MD Moglie Le Quintrec
email: lequintrec-donnette@chu-montpellier.fr

Maxime Teisseyre

Nicole Bec

Emilien Delmont

Ilan Szwarc

Hélène Perrochia

Marie Christine Machet

Anthony Chauvin

Nicolas Mavroudakis

Guillaume Taieb

Luca Lanfranco

Claire Rigothier

Boucraut José

Catalano Concetta

Clair Geneste

Vincent Pernin

Christian Larroque

Jérôme Devaux

Anaïs Beyze

Contactin-1 is a novel target antigen in membranous nephropathy associated with chronic inflammatory demyelinating polyneuropathy

Keywords: Membranous nephropathy, nephrotic syndrome, Contactin-1, Immunoglobulin G type 4, Chronic inflammatory demyelinating polyneuropathy Translational

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION:

Membranous nephropathy (MN) is a rare autoimmune disease affecting the kidney glomerulus and more specifically the podocytes. It is characterized by the subepithelial formation of immune deposits outside the glomerular capillary wall, close to the podocytes. These deposits are responsible for proteinuria and MN is a common cause of nephrotic syndrome in adults with a peak incidence in males aged 50 to 60 years. In around 80% of the cases, MN occurs in the absence of an established cause and is called primary membranous nephropathy. Alternatively, MN occurs in association with a clinical condition -infections (hepatitis B), lupus erythematosus, cancer, or drug intoxication -and is called secondary MN.

In MN, podocyte antigens are the major targets of the autoimmune response. Immunoglobulin G type 4 (IgG4) is the prominent deposited immunoglobulin subclass in MN, while IgG1, IgG2, and IgG3 are more prominent in cases of secondary MN [START_REF] Segawa | IgG subclasses and complement pathway in segmental and global membranous nephropathy[END_REF][START_REF] Ohtani | Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy[END_REF][START_REF] Couser | Primary Membranous Nephropathy[END_REF] . In 2009, Beck et al identified the phospholipase A2 receptor (PLA2R1) as being the main antigenic target involved in human primary MN [START_REF] Beck | M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy[END_REF] . Anti-PLA2R1 antibodies are involved in about 80% of cases of primary MN and anti-Thrombospondin type 1 domain-containing 7 A (THSD7A) in 3-5% of cases [START_REF] Tomas | Thrombospondin type-1 domaincontaining 7A in idiopathic membranous nephropathy[END_REF] . Other autoantibodies, such as anti-neural epidermal growth factor-like 1 protein (NELL-1) and anti-Semaphorine 3B have also recently been reported [START_REF] Sethi | Neural epidermal growth factor-like1 protein (NELL-1) associated membranous nephropathy[END_REF][START_REF] Sethi | Semaphorin 3B-associated membranous nephropathy is a distinct type of disease predominantly present in pediatric patients[END_REF] . However, in about 15% of cases of primary MN, the target antigen remains unknown. Chronic inflammatory demyelinating polyneuropathy (CIDP) is a disease of the peripheral nervous system which has a chronic progressive course over more than 8 weeks. Diagnosis is mostly based on electrophysiological and clinical investigations [START_REF] Kieseier | Immune-mediated neuropathies[END_REF] . The severity of this disease is highly heterogeneous. Auto-antibodies against nodal and paranodal proteins such as neurofascin 155 (NF155), neurofascin 186 (NF186), contactin-associated protein 1 (Caspr1), or contactin-1 (CNTN1), mainly of the IgG4 subclass, have recently been reported in severe cases [START_REF] Querol | Autoantibodies in chronic inflammatory neuropathies: diagnostic and therapeutic implications[END_REF] . These IgG4 autoantibodies are pathogenic and induce the disorganization of the septate-like junctions at the paranodes causing a nodo-paranodopathy [START_REF] Manso | Contactin-1 IgG4 antibodies cause paranode dismantling and conduction defects[END_REF][START_REF] Manso | Anti-Neurofascin-155 IgG4 antibodies prevent paranodal complex formation in vivo[END_REF] . In the case of anti-CNTN1 IgG4, the antibodies have a function-blocking activity and inhibit the interaction between CNTN1 and NF155 at the paranodes [START_REF] Manso | Contactin-1 IgG4 antibodies cause paranode dismantling and conduction defects[END_REF][START_REF] Labasque | Specific contactin N-glycans are implicated in neurofascin binding and autoimmune targeting in peripheral neuropathies[END_REF] . The incidence of these autoantibodies in CIDP is low, varying from 2% in Europe to 10% in Asian countries [START_REF] Cortese | Antibodies to neurofascin, contactin-1, and contactin-associated protein 1 in CIDP: Clinical relevance of IgG isotype[END_REF][START_REF] Delmont | Antibodies against the node of Ranvier: a real-life evaluation of incidence, clinical features and response to treatment based on a prospective analysis of 1500 sera[END_REF][START_REF] Miura | Contactin 1 IgG4 associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia[END_REF] . A few case reports have described the association of MN with anti-CNTN1 related CIDP [START_REF] Cortese | Antibodies to neurofascin, contactin-1, and contactin-associated protein 1 in CIDP: Clinical relevance of IgG isotype[END_REF][START_REF] Delmont | Antibodies against the node of Ranvier: a real-life evaluation of incidence, clinical features and response to treatment based on a prospective analysis of 1500 sera[END_REF][START_REF] Taieb | Neuro-renal syndrome" related to anticontactin-1 antibodies[END_REF][START_REF] Doppler | Destruction of paranodal architecture in inflammatory neuropathy with anti-contactin-1 autoantibodies[END_REF][START_REF] Hashimoto | Chronic Inflammatory Demyelinating Polyneuropathy With Concurrent Membranous Nephropathy: An Anti-paranode and Podocyte Protein Antibody Study and Literature Survey[END_REF] .

To date, nine cases of MN have been described in CIDP patients with anti-CNTN1 IgG4 [START_REF] Cortese | Antibodies to neurofascin, contactin-1, and contactin-associated protein 1 in CIDP: Clinical relevance of IgG isotype[END_REF][START_REF] Delmont | Antibodies against the node of Ranvier: a real-life evaluation of incidence, clinical features and response to treatment based on a prospective analysis of 1500 sera[END_REF][START_REF] Taieb | Neuro-renal syndrome" related to anticontactin-1 antibodies[END_REF][START_REF] Doppler | Destruction of paranodal architecture in inflammatory neuropathy with anti-contactin-1 autoantibodies[END_REF][START_REF] Hashimoto | Chronic Inflammatory Demyelinating Polyneuropathy With Concurrent Membranous Nephropathy: An Anti-paranode and Podocyte Protein Antibody Study and Literature Survey[END_REF] . Although the hypothesis of a shared antigenic target, expressed by nerves and podocytes, has been put forward, no such target has yet been identified.

In this study, we describe CNTN1 as a novel shared target antigen involved in both CIDP and MN. We also present the incidence of combined diseases in a national cohort of CNTN1positive CIDP patients.

RESULTS

Characteristics of patients positive for anti-CNTN1 antibodies with neuro-renal syndrome or isolated CIDP

Since the discovery of the involvement of antibodies directed against the CNTN1 protein in the pathophysiology of inflammatory demyelinating diseases of the peripheral nervous system, 1500 sera from patients with acute polyneuritis or CIDP have been tested in the French reference center of neurologic autoimmune diseases in Marseille. Among these patients, 11 were positive for anti-CNTN1 antibodies (Figure 1) and all were diagnosed with CIDP. The mean age at the onset of CIDP disease was 67 years (range: 39 to 85 years), and the male-to-female ratio was 10: one patient had tobacco exposure, and two were treated for hypertension. All patients had a typical and severe form of CIDP. The median titer of anti-CNTN1 was 1/4450 (1/500 to 1/52700), and the anti-CNTN1 antibody subclasses revealed a predominance of IgG4 subclass (Figure 2). At the time of neurological investigation, five of these 11 patients had kidney injury consisting of proteinuria with or without nephrotic syndrome (Figure 1, Table 1). All of them had a normal serum creatinine. The anti-CNTN1 titers (tested by ELISA assay) appeared higher, although not significantly, in the patients with both CIDP and MN diseases than in those with isolated disease (1/20450 to 1/2300; p<0.064) (Fig S1). Sera anti-PLA2R1 antibodies were negative. The kidney biopsies revealed a stage 1 MN in four patients and a stage 2 MN in one patient. Immunofluorescence showed diffuse granular subepithelial deposits of IgG, mainly composed of IgG4. None of them had a PLA2R1 or TSHD7A positive staining.

Among the five patients with both CIDP and MN three died, as opposed to none of the patients with isolated CIDP disease. Of the three patients who died, one death was due to inhalation pneumonia 3 months after the treatment had ended, one from cardiac arrest resulting from severe dysautonomia, and one from gastric hemorrhage. All three had an uncontrolled neurological disease despite treatment. Of the three patients who did not die, partial remission was achieved for two and complete remission for one.

