François A Giuliano

MD Charles Joussain

MD Pierre Denys

MD Francois Giuliano
email: fgfrancois.giuliano@uvsq.fr

Long Term Effectiveness and Safety of Intracavernosal Botulinum Toxin A as an Add-on Therapy to Phosphosdiesterase Type 5 Inhibitors or Prostaglandin E1 Injections for Erectile Dysfunction

Keywords: OnabotulinumtoxinA, abobotulinumtoxinA, combined treatment, non-responders, pharmacology, duration of action

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

Introduction

There is an unmet medical need for the treatment of erectile dysfunction (ED) in non-responders or, more accurately, insufficient responders to phosphodiesterase type 5 inhibitors (PDE5-Is) [START_REF] Munk | Systematic Review of Oral Combination Therapy for Erectile Dysfunction When Phosphodiesterase Type 5 Inhibitor Monotherapy Fails[END_REF] . To our knowledge, two studies have reported the short-term efficacy of combined therapy involving intracavernosal (IC) injections of botulinum toxin A (BTX-A IC) as an add on to standard therapy in these patients. The first was a randomized placebo-controlled trial that included 24 patients with severe vasculogenic ED who were non-responders to PDE5-Is or IC injections of tri-mix (a custom-prepared mixture of prostaglandin E1 (PGE1) with papaverine and phentolamine) [START_REF] Ghanem | Botulinum Neurotoxin and Its Potential Role in the Treatment of Erectile Dysfunction[END_REF] . At the 4-week follow-up, erectile function and penile rigidity were significantly more improved in the treatment group. The second study was a retrospective, uncontrolled study of 47 patients who were insufficiently responsive to PDE5-Is or PGE1 IC and who received IC injections of abobotulinumtoxinA (Dysport®; Ipsen, Paris, France) 250 or 500U as an add on therapy [START_REF] Giuliano | Safety and Efficacy of Intracavernosal Injections of AbobotulinumtoxinA (Dysport®) as Add on Therapy to Phosphosdiesterase Type 5 Inhibitors or Prostaglandin E1 for Erectile Dysfunction-Case Studies[END_REF] . The results showed a 54% response rate at 6-weeks.

Although the results of both these studies are encouraging, both only assessed the short-term effectiveness and tolerance of BTX-A IC injections (at 4 2 and 6 3 weeks, respectively). More recently, the group who performed the first randomized placebo-controlled trial performed a second randomized placebo-controlled trial also in patients with severe vasculogenic ED, who were non responders to PDE5-Is and to IC injections of tri-mix. This second trial was large, with 176 patients randomized to receive either onabotulinumtoxinA 50 U, 100 U or placebo IC injections. Forty percent of patients were responders, with a maximal improvement at 3 months post BTX-A IC [START_REF] El-Shaer | Intra-cavernous injection of BOTOX® (50 and 100 Units) for treatment of vasculogenic erectile dysfunction: Randomized controlled trial[END_REF] . At 6 months, the effectiveness of onabotulinumtoxinA 100 U was superior to that of onabotulinumtoxinA 50 U. To our knowledge, these are the only data available regarding the effect of onabotulinumtoxinA at 6 months.

It is believed that BTX-A reduces ED by decreasing sympathetic activity within the cavernous and penile nerves that supply the arteries and smooth muscle of the penis [START_REF] Giuliano | Botox for Erectile Dysfunction[END_REF] . This mechanism likely functions by the partial blocking of norepinephrine release of the normal vesicular dependent release of norepinephrine from the sympathetic nerve terminals into the neuromuscular junction [START_REF] Morris | Botulinum neurotoxin A attenuates release of norepinephrine but not NPY from vasoconstrictor neurons[END_REF] . This putative mechanism of action is similar to that of BTX-A intradetrusor injection for the treatment of overactive bladder and neurogenic detrusor overactivity 7 . To initiate voiding, parasympathetic postganglionic nerves release acetylcholine in the neuromuscular synapse which binds with the muscarinic receptor in the detrusor muscle and generates muscle contraction [START_REF] Fowler | The neural control of micturition[END_REF] . BTX-A injected in the detrusor muscle prevents acetylcholine release, reducing detrusor overactivity. Studies have shown that for optimal effectiveness, intradetrusor onabotulinumtoxinA injections should be administered every 5 (200U dose) or 6 months (300U dose) [START_REF] Joussain | Long-term outcomes and risks factors for failure of intradetrusor onabotulinumtoxin A injections for the treatment of refractory neurogenic detrusor overactivity[END_REF] , and the overall median duration of the effect is 9 months 10 . Such data do not currently exist for BTX-A IC injections for the treatment of ED. The primary aim of the present retrospective case series study was therefore to assess the duration of effect of BTX-A IC injection in patients with ED of various etiologies. The secondary aims were to evaluate the type and rate of side effects at the time of BTX-A IC injection and during the follow-up, and the proportion of responders at the 2-month follow-up.

Materials and Methods

We conducted a retrospective, single center study of medical files from consecutive patients who responded insufficiently to pharmacological treatment for ED and who received BTX-A IC as an add-on therapy between October 2017 and November 2020.

Eligibility criteria

Male patients were included if they were aged > 18 years, diagnosed with ED, in a stable heterosexual relationship and had a history of insufficient effectiveness of at least one marketed PDE5-I at the highest approved dose, or PGE1 IC injections with a dose up to 60 µg. Current highest approved doses of PDE5-Is on demand are 100mg for sildenafil and 20 mg for tadalafil or vardenafil, and 5 mg for daily tadalafil. Insufficient effectiveness was defined by an EF domain score of the IIEF < 26 with standard treatment. Exclusion criteria were contra-indications to BTX-A determined by abobotulinumtoxinA and onabotulinumtoxinA monographies [START_REF]Dysport Therapeutic[END_REF]12 . Patients were categorized according to their etiology(ies) and/or risk factor(s) for ED based on their medical history and routine laboratory investigations, including glucose fasting and lipid profiles. Patients with endocrinopathies (apart from diabetes mellitus), sleep apnea or no organic etiology and/or risk factors identified (thus ED of likely psychogenic origin) were categorized as "other". Since our sexual medicine clinic is situated within a hospital that is specialized in the treatment of neurological disorders, the cohort was composed of a large proportion of patients with spinal cord-injury (SCI).

Intracavernosal injection

The intracavernosal injection technique has been described elsewhere [START_REF] Ghanem | Botulinum Neurotoxin and Its Potential Role in the Treatment of Erectile Dysfunction[END_REF] . Briefly, an adjustable penile loop ring was placed at the penoscrotal junction for 30 minutes prior to the injections. Then, 250 or 500 Speywood units of abobotulinumtoxinA or 100 units of onabotulinumtoxinA were injected via a 13mm long 29 ½ G needle: 0.5 ml into each corpus cavernosum. The choice of BTX-A type (abobotulinumtoxinA or onabotulinumtoxinA) was not related to ED severity or to ED risk factors and/or etiologies. The first 32 patients who were administered abobotulinumtoxinA received a dose of 250 units and the following 37 patients received a dose of 500 units to ensure safety, as previously reported [START_REF] Giuliano | Safety and Efficacy of Intracavernosal Injections of AbobotulinumtoxinA (Dysport®) as Add on Therapy to Phosphosdiesterase Type 5 Inhibitors or Prostaglandin E1 for Erectile Dysfunction-Case Studies[END_REF] .

Patient follow-up

Following BTX-A IC injection, patients were advised to attempt sexual intercourse using their usual pharmacological treatment. An early follow-up assessment was scheduled during the 2 nd month after the BTX-A IC injection. Responders (see endpoints) attended a second assessment session during the 7 th month after the BTX-A IC injection or upon their request if the effectiveness decreased.

Endpoints

The primary endpoint was the proportion of responders at the first assessment. A patient was classified as a responder to BTX-A IC if the minimum clinically important difference (MCID) in the IIEF-EF score relative to their baseline severity of ED was achieved. The MCIDs according to ED severity are as follows: mild: 2 points, moderate: 5 points and severe: 7 points [START_REF] Rosen | Minimal clinically important differences in the erectile function domain of the International Index of Erectile Function scale[END_REF] . Baseline severity of ED was measured using the IIEF-EF during treatment with PDE5-Is or PGE1 IC injections but before BTX-A IC, and thus provided a score for the severity of persisting ED with standard treatment. Severity was classified as mild (EF score 17-30), moderate (EF score 11-16) or severe (EF score 0-10) [START_REF] Rosen | Minimal clinically important differences in the erectile function domain of the International Index of Erectile Function scale[END_REF] .

The secondary endpoints were i) side effects identified by patient self-report at the time of the BTX-A IC injection and during the follow-up and ii) effectiveness at the 2-month follow-up. Data from the early assessment (2 months post BTX-A IC) of some patients in the present series treated with abobotulinumtoxinA IC have previously been reported [START_REF] Giuliano | Safety and Efficacy of Intracavernosal Injections of AbobotulinumtoxinA (Dysport®) as Add on Therapy to Phosphosdiesterase Type 5 Inhibitors or Prostaglandin E1 for Erectile Dysfunction-Case Studies[END_REF] .

Statistical analyses

Continuous variables were expressed as means with standard deviations. IIEF-EF score, time elapsed between BTX-A IC and duration of ED were expressed as medians [first-third quartile]. No formal sample size calculation was performed, and all analyses were exploratory. Comparisons of qualitative data were performed using a Fisher's exact test (Prism V5, GraphPad software -La Jolla, CA, USA).

Odds Ratios (OR) were calculated from logistic regressions in both the univariate and multivariate analysis to determine risk factors for non-response to BTX-A IC. (Stata/MP 16.0 (Timberlake -Richmond upon Thames UK). The variables included in the univariate logistic regression at 34 [27-42] days were: age, ED duration, comorbidities, ED treatment prior to BTX-A IC, type of BTX-A, IIEF-EF score at baseline and ED severity. The variables with a p value < 0.05 were entered into a multivariate logistic regression analysis.

Ethics

In accordance with French legislation for retrospective studies, the database was approved by the French Data Protection Authority (Commission Nationale de l'Informatique et des Libertés) under the registration number 2209010v0. Patients' medical files were anonymized, and patients were informed they could deny access to their personal and medical data at any time. Written informed consent was obtained from all patients whose data were included in the study.

