Epac-Rap Signaling Reduces Oxidative Stress in

Download PDF
0 downloads 0 Views 3MB Size Report
Comment
Pharmacologic activation of Epac-Rap signaling by the Epac-selective cAMP .... LZM-007 compared with 20 mg/kg free nonconjugated 8-pCPT-. 2!-O-Me-cAMP.
BASIC RESEARCH

www.jasn.org

Epac-Rap Signaling Reduces Oxidative Stress in the Tubular Epithelium Geurt Stokman,* Yu Qin,* Tijmen H. Booij,* Sreenivasa Ramaiahgari,* Marie Lacombe,† M. Emmy M. Dolman,‡ Kim M.A. van Dorenmalen,‡ Gwendoline J.D. Teske,§ Sandrine Florquin,§ Frank Schwede,| Bob van de Water,* Robbert J. Kok,‡ and Leo S. Price*¶ *Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden, The Netherlands; †LinXis B.V., Amsterdam, The Netherlands; ‡Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands; § Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands; |BIOLOG Life Science Institute, Bremen, Germany; and ¶OcellO BV, Leiden, The Netherlands

ABSTRACT Activation of Rap1 by exchange protein activated by cAMP (Epac) promotes cell adhesion and actin cytoskeletal polarization. Pharmacologic activation of Epac-Rap signaling by the Epac-selective cAMP analog 8-pCPT-29-O-Me-cAMP during ischemia-reperfusion (IR) injury reduces renal failure and application of 8-pCPT-29-O-Me-cAMP promotes renal cell survival during exposure to the nephrotoxicant cisplatin. Here, we found that activation of Epac by 8-pCPT-29-O-Me-cAMP reduced production of reactive oxygen species during reoxygenation after hypoxia by decreasing mitochondrial superoxide production. Epac activation prevented disruption of tubular morphology during diethyl maleate–induced oxidative stress in an organotypic three-dimensional culture assay. In vivo renal targeting of 8-pCPT-29-O-Me-cAMP to proximal tubules using a kidney-selective drug carrier approach resulted in prolonged activation of Rap1 compared with nonconjugated 8-pCPT-29-O-Me-cAMP. Activation of Epac reduced antioxidant signaling during IR injury and prevented tubular epithelial injury, apoptosis, and renal failure. Our data suggest that Epac1 decreases reactive oxygen species production by preventing mitochondrial superoxide formation during IR injury, thus limiting the degree of oxidative stress. These findings indicate a new role for activation of Epac as a therapeutic application in renal injury associated with oxidative stress. J Am Soc Nephrol 25: 1474–1485, 2014. doi: 10.1681/ASN.2013070679

Renal ischemia-reperfusion (IR) injury is an important cause of AKI1 and a significant risk factor for the development of renal dysfunction after kidney transplantation.2 During IR injury, morphologic and functional alterations of the proximal tubular epithelium occur that are linked to the development of renal failure and activation of immune cells via release of proinflammatory cytokines.3 Exchange protein activated by cAMP (Epac) is a guanine nucleotide exchange factor for the small GTPase Rap1.4 Activation of Epac by cAMP or by the Epac-selective cAMP analog 8-pCPT-29-O-MecAMP (also referred to as 007) induces functional activation of Rap1.5 Initial studies showed that Epac-Rap signaling enhances cell adhesion by supporting maturation of cell-cell junctions6,7 and promoting integrin-mediated cell-matrix adhesion.8,9 In 1474

ISSN : 1046-6673/2507-1474

line with these studies, we recently demonstrated that selective activation of Epac reduces proximal tubular epithelial cell (PTEC) detachment during IR injury using in vitro and in vivo models.10 Activation of Epac-Rap was associated with reduced expression of markers for cellular stress in PTECs. In addition,

Received July 1, 2013. Accepted December 2, 2013. G.S. and Y.Q. contributed equally to this work. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Geurt Stokman, Department of Pathology, Academic Medical Center, Meibergdreef 9, L2-111, 1105 AZ Amsterdam, The Netherlands. Email: [email protected] Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 25: 1474–1485, 2014

www.jasn.org

BASIC RESEARCH

in vitro cisplatin-induced apoptosis of PTECs could be significantly reduced by activation of Epac and this was also associated with improved adhesion of cells.11 On the basis of these findings, we hypothesized that activation of Epac-Rap signaling may protect against a common cytotoxic event in these injury models. Unbalanced and uncontrolled production of reactive oxygen species (ROS) is an important mediator of cell injury and occurs during cisplatin nephrotoxicity,12 IR injury,13 and renal fibrosis.14 In renal pathology, intracellular ROS can be produced enzymatically such as by NADPH oxidase (NOX) complexes or derive from dysfunctional mitochondrial activity. Mitochondrial ROS production appears to be the driving force behind hypoxiareoxygenation cell injury15 and cisplatin cytotoxicity.16 Here we studied the role of specific proximal tubular activation of Epac and how this protects against renal injury in both in vitro and in vivo models for IR injury. We found that ROS production during reoxygenation after hypoxia was decreased by activation of Epac. Selective proximal tubular activation of Epac by renal targeting of 8-pCPT-29-O-Me-cAMP conjugated to lysozyme (LZM-007) reduced oxidative stress in an in vivo model for IR injury and significantly decreased IR injury–associated renal failure and tubular damage. Our data show that Epac activation reduces ROS-mediated cellular injury in renal disease and may be a therapeutic strategy for modulation of oxidative stress.

RESULTS Activation of Epac by 8-pCPT-29-O-Me-cAMP Reduces Production of ROS after Hypoxic Cell Stress

We first examined a possible role for Epac-Rap signaling in controlling ROS production in vitro using the conditionally immortalized (IM) PTEC line IM-PTECs, which expresses endogenous Epac1 and shows robust activation of Rap1 upon stimulation with 8-pCPT-29-O-Me-cAMP-AM, a membrane-permeable prodrug of 8-pCPT-29-O-Me-cAMP,17 or forskolin.10,11 IM-PTECs were preloaded with 5-(and-6)carboxy-29,79-dichlorodihydrofluorescein diacetate (DCF-DA), which is converted into fluorescent DCF via oxidation by radicals.18 After hypoxia, reoxygenation was induced by addition of fresh medium to the cells. Reoxygenation induced a rapid increase in probe conversion (Figure 1A), indicating fast accumulation of cytosolic ROS. We did not observe ROS production during hypoxia and in cells that were maintained in medium with a layer of paraffin oil on top (data not shown), demonstrating that ROS production only occurs in cells that undergo reoxygenation after a hypoxic event. Addition of 5 mM 8-pCPT-29-O-Me-cAMP-AM or 10 mM forskolin during reoxygenation significantly reduced ROS production (Figure 1A). The role of ROS production in this assay was confirmed by exposure to antioxidants, 5 mM N-acetyl-cysteine (NAC) and 100 mM resveratrol, which significantly reduced the conversion of carboxy-DCF-DA (Figure 1A). The increase in ROS production was apparent up to approximately 60 minutes after J Am Soc Nephrol 25: 1474–1485, 2014

