Application of ultrasound microbubble contrast to evaluate the effect of sitaxentan on renal microvascular perfusion in beagles undergoing cardiopulmonary bypass

Abstract

BACKGROUND & OBJECTIVE:

We aimed to evaluate the effect of sitaxentan on renal microvascular perfusion via application of ultrasound microbubble contrast.

METHODS:

Male beagles were randomly divided into: Sham, cardiopulmonary bypass (CPB) and sitaxentan-infused (Sit) groups (n = 6). The ascending slope rate (ASR), area under the curve (AUC), derived peak intensity, and time to peak (TTP) were obtained via ultrasound microbubble contrast before CPB (T₁), after 1 h CPB (T₂), at end of CPB (T₃), and 2 h after CPB (T₄).

RESULTS:

Compared with the Sham group, the CPB group had lower ASR of the renal cortex and medulla at T_2 - 4, higher AUC and TTP at T_3 - 4, and lower derived peak intensity at T₄. The ASR at T_2 - 4 in the Sit group was lower, TTP was higher at T_2 - 4, and AUC was higher at T_3 - 4 (P < 0.05). Compared with the CPB group, the Sit group had higher ASR of the renal cortex and medulla at T_3 - 4 and AUC and TTP at T_3 - 4 (P < 0.05). Compared with that at T₁, the ASR of the renal cortex and medulla at T_2 - 4 in the CPB group was lower, and AUC and TTP were higher at T_3 - 4. The ASR of the renal cortex and medulla at T_2 - 4 in the Sit group was lower, TTP was higher at T_2 - 4, and AUC was higher at T₄ (P < 0.05).

CONCLUSIONS:

Ultrasound microbubble contrast could be effectively used to evaluate renal microvascular perfusion peri-CPB in beagles, which was prone to decrease and could be improved via pretreatment with sitaxentan.

Keywords

Ultrasound kidney microcirculation cardiopulmonary bypass endothelin receptor antagonist

1 Introduction

Patients undergoing cardiopulmonary bypass (CPB) are prone to a higher incidence of acute kidney injury (AKI) [1, 2], which accounts for the main cause of perioperative mortality [2, 3]. Studies on AKI following coronary artery bypass graft surgery using the Acute Kidney Injury Network (AKIN) classification have shown that small increases in serum creatinine level (AKIN class 1) increase the risk of end-stage renal disease by 3-fold and that of mortality by nearly 1.5-fold [4]. Therefore, early diagnosis and treatment are key to prevention and treatment [5, 6]. Renal perfusion is closely related to CPB-associated AKI, and all regions of renal perfusion decrease significantly after the initiation of CPB [7, 8]. This may because venous drainage during CPB can generate a negative central venous pressure (CVP). Vacuum-assisted venous drainage with even lower CVP might result in higher flow to maintain circulation, leading to reduced microcirculatory perfusion of the tissue [9]. Endothelin is a potent vasoconstrictor with two receptors: endothelin A receptor and endothelin B receptor. The endothelin A receptor is mainly expressed in smooth muscle cells in renal vessels, antagonists of which can selectively dilate renal vessels and improve renal microcirculation [10 –12]. In contrast, the endothelin B receptor is principally involved in the clearance of endothelin, particularly in the vascular beds of the lung and kidney [13]. Sitaxentan is a kind of selective antagonist of the endothelin A receptor, which may block the vasoconstrictor effects of the receptor while maintaining the vasodilator and clearance functions of the endothelin B receptor [14]. This study aimed to use ultrasound microbubble contrast to perform real-time, non-invasive, and accurate renal microcirculation monitoring to evaluate the effect of high-selective endothelin A receptor antagonist on renal microcirculation in beagles undergoing CPB. Our findings will provide a theoretical basis for clinical medical care.

2 Materials and methods

Our animal protocol was approved by the Animal Care and Use Committee at Affiliated Hospital of Southwest Medical University in accordance with the requirements of the Chinese Animal Care Committee. All protocols that used animals were in compliance with the ARRIVE guidelines (https://arriveguidelines.org). Beagles were healthy, with no heart or lung disease, no anemia and hypoproteinemia, and no lung damage caused by biological and physicochemical substances.

Eighteen male beagles (10–15 kg, aged 2–4 years) were randomly divided into three groups (n = 6): Sham group (thoracotomy followed by 4 h observation), CPB group (2 h CPB followed by 2 h observation), and Sit group (sitaxentan infusion for 1 h prior to 2 h CPB, followed by 2 h observation).

