key: cord-0944678-pndg2qc0
authors: El-Hefnawy, Sally M.; Kasemy, Zeinab A.; Eid, Hanaa A.; Elmadbouh, Ibrahim; Mostafa, Rasha G.; Omar, Thoria A.; Kasem, Heba E.; Ghonaim, Eman M.; Ghonaim, Mohamed M.; Saleh, Amany A.
title: Potential impact of serpin peptidase inhibitor clade (A) member 4 SERPINA4 (rs2093266) and SERPINA5 (rs1955656) genetic variants on COVID-19 induced acute kidney injury
date: 2022-03-11
journal: Meta Gene
DOI: 10.1016/j.mgene.2022.101023
sha: 4e66d4194c8b11e41b580122fe25419deb073263
doc_id: 944678
cord_uid: pndg2qc0

BACKGROUND: SARS-CoV-2 has a number of targets, including the kidneys. Acute Kidney Injury (AKI) might develop in up to a quarter of SARS-CoV-2 patients. In the clinical environment, AKI is linked to a high rate of death and leads to the progression of AKI to chronic renal disease. AIM: We aimed to investigate rs2093266 and rs1955656 polymorphisms in SERPINA4 and SERPINA5 genes, respectively, as risk factors for COVID-19 induced AKI. SUBJECTS AND METHODS: A case-control study included 227 participants who were divided into three groups: 81 healthy volunteers who served as controls, 76 COVID-19 patients without AKI and 70 COVID -19 patients with AKI. The TaqMan assay was used for genotyping the SERPINA4 (rs2093266) and SERPINA5 (rs1955656) polymorphisms by real-time PCR technique. RESULTS: Lymphocytes and eGFR showed a significantly decreasing trend across the three studied groups, while CRP, d-Dimer, ferritin, creatinine, KIM-1and NGAL showed a significantly increasing trend across the three studied groups (P < 0.001). Rs2093266 (AG and AA) genotypes were significant risk factors among non-AKI and AKI groups in comparison to controls. Rs1955656 (AG and AA) were significant risk factors among the AKI group, while AA was the only significant risk factor among the non-AKI group. Recessive, dominant, co-dominant, and over-dominant models for genotype combinations were demonstrated. The GG v AA, GG + AG v AA, and GG v AG + AA models of the rs2093266 were all significant predictors of AKI, whilst only the GG v AA model of the rs1955656 SNP was a significant predictor. The logistic regression model was statistically significant, χ2 = 56.48, p < 0.001. AKI was associated with progressed age (OR = 0.95, 95% CI: 0.91–0.98, p = 0.006), suffering from chronic diseases (OR = 3.25, 95% CI: 1.31–8.01, p = 0.010), increased BMI (OR = 0.89, 95% CI: 0.81–0.98, p = 0.018), immunosuppressive (OR = 4.61, 95% CI: 1.24–17.16, p = 0.022) and rs2093266 (AG + AA) (OR = 3.0, 95% CI: 1.11–8.10, p = 0.030). CONCLUSION: Single nucleotide polymorphisms (rs2093266) at SERPINA4 gene and (rs1955656) at SERPINA5 gene were strongly linked to the development of AKI in COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-caused global pandemic of coronavirus disease 2019 (COVID-19) is rapidly moving from moderate respiratory tract infection to acute respiratory distress syndrome (ARDS) and multiple organ failure, with a high fatality rate . Kidney affection became prevalent in COVID-19, and acute kidney injury (AKI) influences about 20-40% of COVID-19 patients admitted to intensive care in Europe and the USA and is emerging as a potential indicator of disease severity and decreased patient's survival .

The cause of kidney affection in COVID-19 is complex. Different reasons have been proposed for the progression of AKI, with concomitant cardiovascular disease and predisposing factors such as sepsis, dehydration, and nephrotoxic drug as significant J o u r n a l P r e -p r o o f elements (Ronco et al., 2019) . In critically ill patients, sepsis is the most common condition predisposing them to AKI. It affects up to 50% of patients with sepsis and up to 60% of individuals with septic shock (Poukkanen et al., 2013) . Through T cell apoptosis driven by type I interferon, the cytokine storm syndrome (CRS) inhibits the immune system and causes lymphopenia (reduction of CD8 and CD4 T cells) in persons with COVID-19. IFN-stimulated T cells also produce less ATP and undergo apoptosis (Channappanavar et al., 2016) . In COVID-19, the beginning of cytokine storms is linked to a rise in apoptosis in the kidney and lung. Apoptosis decreases the efficiency of virus recognition via receptors by increasing the availability of nucleic acids (Devaux et al., 2020) .