Glomerular expression of CNTN1

We performed immunoblotting of reduced and non-reduced protein extracts from normal human glomeruli with a commercial anti-CNTN1 antibody (Figure 3). Reduced or nonreduced human brain extracts were used as a positive control for CNTN1 blotting.

On both the brain extracts and non-reduced human glomerular extracts a distinctive band of approximately 125-135 kDa was detected which corresponds to the expected molecular weight of CNTN1. The band obtained on the extract of reduced human glomeruli was slightly positive. The presence of CNTN1 was unambiguously confirmed by the identification of seven unique peptides in the above-mentioned immunoblotting band using mass spectrometry.

In order to validate our immunoblotting approach, we performed immunoblotting of nonreduced normal human glomeruli protein extracts with a commercial anti-PLA2R1 antibody.

As described by Beck et al, we found a strongly positive band corresponding to the expected size of PLA2R1 in non-reduced extracts of human glomeruli.

These results show that CNTN1 is expressed in normal kidney glomeruli.

RT-PCR analysis on a podocyte cell line showed that CNTN1 is expressed by podocytes (data not shown).

We also performed immunoblotting for NF155, NF186 and Caspr1 and found that all three were expressed in human glomerular extracts (date not shown).

CNTN1 staining on patient kidney biopsies

We then performed immunofluorescence staining for CNTN1, IgG4, PLA2R1 and TSHD7A on deparaffinized kidney biopsy sections from patients presenting both MN and CIDP, from a patient with anti-PLA2R1-MN, from a patient with membranous lupus nephritis (MLN class V), and from a healthy donor (Figure 4 panel A).

CNTN1 was revealed in the glomeruli from patients presenting both MN and CIDP, but not in the other samples. Neither PLA2R1 nor THSD7A were found in patients with MN and CIDP.

As expected, both IgG4 deposits and PLA2R1 were detected in the glomeruli of the patient with anti-PLA2R1-MN but not CNTN1. Neither CNTN1 nor PLA2R1 were found in the kidney specimen from the MLN class V patient. However, IgG4 deposits were detected. No significant signal was detected using the secondary antibody alone (data not shown).

Confocal microscopic analysis was performed on the four patients with an available deparaffinized kidney-tissue specimen. CNTN1 and IgG4 were colocalized in the glomerular basement membrane (Figure 4 Panel B). As previously described in Beck and al (4), a similar confocal microscopy analysis of a kidney biopsy from a patient with anti-PLA2R1-MN revealed colocalization of PLA2R1 and IgG4 in the glomeruli (data not shown).

These results demonstrate that IgG4 deposits colocalize with CNTN1 in glomeruli from patients with concomitant MN and CIDP, in the same manner as PLA2R1 and IgG4 deposits are colocalized in glomeruli from PLA2R1-MN We then performed immunohistochemistry staining for CNTN1 on deparaffinized kidney biopsies from patients presenting both MN and CIDP, from a patient with anti-PLA2R1-MN, from a patient with membranous lupus nephritis (MLN class V), from a healthy donor, and brain tissue (Figure 5). As expected, CNTN1 was strongly expressed in the brain. As in immunofluorescence, CNTN1 was revealed in the glomeruli from patients presenting both MN and CIDP, but not in the other samples. No significant signal was detected using only the secondary antibody (data not shown).

IgG4 eluted from MN kidney biopsies are reactive against CNTN1 and peripheral nerve

Frozen kidney sections were available for three of the five patients with both anti-CNTN1 positive CIDP and MN. IgG was eluted from these sections using an acidic buffer. Eluted IgG were then tested for CNTN1 reactivity by ELISA. Results of IgG eluted from the three patients were compared to IgG eluted from an anti-PLA2R-positive MN, and to the last washes obtained just before elution (Fig 6 panel A).

As represented in

DISCUSSION

This study is the first to provide evidence of a shared antigenic target -CNTN1 -in patients presenting with combined CIDP and MN. Furthermore, we show a high prevalence of kidney involvement in anti-CNTN1 positive CIDP patients.

The description of anti-CNTN1 antibodies in CIDP confirms the autoimmune origin of this pathology. These antibodies are pathogenic through a function-blocking effect and can block the interaction between CNTN1 and NF155 [START_REF] Manso | Contactin-1 IgG4 antibodies cause paranode dismantling and conduction defects[END_REF][START_REF] Labasque | Specific contactin N-glycans are implicated in neurofascin binding and autoimmune targeting in peripheral neuropathies[END_REF] . The role of CNTN1 in kidney physiopathology has never been studied. We, and other authors, have previously reported cases of patients with anti-CNTN1-related CIPD associated with glomerular proteinuria, and hypothesized about a common potential immunopathologic mechanism. To the best of our knowledge, only one study to date has focused on the expression of CNTN1 in human kidney and showed a low mRNA expression [START_REF] Reid | Identification and characterization of the human cell adhesion molecule contactin[END_REF] .

As for PLA2R1, we demonstrated that CNTN1 is expressed in human glomeruli as an endogenous integral membrane protein of the podocyte but is not revealed in normal conditions as shown by the negative immunofluorescence and immunochemistry in our healthy donor. Reciprocally, PLA2R1 was detectable by immunofluorescence in the podocytes of PLA2R1 patients but not from healthy donors or CNTN1 patients. This suggests that in situ formation of immune complexes is initiated by the binding of circulating antibodies to the antigens, and that a common pathophysiology is induced by an immune response elicited after antigen unmasking. However, the unmasking mechanism remains to be determined. Alternatively, the antibody-mediated clustering of CNTN1 may favor its detection on the podocyte membrane or may increase CNTN1 surface expression. In vivo and in vitro models would be useful to further understand the pathophysiology of kidney involvement in the presence of anti-CNTN1 antibodies.

CNTN1 is a GPI-anchored cell membrane protein expressed on the extracellular side. Its 3D structure has been partially elucidated by both nuclear magnetic resonance and X-ray studies.

The secondary structure of CNTN1 has six disulfide bonds in its extracellular Ig-like domain.

The antibody used for the western blot experiments was raised against the whole native protein expressed in the sf-21 cell line. As it has been suggested for the PLA2R1 protein [START_REF] Beck | M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy[END_REF] , structural modifications resulting from reduction of these disulfide bonds could explain the lower reactivity observed with the commercial antibody in western blots under reduced conditions. The strong expression of CNTN1 in the brain probably explains why the antibody gave a strong signal in human brain extracts despite reduction. As for PLA2R1, CNTN1 was not detected by immunoblot in rodent kidney (data not shown), limiting the use of these models to study the pathogenic function of anti-CNTN1 in kidney.

Anti-CNTN1 and anti-PLA2R1 antibodies predominantly comprise the IgG4 subclass and IgG4 deposits were colocalized with CNTN1 or PLA2R1, respectively, in kidney biopsy.

While IgG4 is the dominant subclass in patients with long-standing anti-PLA2R1-MN (electron microscopy showing stages III/IV), the predominant subclass in early MN (stages I/II by electron microscopy) is IgG1 (20). IgG4-PLA2R1-MN is considered as a renal autoimmune disease. After the formation of immune complexes, complement can be activated by three different pathways [START_REF] Brglez | Complement Pathways in Membranous Nephropathy: Complex and Multifactorial[END_REF] . These processes trigger cell damage which is responsible for proteinuria and end-stage renal failure. In our study, the main subclass of anti-CNTN1 was IgG4, but the IgG4-CNTN1 complex was associated with early-stage MN. This suggests that the onset of the neurological disease precedes MN. The kidney-renal function was normal in all patients on diagnosis of MN/CIDP while in other types of MN, about 30% of the patients have chronic renal failure at time of the diagnosis [START_REF] Donadio | Idiopathic membranous nephropathy: the natural history of untreated patients[END_REF] . CNTN1 is the first common antigen target involved in MN associated with CIDP as we demonstrated that the eluted IgG from the kidney bind CNTN1 at the paranode. As mentioned above, the prevalence of CNTN1-reactive CIDP patients is low in Europe and Asia [START_REF] Cortese | Antibodies to neurofascin, contactin-1, and contactin-associated protein 1 in CIDP: Clinical relevance of IgG isotype[END_REF][START_REF] Miura | Contactin 1 IgG4 associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia[END_REF] .

Interestingly though, the kidney target is common: half (5/11) of the characterized anti-CNTN1 positive CIDP patients had MN, in contrast with the general CIDP population.

Importantly, the CNTN1-reactive patients with MN and CIDP had a higher rate of mortality: three out of the five patients with MN and CIDP died, as opposed to none of those affected with CIDP only. Furthermore, the neurological score of severity was similar for all patients whereas three patients affected by both CIDP and MN had an uncontrolled neurological disease and two of them died of swallowing disorders. This suggests that kidney involvement is more predictive of mortality than the neurological severity score. However, treatment was less intense in the MN group, which could also explain the difference in the mortality rates. Thus, we recommend that CIDP patients be systematically screened for proteinuria, especially those with CNTN1 related CIDP.