Intraperitoneal injection of pentobarbital (25 mg/kg) was followed by dynamic monitoring of vital signs using the Dash 3000 monitor, including invasive blood pressure detected from femoral artery, temperature, heart rate, electrocardiogram, and percutaneous blood oxygen saturation in ears. Anesthesia was maintained after intubation with intravenous infusion of fentanyl (0.2μg/kg/min) and vecuronium (0.2μg/kg/min) after establishment of a peripheral venous channel through the superficial vein of a hind limb. Ventilation parameters were set with 10 ml/kg tidal volume and 16–20 breaths/min of respiratory rate to maintain PETCO₂ at 35–45 mmHg (1 mmHg = 0.133 kPa). We did not use vasoconstrictive medication during CPB or after weaning.

When vital signs were stable, a middle incision was made to expose the major blood vessels, followed with intravenous administration of heparin (3 mg/kg). An aortic and venous cannula was placed in the right atrial appendage after the activated clotting time of whole blood was > 380 seconds; CPB was initiated after activated clotting time was > 480 seconds. St. Thomas cardioplegia (20 ml/kg) was infused 10 min after the initiation of CPB from the root of the aorta with the aortic cross-clamp to ensure a bloodless surgical field and to protect the myocardium [15]. Extra 10 ml/kg St. Thomas cardioplegia was infused every 30 minutes. CPB was performed for 2 h (32–34°C, 70–100 ml/kg/min) with 2 h aortic cross clamping; mean arterial pressure was maintained at 50–70 mmHg, and hemoglobin was maintained at > 70 g/L. The beagles were weaned from CPB after stabilizing the internal environment and hemodynamics and received protamine to neutralize heparin, followed by another 2 h of observation in the CPB and Sit groups. Then, 0.7 mg/kg sitaxentan (B29F7E10070, Shanghai Source Leaf Biotechnology Co., Ltd.) infusion was administrated for 1 h prior to 2 h CPB in the Sit group.

Renal microcirculation was evaluated via ultrasound microbubble contrast, which used the real-time harmonic supersonic imaging conditions with low mechanical index (M 10.06). The Philips iU-22 system (Philips, the Netherlands) and the probe type C5-1 were used at the following four time points: before CPB (T₁), after 1 h CPB (T₂), end of CPB (T₃), and 2 h after CPB (T₄) (Fig. 1). The 1 ml ultrasound microbubble contrast agent, which was a hexafluoride sulphur microbubble (Brac co, Italy) with a microbubble density of 2×10⁸/ml and an average diameter of 2.5μm, was injected into the central vein followed by 5 ml of physiological saline. The data were analyzed using QLAB quantitative analysis software (Philips). Two regions of interest, renal cortex and renal cone, were selected to detect changes in the number of echoes of each pixel and contrast agent microbubbles to obtain the time-intensity curve of renal microvascular perfusion. The relevant quantitative parameters, including ascending slope rate (ASR), area under the curve (AUC), derived peak intensity (DPI), and time to peak (TTP), were obtained after Gamma Fitting I(t)=A*t*exp(-t)+c (Fig. 2) [16, 17].

Fig. 1

Flow scheme of experimental animal procedures. Subjects were divided into three groups: Sham, cardiopulmonary bypass (CPB), and Sit groups. Sitaxentan was infused in the Sit group for 1 h prior to 2 h CPB. Renal cortex and medulla microvascular perfusion parameters were detected at the following four time points: before CPB (T₁), after 1 h CPB (T₂), end of CPB (T₃), and 2 h after CPB (T₄).

Fig. 2

Renal microvascular perfusion parameters. A) Ascending slope rate (ASR), B) derived peak intensity (DPI), C) time to peak (TTP), and D) area under the curve (AUC) were obtained via ultrasound microbubble contrast. The decreased ASR and DPI, prolonged TTP, and increased AUC indicate decrease in renal microvascular perfusion.

2.1 Statistical analysis

Data were analyzed using SPSS 20.0 (IBM). Physiological data were assessed for time effect and treatment using Two-Way ANOVA with repeated measurement. Tukey’s test was used to adjust P value results. Unless otherwise noted, data are presented as the mean or percentage (%). Significance was defined as a two-sided P-value<0.05.

