Romaric Larchera

Anne-Sophie Bargnouxb

Stephanie Badioub

Noemie Besnardc

Vincent Brunotc

Delphine Daubinc

Laura Platonc

Racim Benomarc

Matthieu Amalricc

Anne-Marie Dupuyd

Kada Klouchec

Jean-Paul Cristolb

Acute kidney injury in critical COVID-19 patients: usefulness of urinary biomarkers and kidney proximal tubulopathy

Keywords: NGAL, TIMP-2, IGFBP7, NephroCheck, kidney proximal tubulopathy, Fanconi syndrome, proteinuria, AKI, covid-19, SARS-CoV-2

Tubular injury is the main cause of acute kidney injury (AKI) in critically ill COVID-19 patients. Proximal tubular dysfunction (PTD) and changes in urinary biomarkers, such as NGAL, •[IGFBP7]), could precede AKI. We conducted a prospective cohort study from 2020/03/09 to 2020/05/03, which consecutively included all COVID-19 patients who had at least one urinalysis, to assess the incidence of PTD and AKI, and the effectiveness of PTD, NGAL, and [TIMP-2]•[IGFBP7] in AKI and persistent AKI prediction using the area under the receiver operating characteristic curves (AUCs), Kaplan-Meier methodology (log-rank tests), and Cox models. Among the 60 patients admitted to the ICU with proven COVID-19 (median age: 63-year-old (interquartile range: IQR, 55-74), 45 males (75%), median simplified acute physiology score (SAPS) II: 34 (IQR, 22-47) and median BMI: 25.7 kg/m2 (IQR, 23.3-30.8)) analyzed, PTD was diagnosed in 29 patients (48%), AKI in 33 (55%) and persistent AKI in 20 (33%). Urinary NGAL had the highest AUC for AKI prediction: 0.635 (95%CI: 0.491-0.779) and persistent AKI prediction: 0.681 (95%CI: 0.535-0.826), as compared to PTD and [TIMP-2]•[IGFBP7] (AUCs <0.6). AKI was independently associated with higher SAPSII (HR = 1.04, 95%CI: 1.01-1.06, p = 0.005) and BMI (HR = 1.07, 95%CI: 1.00-1.14, p = 0.04) and persistent AKI with higher SAPSII (HR = 1.03, 95%CI: 1.00-1.06, p = 0.048) and nephrotoxic drug use (HR = 3.88, 95%CI: 1.20-12.5, p = 0.02). In conclusion, in critically ill COVID-19 patients, the incidence of PTD and AKI was relatively high. NGAL was the best urinary biomarker for predicting AKI, but only clinical severity was independently associated with its occurrence.

Patients and methods

Study design and settings

This single-center observational cohort study was conducted at the Intensive Care Medicine Department of Montpellier University Hospital. Investigators prospectively collected clin-ical and biological data for all consecutive critically ill COVID-19 patients admitted to the ICU from 9 March 2020 to 3 May 2020.

Patients were followed up until hospital discharge, then data were analyzed retrospectively.

Patients

All consecutive adult patients who were hospitalized during the study period were screened for ICU admission. SARS-CoV-2 pneumonia was confirmed by RT-PCR on nasopharyngeal swabs or bronchoalveolar lavage, combined with suggestive abnormalities on chest radiography or CT-scan. Patients aged <18-year-old or pregnant and those with suspected COVID-19 not confirmed by SARS-CoV-2 RT-PCR were excluded. Patients on maintenance dialysis, those with stage 4-5 chronic kidney disease [14] at admission, and those who did not undergo urinalysis at admission were also excluded.

Data collection

.

Introduction

Two years after the pandemic, coronavirus disease 2019 (COVID-19) has been reported to be responsible for approximately 18 million deaths worldwide [START_REF] Wang | Estimating excess mortality due to the COVID-19 pandemic: a systematic analysis of COVID-19-related mortality, 2020-21[END_REF]. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to asymptomatic or paucisymptomatic infections as well as severe or critical COVID-19. Among patients admitted to the intensive care unit (ICU) for COVID-19, acute kidney injury (AKI) is a prevalent complication (in approximately one-third of patients), associated with worse outcomes [START_REF] Sullivan | Acute kidney injury in patients hospitalized with COVID-19 from the ISARIC WHO CCP-UK study: a prospective, multicentre cohort study[END_REF][START_REF] Nadim | COVID-19-associated acute kidney injury: consensus report of the 25th acute disease quality initiative (ADQI) workgroup[END_REF][START_REF] Gabarre | Acute kidney injury in critically ill patients with COVID-19[END_REF].

However, the pathophysiology of COVID-19-associated AKI remains incompletely elucidated [START_REF] Nadim | COVID-19-associated acute kidney injury: consensus report of the 25th acute disease quality initiative (ADQI) workgroup[END_REF]. Several hypotheses have been proposed, including kidney tropism and direct damage by the virus, SARS-CoV-2 induced cytokine storm and/or coagulopathy, organ crosstalk, shock, nephrotoxic drugs, and high levels of positive end-expiratory pressure (PEEP) [START_REF] Nadim | COVID-19-associated acute kidney injury: consensus report of the 25th acute disease quality initiative (ADQI) workgroup[END_REF][START_REF] Gabarre | Acute kidney injury in critically ill patients with COVID-19[END_REF]. Although specific kidney diseases have been reported, such as collapsing glomerulopathy, acute tubular necrosis appears to be the predominant renal pathology [START_REF] Gabarre | Acute kidney injury in critically ill patients with COVID-19[END_REF]. Several studies have highlighted kidney proximal tubular dysfunction in COVID-19 patients [START_REF] Aroca-Martínez | Renal tubular dysfunction in COVID-19 patients[END_REF][START_REF] Werion | SARS-CoV-2 causes a specific dysfunction of the kidney proximal tubule[END_REF][START_REF] Kormann | Coronavirus disease 2019: acute fanconi syndrome precedes acute kidney injury[END_REF] and have suggested that it could precede AKI [START_REF] Kormann | Coronavirus disease 2019: acute fanconi syndrome precedes acute kidney injury[END_REF]. Thus, tubular dysfunction could play a critcal role by facilitating an early prediction of COVID-19-associated AKI, as a urinary biomarker of kidney damage or stress [START_REF] Zhou | Urinalysis, but not blood biochemistry, detects the early renal impairment in patients with COVID-19[END_REF]. Kidney damage biomarkers, such as urinary neutrophil gelatinase-associated lipocalin (NGAL) [START_REF] Xu | Elevated neutrophil gelatinase-associated lipocalin is associated with the se-verity of kidney injury and poor prognosis of patients with COVID-19[END_REF][START_REF] He | Incorporation of urinary neu-trophil gelatinase-associated lipocalin and computed tomography quantification to predict acute kidney inju-ry and inhospital death in COVID-19 patients[END_REF][START_REF] Menez | Prognostic significance of urinary biomarkers in patients hospitalized with COVID-19[END_REF][START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF], and kidney stress biomarkers, such as tissue inhibitor of metalloproteinases-2 and insulin-like growth factor binding protein 7 product ([TIMP-2]•[IGFBP7]) [START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF][START_REF] Husain-Syed | Acute kid-ney injury and urinary biomarkers in hospitalized pa-tients with coronavirus disease-2019[END_REF], have been reported, in a few studies, to be good predictors of AKI in COVID-19 patients. However, none of these studies has com-pared their diagnostic effectiveness.

