WO2010020786A1

WO2010020786A1 - Organ protection

Info

Publication number: WO2010020786A1
Application number: PCT/GB2009/002039
Authority: WO
Inventors: Leo Sebastian Price; Bob Van De Water
Original assignee: Universiteit Leiden
Current assignee: Universiteit Leiden
Priority date: 2008-08-21
Filing date: 2009-08-20
Publication date: 2010-02-25
Anticipated expiration: 2011-02-21
Also published as: GB0815315D0

Abstract

The present invention provides compositions, medicaments and methods for use in preventing and/or alleviating the damaging effects of hypoxia and/or reperfusion injury.

Description

ORGAN PROTECTION Field of the Invention

Background of the Invention

Exchange pjoteins directly activated by cAMP (Epacs) are a family of guanine nucleotide exchange factors for Rap GTPases and are activated by direct binding of cAMP. Rap proteins are members of the Ras family of GTPases and include Rapl and Rap2 which are known to control a number of cellular functions, including cell-cell and cell-matrix adhesion.

The development of hypoxia within a tissue may lead to cell damage brought about by, for example, the disruption of the actin cytoskeleton, cell detachment and the induction of apoptosis. Furthermore, the condition known as reperfusion injury refers to damage caused by reperfusion of a hypoxic organ with blood. The precise mechanisms by which reperfusion injury occurs are varied and complex but are known to involve the release of inflammatory factors and free radicals.

Hypoxia related damage and reperfusion (or ischaemic reperfusion) injury is the primary cause of damage to transplanted kidneys and results from the state of hypoxia that develops during cold storage of the donor organ. After periods of cold storage, reperfusion with oxygenated blood (i.e. following connection of the donor organ to the recipients circulation), can lead to inflammation and the induction of oxidative stress pathways.

An essential part of any organ transplantation process is an effective storage protocol which preserves the organ and minimises damage, ensuring the organ is maintained in an optimal morphological and biochemical state from the time of retrieval to the time of transplantation and offers means not only to avoid the insults of initial cold ischaemia, but also the damage which may occur following reperfusion.

By way of example, University of Wisconsin (UW) solution has revolutionised cold ischaemic preservation of solid organs, permitting preservation times of up to 72 hours.

Other diseases and/or conditions in which hypoxic conditions and sudden reperfusion lead to cell damage may include, for example, trauma (particularly head trauma) or stroke. In such cases, the cells of a particular tissue (for example, brain tissue) may be deprived of oxygen and/or nutirents by, for example, a blood clot which blocks or reduces the flow of blood to that tissue. The resulting hypoxia leads to cell death which, depending on the particular tissue involved, may have serious implications for the patient.

In these types of diseases and/or conditions, the damage caused by the limited availability of oxygen and/or nutrients may be further compounded by damage resulting from reperfusion. The mechanisms involved in reperfusion injury occuring as a result of trauma or stroke are as decribed above. It should be noted that owing to the nature of the condition, the damage resulting from localised ischaemic events (such as stroke or trauma) may be referred to as acute cellular degeneration.

In view of the above, there is a drive to develop compositions, medicaments, and methods capable of eliminating, reducing and/or alleviating the damage caused by the development of hypoxic conditions within organs and/or reperfusion of organs. SUMMARY OF THE INVENTION

The present invention concerns exchange proteins directly activated by cAMP (Epacs) and the finding that these are highly expressed in a number of organs. More specifically, the inventors have discovered that compounds which are capable of modulating Epacs activity, may be useful in treating and/or preventing ischaemic events.

Thus, in a first aspect, the present invention provides a use of a compound capable of modulating the activity of Epacs for treating or preventing ischaemia induced injury.

Furthermore, in a second aspect, the present invention provides a use of a compound capable of modulating the activity of Epacs for the manufacture of a medicament for treating or preventing ischaemia induced injury.

Preferably said compound is specific for modulating the activity of Epacs, that is it may not substantially effect the activity of other proteins or molecules, such as protein kinase A (PKA).

It is to be understood that the term "ischaemia induced injury" encompasses diseases and/or conditions which may consist of, begin with, or involve, an ischaemic event or the development of ischaemia. Ischaemic events may, in turn, comprise the development of hypoxic conditions within, for example, cells, tissues and/or organs. Among the diseases and/or conditions which may be regarded as ischaemia induced injuries are those known in the art as "reperfusion injury" and/or "ischaemic reperfusion injury" where reperfusion of a hypoxic tissue and/or organ may lead to damage caused by inflammatory mediators and/or the formation of free radicals. Additionally, ischaemia induced injury may include injury resulting from conditions such as stroke, coma and/or trauma (particularly head trauma) whereby, as a result of, for example, the development of a clot or other such blockage stemming the flow of blood to a tissue/organ, cells of that tissue/organ are deprived of oxygen and/or nutrients, become hypoxic and begin to die or undergo apoptosis. One of skill will appreciate that conditions such as "stroke", trauma and/or coma may be temporary and thus the ischaemia induced injury may be an acute event resulting in localised cell death or degeneration. Furthermore, upon removal or treatment of the cause of the stemmed blood flow, reperfusion of the hypoxic tissue may result in further damage through reperfusion injury. It is to be understood that the term "ischaemia induced injury" encompasses reperfusion injury.

It should also be understood that the term "prevention" may relate to the prophylactic use and/or administration of the medicaments and/or treatment regimes provided by this invention. Furthermore, while the term "treatment" may be held to encompass the act of curing a disease and/or resolving a condition, it may also more generally relate to alleviating and/or reducing the symptoms and/or effects of that disease and/or condition.

As stated above, Epac is a cAMP-dependent exchange factor (activator) of the small GTPase protein "Rap" and, without wishing to be bound by theory, it is thought that compounds capable of modulating Epac activity are useful in the treatment of ischaemia induced injury (such as reperfusion injury) as they modulate Epac-Rap signalling which in turn may prevent disruption of the actin cytoskeleton as well as cell detachment and apoptosis.

