WO2017013270A1 - Use of leptin signaling inhibitor for protecting kidneys from patients having ciliopathy - Google Patents

Abstract

The present invention relates to the use of an inhibitor of the Leptin signaling pathway for protecting kidneys of patients having a ciliopathy.

Description

USE OF LEPTIN SIGNALING INHIBITOR FOR PROTECTING KIDNEYS FROM

PATIENTS HAVING CILIOPATHY

FIELD OF THE INVENTION

The present invention relates to the field of medicine. In particular, it relates to a method of protecting kidneys of patients suffering of ciliopathy associated with obesity.

BACKGROUND OF THE INVENTION

The primary cilium is a key organelle involved in fundamental biological signaling pathways. Defects, of genetic origin, in this ubiquitously expressed, hair-like structure lead to clinically heterogeneous diseases termed ciliopathies.

Ciliopathies include, among others, polycystic kidney diseases, either dominant (ADPKD for autosomal dominant polycystic kidney disease) or recessive (ARPKD for autosomal recessive polycystic kidney disease), Bardet-Biedl syndrome (BBS), Alstrom syndrome, Joubert syndrome,Meckel-Gruber syndrome, and orofaciodigital syndrome 1.

The kidneys are the most commonly affected organ in ciliopathies, displaying pathologies ranging from nonfunctional cystic dysplastic kidneys to an isolated urinary concentration defect. The disorders contributing to this pathology include nephronophithisis, glomerulocystic kidney disease and medullary sponge kidneys. Decreased urinary concentration ability, resulting in polyuria and polydipsia, is the first and most common renal symptom in ciliopathies. The majority of ADPKD, ARPKD and nephronophithisis patients requires renal transplantation, despite the frequency and rate of progression to renal failure varies considerably in other ciliopathies.

Renal failure is a major life-threatening complication associated with ciliopathy. Indeed, it is a major cause of morbidity and mortality with most BBS patients presenting urinary concentrating defects associated with polyuria-polydypsia.

Bardet-Biedl syndrome (BBS) is an iconic member of these ciliopathies and is primarily characterized by obesity, kidney dysfunction, retinal degeneration and Polydactyly.

To date, 20 genes have been identified (BBS 1-20) coding for proteins mostly localized within the primary cilium complex. On a molecular basis, 7 BBS proteins form a protein complex known as the BBSome involved in the intraciliary transport whereas the BBS6, BBS 10 and BBS 12 together with other molecular chaperones form a scaffold chaperon complex assisting the assembly of the BBSome. Despite the high frequency of renal dysfunction in BBS patients, a clear understanding on how inactivating BBS proteins leads to renal dysfunction is still lacking. This is undeniably due to the variable renal phenotypes observed in the human patients and the different Bbs mouse models: ranging from defective planar cell polarity of epithelial cells, cysts and polydipsia/polyuria. The latter phenotype is thought to be linked to a resistance to the antidiuretic hormone Arginine Vasopressin (A VP). In addition, BBS-induced obesity associated with its disturbed hormonal profile, namely hyperleptinemia, is unquestionably impacting renal function. Because of these variable renal phenotypic traits pertaining to the BBS and because obesity impairs renal function, the mechanisms leading to renal failure remain poorly understood.

Alstrom syndrome is also a ciliopathy closely related to BBS, characterized by renal abnormalities, obesity, and insulin resistances.

Therefore, there is a strong and unmet need for treatments in order to protect kidneys in patients having ciliopathies, in particular ciliopathies associated with hyperleptinemia.

In the more general context of obesity and based on several obesity-associated animal models studies, it is well established that chronic hyperleptinemia contribute to the development and progression of chronic kidney diseases (Nasrallah MP et al, 2013, Seminars in Nephrology, 33, 54-65). These studies are pointing to a hypothalamic path of blood pressure increase as the main cause of these chronic kidney diseases development. In this hypothalamic path, the leptin- induced increase in blood pressure is caused by sympathetic nervous system activation in response to stimulation of leptin-receptors of the hypothalamus. Otherwise, two studies have also suggested the possibility of a direct effect of leptin on kidney (Lee MP et al, 2005, Int J Obes., 29, 1395-1401; Han DC et al., 2001, Kidney Int., 59, 1315-1323). Leptin was shown to induce, through kidney's leptin-receptors, the synthesis of TGF-βΙ in glomerular endothelium and the synthesis of collagen in mesanglial cells, two kidney cell-types. From these effects, it was thought that leptin may induce glomerulosclerosis and proteinuria, two renaldisorders. However, these results remain to be confirmed. Thus, according to the state of the art, chronic obesity-related hyperleptinemia contribution to chronic kidney diseases relies on a well- documented major hypothalamic path and a possible accessory, yet to be fully demonstrated, direct kidney path.

Because leptin has been involved in chronic kidney diseases development, the use of leptin- receptor antagonists in a therapeutic strategy has been proposed. According to the state of the art, a major issue has yet to be overcome prior to the use of such antagonists. Indeed, when leptin activates the sympathetic nervous system in the hypothalamus, it results in an increased sympathetic nervous system output to the kidney but also to the brown adipose tissue. The stimulation of the brown adipose tissue by this hypothalamic path leads to a diminution of food- intake and to satiety. On the opposite, the use of leptinreceptor antagonists in the hypothalamus increases body weight (Turner N. et al., 2007, J. Hypertens., 25, 2471-2478). This latter hypothalamic effect is particularly inconvenient in subject which are already suffering from obesity. That's why some authors are insisting on the absolute necessity to develop selective leptinreceptor antagonists prior to consider their clinical use. Even if the hypothalamic-kidney path and the hypothalamic -brown adipose tissue path display different intracellular signaling pathways in hypothalamic cells, they rely on the same receptor and are both located in the hypothalamus, which makes it very difficult to design a leptinreceptor antagonist selective of one path but not of the other. Thus, currently, research of suitable leptinreceptor antagonists for developing chronic kidney diseases treatment in obese subjects seems to have reached an impasse.

SUMMARY OF THE INVENTION

The present invention relates to a method for protecting kidneys in patients having a ciliopathy combined with hyperleptinemia. Ciliopathy is generally associated with a series of moderate developmental and acquired structural defects in the kidneys. The inventors hypothesized and proved that leptin signaling in the kidneys is driving the major deleterious effects in kidneys of subjects having a ciliopathy, whereas it has no or minor impact on kidney with a wild-type subject, obese or not. It could be explained by the combination of ciliopathies renal defect and leptin signaling. In other words, ciliopathies weaken the kidneys and make them more sensitive to hyperleptinemia. In addition, the inventors demonstrated that the leptin signaling pathway in kidney is distinct from that of the hypothalamus. Finally, they identified that an inhibitor of Leptin Receptor (LEPR) is efficient in protecting kidney and then preventing renal failure in subjects having a ciliopathy. Indeed, ciliopathies weaken the kidneys and make them more sensitive to hyperleptinemia, but this deleterious effect on kidneys can be prevented by neutralizing the leptin signaling, especially with a LEPR inhibitor.

The present invention relates to the use of an inhibitor of leptin signaling for use for treating patients having a ciliopathy, preferably a ciliopathy with hyperleptinemia. Preferably, the leptin signaling inhibitor is a leptin Receptor inhibitor. More particularly, it is for use for protecting kidney or preventing kidney damage. The present invention also relates to the use of a leptin signalinginhibitor, preferably a leptin Receptor inhibitor, for the manufacture of a medicament for use for treating patients having a ciliopathy, in particular a ciliopathy with hyperleptinemia, more preferably for use for protecting kidney or preventing kidney damage of patients having a ciliopathy, in particular a ciliopathy with hyperleptinemia. Finally, the present invention relates to a method for treating patients having a ciliopathy, in particular a ciliopathy with hyperleptinemia, comprising administering a therapeutically efficient amount of a leptin signaling inhibitor, preferably a leptin Receptor inhibitor, to said patients, thereby protecting kidney or preventing kidney damage.

In a preferred embodiment, the ciliopathy is selected for the group consisting of Bardet-Biedl syndrome (BBS), Alstrom syndrome, and MORM syndrome. More preferably, the ciliopathy is Bardet-Biedl syndrome (BBS).

In a first embodiment, the Leptin signaling inhibitor is an antibody directed against leptin or LEPR (Leptin Receptor), preferably against LEPR and having an antagonist or neutralizing activity on LEPR.

In a second embodiment, the Leptin signaling inhibitor is a peptide derived from leptin or a mutated leptin and has an antagonist activity on LEPR, or a nucleic acid encoding said peptide or mutated leptin.

In a third embodiment, the Leptin signaling inhibitor is a polypeptide derived from the leptin receptor and having an inhibiting effect on the LEPR activity, such as a soluble polypeptide comprising the extracellular domain of LEPR or a leptin-binding portion thereof or a fibronectin III domain thereof.

In a fourth embodiment, the Leptin signaling inhibitor is an oligonucleotide inhibiting or decreasing the expression of leptin or LEPR, preferably LEPR, such as antisense, miRNA, siRNA or shRNA.

In a fifth embodiment, the Leptin signaling inhibitor is an aptamer specific to leptin or LEPR, preferably of LEPR and having an antagonist activity.

In a sixth embodiment, the leptin signaling inhibitor is a small molecule inhibiting or blocking the leptin signaling pathway, preferably the LEPR activity. Preferably, the Leptin signaling inhibitor is JAK2 inhibitor, preferably ruxolitinib or baricitinib, more preferably ruxolitinib.

In a seventh embodiment, the leptin signaling inhibitor is an immunoadhesin resulting from the fusion of a fragment of the human LEPR to the Fc portion of immunoglobulins.

In a preferred embodiment, the Leptin signaling inhibitor does not cross the Blood Brain Barrier. More preferably, the Leptin signaling inhibitor is targeted to the kidneys or is to be locally administered to the kidneys.

In a preferred embodiment, the Leptin signaling inhibitor is to be administered to said patient when the serum level of leptin increases, in particular above about 12.5 ng/ml. DETAILED DESCRIPTION OF THE INVENTION Patients and diseases

The present invention relates to the use of leptin signaling inhibitor for treating patients having a ciliopathy. More particularly, the ciliopathy is associated with hyperleptinemia. Preferably, the present invention relates to the use of Leptin signaling inhibitor for protecting kidney in patients having a ciliopathy. In particular, the Leptin signaling inhibitor is used for preventing or delaying renal failure or dysfunction.

More preferably, the ciliopathy associated with hyperleptinemia is selected from the group consisting of Bardet-Biedl syndrome (BBS), Alstrom syndrome, and MORM syndrome.

