Klotho and S100A8/A9 as Discriminative Markers between Pre-Renal and Intrinsic Acute Kidney Injury

Early detection and accurate differentiation of the cause of AKI may improve the prognosis of the patient. However, to date, there are few reliable biomarkers that can discriminate between pre-renal and intrinsic AKI. In this study, we determined whether AKI is associated with altered serum and urinary levels of Klotho, S100A8/A9 (an endogenous ligand of toll-like receptor 4), and neutrophil gelatinase-associated lipocalin (NGAL), which may allow differentiation between pre-renal and intrinsic AKI. A volume-depleted pre-renal AKI model was induced in male Sprague Dawley rats fed a low-salt diet (0.03%) without water 96 h before two intraperitoneal (IP) injections of furosemide (20 mg/kg) at a 24 h interval. In contrast, in the cisplatin-induced intrinsic AKI model, animals were given a single IP injection of cisplatin (5 mg/kg). All of the animals were euthanized 72 h after the first IP injection. Serum and urinary levels of Klotho, S100A8/A9, and NGAL were measured using an enzyme-linked immunosorbent assay. We also performed a proof-of-concept cross-sectional study to measure serum and urinary biomarkers in 61 hospitalized patients with established AKI. Compared to the intrinsic AKI group, the pre-renal AKI group showed a marked depression in urinary Klotho levels (13.21±17.32 vs. 72.97±17.96 pg/mL; P = 0.002). In addition, the intrinsic AKI group showed marked elevation of S100A8/A9 levels compared to the pre-renal AKI group (2629.97±598.05 ng/mL vs. 685.09±111.65 ng/mL; P = 0.002 in serum; 3361.11±250.86 ng/mL vs. 741.72±101.96 ng/mL; P = 0.003 in urine). There was no difference in serum and urinary NGAL levels between the pre-renal and intrinsic AKI groups. The proof-of-concept study with the hospitalized AKI patients also demonstrated decreased urinary Klotho in pre-renal AKI patients and increased urinary S100A8/A9 concentrations in intrinsic AKI patients. The attenuation of urinary Klotho and increase in urinary S100A8/A9 may allow differentiation between pre-renal and intrinsic AKI.


Materials and Methods Experimental Animals
Eighteen male Sprague Dawley rats were purchased from Orient Bio (Seongnam City, Gyeonggi Province, Korea). Animals were checked daily for any signs of sickness during the experimental period. All of the animal experiments were performed under the approval of the Institutional Animal Care and Use Committee of the Clinical Research Institute at Gachon University Gil Medical Center, and in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Gachon University Lee Gil Ya Cancer and Diabetes Institute.

Induction of AKI
Rats were distributed evenly into the following three groups (Fig 1). Dong-A ST, Seoul, Korea) was given at 0 h, and a single IP of vehicle (normal saline) was given at 24 h. 3. Control (n = 6; 302-314g; 8 weeks old): Rats had free access to water and food (PicoLab Rodent Diet 20 5053; LabDiet, St. Louis, MO, USA). Two IP injections of vehicle (normal saline) were administered at 0 h and 24 h.
Previously described experimental models of VD and CDDP-induced AKI models were modified in this study [13,14]. All of the animals were placed in metabolic cages, and 24 h urine samples were collected 48-72 h after the first injection with furosemide, CDDP, or normal saline. Rats were fully anesthetized by isoflurane inhalation before blood sampling and phlebotomy. Blood samples were collected from the abdominal aorta at 72 h after the first injection of furosemide, CDDP, or normal saline. After blood sampling, rats were sacrificed by phlebotomy and both kidneys were removed. Kidneys were promptly fixed in 10% buffered formalin solution.

Examination of biomarkers
Enzyme-linked immunosorbent assay. Klotho (Rat Klotho ELISA Kit; Cusabio Biotech, Wuhan, China), S100A8/A9 (Calprotectin ELISA Kit; MyBioSource, San Diego, CA, USA), and NGAL (Lipocalin-2 Rat ELISA Kit; abcam, Cambridge, UK) levels were assessed by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Because variability in urine concentrations could impair the use of a urinary biomarker as a discriminative marker, we corrected biomarker concentrations with reference to the urinary creatinine concentrations.
