WO2020064597A1

WO2020064597A1 - Use of epac1 activators for the treatment of chronic kidney diseases

Info

Publication number: WO2020064597A1
Application number: PCT/EP2019/075468
Authority: WO
Inventors: Frank Lezoualc'h; Christos CHATZIANTONIOU; Panagiotis KAVVADAS
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université Paul Sabatier Toulouse Iii; Sorbonne Université,
Priority date: 2018-09-24
Filing date: 2019-09-23
Publication date: 2020-04-02

Abstract

Chronic Kidney Disease (CKD) is a major burden of public health affecting millions of people around the world. It is rising in prevalence with high public health costs and is associated with a high degree of morbidity and mortality. Here, the inventors determined whether the exchange protein directly activated by cAMP 1 (Epac1) contributed to severe glomurelonephritis (GN) induced by injecting nephrotoxic serum (NTS) in mice. Treatment of GN mice with a pharmacological activator of Epacl, 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3', 5'- cyclic monophosphate (8-pCPT) improved functional and structural renal parameters, and reduced renal inflammation after NTS injection. Taken together, these findings suggest that Epacl activation is an attractive and promising pharmacological intervention for the therapeutic treatment of CKD. Thus the present invention relates to use of Epac1 activators for the treatment of chronic kidney diseases..

Description

USE OF EPAC1 ACTIVATORS FOR THE TREATMENT OF CHRONIC KIDNEY

DISEASES

FIELD OF THE INVENTION:

The present invention relates to use of Epacl activators for the treatment of chronic kidney diseases.

BACKGROUND OF THE INVENTION:

Chronic Kidney Disease (CKD) is a major burden of public health affecting millions of people around the world. It is rising in prevalence with high public health costs and is associated with a high degree of morbidity and mortality. Diabetes and hypertension are the predominant causes of CKD, accounting for approximately 44% and 30% of incident CKD cases. Even though many aspects of the complex mechanisms orchestrating progression of renal disease have been identified, so far there is no specific treatment to slow down or prevent CKD progression.

Cyclic AMP is a universal second messenger, and its production is initiated upon binding of a wide range of extracellular ligands to G protein-coupled receptors and subsequent activation of membrane-bound adenylyl cyclases (ACs), which generate cAMP (Robichaux and Cheng, 2018). Historically, the biological effects of cAMP have been attributed to phosphorylation events catalyzed by the protein kinase A (PKA). Recently, a family of proteins directly activated by cAMP, named Epac was discovered as an important new regulator of cAMP mediated signaling (Robichaux and Cheng, 2018). The Epac family members, Epacl and Epac2, act as guanine exchange factors (GEFs) for the small G-proteins Rapl and Rap2, and function in a PKA-independent manner (de Rooij et a , 1998; Kawasaki et a , 1998; Breckler et a , 2011). In human and rodents Epacl is abundantly expressed in the kidney and is the major Epac isoform expressed in this tissue (de Rooij et a , 1998; Kawasaki et a , 1998; Robichaux and Cheng, 2018).

Although the role of PKA in kidney pathophysiology has been largely studied, the impact of Epac remains relatively enigmatic.

SUMMARY OF THE INVENTION:

The present invention relates to use of Epacl activators for the treatment of chronic kidney diseases. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION: Here, the inventors determined whether the exchange protein directly activated by cAMP 1 (Epacl) contributed to severe glomurelonephritis (GN) induced by injecting nephrotoxic serum (NTS) in mice. They showed that the glomerular expression of Epacl was decreased in mice treated with NTS. Notably, Epacl genetic ablation accelerated the decline of renal function compared with Wild-Type (WT) NTS treated animals. Crescent formation and tubular dilation were increased in Epacl knock-out (KO) versus WT animals indicating that decreased Epacl expression also potentiated the deterioriation of renal structure during the progression of GN. Additionally, Epacl KO mice showed exacerbated renal infiltration of inflammatory cells and interstitial renal fibrosis. Of particular importance, treatment of GN mice with a pharmacological activator of Epacl, 8-(4-Chlorophenylthio)-2'-0- methyladenosine-3', 5'- cyclic monophosphate (8-pCPT) improved functional and structural renal parameters, and reduced renal inflammation after NTS injection. Taken together, these findings suggest that Epacl activation is an attractive and promising pharmacological intervention for the therapeutic treatment of CKD.