This study aimed to assess the incidence of proximal tubular dysfunction and AKI in COVID-19 patients and the effectiveness of urinary biomarkers, including tubular param-eters, NGAL, and [TIMP-2]•[IGFBP7], in predicting COVID-19-associated AKI in ICU patients.

Blood and urine samples were collected upon ICU admission. Samples were immediately processed, except for urinary NGAL, TIMP-2, IGFBP7, and amino acids, which were further processed on urinary samples frozen at -20 C. Almost all chemical lab measurements, including the NGAL test, were performed on a Cobas 8000 instrument using the e802, c702, and c502 modules (Roche Diagnostics, Meylan, France). Plasma creatinine was measured by an isotope dilution mass spectrometry (IDMS) traceable enzymatic method on Cobas 8000 instrument.

Urine TIMP-2 and IGFBP7 levels were measured using a Vitros® 3600 Immunodiagnostic System (Ortho Clinical Diagnostics, Rochester, NY, USA) on urine samples of at least 100 μL. The TIMP-2 and IGFBP7 product ([TIMP-2]•[IGFBP7]), also known as NephroCheck® score, was calculated according to the formula: ([TIMP-2] × [IGFBP7])/1000. Amino acids in the urine samples were quantified by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on an Acquity UPLC system coupled to a XevoTQD (Waters Corporation, Milford, MA, USA). Ion-pairing chromatography was performed on a BEH C18 1.7 μm column (Waters®) at a flow rate of 0.65 mL/min using mobile phases containing tridecafluoroheptanoic acid (Sigma-Aldrich, St. ® Louis, Missouri, USA) and Sigma® calibrators. The amino acid concentration in the urine was normalized to urine creatinine (in mmol/L) and compared to the values of normal subjects (N = 80, without tubulopathy, or COVID-19).

The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula was used to estimate glomerular filtration rate (GFR) [START_REF] Levey | A new equation to estimate glomerular filtration rate[END_REF]. The renal phosphate threshold/GFR (TmP/GFR) ratio was calculated as follows [START_REF] Payne | Renal tubular reabsorption of phosphate (TmP/GFR): indications and interpretation[END_REF]: (i) calculation of the renal tubular reabsorption of phosphate (RTP) using the following formula: RTP = 1-[phosphate clearance/creatinine clearance] × 100, and (ii) if RTP ≤0.86: TmPi/GFR = RTP × serum phosphate (mmol/L) and RTP >0.86: TmPi/GFR = 0.3 × RTP/[1-(0.8 × RTP)] × serum phosphate (mmol/L).

Definitions

Proximal tubular dysfunction was defined as the association of at least two of the following six criteria:

• urinary protein/creatinine ratio >0.2 g/g with urinary albumin to protein ratio <0.5;

• amino acids in the urines;

• urinary levels of β2-microglobulin > 0.3 mg/L [START_REF] Portman | Use of beta 2 mi-croglobulin to diagnose tubulointerstitial renal lesions in children[END_REF];

• hypouricemia (serum uric acid level <220 μmol/L in male and <184 μmol/L in female) with inappropriate uricosuria (fractional excretion of uric acid >10%)

• hypophosphatemia (serum phosphate level <0.81 mmol/L) with inappropriate phosphaturia (fractional excretion of phosphate >20%) or renal phosphate leak (TmP/GFR <0.77 mmol/L [START_REF] Bijvoet | The renal tubular reabsorption of phosphate in thyrotoxicosis[END_REF]);

• normoglycemic glycosuria (glycosuria >0.15 g/L and glycemia <1.80 g/L).AKI was defined according to the 2012 Kidney Disease: Improving Global Outcomes (KDIGO) clinical practice guide-lines [START_REF] Group | KDIGO clinical practice guideline for acute kidney injury[END_REF].

All patients were classified on day seven after ICU admission.

Baseline serum creatinine level was defined as the usual value of serum creatinine level preceding admission within at least 7 days.

Persistent AKI was defined as a steady or higher AKI KDIGO stage and/or persistence of oliguria after day 2 of AKI onset [START_REF] Chawla | Acute kidney disease and renal recovery: consensus report of the acute disease quality initiative (ADQI) 16 workgroup[END_REF].

Outcomes

Hospital and ICU lengths of stay were recorded. Vital status at ICU and hospital discharge were collected, and mortality rates were calculated.

Statistical analysis

Categorical data were described as numbers and percentages and continuous data as medians with the 25th and 75th per-centiles (interquartile range: IQR). Patients were divided into two groups: those with and without AKI. Categorical variables were compared using the Chisquare or Fisher's exact test, and continuous variables were compared using Student's t-test or Wilcoxon's rank-sum test, as appropriate.

The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for AKI prediction were calculated, and receiver operating characteristic (ROC) curves were plotted for proximal tubular dysfunction, [TIMP-2]•[IGFBP7], and urinary NAGL. The area under the ROC curve (AUC) for AKI prediction was calculated. Optimal cutoff values were estimated by maximizing the Youden index. The same calculations were performed for biomarkers of persistent AKI.

The population was then divided into groups according to the cutoff value for each biomarker. Cumulative incidence curves of AKI and persistent AKI were obtained using the Kaplan-Meier method and compared using the log-rank test.

Factors associated with AKI (and persistent AKI) were assessed using univariate and multivariate Cox regression models. A conditional stepwise regression with 0.1 a critical pvalue in the univariate analysis for entry into the multivariable analysis was performed to select the most informative variables. In the case of interactions and correlations between the explanatory variables, only the most clinically relevant variables were included in the final model (for example, we found an interaction between norepinephrine requirements, invasive mechanical ventilation requirements, and SAPSII. Therefore, we included SAPS II in the model because it reflects both the severity of respiratory and hemodynamic failure). In the case of skewed distributions, biomarker values were log-transformed before modeling. The proportional hazard assumption was assessed by inspecting scaled Schoenfeld residuals. The results of the Cox regression models are reported as HR or aHR with 95%CI.

All tests were two-sided, and a p-value less than 0.05 was considered statistically significant. The R software version 4.2.0 (The R Foundation for Statistical Computing, Vienna, Austria) was used for the analyses.

Ethics

The Institutional Review Board of Montpellier University Hospital approved the study protocol (IRB-MTP_2020_05_202000482, date of approval: 20/05/2020) and waived the need for signed patient consent. The study protocol was registered at clinicaltri-als.gov (NCT04393428), and the study was conducted in accordance with the Declaration of Helsinki.

Results

Patients

Among 170 patients admitted to the ICU during the study period, 77 had a proven COVID-19, and 60 (45 males, 75%) were included in the study (Figure 1). The characteristics of the study population at ICU admission and during COVID-19 management are shown in Table 1. The median age and BMI were 63-year-old (IQR: 55-74) and 25.7 kg/m2 (IQR: 23.3, 30.8), respectively, and the median Charlson index was 2 (IQR: 1-4). Importantly, 9 (15%) patients were followed up for chronic kidney disease, and 23 (38%) received a nephrotoxic drug before or immediately after ICU admission.

During the first 24 h of ICU stay, the median SAPS II and worst PaO2/FiO2 were 34 (IQR: 22, 47) and 120 mmHg (IQR: 80, 172), respectively. All patients received oxygen support, and almost half (29 patients, 48%) were intubated and mechanically ventilated. More than half of the patients (n = 33, 55%) required vasopressors. All patients who required vasopressors were mechanically ventilated and received a low dose (<1 μg/kg/min) of norepinephrine, except for two with septic shock. The median daily urine output was 800 mL (IQR: 600-1100 mL).