In one embodiment, the compound capable of modulating the activity of Epacs and which may be used in any of the medicaments, methods and/or compositions described herein, is a cAMP analogue. An exemplary compound useful in the present invention has the structural formula given as Formula (I) below:

and deaza-analogues thereof, wherein:

Ri can be independently H, halogen, azido, alkyl, aryl, amino-alkyl, amido- aryl , OH, O-alkyl, O-aryl, SH, S-alkyl, s-aryl, SeH, Se-alkyl, Se-aryl, amino, NH- alkyl, NH-aryl, N-bisalkyl, N-bisaryl, cycloalkylamino;

R₂ can be independently H, halogen, azido, O-alkyl, S-alkyl, Se-alkyl, NH- alkyl, N-bisalkyl, alkyl-carbamoyl, cycloalkylamino, silyl;

R₃ can be independently H, halogen, OH, azido, amino-alkyl, amido-aryl, O- alkyl, O-aryl, SH, A-alkyl, S-aryl, amino, NH-alkyl, NH-aryl, N-bisalkyl, N-bisaryl, NH-alkyl-carbamoyl, cycloalkylamino; and wherein

R₄ Is O(H) of S(H); and

R₅ is O(H), S(H), amino, H, alkyl, O-alkyl, O-aryl, S-alkyl, S-aryl, NH-alkyl, NH-aryl, N-bisalkyl, N-bisaryl; or

R₄ is O (H), S (H), amino, H, alkyl,O-alkyl,O-aryl, S- alkyl, S-aryl, NH-alkyl, NH-aryl, N-bisalkyl, N-bisaryl; and

R₅ is O(H) or S(H) ; and pharmaceutically acceptable salts, esters, and/or solvates thereof.

In a preferred embodiment, the compound capable of activating Epacs is specific and does so without activating other targets of cAMP. The most prominent target of cAMP is protein kinase A (PKA) and concentrations of cAMP analogues that activate PKA can have serious side effects, particularly when administered systemically. As such, the compounds capable of activating of activating Epacs provided by this invention do not activate PKA.

In this regard, a preferred embodiment of the invention may involve the use of the cAMP analogue 8CPT2'-O-Me-cAMP (otherwise referred to as 007).

Other exemplary compounds useful in the compositions, medicaments and methods described herein are detailed in WO03/104250. One of skill in the art will appreciate that other compounds capable of modulating the activity of Epacs, and in particular those which are capable of modulating Epac-Rap signalling, may also be useful in the treatment and/or prevention of ischaemia induced injury. By way of example, 007 mimetic compounds, structural analogues and the like may be useful in this way.

In one embodiment, the compound capable of activating Epacs is an 8CPT2'- O-Me-cAMP modified to include an acetoxymethyl group. Such a compound may otherwise be known as 8CPT2'-O-Me-cAMP-AM (or "007-AM"). Compounds of this type are particularly advantageous as they are more easily taken up by cells.

In a third aspect, the present invention provides a method of treating or preventing an ischaemia induced injury, said method comprising the step of administering to a patient, a pharmaceutically effective amount of a compound capable of modulating the activity of Epacs.

Insofar as the medicaments and treatment regimes provided by this invention treat or prevent ischaemia induced injury, one of skill will appreciate that they may also be useful in the treatment of human or animal transplant patients. In particular, a patient who has received or will receive a donor organ or organs may be administered a medicament or subjected to a treatment regime provided by this invention prior to undergoing surgery or receiving the donor organ and/or for a period thereafter. In this way, the donor organ may be protected against the damaging effects caused by reperfusion with the recipient's blood.

In addition to the above, one of skill in the art will readily understand that the present invention may find particular application in the field of organ storage and/or organ preservation. In particular, the present invention may be useful in the preparation of compositions known to those skilled in this field as "organ preservation solutions" which are formulated so as to provide a medium in which organs can be stored for prolonged periods of time without becoming hypoxic or incurring substantial damage. The storage of an organ using an organ preservation solution is particularly preferable as the organ may maintain the morphological and biochemical characteristics of the organ at the time of retrieval.

It is to be understood that the term "organ" may be taken to refer to whole organs or to samples, portions or biopsies thereof. Furthermore, the term "organ" may be taken to encompass tissues, cells and/or components of the circulatory system such as veins or arteries which may be grafted to a patient. Typically the subject is a human subject, but other non-human organisms may also receive transplants and as such the compositions of the present invention may also be used in the storage/preservation of non-human animal organs.

Organs which may be protected from ischaemia induced injury, may include, for example, the kidney, liver, heart, lung, pancreas and the like and the organ may be obtained, although not necessarily so, from a cadaver. The human or animal body from which the organ is retrieved shall be referred to hereinafter as the "donor".

In the field of organ transplantation, correct organ storage is essential. Once an organ has been retrieved or removed from a donor, it may be necessary to place the organ into cold storage so as to minimise morphological/biochemical changes and/or cell death. During cold storage, an organ may become hypoxic and susceptible to damage caused by reperfusion with the recipient's blood (i.e. reperfusion injury). As such, there is a drive to provide improved storage conditions.

Accordingly, the fourth aspect of this invention provides a composition for use as an organ preservation solution, wherein the composition comprises a compound capable of modulating the activity of Epacs.

Without wishing to be bound by theory, the present inventors have determined that a number of organs (particularly the kidney and the epithelial tubules cells thereof) exhibit high levels of Epac expression and as such are particularly susceptible to the effects of the compounds described herein which, insofar as they modulate Epac activity, may prevent disruption of the actin cytoskeleton, cell detachment and apoptosis - processes which occur during ischaemia induced injury.

By administering to an organ a composition comprising a compound capable of modulating the activity of Epacs (for example a compound having structural formula (I), a functionally active analogue and/or derivative thereof, 8CPT2'-O-Me- cAMP or cAMP mimetic compounds), it may be possible to minimise damage which, as a result of the hypoxic state of the organ, may occur prior to, during and/or following reperfusion. It is to be understood that the term "administering" may encompass the act of immersing an organ in a composition according to the fourth aspect of this invention. Additionally, or alternatively, the term "administering" may involve perfusing the organ with a composition according to the fourth aspect of this invention and using procedures and apparatus well known in the art. One of skill will appreciate that the organ should be administered a composition according to this invention, as soon as possible after removal from the donor. In this way, it may be possible to ensure that immediately prior to being transplanted, the organ retains the morphological and biochemical features present at the time of retrieval from the donor.