In a preferred embodiment, the ciliopathy is selected Bardet-Biedl syndrome (BBS). In a particular aspect, the patient has one or several mutations in proteins of the BBSome. In a particular embodiment, the patient may have a mutation in BBS 12, BBS 10 and BBS1.

Bardet biedl syndrome (BBS) is characterized by hyperleptinemia combined with obesity associated with developmental defect in of the glomeruli of genetic origin. The treatment according to the present invention is a therapeutic approach which is based on the inhibition and/or repression of the leptin signaling in the podocytes of the kidney glomeruli to prevent or slow down progression towards full-blown kidney dysfunction through specific protection of the podocytes. BBS is an iconic member of the ciliopathies, a group of rare genetic diseases. Besides the BBS, Alstrom syndrome (ALMS) is also characterized by massive obesity and hyperleptinemia together with kidney dysfunctions. Based on the phenotypic similarities between BBS and ALMS, it is seems scientifically justified that the treatment with an inhibitor of the leptin signaling, which targets the adverse effect of hyperleptinemia in the podocytes, irrespective of the genetic mutation, would work in the ALMS condition.

Inhibitor of Leptin Signaling

According to the invention, the leptin signaling inhibitor is a molecule capable of blocking, preventing and/or decreasing the effects of leptin signaling on the kidney. Preferably, the leptin signaling inhibitor is specific of the renal leptin signaling.

The leptin signaling inhibitor can modulate the leptin signaling pathway at any level from the leptin synthesis to its final effects on the kidney. The leptin signaling inhibitor may thus block, prevent or reduce the leptin production, the leptin blood circulation, the leptin receptor (i.e. LEPR) binding, the leptin receptor activity, the leptin receptor expression and the LEPR intracellular transduction. In an embodiment, the leptin signaling inhibitor is a molecule capable of blocking, preventing and/or decreasing the production of leptin. The term "production" as used herein refers to the leptin synthesis and to its secretion. The Leptin is produced primarily in the adipocytes of white adipose tissue. It is also produced by brown adipose tissue, placenta (syncytiotrophoblasts), ovaries, skeletal muscle, stomach (the lower part of the fundic glands), mammary epithelial cells, bone marrow, gastric chief cells and P/Dl cells. Preferably, the leptin signaling inhibitor is a molecule capable of blocking, preventing and/or decreasing the adipocyte leptin production. Accordingly, the leptin signaling inhibitor can be a nucleic acid molecule or oligonucleotide interfering specifically with the expression of leptin.

In another embodiment, the leptin signaling inhibitor is a molecule capable of decreasing the blood level of leptin. Leptin circulates in blood in free form and bound to proteins. Therefore, the leptin signaling inhibitor can be a molecule capable of capturing and retaining the leptin. Alternatively, the leptin signaling inhibitor can be capable of decreasing the stability or half- life of circulating leptin.

In yet another embodiment, the leptin signaling inhibitor is an inhibitor of LEPR, i.e. a molecule capable of blocking, preventing and/or decreasing the expression of LEPR, the binding of leptin to its receptor; any molecule capable of preventing LEPR multimerization or any molecule having an antagonist or neutralizing activity on LEPR.

The Leptin receptor, also called LEPR or OBR, is referenced in UniProtKB/Swiss-Protas P48357, in Gene ID under accession number 3953 or in GeneCard under accession number

GC01P065886. Reference sequences for isoform B considered as the canonical form are disclosed in Genbank under NM_001003679.3 for mRNA and NP_001003679.1 for protein.

Structure of the LEPR receptor is known and is disclosed for instance in WO2013/017830.

Preferably, the inhibitor of LEPR can block, prevent and/or decrease the binding of leptin to its receptor either by binding the blood circulating leptin, or by binding the LEPR.

In a preferred embodiment, the inhibitor of the leptin receptor has no direct effect on leptin, except the effect on its binding to the leptin receptor.

Preferably, a LEPR inhibitor is a molecule capable of decreasing the activity of LEPR by at least 10, 20, 30, 40 or 50 % when compared to the activity in the absence of the inhibitor. The terms "inhibitor", "blocker" and "antagonist" can be used and are interchangeable. The activity can be measured for instance by measuring intracellular signal transduction, for instance by measuring the Scocs3 expression levels and/or STAT3 phosphorylation as detailed in the Examples. In yet another embodiment, the leptin signaling inhibitor is a molecule capable of blocking, preventing and/or decreasing the intracellular signal transduction of LEPR, preferably in the kidney. Leptin-induced renal pathology are specifically mediated by phosphorylation of the serine 727 residue of STAT3 in the glomeruli (cf. figure 6). Preferably, the leptin signaling inhibitor is a molecule capable of blocking, preventing and/or decreasing the serine 727 phosphorylation of STAT3 in the glomeruli.

Antibodies

The leptin signaling inhibitor can be an antibody directed against the leptin or against the LPER, or a fragment or derivative of these antibodies.

Preferably, the leptin signaling inhibitor is an antibody directed against LEPR or a fragment or derivative of this antibody. The antibody, fragment or derivative thereof has a neutralizing or blocking effect on LEPR. It is an antagonist of LEPR.

Several anti-LEPR antibodies have been disclosed in the prior art and are available. For instance, antibodies directed against LEPR or methods for producing such antibodies are disclosed in details in patent applications such as WO 97/19952, WO 97/25425, WO 2005/49655, US 7,612,171, WO2007/080404, and WO2013/017830, the disclosure thereof being incorporated herein by reference. More specifically, the antibody can be selected among the following group ZMC2 as disclosed in WO 2005/49655, the disclosure thereof being incorporated herein by reference. Blocking/neutralizing LEPR antibodies are commercially available (e.g. Abeam, Sigma, Thermo Scientific, Abbiotec) or disclosed in the scientific literature.

In a particular aspect, the LEPR antibody is the rabbit polyclonal antibody of reference number Ab5593 purchased from Abeam or a humanized derivative thereof. Alternatively, the LEPR antibody has the same epitope than the Ab5593 antibody from Abeam or is able to compete with the Ab5593 antibody from Abeam for LEPR binding.

Preferably, the antibody is specific for the extracellular moiety of the LEPR. More specifically, it can be specific for the leptin binding part of the extracellular moiety of the LEPR (i.e., from 467-484 of LEPR). Alternatively, the antibody is able to bind an epitope located in the region 438-525 of LEPR, preferably 463-504, more preferably is able to bind a polypeptide comprising 463-473, 477-487 or 500-504, or it has been prepared by using polypeptides comprising the sequence of the region 438-525 of LEPR, preferably 463-504, more preferably 463-473, 477- 487 or 500-504. More details are provided in WO2013/017830, the disclosure thereof being incorporated herein by reference. As used herein, the terms "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antigen-binding antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In particular, the antibody according to the invention may correspond to a polyclonal antibody, a monoclonal antibody (e.g. a chimeric, humanized or human antibody), a fragment of a polyclonal or monoclonal antibody or a diabody.

In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (V_L) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI , CH2 and CH3, collectively referred to as CH). The variable regions of both light (V_L) and heavy (V_H) chains determine binding recognition and specificity to the antigen. Are also contemplated camelid antibodies, such as heavy-chain antibodies, and fragments and derivatives thereof such (VHH)₂ fragments, nanobodies and sdAb.

The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). They refer to amino acid sequences which, together, define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H- CDR1, H-CDR2, H-CDR3, respectively. Therefore, an antigen-binding site includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

Framework regions (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species, as defined by Kabatei al, 1991 (Kabatei ah, 1991, Sequences of Proteins Of Immunological Interest, National Institute of Health, Bethesda, Md). As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, in particular, 90%, 95% or 100%) to the framework region of naturally occurring human antibody.

The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single amino acid composition, that is directed against a specific antigen and which may be produced by a single clone of B cells or hybridoma, or by recombinant methods. A "humanized antibody" is a chimeric, genetically engineered, antibody in which the CDRs from an antibody, e.g. a mouse antibody, ("donor antibody") are grafted onto a human antibody ("acceptor antibody"). Thus, a humanized antibody is an antibody having CDRs from a donor antibody and variable region framework and constant regions from a human antibody. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.

Antibodies according to the invention may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. The antibodies of this invention can be obtained by producing and culturing hybridomas.

The term "Heavy-chain antibodies" or "HCAbs" refer to immunoglobulins which are devoid of light chains and consist in two heavy chains. These antibodies do not rely upon the association of heavy and light chain variable domains for the formation of the antigen-binding site but instead the variable domain of the heavy polypeptide chains alone naturally form the complete antigen binding site. Each heavy chain comprises a constant region (CH) and a variable domain which enables the binding to a specific antigen, epitope or ligand. As used herein, HCAbs encompass heavy chain antibodies of the camelid-type in which each heavy chain comprises a variable domain called VHH and two constant domains (CH2 and CH3). Such heavy-chain antibodies directed against a specific antigen can be obtained from immunized camelids. Camelids encompass dromedary, camel lama and alpaca. Camelid HCAbs have been described by Hamers-Casterman et al., 1993. Other examples of HCAb are immunoglobulin-like structures (Ig-NAR) from cartilaginous fishes. Heavy-chain antibodies can be humanized using well-known methods.

"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')₂, Fab', Fd, dAb, dsFv, scFv, di-scFvs, sc(Fv)₂, CDRs, VHH, (VHH)₂, diabodies and multi- specific antibodies formed from antibodies fragments.

The term "Fab" denotes an antibody monovalent fragment having a molecular weight of about 50,000 and antigen binding activity, and consisting of the VL, VH, CL and CHI domains which can be obtained by cutting a disulfide bond of the hinge region of the F(ab')₂ fragment.

The Fv fragment is the N-terminal part of the Fab fragment and consists of the variable portions of one light chain and one heavy chain. The term "F(ab')₂" refers to an antibody bivalent fragment having a molecular weight of about 100,000 and antigen binding activity, which comprises two Fab fragments linked by a disulfide bridge at the hinge region.

The term "Fd" refers to an antibody fragment consisting of the V_H and C_HI domains.

The term "dAb" (Ward et al., 1989 Nature341:544-546) refers to a single variable domain antibody, i.e. an antibody fragment which consists of a V_H or V_L domain.

A single chain Fv ("scFv") polypeptide is a covalently linked V_H::V_L heterodimer which is usually expressed from a gene fusion including V_H and V_L encoding genes linked by a peptide- encoding linker. "dsFv" is a V_H: :V_L heterodimer stabilised by a disulfide bond. Divalentand multivalent antibody fragments can form either spontaneously by association of monovalent scFvs such as di-scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc (Fv)₂.

