Circulating Extracellular Vesicle-Propagated microRNA Signature as a Vascular Calcification Factor in Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) accelerates vascular calcification via phenotypic switching of vascular smooth muscle cells (VSMCs). We investigated the roles of circulating small extracellular vesicles (sEVs) between the kidneys and VSMCs and uncovered relevant sEV-propagated microRNAs (miRNAs) and their biological signaling pathways. METHODS AND RESULTS: We established CKD models in rats and mice by adenine-induced tubulointerstitial fibrosis. Cultures of A10 embryonic rat VSMCs showed increased calcification and transcription of osterix (Sp7), osteocalcin (Bglap), and osteopontin (Spp1) when treated with rat CKD serum. sEVs, but not sEV-depleted serum, accelerated calcification in VSMCs. Intraperitoneal administration of a neutral sphingomyelinase and biogenesis/release inhibitor of sEVs, GW4869 (2.5 mg/kg per 2 days), inhibited thoracic aortic calcification in CKD mice under a high-phosphorus diet. GW4869 induced a nearly full recovery of calcification and transcription of osteogenic marker genes. In CKD, the miRNA transcriptome of sEVs revealed a depletion of 4 miRNAs, miR-16-5p, miR-17~92 cluster-originated miR-17-5p/miR-20a-5p, and miR-106b-5p. Their expression decreased in sEVs from CKD patients as kidney function deteriorated. Transfection of VSMCs with each miRNA-mimic mitigated calcification. In silico analyses revealed VEGFA (vascular endothelial growth factor A) as a convergent target of these miRNAs. We found a 16-fold increase in VEGFA transcription in the thoracic aorta of CKD mice under a high-phosphorus diet, which GW4869 reversed. Inhibition of VEGFA-VEGFR2 signaling with sorafenib, fruquintinib, sunitinib, or VEGFR2-targeted siRNA mitigated calcification in VSMCs. Orally administered fruquintinib (2.5 mg/kg per day) for 4 weeks suppressed the transcription of osteogenic marker genes in the mouse aorta. The area under the curve of miR-16-5p, miR-17-5p, 20a-5p, and miR-106b-5p for the prediction of abdominal aortic calcification was 0.7630, 0.7704, 0.7407, and 0.7704, respectively. CONCLUSIONS: The miRNA transcriptomic signature of circulating sEVs uncovered their pathologic role, devoid of the calcification-protective miRNAs that target VEGFA signaling in CKD-driven vascular calcification. These sEV-propagated miRNAs are potential biomarkers and therapeutic targets for vascular calcification.

Drug-Repositioning Screening for Keap1-Nrf2 Binding Inhibitors using Fluorescence Correlation Spectroscopy.

The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 µM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.

Anaemia management and mortality risk in newly visiting patients with chronic kidney disease in Japan: The CKD-ROUTE study.

AIM: To investigate the association between iron deficiency anaemia and mortality risk and assess the changes in anaemia and iron status after primary management by a nephrologist. METHODS: In this prospective cohort study, we stratified 951 non-dialysis chronic kidney disease (CKD) G2-G5 patients newly visiting 16 nephrology centres into four groups according to the presence of anaemia with or without iron deficiency. All-cause mortality, cardiovascular (CV)-related mortality, and a change in anaemia and iron status after specialized primary care were the endpoints evaluated. RESULTS: During a median follow-up time of 19 months, the number of all-cause deaths and CV-related deaths were 56 and 26, respectively. Compared with the control group, the groups with isolated anaemia and iron deficiency anaemia had significantly higher all-cause mortalities (isolated anaemia: hazard ratio (HR), 3.37; 95% confidence intervals (CI), 1.76-6.44; iron deficiency anaemia: HR, 3.11; 95% CI, 1.21-8.01) and CV-related mortalities (isolated anaemia: HR, 3.64; 95% CI, 1.36-9.73; iron deficiency anaemia: HR, 3.86; 95% CI, 1.11-13.41). In the isolated anaemia group, erythropoietin-stimulating agent (ESA) prescriptions significantly increased to approximately 70%. However, in patients with both anaemia and iron deficiency, iron prescriptions only increased to 48.1%. CONCLUSIONS: Iron deficiency anaemia and isolated anaemia were associated with all-cause and CV-related mortality. The absence of relative increase in iron prescriptions suggests that iron deficiency should be accurately assessed and iron supplementation should be appropriately used to manage anaemia in non-dialysis patients with CKD.