Effects of Ulinastatin on Postoperative Renal Function in Patients Undergoing Cardiac Surgery With Cardiopulmonary Bypass: a Prospective Cohort Study

Background: Ulinastatin is a serine protease inhibitor with anti-inammatory effects. Evidence for the effects of ulinastatin on renal outcomes remains sparse in patients receiving cardiac surgery with cardiopulmonary bypass (CPB). Methods: This prospective cohort study evaluated 413 patients aged 18–70 years who underwent cardiac surgery with CPB, from Aug 2008 to Jul 2019 in Fuwai Hospital, Beijing ,China. The ulinastatin group included 135 patients who received intravenous ulinastatin (1×106 U) after induction of anesthesia. The remaining 278 patients without ulinastatin served as the control group. The primary outcome was the rate of new-onset postoperative acute kidney injury (AKI). The secondary outcome was renal replacement therapy(RRT). Serum creatinine, plasma NGAL, and serum IL-6 levels were evaluated and the CSANGAL Score was calculated. In addition, in-hospital mortality, morbidity, adverse outcomes and 10-year follow-up the survival rate was analyzed. Results: Rate of new-onset AKI was signicantly lower in the ulinastatin group than in the control group (20.00% vs. 32.40%, p=0.009). There was no signicant difference of RRT between the two groups (0.00% vs. 2.16%, p=0.09). In-hospital mortality, morbidity, and adverse outcomes were comparable between the two groups except for a signicantly lower incidence of respiratory failure in the ulinastatin group compared with the control group (0.76% vs. 5.40%, p=0.02). The 10-year follow-up survival rates did not differ signicantly between the two groups. Conclusions: Ulinastatin signicantly reduced postope rative AKI and respiratory failure in patients receiving cardiac surgery with CPB. But ulinastatin did not reduce ICU and hospital stay and mortality. Clinical trial registration:


Introduction
Cardiac surgery-associated acute kidney injury (CSA-AKI) is a frequent and severe complication in adult patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) [1] and the second most common cause of AKI in the intensive care unit (ICU). Notably, AKI is independently associated with the increased need for renal replacement therapy (RRT), length of hospital stay, hospitalization cost, and mortality [2]. However, to date, e cacious drugs that can reduce the incidence of AKI and the requirement for RRT remain limited [3]. Prior investigations indicated that CSA-AKI has associations with systemic in ammatory response syndrome (SIRS), renal ischemia-reperfusion injury, renal hypoperfusion, blood transfusion, and nephrotoxic drugs [4]. Moreover, CPB-induced in ammatory responses are associated with kidney dysfunction [4,5].CSA-AKI was de ned according to the KDIGO criteria in the present study. The KDIGO criteria de ne AKI severity according to serum creatinine (SCr) and urinary output. However, SCr increases relatively late after the onset of kidney injury without speci c symptoms, leading to limitations in the SCr-based de nition of AKI. Neutrophil gelatinase-associated lipocalin (NGAL) is one AKI biomarker that shows promising for identifying acute tubular damage, especially in the subclinical AKI stage, and can represent acute tubular damage before the presence of clinical renal dysfunction [6].
Ulinastatin is a glycoprotein puri ed from healthy human urine as a broad spectrum protease inhibitor, which was demonstrated to reduce complications in patients undergoing cardiac surgery with CPB through its anti-in ammatory and antioxidant activities [7]. However, it remains controversial whether ulinastatin has bene ts for renal function and can reduce morbidity and mortality associated with AKI in patients undergoing cardiac surgery procedures with CPB.
The present study aimed to determine the potential effects of ulinastatin on renal function and long-term survival in patients receiving cardiac surgery with CPB.

Patients And Methods
This single-center prospective cohort study was approved by the Ethics Committee of Fuwai Hospital, Patients aged 18-70 years who underwent cardiac surgery with CPB from Aug 2008 to Jul 2019 in Fuwai Hospital, Beijing ,China were enrolled. Patients with preoperative renal dysfunction were excluded. For inclusion criteria, the patients required: diagnosis of coronary heart disease, valvular heart disease, congenital heart disease, or complex heart disease; cardiac surgery necessitating CPB; normal renal function with GFR ≥60 mL/min/1.73 m2, calculated by the Chronic Kidney Disease Epidemiology Collaboration equation; and no kidney transplantation within the past 365 days. Finally, 413 patients were included in the study, and they were divided into the ulinastatin group (n=135) or the control group (n=278) (Figure 1), according to whether the ulinastatin was used during the surgery or not. The participants did not experience any events that resulted in AKI during the preoperative and intraoperative periods, including hypotension, shock, and use of nephrotoxic drugs, which will be excluded from the whole trials.
The primary outcome of the study was the rate of new-onset AKI ,which was de ned by the KDIGO criteria after surgery. The secondary outcome was the need for RRT. In-hospital mortality and morbidity and adverse outcomes were observed. The CSA-NGAL Score is a tubular damage score based on the level of NGAL and a clinical index for early prediction of subclinical AKI. SCr, pNGAL, and serum IL-6 concentrations were detected by ELISA for each sample, in which pNGAL and IL-6 were detected by emulsion enhanced immune turbidimetry assay and double antibody sandwich ELISA, separately.
A 10-year follow-up was achieved by telephone call, outpatient service, face-to-face visit, QQ, or WeChat. The data ascertained by the follow-up team were as follows: onset of death, loss to follow-up, and renal dysfunction; presence or absence of heart failure, hematosepsis, or re-hospitalization for renal dysfunction, RRT, intermittent hemodialysis, or potential nephrotoxic drugs; levels of blood glucose; and results for renal function. All of these items were recorded. In death cases, information on the date and the primary and secondary causes of death were obtained.