The incidence of anti-CNTN1 IgG4 has not been evaluated in isolated MN to date. We did not find anti-CNTN1 antibodies in the nine patients with anti-PLA2R1 and anti-TSHD7A negative isolated MN. This deserves to be explored in larger MN cohorts in the future.

To conclude, CNTN1 is the first discovered target involved in combined MN and anti-CNTN1-related CIDP. It appears to be a common association in CNTN1 positive patients and a relevant marker of disease severity. Further studies are needed to elucidate the pathological significance of these autoantibodies and their role in MN.

METHODS

Serum and kidney-tissue samples

Sera from patients with CNTN1-positive CIDP patients were collected and analyzed. We also collected and analyzed sera from patients with anti-PLAR1 and anti-TSHD71 negative MN.

Written informed consent was obtained from each participant. The study was approved by the Ethics Committee of Montpellier University Hospital (IRB-MTP-2020-01-20200339) and of the Assistance Publique des Hôpitaux de Marseille (PADS19-365). Paraffin-embedded kidney tissue sections and frozen tissue sections were collected from the same patients.

MN was diagnosed by renal biopsy in patients who lacked the features suggestive of secondary MN, such as antinuclear antibodies or hepatitis B antibodies in the serum.

Normal glomeruli were obtained from a kidney that was unsuitable for transplantation because of vascular issues. This sample was exempt from the requirement of informed consent according to the institutional review board's approved use of organs and tissues from deceased donors for research.

Serum anti-CNTN1 screening

Reactivity to CNTN1 and determination of the isotype was performed by flow cytometry as described previously [START_REF] Delmont | Antibodies against the node of Ranvier: a real-life evaluation of incidence, clinical features and response to treatment based on a prospective analysis of 1500 sera[END_REF] and confirm by ELISA (see below).

Brain protein extraction

The whole procedure of brain protein extraction was performed on ice using two surgical pieces of peri-tumoral brain white matter provided by the pathology department. Briefly, the pieces of brain were rinsed several times in cold phosphate buffered saline (PBS) to remove surface contaminants. They were then cut into small pieces with a sterile razor blade and shredded with sterile pliers. The resulting paste was homogenized in an RIPA buffer (Tris 50mM, pH 7.4, NaCl 150mM, NP-40 1%, sodium deoxycholate 0.5%, SDS 0.1%) containing protease inhibitors (ThermoFisher Scientific) in a glass Potter-Elvehjem homogenizer. After a 30-minute incubation on ice, insoluble materials were removed by centrifugation at 10 000 g for 10 min.

Human glomerular extract preparation

Pieces of renal cortex were excised after decapsulation. The glomeruli were prepared according to the method described by [START_REF] Yoshida | Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database[END_REF] . Briefly, cortical tissue was cut into small pieces with a sterile razor blade and shredded with sterile pliers. The resulting paste was rinsed in cold PBS at pH 7.4 and crushed through a 300 µm-inox sieve. At this step, the glomeruli and tubular fragments were not retained. The glomeruli were then isolated and washed twice with cold PBS on a 100 μm-nylon filter (Millipore). The purity of the preparation was checked under a phase-contrast microscope. After centrifugation at 300 g for 5 min, the pellet was resuspended in an equal volume of RIPA buffer containing protease inhibitors (ThermoFisher Scientific). The glomeruli were then manually ground ten times in a glass Potter-Elvehjem homogenizer. Glomerular proteins were extracted on ice for 20 min, with intermittent shaking. The insoluble fraction was removed by centrifugation at 14 000 g for 10 min at 4°C.

Immunoblotting

Protein concentration of both human glomerular extracts and brain extracts were determined using the bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific). For western blot analysis, 20 µg of total proteins in Laemmli buffer (Tris 70mM, pH 6.8, glycerol 10%, SDS 1%, bromophenol blue 0.005%, Bio-Rad) with or without 2-mercaptoethanol (355mM, Sigma) as a reducing agent were loaded per lane, separated on a 4-15 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes using the Transblot Turbo system (Bio-Rad). The membranes were saturated with tris-buffered saline-tween (TBS-T) (Tris 20mM, pH 7.2, NaCl 150mM, Tween 20 0.05%) containing bovine serum albumin (BSA) 2%, at room temperature (RT) for 1 hr to block unspecific binding, and were then incubated with primary antibodies in blocking buffer at 4°C overnight. The primary antibodies used were goat anti-CNTN1 antibody (R&D systems; AF904, 1:400), rabbit anti-PLA2R1 polyclonal IgG (Sigma-Aldrich; HPA012657, 1:1000), anti-NF155 (Eurogenetec; generated against peptide SQKRQQASFPGDRPR; 1:500), anti-NF186 (Cell Signaling Technology; 15034S; 1:500) and anti-Caspr1 (Gift from Dr. Laurence Goutebroze; 1:500). After three washes with TBS-T for 15 min, the membranes were incubated for 1 hr at RT with the proper horseradish peroxidase (HRP)-conjugated secondary antibody (anti-goat 1:10000; Sigma-Aldrich and anti-rabbit 1:2000; Abcam) in blocking buffer. After another three washes with TBS-T for 15 min, the protein bands were detected using HRP substrate (Millipore) emitting chemiluminescence recorded with a ChemiDoc Imaging system (Bio-Rad).

Immunofluorescence studies

The biopsy sections were deparaffinized by three successive baths in Neo-Clear® (Sigma) for 5 min each. The tissue was then rehydrated by submerging the slides for 10 min twice in 100%, 90%, 70%, 50% ethanol successively, and finally in water (5 min). To retrieve the antigen, the sections were immersed in an EDTA buffer at pH 8.5 (Sigma-Aldrich) at 95°C twice for 30 min. After cooling, the sections were rinsed in water, equilibrated in PBS then saturated in PBS-BSA 1mg/ml-Triton X100 0.1% at RT for 1 hr to block unspecific binding.

The tissues were then incubated at 4°C with primary antibodies in blocking buffer overnight. The primary antibodies used were goat anti-CNTN1 (1:500), Alexa Fluor® 488-conjugated mouse anti-PLA2R1 (clone 12-6-5, 1:250, Novus Biologicals), mouse anti-THSD7A (clone CL3778, 1:200, Sigma-Aldrich) and rabbit anti-human IgG4 (clone RM120, 1:500, Novus Biologicals). Following three washes in PBS-Triton X100 0.1% for 5 min, the slides were incubated for 1 hr at RT with proper Alexa Fluor®-conjugated affinity purified donkey secondary antibodies from Jackson Immunoresearch (1:500) in blocking buffer. The sections were washed once with PBS-Triton X100 0.1% for 5 min, twice in PBS for 5 min and briefly in water before being dried and mounted in Mowiol 4-88 (Tris 0.1 M, pH 8.5, Mowiol 4-88 10%, glycerol 25%). The prepared slides were analyzed using an inverted microscope Axiovert 200 M driven by Axiovision software (Carl Zeiss). Colocalization of staining was checked using an inverted confocal laser scanning microscope Leica SP8. Images were processed using ImageJ software.

Immunohistochemistry

High sensitivity and specificity immunohistochemistry was performed using a 3 rd generation polymer detection system -Polink-2 Plus HRP Anti-GOAT DAB Detection Kit (GBI Labs)according to the manufacturer's instructions. Briefly, the sections were deparaffinized, rehydrated and subjected to antigen retrieval as described previously after blocking endogenous peroxidases. The sections were then incubated at 4°C with the primary antibody in blocking buffer overnight. The primary antibody used was goat anti-CNTN1

(1:500). Following three washes in PBS-Triton X100 0.1% for 2 min, the slides were incubated for 30 min at RT with goat antibody enhancer. After another three washes in PBS-Triton X100 0.1% for 2 min, the slides were incubated for 30 min at RT with polymer-HRP for goat.

Diaminobenzidine was used as the chromogen. Finally, the sections were mounted in Mowiol as described previously. The prepared slides were analyzed using an upright microscope (Olympus BX43).

qRT-PCR analysis

Total RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer's instructions and summarized in Supplementary methods.

Mass spectrometry analysis:

Briefly, proteins from SDS-PAGE gel electrophoresis were digested with trypsin. The recovered peptides were separated onto a reverse phase column, detected on a Qexactive HFX, Thermofisher mass spectrometer and identified using the Maxquant software (version 1.6.10.43).