Accordingly, the first object of the present invention relates to a method of treating a chronic kidney disease in a patient in need thereof comprising administering to the patient a therapeutically effective amount of at least one Epacl activator.

As used herein, the term“chronic kidney disease” or“CKD” has its general meaning in the art and refers to a progressive loss in renal function over a period of months or years. CKD is used to classify numerous conditions that affect the kidney, destruction of the renal parenchyma and the loss of functional nephrons or glomeruli. It should be further noted that CKD can result from different causes, but the final pathway remains renal fibrosis. CKD is defined as kidney damage or glomerular filtration rate (GFR) <60 mL/min/l .73 m² for 3 months or more, irrespective of cause. Kidney damage in many kidney diseases can be ascertained by the presence of albuminuria, defined as albumin-to-creatinine ratio >30 mg/g in two of three spot urine specimens. GFR can be estimated from calibrated serum creatinine and estimating equations, such as the Modification of Diet in Renal Disease (MDRD) Study equation or the Cockeroft-Gault formula. Kidney disease severity is classified into five stages according to the level of GFR. Examples of etiology of CKD include, but are not limited to, cardiovascular diseases, hypertension, diabetes, glomerulonephritis, polycystic kidney diseases, and kidney graft rejection. In some , the patient in need thereof suffers from a disease selected from the group consisting of nephropathy (e.g. membranous nephropathy (MN), diabetic nephropathy and hypertensive nephropathy), glomerulonephritis (e.g. membranous glomerulonephritis and membranoproliferative glomerulonephritis (MPGN) such as rapidly progressive glomerulonephritis (RPGN)), interstitial nephritis, lupus nephritis, idiopathic nephrotic syndrome (INS) (e.g. minimal change nephrotic syndrome (MCNS) and focal segmental glomerulosclerosis (FSGS)), obstructive uropathy, polycystic kidney disease (e.g. Autosomal Dominant Polycystic Kidney Disease (ADPKD) and Autosomal Recessive Polycystic Kidney Disease (ARPKD)), cardiovascular diseases, hypertension, diabetes, and kidney graft rejection (e.g. acute and chronic kidney rejection).

As used herein the term“Epac” has its general meaning in the art and refers to the guanine exchange factor (GEF) Epac (Exchange Protein directly Activated by Cyclic AMP). There are two isoforms of Epac, Epacl and Epac2, both consisting of a regulatory region binding directly cAMP and a catalytic region that promotes the exchange of GDP (Guanosine diphosphate) for GTP (Guanosine-5’ -triphosphate) on the Ras-like small GTPases Rapl and Rap2 isoforms). The two isoforms of Epac differ in that Epacl has a single cyclic nucleotide binding (CNB) domain, whereas Epac2 has two CNB domains, called CNB-A and CNB-B, which are located on both sides of the DEP domain (Dishevelled, Egl-lO and Pleckstrin domain). The additional N-terminal CNB domain in Epac2 has a low affinity for cAMP, and its deletion does not affect the regulation of Epac2 in response to agonists (./. de Rooij, H. Rehmann, M. van Triest, et ah, Mechanism of regulation of the Epac family of cAMP -dependent RapGEFs, J. Biol. Chem. 275 (2000) 20829-20836). An exemplary human amino acid sequences is represented by SEQ ID NO:l.