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a V_H domain connected to a V_L domain in the same polypeptide chain (V_H- V_L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementarity domains of another chain and create two antigen-binding sites. The diabody may be mono- or bi-specific.

The term "VHH" refers to an antibody fragment consisting of the V_H domain of camelid heavy- chain antibody. VHH fragments can be produced through recombinant DNA technology in a number of microbial hosts (bacterial, yeast, mould), as described in WO 94/29457. Alternatively, binding domains can be obtained by modification of the VH fragments of classical antibodies by a procedure termed "camelisation", described by Davies et al, 1995. Dimers of VHH fragments, i.e. (VHH)₂, can be generated by fusing two sequences encoding VHH fragments, end to end, e.g. by PCR. Preferably, the (VHH)₂ fragment is monospecific. Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art. (See, e.g., Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)).

General techniques for cloning murine immunoglobulin variable domains have been disclosed, for example, by the publication of Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniques for constructing chimeric antibodies are well known to those of skill in the art. As an example, Leung et al., Hybridoma 13:469 (1994), disclose how they produced an LL2 chimera by combining DNA sequences encoding the Vk and VH domains of LL2 monoclonal antibody, an anti-CD22 antibody, with respective human and IgGl constant region domains. This publication also provides the nucleotide sequences of the LL2 light and heavy chain variable regions, Vk and VH, respectively. Techniques for producing humanized antibodies are disclosed, for example, by Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993).

In one alternative, the phage display technique may be used to generate human antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4: 126-40, incorporated herein by reference). Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as cancer (Dantas-Barbosa et al., 2005). The advantage to constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al. (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Generally, total RNA was obtained from circulating blood lymphocytes (Id.) Recombinant Fab were cloned from the [mu], [gamma] and [kappa] chain antibody repertoires and inserted into a phage display library (Id.) RNAs were converted to cDNAs and used to make Fab cDNA libraries using specific primers against the heavy and light chain immunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97). Library construction was performed according to Andris- Widhopf et al. (2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), l<st>edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporated herein by reference). The final Fab fragments were digested with restriction endonucleases and inserted into the bacteriophage genome to make the phage display library. Such libraries may be screened by standard phage display methods. The skilled artisan will realize that this technique is exemplary only and any known method for making and screening human antibodies or antibody fragments by phage display may be utilized.

In another alternative, transgenic animals that have been genetically engineered to produce human antibodies may be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols as discussed above. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7: 13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994). A non-limiting example of such a system is the XenoMouse(R) (e.g., Green et al., 1999, J. Immunol. Methods 231: 11-23, incorporated herein by reference) from Abgenix (Fremont, Calif.). In the XenoMouse(R) and similar animals, the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.

Antibody fragments which recognize specific epitopes can be generated by known techniques. The antibody fragments are antigen binding portions of an antibody, such as F(ab)2, Fab', Fab, Fv, scFv and the like. Other antibody fragments include, but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab' fragments, which can be generated by reducing disulfide bridges of the F(ab')2 fragments. Alternatively, Fab' expression libraries can be constructed (Huse et al., 1989, Science, 246: 1274-1281) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity.

Inhibitors derived from leptin: peptides, polypeptides and mutated leptins

Alternatively, the leptin signaling inhibitor is a LEPR inhibitor which competes with leptin for the receptor binding. Accordingly the LEPR inhibitor can be a leptin fragment or a mutated leptin.

Several mutated leptins or leptin muteins are well-known in the art for their antagonist effects. For instance, the following patent applications provide examples of such leptin muteins: WO2009/100255, the disclosure thereof being incorporated herein by reference.

In addition, the LEPR inhibitor can be peptides derived from leptin and having an antagonist activity on LEPR are also known in the art, and disclosed for instance in W098/12224, WO2005/110461, WO2006/056987 and WO2011/132189, and WO2010/117785, the disclosure thereof being incorporated herein by reference.

BL-5040 is a drug based on a mutated leptin and is under clinical development (BioLineRx). It is a pegylated leptin antagonist.

Inhibitors derived from leptin receptor

Alternatively, the leptin signaling inhibitor is a polypeptide derived from the leptin receptor. This leptin receptor inhibitor can block, prevent and or decrease the binding of leptin to its receptor either by binding the blood circulating leptin, or by binding the LEPR, preferably by binding the LEPR.

In a preferred embodiment, the polypeptide derived from the leptin receptor is a soluble polypeptide comprising the extracellular domain of LEPR or the leptin-binding portion thereof. For instance, WO2006/53883 discloses antagonist polypeptides derived from the extracellular domain of LEPR, more particularly from the fibronectin III domain of the extracellular domain of LEPR. More particularly, the antagonist polypeptides are able to inhibit leptin induced signaling while the polypeptides are not able to bind leptin.

In a most prefered embodiment, the polypeptide derived from the leptin receptor is BL-5040 (Cheung WW. et al, J Am Soc Nephrol., 2014, 25(1): 119-28).

Optionally, the leptin signaling inhibitor can be a protein fusion comprising a fragment of LEPR and an Fc region of an immunoglobulin, preferably an IgG, especially an IgGl, more preferably a human IgG. The fragment of LEPR is preferably the extracellular domain of LEPR (e.g., 1- 839) or a portion or derivative thereof. Such leptin signaling inhibitors are commercially available (e.g., Sigma, reference L4787). A linker sequence can be added between the fragment of LEPR and the Fc region. Such an inhibitor is generally called Leptin receptor/Fc chimera or immunoadhesin (De Rosa V et al, 2006, J. Clin. Invest, 116(2):447-55; Lam QL et al, 2006, Eur J Immunol, 36(12):3118-30).

Similarly, US 2006/046959 discloses antagonist polypeptides derived from LEPR. Oligonucleotides inhibiting or decreasing the expression of LEPR or Leptin

In a particular embodiment of the invention, the leptin signaling inhibitor is a nucleic acid molecule or oligonucleotide interfering specifically with the expression of key proteins of the leptin signaling pathway. This oligonucleotide can interfere specifically with the leptin or with the LEPR expression, preferably with the LEPR expression, thereby decreasing or suppressing the expression of these proteins. Such nucleic acids are more amply detailed below. Preferably, this nucleic acid is selected from the group consisting of a RNAi, an antisense nucleic acid or a ribozyme. Said nucleic acid can have a sequence from 15 to 50 nucleotides, preferably from 15 to 30 nucleotides. By a "decrease" in expression is meant, for example, a decrease of 30%, 40%, 50%, 70%, 80%, 90% or 95% of the gene expression product.

The term "RNAi" or "interfering RNA" means any RNA that is capable of down-regulating the expression of the targeted protein. It encompasses small interfering RNA (siRNA), double- stranded RNA (dsRNA), single-stranded RNA (ssRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules. RNA interference designates a phenomenon by which dsRNA specifically suppresses expression of a target gene at post-translational level. In normal conditions, RNA interference is initiated by double-stranded RNA molecules (dsRNA) of several thousands of base pair length. In vivo, dsRNA introduced into a cell is cleaved into a mixture of short dsRNA molecules called siRNA. The enzyme that catalyzes the cleavage, Dicer, is an endo-RNase that contains RNase III domains (Bernstein et al., 2001). In mammalian cells, the siRNAs produced by Dicer are 21-23 bp in length, with a 19 or 20 nucleotides duplex sequence, two-nucleotide 3' overhangs and 5'-triphosphate extremities (Elbashir et al., 2001a; Elbashir et al., 2001b; Zamore et al., 2000). A number of patents and patent applications have described, in general terms, the use of siRNA molecules to inhibit gene expression, for example, WO 99/32619, US 20040053876, US 20040102408 and WO 2004/007718.

Antisense nucleic acid can also be used to down-regulate the expression of Leptin or LEPR, preferably LEPR. The antisense nucleic acid can be complementary to all or part of a sense nucleic acid encoding LEPRe.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence, and is thought to interfere with the translation of the target mRNA. Preferably, the antisense nucleic acid is an RNA molecule complementary to a target mRNA encoding LEPR.

Finally, the oligonucleotide can also be a miRNA specific for Leptin or LEPR, preferably LEPR.

Small molecule

Small molecules refer in particular to small organic molecules with a molecular mass <1000 Da. Small molecules and other drug candidates can readily be obtained, for example, from combinatorial and natural product libraries and using methods known to the art, or screening methods for their ability to block, prevent or reduce the leptin signaling pathway, i.e. the leptin production, the leptin blood circulation, the binding of leptin to its receptor (i.e. LEPR), the leptin receptor activity, the leptin receptor expression and the LEPR intracellular transduction. Preferably, small molecules have an LEPR antagonizing activity.

Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). Combinatorial libraries are available or can be prepared according to known synthetic techniques. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, Wash.) and MycoSearch (NC), or are readily producible by methods well known in the art.Furthermore, random peptide libraries, consisting of all possible combinations of amino acids, attached to a solid phase or in solution, may also be used to identify peptides that act as antagonists.

For instance, WO2009/147221 discloses small molecules having an antagonist activity against LEPR (the disclosure thereof being incorporated herein by reference). Small molecules can also target intracellular leptin signaling pathway, for example the proteins JAK1, JAK2, JAK3, STAT3, SOCS3 and ERK1/2, preferably the small molecules inhibit the proteins JAK1, JAK2, JAK3 and/or STAT3, more preferably JAK2 and/or STAT3.

