Abstract
BACKGROUND AND AIMS: Several models have been proposed to describe the pathogenesis of Immunoglobulin A nephropathy (IgAN) and, among them, the multihit model where the gut-microbiota may play an important role. These models explain the pathogenesis of IgAN caused by the production of aberrant IgA, but it is believed that further predisposing factors are present, including immunological, genetic, environmental or nutritional factors. Recently, the role of IL-6 in IgAN pathogenesis is becoming increasingly important. It is essential for the deposition of glomerular immunoglobulin A and the development of renal disease in Cd37-deficient mice, although the pathogenetic mechanisms that determine its increase are not well known. A possible hypothesis emerges from our recent work on genome-wide DNA methylation screening in patients with IgAN, which identified, among other findings, a hypermethylated region comprising Vault 2–1 RNA (VTRNA2-1), a non-RNA coding also known as a precursor of miR-886 (pre-mi-RNA). Consistently, VTRNA2-1 expression was found downregulated in IgAN patients. Here we studied the involvement of the VTRNA2-1/PKR/CREB/IL-6 pathway in IgAN. METHOD: Total RNA were isolated from PBMCs of IgAN patients, transplanted IgAN patients (TP-IgAN), non-IgAN transplanted patients (TP) and healthy subjects (HS). VTRNA2-1, CREB and PKR transcripts were evaluated by RT-PCR. Total and phosphorylated PKR, CREB and Il-6 proteins were evaluated by ELISA. Poly (I: C), a synthetic analogue of dsRNA and Pfizer-BioNTech COVID-19 COMIRNATY vaccine were used to transfect patient PBMCs. PKR inhibitor imoxin (C16) 1 µM was used to stimulate patient PBMCs. RESULTS: Here we confirm that VTRNA2-1 transcript was down-regulated in native and transplanted IgAN subjects compared to HS and non IgAN transplanted patients, with a decrease of 30- and 100-folds, respectively (P < 0.05, and P < 0.0001). IgAN patients with downregulated VTRNA2-1 showed a PKR overactivation (fold increase of phosphorilation of 2.6- in IgAN and 2-folds in TP-IgAN patients; P < 0.05), coherently with the role played by VTRNA2-1 that binds to PKR and inhibits its phosphorylation. Then, we found that PKR causes the activation of CREB, a classical cAMP-inducible CRE-binding factor (fold increase of phosphorilation of 3- in IgAN and 2.67-folds in TP-IgAN patients; P < 0.01). CREB, interacting with a region of the IL-6 promoter, led to IL-6 production. Indeed, in IgAN patients we showed a IL-6 mean increase to 120 pg/mL compared to the respective controls (P < 0.05). Moreover, the IL-6 levels correlated with CREB and PKR phosphorylation (r = 0.97; P = 0.0006 and r = 0.89; P = 0.0064, respectively, for IgAN and TP-IgAN patients). Since PKR is normally activated by bacterial and viral RNA, we hypothesized that these microorganisms can further activate the PKR/CREB/IL-6 pathway leading to an excess of IL-6 production. This may explain both the high levels of IL-6, and infection involvement in the disease, and cases of IgAN associated with COVID-19 infection or with COVID-19 RNA-vaccination, and recent data showing microbiota involvement in IgAN. Effectively, we found that IgAN PMBCs stimulated with RNA poly(I: C) or the COVID-19 RNA-vaccine showed a significant increase in IL-6 levels compared to not-stimulated PBMCs (P < 0.05), supporting the pathogentic role played by viral RNA in IgAN pathogenesis and explaining the cases of IgAN patients developing episodes of macrohematuria after a COVID-19 infection or vaccination. Finally, we showed that the IL-6 secretion can be reduced by the PKR inhibitor imoxin (fold decrease of 5-folds in IgAN and TP-IgAN patients; P < 0.05). CONCLUSION: In conclusion, the discovery of the upregulated VTRNA2-1/PKR/CREB/IL-6 pathway in IgAN patients may provide a new pathogenic mechanism in IgAN and may be useful for the development of novel therapeutic approaches, likely by modulating the VTRNA2-1 methylation level in IgAN patients.