Urinary CXCL10 is Associated with Acute Kidney Injury and Sepsis, and Predicts Mortality in Critically Ill Children


 Background: Acute kidney injury (AKI) biomarkers are often susceptible to confounding factors, limiting their utility as a specific biomarker, in the prediction of AKI, especially in heterogeneous population. The urinary CXC motif chemokine 10 (uCXCL10), as an inflammatory mediator, has been proposed to be a biomarker for AKI in a specific setting. Whether uCXCL10 is associated with AKI and predicts AKI in critically ill patients remains unclear. The aims of the study were to investigate clinical variables potentially associated with uCXCL10 levels and determine the associations of uCXCL10 with AKI, sepsis and PICU mortality in critically ill children, as well as its predictive values of aforementioned issues. Methods: Urinary CXCL10 levels were serially measured in a heterogeneous group of children during the first week after pediatric intensive care unit (PICU) admission. AKI diagnosis was based on the criteria of Kidney Disease: Improving Global Outcomes with serum creatinine and urine output. Sepsis was diagnosed according to surviving sepsis campaign international guidelines for children. Mortality was defined as all-cause death occurring during the PICU stay.Results: Among 342 critically ill children, 52 (15.2%) developed AKI during the first week after PICU admission, and 132 (38.6%) were diagnosed as sepsis and 30 (12.3%) died during PICU stay. Both the initial and peak values of uCXCL10 remained independently associated with AKI with adjusted odds ratios (AORs) of 1.791 (P = 0.010) and 2.002 (P = 0.002), sepsis with AORs of 1.679 (P = 0.003) and 1.752 (P = 0.002), septic AKI with AORs of 3.281 (P <0.001) and 3.172 (P <0.001), and PICU mortality with AORs of 2.779 (P = 0.001) and 3.965 (P <0.001), respectively. The AUCs of the initial uCXCL10 for predicting AKI, sepsis, septic AKI, and PICU mortality were 0.63 (0.53-0.72), 0.62 (0.56-0.68), 0.75 (0.64-0.87), and 0.77 (0.68-0.86), respectively. The AUCs for prediction by using peak uCXCL10 were as follows: AKI 0.65 (0.56-0.75), sepsis 0.63 (0.57-0.69), septic AKI 0.76 (0.65-0.87), and PICU mortality 0.84 (0.76-0.91).Conclusions: Urinary CXCL10 is independently associated with AKI and sepsis, and may be a potential indicator of septic AKI and PICU mortality in critically ill children.


Introduction
Acute kidney injury (AKI) is a common complication in clinical settings and associated with high morbidity and mortality [1][2][3], especially in critically ill patients [4][5][6]. The clinical diagnosis of AKI is delayed and the development of therapeutic strategies to improve outcomes of AKI is greatly impeded.
Studies on AKI have focused on novel injury biomarkers, contributing to earlier diagnosis of AKI. Although many urinary biomarkers have been reported in adults and children [1,7,8], these early biomarkers have not been widely accepted and adopted to clinical practice, especially in children. Strong evidence is still needed to con rm that early urinary biomarkers of AKI have bene cial effects on the clinical outcomes in a general pediatric intensive care unit (PICU) population.
A growing body of evidence demonstrates that many chemokines play an active role in injury and in ammation during AKI development [9,10]. The CXC motif chemokine ligand 10 (CXCL10), also known as IFN-γ-inducible protein of 10 (IP-10), is released by in ammatory cells and various epithelial cells including renal tubular epithelial cells, and is upregulated in kidney tissue exposed to ischemic, nephrotoxic or in ammatory stress [11][12][13][14][15]. It is reported that CXCL10 levels in urine (uCXCL10) were elevated in adult patients with biopsy-proven acute tubular injury after renal transplantation [16] and patients undergoing cardiac surgery [17], and had an association with AKI after hematopoietic cell transplantation (HCT) in children [18]. In addition, a clinical cross-sectional study indicated that urinary CXCL10 were signi cantly higher in patients with documented AKI and performed well in differentiating between adult patients with and without AKI [19]. Taken together, however, researches on the relationship between uCXCL10 and AKI were relatively small and conducted in a speci c setting [16][17][18]. It still remains unclear whether uCXCL10 could be used to discriminate AKI of the critically ill patients, because that AKI biomarkers are often susceptible to confounding factors, limiting their utility as a speci c biomarker, in the prediction of AKI, especially in heterogeneous population. AKI is frequently associated with sepsis, and sepsis is the most common cause of AKI in critically ill patients, which may limit the use of uCXCL10 as a speci c biomarker of AKI in the critically ill patients [5]. Several studies have revealed that serum CXCL10 is early diagnosis of sepsis in adults [20,21]. There has been little previous evidence for the association between uCXCL10 and sepsis. In this study, we rstly investigated clinical variables potentially associated with uCXCL10 levels and determined the associations of uCXCL10 with AKI, sepsis and PICU mortality in critically ill children. Moreover, we evaluated whether uCXCL10 could serve as an early predictor of AKI, septic AKI or mortality in this population, independently of potential confounders.

