The effectiveness of continuous renal replacement therapy in critical COVID-19 patients with cytokine release syndrome: a retrospective, multicenter, descriptive study from Wuhan, China

Background: Coronavirus disease (COVID-19) has spread rapidly since 2019. Approximately 15% of the patients will develop severe complications such as multiple organ disease syndrome related to cytokine release syndrome (CRS). Continuous renal replacement therapy (CRRT) can remove inflammatory cytokines through filtration or adsorption. We evaluated the effectiveness of CRRT in COVID-19 patients with CRS. Methods: This retrospective, multicenter, descriptive study included 83 patients with CRS from three hospitals in Wuhan. Results: In COVID-19 patients with CRS, the fatality rate was even higher in CRRT group (P=0.005). However, inflammatory markers such as C-reactive protein, neutrophil counts, and D-dimer decreased after CRRT (P<0.05). Results of Lasso model showed that tracheotomy (β -1.31) and convalescent plasma (β -1.41) were the protective factors. In contrast, CRRT (β 1.07), respiratory failure (β 1.61), consolidation on lung CT (β 0.48), acute kidney injury (AKI) (β 0.47), and elevated neutrophil count (β 0.02) were the risk factors for death. Conclusions: Our results showed that although CRRT significantly reduced the inflammation, it did not decrease the fatality rate of patients with CRS. Therefore, the choice of CRRT indication, dialysis time and dialysis mode should be more careful and accurate in COVID-19 patients with CRS.

AGING of death in COVID-19, associated with a massive inflammatory response mediated by the CRS [7]. SARS and Middle East respiratory syndrome (MERS) have also been shown to be associated with CRS and are closely linked to mortality [8]. CRS is a systemic inflammatory response that occurs mainly in cases of severe infection and is manifested by a sudden increase in the production of a large number of inflammatory factors, such as interleukin-2 (IL-2), IL-4, IL-6, and monocyte chemoattractant protein-1 [9]. Among these inflammatory cytokines, IL-1 and IL-6 are the main pathogenic factors. The cutoff value of specific inflammatory cytokines for CRS in patients with COVID-19 is yet to be defined. Recent research shows that an IL-6 value greater than 100 pg/mL carries the risk of death, and artificial-liver blood-purification therapy can be considered when IL-6 is five times the normal limit [6,10].
Although there are no standard diagnostic criteria for CRS, some treatments based on managing CRS have been widely applied clinically. These include antagonism at the IL-6 receptor (using the antagonist Tocilizumab), artificial-liver blood-purification therapy that can remove cytokines from the circulation, and continuous renal replacement therapy (CRRT) can remove inflammatory factors such as cytokines from the circulation via filtration and adsorption [11]. Tocilizumab has been proved to be effective in COVID-19 at the initial stages [12,13]. CRRT has been used in the treatment of COVID-19 patients with multiple organ failure, especially when accompanied by refractory CRS, and in some critical patients with acute kidney injury (AKI). One case report suggests that CRRT is initially effective in the treatment of patients with COVID-19 [14]. However, as little data on this topic exists, there is no definitive conclusion regarding the effectiveness of CRRT. In this study, we retrospectively collected and analyzed the clinical data of COVID-19 patients with CRS from three hospitals in Wuhan, to determine the effectiveness of CRRT.

RESULTS
Of the 83 cases included in our study, 67 were classified as critical, and 16 as non-critical. Of the 67 critical patients, 38 cases were treated with CRRT during hospitalization. A total of 45 patients died, and 36 were cured or improved. The outcome of two patients is unknown.

Patients demographics
The mean age of the patients was 67.3±12.6 years. Almost three-quarters (73.5%) of the patients were male; 67 were critical cases, and 13.3% of the patients had a history of smoking. The underlying medical conditions of the patients included hypertension (55.4%), diabetes (24.1%), tumors (6%), and chronic obstructive pulmonary disease (1.2%). Pulmonary CT revealed that 51.7% of the critical patients had consolidation, and 73.3 % of non-critical patients had ground-glass changes in the lungs. The mean peak creatinine value during hospitalization was 133.2 pg/mL (range, 72.8 to 276.7). AKI was present in 60.2%, MODS in 36.1%, and ARDS in 42.2% of the patients. Compared to the non-critical groups, the critical group had more patients with an IL-6 value >4000 pg/mL (29.9% vs.18.8%) ( Table 1).

