Association of endothelial and glycocalyx injury biomarkers with fluid administration, development of acute kidney injury, and 90-day mortality: data from the FINNAKI observational study

Endothelial cells line the luminal side of all blood vessels. Glycocalyx is located on the top of the endothelium. It is a thin (few dozens of nanometers), complex layer with a framework consisting of glycoproteins and proteoglycans between the endothelial cell wall and flowing blood [1, 2]. It plays a key role in regulation of vascular permeability and blood cell–vessel interactions [1‐3]. In critical illness, the body responds to stress by initiating sympathoadrenal activation, which leads to the release of inflammatory mediators, such as interleukin-6 (IL-6), and damage to the endothelium and glycocalyx, resulting in increased vascular permeability [4‐6]. Several plasma biomarkers may be measured as indicators of glycocalyx [4, 7, 8] and endothelial injury [7‐11].

We hypothesized that higher plasma levels of endothelial and glycocalyx biomarkers measured at intensive care unit (ICU) admission would indicate more severe endothelial and glycocalyx injury, more severe capillary leakage, and subsequently, higher amounts of administered fluid. Therefore, we primarily studied the association between glycocalyx (Syndecan-1; SDC-1) and endothelial injury biomarkers (Angiopoietin-2; Ang-2, soluble thrombomodulin; sTM, and vascular adhesion protein-1; VAP-1) and cytokine IL-6 (as marker of the severity of inflammation) and fluid administration among critically ill septic patients. Second, we aimed to study the association of these markers with acute kidney injury (AKI) and 90-day mortality.

This was a post hoc laboratory study of the prospective multicenter observational Finnish Acute Kidney Injury (FINNAKI) cohort study conducted in 17 Finnish intensive care units in 2011–2012 [12]. The Ethics Committee of the Department of Surgery at the Helsinki University Hospital gave nationwide approval for the study (reference number: 18/13/03/02/2010) and accepted the use of deferred consent from the patient or next of kin with written informed consent obtained as soon as possible. The Finnish National Institute of Health and Welfare allowed data collection from medical records of deceased patients to avoid bias in the primary endpoint of the FINNAKI study (incidence and outcome of AKI). The current laboratory study includes only patients who or whose next of kin gave a written informed consent.

The FINNAKI study enrolled all adult emergency ICU admissions to study ICUs of any length and elective post-operative patients with an expected ICU stay longer than 24 h [12]. Exclusion criteria are presented in Additional file 1: Methods section of the 1.2, page 6. The current study included all FINNAKI study patients who were septic at ICU admission, had fluid balance data available for day 1, and a blood sample taken at ICU admission. FINNAKI study was observational and did not include fluid management protocol. Thus, all patients were treated according to the judgement of the treating clinician and the Surviving Sepsis Guidelines in place at that time [13].

We found VAP-1 and IL-6 to be associated with the amount of administered fluid on the first ICU treatment day and other fluid balance-related variables; however, the other investigated markers did not differ according to fluid administration. Only sTM remained associated with the development of AKI after adjusting for known confounders. Finally, Ang-2, SDC-1, and sTM were associated with an increased risk for 90-day mortality, also after adjustments. Our results demonstrate that injury to glycocalyx and endothelium present at ICU admission is associated with an increased risk for AKI and mortality.

We observed roughly comparable plasma biomarker levels to previous studies [8, 22, 23]. Previously, Ang-2 has been shown to correlate with the first 6-h balance, but not with the 24-h balance [24]. Our results corroborate this finding. However, we did not find significant differences in SDC-1 levels in terms of fluid administration or balance, in contrast with a previous study that found SDC-1 to be associated with fluid balance over the first 24 h [25]. That study measured SDC-1 levels on the second ICU day, whereas we obtained samples right after ICU admission. A novel finding in our study was the association of higher IL-6 and lower VAP-1 levels with higher amount of received fluids. As more severely ill patients have higher IL-6 levels [26], and typically receive also more fluids [27], the finding regarding IL-6 was to be expected. The finding regarding VAP-1 is surprising, as one would have expected higher VAP-1 levels with higher fluid administration and severity of illness. However, the transmembrane form of VAP-1 facilitates leukocyte rolling and transmigration, whereas the soluble form acts as a primary serum amine oxidase [28]. Possibly, in the most severely ill septic patients, the shedding of transmembrane form of VAP-1 into soluble form is reduced due to greater needs of the transmembrane form. Moreover, the plasma levels do not necessarily reflect the tissue levels of a biomarker. Finally, dilution of VAP-1 with increasing fluid balance cannot be excluded.

sTM was independently associated with the development of AKI, as in a recent retrospective observational study among 514 septic patients [29] and a multicenter prospective observational study among 80 ICU patients [30]. Moreover, we demonstrated that an increase in sTM levels occurred already before AKI onset, unlike in the largest previous study [29]. Interestingly, we found Ang-2 to be significantly associated with AKI in the univariate analysis but not in the adjusted model. A previous study reported an unadjusted association of AKI defined by the KDIGO creatinine criterion and Ang-2 levels [24]. Higher SDC-1 values have been previously observed to be associated with AKI in a study among 175 septic patients [31]. Albeit the SDC-1 levels in this study were higher among AKI_>12 h patients, SDC-1 was not independently associated with AKI.

