E Guichard
email: emilie.guichard@univ-poitiers.fr

F Autin

INCREASING VEGETABLES PURCHASE WITH A DESCRIPTIVE-NORM MESSAGE

Keywords: Social norms, Descriptive norms, Field study, Cluster randomized crossover design

Exposure to social norms is a popular way to foster healthy food behavior. Testing the robustness of this effect we report a field study assessing the impact of a vegetable-related descriptive norm message on vegetables purchase. The first contribution was to rely on a cluster randomized crossover design: Two canteens were randomly selected to display either a vegetable-related or a neutral-behavior norm message. After a first period of data collection, the displays were reversed for a second period: The number of vegetable portions on the main plate were recorded before, during and after the message display (N = 12.994).

The second contribution was to test the impact of a message describing vegetables as the normative choice beyond the mere selection of vegetables, on the quantity of vegetables purchased in lunches containing some. Results indicated that the vegetable-related norm message led to a sustained probability of choosing vegetables, contrary to a decrease observed in the control condition. Moreover, students who ordered vegetables ordered a higher quantity when exposed to a vegetable-related message than before whereas quantity declined in the control condition. By treating both canteens as experimental and control and by analyzing both the presence and the amount of vegetables, these results extend and strengthen those previously observed, bringing support for the effectiveness of a descriptive norm message in eliciting healthier food behavior.

Changing Behaviors with Social Norms

Social norms define the valued or common behaviors for a given social group [START_REF] Göckeritz | Descriptive normative beliefs and conservation behavior: The moderating roles of personal involvement and injunctive normative beliefs[END_REF]. According to the Focus Theory of Normative Conduct, social norms drive individuals' choices and actions [START_REF] Cialdini | A focus theory of normative conduct: A theoretical refinement and reevaluation of the role of norms in human behavior[END_REF]. The social norms that are prominent in an environment shape people's behavior [START_REF] Schultz | The constructive, destructive, and reconstructive power of social norms[END_REF]. Indeed, literature on health and behavior indicates that individuals use norms as a guide to behave efficiently (general behaviors: Cialdini & Glodstein, 2004; food intake: [START_REF] Higgs | Social influences on eating[END_REF], and that young people's eating habits are linked to the perceived peer norms [START_REF] Stok | The potential of peer social norms to shape food intake in adolescents and young adults: a systematic review of effects and moderators[END_REF][START_REF] Lally | Social norms and diet in adolescents[END_REF][START_REF] Liu | The relationship between social identity, descriptive social norms and eating intentions and behaviors[END_REF][START_REF] Rice | Interactions among perceived norms and attitudes about health-related behaviors in US adolescents[END_REF].

To better understand how people's behaviors are influenced by norms, two different aspects of normative conduct have been distinguished [START_REF] Cialdini | A focus theory of normative conduct: Recycling the concept of norms to reduce littering in public places[END_REF]. First, injunctive norms refer to what should be done, the behavior that is socially approved or disapproved.

Second, descriptive norms refer to what is done by most people. The first type of norms prescribes behavior, while the second describes behavior.

Many public-health campaigns use injunctive norms (e.g. "For your health, no more than 2 alcohol drinks a day, and not every day"). However, injunctive norm messages can actually promote counter-normative behaviors under specific circumstances [START_REF] Rivis | Descriptive norms as an additional predictor in the theory of planned behaviour: A meta-analysis[END_REF][START_REF] Lally | Social norms and diet in adolescents[END_REF]. For example, teenagers exposed to an injunctive norm message prescribing fruit consumption ("a majority of high school students think other high school students should eat sufficient fruit") reported lower fruit intake intentions than a group exposed to no normative information [START_REF] Stok | Don't tell me what I should do, but what others do: The influence of descriptive and injunctive peer norms on fruit consumption in adolescents[END_REF]. Reactance against injunctive messages [START_REF] Brehm | A theory of psychological reactance[END_REF] is especially likely among young people [START_REF] Hong | Psychological reactance: Effects of age and gender[END_REF], for whom healthy behaviors are not valued by their social group [START_REF] Stead | Why healthy eating is bad for young people's health: Identity, belonging and food[END_REF]. Hence, health behaviors interventions based on injunctive message could produce a "boomerang effect", inadvertently leading students to act even less healthy.

Descriptive norms, which describe how people actually behave in a given situation and a given social group [START_REF] Cialdini | Social influence: Compliance and conformity[END_REF], have been found to be especially effective to promote healthy behaviors concerning tobacco [START_REF] Sheikh | Confounding and statistical significance of indirect effects: childhood adversity, education, smoking, and anxious and depressive symptomatology[END_REF], alcohol [START_REF] Neighbors | Targeting misperceptions of descriptive drinking norms: Efficacy of a computer-delivered personalized normative feedback intervention[END_REF], and daily exercises [START_REF] Burger | Changing everyday health behaviors through descriptive norm manipulations[END_REF]. Interestingly, describing how the majority acts influences behavior even in situations where individuals lack the cognitive resources needed for effortful and conscious information processing [START_REF] Jacobson | Bodies obliged and unbound: Differentiated response tendencies for injunctive and descriptive social norms[END_REF]. Indeed, descriptive norms operate as a decision heuristic, indicating what is appropriate in the situation and driving behavior even if individuals are not influenced at a higher cognitive level. Hence, [START_REF] Stok | Don't tell me what I should do, but what others do: The influence of descriptive and injunctive peer norms on fruit consumption in adolescents[END_REF] observed that a descriptive message in favor of fruit intake ("a majority of high school students try to eat sufficient fruit themselves") increased self-reported fruit intake compared to an injunctive norm message, although it did not significantly influence the consumption intention declared by students. These findings are congruent with the idea that a descriptive message can impact behavior without affecting its corresponding cognition [START_REF] Stok | Don't tell me what I should do, but what others do: The influence of descriptive and injunctive peer norms on fruit consumption in adolescents[END_REF]. The positive influence of descriptive norms on food-related behaviors has been consistently observed in laboratory studies [START_REF] Burger | Nutritious or delicious? The effect of descriptive norm information on food choice[END_REF][START_REF] Stok | Don't tell me what I should do, but what others do: The influence of descriptive and injunctive peer norms on fruit consumption in adolescents[END_REF][START_REF] Lapinski | Descriptive norms and involvement in health and environmental behaviors[END_REF].

Increasing Vegetable selection via Descriptive-Norm Messages in Field

Studies

Drawing on these laboratory findings, real-world interventions have been designed to assess the effect of descriptive-norm messages on eating behaviors. [START_REF] Mollen | Healthy and unhealthy social norms and food selection. Findings from a field-experiment[END_REF] were the first to compare the effect of a norm-based message on students' food choices (burger or salad) in a campus food-court. Students were exposed either to a healthy injunctive norm ("Have a tossed salad for lunch!"), a healthy descriptive norm ("Every day more than 150 [name of university] students have tossed salad for lunch here"), an unhealthy descriptive norm ("Every day more than 150 [name of university] students have a burger for lunch here") or no message (control condition). Food choices were recorded through a questionnaire administered after lunch. Among students who correctly reported seeing the message, those exposed to the healthy descriptive norm had higher chances of choosing a salad in comparison to students in the unhealthy or the control condition. The injunctive healthy norm had a significant effect on healthy decisions compared to the unhealthy descriptive norm condition but not to the control condition.

Moving beyond self-reported behavior, [START_REF] Thomas | Using a descriptive social norm to increase vegetable selection in workplace restaurant settings[END_REF] explored the influence of a descriptive norm message ("Most people here choose to eat vegetables with their lunch") on actual food selection. Vegetable purchase was recorded in the cashier point of three workplace restaurants during three 2-week stages: A first no-message stage (preintervention stage), before displaying the descriptive-norm message for two weeks (intervention stage), followed by a 2-week stage without the message (post-intervention stage). The percentage of meals purchased with vegetables significantly increased when the norm message was displayed in comparison to the pre-intervention stage, and this effect persisted after the message removal. The benefit of descriptive norm intervention on actual vegetables selection was recently confirmed in two studies conducted in student canteens [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF]. Relying on a similar three-stage design (i.e., pre-intervention, intervention, post-intervention), [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF] showed that a descriptive-norm message ("Did you know that most students here choose to eat vegetables with their meal") increased the percentage of meals with vegetables in comparison to the pre-intervention stage. This effect was sustained during post-intervention in their first study but in the second.