One of skill in this field will readily appreciate that organ storage is best performed at a reduced temperature such as 2 - 10⁰C and that prior to use the organ may be immersed and/or reperfused with a solution at or close to body temperature, such as about 37°C.

In a further aspect, there is provided a (in vitro) method of protecting an organ against ischaemia induced injury, said method comprising the step of administering a composition according to the fourth aspect of this invention to an organ.

Preferably, the composition is in the form of a solution which may further comprise one or more other components.

Advantageously, the compositions provided by the fourth aspect of this invention may comprise other components such as, for example, those used in solutions such as University of Wisconsin (UW) solution (see US 4,798,824 and 4,879,283, for example), Celsior and Belzer MPS solutions. Other components may include, for example, sources of nutrition and/or acid-base buffers to help maintain the pH of the solution. Typical buffers may be based on phosphates, such as KH₂PO₄. Additionally, or alternatively, the composition may comprise other compounds such as, compounds providing or acting as a source of potassium and/or sodium. Such compounds may be particularly useful when the solution is required to have a certain osmolality.

Other components may include, or be selected from, for example, starch; hydroxyethyl starch; lactobionic acid; sodium and/or potassium gluconate; glucose; CaCl₂; potassium phosphate; EDTA or other metal chelating agent such as chelex magnesium sulphate; raffinose; dextran; recombinant albumin; further agents which protect against ischaemic insult, such as adenosine; antioxidants, such as allopurinol and/or pentafraction. Additional optional components may include antibiotics, such as penicillin; insulin (to aid in glucose uptake) and/or antiinflammatories such as dexanethasone.

Alternatively, and in a further embodiment, the composition and/or those other components to be added thereto, may be in solid form such that they may be reconstituted in a suitable solution (for example water) prior (preferably immediately prior) to use. The composition in its solid form may further comprise the one or more other components mentioned above or, additionally, or alternatively, all or some of these may be present in the solution in which the composition is to be reconstituted.

Detailed Description

The present invention will now be described in detail with reference to the following figures which show:

Figure 1 : 007 Caspase activation is a critical event in the induction of cell death by apoptosis. Caspase activation induced by cisplatin treatment is inhibited by treatment with 007 in primary mouse kidney epithelial cells, demonstrating that 007 protects kidney cells from damage. Cell cycle analysis is an alternative method of measuring cell death by apoptosis. Cell cycle analysis also demonstrates that 007- treatment protects against cisplatin-induced injury.

Figure 2: 007 Microscopy images of control isolated primary proximal tubule cells and cells treated with cisplatin in the presence and absence of 007 demonstrate that 007 treatment protects against cisplatin-induced cell damage. Note rounded, detached and fewer cells in cisplatin-treated cells without 007 (top right) compared to cells treated with 007 (bottom right).

Figure 3: Intra-renal injection of 007 during ischaemia induced injury significantly reduces injury as determined by measurement of plasma urea levels. Plasma samples were taken 24 hours after ischemic insult, which consisted of clamping the renal artery for 25 minutes to restrict blood flow. Reduced ischemic injury in animals treated with 007 was confirmed histologically by a pathologist (not shown).

Figure 4: 007 was injected via the tail vein in mice. After 30 minutes, mice were sacrificed, kidneys were isolated and frozen in liquid nitrogen. Kidneys were homogenised in lysis buffer and active Rap (the target of the 007-Epac complex) was precipitated using a probe specific for the active (GTP-Rap) form. Precipitated protein was resolved by SDS-PAGE and following Western blotting, identified by anti Rapl antibodies. Equivalent amounts of total protein (containing active + inactive Rap) was similarly analysed (lower panel). The results show that Rapl is specifically activated by intravenous 007 injection compared to saline vehicle. Similar results were also obtained with intravenous injection of 20 mg/kg 007 (not shown).

Figure 5: Assay of proportion of proximal tubule epithelial monolayer remaining after chemical hypoxia in the presence and absence of 007. Confluent cells in 6-well dishes were treated with serum-free medium alone (control) or serum-free medium containing deoxyglucose + antimycin A (anoxia) for 60 minutes in the presence ('007') or absence ('no 007') of 007. Cells were washed to remove detached cells and the proportion of remaining attached cells was evaluated.

Figure 6: Effect of 007 on morphological changes induced by hypoxia by oil immersion. Cells were incubated with (C) 50μM 007 or (A+B) vehicle for 30 minutes and then subjected to (B+C) in vitro hypoxia by mineral oil submersion or incubated with (A) serum-free medium for 60 minutes. Cells were fixed directly after hypoxia and the F actin cytoskeleton was stained using rhodamine-conjugated phalloidin. Magnification: 100-fold.

Figure 7: IM-PTEC cells express Epacl and respond to 8-pCPT-2'-O-Me- cAMP by activation of Rap: (a) Conditionally immortalized IM-PTEC were cultured under permissive (per) or restrictive (res) conditions and analyzed for expression of Epacl by Western blotting. Tubulin (tub) was used as a loading control, (b) IM-PTEC were exposed to vehicle (saline) as control, 50μM 8-pCPT-2'-O-Me-cAMP (007), lOμM forskolin or 2.5μM 8-pCPT-2'-O-Me-cAMP-AM (007-AM). Lysates were used for detection of activated GTP-bound Rapl levels by pull down analysis followed by immunoblotting. Total Rapl and Epacl expression was confirmed by western blotting. IM-PTEC were cultured on glass coverslips and stained for (c) Epacl (red) and nuclei (blue), (d) IM-PTEC stained with secondary antibodies only.

(e) ZO-I (green) expression was confirmed by immunofluoresence staining establishing the presence of tight junctions and epithelial phenotype of the IM-PTEC.