Statistical Analysis
For continuous variables, the Shapiro-Wilk test was used to estimate the normal distribution assumption, and the Levene test was applied to assess the equal variance assumption. Differences in these characteristics between the two groups were evaluated by an independent two-sample t-test or one-way ANOVA. Categorical variables were summarized by frequency or percentage and compared by the chisquare test or Fisher's exact test. Inter-group differences in serum IL-6, pNGAL, and SCr were assessed by two-way repeated-measures analysis of variance (ANOVA) with a Bonferroni post-hoc test. Survival analysis was performed using the Kaplan-Meier method and a log-rank test. All statistical analyses were performed using SPSS Version 18.0 software (SPSS Inc., Chicago, IL). Values of p<0.05 were considered statistically signi cant.

Demographic Data
There were no signi cant differences in the demographic characteristics between the two groups, Preoperative diagnoses comparable between the two groups (Table 1).

Perioperative Data
The intraoperative and the postoperative data were comparable between the two groups ( Table 2).  The differences in these characteristics between the two groups were evaluated by an independent two-sample t-test or one-way ANOVA. Categorical variables were summarized as frequency or percentage and compared by the chi-square test or Fisher's exact test.
There was no signi cant differences in the intraoperative and postoperative data (p>0.05).

Renal Function Outcomes
The incidence of AKI in the ulinastatin group was signi cantly lower than that in the control group (20.00% vs. 32.37%, p=0.009)). Furthermore, a signi cant difference was observed between the two groups (p=0.002) about the subclinical acute kidney injury, calculated by CSA-NGAL Scores ( Table 3). Six of 278 patients in the control group and no patients in the ulinastatin group required RRT, but the difference was not signi cant (p=0.09). The postoperative SCr levels did not differ signi cantly between the two groups (p=0.23; Figure 2a), while the postoperative pNGAL levels (p=0.007; Figure 2b) and IL-6 levels (p=0.001; Figure 2c) were signi cantly lower in the ulinastatin group than in the control group. The CSA-NGAL Score was signi cantly lower in the ulinastatin group than in the control group (p=0.002; Table 3), while the KDIGO Score did not differ signi cantly between the two groups (p=0.07; Table 3) during the rst 24 hours after surgery.

Mortality and Other Adverse Outcomes
The in-hospital mortality rate was 1 case (0.74%) in the ulinastatin group and 1 case (0.36%) in the control group, with no signi cant difference between the two groups (p=0.60). In addition, length of ICU stay, length of mechanical ventilation, and length of hospital stay did not differ signi cantly between the two groups. The two groups also showed no signi cant differences in postoperative complications like stroke, postoperative myocardial infarction, cardiac arrest, deep sternal infection, ICU readmission, surgery-associated reoperation, and RRT, IABP, and ECMO (p>0.05; Tables 2 and 4). Notably, the incidence of respiratory failure was signi cantly lower in the ulinastatin group than in the control group (p=0.02; Table 4).