These findings plead in favor of the implementation of norm-based interventions in collective catering sites. And indeed, exposure to social norms is often presented as an easy and efficient way to change individuals' behavior (e.g., [START_REF] Higgs | Social norms and their influence on eating behaviours[END_REF]. This literature is popularized outside the academic world to inform policy making (e.g., [START_REF] Hallsworth | Behavioural government: Using behavioural science to improve how governments make decisions[END_REF][START_REF] Oecd | Tools and Ethics for Applied Behavioural Insights: The BASIC Toolkit[END_REF] and has already been applied in large scale campaigns (e.g., [START_REF] Perkins | Effectiveness of social norms media marketing in reducing drinking and driving: A statewide campaign[END_REF].

Despite this popularity -or maybe because of this popularity -the literature on social norms should tackle the current question of replication and findings robustness that shakes many scientific fields (e.g., [START_REF] Hetherington | Methodological and reporting quality in laboratory studies of human eating behavior[END_REF][START_REF] Pashler | Editors' introduction to the special section on replicability in psychological science: A crisis of confidence?[END_REF]. This paper aims at contributing to this important matter by (1) testing the impact of a descriptive-norm based message on vegetable purchase in a field study with (2) a large sample, a control group with a randomization by canteens (i.e., cluster-randomized crossover design) and an appropriate analytical strategy for such a design.

Methodological Limitations of Field Studies

The extant field studies on norm-based interventions to promote vegetable purchase suffer from two important limitations. The first limitation concerns suboptimal internal validity due to weak designs. One study used a one-group pretest-posttest design [START_REF] Thomas | Using a descriptive social norm to increase vegetable selection in workplace restaurant settings[END_REF] in which vegetable selection was recorded during a pre-intervention stage (i.e., no message displayed), then when the intervention message was displayed, then during a postintervention stage after the message was removed. Because of the lack of a comparison group, no causality conclusion can be drawn about the effect of the intervention on vegetable selection, as any change from pre-to posttest could be due to time trend or individual changes unrelated to the intervention. [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF] used a similar pretest-posttest design with a comparison group. Indeed, the norm-based message was displayed in one canteen while a health message was displayed in another canteen. This however creates a confound between the type of message (norm vs. health) and the canteen, precluding again clear conclusion about whether changes in vegetable selection were caused by the type of message or preexisting differences between the canteens and their customers. [START_REF] Mollen | Healthy and unhealthy social norms and food selection. Findings from a field-experiment[END_REF] used an experimental design with three experimental messages (including descriptive norm) and a control group. However, they relied on self-reported food-choices limited to the participants who correctly recalled the experimental messages displayed in the canteens. This firstly limits their conclusion about the impact of the messages to this particular population. It secondly led to the exclusion of almost 78% of the participants in the experimental groups, resulting in small sample sizes (n ranging from 33 to 42), which reduces the chances that the results reflect a true effect [START_REF] Button | Power failure: why small sample size undermines the reliability of neuroscience[END_REF]. To our knowledge, only one other paper [START_REF] Thorndike | Social norms and financial incentives to promote employees' healthy food choices: A randomized controlled trial[END_REF] presents a randomized controlled trial but the reported results show no significant increase from baseline of healthy food purchase for participants who received social norms feedback compared to the control group. Connected research was conducted on food selection in grocery stores, not restaurants. The field studies on this related behavior however suffer similar limitations, with a confound between stores and type of messages (control vs. norm-based;[START_REF] Payne | Shopper marketing nutrition interventions: Social norms on grocery carts increase produce spending without increasing shopper budgets[END_REF], small samples and results above the conventional level of significance [START_REF] Salmon | Social proof in the supermarket: Promoting healthy choices under low selfcontrol conditions[END_REF], or combining social norm message with another nudge [START_REF] Huitink | Social norm nudges in shopping trolleys to promote vegetable purchases: A quasiexperimental study in a supermarket in a deprived urban area in the Netherlands[END_REF].

The available evidence documenting the effectiveness of using social norms to increase vegetable selection in field studies is not strong. Although great efforts have been made to test the impact of such interventions, more compelling evidence needs to be provided. The current research was specifically designed to address this issue. We recorded a large sample of actual vegetable purchase in a pretest-posttest design implemented in two similar student canteens. To allow for stronger conclusions about causality, canteens were randomly assigned to a vegetable-related norm message and a neutral behavior-related norm message. More specifically, our study relied on a cluster randomized crossover design, in which each message was administered to each cluster in separate periods of time [START_REF] Arnup | Understanding the cluster randomised crossover design: a graphical illustration of the components of variation and a sample size tutorial[END_REF][START_REF] Rietbergen | The design of cluster randomized crossover trials[END_REF]. During the first period, the experimental message was displayed in one canteen (cluster A) while the control message was displayed in the other canteen (Cluster B). During the second period, displays were reversed. We believe that such a stronger design does not only provide more reliable evidence about the effectiveness of a norm-based message to foster vegetable selection, it also provides a more robust test of the social norm theoretical approach. The present research also aimed to extend the extant literature by investigating whether descriptive norms about vegetable purchase can influence not only the selection of vegetables but also the quantity of vegetables purchased.

Of the Extended Impact of Descriptive-Norm Message: from vegetable selection to quantity

Previous studies focused on food selection as behavioral outcome, and assessed whether descriptive norm messages increase self-reported food choice [START_REF] Mollen | Healthy and unhealthy social norms and food selection. Findings from a field-experiment[END_REF] or the percentage of actual meals containing vegetables [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF][START_REF] Thomas | Using a descriptive social norm to increase vegetable selection in workplace restaurant settings[END_REF][START_REF] Thorndike | Social norms and financial incentives to promote employees' healthy food choices: A randomized controlled trial[END_REF]. Nevertheless, an important question remains unanswered: Does a descriptive norm-message lead people who purchase vegetables, to purchase a greater amount of vegetables? Descriptive norms motivate individuals to behave in a certain way by providing information on what is effective or adaptative to do [START_REF] Cialdini | A focus theory of normative conduct: A theoretical refinement and reevaluation of the role of norms in human behavior[END_REF]. If the behavior appears as efficient and adapted, we can expect that more individuals will make the decision to produce the behavior but also that individuals who decided to produce the behavior will produce it more. Accordingly, [START_REF] Agerström | Using descriptive social norms to increase charitable giving: The power of local norms[END_REF] observed that not only a descriptive norm message increased the number of students initiating the expected behavior, here donation, but it also increased the amount of donation. The positive impact of a descriptive norm message about prevalence on both frequency and intensity can also be expected on a health-related behavior. Indeed, health prevention messages often emphasize both the frequency and quantity of behaviours to be performed (e.g., World Health Organization campaign "5 A Day" recommends eating at least 5 portions of fruits and vegetables per day). In doing so, public campaigns suggest that the appropriate behavior is the combination of these two factors. In a field study on food choice at school, [START_REF] Thompson | Self-efficacy and norm measures for lunch fruit and vegetable consumption are reliable and valid among fifth grade students[END_REF] showed a positive correlation between perceived descriptive norms related to the selection of vegetable (e.g. "Most kids eat a serving of cooked vegetables at school lunch") and the amount of vegetables consumed recorded over a week's lunches. We therefore predicted that the descriptive norm message about healthy eating would increase both the frequency of the behavior (i.e., number of meals purchased with vegetables) and the intensity of the behavior (i.e. the amount of vegetables selected).

Overview and Hypotheses

We drew on previous studies in collective catering sites [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF][START_REF] Thomas | Using a descriptive social norm to increase vegetable selection in workplace restaurant settings[END_REF] to conduct a field experiment testing the effect of a descriptive norm message on vegetable purchase in two university canteens. The number of portions of vegetables on the students' main plate (from 0 to 4) was collected during three stages: a pre-intervention stage, an intervention stage (during which a vegetable-related norm message or a neutral behavior-related norm message was displayed) and a post-intervention stage (in which no message was displayed). The type of message was randomly displayed in one or the other canteen during the first period of observation, then crossed during a second period of observation.

We firstly hypothesized that the norm-based intervention would increase the likelihood of purchasing vegetables (presence on the plate versus not). In comparison to the pre-intervention stage, displaying a vegetable-related norm message should increase the number of plates with vegetables. The impact of the message might last after removal [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF][START_REF] Thomas | Using a descriptive social norm to increase vegetable selection in workplace restaurant settings[END_REF] but should be less strong so we expect the number of plates containing vegetables in the post-intervention stage to still be higher than during the pre-intervention stage but to decrease compared to the intervention stage. When the neutral behavior-related norm message was displayed, we did not expect to observe this pattern.

