Qin, Y.
Citation
Qin, Y. (2011, October 18). Cell adhesion signalling in acute renal failure. Retrieved from https://hdl.handle.net/1887/17953
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/17953
Note: To cite this publication please use the final published version (if applicable).
General discussion
DISCUSSION
Defining the role of cell adhesion signalling in the progression of renal cell injury and tissue damage will contribute to a better understanding of disease pathophysiology and may help in the identification of new therapeutic targets. Indeed, regulating tubular epithelial cell adhesion in general or via targeting cell adhesion-associated signalling mediators represents a novel therapeutic strategy which may reduce renal injury and prevent acute renal failure.
A better understanding in ARF-associated cellular response
Proximal tubule epithelial cells (PTECs) are known to be the primary target of renal injury by either ischemic or toxic insult. Mitochondrial dysfunction leads to ATP depletion in PTECs, initiating a number of metabolic alterations. It consequently causes the disruption of the actin cytoskeleton in association with the perturbation of cell-cell and cell-ECM adhesions 1, which in turn contributes to further metabolic defects in these cells 2. Considerable research has been directed to the role of integrins in this process. Here, the research is specifically focused on the downstream signalling pathways underlying these changes and the identification of potential therapeutic strategies.
Cell adhesion/cytoskeleton dynamics
The regulation of cell adhesion and organization of the F-actin cytoskeleton during renal cell injury is generally studied in vitro. Although FA-like signalling complexes are likely to be present in a variety of cells and immuno-electron microscopy has indicated their presence in tubule epithelial cells 3, classical FA contacts have not been studied directly in vivo. Indeed, some have suggested that FAs are a tissue culture artefact and do not exist in vivo 4. We show for the first time the existence of FAK and paxillin containing FAs that are tyrosine phosphorylated and connected to F-actin stress fibers in vivo (chapter 2). Given that FA size and stability is linked to the amount of cytoskeletal contractility in vitro, the presence of FAs and their attachment to F-actin stress fibers in vivo may allow regulation of tension in the proximal tubulus, thereby suggesting a role in regulating tubular pressure and ultrafiltration rate 5, 6. With an in vivo unilateral I/R injury model, we demonstrated that ischemia caused disruption of the F-actin cytoskeleton together with dephosphorylation and restructuring of FAs, while reperfusion results in a rephosphorylation together with F-actin stress fiber formation and enlarged FA size (chapter 2). The dephosphorylation of basolateral associated proteins on tyrosine residues is consistent with other studies that have shown dephosphorylation in vitro 7 and in other organs such as brain and heart 8, 9.
FAK signalling
FAK is central in the structure and signalling of the FA complex and we therefore postulated an essential role of FAK in the reorganization of FAs and the actin cytoskeletal network during I/R injury. FAK phosphorylation observed during the reperfusion period was both temporal and tyrosine residue site-specific (chapter 2). This suggests multiple functions of FAK, such as binding to adaptor proteins, activation of Src kinase and paxillin and an involvement in FA turnover and cell motility that eventually contribute to renal cell injury
and recovery during the reperfusion period. The coordinated and differential phosphorylation of FAK and downstream substrates which occurs prior to renal injury also indicates a requirement for FAK-specific remodelling and FA remodeling in general to drive a cellular stress response that can result in renal tissue injury.
Direct evidence indicating FAK as a mediator for cellular stress and consequent tissue injury was obtained by implementing local proximal tubular FAK knockout in mice which were subjected to renal I/R (chapter 3). Our in vivo results showing that FAK deficiency alleviated tubular damage and KIM-1 expression during I/R injury, suggest that tubular epithelial cell stress is mediated by FAK. Previous studies demonstrated that reactive oxygen species (ROS) mediate FA complex disassembly accompanied by FAK dephosphorylation which weakens FA-mediated cell-matrix adhesion of tubular epithelial cells 10. In our study, FA protein recurrence after oxidative stress-mediated FA complex disassembly was delayed in FAK-deficient renal cells compared with FAK-expressing cells in vitro.
