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MiRNA-486-5p Protects Against Kidney Ischemic Injury and Modifies the Transcriptome in Proximal Tubule and Endothelium

ABSTRACT: Background: Acute kidney injury (AKI) is a common clinical event with high morbidity and mortality. We previously demonstrated that injection of cord blood endothelial colony forming cell (ECFC)-derived exosomes, highly enriched in miRNA(miR)-486-5p, prevented ischemic kidney injury in mice. While preclinical models support involvement of several miRs in AKI, the direct effects of miR-486-5p on injury and the kidney transcriptome are unknown. Objective: We studied effects of miR-486-5p in mice with kidney ischemia-reperfusion (IR) injury, and compared the impact of miR-486-5p and ECFC-derived exosomes on the transcriptome of proximal tubules (PTs) and renal endothelial cells. Methods: Adult male mice were subjected to bilateral kidney ischemia (30 min), and injected via tail vein with or without vehicle, miR-486-5p mimic, ECFC-derived exosomes, or scramble miR at the start of reperfusion. Plasma and tissues were collected 24 hrs after reperfusion, and proximal tubular and endothelial cells were isolated for mRNA analysis by RNA-Seq. Results: In mice with kidney IR, injection of miR-486-5p mimic increased levels of miR-486-5p in proximal tubules and endothelial cells, and significantly improved plasma creatinine, histological injury, neutrophil infiltration, and apoptosis, compared to vehicle treatment. MiR-486-5p inhibited expression of phosphatase and tensin homolog (PTEN), and activated AKT. Similar results were observed with exosomes, but scramble miR had no effect. In proximal tubules, miR-486-5p or exosomes reduced expression of several AKI genes (e.g. Lcn2, Havcr1 and Krt20) and caused alterations in apoptotic genes compared to IR alone. In endothelium, miR-486-5p or exosomes caused milder effect than in PTs but still impacting on expression of injury-related genes. Conclusion: MiR-486-5p protects mice against ischemic kidney injury. Both miR-486-5p and exosomes reduce expression of apoptotic genes in PTs and endothelium. The results suggest that systemic delivery of miR-486-5p has therapeutic potential in ischemic AKI.

ORGANISM(S): Mus musculus

PROVIDER: GSE159701 | GEO | 2023/10/10

REPOSITORIES: GEO

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Project description:Renal hypoxia is widespread in acute kidney injury (AKI) of various aetiologies. Hypoxia adaptation, conferred through the hypoxia-inducible factor (HIF), appears to be insufficient. Here we show that HIF activation in renal tubules through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO) protects from rhabdomyolysis-induced AKI. In this model, histological observations indicate that injury mainly affects proximal convoluted tubules, with 5% necrosis at d1 and 40% necrosis at d2. HIF-1alpha up-regulation in distal tubules reflects renal hypoxia. However, lack of HIF in proximal tubules suggests insufficient adaptation by HIF. AKI in VHL-KO mice leads to prominent HIF activation in all nephron segments, as well as to reduced serum creatinine, serum urea, tubular necrosis, and apoptosis marker caspase-3 protein. At d1 after rhabdomyolysis, when tubular injury is potentially reversible, HIF mediated protection in AKI is associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal as demonstrated by qPCR, pathway enrichment analysis and immunohistochemistry. Together, our data provide evidence for a HIF-orchestrated multi-level shift towards glycolysis as a major mechanism for protection against acute tubular injury. All experiments were carried out in transgenic mice in which selective renal tubular VHL knockout (VHL-KO) was inducible by doxycycline (Reference: Mathia S, Paliege A, Koesters R, Peters H, Neumayer HH, Bachmann S, Rosenberger C. Action of hypoxia-inducible factor in liver and kidney from mice with Pax8-rtTA-based deletion of von Hippel-Lindau protein. Acta Physiol (Oxf). 2013; 207(3):565-76.). Four groups of animals were used: 1) controls: untreated mice; 2) VHL-KO: injected with doxycycline (0.1 mg per 10 g body weight SC), 4 days prior to sacrifice; 3) AKI: rhabdomyolysis; 4) VHL-KO/AKI: doxycycline plus rhabdomyolysis. To induce AKI, 50% glycerol (0.05 ml per 10 g body weight) was injected IM into the left hind limb under isoflurane narcosis. Drinking water was withdrawn between 20 h prior and 24 h after glycerol injection.

Project description:Objectives: Secretion of extracellular vesicles (EV) and associated micro-RNAs (miR) is altered during cellular stress and may serve as biomarkers of organ injury. We hypothesized that measuring changes in urinary levels of EV and miR will predict the onset of acute kidney injury in cardiac surgery patients. Design: Predictive accuracy biomarker study performed in the cohort of the REVAKI-2 trial Setting: Single center ICU between September 2015 and September 2018 Interventions: Intravenous sildenafil citrate 12.5 mg kg-1 over 150 min or dextrose 5% at the commencement of surgery. Measurements and main results: Urine samples were collected before and 24 hours after the procedure from 93 cardiac surgery patients. Urine EV concentrations and size distribution were assessed using NanoSight. EV derivation and levels were measured using flow cytometry. Samples from 10 selected patients were sequenced to detect differentially expressed miR. Verification was performed with advanced TaqMan assays in samples from all patients. Urine EV concentrations significantly increased in patients with AKI after surgery, with the percentage of EV positive for aquaporin-2 and β1-integrin also increasing. Pre surgery podocalyxin-positive EV were significantly lower, and β1-integrin EV werehigher in patients with AKI. The levels of the former correlated with the severity of theinjury. miR-125a-5p was expressed at higher levels in urine from patients with AKI stage 2/3. Levels of miR-10a-5p decreased after surgery in AKI patients; its levels correlated with the severity of the injury. Preoperative levels of podocalyxin EVs and miR-125a-5p had moderate AKI predictive value and, in a logistic model together with ICU lactate levels, offered good (AUC = 80.9%) AKI prediction. Conclusions: Lower levels of podocalyxin-positive EV at baseline predict the severity of post-surgery AKI. Urine EV concentrations and miR expression offer excellent predictive accuracy when combined with commonly measured biomarkers.

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MiRNA-486-5p Protects Against Kidney Ischemic Injury and Modifies the Transcriptome in Proximal Tubule and Endothelium

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