Elevated Urinary Mitochondrial DNA Induces Inflammation and Associated with Acute Kidney Injury in Sars-Cov-2 Infected Renal Allograft Recipient

ABSTRACT

BACKGROUND: Severe acute respiratory coronavirus-2 (SARS-CoV-2) affected multiple organs including kidney. SARSCoV- 2 open reading frame protein 3a induces necroptosis in infected cell leading release of mtDNA which binds to TLR9 and trigger innate immunity which may lead to acute allograft injury. AIM OF THE STUDY To determine the specificity and sensitivity of urinary mitochondrial DNA (umt-DNA) and neutrophil gelatinase-associated lipocalin (NGAL) in predicting COVID-19-associated acute kidney injury (AKI) mitochondrial stress and inflammation. METHOD(S) Live-related RTRs (n = 66) who acquired SARSCoV- 2 infection and were admitted to a COVID hospital were included and subclassified into AKI (N = 19) with >25% spike in serum creatinine level from the pre-COVID-19 serum creatinine level and non-AKI (N = 47) whose serum creatinine value remained stable similar to the baseline value or a rise of < 25% of the baseline values of pre-COVID-19. A 50 ml urine sample was collected and umt-DNA and N-GAL was determined by the RT-PCR and ELISA methods respectively. A 1 x 106 PBMCs were stimulated for 24 hrs. with 1mug/ml of urinary DNA or CpG oligodeoxynucleotide (5) in duplicate. Unstimulated PBMCs served as control. The gene expression of IL-10 IL-6 and MYD88 was analyzed by the RT-PCR and IL-6 IL-10 level in supernatants by the ELISA. RESULT(S) Both the urinary mitochondrial gene ND-1 and NGAL level were significantly higher in AKI group compared to non-AKI. The mean ND-1 gene Ct in AKI group was (19.44 +/- 2.58 a.u) compared to non-AKI (21.77 +/- 3.60;p = 0.013). The normalized ND-1 gene Ct in AKI was (0.79 +/- 0.11 a.u) compared to non- AKI (0.89+0.14;P = 0.007). The median urinary NGAL level in AKI group was (453.53;range, 320.22-725.02, 95% CI) ng/ml compared to non-AKI (212.78;range, 219.80-383.06, 95%CI;p = 0.015). The median urine creatinine normalized uNGAL was 4.78 (0.58-70.39) ng/mg in AKI group compared to 11.26 ng/mg (0.41-329.71) in non-AKI group. The area under curve of ND-1 gene Ct was 0.725, normalized ND-1 Ct was 0.713, and uNGAL was 0.663 and normalized uNGAL was 0.667 for detecting the AKI and mitochondrial stress. The IL-10 gene expression was downregulated in umt-DNA-treated PBMCs compared to control (-3.5 +/- 0.40 vs 1.02 +/- 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 pg/ml;and 200.2 +/- 33.67 vs 47.6 +/- 12.83pg/ml, p = 0.001 respectively. CONCLUSION(S) Urinary mt-DNA quantification can detect the Covid-associated AKI and mitochondrial distress with higher sensitivity than uNGAL in RTRs and induces inflammation in PBMCs.