SEQ ID NO:l >sp | 095398 | RPGF3_HUMAN Rap guanine nucleotide exchange factor 3 OS=Homo sapiens OX=9606 GN=RAPGEF3 PE=1 SV=6

MKVGWPGESCWQVGLAVEDSPALGAPRVGALPDVVPEGTLLNMVLRRMHRPRSCSYQLLL EHQRPSCIQGLRWTPLTNSEESLDFSESLEQASTERVLRAGRQLHRHLLATCPNLIRDRK YHLRLYRQCCSGRELVDGILALGLGVHSRSQVVGICQVLLDEGALCHVKHDWAFQDRDAQ FYRFPGPEPEPVRTHEMEEELAEAVALLSQRGPDALLTVALRKPPGQRTDEELDLIFEEL LHIKAVAHLSNSVKRELAAVLLFEPHSKAGTVLFSQGDKGTSWYI IWKGSVNVVTHGKGL VTTLHEGDDFGQLALVNDAPRAATI ILREDNCHFLRVDKQDFNRI IKDVEAKTMRLEEHG KVVLVLERASQGAGPSRPPTPGRNRYTVMSGTPEKILELLLEAMGPDSSAHDPTETFLSD FLLTHRVFMPSAQLCAALLHHFHVEPAGGSEQERSTYVCNKRQQILRLVSQWVALYGSML HTDPVATSFLQKLSDLVGRDTRLSNLLREQWPERRRCHRLENGCGNASPQMKARNLPVWL PNQDEPLPGSSCAIQVGDKVPYDICRPDHSVLTLQLPVTASVREVMAALAQEDGWTKGQV LVKVNSAGDAIGLQPDARGVATSLGLNERLFVVNPQEVHELIPHPDQLGPTVGSAEGLDL VSAKDLAGQLTDHDWSLFNS IHQVELIHYVLGPQHLRDVTTANLERFMRRFNELQYWVAT ELCLCPVPGPRAQLLRKFIKLAAHLKEQKNLNSFFAVMFGLSNSAI SRLAHTWERLPHKV RKLYSALERLLDPSWNHRVYRLALAKLSPPVIPFMPLLLKDMTFIHEGNHTLVENLINFE KMRMMARAARMLHHCRSHNPVPLSPLRSRVSHLHEDSQVARI STCSEQSLSTRSPASTWA YVQQLKVIDNQRELSRLSRELEP As used herein the term“Epacl activator” refers to any compound that is able to activate or increase the activity or expression of Epac 1. Epac 1 activators are known in the art. Typically, the Epacl activator is a cAMP analog, more preferably a cAMP analog which has no effect on the activity of protein kinase A (PKA) and moderate or no inhibitory effect on Epac2 isoform. Suitable compounds for use in the present invention are the cAMP analogs disclosed in W02003104250. In some embodiments, the Epacl activator is 8-(4-Chlorophenylthio)-2'-0- methyladenosine-3',5'-cyclic monophosphate (also known as 8-pCPT-2'-0-Me-cAMP). Of course, other known or yet to be identified (small molecule) agents capable of activating Epacl, preferably without affecting PKA activity, may also be used.

In some embodiments, the Epacl activator is an activator of Epacl expression. An “activator of expression” refers to a natural or synthetic compound that has a biological effect to increase the expression of a gene or to prevent its degradation. Therefore, an“activator of Epacl expression” denotes a natural or synthetic compound that has a biological effect to increase the expression of Epacl gene or prevent the degradation of Epacl mRNA or protein. In some embodiments, the activator is a nucleic acid that encodes for a Epacl polypeptide (e.g. SEQ ID NO: l) that can be administered to the patient. In some embodiments, the nucleic acid molecule of the present invention is included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. So, a further object of the invention relates to a vector comprising a nucleic acid encoding for a mutated FX polypeptide of the invention. Typically, the vector is a viral vector which is an adeno-associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" means a vector derived from an adeno- associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. Typically, the nucleic acid molecule or the vector of the present invention include "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3’-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”.