Secondly we expected the intervention to increase the amount of vegetables ordered. In comparison to the pre-intervention stage, the vegetable-related norm message should increase the number of vegetable portions on the main plate. Again, the impact of the message should lessen after its removal so we expect the number of vegetable portions to decrease in the post-intervention stage compared to the intervention stage. When the neutral behavior-related norm message was displayed this pattern was not expected. These hypotheses were specified before the data were collected.

Method

Participants

Participants were students purchasing a meal from one of two on-campus canteens.

All aspects of this study were performed in accordance with the ethical standards and we obtained a posteriori ethical approval (CERT-TP n°2020-07-03). A total of 12.994 meal purchases were recorded (47.08% by women, 52.54% by men, 0.38% by a person of undetermined gendergender was categorized on visual appraisal by the observers).

Design

The study took place in the two larger university canteens of the University of Poitiers.

Located in the same area (distant from 0.68 miles) and run by the same public service, they mainly serve students and offer the same self-service meals five days a week. The daily offer includes a main course and two side dishes. At the food counters, a choice between meat and fish is available as well as at least one type of carbs and two types of vegetables. During a first period of observation, one canteen was randomly selected to display the vegetable descriptive-norm message, while the other displayed the neutral behavior descriptive-norm message. During the second period of observation the messages were reversed at both locations. For each period of observation, data was collected during three days (Monday, Tuesday and Thursday) for three consecutive weeks corresponding to three stages: the first week formed the pre-intervention stage, the second week the intervention stage (i.e., messages are displayed), and the last week the post-intervention stage (i.e., after the message removal). Data collection took place from the 20th of November to the 7th of December 2017 for the first period, and from the 22th of January to the 8th of February 2018 for the second one 1 .

Sample size

Small effects have been documented in previous field studies testing the influence of social norm interventions on vegetable purchase in canteens [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF] 1.24 < OR < 1.62, 0.04 < F < 0.09; [START_REF] Thomas | Using a descriptive social norm to increase vegetable selection in workplace restaurant settings[END_REF] 1.2 < OR < 1.4, 0.03 < F < 0.08). Using simulation [START_REF] Reich | Empirical power and sample size calculations for cluster-randomized and cluster-randomized crossover studies[END_REF], we estimated the smallest effect size detectable in our cluster-randomized crossover design with 80% power and a 5% alpha level. The numbers of canteens and periods were fixed at 2 and the smallest canteen-period size to 2800 observations. We set the vegetable purchase baseline rate to be 60% [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF] and the variation in purchase rate across canteens to be of .01. We simulated 500 datasets assuming data would be analyzed using a fixed-effects logistic regression model. The results indicated that we had sufficient power to detect an effect associated with an odd ratio of 1.12.

Material

The messages were conveyed via posters. In the experimental condition, the message described normative vegetable purchase ("Most students here choose to eat vegetables at lunch") whereas in the control condition the norm was about a recycling behavior ("Most students here sort their tray after lunch"). The vegetable-related descriptive norm message was based on the one used by [START_REF] Thomas | Using a descriptive social norm to increase vegetable selection in workplace restaurant settings[END_REF] and [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF] 2 . The posters were printed in colour in two formats: the three biggest (210 mm x 297

1 Beginning the data collection too early in the semester (September) is problematic with respect to the low frequentation of the canteens by new arriving students, and ending it too lately also because students attend less the canteens during exams period (after December, the 15 th ). Moreover, a one-week holiday divides each semester (at the end of October and end of February). Finally, we wanted the type of menu to be relatively constant and then to collect the data in the same season. mm) were displayed in the waiting area of the canteens and the two smallest (148 mm x 210 mm) were placed on each food-selection counter.

Measures

Five observers alternatively stood behind the cashier of both canteens to record the customers' tray content. Data was collected at the lunchtime between 11:50 and 13:00 on every Monday, Tuesday and Thursday. The tray content was recorded using a grid including four items: "meat or fish" (V: meat; P: fish), "portions of vegetables in the main plate" that is the proportion of the plate occupied by vegetables (from 0 = 0% to 4 = 100%), "appetizer" 3 (crudités; half crudités; soup; cold meat; pasta salad), "fruit" (0 to 2). As in other studies, potatoes and lettuce leaves (as decoration) were not counted as vegetables.

To assess inter-rater reliability, the five observers all coded the same trays during a training session before actual data collection. The analysis of the inter-rater agreement indicated satisfactory Cohen Kappa coefficients ranging from 0.73 (starters coding) to 0.83 (gender coding). Moreover, during actual data collection, as menus changed every day, the observers of the two canteens daily reached agreement by phone on the coding for the suggestion of the day.

Data Analyses

A pre-specified generalized linear model with canteen and period fixed effects was used to examine the selection and amount of vegetables depending on the interaction between the stage of intervention and the type of message. Because observations were nested in periods and canteens, the data structure violates the assumption of independence of residuals. To address this issue both fixed-effects models and multi-level models can be performed. A fixed-effects model was preferred over a multi-level model because the latter can produce biased estimates when the number of higher-order units is low [START_REF] Maas | Sufficient sample sizes for multilevel modeling[END_REF][START_REF] Sommet | Keep calm and learn multilevel logistic modeling: A simplified three-step procedure using Stata, R, Mplus, and SPSS[END_REF]. Fixed-effects models control for any canteen-period variation and estimate the within canteen-period impact of the intervention.

To test both our hypotheses on the selection of vegetables and the amount of vegetables, and because the outcome (number of portions of vegetables) was highly skewed with many zeros, we modeled those data with a negative binomial hurdle model [START_REF] Atkins | A tutorial on count regression and zero-altered count models for longitudinal substance use data[END_REF]. The hurdle model combines two simultaneous models: (i) a logistic regression model for zeros vs. non zeros (i.e., presence or absence of vegetables), and (ii) a Poisson regression (or "zero truncated count regression") for the distribution of nonzero values (e.g., number of portions of vegetable in lunches with vegetables). Effect sizes are expressed with an Odd Ratio (OR) for the logistic regression, and with an Incident Rate Ratio (IRR) for the truncated Poisson regression. Values over 1.0 indicate an upper likelihood of having vegetables on the plate, and of having a higher number of portions on the plate.

We hypothesized that when the message is related to vegetable-purchase norm the presence of vegetables is more likely and the number of portions is higher during the intervention stage than before and decreases after the intervention without falling back to the baseline point (i.e., a moderate lasting impact of the intervention). To test this prediction, we created two orthogonal contrasts coding the stages. The contrast of interest compared the pre-intervention stage with the intervention stage (pre-intervention coded -0.5, intervention coded +0.5, post-intervention coded 0). The contrast testing the residual variance compared the post-intervention stage (coded 2/3) to the two other stages (pre-intervention and intervention both coded -1/3). The type of message was also contrast coded (-0.5 for the control message, +0.5 for the vegetable-related norm message). A pattern consistent with our hypothesis would result in a significant interaction between the type of message and the first contrast coding the stagesthe vegetable purchase should increase from the pre-to the intervention stage, especially in the vegetable-related norm condition. Simultaneously, the type of message should not significantly interact with the second contrast coding the stages. Indeed, in both conditions we expect the post-intervention stage to not significantly differ from the two previous stages: In the vegetable-related norm condition because we expect the purchase of vegetables to decrease after the intervention without falling back to pre-intervention level then placing the post-intervention between the two other stages; in the control condition no differences are expected between stages.

Results

Table 1

Observed number (and percentage) of meals as a function of the number of portions of vegetables purchased, the type of message displayed, during each stage of intervention. Data are available at https://osf.io/5qgk3/?view_only=dbe1fa96d0894314aa7dcb68078b0825. Details about the observed meals across the three stages and the two conditions are presented in Table 1.

The logistic part of the model testing the presence vs. absence of vegetable on the plates showed, as expected, a significant interaction between the message type and the contrast of interest comparing the pre-intervention and the intervention stage, OR = 1.23, p = .016, 95% CI = [1.04, 1.46] (see Figure 1). Simple slopes analyses showed that students in the control condition were 17% less likely to purchase vegetables during the intervention stage than before, OR = .83, p = .003, 95% CI [0.74, 0.94]. Students exposed to the vegetable-related norm message did not show this decrease, as their chances to purchase vegetables did not significantly change from the baseline to the intervention stage, OR = 1.03, p = .671, 95% CI [0.91, 1.16]. As expected also, the interaction between the message type and the residual variance contrast comparing the post-intervention stage with the two previous stages was not significant, OR = 0.93, p = .346, 95% CI [0.80, 1.08]. However, contrary to expectations, results indicated that on average across message type, the likelihood of purchasing vegetables was higher in the post-intervention stage than in the combined two previous stages, OR = 1.16, p < .001, 95% CI [1.08,1.26].