3 Results

Compared with that of the Sham group, the ASR of the renal cortex and medulla at T_2 - 4 in the CPB group was lower, AUC and TTP were higher at T_3 - 4, and DPI was lower at T₄. Compared with that of Sham group, the ASR at T_2 - 4 in the Sit group was lower, TTP was higher at T_2 - 4, and AUC was higher at T_3 - 4 (P < 0.05) (Figs. 3 4). Compared with that of the CPB group, the ASR of the renal cortex and medulla at T_3 - 4 in the Sit group was higher, and AUC and TTP were higher at T_3 - 4 (P < 0.05) (Figs. 3 4). Compared with that at T₁, the ASR of the renal cortex and medulla at T_2 - 4 in the CPB group was lower, and AUC and TTP were higher at T_3 - 4. The ASR of the renal cortex and medulla at T_2 - 4 in the Sit group was lower, TTP was higher at T_2 - 4, and AUC was higher at T₄ (P < 0.05) (Figs. 3 4).

Fig. 3

Changes in renal cortex microvascular perfusion. A) Ascending slope rate (ASR), B) area under the curve (AUC), C) derived peak intensity (DPI), and D) time to peak (TTP). *, P < 0.05 vs. T1; #, P < 0.05 vs. Sham; &, P < 0.05 vs. CPB.

Fig. 4

Changes in renal medulla microvascular perfusion. A) Ascending slope rate (ASR), B) area under the curve (AUC), C) derived peak intensity (DPI), and D) time to peak (TTP). *, P < 0.05 vs. T1; #, P < 0.05 vs. Sham; &, P < 0.05 vs. CPB.

4 Discussion

AKI is a serious complication of cardiac surgery, affecting a considerable proportion of patients and increasing postoperative morbidity and mortality [18]. In previous studies, a tracer was administrated to image renal radionuclides and renal blood flow. However, this could not clearly distinguish the renal medulla and cortex blood flow and, most concerningly, exhibits radioactivity [19]. Computed tomography and magnetic resonance imaging perfusion imaging can dynamically assess renal blood flow, but such methods require large, expensive equipment and facilities [20, 21]. Ultrasound microbubble contrast can non-invasively and accurately monitor renal microvascular perfusion in real-time. The method is easy and fast to perform, there is no radiation or renal toxicity, and it can achieve the same evaluation efficiency as radionuclide imaging [8, 22]. Decrease of ASR and DPI, prolonged TTP, increase of AUC indicate decrease of renal microvascular perfusion [8, 16].

Kidney function depends on oxygen delivery, especially under the conditions of nonpulsatile flow generated by CPB. As a result of its blood supply, the kidney enters the hypoxic state under conditions of progressive acute anemia much earlier than the intestine and the heart [23]. Lannemyr et al. demonstrated that renal DO₂ is decreased by 20% following hemodilution and vasoconstriction during CPB, and there is an increase in renal oxygen extraction of up to 45%, which indicates a renal oxygen supply/demand mismatch [24]. Maintaining DO₂ levels above the recommended threshold during CPB improves physiological and clinical outcomes and can ensure balance between the supply and demand of renal oxygen, which has been described as goal-directed perfusion [25 –28]. A recent systematic review and meta-analysis detailed the goal-directed perfusion strategy during CPB and reported that AKI stage 1 is evidently reduced, which reduces overall AKI incidence [29]. In the present study, the peri-CPB average arterial pressure was maintained above 60 mmHg, which was above the normal perfusion pressure previously identified [30, 31]. However, although this experiment met the requirements of goal-directed perfusion, AKI occurred. Ultrasound microbubble contrast showed significantly decreased ASR and DPI, prolonged TTP, and increased AUC, which indicated the deterioration of perfusion in the renal cortex and medulla. These results were in accordance with those reported from previous studies [32, 33]. The reason may be that nonpulsatile perfusion of CPB is non-physiological, and renal microvascular perfusion may also be reduced even under normal perfusion pressure [34]. The underlying cause of the changes should be investigated in detail in future studies. Sitaxentan is a kind of selective antagonist of the endothelin A receptor which is hypothesized to offer an advantage over endothelin A/endothelin B receptor blockade by allowing the endothelin B receptor in endothelial cells to release the vasodilators nitric oxide and prostacyclin and clear ET-1, while suppressing the vasoconstrictor and proliferative actions of the endothelin A receptor [35]. We selected the dosage of IV infusion of sitaxentan (0.7 mg/kg) according to a previous study [36], and administration was carried out 1 h before CPB in accordance with pre-experimental results [8]. The results of the present study showed that ASR increased and AUC and TTP decreased as detected by ultrasound microbubble contrast after pre-CPB administration of sitaxentan, which indicated improvement of renal microvascular perfusion. Barst et al. [37] reported that endothelin A might reduce renal blood flow and glomerular filtration rate without decreasing systemic arterial pressure, which could be reversed by its antagonists; these results support our study. Whether sitaxentan plays a protective effect in renal function via improvement of renal microcirculation remains unknown and requires further study. In summary, sitaxentan could improve renal microcirculation in beagles during CPB; however, the extent of potential improvements remains to be further studied.