Figure 1. Activation of Epac reduces ROS production after hypoxia. ROS production is determined during reoxygenation (recovery) after 60 minutes of hypoxia by measuring carboxylDCF-DA activation using an automated fluorescence plate reader at 5 minutes after the start of reoxygenation. (A) Cells maintained in medium under normoxic conditions show no significant increase in ROS production, whereas cells that are subjected to 60 minutes of hypoxia show rapid production of ROS during reoxygenation. Addition of 5 mM 8-pCPT-29-O-Me-cAMP-AM (007-AM), 10 mM forskolin, or the antioxidants NAC (5 mM) and resveratrol (100 mM) reduces ROS production during reoxygenation. Data are expressed as the mean relative increase of the specific fluorescence (n=6, except n=3 for NAC and resveratrol). (B) ROS production at 60 minutes after start of the measurement is expressed as a percentage of the control group. Data are expressed as the mean6SEM. *P,0.05; **P,0.01. N, normoxic conditions; R, reoxygenation.

the start of reoxygenation, indicating that ROS production is a rapid response of cells that are subjected to reoxygenation after hypoxia. At 60 minutes of reoxygenation, 8-pCPT-29-O-MecAMP-AM and forskolin significantly reduced ROS production by 50% compared with controls (Figure 1B). These results show that Epac activation affects accumulation of cytosolic ROS during reoxygenation after hypoxia. Epac activation by Epac and Renal Oxidative Stress

1475

BASIC RESEARCH

www.jasn.org

8-pCPT-29-O-Me-cAMP has previously been implicated in NOX inhibition.19 The NOX inhibitor diphenyleneiodonium (10 mM) did not result in decreased ROS production (data not shown), suggesting that Epac activation by 8-pCPT-29-O-MecAMP in our model does not involve inhibition of NOX. Hypoxia-Induced Mitochondrial ROS Production Is Decreased by Exposure to 8-pCPT-29-O-Me-cAMP

Mitochondrial generation of superoxide and other ROS is considered the principal mechanism underlying initiation of cellular oxidative stress in IR injury.13,15 We therefore examined whether mitochondrial ROS production was involved by detecting oxide radical formation using the superoxide activated probe MitoSOX Red.20 As a positive control, cells were incubated with 25 mM antimycin A, a mitochondrial complex III inhibitor, which induced significant mitochondrial ROS production (Figure 2A). Cells subjected to hypoxia followed by reoxygenation for 30 minutes showed increased mitochondrial ROS production compared with normoxic controls (Figure 2, B and C). Addition of 5 mM 8-pCPT-29-O-Me-cAMP-AM or 10 mM forskolin reduced MitoSOX Red staining during reoxygenation indicating a reduction in mitochondrial ROS production (Figure 2, B and C). Similarly, addition of 5 mM NAC and 100 mM resveratrol prevented mitochondrial ROS formation (Figure 2, B and C). To examine the role of functional Epac-Rap signaling on mitochondrial ROS production, cells were subjected to hypoxia and exposed to the pharmacologic Epac1/2 inhibitors HCJ019721 or ESI-0922 (both at 10 mM) during reoxygenation (Figure 2D). Both Epac inhibitors increased MitoSOX Red fluorescence intensity and resulted in detachment of cells during reoxygenation. Stimulation with 8-pCPT-29-O-Me-cAMP-AM did not reduce MitoSOX Red staining or cell detachment during reoxygenation and exposure to HCJ0197 or ESI-09. On the basis of these results, we propose that activation of Epac-Rap signaling dampens oxidative stress by reducing mitochondrial ROS. Oxidative Stress–Induced Tubular Disintegration Can Be Prevented by 8-pCPT-29-O-Me-cAMP-AM

Because hypoxia-reoxygenation results in an acute increase in ROS production, we also investigated the effect of Epac activation in a model for progressive, long-term oxidative stress. For this purpose, IM-PTECs were cultured for 5 days in Matrigel-collagen gels. During this period, tubulogenic outgrowth of IM-PTECs resulted in the formation of a branched tubular network (Figure 3B). Overnight exposure to diethyl maleate (DEM), a compound known to provoke oxidative stress by depletion of glutathione,23 had clear morphologic effects on the integrity of the network. DEM induced mitochondrial ROS accumulation (Figure 3A), decreased the size of tubule-forming objects (Figure 3C), and reduced the total length of tubules formed per object (Figure 3D) compared with controls. Coexposure to the antioxidant NAC did not result in complete protection against tubular damage (Figure 3, B–D). Addition of 8-pCPT-29-O-Me-cAMP-AM reduced DEM-induced tubular damage (Figure 3, B–D), 1476

Journal of the American Society of Nephrology

demonstrating that activation of Epac provides protection against morphologic alterations in oxidative stress–induced cell injury. Interestingly, exposure to forskolin, a nonselective activator of Epac, decreased object size and tubule length under control conditions and did not provide any beneficial effect on DEM-induced damage (Figure 3, B–D). These findings show that selective Epac activation is cytoprotective against both acute and chronic exposure to oxidative stress. Selective and Prolonged Rap1 Activation in the Kidney after Systemic Administration of 8-pCPT-29-O-Me-cAMP Conjugated to Lysozyme

Although local intrarenal administration of 8-pCPT-29-O-MecAMP is a feasible experimental approach to activate tubular Epac-Rap signaling,10 systemic administration and renal targeting is preferable in the context of a renal ischemia model. To this end, an 8-pCPT-29-O-Me-cAMP- lysozyme conjugate was generated (Figure 4A). Conjugation of bioactive compounds to the low molecular mass protein lysozyme (14 kD) leads to efficient and selective proximal tubular uptake of the compound via megalin-dependent endocytosis of the drug-protein conjugate from the ultrafiltrate.24 Systemic administration of nonconjugated 8-pCPT-29-O-MecAMP (20 or 50 mg/kg bodyweight) or LZM-007 conjugate (40 mg/kg bodyweight) resulted in increased Rap1 activity compared with vehicle-treated controls within 2 hours after intravenous injection (Figure 4, B and C). Because the lysozyme conjugate contains 40 mg of 8-pCPT-29-O-Me-cAMP per milligram of conjugate, a 12.5-fold reduction in total 8-pCPT-29-O-MecAMP could be achieved to induce Rap1 activation by 40 mg/kg LZM-007 compared with 20 mg/kg free nonconjugated 8-pCPT29-O-Me-cAMP. In addition, kidneys were collected at 3, 6, 12, and 24 hours after administration of either 20 mg/kg 8-pCPT-29-O-MecAMP or 40 mg/kg LZM-007. Renal Rap1 activation peaked at 3 hours after 8-pCPT-29-O-Me-cAMP administration but returned to control levels at later time points. In contrast, Rap1 activity was significantly increased for up to 24 hours in kidneys of animals that received LZM-007 (Figure 5, A and B), which is in agreement with the prolonged renal residence of this type of LZM-drug conjugate.25 No significant Rap1 activation was detected in liver, heart, or lung tissue after LZM-007 administration (data not shown). Biodistribution of the LZM-007 conjugate was determined by immunostaining for chicken lysozyme. As previously demonstrated,26 we detected accumulation of this lysozyme ortholog after administration of the conjugate in most proximal tubules located in the cortex and corticomedullary area of the kidney (Figure 5C). Lung, brain, liver, and heart tissue obtained from the same animals did not stain positive for lysozyme (Figure 5D). These findings suggest that LZM-007 after systemic administration is filtered by the glomerulus and taken up by the proximal tubular epithelium, leading to prolonged activation of Rap1 compared with nonconjugated 8-pCPT-29-O-Me-cAMP. J Am Soc Nephrol 25: 1474–1485, 2014

www.jasn.org

BASIC RESEARCH

Differential Antioxidant Response in Proximal Tubules during Early Reperfusion after Epac Activation