(f) Cells cultured on normal conditions (control, upper panel) or subjected to 60 minutes of hypoxia (lower panel) were stained for HIF- lα (green), F actin (red) and nuclei (blue). Note translocation of HIFl α to nucleus in response to hypoxia. Original magnification of microscopy images: 60Ox) Figure 8: Epithelial monolayer disruption during hypoxia is reduced by 8- pCPT-2'-O-Me-cAMP: (a) (left) Cells were pretreated with vehicle (control), 8- pCPT-2'-O-Me-cAMP (007), 8-pCPT-2'-O-Me-cAMP-AM (007-AM) or forskolin prior to start of hypoxia. Cells were fixed and stained with rhodamine-conjugated phalloidin at the indicated time points and imaged. Monolayer disruption was quantified using image analysis. Data are expressed in arbitrary units (mean±SEM), whereby monolayer disruption of control cells was set to the values 0 and 1 representing 0 and 75 minutes after the start of hypoxia, respectively. *P=0.048, **P=0.0175. (right) Representative images are shown for cells subjected to normoxic conditions and 75 minutes of hypoxia. Arrow heads indicate examples of sites of monolayer disruption. Box outlines indicate image area shown as detail below. Original magnification: 2Ox.

(b) Barrier function was determined by measuring transepithelial electrical resistance (TER). Data were normalized for the TER at steady state. Monolayers composed of control cells (white bars) display a reduction in barrier function which can be significantly reduced by treatment of cells with pCPT-2'-O-Me-cAMP-AM (black bars). Data are expressed as mean±SEM, *P=0.0008, and are composed of the combined results of three independent experiments.

Figure 9: 8-pCPT-2'-O-Me-cAMP reduces redistribution of beta-catenin during hypoxia: IM-PTEC were treated with 2.5μM 8-pCPT-2'-O-Me-cAMP-AM (007-AM) or vehicle for 30 minutes and subjected to 60 minutes of hypoxia or maintained under normal culture conditions. After 60 minutes, cells were fixed directly. Cells were stained for beta-catenin (green), ZO-I (red), f-actin (purple) and counterstained with Hoechst to visualize nuclei. Original magnification: 60Ox.

Figure 10: Protection of focal adhesion complexes during hypoxia by 8-pCPT- 2'-O-Me-cAMP: IM-PTEC cells were treated with 2.5μM 8-pCPT-2'-O-Me-cAMP- AM (007-AM) or vehicle for 30 minutes and subjected to 60 minutes of hypoxia or maintained under normal culture conditions. After 60 minutes cells were fixed and stained for phosph- (pY118) paxillin (green), talin (blue), f-actin (red). Original magnification. Cells cultured under normoxic conditions either with or without prior 8-pCPT-2'-O-Me-cAMP-AM exposure display thick focal adhesion complexes near the site of the cell-cell junction but also fibrillar adhesions which associate with actin fibers. Cells subjected to hypoxia lose the vast majority of their focal adhesion complexes and fibrillar adhesions. Cells treated with 8-pCPT-2'-O-Me-cAMP-AM and subjected to 60 minutes of hypoxia maintain smaller focal adhesions at the site of cell-cell junctions and small fibrillar adhesions. Original magnification 60Ox.

Figure 11 : Intrarenal administration of 8-pCPT-2'-O-Me-cAMP activates Rapl and reduces renal failure during IR injury: Kidney sections were stained for (a and b) Epacl expression or (c) labeled with secondary antibodies only, (d) GTP-Rapl pull down analyses demonstrated that treatment with 8-pCPT-2'-O-Me-cAMP (007) induced Rap activation. In the lower part of the figure is a representative western blot image with samples from saline (-) and 8-pCPT-2'-O-Me-cAMP (+) treated kidneys. Rap activation was quantified by densitometric analysis (upper part) and expressed as the amount of active Rapl over total Rapl. In a repeat experiment similar results were obtained. Data are expressed as mean±SEM (*P=0.0286). (e) Plasma samples were collected 24 hours after surgery from sham operated and IR animals. Plasma urea was measured and expressed in mM. Plasma samples from non-operated animals were included in the analysis to demonstrate the mild effect of capsular punction and sham surgery on renal function. Treatment with 8-pCPT-2'-O-Me-cAMP (007) significantly reduced plasma urea during IR injury compared to saline treated controls (*P=0.02). Data are expressed as mean±SEM.

Figure 12: Tubular epithelial cell injury following ischemia is reduced after 8- pCPT-2'-O-Me-cAMP treatment: (a) Clusterin-α expression in kidney sections was quantified using immunostainings and image analysis software. Representative images of each group are shown. Clusterin-α expression in saline treated animals was increased after ischemia (left hand micrographs - see arrows and white bars in bar chart), compared to sham operated animals. Treatment with 8-pCPT-2'-O-Me-cAMP (007) significantly reduced clusterin-α expression (right hand micrographs and black bars in bar chart). Data are expressed as mean±SEM. Original magnification: 20Ox. (b) Paraffin embedded tissue sections were stained for beta catenin (brown) and counterstained with hematoxylin (blue). Beta catenin is localized at the cell membrane in tissue from sham operated animals. During IR injury, beta catenin shows an irregular, more pronounced staining pattern in saline treated control animals, whereas its expression in 8-pCPT-2'-O-Me-cAMP (007)- treated animals showed an expression pattern that is more equal to that found in shams. Original magnification: 400x. Materials and methods Antibodies and reagents

For Epacl detection the mouse monoclonal 5D3 was used for western blot analysis (11) and the rabbit polyclonal 2293 for immunostainings (9). A goat-anti- Rapl-IgG, goat-anti-clusterin (M- 18) and a rabbit-anti-beta actin-IgG were purchased from Santa Cruz Biotech (San Cruz, CA). The rabbit-anti-ZO-1-IgG was from Zymed (Burlington, NC), the antibody to pY118-paxillin was from Cell Signaling (Danvers, MA), to beta-catenin from BD Biosciences (San Jose, CA). The mouse-anti-talin and mouse-anti-tubulin antibodies were purchased from Sigma (St.Louis, MO). The mouse-anti-HIFla antibody was from Abeam (Cambridge, UK). Secondary antibodies conjugated to HRP were from Jackson Immunoresearch (Newmarket, UK); antibodies conjugated to Alexa-488 and Cy3 and rhodamine + Alexa-644 conjugated phalloidin were from Invitrogen (Breda, The Netherlands). Forskolin was purchased from Calbiochem (Nottingham, UK); 8-pCPT-2'-O-Me-cAMP and 8-pCPT-2'-O-Me- cAMP-AM (12) were from BIOLOG (Bremen, Germany). Animals and experimental IR model