Funding

This study was financially supported by Doctoral Research Initiation Fund of Affiliated Hospital of Southwest Medical University (19022).

References

Kourliouros Antonios , Valencia Oswaldo , Phillips Simon

et al. Low cardiopulmonary bypass perfusion temperatures are associated with acute kidney injury following coronary artery bypass surgery[J]. Eur J Cardiothorac Surg. 2010;37:704–9.

Deferrari Giacomo , Bonanni Alice , Bruschi Maurizio et al. Remote ischaemic preconditioning for renal and cardiac protection in adult patients undergoing cardiac surgery with cardiopulmonary bypass: systematic review and meta-analysis of randomized controlled trials[J]. Nephrol. Dial. Transplant. 2018;33:813–824.

Carreno Joseph , Smiraglia Tori , Hunter Christopher et al. Comparative incidence and excess risk of acute kidney injury in hospitalised patients receiving vancomycin and piperacillin/tazobactam in combination or as monotherapy[J]. Int. J. Antimicrob. Agents. 2018;52:643–650.

Rydén

, Sartipy

, Evans

et al. Acute kidney injury after coronary artery bypass grafting and long-term risk of end-stage renal disease. Circulation. 2014;130(23):2005–11.

Pan Chuanliang , Liu Jianping and Hu Xing . [Effect of goal-directed therapy bundle based on PiCCO parameters to the prevention and treatment of acute kidney injury in patients after cardiopulmonary bypass cardiac operation:a prospective observational study][J]. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2019;31:731–736.

Kashani Kianoush , Rosner Mitchell Howard , Haase Michael et al. Quality Improvement Goals for Acute Kidney Injury[J]. Clin J Am Soc Nephrol. 2019;14:941–953.

Graßler Angelika , Bauernschmitt Robert , Guthoff Irene et al. Effects of pulsatile minimal invasive extracorporeal circulation on fibrinolysis and organ protection in adult cardiac surgery-a prospective randomized trial[J]. J Thorac Dis. 1453;11:S1453–S1463.

Bai Yiping , Zhang Yabing , Yang Shuting et al. Protective effect of vascular endothelial growth factor against cardiopulmonary bypass-associated acute kidney injury in beagles[J]. Exp Ther Med. 2018;15:963–969.

Saemann

and Wenzel

. Cutaneous microcirculation during operations with a cardiopulmonary bypass. Clin Hemorheol Microcirc. 2018;69(1-2):13–21.

10.

Regal Jean

, Lund Jenna

, Wing Cameron

et al. Interactions between the complement and endothelin systems in normal pregnancy and following placental ischemia[J]. Mol. Immunol. 2019;114:10–18.

11.

Palygin Oleg , Miller Bradley

, Nishijima Yoshinori et al. Endothelin receptor A and p66Shc regulate spontaneous Ca oscillations in smooth muscle cells controlling renal arterial spontaneous motion[J]. FASEB J. 2019;33:2636–2645.

12.

Larivière Richard , Gauthier-Bastien Alexandra , Ung Roth-Visal et al. Endothelin type A receptor blockade reduces vascular calcification and inflammation in rats with chronic kidney disease[J]. J. Hypertens. 2017;35:376–384.

13.

Benigni

and Remuzzi

. Endothelin antagonists. Lancet. 1999;353:133–138.

14.

Newman

. Treatment of primary pulmonary hypertension-the next generation. N Engl J Med. 2002;346:933–935.

15.

Lazar

. Commentary: The role of del Nido cardioplegia in adult cardiac surgery: The jury is still out. J Thorac Cardiovasc Surg. 2021;162(2):523–525.

16.

Amadori Michele , Barone Domenico , Scarpi Emanuela et al. Dynamic contrast-enhanced ultrasonography (D-CEUS) for the early prediction of bevacizumab efficacy in patients with metastatic colorectal cancer[J]. Eur Radiol. 2018;28:2969–2978.

17.

Dong Yi , Wang Wen-Ping , Lin Pan et al. Assessment of renal perfusion with contrast-enhanced ultrasound: Preliminary results in early diabetic nephropathies[J]. Clin. Hemorheol. Microcirc. 2016;62:229–38.

18.

