Acute Kidney Injuries in different stages of COVID-19: An analysis on patients without prior kidney disease

Background Kidney involvement in COVID-19 may manifest as acute kidney injury (AKI). This study aimed to analyze and compare AKIs in different stages of COVID-19. 1056 hospitalized COVID-19 patients were retrospectively evaluated and 383 of them met the inclusion criteria. Eighty-nine patients who developed AKI, but didn’t have prior kidney diseases were involved in the nal analysis. Patients were classied into three groups, those who had AKI on admission, those who developed AKI in the rst week and those who developed AKI starting from the 7 th day. Electrolytes, acid-base status and changes in the inammatory markers were compared.


Introduction
The clinical course of Coronavirus Disease 19 (COVID-19) patients who needed hospital admission might be examined in 3 consecutive stages: stage 1 as the early infection period ( rst 3 days after being infected by the virus) stage 2 as the intermediate period (until 7 th day of the illness) with pulmonary involvement and stage 3 as the systemic hyper-in ammation phase. Stage 3 is generally accepted to start within the 2nd week of disease course [1]. While COVID-19 is mainly a respiratory illness, kidneys may also be involved. Multiple pathologic mechanisms have been proposed to explain the cause of kidney involvement including uid balance disturbances, angiotensin II pathway activation, endotheliitis with intravascular coagulation, lung-kidney and heart-kidney cross talks, cytokine release syndrome and drug nephrotoxicity [2,3].
Kidney involvement in COVID-19 can be manifested as acute kidney injury (AKI). Previous studies generally evaluated all forms of AKIs together. However, AKIs may have different characteristics depending on the timing and etiologies. This study aims to analyze the characteristics of AKI in different phases of the disease among hospitalized COVID-19 patients without prior kidney diseases.

Materials And Methods
Setting Patients who were admitted to the designated COVID wards in Cerrahpasa Medical Faculty, a tertiary healthcare center, between 15th March and 1st July 2020 were retrospectively analyzed. This period has been the rst wave of the pandemic and symptomatic patients were admitted immediately.
Hospitalized COVID-19 patients whose disease status was con rmed by a real-time polymerase chain reaction (RT-PCR) test for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-Cov-2) were involved in the study. Kidney transplant patients and those who were younger than 18 years old were excluded from the study. As glomerular ltration rate (GFR) below 60 mL/min/1.73 m 2 was already shown to be related to mortality [4], these patients were also excluded. (Figure-1).

De nitions
To de ne AKI, Kidney Disease Improving Global Outcomes (KDIGO) criteria were used; an absolute increase of 0.3 mg/dl in creatinine levels in 48 hours or 50% increase in creatinine levels in the last 7 days or when urine output is less than 0.5 mL/kg/h for the previous 6 hours. [5].
We observed the progression of creatinine values in all patients who were admitted with COVID-19 diagnosis. In patients with an increase in creatinine levels, we directly applied KDIGO criteria. The rst calculated creatinine level after being admitted to hospital was taken as the baseline creatinine level for these patients. For patients with a decrease in their creatinine levels following hospital admission, KDIGO criteria were applied according to patients' previous creatinine levels. When there was no previous data 7 to 365 days prior to hospital admission, baseline creatinine levels were backwards calculated using the MDRD 75 formula [5,6].
Stage of the AKI was also de ned according to KDIGO criteria; 1.5 -1.9 times baseline creatinine or 0.3 mg/dl absolute increase as stage 1 AKI; 2.0 -2.9 times baseline creatinine as stage 2 AKI and more than 3.0 times baseline creatinine or increase to more than 4.0 mg/dL as stage 3 AKI.
Estimated glomerular ltration rate (eGFR) was used to de ne the kidney functions and it was calculated by Chronic Kidney Diseases Epidemiology Collaboration (CKD-EPI) formula.
We de ned three groups on the basis of the timing of AKI; those seen on admission, those developed in the 1st week and those developed after the 1 st week.

Severity of COVID-19
Clinical picture of COVID-19 patients were classi ed according to a scale that included following categories: Mild (symptoms of upper respiratory tract infection or digestive symptoms), moderate (pneumonia without hypoxemia), severe (pneumonia with hypoxemia) and critical (acute respiratory distress syndrome, shock) [7].

Acquisition of Data and Statistical Analysis
Hospital electronic health records and patient les were used to collect the data. Admission day to the COVID ward was accepted as the day zero of the patient follow-up. Data were expressed as means ± standard deviation. Continuous variables were compared by independent samples t-test. Categorical variables were compared either by Pearson chi-square or Fisher's exact test. For the comparison of three groups that were created according to the timing of AKI, ANOVA test was performed. Tukey HSD test was used for post-hoc analysis. All tests were applied using SPSS for Windows, version 22.0 software (SPSS Inc. Chicago, IL, USA). P values less than 0.05 were accepted as statistically signi cant.