Histological analysis. To assess the light microscopic appearance, 4μm thick paraffin sections were subjected to hematoxylin and eosin (H&E) staining. H&E-stained kidney sections were photographed by one of the investigators blinded to the experimental protocol using an Olympus BX51 microscope. Previously described semi-quantitative pathological scoring system was used with minor modification [15,16]. The severity of renal damage was scored with a grading system (0, 1, 2, 3): 0 = no visible lesions, normal or near normal kidney morphology; 1 = mild dilation in some tubules, cell swelling, luminal debris (cast), and nuclear condensation, partial loss of brush border membranes in < 1/3 tubules in high field; 2 = obvious dilation of many tubules, loss of brush border membranes, nuclear loss, and cast in < 2/3 tubules in high field; 3 = severe dilation of most tubules, total loss of brush border membranes, and nuclear loss in > 2/3 tubules in high field. Fifty fields (400×) were counted for cortex and outer stripe of outer medulla in each kidney section. The total score for each kidney was calculated by addition of all scores from 100 fields with a maximum score of 300.
Confocal microscopy. Confocal microscopy was performed with a LSM 700 laser scanning confocal microscope (Carl Zeiss; Jena, Germany). After blocking with 5% BSA solution for 1h, sections were incubated at 4°C overnight with the following primary antibodies

Human Study
Study Design and Participants. This study was performed as a cross-sectional study. Adult (aged over 18 years old) patients with AKI who had been admitted to the general ward or intensive care units in Gachon University Gil Medical Center between January and December 2014 were eligible for enrollment. AKI was defined as a rapid (within 48 h) decline in renal function, designated as an absolute increase in serum creatinine of at least 0.3 mg/dL, a percentage increase in serum creatinine of at least 50%, or documented oliguria of less than 0.5 mL/kg/h for more than 6 h [17]. Patients were excluded for post-renal obstruction as a cause of AKI, previous history of RRT, or renal transplantation. Patients were considered to have prerenal AKI when the rise in serum creatinine concentration had been caused by factors that reduce renal perfusion, such as dehydration, bleeding, hepatorenal syndrome, and cardiorenal syndrome, and when creatinine recovered rapidly to baseline after volume repletion within 3 days. Recovery from AKI was defined by the improvement of creatinine to baseline levels in patients who have followed this hospital with known baseline creatinine. In case of in-hospital AKI, reverse decrease of creatinine from AKIN classification (absolute decrease in serum creatinine of at least 0.3mg/dL or a percent decrease in serum creatinine of at least 50%) or to the level of before AKI occurrence was considered to recover from AKI. In addition, patients with pre-renal AKI had no previous exposure history to insults that result in intrinsic kidney injury. Patients were considered to have intrinsic AKI when the increased serum creatinine concentration did not respond to fluid replacement within 3 days and/or was associated with nephrotoxic events, such as hypotension, sepsis, interstitial nephritis, contrast-induced AKI, and glomerulonephritis.
The study was approved by the Institutional Review Board at the Gachon University Gil Medical Center (GAIRB2014-202). Written informed consent was obtained from all of the patients. The biospecimens and data used were provided by Gachon University Gil Medical Center Bio Bank (No: GBB2014-04).
Parameters. We obtained the following demographic data: age, gender, comorbid conditions (diabetes, hypertension, cardiovascular disease), and medication history (ACE inhibitors/ ARBs, statins, β-blockers, calcium channel blockers, diuretics, aspirin). AKI severity by the Risk, Injury, Failure, Loss, and End-stage kidney (RIFLE) and Acute Kidney Injury Network (AKIN) criteria were estimated on the day of the serum and urine sample. Serum and urinary Klotho (Human soluble α-Klotho Assay Kit-IBL; IBL, Gunma, Japan), S100A8/A9 (PhiCal Calprotectin ELISA Kit; Immundiagnostik AG, Bensheim, Germany), and NGAL (Lipocalin-2 Human ELISA Kit; abcam, Cambridge, UK) levels were assessed by ELISA according to the manufacturer's instructions. Biomarker concentrations were corrected with reference to the urinary creatinine concentrations.