Results

One hundred and thirty-one patients with ED were treated with BTX-A IC during the study period: data from 123 files were analyzed (inclusion flow chart is shown in figure 1). Mean age was 53 ± 14 years and median duration of ED prior to BTX-A IC was 5 [2-10] years. The risk factors and etiologies for ED were as follows: cardio-metabolic for 49 (39.8%) patients, spinal cord injury for 50 (40.7%) patients, radical prostatectomy for 27 (22%) patients and for 19 (14.4%) patients categorized as "other", no organic etiology or risk factor was identified for the majority. Prior to BTX-A IC, 77% of patients were treated with highest dose PDE5-Is and 29% by PGE1 IC injections (mean dose: 38 ± 18 µg). Eight patients (6%) were treated with PDE5-Is combined with PGE1 IC injections. Median baseline IIEF-EF score (i.e. with standard treatment) was 11 [6-17] points prior to BTX-A IC; 35 (%) patients had mild, 26 (%) had moderate and 59 (48%) had severe ED. Fifty-eight patients (47%) were treated with onabotulinumtoxinA 100 U and 65 patients (53%) with abobotulinumtoxinA (32 patients (26%) with 250 Speywood U and 33 patients (27%) with 500 Speywood U).

Early assessment

Ninety-six patients underwent early assessment, 34 [27-42] days following BTX-A IC. Of these, 53 (55.2%) were classed as responders, with an increase in median IIEF-EF score from 17 [10-19] to 26 [22-28] points (table 1). The response rate was 90% in those with mild baseline ED, 50% in those with moderate ED and 33% in those with severe with ED; and was 56.8% in patients treated with PDE5-Is and 51.9% in patients treated with PGE1-IC injections (table 2). Of the 39 patients treated by PDE5-Is, none switched to PGE1 IC injections. Of the 11 patients treated by PGE1 IC injections, the dose of PGE1 was reduced for 5, and one patient switched to PDE5-Is. The response rate according to ED risk factors and/or etiologies is shown in figure 2. Response rate was lower in patients with post-radical prostatectomy and SCI compared to patients with other comorbidities (p=0.002 and p=0.0045 respectively). There was no difference in the proportion of responders between abobotulinumtoxinA (250 or 500 U) and onabotulinumtoxinA 100 U (47% and 54% respectively, p=0.67) (table 2). The univariate logistic regression identified the following factors as associated with the response to BTX-A IC: other comorbidities (p=0.01), SCI (p=0.047), post-prostatectomy (p=0.04) and ED severity (p<0.01).

EF-score at baseline was also identified but is has the same signification as ED severity and thus was not selected for multivariate analysis. After multivariate analysis, only ED severity was identified as a risk factor OR=0.3, IC(95%) = [0.16 -0.56] for non-response to BTX-A IC(table 3).

Late assessment

Among all the patients treated, 46 (41%) were categorized as responders at the late assessment (median 5.9 [3.9 -8.1] months post BTX-A-IC). Of the 42 initial responders (i.e. among those who underwent early assessment and who were re-assessed at the late assessment), 32 remained responders (76%). Three non-responder patients at the early visit were responders at the late visit.

The effectiveness of the treatment did not decrease significantly from the early to the late assessment in any of the ED risk factor and/or etiology groups (figure 2). The decrease in effectiveness over time was not different between the types of BTX-A IC (figure 3). The univariate analysis did not show any association between the type of standard treatment for ED, type of BTX-A or ED severity, and response to BTX-A IC.

Side effects

One patient reported mild penile pain during BTX-A IC injection and a second patient reported mild penile pain for 3 days following BTX-A IC, with no need for pain medication. No systemic effects (generalized muscle weakness, dysphonia, diplopia, dysphagia and respiratory function impairment [START_REF] Ruet | Single-fiber electromyography analysis of botulinum toxin diffusion in patients with fatigue and pseudobotulism[END_REF] were reported.

Discussion

This retrospective case study confirmed that BTX-A IC is an effective add-on therapy to PDE5-Is or PGE1 IC injection, with an effect that lasted for at least 6 months: 41 % of patients with ED who responded insufficiently to PDE5-Is or PGE1 IC injection responded at 6 months with BTX-A IC. In addition, both abobotulinumtoxinA (250 and 500 U) and onabotulinumtoxinA 100U IC appeared to be safe. The only side effect reported was mild pain during or after the IC injection in less than 1% of patients; no systemic side effects of BTX-A were reported. Such a high level of safety was also found in a study of onabotulinumtoxinA 100U injection in the detrusor muscle (also smooth muscle) in patients with neurogenic detrusor activity caused by multiple sclerosis [START_REF] Tullman | Low-dose onabotulinumtoxinA improves urinary symptoms in noncatheterizing patients with MS[END_REF] .

The primary endpoint of this study was a clinically meaningful improvement in the IIEF-EF score. This is commonly referred to as the minimal clinically important difference (MCID), and has been defined as the smallest change in a score in the domain of interest that patients perceive as beneficial and that would indicate a need for change in the patient's treatment, in the absence of side effects [START_REF] Jaeschke | Measurement of health status. Ascertaining the minimal clinically important difference[END_REF] . The MCID was achieved in 41% of patients at 6 months. At the time of writing, 22 of the 46 responder patients at 6 months had undergone further follow-up visits. In 18 (82%) of these patients, the response to BTX-A IC was sustained up to 11.2 [9.2 -19.4] months post BTX-A IC (data not shown).

Not surprisingly, the short-term response rate to BTX-A IC was higher in patients with mild baseline ED. In patients with ED caused by radical prostatectomy or SCI, BTX-A IC was less effective than in patients with ED associated with cardio-metabolic conditions (i.e. cardiovascular diseases and diabetes or other comorbidities. However, baseline ED (i.e. ED with standard treatment) was more severe in patients with radical prostatectomy or SCI than in those with cardio-metabolic conditions or other comorbidities. The response to BTX-A IC therefore seemed more influenced by the severity of the ED than by its etiology. However, this result was not confirmed at the late follow-up evaluation, probably due to a lack of statistical power resulting from the retrospective nature of the study. Of note is that most of the patients in the "other comorbidity(ies)" group who responded insufficiently to PDE5-Is or PGE1 IC injection had ED of psychogenic origin with no identified organic risk factors for ED. PDE5-Is is the first-line treatment for ED caused by either psychogenic or organic factors, however around 25 to 35% of patients do not respond to this treatment [START_REF] Hatzimouratidis | Non-responders to phosphodiesterase type 5 inhibitors: is there a second chance?[END_REF][START_REF] Huang | Phosphodiesterase-5 (PDE5) Inhibitors In the Management of Erectile Dysfunction[END_REF][START_REF] Yuan | Comparative effectiveness and safety of oral phosphodiesterase type 5 inhibitors for erectile dysfunction: A systematic review and network meta-analysis[END_REF] . Second-line therapy for these nonresponders consists of local pharmacological treatment [START_REF] Montorsi | Summary of the recommendations on sexual dysfunctions in men[END_REF] . It has been postulated that BTX-A IC has a chemical sympathectomy-like effect on the cavernosal nerve; by reducing the anti-erectile effect of cavernosal sympathetic innervation [START_REF] Dean | Physiology of penile erection and pathophysiology of erectile dysfunction[END_REF][START_REF] Andersson | Sympathetic pathways and adrenergic innervation of the penis[END_REF] , BTX-A IC enhances the pro-erectile effect of PDE5-Is or PGE1 ICI [START_REF] Giuliano | Safety and Efficacy of Intracavernosal Injections of AbobotulinumtoxinA (Dysport®) as Add on Therapy to Phosphosdiesterase Type 5 Inhibitors or Prostaglandin E1 for Erectile Dysfunction-Case Studies[END_REF] . In the case of ED of vascular origin, it has been proposed that BTX-A IC increases the cavernosal expression of VEGF and CD31 which could be involved in vasodilation and endothelial cell proliferation [START_REF] El-Shaer | Intra-cavernous injection of BOTOX® (50 and 100 Units) for treatment of vasculogenic erectile dysfunction: Randomized controlled trial[END_REF] .

Three preparations of botulinum toxin A (BTX-A) are commercially available and approved by the United States Food and Drug Administration and European Medicines Agency: onabotulinumtoxinA, abobotulinumtoxinA and incobotulinumtoxinA (Xeomin®, NT 201; Merz Pharmaceuticals GmbH, Frankfurt, Germany). These preparations differ from each other and are not interchangeable. The main difference between onabotulinumtoxinA and the other two types is the purification procedure.

OnabotulinumtoxinA is purified by repeated precipitation and re-dissolution, whereas abobotulinumtoxinA is purified by a column separation method [START_REF] Walker | Comparison and overview of currently available neurotoxins[END_REF] . Dose ratios between onabotulinumtoxinA and abobotulinumtoxinA are the subject of debate [START_REF] Walker | Comparison and overview of currently available neurotoxins[END_REF] , nevertheless, in the present study the effectiveness was not influenced by the type of BTX-A, either with a ratio of 2.5 or a ratio of 5 between ona and abo-botulinumtoxinA. In addition, we have recently found similar results for the early (2 months) effectiveness of incobotulinumtoxinA 100U IC in 35 patients using the same treatment paradigm (unpublished data).

As described in the introduction, the only large, randomized placebo-controlled clinical trial in patients with severe vasculogenic ED suggested that the duration of effectiveness of onabotulinumtoxinA was dose-dependent [START_REF] El-Shaer | Intra-cavernous injection of BOTOX® (50 and 100 Units) for treatment of vasculogenic erectile dysfunction: Randomized controlled trial[END_REF] . Similar findings have been reported for intradetrusor injections of BTX-A to treat neurogenic detrusor overactivity. In the present study, there was a trend towards a more pronounced decrease in effectiveness for abobotulinumtoxinA 250 U (relative decrease in effectiveness of 46% over time) when compared to 500 U (relative decrease in effectiveness of 28% over time). It is unlikely that this was influenced by severity, since around 50% of patients in each group had severe baseline ED (i.e.

ED with standard treatment) (data not shown). The lack of statistical significance may be due to the small sample size.

The main limitation of this study is that it was uncontrolled and retrospective. However, the large randomized, placebo-controlled trial described above found no placebo effect in patients with severe ED of vascular etiology treated by BTX-A IC [START_REF] El-Shaer | Intra-cavernous injection of BOTOX® (50 and 100 Units) for treatment of vasculogenic erectile dysfunction: Randomized controlled trial[END_REF] .

