SARS-CoV-2 infection alters cellular RNA content. Cellular RNAs are chemically modified and eventually degraded, depositing modified nucleosides into extracellular fluids such as serum and urine. Here we searched for COVID-19-specific changes in modified nucleoside levels contained in serum and urine of 308 COVID-19 patients using liquid chromatography-mass spectrometry (LC-MS). We found that two modified nucleosides, N6-threonylcarbamoyladenosine (t6A) and 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), were elevated in serum and urine of COVID-19 patients. Moreover, these levels were associated with symptom severity and decreased upon recovery from COVID-19. In addition, the elevation of similarly modified nucleosides was observed regardless of COVID-19 variants. These findings illuminate specific modified RNA nucleosides in the extracellular fluids as biomarkers for COVID-19 infection and severity.

Objective: Advances in medical science have made it possible to control diseases such as cancer, autoimmune diseases, and infectious diseases, extending the lifespan of affected people around the world. On the other hand, the number of patients with Chronic Kidney Disease (CKD) is increasing worldwide, and hypertension is a key factor. Suppression of CKD is important for extending healthy life expectancy. In this study, we will clarify the effects of the interaction between hypertension and atherosclerosis on renal fibrosis and aging.

Objective:The epithelial sodium channel (ENaC) in the kidney plays pivotal roles in blood pressure regulation, and the γ subunit is activated by extracellular serine proteases. In proteinuric renal diseases, plasmin filtered through injured glomeruli proteolytically activates γENaC. In addition, filtered plasmin directly causes podocyte injury. We previously reported that Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet developed severe hypertension and proteinuria together with γENaC activation, and that a synthetic serine protease inhibitor, camostat mesilate, mitigated these changes. However, the role of plasmin in DS rats remains unclear. In this study, we evaluated the relationship between plasmin and hypertension as well as glomerular injury and the effects of plasmin inhibitors in DS rats.Design and method:Five-week-old male DS rats were divided into normal-salt (NS) diet, HS diet, and HS + plasmin inhibitors [tranexamic acid (TA, 2 mg/mL TA in drinking water) and synthetic plasmin inhibitor YO-2 (4 mg/kg/day, intraperitoneal injection)] groups. After systolic blood pressure measurement and 24-h urine collection over time for 5 weeks, the rats were sacrificed for biochemical examination.Results:The HS group displayed severe hypertension and proteinuria together with activation of plasmin in urine and γENaC in the kidney, which were not attenuated by TA. On the other hand, YO-2 mitigated both hypertension and proteinuria [SBP (mmHg): NS, 130.8 ± 7.5; HS, 209.0 ± 14.2; HS+YO-2, 183.8 ± 8.4; Urinary protein (mg/day): NS, 15.4 ± 3.2; HS 259.0 ± 129.3; HS+YO-2, 94.5 ± 31.5]. YO-2 inhibited the attachment of plasmin(ogen) to podocytes and alleviated podocyte injury by reducing glomerular apoptotic cells. Furthermore, YO-2 suppressed the upregulation of protease-activated receptor-1 and phosphorylated ERK1/2 as well as mRNA expression of inflammatory and pro-fibrotic cytokines in the kidney.Conclusion:These results indicate that plasmin plays important roles in the development of salt-sensitive hypertension and glomerular injuries in a rat model of hypertension, and suggest that plasmin inhibition could be a potential therapeutic strategy against salt-sensitive hypertension.

Objective:The number of patients with chronic kidney disease (CKD) continues to rise by population aging. Moreover, CKD patients progress to end-stage renal failure and require renal replacement therapy. Many cohort studies have shown that impaired renal function itself is a risk factor for the development of cardiovascular disease, rather than comorbidities such as diabetes or hypertension. However, the molecular mechanisms are not fully understood. We focused on chemical modifications in RNA and their pathophysiological significance. Previously, we have shown that modified nucleosides, the metabolites of modified RNA, have unique physiological effects. For example, we reported that N6-methyladenosine is a true ligand for the A3 adenosine receptor, and that 1-methylguanosine promotes Zika virus replication in the host. In addition, it was found that a number of modified nucleosides are present in serum and excreted into extracellular spaces like urine. Therefore, we hypothesized that the decreased urinary excretion of modified nucleosides upon renal failure may cause abnormally high levels of modified nucleosides in the blood, which may exert detrimental effects on the heart and vasculature.Methods and Results:First, serum samples were collected from 210 CKD patients, and the correlation between serum modified nucleoside levels and eGFR was investigated. The results showed that several modified nucleosides such as 5-methylcytidine and pseudouridine were inversely correlated with renal function (r2 = -0.76, p < 0.0001; r2 = -0.78, p < 0.0001, respectively). Moreover, we found that 1-methylinosine was inversely correlated with renal function and positively correlated with BNP, a typical marker for heart failure (r2 = -0.82, p < 0.0001 vs. eGFR; r2 = 0.41, p = 0.010 vs. BNP). Next, to explore the possibility of cardiovascular injury associated with this elevation of 1-methylinosine, we examined its effect on vascular endothelial cells. We found that the addition of 1-methylinosine significantly increased cell proliferation using cultured human aortic endothelial cells (p = 0.004, n = 6).Conclusions:Through this study, we found that multiple modified nucleosides are significantly elevated in serum of CKD patients. Furthermore, the elevation of 1-methylinosine in serum can induce abnormal proliferative signals in vascular endothelial cells, which may thereby lead to endothelial damage. In conclusion, these results suggest that the elevated blood levels of modified nucleosides could be a novel marker for vascular injury, and might become a potential target for drug intervention in the cardio-renal interaction.