Statistical analyses. Continuous data are reported as means ± standard deviation (SD) unless otherwise specified. Categorical data are reported as absolute values and percentage. Non-parametric tests of significance were performed. The Kruskal-Wallis test with Dunn's multiple comparisons was used when comparing three groups. The Mann-Whitney U-test for continuous variable and Fisher's exact test for categorical variables were used when comparing two groups. Statistical analyses were performed using the GraphPad Prism software (ver. 6.0; GraphPad, San Diego, CA, USA). The significance level was set at P < 0.05.
Histology and semiquantitative pathologic score of AKI models. H&E-stained kidney sections showed normal histology in the control group and near normal or mild brush border loss in the VD group (Fig 3(a)). On the other hand, the CDDP group showed loss of brush border membranes, obvious dilation of many tubules, nuclear loss and interstitial inflammatory infiltration. Index of histological damage was higher in the CDDP group than the control and VD group (Control, 11.3±2.6; VD, 13.8±3.4; CDDP, 33.8±4.5; P = 0.0016 ; Fig 3(b)).
Semiquantitative immunoblotting and renal expression of Klotho, S100A8/A9, and NGAL in pre-renal and intrinsic AKI models. As shown in Fig 4(a), the semiquantitative immunoblotting of the kidney homogenates showed a significant decrease in renal klotho protein in VD group (100% of those in control group; 48% of those in VD group; 74% of those in CDDP group, respectively) compared with the control and CDDP groups. Immunohistochemical analysis also showed decreased renal Klotho labeling in kidneys from VD group. As can be seen in Fig 4(b), S100A8/A9 in the CDDP group significantly increased (100% of those in control group; 83% of those in VD group; 220% of those in CDDP group, respectively) compared with that in the control and VD groups. This result was confirmed by immunohistochemistry showing increased labeling of renal S100A8/A9 in kidneys from CDDP group. As shown in Fig  4(c), NGAL protein levels and renal NGAL expression did not appear to differ between the VD and CDDP groups. These results were consistent with the ELISA results. S100A8/A9 expression in inflammatory cells in intrinsic AKI models. To evaluate the mechanism underlying the elevation of S100A8/A9 in the intrinsic AKI group, we examined inflammatory cells in an immunofluorescence study. As expected, intra-renal expression of S100A8/A9 was found more often in the CDDP group which might reflect the inflammatory damage in this group. In addition, intra renal infiltration of CD 15 + neutrophils and CD 68 + macrophages were found frequently in the CDDP group, and the area of distribution was consistent with that of S100A8/A9 (Fig 5(a) and 5(b)). These findings also suggest that increased S100A8/A9 expression reflects the infiltration of inflammatory cells in intrinsic AKI. These inflammatory cells were detectable in the glomerular and tubule-interstitial space.

Clinical study
Patient characteristics. In total, 61 patients were enrolled in the study. Of them, 42 patients had pre-renal AKI and 19 had intrinsic AKI ( Table 2). The severity of AKI, as measured by the RIFLE and AKIN criteria, did not significantly different between the pre-renal and intrinsic AKI group (P = 0.121 by RIFLE criteria, P = 0.172 by AKIN criteria). Serum creatinine was not different between the groups (3.3±2.4 mg/dL in pre-renal AKI, 2.6±1.3 mg/dL in intrinsic AKI; P = 0.296). However, BUN levels were higher in pre-renal AKI than intrinsic AKI patients (59.7±31.1 mg/dL in pre-renal AKI, 42.6±20.4 mg/dL in intrinsic AKI; P = 0.049). Age, gender, and other laboratory findings were not significantly different between the groups. Calcium channel blocker use was more common in intrinsic than pre-renal AKI patients (P = 0.042). Other medications were prescribed similarly in both groups.