Conclusions

This study provides preliminary evidence to support the use of BTX-A IC as an add-on therapy to treat ED in insufficient responders to PDE5-Is or PGE1 ICIs: the duration of action was at least 6 months in 41% of patients with ED of varying etiologies and the treatment was safe. These results justify further assessment of the effectiveness and safety of BTX-A IC in randomized clinical trials. Such clinical trials are particularly important to determine the effectiveness of this add-on treatment in difficult to treat patients with ED of various etiologies, including radical prostatectomy. Table 2: ED severity according to IIEF-EF [START_REF] Rosen | Minimal clinically important differences in the erectile function domain of the International Index of Erectile Function scale[END_REF] , risk factors and etiologies of ED, treatment prior to BTX-A IC, type of botulinum toxin A IC delivered and IIEF-EF at baseline and time from injection to early assessment according to the response to botulinum toxin A (BTX-A) IC as an add on to their current pharmacological treatment in 123 consecutive patients who were insufficient responders at baseline to phosphodiesterase type 5 inhibitors or PGE1 intracavernosal injections and assessed at least once post-BTX-A IC.

Figure 1 : 2 C

 12 Figure 1: Flow chart of patients treated by BTX-A IC 1

Early assessment at 34 [27-42] days post-BTX-A IC in 96 patients ED severity according to IIEF-EF 13

		Severe (n=42)	Moderate (n=24)	Mild (n=30)
	IIEF-EF at baseline (median [1 st quartile -3 rd quartile])	6 [6-7]	13 [12-14]	19 [18-20]
	IIEF-EF post BTX-A IC (median [1 st quartile -3 rd quartile)	9 [6-17]	18 [12.-24]	26 [23-28]
	Increase in IIEF-EF (median [1 st quartile -3 rd quartile)	2 [0-8.5]	5 [-1-11]	6 [3-9]
	Responders (n (%))	14 (33.3%)	12 (50%)	27 (90%)

Table 1 :

 1 IIEF-EF at baseline and post BTX-A IC, increase in IIEF-EF and responder rate according to ED severity in 96 consecutive patients who were insufficient responders at baseline to phosphodiesterase type 5 inhibitors or PGE1 IC injections assessed 34 [27-42] days post-BTX-A IC as an add on therapy to their current pharmacological treatment.

	Early assessment post-BTX-A IC in 96 patients
	Responders (n=53)	Non responders (n=43)

mean dose PGE1 (mean +/-standard deviation) µg

	EF post injection (median [1st quartile -3rd quartile)	26 [22-28]	8 [6-12]
	Time between injection and assessment (median [1st quartile -	33 [27-42]	36 [28-43.5]
	3rd quartile]) days
		36.4 +/-18.2	38.5 +/-19.1
	PDE5-I + PGE1 n (%)	3 (5.7)	2 (4.7)
	Botulinum toxin type
	onabotulinumtoxinA 100 U n (%)	24 (45.3)	17 (39.5)
	abobotulinumtoxinA 250 Speywood U n (%)	15 (28.3)	13 (30.2)
	abobotulinumtoxinA 500 Speywood U n (%)	14 (26.4)	13 (30.2)
	BTX-A ICI result
	EF at baseline (median [1st quartile -3rd quartile])	17 [10-19]	7 [6-11 .5]


Karine Delord
email: karine.delord@cebc.cnrs.fr

Timothée Poupart

Nicolas Gasco

Henri Weimerskirch

Christophe Barbraud

Timothee Poupart

Jean-Baptiste Thiebot

Jérémy Demay

Rémi Bigonneau

Romain Bazire

Hélène Le Berre

Marine Quintin

Marine Devaud

Chloé Tanton

Jérémy Dechartre

Anthony Le

First evidence of migration across the South Pacific in endangered Amsterdam albatross and conservation implications

Keywords: Diomedea amsterdamensis, National Plan of Actions, juvenile dispersal, immature at sea distribution, international conservation responsibility

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

Introduction

Many seabirds undergo long-distance migrations between their breeding and wintering areas. This is particularly the case to reach upwelling systems: across North Pacific [START_REF] Frederiksen | Multicolony tracking reveals the winter distribution of a pelagic seabird on an ocean basin scale[END_REF][START_REF] Gaston | First evidence of east-west migration across the North Pacific in a marine bird[END_REF], across South Pacific from Australia to the Humboldt Current region [START_REF] Brooke | Albatrosses and petrels across the world[END_REF] or across South Atlantic to the Benguela/Agulhas Current region [START_REF] Phillips | Summer distribution and migration of nonbreeding albatrosses: individual consistencies and implications for conservation[END_REF]. As oceanic migrants albatrosses are amongst the most globally-threatened species and have direct interaction with fishing vessels, fisheries bycatch is one of the major conservation issue worldwide [START_REF] Croxall | Seabird conservation status, threats and priority actions: a global assessment[END_REF][START_REF] Phillips | The conservation status and priorities for albatrosses and large petrels[END_REF][START_REF] Good | National Plans of Action (NPOAs) for reducing seabird bycatch: Developing best practice for assessing and managing fisheries impacts[END_REF].

Albatrosses are long-lived species (i.e. high adult survival rates, delayed sexual maturity and low fecundity) and large-sized species (i.e. Diomedea spp.) breed biennially if successful in fledging a chick [START_REF] Warham | The petrels: their ecology and breeding systems[END_REF]. Given these extreme life-history traits, population trajectories are highly sensitive to changes in adult mortality. Albatrosses have a wide at sea distribution and are therefore potentially threatened by a multitude of fisheries in national and international waters [START_REF] Phillips | Summer distribution and migration of nonbreeding albatrosses: individual consistencies and implications for conservation[END_REF][START_REF] Baker | A global assessment of the impact of fisheries-related mortality on shy and whitecapped albatrosses: Conservation implications[END_REF][START_REF] Zador | Population impacts of endangered short-tailed albatross bycatch in the Alaskan trawl fishery[END_REF][START_REF] Delord | Areas of importance for seabirds tracked from French southern territories, and recommendations for conservation[END_REF][START_REF] Jiménez | Bycatch of great albatrosses in pelagic longline fisheries in the southwest Atlantic: Contributing factors and implications for management[END_REF]. Naïve juvenile albatrosses disperse widely and may have distinct routes and use different areas compared to adults [START_REF] Weimerskirch | Lifetime foraging patterns of the wandering albatross: life on the move![END_REF][START_REF] De Grissac | Contrasting movement strategies among juvenile albatrosses and petrels[END_REF][START_REF] Orben | Ontogenetic changes in at-sea distributions of immature short-tailed albatrosses Phoebastria albatrus[END_REF].

Among the albatrosses the Amsterdam albatross (Diomedea amsterdamensis) is listed amongthe most endangered species due to a very small population (~ 300-350 individuals; Barbraud et al. unpublished data) and being endemic to a single isolated island in the southern Indian Ocean, Amsterdam Island. It is one of the world's rarest birds. Very sensitive to additional mortality [START_REF] Weimerskirch | Population dynamics of Wandering Albatross Diomedea exulans and Amsterdam Albatross D. amsterdamensis in the Indian Ocean and their relationship with long-line fisheries: conservation implications[END_REF][START_REF] Rivalan | Combined impact of longline fisheries and climate on the persistence of the Amsterdam albatross[END_REF], the Amsterdam albatross is identified as a priority species to protect, especially at risk due to bycatch [START_REF] Abraham | Assessment of the risk of surface longline fisheries in the Southern Hemisphere to albatrosses and petrels, for 2016[END_REF], and has benefited from two successive National Plans of Actions since 2011 (NPOA 1 st : 2011-2015, 2 nd : 2018-2027). The Amsterdam albatross is a dispersive migrant known to undertake long-distance movements from eastern to western South Indian Ocean, occasionally reaching South Atlantic and therefore, crossing many fishing areas [START_REF] De Grissac | Contrasting movement strategies among juvenile albatrosses and petrels[END_REF][START_REF] Thiebot | Stage-dependent distribution of the critically endangered Amsterdam albatross in relation to Economic Exclusive Zones[END_REF]. To our knowledge no observation of incidental catch has been reported for this species. Yet this does not imply that it is not happening especially i) in fisheries without onboard observers dedicated to bycatch or not required to report bycatch or ring recoveries, or in Illegal, Unreported and Unregulated (IUU) fisheries, ii) when low percentage of hauled hooks (<5%) are being surveyed by bycatch-dedicated observers onboard vessels [START_REF] Huang | Bycatch of high sea longline fisheries and measures taken by Taiwan: Actions and challenges[END_REF], and iii) due to identification issues.

During last decades, bycatch estimates of hundreds of albatrosses were reported in the South Indian Ocean [START_REF] Huang | Bycatch and discards by Taiwanese large-scale tuna longline fleets in the Indian Ocean[END_REF] and included several species at risk across the Southern Hemisphere [START_REF] Abraham | Assessment of the risk of surface longline fisheries in the Southern Hemisphere to albatrosses and petrels, for 2016[END_REF].

As part of a multi-year survey evaluating juvenile dispersal and immature at-sea distribution linked to the current NPOA framework for the Amsterdam albatross, the present study reports the first evidence from a single individual that Amsterdam albatross can wander for extensive periods outside the Indian Ocean, in the Pacific Ocean. This unprecedented, unexpected and novel finding is discussed in terms of overlap risk with fisheries and conservation issues.

Materials and methods

Study species and data loggers

Amsterdam Island (37° 50' S; 77° 33' E) is located in the subtropical part of the southern Indian Ocean.

In this oceanic area, the southern subtropical front (SSTF) delimits the warmer subtropical from the colder sub-Antarctic waters [START_REF] Im | Southern Ocean fronts from the Greenwich meridian[END_REF]. The Amsterdam albatross, like other great albatrosses, is a biennial breeder [START_REF] Jp | Un nouvel albatros Diomedea amsterdamensis n. sp. découvert sur l'île Amsterdam (37°50[END_REF][START_REF] Jouventin | Breeding biology and current status of the Amsterdam Island Albatross Diomedea amsterdamensis[END_REF], with high survival during juvenile, immature and adult phase [START_REF] Rivalan | Combined impact of longline fisheries and climate on the persistence of the Amsterdam albatross[END_REF]. Juvenile birds fledge and migrate independently from the adults in January. Immature birds may visit the colony when they are 4-7 yrs old, but generally only start breeding at 9 yrs old [START_REF] Weimerskirch | Population dynamics of Wandering Albatross Diomedea exulans and Amsterdam Albatross D. amsterdamensis in the Indian Ocean and their relationship with long-line fisheries: conservation implications[END_REF].