As used herein, the term "effective amount" refers to a quantity sufficient of the Epacl activator to achieve treatment of CKD. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. In some embodiments, an effective amount of the Epacl activator for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Typically, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In some embodiments, a single dosage of peptide ranges from 0.1-10,000 micrograms per kg body weight. In some embodiments, aromatic- cationic peptide concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.

Typically, the Epacl activator is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Typically, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Sterile injectable solutions are prepared by incorporating the Epacl activator in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Epacl is downregulated in GN and Epacl gene deletion worsens the progression of NTS-induced GN, monitored by body weight increase. (A) Upper panel. Representative immunoblots of Epacl and Epac2 isoforms. The GAPDH (glyceraldehyde-3- phosphate dehydrogenase) is the loading control. Graph. Quantification of Epacl protein in wild-type (WT) glomeruli mice subjected or not to NTS injection. Epacl expression was determined at day 15. (B) Body weight increase. n=6 to 7 per group. *: p < 0.05, **:p < 0.01, vs indicated values (ANOVA). n=7 mice per group. D, number of days after NTS injection.

Figure 2. Epacl genetic inhibition accelerates deterioration of renal function and has a deleterious effect on maintenance of renal structure in the experimental model of NTS-induced GN. Renal function was evaluated by proteinuria (A) and BUN (B). NTS- induced alterations of renal structure was more important in Epacl KO mice because they developed more glomerular crescents (C) and tubular dilation compared with WT treated NTS mice (D). Crescents are expressed as the percentage of glomeruli presenting cellular crescents. n=7 mice per group. *: p < 0.05, **:p < 0.01, ***:p < 0.001, vs indicated values (ANOVA). D, number of days after NTS injection.

Figure 3. Infiltration of macrophage and T cells are highly increased in the kidney of Epacl KO mice. (A) Evaluation of the percentage of positive area for F4/80 and (B) CD3e staining at day 15 in WT and Epacl KO kidney treated or not with NTS. n=7 mice per group.*: p < 0.05, **:p < 0.01, ***:p < 0.001, vs control or WT+NTS as indicated (ANOVA).

Figure 4. Epacl gene deletion dramatically increases expression of renal pro- inflammatory markers. (A-C) Mice were treated and measurements of mRNA of pro- inflammatory markers were made at day 15. n=7 mice per group. *: p < 0.05, **:p < 0.0l, ***:p

< 0.001, vs control or WT+NTS as indicated (ANOVA).

Figure 5. Fibrotic and renal cell stress markers are increased in Epacl KO mice after induction of NTS GN. (A, B) Fibrotic and (C, D) renal cell stress markers were determined by quantitative PCR as described in methods. n=7 mice per group.*: p < 0.05, **:p

< 0.01, ***:p < 0.001, vs control or WT+NTS as indicated (ANOVA).

Figure 6. Deletion of Epacl increases renal fibrosis assessed by Sirius red coloration. Quantification of fibrillar collagen at day 15. n=7 mice per group.*: p < 0.05, **:p

< 0.01, vs control or WT+NTS as indicated (ANOVA).

Figure 7. Epacl pharmacological activation protects against NTS-induced GN. (A) Body weight evolution and (B, C) renal function parameters (proteinuria and serum creatinine). n=8 or 4 mice for NTS or PBS groups, respectively.*: p < 0.05, **:p < 0.01, vs control or 8- pCPT+NTS as indicated (ANOVA).

Figure 8. Activation of Epacl with 8-pCPT preserves renal structure and reduces renal macrophage infiltration. (A)Renal structure was preserved in NTS injected mice treated with 8-pCPT, because they developed fewer glomerular crescents. (B) Percentage of positive area for F4/80. n=4 mice per group.*: p < 0.05.

* Mini pumps were implanted 1 day after the final injection of NTS; these pumps are active and start delivering their content 24h after implantation.

