Dysregulation of Principal Cell miRNAs Facilitates Epigenetic Regulation of AQP2 and Results in Nephrogenic Diabetes Insipidus

Abstract

BackgroundMicroRNAs (miRNAs), formed by cleavage of pre-microRNA by the endoribonuclease Dicer, are critical modulators of cell function by post-transcriptionally regulating gene expression.MethodsSelective ablation of Dicer in AQP2-expressing cells (DicerAQP2Cre+ mice) was used to investigate the role of miRNAs in the kidney collecting duct of mice.ResultsThe mice had severe polyuria and nephrogenic diabetes insipidus, potentially due to greatly reduced AQP2 and AQP4 levels. Although epithelial sodium channel levels were decreased in cortex and increased in inner medulla, amiloride-sensitive sodium reabsorption was equivalent in DicerAQP2Cre+ mice and controls. Small-RNA sequencing and proteomic analysis revealed 31 and 178 significantly regulated miRNAs and proteins, respectively. Integrated bioinformatic analysis of the miRNAome and proteome suggested alterations in the epigenetic machinery and various transcription factors regulating AQP2 expression in DicerAQP2Cre+ mice. The expression profile and function of three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) whose predicted targets were involved in epigenetic control (Phf2, Kdm5c, and Kdm4a) or transcriptional regulation (GATA3, GATA2, and ELF3) of AQP2 were validated. Luciferase assays could not demonstrate direct interaction of AQP2 or the three potential transcription factors with miR-7688-5p, miR-8114, and miR-409–3p. However, transfection of respective miRNA mimics reduced AQP2 expression. Chromatin immunoprecipitation assays demonstrated decreased Phf2 and significantly increased Kdm5c interactions at the Aqp2 gene promoter in DicerAQP2Cre+ mice, resulting in decreased RNA Pol II association.ConclusionsNovel evidence indicates miRNA-mediated epigenetic regulation of AQP2 expression.

Role of microRNAs in aquaporin 2 regulation

“…Indeed, by comparing the proteome and the miRNome of the inner medulla from AQP2 CRE :Dicer floxed cKO mice we identified and validated in-vitro three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) regulators of crucial partners of the epigenetic machinery (Kdm5c, PHF2, and Kdm4a) involved on AQP2 promoter chromatin assembly (Fig. 1) and on known AQP2 transcription factors (GATA3, GATA2, and ELF3) [54 ▪▪ ]. However, none of the three miRNAs showed direct binding with AQP2 mRNA.…”

Updates and Perspectives on Aquaporin-2 and Water Balance Disorders

“…Ranieri et al find that calcium-sensing receptor (CaSR) signaling reduces AQP2 abundance via miRNA-137 [ 119 ]. In mice, ablation of Dicer, which is required for miRNA maturation, induces NDI [ 120 ]. These findings implicate AQP2-targeting miRNAs as a therapeutic target in water balance disorders, including both dehydration and water retention.…”

Genetic deletion of the nuclear factor of activated T cells 5 in collecting duct principal cells causes nephrogenic diabetes insipidus

“…These effects are driven by a complex intracellular signaling response involving activation of various protein kinases 3 and AQP2 phosphorylation, 5 changes in AQP2 ubiquitylation 6,7 and cytoskeletal remodeling 3 . Long‐term, in addition to altering AQP2 protein stability, 8 AVP increases AQP2 protein synthesis by transcriptional and posttranscriptional mechanisms 9–12 . These long‐term effects are a consequence of AVP‐dependent V2R signaling, but the enhanced medullary osmolality subsequent to AVP may also play an additional role.…”