Figure 1.

Predicted probability of purchasing vegetables (vs. no vegetable) as a function of the type of message and the stages, within canteens and periods of observation.

The positive count model testing the amount of vegetables among those who purchased some revealed a significant interaction between the message type and the contrast of interest comparing the pre-and intervention stage, IRR = 2.12; p < .001, 95% CI = [1.69, 2.67] (see Figure 2). Among students who purchased vegetables in the control condition, the amount of vegetable purchased during the intervention stage is 0.26 times the amount before the intervention, IRR = 0.74, p < .001, 95% CI [0.63, 0.87]. Among students who purchased vegetables in the vegetable descriptive-norm condition, the amount of vegetable purchased increased by 1.57 times from the pre-to the intervention stage, IRR = 1.57, p < .001, 95% CI [1.38,1.84]. The interaction between the type of message and the residual variance contrast was not significant, IRR = 0.94, p = .59, 95% CI [0.77,1.16].

Consistent with the hypothesis, on average across message type, the level of purchase in the post-intervention stage did not significantly differ from the level of purchase in both the pre-and intervention stage, IRR = 0.90, p = .055, 95% CI [0.81, 1.002]. Predicted means of portions of vegetable in plates containing vegetable as a function of the type of message and the stages, within canteens and periods of observation.

Discussion

This field study drew on previous studies testing the effect of a vegetable-related descriptive norm messages on vegetable purchase. This literature suggested that simply describing that most people eat vegetables can increase healthy food selection. However, this literature suffered from some methodological shortcomingsabsence of a control group, confounds between restaurants and conditions, small sample sizesthat limited the conclusions. To address these limitations, we implemented a design with a vegetable-related norm message versus a control message randomly displayed in two canteens. The display was crossed over canteens in two different periods to avoid confound and the canteens and periods variations were accounted for in the analytical strategy. Vegetable purchase was recorded from over 12,900 meals to test whether the increase in the number of plates containing vegetables induced by the display of a norm-based message could be replicated.

The results showed that the vegetable-related norm message led to a sustained number of plates containing vegetables, contrary to the decrease observed in the control condition. Though we expected an increase in purchase rather than a non-decrease, the results do show a more positive trend in the vegetable-related norm condition than in the neutral norm condition and is therefore consistent with the hypothesis. The vegetable-related norm message seems to have shielded students from a decline. This unexpected decline in the control condition could be due to the specificities of the menus offered at the times of observation, which could as well be an explanation for the unexpected overall increase in vegetable selection in the post-intervention stage. Importantly however, the daily menus were identical in both canteens so the more positive trend observed in the vegetable-related norm condition compared to the control condition can not be explained by differences in menus. This study therefore brings evidence consistent with the previous field studies

showing that a message describing vegetable purchase as produced by a majority of their social group can positively influence students' choice to order vegetables [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF].

A second goal of this research was to test the extended influence of a norm-based message beyond the mere selection of vegetables, by recording the quantity of vegetables (i.e., number of portions) purchased by those who selected vegetables. The results showed that students who ordered vegetables ordered a higher quantity when surrounded by a vegetable-related norm message than before, whereas the quantity decreased from the preto the intervention stage in the control condition. The increase observed when the vegetablerelated message was displayed was however not sustained after its removal, which is consistent with other field studies [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF]. This finding brings new insight about the impact of descriptive norms. Although the message did not directly mention quantity ("most students here choose to eat vegetables"), it influenced the number of portions purchased. This is especially important for health-related behavior as in many cases a healthy behavior implies not only to produce a behavior but also to produce it with a certain intensity. More research should investigate which type of messageeither describing the prevalence of the behavior or describing the quantitymost effectively influences both the frequency and intensity of subsequent healthy behavior. It would be interesting to compare the impact of a "frequency and quantity related message" ("Most students here, choose to eat 2 portions of vegetable with their lunch") that proved its effectiveness [START_REF] Agerström | Using descriptive social norms to increase charitable giving: The power of local norms[END_REF], a "frequency related message" ("Most students here, choose to eat vegetables with their lunch") a "quantity related message" ("Students purchased 2 portions of vegetables when they choose to eat some") and a control message. The fact that a message about the prevalence of the behavior in the social group makes the behavior more likely to be produced both in terms of frequency (selection of vegetable) and intensity (amount of vegetables) is consistent with the theoretical claim of the social norm approach that descriptive norms inform about the efficiency of a behavior, as an efficient behavior should be both produced more often and/or more intensively [START_REF] Cialdini | A focus theory of normative conduct: A theoretical refinement and reevaluation of the role of norms in human behavior[END_REF]. To design more effective policy campaign message, it could be important to ensure that the quantity selected be displayed explicitly.

Despite these contributions, the present field study has several limitations. The first limit, outlined by the unexpected results (decreasing selection of vegetables in the control condition, overall higher selection in the post-intervention stage), lies in the short duration of observation. Data collection was conducted for only 3 days at each stage (pre-, intervention, post-intervention), which led to a measure of behavior that is sensitive to temporary and random external influences such as variations in menus, or in specificities of individuals attending the canteens from one day to another. The presence of a control group subjected to the same random influences limits the risk of erroneous conclusions about the effectiveness of the intervention but future studies would benefit from longer observation duration to better estimate the behavioral tendencies. Longer observation duration would also offer the possibility to further investigate the immediate effect of the message by increasing exposure and its mid-term influence after removal.

Second, as often in observational studies with large samples (e.g., [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF][START_REF] Thomas | Using a descriptive social norm to increase vegetable selection in workplace restaurant settings[END_REF] it was not possible to follow individual students across the stages of the experiment due to technical limitations. Consequently, we could neither assess the behavioral change within students nor account for between individual variations. This leaves open the question of whether the vegetable-related norm message made people who previously did not order vegetables to start doing so and/or reinforced the behavior among people who already produced it. Future research would benefit from a longitudinal design to establish how descriptive norms can lead to within-individuals changes and whether some individuals are more sensitive to the message than others.

Third, both messages displayed in this study presented a descriptive norm, one about vegetable purchase, the other about sorting one's tray. This procedure was chosen to rule out the idea that merely activating a descriptive norm is enough to increase vegetable purchase.

However, this leaves the possibility that it is the mere mention of vegetables that increased purchase. Other field studies used a comparison message mentioning vegetables but not based on a descriptive norm (e.g., health benefits of vegetables, [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF]; injunctive or unhealthy norms, [START_REF] Mollen | Healthy and unhealthy social norms and food selection. Findings from a field-experiment[END_REF]. Future studies could use two comparison messages, one mentioning vegetables, the other displaying a descriptive norm on a different topic to further test the effectiveness of the present vegetable-related descriptive norm message.

Fourth, vegetable purchase was collected, as in previous studies [START_REF] Collins | Two observational studies examining the effect of a social norm and a health message on the purchase of vegetables in student canteen settings[END_REF][START_REF] Mollen | Healthy and unhealthy social norms and food selection. Findings from a field-experiment[END_REF][START_REF] Thomas | Using a descriptive social norm to increase vegetable selection in workplace restaurant settings[END_REF], but actual consumption was not measured.

Hence, the results indicated that vegetable descriptive norm message led students to sustainably order vegetables and in greater quantity, but no conclusion can be drawn about actual consumption. Future research is needed to assess the amount of vegetables left on the plate.

Finally, two factors could have minimized the impact of our intervention on vegetable purchase. First, as in previous studies, the posters were placed on the service counters, being then accessible to the attention at the very moment students choose their meal.

Nevertheless, we did not assess whether participants actually saw the posters. Given that the influence of the message displayed in the posters was observed to be effective only among students who saw them [START_REF] Mollen | Healthy and unhealthy social norms and food selection. Findings from a field-experiment[END_REF], our results might underestimate the effect of the message on vegetable purchase by including data from students who did not see the posters. Second, the two canteens were located on the same campus and students could have attended both canteens, either in the same period, or in the two periods separated by a two-month delay. In both cases, if some students did attend both canteens, it could weaken the difference between the control and experimental conditions. The potential contamination between conditions could have led to an underestimation of the impact of the vegetable norm intervention relatively to the control intervention but it can hardly account for the observed interactions.

In conclusion, the original cluster randomized cross-over design, in which both messages (Control, Experimental) are administered to each cluster (Canteen A, Canteen B), combined with the statistical control for canteen and period variations, provides strong evidence that displaying a vegetable-related descriptive norm message in canteens is effective in increasing vegetable purchase. This research strengthens the scientific literature fueling theory-and evidence-based behavior change interventions. Although more research is needed to assess actual consumption, social norms messages seem to be a promising lever for public authorities to set up cost-efficient campaigns.

