The effect of targeting Tie2 on hemorrhagic shock-induced renal perfusion disturbances in rats

Abstract

Background: Hemorrhagic shock is associated with acute kidney injury and increased mortality. Targeting the endothelial angiopoietin/Tie2 system, which regulates endothelial permeability, previously reduced hemorrhagic shock-induced vascular leakage. We hypothesized that as a consequence of vascular leakage, renal perfusion and function is impaired and that activating Tie2 restores renal perfusion and function.

Methods: Rats underwent 1 h of hemorrhagic shock and were treated with either vasculotide or PBS as control, followed by fluid resuscitation for 4 h. Microcirculatory perfusion was measured in the renal cortex and cremaster muscle using contrast echography and intravital microscopy, respectively. Changes in the angiopoietin/Tie2 system and renal injury markers were measured in plasma and on protein and mRNA level in renal tissue. Renal edema formation was determined by wet/dry weight ratios and renal structure by histological analysis.

Results: Hemorrhagic shock significantly decreased renal perfusion (240 ± 138 to 51 ± 40, p < 0.0001) and cremaster perfusion (12 ± 2 to 5 ± 2 perfused vessels, p < 0.0001) compared to baseline values. Fluid resuscitation partially restored both perfusion parameters, but both remained below baseline values (renal perfusion 120 ± 58, p = 0.08, cremaster perfusion 7 ± 2 perfused vessels, p < 0.0001 compared to baseline). Hemorrhagic shock increased circulating angiopoietin-1 (p < 0.0001), angiopoietin-2 (p < 0.0001) and soluble Tie2 (p = 0.05), of which angiopoietin-2 elevation was associated with renal edema formation (r = 0.81, p < 0.0001). Hemorrhagic shock induced renal injury, as assessed by increased levels of plasma neutrophil gelatinase-associated lipocalin (NGAL: p < 0.05), kidney injury marker-1 (KIM-1; p < 0.01) and creatinine (p < 0.05). Vasculotide did not improve renal perfusion (p > 0.9 at all time points) or reduce renal injury (NGAL p = 0.26, KIM-1 p = 0.78, creatinine p > 0.9, renal edema p = 0.08), but temporarily improved cremaster perfusion at 3 h following start of fluid resuscitation compared to untreated rats (resuscitation + 3 h: 11 ± 3 vs 8 ± 3 perfused vessels, p < 0.05).

Conclusion: Hemorrhagic shock-induced renal impairment cannot be restored by standard fluid resuscitation, nor by activation of Tie2. Future treatment strategies should focus on reducing angiopoietin-2 levels or on activating Tie2 via an alternative strategy.

Keywords: Acute kidney injury; Contrast-enhanced ultrasonography; Endothelium; Hemorrhage; Microcirculatory perfusion; Vascular leakage.

Fig. 1

Experimental protocol. Schematic overview of experimental protocol (a). Hemorrhagic shock was induced by pressure-controlled blood withdrawal, and mean arterial pressure (MAP) was maintained for 1 h at 30 mmHg (shock). After 1 h of shock, animals were resuscitated with fluids (R), which was paralleled by administration of vasculotide (VT) as treatment or PBS as control, and monitored for 4 consecutive hours (b, c). Renal and cremaster perfusion measurements were performed directly after the surgical preparation (baseline), 30 min after shock induction (0.5 h HS), 1 h after shock induction (1 h HS), and 30 min (R + 0.5 h), 1 h (R + 1 h), 2 h (R + 2 h), 3 h (R + 3 h) and 4 h (R + 4 h) after start of fluid resuscitation. Plasma was collected at baseline and before killing (4 h after starting fluid resuscitation). Rats were killed and kidneys were isolated for additional molecular analyses and determination of edema formation. d, e An example of renal perfusion analysis. Regions of interest were drawn in the cortex of the kidney (d). The estimate of perfusion was calculated as the product of microvascular blood volume A and microvascular filling velocity β (e). Two-way ANOVA with Bonferroni post hoc analyses, *P < 0.05 HS group compared to baseline; ^#P < 0.05 HS + VT vs. HS group. Data represent mean ± SD, n = 13

Fig. 2

Cremaster and renal perfusion following hemorrhagic shock and fluid resuscitation. Continuously perfused vessels (a), non-perfused (b) and intermittently perfused vessels (c) in rat cremaster muscle using intravital microscopy. Renal microvascular blood volume A (d), microvascular filling velocity β (e) and estimate of renal perfusion (A*β; f) as assessed by contrast-enhanced ultrasound echography in rats during and after hemorrhagic shock and fluid resuscitation as control (HS; black line) or with vasculotide treatment (HS + VT; grey dotted line). Two-way ANOVA with Bonferroni post hoc analyses, *P < 0.05 HS group compared to baseline; ^#P < 0.05 HS + VT vs. HS group. Data represent mean ± SD, n = 13

Fig. 3

Expression levels of the angiopoietin/Tie2 system. Plasma levels of soluble Tie2 (a), angiopoietin-1 (b) and angiopoietin-2 (c), measured at baseline (white circles) and following hemorrhagic shock and fluid resuscitation (HS; black circles, HS + VT; grey circles). Renal gene expression of Tie2 (d) and renal protein expression of total Tie2 (e) measured at 4 h after fluid resuscitation (HS; black circles, HS + VT; grey circles). Data represent mean ± SD. Plasma: Kruskal–Wallis with Dunn’s analyses. Gene and protein expression: Student’s T-test. *P < 0.05 HS group compared to baseline; ^#P < 0.05 HS + VT vs. HS group. VT; vasculotide

Fig. 4

Expression levels of renal injury markers. Plasma levels of NGAL (a), KIM-1 (b) and creatinine (c), measured at baseline (white circles) and following hemorrhagic shock and fluid resuscitation (HS; black circles, HS + VT; grey circles). Urinary levels of NGAL (d) and KIM-1 (e), measured following hemorrhagic shock and fluid resuscitation (HS; black circles, HS + VT, grey circles). Renal gene expression of NGAL (f) and KIM-1 (g), and renal protein expression of NGAL (h) measured following hemorrhagic shock and fluid resuscitation (HS; black circles, HS + VT; grey circles). Microphotographs (original magnification × 20) of PAS-d stained renal tissue sections from untreated HS rats (i) and VT-treated HS rats (j), showing similar degrees of ischemic tubular injury, as evidenced by tubular dilation and loss of brush borders, tubular injury scores represented in graph (k). Data represent mean ± SD. Plasma: Kruskal–Wallis with Dunn’s analyses. Gene and protein expression: Student’s T-test. *P < 0.05 HS group compared to baseline; # P < 0.05 HS + VT vs. HS group. VT vasculotide, NGAL neutrophil gelatinase-associated lipocalin, KIM-1 kidney injury marker-1