At ICU admission, the median C-reactive protein (CRP) level and lymphocyte count were 136 mg/L (IQR: 67, 215) and 870 cells/mm3 (IQR: 570, 885), respectively. The median serum creatinine levels at admission and at baseline were 81 μmol/L (IQR: 59, 103) and 74 μmol/L (IQR: 57, 79), respectively.

Proximal tubular dysfunction was diagnosed in 29 COVID-19 patients (48%) upon ICU admission. Most patients had partial Fanconi syndrome, as only 3 (5%) presented with 6/6 features of proximal tubular dysfunction on urinalyses and 8 (13%) with 5. The median values of urinary NGAL and [TIMP-2]•[IGFBP7] were 56 ng/mL (IQR:50-88) and 0.73 (IQR:0.32, 1.59), respectively (Table 1).

Features of ICU admission urinalyses are summarized in Figure 2.

During the ICU stay, more than half of the patients (33 patients, 55%) developed AKI according to the 2012 KDIGO classification. Among them, 20 patients (33%) developed persistent AKI, KDIGO stage 1 in 2 patients (10%), stage 2 in 7 (35%), and stage 3 in 11 (55%); renal replacement therapy (RRT) was initiated in 6 patients (18%) (Figure 1). Interestingly, of the 27 patients without AKI, 11 had elevated NGAL, 11 had elevated [TIMP-2]-[IGFBP7] and 13 had PTD.

The risks for AKI and persistent AKI were estimated at 55.7% (confidence interval at 95%:95%CI: 41.0 -66.6) and 33.3% (95%CI: 20.3 -44.3), respectively (Figure 3).

Outcomes

The median lengths of stay in the ICU and hospital were 9 days (IQR: 6, 15) and 12 days (IQR: 8, 23), respectively. Thirty-three patients (22%) died during the hospital stay, and among those discharged alive, 5 patients (8%) still required RRT.

Urinalysis for AKI and persistent AKI prediction

At ICU admission, the AUCs for AKI prediction of NGAL, [TIMP-2]•[IGFBP7], and proximal tubular dysfunction were 0.619 (95%CI: 0.479 -0.759), 0.593 (95% CI: 0.445 -0.740), and 0.470 (95%CI: 0.325 -0.614), respectively. The AUCs for persistent AKI prediction for these three biomarkers were 0.681 (95%CI: 0.535 -0.826), 0.569 (95%CI: 0.405 -0.733), and 0.547 (95%CI: 0.395 -0.699), respectively. ROC curves are displayed in Figure 4, and Table 2 summarizes the effectiveness of the biomarkers of kidney stress, kidney damage, and proximal tubular dysfunction in predicting AKI and persistent AKI.

Urinary NGAL had the highest sensitivity (66.7%) and NPV (59.3%) for AKI prediction.

[TIMP-2]•[IGFBP7] had the highest PPV (67.7%), whereas the presence of four or more signs of proximal tubular dysfunction had the highest specificity (96.3%) for AKI prediction (Appendix A1).

In addition, proximal tubular dysfunction showed the highest sensitivity (85.0%) for persistent AKI prediction but the lowest specificity (33.5%). In contrast, NGAL showed the highest specificity (67.5%) and sensitivity (65%). The NPVs ranged from 76.7 to 85.7%, while all factors had low PPVs (≤50%) for predicting persistent AKI.

Interestingly, only urinary NGAL was able to discriminate between patients with AKI and persistent AKI on day 7 (p = 0.02 and p = 0.03, respectively) (Figure 5). On the contrary, the incidence of AKI events was not significantly different in patients with [TIMP-2]•[IGFBP7] ≥ 0.7 (p = 0.08 and p = 0.2), in those who presented with proximal tubular dysfunction (p = 0.7 and p = 0.6), or with a urine protein/creatinine ratio (UPCR) ≥50 mg/g (p = 0.4 and p = 0.6) (Appendices A2-A5).

Factors associated with AKI and persistent AKI

In the univariate analysis patients with AKI had higher value of BMI (hazard ratio: HR = 1.06, 95%CI: 1 -1.13, p = 0.034), baseline creatinine (HR = 1.02, 95%CI: 1.01 -1.03, p = 0.003), Charlson index (HR = 1.15, 95%CI: 1.03 -1.29, p = 0.011), and SAPS II (HR = 1.04, 95%CI: 1.03 -1.06, p < 0.001), and lower PaO2/FiO2 ratio (HR = 0.99, 95%CI: 0.982 -0.996, p = 0.002) and lymphocyte count (HR = 0.06, 95%CI: 0.01 -0.31, p < 0.001), and history of chronic kidney disease (HR = 3.99, 95%CI: 1.77 -8.99, p < 0.001). Moreover, AKI was associated with the need for invasive mechanical ventilation (HR = 4.09, 95%CI: 1.88 -8.88, p < 0.001) or norepinephrine (HR = 2.79, 95%CI: 1.76 -4.42, p < 0.001). Importantly, NGAL levels ≥53 ng/mL were associated with AKI (HR = 2.27, 95%CI: 1.10 -4.69, p = 0.027), whereas [TIMP-2]•[IGFBP7] (HR = 1.89, 95%CI: 0.927 -3.85, p = 0.08) were not.

The variables associated with persistent AKI in the univariate analysis were similar to those associated with AKI. However, we found that AKI was independently associated only with higher SAPS II (adjusted HR: aHR = 1.04, 95%CI: 1.01-1.06, p = 0.005) and higher BMI (aHR = 1.07, 95%CI: 1.00-1.14, p = 0.04) by multivariable analysis.

In the multivariable analysis, only higher SAPS II (aHR = 1.03, 95%CI: 1.00-1.06, p = 0.048) and nephrotoxic treatment before ICU admission (aHR = 3.88, 95%CI: 1.20-12.5, p = 0.02) were associated with persistent AKI.

The results of univariate and multivariable analyses are presented in Table 3.

Discussion

Here, we report the results of a cohort study including 60 critically ill patients with COVID-19, showing that 48% presented with proximal tubular dysfunction, 55% had AKI, and 33% had persistent AKI. In patients with COVID-19, urinary NGAL on ICU admission had a better effectiveness in predicting AKI and persistent AKI on day 7 than [TIMP-2]•[IGFBP7] and proximal tubular dysfunction. However, multivariable analyses showed that only increased SAPS II and BMI and use of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB) were independently associated with AKI or persistent AKI.

