The Role Of ISG20-mediated Degradation Of Oxidized RNA In Acute Kidney Injury

Excessive reactive oxygen species(ROS)can induce oxidative stress,leading to DNA,RNA,lipid,and protein oxidative damage and ultimately cell death.Compared to the extensive research on DNA,lipid and protein oxidation in various diseases,RNA oxidation has received little attention.In fact,RNA is more vulnerable to oxidation than other cellular components.Cells contain a significantly higher RNA content than DNA,and RNA primarily exists in single stranded form,lacking the protection of histones,resulting in RNA being more sensitive to oxidative stress than DNA.Whether under normal condition or oxidative stress,the level of intracellular RNA oxidation is much higher than that of DNA oxidation.Emerging evidence indicates that RNA oxidation is linked to a range of diseases and pathological conditions,such as neurodegenerative diseases,diabetes,heart failure,hemochromatosis,and β-cell destruction.Acute kidney injury(AKI)stands as a prevalent clinical kidney disease and a significant global public health concern due to its high morbidity and mortality rates.Other than dialysis,no therapeutic interventions reliably enhance survival,limit injury,or expedite recovery.Oxidative stress plays a crucial role in AKI,and is involved in the major pathogenesis of AKI,such as acute renal tubular cell death,hemodynamic changes,and inflammation.Consequently,oxidative stress is regarded as a central exacerbating factor in the progression of AKI.Increased levels of ROS and oxidative stress in AKI may contribute to the production of RNA oxidation.However,it is still unclear whether RNA oxidation is involved in the pathogenesis of AKI.Importantly,the mechanisms of how cells handle oxidized RNA have not been identified in the kidney so far.Therefore,identifying the contribution of RNA oxidation to AKI and the key regulators of RNA oxidation in kidney injuries may provide clues to develop new therapeutic strategies for patients with kidney disease.RNA degradation is believed to play a major role in eliminating oxidized RNA.RNAs are generally degraded by exoribonucleases,and 3’-5’ exoribonucleases are key enzymes in the degradation of superfluous or aberrant RNAs.A deeper research of the pathway of exonucleasemediated oxidized RNA degradation can contribute to a better understanding of the pathogenesis of diseases linked to oxidative stress.Nuclear 3’-5’ exoribonuclease interferon stimulated gene 20(ISG20),belongs to the DEDDh subgroup of the DEDD exonuclease superfamily,and exerts a strong substrate preference of single-stranded RNA(ssRNA)over single-stranded DNA(ssDNA).Previously,ISG20 related research mainly focused on infectious diseases,especially its antiviral activity in degrading viral RNA and hindering viral replication.The key question is whether ISG20,as a 3’-5’ exoribonuclease,participates in oxidative RNA degradation and regulates renal tubular epithelial cell damage in AKI.To address the above questions,this study analyzed the correlation between oxidized RNA and AKI at the levels of clinical,animal and cell models.The deleterious effect of oxidized RNA on renal tubular epithelial cells was determined by using oxidative RNA overload experiments.RNA sequencing was used to screen ISG20 as a significantly increased exoribonuclease in the kidneys from IRI mice.In vitro and in vivo methods such as tubulespecific ISG20 knockout mice and enzyme activity mutants were used to explore the role and mechanism of ISG20 in mediating oxidative RNA degradation and regulating cell damage in AKI,and AKI intervention strategies targeting ISG20-handled RNA oxidation were carried out.This study yielded four main conclusions:1.RNA oxidation is involved in the progression and development of acute kidney injury.Using various oxidized RNA detection methods such as immunohistochemistry staining,immunoenzyme-linked immunosorbent assay,and RNA dot hybridization,this study found that the levels of oxidized RNA in the nuclei of renal tubular epithelial cells were significantly increased in patients with acute tubular necrosis(ATN)and in mice of ischemia-reperfusion(I/R)-and