Previous study during the first months at sea of juveniles Amsterdam albatross showed an initial rapid movement taking all individuals away from the vicinity of their native colony. Then, they dispersed at large-scale in the entire southern Indian Ocean (15° to 135° E, ~ 4 500 km from the colony), through a large range of latitudinal gradient (27° to 47° S) similarly to the adults during their sabbatical period [START_REF] De Grissac | Contrasting movement strategies among juvenile albatrosses and petrels[END_REF][START_REF] Thiebot | Stage-dependent distribution of the critically endangered Amsterdam albatross in relation to Economic Exclusive Zones[END_REF].

The main objective of the ongoing NPOA is to improve the conservation status of the species by removing or reducing the threats on breeding success and survival rates for the species. Planned actions concern specific objectives regarding threats on land (i.e. pathogen contamination, limitation or eradication of introduced invasive species) and at sea (i.e. at-sea distribution of different stages, habitat modelling and projection, overlap risk assessment with fisheries, implementation of the most efficient bycatch mitigation measures). Under this NPOA framework, a study was launched to gain additional knowledge on the at-sea distribution of the immature stage (between fledging and the first return on land), a period for which only very limited data exist [START_REF] De Grissac | Contrasting movement strategies among juvenile albatrosses and petrels[END_REF][START_REF] Thiebot | Stage-dependent distribution of the critically endangered Amsterdam albatross in relation to Economic Exclusive Zones[END_REF]. Hence, the study of juvenile birds at-sea movements was completed using Global Location Sensing (GLS; Mk15-British Antarctic Survey). GLSs record the ambient light level every 10 min, from which local sunrise and sunset hours can be inferred allowing estimating location every 12 h [START_REF] Wilson | Estimation of location: global coverage using light intensity[END_REF]. Locations based on light-based archival tags are associated with spatial error estimates (~200 km; [START_REF] Phillips | Accuracy of geolocation estimates for flying seabirds[END_REF]). Beside the accuracy of those loggers, broad-scale movements can be confidently identified and allowed us to track the birds for prolonged periods with a minimal disturbance [START_REF] Weimerskirch | Lifetime foraging patterns of the wandering albatross: life on the move![END_REF]. The birds were tagged as chick (n = 21) in 2011 before leaving the colony for the first time. To date, at year t+9, we have retrieved 12 of the 21 GLS loggers deployed (recovery rate ~57%) from which 10 individual tracks were estimated (2 loggers failed to record data), thereby allowing to extend the study of juvenile and immature stages over a period up to ~ 3.1 yrs after fledging. The present study focuses on one individual which travelled outside the Indian Ocean, revealing a new migratory pathway and bringing additional information about fisheries overlap.

Data and processing

Fishing data

To describe the overlap between the distributions of the tracked bird and fishing effort (i.e. longline and trawl), we collated the data available from the relevant regional fisheries management organisations (RFMOs; Table 1, Fig. 1,Appendix A) and the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR). Note that some RFMO fishing effort datasets are very incomplete (particularly in the Indian Ocean) and can thus result in biased estimates of overlap, particularly when comparing across different RFMOs [START_REF] Francis | Estimation of fishing effort in the Southern Hemisphere[END_REF]. To assess the association of albatrosses with fishing vessels [START_REF] Orben | Across borders: External factors and prior behaviour influence North Pacific albatross associations with fishing vessels[END_REF] we used Global Fishing Watch (GFW) available datasets for assessing fisheries interactions by estimating daily overlap between fishing activity (presence and intensity) and bird distribution (twice daily locations), i.e. a proxy of being at risk to incidental bycatch (Appendix A).

Fishery observer data

The seabird observation data collected during fishing activities by the scientific onboard observers [START_REF] Martin | Data Curation, Fisheries, and Ecosystembased Management: the Case Study of the Pecheker Database[END_REF] were used to identify direct interactions through photo-identification of individuals ringed as part of an ongoing long-term monitoring capture-recapture study (CEBC dataset; see [START_REF] Rivalan | Combined impact of longline fisheries and climate on the persistence of the Amsterdam albatross[END_REF]). Observer data were collected under the French Southern Ocean Fishery Observer Program [START_REF] Gasco | Contributions to marine science by fishery observers in the French EEZ of Kerguelen[END_REF] and entered on board into an electronic logbook designed by the Muséum National d'Histoire Naturelle de Paris (MNHN) and then uploaded in an 11G Oracle database named "PECHEKER" hosted and managed by MNHN [START_REF] Martin | Data Curation, Fisheries, and Ecosystembased Management: the Case Study of the Pecheker Database[END_REF].

Analysis of light data

We used the FLightR R package [START_REF] Rakhimberdiev | FLightR: an R package for reconstructing animal paths from solar geolocation loggers[END_REF][START_REF] Lisovski | Light-level geolocator analyses: A user's guide[END_REF] to determine post-fledging migration movements and timing.

FLightR uses the slope of the light curve around twilight to estimate locations. We defined twilights with the preprocessLight function followed by an additional automated screening using the twilightEdit function, in the TwGeos [START_REF] Wotherspoon | TwGeos: basic data processing for light-level geolocation archival tags[END_REF] R package (Appendix A).

Overlap with fisheries

The number of GLS locations situated in Highs Seas, RFMOs and Exclusives Economic Zones (EEZs) areas were counted using R software (version 4.0.4) in order to assess the percentage of the entire movement in each area. Then, the GLS locations of the albatross were aggregated in visited cells, according to the spatial accuracy of the fishing data. This spatial overlap based on the common centroids of the cells was completed by a temporal join to assess the concomitant use of the same cell by the bird and the fishing vessels (e.g. proxy of being at risk to incidental bycatch, see Appendix A).

Results

Out of the ten juvenile individuals with recorded tracks, nine distributed entirely in the southern Indian Ocean during their first years at sea (not illustrated, see [START_REF] Thiebot | Stage-dependent distribution of the critically endangered Amsterdam albatross in relation to Economic Exclusive Zones[END_REF]). However, surprisingly, one travelled outside the Indian Ocean, providing the first evidence that Amsterdam albatross can wander in the Pacific Ocean (Fig. 1). Before its GLS tag stopped (1176 days after leaving the colony), this individual travelled a total distance of ~185 800 km and went as far as 12 915 km away from its colony. During its first three years at sea, this individual crossed two Ocean basins: the Indian Ocean and the Pacific Ocean. It distributed mainly in the southern Indian Ocean (~66% of locations), but in a totally unexpected way it also spent a large part of its time in the southern Pacific Ocean (~34%), the rest being distributed among the global Southern Ocean (<~1%). The bird spent its first 793 days (06/12/2011 to 06/02/2014) in the Indian Ocean basin and the next 383 days in the Pacific Ocean basin (06/02/2014 to 24/02/2015). The latitudinal range expanded between -22.8°S to -54.8°S. As the device stopped recording off the Chilean coast (10 696 km away from Amsterdam Island), the return journey to the colony was not completed (Fig. 1).

The bird distributed mainly in high seas (78% of the locations) and within 13 EEZs (22% of locations; Appendix Table S1 &S2). In high seas, its distribution overlapped with seven RFMOs (3 targeting tunalike species and 3 targeting non-tuna-like species; Table 1, Appendix Table S3, Fig. 1 &2). Overlaps occurred in the southern Indian Ocean within IOTC (~62%) and SIOFA (~48%) areas, and in Pacific Ocean RFMOs (SPRFMO: 24%, WCPFC: 2% and IATTC: 28%) areas.

The French EEZs was the most overlapping country with 5 EEZs representing up to 41% of the bird's distribution (Fig. A2), followed by Chile (2 EEZs, ~38%), South Africa (2 EEZs, ~15%), and New Zealand (~1.8%).

While SIOFA was created only recently (agreement signed in 2006) and entered into force in 2012 the overlap between fishing effort and bird location could not be estimated due to data inaccessibility.

Overlap with fishing effort

This Amsterdam albatross movement encompassed 143 cells on the RFMOs grid (3 340 661 km²) and 451 cells on the GFW grid (1 383 682 km²). These cells were spatially overlapping with fishery areas for 82% and 66% respectively.

The spatio-temporal overlap with the monthly RFMO fishing data showed no concomitant use of the same cell with trawling (data available only for SPRFMO and CCAMLR). However, it happened with longlining for 120 cells visits (36%, Fig. 3a). These potential interactions happened primarily in IOTC (95 cells visits, 79%) and to a lesser extent in IATTC (12 cells visits, 10%), CCAMLR (9 cells visits, 7.5%) and WCPFC (4 cells visits, 3.6%). In time, this overlap spanned almost all the trip duration, occurring on 27 months out of the 39 recorded. It also included all the months of the year. The corresponding fishing effort was 7 655 856, 920 785, 2 068 290 and 7 041 deployed hooks respectively in IOTC, IATTC, CCAMLR and WCPFC.

The spatio-temporal overlap with the daily GFW data showed concomitant use of the same cell with fishing vessels detected during the same day for 11 cells visits (0.7%, Fig. 3b) in 2012. These potential interactions happened primarily in IOTC / SIOFA (7 cells visits, 63.6%) in May, June, November and December with maximum fishing effort estimated at 24 hours. CCAMLR was also concerned to a lesser extent with 4 cells visits (36.3%) in April, with maximum fishing effort estimated at 22 hours.

Fishery observer data

Sixteen observations of Amsterdam albatrosses were reported for fifteen different individuals, during 2018-2021 (28 Feb -6 Apr) and from the stern of a pot fishery vessel targeting the Saint Paul rock lobster (Jasus paulensis). All the observed individuals were immature birds (aged between 2 and 11 yrs) that never attempted to breed, except one breeding adult (aged 15 yrs). They were all observed within the Amsterdam and Saint Paul EEZ. Observations from on-board observers provide evidence of direct interactions with fishing vessels and instances of prey consumption (offals) related to fishing activity (Appendix Fig. A1).