Study design
This was a prospective study conducted from September to December 2016 and from December 2017 to January 2018 in the PICU and critically ill children aged between 1 month and 16 years were eligible for enrollment in the study. Exclusion criteria included: known congenital abnormality of the kidney and a failure to collect urine samples before discharge from the PICU or death. This study was approved by the Institutional Review Board at the Children's Hospital of Soochow University and performed in accordance with the Declaration of Helsinki. Informed parental consent was obtained on admission.

Clinical data collection
We reviewed and recorded demographic and clinical characteristics, including age, body weight, gender, clinical diagnosis, the use of mechanical ventilation (MV), hemo ltration, furosemide, antibiotics, steroids and inotrope during PICU stay. The diagnosis of multiple organ dysfunction syndrome (MODS), shock, and disseminated intravascular coagulation (DIC) that developed during the PICU stay were de ned clinically and diagnosed by the attending PICU physicians as the criteria described previously [22]. The duration of MV and the length of PICU stay were also recorded. In addition, the pediatric risk of mortality III (PRISM III) score was calculated within 24 h after PICU admission to assess the severity of illness in the critically ill children, as descripted previously [23].

Diagnosis of AKI
The diagnosis of pediatric AKI developed during the rst 7 days after PICU admission was based on the increase of serum creatinine (SCr) and/or the reduction of the urine output according to the criteria of Kidney Disease: Improving Global Outcome (KDIGO) [24]. The baseline SCr was de ned as the lowest level obtained within 3 months prior to PICU admission. If the baseline SCr value was not available, admission SCr was used. For the critically ill children with SCr ≥ 1.2 mg/dL (106.0 µmol/L) at PICU admission, the lowest value of SCr within 2 weeks during the PICU stay was considered as the baseline SCr, which was in accordance with our previous studies [23,25]. The SCr level was routinely measured daily during the rst week after PICU admission, followed by measurement every 48-72 hours during the PICU stay.

Diagnosis of sepsis
Sepsis was diagnosed by a chief physician and two attending physicians specialized in pediatric critical care medicine, who were completely blinded to the urinary biomarker results, retrospectively, in accordance with the international guidelines for management of septic shock and sepsis-associated organ dysfunction in children (Surviving Sepsis Campaign International Guidelines) [26].

Clinical outcomes
The primary outcome measure was PICU mortality de ned as all-cause death occurring during the PICU stay, including death resulting from withdrawal of therapy.

Measurement of urinary CXCL10
Urine samples were collected at predetermined time points for all children: within 24 h after PICU admission, followed by every 48-72 h within the rst week of the PICU stay. All samples were immediately stored at -80 °C until testing. The urine samples were centrifuged for 15 min at 1500 × g at 4 °C, and the supernatants were used for measurement of uCXCL10 and urine creatinine (uCr). The concentration of uCXCL10 was measured by means of ELISA (human CXCL10/IP-10 Duo Set ELISA DIP100, R&D Systems, USA). The minimum detectable level of CXCL10 was < 1.67 pg/mL, and the coe cient of variation of intra-assay and inter-assay were less than 5 and 10%, respectively. The uCXCL10 for analysis were adjusted by uCr (ng/mg uCr). The concentration of uCr was determined by sarcosine oxidase assay using an automatic biochemical analyzer (Hitachi 7600, Tokyo, Japan).