In vivo ROS production resulting from mitochondrial dysfunction occurs rapidly during reperfusion.27 To examine the effect of LZM-007 treatment on cellular responses to oxidative stress in a model for bilateral renal IR injury, we treated animals with 40 mg/kg LZM-007 or vehicle 3 hours before bilateral clamping of the renal pedicles. Animals were euthanized after 1 hour of reperfusion. Nuclear factor erythroid 2–related factor2 (Nrf2) controls expression of cytoprotective proteins such as heme oxygenase-1 (HO-1) and a variety of enzymes involved in the antioxidant response, including g-glutamylcysteine synthetase (g-GCS). Staining for Nrf2 showed a decreased number of positive nuclei during IR injury in tubules in the corticomedullary area of LZM-007–treated animals compared with controls (Figure 6A). The enzyme g-GCS is involved in glutathione synthesis. In sham-operated kidneys, g-GCS was predominantly localized in the brush border of proximal tubules in both vehicle and LZM-007–treated animals (Figure 6B, upper panel) and glomeruli. IR injury led to decreased cytoplasmic staining intensity and loss of brush border–associated g-GCS in control kidneys. Both staining intensity and localization were protected in LZM-007–treated animals (Figure 6B, lower panel). The expression of HO-1 was not yet detectable at this time point in both groups (data not shown). These data suggest that ROS-induced activation of the Nrf2 pathway is decreased after Epac activation and loss of tubular g-GCS is prevented. Figure 2. Mitochondrial superoxide generation after hypoxia is reduced by activation of Epac1. (A) Exposure to the mitochondrial complex III inhibitor antimycin A results in increased MitoSOX Red labeling (red) of IM-PTECs, indicating accumulation of mitochondrial superoxide. Nuclei are counterstained (blue). (B) Representative examples of images taken from normoxic controls or cells after 30 minutes of reoxygenation (re-ox) as used for analysis. MitoSOX Red labeling are shown in red, and counterstained nuclei are in blue. (C) MitoSOX Red labeling is quantified using digital image analysis. The area of high intensity–labeled mitochondria is measured per field and adjusted for the number of nuclei. Thirty minutes of reoxygenation after 60 minutes of hypoxia significantly increases superoxide production (MitoSOX Red labeling) compared with normoxic controls. Exposure of cells to 8-pCPT-29-O-Me-cAMP-AM (007-AM) or forskolin reduces superoxide accumulation after hypoxia. (D) Representative examples of images taken from normoxic controls or cells after 30 minutes of reoxygenation. Cells are treated with vehicle (left panel) or 8-pCPT-29-O-Me-cAMP-AM (007-AM, right panel). Data are expressed as mean6SEM. *P,0.05; **P,0.01. re-ox, reoxygenation. Original magnification, 360.

J Am Soc Nephrol 25: 1474–1485, 2014

Proximal Tubule–Specific Activation of Epac Reduces Renal Failure and Tissue Injury during IR Injury

To examine the effect of proximal tubular Epac-Rap activation on IR-induced renal failure and tissue damage, we injected animals with LZM-007 3 hours before induction of bilateral IR injury and euthanized animals after 1 or 3 days. Compared with vehicletreated controls, mice treated with LZM-007 showed a significant reduction in plasma urea and a trend toward reduced plasma creatinine levels at day 1 after ischemia (Figure 7, A and B). Tubular injury, determined by staining

Epac and Renal Oxidative Stress

1477

BASIC RESEARCH

www.jasn.org

for clusterin-a, was significantly reduced in kidneys of mice treated with LZM-007 (Figure 7, C and D). Histopathology was scored using a semiquantitative grading system and confirmed a decrease in tubular injury during IR injury in mice treated with LZM-007 compared with controls (Figure 7E). To rule out any effect of lysozyme itself on renal function and tubular injury during IR injury, we administered 40 mg/kg lysozyme to mice. We found that plasma urea, plasma creatinine, and histopathologic scoring of tubular injury were not significantly different between vehicle- and lysozyme-treated animals (Figure 7F). HO-1 expression was used to evaluate the Nrf2-mediated antioxidant response in proximal tubules. Treatment with LZM-007 significantly reduced HO-1 expression by proximal tubules compared with that in control animals (Figure 8A), suggesting that, in combination with improved renal function, activation of Epac-Rap signaling decreased the degree of oxidative stress during IR injury. In addition, we detected significantly fewer apoptotic tubular epithelial cells during IR injury in LZM-007–treated animals (Figure 8B). Consistent with a decrease in tubular injury after treatment with LZM-007, the number of granulocytes that infiltrated the tubulointerstitium was significantly lower compared with controls (Figure 8C). These findings suggest that renal targeting of Epac decreases oxidative stress during IR injury and is associated with improved renal function and prevention of tubular injury.

DISCUSSION

In our previous studies, we demonstrated that pharmacologic activation of Epac-Rap signaling provides protection against cell detachment during IR injury10 and apoptosis during exposure to cisplatin.11 Because excessive generation of ROS by mitochondria is a common feature in a diverse array of renal pathologies including IR injury and cisplatin nephrotoxicity, we hypothesized that Epac-Rap signaling influences mitochondrial function during injury. Although antioxidant therapy has been successfully applied in experimental models for renal injury,28,29 the clinical effects in patients with kidney disease are less well characterized and do not show the same potential as observed in experimental

Figure 3. 8-pCPT-29-O-Me-cAMP-AM reduces tubular disintegration during oxidative stress in 3D culture. IM-PTECs are cultured for 5 days in a Matrigel-collagen matrix during which a branched tubular network is formed. (A) Cells are exposed

1478

Journal of the American Society of Nephrology

overnight to 0.15 mM DEM, stained for 30 minutes with 1 mM MitoSOX Red (red) and nuclei are counterstained with Hoechst (blue). (B) Cells are exposed overnight to 0.15 mM DEM to induce cellular oxidative stress and/or 5 mM 8-pCPT-29-O-Me-cAMP-AM (007-AM), 10 mM forskolin, or 5 mM NAC. Control cells are treated with vehicle solution only. Cells are fixed and stained for F-actin (red) and nuclei (blue) using phalloidin and Hoechst, respectively. The size of each object (C) and the mean total length of all tubules per object (D) are determined by image analysis software and expressed as pixels per object. Data are presented as mean6SEM. *P,0.05; **P,0.01. Original magnification, 360 in A; 320 in B. FOR, forskolin.