Elmistekawy

, McDonald

, Hudson

et al. Clinical impact of mild acute kidney injury after cardiac surgery. Ann Thorac Surg. 2014;98(3):815–22.

19.

Keramida Georgia , James Jacqueline

, Prescott Mary

et al. Pitfalls and Limitations of Radionuclide Renal Imaging in Adults[J]. Semin Nucl Med. 2015;45:428–39.

20.

Saad Ahmed , Herrmann Sandra

M S

, Eirin Alfonso et al. Phase 2a Clinical Trial of Mitochondrial Protection (Elamipretide) During Stent Revascularization in Patients With Atherosclerotic Renal Artery Stenosis[J]. Circ Cardiovasc Interv. 2017;10: undefined.

21.

Grenier

, Cornelis

, Le Bras

et al. Perfusion imaging in renal diseases[J]. Diagn Interv Imaging. 2013;94:1313–22.

22.

Ciccone

M M

, Cortese

, Fiorella

et al. The clinical role of contrast-enhanced ultrasound in the evaluation of renal artery stenosis and diagnostic superiority as compared to traditional echo-color-Doppler flow imaging[J]. Int Angiol. 2011;30:135–9.

23.

van Bommel

, Siegemund

, Henny Ch

et al. Heart, kidney, and intestine have different tolerances for anemia. Transl Res. 2008;151(2):110–7.

24.

Lannemyr

, Bragadottir

, Krumbholz

et al. Effects of Cardiopulmonary Bypass on Renal Perfusion, Filtration, and Oxygenation in Patients Undergoing Cardiac Surgery. Anesthesiology. 2017;126(2):205–213.

25.

Ranucci

, Johnson

, Willcox

et al. Goal-directed perfusion to reduce acute kidney injury: A randomized trial. J Thorac Cardiovasc Surg. 2018;156:1918–1927.e2.

26.

Srey

, Rance

, Shapeton

et al. A quick reference tool for goal-directed perfusion in cardiac surgery. J Extra Corpor Technol. 2019;51:172–4.

27.

Magruder

, Crawford

, Harness

et al. A pilot goal-directed perfusion initiative is associated with less acute kidney injury after cardiac surgery. J Thorac Cardiovasc Surg. 2017;153:118–125.e1.

28.

Ranucci

, Romitti

, Isgrò

, et al. Oxygen delivery during cardiopulmonary bypass and acute renal failure after coronary operations. Ann Thorac Surg. 2005;80:2213–20.

29.

Gao

, Liu

, Zhang

et al. Goal-directed perfusion for reducing acute kidney injury in cardiac surgery: A systematic review and meta-analysis. Perfusion. 2023;38(3):591–599.

30.

Hirao

, Minakata

, Masumoto

et al. Recombinant human soluble thrombomodulin prevents acute lung injury in a rat cardiopulmonary bypass model[J]. J Thorac Cardiovasc Surg. 2017;154(6):1973–1983.e1.

31.

, Zhang

, Xie

et al. Role of PI3K/Akt/NF-κB and GSK-3β pathways in the rat model of cardiopulmonary bypass-related lung injury[J]. Biomed Pharmacother. 2018;106:747–754.

32.

Marrazzo Francesco , Spina Stefano , Zadek Francesco et al. Protocol of a randomised controlled trial in cardiac surgical patients with endothelial dysfunction aimed to prevent postoperative acute kidney injury by administering nitric oxide gas[J]. BMJ Open. 2019;9:e026848.

33.

Lankadeva Yugeesh

, Cochrane Andrew

, Marino Bruno et al. Strategies that improve renal medullary oxygenation during experimental cardiopulmonary bypass may mitigate postoperative acute kidney injury[J]. Kidney Int. 2019;95:1338–1346.

34.

O’Neil

, Alie

, Guo

, et al. Microvascular Responsiveness to Pulsatile and Nonpulsatile Flow During Cardiopulmonary Bypass[J]. Ann Thorac Surg. 2018;105(6):1745–1753.

35.

Galie

, Manes

and Branzi

. The endothelin system in pulmonary arterial hypertension. Cardiovasc Res. 2004;61227–237.

36.

Ikonomidis

, Hilton

, Payne

et al. Selective endothelin-A receptor inhibition after cardiac surgery: a safety and feasibility study[J]. Ann Thorac Surg. 2007;83(6):2153–60.

37.

Barst

, Langleben

, Frost

et al. Sitaxsentan therapy for pulmonary arterial hypertension[J]. Am J Respir Crit Care Med. 2004;169(4):441–447.