Statistical analysis
All data were analyzed using SPSS statistical software Version 22. The assumptions of normality and homogeneity of variance were checked. Continuous data were presented as median and interquartile range (IQR), as they were not-normally distributed. Categorical data were presented as counts and percentage. To compare differences among groups, the Mann-Whitney U test or Kruskal-Wallis H test was used for continuous variables and the Chi-Square test or Fisher's exact test was used for categoric variables, as appropriate. Spearman's correlation tests were performed to investigate correlations between uCXCL10 and clinical variables. To determine clinical variables potentially associated with the levels of uCXCL10, variables with P < 0.05 in Spearman's test were analyzed by the generalized linear model (GLM), and the beta coe cient (β), standard error (SE) and odds ratio (OR) with a 95% con dence interval (CI) were calculated. Continuous variables were log-transformed (base10) to meet approximate normality. Multicollinearity of variables was evaluated via tolerance and variance in ation factor (VIF), and tolerance ≤ 0.5 and the VIF value ≥ 2 indicated the presence of multicollinearity. Univariate and multivariate logistic regression analysis were used to evaluate the association between uCXCL10 and AKI, sepsis, septic AKI and mortality. The predictive values of uCXCL10 for AKI, sepsis, septic AKI and mortality were assessed by the receiver operating characteristic (ROC) curve and area under the curve (AUC). The sensitivity, speci city and Youden index were also calculated, and the optimal cutoff values were determined by the maximum Youden index. A 2-tailed P < 0.05 was considered signi cant.

Patient characteristics
A total of 351 critically ill children admitted to the PICU during the study period, 9 were excluded: including 1 child who was admitted to the PICU at age < 1 month, 4 children who had a failure in collecting urine samples before discharge from the PICU or death, and 4 children with multiple PICU admission during one hospitalization and only the last admission to PICU was used for analysis. Admission diagnoses included respiratory diseases (42.4%), neurological diseases (16.1%), hematologic/oncologic diseases (9.9%), gastrointestinal diseases (9.7%), trauma or postoperative acute care (8.8%) and others (13.1%) Among the 342 critically ill children, 52 (15.2%) developed AKI during the rst week after admission, including 23 with KDIGO stage 1, 14 with stage 2, and 15 with stage 3. One hundred and thirty-two (38.6%) critically ill children were diagnosed with sepsis during the PICU stay. A Comparison of demographic and clinical characteristics between patients with and without AKI or sepsis are presented in Table 1. The uCXCL10 level from the rst 24 h after admission to PICU was denoted as the initial uCXCL10. The highest uCXCL10 level among all collected samples during the rst 7 days after PICU admission was denoted the peak uCXCL10. The initial and the peak values of uCXCL10 were used for analysis.

Correlation of urinary CXCL10 levels with clinical variables
All variables in Table 1 were analyzed for association with uCXCL10. Spearman's correlation analysis showed that both the initial and peak levels of uCXCL10 were signi cantly correlated with age, body weight, PRISM III score, AKI, AKI stage, sepsis, MODS, Shock/DIC, MV, duration of MV, the use of furosemide, steroid and antibiotic (P < 0.05).
Variables with P < 0.05 under the Spearman's test were included in the GLM to con rm factors signi cantly associated with the uCXCL10 levels after checking the multicollinearity. As listed in Table 2, the initial uCXCL10 was independently associated with body weight (P = 0.020), PRISM III score (P = 0.007), sepsis (P = 0.026) and AKI stage (P = 0.047); the peak uCXCL10 was independently associated with body weight (P = 0.003), PRISM III score (P = 0.007), sepsis (P = 0.038), AKI stage (P = 0.006) and Shock/DIC (P = 0.046). Association between urinary CXCL10 and AKI The initial and peak uCXCL10 levels were signi cantly higher in the AKI than in the non-AKI ( Table 1). Comparisons of the initial and peak uCXCL10 levels among different stages of AKI in critically ill children are displayed in Additional le 2: Figure. S1 (a, b).