Our study corroborates previous results on the associations between higher SDC-1, Ang-2 and sTM plasma levels and the severity of critical illness and mortality [9, 22, 25, 31‐33]. A multicenter trial in acute respiratory distress syndrome (ARDS) patients reported a positive correlation between sTM plasma levels and mortality, but found no difference in sTM levels between conservative and liberal fluid management groups [34]. Moreover, a recent study comparing early goal-directed therapy, standard therapy, and protocolized therapy found no difference in endothelial injury biomarkers including sTM and Ang-2 between the treatment arms, but higher Ang-2 and STM plasma levels were associated with 60-day mortality [32]. We found an independent association between Ang-2, sTM, and SDC-1 and 90-day mortality. Moreover, patients who had high levels of all these three markers had a fivefold adjusted OR for 90-day mortality compared to those in whom all these markers were low.

Given the multiple roles of the glycocalyx and endothelium in regulating vascular permeability and leukocyte adhesion [1‐4], our results on increased mortality are biologically plausible. A recent animal study in experimental sepsis found fluid resuscitation (compared to no resuscitation) to increase atrial natriuretic peptide (ANP) levels and lead to glycocalyx injury [35]. Furthermore, fluid resuscitation group exhibited higher vasopressor requirements, potentially due to glycocalyx damage [35]. Additionally, hypervolemia has been found to increase glycocalyx shedding in an experimental volume loading study in surgical patients [36]. Taken together, these data suggest that some of the typical interventions used in sepsis resuscitation may aggravate glycocalyx and endothelial injury. Our results add to the increasing body of evidence highlighting the importance of endothelial and glycocalyx injury in the pathway of adverse outcomes and call for studies assessing novel means in avoiding and alleviating injury to endothelium and glycocalyx.

Our study has some strengths and limitations. This was a post hoc study from a large prospective multicenter study with good generalizability to other critically ill septic patient cohorts. Moreover, we used the full set of KDIGO AKI criteria, and collected detailed and comprehensive data about clinical variables and risk factors for AKI. Most of the previous studies have used only the creatinine criterion for AKI [22‐24, 31]. As a limitation, first, our study collected data about fluid administration and balance in 24-h blocks, which may not be granular enough to distinguish the fluid resuscitation phase. However, we extracted the fluid administration data of 44 patients in 12-h blocks from the ICU data management system as a sensitivity analysis, and found it to correlate well with the ICU admission day fluid administration normalized to the duration of observation period. Second, we did not have data regarding the pre-ICU fluid therapy, which may have affected plasma biomarker levels at ICU admission and may explain why we could not show a connection between other biomarkers than VAP-1 and IL-6 and fluid load. However, some studies have suffered from the same limitation [25]. Third, we had to exclude 36 deceased patients from the analysis because of absent consent. Nevertheless, percentage of these patients of the cohort was low (5.5%), and thus, unlikely to cause significant bias. Fourth, this study was observational and had no protocol concerning hemodynamic monitoring or fluid responsiveness. Therefore, hemodynamic monitoring was performed and fluids were administered according to the treating clinician and standards of each participating ICU following the Surviving Sepsis Guidelines [13] in place that time. Fifth, we defined sepsis according to the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) definition [14]. Due to the structure of data collection, classifying patients retrospectively according to the sepsis-3 definition was not possible. Sixth, fluid balance was calculated with inputs and outputs since weighting of all study participants was not possible. However, it is not obvious that regular body weight measures would be more precise to estimate fluid balance than fluid balance calculations [37]. Seventh, we used data-driven approach supplemented with clinical discretion in selecting confounders for the logistic regression analyses, albeit a propensity score analysis might have been able to mitigate confounding better [38]. Finally, some unknown confounders might have been missing.

We found VAP-1 and IL-6, but not the other markers, to be associated with fluid administration on the first ICU day. Supporting previous results, sTM was independently associated with the development of AKI. Finally, Ang-2, sTM, and SDC-1 were independently associated with an increased risk for 90-day mortality. These findings support the importance of endothelial injury in the pathophysiology of sepsis-associated AKI and highlight the role of the glycocalyx and endothelium in the pathway of adverse outcomes.