Citation
Alderliesten, M. C. (2009, February 11). Focal adhesion signaling in acute renal failure. LACDR, Division of Toxicology, Faculty of Science, Leiden University.
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General discussion and conclusions
Acute renal failure (ARF) is characterized by abrupt decline in renal function. When the insult that causes ARF is severe it can lead to permanent renal injury and chronic renal failure1-3. Currently, therapy is non-specific and supportive and mortality remains high4;5. A good and complete understanding of the cellular and molecular processes involved in both the injury and the recovery phase is important for designing useful treatments for ARF6-9. Accumulating evidence indicates that loss of renal cell adhesion is important in the pathogenesis of ARF. The studies described in this thesis aimed at investigating the role of cell adhesion to the extracellular matrix (ECM) mediated by signaling and scaffolding protein focal adhesion kinase (FAK) during renal ischemia/reperfusion (I/R) injury and recovery. In this last chapter the most important findings and subsequent implications for further research will be discussed.
Focal adhesions: cell cultures versus 3D in vivo environment
Focal adhesions (FAs) are sites where integrins and ECM mediated adhesion compo- nents are linked to the actin cytoskeleton. Integrins cluster when bound to their extracel- lular components and become activated, providing binding places for FA proteins that are subsequently recruited to these FAs10-13. The FA components are diverse and include kinases like FAK and Src, which are involved in the activation or modulation of down- stream signaling pathways, and structural adaptor proteins (i.e. paxillin) that bind proteins and function as a scaffold, and F-actin binding proteins which connect the FAs to the cytoskeleton12-14;14;15;15. In previous work we have shown that FAs are important structures in cultured renal cell lines. FAK dephosphorylation and dissolution of the FAs preceded chemically induced apoptosis in renal epithelial cells16. Furthermore introducing a domi- nant negative of FAK (i.e. FAT) construct in the porcine proximal tubule cell line LLC-Pk1 potentiated chemically induced apoptosis17. The changes in the organization of the FAs correspond to alterations in downstream signaling affecting survival and adhesion. There- fore intact FA signaling is important in kidney cells to maintain tissue homeostasis. One of the drawbacks of these in vitro studies is that they investigated FAs in cultured cells in a 2D environment on ECM-coated surfaces. Whether FAs are artifacts of 2D culturing or in fact present in vivo has been a subject of research for years. Several studies show that fibroblasts do form adhesions in 3D matrix surroundings but that they are not the same as their 2D counterparts15;18;19. Whether this is the case for FA-like structures in renal cells in vivo is unknown. For the first time we show in chapter 2 FA-like structures in renal epi- thelial cells in vivo. These structures, rich in tyrosine phosphorylation, were present at the basolateral membrane of the renal tubular epithelial cells. They contained FAK, paxillin,
vinculin, talin and were connected to F-actin stress fibers, just as
6
in vitro FAs.General Chapter 6
GENERAL DISCUSSION AND
CONCLUSIONS
discussion and conclusions
Acute renal failure (ARF) is characterized by abrupt decline in renal function. When the insult that causes ARF is severe it can lead to permanent renal injury and chronic renal failure1-3. Currently, therapy is non-specific and supportive and mortality remains high4;5. A good and complete understanding of the cellular and molecular processes involved both during the injury and the recovery phase is important for designing useful treatments for ARF6-9. Accumulating evidence indicates that loss of renal cell adhesion is important in the pathogenesis of ARF. The studies described in this thesis aimed at investigating the role of cell adhesion to the extracellular matrix (ECM) mediated by signaling and scaffolding protein focal adhesion kinase (FAK) during renal ischemia/reperfusion (I/R) injury and recovery. In this last chapter the most important findings and subsequent implications for further research will be discussed.
Focal adhesions: cell cultures versus 3D in vivo environment
Focal adhesions (FAs) are sites where integrins and ECM mediated adhesion compo- nents are linked to the actin cytoskeleton. Integrins cluster when bound to their extracel- lular components and become activated, providing binding places for FA proteins that are subsequently recruited to these FAs10-13. The FAs components are diverse and include ki- nases like FAK and Src, which are involved in the activation or modulation of downstream signaling pathways, and structural adaptor proteins (i.e. paxillin) that bind other proteins and function as a scaffold and F-actin binding proteins which connect the FAs with the cytoskeleton12-14;14;15;15. In previous work we have shown that FAs are important structures in cultured renal cell lines. FAK dephosphorylation and dissolution of the FAs preceded chemically induced apoptosis in renal epithelial cells16. Furthermore introducing a domi- nant negative of FAK (i.e. FAT) construct in the porcine proximal tubule cell line LLC-Pk1 potentiated chemically induced apoptosis17. The changes in the organization of the FAs correspond to alterations in downstream signaling affecting survival and adhesion. There- fore intact FA signaling is important in kidney cells to maintain tissue homeostasis. One of the drawbacks of these in vitro studies is that they investigated FAs in cultured cells in a 2D environment on ECM-coated surfaces. Whether FAs are artifacts of 2D culturing or in fact present in vivo has been a subject of research for years. Several studies show that fibroblasts do form adhesions in 3D matrix surroundings but that they are not the same as their 2D counterparts15;18;19. Whether this is the case for FA-like structures in renal cells in vivo is unknown. For the first time we show in chapter 2 that FA-like structures in renal epithelial cells in vivo. These structures, rich in tyrosine phosphorylation, were present at the basolateral membrane of the renal tubular epithelial cells. They contained FAK, paxillin, vinculin, talin and were connected to F-actin stress fibers, just as in vitro FAs.
