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Novel Damage Biomarkers of Sepsis-Related Acute Kidney Injury

Sepsis-related acute kidney injury (AKI) is one of the most common complications of sepsis at the intensive care unit (ICU) with more adverse mortality rates. The early diagnosis and reliable monitoring of sepsis-related AKI are essential in achieving a favorable outcome. Novel serum and urinary biomarkers could yield valuable information during this process. Regarding the widely used Kidney Disease Improving Global Outcomes (KDIGO) classifications, the diagnosis of AKI is still based on the increase of serum creatinine levels and the decrease of urine output; however, these parameters have limitations in reflecting the extent of kidney damage, therefore more sensitive and specific laboratory biomarkers are needed for the early diagnosis and prognosis of sepsis-related AKI. Regarding this, several serum parameters are discussed in this review including presepsin and the most important actin scavenger proteins (gelsolin, Gc-globulin) while other urinary markers are also examined including cell cycle arrest biomarkers, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), Cystatin C and actin. Novel biomarkers of sepsis-related AKI could facilitate the early diagnosis and monitoring of sepsis-related AKI.


INTRODUCTION
Sepsis is a complex clinical syndrome with increasing incidence and unfavorable mortality rates which still poses a significant challenge in intensive care despite the availability of advanced treatment methods [1][2][3]. As stated in the latest Sepsis-3 definitions, sepsis is a lifethreatening organ dysfunction caused by a dysregulated host response to infection [4].

CONVENTIONAL SEPSIS BIOMARKERS
Timely diagnosis along with effective causal and supportive treatment of sepsis are essential for achieving adequate recovery. Although not being part of the Sepsis-3 definitions, serum high-sensitivity C-reactive protein (CRP) and procalcitonin (PCT) are still widely used inflammatory markers in the clinical evaluation of sepsis [4,5]. CRP is a non-specific inflammatory marker; thus, it can be elevated in various acute and chronic diseases (e.g. autoimmune disorders, trauma, malignancies) besides sepsis [6][7][8]. However, despite CRP being a general inflammatory marker, its mostly inversely proportional relation to albumin -namely the CRP:albumin ratio -has already been investigated in numerous clinical conditions (e.g. sepsis, pancreatitis, coronary artery disease, malignancies) [9][10][11]. CRP reaches its peak levels 48 hours after the start of the inflammatory process and significantly elevated CRP levels also have a moderate correlation with the severity of sepsis [6,12]. Compared to CRP, PCT concentrations increase 4-6 hours after the onset of infection while PCT also showed better performance regarding the diagnosis and mortality prediction of sepsis [8,13,14]. However, exclusively fungal or viral infections do not elevate PCT, yet other inflammatory conditions (e.g. extensive tissue injury, pancreatitis) besides infection could result in slightly increased PCT levels [14,15]. Furthermore, the antagonistic changes in PCT and albumin were also investigated in sepsis-related AKI [16].
Besides hs-CRP and PCT, more than 200 novel (mostly serum) sepsis biomarkers have been evaluated, yet no single marker was sensitive or specific enough for accurately diagnosing sepsis [17,18]. However, a multi-marker approach including various promising sepsis biomarkers (e.g. presepsin, IL-6) may provide useful information regarding the early diagnosis of sepsis.

Presepsin
Presepsin (PSEP) is the soluble N-terminal fragment (MW=13 kDa) of the cluster of differentiation (CD) marker protein CD14 (MW=55 kDa), which is the receptor for lipopolysaccharide (LPS) and LPS-binding protein complexes [19,20]. Compared to PCT, PSEP has an even more rapid response time of 2-4 hours after the onset of infection, while PSEP was also deemed valuable for the early diagnosis and prognosis of sepsis in contrast to other conditions (e.g. trauma, burn injury, major surgical operations) [21,22]. PSEP has varying diagnostic cut-off values among 400-600 pg/mL for sepsis, yet there is a concern that PSEP concentrations are affected by kidney function [23][24][25]. It is presumed that PSEP is filtered by the glomeruli, then reabsorbed and catabolized within proximal tubular cells. Several studies reported increasing PSEP levels as kidney function decreases (e.g. during chronic kidney disease or sepsis-related AKI) [26][27][28]. However, PSEP -along with hs-CRP and PCT -could be removed from the circulation Dániel Ragán, Zoltán Horváth-Szalai, Balázs Szirmay, Diána Mühl Novel damage biomarkers of sepsis-related acute kidney injury using different modalities of renal replacement therapy (RRT), therefore potentially causing falsely low inflammatory marker levels [28,29].