EXAMPLE:

Methods

Reagents and antibodies. The Epacl agonist 8-(4-Chlorophenylthio)-2'-0- methyladenosine-3', 5'- cyclic monophosphate (8-pCPT) was purchased from BioLog (Bremen, Germany). Antibodies and their suppliers were: anti-GAPDH from Cell Signaling, anti-Epacl from Abeam, anti-F4/80 from AbD Serotec, and anti-CD3 from DakoFrance. Animal and protocols. All mice were handled in strict accordance with good animal practice as defined by the relevant national animal welfare bodies of France, and all animal work was approved by the appropriate committee of the National Institute for Health and Medical Research and the Pierre and Marie Curie University (Paris, France). Animals were housed at constant temperature with access to water and food ad libitum. Epacl -deficient mice (Epacl KO) have been engineered in our laboratory as previously described (Laurent et al., 2015). Glomerulonephritis (GN) was induced by intravenous injection of decomplementated nephrotoxic serum (NTS) prepared as previously described (Guerrot et al., 2011). Three-month- old Epacl female mice (C57BL/6-SV129 background) and their wild-type littermates were used to induce a passive NTS-GN by intravenous administration of 20 mΐ NTS per gram of body weight over 2 consecutive days. Mice were euthanized 15 days after NTS administration. Control mice were injected with PBS. In a separate study, 3 month-old C57B16 female mice were injected with NTS (17 pl/gBW for 2 consecutive days). The day after the last injection, osmotic mini-pumps (Alzet model 1002) were implanted subcutaneously in mice anaesthetized with isoflurane (1%). Pumps were filled with either 8-pCPT or sterile PBS and were set to deliver 8-pCPT at 3 mM (1.5 mg)/mouse/day. Mice were then subsequently sacrificed.

Renal Function. Urine samples were collected on days 0, 4, 8, 12, and 15, and blood samples were collected before euthanasia. Proteinuria was measured with a Konelab automater (Thermo Fisher Scientific, Waltham, MA) and normalized to urine creatinine. Blood urea nitrogen (BUN) and plasma creatinine levels were measured with an enzymatic method (Konelab automater) and expressed in millimoles per liter and micromolar, respectively.

Histology. Kidneys were fixed in 4% formalin solution and embedded in paraffin; 4-m m sections were stained with Masson Trichrome for histologic evaluation. Interstitial fibrosis was assessed semi-quantitatively on Sirius red-stained paraffin sections at magnification of 200 using computer-based morphometric analysis software (Analysis; Olympus) as previously described (Abed et al., 2014). Crescent formation and tubular dilation were evaluated blinded on coded slides as already reported (Kerroch et al., 2012). Immunohistochemistry for F4/80 and CD3 was peformed as previously described (Kerroch et al., 2016).

Western Blot analysis. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were incubated with the primary antibody and followed by incubation with peroxidase-conjugated secondary antibodies (Bio-Rad). Membranes were revealed with a ECL Plus Prime (Amersham Biosciences). Signals were quantified by densitometry using the Image Lab software (Bio-Rad). Total RNA isolation and real-time quantitative RT-PCR. Total RNA was extracted from kidneys using TRIzol reagent (Euromedex). RNA quality was checked by control of OD at 260 and 280 nm. cDNA was synthesized from 1 mg RNA using the Fermentas H Minus First- Strand cDNA Synthesis Kit according to the manufacturer’s instructions. Quantitative PCR experiments were performed as previously described (Abed et a , 2014). Each sample was run in triplicate, and analysis of relative gene expression was done by using the 2-AACT method. Results are expressed in graphs as arbitrary units, which represent the ratio of the target gene to the internal control gene (HPRT).

Data analysis. Values are expressed as mean±SEM. Data were analyzed using oneway ANOVA followed by the protected least significant difference Fisher test of the Statview software package.