The role of CRRT in critical COVID-19 patients with CRS
Over half of the critical patients (38/67) were treated with CRRT. Those who received CRRT had a lower blood platelets (PLT) (P=0.023), and lower albumin (P=0.041), a higher peak creatinine (P=0.018), and a greater incidence of MODS (P<0.001) than those who did not receive CRRT. More patients receiving CRRT also underwent tracheal cannulation (P=0.003) or invasive mechanical ventilation (P=0.002). Most patients who received CRRT (64.7%) had pulmonary consolidations on CT (Table 2). Unexpectedly, mortality was higher in the CRRT group than that in the non-CRRT group (P=0.005).
Compared to the non-CRRT group, the CRRT group had more patients with an IL-6 value >4000 pg/mL (24.1% vs. 34.2%). The blood oxygen saturation in patients who received CRRT was lower than in the non-CRRT group AGING   Table 2). Compared with the non-CRRT patients, the patients in the CRRT group appeared to be in a worse condition, although there was no statistical difference in the indicators mentioned above.
For the 38 patients treated with CRRT, the changes of inflammation-related indicators before and after CRRT were compared. These included the count and percentage of neutrophils, WBC counts, lymphocytes counts, and levels of IL-6, CRP, D-dimer, and PCT. The analysis showed that after CRRT, WBC counts (P=0.039), neutrophil counts (P=0.014), CRP (P=0.049), D-dimer (P=0.006) all declined significantly from the values before CRRT. However, lymphocytes, PCT and IL-6 did not change significantly (Table 3).
Logistic regression analysis of 66 critical patients further showed that respiratory failure was an independent risk factor for death (odds ratio [OR], 0.06; 95% confidence interval [CI], 0.01-0.38, P<0.001), while tracheotomy was found to be an independent protective factor for death (OR, 68.72; 95% CI, 4.81-10404.57, P<0.001). Therefore, it is suggested that timely tracheotomy should be performed in critically ill patients with respiratory failure (Table 5).

DISCUSSION
In this study, we found that the fatality rate of critical COVID-19 patients with CRS who received CRRT was AGING   Table 4, these variables such as cough, dyspnea, hormone treatment, Invasive mechanical ventilation, acute myocardial injury, WBC count (10 9 /L), Neutrophil count (10 9 /L), Lymphocyte count (10 9 /L), Albumin (g/L), LDH (U/L), maximum creatinine value during hospitalization(μmol/L), CRP (mg/L) and D-dimer (ug/mL) were also included into lasso model. However, their coefficients were shank to 0 by the model due to their negligible effects, therefore these variables were not included in the table. higher than that of those who did not. This means that the clearance of inflammatory factors by CRRT did not improve the prognosis of CRS. Furthermore, the results from Lasso analysis also showed that CRRT was a dependent risk factor for death. The result may not have been expected, and is not consistent with some reports in the literature. For example, several case reports suggest that CRRT can improve the prognosis and may be a potential treatment for COVID-19 [15][16][17]. However, the results of Fominskiy E et al. also show that CRRT does not reduce the mortality of patients with COVID-19, and CRRT also carries a higher risk of in-hospital AGING death [18]. Therefore, whether CRRT can improve the prognosis of critical COVID-19 patients with CRS is still a controversial question.
In our study, the patients in the CRRT group were more seriously ill. Compared with the non-CRRT patients, they had a higher level of peak creatinine and had a higher incidence of MODS or ARDS. In addition, the unsuitable choice of dialysis timings or dialysis mode, and dialysisrelated complications, such as bleeding caused by anticoagulation, might contribute to the high fatality rate in the CRRT group [19]. The therapeutic effect of CRRT is reduced when IL-6 suddenly increases to more than 100 pg/mL or when other complications are present [6,20]. Therefore, when COVID-19 patients develop CRS, the use of CRRT should not be delayed. As currently recommended for interventional treatment of artificialliver blood-purification, when IL-6 is five times the normal limit, the application of CRRT should be considered. If the characteristics of our cases and the current literature are considered together, IL-6 values greater than 100pg/ml could be an important critical index for intervention. However, the determination of the specific cutoff value of IL-6 for early intervention with CRRT requires further investigation [10]. Although CRRT did not improve the survival of critical COVID-19 patients with CRS, some inflammatory markers such as the CRP, D-dimer, WBC counts, and neutrophil counts decreased significantly after CRRT. It is suggested that CRRT can reduce inflammation in such patients.
The results from Lasso analysis indicated that AKI is a risk factor for death in critical COVID-19 patients with CRS. This result is consistent with the conclusion of a study conducted by Tongji Hospital, another hospital focused on the treatment of COVID-19 patients in Wuhan [21]. The incidence of AKI in our study was much higher than previously reported for ICU patients [2]. It is suggested that in addition to the lung, the kidney is also one of the most frequently affected organs in COVID-19 infections. Recent studies have confirmed that the expression of angiotensin-converting enzyme 2, a cell entry receptor of SARS-CoV-2, is very high in the kidney [22,23]. In addition, the changes in hemodynamics and the damage done by inflammatory cytokines to the kidneys are also the reason for the high incidence of AKI in COVID-19 patients [24,25]. So, the urinary system is a potential route for SARS-CoV-2 infection. In addition, the Lasso analysis suggests that consolidation on lung CT and elevated neutrophils also increase the risk of death.
The incidence of respiratory failure is higher in patients with critical COVID-19 (73.5%), and multiple regression analysis also confirms that respiratory failure is a risk factor for death. Tracheotomy was found to be protective for death among critical COVID-19 patients with CRS, indicating that patients with respiratory failure should undergo tracheotomy and invasive ventilation at the correct time.
In terms of treatment, in addition to CRRT and invasive ventilation, many other methods are used to treat these seriously ill patients. These treatments include convalescent plasma, hormones, globulin, and Chinese medicine, but the benefits are also controversial. In our study, convalescent plasma treatment might have been beneficial for critical COVID-19 patients with CRS, according to Lasso analysis. Hormones and globulin were commonly used treatments in severe SARS or MERS patients in the past. Over 78.3% of the critical patients in this study received hormone therapy for the treatment of COVID-19. Though the treatment with hormones was not found to be either a protective or a risk factor in this study, it should be carefully considered [26].
Regarding Chinese medicine, we did not see any protective factors in our results, which may be inconsistent with some reports [27]. The reason may be that most Chinese medicines are used in patients with mild or moderate symptoms, while most COVID-19 patients with CRS have severe or critical symptoms, and fewer of them are treated with Chinese medicine.
Our study has several limitations. First, though we have compiled cases from three hospitals that mainly treated patients with COVID-19, the sample size is still comparatively small. Second, although some confounding factors were compensated for, the influences of some unknown/unavailable factors cannot be completely excluded. Third, the medical records of two patients are incomplete, and the prognosis is unknown, resulting in missing data.
In conclusion, the fatality rate of CRRT patients did not decrease as expected, and even had an opposite trend in our study. The decision whether and how to use CRRT in COVID-19 patients with CRS should be carefully assessed. In the future, more studies and larger sample size are needed to evaluate the effect of CRRT on COVID-19 patients with CRS. Convalescent plasma therapy might be clinically considered in critical COVID-19 patients, and tracheotomy could be recommended for those who developed respiratory failure.