Apoptosis-related genes, such as serpin peptidase inhibitor clade A (alpha-1 antiproteinase, antitrypsin) member 4 (SERPINA4) and serpin peptidase inhibitor clade A member 5 (SERPINA5), and salt-inducible kinase family 3 (SIK3), are good candidates for AKI because apoptosis appears to be a key mechanism in AKI (Vilander et al., 2017) . The serpins (serine protease inhibitors) are a family of endogenous proteins of molecular weight about 40-60 kDa. They are classified into 16 clades (A-P) (Kelly-robinson et al., 2021) . Clade A serpins are plasma, antitrypsin-like proteins and encoded by genes found on chromosome 14 (Mkaouar et al., 2019) .

The SNP rs2093266 is found in the SERPINA4 gene that encodes kallistatin, a serine proteinase inhibitor with many regulatory functions in biological processes (Vilander et al., 2017) . Kallistatin has two functional domains: an active site and a heparinbinding site, both of which influence a variety of signaling and metabolic pathways.

It contains anti-inflammatory, antioxidant, vasodilator, and angiogenesis inhibitory properties and its apoptosis-related actions (Chao et al., 2016) . SERPINA5 is a 57 kDa glycoprotein and is identified as a protein C inhibitor (PCI). PCI is a heparindependent serpin that exhibits both pro-and anti-coagulant actions and is involved in various anti-inflammatory and anti-coagulant mechanisms (Kelly-robinson et al., 2021) .

There is a lot of diversity in disease behavior among COVID-19 infected individuals; a multifactorial analysis might help uncover the probable risk factors for AKI induction in COVID-19 patients. In order to estimate the COVID-19 patients' genetic predisposition to develop AKI and as per the previous analyses of Vilander et al., J o u r n a l P r e -p r o o f and rs1955656 of SERPINA5 were related to the risk of AKI in severely ill patients with septic shock. Also, both SERPINA4 and SERPINA5 have anti-inflammatory and apoptosis-related activities (Chao et al., 2016 and Kelly-robinson et al., 2021) . Accordingly, we aimed to evaluate the link between genetic polymorphisms in the apoptosis-related genes SERPINA4 (rs2093266) and SERPINA5 (rs1955656) and AKI risk COVID-19 patients. Patients having a positive Reverse Transcription polymerase chain reaction (RT-PCR) for SARS-COV-2 utilizing a nasopharyngeal swab specimen were included in the COVID-19 patients' both groups. COVID-19 patients who developed AKI during hospitalization were included in group3. On the other hand patients with end-stage kidney disease, kidney transplantation, pregnancy and patients with AKI on admission due to any other reason were excluded from the study. Negative RT-PCR patients and patients with a proved other concurrent acute illness were also excluded from the study. At the same time, control subjects were confirmed to be negative by RT-PCR for COVID-19.

Our study comprised adult patients with mild, moderate, severe, and critical COVID-19 infection, as defined by World Health Organization (WHO) criteria for COVID-19 clinical care (World Health Organization, 2021) The KDIGO (Kidney Disease Improving Global Outcomes) criteria will be used to define AKI based on both urinary output and serum creatinine (KDIGO, 2012) . For patients without a baseline creatinine, we used the hospital admission creatinine as a baseline, and eGFR was calculated using the MDRD ml/min per 1.73 m 2 equation.

Inflammatory indicators such as serum ferritin, C-reactive protein (CRP), D-dimer, J o u r n a l P r e -p r o o f and markers of AKI such as kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) are all evaluated in the lab, as well as a complete blood count (CBC) with differential. The chest imaging of these individuals was evaluated using X-rays and computed tomography.

Before blood samples were taken, all individuals completed a permission form authorized by "the Local Ethics & Human Rights committee in Research at Faculty of Medicine, Menoufia University."

Six millilitres of fresh venous blood were drawn using sterile venipuncture. Two millilitres of blood were transferred to an EDTA tube and separated into two aliquots, one for CBC and the other for DNA extraction for further SNP analysis. 1.8 mL was transferred to a sodium citrate tube for the D-dimer assay. The remaining 2.2 ml was transported to a plain tube and centrifuged for 20 minutes at 4000 rpm; the serum was then frozen at -20 °C until serum ferritin, C-reactive protein (CRP), and KIM-1 & NGAL were assayed.