This prompted us to think that these in vivo structures are bonafide FAs, although we can not exclude that they may differ to the FAs observed in vitro with respect to qualitative and quantitative protein composition and signaling processes. These questions should be addressed in further research. Using both 3D and in vivo models and state of the art microscopy techniques can further elucidate the properties and dynamics of the FA like structures in vivo. Furthermore it should be kept in mind that cell type and thus cell func- tion is an important factor in FA composition and formation, both in vitro and in vivo, and that results from experiments with mesenchymal cells like fibroblasts can not directly be extrapolated to epithelial cells like the renal proximal tubule cells20;21.
What could be the function of the FA-like structures found at the basolateral side of the renal proximal tubules? Differentiated cells are in a constant state of isometric tension, an exertion of force equal to that of the local ECM by the cells22. This tension prevents changes in cellular architecture that would disrupt normal tissue organization22;23. FAs are considered mechanosensors because of their physical link between the ECM and the F-actin cytoskeleton24. Mechanical stress can lead to activation of FAK and is associated with the formation of a FAK-Src signaling complex. In our hands we see phosphorylation of FAK at its 397 tyrosine residue, under normal conditions both in vivo and in vitro, which is indicative for activation of FAK. FAs grow when physical force is applied to the cell by, for example, stretching24;25. Likewise if the force is decreased, the FAs will disassemble
26;27. Renal tubules are under constant pressure of the passing fluid this can inevitably lead to the formation of large FAs and the appearance of stress fibers in vivo at the base- ment membrane. In addition, cells are able to sense the rigidity of the surrounding ECM.
The organization and rigidity of the ECM appears to play a role in the formation and maintenance of FAs. Rigid substrates show more robust adhesions than the more flexible substrates28;29 presumably because rigid substrates are more capable of resisting the con- tractile forces of the cell. It is possible that the continuous pressure asks for a rigid ECM further influencing the size of the FAs. The kidney can indeed be considered a rigid organ, rich in ECM components associated with the basement membrane of the proximal tubule cells30. Furthermore it is possible that the renal tubule cells use the tension and strong in- teraction with the ECM to regulate the pressure in the tubules. This may well be regulated by angiotensin-dependent signaling where an increase in angiotensin causes phosphory- lation and thereby activation of FAK31;32. Increase of FAK activation might strengthen the FAs and increase the pressure of the passing fluid in the proximal tubule.
FAK signaling mediated cellular process
As mentioned before FAs are important structures involved in many crucial cellular pro- cesses like adhesion, survival, proliferation and migration. FAK is a non-receptor protein tyrosine kinase and considered to be an crucial component of the FAs. In chapter 5 we deleted the FAK gene from primary cultured renal cells which resulted in an increase in the size of FAs and impaired spreading of cells on collagen-coated dishes. During spread- ing the F-actin cytoskeleton was less well organized in the FAK knockout cells compared to the wildtype renal cells. This can be attributed to perturbed FA organization due to a loss of FAK. FAK is a key determinant that controls FA turnover and necessary for FA disassembly; furthermore it is involved in proper cytoskeleton organization. Deletion of FAK influences the downstream signaling and adhesion. From our data and that others it seems that FAK is dispensable for adhesion, proliferation and survival. Rather FAK controls cytoskeletal dynamics and FA disassembly. Without FAK epithelial cells are per- turbed in their rate and extent of spreading. Mechanistically this can be due to decreased phosphorylation of the GTPase activating protein p190RhoGAP yielding increased Rho/
RhoKinase (ROCK) signaling and inefficient recruitment of the paxillin kinase linker-PAK interacting exchange factor activated kinase (PKL-PIX-PAK) complex to the FAs as is shown by schrober etal.33;34. Increased activation of Rho/ROCK sinaling can lead to in- creased stress fiber formation and FA stabilization, thereby impairing cell spreading34
20;21;35. It is noteworthy to mention that FAKs specific functions may vary both with changes in the microenvironment and inherent differences in cell type. This is supported by fak-null mediated basement membrane assembly defects observed in cerebellum36 but not in em- bryonic fibroblasts37 or skin epidermis33. Furthermore fak null fibroblasts or keratinocytes show increased FA size and elaborate stress fibers that impair migration, whereas FAK deficient HeLa cells display elevated Rac in stead of Rho activity and enhanced migration
37-40. In addition, fak-null endothelial cells spread poorly and display aberrant cytoskeletal organization but surprisingly, they showed no obvious perturbations in polarized migra- tion during vascularigenesis38. Finally FAK deficient fruitflies which do not express pyk2 are surprisingly viable and fertile and show no defects in either integrin function or cell migration 41.