In a particular embodiment, the small molecule is a STAT3 inhibitor, preferably selected from the group consisting of napabucasin (WO2011116399, WO2009/036101), LY-5 (WO/2011/066263), nifuroxazide (Nelson EA. et al, Blood. 2008, 112(13):5095-102), pyrimethamine (Takakura A. et al, Hum Mol Genet., 2011, 20(21):4143-54), anatabine (Paris D. et al, Eur J Pharmacol., 2013, 698(1-3): 145-53), and apratoxin A (Liu Y. et al, Mol Pharmacol., 2009, 76(1):91-104), even more preferably the small molecule is napabucasin. For instance, WO2010004761, US2015065526, WO2008070740, WO2014205416, WO2011081205, WO2012159107, discloses other small molecules having an inhibitory activities against STAT3 (the disclosure thereof being incorporated herein by reference). In another particular embodiment, the small molecule is a JAK1, JAK2 and/or JAK3 inhibitor, preferably selected from the group consisting of ruxolitinib (Verstovsek S. et al, N Engl J Med, 2010, 363: 1117-27), baricitinib (Shi JG. et al, J Clin Pharmacol., 2014, 54(12): 1354-61), momelotinib (Tyner JW. et al, Blood., 2010, 115(25):5232-40), pacritinib (Derenzini E. et al, Expert Opin Investig Drugs., 2013, 22(6):775-85), gandotinib (Ma L. et al, Blood Cancer J., 2013, 3:el09.), fedratinib (Zhou T. et al, Leukemia., 2014, 28(2):404-7), degrasyns (Kapuria V. et al, Cell Signal., 2011, 23(12):2076-85), AG490 (De Vos J., Br J Haematol., 2000, 109(4):823-8), AT-9283 (Dawson M. et al, British Journal of Haematology, 150, 46-57), BMS-911543 (Purandare AV. et al, Leukemia, 2012, 26, 280-288), NS-018 (Nakaya et al, Blood Cancer Journal, 2014, 4, el 74), AC-410 (Fridman J. et al, Journal of Investigative Dermatology, 2011, 131, 1838-1844), BVB808 (Weigert O. et al, The Journal of Experimental Medicine, 2012, 209(2):259-273.), CEP-33779 (Seavey MM. et al, Mol Cancer Ther., 2012, l l(4):984-93), and NMS-P953 (Brasca MG. et al, Bioorg Med Chem., 2014, 22(17):4998- 5012), even more preferably the small molecule is ruxolitinib or baricitinib, still more preferably ruxolitinib. For instance, WO2012078574, WO2015061665, WO2011013785, WO2009017714, WO2008114812, WO2011028864, WO2009085913 WO2011112662, WO2012069202, WO2011101161, WO2012146657, WO2010020905, WO2007115269, WO2011097087, WO2012037132, WO2010141062, WO2007061764 disclose other small molecules having an inhibitory activities against JAK1, JAK2 and/or JAK3 (the disclosure thereof being incorporated herein by reference). In one embodiment, the inhibitor is specific for JAK2 in comparison with JAK3. In one embodiment, the inhibitor is specific for JAK2 in comparison with JAK1. Aptamer

In a particular embodiment of the invention, the leptin signaling inhibitor is an aptamer. In the context of the invention, the term "aptamer" means a molecule of nucleic acid or a peptide able to bind to leptin or to LEPR, preferably to LEPR. The aptamers are nucleic acids, preferably RNA, generally comprising between 5 and 120 nucleotides (Osborne et al., 1997, CurrOpinChem Biol. 1, 5-9). It refers to a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., Science, 1990, 249(4968):505-10.

In a preferred embodiment, the LEPR inhibitor can be selected in order to be unable to cross the BBB (blood brain barrier). Indeed, such an inhibitor may prevent any side effect due to the effect of the inhibitor on the hypothalamus. In this context, the inhibitor can be preferably an antibody or a nanobody.

In a particular embodiment, the LEPR inhibitor is targeted to the kidney. For instance, the LEPR inhibitor is linked to a molecule having specificity to a kidney protein, e.g. an antibody or a derivative thereof. Especially, the inhibitor can be a bispecific antibody or nanobody having both a targeting moiety and a LEPR binding moiety responsible for the antagonist activity. Kidney targeting can also be achieved with a group of peptides known as Low Molecular Proteins (LMWP) with as suitable examples lysozyme, cytochrome C and apro- tein. These LMWPs are specially targeted at the kidneys. They are rapidly cleared out of the bloodstream by glomerular filtration and then quantitatively reabsorbed in the proximal tubular cells, whereafter they are broken down to amino acids by the lysosomes. (Franssen et al, J Med Chem, 1992, 35, 1246-1259; Kok et al. J PharmacolExpTher. 1999, 288, 281-5, the disclosure thereof being incorporated herein by reference). Other strategies for specific drug delivery to kidneys are disclosed in EP953357, by usinge-polylysine conjugatesas disclosed in US2012122788, by saccharides as disclosed in WO2006/104530 or Shirota et al. (J PharmacolExpTher. 2001, 299, 459-67), with a bioabsorbable materialstransiently accumulating in a kidney (US2012196807), by kidney-targeting peptides as disclosed in WO2007/046818 or US20050074812, by engineered nanoparticle (US2012148488), the disclosure of these references being incorporated herein by reference. Kidney-targeting peptides are disclosed in WO2007/046818. In certain embodiments, the targeting agent is an aptamer that specifically binds to kidney cells. Dosage and Regimen

In a more general aspect, the present invention relates to a leptin signaling inhibitor, preferably a LEPR inhibitor, for use as a drug or to the use of a leptin signaling inhibitor, preferably a LEPR inhibitor, for the manufacture of a drug, or a pharmaceutical composition comprising a leptin signaling inhibitor, preferably a LEPR inhibitor, and optionally a pharmaceutically acceptable carrier or excipient.

The leptin signaling inhibitor, preferably LEPR inhibitor, is used for treating patients having a ciliopathy, especially a ciliopathy associated with hyperleptinemia or a ciliopathy associated with obesity.

Preferably, the treatment is a preventive treatment aiming at protecting kidney in patients. In particular, the treatment is aiming at preventing, delaying or decreasing renal dysfunctionor failure.

The leptin signaling inhibitor, preferably LEPR inhibitor, is administered in a therapeutically effective amount. By "effective amount", it is meant the quantity of the pharmaceutical composition of the invention that prevents, removes or reduces the deleterious effects of leptin on kidneys. It is understood that the administered dose may be adapted by those skilled in the art according to the patient, the pathology, the mode of administration, etc. More particularly, by "therapeutically efficient amount of leptin signaling inhibitor, preferably LEPR inhibitor," is intended the amount that is sufficient to protect kidneys.

The leptin signaling inhibitor, preferably LEPR inhibitor, is preferably administered to the patients when the serum level of leptin increases, i.e. reaches a level higher than the "normal" level, preferably at least about 10%, 20%, 30%, 40% higher than the "normal" level. As used herein, the "normal" level refers to the average level of the population, preferably a population of same ethnicity and/or same sex and/or same BMI. For instance, the leptin signaling inhibitor, preferably LEPR inhibitor, administration can begin from above about 12.5 ng/ml.The pharmaceutical compositions of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, or subcutaneous administration and the like. In a particular embodiment, the leptin signaling inhibitor, preferably LEPR inhibitor, is administered locally in or near kidneys.

The pharmaceutical composition comprising the leptin signaling inhibitor, preferably LEPR inhibitor, is formulated in accordance with standard pharmaceutical practice (Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art.

Pharmaceutical compositions according to the invention may be formulated to release the leptin signaling inhibitor, preferably LEPR inhibitor, substantially immediately upon administration or at any predetermined time or time period after administration.

Pharmaceutical compositions according to the invention can comprise leptin signaling inhibitor, preferably LEPR inhibitor, associated with pharmaceutically acceptable excipients and/or carriers. These excipients and/or carriers are chosen according to the form of administration as described above. Finally, the leptin signaling inhibitor, preferably LEPR inhibitor, can be used in combination with any additional drug.

IL-23 as a biomarker

The inventors propose to use IL-23 as a biomarker. Indeed, the level of IL-23 can be indicative to glomerular activity. Then, when the IL-23 level increases, it is indicative of a deterioration of the glomerular activity. Therefore, IL-23 can be used to assess the efficiency of the treatment by the Leptin signaling inhibitor or the necessity to begin such a treatment.

The determination of the level of IL-23 is easier, rapid and more convenient for the following of the glomerular activity through the determination of the creatinine clearance or the albuminuria.

The invention therefore relates to the use of IL-23 as a biomarker of the efficiency of the treatment of the ciliopathy with hyperleptinemia, especially Bardet-Biedl syndrome (BBS), Alstrom syndrome,or MORM syndrome, by the Leptin signaling inhibitor. Indeed, the efficiency can be deduced when the level of IL-23 decreases. Indeed, a decreasing or decreased level of IL-23 is indicative of the efficiency of the treatment.

It also relates to the use of IL-23 as a biomarker for determining the beginning of the treatment of the ciliopathy with hyperleptinemia, especially Bardet-Biedl syndrome (BBS), Alstrom syndrome,or MORM syndrome, by the Leptin signaling inhibitor. Indeed, an increasing or increased level of IL-23 is indicative of an appropriate timer for beginning the treatment. The method comprises providing a biological sample from the patient, determining the level of IL-23 and comparing the level of IL-23 with a level of reference.

The biological sample can be a urine sample or a blood, serum or plasma sample.

The level of IL-23 can be determined by any method available and well-known to the person skilled in the art, for instance with anti-IL-23 antibodies by ELISA.

In the context of the determination of the treatment efficiency, the level of reference can be for instance the level of IL-23 at the beginning of the treatment or before the beginning of the treatment with the Leptin signaling inhibitor.

In the context of the determination of the beginning of the treatment, the level of reference can be a IL-23 threshold from which it is advised to begin the treatment. Alternatively, the level of reference can be a baseline level, corresponding to the IL-23 level in a healthy subject or to the IL-23 level when the glomerular activity is normal.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 3-month-old renal phenotype Bbsl2^v~ fed on chow diet, (a) 3-month-old average body weight of mice with indicated genotype (n = 12). (b) Normalized renal mRNA expression of Bbsl2 (n = 5). Gapdh was used for normalization, (c) Histological representation of Hematoxylin and Eosin (H&E) stained kidney sections. Scale bar: 150μιη. (d) Transmission electron microscopy (T.E.M.) pictures at lower (top panels) and higher magnification (bottom panels) of the glomerulus. BS: Bowman's space, P: Podocyte, PI: Podocyte primary foot process, P2: Podocyte secondary foot process, C: capillary loop, E: endothelium, F: fenestrations. Scale bar: 5 μιη. (e) Measured albuminuria levels in 24-hour urine collection (n=8). (f) Calculated creatinine clearance of the same mice (n = 8). (g, h) Scanning electron microscopy (S.E.M.) pictures of epithelial cells in the proximal convoluted tubule (PCT) and in the collecting duct (CD). Scale bars: 2 μιη. (i) Measured urinary volumes over 24-hour period in fluid-deprived mice (n = 12). j) Urinary osmolality of harvested urine (n = 10 per group), (k) Circulating concentrations of the adipokine Leptin in the same mice (n = 6). Data are expressed as means + s.e.m. *P < 0.05.