  Figure 2.

Observations made during the first stage of our study confirmed the accuracy of the descriptive norm message (48% of trays contained vegetables).

Acknowledgements :

We would like to acknowledge the CROUS of Poitiers, the director Mariannig Hall and the staff of the two canteens for granting us access to the canteens. We would like to thank Noëmie Mocquant, Aurélie Ribeiro, Thomas Olivet, Maximilien Nédélec and Coralie Botalla for assisting with data collection.

Authors contributions:

Emilie Guichard, Jean-Claude Croizet and Stephane Jouffre designed the study. Emilie Guichard supervised and carried out the data collection. Emilie Guichard and Frédérique Autin performed the analyses. Emilie Guichard, Frédérique Autin, and Stephane Jouffre drafted the manuscript. Emilie Guichard, Frédérique Autin, Jean-Claude Croizet, and Stephane Jouffre provided substantial comments and all approved the final version of the manuscript.


J Klein

J Gonzalez

J Duchene

L Esposito

Jean-Philippe Pradère

Eric Neau

C Delage

Denis Calise

A Ahluwalia

Pierre Carayon

Gonzalez

Pradère Jp

Delage

Pesquero Jb

Schanstra Jp
email: peter.schanstra@inserm.fr

Bascands Jl
email: loup.bascands@inserm.fr

Delayed blockade of the kinin B1 receptor reduces renal

Keywords: kidney disease, bradykinin, unilateral ureteral obstruction, chemokine, reversion

The incidence of chronic kidney disease (CKD) leading to end-stage renal disease (ESRD) has significantly increased and may reach epidemic proportions over the next decade [START_REF] Nahas | Chronic kidney disease: the global challenge[END_REF]. Regardless of the initial insult, the progression of most forms of renal disease results in tubulointerstitial fibrosis, which is the main histological hallmark of CKD [START_REF] Klahr | The progression of renal disease[END_REF]. The presence of fibrosis in CKD is closely correlated to the future appearance of renal failure and has therefore been associated with poor long-term prognosis [START_REF] Nath | Tubulointerstitial changes as a major determinant in the progression of renal damage[END_REF]. Interstitial fibrosis is characterized by the progressive accumulation of extracellular matrix (ECM) proteins in the tubulointerstitial compartment. A multitude of events and factors (4) were identified to be involved in the development of renal fibrosis, potentially leading to new antifibrotic strategies and compounds [START_REF] Boor | Treatment targets in renal fibrosis[END_REF][START_REF] Nguyen | Bone Morphogenetic Protein-7 and Connective Tissue Growth Factor: Novel Targets for Treatment of Renal Fibrosis?[END_REF](7). However in human, blockade of the renin-angiotensin aldosterone system remains the only effective therapy [START_REF] Nahas | Chronic kidney disease: the global challenge[END_REF]. These therapies only slow down the progression towards ESRD and alternative molecules or therapies are still necessary.

As a general rule the acute inflammatory response protects against infection and injury, however the chronicity of inflammation is often deleterious [START_REF] Nathan | Points of control in inflammation[END_REF]. Chronic inflammation is a key event in CKD that is mainly characterized by monocyte/macrophage accumulation in the renal interstitium and is well correlated with the progression of CKD [START_REF] Kluth | Multiple facets of macrophages in renal injury[END_REF][START_REF] Eddy | Progression in chronic kidney disease[END_REF]. Consequently, any strategy or agent able to limit or attenuate chronic renal inflammation should significantly slow down the rate of progression of CKD.

Lys-bradykinin and Lys-des-Arg 9 -bradykinin are peptides intimately linked to inflammation [START_REF] Leeb-Lundberg | International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences[END_REF]. These kinin peptides interact with two different G-protein-coupled receptors: the bradykinin B2 receptor (B2R) and the B1R, respectively [START_REF] Leeb-Lundberg | International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences[END_REF]. Constitutively expressed in most tissues, the B2R has been shown to mediate most of the physiological actions of kinins. The role of the B2R has been studied in experimental CKD. We and others have observed that blockade of the B2R increased experimental renal fibrosis [START_REF] Schanstra | In vivo bradykinin B2 receptor activation reduces renal fibrosis[END_REF][START_REF] Pawluczyk | The role of bradykinin in the antifibrotic actions of perindoprilat on human mesangial cells[END_REF][START_REF] Okada | Bradykinin decreases plasminogen activator inhibitor-1 expression and facilitates matrix degradation in the renal tubulointerstitium under angiotensinconverting enzyme blockade[END_REF][START_REF] Seccia | The renal antifibrotic effects of angiotensin-converting enzyme inhibition involve bradykinin B2 receptor activation in angiotensin IIdependent hypertension[END_REF], but the ubiquitous expression of the B2R does not make it an ideal target.

In contrast, the kinin B1R, which is hardly detectable under physiological conditions, is overexpressed under inflammatory conditions in a variety of different tissues [START_REF] Leeb-Lundberg | International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences[END_REF] and in turn stimulates inflammation upon activation. These data suggest that the B1R might be a more suitable target in chronic inflammatory renal disease.

A large number of in vitro and in vivo studies have shown that B1R induction is controlled by many pro-inflammatory cytokines and growth factors including IL1-ß, TNFα, Interferon gamma, as well as EGF [START_REF] Leeb-Lundberg | International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences[END_REF]. There is now clear evidence that induction of the B1R by many of these factors involves activation of transcription factor NF-κB and, conversely, B1R stimulation activates NF-κB [START_REF] Leeb-Lundberg | International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences[END_REF][START_REF] Marin-Castano | Differential induction of functional B1bradykinin receptors along the rat nephron in endotoxin induced inflammation[END_REF]. The activated B1R stimulates release of TNFα and IL-1 [START_REF] Tiffany | Bradykinin stimulates tumor necrosis factor and interleukin-1 release from macrophages[END_REF] and is also involved in leukocyte accumulation and activation [START_REF] Ahluwalia | Involvement of bradykinin B1 receptors in the polymorphonuclear leukocyte accumulation induced by IL-1 beta in vivo in the mouse[END_REF][START_REF] Bockmann | Kinins and kinin receptors: importance for the activation of leukocytes[END_REF]. The B1R is known to be expressed on macrophages [START_REF] Ricupero | Des-Arg(10)-kallidin engagement of the B1 receptor stimulates type I collagen synthesis via stabilization of connective tissue growth factor mRNA[END_REF] and on fibroblasts in vitro [START_REF] Ricupero | Des-Arg(10)-kallidin engagement of the B1 receptor stimulates type I collagen synthesis via stabilization of connective tissue growth factor mRNA[END_REF]. Furthermore we have previously shown in vivo that experimental inflammation induces functional B1R expression in renal epithelial cells [START_REF] Marin-Castano | Differential induction of functional B1bradykinin receptors along the rat nephron in endotoxin induced inflammation[END_REF][START_REF] Schanstra | Bradykinin B(1) receptor-mediated changes in renal hemodynamics during endotoxin-induced inflammation[END_REF]. Its role in acute renal disease (ischemiareperfusion) has been studied, but yielded contradictory results most probably due to compensation in the genetically engineered B1R, B2R single and B1R/B2R double knockout mice [START_REF] Kakoki | Bradykinin B1 and B2 receptors both have protective roles in renal ischemia/reperfusion injury[END_REF][START_REF] Wang | Influence of bradykinin B1 and B2 receptors in the immune response triggered by renal ischemia-reperfusion injury[END_REF]. However, to the best of our knowledge, the role of the B1R in chronic renal inflammation and fibrosis has never been studied.

Taken together, specific induction of the B1R in renal inflamed tissues, its role in inflammation and the role of chronic inflammation in renal fibrosis led us to hypothesize that blocking the B1R could be an efficient approach to control the progression of CKD.

MATERIALS AND METHODS

Drug

B1R-antagonist SSR240612 was synthesized at Sanofi-Aventis R&D Montpellier-France (24). For in vivo experiments this compound was dissolved in water containing 2% dimethylsulfoxide (DMSO) to obtain a 1 g/L solution. The SSR240612 solution was diluted with distilled water and administered by gavage at a dose of 10 mg/kg/d. Final DMSO concentration was 0.01%.