CONVENTIONAL MARKERS OF SEPSIS-RELATED AKI
AKI refers to an abrupt decrease in kidney function resulting in the retention of numerous waste products and the dysregulation of extracellular volume [30,31]. The Kidney Disease Improving Global Outcomes (KDIGO) classification is widely used for the diagnosis of AKI based on the increase in serum creatinine (sCr) levels and the decrease of urine output, both of which are kidney function markers [32]. Urine output often decreases before the elevation of sCr concentration could be detected, yet not all reductions in urine output indicate AKI. Unfortunately, due to the non-linear relationship between glomerular filtration rate (GFR) and sCr, the increase of creatinine could be overlooked in the early phase of AKI. Therefore, the changes in these parameters do not reflect the extent of kidney damage, hence more sensitive and specific laboratory biomarkers are needed for the early diagnosis and prediction of AKI [33,34].
So far, several novel biomarkers have been investigated to improve early diagnosis and prognosis of sepsis-related AKI [34,35]. However, the clinical use of the studied biomarkers remains unclear due to heterogeneity of AKI itself and the limitations of novel AKI biomarkers. Regarding this, several biomarkers are discussed including tissue inhibitor of metalloproteinases-2 (TIMP-2), insulin-like growth factor-binding protein 7 (IGFBP7), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and Cystatin C (CysC) while urinary actin and the most important proteins of the actin scavenger system (gelsolin, Gc-globulin) are also presented in this review.

Pre-injury phase stress markers
It has been proposed that the development of AKI is mostly preceded by a so-called acute kidney stress (AKS) phase which can occur due to several sources of renal insults (e.g. hypoperfusion, nephrotoxic drugs, cytokines, reactive oxygen species). The expression of several cell growth regulating proteins including TIMP-2 (MW=21 kDa) and IGFBP7 (MW=29 kDa) could be upregulated in the tubular system as a consequence of kidney stress, thus leading to G1 cell cycle arrest through the induction of several apoptotic pathways. In the case of prolonged kidney stress, the persistent urinary increase of these biomarkers could also indicate apoptotic tubular injury, thereby signaling the early development of AKI. Urine measurements of TIMP-2 and IGFBP7 were found useful in the early diagnosis and prognosis of AKI according to several multicentric studies. However, these biomarkers did not prove to be beneficial in patients with more severe stages of AKI while false positive results could also occur frequently in lowrisk patients [36][37][38].

Tubular markers of AKI progression
NGAL is a glycoprotein (MW=25 kDa) of the lipid carrier protein superfamily expressed mostly on the surface of neutrophils while also being scarcely present in other cell types (e.g. nephrocytes, hepatocytes). NGAL is filtered through the glomeruli and reabsorbed in the proximal tubules under normal circumstances. NGAL expression is rapidly upregulated if the tubular system is affected during ischemic or nephrotoxic renal injury, hence NGAL is detectable in the urine as early as 3 hours after the onset of kidney damage. Plasma NGAL levels seem to have a stronger correlation with absolute neutrophil count than proinflammatory cytokines, Dániel Ragán, Zoltán Horváth-Szalai, Balázs Szirmay, Diána Mühl Novel damage biomarkers of sepsis-related acute kidney injury therefore lower plasma NGAL levels could still occur due to neutropenia in patients with systemic inflammation [39]. However, urine NGAL levels may also be significantly increased in AKI patients with neutropenia if the tubular system is affected. As NGAL also has an antimicrobial effect by binding siderophores, it was also found valuable along with hepcidin -a main regulator of iron homeostasis -in the prognosis of sepsisrelated AKI. All in all, urinary NGAL elevation could reflect the decreased reabsorption of filtered NGAL due to the dysfunction or injury of the proximal tubules while its expression could also be upregulated in the tubular system during sepsis-related AKI [40][41][42][43].
KIM-1 is a transmembrane glycoprotein (MW= 39 kDa) containing extracellular immunoglobulin and mucin domains having a low expression in the kidney under physiological conditions. However, it is upregulated after ischemia-reperfusion injury, especially in proliferating de-differentiated proximal tubular epithelial cells 48 hours after injury. KIM-1 appears to be a highly sensitive marker of AKI in non-cardiac surgical patients as well as after cardiac surgery. Persistent serum KIM-1 elevation indicates ongoing tubular injury, potentially increasing the risk for the development of chronic kidney disease, while urinary KIM-1 also shows a similar correlation to kidney injury [44][45][46][47].
CysC (MW=13 kDa) is constantly produced by all nucleated cells, filtered by the glomeruli, then mostly reabsorbed and catabolized in the proximal tubular cells. The measurement of serum CysC was found to be preferable to sCr in patients after non-traumatic and traumatic amputation while also being superior in predicting cardiovascular events and mortality in elderly patients [48][49][50]. However, as CysC is a more reliable kidney function marker compared with sCr, it seems to be influenced by old age, large doses of corticosteroids, conditions affecting the thyroid gland, inflammation and malignancies. As the changes in sCr concentrations have some limitations, especially late in the clinical course of ICU patients, urine CysC seems to be unaffected by several non-renal factors affecting creatinine levels in sepsis-related AKI [50][51][52][53].