Figure 2 Structural defects associated with an activated UPR are present in the Bbsl2^v~ kidney tubules, (a) Transmission electron microscopy (T.E.M.) pictures of the proximal convoluted tubule. L: lumen, (b) Higher magnified pictures of the epithelial cells: mitochondria circled in red, P: cell processes yellow arrows, BM: basolateral membrane, BM_E: basement membrane of endothelium and S: supporting tissue, (c) 3D pictures of immunostained sections with tight junctions detected with anti-ZO-1 and mitochondria with anti-COXIV. (d) T.E.M. pictures at different magnifications of the epithelial cells in the collecting duct for the indicated genotypes. L: lumen, (e) Kidney sections immunostained for Apical Aquaporin 2 (AQP2) in green and basolateral Aquaporin 3 (AQP3) in red, with nuclei counterstained with DAPI. L: Lumen. Scale bars: 10 μιη. (f, g) Renal protein level for AVPR2 and AQP2 presented as the ratio of protein of interest over β-Tubulin in total kidney extracts, (h) Plasma levels of anti-diuretic hormone Arginine Vasopressin (A VP) in mice after indicated time of fluid deprivation, (n =6). (i) Normalized renal mRNA expression of unfolded protein response (UPR)-related genes in kidney extracts. Gapdh was used for normalization, (n = 6). Data are expressed as means + s.e.m. *P < 0.05.

Figure 3 Effects of diet- induced obesity on Bbsl2^v~ renal phenotype at 16 weeks, (a) Average body weight of 16- week-old mice with indicated genotype after 4 weeks of high fat/high glucose diet (n = 6). Corresponding plasma levels of Leptin (b), creatinine (c), 11-6 (d) and MCP- 1 (e) (n = 6). (f) Representative photographs of Toluidin blue stained sections. Scale bars: 50 μιη in top panels and 20 μιη in bottom ones, (g) Normalized renal mRNA expression of Caspases and inflammatory markers in total kidney extracts. Gapdh was used for normalization, (n = 6). Data are expressed as means + s.e.m. *P < 0.05.

Figure 4 Inflammation and apoptosis impair kidney function in obese, 6-month-old Bbsl2^v~ mice. Average body weight of 6-month-old mice with indicated genotype fed on HF/HG diet (n = 6). Corresponding plasma levels of Leptin (b), creatinine (c), 11-6 (d) and MCP-1 (e) (n = 6). (f) Normalized renal mRNA expression of inflammatory markers in total kidney extracts. Gapdh was used for normalization, (n = 4). (g) Representative 3D image of kidneys sections immunostained for Μφ with a CD68 antibody (red). Nuclei were counterstained with DAPI (blue), (h) Representative pictures of kidneys sections immunostained for tight -junctions (ZO- 1 in red) and effector Caspase3 (green). Nuclei were counterstained with DAPI (blue). Scale bars: 20 μιη. (i) Representative pictures of TUNEL-stained kidney sections. Scale bars: 50 μιη. j) Normalized renal mRNA expression of Socs3 in total kidney extracts of the 6-month-old mice. Gapdh was used for normalization, (n = 4). (k) Representative 3D image of a glomerulus immunostained for STAT3 (red) and LEPR (green) generated with Zeiss Zen 2012 program. Data are expressed as means + s.e.m. *P < 0.05.

Figure 5 Effects of Leptin injections on renal phenotype of 8-week-old mice, (a) Corresponding plasma levels of creatinine from the treated mice (n = 6). (b) Normalized renal mRNA expression of inflammatory markers in total kidney extracts. Gapdh was used for normalization. (n = 4). H&E stained histological sections of the glomeruli (c) and of the tubules (d) following the Leptin injection with the indicated genotype Scale bars: 10 and 25μιη respectively, (e) Representative TUNEL-stained pictures of kidney sections from the Leptin injected mice. Scale bars: 40 μιη. (f) Representative 3D images of glomeruli immunostained for Μφ (CD68 in red) and nuclei in blue, (g) Top panel, representative 3D images of glomeruli immunostained for Μφ (CD68 in red) and nuclei in blue from Bbsl^v~ with or without 11 -day leptin injection. Bottom panel: toluidine blue stained histological sections correlating with the immunostained section of the top panel, (h) Circulating plasma leptin levels in mice with or without 11-day- leptin injections for the indicated groups (n = 4-6). (i) Top panel, representative 3D images of glomeruli immunostained for Μφ (CD68 in red) and nuclei in blue from BbsKfl^ Pod-Cre^+,~ with or without 11 -day leptin injection. Bottom panel: toluidine blue stained histological sections correlating with the immunostained section of the top panel, (j) Top panel, representative 3D images of glomeruli immunostained for Μφ (CD68 in red) and nuclei in blue from BbsKfl^ LPJ¹^; Pod-Cre^+,~ with or without 11-day leptin injection. Bottom panel: toluidine blue stained histological sections correlating with the immunostained section of the top panel, (k) Normalized renal expression of IL23 in total kidney extracts from mice with the indicated genotype (X-axis) with or without Leptin (figure legend) (n = 4 per group). Gapdh was used for normalization. Data are expressed as means + s.e.m. *P < 0.05. Scale bar for all images where not indicated otherwise: 10 μιη.

Figure 6 Neutralizing Leptin signaling preserves kidney function in the Bbsl2^v~ mice.

(a) Normalized renal mRNA expression of Socs3 in total kidney extracts of the Leptin injected mice. Gapdh was used for normalization, (n = 4). Data are expressed as means + s.e.m. *P < 0.05. (b-d) Ratio of grayscale intensities for immunodected bands of STAT3-P (Ser727) over STAT3, p-NFkB-p65/NFKB-p65 and SOCS3/p-TUB respectively of the Leptin injected mice.. (e) Representative pictures of kidneys sections from Leptin injected mice immunostained for STAT3-P (Ser727) (green) and STAT3 (red). Nuclei are counterstained with DAPI (blue), (f) Average body weight of 9-week-old mice on chow-diet which received a daily injection of Leptin and neutralizing LEPR antibody (LEPR Ab.) and (g) the corresponding plasma levels of Leptin (n = 6). (h) Representative pictures of kidneys sections from Leptin and LEPR Ab. injected mice immunostained for STAT3-P (Ser727) (green) and STAT3 (red), (j) Corresponding average plasma levels of Creatinine from the Leptin and LEPR injected mice, (i) H&E stained histological sections of the glomeruli from the Leptin and LEPR injected mice. Scale bars: ΙΟμιη. (k) Normalized renal mRNA expression of Socs3 in total kidney extracts of the Leptin and LEPR Ab. injected mice. (1) Normalized renal expression of IL23 in total kidney extracts from Bbsl2^v~ mice injected following the same experimental procedure with the indicated injected antibodies (X-axis) with or without Leptin (figure legend), (n = 3 per group). Gapdh was used for normalization, (n = 4). Data are expressed as means + s.e.m. *P < 0.05. Figure 7. Immunodetection of JAK1 and JAK2 isoforms in the glomeruli of adult mouse kidneys depicting the presence of JAK2 rather than JAK1. JAK1 or JAK2 in green, Leptin receptor in red and DAPI in blue.

EXAMPLES

Therapeutic strategies to block renal leptin signaling prevent progressive renal dysfunction in Bardet Biedl syndrome models

Bardet-Biedl syndrome (BBS) is a ciliopathy disorder whose clinical features includes renal failure although the mechanism for this remains unknown. We utilisedfib5^,i2^"/" mice to identify the mechanism behind BBS-mediated renal dysfunction with the hope of thereby identifying a strategy to prevent it. Non-obese Bbsl2^v~ mice displayed generalized mild renal structural defects with an unfolded protein response in ciliated epithelial cells. Onset of obesity was associated with progressive plasma leptin elevation which in turn correlated with renal function deterioration with increased lesions, glomerular macrophage infiltration and inflammation,.; this adverse renal effect was reproduced by leptin administration to non-obese Bbsl2^v~ as well as to Bbsl^v~, podocyte-specific BbslO knockout mice (Bbslff^; Pod-Cre^+/~) mice. Leptin- induced renal pathology was specifically mediated by phosphorylation of the serine727 residue of STAT3 in the glomeruli and associated with IL23 production.. Notably, cell-specific leptin receptor inactivation in the BbslO-deficient podocytes protected the kidney against the deleterious effects of hyperleptinemia as did the administration of a neutralizing leptin receptor antibody. Hence therapeutic strategies to block renal leptin signaling could help protect BBS individuals from severe renal complications.

RESULTS

Renal phenotype in non-obese, chow-fed Bbsl2^~' ' mice

The inventors started by studying 12-week-old, non-obese Bbsl2^v~ mice (Fig.la) with no detectable Bbsl2 expression in the kidneys (Fig. lb). These mice exhibit neither kidney enlargement nor cystogenic lesion (Fig. lc). Structural analysis of the glomerular filter highlighted significant general size reduction (Fig. Id) with the podocytes missing primary and secondary structures (Fig. Id, lower panel). This defect was correlated with an increase in microalbuminuria levels (Fig. le). Renal function was maintained as depicted by similar creatinine clearances between the tested groups (Fig. If). Subsequent electron microscopy analysis of the epithelial cells in the proximal convoluted tubule (P.C.T.) and collecting duct (CD.) showed normal ciliated epithelial cells, irrespective of the genotype (Fig. lg, h). The common BBS patient's phenotype of polyuria was on the other hand recapitulated in the Bbsl2^{~ A} mice as 24-hour urinary volumes were increased (Fig. li) and urinary osmolality was decreased (Fig. lj). The Renin and Angiotensin II hormone system also involved in regulating fluid balance as well as the classical electrolytes and macromolecules: Chloride (CI ), Phosphate (P ), Potassium (K⁺) and Calcium (Ca²⁺) ions, urea and total protein concentrations were all similar between WT and Bbsl2^v~ animals. Moreover, no Caspase activity or TUNEL positive apoptotic nuclei were detected in Bbsl2^v~ kidneys indicating absence of cell death at 3 months. Of note, although the mice were not obese, they anyway displayed increase circulating plasma leptin concentrations (Fig. Ik).

Cortical collecting duct epithelial cells display adaptive structural modifications

The Bbsl2^v~ PCT epithelial cells showed no major structural defect (Fig. 2a) with the epithelial cells properly polarized and well-organized in monolayers. However, at higher magnification, the basement membrane, the supporting tissue and the basement membrane (BM_E) were abnormal (Fig. 2b, blue arrows). The basolateral membranes of these cells (Fig. 2b, yellow arrows) were significantly thicker in Bbsl2^v~ with longer and denser cellular processes pushing away the mitochondria from the basolateral side (Fig.2B, red circles); an effect also observed by immunodetection (assessed by COX-IV staining) (Fig. 2C). On the other hand, the Na⁺ pump (ATP1A1) relying on the adjacent mitochondrial ATP retained its basolateral localization and no difference in expression levels of key epithelial genes involved in Na⁺ /H⁺/K⁺ urinary balance (Atplal, Nedcl4l, Nr3c2, ENaC and Sgkl ) as well as in protein contents of the ATP1A1 and epithelial Na⁺ channel isoforms a and β (ENaCa, β) were found.