It is noteworthy to mention that the reported functions of FAK vary significantly and are sometimes contrary to each other. These discrepancies most likely result from the differences in cell type and microenvironment employed in the different experimental models. For instance, FAK-null fibroblasts or keratinocytes show enlarged FAs and elaborate stress fibers that impair migration 11, 12, whereas deficient FAK activation in HeLa cells induces elevated Rac instead of Rho activity as well as enhanced dynamic membrane protrusions 13. In vitro studies provided evidence indicating that FAK mediates cell survival via NFκB, MEK/ERK or PI3K/Akt-1 signalling pathways as well as maintaining FA organization in several cell lines 14-16. Regarding the in vivo studies, a recent study employing inducible, myocyte- specific knockdown of FAK demonstrates a role for FAK in NF-κB dependent cell survival following myocardial infarction and the reduction of infarction size in control animals compared to mice with FAK deficient myocytes 17. However, our findings indicate a role for FAK in the initiation of tubular epithelial cell stress and FA protein tyrosine phosphorylation alongside the control of dynamic FA re-organization, which together mediate I/R-induced renal injury (chapter 2 and 3). The discrepancy may also suggest an organ-specific function of FAK.
Stress signalling pathways
Several stress-related signalling pathways are involved in cellular and tissue injury during I/R or nephrotoxicity, including the Keap1/Nrf2 pathway, NFκB pathway and MAPK pathways – pathways which are also associated with FA signalling 18-21.
During I/R injury, the reactive oxygen species (ROS) that are formed directly after ischemia initiate the activation of both Keap1/Nrf2 and NFκB pathways. Activation of the transcription factor Nrf2 leads to the expression of various anti-oxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductases (NQO) and glutamate cysteine ligase catalytic subunit (GLGC). Keap1, the negative regulator of Nrf2, has been found at FAs in epithelial cells 22. This suggests a working model whereby the disassembly of FAs during ischemia or nephrotoxicity impairs either localization or dynamics of Keap1 at FAs, leading to the activation of Nrf2 and production of anti-oxidant enzymes which protect cells against ROS after ischemia. Indeed Nrf2 has been identified as protective during both ischemic and nephrotoxic acute kidney injury in mice 23. We have shown an increase of HO-
1 protein level in mouse kidney after ischemic injury, indicating that oxidative stress accompanied renal tubular damage (chapter 4). The gene expression profiles of primary rat PTECs after MI-induced ATP depletion also showed a strong up-regulation of metallothioneines, small cysteine-rich metal-binding proteins that mediate the detoxification of toxic metals and are induced as part of a general response to oxidative stress via the Nrf2 transcription factor 24, 25 (chapter 6).
NFκB is a pleiotropic transcription factor which regulates diverse biological processes including cell proliferation, survival and apoptosis. The NFκB pathway has been implicated in cytokine production and the subsequent infiltration of immune cells leading to the aggravation of renal injury after ischemia/reperfusion 26. Recent studies demonstrated that the activation of NFκB is impaired in FAK deficient fibroblasts, suggesting the involvement of FAK in the regulation of NFκB 27. In our hands however, although NF-κB-mediated expression of MCP-1 and IL-6 28 increased in renal cells after H2O2 exposure, this was independent of FAK expression (chapter 3). Furthermore, no significant difference was found in vivo between FAKloxP/loxP and FAKΔloxP/ΔloxP animals, regarding the infiltration of leukocytes and macrophages after ischemia.
The MAPK family members p38, JNK and ERK are known to be activated during renal injury either by I/R-associated oxidative stress or nephrotoxicants 29, 30. Activation of ERK1/2 and p38 have been associated with the increase in expression of pro-inflammatory cytokines such as TNFα during renal injury 31, 32. Our gene profiling results also indicated that several heat shock proteins were significantly up-regulated in response to MI-induced ATP depletion (chapter 6). These are important downstream effectors that mediate stress signalling to transcription factors which respond by initiating diverse nuclear events and ultimately induce cell injury.
Activation of the ERK pathway, commonly by EGFR activation or ROS formation, has been known to contribute to the modulation of protein tyrosine phosphorylation 33, 34. In vitro ERK activation increases phosphorylation of paxillin promoting the association between paxillin and FAK, while inhibition of ERK resulted in disruption of the complex and dephosphorylation of FAK 33, 35. In vivo ERK activation during reperfusion occurs at an early time-point prior to phosphorylation of FAK and paxillin 36, 37, suggesting that the rapid activation of ERK in the reperfusion period stimulates tyrosine phosphorylation of FA proteins thereby recruiting FAs to the signalling response to I/R. Consistent with this, we also showed an ERK-dependent reorganization of FAs and F-actin stress fibers during I/R injury and regeneration whereas inhibition of ERK blocked these events (chapter 2).