Confocal microscopy techniques like FRET, FRAP and FLIP can be used to further elu- cidate the role of FAK in FA maintenance and turnover in renal cells. In addition tubulo- genesis studies can be useful to elucidate the role of FAK in migration and proliferation in 3D environment. This can provide novel insights in recovery of injured tubules but also nephrogenesis and normal kidney tissue homeostasis.
Differential phosphorylation of focal adhesions during renal cell injury The proximal tubule epithelial cells are known to be the primary target of renal injury.
One of the earliest events in the pathophysiology at a cellular level is the disruption of the actin cytoskeleton in the proximal tubule epithelial cells. This disruption has been as- sociated with other events like loss of polarity and perturbation of cell-cell and cell-ECM adhesions7;42-45. However knowledge about the downstream signaling pathways underly- ing these changes remain unclear since most research has been focused on the role of integrins in this matter. To study the changes in cell adhesion during I/R and regeneration;
we used both an in vitro chemical anoxia and recovery model and an in vivo unilateral I/R model. We show that protein tyrosine phosphorylation was lost both in vivo as well as in vitro directly after ischemia or chemical anoxia respectively, and was associated with disruption of the FAs and the cytoskeleton (Chapter 2 and 5). During reperfusion, total phosphorylation increased in conjunction with an increase in FA size and F-actin stress fiber formation at later timepoints. Because 30 min of ischemia does not cause significant cell death or cell detachment it can be suggested that the changes in FA phosphorylation and cytoskeleton organization that take place after injury do not necessarily result in cell death and are part of sublethal injury and/or responses to such injury. In several studies both in humans and animals, viable cells were found in the urine after ARF46-48. Although in our studies we did not determine exfoliation of PTCs in the urine of rats after I/R it is likely that the early changes we found at the FAs and cytoskeleton after ischemia are involved in cell detachment rather than cell death.
Our data indicate a role for FAK and FAs during I/R induced injury and recovery. The phosphorylation status of FAK changed over time during I/R, resulting in activation of dis- tinct downstream signaling pathways that can dynamically reorganize the FAs and actin cytoskeleton. Also we show in chapter 4 that FAK-deleted renal cells are less susceptible to I/R induced injury in vivo. Although deletion of FAK in vitro did not affect renal cell injury induced by chemical anoxia, recovery of the FAs after ATP depletion was delayed in the FAK deleted cells. In addition, in chapter 3 we show an ERK-dependent reorganization of FAs and F-actin stress fibers during I/R injury and regeneration. Inhibition of the MEK/ERK pathway with a specific MEK inhibitor attenuated de- and enhanced re-phosphorylation of FAK and dissolution of FAs in conjunction with a decrease in renal injury. Several mecha- nisms by which FAK affects cell survival can be postulated. Firstly FAK can be involved in one or more direct signaling pathways that affect FA structuring and subsequently influ- encing cell adhesion, survival and migration. This affects the injury and recovery process.
Secondly FAK can indirectly be involved in tissue maintenance and cell survival by affect- ing a stress response and modulating inflammation of the injured tissue.
The FAK phosphorylation observed during reperfusion was both temporal and tyrosine
residue specific. This suggests multiple functions for FAK during reperfusion with pos- sible consequences for the outcome of regeneration processes after I/R injury. Early dur- ing reperfusion a pathway can be active where the FAs have to be remodeled involving an active kinase domain and paxillin and Src-kinase. Later during reperfusion migratory processes and JNK activation can be involved in cell death and regeneration of surviving cells. Therefore we propose that the coordinated and differential phosphorylation of FAK and downstream substrates may indicate a requirement for reorganization of FAs and the actin cytoskeletal network early after I/R and drives a cellular stress response that results in renal tissue injury. As mentioned above inhibition of MEK1/2 protects against I/R induced renal injury and maintains FAK phosphorylation in a basal level. It is well known that various biochemical perturbations cause stress signaling. The family of mitogen-acti- vated protein kinases, including p38, JNK and ERK are activated during I/R. In vitro, ERK activation is associated with increased phosphorylation of paxillin, resulting in increased association between paxillin and FAK, while inhibition of ERK resulted in disruption of the complex and dephosphorylation of FAK49-52. Since ERK is activated early during reperfu- sion prior to FAK and paxillin phosphorylation in vivo53;54, ERK may well be involved in FA organization by activating signaling pathways leading to phosphorylation of FA proteins on tyrosine residues, including FA-associated proteins55. Activation of the ERK pathway can occur in response to EGFR activation or ROS formation and has been known to contribute to the modulation of protein tyrosine phosphorylation, suggesting that the rapid activation of ERK in the reperfusion period further stimulates tyrosine phosphorylation.