Discussion

This is the first evidence that Amsterdam albatross wander for extended periods outside the Indian Ocean, in the Pacific Ocean (except short visits in the southeastern part of the Atlantic Ocean; [START_REF] Thiebot | Stage-dependent distribution of the critically endangered Amsterdam albatross in relation to Economic Exclusive Zones[END_REF]). This novel finding changes the scope of coverage (e.g. spatial and temporal extent) and conservation strategy for the species. Unexpectedly the South Pacific RFMOs were largely at risk of overlap. Multi-year tracking datasets of immature birds of this extremely rare species is an important input to fisheries management processes, highlighting new fisheries that were not considered to pose risk to this species until now. Furthermore, our results clearly evidenced for the first-time direct interactions with fishing vessels during the immature stage.

Even if the way back to Amsterdam Island was not recorded, it is likely that this bird went back to its birth colony following the prevailing winds of the Antarctic circumpolar current belt, as wandering albatrosses D. exulans from the South Indian do [START_REF] Weimerskirch | Lifetime foraging patterns of the wandering albatross: life on the move![END_REF]. Therefore, we strongly suspect that Amsterdam albatrosses may also cross the South Atlantic to complete a circumpolar navigation, and potentially use all the RFMOs of the Southern Ocean (ICCAT, SEAFO or more of the CCCAMLR areas). This unique migratory behaviour is unprecedented for the Amsterdam albatross either because it may be very rare, or alternatively, because only few juvenile individuals (n = 10) have been successfully tracked over several years. This raises the possibility of the existence of an unrevealed or novel migratory pathway unknown for this species. Although this finding concerns a single bird to date, it represents 10% of the individuals tracked so far.

As previously described for juveniles Amsterdam albatross in their first year, this bird distributed mainly within the South Indian during its first years at sea after fledging [START_REF] Thiebot | Stage-dependent distribution of the critically endangered Amsterdam albatross in relation to Economic Exclusive Zones[END_REF]. French, South African and Australian EEZs were previously identified as important foraging areas for this species [START_REF] Thiebot | Stage-dependent distribution of the critically endangered Amsterdam albatross in relation to Economic Exclusive Zones[END_REF], but new concerned EEZs have arisen from this study: Chilean and New Zealand EEZs. The finding of this unrevealed migratory pathway highlights that the conservation of this species goes far beyond France, which already provides a high level of protection within its EEZs particularly for the French Southern and Antarctic Lands [START_REF] Claudet | Critical gaps in the protection of the second largest exclusive economic zone in the world[END_REF], and the South Indian. This individual distributed largely over the high seas overlapping mainly with two RFMOs areas (i.e. IOTC and SIOFA, and overlaying marginally with the Indian part of the SEAFO). Nonetheless, in a completely unexpected way the South Pacific RFMOs were also largely overlaid (i.e. SPRFMO, IATTC and WCPFC). This reinforces the need for the application of best practices for bycatch mitigation measures and fisheries observer coverage to be mandated and enforced at a global scale to ensure no negative effects on this endangered population.

Despite the contrasted results of overlap analysis between RFMOs and GFW data, both approaches conducted on a partial migration of a single individual confirmed the potential for interactions with fisheries in different RFMOs. Nonetheless, some RFMO fishing effort datasets are known to be very incomplete (namely in the Indian Ocean) resulting in a biased estimate of overlap. The approach using GFW data should be important to consider in order to complement and refine the overlap estimations, especially concerning specific fisheries (i.e. South Pacific trawl activity occurs in EEZS while SPRFMO covers trawling data only in the high seas).

The Amsterdam albatross is identified as a priority species to protect, especially at risk due to bycatch [START_REF] Abraham | Assessment of the risk of surface longline fisheries in the Southern Hemisphere to albatrosses and petrels, for 2016[END_REF] with breeding adults foraging in a dense boat seascape (i.e. including density of fishing vessels [START_REF] Corbeau | Fine-scale interactions between boats and large albatrosses indicate variable susceptibility to bycatch risk according to species and populations[END_REF]).

Even if fine scale analysis between breeding adults tracked with GPS and AIS data seemed to reveal low interactions with fishing vessels [START_REF] Corbeau | Fine-scale interactions between boats and large albatrosses indicate variable susceptibility to bycatch risk according to species and populations[END_REF], and especially for juvenile Amsterdam albatrosses [START_REF] Weimerskirch | Ocean sentinel albatrosses locate illegal vessels and provide the first estimate of the extent of nondeclared fishing[END_REF], our results show clear evidence of direct interactions with fishing vessels during the immature stage. This suggests that interactions are likely underestimated, given the opportunistic aspect of these data, the elusiveness of this species and its identification difficulty. Further study on the juveniles' dispersal at sea with GLS loggers are needed to improve the knowledge on their spatial distribution and exposition to fisheries interactions. Additional deployments are planned during the ongoing NPOA.

We reported the first evidence of an immature of the Amsterdam albatross crossing the Pacific Ocean and estimated the relative importance of national jurisdictions and high seas areas for this individual. This bird made extensive use of the high seas, reinforcing the stake each country has in the management of biodiversity in international waters. While in the high seas, this individual, in each ocean basin, spent the most time within the areas of competence of the five RFMOs targeting tuna and nontuna species. By making explicit the relative responsibilities that each country and RFMO has for the management of shared biodiversity [START_REF] Beal | Global political responsibility for the conservation of albatrosses and large petrels[END_REF] and by providing new evidence for direct interactions with fishing vessels, this work provides novel and important information for the conservation and management of this endangered species at the appropriate scale in the marine realm.

Fig. 2

 2 Fig. 2 Distribution of the different levels of overlap between at sea distribution of one Amsterdam

Fig. 3

 3 Fig. 3 Overlap between an immature of Amsterdam albatross Diomedea amsterdamensis tracked up to

  Fig 3a 262

Table 1

 1 Summary of regional fisheries management organisations characteristics competent in the high seas overlapping with an Amsterdam albatross tracked during its first 3 yrs at sea At sea track of a transoceanic immature Amsterdam albatross Diomedea amsterdamensis after fledging from Amsterdam Island, southern Indian Ocean, tracked up to age ~3 yrs. The first part of the

	Ocean basin	Organisation		Targeted species	Gear type	Fishing effort data availability
	Indian Ocean	Indian	Ocean	Tuna	Tunas	Longline	https://www.iotc.org/data/datasets/latest/CELongline
		Commission (IOTC)
	Indian Ocean	Southern Indian Ocean	Non-tunas	Longline, trawl Not considered (data not available)
		Fisheries	Agreement
		(SIOFA)
	Pacific Ocean	Western and Central	Tunas	Longline	https://www.wcpfc.int/wcpfc-public-domain-
		Pacific		Fisheries			aggregated-catcheffort-data-download-page
		Commission (WCPFC)
	Pacific Ocean	InterAmerican	Tropical	Tunas	Longline	https://www.iattc.org/PublicDomainData/IATTC-
		Tuna Commission (IATTC)			Catch-by-species1.htm
	Pacific Ocean	South Pacific Regional	Non-tunas	Longline, trawl https://www.sprfmo.int/data/catch-information/
		Management Organisation
		(SPRFMO)
	Atlantic Ocean	International Commission	Tunas	Longline	Not considered (no overlap)
		for the Conservation of
		Atlantic Tuna (ICCAT)
	Atlantic Ocean	South	East	Atlantic	Non-tunas	Longline, trawl Not considered (weak overlap)
		Fisheries	Organisation
		(SEAFO)

Marine Resources (CCAMLR). The different Exclusive Economic Zones (EEZs) are also presented (white areas). The breeding colony on the Amsterdam Island is denoted by a green dot, the red dot indicated the last available location.

Acknowledgements

This study is a contribution to the National Plan of Actions for Amsterdam albatross. This monitoring program was supported financially and logistically by the French Polar Institute IPEV (program 109, resp. Henri Weimerskirch), the Zone Atelier Antarctique (CNRS-INEE), Terres We thank the Museum National d'Histoire Naturelle (


Camille Cohen

Séverine Coulon

Kanit Bhukhai

Antoine Neuraz

Michael Dussiot

Guillemette Fouquet

Marie-Bénédicte Le Stang

Martin Flamant

François Vrtovsnik

Aurélie Hummel

Bertrand Knebelmann

Laurent Mesnard

Eric Rondeau

Thiago T Maciel

Fabrizia Favale

Nicole Casadevall

Thao Nguyen-Khoa

Stéphane Moutereau

Christophe Legendre

Marc Benhamou

Renato C Monteiro

Olivier Hermine
email: ohermine@gmail.com

Khalil El Karoui
email: khalil.el-karoui@aphp.fr.

Ivan C Moura

Pr Olivier Hermine

Dr El Khalil

Karoui

Erythrocytosis associated with IgA nephropathy

Keywords: IgA, IgA nephropathy, erythrocytosis, polycythemia, Textword count: 3872

Research in context

Evidence before this study Erythropoiesis is a tightly regulated mechanism allowing the production of red blood cells to supply oxygen to the tissues. When some of the regulation mechanisms are not fully functional, it can lead to anemia (decreased hemoglobin levels) or erythrocytosis (increased hemoglobin levels).

Erythrocytosis is a hematological disorder characterized by hemoglobin elevation usually related, in adults, to hematopoietic stem cell somatic mutations. However, some patients do not present known specific mutations like JAK2, and other etiologies of erythrocytosis remain to be identified. Interestingly, we previously reported that polymeric IgA1 (antibodies that usually control mucosal infections) are able to stimulate erythropoiesis in vitro and in vivo in IgA1 humanized mice. Thus, we hypothesized that polymeric IgA could also induce erythrocytosis in human.

Added value of this study

Here, we describe 6 patients presenting erythrocytosis associated with IgA nephropathy (IgAN), a renal disease known to be associated with higher polymeric and/or galactosylation deficiency (Gd) IgA1 rate. Circulating IgA1 were frequently polymeric and/or Gd-IgA1.

Moreover, these IgA1 increased the sensitivity of erythroid progenitors to Epo in vitro.

Lastly, in large chronic kidney disease cohorts, IgAN was an independent factor associated to higher hemoglobin levels, and higher risk of developing erythrocytosis.

Implication of all the available evidences

This study reveals that polymeric IgA1 might be a regulator of erythropoiesis in humans, and that dysregulation of polymeric/monomeric IgA1 ratio or Gd-IgA1 rate can drive erythrocytosis.

Given the frequent elevation of pIgA1 in IgAN patients, we propose that IgAN should be evoked in case of unexplained erythrocytosis.