IgG elution from kidney-tissue specimens

IgG was eluted from a pool of frozen remnant cores of human anti-PLA2R1 positive MN samples (n=5); and from three independent concomitant MN + CIDP samples. Briefly, frozen remnant cores of human non-fixed biopsy specimens were sliced into 20-µm sections and pooled in a 1.5 mL microtube (according to pathological and clinical diagnosis) to obtain sufficient eluted IgG for analysis. After three washes in PBS, the remaining paste was centrifugated for 2 min at 12500 rpm to eliminate residual OCT compounds, blood and external contaminants. The last wash was kept as a negative control. 50 μl of elution buffer, pH 2.8 (Thermofisher Scientific), was incubated with each sample for 5 min and collected after centrifugation. The eluate was neutralized with 5 μL of neutralization buffer (Tris 1M pH 8.5). A second and a third eluate resulting from the same procedure was obtained and then neutralized.

The reactivity of the eluted IgG was tested by ELISA as described in (24). Briefly, polystyrene microwells were coated with 25 ng of human recombinant CNTN1 (Sino Biological Inc) in PBS at 4°C overnight. The plates were then blocked with 0.5% casein sodium in PBS and 0.05% Tween-20 at RT for 1 hr and incubated with biopsy eluate diluted 1:10, 1:20, 1:50, 1:100, 1:500, 1:1000 and 1:5000 in blocking solution, at RT for 1 hr. HRPconjugated donkey antibody to human IgG (dilution, 1:2000; Jackson ImmunoResearch), or IgG subclasses (dilution, 1:1000; Thermofisher Scientific) was added, and the plates were developed with SIGMAFAST OPD tablets (Sigma-Aldrich). The plates were washed between each incubation with PBS, and subsequently read at 495 nm (ELx800, Biotek). Positive results were defined as those showing optical density (OD) values above the OD mean values of healthy controls plus three standard deviations.

The specificity of eluted IgG from anti-PLA2R1 positive MN biopsy specimens was confirmed by indirect immunofluorescence on PLA2R1-expressing cells (Euroimmun). 20 μL of 1:10 or 1:100 diluted eluate was incubated with slide-adhesive PLA2R1-expressing or nonexpressing cells for 30 min at RT. The slides were then washed in PBS-Tween 1% for 5 min.

Then, 25 μL of fluorescein-labelled anti-human immunoglobulin was incubated with each reaction field for 30 min in the dark. After incubation, the slides were washed in PBS-Tween for 5 min. 10 μL of glycerol/PBS was applied on each reaction field and a cover glass was added. The prepared slides were analyzed using an inverted microscope Axiovert 200 M driven by Axiovision software (Carl Zeiss).

Eluted IgG were also tested on teased sciatic nerve fibers from male C57BL/6J mice as previously described (20). Briefly, the teased fibers were permeabilized by immersion in -20°C acetone for 10 min, blocked at RT for 1 hr with PBS containing 5% fish skin gelatin and 0.1% We investigated kidney biopsies from five patients positive for anti-contactin 1 (CNTN1) IgG4 antibodies and presenting combined MN and CIDP and use control MN biopsies. We screened the presence of contactine in normal glomerulous, eluted IgG from biopsy sections against CNTN1 and nerve tissue.

MN can be associated with chronic inflammatory demyelinating polyneuropathy (CIDP). A common antigenic target has yet to be identified CNTN1 as a shared antigen in both diseases?

  Figure 6 panel A, eluted IgG from kidney biopsies of CIDP-MN patients recognized CNTN1, but not the IgG eluted from an anti-PLA2R1-MN kidney biopsy, or washes before the acidic elution. For all patients, IgG4 was the main representative eluted subclass of IgG (Fig 6 panel B). This is corroborated by the observation that the IgG deposits from the patients' kidneys were IgG4 raised against CNTN1 (as illustrated in Fig 4 Panel B). Because antibodies may recognize distinct forms of CNTN1 expressed in the kidney or neurons, we investigated whether the IgG eluted from kidney were able to target neuronal CNTN1. Fig 6 Panel C shows that eluted IgG from patient 2 were able to bind paranodal tissue and colocalized with CNTN1, thus demonstrating that these antibodies target CNTN1 in both tissues.

  Figures, Legends:

Figure 1 :

 1 Figure 1: Flow chart

Figure 2 :

 2 Figure 2: Diagnosis of anti-CNTN1 and IgG subclasses by flow cytometry using HEK transfected with human CNTN1 Reactivity to CNTN1 and antibody isotype were determined by flow cytometry using HEK transfected with human CNTN1 and represented as mean fluorescence intensity (MFI).

Figure 3 :

 3 Figure 3: CNTN1 is expressed in human glomeruli detected by Immunoblot Immunoblot of reduced (+) or non-reduced (-) human brain extract or normal human glomerular extract with an anti-CNTN1 or an anti-PLA2R1 antibody. Molecular weights are shown on the left in kDa. The arrow shows the band of interest. Ab: antibody; CNTN1: contactin 1; 2-ME: b-mercaptoethanol; HBE: human brain extract; MW: molecular weight; PLA2R1: phospholipase A2 receptor

Fig 4 :

 4 Fig 4: Panel A Immunofluorescence staining for IgG4, CNTN1, PLA2R1 and TSHD7A on deparaffinized kidney biopsy sections from patients presenting both MN and CIDP, from a patient with anti-PLA2R1-MN, from a patient with membranous lupus nephritis (LN class V), and from a healthy donor. No significant signal was detected using the secondary antibody alone.

Figure 4 :

 4 Figure 4: Panel B: CNTN1 and IgG4 deposits are colocalized in glomeruli from four patients with MN associated with CIDP. Each column represents a single patient. A: Immunofluorescence staining for IgG4, CNTN1, PLA2R1 and TSHD7A on deparaffinized kidney biopsy sections from patients presenting both MN and CIDP, from a patient with anti-PLA2R1-MN, from a patient with membranous lupus nephritis (LN class V), and from a healthy donor B: CNTN1 (green) and IgG4 (red) staining and their colocalization (merge) were examined by confocal microscopy. Scale bar: 20 μm. CIDP: chronic inflammatory demyelinating polyneuropathy; CNTN1: contactin 1; IgG4: Immunoglobulin G of subclass 4; MN: primary membranous nephropathy

Figure 5 :

 5 Figure 5: Immunochemistry staining for CNTN1 on deparaffinized sections. Panels A and B: kidney biopsy sections from patients presenting both MN and CIDP (patient 1). Panel C: kidney biopsy section from a patient with anti-PLA2R1-MN. Panel D: kidney biopsy section from a patient with membranous lupus nephritis (LN class V). Panel E: kidney biopsy section from a healthy donor. Panel F: Brain section.

Figure 6 :

 6 Figure 6: IgG Eluted from kidney biopsies are raised against CNTN1. A: Serial dilutions of eluted IgG from kidney biopsies of CNTN1 positive patients (Patients 2, 3 and 5) or PLA2R1 positive patients (PLA2R1) were tested by ELISA against CNTN1. As control, the last wash before elution was tested (wash). Only the eluate from CNTN1 positive biopsies reacted toward CNTN1. B: The subclass of eluted IgG was determined by ELISA against CNTN1. IgG4 appeared as the predominant isotype. Asterisks denotes the significant reactivities. C: Mouse teased sciatic nerve fibers were incubated with eluted IgG from CNTN1 or PLA2R patients' biopsies (green) and immunostained with antibodies against CNTN1 (red). Eluted IgG from CNTN1 positive biopsies deposited at paranodes. Scale bar: 10 µm. CNTN1: contactin 1. OD: optic density. Pat: patient. PLA2R : phospholipase A2 receptor.

HYPOTHESISContactin- 1

 1 is a novel target antigen in membranous nephropathy associated with chronic inflammatory demyelinating polyneuropathy.RESULTS50% (5/11) of patients with anti-CNTN1 positive CIDP have a MN.Confocal microscopic analysis showed the presence and colocalization of CNTN1 and IgG4 on the glomerular basement membrane of our patients. 1 st discovered target involved in combined MN and anti-CNTN1-related CIDPLe Quintrec et al, 2021Western blot revealed CNTN1 expression in normal kidney glomeruli.

ACKNOWLEDGEMENTS

Versna Brglez and Barbara Seitz-Polski (immunology department of Nice) for given us sera from patients with PLA2R negative MN w/o CIDP, Claire Geneste, nephrology department Tours and Stephane Beltran neurology department, Tours to give information from a patient, Claire Royer department of pathology for given us the biopsy materiel, Erasme ULB, Concetta Cattalano, Erasme ULB for clinical information of one patient and Viviane Gnemmi, for give the biopsy control of THSD7A + PLA2R +, Mass spectrometry experiments were carried out using the facilities of the Montpellier Proteomics Platform (PPM, BioCampus Montpellier).

AUTHOR CONTRIBUTION

MLQ, NB, CL, AB and JD designed the study, NB, CL, JD, AB did the experiments, AB , MLQ, JD, NB, CL, , MT wrote the manuscript, ED, VP, AC, IS, MCM, HP, NM, GT, LF, CR participated to the study, collected data from patients and biopsies and revised the manuscript.