J Am Soc Nephrol 25: 1474–1485, 2014

www.jasn.org

Figure 4. Systemic and intrarenal administration of 8-pCPT-29O-Me-cAMP and renal Rap1 activation. (A) Chemical structure of the 8-pCPT-29-O-Me-cAMP–Lx Linker conjugate. (B) Representative immunoblots of in vivo Rap1 pull-down analyses for intravenous administration of LZM-007 (top panels), unconjugated 8-pCPT-29-O-Me-cAMP (007, middle panels), or intrarenal administration of 8-pCPT-29-O-Me-cAMP (007, bottom panels). (C) Densitometric analysis of Rap1 pull-down assays quantified as active GTP-bound Rap1 over total Rap1. Data are expressed as mean6SEM. *P,0.05; **P,0.01. a.u., arbitrary unit; i.v., intravenous.

J Am Soc Nephrol 25: 1474–1485, 2014

BASIC RESEARCH

studies. For example, studies concerning administration of the antioxidant NAC showed responses varying from slightly beneficial to responses without any significant clinical effect.30,31 Although NAC does reduce oxidative stress, presumably by replenishment of glutathione, it does not act on the source of ROS production, being mitochondrial dysfunction. Regulation of pathways that mediate mitochondrial function or dysfunction may provide better protection against oxidative stress. A possible link between Epac and regulation of mitochondrial metabolism was previously suggested32 and may be reflected by the mitochondrial localization of Epac.33 A recent study by Park et al.34 demonstrated that inhibition of cAMP phosphodiesterases by resveratrol promotes mitochondrial function in skeletal muscle cells via Epac1 through an indirect interaction with AMP-activated protein kinase and peroxisome proliferatoractivated receptor g coactivator-1a. Mukai et al.35 reported that high glucose that results in mitochondrial ROS production in pancreatic b cells36 was decreased by stimulation to the glucagon-like peptide-1 receptor ligand exendin-4 in an Epacdependent fashion. A similar effect was obtained using 8-pCPT-29-O-Me-cAMP. Although their results did not implicate Epac, Reedquist et al.37 demonstrated that Rap1 signaling was required for Ras-induced ROS in T lymphocytes. These studies, in combination with our results, suggest an unexpected role for Epac-Rap signaling in mediating mitochondrial function and ROS production upon cellular injury or stress. Whether Epac-Rap signaling prevents mitochondrial ROS production during IR injury directly or indirectly via its effect on improved cell adhesion remains to be investigated. Using acute in vitro models for IR injury, we found that ROS production occurs rapidly during recovery from hypoxia and is associated with increased superoxide accumulation in mitochondria. To examine the effect of Epac activation during longterm exposure to ROS and to study its effects on morphologic alterations, we developed a three-dimensional (3D) culture assay combined with DEM-induced oxidative stress. DEM is conjugated to glutathione by glutathione S-transferase, resulting in loss of the cellular antioxidant capacity. Although forskolin significantly decreased ROS formation in monolayer assays, we found that forskolin did not protect against disruption of tubular network during exposure to DEM. We previously established that 8-pCPT-29-O-Me-cAMP-AM selectively activates Epac, whereas forskolin induces activation of Epac as well as protein kinase A (PKA).10,11 The effect of long-term activation of PKA by forskolin may antagonize the protective outcome we observed in the acute exposures in monolayer assays and reflect PKAmediated differences in gene expression. In this study, we used ESI-0922 and HJC0197,21 two novel identified Epac1/2 inhibitors. A recent publication shows that ESI-09 and HJC0197 do not act as selective inhibitors for both Epac isoforms, but might also inhibit RapGEF6 activity.38 In addition, both compounds appear to have protein denaturing properties but only at higher concentrations than those used in our study (10 mM for both compounds). Epac and Renal Oxidative Stress

1479

BASIC RESEARCH

www.jasn.org

We determined the effect of Epac activation on in vivo ROS production and cell stress activity by examining established markers for ROS-induced pathway activation. Nrf2 protein levels increase in the presence of ROS, whereas treatment with NAC reduces Nrf2 expression after hypoxia.39 In line with this, we found that treatment with LZM-007 decreased nuclear Nrf2 translocation during IR injury compared with control-treated animals. The expression of HO-1 is dependent on Nrf2-mediated transcriptional activation; the number of HO-1–expressing tubular epithelial cells in the corticomedullary area was significantly decreased in LZM-007–treated animals, reflecting a reduction in Nrf2-mediated transcription upon activation of Epac. In addition, LZM-007 treatment prevented redistribution of g-GCS from the apical membrane and decreased staining intensity in proximal tubules during IR injury, suggesting that Epac activation may also act on maintaining glutathione synthesis. The proximal tubular epithelium plays an important role in the pathogenesis of IR injury. In the tubular compartment, it is the proximal tubules that are most affected by IR injury and are crucially involved in activation of the inflammatory response.40 In our previous study, we used intrarenal administration of 8pCPT-29-O-Me-cAMP to activate Epac.10 Epac is highly expressed in the kidney but expression is also described in endothelial cells and other organs.4 To limit Epac activation to proximal tubules in combination with systemic administration, we used a protein-based carrier system in which 8pCPT-29-O-Me-cAMP was conjugated to chicken egg white lysozyme via a platinum (II)–based linker molecule (Lx). This approach is dependent on glomerular filtration of the low molecular weight protein lysozyme followed by proximal tubular uptake by megalin.24 Lysozyme-drug complexes have high selectivity for proximal tubular uptake; in accordance with previous studies, we were unable to find any significant labeling for chicken lysozyme in heart, lung, liver, and brain tissue, whereas proximal tubules stained positive in kidney tissue (Figure 5, C and D). Lysozyme itself also has no significant effect on the outcome of IR injury (Figure 7F). Renal targeting of 8-pCPT-29-O-Me-cAMP therefore results in prolonged activation of the Epac target, allowing a reduced systemic dose and reduced bioavailability in nonrenal tissues. In this study, we found a clear and unexpected role for EpacRap signaling in controlling oxidative stress in PTECs during Figure 5. Lysozyme-conjugated 8-pCPT-29-O-Me-cAMP selectively activates prolonged Epac-Rap signaling in proximal tubules. (A and B) Mice are treated with 20 mg/kg bodyweight unconjugated 8-pCPT-29-O-Me-cAMP, 40 mg/kg bodyweight LZM-007 (corresponding to 1.6 mg/kg 007), or vehicle (PBS) via intravenous injection. Kidneys are collected after 3, 6, 12, and 24 hours after administration and analyzed for activation of Rap1. (A) Active (GTP-)Rap1 and total Rap1 detection by Western blotting. (B) Densitometric analysis of Western blots. The amount of GTPRap1 over the total amount of Rap1 is quantified for vehicle controls (white bars), unconjugated 8-pCPT-29-O-Me-cAMP (gray bars), and LZM-007 (black bars) and expressed as arbitrary units. (C) Chicken lysozyme is used for the 8-pCPT-29-O-Me-cAMP

