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Critical roles of cytokine storm and secondary bacterial infection in acute kidney injury development in COVID‐19: A multi‐center retrospective cohort study

Abstract Acute kidney injury (AKI) may develop in patients with coronavirus disease 2019 (COVID‐19) and is associated with in‐hospital death. We investigated the incidence of AKI in 223 hospitalized COVID‐19 patients and analyzed the influence factors of AKI. The incidence of cytokine storm syndrome and its correlation with other clinicopathologic variables were also investigated. We retrospectively enrolled adult patients with virologically confirmed COVID‐19 who were hospitalized at three hospitals in Wuhan and Guizhou, China between February 13, 2020, and April 8, 2020. We included 124 patients with moderate COVID‐19 and 99 with severe COVID‐19. AKI was present in 35 (15.7%) patients. The incidence of AKI was 30.3% for severe COVID‐19 and 4.0% for moderate COVID‐19 (p < 0.001). Furthermore, cytokine storm was found in 30 (13.5%) patients and only found in the severe group. Kidney injury at admission (odds ratio [OR]: 3.132, 95% confidence interval [CI]: 1.150–8.527; p = 0.025), cytokine storm (OR: 4.234, 95% CI: 1.361–13.171; p = 0.013), and acute respiratory distress syndrome (ARDS) (OR: 7.684, 95% CI: 2.622–22.523; p < 0.001) were influence factors of AKI. Seventeen (48.6%) patients who received invasive mechanical ventilation developed AKI, of whom 64.7% (11/17) died. Up to 86.7% of AKI patients with cytokine storms may develop a secondary bacterial infection. The leukocyte counts were significantly higher in AKI patients with cytokine storm than in those without (13.0 × 10⁹/L, interquartile range [IQR] 11.3 vs. 8.3 × 10⁹/L, IQR 7.5, p = 0.005). Approximately 1/6 patients with COVID‐19 eventually develop AKI. Kidney injury at admission, cytokine storm and ARDS are influence factors of AKI. Cytokine storm and secondary bacterial infections may be responsible for AKI development in COVID‐19 patients.

Special Project for Significant New Drug Research and Development in the Major National Science and Technology Projects of China (2020ZX09201007); National Natural Science Foundation (grants 81772046 and 81971816) 2.622-22.523; p < 0.001) were influence factors of AKI. Seventeen (48.6%) patients who received invasive mechanical ventilation developed AKI, of whom 64.7% (11/17) died. Up to 86.7% of AKI patients with cytokine storms may develop a secondary bacterial infection. The leukocyte counts were significantly higher in AKI patients with cytokine storm than in those without (13. pandemic has so far spread to more than 200 countries in the world, infected more than three million persons globally, and caused more than 210,000 deaths globally, 1 surpassing the previous severe acute respiratory syndrome (SARS) epidemic and posing a severe worldwide public health threat. 2 Coronavirus disease 2019 , caused by SARS-CoV-2 has prominent respiratory involvement characterized by the symptoms of viral pneumonia such as fever, fatigue, dry cough, and lymphopenia. 3 Although the lungs are considered as the major targeted organ by SARS-CoV-2 with diffuse alveolar damage, SARS-CoV-2 may also cause damage to other organs such as the heart, liver, and kidneys. Patients could eventually succumb to multiple organ failure, shock, acute respiratory distress syndrome (ARDS), heart failure, and renal failure. [4][5][6] SARS-CoV-2 has been shown to directly attack the kidneys, possibly by binding to converting enzyme-2 (ACE2) on the renal cell membrane. 7 The presence of ACE2 in extrapulmonary tissues could explain the multiorgan dysfunction caused by COVID-19. The kidneys have one of the highest content of ACE2, which SARS-CoV-2 spike protein directly recognizes and binds to. 8 A postmortem study found that clusters of coronavirus particles with distinctive spikes were in the tubularepithelium and podocytes. Diffuse proximal tubule injury with loss of brushborder, and even frank necrosis were observed in COVID-19 cases. 9 A recent meta-analysis showed that COVID-19 patients with severe acute kidney injury (AKI) were at significant risk for mortality (relative risk: 4.19, 95% confidence interval [CI]: 3.31−5.31). 10 In 701 patients with COVID-19 from Wuhan, the epicenter of SARS-CoV-2 outbreak, Cheng et al. found that 5.1% of the patients developed AKI, which was associated with a significantly elevated risk of in-hospital death. 11 A prominent feature of COVID-19 in a sizable proportion of SARS-CoV-2 infected persons is development of ARDS. Elevated production of proinflammatory cytokines/chemokines or even hypercytokinemia, also known as cytokine storm, may develop in SARS-CoV-2 infections and contributes to acute lung injury and development of ARDS. 12,13 And bacterial infection may also aggravate the patient's condition and increase the difficulty of treatment. 14,15 Exaggerated, excessive synthesis of interleukin 6 (IL-6) leads to an acute severe systemic inflammatory response and also can activate the coagulation pathway and vascular endothelial cells, leading to multiorgan dysfunction within a short time. 16,17 In our clinical experience, the sudden massive surge in IL-6 levels makes it very difficult for patients with COVID-19 to recover from kidney injury.
However, currently, there is no study on the role of cytokine storm in kidney injury.
In the current retrospective study, we investigated the incidence of AKI in hospitalized patients with COVID-19 from three medical centers in and beyond Wuhan and analyzed the influence factors of AKI in COVID-19 patients. We further studied the incidence of cytokine storm syndrome and its correlation with other clinicopathologic variables. The study protocol was approved by the Chinese ethics committees of the authors' affiliated institutions (No. 2020020 and No. 2020020). Due to the retrospective nature of the study, informed consent was waived by the ethics committees. Patients provided written informed consent to the collection of their urine samples.
Patient data were anonymized in this paper.