Other stress related signaling pathways activated during I/R can also involve FAK signal- ing. Firstly, reactive oxygen species (ROS) formation occurs directly after ischemia. Upon ROS formation specific pathways leading to transcription factor activation are initiated i.e.
the Keap1/Nrf2 pathway and the NFκB pathway. Nrf2 activation leads to the expression of various anti-oxidant enzymes amongst which are hemeoxygenase-1 NQO and GLGC.
Interestingly, Keap1, the negative regulator of transcription factor Nrf2, is found in the FAs in epithelial cells56;57. We suggest that in the absence of FAK affects either localization or dynamics of Keap1 at the FAs. This may lead to the activation of Nrf2 and an induction of anti-oxidant enzymes. In such a condition the FAK deleted cells are likely to be protected against ROS after ischemia.
Alternatively FAK deletion affects the NFκB signaling in renal cells. NFκB is a pleiotropic transcription factor implicated in the regulation of diverse biological phenomena including apoptosis, cell survival, cell growth and the cellular responses to stress, hypoxia and isch- emia. NFκB is activated by many of the signaling pathways and in response to oxidative stress58. It has been shown that blockade of NFκB reduces infarct size in the murine heart after I/R and attenuates renal I/R injury in rats, implicating NFκB as a major determinant
of cell faith after I/R59. The activation of NFκB is impaired in FAK deficient fibroblasts60;61 suggesting that FAK deletion can also protect against renal I/R injury, by inhibition of the NFκB pathway. Some studies report a connection between NFκB en the ERK pathway.
Furthermore other possible stress related pathways can be considered to have a role dur- ing I/R injury. The MAPK family member p38 is also known to be activated during renal injury51;62-64. Just as ERK1/2, p38 is also involved in the production of cytokines like TNF-α and pharmacological inhibition of p38 resulted in a substantial reduction in cisplatin neph- rotoxicity in vivo. In addition inhibition of JNK activation by preconditioning also decreased renal injury 63.
The NFκB and MAPK pathways are known to lead to cytokine production and therefore the attraction of immune cells that are implicated in the aggravation of I/R injury. When these pathways are impaired, less immune cells are likely to infiltrate the damaged area and are not able to execute their damaging functions. Other studies have shown a link between cytokine production and FAK activity. FAK is necessary for IL-6 production in a Src-independent MAPK pathway with potential influence to inflammation 65. We found a decrease in immune cell infiltration after I/R when FAK is deleted. This would fit a model whereby deletion of FAK decreases stress-induced cytokine production either via a NFκB dependent or independent way thereby inhibiting the infiltration of immune cells into the damaged area. Also ERK1/2 activation has been associated with and increase in the proinflammatory cytokines like TNFα. Blocking the production of TNFα decreased the number of infiltrating leukocytes with functional and histologic protection in a cisplatin induced renal injury model 64;66.
The I/R model is one of several models used to study renal injury. The anti-tumour agent cisplatin is a known nephrotoxicant and also used as model for acute renal failure just as S-(1,2-dichlorovinyl)-L-cysteine (DCVC) a model nephrotoxicant. All models induce renal injury in their own manner thereby activating the same but also very distinct stress related pathways. Exposure of heterozygous FAK knockout mice to DCVC shows a clear protec- tion of FAK against the induced renal injury. However exposure of the same mice to cis- platin shows more an aggravation of the induced injury just as we showed in the proximal tubule specific FAK knockout mice exposed to I/R. One of the main differences between the I/R and cisplatin model and the DCVC model is the timing. DCVC causes injury after 6 hours whereas I/R injury is only visible after 24 h and cisplatin injury only after 3 days.
In the DCVC model it is rather intrinsic PTC injury than the immune response component, causing infiltration of immune cells, that is a factor in the cause of renal failure. Yet in the cisplatin and I/R injury model the immune response is activated and has caused the infil- tration of immune cells like macrophages that worsen the injury 64;66.
All together with our own results and published work suggest that direct cytotoxicity as
studied in vitro is only one component of a complex mechanism of renal injury in vivo. This makes in vivo studies of ARF very important for further understanding and for the design of proper therapies.
Regeneration
In contrast to the heart or brain, the kidney can completely recover from an ischemic or toxic insult that results in cell death or cell detachment67. In addition, many cells show changes in morphology that do not or have not resulted in either of these end stages but are sublethally injured. When the kidney recovers from acute injury it relies on a sequence of events that include cell spreading and possibly migration to cover the exposed areas of the basement membrane67. In this thesis we show that the proximal tubules were regener- ated almost completely two weeks after a more severe ischemic insult in conjunction with similar phosphorylation-dependent dynamic reorganization of FAs and F-actin cytoskel- etal network as seen after mild renal injury. This suggests that the changes in the status of FAK phosphorylation and actin dynamics are important in this regeneration process.