Eight week old wild type male C57BL/6 mice were purchased from Charles River (Maastricht, The Netherlands). Mice were anesthetized using Dormicum (Roche, Woerden, The Netherlands) and Hypnorm (Vetapharma, Leeds, UK). Both renal pedicles were clamped for 25 minutes using B-2 vascular clamps (S&T AG, Neuhausen, Switzerland). Intrarenal treatment with 8-pCPT-2'-O-Me-cAMP or vehicle (saline) was performed using two 20μl administrations in each renal pole during ischemia (n=8 per group). All animals received one post-operative dose of buprenorfϊne (subcutaneous, 0.15mg/kg, Shering-Plough, Brussels, Belgium). Shams (n=6 per group) received identical treatment without clamping of the renal arteries. All animals were sacrificed the next day. Blood samples were collected by heart puncture and transferred to heparin-coated containers containing separation gels (BD, Alphen a/d Rijn, The Netherlands). Both kidneys were removed and fixed in 4% formaldehyde or snap-frozen in liquid nitrogen. All experimental procedures were approved by the Animal Care and Use Committee of Leiden University, the Netherlands. Histology, renal function and immunohistochemistry

Renal tissue was fixed in 4% formaldehyde for 24 hours and embedded in paraffin in a routine fashion. Four μm thick sections were cut and used for all stainings. To determine tubular damage, sections were pretreated with α-amylase (Sigma), stained with periodic acid-Schiff reagens (PAS/D) and counterstained using hematoxylin. To determine renal function, plasma samples were analyzed using a Reflotron and urea-specific test strips (Roche Diagnostics, Almere, The Netherlands). Tubular damage was scored semi-quantitatively on 10 non-overlapping fields in the corticomedullary area as described previously (13) using the criteria tubular dilatation, brush border shedding, cast deposition and epithelial necrosis on a scale from 0 - 5 based on the percentage of tubules involved: 0 = no tubular damage; 1 = <10%; 2 = 11 - 25%; 3 = 26 - 50%; 4 = 51 - 75%; 5 = 76 - 100% of tubules. For the beta catenin immunostainings, tissue sections were dewaxed and blocked with normal goat serum (Jackson). Primary antibodies were labeled with HRP-conjugated secondary antibody. Visualization was performed using 3,3'-diaminobenzidine and sections were counterstained with hematoxylin. Sections were imaged using a Leica DM6000B light microscope (Rijswijk, The Netherlands). Immunoblotting

Cells were lysed in the above mentioned lysisbuffer supplemented with protease inhibitor cocktail II (Sigma-Aldrich), sodium fluoride and vanadate. After centrifugation, supernatants were boiled for 5 minutes in Laemmli samplebuffer containing β-mercaptoethanol, then subjected to protein separation and blotted on Immobilon-P (Millipore, Amsterdam, The Netherlands). Immunoblots were blocked in Tris-buffered saline with 5% (w/v) bovine serum albumin 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). Rapl-GTP pull down assay on cell - and tissue Iysates

To determine in vitro Rapl activation, cells were lysed for 15 minutes in a lysisbuffer containing 10% glycerol, 1% Nonidet P40, 50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 2.5 mM MgC12 supplemented with lμM aprotonin and 2 μM leupeptide. To determine in vivo renal Rapl activation, ten lOμm thick cryosections per sample were used for analysis. Sections were incubated with lysisbuffer for 30 minutes at 4°C. Lysates were centrifuged and the supernatants were incubated with Gluthation Sepharose 4B (Roche) beads coated with RaIGDS-RBD fusion protein as described previously (14). Samples were then used for Rapl immunoblotting as described below.

TEC were isolated from Immorto mice as described previously (13) labeled with antibodies to neprilsyin/CDlO and aquaporin 4 combined, as markers for proximal tubular epithelium (15, 16) and sorted by flow cytometry on a FacsAria cell sorter (BD Biosciences). Cells were grown in HK-2 medium (DMEM/F12 medium (Invitrogen) with 5% fetal bovine serum (Hyclone, Etten-Leur, The Netherlands), 5 μg/ml insulin and transferrin, 5 ng/ml sodium selenite (Roche), 20 ng/m tri-iodo- thyrionine (Sigma Aldrich), 50 ng/ml hydrocortisone (Sigma Aldrich) and 5 ng/ml prostaglandin El (Sigma Aldrich) with L-glutamine and antibiotics (both from Invitrogen) and mouse interferon-γ (IFN-γ, 1 ng/ml, R&D)) at 33⁰C in 5% CO2 and 95% air. From this cell population, monoclonal cell lines were generated by limiting dilution and examined for down regulation of SV40 activity during restrictive conditions by immunofluoresence (data not shown) with recurrence of the cobble stone-like morphology (data not shown) and megalin/gp330 transcription (data not shown). One clone was used for all experiments described below and named IM- PTEC hereafter.

Cells were grown in flasks at restrictive conditions for 7 days, passed to the appropriate assay plates at high density and cultured for an additional two days. Cells were briefly serum-starved in DMEM/F12 for 2 hours. Before being subjected to hypoxia, cells were pre-treated with 50μM 8-pCPT-2'-O-Me-cAMP, 2.5μM 8-pCPT- 2'-O-Me-cAMP-AM, lOμM forskolin or vehicle for 30 minutes. Hypoxia was induced by submersion of the monolayer in paraffin oil (Bufa, Uitgeest, The Netherlands) for 60 minutes as described previously (13). To remove the oil layer, cells were washed once with excess ice-cold PBS whereby the top oil layer was aspirated first followed by the lower PBS layer. Cells on coverslips were fixed directly by adding 4% formaldehyde solution under the oil layer. Cells were washed with PBS after 10 minutes and processed for staining. In vitro hypoxia models

Chemical hypoxia: IM-PTEC cells were cultured under restrictive conditions for 7 days prior to testing. Cells were briefly serum-starved in DMEM/F12 for 2 hours. Hypoxia was induced chemically by treatment for 60 minutes with deoxyglucose (1OmM) and antimycin A (10 uM) in the presence or absence of 007 (100 μM). Cells were washed twice to remove dead and floating cells and the remaining cells were determined by Bradford assay.