1480

Journal of the American Society of Nephrology

conjugation to ULS. Specific immunostainings were performed for detection of chicken lysozyme as indication for uptake of LZM-007. Proximal tubules showed specific staining (brown) at the apical membrane (indicated by arrows) or a distinct cytosolic staining pattern (boxed area, indicated by arrow head) suggesting endocytic uptake. (D) Lung, brain, liver, and heart tissue sections from vehicle and LZM-007–treated mice do not stain positive for lysozyme. Nuclei are counterstained with hematoxylin (blue). Data are expressed as the mean6SEM. *P,0.05; **P,0.01. a.u., arbitrary unit. Original magnification, 320 in D.

J Am Soc Nephrol 25: 1474–1485, 2014

www.jasn.org

BASIC RESEARCH

IR injury, most likely by reducing mitochondrial ROS production as found in our in vitro assays. Therapeutic activation of Epac-Rap signaling may thus improve the clinical outcome of renal disease associated with oxidative stress.

CONCISE METHODS Reagents

Figure 6. Activation of Epac-Rap signaling affects early expression cytoprotective protein expression. Immunostaining and quantitative analysis for Nrf2(A) and g-GCS (B) on kidney tissue from animals subjected to sham-surgery and IR. Tissue is collected at 1 hour after surgery. (A) Nuclear staining for Nrf2 is almost absent in kidneys from sham-operated animals but increases during IR injury. Examples of Nrf2-positive nuclei are indicated by white arrows. The number of positive nuclei in tubular epithelial cells is counted per HPF on kidney sections from animals subjected to IR injury. (B) Staining for the modifier subunit of g-GCS is present in brush borders of tubules located in the corticomedullary area of sham-operated kidneys. After IR injury, the apical distribution and intensity of the staining are decreased in kidneys from vehicle-treated animals and protected in animals treated with 40 mg/kg LZM-007. Negative controls (ctrl) are incubated with secondary antibodies only. Nuclei in B are counterstained with methylene green. All data are expressed as mean6SEM. *P,0.05; **P,0.01. Original magnification, 320.

J Am Soc Nephrol 25: 1474–1485, 2014

Diethylmaleate, antimycin A, diphenylene iodonium, rhodamine-conjugated phalloidin, Hoechst 33258 and 33342, forskolin, NAC, and resveratrol were purchased from Sigma-Aldrich (St. Louis, MO). 8-pCPT-29-O-Me-cAMP, 8-pCPT-29-OMe-cAMP-AM, HJC0197, and ESI-09 were from Biolog (Bremen, Germany). Paraffinum liquidum (110–230 mPa) was from Spruyt Hillen (IJsselstein, The Netherlands). Matrigel was from BD Biosciences (Erembodegem, Belgium). All cell culture reagents and rat tail collagen were from Invitrogen (Bleiswijk, The Netherlands). Buffered formaldehyde solutions were from Added Pharma (Oss, The Netherlands). LZM-007 was coupled to chicken egg white lysozyme (Sigma-Aldrich) via the Lx linker (LinXis B.V., Breda, The Netherlands) as described previously for other conjugates,25,26,41,42 resulting in a LZM-drug conjugate with a 1.1:1 drug/LZM molar ratio. Rabbit anti-Rap1 (clone 121), goat anticlusterin (M-18), rabbit anti-Nrf2 (C-20), rabbit anti–g-GCS modifier subunit, and mouse anti– b-actin-IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit anti-cleaved caspase-3 was from Cell Signaling Technology (Danvers, MA). The rabbit anti– HO-1 (SPA-895) antibody was purchased from Enzo Lifesciences (Zandhoven, Belgium). The rat anti–Gr-1 was from BD Biosciences. Texas Red–labeled donkey anti-goat is from Invitrogen. Horseradish peroxidase–conjugated secondary antibodies are from DAKO (Glostrup, Denmark).

Cell Culture and 3D Assay Conditionally immortalized IM-PTECs were cultured as previously described.10 All experiments were performed using cells that had been cultured under restrictive conditions for at least 7 days. For 3D culture assays, IM-PTECs were cultured under restrictive conditions for 2 days in normal culture dishes prior to “in gel” 3D culture in 96 wells mClear imaging plates (Greiner Bio-One, Alphen a/d Rijn, The Netherlands) using a Matrigel-collagen gel Epac and Renal Oxidative Stress

1481

BASIC RESEARCH

www.jasn.org

mix as previously described.43 To prevent adhesion and monolayer formation to the plate bottom, a 35-ml base gel layer was created in each well, on top of which a 30-ml cell-gel suspension was added. After 24 hours, cells were stimulated with hepatocyte growth factor and TGF-b1 (R&D Systems, Abingdon, UK). Cells were cultured for an additional 4 days.

Cellular ROS Production

Figure 7. LZM-007 prevents renal failure and tubular injury during IR injury. Renal function of animals treated with vehicle (white bars) or LZM-007 (black bars) is determined by measuring plasma urea (A) and plasma creatinine (B). (C and D) Tubular injury is determined by staining for marker expression of clusterin-a. (C) Clusterin-a stainings on kidney sections from animals treated with vehicle (white bars) or LZM-007 (black bars) are quantified using digital image analysis and expressed as percentage of positive staining per field. (D) Representative images of immunostainings used for clusterin-a quantification. Clusterin-a is stained in red, and nuclei are counterstained in blue. (E) Representative examples of periodic acid-Schiff reagent–stained tissue sections (left) and histopathology scoring of tubular injury of mice treated with vehicle (white bars) or LZM-007 (black bars) (right). (F) Renal function of animals treated with vehicle (white bars) or lysozyme (gray bars) is determined by measuring plasma urea (left) or plasma creatinine (middle). Histopathology scoring of tubular injury (right) of animals treated with vehicle (white bars) or lysozyme (gray bars). Data are expressed as mean6SEM. *P,0.05. Original magnification, 310 in C and D.