Results
A total of 1056 patients were admitted in this speci ed period. 427 patients were con rmed by RT-PCR. 104 of the PCR con rmed COVID-19 patients experienced AKI (24,3%). 89 patients who developed AKI with an eGFR of over 60 ml/min/1.73 m 2 were included in the nal analysis ( Figure-1). Patients who were 62,4 ± 14,2 years old and there was a male predominance (67 males, 75%).
Twenty-nine (32%) of the patients had AKI on admission. 33 of them (37%) developed AKI during the rst week of admission and 27 patients (30%) developed AKI after the 1 st week. For patients who developed AKI later than hospital admission date, AKI developed on the 6.7th ± 5.4th day of the admission.

Urine analysis
Urine analysis was available in a total of 35 patients. Hematuria was the most prominent nding, which was seen in 21 of them. Proteinuria was documented in 9 patients and they were all 1+ semiquantitavely.
Proteinuria was going along with hematuria in 7 patients while two patients had isolated proteinuria.

Treatment modalities
Although there is no speci c validated treatment for COVID-19 yet, some antiviral therapies were applied in accordance with the ministry of health (MoH) treatment guidelines. These include different combinations of hydroxychloroquine, favipiravir and lopinavir. Anti IL-6 receptor antibody tocilizumab or steroids were used in patients who had high in ammatory response. Low-molecular-weight heparin were prescribed for all patients in line with the MoH guidelines [8]. Continuous renal replacement therapy (CRRT) in ICU setting was performed with Prisma ex® system in a citrate anti-coagulated circuit, aiming a blood ow of around 20 mL/kg/hour.

Comparison according to the timing of AKI
Patients of the three groups (AKI on admission, AKI in the 1 st week, AKI after the 1 st week) were in similar age and had similar baseline mean arterial pressure, creatinine and hemoglobin levels. Co-morbidities such as diabetes, hypertension, malignancies and ischemic heart diseases/heart failure were also similar between three groups. CRP and D-dimer levels on admission didn't differ between the groups. Patients who had AKI on admission day had higher initial uric acid levels. All initial laboratory values of the patients can be found in While there were no signi cant differences between the initial in ammatory markers of the three groups, comparison of changes put forth signi cant differences. Nadir lymphocyte counts were signi cantly lower while peak CRP and peak D-dimer levels were signi cantly higher for patients who developed AKI later in the disease course (Table-3). Although it couldn't reach the statistical signi cance, peak ferritin levels were also higher for patients who developed AKI later.
Sodium, chlorine and potassium abnormalities were more common in patients who developed AKI later (Table-3).
Treatment modalities were similar for all groups. RRT had to be performed in 6 patients who developed AKI later (2 among the 1 st week AKIs and 4 among the AKIs developed after the 1 st week) but none of the patients who had AKI on admission needed RRT.

Comparison between survivors and non-survivors
Duration of hospital stay was not different for survivors and non-survivors. Those who died were older. Patients who survived and who didn't had similar rates of diabetes or hypertension, while concomitant malignancies were more frequent in patients who died (Table-4).
AKI had 24.7% mortality in our patients who had eGFRs above 60 ml/min/1.73 m 2 . AKI developed later in non-survivors and it lasted longer. Non-survivors had signi cantly higher initial CRP, LDH, ferritin and Ddimer levels while their hemoglobin and lymphocyte counts were signi cantly lower (Table-4).
Patients who died had more secondary bacterial infections (OR: 3.5 ; 95%CI: 1.9 -6.4). However, ferritin levels, as a marker of in ammation, were similar in patients who had secondary bacterial infections and in those who hadn't (n=24; 1120 ±691 vs n=62; 976 ± 109; p=0.548). Urea-to-creatinine ratios checked both on the day of AKI and on the day of worst kidney function, were higher in patients who died (p=0,02 and p=0,000 respectively).