Both in vivo and in vitro studies demonstrate the increased phosphorylation and thus activation of JNK during acute renal injury caused by various insults 38, 39. The inhibition of JNK by either a specific inhibitor SP 600125 or an antisense oligonucleotide targeted to JNK1 showed protection against renal cell apoptosis 39, 40. The gene expression profiling on primary rat PTECs also revealed strong activation of JNK signalling and up-regulated expression of the JNK interacting protein (Mapk8ip3) after MI-induced ATP depletion (chapter 6). Activation of the stress kinase JNK is associated with cell adhesion modifications during renal injury as well as the cellular stress response to injury 38. A previous study demonstrated that activated (phosphorylated) JNK localizes at FAs 39, which is probably mediated through FAK. The notion of a JNK-FAK interaction is further
supported by the observation that FAK and JNK physically associate via the adaptor protein JIP3 41. In our study, FAK deficiency suppressed oxidative stress-induced activation of JNK in renal cells (chapter 3). This is consistent with a model whereby the protection against renal injury is due to reduced JNK activation 39.
Protein tyrosine phosphorylation (pTyr)
Protein (de)phosphorylation is a common event that drives signalling cascades and cellular responses during the progression of acute renal cell injury and recovery. Protein tyrosine phosphorylation events at the FAs were lost directly after the ischemic event, which coincided with a disruption of the FA structures and the F-actin network (chapter 2). During the reperfusion period a biphasic protein tyrosine re-phosphorylation wave was observed in association with an increase in FA size and F-actin stress fibers formation. Therefore the changes in pTyr may determine the outcome of renal cell injury 39, and the involved kinases and phosphatases can be potential targets for therapeutic intervention in acute renal failure.
Our microarray analysis in chapter 6 also identified several genes coding protein tyrosine phosphatase (PTP) and protein tyrosine kinase (PTK) members that were differentially regulated in response to MI treatment. The most up-regulated gene Ptprr identified a protein tyrosine phosphatase PTPRR, which is an inactivator of MAPKs such as ERK1/2, which was found important in mediating the renal ischemic injury in rats (chapter 2). Thus activation of PTPRR may have a therapeutic potential, offering a selective regulation of MAP kinase signalling and subsequent cell stress.
Potential therapeutic strategies based on cell adhesion regulation Cell adhesion in general as a target
The maintenance of cell adhesions is important for cell survival, whereas loss of cell adhesion results in the onset of apoptosis 42, 43. In vivo, renal injury is associated with loss of cell adhesion and apoptosis, which both appear important in the pathogenesis of acute renal failure 44. Loss of matrix adhesion is thought to lead to exfoliation of tubular epithelial cells in the urine and has been described in patients with ARF and in animal models 45. Loss of cell-cell adhesion has also been linked to renal failure in patients undergoing allograft transplantation 46. The in vitro ATP depletion model combined with a transcriptomics analysis identified significant alterations in genes and pathways that are important in maintaining proper cell adhesions (chapter 6). Therefore regulation of cell adhesion can be the basis for therapeutic strategies to reduce the extent of injury in kidney disease and transplantation. Two distinct strategies have been investigated in this thesis: stabilization of adhesion complex by reducing dynamics (chapter 2 and 3) and pharmacological activation of cell adhesion-enhancing signalling pathways (chapter 4 and 5).
Stabilization of FA by reducing dynamics
Our study in chapter 2 revealed a dynamic dissolution and re-structuring of FAs and the F- actin cytoskeleton during renal I/R injury. The MEK/ERK pathway is activated preceding protein tyrosine phosphorylation during the reperfusion period and is also linked to renal
injury 36, 37, 47. We have shown that reperfusion of the kidney resulted in early ERK activation and subsequent phosphorylation of FAK, paxillin and Src (chapter 2). In cell culture models it is well established that increased activation of FA-associated kinases, such as FAK and Src increases turnover of FA structures 37. Therefore the pronounced increase in tyrosine phosphorylation early in the reperfusion period (1 h) most likely affects the turnover of the FA complexes, resulting in their dissolution. ERK inhibition with U0126, a specific MEK inhibitor, blocked tyrosine phosphorylation and dissolution of FAs in conjunction with decreased renal injury (chapter 2). Likewise, adenovirus-mediated antisense ERK2 gene therapy attenuated chronic allograft nephropathy, thereby protecting against ARF 48 and pretreatment of mice with U0126 reduced tissue damage and improved renal function after cisplatin treatment 31. Together these data suggest that inhibition of the MEK/ERK pathway and/or a specific protein tyrosine kinase during the ischemic period may be potential therapeutic means to protect against renal failure caused by ischemic insults.