Association between urinary CXCL10 and sepsis
The sepsis group in critically ill children had higher initial and peak levels of uCXCL10 than the nonsepsis group ( Table 1). Comparisons of the initial and peak uCXCL10 levels between non-sepsis and sepsis in critically ill children are displayed in Additional le 3: Figure. 2 (a, b). To identify the association between uCXCL10 and sepsis, univariate and multivariate logistic regression analysis were performed. As shown in Table 3 and Figure.1 (a, b), the initial and peak uCXCL10 were both independently associated with sepsis (initial: AOR = 1.679, 95% CI 1.189-2.371, P = 0.003; peak: AOR = 1.752, 95% CI 1.231-2.494, P = 0.002) after adjustment for body weight, PRISM III score and AKI stage. The AUCs of the initial and peak uCXCL10 in predicting sepsis were 0.62 (0.56-0.68) and 0.63 (0.57-0.69), respectively.
Association between urinary CXCL10 and septic AKI Since the initial and peak uCXCL10 levels were both independently associated AKI and sepsis (Table 2), the relationship between uCXCL10 and septic AKI was further investigated. Children were divided into four groups according to the presence/absence of AKI and sepsis: sepsis/AKI (septic AKI, n = 22), non-AKI/sepsis (n = 110), AKI/non-sepsis (n = 30), non-sepsis/non-AKI (n = 180). As displayed in Figure. 2 (a, b), both the initial and peak uCXCL10 levels in septic AKI group were the highest. Comparison of clinical characteristics among four groups according to the status of AKI and sepsis are shown in Additional le 1: Table S1.
Univariate and multivariate logistic regression analysis were used to determine the association between uCXCL10 and septic AKI (n = 342). As shown in Table 3 and Figure.1 (a, b), the initial and peak uCXCL10 were both independently associated with septic AKI after adjustment for body weight and PRISM III (initial: AOR = 3.281, 95% CI 1.788-6.018, P < 0.001; peak: AOR = 3.172, 95% CI 1.735-5.799, P < 0.001). The AUCs of the initial and peak uCXCL10 in predicting septic AKI were 0.75 (95% CI 0.64-0.87) and 0.76 (95% CI 0.65-0.87). The ROC curves of the initial and peak uCXCL10 and PRISM III for predicting septic AKI are displayed in Figure. 3a.
In addition, univariate and multivariate logistic regression analysis indicated that the uCXCL10 was robustly associated with septic AKI in the critically ill children with sepsis (n = 132), as shown in Additional le 1: Table S2. The performances of the initial and peak uCXCL10 in predicting septic AKI were 0.72 (95% CI 0.59-0.85) and 0.73 (95% CI 0.60-0.85), respectively, in this population in Additional le 1: Table S2.

Association between urinary CXCL10 and mortality
A comparison of demographic and clinical characteristics between survivors and non-survivors is shown in Additional le 1: Table S3. Out of the 342 children, 30 (8.8%) died during PICU stay. The values of uCXCL10 were signi cantly higher in those who died within PICU than in survivors as displayed in Additional le 4: Figure. 3 (a, b). In order to explore whether uCXCL10 levels were independently associated with mortality in critically ill children, univariate and multivariate logistic regression analysis were performed. After adjustment for body weight, PRISM III score, sepsis and AKI stage, the initial and peak uCXCL10 levels remained independently associated with mortality, with adjusted odds ratios of 2.779 (95% CI 1.487-5.195, P = 0.001) and 3.965 (95% CI 2.037-7.717, P < 0.001) as listed in Table 3 and Figure.1(a, b).
The initial and peak uCXCL10 displayed AUCs of 0.77 (95% CI 0.68-0.86) and 0.84 (95% CI 0.76-0.91), and PRISM III score displayed the AUC of 0.83 (95% CI 0.75-0.91) in predicting mortality. The ROC curves of the initial and peak uCXCL10 and PRISM III for predicting mortality are shown in Figure. 3b.