Hypoxia by oil immersion: IM-PTEC cells were cultured as for chemical hypoxia. Cells were incubated with 50μM 007 or vehicle for 30 minutes. The medium was then removed and a layer of mineral oil was placed over the cells for 60 minutes to deprive the cells of atmospheric oxygen. Cells were then fixed and stained with rhodamine phalloidin to visualise the actin cytoskeleton and photographed using a Nikon inverted epifluorescence microscope. Cisplatin treatment

IM-PTEC cells were cultured under restrictive conditions for 7 days prior to testing. Cells were then treated with 25 μM cisplatin for 24 hours in HK-2 medium in the presence or absence of 007 (lOOμM). cells were then photographed and processed for cell cycle analysis and analysis of caspase activation as previously described (Imamdi et al, JPET 2004; 311 :892-903). Cell and cryosection immunofluorescence staining

Formaldehyde fixed cells on glass coverslips were permeabilized and blocked in PBS containing 0.05% Triton X-100 (Sigma) and 0.5% bovine serum albumin (TBP). All antibodies were diluted in TBP. Ten μm cryosections were used for immunostainings. Sections were dried to air and fixed in 4% buffered formaldehyde. Sections were permeabilized in 0.2% Triton X-100 in PBS and blocked with 5% normal horse serum (Jackson) in PBS with 0.05% Triton X-100. All antibodies were diluted in PBS with 0.05% Triton X-100. Cells and sections were counterstained with Hoechst 33342 dye. Coverslips and sections were mounted with Aqua-poly/mount (Polysciences, Eppelheim, Germany). Stainings were imaged using a Nikon E600 epifluorescence microscope (Nikon, Tokyo, Japan), except focal adhesion stainings on a Bio-Rad Radiance 2100 confocal laser scanning microscope (Bio-Rad, Hercules, CA) using a 6Ox Plan Apo NA 1.4 objective lens (Nikon). Monolayer analysis, image analysis and signal quantification

IM-PTEC were cultured in 24 well plates, exposed to 8-pCPT-2'-O-Me- cAMP, 8-pCPT-2'-O-Me-cAMP -AM and forskolin and subjected to hypoxia in duplo as described above. Cells were fixed and stained with rhodamin-phalloidin at 15 minutes intervals. Plates were imaged using a BD Pathway 855 high-content bioimager (BD Biosciences) using a long- working distance objective lens (2Ox magnification). Six images per well were made. Phalloidin staining was analyzed using Image-Pro Plus vό.l analysis software (MediaCybernetics, Gleichen, Germany) by quantification of the fluorescent signal per field. Data from control cells was set at 0 and 1 representing minimal and maximal monolayer disruption respectively. Values from stimulated cells where expressed accordingly.

Clusterin expression was quantified by using five 2Ox magnifications per stained cryosection. Clusterin expression was expressed as the percentage of the area with positive signal per total field in the corticomedullary region of the kidney using Image-Pro Plus vό.l analysis software. Epithelial barrier function measurement

Epithelial barrier function was determined using the electric cell-substrate impedance sensing (ECIS) method on an ECIS 1600R using 8Wl OE electrode array slides (Applied Biophysics, Troy, NY). All measurements were performed using 400Hz frequency. Cells were subjected to pre-stimulation and hypoxia as described above. After 60 minutes, an equal volume of DMEM/F12 medium was added to the cells submerged in paraffin oil, enabling re-continuation of the ECIS measurement. Barrier function was determined before and during pre-stimulation and directly after recovery from hypoxia. Statistical analyses

Results are expressed as mean ± standard error of the mean (SEM). Data were tested for normality using the Kolmogorov-Smirnow test and analyzed using an unpaired t test. Tubular injury scores were analyzed using the non-parametric Mann- Whitney U Test. Values of P<0.05 were considered statistically significant. All statistical analyses were performed using Graphpad Prism4 (GraphPad Software, San Diego, California, USA). Results

007 treatment inhibits cell injury in vitro following insult.

Treatment of proximal tubule cells with cisplatin induces cellular damage via induction of reactive oxygen, DNA modification and other unknown mechanisms, leading to apoptosis. Simultaneous treatment of cells with 007 attenuates cisplatin- induced injury as measured by cell cycle analysis and caspase activation. See figure 1.

Bright field microscopic analysis also shows that 007 treatment protects against cisplatin injury, namely cell rounding, detachment and fragmentation. See figure 2. In a murine in vivo model of renal ischaemia, intrarenal administration of 007 during ischemic injury significantly reduces injury as determined by measurement of plasma urea levels. The reduced ischemic injury in animals treated with 007 was confirmed histologically by a pathologist. See figure 3.

Intra venous administration of 007 also induces activation of Rap in kidney tissue.

This indicates that i.v. administration will also be effective at protecting against ischaemic renal injury. See figure 4. Future experiments will determine optimal concentration required and degree of protection following i.v. administration of 007.

Assay of proportion of proximal tubule epithelial monolayer remaining after chemical hypoxia in the presence and absence of 007.

In the in vivo situation, ischemia-reperfusion injury results in apoptosis of tubular epithelial cells and detachment and subsequent loss into the urine. An in vitro model of this is chemical-induced anoxia whereby ATP production and oxygen metabolism are inhibited chemically. Chemical-induced anoxia of immortalized mouse PTECs resulted in cell rounding and detachment of 60% of cells (see upper panel of figure 4). The detachment of epithelial cells was largely prevented by simultaneous incubation with 007.

In a second in viro model of hypoxia, where cells are deprived of atmospheric oxygen with a layer of mineral oil, the actin cytoskeleton remodels and cells begin to loose cell-cell contacts and undergo cell rounding - an indication of weakening in cell-matrix contacts. Pre-treatment of IM-PTEC cells with 007 largely prevented these changes from taking place.

Epac-Rap signaling can be induced in conditionally immortalized proximal tubular epithelial cells (IM-PTEC).