During reperfusion, ECM components are excreted by renal cells. Because the cells form FAs and the size and formation of these FAs are matrix dependent we can suggest that the increase in FAs after 1 week is caused by an increase and different composition of the ECM components. Furthermore migration and differentiation as well as proliferation involve cell-ECM contacts to be highly active, which is associated with FAK activation.
In vitro we show that FAK knockout primary renal cells recover differently from ATP deple- tion compared to the wildtype renal cells. The recovery of phosphorylated FAs together with stress fiber formation, as seen in unaffected cells, is delayed. However, it does occur slightly later in the recovery process. This suggests an important role for FAK in the re- covery process. Myosin II has been demonstrated to play an important role in organizing basal actin structures. It has been shown that myosin II inactivation occurs rapidly and precedes dissociation of myosin II from actin stress fibers during ATP depletion. Myosin II activation and stress fiber formation were found to be Rho-associated during recovery.
Myosin II based contractility plays an important role in actin stress fiber formation. Loss of mysosin II contractility disrupts actin stress fiber formation that could lead to cell de- tachment. This stress fiber formation is ROCK dependent44;68;69. Tension increases when myosin is activated, stress fibers are formed and FAs increase. We hypothesize that when FAK is deleted this Rho-ROCK myosin pathway is impaired causing delayed recovery of the stress fibers and FAs after ATP depletion.
We suggest that during the initial injury phase in vivo FAK is involved in stress signaling, thereby aggravating the injury induced by ischemia. However, during the regeneration process FAK can be important for successful and fast recovery of the renal proximal
tubule lining and thereby restoring renal function. This dual role for FAK in I/R should be subject of study in both in vivo and in vitro models to further understand the cellular and molecular mechanisms of renal I/R.
Therapy
Renal injury caused by I/R can be divided into several phases, all providing their own pos- sible targets for therapy. We show that the FAs are involved in I/R injury.
The results presented in this thesis suggest that FAK has multiple functions during ARF, mostly depending on the cause of injury, but also on the phase of acute renal injury. Dur- ing the initial phase of injury and first hours of reperfusion we have shown that FAK signal- ing is involved in cellular injury. Inhibition of FAK signaling or prevention of changes in FAK signaling can be useful to avert renal damage caused either by loss of adhesion signaling and cell detachment or by the infiltrating immune cells and ROS formation. However, initial results of in vitro recovery experiments show that deletion of FAK impairs the recovery of the FAs after injury. This is consistent with the results described by us and others where FAK is implicated in survival, migration and proliferation.
These results raise the important question whether FAK is an attractive drug target and if so, how and when FAK should be inhibited during ARF. A consensus has emerged that kinase modulators will be effective treatments for various diseases. However, most kinases have an unfavorably high degree of structural conservation within key domains of all protein kinases. This makes it difficult to generate a highly selective small molecule protein kinase inhibitor. Furthermore, in concurrence to our observations, modulation of a protein kinase could in one system be beneficial while proving deleterious in another.
Finally, toxicity with long-term use is a concern, because many kinases not only play a role in pathogenesis but also function in pathways that regulate the most basic of normal cellular processes.
The FAK protein contains multiple domains each important in activation of specific or related pathways. Kinase inhibitors can block FAK induced phosphorylation of down- stream targets. It should be considered that other proteins and kinases are able to com- pensate for the loss of one or more functions of FAK. We do know that in vivo in a rodent model the loss of FAK in the kidney does not directly result in injury however this deletion is partial and no data are available about the long term effect. Very specific cell type de- livery is difficult but might be necessary to ablate potential side effects. Other cells that can be targeted, like neurons and vascular cells might worsen the condition when FAK is deleted or inhibited.
As we have shown in this thesis, ERK inhibition is also beneficial for the outcome of ARF.
Furthermore inhibition of the inflammatory response might also be good.
Conclusions and future perspectives
In closing, our analysis of FAK and FAK dynamics in renal epithelial cells both in vivo and in vitro has provided new insights and strengthened prior notions as to how FAK activity converges on the constellation of pathways that regulate cytoskeletal dynamics and bal- ance FA assembly and disassembly in cells. Our findings are particularly interesting in the context of in vivo studies that show that FAK is important in tissue homeostasis. FAK dele- tion can not only lead to a better outcome after injury in vivo, possibly by perturbing initial stress signaling pathways, but can also lead to impaired regeneration after injury. These new findings provide fertile ground for future investigations in this area.
Furthermore we show that primary renal cells in culture are a good model to study renal cell injury after simulated I/R and that they resemble in many ways the in vivo situation better than the widely used cell lines. For future research 3D culturing of these primary cells can give further insight in renal tubule development and the dynamics of the FAs during this development, but also the renal tubule cell response after injury. Using these in vitro models together with state of the art microscopy techniques can further unravel the mechanism of renal cell injury on a molecular level. In addition, siRNA screens can be used to find other targets that are involved in the maintenance of tissue and cell homeo- stasis during renal cell injury.
References
Bonventre JV: Mechanisms of ischemic acute renal failure. Kidney Int 1993, 43:1160- 1.
1178.