At the beginning of the pandemic, proteinuria, hematuria, and leukocyturia have been investigated as biomarkers for the early recognition of COVID-19-associated AKI [8, p.19], and proteinuria has been reported to be the most interesting [START_REF] Mohamed | Proteinuria in COVID-19[END_REF][START_REF] Chaudhri | Association of proteinuria and hematuria with acute kidney injury and mortality in hospitalized patients with COVID-19[END_REF][START_REF] Chan | AKI in hospitalized patients with COVID-19[END_REF]. However, its effectiveness in diagnosing COVID-19-associated AKI remains controversial because most patients infected with SARS-CoV-2 have proteinuria [START_REF] Mohamed | Proteinuria in COVID-19[END_REF][START_REF] Flammia | Renal surgery for kidney cancer: is preoperative proteinuria a predictor of functional and survival outcomes after sur-gery? A systematic review of the literature[END_REF]. A specific kidney proximal tubular dysfunction (Fanconi syndrome) associated with SARS-CoV-2 infection has been reported to be the primum movens of COVID-19 associated AKI [START_REF] Kormann | Coronavirus disease 2019: acute fanconi syndrome precedes acute kidney injury[END_REF] and is associated with poor prognosis [START_REF] Werion | SARS-CoV-2 causes a specific dysfunction of the kidney proximal tubule[END_REF]. In accordance with previous studies [START_REF] Aroca-Martínez | Renal tubular dysfunction in COVID-19 patients[END_REF][START_REF] Werion | SARS-CoV-2 causes a specific dysfunction of the kidney proximal tubule[END_REF][START_REF] Kormann | Coronavirus disease 2019: acute fanconi syndrome precedes acute kidney injury[END_REF], approximately half of our patients had Fanconi syndrome; however, its occurrence poorly predicted AKI and persistent AKI. Like HIV, SARS-CoV-2 attacks the kidney in different ways [START_REF] Reynes | Tubular and glomerular proteinuria in HIVinfected adults with estimated glomerular filtration rate ≥60 ml/min per 1.73 m2[END_REF]. Different patterns have been reported: (i) nephrotic proteinuria suggestive of COVID-19-associated nephropathy (COVAN), (ii) acute glomerular injury with non-nephrotic proteinuria, and (iii) acute tubular injury associated with tubular proteinuria related to proximal tubulopathy [START_REF] Gabarre | Acute kidney injury in critically ill patients with COVID-19[END_REF][START_REF] Mohamed | Proteinuria in COVID-19[END_REF]. Alterations in renal function may be associated with any of these clinical profiles.

The use of kidney stress and damage biomarkers as predictors of COVID-19-associated AKI has been suggested previously [START_REF] Xu | Elevated neutrophil gelatinase-associated lipocalin is associated with the se-verity of kidney injury and poor prognosis of patients with COVID-19[END_REF][START_REF] He | Incorporation of urinary neu-trophil gelatinase-associated lipocalin and computed tomography quantification to predict acute kidney inju-ry and inhospital death in COVID-19 patients[END_REF][START_REF] Menez | Prognostic significance of urinary biomarkers in patients hospitalized with COVID-19[END_REF][START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF][START_REF] Husain-Syed | Acute kid-ney injury and urinary biomarkers in hospitalized pa-tients with coronavirus disease-2019[END_REF]. This increase may reflect acute tubule injury that precedes loss of renal function [START_REF] Kellum | Diagnostic criteria for acute kidney injury: present and future[END_REF]. Two studies [START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF][START_REF] Husain-Syed | Acute kid-ney injury and urinary biomarkers in hospitalized pa-tients with coronavirus disease-2019[END_REF] assessing [TIMP-2]•[IGFBP7] for the early recognition of COVID-19-associated AKI reported conflicting results. Husain-Syed et al. [START_REF] Husain-Syed | Acute kid-ney injury and urinary biomarkers in hospitalized pa-tients with coronavirus disease-2019[END_REF] found that higher levels of [TIMP-2]•[IGFBP7] were associated with worse outcomes (need for RRT and death). Casas-Aparicio et al. [START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF] have reported the effectiveness of [TIMP-2]•[IGFBP7] in predicting COVID-19-associated AKI. However, our results demonstrated the effectiveness of NGAL in predicting early COVID-19-associated AKI [START_REF] Xu | Elevated neutrophil gelatinase-associated lipocalin is associated with the se-verity of kidney injury and poor prognosis of patients with COVID-19[END_REF][START_REF] He | Incorporation of urinary neu-trophil gelatinase-associated lipocalin and computed tomography quantification to predict acute kidney inju-ry and inhospital death in COVID-19 patients[END_REF][START_REF] Menez | Prognostic significance of urinary biomarkers in patients hospitalized with COVID-19[END_REF][START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF] and that NGAL outperformed [TIMP-2]•[IGFBP7] [START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF]. NGAL has also been reported as a prognostic factor for RRT requirement and death in COVID-19 patients [START_REF] Menez | Prognostic significance of urinary biomarkers in patients hospitalized with COVID-19[END_REF][START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF][START_REF] Bezerra | Urinary tubular biomarkers as predictors of death in critically ill patients with COVID-19[END_REF]. Of note, our NGAL threshold of 53 ng/mL is in line with those reported previously (45 ng/mL) using an enzyme-linked immunosorbent assay (ELISA) [START_REF] Xu | Elevated neutrophil gelatinase-associated lipocalin is associated with the se-verity of kidney injury and poor prognosis of patients with COVID-19[END_REF][START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF], whereas thresholds of 118.8 or 150 ng/mL have been reported using NGAL dipstick [START_REF] Xu | Elevated neutrophil gelatinase-associated lipocalin is associated with the se-verity of kidney injury and poor prognosis of patients with COVID-19[END_REF][START_REF] He | Incorporation of urinary neu-trophil gelatinase-associated lipocalin and computed tomography quantification to predict acute kidney inju-ry and inhospital death in COVID-19 patients[END_REF][START_REF] Bezerra | Urinary tubular biomarkers as predictors of death in critically ill patients with COVID-19[END_REF].

In our patients, the occurrence of AKI was independently associated only with BMI [START_REF] Chetboun | BMI and pneumonia outcomes in critically ill COVID-19 patients: an international multicenter study[END_REF], ACEI or ARB use [START_REF] Cai | Risk factors for acute kidney injury in adult patients with COVID-19: a systematic review and meta-analysis[END_REF][START_REF] Oussalah | Long-term ACE inhibitor/ARB use is associated with severe renal dys-function and acute kidney injury in patients with severe COVID-19: results from a referral center cohort in the northeast of France[END_REF], and most importantly with COVID-19 severity [START_REF] Redant | Severe acute respiratory syndrome coronavirus-2associated acute kidney injury: a narrative review focused upon pathophysiology[END_REF][START_REF] Ottolina | Acute kidney in-jury (AKI) in patients with covid-19 infection is associated with ventilatory management with elevated positive endexpiratory pressure (PEEP)[END_REF] whereas elevation of tubular injury biomarkers was associated with AKI only in univariate analysis. Indeed, NGAL elevation is most likely related to the activation of the immune system [START_REF] Serwin | Renal and inflammation markersrenalase, cystatin C, and NGAL levels in asymptomatic and symptomatic SARS-CoV-2 infection in a one-month follow-up study[END_REF] explaining that urinary biomarkers, including NGAL, are not independently associated with AKI [START_REF] Xu | Elevated neutrophil gelatinase-associated lipocalin is associated with the se-verity of kidney injury and poor prognosis of patients with COVID-19[END_REF][START_REF] He | Incorporation of urinary neu-trophil gelatinase-associated lipocalin and computed tomography quantification to predict acute kidney inju-ry and inhospital death in COVID-19 patients[END_REF][START_REF] Menez | Prognostic significance of urinary biomarkers in patients hospitalized with COVID-19[END_REF][START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF][START_REF] Bezerra | Urinary tubular biomarkers as predictors of death in critically ill patients with COVID-19[END_REF]. In the same vein, there is an association between elevated NGAL and mortality in COVID-19 patients, confirming its link with inflammation and severity [START_REF] Xu | Elevated neutrophil gelatinase-associated lipocalin is associated with the se-verity of kidney injury and poor prognosis of patients with COVID-19[END_REF][START_REF] He | Incorporation of urinary neu-trophil gelatinase-associated lipocalin and computed tomography quantification to predict acute kidney inju-ry and inhospital death in COVID-19 patients[END_REF] and also the role of inflammation in the development of AKI [START_REF] Saygili | Clinical and sub-clinical acute kidney injury in children with mild-to-moderate COVID-19[END_REF][START_REF] Kerget | Evaluation of the relationship between KIM-1 and suPAR levels and clinical se-verity in COVID-19 patients: a different perspective on suPAR[END_REF].