Discussion/conclusion
Different studies reported variable AKI incidences in COVID-19 [9][10][11][12]. In the consensus report of Acute Disease Quality Initiative, AKI incidence was reported to be around 20% for hospitalized patients [13]. Same report underlines that AKI may develop in 50% of the patients who needed ICU support. AKI has been proposed as a poor prognostic factor for COVID-19 [14]. In a meta-analysis, it was found that 52% of patients who developed AKI had died [15]. Another study showed that, chronic kidney disease and male sex were independent predictors of AKI severity [16]. However, AKI studies solely analyzing patients with normal kidney functions are scarce. In this study, we focused on the prognosis of AKI of otherwise normal kidneys by excluding patients whose eGFRs were below 60 ml/min/1.73 m 2 . Overall mortality was calculated as 24.7% in this group.
Consequences of all AKIs in COVID-19 might not be the same. As COVID-19 is a febrile illness and patients are experiencing gastrointestinal disturbances, pre-renal AKI is somewhat expected upon admission and should be transient. There may still be AKIs related to other etiologies on admission, and this may be because of differences in the severity of the disease or relatively late referrals of some patients. On admission AKIs were mainly transient pre-renal AKIs (41%) that were responsive to uid therapy in 48 hours. This decreased to 30% for rst week AKIs and to 3% for AKIs after the 1 st week. It may not possible to differentiate between coagulopathy and cytokine release as both pathologies may be intertwined with each other [17,18]. When high levels of ferritin (>750 ng/mL) and D-dimer (>5 mg/L) were taken together, in ammation-mediated injury was around 27.5% for on admission AKIs. This increased to 39.3% for rst week AKIs and it was 59.2% for patients who experienced AKI starting from the second week. Severe or critical COVID-19 was more common in patients who developed AKI later. The mortality of patients who experienced AKI in the early period was 13.7%, and this increased to 44% for patients who had AKI after the 7 th day.
It may be di cult to nd the exact etiology of AKI in the course of COVID-19. Kidney biopsies may give some clues. Direct virulence of SARS-CoV-2 may be responsible for kidney involvement with acute tubular injury and podocytopathies [19][20][21]. In a report of kidney biopsies in COVID-19 patients, podocytopathies and tubulo-interstitial diseases were main ndings while immune mediated glomerular diseases were also found [22]. That study didn't detect virus particles in the kidney. Another study of kidney biopsies on a series of 10 patients found acute tubular necrosis as the leading pathology of AKI. Myoglobin casts as well as thrombotic microangiopathy were also reported [23]. We didn't perform kidney biopsies, as it was neither clinically indicated nor would change treatment modalities in the vast majority of our patients. It's known from before that immune system dysregulation, complement system activation and hypercoagulopathy were all linked with each other [24]. It may not be always possible to de ne which has started before and caused the others. That is why, AKIs in patients either with increasing D-dimer levels or cytokine release syndrome that manifests with increasing levels of ferritin might be associated with the hyper-in ammation state of COVID- 19 [25]..
Patients who develop AKI later had higher peak CRP, D-dimer and ferritin levels. Such higher in ammatory response may point out that later AKI is more immune-mediated. Although secondary bacterial infections could be a confounding factor, ferritin levels, as a marker of in ammatory response in patients with or without secondary bacterial infections didn't differ.
Drug induced nephrotoxicity should not be overlooked in AKIs that develop later. Drugs that resulted in AKI in our patients were non-steroidal anti-in ammatory drugs, antibiotics (e.g. aminoglycosides) and contrast agents that were used for computer tomography scans. Apart from transient pre-renal AKIs, in ammation related AKIs and drug toxicities; we chose not to speculate about other etiologies as that would lead to erroneous interpretations Hyponatremia, and hypochloremia were common electrolyte abnormalities in COVID-19 patients who had AKIs, but they were at a similar rate for survivors and non-survivors. Hypernatremia tended to develop later and this might be related to hypertonic enteral feeding formulas, saline uid administrations or steroid use [26]. Mortality was increased in patients who had hypernatremia or hyperchloremia.
Both hyperpohospatemia and hypophosphatemia were more common in patients who died. Hyperphosphatemia mainly develops as a consequence of GFR loss in patients who have AKI. On the other hand, tubular injury, anti-acid drugs, malnutrition, respiratory alkalosis or CRRTs may be responsible factors for the development of hypophosphatemia. Negative impact of hypophosphatemia on prognosis might be a consequence of decreased diaphragmatic contractility [27].
Our ndings showed that high urea-to-creatinine ratio could be a marker of poor prognosis. These patients might have higher serum urea levels that point out to higher catabolic state and they may also have relatively lower creatinine levels, which is indicative of reduced muscle mass.
There are some limitations of our study. Firstly, due to the retrospective nature of the study, urine analysis and urinary imaging studies were not available for all patients. Sample size is relatively small, and this is because of including only PCR con rmed patients who have eGFRs of over 60 ml/min/1.73 m 2 . Due to reasons stated above, kidney biopsies, which might have given more information about etiologies, were not performed.
In conclusion, AKI in COVID-19 is not of one kind. When developed, AKI should be evaluated in conjunction with the disease stage. Early AKI tends to be more transient and may be more responsive to uid resuscitation. However, AKIs that develop later are more immune-related and have worse prognosis. Patients who develop later AKIs are also more prone to electrolyte abnormalities.
All participants provided written informed consent for their data to be used anonymously for research purposes.

Con ict of Interest Statement
The authors have no con icts of interest to declare 25.Ahmadian E, Khatibi SMH, Soo yani SR, Abediazar S, Shoja MM, Ardalan M, Vahed SZ. COVID-19 and kidney injurt: pathophysiology and molecular mechanisms.