Results

Loss of Epacl expression accelerates the progression of renal disease

Epacl was mainly expressed in the glomeruli of wild type (WT) mice and was downregulated after NTS treatment whereas Epac2 was barely detectable in basal and disease conditions (Figure 1A). To investigate the functional relevance of Epacl downregulation in experimental GN, we used Epacl KO mice that were subjected or not to NTS injections. Epacl deletion did not generate renal defects in mouse in basal conditions (Figure IB) with no compensation with Epac2 (Figure 1A). As expected, NTS administration progressively degraded renal function as evidenced by the values of body weight increase (Figure IB), proteinuria and BUN (Figures 2A and 2B). The sharp decline of renal function was accompanied with renal structure abnormalities as shown by increased crescent formation and tubular dilation (Figures 2C and 2D) in WT animals. Importantly, Epacl genetic ablation accelerated the decline of renal function compared with WT NTS treated animals (Figures IB, 2A). In accordance, at day 15, BUN, crescent formation and tubular dilation were significantly increased in Epacl KO versus WT mice (Figures 2B-D). Thus, Epacl deficiency does not cause baseline renal abnormalities but remarkably potentiates the deterioration of renal structure and function during the progression of NTS-GN.

Epacl KO mice show increased inflammation and fibrosis after NTS Injection

Macrophages and T-lymphocyte play a crucial role in the onset and the progression of crescentic GN (Maria and Davidson, 2017). F4/80 and CD3 immunostainings showed a dramatic increase in macrophage and T lymphocyte infiltration in the renal cortex of Epacl KO mice compared with WT mice after 15 days of NTS administration (Figures 3A and 3B). Consistently, upregulation of VCAM-l, crucial for monocyte adhesion, was largely increased in Epacl KO NTS mice (Figure 4A). Furthermore, other important pro-inflammatory genes including MCP-l and TNF-a was highly increased in Epacl KO NTS mice 15 days post-NTS injection (Figures 4B and 4C). As expected, a pro-fibrotic agent (TGF-b, Figure 5A), a fibrotic marker (Collagen I, Figure 5B) and a typical marker of renal tubular damage, KIM1 (Figure 5C) were highly increased in Epacl KO NTS mice. These effects were associated with podocyte injury as evidenced by the down-regulation of the podocyte-specific marker, WT1 (Figure 5D). Finally, quantification of Sirius red colorations confirmed that interstitial collagen accumulation was potentiated (Figure 6). Thus, decreased Epacl expression markedly potentiated renal inflammation and interstitial fibrosis in NTS-GN.

Pharmacological activation of Epacl delays the development of NTS-GN

We next sought to determine whether Epacl activation could have beneficial effects on renal function after the onset of the disease. To this end, at day 3 of the protocol WT C57BL/6 mice were treated with either the Epacl selective agonist, 8-pCPT (1.5 mg/mouse/day), or vehicle until the end of experiments (day 15). 8-pCPT treatment attenuated NTS-induced increase of body weight, proteinuria and creatininemia (Figures 7A-C). This functional protection was accompanied with a preservation of renal structure and decreased numbers of crescents (Figure 8A). F4/80 immunostaining showed limited macrophage infiltration in the renal cortex of 8-pCPT treated mice (Figure 8B) and further confirmed the beneficial effect of Epacl activation. Thus, activating Epacl protects against the progression of the NTS-GN.

Conclusion:

In conclusion, Epacl appears as an interesting target for therapy of CKD. Here, we have shown that genetic inhibition of Epacl is critical for and accelerates the progression of nephropathy. Inversely, Epacl pharmacological activation blunts the progression of experimental GN. Our study represents a“proof-of-concept” for the therapeutic effectiveness of activating Epac 1 in CKD using small-molecule pharmacotherapy.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Abed A, Toubas J, Kavvadas P, Authier F, Cathelin D, Alfieri C, Boffa JJ, Dussaule JC, Chatziantoniou C, Chadjichristos CE: Targeting connexin 43 protects against the progression of experimental chronic kidney disease in mice. Kidney Int. 86: 768-779, 2014. Breckler M, Berthouze M, Laurent AC, Crozatier B, Morel E, Lezoualc'h F. Rap-linked cAMP signaling Epac proteins: compartmentation, functioning and disease implications. Cell Signal. 23:1257-66, 2011.

de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL. Epac is a Rapl guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396:474-7, 1998

Eckardt KU, Coresh J, Devuyst O, Johnson RJ, Kottgen A, Levey AS, et al. Evolving importance of kidney disease: from subspecialty to global health burden. Lancet. 382:158-69, 2013.