Bonventre JV, Weinberg JM: Recent advances in the pathophysiology of ischemic 2.
acute renal failure. J Am Soc Nephrol 2003, 14:2199-2210.
Weinberg JM: The cell biology of ischemic renal injury. Kidney Int 1991, 39:476-500.
3.
Marshall MR: Current status of dosing and quantification of acute renal replace- 4.
ment therapy. Part 2: dosing paradigms and clinical implementation. Nephrology (Carlton ) 2006, 11:181-191.
Schiffl H: Dosing pattern of renal replacement therapy in acute renal failure: current 5.
status and future directions. Eur J Med Res 2006, 11:178-182.
Goligorsky MS, Lieberthal W, Racusen L, Simon EE: Integrin receptors in renal tubu- 6.
lar epithelium: new insights into pathophysiology of acute renal failure. Am J Physiol 1993, 264:F1-F8.
Molina A, Ubeda M, Escribese MM, Garcia-Bermejo L, Sancho D, de Lema GP, Liano 7.
F, Cabanas C, Sanchez-Madrid F, Mampaso F: Renal ischemia/reperfusion injury:
functional tissue preservation by anti-activated {beta}1 integrin therapy. J Am Soc Nephrol 2005, 16:374-382.
Weinberg JM, Venkatachalam MA, Roeser NF, Senter RA, Nissim I: Energetic deter- 8.
minants of tyrosine phosphorylation of focal adhesion proteins during hypoxia/reoxy- genation of kidney proximal tubules. Am J Pathol 2001, 158:2153-2164.
Zuk A, Bonventre JV, Brown D, Matlin KS: Polarity, integrin, and extracellular matrix 9.
dynamics in the postischemic rat kidney. Am J Physiol 1998, 275:C711-C731.
Juliano RL, Aplin AE, Howe AK, Short S, Lee JW, Alahari S: Integrin regulation of re- 10.
ceptor tyrosine kinase and G protein-coupled receptor signaling to mitogen-activated protein kinases. Methods Enzymol 2001, 333:151-163.
Kagami S, Kondo S, Urushihara M, Loster K, Reutter W, Saijo T, Kitamura A, Ko- 11.
bayashi S, Kuroda Y: Overexpression of alpha1beta1 integrin directly affects rat me- sangial cell behavior. Kidney Int 2000, 58:1088-1097.
Reddig PJ, Juliano RL: Clinging to life: cell to matrix adhesion and cell survival. Can- 12.
cer Metastasis Rev 2005, 24:425-439.
Sepulveda JL, Gkretsi V, Wu C: Assembly and signaling of adhesion complexes. Curr 13.
Top Dev Biol 2005, 68:183-225.
DeMali KA, Wennerberg K, Burridge K: Integrin signaling to the actin cytoskeleton.
14.
Curr Opin Cell Biol 2003, 15:572-582.
Wozniak MA, Modzelewska K, Kwong L, Keely PJ: Focal adhesion regulation of cell 15.
behavior. Biochim Biophys Acta 2004, 1692:103-119.
van de WB, Nagelkerke JF, Stevens JL: Dephosphorylation of focal adhesion kinase 16.
(FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells. J Biol Chem 1999, 274:13328-13337.
van de WB, Houtepen F, Huigsloot M, Tijdens IB: Suppression of chemically induced 17.
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.
Cukierman E, Pankov R, Stevens DR, Yamada KM: Taking cell-matrix adhesions to 18.
the third dimension. Science 2001, 294:1708-1712.
Grinnell F, Ho CH, Tamariz E, Lee DJ, Skuta G: Dendritic fibroblasts in three-dimen- 19.
sional collagen matrices. Mol Biol Cell 2003, 14:384-395.
Burridge K, Chrzanowska-Wodnicka M: Focal adhesions, contractility, and signaling.
20.
Annu Rev Cell Dev Biol 1996, 12:463-518.
Burridge K, Chrzanowska-Wodnicka M, Zhong C: Focal adhesion assembly. Trends 21.
Cell Biol 1997, 7:342-347.
Chicurel ME, Singer RH, Meyer CJ, Ingber DE: Integrin binding and mechanical ten- 22.
sion induce movement of mRNA and ribosomes to focal adhesions. Nature 1998, 392:730-733.
Geiger B, Bershadsky A, Pankov R, Yamada KM: Transmembrane crosstalk between 23.
the extracellular matrix--cytoskeleton crosstalk. Nat Rev Mol Cell Biol 2001, 2:793- 805.
Riveline D, Zamir E, Balaban NQ, Schwarz US, Ishizaki T, Narumiya S, Kam Z, Gei- 24.
ger B, Bershadsky AD: Focal contacts as mechanosensors: externally applied lo- cal mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. J Cell Biol 2001, 153:1175-1186.
Sawada Y, Sheetz MP: Force transduction by Triton cytoskeletons. J Cell Biol 2002, 25.
156:609-615.