1482

Journal of the American Society of Nephrology

To detect ROS production cells were cultured in black 96-well mClear plates (Greiner Bio-One) and loaded with 40 mM DCF-DA (Invitrogen) in phenol red–free culture medium for 1 hour. After washing in PBS, cells were maintained in serum-free medium without phenol red or under paraffin oil for 60 minutes. Serum-free medium without phenol red and supplemented with compounds (as indicated in the Results) was added to the wells to induce reoxygenation. Fluorescence intensity (480 nm excitation, 520 nm emission) was measured on an automated fluorescence plate reader (FLUOstar OPTIMA; BMG Labtech, Ortenberg, Germany) at 37°C at 2-minute intervals for a total of 2 hours. Data were normalized by plotting values against the starting value. Mitochondrial dysfunction and superoxide production were determined by loading cells with MitoSOX Red (Invitrogen). Cells cultured on glass coverslips were subjected to hypoxia as described above and 1 mM MitoSOX Red was added to the medium during reoxygenation for 30 minutes. Cells were washed with PBS and fixed in 4% formaldehyde. Cells were then washed in PBS with 0.1% (v/v) Triton X-100, counterstained with 2 mg/ml Hoechst 33258, and mounted with Aqua-Polymount (Polysciences, Eppelheim, Germany). Stainings were imaged using a Nikon E600 epifluorescence microscope (Nikon Instruments Europe, Amstelveen, The Netherlands) and a Coolsnap CCD camera (Roper Scientific, Ottobrunn, Germany). Images were analyzed using Image-Pro Plus software (version 6.2; Media Cybernetics, Marlow, UK).

Chronic Oxidative Stress Assay and 3D Imaging IM-PTECs were cultured in 3D gel matrices for 5 days as described above. After establishment of tubular structures, cells were exposed to 0.15 mM diethylmaleate in culture medium supplemented with 8-pCPT-29-O-Me-cAMP-AM, forskolin, NAC, or appropriate vehicle solutions. After 24 hours, cells were fixed in 4% formaldehyde

J Am Soc Nephrol 25: 1474–1485, 2014

www.jasn.org

BASIC RESEARCH

analysis software (OcellO, Leiden, The Netherlands) and KNIME and expressed as the mean sum of the total tubule lengths per object.

Mice and IR Injury Eight-week-old wild-type male C57BL/6 mice were purchased from Janvier SAS (Le Genest Saint Isle, France). 8-pCPT-29-O-Me-cAMP, LZM-007, or lysozyme were dissolved in sterile PBS and administered at the times indicated in the text by tail vein injection in a total volume 100 ml, control animals were injected with PBS only. Kidneys were collected at the indicated time points. Mice (n=10 per group) were anesthetized using Dormicum (Roche, Woerden, The Netherlands) and Hypnorm (Vetapharma, Leeds, UK). Bilateral renal ischemia was induced by clamping of the renal pedicles for 35 minutes using B-2 vascular clamps (S&T AG, Neuhausen, Switzerland) during which the animals were kept at 32°C in a temperature-controlled environment. All animals received one postoperative dose of buprenorphine (subcutaneous, 0.15 mg/kg; Schering-Plough, Brussels, Belgium). Shams (n=6 per group) received identical treatment without clamping of the renal arteries. The lysozyme control experiment used sham animals (n=3 per group) and mice with IR injury (n=6 per group). Animals were allowed to recover overnight and temperature was maintained at 28°C. Animals were sacrificed at 1 hour, day 1, or day 3 after ischemia. Blood samples were collected by heart puncture and transferred to heparin-coated containers containing separation gels (BD Biosciences). Both kidneys were removed and fixed in 4% formaldehyde or snapfrozen in liquid nitrogen. For intrarenal administration, animals were anesthetized as described above. We injected 30 or 60 mg 8-pCPT-29-OMe-cAMP in saline under the capsule into both renal poles (20 ml per injection) during clamping of the renal pedicle. Kidneys were collected after 30 minutes. All experimental procedures were approved by the Animal Care and Use Committee of Leiden University. Figure 8. Oxidative stress, apoptosis, and inflammatory cell influx during IR injury are decreased after LZM-007 treatment. Scoring and representative examples of immunostainings for HO-1 (A), cleaved caspase-3 (B), and the granulocytic Gr-1 epitope (C). Images are from kidney sections collected at day 1 after ischemia. Positive cells are counted per HPF in the corticomedullary area of kidneys from mice treated with vehicle (white bars) or LZM-007 (black bars). Data are expressed as the mean6SEM. *P,0.05; **P,0.01. TEC, tubular epithelial cell.

containing rhodamine-phalloidin and Hoechst 33258. To demonstrate mitochondrial ROS production, cells were labeled with 1 mM MitoSOX Red for 30 minutes and fixed in 4% formaldehyde containing Hoechst 33258. Plates were imaged using a BD Pathway 855 high content bioimager (BD Biosciences) using a long-working distance objective lens (34 magnification) and Z-stacks were compressed as single collapsed images composed of 333 image montages. Alternatively, whole gels were mounted on glass slides using Aqua-Poly/Mount (Brunschwig, Amsterdam, The Netherlands) and imaged by epifluorescence microscopy. Tubule length was calculated after image processing of the F-actin stain using watershed masked clustering segmentation44 in Konstanz Information Miner Open Source Software (KNIME.com AG, Zürich, Switzerland). Quantification of tubule length was performed by OcellO using 3D image J Am Soc Nephrol 25: 1474–1485, 2014

Immunofluorescence and Immunohistochemistry For immunofluorescence stainings, cryosections (10-mm thick) were cut and briefly fixed with 4% paraformaldehyde in PBS (clusterin-a) or ice-cold acetone (Nrf2) for 10 minutes. Sections were permeabilized with 0.2% Triton X-100 for 15 minutes and blocked with Ultra V Block (Labvision, Fremont, CA) for 30 minutes. Sections were incubated overnight at 4°C with anti-clusterin or anti-Nrf2 antibody in antibody diluent (ScyTek, Logan, UT). FITC (Nrf2) or Texas Red (clusterin-a) labeled secondary antibodies were incubated for 30 minutes. Sections were counterstained with 2 mg/ml Hoechst 33342 in PBS for 15 minutes and mounted with Aqua-Poly/Mount (Brunschwig). Formaldehyde-fixed kidneys were embedded in paraffin in a routine fashion at the Leiden University Medical Center Department of Pathology. Sections (4-mm thick) were used for all stainings. After dewaxing, sections were treated with 0.3% hydrogen peroxide in methanol for 15 minutes. For cleaved caspase-3 and HO-1 stainings, sections were boiled for 10 minutes in a citrate buffer (pH 6.0), for the Gr-1 staining incubated in a pepsin buffer (0.01 U/ml) at 37°C for 15 minutes. Sections were blocked with 5% normal goat serum for 30 minutes and incubated with primary antibodies overnight at 4°C. Sections were incubated with horseradish peroxidase–conjugated secondary antibodies for 30 minutes. Antibodies and serum were diluted in PBS. Sections were stained with 3,3-diaminobenzidine and Epac and Renal Oxidative Stress

1483

BASIC RESEARCH

www.jasn.org

alternatively nuclei were counterstained with hematoxylin or methylene green.