Aghilès Hamroun
email: aghiles.hamroun@inserm.fr

Jean Baptiste Gibier

Mehdi Maanaoui

Arnaud Lionet

M.D Viviane Gnemmi

Sébastien Bouyé

Jean-Christophe Christophe Fantoni

Benoît Averland

M.D Corinne Antoine

Rémi Lenain

Jean-Baptiste Gibier

M.D Marc Hazzan

François Provôt

Claude Huriez

Successful Reuse of Kidney Graft After Early Recurrence of Primary Focal and Segmental Glomerulosclerosis

Keywords: transplantation, primary focal and segmental glomerulosclerosis, transplantectomy, nephrotic syndrome, recurrence, kidney graft reuse

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Primary FSGS (pFSGS) is at high risk of recurrence after transplantation. [START_REF] Kienzl-Wagner | Disease Recurrence-The Sword of Damocles in Kidney Transplantation for Primary Focal Segmental Glomerulosclerosis[END_REF] Recent data estimates this probability at around one in three cases, [START_REF] Uffing | Recurrence of FSGS after Kidney Transplantation in Adults[END_REF] and more than 80% when first kidney transplantation had already failed due to pFSGS recurrence. [START_REF] Kienzl-Wagner | Disease Recurrence-The Sword of Damocles in Kidney Transplantation for Primary Focal Segmental Glomerulosclerosis[END_REF] Currently, there is no undisputed treatment for recurrent pFSGS after kidney transplantation. [START_REF] Morin | Management of post-transplant recurrent focal and segmental glomerulosclerosis[END_REF] Nevertheless, intensive multitargeted management seems necessary, combining high dose corticosteroids (1 mg/kg/day), intravenous (IV) cyclosporine, plasma exchanges or immunoadsorption +/-rituximab. [START_REF] Kienzl-Wagner | Disease Recurrence-The Sword of Damocles in Kidney Transplantation for Primary Focal Segmental Glomerulosclerosis[END_REF][START_REF] Morin | Management of post-transplant recurrent focal and segmental glomerulosclerosis[END_REF] Despite all these therapeutics, graft failure occurs in nearly 40% at 5 years post-transplantation, [START_REF] Uffing | Recurrence of FSGS after Kidney Transplantation in Adults[END_REF] and kidney graft removal may be discussed in case of severe refractory nephrotic syndrome. In the context of organ shortage, the question of kidney graft reuse may arise. [START_REF] Kienzl-Wagner | Disease Recurrence-The Sword of Damocles in Kidney Transplantation for Primary Focal Segmental Glomerulosclerosis[END_REF][START_REF] Kienzl-Wagner | Successful management of recurrent focal segmental glomerulosclerosis[END_REF] To our knowledge, there are only two case reports of kidney transplant transfer after early pFSGS recurrence. [START_REF] Kienzl-Wagner | Successful management of recurrent focal segmental glomerulosclerosis[END_REF][START_REF] Gallon | Resolution of Recurrent Focal Segmental Glomerulosclerosis after Retransplantation[END_REF] In these two cases, the indication for transplant nephrectomy and kidney graft reuse were made very early, over the first month after transplantation. Some authors have also suggested that this procedure could be performed even later, but there is currently no data supporting this hypothesis. [START_REF] Kienzl-Wagner | Disease Recurrence-The Sword of Damocles in Kidney Transplantation for Primary Focal Segmental Glomerulosclerosis[END_REF] We thus describe the first case of successful kidney graft reuse several months after initial transplantation, in the context of early refractory pFSGS recurrence.

CASE REPORT

A 23-year-old patient followed for kidney failure secondary to pFSGS received a first kidney transplant from a deceased donor, 14 months after dialysis initiation. The donor was a 32year-old man, with no medical history and normal kidney function before organ procurement (serum creatinine 0.7 mg/dl). The patient's native kidneys were left in place, since he became anuric a few months after start of dialysis. Induction immunosuppressive therapy consisted of thymoglobulin, followed by maintenance therapy combining corticosteroids, mofetil mycophenolate, and tacrolimus. Diuresis exceeded 2 liters over the first 24 hours, and serum creatinine levels dropped from 6.6 to 1.6 mg/dl over the first week (Figure 1A). Unfortunately, abundant proteinuria was discovered on day 3, followed by the diagnosis of nephrotic syndrome (serum albumin 2.6 g/dl and urinary protein-to-creatinine ratio -uPCR -8.1 g/g at day 7). Kidney graft biopsy performed on day 10 confirmed the diagnosis of pFSGS recurrence, showing early FSGS lesions on 3 glomeruli and severe podocyte foot process effacement by electronical microscopy (Figure S1). The patient thus benefited from a treatment combining high-dose corticosteroids, plasma exchanges, and IV cyclosporine for 14 days. However, the persisting severe nephrotic syndrome (uPCR 10g/g) justified treatment continuation by immunoadsorption for a total of 14 sessions, every 48-72h for a month. Given the absence of treatment response, this was completed by two administrations of rituximab (1g) 15 days apart (12 and 14 weeks after transplantation). Three months later, evolution was still unfavorable with persisting severe nephrotic syndrome (serum albumin 2.0 g/l, uPCR 6.3 g/g) and progressive deterioration of kidney function (serum creatinine 2.7 mg/dl). A second graft biopsy was performed at 6 months and showed growing FSGS lesions associated with diffuse foot process effacement, podocyte microvillous transformation and a mild increase of interstitial and tubular atrophy (Figure S1).

Given the lack of response to all available therapies, iatrogenic risks, and major negative impact on quality of life, the patient himself requested transplant nephrectomy. Considering the two previous cases, [START_REF] Kienzl-Wagner | Successful management of recurrent focal segmental glomerulosclerosis[END_REF][START_REF] Gallon | Resolution of Recurrent Focal Segmental Glomerulosclerosis after Retransplantation[END_REF] and the reassuring histological findings, we suggested potential reuse of the kidney graft for a second recipient who already consented to transplants using kidneys at risk for discard (such as a kidney donated after cardiac death or an HCV-infected kidney), after approval from Biomedicine Agency (French organ transplant regulatory authority). In order to avoid any confounding factor in post-transplantation follow-up, we set the following selection criteria for the waitlist at our center: patient aged over 60, non-immunized, ideally anuric or without history of glomerular nephropathy at risk of recurrence, without advanced atherosclerosis (especially of the iliac vessels) and with the same blood group as the donor.

After obtaining written consent from patient 1, he underwent transplant nephrectomy eight months after initial transplantation. The kidney graft could thus be re-implanted in a 78-year-old man who had been on dialysis for two years following bi-nephrectomy in a context of bilateral clear cell carcinoma. Given his age and low risk immunological profile, he underwent induction with basiliximab, followed by maintenance therapy of tacrolimus, corticosteroids and mofetil mycophenolate. Outcome was rapidly favorable with immediate diuresis of 1800 ml over first 24 hours, a serum creatinine level dropping from 7.4 to 1.9 mg/l and uPCR from 10.2 g/g to 1.04 g/g over the first two weeks (Figure 1B). The early post-transplant period was marked by surgical treatment of obstructive lymphocele at week 8 and acute T-cell rejection at week 12 benefiting from three boluses of corticosteroids (then gradual decrease).

One year after retransplantation, kidney function is still excellent with serum creatinine at 1.2 mg/dl (estimated glomerular filtration rate [eGFR] of 62 ml/min/1.73m 2 ) and proteinuria resolution (uPCR of 0.1 g/g without any recourse to renin-angiotensin-aldosterone system blockers). Compared to day 0 kidney graft biopsy, systematic biopsies performed at month 4 and month 12 exhibited similar FSGS lesions by light microscopy but partial recovery of foot process effacement and substantial decrease of podocyte microvillous transformation (Figure 2).

Interestingly, some calcifications and resorption signs were also seen within the hyaline deposits of FSGS lesions. Meanwhile, patient 1 returned to dialysis. He experienced rapid resolution of clinical symptoms and biological normalization (serum albumin 4.0 g/dl at 6 months).

DISCUSSION

Here, we report the third description of successful kidney graft reuse in case of early refractory pFSGS recurrence after kidney transplantation. However, unlike the two previous reports, this procedure has been effective despite the long delay between first transplantation and graft reuse. [START_REF] Kienzl-Wagner | Successful management of recurrent focal segmental glomerulosclerosis[END_REF][START_REF] Gallon | Resolution of Recurrent Focal Segmental Glomerulosclerosis after Retransplantation[END_REF] In the first observation of Gallon et al in 2012, [START_REF] Gallon | Resolution of Recurrent Focal Segmental Glomerulosclerosis after Retransplantation[END_REF] the kidney graft was removed from a related living donor and graft reuse for a second recipient took place within the first two weeks.