INTRODUCTION

Erythropoiesis is a tightly regulated process that adapts the production of mature erythrocytes to supply organs with oxygen. This process relies essentially on the level of circulating erythropoietin (Epo) which acts on erythroid progenitors/precursors to regulate their survival, proliferation and, to a lesser extent, differentiation [START_REF] Weiss | Anemia of chronic disease[END_REF][START_REF] Camaschella | Iron-deficiency anemia[END_REF].

Overproduction of erythrocytes can lead to polycythemia/erythrocytosis (PE) [START_REF] Arber | The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia[END_REF]. Somatic mutations occurring in hematopoietic progenitors (e.g. JAK2 V617F ) and, rarely, other mutations that induce hypersensitivity to Epo, lead to dysregulated erythropoiesis and are the most frequent etiologies of PE. Other causes include inappropriate production of Epo linked to kidney tumor, hypoxia or abnormal response to hypoxia. However, in numerous cases no etiology can be identified [START_REF] Camps | Gene panel sequencing improves the diagnostic work-up of patients with idiopathic erythrocytosis and identifies new mutations[END_REF].

We previously reported that polymeric IgA1 (pIgA1) interact with transferrin receptor 1 (TfR1/CD71) expressed in erythroid progenitors. Expression of human IgA1 or treatment of wild-type mice with pIgA1 accelerated recovery from acute anemia. Mechanistically, in vitro, pIgA1/TfR1 interaction increases the sensitivity of erythroid progenitors/precursors to Epo [START_REF] Moura | Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy[END_REF][START_REF] Coulon | Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia[END_REF], by inducing activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. Given the role of pIgA1 in erythropoiesis regulation in these in vivo and in vitro models, we hypothesized that pIgA1 could also be involved in regulation of erythropoiesis in human. To test this hypothesis, we took advantage of patients with a pathology related to increased pIgA1 levels, i.e IgA nephropathy (IgAN).

IgA nephropathy (IgAN) is the most common primary glomerulonephritis. The pathogenesis of the disease includes the aberrant galactosylation of pIgA1 in the hinge region, which results in elevated serum levels of galactose-deficient polymeric IgA1 (Gd-pIgA1). These antibodies are recognized by glycan-specific IgA and IgG autoantibodies, leading to the formation of immune complexes of IgG-pIgA1. For unclear reasons, the kidney is the main target of these immune complexes. The glomerular lesions in IgAN are related to the deposition of these pathogenic pIgA1 immune complexes in kidney mesangium [START_REF] Lai | IgA nephropathy[END_REF][START_REF] Robert | Molecular Insights into the Pathogenesis of IgA Nephropathy[END_REF]. These deposits induce the proliferation of mesangial cells, increased synthesis and deposition of extracellular matrix and variable infiltration of inflammatory cells, leading to glomerulosclerosis, tubulointerstitial fibrosis and kidney failure. Although the role of different receptors for pIgA1 in mesangial cells is still evoked, several studies have identified the TfR1 as a key receptor for binding pIgA1-containing immune complexes [START_REF] Lai | IgA nephropathy[END_REF][START_REF] Robert | Molecular Insights into the Pathogenesis of IgA Nephropathy[END_REF]. The impact of the interaction pIgA1/TfR1 in other cells/tissues in IgAN patients is still unknown.

In the present study, we identified 6 patients with IgAN and unexplained PE, despite extensive explorations. We demonstrate that pIgA1 and Gd-IgA1 were elevated in these patients, and that circulating IgA1 increase erythroid progenitor sensitivity to Epo, explaining the pathophysiology of this syndrome. Further, in agreement to our hypothesis, in large cohorts of patients with various chronic kidney disease (CKD) etiologies, IgAN was an independent factor associated with higher steady-state Hb level and anemia recovery after kidney transplantation, suggesting that pIgA1 is a regulator of erythropoiesis in human.

METHODS

Patients and cohort analysis

Human subjects

Patients with biopsy-proven IgAN and unexplained polycythemia/erythrocytosis according to biological WHO criteria (3) (IgAN-PE) were identified by retrospective analysis of medical records from 2 tertiary nephrology centers in Paris (Necker, Tenon). Serum and DNA extracted from PBMC were obtained after written informed consent and local ethics committee approval. Control sera were from IgAN patients with normal hematocrit (HCT), and healthy volunteers. Circulating Epo and soluble TfR1 (sTfR1, Bio-Techne 2474-TR-050) were quantified by enzyme-linked immunosorbent assay (ELISA) and immunoturbidimetric methods, respectively. Estimated glomerular filtration rate (eGFR) was calculated according to CKD-Epi formula [START_REF] Levey | A new equation to estimate glomerular filtration rate[END_REF].

Cohorts

Two multicentric prospective cohorts of CKD patients were studied, (i) the NephroTest study, that enrolled 1993 adult patients with iterative mGFR and written informed consent [START_REF] Moranne | Timing of onset of CKD-related metabolic complications[END_REF], (ii) the DIVAT (Données Informatisées et VAlidées en Transplantation) cohort, which includes 2600 renal transplant patients from 2005 to 2015 [START_REF] Chapal | A useful scoring system for the prediction and management of delayed graft function following kidney transplantation from cadaveric donors[END_REF]. From the NephroTest study, 696 patients with biopsy-proven glomerulonephritis (GN) were selected to limit heterogeneity of the cohort that frequently includes patients with no histological diagnosis. We also excluded patients with autoimmune diseases who receive immunosuppressive drugs known to reduce Hb levels.

Cellular assays

Human CD34+ cord blood cells were cultured on methylcellulose (Methocult SF H4236, stemcell technologies, Vancouver, Canada) with interleukin-3 (IL-3), IL-6, SCF and Epo (0.05 or 1 U/mL) in the presence of sera from IgAN-PE or IgAN patients or healthy volunteers. Depletion of IgA1 was performed as previously described [START_REF] Coulon | Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia[END_REF]. Briefly, sera were passed through a Jaccalin column to catch IgA1. We obtained IgA1-depleted serum on one hand, and purified IgA1 after elution of the column on the other hand. BFU-E-derived colonies in the presence of depleted and non-depleted sera were counted at day 14.

Binding assays

UT 7 cell line were a gift from Dr Patrick Mayeux, at Cochin institute [START_REF] Komatsu | Establishment and characterization of an erythropoietin-dependent subline, UT-7/Epo, derived from human leukemia cell line, UT-7[END_REF]. Briefly, UT7 cells (0.25 x 10 6 ) were pre-incubated with 1 mg/mL of human IgG for 30 min on ice to block IgG receptors. IgA1 binding was examined by FACS by an indirect immunofluorescence assay, using a biotinylated anti human IgA1 (Southern Biotech, ref 2052-08 ), followed by a streptavidin-APC (BD pharmigen ref 554067) [START_REF] Berthelot | Transglutaminase is essential for IgA nephropathy development acting through IgA receptors[END_REF]. For inhibition studies, cells were preincubated with sTfR1 (Bio-Techne, 2474-TR-050). Data were analyzed with FlowJo software (FlowJo LLC, Ashland, Oregon)

Biochemical assays

Quantification of IgA in serum was performed using Architect c16000® analyzer (Abbott) with an immunoturbidimetric method. Immunobloting for monomeric and polymeric IgA1 was performed as described [START_REF] Oruc | IgA Structure Variations Associate with Immune Stimulations and IgA Mesangial Deposition[END_REF]. Briefly, 1 μg plasma IgA or purified IgA mAbs were prepared without reducing agent and loaded on 10% polyacrylamide gels. Proteins were transferred to PVDF membranes. The human α-chain was detected using a purified mouse anti human IgA1/IgA2 (BD Pharmingen ref 555886) followed by an HRP-conjugated donkey anti mouse secondary antibody (Jackson immuno, ref 715-035-150).

Quantification of serum galactose-deficient IgA1 was evaluated by ELISA (Gd-IgA1 KM 55 kit, IBL-Japan), following manufacturer's instructions.

Mice housing and experiments

Alpha1 knock-in transgenic mice were previously described [START_REF] Coulon | Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia[END_REF]. The α1KI mice feature insertion of the human Cα1 gene downstream of the endogenous JH region in the IgH locus, replacing IgM expression by IgA with human Cα1-constant regions. These animals were bred into a BALB/c background and compared with wild-type animals from similar backgrounds.

Mice were fed ad libitum and housed at constant ambient temperature in a 12-h light, 12-h dark cycle. Animal procedures were approved by the "Services Vétérinaires de la Préfecture de Police de Paris", by the "Ministère de l'Enseignement Supérieur de la Recherche et de l'Innovation" and by the ethical committee of the Université de Paris. Hematocrit was measured in mice using a MS9-5 (MS Laboratory). The Epo concentration was measured by ELISA (Quantikine IVD, R&D system) following the manufacturer's recommendations.

Reagent validation

Primary human CD34+ cord blood cells and UT7 cell line were previously validated (RRID).

Cell line and antibody validation references are provided in Supplemental Table 1.

Statistical analysis

For in vitro experiments, statistical analyses were performed with GraphPad Prism Software version 5.0. Data are expressed as mean ± SEM of n determinations unless noted otherwise.

Mann-Whitney test was used to compare two groups. Differences were considered significant at a P value <0.05 (*), <0.01 (**) or < 0.001 (***).

For statistical analysis of clinical cohorts, we used the Kruskal-Wallis test for quantitative covariables, the Pearson's chi-square test for qualitative co-variables. We performed a multivariate linear regression with a bidirectional stepwise selection of covariates. The initial model included all the covariates associated with Hb level (P< 0.05) in a bivariate analysis.

Analyses were performed using R Statistical software version 3.3.2.

Ethics

Written informed consent was obtained from each patient and controls. Ethic committee approval was obtained from the LabEx GR-Ex (N° DC-2016-2618, Imagine Institute, Paris, France).

Role of funding source

The funders had no role in study design, data collection, data analyses, interpretation, or writing of report.

RESULTS

CASE REPORTS

Patient1

A 20-year-old man was referred for microscopic hematuria and hypertension. Serum creatinine was 50µmol/L, without proteinuria. He presented no past medical history. Kidney biopsy revealed mild proliferative mesangial glomerulonephritis, with mesangial IgA and C3 deposits, confirming IgAN. Hemoglobin (Hb) level was 16.3 g/dL, and hematocrit (HCT) was 48%, but the criteria for PE were not reached. During follow-up, angiotensin receptor blocker (ARB) treatment was introduced to control new onset proteinuria (1g/day), and Hb levels progressively increase (17,5g/dL). At the age of 64, despite renal failure occurrence with a serum creatinine at 162µM (measured GFR (mGFR) of 37.8 mL/min/1.73m 2 ) and a stable proteinuria of 0.7g/day, the patient developed PE (Hb 18.6g/dL, HCT 55.5%) with normal leukocyte and platelet counts and no splenomegaly.