Our observations corroborate the existence of a continuum in the spectrum of COVID-19 associated AKI [START_REF] Gabarre | Acute kidney injury in critically ill patients with COVID-19[END_REF][START_REF] Mohamed | Proteinuria in COVID-19[END_REF][START_REF] Luther | COVID-19 patients in intensive care develop predominantly oliguric acute kidney injury[END_REF]. Indeed, some patients may present only subclinical AKI syndrome characterized by significant tubule injury (particularly, changes in the protein endocytosis machinery in proximal tubule epithelial cells) without changes in the glomerular filtration rate [START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF][START_REF] Saygili | Clinical and sub-clinical acute kidney injury in children with mild-to-moderate COVID-19[END_REF][START_REF] Silva-Aguiar | Subclinical acute kidney injury in COVID-19: possible mechanisms and future perspectives[END_REF][START_REF] Sun | Subclinical acute kidney injury in COVID-19 patients: a retrospective cohort study[END_REF] while others may exhibit AKI related to various mechanisms [START_REF] Gabarre | Acute kidney injury in critically ill patients with COVID-19[END_REF][START_REF] Mohamed | Proteinuria in COVID-19[END_REF][START_REF] Luther | COVID-19 patients in intensive care develop predominantly oliguric acute kidney injury[END_REF]. This approach may explain why we found that proximal tubular dysfunction was a sensitive marker for detecting persistent AKI, but its specificity was relatively low and, conversely, it appeared less sensitive for identifying transient AKI. In contrast, biomarkers of kidney damage, particularly NGAL, more clearly signified renal aggression, whatever the cause, likely to lead to AKI, and consequently its performance in predicting such cases was better. Thus, our results and those of others [START_REF] Casas-Aparicio | Role of urinary kidney stress biomarkers for early recognition of subclinical acute kid-ney injury in critically ill COVID-19 patients[END_REF][START_REF] Saygili | Clinical and sub-clinical acute kidney injury in children with mild-to-moderate COVID-19[END_REF][START_REF] Silva-Aguiar | Subclinical acute kidney injury in COVID-19: possible mechanisms and future perspectives[END_REF][START_REF] Sun | Subclinical acute kidney injury in COVID-19 patients: a retrospective cohort study[END_REF] suggest to use urinary biomarkers to better define renal involvement in COVID-19, reflecting the heterogeneity in the clinical presentation and severity and including subclinical AKI which concerned almost 20% of our cohort.

Our study had some limitations. First, our conclusions were limited by the relatively small cohort size. Despite its prospective design, urinalyses were performed at the physician's discretion, and urine samples were not collected from 14 patients. Moreover, the monocentric design of the study could limit the generalizability of the results. Second, our data were collected during the first wave of the pandemic; consequently, our results should not be generalized to patients infected with novel SARS-CoV-2 variants [START_REF] Sullivan | Acute kidney injury in patients hospitalized with COVID-19 from the ISARIC WHO CCP-UK study: a prospective, multicentre cohort study[END_REF]. Finally, interesting factors such as biomarkers of tubular function, Cystatin C (CysC) [START_REF] Serwin | Renal and inflammation markersrenalase, cystatin C, and NGAL levels in asymptomatic and symptomatic SARS-CoV-2 infection in a one-month follow-up study[END_REF][START_REF] Shakked | Early prediction of COVID-19associated acute kidney injury: are serum NGAL and serum cystatin C levels better than serum creatinine?[END_REF], liver-type fatty acid-binding protein (L-FABP) and α1-Microglobulin (α1M) [START_REF] Su | The role of kidney injury bio-markers in COVID-19[END_REF] were not included in our analyses. Similarly, the combination of biomarkers with quantification of lung damage on chest CT [START_REF] He | Incorporation of urinary neu-trophil gelatinase-associated lipocalin and computed tomography quantification to predict acute kidney inju-ry and inhospital death in COVID-19 patients[END_REF] or with Doppler measurement of the renal resistive index [START_REF] Cruz | Renal resistive index as a predictor of acute kidney injury and mortality in COVID-19 critically ill patients[END_REF][START_REF] Garnier | Reversibility of acute kidney injury in medical ICU patients: predictability performance of urinary tissue inhibitor of metalloproteinase-2 x insulin-like growth factor-binding protein 7 and renal resistive index[END_REF] are promising tools that were not investigated in our study. They may help build more informative models for COVID-19-associated AKI, which is a complex phenomenon involving many mechanisms [START_REF] Redant | Severe acute respiratory syndrome coronavirus-2associated acute kidney injury: a narrative review focused upon pathophysiology[END_REF].

In conclusion, kidney proximal tubular dysfunction, AKI, and persistent AKI are common complications in critically ill COVID-19 patients. Disease severity, higher BMI, and use of ACEI or ARBs before ICU admission were independently associated with COVID-19associated AKI. Comparing various uri-nary biomarkers, including proximal tubular dysfunction and [TIMP-2]•[IGFBP7], NGAL had the best predictive effectiveness for AKI and persistent AKI associated with COVID-19.Early detection of COVID-19-associated AKI cannot be approached in a 'one-size-fits-all' manner because of its complex pathophysiology. However, urinary NGAL levels may help physicians diagnose AKI early in combination with clinical information. Further larger multicenter studies are needed to confirm our findings.

Acknowledgments

The authors acknowledge Nils Kuster, an esteemed colleague who passed away on 22 December 2020, during the completion of this work, for his teaching and assistance in statistics.

Funding

The author(s) reported there is no funding associated with the work featured in this article.

Data availability statement

The authors consented to sharing the collected data with others. The raw data supporting the conclusions of this article will be made available by the authors without undue res-ervation. Data will be available immediately after the main publication and indefinitely thereafter.

Disclosure statement

This research received no external funding; however, Roche Diagnostics and Ortho Clinical Diagnostics provided the reactive, calibrator, and quality control materials for this study. These laboratories had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results. No potential conflict of interest was reported by the author(s).


Marc Guyon

Laure Meunier

DIFFERENT KINDS OF REPAIRS AND MAINTENANCE ON A 2 ND CENTURY AD WRECK: THE LYON

Keywords: Caulking, Gallo-Roman, maintenance, shipwreck, repair Résumé Calfatage, gallo-romain, entretien, naufrage, réparation

Different kinds of repairs and maintenance on a 2nd century ad wreck: the Lyon Saint-Georges 4 barge

From October 2002 to June 2004, 16 wrecks dated to between the 1 st century AD and the 18 th century were excavated on the ancient right bank of the River Saône in Lyon, France. The Institute for Preventive Archaeological Research (INRAP) at the request of the Ministry of Culture carried out the excavations of this harbour site, located on the future Parc Saint Georges' car park. Grégoire Ayala was the manager of this huge archaeological intervention and Marc Guyon was responsible for the excavation of the wrecks. Operations took place within a cofferdam following traditional methods of terrestrial archaeology on an area of approximately 4000 m 2 , to a depth of 10 m [START_REF] Ayala | Lyon Saint-Georges (France): la fouille d'une berge de Saône[END_REF].