Large FAs normally do not undergo rapid turnover, tending to be involved passively in anchorage 49. Therefore reduced FA dynamics during cellular stress may contribute to stable FA formation and consequent renal cell protection. There is increasing evidence that JNK plays an important role in controlling FA dynamics 50. In addition to crosstalk between FAs and transcription factors and anti-apoptotic proteins 51, 52, the FA adaptor protein paxillin has been identified as a downstream substrate of JNK 53, suggesting a role for JNK in the regulation of adhesion and cell migration. Phosphorylation of paxillin serine residue 178 by JNK promotes the association of paxillin and FAK and is required for cell migration through controlling FA dynamics 54. Our in vitro studies demonstrated that the dynamic turnover of FAs upon oxidative stress is impaired in FAK-deficient renal cells, which coincided with a reduced activation of the stress kinase JNK and the subsequent phosphorylation of paxillin at Ser178, suggesting stabilized FAs and diminished cellular stress (chapter 3). Importantly, in vivo proximal tubule-specific FAK knockout alleviated I/R-induced tubular injury and KIM-1 expression, a marker of proximal tubular cell stress. This protection may be the result of both reduced FA turnover and reduced downstream cellular stress signalling as a direct result of FAK deficiency in PTECs. Therefore the FAK/JNK/paxillin linkage may represent a potential target for therapeutic intervention in acute renal failure.
Strengthening cell adhesions by pharmacological Epac activation
A more direct and applicable strategy for renoprotection is pharmacologically activating pathways that are able to enhance cell adhesions in the kidney. The activation of the small GTPase Rap1 by guanine nucleotide exchange factors (Rap1GEFs) such as C3G, PDZ-GEF and Epac promotes both cadherin-mediated cell-cell adhesion 55 and clustering of integrins thereby enhancing cell-matrix adhesion 56. Among these endogenous Rap1 activators, the cAMP effector Epac1 is exclusively highly expressed in kidney and enriched in brush border membrane of proximal tubular epithelium, providing a site-specific drug target. The Epac- selective cAMP analogue 8-pCPT-2’-O-Me-cAMP and its AM-derivative 57, 58 were used to assess the effect of active Epac-Rap signalling on both in vitro and in vivo renal injury.
We demonstrated that Epac activation preserved cell adhesion and maintained barrier function of the epithelial monolayer during in vitro hypoxia (chapter 4), which is in accordance with previous findings describing the capacity of Epac to enhance endothelial and
epithelial barrier function 59, 60. The inability of 8-pCPT-2’-O-Me-cAMP-AM to prevent tight junction disassembly in this model is consistent with previous findings that Rap activation in epithelial cells stabilizes adherens junctions but not tight junctions 55, 61. In our in vivo experiments, intrarenal administration of 8-pCPT-2’-O-Me-cAMP reduced renal failure in a mouse I/R model, accompanied by decreased expression of the tubular cell stress marker clusterin-α, and lateral β-catenin expression indicative of sustained tubular barrier function after ischemia (chapter 4). We also found decreased intraluminal obstruction in 8-pCPT-2’- O-Me-cAMP-treated kidneys following ischemia. The Epac-mediated cell junction and FA stabilization may contribute to these in vivo protections, however direct evidence is required to show that there is indeed a causal relationship.
In addition to enhanced cell-cell adhesion, an important cellular response to cAMP- induced Epac-Rap signalling is enhanced cell-matrix adhesion 62. Both cell-cell and cell- matrix adhesions provide survival signals to the cell 43, 63. The effect of Epac activation on preventing the onset of cell apoptosis was demonstrated in a cisplatin nephrotoxicity model (chapter 5). Both adherens and tight junctions were preserved by 8-pCPT-2’-O-Me-cAMP after cisplatin treatment, suggesting that enhanced cell adhesion may underlie the cytoprotective effect of 8-pCPT-2’-O-Me-cAMP treatment. The fact that the Epac-selective analogue protected cell adhesions in the cisplatin model is remarkable and presents a mechanism by which injury can be prevented. This is particularly significant since it is assumed that cisplatin-induced tubular cell injury is predominantly caused by DNA damage.