The nephelometric approach was utilized to determine CRP using Mispa-i2 (Agape Diagnostics, Switzerland). Using a chemoilumincence immunoassay, the Architect plus i1000SR immunoassay analyzer was utilized to assess serum ferritin (Abbott, Illinois, USA).

The D-dimer was measured using the stago (STA Compact Max Analyzer, Fully Automated Coagulation System) (Diagnostic Stago, France). WBCs and lymphocytes were measured using the Sysmex XN-1000 Automated Hematology Analyzer (Sysmex Corporation, Kobe, Japan). Quantikine, Canada, USA, supplied human enzyme-linked immunosorbent assay (ELISA) kits for evaluating blood levels of KIM-1 and NGAL. A quantitative sandwich enzyme immunoassay was utilized by the researchers. The primers, probes, and Master Mix were also provided by Thermo Scientific (40X). seconds at 50°C for primer annealing, and 1.5 minutes at 72°C for primer extension), followed by 1 minute at 72°C for the final extension step.

The ABI 7500 real-time PCR software, version 2.0.1, was used to analyse the results.

( Figure 1 a & b).

SPSS version 28.0 [SPSS Inc., Chicago, IL, USA] was used to conduct the analyses.

The significance of the connection between two categories was determined using participants. In every case, in our study we included a total sample size of 227 which was sufficient for the primary outcome and regression analysis.

Age and BMI were significantly higher among AKI than other groups (P<0.001).

Chronic diseases were more prevalent in the AKI group (74.3%) than COVID non-AKI group (27.6%) (P<0.001). Fever and Dyspnea were more common in the AKI group, while muscle ache, abdominal pain, and anorexia were more prevalent in the non-AKI group (P<0.05). Death was reported among 55.7% of the AKI group versus 5.3% among the Non-AKI group (P<0.001) ( Table 1) .

Lymphocytes and eGFR showed a significantly decreasing trend across the three studied groups, while CRP, d-Dimer, ferritin, creatinine, KIM-1and NGAL showed a significantly increasing trend across the three studied groups (P<0.001) ( Table 2 ).

Hardy-Weinberg Equilibrium calculation should non-significance for rs1955656, and rs205 (P>0.05) among all studied groups except rs1955656 showed weak significance among AKI group (P=0.038) (Table 3) .

Rs2093266 (AG and AA) genotypes were significant risk factors among non-AKI and AKI groups compared to controls. Rs1955656 (AG and AA) were significant risk factors among the AKI group, while AA was the only significant risk factor among the non-AKI group (P<0.001) ( Table 4) .

Up on comparing different gene models between the AKI and the non-AKI groups, recessive, dominant, co-dominant, and over-dominant models for genotype combinations were demonstrated. The GG v AA, GG+AG v AA, and GG v AG+AA models of the rs2093266 were all significant predictors of AKI, while only the GG v AA model of the rs1955656 SNP was a significant predictor (Table 5 ).

In our sample, the potential predictors of AKI in COVID-19 patients in comparison to

No AKI group were analyzed. The potential predictors for AKI development were

Age, co-morbidities, BMI, rs2093266 (AG+AA) and being on immunosuppressive.

With age adjustment, it was evident that co-morbidities were the major risk factors of (Table 6 ).

Despite the specific cause of kidney affection in COVID-19 being indistinct, it is commonly associated with sepsis (Zaim et al., 2020) . Also, AKI in COVID-19 patients may occur due to systemic endothelial injury, and extensive production of cytokines and inflammatory mediators in CRS may cause renal tubular cell injury.

Medications or hyperventilation-stimulated rhabdomyolysis can similarly participate in COVID-19-induced AKI (Ertuğlu et al., 2020).

Serpins are a family of circulatory proteins that control proteases enrolled in coagulation, inflammation, and immune response (Gatto et al., 2013) . Also, SERPINA5 or PCI induced expression in mice lungs displayed a marked reduction in proinflammatory cytokines like necrosis factor-alpha (TNF-α) and reduced coagulation activity in lung tissues (Nishii et al., 2006) . All may explain our J o u r n a l P r e -p r o o f finding of an association of SERPINA5 rs1955656 and SERPINA4 rs2093266 variants with the risk of COVID-19.

Our current analysis revealed an association of rs1955656 and rs2093266 with the risk of AKI patients among COVID-19 patients. Additionally, rs2093266 was found as an independent predictor of AKI progression.