The Lyon Saint-Georges 4 (LSG4) wreck belongs to a set of six Gallo-Roman boats discovered in 2004. A complete architectural study was conducted and published in 2010 [START_REF] Rieth | Epave EP[END_REF], but lack of time and the impossibility of dismantling prevented an in-depth survey. The three most complete wrecks were removed and stored in a lake near Lyon to await project funding. This occurred in 2013 and would ultimately lead to the launch of the LSG4 project, which was designed to run for several years and involved: the raising of the wreck from the water (as with AR3); the consolidation of the waterlogged wood; restoration of the ensemble; the creation of a suitable presentation environment; and the conduct of a complete archaeological study. This large cargo boat could carry a load of up to 53 tons. A full reconstitution of this boat would reach 28 m in length and nearly 5 m in width, however only 15 m in length are preserved. The other parts of the wreck are still under earth, protected by the houses and streets of Old Lyon.

Eight weeks were necessary to remove LSG4 from the car park, but five months were needed to conduct a complete dismantling and to prepare it for PEG consolidation once the sections were raised and transported to the ARC-Nucléart laboratory in Grenoble, France.

The second phase of the conservation campaign began in January 2014. The dismantling took three weeks per section. Elements were stored on shelves by architectural type (flat bottom, sides and ribs) and consolidated in a PEG 2000 solution for at least eight months. The various elements were then freeze-dried using two freeze-dryers. It took between two to six weeks for a cycle and 18 months for the whole shipwreck. Since the end of 2016, it has been stored at the ARC-Nucleart lab, waiting for an exhibition space in the Lugdunum museum to be ready in 2024. Time was thus available for further analysis of the various materials and data recovered leading to new archaeo logical information.

A COLLABORATION OF CONSERVATORS AND ARCHAEOLOGISTS IN COORDINATED RESEARCH TO RECOVER MORE ARCHAEOLOGICAL DATA

Complete coordinated dismantling is a real opportunity for the acquisition of all kinds of archaeological data, only 25 % of which is available when the wreck is not dismantled. (This estimate is based upon the notion of a section still on the excavation site and studied over three days without special equipment and only partial dismantling, versus a complete dismantling with the intervention of conservators). The removal of all frames gave access to the flat bottom and led to a completion of the original plan that had been recorded during discovery in 2003. This initial plan had left many questions unanswered concerning the assembly of planks in the same strake, and had provided no view of the underneath. Dismantling allowed among other things the drawing and recording of all the surfaces of the constituent elements and both sides of the flat bottom once cleaned.

REPAIRS AND MAINTENANCE

After dismantling it became apparent that two planks of the flat bottom had been repaired (Guyon forthcoming). They cracked during installation, and so the carpenters chose to repair and consolidate them by transverse nailing (fig. 1) on the plank BS15, section 4. Another repair involved the replacement of a frame timber. This is visible because of the presence of a lot of small pegs, although this was very difficult to perceive before the wood was freeze-dried. We observed oblique alignments of three pegs (fig. 2), which exactly reproduced the alignment of nailing on the upper surface of the flat bottom under VP7. Once the old frame had been removed, the carpenters filled the holes left by the nails with the pegs before reattaching a new frame. During the archaeological excavations, no pegs were seen on the surface of the flat bottom: it was only during dismantling that these pegs were noticed and recorded. A further search for pegs was carried out after freeze-drying because during this operation a slight shrinking of the wood takes place making the pegs more easily visible.

Another repair can be seen involving a frame timber. A hole had appeared in plank BS15, section 4, of the flat bottom because of a knot being removed, hence causing a potential leak. This hole was filled with caulking textiles held in place by a small wooden plate (fig. 3) [START_REF] Rieth | Epave EP[END_REF].

Another repair was made using a piece of wood embedded in a frame timber. This was first thought to be a waterproofing repair but as nothing was visible on the flat bottom after its removal, the floor timber must have had a fault at this point and the carpenters preferred to remove it during construction. Anyway, this rectangular piece is not from the same wood.

A major repair took place at the front of the boat. Several lead strips, originally from ancient piping, are present [START_REF] Rieth | Epave EP[END_REF]). After removal of these lead strips, we saw that the widest strake had been repaired with many softwood wedges. This repair might have been as a result of operational wear on the plank, which is about 70 cm wide and had split naturally in its centre, which includes the middle of the tree, a fragile area from the outset. Alternatively the repair may have been necessary after a frontal impact, requiring a serious intervention including the cutting of many frames.

At least nine wedges were found in the large crack in the central strake. Other wedges were present but have since disappeared because this part of the boat experienced a number of problems, particularly during its removal from the excavation site when parts of the flat bottom cracked during extraction. Once the wedges had been inserted, in order to make the repair as waterproof as possible, cloth coated with pitch was placed over the wedges before laying the lead strips. We clearly distinguished the weave of the textile used impressed in negative on the underside of the lead plate.

Another repair filled a gap due to the missing part of the bilge, with a knee having a protuberance forming a heel on its lower face. Thanks to the dismantling it was also possible to observe manufacturing marks from a nail header on the underside of the nail heads.

Similar observations have also been made on various shipwrecks, in particular on the Chalon-sur-Saône wreck (France), the Bevaix barge (Swizerland) and the Arles-Rhône 3 (France). These marks are not exclusive to nails for shipbuilding, but are also found on Roman shoe nails, for example. They could be interpreted as motifs recording the production of a particular blacksmith or blacksmith's workshop.

CAULKING

The waterproofing of the boat was made with textiles impregnated with pitch. During the manufacture of the flat bottom planks, carpenters gave a particular shape to the profi le of the edge. This profi le, once the two planks were positioned against each other, formed an inverted Y, with a slight protrusion downwards to keep the caulking material in place without any other means to secure it. The initial profi le of the edge was axe cut. When replacing the waterproofi ng, the protrusion disappeared because, in order to remove it, a gouge had to be used to pull the pitch cloth out. Use of this tool eliminated the protrusion on the edge, thus allowing us to record precisely where the waterproofi ng had been replaced.

During the dismantling of the barge 26 m of caulking material was removed for further study. It mainly came from the space between the bilge and the side. At fi rst, it looked like a piece of wood, but after a more detailed examination the textiles could be seen. During our research we were faced with a new challenge as to how we might carefully unfold this material and how this new data could be exploited to answer historical questions.

Indeed, Roman nautical textiles are quite rare artefacts and previous studies have focused on the textile characteristics [START_REF] Médard | Les tissus associés à la construction des chalands gallo-romains du Parc Saint-Georges (Lyon), i er -iii e siècles ap[END_REF](Médard , 2010a(Médard , 2010b[START_REF] Médard | Les tissus associés à la construction des chalands gallo-romains du Parc Saint-Georges (Lyon), i er -iii e siècles ap[END_REF](Médard , 2014[START_REF] Médard | Les tissus associés à la construction des chalands gallo-romains du Parc Saint-Georges (Lyon), i er -iii e siècles ap[END_REF][START_REF] Schoefer | Textiles issus des fouilles des berges de la Saône, Saint-Georges, Lyon -2003[END_REF]). Our aim was to link these textiles with the barge, to understand how the caulker did his job, and which techniques were employed.

