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Abstract: SA-PO278

Identification of RNAs in Urinary Extracellular Vesicles for Detecting Renal Nrf2 Activation

Session Information

Category: Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)

  • 2000 Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)

Authors

  • Takayama, Haruka, Labolatory of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
  • Sonoda, Hiroko, Labolatory of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
  • Higashijima, Yoshiki, Institute for Promotion of Tenure Track, University of Miyazaki, Miyazaki, Japan
  • Tanaka, Ayae, Labolatory of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
  • Ikeda, Masahiro, Labolatory of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
Background

NF-E2-related factor 2 (Nrf2), a master transcription factor, is known to be activated in response to oxidative stress, leading to producing antioxidant molecules. Renal oxidative stress condition can be estimated by measuring oxidative by-products and reactive oxygen species in urine. On the other hand, since activation of antioxidant molecules may mask renal oxidative stress condition, detecting renal Nrf2 activation is likely helpful for early detection of renal oxidative stress. However, little is known about the non-invasive detection method. Urinary extracellular vesicles (uEVs) released from renal epithelial cells contain RNAs that may serve as biomarkers for renal diseases. Here, we examined RNAs in uEVs after administration of bardoxolone methyl (BARD), an Nrf2 activator, in rats.

Methods

Male SD rats were randomly divided into two groups: the BARD (10 mg/kg intraperitoneally) and vehicle (75% corn oil/25% DMSO) groups. The urine was collected for 6 hrs after the administration, and uEVs were isolated. Kidneys were also obtained at 6 hrs post-treatment. RNAs were extracted from uEVs and kidneys and analyzed by next-generation sequencing and microarray techniques.

Results

Based on the blood and urine tests, BARD administration did not impair renal function. Microarray analyses revealed that 19 genes in the kidney and 211 genes in uEVs were significantly altered after BARD administration. Next-generation sequencing identified 958 genes that were significantly changed by BARD treatment in the uEVs. Of these genes, only two genes, Pir and Ephx1 were commonly increased in the kidneys and uEVs (Pir: > 3-fold, Ephx1: approximately 2-fold). Since the increase in Pir was higher than in Ephx1, we further investigated Pir as a potential biomarker for detecting Nrf2 activation. In situ hybridization revealed that BARD-treatment increased Pir expression in the proximal and distal tubules of the renal cortex. Moreover, we established the pre-amplification method of RNAs in uEVs and successfully detected Pir in uEVs by PCR.

Conclusion

Theoretically, Nrf2 is activated before the early onset of kidney diseases or under pre-oxidative stress condition. Thus, detecting Nrf2 activation in the kidney by measuring Pir in uEVs could be a potential tool to detect early kidney diseases.