Fazal L, Laudette M, Paula-Gomes S, Pons S, Conte C, Tortosa F, Sicard P, Sainte- Marie Y, Bisserier M, Lairez O, Lucas A, Roy J, Ghaleh B, Fauconnier J, Mialet-Perez J, Lezoualc'h F. Multifunctional Mitochondrial Epacl Controls Myocardial Cell Death. Circ Res. 120:645-657, 2017.

Guerrot D, Kerroch M, Placier S, Vandermeersch S, Trivin C, Mael-Ainin M, Chatziantoniou C, Dussaule JC.. Discoidin Domain Receptor 1 Is a Major Mediator of Inflammation and Fibrosis in Obstructive Nephropathy. Am J Pathol. 179 :83— 91, 2011.

Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, Graybiel AM. A family of cAMP-binding proteins that directly activate Rapl. Science 282:2275-9, 1998.

Kerroch M, Alfieri C, Dorison A, Boffa JJ, Chatziantoniou C, Dussaule JC. Protective effects of genetic inhibition of Discoidin Domain Receptor 1 in experimental renal disease. Sci Rep. 6:21262, 2016.

Kerroch M, Guerrot D, Vandermeersch S, Placier S, Mesnard L, Jouanneau C, Rondeau E, Ronco P, Boffa JJ, Chatziantoniou C, Dussaule JC: Genetic inhibition of discoidin domain receptor 1 protects mice against crescentic glomerulonephritis. FASEB J 26: 4079-4091, 2012.

Laurent AC, Bisserier M, Lucas A, Tortosa F, Roumieux M, De Regibus A, Swiader A, Sainte-Marie Y, Heymes C, Vindis C, Lezoualc'h F. Exchange protein directly activated by cAMP 1 promotes autophagy during cardiomyocyte hypertrophy. Cardiovasc Res. 105:55-64, 2015.

Maria NI, Davidson A. Renal Macrophages and Dendritic Cells in SLE Nephritis. Curr Rheumatol Rep. 19:81. 2017.

Robichaux WG 3rd, Cheng X. Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development. Physiol Rev 98:919-1053, 2018.

Claims

CLAIMS:

1. A method of treating a chronic kidney disease (CKD) in a patient in need thereof comprising administering to the patient a therapeutically effective amount of at least one Epacl activator.

2. The method of claim 1 wherein the patient suffers from a disease selected from the group consisting of nephropathy (e.g. membranous nephropathy (MN), diabetic nephropathy and hypertensive nephropathy), glomerulonephritis (e.g. membranous glomerulonephritis and membranoproliferative glomerulonephritis (MPGN) such as rapidly progressive glomerulonephritis (RPGN)), interstitial nephritis, lupus nephritis, idiopathic nephrotic syndrome (INS) (e.g. minimal change nephrotic syndrome (MCNS) and focal segmental glomerulosclerosis (FSGS)), obstructive uropathy, polycystic kidney disease (e.g. Autosomal Dominant Polycystic Kidney Disease (ADPKD) and Autosomal Recessive Polycystic Kidney Disease (ARPKD)), cardiovascular diseases, hypertension, diabetes, and kidney graft rejection (e.g. acute and chronic kidney rejection).

3. The method of claim 1 wherein the Epacl activator is a cAMP analog.

4. The method of claim 1 wherein the Epacl activator is 8-(4-Chlorophenylthio)-2'-0- methyladenosine-3',5'-cyclic monophosphate.

5. The method of claim 1 wherein the Epacl activator is an activator of Epacl expression.

6. The method of claim 5 wherein the activator is a nucleic acid that encodes for a Epacl polypeptide.