Pull-Down Analyses and Western Blotting

To determine in vivo renal Rap1 activation, 10 cryosections (10-mm thick) per sample were used for analysis. Sections were incubated with a lysis buffer containing 10% glycerol, 1% Nonidet P40, 50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 2.5 mM MgCl2 supplemented with 1 mM aprotonin, and 2 mM leupeptide for 30 minutes at 4°C. Lysates were centrifuged and the supernatants were incubated with Glutathione Sepharose 4B (Roche) beads coated with RalGDS-RBD fusion protein as previously described.45 A 63 Laemmli sample buffer containing b-mercaptoethanol solution was added to the samples before boiling for 5 minutes followed by protein separation and blotting on Immobilon-P (Millipore, Amsterdam, The Netherlands). Immunoblots were blocked in Tris-buffered saline with 5% (w/v) BSA and incubated overnight with primary antibodies. For detection, immunoblots were incubated with peroxidase-conjugated secondary antibodies and the presence of proteins was visualized using ECL+ (Amersham, Little Chalfont, UK) on a Typhoon imager (GE Healthcare, Diegem, Belgium).

Renal Function, Immunostaining, and Histopathology Scoring Plasma urea and creatinine were measured using CREA plus (Roche Diagnostics, Almere, The Netherlands) at the Laboratory for Clinical Chemistry (Amsterdam, The Netherlands) following standard protocols. Clusterin-a staining was measured using Image-Pro Plus software. The area of specific staining was determined per field (310 magnification) in the corticomedullary region of the kidney. Cells positive for HO-1, caspase-3, and Gr-1 were counted per high power field (HPF) (340 magnification) of the corticomedullary region of the kidney. g-GCS–positive tubules were counted and expressed as the percentage of all tubules present per HPF in the corticomedullary area. For the Nrf2 staining, positive nuclei were counted per HPF. For histopathology scoring, sections were stained with periodic acid– Schiff reagents after diastase digestion. Injury to tubuli was determined by quantifying the percentage of affected tubules per 10 HPF in the corticomedullary region according to the following criteria: tubular dilation, epithelial necrosis, cast deposition, and loss of brush border. Injury was graded on a scale from 0 to 5: 0, 0%; 1, ,10%; 2, 10%–25%; 3, 25%–50%; 4, 50%–75%; and 5, .75%.

Statistical Analyses Results are expressed as the mean6SEM. Data were tested for normality using the Kolmogorov-Smirnov test and analyzed using an unpaired t test. P values #0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism 4 software (GraphPad Software, Inc., San Diego, CA).

ACKNOWLEDGMENTS The authors thank Dr. Holger Rehman and Dr. Hans Bos for useful discussions regarding devising the strategy for generating LZM-007.

1484

Journal of the American Society of Nephrology

G.S. was supported by a grant from the Dutch Kidney Foundation. Y.Q. was supported by a scholarship from the China Scholarship Council. L.S.P. and S.R. were supported by a grant from The NetherlandsToxicogenomicsConsortium ofThe NetherlandsGenomics Initiative.

DISCLOSURES None.

REFERENCES 1. Thadhani R, Pascual M, Bonventre JV: Acute renal failure. N Engl J Med 334: 1448–1460, 1996 2. Chapman JR, O’Connell PJ, Nankivell BJ: Chronic renal allograft dysfunction. J Am Soc Nephrol 16: 3015–3026, 2005 3. Bonventre JV, Yang L: Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest 121: 4210–4221, 2011 4. de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL; de RJ: Epac is a Rap1 guanine-nucleotideexchange factor directly activated by cyclic AMP. Nature 396: 474–477, 1998 5. Enserink JM, Christensen AE, de Rooij J, van Triest M, Schwede F, Genieser HG, Døskeland SO, Blank JL, Bos JL: A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK. Nat Cell Biol 4: 901–906, 2002 6. Price LS, Hajdo-Milasinovic A, Zhao J, Zwartkruis FJ, Collard JG, Bos JL: Rap1 regulates E-cadherin-mediated cell-cell adhesion. J Biol Chem 279: 35127–35132, 2004 7. Kooistra MR, Corada M, Dejana E, Bos JL: Epac1 regulates integrity of endothelial cell junctions through VE-cadherin. FEBS Lett 579: 4966– 4972, 2005 8. Enserink JM, Price LS, Methi T, Mahic M, Sonnenberg A, Bos JL, Taskén K: The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin. J Biol Chem 279: 44889–44896, 2004 9. Rangarajan S, Enserink JM, Kuiperij HB, de Rooij J, Price LS, Schwede F, Bos JL: Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor. J Cell Biol 160: 487–493, 2003 10. Stokman G, Qin Y, Genieser HG, Schwede F, de Heer E, Bos JL, Bajema IM, van de Water B, Price LS: Epac-Rap signaling reduces cellular stress and ischemia-induced kidney failure. J Am Soc Nephrol 22: 859–872, 2011 11. Qin Y, Stokman G, Yan K, Ramaiahgari S, Verbeek F, deGraauw M, van de Water B, Price LS: cAMP signalling protects proximal tubular epithelial cells from cisplatin-induced apoptosis via activation of Epac. Br J Pharmacol 165: 1137–1150, 2012 12. Pabla N, Dong Z: Cisplatin nephrotoxicity: Mechanisms and renoprotective strategies. Kidney Int 73: 994–1007, 2008 13. Noiri E, Nakao A, Uchida K, Tsukahara H, Ohno M, Fujita T, Brodsky S, Goligorsky MS: Oxidative and nitrosative stress in acute renal ischemia. Am J Physiol Renal Physiol 281: F948–F957, 2001 14. Dendooven A, Ishola DA Jr, Nguyen TQ, Van der Giezen DM, Kok RJ, Goldschmeding R, Joles JA: Oxidative stress in obstructive nephropathy. Int J Exp Pathol 92: 202–210, 2011 15. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT: Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci U S A 95: 11715–11720, 1998 16. Santos NA, Catão CS, Martins NM, Curti C, Bianchi ML, Santos AC: Cisplatin-induced nephrotoxicity is associated with oxidative stress,