Patient 2

A 63-year-old man with no past medical history was referred for microscopic hematuria, hypertension, proteinuria (4g/day), and a serum creatinine 230µmol/L (estimated (e)GFR of 25 mL/min/1.73m 2 ). Kidney biopsy revealed mesangial glomerulonephritis with mesangial thickening but without proliferation or segmental lesions. Interstitial fibrosis was observed in approximately 20% of the biopsy. Immunofluorescence (IF) confirmed IgAN. Hb level was 12.9 g/dL and HCT 37.9%. Angiotensin conversion enzyme inhibitor (ACEi), ARB and thiazide treatments were introduced. A progressive increase of Hb levels occurred, and at the age of 74, he developed PE with Hb17.8g/dL and HCT 54.6%. Leukocyte and platelet counts were normal. No splenomegaly was evidenced. Renal function was altered with mGFR 28mL/min/1.73m 2 . After 12 years of follow-up, serum creatinine was 171µM and proteinuria 0.2g/day.

Patient 3

A 26-year-old man was referred for microscopic hematuria and proteinuria (2.3g/day), with normal renal function (serum creatinine 88µmol/L, eGFR 91ml/min/1.73m 2 ). Kidney biopsy revealed mesangial glomerulonephritis without proliferation. IF confirmed IgAN. Hb was 15.1 g/dL and HCT 45%. ACEi (but no diuretic) treatment was started with significant reduction of proteinuria. At the age of 69, while renal function started to be altered with mGFR 59mL/min/1.73m2, he developed PE (Hb18.5 g/dL, HCT55%). Leukocyte and platelet counts were normal. After 44 years of follow-up, serum creatinine was 128µM and proteinuria 0.8g/day. Description of 3 other patients is available in supplementary information.

The 6 IgAN patients presented here (Table 1) developed a late-onset PE during follow-up, while paradoxically their mGFR decreased. Physical examinations did not show any evidence for myeloproliferative disease (e.g. no splenomegaly) or cerebellum hemangioblastoma.

Renal and hepatic ultrasounds were normal, as well as chest X-ray. All patients had no history of smoking, normal levels of Hb oxygen saturation, and normal Epo levels without JAK2 mutation. No patient developed myeloproliferative neoplasms or thrombotic complications.

No red cell mass studies were available. The patients did not receive immunosuppressive therapy during the course of their renal disease. IgAN patients frequently have increased Gd-IgA1 and polymeric/monomeric IgA1 (pIgA1/mIgA1) ratio [START_REF] Valentijn | Circulating and mesangial secretory component-binding IgA-1 in primary IgA nephropathy[END_REF], and pIgA1 induce a hypersensitivity to Epo of erythroid progenitors through activation of TfR1 in mice [START_REF] Coulon | Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia[END_REF]. We thus hypothesized that pIgA1 from IgAN patients could stimulate erythropoiesis in humans.

Cohort study

We first compared PE prevalence and Hb levels in large cohorts of CKD patients. In the Nephrotest study [START_REF] Moranne | Timing of onset of CKD-related metabolic complications[END_REF], a large cohort study of patients with CKD of different origins, we delineated 4 patient groups based on CKD etiology that included IgAN, other glomerulonephritis (GN), polycystic kidney disease (PKD), and diabetes nephropathy (DN) (Table S2). There was a higher prevalence of PE in IgAN patients according to WHO criteria (3), since 6 out of 171 IgAN patients (3.5%) presented with PE compared with 2/117 PKD, 0/179 other GN and 0/229 DN patients (p=0.004). Overall, Hb level was higher in IgAN patients than in patients with PKD, diabetes and other GN (p <0.0001). The multivariate analysis including age, sex, mGFR, Epo or iron supplementation as covariates showed that IgAN is an independent factor associated with higher Hb levels (p=0.01) (Table S3 and Fig. 1a). Circulating Epo levels were similar in IgAN-PE to that of other CKD patients (Supplemental Figure S1). Collectively, these data support a role for IgA1 in erythropoiesis in humans.

We then compared the Hb recovery status of IgAN versus other CKD patients following kidney transplantation since anemia occurs in the early post-renal transplant period [START_REF] Martinez | High dose epoetin beta in the first weeks following renal transplantation and delayed graft function: Results of the Neo-PDGF Study[END_REF] and pIgA accelerate recovery from anemia in mice [START_REF] Coulon | Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia[END_REF]. In the DIVAT post-transplant cohort Hb levels at 3 different time points (Month (M) 0, day of renal transplantation, M3 and M12 posttransplantation) were available for 2600 patients including 271 IgAN (10.4%), 447 PKD (17.2%) and 248 DN (9.5%) patients. At M0, only male gender and PKD were associated with Hb level by multivariate analysis. The mean change of Hb level at M3 and M12 compared to M0 was higher in IgAN compared to DN and other groups (ΔHb M3-M0=0.87, -0.06 and 0.22g/dL respectively; ΔHb M12-M0=1.36, 0.55 and 0.71g/dL, respectively) (Fig. 1b). Moreover, twelve months after transplantation, IgAN patients presented higher Hb level compared with other patients (13.1 vs 12.7g/dL, p=0.001). Taken together, these data demonstrate that Hb recovery after transplantation is faster in IgAN patients.

Patient study

Given these epidemiological data, we further explored at the cellular level whether IgAN-PE observed in the 6 patients presented in case reports involved pIgA1. Since we noted that Epo levels were similar in IgAN patients and controls (Supplemental Figure S1) despite elevation of hematocrit in IgAN patients, we studied a mouse model of IgAN which overexpresses human pIgA1, the α1KI mice [START_REF] Coulon | Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia[END_REF]. Similar to IgAN patients, we observed an elevation of hematocrit in these mice, with no difference in Epo levels (Supplemental Figure S2 andS3).

These results suggested that elevated circulating IgA1 could amplify erythropoiesis in vivo with no significant impact in Epo levels, as observed in IgAN patients. To further explore the disease mechanisms in the 6 IgAN patients with PE, we then studied the role of IgA1 in these patients. To this aim, we plated CD34 + cord blood cells with serum from IgAN-PE patients, or control individuals (IgAN without PE with eGFR 26-86 mL/mn/1.73m 2 and normal ferritin levels, and healthy controls), in semisolid medium with a cytokine cocktail allowing erythroid progenitor proliferation, survival and differentiation. At suboptimal Epo concentration (0.05 U/mL), the number of erythroid burst-forming unit (BFU-E)-derived colonies was increased in the presence of IgAN-PE versus control or IgAN serum (Fig. 2a). Removal of IgA1 from IgAN-PE serum decreased BFU-E-derived colony number to that observed with serum from healthy volunteers, while adding back the corresponding IgA1 to depleted IgAN-PE serum restored this number to its initial values (Fig. 2a). We then sought to understand the peculiarities of IgA from IgAN-PE patients that could explain these data. Serum IgA concentration showed no significant difference between IgAN-PE, IgAN and controls despite a trend towards a higher concentration for IgAN and IgAN-PE in this limited cohort (Fig. 2b).

Immunoblot analysis of whole serum for total IgA (IgA1 and IgA2) did not show a higher pIgA/mIgA ratio (Fig. 2c). However, when serum IgA1 was first Jacalin-purified before immunoblot analysis, we observed an increased pIgA1/mIgA1 ratio in IgAN-PE serum (Fig. 2d). Moreover, quantification of galactose-deficient IgA1 (Gd-IgA1) by KM55 mAb based ELISA in serum from IgAN-PE, IgAN patients and healthy controls showed an elevation of Gd-IgA1 in IgAN-PE patients (Fig. 2e).

We previously reported the role of pIgA1-TfR1 interaction in erythropoiesis stimulation in transgenic mice [START_REF] Coulon | Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia[END_REF]. To test the hypothesis that a similar mechanism is involved in our present human study, we examined IgA1 binding to TfR1 on UT7 cells (a human erythroid cell line expressing TfR1) as compared to IgA1 from healthy volunteers and from IgAN patients without PE. These experiments showed that IgA1 purified from IgAN-PE serum bound UT7 cells to a higher extent than IgA1 from IgAN patients without PE and healthy volunteers (Figure S4). Moreover, preincubation of IgA1 with soluble TfR1 inhibited this binding, suggesting that it was TfR1-dependent. Although the low number of samples precludes to reach definitive conclusions, these results suggest that IgA1-TfR1 interaction may be involved in erythropoiesis of IgAN-PE patients.

Overall, in line with previous data in mice [START_REF] Coulon | Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia[END_REF], these results demonstrate that IgA1 from IgAN-PE patients mediate hypersensitivity to Epo of erythroid progenitor cells.

DISCUSSION

Here we describe a new etiology of human PE in a subpopulation of IgAN patients related to an IgA1-dependent hypersensitivity to Epo of erythroid progenitors. This syndrome appears among IgAN patients with the highest pIgA1/mIgA1 ratio, and was suspected in 3.5% IgAN patients. In support for the role of pIgA1 in erythropoiesis stimulation, epidemiological data showed slightly elevated Hb levels in IgAN patients.

In a large multicentric cohort of CKD patients, we report that IgAN is associated with a higher frequency of PE and, overall, with a higher Hb level than other kidney diseases.