Conditionally immortalized PTECs were used to model renal epithelium. Culturing these cells at restrictive conditions resulted in a complete loss of SV40 expression (data not shown) and was accompanied by the acquisition of an epithelial phenotype, associated with characteristic localization of the tight junction protein zona occludens-1 (ZO-I) (figure IF). Interestingly, loss of SV40 expression was associated with upregulation of Epac expression (figure Ia). Epac expression by IM- PTEC was also confirmed by immunofluorescence staining (figure Id). Therefore, for subsequent experiments, cells cultured under restrictive (SV40 negative) conditions were used. To determine whether IM-PTEC cells are capable of functional Epac-Rap signaling, cells were exposed to 8-pCPT-2'-O-Me-cAMP, forskolin and 8-pCPT-2'- O-Me-cAMP-AM (figure Ic). A Rap pull down analysis was performed to determine the level of active, GTP-bound Rapl. Here, 8-pCPT-2'-O-Me-cAMP induced a modest increase in active Rapl. 8-pCPT-2'-O-Me-cAMP-AM, which has a higher uptake in cells (ref), showed increased Rapl activation, similar to that of forskolin.

To mimic IR injury in vitro, cells were submerged in paraffin oil. This hypoxia model has been demonstrated to induce pro-inflammatory cytokine expression and leads to cellular ATP depletion (13). hi addition, 60 minutes of hypoxia led to cytoplasmic HIF- lα stabilization, cytoskeletal remodeling and loss of cell-cell contacts in IM-PTEC (figure If).

Epac-Rap activation by 8-pCPT-2'-O-Me-cAMP-AM reduces monolayer disruption and protects the tubular barrier function during in vitro hypoxia

In vitro, hypoxia affects the actin cytoskeleton in TECs, causes a disruption of the epithelial monolayer and, during prolonged hypoxia, results in detachment of cells. Previous experiments showed an effect of Epac-Rap signalling on both cell-cell and cell-extracellular matrix adhesions (refs). We therefore tested whether activation of Epac-Rap signalling could prevent hypoxia-induced damage to the epithelial monolayer. Cells in 96-well plates were exposed to 8-pCPT-2'-O-Me-cAMP, 8- pCPT-2'-O-Me-cAMP-AM, forskolin or vehicle for 30 minutes prior to induction of hypoxia. Cells were fixed at regular intervals durig hypoxiaand stained for f-actin. The plates were imaged using automated fluorescent microscopy and f-actin distribution was used to determine monolayer disruption. In control hypoxic cells, there was a prominent induction of actin stress fibre formation, cell contraction and loss of cell-cell contact resulting in a progressive disruption of the epithelial monolayer. Pre-treatment with 8-pCPT-2'-O-Me-cAMP-AM and forskolin significantly reduced the process of epithelial disruption (figure 2a). Pre-treatment with 8-pCPT-2'-O-Me-cAMP did not result in significant protection against monolayer disruption. This may have been due to the lower uptake of 8-pCPT-2'-O- Me-cAMP and consequent weaker activation of Rap compared to the AM-ester of this cAMP analog (figure IB). These results suggest that activation of Epac-Rap signalling preserves the integrity of the epithelial monolayer in response to hypoxic injury in vitro. An important feature of the tubular epithelium is its barrier function which limits passive diffusion of solutes across the tubular lining. We performed transepithelial electrical resistance (TER) measurements to determine whether the protective effect of 8-pCPT-2'-O-Me-cAMP-AM on hypoxia-induced disruption of the monolayer also resulted in improvement of the epithelial barrier function. Stimulation with 8-pCPT-2'-O-Me-cAMP-AM prior to hypoxia increased the TER compared to controls (figure 2B), indicating that barrier function was enhanced in non-injured cells. A sixty minute hypoxic insult reduced the TER of control treated cells by approximately 20% compared to steady state conditions. This reduction was significantly inhibited by pre-treatment with 8-pCPT-2'-O-Me-cAMP-AM, resulting in a TER level equivalent to that found under steady state conditions. We conclude from these results that that pre-treatment with 8-pCPT-2'-O-Me-cAMP-AM preserves the barrier function of the epithelial monolayer exposed to a hypoxia. Exposure to 8-pCPT-2'-O-Me-cAMP-AM prevents loss of epithelial adherens junctions and focal adhesions during in vitro hypoxia

To determine whether the protective effect of 8-pCPT-2'-O-Me-cAMP-AM on IM-PTECs during hypoxia may be due to an effect on cell-cell junctions, cells were stained for the adherens junction protein beta catenin and the tight junction protein ZO-I (figure 3). Under normal conditions, IM-PTEC exposed to vehicle or 8-pCPT- 2'-0-Me-cAMP-AM display pronounced ZO-I and beta catenin staining at the cell membrane. After 60 minutes of hypoxia, both ZO-I and beta catenin localisation were disrupted. However, treatment with 8-pCPT-2'-O-Me-cAMP-AM before hypoxia reduced loss of beta catenin from the plasma membrane but did not prevent loss of ZO-I. This suggests that the preservation of cell-cell junctions by 8-pCPT-2'-O-Me- cAMP-AM is a consequence of stabilization of adherens junctions.

To determine whether stimulation of the Epac-Rap signaling pathway by 8- pCPT-2'-0-Me-cAMP-AM also affected cell-matrix adhesion during hypoxia, cells were stained for phosphorylated paxillin, which is present in focal adhesion complexes. Hypoxia led to an almost complete loss phosphorylated paxillin (figure 4). Although a decrease in the number and size of phospho-paxillin puncta was also observed in cells treated with 8-pCPT-2'-O-Me-cAMP-AM, phosphorylated paxillin was preserved at focal adhesions in the proximity of the of cell-cell junctions. These findings suggest that Epac-Rap activation during hypoxia prevents monolayer disruption by promoting matrix adhesion at the perimeter of cells and enhancing the stability of cell-cell contacts

Intrarenal administration of 8-pCPT-2'-O-Me-cAMP induces activation of Rapl and protects against loss of renal function during IR injury

To explore the significance of the Epac-Rap pathway as a therapeutic target in ischemia-reperfusion injury and the potential of 8-pCPT-2'-O-Me-cAMP as a prototype drug, we tested the effect of 8-pCPT-2'-O-Me-cAMP administration in a mouse model for IR injury. In accordance with previous studies in humans and rats, in the mouse kidney, Epac is expressed by the tubular epithelium in most segments of the nephron. These include the proximal tubules (figure 5a), glomerular parietal and visceral epithelial cells (figure 5b), supporting the idea that 8-pCPT-2'-O-Me-cAMP has the potential to activate Epac-Rap signalling in vivo. Although previously observed in umbilical cord endothelial cells, we did not detect Epac expression by capillary endothelium.