Vilander et al., in their analysis, reported a similar finding, as both SNPs rs2093266 of SERPINA4 and rs1955656 of SERPINA5 were found to be related to the risk of AKI in severely ill patients with septic shock (Vilander et al., 2017) . On the same line, Frank and colleagues, in their study, showed rs2093266 and rs1955656 as significant risk factors of AKI in ICUs admitted patients with septic shock (Frank et al., 2012) .

AKI triggered by a complex interaction between inflammation, cell death, and microvascular dysfunction as microemboli or microthrombi as part of the coagulopathy state (Kellum et al., 2020) . Progression of AKI in COVID-19 may also be related to SARS-CoV-2-related immune responses like CRS . Additionally, AKI may develop due to endothelial damage, enhanced vascular permeability, and renal inflammatory reactions (Ertuğlu et al., 2020) . COVID-19associated endotheliitis enhances organ damage, and thrombosis and systemic endotheliitis may clarify multi-organ injury in severe COVID-19 cases (Nägele et al., 2020) . Moreover, pulmonary endotheliitis and microvascular dysfunction may explain the occurrence of severe hypoxia despite relatively conserved lung mechanics in COVID-19 (Gattinoni et al., 2020) .

inducing eNOS and NO production. Moreover, SERPINA4 exhaustion enhances glomerular endothelial cell loss (Chao et al., 2018) . SERPINA4 prevents vascular and organ injury by suppressing inflammation, apoptosis, and oxidative stress.

Furthermore, it had a positive association with the anticoagulation markers and a negative correlation with the inflammatory markers (Lin et al., 2013) . SERPINA4

administration enhances renal performance in rats (Shen et al., 2008) . It has been correlated with renal dysfunction in diabetic patients (Jenkins et al., 2010) and shown J o u r n a l P r e -p r o o f to have a protective role in diabetic nephropathy (Yiu et al., 2016) . It inhibits TNF-α enhanced apoptosis in endothelial cells (Shen et al., 2010) .

Additionally, SERPINA5 or PCI in blood was diminished in patients with hypercoagulopathy, sate-like pulmonary embolism. PCI suppresses circulatory kallikrein, which induces vascular inflammation and enhances vascular permeability (Suzuki, 2008) . SERPINA5 or PCI is expressed in renal tubular cells (Radtke et al., 1994) and

has been correlated with metastasis in renal cell carcinoma (Wakita et al., 2004) .

The age is a potential predictor to development of AKI, but when adjusted it was evident that co-morbidities were the major risk factors of AKI development (OR= This is in agreement with Naser et al., 2021 who concluded that age is a major potential predictor of AKI-induced COVID-19 compared to non-AKI-induced COVID-19 patients. As concluded by Kane-Gill et al., 2015 and Coca et al., 2011 indicates that age is a major potential predictor of AKI compared to patients without AKI. Acute kidney injury is strongly associated with advanced age, and the recent rise in AKI incidence is linked to the growing size of the elderly population (Coca et al., 2011; Ishani et al., 2009; Feest et al., 1993) . Elderly patients are at risk of AKI due to decreased renal reserve and altered kidney function inhibiting kidney function recovery following acute injury (Himmelfarb, 2009 ).

Our results are concurrent with previous analyses, verifying a relationship of COVID-19 severity, progression, and unfavorable consequence with the presence of comorbidities as diabetes mellitus , higher body mass index, and chronic pulmonary disease (L. Xu et al., 2020) .

We realize the limitations of a relatively small sample size, as well as the difficulties of making additional inferences about the relationships between these SNPs and other study indices. Nonetheless, our findings are significant in biomedical research since they demonstrate the ability of the SERPINA4 gene polymorphism rs2093266 to predict COVID-19-related AKI.

J o u r n a l P r e -p r o o f

Based on the results of the current analysis, we can conclude that the genetic variants rs2093266 of SERPINA4 gene and rs1955656 of SERPINA5 might participate in the pathogenesis of COVID-19 and were strongly linked to the development of AKI in COVID-19 patients, specifically rs2093266 (AG+AA) was found as independent predictors of AKI progression.

We appreciate the participation of all patients and controls in this study. We also appreciate Dr. Eman Bader (Central Laboratory Manager) and coworkers from Menoufia University's Faculty of Medicine's Central Laboratory for making this assay possible.

Conflict of interest: All authors declare no conflicts of interest.

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