Polyuria in BBS correlates with an activated unfolded protein response (UPR) in renal epithelial cells

Next, to understand the origin of water wasting in Bbsl2^v~ mice, the inventors studied the A VP- responsive of CD epithelial cells. These cells were also correctly polarized (Fig. 2d) without detectable structural defect. Congruently, correct apical targeting of Aquaporin 2 (AQP2) to the luminal side and of Aquaporin 3 (AQP3) to the basolateral side were observed in WT and Bbsl2^v~ mice (Fig. 2e). However, they found a significant reduction of AQP2 and Arginine Vasopressin receptor 2 (AVPR2) expression levels in Bbsl2^v~ mice (Fig. 2f, g). This decrease in AVPR2 correlated with an AVP-resistant status as depicted by constant higher levels of circulating AVP levels in the Bbsl2^v~ mice, regardless of their fluid consumption status (Fig. 2i). As BBS 12 inactivation impairs protein targeting to the primary cilium and hence, causes an accumulation of proteins in the endoplasmic reticulum (ER), the inventors verified if the decrease in AQP2 and AVPR2 could be link to an ER stress triggering an unfolded protein response (UPR). Key UPR genes, namely Bip, ATF4 and CaspaseH were significantly increased in Bbsl2^v~ total kidney extracts (Fig. 2i); an effect even more pronounced in purified renal epithelial cell extracts including the transcription factor Chop 10.

One month of high fat/high glucose (HF/HG) diet severely exacerbates renal defects in Bbsl2^~/~ mice

In order to assess the secondary effects of BBS-mediated obesity on renal function, the inventors fed 12-week-old male Bbsl2^v~ and WT mice with a HF/HG diet for 4 weeks. The Bbsl2^v~ mice became significantly fatter compared to their WT controls (Fig. 3a) and displayed hyperleptinemia (~ 80ng/mL) (Fig. 3b) without hyperglycemia. Plasma creatinine levels were unchanged (Fig. 3c) but signs of systemic inflammation were detected such as increased circulating levels of the pro-inflammatory cytokine, IL-6 (~2pg/mL; Fig. 3d). The circulating levels of the monocyte chemoattractant protein-1 (MCP-1), one of the key chemokines that regulates migration and infiltration of monocytes/macrophages (Μφ), were at this point unchanged (Fig. 3e). Yet, histological analysis highlighted severe dilated tubular lesions in the Bbsl2^v~ kidneys (Fig. 3f) associated with local upregulation of Mcp-1, inducible nitric oxide synthase (iNos), a pro-inflammatory enzyme expressed by Th-1 activated Μφ (Fig. 3g) and Caspasel2 expression.

Prolonged HF/HG diet leads to inflammation-mediated apoptosis in Bbsl2^~/~ kidneys Next, the inventors kept the mice on the HF/HG diet for 6 months and, under these diet conditions, both WT and Bbsl2^v~ mice became obese; Bbsl2^v~ mice were significantly fatter (Fig. 4a). Both groups showed elevated levels of circulating Leptin with Bbsl2^v~ mice presenting severe hyperleptinemia (~150ng/mL; Fig. 4b) and increased plasma creatinine indicating renal dysfunction (Fig.4c). Systemic inflammation was, this time, prominent in Bbsl2 as circulating levels of IL-6 (~ 4pg/mL; Fig. 4d) and MCP-1 (~ 120pg/mL; Fig. 4e) were increased. Inflammation was also conspicuous in the Bbsl2^v~ kidneys with Mcp-1 and iNos also significantly higher (Fig. 4f). This correlated with high levels of Μφ infiltration in the Bbsl2^v~ kidneys (Fig. 4g), increased expression levels of Caspase 12 and the effector Caspase 3 (Fig. 4h) as well as full-blown apoptosis (Fig. 4i). In addition, the Suppressor of Cytokine Signaling 3 (Socs3) whose expression is known to correlate with the severity of inflammation, kept increasing with time (Fig. 4j). Leptin injections recapitulated Μφ infiltration in both young, non-obese Bbsl2^~/~ and Bbsome BbsV^' mice

The inventors identified a positive correlation between plasma leptin concentrations and renal defects in Bbsl2^v~ mice; i.e. the progressive renal defects were mirrored by increasing plasma leptin concentrations. As the leptin receptor is renally-expressed and co-localizes with STAT3 in the glomeruli (Fig. 4k), the inventors hypothesized that excessive renal leptin signaling might underlie renal failure in Bbsl2^v~. To test this hypothesis, the inventors injected leptin intraperitoneally in non-obese 8-week-old male Bbsl2^v~ mice for 11 days. No significant weight difference was detected between the groups at the end of the leptin treatment although plasma leptin concentrations were substantially increased post-treatment. Notably, Bbsl2^v~ mice developed higher plasma creatinine post-leptin treatment, consistent with a negative effect of leptin on renal function (Fig. 5a). Renal IL-6 and Mcp-1 expression were both increased post- leptin treatment (Fig. 5b) and glomerular morphological defects plus tubular dilation (Fig. 5c, d) were observed in the Bbsl2^v~ kidneys. TUNEL-positive apoptotic cells were detected in leptin-treated Bbsl2^v~ mice (Fig.5e) and immunofluorescence imaging demonstrated Μφ infiltration (Fig.5f) in the glomerular region. As leptin signaling in the hypothalamus is mediated by phosphorylation of STAT3 on tyrosine residue 705 (STAT3-P (Tyr705)) resulting in activation of Socs3 transcription, the inventors immuno- stained for STAT3 and STAT3-P (Tyr705) in the leptin-treated kidneys. STAT3 but not the STAT3-P (Tyr705) form was readily detected in the glomerular region, suggesting that leptin signaling in the kidney is different to the hypothalamus.

Next, to ensure the observed renal phenotype was not limited to BBS 12 mutation only, the inventors extended the findings to the Bbsl knockout mouse model (Bbsl ^). Using this model they confirmed that the deleterious effect of leptin on the glomeruli was a shared phenotype of multiple BBS models; in leptin-treated Bbsl '^' mice increased Μφ infiltration (Fig. 5G, upper panel) again correlated with critical structural defects in the glomerular region (Fig. 5G, lower panel) after 11 days of leptin treatment.

Podocyte specific deletion of the leptin receptor prevents glomerular pathology in BbslO- deprived podocytes

The inventors used a conditional knockout model for the BbslO gene (BbslO^) to further validate that glomerular leptin signaling was deleterious for BBS-affected kidneys. To specifically inactive BbslO in the podocytes, BbslO^¹ mice were bred with the Podocin-Cre mouse line to obtain a line of BbslO^; Pod-Cre^+/~. Specific Cre activity was assessed by crossing the Pod-Cre mice with the RosaTomato/eGFP (Gt (ROSA) 26Sortm4 (ACTB-tdTomato,- EGFP) _mi_ce Specific green fluorescence was detected in the glomeruli of the Pod-Cre^+/~; Gt (ROSA) 26Sortm4 (ACTB-tdTomato,-EGFP) LUO/J _{mice indicating spe}cific Cre-mediated excision in the podocytes when mice are bred with the Pod-Cre mice. Subsequently, immunodetection of BBS 10 was negative in Bbslffi^; Pod-Cre ^+/"glomeruli indicating deletion of BBS 10 in the podocytes. On transmission EM analysis podocyte-specific BbslO inactivation impaired the development of both primary and secondary podocyte structures in a similar fashion as seen in the constitutive Bbsl0^v~ knockout mice. Following 11 days of leptin treatment, elevated plasma leptin concentrations (Fig. 5h) and renal Μφ infiltration (Fig. 5i, top panel) were readily detectable in the Bbslff^; Pod-Cre^+/~ kidneys which again correlated with severe structural defects in the glomeruli(Fig5i, lower panel). To confirm that local leptin signaling was driving the injurious phenotype on the weakened BBS glomeruli, the inventors bred the BbslO^; Pod- Cre^+/~ mice with a leptin receptor floxed mouse model (LPJ¹^). The resulting Bbslff^; LPJ¹^; Pod Cre^+/~ mice lacking both BBS 10 and leptin receptor in their podocytes received 11 days of leptin treatment resulting in high serum leptin (Fig. 5h), but notably no Μφ infiltration was seen in the glomerular region (Fig. 5j, top panel) and there was an absence of severe structural defects of the glomeruli (Fig. 5j, bottom panel). Very recently, Goto et al. (2015, International Immunology, 28, 197-208) demonstrated that the pro-inflammatory leptin signaling in the podocytes was mediated by the secretion of IL23 which prompted the inventors to check and use the IL23 expression levels as a biological readout for leptin signaling in the BBS mice. Interestingly, leptin injection in either Bbsl^v~, or Bbsl2^v~ or Bbslff^ ;Pod-Cre^+/~ induced a significant increase in IL23 levels compared to control saline injection (Fig. 5K), an effect which was not observed when the leptin receptor was specifically inactivated in the podocytes (BbsK ^; LR^R^ Pod-Cre^+/~) hence proving that it is the podocytes which is driving the proinflammatory response upon leptin signaling in the kidney.

Neutralizing leptin-mediated phosphorylation of STAT3 Serine 727 residue protects BBS kidneys

During the acute hyperleptinemia induced by leptin injections, the inventors observed a decrease in renal Socs3 expression levels (Fig. 6a) that strongly contrasted with the observed increase over time in renal Socs3 expression levels as Bbsl2^v~ mice developed obesity (Fig. 4j). Depletion of Socs3 with a concomitant Mcp-1 increase has previously been attributed to a hypermethylation of the CpG islands inside the Socs3 promoter driven by a STAT3-NKB-p65 interaction; a pathway linked to the phosphorylation of STAT3 on Serine727 (STAT3-P (Ser727)) rather than on Tyrosine705 (STAT3-P (Tyr705)). This prompted the inventors to check in BbsI2 ^A mice for the presence of STAT3-P (Ser727), p-NFKB-p65 and SOCS3 following l ldays of leptin injections (Fig. 6b-c). A significant increase in the ratio of STAT3- P (Ser727) to total STAT3, and ratio of p-NFKB-p65 to total NFi B-p65 together with a decrease in level of SOCS3 protein was observed (Fig. 6d). In parallel, strong nuclear immunostaining for STAT3-P (Ser727) was found in the leptin-treated Bbsl2^v~ kidneys.