Discussion
This study prospectively assessed uCXCL10 in a typical heterogeneous PICU population. Our dada revealed that elevated uCXCL10 was independently associated with AKI and sepsis after adjustment for confounding factors, and uCXCL10 could be a potential indicator of septic AKI and PICU mortality in critically ill children.
In normal condition, CXCL10 is expressed at low levels in the kidney, but their expression levels are upregulated in renal tubular cells after insult, involving in the pathogenesis of AKI [11,12,27]. Higher uCXCL10 levels were found among the critically ill children with AKI in our study, which was in consistent with a prospective nested case-control study performed by Ho J et al [17]. They compared urinary proteomes of adults before, during, and after cardiopulmonary bypass surgery, discovering that uCXCL10 levels were upgraded in patients with AKI postoperatively [17]. In a recent study on urinary biomarkers of AKI, uCXCL10 was discovered and validated to be associated with AKI in children after HCT [18]. However, these data are limited to small studies originally designed for AKI prediction in a speci c setting. Our results con rmed the signi cant association of uCXCL10 with AKI regardless of illness severity and the presence of sepsis, in critically ill children.
The discriminative power of uCXCL10 for AKI in our study was less well comparing with the study of Vaidya et al [19]. Our result suggested that uCXCL10 may not be a good predictor of AKI in critically ill children. Different from the patients without AKI in the present study, 102 individuals without AKI in previous study were from 3 types: 50 healthy volunteers, 39 patients undergoing cardiac catheterization and 13 patients admitted to the intensive care unit [19]. The healthy volunteers accounted for half of those who were non-AKI, which may partly explain the discrepancy. In critically ill patients, AKI development is characterized with heterogeneity, and the predictive value of AKI is highly dependent on the underlying conditions. Our study indicates that although it is undisputed that uCXCL10 is associated with kidney injury, similar association has been identi ed between uCXCL10 and sepsis, which makes uCXCL10 measurements generally a poor diagnostic tool in complex contexts in critically ill children. Moreover, the levels of uCXCL10 were in uenced by age and body weight, which might be another explanation for the poor performance in the clinical utility of uCXCL10 as an AKI biomarker in PICU population.
Sepsis is a well-known main cause of AKI in critically ill patients [5,28]. The CXCL10 has been reported to regulate the pathogenesis of sepsis [29,30]. In our present study, uCXCL10 levels were higher in critically ill children with sepsis, suggesting that the levels of uCXCL10 were in uenced by in ammation and infection. Urinary CXCL10 remained independently associated with both sepsis and AKI, indicating that the increases of uCXCL10 due to AKI and sepsis are additive. These results were supported by animal research in which a marked upregulation of CXCL10 was detected within kidney in septic acute renal failure model [31].
In this study, uCXCL10 had an ability to discriminate septic AKI in all critically ill children and in children with sepsis. These ndings suggested that uCXCL10 could be diagnostic of septic AKI. Considering that septic AKI is a frequent complication in critically ill patients, and associated with higher risk of in-hospital mortality [28,32], the identi cation of early biomarkers of septic AKI is extremely important. Urinary biomarker neutrophil gelatinase-associated lipocalin and cell cycle biomarkers of tissue inhibitor of metalloproteinases-2 and insulin-like growth factor-binding protein 7 are the frequently investigated and have been proved to predict septic AKI in patients with sepsis [32][33][34]. Our nding emphasizes that uCXCL10, as any other biomarker, must be interpreted in the speci c clinical context. Although, to our knowledge, this is the rst report about the relationship of uCXCL10 and septic AKI, this was a single cohort study and 22 (6.4%) critically ill children were septic AKI, a multi-center study with more cases would be needed to con rm our ndings. Additional studies are also required to delineate the speci c mechanism of CXCL10 in septic and non-septic AKI.
As uCXCL10 has been related to different conditions of in ammation and injury and proven as a reliable marker for septic AKI, it has been consequentially evaluated as a prognostic marker. Our study rstly proved that uCXCL10 levels had independent associations with PICU mortality in critically ill patients. Clinical studies about the association between CXCL10 and mortality have been limited and predominantly investigated in blood rather than in urine of adult patients [21,35,36]. As regards to CXCL10 in urine, a pilot study conducted in patients with ischemic AKI suggested that uCXCL10 might predict renal functional outcome at various times along the course of ischemic AKI and predict mortality of AKI patients within 3 months [37]. However, in adult patients with established AKI, uCXCL10 was a good biomarker of AKI as mentioned earlier, but it was not predictive of in-hospital mortality [19]. Both AKI and sepsis are established risk factors for mortality, and AKI and sepsis increase mortality synergistically [2,38]. In our study, patients with septic AKI had a similar mortality compared to non-septic AKI, suggesting that non-septic etiologies may also contribute to the high mortality rate in AKI patients. The relationship between elevated uCXCL10 levels and increased risk of all-cause PICU death in our cohort was independent of the presence of sepsis and/or AKI and illness severity assessed by the PRISM III score, indicating that uCXCL10 is not simply associated with AKI and sepsis, but also a potential indicator of PICU mortality in critically ill children. Thus, elevated uCXCL10 levels in ICU patients should not be simply viewed as a risk indicator for AKI but rather should prompt a thorough investigation for coexisting conditions, which will ultimately determine the prognosis of the ICU patients. Furthermore, the predictive abilities of the peak uCXCL10 for PICU mortality was better than the initial uCXCL10. These results suggest that dynamic monitoring of uCXCL10 levels upon PICU admission is valuable for clinicians in guiding preventive strategies and improving prognosis.
There are some limitations in this study. First, the AKI incidence may be underestimated when SCr at PICU admission was considered as a baseline, given that the majority of critically ill children did not have baseline SCr prior to PICU admission. In consistent with our previous studies [23,25], the lowest SCr value within 2 weeks during the PICU stay was used as a baseline for patients with elevated SCr ≥ 106.1 µmol/L at PICU admission, which, however, has not been validated in critically ill children.
Nevertheless, a previous study suggests that the lowest SCr within the rst week in the ICU better approximates the true baseline distribution and leads to more accurate diagnosis of AKI, as compared with the estimation methods of back-calculating baseline SCr [39]. Second, although it is a challenge to evaluate the performance of biological indicators on the diagnosis of septic AKI based on the recommendations applying to children [26,32] and the relevance of this nding of uCXCL10 in predicting septic AKI is limited by the small sample size, the diagnostic accuracy of uCXCL10 persisted in all critically ill children and in a subgroup analysis of children with sepsis. Third, we did not perform an etiological analysis for developing AKI. Since our study was carried out in a general and mixed PICU population, it was di cult to distinguish the exact causes of AKI from the existence of complex comorbidities. Fourth, it is unclear whether uCXCL10 is truly causatively involved in pathophysiologic mechanisms of underlying conditions resulting in high mortality or whether it re ects general in ammation and injury in critical illness. Further studies are required for understanding of the biochemical properties and regulatory mechanisms of uCXCL10 in critical illness. Fifth, the single-center study design represents a main limitation. Although we add novel insights to the literature, we did not compare other kidney injury biomarkers, and could not prove the statistical superiority of initial uCXCL10 value overall other standard parameters in early predicting adverse outcome. A multi-center study with a larger quantity of samples is necessary for further validation.