Furthermore, we demonstrated activation of Epac in vivo by 8-pCPT-2’-O-Me-cAMP treatment (chapter 4) giving the green light for future drug development towards this target.
The study therefore provides a novel strategy for reducing the nephrotoxicity associated with cisplatin-based cancer chemotherapy and at the same time gives mechanistic insight into the mode of action. However, Epac also mediates the cAMP-dependent inhibition of the apical membrane protein NHE3 – an effect which would appear to be independent of cell adhesion
64. Further work is required to establish whether 8-pCPT-2’-O-Me-cAMP (also) influences cisplatin-induced apoptosis via effects on NHE3 function.
These studies show a direct protective effect of the compound on the tubular epithelium (chapter 4 and 5). In addition to reducing nephrotoxicity associated with chemotherapy regimens, another application may be pharmacological activation of Epac in isolated perfused kidneys to reduce ischaemic injury associated with transplantation, thus improving renograft survival and function. This study is unique in that it utilizes a selective activator of a physiological process (adhesion), contrasting with other studies that have focused on kinase inhibitors to block stress signalling. Therefore pharmacological modulation of cell adhesion as a potential therapeutic strategy is altogether different in terms of mechanism, therapeutic approach and feasibility for eventual therapy and poses reduced risks of the adverse drug responses which are often associated with off-target effects of inhibitors.
Further perspectives
The findings in chapter 2 and 3 point to an interplay between FAK, MAPK and control of FA dynamics as well as the stress response during tubular epithelial injury. Thus pharmacological modulation of FAK activity in the kidney may represent a valid strategy to
protect against I/R-induced renal failure. Indeed several FAK inhibitors have been studied for their potential as anticancer therapy and may be ‘repurposed’ to this end 65.
We have demonstrated that a cAMP analogue, which stabilizes cell adhesions via Epac- Rap signalling, protects against ischemic or nephtotoxic acute renal (cell) injury (chapter 4 and 5). A follow-up study is underway using a renal-targeting drug delivery approach, to develop Epac-selective analogues with prolonged retention and activity in renal tubules. In addition to reducing potential non-renal off-target effects, prolonged retention is predicted to increase the clinical potential of these compounds. We also demonstrated that the protection was accompanied by a reduced cellular stress (chapter 4). Previous studies demonstrated that oxidative stress (e.g. ROS) is involved in cell adhesion perturbation and cell death 10, 66, while antioxidants were found to be effective in the prevention or therapy of acute kidney injury 67,
68. Future studies will investigate the link between Epac-Rap activation and oxidative stress- associated pathways during acute renal injury.
The IM-PTEC cells display many of the features of primary PTECs with the practical advantages of a cell line, although its rodent origin limits direct translation to the clinical situation. Therefore similar PTEC cell lines with human origin are required for an efficient bench-to-bedside translation.
Several cellular and animal models were applied in this thesis. Although the in vitro cell injury models have been well developed and provide powerful tools for examining the cellular mechanisms of acute renal injury, the discrepancy between cell culture and in vivo microenvironment makes animal studies irreplaceable for translation to the clinical situation.
Several studies suggest that 3D cell culture models may provide a useful alternative to 2D in vitro models to study adhesion 69, 70 and may also be applied to the study of renal ischemia/reperfusion injury.
Considering the fact that ischemic and nephrotoxic ARF share many pathophysiological features and both show a cytoprotective effect of enhancement of adhesion, a comparison of microarray data from in vitro ATP depletion (chapter 6) and cisplatin models may also help to identify the signalling pathways and thus novel drug targets responsible for Epac-mediated protection. A siRNA-based screen of candidate genes identified in microarray analyses, followed by functional validation with pharmacological or biological manipulation would be used to validate novel targets and would provide deeper insights into the cellular mechanisms which may hold the key to future clinical intervention to prevent ischemic and nephrotoxic renal injury.
References
1. Molitoris BA. Actin cytoskeleton in ischemic acute renal failure. Kidney Int 2004; 66: 871- 883.
2. Schafer ZT, Grassian AR, Song L, et al.
Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment.
Nature 2009; 461: 109-113.
3. Lo SH, Yu QC, Degenstein L, et al.
Progressive kidney degeneration in mice lacking tensin. J Cell Biol 1997; 136: 1349- 1361.