As leptin signaling appeared to be the trigger for the signaling cascade leading to renal failure in BBS mice, the inventors intend to prevent the phosphorylation of STAT3 in Bbsl2^v~ mice by inhibiting the binding of leptin to its renal receptor. This time, leptin was co-injected with a neutralizing antibody to the leptin receptor (LEPR). No significant weight difference was observed between the groups at the end of the leptin/anti-LEPR combined treatment (Fig. 6f) although circulating leptin concentrations was as expected increased in both groups, albeit to a greater extent in the Bbsl2^v~ mice (Fig. 6g). In the presence of anti-LEPR treatment, almost no STAT3-P (Ser727) was detected in the glomeruli (Fig. 6h) and the plasma creatinine remained similar between groups (Fig. j). Moreover, severe structural defects were absent in the glomeruli (Fig. 6i), and no reduction was detected in renal Socs3 mRNA levels (Fig. 6k), thereby contrasting with the reduced Socs3 and associated high pro-inflammatory markers and TUNEL-positive cells in the leptin-alone treated Bbsl2^v~ kidneys. Finally, Bbsl2^v~ mice were injected either with leptin or vehicle with either a control rabbit IgG or the neutralizing Leptin receptor antibody followed by the measurement of the renal IL23 expression levels (Fig. 6L). A significant increase in IL23 expression was measured in the kidneys of the mice that received leptin with the control rabbit IgG which did not occur when injected with the LR neutralizing antibody, hence validating the efficacy of this antibody to protect the kidneys of the Bbsl2^v~ mice from leptin-mediated pro-inflammatory response.

Overall, these results demonstrate that blocking renal leptin signaling is effective in protecting BBS kidneys against deteriorating renal function.

The inventors have shown (Figure 7) that the isoform JAK2 is expressed in glomeruli whereas JAK1 is only very weakly expressed. The presence of JAK2 in glomeruli proves its involvement in the renal dysfunctions related to leptin pathway.

DISCUSSION

Renal failure is a major clinical problem in BBS patients, in which it is a leading cause of death. No specific interventions are available to prevent progression of renal dysfunction in BBS with development of treatments hindered by a lack of insight into the exact mechanisms underlying renal disease in this syndrome. These results highlight the complex multimodal mechanism that contribute to renal disease in BBS, with primary defects in renal epithelial cells mediated by a lack of BBSome function in these cells, interacting with the deleterious renal effects of high levels of circulating leptin consequent upon BBS-mediated obesity. These important mechanistic insights provide the first opportunities to develop specific targeted treatments to prevent progression of renal dysfunction in BBS patients.

The findings first demonstrated that non-obese Bbsl2^v~ mice already exhibit polyuria and albuminuria due to a decrease in AVPR2 and AQP2 expression levels in renal epithelial cells, due to UPR activation consequent upon BBS-mediated impaired intracellular protein trafficking. As previously seen in photoreceptors, BBS 12 inactivation impairs the traffic of ciliary proteins from the renal epithelial cell towards the primary cilium resulting in ciliary proteins accumulating in the ER thereby activating the UPR. Although, the findings showed the UPR was activated in Bbsl2^v~ renal epithelial cells, this was not associated with the massive apoptosis seen in photoreceptors when protein traffic is markedly blocked by BBSome inactivation. This may be because the cilium in the renal epithelial cell is primarily a detection device and is less involved in protein transport when compared to cilium function in photoreceptors. Nevertheless, this renal cell mild UPR due to impaired cilium function in the Bbsl2^_/" kidney reduced AVPR2 expression thus causing the observed AVP resistance (Fig. 2h). The study showed that the glomerular defect and associated albuminuria was not due to apoptosis in mature kidneys as TUNEL assays showed absence of apoptosis in Bbsl2^v~ kidneys at 12 weeks of age.

These findings strongly suggest a developmental origin for the observed initial renal defect in Bbsl2 ^v~ prior to the onset of obesity and hyperleptinaemia. This hypothesis is supported by the fact that the immature podocyte during development carries a primary cilium, which subsequently disappears. Inactivation of the ciliary BBS proteins might impair natural podocyte development.

BBSome inactivation whether induced by Bbsl2, BBS1 or BBS10 inactivation causes progressive acquired structural defects throughout the nephron. Although impacting renal function to a modest degree, our findings highlight that BBSome inactivation in the kidneys by itself does not result in severe renal dysfunction and failure. Instead, it is the interaction of obesity, and more specifically obesity-associated hyperleptinemia, with primary BBSome- induced renal defects, that is critical to renal failure in BBS. Notably, this study revealed a close concordance between rising plasma leptin levels and progressive renal pathology including pro-inflammatory Μφ infiltration, leading to the critical hypothesis that leptin signaling was central to progressive renal dysfunction in BBS. Key support for this hypothesis was provided by evidence of progressive renal pathology in response to leptin administration to non-obese Bbs l2 mice. In addition to its hormonal role, leptin also acts as an inflammatory cytokine, modulating humoral and cell-mediated immune responses through STAT3 phosphorylation.

With 19 BBS genes identified and to prove that the deleterious effect of leptin is not unique to the Bbsl2^v~ model, the hypothesis was also tested in Bbsl ^ mice, with Bbsl being the most frequently mutated gene in human subjects with BBS and whose gene product is part of the BBSome complex, as well as in podocyte-specific BbslO^v~ mice. Notably, the same glomerular phenotype in response to leptin treatment was observed in both Bbsl '^' and Bbslffi^; Pod-Cre^+/~ mice. Thus the negative effect of elevated leptin mediates on function of the BBS-affected kidney appears to be a generalised phenomena not dependent on the actual BBS mutation causing the phenotype.

As leptin is a pleiotropic adipokine known to modulate immune responses, the inventors next investigate the target cells on which the excess circulating levels of leptin were acting to promote renal deterioration. Using the conditional Bbslff^ mouse model, they generated a double-conditional KO mouse model for both BbslO and the leptin receptor in the podocytes {Bbslffi^; LR^fi; Pod-Cre^+/~) which, when injected with leptin, failed to develop glomerular lesions (Fig. 5i, j). These findings clearly demonstrated that it is the local glomerular leptin- signaling pathway that is critical to renal dysfunction. These data also pointed to a key role of Μφ activation and infiltration characterized by the synthesis and secretion of IL-6 and iNOS in that paralleled the increasing obese phenotype. Intriguingly, Socs3 and Mcp-1 levels were inversely regulated by leptin treatment in the non-obese Bbsl2^v~ kidneys; i.e. Socs3 was decreased whereas Mcpl was increased (Fig. 5e and k, respectively). Leptin-mediated inhibition of Socs3 has already been described in adipose tissue where leptin signaling was fueling inflammation. The inventors demonstrated the existence of a differential regulation by leptin of STAT3 with phosphorylation on the Ser727 residue of STAT3-P (Ser727) rather than on the Tyr705 residue STAT3-P (Tyr705). This increase in STAT3-P (Ser727) was associated with Socs3 repression and Mcp-1 upregulation. Upon analysis of the leptin-treated Bbsl2^v~ kidneys, the same pattern with phosphorylation of STAT3 on the Ser727 residue was observed. The observed decrease of Socs3 followed by the upregulation of Mcp-1 is likely involved in recruitment and activation of infiltrating renal Μφ upon leptin treatment, consistent with previous reports of Μφ being a major cause of renal injury. Notably, the injection of neutralizing anti-LEPR antibody was efficient in preventing all these deleterious renal effects of leptin (Fig. 6g) thereby preventing progressive renal failure in the leptin-treated BBS mice. Hypothalamic leptin resistance has historically been thought to be the leading cause for the obese phenotype in BBS, although recent data has challenged this hypothesis45. The inventors put in evidence that preobese Bbs4^v~ mice respond to leptin. A similar finding was observed with preserved leptin-mediated phosphorylation of STAT3 Tyr705 in hypothalamic extracts of 9-week-old pre-obese Bbsl2^v~. Of interest, Bbsl2^v~ mice had significantly higher levels of hypothalamic STAT3-P (Tyr 705) proving that prior to development of obesity not only can the Bbsl2^v~ hypothalamus detect leptin, it can also exhibit a graded response to the higher circulating levels of leptin in the Bbsl2^v~ mice. Hence, hypothalamic and renal leptin resistance may not be an all or none phenomena but instead reflect relative resistance of key STAT3 residues to leptin-mediated phosphorylation, with other residues remaining relatively sensitive to leptin-mediated phosphorylation, and thereby mediating the deleterious effects of leptin even in the presence of ostensible 'leptin resistance'. This would parallel the findings in insulin resistance states where the anabolic effects of insulin signaling are relatively preserved by contrast to its metabolic effects, thereby potentially explaining the increased cancer risk in insulin resistance states.

The high circulating leptin levels associated with increased adipose leptin secretion in BBS is also linked to enhanced adipogenic differentiation, potentially leading to a vicious positive feedback cycle that results in ever higher leptin levels. As shown here, these increased leptin levels are a leading contributor to BBS-associated renal failure and hence as an alternative to blocking renal leptin signaling, another strategy to protect BBS kidneys could be to reduce adipose leptin secretion. Leptin secretion is directly proportional to the amount of glucose adipose tissue absorbs. In BBS, adipose tissue has an increased propensity to absorb glucose, and hence it also secretes more leptin. Interestingly, in a previous study the inventors have fed obese Bbsl2^v~ mice with a high fat diet (HFD). In retrospect, comparison of the leptin concentrations between HFD-fed and chow-fed mice, underline a significant drop in circulating leptin concentrations in the HFD-fed Bbsl2^v~ mice. Hence feeding Bbsl2^v~ mice with a HFD is able to decrease leptin concentrations, as shown previously in rats. If this applies to human BBS, then restriction of dietary carbohydrate and a corresponding increase in dietary lipid content, could be beneficial for lowering leptin levels and preserving renal function in BBS subjects.

In conclusion, the inventors demonstrated, using mouse models for three different BBS genes that Bbs inactivation in the kidneys weakens them thereby making them sensitive to the deleterious effects of hyperleptinemia that is a feature of BBS-mediated obesity. The inventors hereby provide the first evidence that BBS-mediated renal failure may in fact be preventable by inhibiting leptin signaling in the kidney or by reducing adipocyte leptin production. This could thereby represent a major therapeutic avenue to preserve kidney function in BBS patients.

METHODS

Animal husbandry

The Bbsl2 ^A mice (Bbsl2^tmL1Vmar; Mouse Genome Informatics Identity number: MGI: 5444297 were previously described). BbslO floxed mice were generated at the Mouse Clinic Institute of Strasbourg. Podocin- Cre mouse line and the Leptin Receptor floxed mouse line (Strain name: B6.Cg-Tg (NPHS2-cre) 295Lbh/J; Stock number: 008205 and Strain name: B6.129P2- Lepr^tmlRck/J; Stock number: 008327 respectively) were purchased at Jackson laboratories. BbsP ^7" mice. For this study, mice were on a C57/BL6 genetic background.