Until now, caulking textiles have been chemically unfolded by removing the pitch, leaving the cloth in a clean and fl at condition for archaeological study [START_REF] Plantec | Cinq ensembles de tissus gallo-romains de calfatage[END_REF][START_REF] Schoefer | Textiles issus des fouilles des berges de la Saône, Saint-Georges, Lyon -2003[END_REF]). Attempts to follow the previous procedure led to the destruction of the samples because of the very high pitch content, which had completely impregnated the textile. This unsatisfying state of affairs encouraged us to elaborate a new protocol, which began fi rst with the question of the link between the material and the barge, and secondly how to record these operations, since each stage, from beginning to end, is a defi nitive transition with no possible return. The functional specifi cations of this new protocol were: to be efficient and fast; to limit waste, costs and toxicity for the operator; and still respect the textile. Organic solvents attack fi bres, which become more fragile when treated this way and then tear at the folds. The new protocol used hot water to make the pitch plastic enough to allow the textile to be unfolded. With this technique, larger pieces of textile can be recovered (Meunier, Guyon forthcoming). Observation of impregnated textiles is more diffi cult because the pitch is not removed with the new method, but this technique allows for complementary recording.

NEW FINDS FOR ARCHAEOLOGY

During the unfolding of the textiles, a clue appeared to identify the waterproofi ng technique used on the Lyon Saint-Georges 4 shipwreck. Normally on the Rhône-Saône axis between the 1 st to the 3 rd century AD luting was used. This technique applies the waterproofi ng material in the course of boat construction, between the seams of timbers as they are being assembled. A very good example of this technique using pitched textiles, as on the Lyon Saint-Georges 4, has been recorded on the Arles-Rhône 3 shipwreck (Marlier 2014, p. 114). Marlier has shown that in this case large amounts of waterproofi ng material extend inside the bottom and that it can reach 6.5 cm outwith the seal between the bilge and the side. Excess waterproofi ng material can regularly be observed inside the boat. A lining was put over this to avoid the necessity of dealing with it. In the Arles-Rhône 3, the form of the seal was an inverted V with no protrusion. This can be explained by the use of the luting technique. Pitched textile was nailed during the construction, helping to keep it in place. This shipwreck, through her characteristics, date (1 st century AD) and location, can be compared to Lyon Saint-Georges 4. Here, no excess waterproofi ng material has been observed inside the shipwreck. The profi le of the seals between the planks, the inverted Y, stops before reaching the other, interior side. This means that waterproofi ng material simply cannot physically escape beyond the seal. Moreover, while unfolding the pitched textiles, no lack of textile has been observed in the pieces located in the upper part of the seal, meaning that the excess textile extending beyond the seam might have been cut. And the protrusion at the bottom of the edge helps to keep the waterproofi ng material in place. All this led to the hypothesis of caulking instead of luting on this barge. This hypothesis was confi rmed when we found an example from the area between the bilge and the side, in which a nail was trapped in the pitched textile (a second nail has also been found, showing this was not a unique example): the nail did not pierce the cloth (fi g. 4). The textile is wrapped around the nail, and the fact that the textile is not pinned by the nail signifies that the waterproofing material was placed after nailing. The textile does not run perfectly to the end of the gap, meaning that there was no way of checking that it did. This shows that the waterproofing was done after the complete construction of the boat, and this process is caulking.

Through exhaustive unfolding work, new perspectives and new data appeared. Nearly 3 m 2 of Roman textiles have been recovered from the Lyon Saint-Georges, the most from one boat in Europe. Some vegetal textiles are part of this material, but they represent a small part of the discoveries, which are mainly animal fibres (essentially sheep and goat: it is very difficult to differentiate them). As regards the vegetal fibres, only two types of weaving are present: tabby and twill. This latter has been identified as a technical fabric already known in the 3 rd century as a support for sewing metal elements to form Roman armour (personal communication with J.P. Wild, April 2018, about his finds on the Vindolanda site). These textiles are of course reused, and we tried to find some clue as to their previous utilisation and to understand if they were specifically chosen for this task.

Nautical textiles from ancient shipwreck have been found before now. The textiles coating the hulls of the Madrague de Giens shipwreck from the 1 st century BC (Tchernia, Pomey, Hesnard 1978, p. 85) and the Nemi ships from the 1 st century AD (Ucelli 1950, p. 265-268) were both woollen, and the thickness of the fibres used on the Nemi ships suggested a very high quality. However, no ancient source records this process. The fabrics we have found were tabby and were used between the planks of the hull and an external protective layer of lead sheeting. This indicates a specific production for that purpose, and a highly controlled quality. The use of wool in this location is a calculated choice, and after 2000 years it has come down to us, conserved by the resin that impregnated it. But these textiles were not the only ones used onboard. Sails and tarpaulin were also present, even if remains from ancient times are scarce. Black [START_REF] Black | Where have all the sails gone?[END_REF] assumed that since no piece of sail has been found in shipwreck excavations in the Mediterranean area, and since antique sources only indicate vegetal fibres as being used for that purpose, the material has not survived the ravages of time either underground or underwater. But with the tabbies of the Nemi and the Madrague de Giens, we have proof that nautical textiles have survived the ages, at least those made from wool when impregnated with resin, which are the same conditions as the pitched textiles used to waterproof the Lyon Saint-Georges' shipwrecks, as well as those of Arles-Rhône and Tolozan.