4. Burridge K, Nuckolls G, Otey C, et al. Actin- membrane interaction in focal adhesions. Cell Differ Dev 1990; 32: 337-342.
5. de Rooij J, Kerstens A, Danuser G, et al.
Integrin-dependent actomyosin contraction regulates epithelial cell scattering. J Cell Biol 2005; 171: 153-164.
6. Goffin JM, Pittet P, Csucs G, et al. Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers. J Cell Biol 2006; 172: 259-268.
7. Weinberg JM, Venkatachalam MA, Roeser NF, et al. Energetic determinants of tyrosine phosphorylation of focal adhesion proteins during hypoxia/reoxygenation of kidney proximal tubules. Am J Pathol 2001; 158:
2153-2164.
8. Zalewska T, Ziemka-Nalecz M, Domanska- Janik K. Transient forebrain ischemia effects interaction of Src, FAK, and PYK2 with the NR2B subunit of N-methyl-D-aspartate receptor in gerbil hippocampus. Brain Res 2005;
1042: 214-223.
9. Rebas E, Lachowicz L, Mussur M, et al. The activity of protein tyrosine kinases of rat heart after ischemia and reperfusion. Med Sci Monit 2001; 7: 884-888.
10. Saenz-Morales D, Escribese MM, Stamatakis K, et al. Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress. Exp Cell Res 2006; 312: 3711-3727.
11. Sieg DJ, Hauck CR, Schlaepfer DD.
Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. J Cell Sci 1999; 112 ( Pt 16): 2677-2691.
12. Schober M, Raghavan S, Nikolova M, et al.
Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics. J Cell Biol 2007; 176: 667-680.
13. Bai CY, Ohsugi M, Abe Y, et al. ZRP-1 controls Rho GTPase-mediated actin reorganization by localizing at cell-matrix and cell-cell adhesions. J Cell Sci 2007; 120: 2828- 2837.
14. Bouchard V, Demers MJ, Thibodeau S, et al.
Fak/Src signaling in human intestinal epithelial cell survival and anoikis: differentiation state- specific uncoupling with the PI3-K/Akt-1 and MEK/Erk pathways. J Cell Physiol 2007; 212:
717-728.
15. van de Water B, Houtepen F, Huigsloot M, et al. Suppression of chemically induced apoptosis but not necrosis of renal proximal tubular epithelial (LLC-PK1) cells by focal adhesion kinase (FAK). Role of FAK in maintaining focal adhesion organization after acute renal cell injury. J Biol Chem 2001; 276:
36183-36193.
16. Huang D, Khoe M, Befekadu M, et al.
Focal adhesion kinase mediates cell survival via NF-kappaB and ERK signaling pathways.
AmJPhysiol Cell Physiol 2007; 292: C1339- C1352.
17. Hakim ZS, DiMichele LA, Rojas M, et al.
FAK regulates cardiomyocyte survival following ischemia/reperfusion. J Mol Cell Cardiol 2009; 46: 241-248.
18. Renshaw MW, Price LS, Schwartz MA.
Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase. J Cell Biol 1999; 147:
611-618.
19. Petzold T, Orr AW, Hahn C, et al. Focal adhesion kinase modulates activation of NF- kappaB by flow in endothelial cells. Am J Physiol Cell Physiol 2009; 297: C814-822.
20. Velichkova M, Hasson T. Keap1 in adhesion complexes. Cell Motil Cytoskeleton 2003; 56: 109-119.
21. van de Water B, de Graauw M, Le Devedec S, et al. Cellular stress responses and molecular mechanisms of nephrotoxicity. Toxicol Lett 2006; 162: 83-93.
22. Jaiswal AK. Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic Biol Med 2004; 36: 1199-1207.
23. Liu M, Grigoryev DN, Crow MT, et al.
Transcription factor Nrf2 is protective during ischemic and nephrotoxic acute kidney injury in mice. Kidney Int 2009; 76: 277-285.
24. Miles AT, Hawksworth GM, Beattie JH, et al. Induction, regulation, degradation, and biological significance of mammalian metallothioneins. Crit Rev Biochem Mol Biol 2000; 35: 35-70.
25. Wilmes A, Crean D, Aydin S, et al.
Identification and dissection of the Nrf2 mediated oxidative stress pathway in human renal proximal tubule toxicity. Toxicol In Vitro 2011; 25: 613-622.