Discussion
CSA-AKI is a severe complication in adult patients undergoing cardiac surgery with CPB, resulting in prolonged ICU stay and increased short-term and long-term morbidity [8]. Several processes to reduce CSA-AKI incidence have been established, and the e cacy of ulinastatin has been examined in recent years [3,9]. In the present study, we found that ulinastatin signi cantly reduced the incidence of CSA-AKI diagnosed according to the KDIGO criteria. In our study, the incidences of AKI were 20.00% in the ulinastatin group and 32.40% in the control group, consistent with the ndings in recent studies [3]. Nakanishi et al. [10] demonstrated that ulinastatin administered before CPB could decrease postoperative elevation of IL-6 and IL-8 in patients undergoing cardiac surgery with CPB, while Song et al. [11] found that ulinastatin has no effect on attenuation of IL-6, TNF-α, and improvements in renal function. Wang et al. [12]. Indicated that ulinastatin could reduce in ammation and dose dependently attenuate CPBinduced kidney injury in infant piglets. Wan et al. [13] performed a propensity score-matched study and showed that administration of ulinastatin during CPB is associated with a lower incidence of CSA-AKI.
Other studies have indicated that ulinastatin can inhibit polymorphonuclear neutrophils, which is helpful to reduce the risk of bleeding and the requirement for blood transfusion after surgery [14,15]. Although AKI is a complex pathophysiological process rather than a speci c syndrome, especially following cardiac surgery[16], SIRS arising after cardiac surgery plays a pivotal role in AKI development [17,18]. The pathogenesis of SIRS is associated with multiple factors, including surgical trauma, transfusion, hypothermia, blood loss, ischemia-reperfusion injury, immune system activation, and endotoxemia [19]. Ulinastatin has anti-in ammatory properties by inhibiting the activation of various in ammatory pathways [14,20,21]. Thus, serum IL-6 levels were evaluated in this study and found to be signi cantly lower in the ulinastatin group compared with the control group (p=0.001; Figure 2c). The effect of ulinastatin on reducing CSA-AKI is considered to involve the inhibition of in ammation.
Evidence suggests that the KDIGO criteria are superior to other criteria, but have less sensitivity to detect early-stage tubular damage. The KDIGO criteria are based on the SCr level for diagnosis of AKI, and changes in SCr often occur 48 hours to 7 days after the original damage. Furthermore, SCr levels are affected by many related factors, and rescue interventions may be too late to prevent early perioperative AKI. These limitations drive us to search for new biomarkers that re ect early AKI. In addition to the KDIGO criteria, pNGAL, a newly discovered biomarker, was tested to predict kidney injury and prove previous ndings [22]. NGAL plays a role in the early detection of AKI and can be upregulated at an earlier stage than elevation of SCr to meet the KDIGO criteria. NGAL in plasma and urine is upregulated when acute tubular damage occurs, but pNGAL is detectable earlier than uNGAL when tubular damage occurs. In a previous study, pNGAL was used to predict AKI based on the CSA NGAL Score, and the results were proven to be similar to those based on the KDIGO Score[6]. According to the KDIGO criteria and CSA-NGAL Score criteria, the incidences of CSA-AKI were similar in the present study. However, according to the CSA-NGAL Score criteria, 48 (35.60%) patients in the ulinastatin group and 77 (27.70%) patients in the control group were in the region of possible tubular damage, so-called subclinical AKI that is a separate entity and provides a warning to trigger awareness and appropriate clinical therapeutic modi cations [6].
Respiratory failure in the present study was de ned as prolonged ventilation support for more than 48 hours. The ndings demonstrated that ulinastatin could reduce the incidence of respiratory failure, consistent with previous studies [23]. Xu et al. [24] suggested that ulinastatin inhibits the release of proin ammatory cytokines and PMNE, reduces pulmonary injury, and improves pulmonary function after CPB, thereby shortening the intubation time and length of ICU stay. The lung-protective effect of ulinastatin is dose dependent and a lower-than-effective dose could lead to negative results.
The survival rate after ten years of follow-up did not differ signi cantly between the two groups.
According to previous studies, the effect of ulinastatin may be diminished depending on the duration of administration [25], which is ascribed to the fact that ulinastatin is administered through a bolus injection rather than a continuous infusion [26]. Another reason is that the half-life of ulinastatin is about 40 minutes in healthy adults [27], while the duration of CPB is often longer than 40 minutes and the peak of IL-6 occurs at 4-6 hours postoperatively [28]. A meta-analysis showed that ulinastatin could reduce in ammatory cytokines but does not affect hospital mortality [29].
There are some limitations to the present study. First, the study was designed as a single-center prospective cohort design with less power than a randomized controlled trial. The rationale lay in the sparse evidence concerning the effect of ulinastatin on renal outcomes and the nature of a pilot study. It was in accordance with ethical considerations to conduct an observational study rather than an interventional study. Second, there were relatively high rates of loss to follow-up, comprising 14.20% (19/134) in the ulinastatin group and 14.10% (39/277) in the control group. It was a great challenge to achieve follow-up for as long as ten years. Nevertheless, the rates of loss to follow-up were comparable between the two groups and associated with less bias. Third, only SCr was evaluated for the AKI criteria in the present study. The urine output in the KDIGO criteria requires a state of oliguria or anuria lasting for more than 6, 12, or 24 hours, which was infeasible in postoperative ICU settings. For ethical considerations, oliguria and anuria would not be observed for such a long period and should be treated according to the institutional protocol promptly. Furthermore, assessment of AKI by SCr alone has been widely accepted in studies such as the BART study [30] and the ATACAS study [8].
In the present study, the SCr, pNGAL, and serum IL-6 were detected, and CSA-AKI incidences according to the KDIGO criteria and CSA-NGAL Score criteria were analyzed, of which differed signi cantly between the two groups, except for SCr. Meanwhile, respiratory failure in the ulinastatin group was considerably lower than that in the control group. In summary, ulinastatin can signi cantly reduce the incidence of early postoperative AKI during cardiac surgery with CPB without affecting the long-term survival rate.
Declarations Figure 1 Scheme of patient recruitment. Ulinastatin group: patients with ulinastatin administration; Control group: patients without ulinastatin administration. Figure 2