Tissue Kallikrein Exacerbating Sepsis-Induced Endothelial Hyperpermeability is Highly Predictive of Severity and Mortality in Sepsis

Figures & data

Table 1 Demographic and Clinical Characteristics of Study Population

Characteristics	Controls	SIRS	Sepsis	Septic Shock	P value^A
	(n=76)	(n=42)	(n=62)	(n=88)
Male, n (%)	27 (36%)	26 (62%)	35 (56%)	48 (55%)	0.030*
Age, year	38±14	52±18	53±17	57±17	<0.001**
CCVD, n (%)	10 (13%)	11 (26%)	15 (24%)	18 (20%)	<0.001**
Infection site, n(%)
Lung	–	24 (57%)	33 (54%)	36 (41%)	–
Abdomen	–	8 (20%)	16 (25%)	29 (33%)	–
Urinary tract	–	3 (8%)	6 (10%)	13 (15%)	–
Skin and soft tissue	–	3 (6%)	2 (3%)	2 (2%)	–
Blood	–	3 (6%)	4 (6%)	1 (1%)	–
Others	–	1 (3%)	2 (3%)	6 (7%)	–
Laboratory values
TK levels, mg/l	0.288±0.097	0.334±0.148	0.495±0.170	0.531±0.188	<0.001**
WBC, 10^9/L	9.7±3.5	13.8±7.1	16.2±8.1	15.3±8.6	<0.001**
Platelets, 10^9/L	252±127	208±73	117±73	113±91	<0.001**
Creatinine, umol/L	75±50	250±377	225±247	214±140	<0.001**
CRP, mg/L	8.9±20.9	122±77	160±104	191±259	<0.001**
PCT, ng/mL	1.6±9.2	13.2±19.2	32.4±36.9	47.5±38.7	<0.001**
IL-6, pg/mL	78±299	278±451	1247±1759	2111±1962	<0.001**
PO2/FiO2, mmHg	-	336±68	224±56	198±48	-
Lactate, mmol/L	1.909±0.757	2.118±0.824	4.119±2.492	6.522±4.778	<0.001**
ICU parameters
MAP, mmHg	89±16	97±17	97±17	61±8	<0.001**
ICU days	-	8±3	7±5	12±6	-
Catecholamine days	-	0±0	2±1	5±3	-
Ventilation days	-	2±1	5±2	7±5	-
CRRT days	-	0±0	1±0.8	2±1	-
APACHE II score	-	7±4	18±6	26±8	-
SOFA score	-	1±1	8±3	13±6	-
28-days mortality, n(%)	0 (0)	8 (19%)	30 (48%)	61 (69%)	<0.001**

Notes: Data are presented as n (%) for categorical variables and mean ± standard deviation of the mean (SD) for continuous variables with normal distributions. A groupwise comparison testing performed using χ2 test for categorical variables and One-way analysis of variance (ANOVA) for continuous variables with normal distributions. ^Ap-values for the difference among controls, SIRS patients and patients with sepsis and septic shock, tested with one-way analysis of variance. *P<0.05; **P<0.01.

Abbreviations: CCVD, cardio-cerebrovascular disease; TK, tissue kallikrein; CRP, C-reactive protein; PCT, procalcitonin; IL-6, interleukin 6; PO2, partial pressure of O2; FiO2, fraction of inspiration O2; ICU, intensive care unit; MAP, mean arterial pressure; CRRT, continuous renal replacement therapy; APACHE, acute physiology and chronic health evaluation; SOFA, sequential organ failure assessment.

Table 2 ROC Curves in Assessing the Statue of Sepsis from the 1st Day to 7th Day

Variables	Sensitivity (%)	Specificity (%)	AUC (95% CI)	Youden Index
TK (Day 1)	88.00	76.74	0.878 (0.842–0.908)	0.647
TK+PCT (Day 1)	91.64	89.15	0.948 (0.921–0.967)	0.808
TK (Day 3)	67.03	80.00	0.788 (0.736–0.834)	0.47
TK+PCT (Day 3)	80.00	79.41	0.863 (0.817–0.902)	0.594
TK (Day 7)	81.48	77.5	0.859 (0.811–0.898)	0.59
TK+PCT (Day 7)	85	81.72	0.901 (0.847–0.941)	0.667

Abbreviations: AUC, area under the curve; TK, tissue kallikrein; PCT, procalcitonin; TK+PCT, combination of TK and PCT for assessment of sepsis; Day 1, the first day after clinical onset of sepsis on the ICU; Day 3, 3 days after diagnosis; Day 7, 7 days after diagnosis.