Actin
Actin is a multifunctional globular protein (MW=42 kDa) existing in monomeric/globular (G-actin) and in polymeric/filamentous (F-actin) forms. In acute tissue injuries the released extracellular actin is found to be highly toxic in the circulation due to its spontaneous polymerizing tendency causing unfavorable effects on the coagulation system. Gelsolin and Gc-globulin are the most important proteins of the so-called actin scavenger system which is responsible for binding and depolymerizing extracellular actin in the circulation, thus making the urinary appearance of these protein complexes unlikely [54][55][56][57]. However, an earlier study indicates that actin could be detected in the urine of kidney transplant patients with sustained AKI [58].
Recently published data suggest that urinary actin (u-actin) could be a complementary diagnostic biomarker to sCr in sepsis-related AKI while higher u-actin levels also seemed to reflect the severity of AKI [59]. Significantly different admission u-actin levels were found between 24 control and 60 septic patients (median: 0.78 vs. 3.98 µg/L, p<0.001), while samples from 17 septic non-AKI and 43 sepsis-related AKI patients also showed differences (median: 1.27 vs. 9.52 µg/L, p<0.001) (Fig 1). Admission u-actin levels were even more elevated in 43 patients with sepsis-related AKI and were in good agreement with the severity of AKI stages (median: 3.16 vs. 10.78 vs. 11.55 µg/L, p<0.05) (Fig 2). This tendency remained the same when referring u-actin to urine creatinine. Parameters of first-day Dániel Ragán, Zoltán Horváth-Szalai, Balázs Szirmay, Diána Mühl Novel damage biomarkers of sepsis-related acute kidney injury septic patient samples could discriminate AKI from non-AKI state (AUC ROC, p<0.001): u-actin: 0.876; se-creatinine: 0.875. Derived cutoff value for u-actin was 2.63 µg/L (sensitivity: 86.0%, specificity: 82.4%). Although this study has several limitations, u-actin showed moderate correlation with other urinary parameters. Furthermore, extremely elevated u-actin levels were found in sepsis-related AKI patients with RRT requirement. Despite actin being bound to the actin scavenger proteins (gelsolin, Gcglobulin) in the circulation, u-actin could appear in the urine due to both severe glomerular and/ or tubular injury, so it seems that the elevation of u-actin indicates severe kidney injury ( Figure  1, Figure 2).

Gelsolin
Gelsolin (GSN) is a multifunctional protein existing in three different isoforms. Secreted or plasma GSN (MW=83 kDa) is an essential component of the extracellular actin scavenger system [55,60,61]. Besides actin, GSN may also bind to pro-inflammatory mediators and bacterial wall components (lipoteichoic acid, LPS). Since GSN seems to have a protective role in the body (e.g. by depolymerizing actin filaments in the circulation), a growing body of evidence indicates decreasing GSN levels in various clinical conditions (e.g. severe sepsis, multiple organ dysfunction syndrome (MODS), extensive trauma, acute liver failure, myocardial infarction) [62][63][64]. Our previous study showed as well that the increase of serum actin was inversely proportional to the amounts of serum GSN which was associated with increased mortality rate [65]. Furthermore, the simultaneous measurement of other inflammatory parameters and GSN levels may provide valuable information in the management of critically ill patients.