The second report by Kienzl-Wagner in 2018 described the successful reuse of a deceased donor transplant, four weeks after pediatric transplantation that failed due to early and severe pFSGS recurrence. [START_REF] Kienzl-Wagner | Successful management of recurrent focal segmental glomerulosclerosis[END_REF] The authors thus suggested the possibility of further extending the delay between initial kidney transplantation and rescue procedure. [START_REF] Kienzl-Wagner | Disease Recurrence-The Sword of Damocles in Kidney Transplantation for Primary Focal Segmental Glomerulosclerosis[END_REF][START_REF] Kienzl-Wagner | Successful management of recurrent focal segmental glomerulosclerosis[END_REF] However, in the absence of detailed histological data, and with the relatively short timeline (only four weeks), there was no evidence to confirm this hypothesis. Our clinical case is distinguished primarily by the very late delay in the transfer procedure, more than eight months after initial transplantation. Moreover, details on clinical, biological and histological features and favorable outcome described in our report provide new insights to support the authors' proposition. Furthermore, these findings may relate to other primary nephropathy that can be also associated with early graft demise, as previously described in thrombotic microangiopathy for example. [START_REF] Kamar | Successful retransplantation of a kidney allograft affected by thrombotic microangiopathy into a second transplant recipient[END_REF] Gallon et al highlighted the early recovery of histological lesions after retransplantation, suggesting a potential for reversibility before the scar formation process. The repeated histological data in our case also confirm that foot process effacement and podocyte microvillous transformation are reversible lesions when the primary source of injury is treated. This potential for early recovery of foot process effacement is similar to that reported after bariatric surgery in obesity-related FSGS. [START_REF] Xu | Obesity-related glomerulopathy: pathogenesis, pathologic, clinical characteristics and treatment[END_REF] Interestingly, we did not observe any correlation between the number of FSGS lesions and the progressive spontaneous resolution of proteinuria, whereas the latter was related to the degree of foot process effacement. Finally, the finding of numerous calcifications within FSGS lesions also pointed toward a chronic nature for these abnormalities. [START_REF] Bertelli | Molecular and Cellular Mechanisms for Proteinuria in Minimal Change Disease[END_REF] Unlike in the previous reports, the question of transplant nephrectomy did not arise earlier, because our patient was willing to try all available therapies. Indeed, transplant nephrectomy was viewed as a double penalty, combining failure of present transplantation and the faint hope of a future one whilst no effective therapy was available. The possibility for kidney transplant reuse has been only rarely described in the literature, [START_REF] Tseng | Successful reuse of a transplanted kidney 9 years after initial transplantation: 4-year follow-up[END_REF] and some authors even questioned its ethical and scientific validity. [START_REF] Graetz | Expansion of the organ donor pool by the reuse of a previously transplanted kidney-is this ethically and scientifically valid?[END_REF] Given his age, the expected median time to transplantation for the second recipient would have been around 27 months, with a 95%-chance of getting a kidney from an over 70-year-old donor. [START_REF]Annual Report of the French Transplantation Activity -2019[END_REF] Under these conditions, we could anticipate a probability of 25-30% delayed graft function, 65% one year-eGFR < 45ml/min/1.73m 2 , and 15% graft failure during the first year. The expected waitlist duration is what initially motivated the patient to consent to any derogatory procedure. The reassuring 6-month biopsy and the expected alternative scenario for this old recipient made us confident about the care option which was offered to him. The procedure was ultimately performed after obtaining written consent from patient 1, whose loss was partially mitigated by the prospect of reusing the kidney graft for another recipient. There is currently no formal contraindication to another transplantation, since early recurrence is not mandatory (approximately 20% recurrence-free) and new molecules seem promising in this indication. [START_REF] Colucci | Ofatumumab rescue treatment in post-transplant recurrence of focal segmental glomerulosclerosis[END_REF][START_REF] Solomon | Ofatumumab in post-transplantation recurrence of focal segmental glomerulosclerosis in a child[END_REF] However, after careful consideration, he preferred to delay any re-enrollment.

In the current context of organ shortage, it appears essential to avoid any organ waste. [START_REF] Bensouda | Optimizing Use of Deceased Donor Kidneys: Organ Waste While We Continue to Wait[END_REF][START_REF] Cooper | Report of National Kidney Foundation Consensus Conference to Decrease Kidney Discards[END_REF] In case of early, severe and resistant pFSGS recurrence after transplantation, kidney graft reuse should be considered. By not systematically defaulting to a new transplantation, reuse may open up prospects for patients facing recurrent pFSGS, whilst informing them of the potential need for this unusual valuable rescue procedure.

Legends to figures

Figure 1 .

 1 Figure 1. Evolution of serum creatinine, albumin levels and urinary protein-to-creatinine ratio

Figure 2 .

 2 Figure 2. Comparative pathological findings in three sequential kidney biopsies performed

Article Information

Support: None.

Financial Disclosure: The authors declare that they have no relevant financial interests.

Data Sharing: Data supporting the findings of this study are available from corresponding author (AH) upon reasonable request.

Patient Protections: The authors declare that they have obtained consent from each patient reported in this article for use of their medical records for research and that this work was performed under approval of the hospital ethics committee.


Marie-Charlotte Trojani

Sabine Santucci-Darmanin

Véronique Breuil

Georges F Carle

Dr Valérie Pierrefite-Carle

Autophagy and bone diseases

Keywords:

Autophagy is a ubiquitous cellular process, allowing the removal and recycling of damaged proteins and organelles. At the basal level, this process plays a role in quality control, thus participating in cellular homeostasis. Autophagy can also be induced by various stresses, such as nutrient deprivation or hypoxia, to allow the cell to survive until conditions improve. In recent years, the role of this process has been widely studied in many pathologies such as neurodegenerative diseases or cancers. In bone tissue, various studies have shown that autophagy is involved in the survival, differentiation and activity of osteoblasts, osteocytes and osteoclasts. The evolution of this knowledge has led to the identification of new molecular pathophysiological mechanisms in bone pathologies. This review reports the current state of knowledge on the role of autophagy in 4 bone diseases: osteoporosis, which seems to be associated with a decrease in autophagy, osteopetrosis and Paget's disease where the course of the autophagic process is disturbed, and finally osteosarcoma where autophagy seems to play a protumoral role. A better understanding of the involvement of autophagy in these pathologies should eventually lead to the identification of new potential therapeutic targets.

Autophagy

Autophagy mechanism

Macroautophagy, commonly referred to as autophagy, is a ubiquitous cellular mechanism for the removal and recycling of damaged proteins and organelles [START_REF] Mizushima | Autophagy fights disease through cellular selfdigestion[END_REF]. At the basal level, this mechanism plays a key role in cellular quality control and homeostasis. Autophagy can also be induced in response to various stresses such as nutrient deprivation, oxidative stress, or hypoxia, to allow cell survival. Finally, under certain conditions, excessive autophagy can lead to cell death [START_REF] Mizushima | Autophagy fights disease through cellular selfdigestion[END_REF]. From a molecular point of view, in the presence of nutrients, autophagy is negatively regulated by the mTOR (mammalian target of rapamycin) complex. When the cell is deprived of nutrients or in any other situation of cellular stress, mTOR is inactivated. This inactivation leads to the activation of Unc-51 like autophagy activating kinase (ULK1) and then to the recruitment of a multiprotein complex called Pi3K class III complex, which induces autophagy [START_REF] Mizushima | Autophagy fights disease through cellular selfdigestion[END_REF]. The autophagic process begins in the cytoplasm, where, under the combined action of different autophagy-related genes (ATGs), a double membrane called phagophore is formed. This phagophore encircles the damaged proteins and organelles and then closes, forming a double-membrane vesicle called an autophagosome. During this process, a cytosolic protein called ATG8 or LC3-I is lipidated and inserted into the double membrane of the autophagosome as LC3-II. Thus LC3-II is considered a marker of autophagosomes. The cellular material to be degraded is sequestered in the autophagosome by the action of specific autophagy receptors including the SQSTM1/p62 (p62) protein, which is considered a substrate of autophagy [START_REF] Mizushima | Autophagy: renovation of cells and tissues[END_REF].

Subsequently, the lysosome fuses with the autophagosome, forming an autolysosome. The action of the lysosomal V-ATPase then leads to acidification of the vesicle resulting in degradation and subsequent recycling of material (Figure 1). Autophagy is a dynamic process, in which autophagosomes are constantly formed and then degraded, and is therefore referred to as autophagic flux. Thus, a decrease in LC3-II expression combined with an increase in p62 expression generally reflects a decrease in autophagy. On the other hand, an increase in LC3-II may reflect either an induction of autophagy or an inhibition of autophagosome maturation, due for example to a defect in lysosomal activity.

The aim of this review is to summarize the main results concerning the role of autophagy in bone tissue and bone pathologies.