Study design
In this retrospective, multi-center study, we analyzed results from 83 patients diagnosed with COVID-19 and CRS from December 2019 to July 2020, at three participating hospitals (Wuhan Union hospital, Wuhan AGING Jinyintan Hospital, and Wuhan First Hospital). The following inclusion criteria were applied: a laboratory diagnosis of COVID-19 (a positive throat swab nucleic acid test or positive serum COVID-19 specific antibody test), and a peak IL-6 value >100 pg/mL, or a peak IL-6 value of 50-100 pg/mL with concurrent ARDS or multiple organ disease syndrome (MODS is considered to be critical when respiratory failure requires mechanical ventilation, or the patient experiences shock or multiple organ failure and is transferred to the intensive care unit. After careful evaluation, all the patients included in this study were classified as severe and critical cases and therefore were divided into critical and non-critical groups.
AKI was defined as an increase in serum creatinine by 26.52 mmol/L within 48 hours or by more than 50% from the baseline within 7 days [28].

Data collection
After careful review, data, including demographics, clinical characteristics, laboratory and radiological examinations and treatments, were extracted from the patients' medical records. Patients were classified as critical or non-critical, according to the criteria described in the definitions sections above. We retrieved the values of relevant CRRT indicators obtained before and after CRRT. All laboratory examinations of patients were carried out by trained physicians.

Statistical analyses
Categorical variables were presented as numbers and proportions, and the difference between the groups was determined using the chi-square test or Fisher's exact test. Continuous variables were presented as mean (SD) or median [interquartile (IQR)], and differences between the groups were determined using a twosample t-test or Wilcoxon rank-sum test. Lasso and accurate logistic analysis were conducted to identify the factors related to death. The explanatory variables included treatments (CRRT, tracheotomy, convalescent plasma), respiratory failure, consolidation on lung CT, AKI, elevated neutrophil percentage, cough, dyspnea, hormone treatment, invasive mechanical ventilation, acute myocardial injury, White blood cell (WBC) count (x 10 9 /L), neutrophil count (x 10 9 /L), lymphocyte count (x 10 9 /L), albumin (g/L), lactate dehydrogenase (LDH) (U/L), maximum creatinine value during hospitalization (μmol/L), C-reactive protein (CRP) (mg/L) and D-dimer (ug/mL) levels. Statistical significance was set as twosided with P < 0.05. All the analyses were conducted using R software (version 3.6.2, R Foundation), and the Lasso and acute logistic analysis were performed with the glmnet and logistf package.

Data availability statement
The data underlying this article are available in the article and its online supplementary material.

AUTHOR CONTRIBUTIONS
JX designed the study and HX collected the data. HX and BS drafted the manuscript. JZ and YZ performed the statistical analysis. JX and JZ revised the manuscript. All the authors have reviewed and approved the final version of the manuscript.