All animals were housed in a temperature and humidity controlled facility, with a 12h-light/12h- dark cycle fed with chow diet (LM-485; Harlan Teklad Premier Laboratory Diets) and tap water ad libitum. For obesity inducing diet, mice were fed ad libitum with Harlan, Teklad Custom Research Diet TD08811 with 47.6% carbohydrate and 23.2% fat. The experiments were approved by the appropriate local ethics committee.

Water deprivation and urine collection

12-week-old mice were used. The 24h-diuresis quantification and collection was achieved using the Tecniplast® diuresis cage (#01-288-2D, Tecniplast®, USA). Mice were undisturbed for 6 days after entering the cage for habituation. Cages were kept in a calm room, with a 12h- light/12h-dark cycle. Animals had access to chow diet food, and to water ad libitum before the beginning of water deprivation experiments. Water was withdrawn on the morning of the seventh day. Food access was maintained ad libitum. The experiment was stopped 24h later, diuresis was quantified and then mice were sacrificed in order to collect blood and organs. Weight was measured at the beginning and at the end of water deprivation for each mouse in order to quantify weight loss. Blood and urine were immediately centrifuged after collection and kept frozen at -80°C until analyzed.

Biochemical assays

Analyses were performed on the 24h-urine volume, collected under water deprivation conditions. Blood jugulo-carotidal collection was achieved by decapitation. Urinary osmolarity was measured at the MCI on 20μ1 of sample on an Osmometer (Fiske associates). Blood was collected in a heparin-coated tube after decapitation. Plasma and urinary creatinine contents were measured with Creatinine (serum) Assay Kit and Creatinine Assay Kit (Catalog #: 7000460, 500701; Cayman Chemical). After centrifugation at 1,000 g for 5 min at 4°C, the plasma was used according to manufacturer's instructions. Plasma leptin, A VP, Renin, Angiotensin II Aldosterone , Π-6, MCP-1 and Microalbuminuria levels were measured using the mouse Leptin ELISA kit (Catalog: #: EZML-82K, Millipore, Billerica, Massachusetts, USA), Arg8- Vasopressin ELISA kit (Catalog #: KA0301, Abnova .Inc.), mouse Renin ELISA kit (Catalog #: NB-E 20271 ; NOVATEINBIO, USA), Angiotensin-II EIA kit (Catalog #: EK- 002-12; Phoenix Pharmaceuticals, INC), Aldosterone EIA kit Monoclonal (Catalog #: 10004377; Cayman Chemical), mouse Microalbumin ELISA kit (Catalog: #KT-343, Kamiya Biomedical Company, Thousand Oaks, California, USA) and Mouse IL-6 ELISA Kit (Catalog: #EZMIL6, Millipore). Glycemia was determined with tail blood using the STAT STRIP Xpress glucometer (Nova Biomedical, UK).

Immunofluorescence and TUNEL Assay

For immunofluorescence and TUNEL assays, kidneys freshly sampled were included in Optimal Cutting Temperature Compound™ (OCT™, Catalog* 4583, Tissue-Tek® OCT™, Sakura® Finetek, Torrance, California, USA) and cryosections of 7μιη were cut with Cryostat Leica CM1950. Cryosections were washed with lx PBS and fixated in 4% formaldehyde solution for 15 min (Catalog #: F555-4L, Sigma- Aldrich, Saint-Louis, Missouri, USA) and then permeabilized with 0.02% SDS-PBS for 30 seconds. Blocking solution was 5%-Bovine Serum Albumin (BSA) in PBS. Primary antibodies were diluted in blocking solution and incubated overnight and indicated secondary antibodies were diluted in PBS for 30 minutes. The TUNEL assays were performed using an In Situ Cell Death Detection kit (Catalog #: 11684795910; Roche Applied Science, Penzberg, Germany) according to the manufacturer's procedure. Nuclei were counterstained with Hoechst (Catalog #: D1306, Invitrogen, Carlsbad, California, USA ). Slides were then mounted with Vectashield® Mounting Medium (Catalog #: H- 1000, Vector Laboratories, Burlingame, California, USA). Images were acquired and analyzed with Zeiss Imager.Z2 microscope equipped with either Zeiss Axio Vision or Zeiss ZEN 2012 software (Carl Zeiss Inc., Oberkochen, Germany).

In vivo Leptin treatment

Mice were treated at 8 weeks. Mice were injected 11 days with 20μg of intraperitoneal recombinant Leptin, diluted in NaCl 0.9% (Leptin mouse recombinant, ALX-201-035-M001, Enzo Life Science Inc., Framingdale, New York, USA). Mice were weighed before each injection and Leptin was injected at 5:00p.m. For the neutralizing Leptin receptor antibody experiments, 8-week-old male mice were injected with a combination of 20μg recombinant Leptin and 4μg/Kg body weight as previously described of a Leptin/Obese receptor (OBR/LEPR) antibody (Rabbit Anti-Mouse OBR antiserum 1; Catalog #: OBR11-S) for 11 days prior to euthanasia and analysis. Rabbit IgG antibody was used as control (Catalog #:

20009-1-200).

Statistical analysis

Data are reported as mean + SEM. Results were analyzed using a two-tailed student's t test using GraphPad Prism software. Significance was accepted at P < 0.05.

List of primary and secondary antibodies used in this study

Primary Antibody Host Manufacturer Catalog #

AVPR2 Rabbit polyclonal AbDserotec 9536-0004

AQP2 (H-40) Rabbit polyclonal Santa cruz sc-28629

AQP3 (C-18) Goat polyclonal Santa cruz sc-9885

ATP1A1 Rabbit polyclonal Proteintech 14418-1-AP

AVPR2 Rabbit polyclonal Abeam abl3149

CD68 Rat monoclonal Abeam Ab53444

Leptin Receptor Rabbit polyclonal Abeam Ab5593

STAT3 Mouse monoclonal Abeam Ab50761

STAT3-P (Tyr705) XP®Rabbit mAb Cell Signaling 9145

STAT3-P(Ser727) Rabbit polyclonal Cell Signaling 9134

β-Tubulin Mouse monoclonal Euromedex 1TUB-2A2

COX-IV Rabbit monoclonal Cell signaling 4753

ENaC Rabbit polyclonal Proteintech 10924-2- AP

ENaC β Rabbit polyclonal Proteintech 14134-1-AP

GFAP Rabbit polyclonal Sigma G 9269

ZO-1 Mouse monoclonal Invitrogen 339100

iNOS Rabbit polyclonal Cell Signaling 2977

MCP-1 Mouse monoclonal Genetex GTX60582

H00166379-

BBS12 Mouse polyclonal Abnova B01P

IL-6 Rat monoclonal Genetex GTX15816

NF-KB p65 (D14E12) Rabbit monoclonal Cell Signaling 8242

Phospho-NF-κΒ p65

(Ser536) Mouse monoclonal Cell Signaling 3036

mAb3344

JAK1 Rabbit monoclonal Cell Signaling (6G4)

mAb3230

JAK2 Rabbit monoclonal Cell Signaling (D2E12)

Secondary Antibody host Dye labeling Manufacturer Catalog #

Donkey anti-rabbit FITC Santa Cruz sc-2090

Molecular

Goat anti-rat Alexa Fluor 594 Probes A-21213

Molecular

Goat anti-mouse Alexa Fluor 594 Probes A- 11032

Chicken anti-rabbit HRP Santa Cruz sc-2955

Goat anti-mouse HRP Santa Cruz sc-2060

Claims

1- A Leptin signaling inhibitor for use for treating patients having a ciliopathy with hyperleptinemia.

2- The Leptin signaling inhibitor for use according to claim 1, wherein said Leptin signaling inhibitor is a Leptin receptor (LEPR) inhibitor.

3- The Leptin signaling inhibitor for use according to claim 1 or 2, for use for protecting kidney or preventing kidney damage.

4- The Leptin signaling inhibitor for use according to anyone of claims 1 to 3, wherein the ciliopathy is selected for the group consisting of Bardet-Biedl syndrome (BBS), Alstrom syndrome,and MORM syndrome.

5- The Leptin signaling inhibitor for use according to claim 4, wherein the ciliopathy is Bardet-Biedl syndrome (BBS).

6- The Leptin signaling inhibitor for use according to any one of claims 1 to 5, wherein the Leptin signaling inhibitor is an antibody directed against Leptin or LEPR, preferably against LEPR, and having an antagonist or neutralizing activity on LEPR.

7- The Leptin signaling inhibitor for use according to any one of claims 1 to 5, wherein the Leptin signaling inhibitor is a peptide derived from leptin or a mutated leptin and has an antagonist activity on LEPR, or a nucleic acid encoding said peptide or mutated leptin.

8- The Leptin signaling inhibitor for use according to any one of claims 1 to 5, wherein the Leptin signaling inhibitor is a polypeptide derived from the leptin receptor and having an inhibiting effect on the LEPR activity, such as a soluble polypeptide comprising the extracellular domain of LEPR or a leptin-binding portion thereof or a fibronectin III domain thereof.

9- The Leptin signaling inhibitor for use according to any one of claims 1 to 5, wherein the Leptin signaling inhibitor is an oligonucleotide inhibiting or decreasing the expression of Leptin or LEPR, preferably LEPR, such as antisense, miRNA, siRNA or shRNA. 10- The Leptin signaling inhibitor for use according to any one of claims 1 to 5, wherein the Leptin signaling inhibitor is an aptamer specific to Leptin or LEPR, preferably specific of LEPR and having an antagonist activity.

11- The Leptin signaling inhibitor for use according to any one of claims 1 to 5, wherein the Leptin signaling inhibitor is a small molecule inhibiting or blocking the Leptin signaling pathway, preferably the LEPR activity.

12- The Leptin signaling inhibitor for use according to claim 11, wherein the Leptin signaling inhibitor is JAK2 inhibitor, preferably ruxolitinib or baricitinib, more preferably ruxolitinib.

13- The Leptin signaling inhibitor for use according to any one of claims 1 to 5, wherein the Leptin signaling inhibitor is a protein fusion comprising a fragment of LEPR and an Fc region of an immunoglobulin, preferably an extracellular fragment of LEPR and an Fc region of an IgGl.

14- The Leptin signaling inhibitor for use according to any one of claims 1 to 13, wherein the Leptin signaling inhibitor does not cross the Blood Brain Barrier.

15- The Leptin signaling inhibitor for use according to any one of claims 1 to 14, wherein the Leptin signaling inhibitor is targeted to the kidney.

16- The Leptin signaling inhibitor for use according to any one of claims 1 to 15, wherein the Leptin signaling inhibitor is to be administered to said patient when the serum level of leptin increases, preferably above 12,5 ng/ml.