Higher Post-COVID-19 Urinary Mitochondrial DNA Level: Serves a Biomarker of Mitochondrial Distress and Inducer of Inflammatory Cytokines Secretion in Peripheral Blood Mononuclear Cells (PBMCs)

ABSTRACT

Background: Severe acute respiratory corona virus-2(SARS-CoV-2) affected multiple organs including Kidney. SARS-CoV-2 open reading frame protein 3a induces necroptosis in infected cell leading release of mt-DNA, which binds to TLR9 and trigger innate immunity, which may lead to acute allograft injury. Method(s) Sixty-six live related renal allograft recipient previously hospitalized with SARS-CoV-2 infection were recruited after 2-3week of discharge. Patients were categorized either in non-AKI(n=47) or AKI(n=19) group, if hospitalization serum creatinine level was >30% of preCovid serum creatinine. A 50ml urine sample was collected for the umt-DNA gene NADH-ubiquinone oxidoreductase chain1(ND-1) and nuclear 36B4 gene quantification by RT-PCR and urine N-GAL measurement by ELISA. A 10ml blood sample from 10healthy volunteers was collected for PBMCs isolation. A 1x106 PBMCs were stimulated for 24hrs. with 1mug/ml of urinary DNA or CpG oligodeoxynucleotide(5'-tcgtcgttttcggcgcgcgccg-3') in duplicate. Unstimulated PBMCs served as control. The gene expression of IL-10, IL-6, MYD88 was analyzed by the RT-PCR and IL-6, IL-10 level in supernatants by the ELISA. Result(s) The precovid creatinine in non-AKI vs AKI patient was 1.06+/-0.20vs0.97+/-0.27, p=.14;at hospitalization 1.27+/-0.18vs1.84 +/-0.37, p<.001;at discharge 1.09+/-0.20vs1.11+/-0.32mg/dl, p=0.73. The mean ND-1 gene Ct in non-AKI vs AKI was 21.77+/-3.60vs19.44+/-2.58a.u, p=.013. The normalized ND-1 Ct in non-AKI vs AKI was 0.89+0.14vs0.79+/-0.11a.u, P=0.007. The median urinary N-GAL level in non-AKI vs AKI group was 212.78 (range, 219.8-383.06) vs 453.5 (range, 320.2-725.02;p=0.015) ng/ml. The area under curve of ND-1 Ct gene was 0.73, normalized ND-1 Ct was 0.71, uNGAL was 0.66 and normalized uNGAL was 0.68 for detecting the AKI. The IL-10 gene expression was downregulated in umt-DNA treated PBMCs compared to control (-3.5+/-0.40vs1.02+/-0.02, p<0.001). IL-6 and Myd88 gene expression was upregulated. The culture supernatant IL-10 and IL-6 level in umt-DNA treatment PBMCs vs control was 10.65+/-2.02 vs 30.3+/-5.47, p=0.001;and 200.2+/-33.67 vs 47.6+/-12.83, p=0.001 respectively. Conclusion(s) Quantification of umt-DNA can detect the post covid19 mitochondrial distress with higher sensitivity compare to uNGAL. umt-DNA induces robust inflammatory response in PBMCs may exacerbate the post-Covid19 allograft injury.