26. Donnahoo KK, Meldrum DR, Shenkar R, et al. Early renal ischemia, with or without reperfusion, activates NFkappaB and increases TNF-alpha bioactivity in the kidney. J Urol 2000; 163: 1328-1332.
27. Huang D, Khoe M, Befekadu M, et al.
Focal adhesion kinase mediates cell survival via NF-kappaB and ERK signaling pathways. Am J Physiol Cell Physiol 2007; 292: C1339-C1352.
28. Sanz AB, Sanchez-Nino MD, Ramos AM, et al. NF-kappaB in renal inflammation. J Am Soc Nephrol 2010; 21: 1254-1262.
29. Dong J, Ramachandiran S, Tikoo K, et al.
EGFR-independent activation of p38 MAPK and EGFR-dependent activation of ERK1/2 are required for ROS-induced renal cell death. Am J Physiol Renal Physiol 2004; 287: F1049-1058.
30. Kunduzova OR, Bianchi P, Pizzinat N, et al.
Regulation of JNK/ERK activation, cell apoptosis, and tissue regeneration by monoamine oxidases after renal ischemia- reperfusion. FASEB J 2002; 16: 1129-1131.
31. Jo SK, Cho WY, Sung SA, et al. MEK inhibitor, U0126, attenuates cisplatin-induced renal injury by decreasing inflammation and apoptosis. Kidney Int 2005; 67: 458-466.
32. Meldrum KK, Meldrum DR, Hile KL, et al.
p38 MAPK mediates renal tubular cell TNF- alpha production and TNF-alpha-dependent apoptosis during simulated ischemia. Am J Physiol Cell Physiol 2001; 281: C563-570.
33. Liu ZX, Yu CF, Nickel C, et al. Hepatocyte growth factor induces ERK-dependent paxillin phosphorylation and regulates paxillin-focal adhesion kinase association. J Biol Chem 2002;
277: 10452-10458.
34. Kwon DS, Kwon CH, Kim JH, et al. Signal transduction of MEK/ERK and PI3K/Akt activation by hypoxia/reoxygenation in renal epithelial cells. Eur J Cell Biol 2006; 85: 1189- 1199.
35. Park KM, Chen A, Bonventre JV.
Prevention of kidney ischemia/reperfusion- induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment. J Biol Chem 2001; 276: 11870- 11876.
36. Ishibe S, Joly D, Liu ZX, et al. Paxillin serves as an ERK-regulated scaffold for coordinating FAK and Rac activation in epithelial morphogenesis. Mol Cell 2004; 16:
257-267.
37. Webb DJ, Donais K, Whitmore LA, et al.
FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat Cell Biol 2004; 6: 154-161.
38. De Borst MH, Prakash J, Melenhorst WB, et al. Glomerular and tubular induction of the transcription factor c-Jun in human renal disease. J Pathol 2007; 213: 219-228.
39. de Graauw M, Tijdens I, Cramer R, et al.
Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis. J Biol Chem 2005; 280: 29885-29898.
40. Garay M, Gaarde W, Monia BP, et al.
Inhibition of hypoxia/reoxygenation-induced apoptosis by an antisense oligonucleotide targeted to JNK1 in human kidney cells.
Biochem Pharmacol 2000; 59: 1033-1043.
41. Takino T, Nakada M, Miyamori H, et al.
JSAP1/JIP3 cooperates with focal adhesion kinase to regulate c-Jun N-terminal kinase and cell migration. J Biol Chem 2005; 280: 37772- 37781.
42. Cordes N. Integrin-mediated cell-matrix interactions for prosurvival and antiapoptotic signaling after genotoxic injury. Cancer Lett 2006; 242: 11-19.
43. Meredith JE, Jr., Fazeli B, Schwartz MA.
The extracellular matrix as a cell survival factor.
Mol Biol Cell 1993; 4: 953-961.
44. Thadhani R, Pascual M, Bonventre JV.
Acute renal failure. N Engl J Med 1996; 334:
1448-1460.
45. Racusen LC, Fivush BA, Li YL, et al.
Dissociation of tubular cell detachment and tubular cell death in clinical and experimental
"acute tubular necrosis". Lab Invest 1991; 64:
546-556.
46. Kwon O, Nelson WJ, Sibley R, et al.
Backleak, tight junctions, and cell- cell adhesion in postischemic injury to the renal allograft. J Clin Invest 1998; 101: 2054-2064.