Gc-globulin
Gc-globulin (MW=52-59 kDa) has 3 major isoforms (Gc1f, Gc1s, Gc2) while also being a member of the albuminoid superfamily, which consists of several transport proteins in the circulation including albumin, alpha-fetoprotein and afamin [66]. Albumin is the most abundant human serum protein acting as a transporter of endogenous and exogenous substances including thyroxine, fatty acids and drugs. Gc-globulin is mainly produced by the liver with a reference range of 300-600 mg/L, yet severely decreased levels of 50-120 mg/L were observed in acute injury or sepsis. Gc-globulin is involved in the vitamin D biosynthesis process by binding and transporting vitamin D metabolites while it also plays an important role in modulating cells of the adaptive immune response [67,68]. Under physiological conditions, Gc-globulin is filtered freely through the glomeruli, then reabsorbed and catabolized by proximal tubular epithelial cells resulting only in a trace urinary excretion. Therefore, tubular kidney damage is expected to result in increased urinary Gc-globulin concentrations [69,70]. There is only scarce data on urinary appearance of Gc-globulin in sepsis, yet this marker was already investigated in other conditions including endometriosis, diabetic nephropathy and contrast-induced nephropathy as well [67,71]. Recently, our research group started investigating urinary Gc-globulin/urine creatinine (u-Gc-globulin/u-Cr) levels in sepsis-related AKI by conducting a small sample size pilot study. Compared to 6 control patients, significantly elevated admission u-Gc-globulin/u-Cr levels were found in 9 septic and 12 sepsis-related AKI patients (median: 1.8 vs. 21.5 vs. 136.7 µg/mmol, p<0.001) (Figure 3).

SUMMARY OF NOVEL BIOMARKERS IN SEPSIS-RELATED AKI
Despite their limitations and the heterogeneity of AKI itself, the discussed laboratory markers yield valuable information regarding the early diagnosis and effective prognosis of sepsis-related AKI.
Most widely known laboratory markers including TIMP-2xIGFBP-7, NGAL, KIM-1 and Cystatin C can be measured using commercially available diagnostic assays, thereby providing accurate results with a short turnaround time (less than 1 hour). Serum GSN was measured using an automated immune turbidimetric assay developed in our laboratory with a short turnaround time (less than 1 hour) as well, yet this measurement method is not yet commercially available.
However, the measurement of u-actin and u-Gc-globulin/u-creatinine was carried out using quantitative Western blot techniques, hence the routine clinical utility of these biomarkers is questionable. Therefore, our research group is currently working on the development of more rapid and efficient ELISA methods for measuring u-actin and u-Gc-globulin/u-creatinine. All of the discussed laboratory markers are shown in Table 1.

CONCLUSION
The early diagnosis and effective therapy of sepsis and sepsis-related AKI are essential for a successful recovery. However, the currently used biomarkers (sepsis: PCT, hs-CRP; AKI: secreatinine, urine output) show several limitations regarding the diagnosis and prognosis of sepsis and AKI, hence investigating novel laboratory markers may prove to be beneficial in achieving a more favorable outcome. Most of the discussed AKI biomarkers provide valuable information regarding the injury of the tubular system, yet the monitoring of serum and urinary actin levels along with measuring the proteins of the actin-scavenger system (GSN, Gc-globulin) could yield more complex information for the assessment of overall renal damage involving both glomerular and tubular sources of kidney injury. Moreover, the increase of these markers could yield valuable information regarding the need for the early initiation of RRT, thus potentially attenuating renal injury and improving the outcome of sepsis-related AKI. Urinary markers provide a non-invasive tool for monitoring of inflammatory conditions. Since sepsis-related AKI is a heterogeneous clinical syndrome, it seems that the measurement of a single marker alone would be insufficient for accurate diagnostic and monitoring purposes. Therefore, a multi-marker approach involving various serum and urinary biomarkers and the complex evaluation of the clinical parameters should improve patient management at the ICU.

Acknowledgements
The present research was funded by the 2020-4.1.1.-TKP2020 project (Thematic Excellence Program 2020 -National Excellence Subprogram of the Hungarian Ministry for Innovation and Technology) and partially by the Human Reproduction National Laboratory as part of the "Establishment of National Laboratories 2020" program. Zoltán Horváth-Szalai was supported by the University of Pécs, Medical School, Hungary grant (KA-2019-36). We express our special thanks for the invaluable help of the nurses and the colleagues from every participating institute.

Ethical statement
The authors state that they have obtained institutional review board approval from the Regional Research Ethics Committee of the University of Pécs (no. 4327.316-2900/KK15/ 2011) conforming to the 7th revision of the Helsinki Declarations (2013) for the research described. Verbal and written informed consent were obtained from the patients for the inclusion of their medical and treatment history within this work.