The study was conducted at Chung-Ang Jeil Hospital (CAJH), a secondary hospital in Jincheon County, Chungbuk Province, South Korea, and the surrounding areas, with a population of approximately 200,000.Since the World Health Organization declared COVID-19 a pandemic on January 30, 2020, South Korea has implemented an active surveillance system that works closely with private healthcare facilities. The system required all individuals with COVID-19 symptoms or epidemiological links to be tested for COVID-19. All confirmed patients with COVID-19 with ESRD on HD were sent to designated health facilities for proper medical care and isolated maintenance of HD.

Between December 21, 2021, and June 30, 2023, 1058 patients with COVID-19 (aged ≥ 18 years, males: 509, females: 549) were referred to CAJH by the Provincial Ministry of Public Health. The diagnosis of COVID-19 was based on nasopharyngeal swab positivity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by polymerase chain reaction or rapid antigen testing for suspected cases that met the World Health Organization clinical criteria or had epidemiological links. Of the 1058 patients with COVID-19, 120 underwent ESRD-demanding HD.

Three groups were identified for the study. The COVID group (n = 17) comprised patients with confirmed COVID-19 but with normal renal function (estimated glomerular filtration rate > 60 mL/min/1.73m2 by the Chronic Kidney Disease Epidemiology Collaboration equation, 2021). The ESRD group (n = 18) comprised patients who regularly underwent HD at the CAJH for > sixmonths without evidence of SARS-CoV-2 infection. Patients with a previous SARS-CoV-2 infection within six months were excluded from this study.

In the context of a pilot study with sample sizes approximating 20 [11, 12], the recruitment strategy entailed disseminating study information to every 15th COVID-19 patient exhibiting normal renal function and every 7th patient with ESRD. Of these, 17 individuals in the COVID group and 18 in the COVID-ESRD group consented to partake in the research, providing written informed consent. Additionally, 18 ESRD patients undergoing hemodialysis (HD) at CAJH with no prior COVID-19 infection were randomly selected to participate.

This study was approved by the Public Institutional Review Board of the Ministry of Health and Welfare of South Korea (http://irb.or.kr/menu02/summary.aspx, approval no: P01-202308–01-030). It was conducted using the principles of the Declaration of Helsinki. Written informed consent was obtained from all the participants.

Total oxidative stress (TOS) and total antioxidant status (TAS) assays were outsourced to EONE Laboratories (Incheon, South Korea: https://www.eonelab.co.kr/institution/certi_list.asp). The commercial assay kits of TOS and TAS were from Rel Assay Diagnostics, Dusseldorf, Germany (https://www.relassay.com/products). The principles of the assay for TOS and TAS are as follows:

Oxidants in the sample oxidize the ferrous ion-chelator complex to ferric ion, which forms a colored complex with chromogen in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the sample's total amount of oxidant molecules. The assay is calibrated with hydrogen peroxide, and the results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (μmolH2O2 Eq./L).

The Fenton reaction generates the hydroxyl radical that reacts with the colorless substrate O-dianisidine to produce the dianisyl radical, which has a bright yellowish-brown color. Upon adding a plasma sample, the oxidative reactions triggered by the hydroxyl radicals present in the reaction mix are suppressed by the antioxidant components of the plasma. This suppression prevents color change, thereby providing an adequate measure of the total antioxidant capacity of the plasma. The assay results are expressed as mmol Trolox Eq./L.

The oxidativestress index (OSI) was calculated from the following equation:

$$\mathrm=TOS\left(\mu mol _2_2 Equiv./L\right)/\left(TAS\times10\right)\left(mmol Trolox Equiv./L\right).$$

After reviewing the electronic medical records, data for the following variables were collected: age, sex, body mass index (BMI), and comorbidities of diabetes mellitus.

Categorical variables were expressed using frequencies and percentages, while continuous variables were described using means and standard deviations. The chi-squared test was used for categorical variables to investigate the significant difference among the three groups.

Analysis of covariance (ANCOVA) was conducted to examine the group mean differences while controlling for covariates that affect oxidative stress, including age, gender, obesity, and diabetes [13,14,15,16]. Transformations were applied to TOS, TAS, and OSI due to their non-normal distribution and lack of homoscedasticity,precisely, logarithmic transformations for TAS and OSI and a square root transformation for TOS. The Shapiro–Wilk and Levene's tests confirmed the normal distribution and homogeneity of variances of the residuals, respectively [17, 18]. Following these transformations, ANCOVA was reapplied to assess the mean differences between the COVID-ESRD, COVID, and ESRD groups, adjusted for the covariates. A post-hoc Tukey's Honestly Significant Difference (HSD) test was then performed for in-depth pairwise group comparisons [19].

Statistical significance was defined as a two-sided p-value of < 0.05. All statistical analyses were conducted using R Statistical Software (version 4.1.2).