47. Saenz-Morales D, Conde E, Escribese MM, et al. ERK1/2 mediates cytoskeleton and focal adhesion impairment in proximal epithelial cells after renal ischemia. Cell Physiol Biochem 2009; 23: 285-294.
48. Gong N, Dong C, Chen Z, et al.
Adenovirus-mediated antisense-ERK2 gene therapy attenuates chronic allograft nephropathy.
Transplant Proc 2006; 38: 3228-3230.
49. Sabbatini M, Santillo M, Pisani A, et al.
Inhibition of Ras/ERK1/2 signaling protects against postischemic renal injury. Am J Physiol Renal Physiol 2006; 290: F1408-1415.
50. Huang C, Rajfur Z, Borchers C, et al. JNK phosphorylates paxillin and regulates cell migration. Nature 2003; 424: 219-223.
51. Gupta S, Barrett T, Whitmarsh AJ, et al.
Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J 1996; 15: 2760-2770.
52. Yamamoto K, Ichijo H, Korsmeyer SJ.
BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol Cell Biol 1999; 19: 8469-8478.
53. Huang CY, Ayliffe MA, Timmis JN. Direct measurement of the transfer rate of chloroplast DNA into the nucleus. Nature 2003; 422: 72-76.
54. Huang C, Jacobson K, Schaller MD. A role for JNK-paxillin signaling in cell migration.
Cell Cycle 2004; 3: 4-6.
55. Price LS, Hajdo-Milasinovic A, Zhao J, et al. Rap1 regulates E-cadherin-mediated cell- cell adhesion. J Biol Chem 2004; 279: 35127- 35132.
56. Rangarajan S, Enserink JM, Kuiperij HB, et al. Cyclic AMP induces integrin-mediated cell
adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor. J Cell Biol 2003; 160: 487-493.
57. Vliem MJ, Ponsioen B, Schwede F, et al. 8- pCPT-2'-O-Me-cAMP-AM: an improved Epac- selective cAMP analogue. Chembiochem 2008;
9: 2052-2054.
58. Enserink JM, Christensen AE, de Rooij J, et al. A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK. Nat Cell Biol 2002; 4: 901-906.
59. Kooistra MR, Corada M, Dejana E, et al.
Epac1 regulates integrity of endothelial cell junctions through VE-cadherin. FEBS Lett 2005;
579: 4966-4972.
60. Guo X, Rao JN, Liu L, et al. Regulation of adherens junctions and epithelial paracellular permeability: a novel function for polyamines.
Am J Physiol Cell Physiol 2003; 285: C1174- 1187.
61. Hogan C, Serpente N, Cogram P, et al.
Rap1 regulates the formation of E-cadherin- based cell-cell contacts. Mol Cell Biol 2004; 24:
6690-6700.
62. Bos JL. Epac: a new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol 2003; 4: 733-738.
63. Kantak SS, Kramer RH. E-cadherin regulates anchorage-independent growth and survival in oral squamous cell carcinoma cells.
J Biol Chem 1998; 273: 16953-16961.
64. Honegger KJ, Capuano P, Winter C, et al.
Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC).
Proc Natl Acad Sci U S A 2006; 103: 803-808.
65. Shi Q, Hjelmeland AB, Keir ST, et al. A novel low-molecular weight inhibitor of focal adhesion kinase, TAE226, inhibits glioma growth. Mol Carcinog 2007; 46: 488-496.
66. Chirino YI, Pedraza-Chaverri J. Role of oxidative and nitrosative stress in cisplatin- induced nephrotoxicity. Exp Toxicol Pathol 2009; 61: 223-242.
67. Koyner JL, Sher Ali R, Murray PT.
Antioxidants. Do they have a place in the prevention or therapy of acute kidney injury?
Nephron Exp Nephrol 2008; 109: e109-117.
68. Pan H, Mukhopadhyay P, Rajesh M, et al.
Cannabidiol attenuates cisplatin-induced nephrotoxicity by decreasing oxidative/nitrosative stress, inflammation, and
cell death. J Pharmacol Exp Ther 2009; 328:
708-714.
69. Grinnell F, Ho CH, Tamariz E, et al.
Dendritic fibroblasts in three-dimensional collagen matrices. Mol Biol Cell 2003; 14: 384- 395.
70. Cukierman E, Pankov R, Stevens DR, et al.
Taking cell-matrix adhesions to the third dimension. Science 2001; 294: 1708-1712.