Balaban NQ, Schwarz US, Riveline D, Goichberg P, Tzur G, Sabanay I, Mahalu D, 26.
Safran S, Bershadsky A, Addadi L, Geiger B: Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat Cell Biol 2001, 3:466-472.
Chen CS, Alonso JL, Ostuni E, Whitesides GM, Ingber DE: Cell shape provides 27.
global control of focal adhesion assembly. Biochem Biophys Res Commun 2003, 307:355-361.
Guo WH, Frey MT, Burnham NA, Wang YL: Substrate rigidity regulates the formation 28.
and maintenance of tissues. Biophys J 2006, 90:2213-2220.
Pelham RJ, Jr., Wang Y: Cell locomotion and focal adhesions are regulated by sub- 29.
strate flexibility. Proc Natl Acad Sci U S A 1997, 94:13661-13665.
Carlson EC, Chatterjee SN: Ultrastructure of isolated basement membranes in the 30.
acellular human renal cortex. Ren Physiol 1983, 6:197-208.
Berk BC, Corson MA: Angiotensin II signal transduction in vascular smooth muscle:
31.
role of tyrosine kinases. Circ Res 1997, 80:607-616.
Montiel M, de la Blanca EP, Jimenez E: Angiotensin II induces focal adhesion kinase/
32.
paxillin phosphorylation and cell migration in human umbilical vein endothelial cells.
Biochem Biophys Res Commun 2005, 327:971-978.
Schober M, Raghavan S, Nikolova M, Polak L, Pasolli HA, Beggs HE, Reichardt LF, 33.
Fuchs E: Focal adhesion kinase modulates tension signaling to control actin and fo- cal adhesion dynamics. J Cell Biol 2007, 176:667-680.
Sutton TA, Mang HE, Atkinson SJ: Rho-kinase regulates myosin II activation in 34.
MDCK cells during recovery after ATP depletion. Am J Physiol Renal Physiol 2001, 281:F810-F818.
Arthur WT, Burridge K: RhoA inactivation by p190RhoGAP regulates cell spreading 35.
and migration by promoting membrane protrusion and polarity. Mol Biol Cell 2001, 12:2711-2720.
Beggs HE, Schahin-Reed D, Zang K, Goebbels S, Nave KA, Gorski J, Jones KR, 36.
Sretavan D, Reichardt LF: FAK deficiency in cells contributing to the basal lamina results in cortical abnormalities resembling congenital muscular dystrophies. Neuron 2003, 40:501-514.
Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto 37.
J, Okada M, Yamamoto T: Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 1995, 377:539-544.
Braren R, Hu H, Kim YH, Beggs HE, Reichardt LF, Wang R: Endothelial FAK is es- 38.
sential for vascular network stability, cell survival, and lamellipodial formation. J Cell Biol 2006, 172:151-162.
Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, Parsons JT, Horwitz AF:
39.
FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly.
Nat Cell Biol 2004, 6:154-161.
Yano H, Mazaki Y, Kurokawa K, Hanks SK, Matsuda M, Sabe H: Roles played by a 40.
subset of integrin signaling molecules in cadherin-based cell-cell adhesion. J Cell Biol 2004, 166:283-295.
Grabbe C, Zervas CG, Hunter T, Brown NH, Palmer RH: Focal adhesion kinase is not 41.
required for integrin function or viability in Drosophila. Development 2004, 131:5795- 5805.
Gailit J, Colflesh D, Rabiner I, Simone J, Goligorsky MS: Redistribution and dysfunc- 42.
tion of integrins in cultured renal epithelial cells exposed to oxidative stress. Am J Physiol 1993, 264:F149-F157.
Molitoris BA, Marrs J: The role of cell adhesion molecules in ischemic acute renal 43.
failure. Am J Med 1999, 106:583-592.
Molitoris BA: Actin cytoskeleton in ischemic acute renal failure. Kidney Int 2004, 44.
66:871-883.
Zuk A, Bonventre JV, Brown D, Matlin KS: Polarity, integrin, and extracellular matrix 45.
dynamics in the postischemic rat kidney. Am J Physiol 1998, 275:C711-C731.
Racusen LC, Fivush BA, Li YL, Slatnik I, Solez K: Dissociation of tubular cell detach- 46.
ment and tubular cell death in clinical and experimental “acute tubular necrosis”. Lab Invest 1991, 64:546-556.
Racusen LC, Solez K: Ideas in pathology. Exfoliation of renal tubular cells. Mod Pathol 47.
1991, 4:368-370.
Racusen LC: Tubular injury in human kidneys: pathologic findings and pathogenic 48.
mechanisms. Clin Investig 1993, 71:858-860.
DiMari J, Megyesi J, Udvarhelyi N, Price P, Davis R, Safirstein R: N-acetyl cysteine 49.
ameliorates ischemic renal failure. Am J Physiol 1997, 272:F292-F298.