Animals

Mice invalidated for the B1R (B1-/-) were obtained as described previously [START_REF] Pesquero | Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors[END_REF]. Briefly, mice were generated by gene targeting on a mixed genetic background (129/SvJ x C57Bl/6J) and backcrossed ten times to C57Bl/6J (Harlan) as previously reported [START_REF] Mori | Kinin B1 receptor deficiency leads to leptin hypersensitivity and resistance to obesity[END_REF]. We used C57Bl/6J (Harlan) as their wild type (B1+/+) control littermates. The mice were housed in a pathogen-free environment. All experiments reported were conducted in accordance with the NIH guide for the care and use of laboratory animals and were approved by a local animal care and use committee.

Unilateral Ureteral Obstruction (UUO) B1+/+ and B1-/-male mice of 8 weeks of age were used for these experiments. The unilateral ureteral ligation was performed as previously described [START_REF] Schanstra | In vivo bradykinin B2 receptor activation reduces renal fibrosis[END_REF]. Briefly, under oxygen-isoflurane anesthesia and through a longitudinal, left abdominal incision, the ureter was exposed and ligated with a 6/0 nylon thread at the uretero-pelvic junction. In sham operations, the ureter was exposed but not ligated and repositioned. Mice were maintained on a standard mouse chow and tap water. Treatments with the B1 receptor antagonist were initiated either 1 day before obstruction or 3 days after and continued throughout the time of obstruction. A control group received only the vehicle (0.01% DMSO solution). At the end of the different protocols, mice were sacrificed, and the kidneys were removed and divided in different parts according to the different protocols employed.

Cell culture HEK293T (Human Embryonic Kidney) cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, GIBCO) without pyruvate and with 4.5g/L glucose and 10% fetal calf serum at 37°C, 5% CO2. Cells were seeded in 6-well plates and were transiently transfected with either a pcDNA3 plasmid containing the GFP gene (Green Fluorescent Protein, pGFP) or a pcDNA3 plasmid containing the gene encoding for the human B1 receptor (phB1R) (#BDKB10TN00; Missouri S&T cDNA resource center) using JetPEI transfection reagent (Ozyme). Two days later, cells were pretreated or not with the peptidic antagonist des-Arg 10 , Leu 9 kallidin (10 -6 M) (NeoMPS) for 15 minutes and then treated with the human B1 receptor agonist des-Arg 10 kallidin (10 -6 M) (NeoMPS) for 4 hours.

Bone marrow transplantation

Bone marrow transplantation was performed as previously described [START_REF] De Winther | Bone marrow transplantations to study gene function in hematopoietic cells[END_REF]. Briefly, 8 week-old B1+/+ male recipient mice were lethally irradiated with 10 Gy and put on acidified water (pH2) with antibiotics (neomycin 100 mg/L and polymyxin B sulphate 60000 U/L, Gibco) until 4 weeks after the transfer. Bone marrow cells were isolated from femurs and tibias of 12 week-old B1+/+ or B1-/-donor mice by flushing with PBS. Cells were then passed trough a 21G needle, centrifuged (250xg, 5 min, 4°C) and resuspended in RPMI 1640 (Gibco), 2% FCS, 5 U/mL heparin. Recipient mice received 5x10 6 bone marrow cells in 100 µL by tail vein injection. UUO was performed 4 weeks after transplantation. At the time of sacrifice peritoneal macrophages were isolated from abdominal cavity by peritoneal lavage and blood was harvested. Haematologic analysis was performed by counting red blood cells, platelets, total leucocytes, neutrophils, monocytes and lymphocytes using a Micros-60CS/18 automated counter (ABX-diagnostics). The genomic DNA was extracted and the genotype was determined by PCR to verify the reconstitution of bone marrow after transplantation.

Histological analysis and immunohistochemistry

Four-micrometer paraffin-embedded sections were cut and used for routine staining (hematoxylin-eosin and periodic acid-Schiff (PAS) staining) and immunohistochemistry. Sections were first de-waxed in toluene and rehydrated through a series of graded ethanol washes before endogenous peroxidase blockage. Specific primary antibodies were incubated (1 hour at room temperature) for the detection of collagen type III (1/500) (Interchim), of F4/80 positive inflammatory cells (macrophages) (anti-mouse F4/80,1/250) (RM2900; Caltag laboratories Inc., Burlingame, California, USA) and α-smooth muscle actin (α-SMA, Dako Epos method, U7033; Dako S.A., Trapppes, France). For visualization we used the Dako Envision system. Sections were finally counterstained with hematoxylin. Negative controls for the immunohistochemical procedures included substitution of the primary antibody with nonimmune sera.

Histomorphometric analyses were performed as previously described ( 12) using a commercially available image-analysis software which allows rebuilding of a kidney section from adjacent individual captures (Explora Nova Mosaïc software, La Rochelle, France).

Isolation of RNA

Total RNA was isolated from mouse tissue or cells using Qiagen RNeasy Mini kit, eluted in 20 µl RNase-free water and treated by DNase (TURBO DNA-free kit, Ambion) according to the manufacturer's protocol. 1.5 µl of this solution was used for quantitation by a NanoDrop instrument (ND-1000 spectrophometer).

Quantification of gene expression by real-time quantitative PCR

Real-time PCR was performed using the ABI PRISM 7900 HT. PCR amplification was performed in a total volume of 25 µl containing 25 ng of cDNA sample, 300 nM of forward and reverse primer and 12.5 µl of Power SYBR Green PCR Master Mix (Applied Biosystems). The reaction mixture was preheated at 95°C for 10 min, followed by 40 cycles at 95°c for 15 s and 60°C for 1 min. In our experimental models the most stable mouse housekeeping gene was the 18S ribosomal RNA (18S). The primers used in this study are listed in Table 1.

Statistical analysis

Data are expressed as mean plus or minus SD. ANOVA with post hoc Tukey alpha test was performed for comparison between the different groups. P values less than 0.05 were considered statistically significant.

RESULTS

Renal B1 receptor expression is induced in mice during unilateral ureteral obstruction (UUO).

We have used the in vivo model of UUO-induced renal fibrosis. This model has the advantage to mimic, in an accelerated manner, the main stages leading to interstitial fibrosis: macrophage infiltration, myofibroblast appearance and ECM-accumulation [START_REF] Bascands | Obstructive nephropathy: insights from genetically engineered animals[END_REF]. Mice were subjected to UUO and kidneys were harvested at different time points after obstruction (figure 1). Macrophage infiltration was quantified by immunohistological staining for F4/80, and B1R mRNA expression was analyzed by real-time PCR. F4/80 positive cells infiltration started 24 h after UUO and their number clearly increased all along the progression of the pathology (figure 1A). 24 h of UUO induced a slight increase in B1R mRNA expression, which became significant at day 3 and 8 (figure 1B). This induction was restricted to the obstructed kidney since UUO did not induce B1R mRNA expression in the controlateral kidney nor in other tissue including aorta, brain, mesenteric tissue, or lung (figure 1C).

Genetic and pharmacological blockade of the B1 receptor reduce significantly the development of UUO-induced renal fibrosis.

We next assessed the effect of B1R blockade on the development of renal fibrosis. Interstitial collagen accumulation was studied by histomorphometric analysis in Sirius redstained renal sections as an index of the fibrotic response to UUO of B1-/-, B1+/+ and mice pretreated with a B1R antagonist (B1Ra, 10mg/kg/d) (figure 2A-B). After 8 days of UUO, interstitial fibrosis was significantly reduced in B1-/-(figure 2A) and mice pretreated with the B1Ra (figure 2B) compared to the B1+/+ group or wild-type mice treated with the vehicle only. In the different control groups (sham) no significant difference in interstitial fibrosis was observed.

B1 receptor blockade decreases inflammatory cell infiltration and myofibroblast appearance.

In the tubulointerstitium of the control groups (sham) of both B1+/+ and B1-/-we found a very low level of F4/80 positive macrophages (figure 2C-D) and α-SMA positive myofibroblasts (figure 2E-F). As expected, UUO induced a significant increase in these renal fibrosis markers. Very interestingly, this increase was prevented by the genetic and pharmacological blockade of the B1R (figure 2C-F).

Delayed B1R antagonist treatment blunts the development of UUO-induced renal fibrosis.

To more closely mimic the clinical situation, we investigated whether B1R blockade would result in improvement of established renal fibrosis and inflammation. We thus studied the effect of delayed B1R blockade on UUO-induced fibrosis. Three days of UUO resulted in significant B1R expression (figure 1B) and kidney sections were also already strongly positive for macrophages, myofibroblasts and collagen accumulation (figure 3A, black line). We therefore decided to start B1Ra treatment 3 days after UUO (figure 3A, arrow). B1Ra halted F4/80 positive macrophage infiltration and this was associated with a significant decrease in interstitial myofibroblasts and fibrosis (figure 3A, blue line). This effect was confirmed at the mRNA level, since we found a significant decrease in collagen type I, III and IV mRNA expression (figure 3B). Treatment with B1Ra did not alter B2R mRNA expression (not shown).