cisplatin-induced AKI models.There exists a positive correlation between the level of renal oxidized RNA and the indicators of kidney damage such as serum creatinine and urea nitrogen.Furthermore,human renal tubular epithelial cells HK-2 were treated with oxidized RNA overload,confirming that oxidized RNA can cause apoptosis and death of renal tubular epithelial cells.These findings represent the first concrete evidence clarifying the correlation between RNA oxidative damage and AKI,providing the direct evidence for the involvement of RNA oxidation in the progression and development of AKI.2.ISG20 is a key regulatory factor for RNA oxidation in AKI.Through RNA sequencing and in vitro and in vivo expression analysis in AKI mouse kidneys,ISG20 was identified as an exoribonuclease with significantly altered expression in AKI.To further validate the significance of ISG20 in the context of AKI,we utilized tubulespecific ISG20 knockout mice and introduced ISG20-specific small interfering RNA(siRNA)into HK-2 cells.These experimental approaches confirm that ISG20 knockout and inhibition of ISG20 expression can aggravate renal damage,renal function loss of mice,and RNA oxidation and cell death of renal tubular epithelial cells under AKI conditions,indicating that ISG20 is the key exoribonuclease in AKI to regulate RNA oxidation.3.ISG20 mediates oxidative RNA degradation and thereby inhibits AKI renal endoplasmic reticulum stress/UPR/cell apoptosis pathway activation.HK-2 cells infected with human ISG20 and enzyme active mutant ISG20D94G adenovirus vectors were used to establish cell models of hypoxia/reoxygenation(H/R),cisplatin,and oxidative RNA overload.The results indicated that overexpression of wild-type ISG20 can significantly reduce RNA oxidation induced by AKI insults and thereby reduce protein aggresomes accumulation,inhibit the activation of unfolded protein response(UPR)signaling molecules and cell apoptosis,while overexpressing ISG20D94G lose these effects.These data indicate that ISG20 relies on its 3’-5’ exoribonuclease activity to restrict RNA oxidative damage induced by AKI insults;ISG20 inhibits the activation of endoplasmic reticulum stress/UPR/cell apoptosis pathway in AKI kidneys by degrading oxidized RNA,which is a key molecular mechanism by which ISG20 regulates renal tubular epithelial cell damage in AKI.4.Targeting ISG20-mediated oxidative RNA degradation is a potential therapeutic strategy for acute kidney injury.Recombinant adeno associated virus 9(AAV9)vectors harboring murine ISG20(AAVmISG20)were delivered into the kidney via intrarenal gene delivery.Then,I/R-induced AKI model was established to evaluate the therapeutic effect of AKI intervention strategies targeting ISG20-handled RNA oxidation,which demonstrated that overexpression of ISG20 can significantly ameliorate renal function loss and damage,prevent renal oxidized RNA accumulation and activation of endoplasmic reticulum stress/UPR/cell apoptosis pathway in AKI mice.In all,targeting ISG20-handled RNA oxidation may be a novel therapeutic strategy for AKI.In summary,this study provides direct evidence that oxidized RNA leads to the damage of renal tubular epithelial cells,clarifies the correlation between RNA oxidation and AKI.ISG20 is screened and identified as a novel regulatory factor for RNA oxidation in AKI.The molecular mechanism that ISG20 mediates oxidized RNA degradation,reduces protein aggresomes accumulation and the subsequent activation of endoplasmic reticulum stress/UPR/apoptosis pathway is elucidated.The effectiveness of a new strategy for AKI prevention and treatment targeting ISG20-handled RNA oxidation using adeno-associated virus vectors is also evaluated.This study innovatively incorporates the concept of RNA oxidative damage into AKI-related research,expands the novel biological functions of ISG20 and proposes a new theory of the scavenging of RNA oxidative damage.Furthermore,it offers fresh targets and novel ideas for the prevention and treatment of AKI.Consequently,this research holds significant theoretical importance and potential practical value for addressing oxidative stress-related diseases,including AKI.