Autophagy and bone tissue

The role of autophagy in bone tissue was still unknown ten years ago [START_REF] Mizushima | Autophagy: renovation of cells and tissues[END_REF]. However, the observation that most of the bone-promoting molecules such as vitamin D, estradiol, or bisphosphonates were known as autophagy inducers, suggested an important function of this process for bone quality [START_REF] Pierrefite-Carle | Autophagy in bone: Self-eating to stay in balance[END_REF]. In the last decade, our understanding of the autophagy role in bone tissue has significantly increased. It has been shown that autophagy is involved in the survival and differentiation of osteoblasts (OB) and osteocytes (OST) [START_REF] Liu | Suppression of autophagy by FIP200 deletion leads to osteopenia in mice through the inhibition of osteoblast terminal differentiation[END_REF], and that this process is required for bone mineralization [START_REF] Nollet | Autophagy in osteoblasts is involved in mineralization and bone homeostasis[END_REF]. With regard to osteoclasts (OC), autophagy is involved in their differentiation [START_REF] Zhao | Autophagy regulates hypoxia-induced osteoclastogenesis through the HIF-1α/BNIP3 signaling pathway[END_REF] and some autophagy proteins are required for bone resorption, notably through their involvement in the brush border establishment and in the secretory activity of OCs [START_REF] Deselm | Autophagy proteins regulate the secretory component of osteoclastic bone resorption[END_REF].

Autophagy role in bone pathologies

Osteoporosis

Osteoporosis (OP) is a diffuse skeletal pathology characterized by major bone fragility that affects one in 3 women after menopause and one in 6 men after 50 years of age [START_REF] Sànchez-Riera | Osteoporosis and fragility fractures[END_REF]. Multiple molecular mechanisms are involved in postmenopausal OP, but two crucial factors for disease onset are the decrease in estrogen and the increase in oxidative stress [START_REF] Almeida | Basic biology of skeletal aging: role of stress response pathways[END_REF]10]. Interestingly, estrogen appears to induce autophagy in bone cells [START_REF] Gavali | Estrogen enhances human osteoblast survival and function via promotion of autophagy[END_REF][START_REF] Cheng | Oestrogenαactivated autophagy has a negative effect on the antiαosteoclastogenic function of oestrogen[END_REF] while oxidative stress is generated by autophagy deficiency.

In addition, a pan-genomic link has been observed between autophagy genes, human height and OP, suggesting that autophagy is involved in this pathology [START_REF] Pan | The regulation-of-autophagy pathway may influence Chinese stature variation: evidence from elder adults[END_REF][START_REF] Zhang | Pathway-based genome-wide association analysis identified the importance of regulation-of-autophagy pathway for ultradistal radius BMD[END_REF]. In addition, several studies also support a role for autophagy in OP: (i) OB and OST from aged mice exhibit a decreased autophagy [START_REF] Chen | Decreased activity of osteocyte autophagy with aging may contribute to the bone loss in senile population[END_REF][START_REF] Camuzard | Sex-specific autophagy modulation in osteoblastic lineage: a critical function to counteract bone loss in female[END_REF]; (ii) autophagy increases in OST after ovariectomy, likely counterbalancing estrogen loss and oxidative stress [START_REF] Camuzard | Sex-specific autophagy modulation in osteoblastic lineage: a critical function to counteract bone loss in female[END_REF][START_REF] Yang | Increased activity of osteocyte autophagy in ovariectomized rats and its correlation with oxidative stress status and bone loss[END_REF]; (iii) autophagy activation in OST through treatment with the mTOR inhibitor rapamycin reduces OP severity [START_REF] Luo | Rapamycin reduces severity of senile osteoporosis by activating osteocyte autophagy[END_REF]; (iv) bone loss related with aging and estrogen deprivation is exacerbated by an autophagy deficiency in OB [START_REF] Camuzard | Sex-specific autophagy modulation in osteoblastic lineage: a critical function to counteract bone loss in female[END_REF]. All these results show that autophagy induction in OB and OST can compensate the deleterious effects of estrogen drop in OP, suggesting that this pathway represents a promising target for this pathology.

A link between deficient hematopoietic autophagy and OP has recently been evidenced in humans [START_REF] Yuan | Deterioration of hematopoietic autophagy is linked to osteoporosis[END_REF]. Moreover, a drastic bone loss along with an imbalance in OST homeostasis was observed in mice after Atg7 autophagy gene inactivation in hematopoietic cells [START_REF] Yuan | Deterioration of hematopoietic autophagy is linked to osteoporosis[END_REF]. This phenotype was associated with the disappearance of H-vessels, connecting OST and hematopoietic cells. In summary, autophagy induction in hematopoietic cells or in OB/OST could represent a new path in OP treatment (Figure 2A). Some molecules already commercially available are known to modulate autophagy. For example, everolimus, an immunosuppressant that induces autophagy by inhibiting mTOR, decreases bone loss in a postmenopausal osteoporosis model in mice [START_REF] Kneissel | Everolimus suppresses cancellous bone loss, bone resorption, and cathepsin K expression by osteoclasts[END_REF]. In humans, coupling an autophagy inducer such as everolimus or resveratrol to a compound with bone tropism could be used to stimulate autophagy specifically in the bone microenvironment.

In corticoid-induced OP, in addition to the decrease in bone formation that is the main mechanism involved in the pathology, there is an increase in resorption related to a prolongation of OC life [START_REF] Jia | Glucocorticoids act directly on osteoclasts to increase their life span and reduce bone density[END_REF][START_REF] Kim | Glucocorticoids suppress bone formation via the osteoclast[END_REF]. Although a relationship between autophagy and glucocorticoid treatment has already been established [START_REF] Lin | Inactivation of autophagy ameliorates glucocorticoidinduced and ovariectomy-induced bone loss[END_REF], the underlying molecular mechanism has only very recently been identified, and involves the PI3K/Akt/mTOR autophagic signaling pathway in primary OCs [START_REF] Fu | Glucocorticoids Enhanced Osteoclast Autophagy Through the PI3K/Akt/mTOR Signaling Pathway[END_REF]. Thus, both in vitro and in vivo, glucocorticoid-treated OCs display an increased autophagic activity, suggesting that this pathway could be therapeutic target for corticoid-induced OP.

Osteopetrosis

This heterogeneous genetically inherited bone pathology, also called "marble bone disease", results from OC function alteration. Its severity ranges from mild to a lethal form such as infantile malignant osteopetrosis (IMO) also called autosomal recessive osteopetrosis. This last form of osteopetrosis is characterized by an increased bone mass due to the OC resorption defect. Without bone marrow transplantation, which remains the only treatment to date, this disease is lethal in children [START_REF] Shadur | Successful hematopoietic stem cell transplantation for osteopetrosis using reduced intensity conditioning[END_REF].

The TCIRG1 gene, encoding the a3 subunit of V-ATPase, displays the large majority of mutations described in IMO patients although some mutations are also found in the CLC-7 gene or OSTM1 [START_REF] Frattini | Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis[END_REF][START_REF] Sobacchi | The mutational spectrum of human malignant autosomal recessive osteopetrosis[END_REF]. The resorption activity in OC is affected by these mutations through lysosomal dysfunction. Several autophagy proteins have been shown to participate in the relocalization of this organelle to the brush border where the resorption takes place [START_REF] Deselm | Autophagy proteins regulate the secretory component of osteoclastic bone resorption[END_REF]. Moreover, lysosomes also play a major role in the final step of autophagy, allowing degradation and recycling of autophagosome content. Thus, autophagy appears to play a complex role in this rare bone pathology.

A defect in autophagy, characterized by a reduced LC3-II expression and an increased in p62 protein level, has been observed in OC of the osteopetrotic oc/oc mouse, a mouse model of IMO, carrying a homozygous mutation in the TCIRG1 gene [START_REF] Ochotny | The R740S mutation in the V-ATPase a3 subunit results in osteoclast apoptosis and defective early-stage autophagy[END_REF]. In addition, mice models carrying OSTM1 or CLC-7 mutations exhibit, on top of an osteoporotic phenotype, a rapid neurodegeneration with an increased LC3-II expression in neurons [START_REF] Héraud | Severe Neurodegeneration with Impaired Autophagy Mechanism Triggered by Ostm1 Deficiency[END_REF][START_REF] Wartosch | Lysosomal degradation of endocytosed proteins depends on the chloride transport protein ClC-7[END_REF], suggesting a disrupted autophagic flux.