Other nautical textiles have been found, mainly in Egypt, where the climate is more favourable to their conservation. These includes examples from Berenike, 1 st to 5 th century AD [START_REF] Wild | Sails from the Roman Port of Berenike[END_REF], Quseir al-Qadim from 1 st to 2 nd century AD [START_REF] Handley | Textiles: a preliminary report[END_REF], Myos Hormos 1 st century or early 2 nd century AD [START_REF] Whitewright | Roman Rigging Material from the Red Sea Port of Myos Hormos[END_REF] and Thebes, from an Egyptian mummy conserved in Lyon, France, dated to the 2 nd century AD (Schoefer, Cotta, Beentjes1987;[START_REF] Rougé | La momie contenait-elle les fragments d'une voile?[END_REF][START_REF] Wild | Sails from the Roman Port of Berenike[END_REF]. All these textiles are from vegetal fibres. Those from Berenike were compared with those from the mummy by J.P. Wild, who identified several pieces as reinforcement strips, often seen as dividing lines on representations of traditional sails, forming a grid pattern where the horizontal widths of fabric intersect with brail lines, to which brail rings were sewn. Parts of these brail rings were found in Myos Hormos, Thebes and Quseir, thus helping to identify the strips on which they were sewn. In Quseir, the amount of material (2,455 fragments of Roman textiles) and the logic of utilisation by spatial location led Handley to assume that textiles remained in the place they were first used until eventual deposition, therefore, in a boat repair yard the cloth would come from a torn sail (Handley 2011, p. 323). This led us to reassess both new and old finds from the Lyon Saint-Georges shipwrecks. While making a catalogue of the nearly 150 pieces of textiles [START_REF] Meunier | Histoire des techniques d'étanchéification des bateaux fluviaux à coque en bois à l'époque romaine en Europe[END_REF], it quickly appeared that some analogies could be made with the ancient nautical textiles from Nemi. The finest textile recovered was the fourth sample, a weft-faced tabby, with a count of 21/2x35 (warp/weft) threads in each direction and made of fibres with a 16μ range. This is a technical fabric that has been specifically chosen for the purpose by the Romans with no random element. Fifteen similar examples can be found in the collection of Lyon Saint-Georges, with an average fibre diameter of maximum 20μ and the same tabby aspect and z/z spun (fig. 5). Figure 5 is an example of this warp-faced tabby, with regular bundles of threads clearly designed to attach something to the fabric. The number of threads is only available for a few textiles because the survey is still ongoing, but it is between 24-28/10-12 and 90-110/16. Thread diameters are between 0.1 to 0.3 mm. Six textiles with these characteristics are also present in the collection of Arles-Rhône 3 barge [START_REF] Médard | Les tissus associés à la construction des chalands gallo-romains du Parc Saint-Georges (Lyon), i er -iii e siècles ap[END_REF]. In Lyon, three strips can be compared to those from Quseir, Berenike and Thebes, with a width of 4 cm, 3.77 cm and 4.5 cm. Even if the materials used are different -wool for the French textiles and vegetal fibres for the Egyptian -the textiles look the same, are from the same nautical context, and were adapted from material available in their geographical area. Previous surveys by Médard on half of the Lyon Saint-Georges textiles [START_REF] Médard | Les tissus associés à la construction des chalands gallo-romains du Parc Saint-Georges (Lyon), i er -iii e siècles ap[END_REF](Médard , 2010a(Médard , 2010b(Médard , 2014[START_REF] Médard | Les tissus associés à la construction des chalands gallo-romains du Parc Saint-Georges (Lyon), i er -iii e siècles ap[END_REF] provided precious indications as to the technical aspect of the textiles and their fabrication, but made no real link with the nautical world. Médard's experience in the study of clothing and domestic fabrics has convinced her that there are no sewn remains from that particular domain. Another detail to be noticed is the impregnation of the textiles with ochre, which cannot be dissolved in water or organic solvents, in 45 examples (also visible in fig. 5). This cannot be due to the burial phase, since the soil consisted of sand, or from the storage lake, because then all the textiles would have been impregnated. This can be interpreted in the light of nautical practices as a well-known technique recorded since antiquity: sails used to be treated to improve their technical performance and to avoid rotting. This technique has been successfully applied to the woollen sails of the Skuldelev ship replicas [START_REF] Cooke | Viking woollen square-sails and fabric cover factor[END_REF] and also the sails of the Gyptis, a sailing replica of a 6 th -century BC Greek sewn boat [START_REF] Pomey | The Prôtis Project: the Gyptis sailing trials[END_REF], after two years because of the rotting of the sail provoked by dry/wet cycles (P. Poveda, personal commu nication). The textile is boiled in a mixture of seawater, grease and ochre, then rinsed in the sea and naturally dried. Similar aspects are present in the Arles-Rhône 3 collection. All these observations lead us to the conclusion that nautical textiles and certainly sailcloth were reused to waterproof the boats of Lyon and Arles (fig. 6). All of this survey is still ongoing and a doctoral thesis will begin next September focusing on the subject. A scientific publication on the new discoveries made during the conservation of Lyon Saint-Georges 4 is also in preparation.

While working on the newly found textiles, we had the opportunity to go and see the hemp sails of the Jeanne-Elisabeth, wrecked in 1755 near Palavas-les-Flots, France [START_REF] Bartoš | The Sail of the Swedish Merchantman Jeanne-Elisabeth[END_REF]. Comparison with some of the nautical textiles conserved in Lyon showed the same reinforcement strip, with an approximate width of 5 cm, and with the same sewing points as on the LSG8 shipwreck, dated to AD 55. They are different on each side of the strip: one with wide stitches, spaced and with a triple twisted thread, and invisible sewing on the other side (fig. 4). This would seem to imply that we have a technological continuity in sail manufacture between AD 55 and 1755, that is, over 17 centuries. Given that nautical techniques are known to be conservative, this could be possible.

RECORDING THE DIFFERENT STEPS

Several complementary techniques have been used to record the different steps of the protocol. First, photogrammetry cover age of the caulking material was made in order to retain a 3D memory of how it was before and to provide documentation for recording. Thereafter, a GoPro film was made while unfolding the textiles, and a map of the different pieces of textiles was drawn. This was a transitional stage, where observation could be conducted.

This stage was also recorded, and a relief map was made to understand how the textile was folded to be inserted between the planks. The folds are still visible and can be read by transferring the relief map into a monochrome picture. Through this technique, we compared the ways of folding the textiles. We wanted to know if it was always done in the same way or not, so that we might understand the actions involved in doing this task. We can also compare the textile type and the location of the samples to know if there is a link between the space to be filled and the textile used. This seemed to be correct, since the textiles used between the side and the flat bottom were often thicker and their preparation looked rougher, in that, the pieces used were less regular and transformed for this aim, than between the flat bottom planks, where the border was torn. These small pieces showed reduced dimensions to fit the space to be filled, in contrast with the space between the flat bottom and the side, which is wider. It seems logical to quickly fill this gap with a thicker textile, which would allow for faster work and which appears more suitable for this location.

All these textiles can provide exceptional information on Roman techniques of caulking, of course, but also about textiles themselves. The way the caulker operated is still not well understood, but several avenues are currently being explored. We already have more than 2 m 2 of unfolded textile, from nearly 6 linear metres of material, and there are still 23 m waiting to be done. There is a great potential here to make a reference set of Roman textiles. This is what is planned for the near future.

CONCLUSION

This work has shown the complementarity of, and the necessity of collaboration between the archaeologist and the conservator. The conservator's know-how can shed new light on material and add to the understanding of artefacts. Repairs are small but can provide a lot of information regarding the maintenance applied to the shipwreck during its active life. Through archaeodendrochronology of these remains, we hope to know how long this boat was in use. For textiles, the conservation process has allowed us to recover information about textiles used to waterproof barges on the Rhône, showing that they originated within a closed system proper to the shipyard itself, even if complementary observations still need to be conducted.

Textiles are more intimately linked to the artefact in which they feature, and are not just add-ons.

Fig. 1 :

 1 Fig. 1: A crack in plank BS15, section 4 (M. Guyon, Inrap).

Fig. 2 :

 2 Fig. 2: Pegs on the upper face of the fl at bottom under VP7 (M. Guyon, Inrap).

Fig. 3 :

 3 Fig. 3: Wooden plate on plank BS15, section 4 of the fl at bottom (M. Guyon, Inrap).

Fig. 4 :

 4 Fig. 4: Waterproofing material surrounds a nail (M. Guyon, Inrap).

Fig. 5 :

 5 Fig. 5: Warp-faced tabby from Lyon Saint-Georges 4, comparable to the Nemi's finest textiles (L. Meunier, IFIRES).

Fig. 6 :

 6 Fig. 6: Example of the material that helped connect these finds to a nautical context: two elements sewn together at right angles; two deliberately made circular holes of the same diameter; and a very thick (2.2 mm diam.) vegetalfibre S2z sewing thread.

ACKNOWLEDGMENTS

Thanks are due to the Lugdunum Museum (Lyon, France) for allowing the study of the textiles from the Lyon Saint-Georges shipwrecks, to Inrap (Institut National de Recherches Archéologiques Préventives) for archaeological support, to ARC Nucléart (Grenoble, France) for technical support and access to the textiles, and to LPA (Lyon Parc Auto) for funding.


Cornelis Jacominus Van Der Beek

Giancarlo Rizza

Marcin Konczykowski

Pierre Fertey

Isabelle Monnet

Ryuji Okazaki

Terukazu Kato

Kenishiro Hashimoto

M Shimozawa

Hiroaki Shishido

Disorder and flux pinning in superconducting pnictide single crystals

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