: I. Relative importance of proximal vs. distal tubular injury.
Am J Physiol 275
: F623
–F631, 1998[Medline]
Witzgall R, Brown D, Schwarz C, Bonventre JV: Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney: Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells.
J Clin Invest 93
: 2175
–2188, 1994[Medline]
Safirstein R: Gene expression in nephrotoxic and ischemic acute renal failure.
J Am Soc Nephrol 4
: 1387
–1395, 1994[Abstract]
Ueda N, Kaushal GP, Shah SV: Recent advances in understanding mechanisms of renal tubular injury.
Adv Ren Replace Ther 4
: 17
–24, 1997[Medline]
Walker LM, York JL, Imam SZ, Ali SF, Muldrew KL, Mayeux PR: Oxidative stress and reactive nitrogen species generation during renal ischemia.
Toxicol Sci 63
: 143
–148, 2001[Abstract/Free Full Text]
Chien CT, Lee PH, Chen CF, Ma MC, Lai MK, Hsu SM: De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion.
J Am Soc Nephrol 12
: 973
–982, 2001[Abstract/Free Full Text]
Berger NA: Poly(ADP-ribose) in the cellular response to DNA damage.
Radiat Res 101
: 4
–15, 1985[Medline]
Zheng J, Devalaraja-Narashimha K, Singaravelu K, Padanilam BJ: Poly(ADP-ribose) polymerase-1 gene ablation protects mice from ischemic renal injury.
Am J Physiol Renal Physiol 288
: F387
–F398, 2005[Abstract/Free Full Text]
Martin DR, Lewington AJ, Hammerman MR, Padanilam BJ: Inhibition of poly(ADP-ribose) polymerase attenuates ischemic renal injury in rats.
Am J Physiol Regul Integr Comp Physiol 279
: R1834
–R1840, 2000[Abstract/Free Full Text]
Filipovic DM, Meng X, Reeves WB: Inhibition of PARP prevents oxidant-induced necrosis but not apoptosis in LLC-PK1 cells.
Am J Physiol 277
: F428
–F436, 1999[Medline]
Paschen W, Olah L, Mies G: Effect of transient focal ischemia of mouse brain on energy state and NAD levels: No evidence that NAD depletion plays a major role in secondary disturbances of energy metabolism.
J Neurochem 75
: 1675
–1680, 2000[CrossRef][Medline]
Wright SC, Wei QS, Kinder DH, Larrick JW: Biochemical pathways of apoptosis: Nicotinamide adenine dinucleotide-deficient cells are resistant to tumor necrosis factor or ultraviolet light activation of the 24-kD apoptotic protease and DNA fragmentation.
J Exp Med 183
: 463
–471, 1996[Abstract/Free Full Text]
Bagnasco S, Good D, Balaban R, Burg M: Lactate production in isolated segments of the rat nephron.
Am J Physiol 248
: F522
–F526, 1985[Medline]
Mohr S, Stamler JS, Brune B: Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents.
FEBS Lett 348
: 223
–227, 1994[CrossRef][Medline]
Zhang J, Snyder SH: Nitric oxide stimulates auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase.
Proc Natl Acad Sci U S A 89
: 9382
–9385, 1992[Abstract/Free Full Text]
Dimmeler S, Lottspeich F, Brune B: Nitric oxide causes ADP-ribosylation and inhibition of glyceraldehyde-3-phosphate dehydrogenase.
J Biol Chem 267
: 16771
–16774, 1992[Abstract/Free Full Text]
Du X, Matsumura T, Edelstein D, Rossetti L, Zsengeller Z, Szabo C, Brownlee M: Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells.
J Clin Invest 112
: 1049
–1057, 2003[CrossRef][Medline]
Gobeil S, Boucher CC, Nadeau D, Poirier GG: Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): Implication of lysosomal proteases.
Cell Death Differ 8
: 588
–594, 2001[CrossRef][Medline]
Yoon YS, Kim JW, Kang KW, Kim YS, Choi KH, Joe CO: Poly(ADP-ribosyl)ation of histone H1 correlates with internucleosomal DNA fragmentation during apoptosis.
J Biol Chem 271
: 9129
–9134, 1996[Abstract/Free Full Text]
Lash LH, Tokarz JJ, Chen Z, Pedrosi BM, Woods EB: ATP depletion by iodoacetate and cyanide in renal distal tubular cells.
J Pharmacol Exp Ther 276
: 194
–205, 1996[Abstract/Free Full Text]
Lemasters JJ, DiGuiseppi J, Nieminen AL, Herman B: Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes.
Nature 325
: 78
–81, 1987[CrossRef][Medline]
Hernandez-Fonseca K, Cardenas-Rodriguez N, Pedraza-Chaverri J, Massieu L: Calcium-dependent production of reactive oxygen species is involved in neuronal damage induced during glycolysis inhibition in cultured hippocampal neurons.
J Neurosci Res 86
: 1768
–1780, 2008[CrossRef][Medline]
Hurst RD, Azam S, Hurst A, Clark JB: Nitric-oxide-induced inhibition of glyceraldehyde-3-phosphate dehydrogenase may mediate reduced endothelial cell monolayer integrity in an in vitro model blood-brain barrier.