Discussion
We showed that decreased urinary Klotho and increased urinary S100A8/A9 levels may be helpful in differentiating between pre-renal and intrinsic AKI. The urinary Klotho/creatinine ratio was decreased in a VD-induced AKI animal model and pre-renal AKI patients. The urinary S100A8/A9/creatinine ratio was elevated in a CDDP-induced AKI animal model and intrinsic AKI patients. Renal Klotho expression was decreased in a VD animal model, and renal S100A8/A9 expression was increased in a CDDP-induced intrinsic AKI model. Renal S100A8/A9 expression in the intrinsic AKI model may be related to the infiltration of neutrophils and macrophages. A defect in the klotho gene results in a syndrome similar to aging [18], whereas overexpression of the klotho gene extends life span [19]. Klotho is expressed in the kidney, brain [18], pituitary, parathyroid [20], pancreas, ovary, testis, and placenta [18]. After its discovery in 1997 [18], subsequent studies have demonstrated various functions of the Klotho protein including anti-oxidation [21], anti-apoptosis, anti-senescence [22], promotion of angiogenesis [23], and inhibition of fibrogenesis [24]. In addition, full-length Klotho, a single-pass transmembrane protein, functions as a co-receptor for fibroblast growth factor-23, thus inhibiting phosphate absorption and promoting phosphaturia by inhibiting 1,25(OH) 2 vitamin D3 synthesis, which may contribute to the prevention of vascular calcification [25,26]. Soluble Klotho modulates Na-phosphate co-transporter-2a [25], renal epithelial calcium channel [27], and the renal outer medullary potassium channel 1 [28]. Animal studies have shown transient renal Klotho deficiency in AKI under a variety of conditions, including hypovolemia [8], nephrotoxins such as CDDP and folic acid [29], ischemia-reperfusion injury [30], lipopolysaccharides [8], and ureteric obstruction [24]. However, there is limited data on whether Klotho may differentiate functional loss from structural damage in the kidney.
Our study demonstrated that serum and urinary Klotho levels significantly decreased in the VD-induced AKI model versus the CDDP-induced AKI model. In the proof-of-concept human study, urinary Klotho concentrations and the urinary Klotho/creatinine ratio were also decreased in the intrinsic AKI group compared to the pre-renal AKI group. These findings suggest that decreased urinary Klotho and the Klotho/creatinine ratio may represent loss of renal function. The results of this study are in accordance with previous observations, showing downregulation of Klotho expression in dehydration [31]. However, the current study differed from a study that showed downregulation of Klotho in a CDDP-induced AKI animal model [29]. That study did not measure serum or urinary Klotho, but only reported renal Klotho protein expression and KL mRNA transcripts. Moreover, only a mouse model was examined. Further studies are needed to determine whether the temporal changes in urinary Klotho during the time course of AKI may alter these findings, and whether the degree of urinary Klotho could reflect the degree of functional loss. S100A8/A9 is calcium-and zinc-binding protein, consisting of a heterotetramer of S100A8 and S100A9 [32]. It is predominantly derived from neutrophils [33], but can also be produced by monocytes, activated macrophages, dendritic cells, and mucosal squamous epithelium [34]. S100A8/A9 is an activator (mediator protein) of the innate immune system and an amplifier of inflammatory activity via activation of Toll-like receptor 4 [35]. S100A8/A9 is an excellent biomarker of inflammatory processes, such as rheumatoid arthritis and juvenile idiopathic arthritis [36]. In addition, elevated S100A8/A9 plasma concentrations are an independent risk predictor for cardiovascular events in both healthy individuals [37] and patients with acute coronary syndrome [38]. It is also useful as a candidate for the detection of unstable versus stable coronary artery plaques [39]. Increased expression of S100A8/A9 has also been recently identified in various human cancers [40,41]. In the field of gastroenterology, fecal S100A8/A9 is a well-known biomarker for differentiating between inflammatory bowel disease and irritable bowel syndrome (a functional disorder without inflammation). Because there is no epithelial inflammation in irritable bowel syndrome, fecal S100A8/A9 concentrations are low. However, they are highly elevated in inflammatory bowel disease. Based on the absence of inflammatory damage in pre-renal AKI, it may be that the S100A8/A9 level does not increase in pre-renal AKI, whereas it is elevated in intrinsic AKI.