Delayed B1R antagonist treatment blunts CTGF, CCL2 and CCL7 mRNA expression.

To better understand the mechanism by which B1R blockade is reducing renal fibrosis, we analyzed mRNA expression of the profibrotic cytokines TGFβ and CTGF, and two chemokines shown to be critical for monocyte/macrophage recruitment, CCL2 (MCP-1) and CCL7 (MCP-3) [START_REF] Tsou | Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites[END_REF][START_REF] Segerer | Chemokines, chemokine receptors, and renal disease: from basic science to pathophysiologic and therapeutic studies[END_REF]. As shown in figure 4, the UUO-induced expression of CTGF, CCL2 and CCL7 was blunted by delayed oral B1Ra administration. However, the treatment did not significantly affect TGFβ.

Activation of the B1R directly induces CTGF, CCL2 and CCL7 mRNA expression in vitro.

To investigate whether the B1R is able to directly stimulate cytokine and chemokine expression, we used a B1R-transfected HEK293 cell line, which is a classical cellular model commonly used to evaluate the effects of the B1R (24, 31). We analyzed expression of CCL2, CCL7 and CTGF in these cells transfected with a GFP containing plasmid (pGFP), as a control, or a human B1 receptor containing plasmid (phB1R) (figure 5). Cells were treated either with the human B1R agonist, des-Arg 10 kallidin (1µM), or the peptidic B1 receptor antagonist, des-Arg 10 , Leu 9 kallidin (1µM). Treatments with either B1R-agonist or -antagonist alone were without effect on chemokine expression in control GFP transfected cells. In B1R overexpressing cells, the B1R agonist induced rapid expression (4h) of CCL2, CCL7 and CTGF, and this effect was blunted by B1Ra treatment. No effect was observed on TGFβ expression (not shown).

Specific deletion of the B1R on blood circulating cells does not affect the development of UUO-induced fibrosis.

As the B1R is expressed on infiltrating macrophages as well as on resident renal cells in the inflamed kidney we investigated whether specific B1R knockout on blood circulating cells (including inflammatory cells) modifies the development of UUO-induced fibrosis in B1+/+ mice. In order to verify that the mice were successfully reconstituted with bone marrow, peritoneal macrophages and blood of the recipient mice were harvested before the sacrifice, counted and genotyped. As shown in figure 6A, no difference was observed in the number of circulating hematopoietic cells in wild-type mice with B1+/+ or B1-/-marrow. Moreover, in wild-type mice reconstituted with B1-/-marrow, the B1R mRNA corresponding band was not detected in the blood as well as in peritoneal macrophages (figure 6B). These results demonstrated that the mice were successfully reconstituted and that blood-circulating cells were primarily from the donor. B1-/-bone marrow (B1-/-Bm) transplanted wild-type mice subjected to 8 days UUO did not display significant differences in chemokine mRNA expression (CCL2, CCL7), macrophage infiltration (F4/80 immunohistochemistry) and fibrosis development (Sirius Red) compared to obstructed B1+/+Bm transplanted mice (figure 6C). These results strongly support the hypothesis that the effect of B1R blockade on interstitial fibrosis is mainly mediated by resident renal cells, most likely by reducing chemokine expression and subsequently reduced inflammatory cell recruitment.

DISCUSSION

The main characteristics of most forms of chronic kidney disease (CKD) are persistent inflammation and tubulointerstitial fibrosis which are strong prognostic factors for progression towards ESRD [START_REF] Klahr | The progression of renal disease[END_REF][START_REF] Nath | Tubulointerstitial changes as a major determinant in the progression of renal damage[END_REF][START_REF] Becker | The role of tubulointerstitial injury in chronic renal failure[END_REF]. As recently reviewed [START_REF] Kovesdy | Novel targets and new potential: developments in the treatment of inflammation in chronic kidney disease[END_REF], chronic inflammation is one of the novel risk factors contributing to the increased mortality seen in CKD patients. Therefore it is hypothesized that reduction of renal interstitial inflammation has the potential to reduce the progression of interstitial fibrosis and thus prevent ESRD (7, 34). To the best of our knowledge, even if a number of promising strategies are emerging (7, 34), we still do not have effective therapeutic strategies or molecules able to specifically target and reduce chronic renal inflammation without blocking other important pathophysiological functions.

We present here data that support the hypothesis that specific blockade of the kinin B1 receptor might be a promising strategy to reduce renal chronic inflammation and subsequently blunt the development of renal fibrosis. Several lines of evidence support this hypothesis. We first demonstrated that the B1R contributed to the progression of UUO-induced renal inflammation and fibrosis. We next showed that delayed treatment with an orally-active nonpeptide B1R antagonist in the UUO model significantly decreased the progression of established renal fibrosis. We further showed, using the UUO model associated to bone marrow transplantation experiments and in vitro studies, that this effect was partly mediated via a mechanism involving the inhibition of CTGF and chemokine expression by resident renal cells. To our knowledge this is the first in vivo study reporting that B1R blockade could become an efficient antifibrotic strategy.

We first demonstrated that during UUO, B1R mRNA expression was significantly increased in the obstructed kidney. Very interestingly, this induction seemed to be restricted to the site of the pathology since there was no marked difference of B1R expression in other tissues. The effects of genetic ablation of the B1R (B1-/-) showed reduced macrophage recruitment associated with a reduction in interstitial fibrosis. This suggests that B1R blockade is modifying the inflammatory response. This was consistent with the attenuated inflammatory response observed in B1-/-mice in different inflammatory models including pleurisy, paw edema [START_REF] Pesquero | Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors[END_REF], as well as in a model of intestinal ischemia and reperfusion injury [START_REF] Souza | Role of bradykinin B2 and B1 receptors in the local, remote, and systemic inflammatory responses that follow intestinal ischemia and reperfusion injury[END_REF].

It is well known that compensatory mechanisms occur in genetically engineered animals. We have previously reported increased renal B1R expression in B2-/-mice [START_REF] Marin-Castano | Induction of functional bradykinin b(1)receptors in normotensive rats and mice under chronic Angiotensin-converting enzyme inhibitor treatment[END_REF].

Similarly, increased renal B2R mRNA expression in B1-/-mice has been reported [START_REF] Seguin | Hemodynamic and renal involvement of B1 and B2 kinin receptors during the acute phase of endotoxin shock in mice[END_REF]. We have also shown that in vivo B2R activation reduces UUO-induced renal fibrosis [START_REF] Schanstra | In vivo bradykinin B2 receptor activation reduces renal fibrosis[END_REF]. Therefore it is possible that the B2R is involved in the effects observed in B1-/-mice. However, B1R antagonist treatment, mimicking the effects observed in B1-/-mice did not induce renal B2R mRNA expression. Thus, the reduction in UUO-induced renal fibrosis observed B1-/-mice is most likely not due to the modification of kinin B2R expression.

How does B1R blockade reduces the inflammatory response in this model of renal fibrosis? As shown by a number of studies, interstitial inflammation mediated by macrophages constitutes an early and major event in response to UUO [START_REF] Chevalier | Obstructive nephropathy: towards biomarker discovery and gene therapy[END_REF][START_REF] Sung | Reduction of renal fibrosis as a result of liposome encapsulated clodronate induced macrophage depletion after unilateral ureteral obstruction in rats[END_REF]. The precise molecular mechanism of the development of renal fibrosis is not yet fully elucidated, but key mediators including the major profibrotic cytokines TGF-ß and CTGF were clearly identified [START_REF] Iwano | Mechanisms of tubulointerstitial fibrosis[END_REF][START_REF] Bascands | Obstructive nephropathy: insights from genetically engineered animals[END_REF][START_REF] Border | TGF-beta in kidney fibrosis: a target for gene therapy[END_REF][START_REF] Okada | Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis[END_REF]. As expected, we observed an increased TGF-ß and CTGF mRNA expression in UUO-induced fibrosis. Interestingly, the blockade of the B1R was not associated with a decrease in TGFβ mRNA expression, but reduced its downstream mediator CTGF. This observation confirms in vitro experiments reporting that activation of the B1R stabilized CTGF mRNA, without modifying TGFβ expression [START_REF] Ricupero | Des-Arg(10)-kallidin engagement of the B1 receptor stimulates type I collagen synthesis via stabilization of connective tissue growth factor mRNA[END_REF]. In addition, this is the first demonstration that B1R antagonism reduces CTGF mRNA expression in vivo. Furthermore a number of chemokines are involved in renal monocyte/macrophage infiltration [START_REF] Kluth | Multiple facets of macrophages in renal injury[END_REF]. The role of CCL2 (MCP-1) (30) and even more recently the role of CCL7 (MCP-3) (29) were clearly established in this process. Our data showed reduced renal expression of these two chemokines in response to B1Ra treatment.