Finally, the PLEKHM1 gene, which is implicated in an intermediate type of human osteopetrosis and in osteoclastic vesicular trafficking in the incisorless rat model [START_REF] Van Wesenbeeck | Localization of the gene causing the osteopetrotic phenotype in the incisors absent (ia) rat on chromosome 10q32.1[END_REF][START_REF] Bo | Characterization of a Relatively Malignant Form of Osteopetrosis Caused by a Novel Mutation in the PLEKHM1 Gene[END_REF], plays also a role in the regulation of the endosomal/autophagic pathway through the binding to LC3 and Rab7/HOPS proteins [START_REF] Tabata | Rubicon and PLEKHM1 negatively regulate the endocytic/autophagic pathway via a novel Rab7-binding domain[END_REF][START_REF] Mcewan | PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins[END_REF].

In summary, most of osteopetrosis gene mutations affecting OC endosomal and lysosomal trafficking, appear to be also associated with neuronal and OC autophagy regulation (Figure 2B).

Paget disease of bone

Disorganized bone formation as well as an elevated bone resorption are the characteristics of Paget's disease of bone (PDB). Although most patients affected by this chronic focal disease are asymptomatic, joint or neurological complications can be observed due to fissures, fractures, and bone deformities. An increase of bone remodeling is observed, associated with the presence of "hyperactive" giant OCs with many nuclei [START_REF] Reddy | Cell biology of Paget's disease[END_REF]. Inclusion bodies composed of aggregates of ubiquitinated or misfolded proteins are also present in Pagetic OCs [START_REF] Helfrich | Genetics and aetiology of Pagetic disorders of bone[END_REF]. It seems that environmental and genetic factors are involved in PDB onset. Mutations in the p62/SQSTM1 gene are observed in 40% of familial cases and 10% of sporadic cases. Besides its role as an autophagy receptor, p62 has a key function in NF-kB signaling and apoptosis during osteoclastogenesis [START_REF] Alonso | Clinical and Genetic Advances in Paget's Disease of Bone: a Review[END_REF]. The ubiquitin-associated domain (UBA) of the p62 gene is the main target of mutations [START_REF] Alonso | Clinical and Genetic Advances in Paget's Disease of Bone: a Review[END_REF] and a point mutation in this domain appears sufficient to trigger PDB-like pathology in mice in which OC exhibit an increased autophagy level [START_REF] Daroszewska | A point mutation in the ubiquitin-associated domain of SQSMT1 is sufficient to cause a Paget's disease-like disorder in mice[END_REF]. Nevertheless, a study on OC derived from PDB patients suggests an autophagy flux inhibition combined with a build-up of undegraded autophagosomes [START_REF] Mcmanus | Autophagy and 3-Phosphoinositide-Dependent Kinase 1 (PDK1)-Related Kinome in Pagetic Osteoclasts[END_REF], potentially explaining the presence of inclusion bodies. The study of Usategui-Martín et al. strengthens the previous results as transfection of a p62 gene carrying one of the PDB mutations identified in patients results in autophagosome maturation inhibition [START_REF] Usategui-Martín | A mutation in p62 protein (p. R321C), associated to Paget's disease of bone, causes a blockade of autophagy and an activation of NF-kB pathway[END_REF]. An inhibition of cell proliferation via the NFk-B pathway was also associated with this mutation [START_REF] Usategui-Martín | A mutation in p62 protein (p. R321C), associated to Paget's disease of bone, causes a blockade of autophagy and an activation of NF-kB pathway[END_REF]. In summary, these experimental results suggest an autophagy deregulation in PDB, which is further supported by ATG gene polymorphisms association with this pathology [START_REF] Usategui-Martín | Polymorphisms in Autophagy Genes Are Associated with Paget Disease of Bone[END_REF] and the implication of optineurin (OPTN), another autophagy receptor, as a predisposing gene for this disease [START_REF] Albagha | Genome-wide association study identifies variants at CSF1, OPTN and TNFRSF11A as genetic risk factors for Paget's disease of bone[END_REF]. More recently, the OATL1 gene, implicated in autophagosome maturation [START_REF] Itoh | OATL1, a novel autophagosome-resident Rab33B-GAP, regulates autophagosomal maturation[END_REF], appears to be also involved in PDB [START_REF] Alonso | Clinical and Genetic Advances in Paget's Disease of Bone: a Review[END_REF]. Altogether, these various studies, associated with the role of autophagy/ATG proteins in OC differentiation and function, strongly support the hypothesis of autophagy involvement in PDB (Figure 2C).

Osteosarcoma

Osteosarcoma (OS) is a malignant bone tumor, primarily of osteoblastic origin, with a peak incidence in childhood and adolescence. The lungs are the most common site of metastasis and the 5-year survival of patients with metastases is approximately 20%. From a genetic perspective, OS has major genomic complexity. Among the tumor suppressor genes frequently inactivated in OS, some such as RB1, PTEN or TP53 regulate autophagy. In parallel, various oncogenes that can be activated in OS also regulate autophagy. These data suggest a deregulation of autophagy in OS [START_REF] Camuzard | Role of autophagy in osteosarcoma[END_REF].

A dual role of autophagy is observed in OS as in other tumor types. OS tumor cells use autophagy as a survival pathway for treatment resistance, proliferation and cancer stem cell protection [START_REF] Camuzard | Role of autophagy in osteosarcoma[END_REF]. Thus, it seems interesting to specifically block autophagy in these cells, in order to potentiate the efficacy of classical therapies. For this purpose, we dispose of autophagy inhibiting molecules, such as hydroxychloroquine (late inhibitor) or VPS34 inhibitors (early inhibitors). Numerous clinical trials are underway, combining conventional chemotherapies with autophagy modulators [START_REF] Niu | Insight Into the Role of Autophagy in Osteosarcoma and Its Therapeutic Implication[END_REF]. However, autophagy can also lead to cancer cell death, although the conditions required to transform prosurvival autophagy into a death process are not yet elucidated [START_REF] Camuzard | Role of autophagy in osteosarcoma[END_REF].

Conclusion

Over the past decade, our knowledge regarding the role of autophagy in bone tissue and bone pathology has significantly increased. Modulation of autophagy has emerged as an exciting new avenue of research for the development of new therapeutic strategies.

In postmenopausal OP, the body of preclinical work in animal models suggests that a decrease in autophagy in OB/OST participates in the development of this pathology, although definitive proof in humans remains to be established. Understanding the reason for this age-related decrease remains an important issue as these data could help identify new therapeutic molecules. In this context, an agerelated increase in the expression of Rubicon, a negative regulator of autophagy, has been observed in different tissues in mice. Although the age-dependent expression level of Rubicon has not yet been studied in bone, it has already been shown that inactivation of this gene restores autophagy, thus reducing age-related phenotypes [START_REF] Nakamura | Suppression of autophagic activity by Rubicon is a[END_REF]. Thus, stimulating autophagy in the bone microenvironment appears to be a promising strategy for the treatment of OP. In osteopetrosis and PDB, the autophagic process is disturbed either indirectly by mutations affecting lysosomal function (osteopetrosis) or directly by mutations affecting autophagy receptors (PDB). In both cases, it is likely that autophagy perturbation contributes to the pathology by affecting osteoclastic function.

Finally, in osteosarcoma as in other tumor types, the role of autophagy is complex but tumor cells generally use this process to resist the multiple stresses to which they are subjected. In addition to blocking autophagy, identifying the molecular mechanisms that allow switching from pro-survival autophagy to cell death could lead to the identification of new treatment pathways.

In conclusion, we are at the beginning of deciphering the role of autophagy in bone tissue and bone pathology. Continued studies in this area offer the prospect of new therapeutic solutions in a wide spectrum of pathologies for which options are still sometimes limited. In response to cellular stress, mTOR is inactivated. Autophagy is then triggered by the action of the ULK1 complex and the PI3K class III complex. A double membrane called phagophore is formed in the cytosol, and encloses damaged organelles, aggregates and proteins. The phagophore then closes, leading to the formation of an autophagosome. During this process, a cytosolic protein named LC3-I is lipidated and inserted into the double membrane as LC3-II. The p62 protein acts as a receptor for autophagy by binding the ubiquitinated proteins on the one hand and the LC3-II protein on the other. Finally, the autophagosome fuses with the lysosome to degrade, thanks to the action of V-ATPase, the contents which will be recycled. PE : Phosphatidylethanolamine.

Figure 1 :

 1 Figure 1: Autophagy mechanism. The mechanism of autophagy and the main molecular complexes

Figure 2 :

 2 Figure 2 : Autophagy in bone pathologies. (A) Development of osteoporosis is associated with decreased autophagy with age in OB, OST, and hematopoietic stem cells (HSCs). (B) Mutations identified in osteopetrosis (OMI) induce an autophagy defect or an autophagic flux disturbance in OC, resulting in decreased resorption. (C) In Paget's disease of bone, mutations in p62, or OPTN could promote inhibition of autophagic flux and the appearance of inclusion bodies. This autophagy defect could be involved in the development of PDB, especially through the role of autophagy and ATG proteins in the differentiation and function of OC. VS: vessels.

Acknowledgments:

The authors thank the French Society of Rheumatology (SFR) and the France Cancer association.