J Am Soc Nephrol 25: 1474–1485, 2014

www.jasn.org

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Arch Toxicol 81: 495–504, 2007 Vliem MJ, Ponsioen B, Schwede F, Pannekoek WJ, Riedl J, Kooistra MR, Jalink K, Genieser HG, Bos JL, Rehmann H: 8-pCPT-29-O-Me-cAMPAM: An improved Epac-selective cAMP analogue. ChemBioChem 9: 2052–2054, 2008 Wang Y, Klein JD, Blount MA, Martin CF, Kent KJ, Pech V, Wall SM, Sands JM: Epac regulates UT-A1 to increase urea transport in inner medullary collecting ducts. J Am Soc Nephrol 20: 2018–2024, 2009 Fang F, Liu GC, Kim C, Yassa R, Zhou J, Scholey JW: Adiponectin attenuates angiotensin II-induced oxidative stress in renal tubular cells through AMPK and cAMP-Epac signal transduction pathways. Am J Physiol Renal Physiol 304: F1366–F1374, 2013 Mukhopadhyay P, Rajesh M, Haskó G, Hawkins BJ, Madesh M, Pacher P: Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. Nat Protoc 2: 2295–2301, 2007 Chen H, Tsalkova T, Mei FC, Hu Y, Cheng X, Zhou J: 5-Cyano-6-oxo-1,6dihydro-pyrimidines as potent antagonists targeting exchange proteins directly activated by cAMP. Bioorg Med Chem Lett 22: 4038–4043, 2012 Almahariq M, Tsalkova T, Mei FC, Chen H, Zhou J, Sastry SK, Schwede F, Cheng X: A novel EPAC-specific inhibitor suppresses pancreatic cancer cell migration and invasion. Mol Pharmacol 83: 122–128, 2013 Hidaka T, Furuno H, Inokuchi T, Ogura R: Effects of diethyl maleate (DEM), a glutathione depletor, on prostaglandin synthesis in the isolated perfused spleen of rabbits. Arch Toxicol 64: 103–108, 1990 Dolman ME, Harmsen S, Storm G, Hennink WE, Kok RJ: Drug targeting to the kidney: Advances in the active targeting of therapeutics to proximal tubular cells. Adv Drug Deliv Rev 62: 1344–1357, 2010 Dolman ME, van Dorenmalen KM, Pieters EH, Lacombe M, Pato J, Storm G, Hennink WE, Kok RJ: Imatinib-ULS-lysozyme: A proximal tubular cell-targeted conjugate of imatinib for the treatment of renal diseases. J Control Release 157: 461–468, 2012 Prakash J, de Borst MH, Lacombe M, Opdam F, Klok PA, van Goor H, Meijer DK, Moolenaar F, Poelstra K, Kok RJ: Inhibition of renal rho kinase attenuates ischemia/reperfusion-induced injury. J Am Soc Nephrol 19: 2086–2097, 2008 Hall AM, Crawford C, Unwin RJ, Duchen MR, Peppiatt-Wildman CM: Multiphoton imaging of the functioning kidney. J Am Soc Nephrol 22: 1297–1304, 2011 DiMari J, Megyesi J, Udvarhelyi N, Price P, Davis R, Safirstein R: N-acetyl cysteine ameliorates ischemic renal failure. Am J Physiol 272: F292–F298, 1997 Heyman SN, Goldfarb M, Shina A, Karmeli F, Rosen S: N-acetylcysteine ameliorates renal microcirculation: Studies in rats. Kidney Int 63: 634– 641, 2003 Adabag AS, Ishani A, Koneswaran S, Johnson DJ, Kelly RF, Ward HB, McFalls EO, Bloomfield HE, Chandrashekhar Y: Utility of N-acetylcysteine to prevent acute kidney injury after cardiac surgery: A randomized controlled trial. Am Heart J 155: 1143–1149, 2008 Hilmi IA, Peng Z, Planinsic RM, Damian D, Dai F, Tyurina YY, Kagan VE, Kellum JA: N-acetylcysteine does not prevent hepatorenal

J Am Soc Nephrol 25: 1474–1485, 2014

32.

33.

34.

35.

36.

37.

38. 39.

40. 41.

42.

43.

44.

45.

BASIC RESEARCH

ischaemia-reperfusion injury in patients undergoing orthotopic liver transplantation. Nephrol Dial Transplant 25: 2328–2333, 2010 Holz GG: Epac: A new cAMP-binding protein in support of glucagonlike peptide-1 receptor-mediated signal transduction in the pancreatic beta-cell. Diabetes 53: 5–13, 2004 Qiao J, Mei FC, Popov VL, Vergara LA, Cheng X: Cell cycle-dependent subcellular localization of exchange factor directly activated by cAMP. J Biol Chem 277: 26581–26586, 2002 Park SJ, Ahmad F, Philp A, Baar K, Williams T, Luo H, Ke H, Rehmann H, Taussig R, Brown AL, Kim MK, Beaven MA, Burgin AB, Manganiello V, Chung JH: Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases. Cell 148: 421–433, 2012 Mukai E, Fujimoto S, Sato H, Oneyama C, Kominato R, Sato Y, Sasaki M, Nishi Y, Okada M, Inagaki N: Exendin-4 suppresses SRC activation and reactive oxygen species production in diabetic Goto-Kakizaki rat islets in an Epac-dependent manner. Diabetes 60: 218–226, 2011 Bindokas VP, Kuznetsov A, Sreenan S, Polonsky KS, Roe MW, Philipson LH: Visualizing superoxide production in normal and diabetic rat islets of Langerhans. J Biol Chem 278: 9796–9801, 2003 Reedquist KA, Ross E, Koop EA, Wolthuis RM, Zwartkruis FJ, van Kooyk Y, Salmon M, Buckley CD, Bos JL: The small GTPase, Rap1, mediates CD31-induced integrin adhesion. J Cell Biol 148: 1151–1158, 2000 Rehmann H: Epac-inhibitors: Facts and artefacts. Sci Rep 3: 3032, 2013 Leonard MO, Kieran NE, Howell K, Burne MJ, Varadarajan R, Dhakshinamoorthy S, Porter AG, O’Farrelly C, Rabb H, Taylor CT: Reoxygenation-specific activation of the antioxidant transcription factor Nrf2 mediates cytoprotective gene expression in ischemia-reperfusion injury. FASEB J 20: 2624–2626, 2006 Bonventre JV, Zuk A: Ischemic acute renal failure: An inflammatory disease? Kidney Int 66: 480–485, 2004 Fretz MM, Dolman ME, Lacombe M, Prakash J, Nguyen TQ, Goldschmeding R, Pato J, Storm G, Hennink WE, Kok RJ: Intervention in growth factor activated signaling pathways by renally targeted kinase inhibitors. J Control Release 132: 200–207, 2008 Prakash J, Sandovici M, Saluja V, Lacombe M, Schaapveld RQ, de Borst MH, van Goor H, Henning RH, Proost JH, Moolenaar F, Këri G, Meijer DK, Poelstra K, Kok RJ: Intracellular delivery of the p38 mitogen-activated protein kinase inhibitor SB202190 [4-(4-fluorophenyl)2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] in renal tubular cells: A novel strategy to treat renal fibrosis. J Pharmacol Exp Ther 319: 8– 19, 2006 Zhang Y, Moerkens M, Ramaiahgari S, de Bont H, Price L, Meerman J, van de Water B: Elevated insulin-like growth factor 1 receptor signaling induces antiestrogen resistance through the MAPK/ERK and PI3K/Akt signaling routes. Breast Cancer Res 13: R52, 2011 Yan K, Verbeek FJ: Segmentation for high-throughput image analysis: Watershed masked clustering. In: ISoLA 2012, Part II, LNCS 7610, edited by Margaria T, Steffen B, Merten M, Berlin, Heidelberg, Springer, 2012, pp 25–41 Franke B, Akkerman JW, Bos JL: Rapid Ca2+-mediated activation of Rap1 in human platelets. EMBO J 16: 252–259, 1997

Epac and Renal Oxidative Stress

1485