The next issue was to determine whether the B1R is directly involved in this attenuated chemokine response observed in vivo. To verify this hypothesis we used human embryonic kidney (HEK) cells transfected with the human B1R to study the effect of B1R stimulation on CTGF and chemokine expression. We observed that B1R stimulation is able to rapidly (4h) induce a significant increase in CTGF, CCL2 and CCL7 mRNA expression, which was abolished by a specific B1R antagonist. Although the mechanism by which the B1R stimulates CTGF mRNA overexpression has been previously shown to be mediated by mRNA stabilization [START_REF] Ricupero | Des-Arg(10)-kallidin engagement of the B1 receptor stimulates type I collagen synthesis via stabilization of connective tissue growth factor mRNA[END_REF], the effects of B1R activation on CCL2 and CCL7 mRNA expression most probably involves NF-κB as it was shown that the B1R activates this transcription factor [START_REF] Schanstra | The B1agonist [des-Arg10]-kallidin activates transcription factor NF-kappaB and induces homologous upregulation of the bradykinin B1-receptor in cultured human lung fibroblasts[END_REF]. In addition, down regulation of CCL2 and CCL7 expression was observed by inhibition of NF-κB [START_REF] Cuaz-Perolin | Antiinflammatory and antiatherogenic effects of the NF-kappaB inhibitor acetyl-11-keto-beta-boswellic acid in LPS-challenged ApoE-/-mice[END_REF][START_REF] Wuyts | Involvement of p38 MAPK, JNK, p42/p44 ERK and NF-kappaB in IL-1beta-induced chemokine release in human airway smooth muscle cells[END_REF]. These results thus suggest that direct stimulation of CTGF and chemokine expression by the B1R could be involved in the pro-inflammatory and pro-fibrotic actions of the B1R, although the identity of the cell type involved in this effect remains to be determined. Almost any renal cell type can express functional chemokines in vivo [START_REF] Segerer | Chemokines, chemokine receptors, and renal disease: from basic science to pathophysiologic and therapeutic studies[END_REF] and the B1R is potentially expressed on infiltrating macrophages and also on resident renal cells in the inflamed kidney. We were not able to perform co-localization experiments, as antibodies against the mouse B1R are not commercially available. Therefore, we carried out bone marrow transplantation experiments to clarify whether decreased expression of CTGF and chemokines induced by B1Ra treatment could be mediated by infiltrating inflammatory or resident renal cells, or both. We performed UUO in wild-type mice reconstituted with either B1+/+ bone marrow or bone marrow lacking the B1R gene (B1-/-). Eight days after UUO, there was no difference between mice with B1+/+ or B1-/-bone marrow, showing that the absence of the B1R on monocytes/macrophages was without effect on inflammatory cell infiltration, chemokine expression and interstitial fibrosis. Thus, although the B1R is also expressed on monocytes/macrophages [START_REF] Leeb-Lundberg | International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences[END_REF], these apparently did not participate in the anti-inflammatory and anti-fibrotic effects of B1R antagonism. These data strongly suggested that the beneficial effect of the B1R blockade was mainly mediated by resident renal cells.

As recently reviewed, a number of experimental data showed that the progression of renal fibrosis is a reversible process [START_REF] Chatziantoniou | Is kidney injury a reversible process?[END_REF]. Our data showed that, in UUO-induced fibrosis, delayed B1Ra administration leads to reversion of fibrosis. However, we must remain cautious in our interpretation because the UUO model is an accelerated model of interstitial fibrosis. We are currently validating these data on chronic kidney disease models where interstitial fibrosis appears more progressively.

Because of its inducible character, mainly localized at the site of inflammation, it has been suggested that blocking the B1R will be potentially without significant side effects. This has drawn attention to the B1R as a new therapeutic target for the treatment of pathologies related to chronic inflammation such as airway inflammation, diabetic neuropathy, arthritis and chronic and neuropathic pain [START_REF] Campos | Non-peptide antagonists for kinin B1 receptors: new insights into their therapeutic potential for the management of inflammation and pain[END_REF]. An additional advantage over other potential antifibrotic agents is that the B1R antagonist is already orally available (24).

Because failure of many organs (lung [START_REF] Meltzer | Idiopathic pulmonary fibrosis[END_REF], liver (48), heart [START_REF] Diez | Mechanisms of disease: pathologic structural remodeling is more than adaptive hypertrophy in hypertensive heart disease[END_REF]) is often associated to the development of fibrosis, our study should be considered as a "proof of concept" of the therapeutic potential associated with B1R blockade. grants from Sanofi-Aventis. J. Duchene is supported by a Basic Science Fellowship of the Barts and the London Charity. P Carayon is an employee of Sanofi Aventis.

TECHNICAL HELP

We thank S Schaak for advice and help.

Primer sequences used for real-time PCR.

Figure 6

Absence of B1R in bone marrow-derived cells does not affect the development of fibrosis during UUO in wild-type mice. The successful reconstitution of wild-type mice with either wild-type (B1+/+) or B1 knockout (B1-/-) bone marrow was confirmed by counting the number of circulating hematopoietic cells (A) and by genotyping blood cells (B, upper panel) and peritoneal macrophages using PCR (B, lower panel). The effect of 8 days UUO in wildtype mice with either B1+/+ or B1-/-bone marrow (Bm) transplantation was assessed by measurement of CCL2 and CCL7 mRNA levels and by immuno-histological staining for F4/80 and collagen (sirius red). *p<0,05 versus sham operated mice (white bar). n=7/group.

Figure 1 B1R

 1 Figure 1 B1R mRNA expression is induced locally in the kidney by UUO. Kinetics of macrophage infiltration was quantified by immunohistological staining for F4/80 (A) and B1R mRNA expression was analyzed by real-time PCR expression (B) during UUO. B1R mRNA expression was quantified by real-time PCR in aorta, brain, mesenteric tissue, lung and controlateral kidney of mice submitted to 8 days UUO (grey bars) compared to sham operated mice (white bars) (C). *p<0.05 versus time 0. n= 10/group.

Figure 2

 2 Figure 2Genetic and pharmacological blockade of the B1R reduce UUO-induced renal inflammation and fibrosis. The effect of B1R knockout (B1-/-) (A, C, E) or B1R antagonist (B1Ra) treatment (B, D, F) after 8 days UUO was assessed by comparison to wild-type mice (B1+/+) or vehicle treated mice respectively. Collagen accumulation was quantified by sirius red staining (A, B), macrophage infiltration by anti-F4/80 immunochemistry (C, D) and myofibroblast appearance by anti-α-smooth muscle actin (α-SMA) immunochemistry (E, F). Pictures display representative areas of kidneys from vehicle-treated or B1Ra-treated, sham or obstructed mice. *p<0.05 versus sham. #p<0.05 versus UUO 8 days. n= 10/group.

Figure 3 UUO

 3 Figure 3

Figure 4

 4 Figure 4 Delayed administration of B1R antagonist blunts UUO-induced chemokine and cytokine overexpression. We analyzed mRNA expression of the profibrotic cytokines (TGFβ, CTGF) and chemokines (CCL2, CCL7) by real-time PCR during UUO in vehicle-treated mice (open bars) or in B1R antagonist (B1Ra) treated mice (filled bars). *p<0.05 versus sham operated mice. #p<0.05 versus UUO 8 days vehicle-treated mice. n= 10/group.

Figure 5 B1R

 5 Figure 5B1R stimulation induces CCL2, CCL7 and CTGF overexpression in HEK293 cells. CCL2, CCL7 and CTGF mRNA expression analysis by real-time PCR in HEK293T cells transfected with GFP (pGFP) or human B1 receptor (phB1R). Cells were pretreated with B1R antagonist (B1Ra) (des-Arg 10 , Leu 9 kallidin 10 -6 M) for 15 min and then treated with B1 receptor agonist (B1R ago) (des-Arg 10 kallidin 10 -6 M) for 4 hours. *p<0.05 versus unstimulated cells. #p<0.05 versus B1R agonist alone. n=3.

ACKNOWLEDGMENTS

This study was mainly funded by INSERM and University Paul Sabatier Toulouse III and by