Kunduzova OR, Bianchi P, Pizzinat N, Escourrou G, Seguelas MH, Parini A, Cam- 50.
bon C: Regulation of JNK/ERK activation, cell apoptosis, and tissue regeneration by monoamine oxidases after renal ischemia-reperfusion. FASEB J 2002, 16:1129- 1131.
Park KM, Kramers C, Vayssier-Taussat M, Chen A, Bonventre JV: Prevention of kid- 51.
ney ischemia/reperfusion-induced functional injury, MAPK and MAPK kinase activa- tion, and inflammation by remote transient ureteral obstruction. J Biol Chem 2002, 277:2040-2049.
Pombo CM, Bonventre JV, Avruch J, Woodgett JR, Kyriakis JM, Force T: The stress- 52.
activated protein kinases are major c-Jun amino-terminal kinases activated by isch- emia and reperfusion. J Biol Chem 1994, 269:26546-26551.
Ishibe S, Joly D, Liu ZX, Cantley LG: Paxillin serves as an ERK-regulated scaffold 53.
for coordinating FAK and Rac activation in epithelial morphogenesis. Mol Cell 2004, 16:257-267.
Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, Parsons JT, Horwitz AF:
54.
FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly.
Nat Cell Biol 2004, 6:154-161.
Xing Z, Chen HC, Nowlen JK, Taylor SJ, Shalloway D, Guan JL: Direct interaction of 55.
v-Src with the focal adhesion kinase mediated by the Src SH2 domain. Mol Biol Cell 1994, 5:413-421.
Bloom D, Dhakshinamoorthy S, Jaiswal AK: Site-directed mutagenesis of cysteine 56.
to serine in the DNA binding region of Nrf2 decreases its capacity to upregulate anti- oxidant response element-mediated expression and antioxidant induction of NAD(P) H:quinone oxidoreductase1 gene. Oncogene 2002, 21:2191-2200.
Jaiswal AK: Nrf2 signaling in coordinated activation of antioxidant gene expression.
57.
Free Radic Biol Med 2004, 36:1199-1207.
Donnahoo KK, Meldrum DR, Shenkar R, Chung CS, Abraham E, Harken AH: Early 58.
renal ischemia, with or without reperfusion, activates NFkappaB and increases TNF- alpha bioactivity in the kidney. J Urol 2000, 163:1328-1332.
Cao CC, Ding XQ, Ou ZL, Liu CF, Li P, Wang L, Zhu CF:
59. In vivo transfection of NF-
kappaB decoy oligodeoxynucleotides attenuate renal ischemia/reperfusion injury in rats. Kidney Int 2004, 65:834-845.
Funakoshi-Tago M, Sonoda Y, Tanaka S, Hashimoto K, Tago K, Tominaga S, Kasa- 60.
hara T: Tumor necrosis factor-induced nuclear factor kappaB activation is impaired in focal adhesion kinase-deficient fibroblasts. J Biol Chem 2003, 278:29359-29365.
Huang D, Khoe M, Befekadu M, Chung S, Takata Y, Ilic D, Bryer-Ash M: Focal adhe- 61.
sion kinase mediates cell survival via NF-kappaB and ERK signaling pathways. Am J Physiol Cell Physiol 2007, 292:C1339-C1352.
Meldrum KK, Meldrum DR, Hile KL, Yerkes EB, Ayala A, Cain MP, Rink RC, Ca- 62.
sale AJ, Kaefer MA: 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-C570.
Park KM, Chen A, Bonventre JV: Prevention of kidney ischemia/reperfusion-induced 63.
functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pre- treatment. J Biol Chem 2001, 276:11870-11876.
Ramesh G, Reeves WB: p38 MAP kinase inhibition ameliorates cisplatin nephrotoxic- 64.
ity in mice. Am J Physiol Renal Physiol 2005, 289:F166-F174.
Schlaepfer DD, Hou S, Lim ST, Tomar A, Yu H, Lim Y, Hanson DA, Uryu SA, Molina 65.
J, Mitra SK: Tumor necrosis factor-alpha stimulates focal adhesion kinase activity re- quired for mitogen-activated kinase-associated interleukin 6 expression. J Biol Chem 2007, 282:17450-17459.
Ramesh G, Reeves WB: TNF-alpha mediates chemokine and cytokine expression 66.
and renal injury in cisplatin nephrotoxicity. J Clin Invest 2002, 110:835-842.
Bonventre JV: Dedifferentiation and proliferation of surviving epithelial cells in acute 67.
renal failure. J Am Soc Nephrol 2003, 14 Suppl 1:S55-S61.
Hallett MA, Dagher PC, Atkinson SJ: Rho GTPases show differential sensitivity to 68.
nucleotide triphosphate depletion in a model of ischemic cell injury. Am J Physiol Cell
Physiol 2003, 285:C129-C138.
Prahalad P, Calvo I, Waechter H, Matthews JB, Zuk A, Matlin KS: Regulation of MDCK 69.
cell-substratum adhesion by RhoA and myosin light chain kinase after ATP depletion.
Am J Physiol Cell Physiol 2004, 286:C693-C707.