Conclusions
Urinary CXCL10 levels are independently associated with increased risk for AKI, sepsis, septic AKI and PICU mortality even after adjustment for confounding factors. A higher uCXCL10 may be predictive of septic AKI and PICU mortality in critically ill children. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Figure 1 The correlation between uCXCL10 and AKI, sepsis, septic AKI and mortality in multivariate logistic regression. Adjusted odds ratio: AKI, after adjustment for body weight, PRISM III score and sepsis. Sepsis, after adjustment for body weight, PRISM III score and AKI stage. Septic AKI, after adjustment for body weight and PRISM III score. Mortality, after adjustment for body weight, PRISM III score, sepsis and AKI stage. a Initial uCXCL10, b Peak uCXCL10. AKI acute kidney injury, PRISM III pediatric risk of mortality III, uCXCL10 urinary CXCL10.

Figure 1
The correlation between uCXCL10 and AKI, sepsis, septic AKI and mortality in multivariate logistic regression. Adjusted odds ratio: AKI, after adjustment for body weight, PRISM III score and sepsis. Sepsis, after adjustment for body weight, PRISM III score and AKI stage. Septic AKI, after adjustment for body weight and PRISM III score. Mortality, after adjustment for body weight, PRISM III score, sepsis and AKI stage. a Initial uCXCL10, b Peak uCXCL10. AKI acute kidney injury, PRISM III pediatric risk of mortality III, uCXCL10 urinary CXCL10.

Figure 2
Page 22/23 Median uCXCL10 differences among children with and without AKI and with and without sepsis. a Initial uCXCL10, b Peak uCXCL10. AKI acute kidney injury, uCXCL10 urinary CXCL10, uCr urinary creatinine.

Figure 2
Median uCXCL10 differences among children with and without AKI and with and without sepsis. a Initial uCXCL10, b Peak uCXCL10. AKI acute kidney injury, uCXCL10 urinary CXCL10, uCr urinary creatinine.  ROC curves of PRISM III and uCXCL10 to predict septic AKI and mortality.