Mice were treated with 8-pCPT-2'-O-Me-cAMP by intrarenal administration. To demonstrate that this approach effectively results in Rapl activation, kidneys of mice were clamped, injected with 8-pCPT-2'-O-Me-cAMP or saline (vehicle) and collected 30 minutes after injection. Rapl -GTP pull down analysis performed on tissue cryosections showed that injection of 8-pCPT-2'-O-Me-cAMP led to a significant increase in whole kidney Rapl activation (figure 5d). These findings demonstrate that intrarenal 8-pCPT-2'-O-Me-cAMP treatment is effective in activating Epac-Rap signaling and that this activation is most likely confined to the tubular epithelium as these cells form the vast majority of Epac expressing cells in the mouse kidney.

We next examined whether activation of Epac-Rap signaling by 8-pCPT-2'-O- Me-cAMP treatment can affect the pathogenesis associated with IR injury. To do this, both renal pedicles of mice were clamped for 25 minutes. Directly after placement of the vascular clips, 8-pCPT-2'-O-Me-cAMP or saline were administered. Animals were sacrificed 24 hours after ischemia and plasma urea levels were measured as an indicator of renal function. Animals subjected to IR injury and treated with saline showed a significant increase in plasma urea levels compared to sham operated animals (figure 5e). When animals were treated with 8-pCPT-2'-O-Me- cAMP during ischemia, a significant reduction of plasma urea was measured, suggesting that activation of Epac-Rap signaling in vivo reduced renal failure during IR injury.

Treatment with 8-pCPT-2'-O-Me-cAMP during IR injury prevents tubular epithelial cell stress

Clusterin (apolipoprotein J) is thought to be involved in cellular stress as a regulator of apoptosis (18). It has been shown to be an early cellular marker of tubular epithelial damage (19) and can be used as a urinary marker for tubular injury (20). Immunostainings for clusterin-α showed low reactivity on tissue sections of sham operated animals (figure 6a). Ischemic tissue showed increased clusterin-α expression which was predominantly present inside the tubular lumen although some cells in the epithelial lining also stained positive. Quantification of the immunostainings demonstrated that kidney tissue from mice treated with 8-pCPT-2'-O-Me-cAMP had a lower level of clusterin-α expression during IR injury than mice from the control group suggesting that treatment with 8-pCPT-2'-O-Me-cAMP reduces tubular epithelial cell stress during IR injury.

Although scoring of PAS/D-stained tissue sections for histological parameters of damage revealed increased tissue damage as a result of IR injury, we were unable to detect a significant reduction in damage by pre-treatment with 8-pCPT-2'-O-Me- cAMP. This suggests a more subtle or subcellular mechanism of protection. Since our in vitro experiments demonstrated that 8-pCPT-2'-O-Me-cAMP preserved epithelial cell-cell contacts during hypoxia, we examined whether there was also an effect on adherens junctions in vivo. To do this we stained for beta catenin expression. Beta catenin is a vital component of adherens junctions, and its cytoplasmic localization has been found to correlate with tubular epithelial dedifferentiation and represents an early risk factor for epithelial to mesenchymal transition in kidney allografts (21, 22). Tissue from both groups of sham operated animals showed a distinct lateral membrane staining pattern (figure 6b). Sections from saline treated ischemic kidneys showed an irregular and more cytoplasmic staining pattern. In contrast to this, localization of beta catenin in 8-pCPT-2'-O-Me-cAMP treated ischemic kidneys resembled the pattern found in sham operated controls. References 1. Abuelo, JG: Normotensive ischemic acute renal failure. N Engl J Med, 357: 797-

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Claims

1. A compound capable of modulating the activity of Epacs for treating or preventing ischaemia induced injury.

2. The use of a compound capable of modulating the activity of Epacs for the manufacture of a medicament for treating or preventing ischaemia induced injury.

3. The compound of claim 1 or use of claim 2, wherein the ischaemia induced injury is reperfusion injury and/or ischaemic reperfusion injury.

4. The compound or use of any preceding claim wherein the ischaemia induced injury is an acute event arising from stroke, trauma and/or coma.

5. The compound or use of any preceding claim, wherein the compound capable of modulating the activity of Epacs is a cAMP analogue.

6. The compound or use of any preceding claim, wherein the compound does not substantially activate protein kinase A (PKA).

7. The compound or use of any preceding claim, wherein the compound capable of modulating the activity of Epacs has the structural formula given as Formula (I) below:

and deaza-analogues thereof, wherein:

R₁ can be independently H, halogen, azido, alkyl, aryl, amino-alkyl, amido- aryl , OH, O-alkyl, O-aryl, SH, S-alkyl, s-aryl, SeH, Se-alkyl, Se-aryl, amino, NH- alkyl, NH-aryl, N-bisalkyl, N-bisaryl, cycloalkylamino;

R₄ Is O(H) of S(H); and

8. The compound or use of any preceding claim, wherein the compound capable of modulating the activity of Epacs is the cAMP analogue 8CPT2'-O-Me-cAMP or an analogue or derivative thereof.

9. The compound or use of claim 1-7, wherein the compound capable of modulating the activity of Epacs is the cAMP analogue 8CPT2'-O-Me-cAMP-AM.

10. A composition for use as an organ preservation solution, wherein the composition comprises a compound capable of modulating the activity of Epacs.

11. A composition according to claim 10 wherein the compound is a compound according to any one of claims 7-9.

12. An in vitro method of protecting an organ against the effects of ischaemia induced injury, said method comprising the step of administering a composition according to either of claims 10 or 11 to an organ.