| Data collection
We reviewed the clinical charts, nursing records, laboratory results, and chest computed tomography scans of all patients using the hospitals' electronic records system. Epidemiological, clinical, imaging, serological records, and treatment and outcomes data were collected. To ensure accuracy, four researchers independently reviewed and checked the data form.

| Enzyme-linked immunosorbent assay (ELISA)
Thirty-one urine samples were collected from COVID-19 patients without a history of kidney disease or abnormality of urinary sediment test at admission. The urine samples were centrifuged at 1000 g for 5 min and the supernatants were stored at −20°C for 1 week, and then stored at −80°C. The tubular injury marker, human neutrophil gelatinase-associated lipocalin (NGAL) (ab119600; Abcam PLC) and kidney injury factor-1 (KIM-1) (ab235081; Abcam PLC), and the glomerular injury marker-urinary transferrin (u-TF) (ab217780; Abcam PLC), and urinary microalbumin (ab108788; Abcam PLC) were quantified by ELISA according to the manufacturer's instructions.
Urine samples were diluted in 1:50 and 1:5000 before an ELISA assay to fit the concentrations of respective proteins in the linear range of the standard curve. All the detection results were expressed in absolute terms and also normalized against the urinary creatinine concentration.23

| Statistical analysis
The primary outcome of the study was the incidence of AKI in patients with COIVD-19. Statistical analysis was done with SPSS 23 (SPSS Inc.).
Analyses were descriptive in nature. Summary tabulations included the number of observations; median, interquartile range (IQR) for continuous variables; number and percentage per category for categorical data.
Categorical data were compared using the χ 2 test. Continuous data were compared using Student's t test. Nonnormal distributed continuous data were compared using the Mann-Whitney-Wilcoxon test. Risk factors of AKI were identified by univariate and multivariate analysis and the factors with p < 0.1 entered the multivariate analysis. All p values were two-tailed and p < 0.05 indicated a significant statistical difference. Association rule analysis: support ≥10%, credibility ≥80%, Apriori algorithm.

| Patient demographic and baseline data
This study included a total of 223 patients, 52 patients from Zhaongnan Hospital of Wuhan University, 70 from Leishenshan Hospital, and 101 patients from Jiangjunshan Hospital. The study flowchart is shown in Figure 1, patient demographic and baseline characteristics in Table 1 21.9%, p < 0.001) ( Table 3) Table).

| Influence factors of AKI in COVID-19
Our univariate analysis showed that older and male patients, patients with major chronic diseases, including hypertension and cardiovascular disease, were at significantly increased risk for in-hospital AKI (Table 4). Other AKI risks included COVID severity, lymphopenia, leukocytosis, and elevated CRP, D-dimer, IL-6, kidney injury at admission, and development of cytokine storm and ARDS.
Our logistic regression multivariate analysis revealed that female patients were an independent negative risk factor in the occurrence of AKI (OR: 0.288, 95% CI: 0.095-0.869; p = 0.027). Evidence of kidney injury at admission was associated with more than a threefold increased risk of AKI (OR: 3.132, 95% CI: 1.150-8.527; p = 0.025).

| Cytokine storm syndrome
The median baseline levels of IL-6 were 5.8 pg/ml (IQR: 60.5) in the study cohort and were significantly higher in patients with severe  (Table 3). Furthermore, our association rule analysis showed that cytokine storm exhibited a strong correlation and collinearity with leukocyte counts, lymphocyte counts, IL-6 levels, D-dimer, and positive contact history ( Figure 2). Similar results were observed when we analyzed AKI and newly emerging kidney disease in COVID-19 patients (Table 6).       The cytokine storm occurred 3 days before AKI, and patients died or were discharged on average 6 days after AKI. AKI, acute kidney injury makes the study less susceptible to the confounding factor of medical crowding. Our case ratio of moderate and severe patients is 1.25:1, which makes the attribution analysis of AKI more rational.

| Secondary bacterial infection
Our study has some limitations. First, we underestimated the impact of cytokine storms throughout the study because our diagnostic criteria for cytokine storms were conservative. Second, as limited data were available on the proportion of CRRT in patients with AKI, its impact on AKI was not studied. Last, interpretation of our findings might be limited by the small sample size. However, by including all adult patients in the three designated hospitals for COVID-19, we believe our study population is representative of cases diagnosed and treated in China. In