Notably, this effect was independent from other factors modulating erythropoiesis such as age, sex, or GFR. Although the Hb level was only slightly increased in IgAN patients, these epidemiological data revealed that erythropoiesis is stimulated in these patients in steady state conditions. Moreover, we show in a cohort of kidney transplant patients that IgAN is associated with a faster post-transplant anemia recovery. This suggests that the stimulated erythropoiesis occurring in IgAN patients is beneficial when these patients undergo renal transplantation. Besides, these analyses in transplant patients underscore that immunosuppression is not sufficient to inhibit the IgA-related erythropoiesis stimulation after transplantation (similarly to recurrence of IgA deposits observed in 20-25% cases [START_REF] Uffing | Recurrence of IgA Nephropathy after Kidney Transplantation in Adults[END_REF]). Since increased levels of pIgA1 immune complexes are observed in IgAN [START_REF] Tomana | Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies[END_REF], these data support in human the role of pIgA1 previously observed in three humanized mouse models of anemia which showed that pIgA1 may control erythropoiesis and allow rapid anemia recovery [START_REF] Coulon | Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia[END_REF]. Indeed, we have previously shown in a detailed mechanistic study that human pIgA1 drive hematocrit elevation via pIgA1 binding to TfR1. In this model, deficiency of the J chain (leading to an absence of pIgA1) significantly reduces hematocrit elevation [START_REF] Coulon | Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia[END_REF] . Interestingly, we previously observed in vitro that both IgA1 polymerization and IgA1 hypogalactosylation modulate IgA1 binding to TfR1 in cellular models [START_REF] Moura | Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy[END_REF]. Considering the role of Gd-IgA1 in IgAN pathophysiology [START_REF] Lai | IgA nephropathy[END_REF], we evaluated Gd-IgA1 in patients with IgAN-PE and controls. We observed that Gd-IgA1 were elevated in patients with IgAN-PE, compared to IgAN without PE and healthy controls (Figure 2e). This result suggests that patients with IgAN-PE have markers of dysregulated IgAN galactosylation. Overall, our results show that IgAN-PE patients have elevated serum polymeric IgA1 (Figure 2d) and Gd-IgA1 (Figure 2e) that support increased BFU-E number in vitro (Figure 2a).

Whether Gd-IgA1 were preferentially in monomeric or polymeric form was not technically evaluable, given the lack of enough residual volume of serum samples needed to perform HPLC studies in this human study. Thus, while pIgA1 plays a major role in erythropoiesis amplification in vivo through TfR1 interaction, the impact of another, non-exclusive mechanism of erythropoiesis stimulation involving Gd-IgA1 in humanized mice and humans remains to be studied. In particular, whether it is the Gd-pIgA1 within pIgA1 that is the main factor responsible for IgA1 involvement in IgAN-PE will need full attention in a future dedicated study.

The production of Hb physiologic levels is a tightly regulated process which could depend of several factors, including age, renal function, or iron status. Patients with IgAN and PKD could have fewer comorbidities than patients with other causes of GN, since they usually present with an early, mainly kidney-specific disease. However, our multivariate analyses suggest that the highest Hb levels observed in IgAN are not dependent of associated factors such as age, mGFR or iron status. Moreover, regarding IgAN-PE patients, we developed in vitro studies to better describe the pathophysiology of this syndrome, which clearly demonstrate the role of IgA1 in erythropoiesis activation.

Our study demonstrates in humans the role of IgA1 in unexplained cases of IgAN-PE. To our knowledge, only one case of IgAN with unexplained PE has been previously reported [START_REF] Mahesh | Idiopathic erythrocytosis in IgA nephropathy[END_REF].

However, this new pathophysiological mechanism is probably not restricted to IgAN patients, since elevated pIgA1/mIgA1 and Gd-IgA1 ratio has been also described during chronic liver disease or IgA myeloma [START_REF] Woof | The function of immunoglobulin A in immunity[END_REF]. Moreover, in familial forms of IgAN, increased pIgA1/mIgA1 ratio has been observed in both patients and unaffected relatives [START_REF] Schena | Abnormalities of the IgA immune system in members of unrelated pedigrees from patients with IgA nephropathy[END_REF]. Therefore, our report suggests that the potential role of IgA1 should be considered in sporadic and familial forms of unexplained polycythemia (4) with no JAK2 mutations, normal/subnormal Epo levels and no obvious secondary polycythemia etiology. Unfortunately, analysis of pIgA1/mIgA1 ratio and serum Gd-IgA1 is not available in routine practice, calling for further studies to develop a simple diagnosis test allowing to identify PE cases, which may be dependent on this mechanism. In the meantime, renal explorations of patients with unexplained PE, including kidney ultrasound, renal function evaluation, and urinalysis with search for proteinuria and haematuria should be proposed, followed, in cases of high suspicion of IgAN, by kidney biopsy.

Interestingly, Epo levels were similar in IgAN-PE and other CKD patients (Supplemental Figure S1), as previously observed in some patients with JAK2 mutation and "normal" Epo level [START_REF] Ancochea | The role of serum erythropoietin level and jak2 v617f allele burden in the diagnosis of polycythaemia vera[END_REF]. Regulation of erythropoiesis in CKD is poorly understood. Actually, Epo levels are not correlated with Hb in non-anemic CKD patients, and may be regulated by other factors frequent in CKD, such as iron deficiency, obesity or inflammation [START_REF] Mercadal | Timing and Determinants of Erythropoietin Deficiency in Chronic Kidney Disease[END_REF]. The exact regulation of Epo levels in IgAN-PE patients remains to be studied. However, we anticipate that these normal Epo levels could participate to erythropoiesis stimulation in IgAN patients, since pIgA1 increases the sensitivity to Epo-dependent stimulation of erythropoiesis.

Most IgAN patients are currently treated with angiotensin converting enzyme inhibitors (ACEi) or angiotensin II receptor blockers (ARB) [START_REF] Berthoux | Predicting the risk for dialysis or death in IgA nephropathy[END_REF]. Actually, these drugs have a major role in inhibiting renin angiotensin system (RAS) activation in IgAN, leading to reduction of intraglomerular pressure, proteinuria, and CKD progression [START_REF] Rauen | Intensive Supportive Care plus Immunosuppression in IgA Nephropathy[END_REF]. Interestingly, RAS activation has a positive effect on erythropoiesis [START_REF] Vlahakos | The role of the renin-angiotensin system in the regulation of erythropoiesis[END_REF]. Moreover, RAS inhibition may even control Epo-dependent polycythemia [START_REF] Vlahakos | The role of the renin-angiotensin system in the regulation of erythropoiesis[END_REF]. Consequently, we can hypothesize that IgA1amplified erythropoiesis stimulation might be attenuated by RAS inhibition in IgAN patients.

As shown in our case reports, 6 patients developed erythrocytosis while they were treated with RAS blockers. This suggests that this syndrome could be even more pronounced in the absence of RAS blockers. In addition, the limiting effect of RAS blockers on erythrocytosis may explain why IgAN-PE was mainly observed in patients with long-term diagnosis of IgAN, since the effect of RAS inhibition may decline after long-term blockade [START_REF] Garg | Overview of randomized trials of angiotensin-converting enzyme inhibitors on mortality and morbidity in patients with heart failure. Collaborative Group on ACE Inhibitor Trials[END_REF]. In fact, aldosterone breakthrough (e.g., paradoxical elevation of aldosterone levels after long-term RAS inhibition) has been largely documented in chronic kidney disease patients [START_REF] Moranne | Determinants and changes associated with aldosterone breakthrough after angiotensin II receptor blockade in patients with type 2 diabetes with overt nephropathy[END_REF].

Alternatively, we cannot rule out that other additional hematological factors associated with age (such as acquisition of somatic clonal hematopoiesis) may impact the evolution of polycythemia in our patients [START_REF] Wouters | Erythrocytosis in the general population: clinical characteristics and association with clonal hematopoiesis[END_REF]. Lastly, we cannot exclude a recruitment bias in patients with advanced CKD and elevated levels of Hb, a very unusual event in CKD patients. This bias is limited by our epidemiological study showing that 3.5% of patients with IgAN in the Nephrotest study have PE according to WHO criteria. This study has several limitations. We have shown the role of IgA1 only in patients with PE, and not in the whole cohort of IgAN patients. Moreover, in IgAN patients, we did not study the molecular consequences and the signaling pathways induced by the interaction of IgA1 with TfR1. However, our in vitro data shows the impact of IgA1 on the number of erythroid burst-forming unit (BFU-E)-derived colonies, which is in line with our epidemiological observations. Finally, this work confirms our previous report showing that IgA1 is a regulator of erythropoiesis. In support, IgAN patients presented higher Hb levels and better Hb recovery after transplantation than other CKD etiologies. Further in vitro studies will be necessary to decipher the molecular mechanisms involved in IgA1 promotion of a hypersensitivity to Epo.

Contributors

Declaration of interest

No conflict of interest to disclose

  CC, SC, KB, MD, KEK, IM and OH designed the study, collected the data, verified the data and performed in vitro experiments. CC and AN performed the statistical analysis. MBLS, GF, MF, FV, AH, BK, LM, ER, TTM, FF, NC, TNK, SM, CL, RCM, collected the data. CC, SC, MB, OH, KEK and IC drafted the manuscript and made the figures. All authors approved the final version of the manuscript. We dedicate this work to Ivan C Moura, whose scientific contribution has greatly improved the comprehension of pathophysiology of IgA-related diseases in humans.

Figure 1 :Figure 2 :

 12 Figure 1: CKD cohort analysis.

Table 1 : Clinical, demographical and biological data from the six patients with IgAN-PE.

 1 Normal measurements: Epo: 1.4<N<13.7 mIU/L; sTfR: 1.9<N<4.4 mg/L. Abbreviations: IgAN: IgA nephropathy; GFR: glomerular filtration rate; Epo: erythropoietin, ACEi: angiotensin conversion enzyme inhibitor; ARB: angiotensin receptor blocker.

	Patient #	age at polycythemia diagnosis	maximum hematocrit (%)	maximum hemoglobin (g/dL)	measured eGFR (mL/min/1.73m2)	proteinuria (g/24h)	EPO level (mUI/L)	pulsed oxygen saturation (%)	Ferritin (µg/L)	transferrin (%) saturation coefficient	sTfR (mg/L) ACEi or ARB
	1	64	55.5	18.6	43	0.7	9.9	98	102	23	3.8	ARB
	2	74	54.6	17.8	28	0.2	8.4	96	61	22	3.23	ACEi and ARB
	3	69	55.0	18.5	59	0.7	8.4	96	181	25	6.53	ACEi
	4	45	54.3	18	83	0.4	5.8	97	236	23	3.77	ARB
	5	61	56.4	18.4	53	0.2	6.8	98	201	22	2.2	ARB
	6	26	59	18.5	27	1.1	6.8	97	153	24	4.42

Acknowledgments

We thank all the patients involved in this study. Funding: this work was supported by INSERM (French national institute for biomedical research) and Imagine Institute (Paris, France) Data sharing Statement Data are available upon reasonable request

Funding This work was supported by INSERM (French national institute for health and medical research), Labex GRex and Imagine Institute (Paris, France)