Figure 1 The mean plasma tissue kallikrein (TK) levels in controls and septic patients. TK levels in controls (76 controls and 42 SIRS patients) and septic patients who were admitted to ICU (62 septic patients and 88 patients with septic shock) on the day of diagnosis (Day 1), 3 days after diagnosis (Day 3), and 7 days after diagnosis (Day 7) are displayed. Data were presented as mean ± standard deviation of the mean (SD). One-way analysis of variance was used to indicate statistical difference. ****P<0.0001.

Figure 2 Diagnostic significance of TK on sepsis. ROC curves were plotted and pairwise comparisons were made among TK, CRP, WBC, PCT, IL6, and combination of TK and PCT for assessment of sepsis on the day of diagnosis (A–C), 3 days after diagnosis (D–F), and 7 days after diagnosis (G–I). Area under the curve (AUC) is shown in (A and B), (D and E), and (G and H) groups.

Table 3 Correlations Between TK Levels and General Variable in Septic Patients

Variable	Correlations	P value
Age	0.152	0.071
CRP	−0.038	0.673
PCT	−0.176	0.039*
IL-6	0.087	0.333
WBC	0.047	0.585
MAP	−0.156	0.007**
HCT	−0.16	0.059
Platelets	−0.06	0.482
ALT	0.181	0.032*
AST	0.281	<0.001**
Albumin	−0.021	0.808
BUN	0.022	0.799
Creatinine	−0.115	0.177
PO2	−0.057	0.515
Lactate	0.202	0.018*

Note: *P<0.05, **P<0.001.

Abbreviations: CRP, C-reactive protein; PCT, procalcitonin; MAP, mean arterial pressure; HCT, hematocrit; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urine nitrogen; PO2, partial pressure of O2; SOFA, sequential organ failure assessment; APACHE, acute physiology and chronic health evaluation.

Figure 3 Tissue kallikrein (TK) levels predicted the severity of sepsis. (A) Mean plasma TK levels in controls and patients with SIRS, sepsis, and septic shock. Data are expressed as mean ± standard deviation of the mean (SD). One-way analysis of variance was used to indicate statistical difference. ****P < 0.0001. (B) Correlation between TK levels and SOFA score. TK levels and SOFA score were highly correlative by bivariate correlation (R=0.311, P<0.001). (C) Correlation between TK levels and APACHE II score. TK levels and APACHE II score were highly correlative by bivariate correlation (R=0.311, P<0.001).

Figure 4 Kaplan–Meier survival curves based on plasma tissue kallikrein (TK) level. 1 = quartile 1; 2 = quartile 2; 3 = quartile 3; 4 = quartile 4. Compared with Quartile 1, Quartile 2 (24%, 26 days vs 8%, 27 days, Log rank test, p =0.079); Quartile 3 (33%, 24 days vs 8%, 27 days, Log rank test, p=0.008), and Quartile 4 (89%, 11 days vs 8%, 27 days, logrank test, p < 0.001) had higher risk of death and shorter event-free survival time.

Figure 5 Tissue kallikrein (TK) exacerbated sepsis-induced endothelial hyperpermeability. TK treatment and Kallistatin treatment of HUVECs with LPS-induced sepsis model resulted in endothelial hyperpermeability (A) Real time electric cell–substrate impedance sensing (ECIS) measurements were conducted using normalized transcellular electrical resistance (TER) to record HUVECs permeability from 3 independent experiments. The lines denote the mean normalized resistance TERs ± standard deviation of the mean (SD). (B) Mean normalized TERs were measured from 3 independent experiments using ECIS. ****P < 0.0001 versus control; ^#### P < 0.0001 versus sepsis by two-tailed t-test. (C–F) Protein expressions of TK, ZO-1, and VE-cadherin were analyzed by Western blotting. Representative immunoblots were presented in the bar charts. The quantified OD of bands was corrected for the corresponding GAPDH. Data were presented as the mean ± standard deviation of the mean (SD). ****P < 0.0001 versus control; ^#P < 0.05 versus sepsis; ^##P < 0.01 versus sepsis; ^####P < 0.0001 versus sepsis by two-tailed t-test.

Figure 6 Experimental Design. Note: Created with BioRender.com.