Vasopressin-regulated miRNAs and AQP2-targeting miRNAs in kidney collecting duct cells

Mature microRNA (miRNA) acts as an important posttranscriptional regulator. We aimed to profile vasopressin-responsive miRNAs in kidney inner medullary collecting duct cells and to identify aquaporin-2 (AQP2)-targeting miRNAs. Microarray chip assay was carried out in inner medullary collecting duct tubule suspensions from rat kidneys in the absence or presence of desmopressin (dDAVP) stimulation (10^-9 M, 2 h). The results demonstrated 19 miRNAs, including both precursor and mature miRNAs, as potential candidates that showed significant changes in expression after dDAVP stimulation (P< 0.05). Nine mature miRNAs exhibiting >1.3-fold changes in expression on the microarray (miR-127, miR-1, miR- 873, miR-16, miR-206, miR-678, miR-496, miR-298, and miR- 463) were further examined by quantitative real-time PCR, and target genes of the selected miRNAs were predicted. Next, to identify AQP2-targeting miRNAs, in silico analysis was performed. Four miRNAs (miR-32, miR-137, miR-216a, and miR- 216b) target the 3′-untranslated region of rat AQP2 mRNA. Target seed regions of miR-32 and miR-137 were also conserved in the 3′-untranslated region of mouse AQP2 mRNA. Quantitative realtime PCR and immunoblot analysis demonstrated that dDAVPinduced AQP2 expression was significantly attenuated in mpkCCDc14 cells when cells were transfected with miRNA mimics of miR-32 or miR-137. Moreover, luciferase reporter assay demonstrated a significant decrease of AQP2 translation in mpkCCDc14 cells transfected with miRNA mimics of miR-32 or miR-137. The present study provides novel insights into the regulation of AQP2 by RNA interference; however, vasopressin-regulated miRNAs did not include miR-32 or miR-137, indicating that the interaction of miRNAs with the AQP2 regulatory pathway requires further analysis.