Brain Res 894
: 181
–188, 2001[CrossRef][Medline]
Shanley PF, Brezis M, Spokes K, Silva P, Epstein FH, Rosen S: Differential responsiveness of proximal tubule segments to metabolic inhibitors in the isolated perfused rat kidney.
Am J Kidney Dis 7
: 76
–83, 1986[Medline]
Fukuhara Y, Yamamoto S, Yano F, Orita Y, Fujiwara Y, Ueda N, Kamada T, Noguchi T, Tanaka T: Changes in activities and mRNA levels of glycolytic enzymes of ischemia-reperfused rat kidney.
Contrib Nephrol 95
: 222
–228, 1991[Medline]
Ruegg CE, Mandel LJ: Bulk isolation of renal PCT and PST: II. Differential responses to anoxia or hypoxia.
Am J Physiol 259
: F176
–F185, 1990[Medline]
Vandewalle A, Wirthensohn G, Heidrich HG, Guder WG: Distribution of hexokinase and phosphoenolpyruvate carboxykinase along the rabbit nephron.
Am J Physiol 240
: F492
–F500, 1981[Medline]
Knight RJ, Kofoed KF, Schelbert HR, Buxton DB: Inhibition of glyceraldehyde-3-phosphate dehydrogenase in post-ischaemic myocardium.
Cardiovasc Res 32
: 1016
–1023, 1996[Abstract/Free Full Text]
Gstraunthaler G, Seppi T, Pfaller W: Impact of culture conditions, culture media volumes, and glucose content on metabolic properties of renal epithelial cell cultures: Are renal cells in tissue culture hypoxic?
Cell Physiol Biochem 9
: 150
–172, 1999[CrossRef][Medline]
Ruegg CE, Mandel LJ: Bulk isolation of renal PCT and PST: I. Glucose-dependent metabolic differences.
Am J Physiol 259
: F164
–F175, 1990[Medline]
Didlake R, Kirchner KA, Lewin J, Bower JD, Markov AK: Attenuation of ischemic renal injury with fructose 1,6-diphosphate.
J Surg Res 47
: 220
–226, 1989[Medline]
Kacser H, Burns JA: Molecular democracy: Who shares the controls?
Biochem Soc Trans 7
: 1149
–1160, 1979[Medline]
Polosukhina D, Singaravelu K, Padanilam BJ: Activation of protein kinase C isozymes protects LLCPK1 cells from H2O2 induced necrotic cell death.
Am J Nephrol 23
: 380
–389, 2003[CrossRef][Medline]
Kim BS, Chen J, Weinstein T, Noiri E, Goligorsky MS: VEGF expression in hypoxia and hyperglycemia: Reciprocal effect on branching angiogenesis in epithelial-endothelial co-cultures.
J Am Soc Nephrol 13
: 2027
–2036, 2002[Abstract/Free Full Text]
Saikumar P, Dong Z, Patel Y, Hall K, Hopfer U, Weinberg JM, Venkatachalam MA: Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.
Oncogene 17
: 3401
–3415, 1998[CrossRef][Medline]
Wei Q, Alam MM, Wang MH, Yu F, Dong Z: Bid activation in kidney cells following ATP depletion in vitro and ischemia in vivo.
Am J Physiol Renal Physiol 286
: F803
–F809, 2004[Abstract/Free Full Text]
Tentori L, Leonetti C, Scarsella M, D’Amati G, Vergati M, Portarena I, Xu W, Kalish V, Zupi G, Zhang J, Graziani G: Systemic administration of GPI 15427, a novel poly(ADP-ribose) polymerase-1 inhibitor, increases the antitumor activity of temozolomide against intracranial melanoma, glioma, lymphoma.
Clin Cancer Res 9
: 5370
–5379, 2003[Abstract/Free Full Text]
Di Paola R, Mazzon E, Xu W, Genovese T, Ferrraris D, Muia C, Crisafulli C, Zhang J, Cuzzocrea S: Treatment with PARP-1 inhibitors, GPI 15427 or GPI 16539, ameliorates intestinal damage in rat models of colitis and shock.
Eur J Pharmacol 527
: 163
–171, 2005[CrossRef][Medline]
Tao Y, Howlett A, Klein C: Nitric oxide regulation of glyceraldehyde-3-phosphate dehydrogenase activity in Dictyostelium discoideum cells and lysates.
Eur J Biochem 224
: 447
–454, 1994[Medline]
Zhang J: Use of biotinylated NAD to label and purify ADP-ribosylated proteins.
Methods Enzymol 280
: 255
–265, 1997[Medline]
Smith S, Giriat I, Schmitt A, de Lange T: Tankyrase, a poly(ADP-ribose) polymerase at human telomeres.
Science 282
: 1484
–1487, 1998[Abstract/Free Full Text]
Ditsworth D, Zong WX, Thompson CB: Activation of poly(ADP)-ribose polymerase (PARP-1) induces release of the pro-inflammatory mediator HMGB1 from the nucleus.
J Biol Chem 282
: 17845
–17854, 2007[Abstract/Free Full Text]
Uliasz TF, Hewett SJ: A microtiter Trypan blue absorbance assay for the quantitative determination of excitotoxic neuronal injury in cell culture.
J Neurosci Methods 100
: 157
–163, 2000[CrossRef][Medline]
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Abstract
Introduction
P < 0.02 versus control; *
