Insights into the Regulation of Collecting Duct Homeostasis by Small Noncoding RNAs
Authors: Yu Leng Phua, Jacqueline Ho
Journal: Journal of the American Society of Nephrology (2017)

Tubulointerstitial fibrosis is a common, convergent hallmark of progression to renal failure in many forms of CKD. It is characterized by inflammation, tubular injury, loss of vascular integrity, and proliferation of fibroblasts and myofibroblasts, leading to excessive secretion of extracellular matrix into the surrounding interstititum.1 Understanding the mechanisms responsible for the initiation and progression of tubulointerstitial fibrosis is a promising strategy to identify avenues for novel renal therapies for the improvement of outcomes for patients with CKD. MicroRNAs (miRNAs) are small noncoding RNAs that regulate and fine-tune gene expression at the post-transcriptional or post-translational level.2 The formation and maturation of mature miRNAs requires a series of coordinated processing steps mediated by several critical endoribonuclease enzyme complexes. Biogenesis of miRNAs begins in the nucleus with the transcription of the primary miRNA transcript from the genomic locus by RNA polymerase II.2 Primary miRNAs are subsequently processed by the nuclear RNase III Drosha-Dgcr8 complex, releasing hairpin-shaped precursor miRNAs, which are then exported from the nucleus into the cytoplasm via Exportin 5.2 Finally, Dicer processes the precursor miRNA into mature miRNA, ready for loading into the RNA-induced silencing complex, which includes the argonaute subunit.2 The target mRNA is recognized on the basis of consensus Watson–Crick base pairing in the miRNA seed region (nucleotides 2–8), and the RNA-induced silencing complex either catalyzes the degradation of its target or inhibits the translation.2 Mutations in DICER1 or DROSHA have been identified and implicated in a wide spectrum of disease, including the human cancers pleuropulmonary blastoma, Sertoli–Leydig cell tumor, cystic nephroma, and Wilms tumor.3 A requirement for miRNAs in both renal development and disease has been demonstrated in several mouse models. Conditional ablation of Dicer in the nephron progenitor or ureteric bud lineages during renal development resulted in severe renal hypodysplasia, as characterized by overt progenitor loss due to aberrant apoptosis, and collecting duct cyst formation due to attenuated cilium length, respectively.4,5 Moreover, conditional ablation of Dicer or Drosha in terminally differentiated glomerular podocytes results in podocyte foot process effacement, marked proteinuria, and early postnatal death.6–9 Interestingly, conditional ablation of Dicer-dependent miRNA biogenesis in proximal tubules had no adverse phenotypic changes, but instead was protective from renal injury.10 Together, these studies suggest that targeting and modulation of miRNAs represent an attractive avenue for novel therapies for patients with CKD. Indeed, silencing of miR-21 and miR-132 have shown promise as candidate targets for antifibrotic therapies in mouse models.11,12 In this issue of the Journal of the American Society of Nephrology, Hajarnis et al.13 address the functional roles of miRNAs in postnatal collecting duct homeostasis and disease pathogenesis. Using three distinct mouse models, Hajarnis et al.13 demonstrate that postnatal inactivation of miRNA biogenesis in the mature collecting duct (Pkhd1Cre; Dicerfl/fl, Pkhd1Cre; Dgcr8fl/fl, Pkhd1Cre; Ago2fl/fl; Ago1/3/4 Knockout) elicits a striking inflammatory and profibrotic response. The onset of global tubular injury is characterized by Kim1 and Ngal expression, partial collecting duct epithelial to mesenchymal transition (EMT), tubulointerstitial fibrosis, and reduced renal function, leading to renal failure and death. The consistency of the fibrotic phenotype observed in all three mouse models suggests that this is primarily the result of loss of miRNAs in the collecting duct lineage, rather than that of other Dicer-dependent small noncoding RNAs. The next major challenge posed by this work is the identification of the specific miRNAs that determine disease pathogenesis, because the three transgenic manipulations used in these studies produce global miRNA loss in the collecting duct. By carrying out miRNA expression profiling of microdissected nephron segments, Hajarnis et al.13 take an important step by elucidating the specific clusters of miRNAs expressed in different epithelial segments of the nephron. They found that miR-200c is highly enriched in collecting ducts when compared with proximal tubules. miR-200c belongs to a group of miRNAs in the miR-200 family that consists of miRs-200a, 200b, 200c, 141, and 429 (which are transcribed in two clusters). These two clusters were identified as epithelial-specific miRNA clusters, on the basis of the evaluation of miRNA expression profiles from 59 epithelial and mesenchymal cell lines of the drug screening panel, maintained by the National Cancer Institute.14 The miRs-200 class has been implicated in EMT. Functionally, inhibition of miRs-200 in epithelial cells through locked nucleic acid anti-miRs induced EMT with loss of E-cadherin. These observations were attributed to the loss of miRs-200 repression of Zeb1 and Zeb2, negative regulators of E-cadherin.14 Hajarnis et al.13 provide evidence for partial collecting duct EMT on the basis of localized expression of the mesenchymal markers, Vimentin and Snail2, in a subset of collecting ducts after a global loss of miRNAs; although lineage tracing did not show transdifferentiation of collecting ducts into myofibroblasts. Given that partial EMT has previously been proposed as a potential driver of renal fibrosis, Hajarnis et al.13 suggest that loss of miRNAs, particularly miR-200c, results in partial EMT in collecting ducts, causing dysregulation of adjacent interstitial cells and eventual progression to tubulointerstitial fibrosis. Although the in vitro data are consistent with the idea that loss of miR-200c drives the collecting duct partial EMT, it remains unclear whether loss of miR-200c (or its two parent clusters) in vivo is sufficient to initiate tubulointerstitial fibrosis. Other epithelial regulatory miRNAs, such as miR-9, that would otherwise repress E-cadherin are also probably compromised in these models. Indeed, partial EMT was only observed in a subset of collecting ducts. The heterogeneity may reflect miRNA-mediated activity opposing EMT, or other intrinsic responses to loss of miRNA regulation in the collecting ducts. As an alternative interpretation, it is tempting to speculate that collecting ducts undergoing partial EMT could in fact represent a snapshot of dedifferentiating tubules that could contribute toward renal repair.1 Regardless, the exhaustive work of Hajarnis et al.13 using these miRNA biogenesis mouse models represent the first study to elucidate the importance of miRNA biology in postnatal collecting duct homeostasis, and provides information that will aid the identification of potential antifibrotic miRNA candidates. Disclosures None.