Association of urinary mitochondrial DNA with COVID-19 related acute kidney injury, mitochondrial stress, and inflammation in renal transplant recipients

ABSTRACT

Introduction: ACE-receptors are profusely expressed in the renal cell, making it highly susceptible for severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) infection. After entering the cells, the virus induces high levels of cytokines, chemokines, and inflammatory responses, resulting neutrophilic infiltration, activation, and profuse reactive oxygen species (ROS) formation, leading to cellular necrosis and acute tubular injury. Proximal convoluted tube cell are rich in mitochondria and susceptible for developing acute kidney injury (AKI) due to mitochondrial stress. Early detection of AKI may helpful in its management, limiting the severity, avoiding nephrotoxic medicines and modifying the drug dose depending on renal function. Therefore, in the current study, we have determined the utility of urinary mitochondrial DNA (umt-DNA) and neutrophil gelatinase-associated lipocalin (NGAL) in predicting COVID-19-associated acute kidney injury (AKI) and mitochondrial stress and demonstrated the inflammatory response of urinary mt-DNA. Method(s) Live-related RTRs(n=66), who acquired SARS-CoV-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 50ml urine sample was collected and umt-DNA and N-GAL was determined by the RT-PCR and ELISA methods respectively. A 10ml blood sample from 10 healthy volunteers was also collected for PBMC isolation and inflammatory response demonstration. A 1x106 PBMC was stimulated for 24hrs. with 1microg/ml of urinary DNA or TLR9 agonist 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) Both the urinary mitochondrial gene ND-1 and NGAL level was 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+/-0.40vs1.02+/-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 pg/ml respectively. Conclusion(s) Urinary mt-DNA quantification can detect the Covid19 associated AKI and mitochondrial distress with higher sensitivity than uNGAL in RTRs. Urinary mt-DNA also induces a robust inflammatory response in PBMCs, which may exacerbate the Covid19 associated allograft injury. No conflict of interestCopyright © 2023