In the present study, animal experiments indicated that serum and urinary S100A8/A9 concentrations were elevated in CDDP-induced AKI versus the control groups, whereas there was no significant change in the VD group. In the proof-of-concept human study, the urinary S100A9/A9/creatinine ratio was also elevated in the intrinsic AKI group versus the pre-renal AKI group. Immunohistochemistry and confocal microscopy findings suggested that S100A8/ A9 expression was increased in intrinsic AKI, and its expression was from the recruited inflammatory cells, including neutrophils and macrophages at the glomerular and tubule-interstitial space. These findings suggested that the increased urinary S100A8/A9/creatinine ratio represented structural damage and enhanced inflammatory activity of the kidney. Larger multicenter studies are needed for further validation of the use of S100A8/A9 in heterogeneous patient populations and for defining cut-off values for differential diagnosis and outcomes of AKI. Our results were consistent with those of other studies suggesting that urinary S100A8/A9 concentrations can be used to differentiate between intrinsic and pre-renal AKI [12,42].
Human NGAL was originally recognized as being bound to gelatinase in specific granules of the neutrophil. NGAL is synthesized during granulocyte maturation in the bone marrow [43], and is normally expressed in several human tissues, including the kidneys, lungs, prostate, uterus, stomach, and colon [44]. NGAL expression is induced markedly in injured epithelia in the setting of inflammation or malignancy [45]. NGAL is one of seven genes that show increased expression within the first several hours following ischemic or CDDP-induced nephrotoxic renal injury in animal models [46]. NGAL protein is highly expressed in kidney proximal tubule cells after ischemic or CDDP-induced renal injury and readily detected in the blood and urine shortly after AKI.
NGAL is one of the most widely studied and most promising biomarkers of AKI to date. It has been investigated in various clinical settings of AKI, such as after cardiopulmonary bypass and cardiac surgery [47], sepsis-associated AKI [48], contrast-induced AKI [49], in critically ill patients [50], in patients admitted to the emergency department [51], and AKI following kidney transplantation [52]. Moreover, previous studies showed that urinary NGAL discriminates intrinsic AKI from normal renal function, pre-renal AKI, and CKD in the emergency department [51] and effectively distinguishes pre-renal from intrinsic AKI in all hospitalized patients [11].
In the present study, serum NGAL and urinary NGAL/creatinine were higher in the intrinsic AKI animal model; however, serum NGAL was higher in pre-renal AKI patients. We suggest that this may be affected by the somewhat higher serum creatinine concentration among pre-renal AKI patients than intrinsic AKI, although it was not statistically significant. In addition, the proportion of stage 3 AKI patients by the AKIN criteria also tended to be higher in prerenal AKI than intrinsic AKI. Because previous reports have shown that urinary NGAL is correlated with the degree of renal damage [47], this different degree of renal dysfunction may affect the level of NGAL in both types of AKI patients. Further larger-scale studies are needed to determine the discriminative role of NGAL in AKI patients.
The attenuation of urinary Klotho and increase in urinary S100A8/A9 may contribute to discriminating between pre-renal and intrinsic AKI. The increased S100A8/A9 may indicate an activated immune response and inflammation in intrinsic AKI. Further studies are needed to assess whether biomarker combinations are likely to improve our ability to predict the cause of AKI and its outcomes and to validate the sensitivity and specificity of these biomarker combinations in larger, heterogeneous patient populations.