Probes for INS

Fibrosis is a relentlessly progressive and irreversible cause of organ damage, as in chronic kidney disease (CKD), but its underlying mechanisms remain elusive. We found that a circular RNA, circPTPN14, is highly expressed in human kidneys with biopsy-proved chronic interstitial fibrosis, mouse kidneys subjected to ischemia/reperfusion (IR) or unilateral ureteral obstruction (UUO), and TGFβ1-stimulated renal tubule epithelial cells (TECs). The intrarenal injection of circPTPN14 shRNA alleviated the progression of fibrosis in kidneys subjected to IR or UUO. Knockdown of circPTPN14 in TECs inhibited TGFβ1-induced expression of profibrotic genes, whereas overexpressing circPTPN14 increased the profibrotic effect of TGFβ1. The profibrotic action of circPTPN14 was ascribed to an increase in MYC transcription. The binding of circPTPN14 to the KH3 and KH4 domains of far upstream element (FUSE) binding protein 1 (FUBP1) enhanced the interaction between FUBP1 and FUSE domain, which was required for the initiation of MYC transcription. In human kidneys (n = 30) with biopsy-proved chronic interstitial fibrosis, the expression of circPTPN14 positively correlated with MYC expression. Taken together these studies show a novel mechanism in the pathogenesis of renal fibrosis, mediated by circPTPN14, which can be a target in the diagnosis and treatment of CKD.

Mammalian nephrons arise from a population of nephron progenitor cells (NPCs) expressing the master transcription factor, WT1, which is crucial for NPC proliferation, migration, and differentiation. In humans, biallelic loss of WT1 precludes nephrogenesis and leads to formation of Wilms tumor precursor lesions. We hypothesize that WT1 normally primes the NPC for nephrogenesis by inducing expression of NPC-specific DNA-repair genes that protect the genome. We analyzed transcript levels for a panel of DNA-repair genes in E17.5 vs adult mouse kidneys and noted seven that were increased >20-fold. We then isolated d1(+) NPCs from E17.5 kidneys and found that only one, Neil3, was enriched. RNAscope ISH of E17.5 mouse kidneys showed increased Neil3 expression in the nephrogenic zone vs mature nephron structures. To determine whether Neil3-expression is WT1-dependent, we knocked down Wt1 in d1(+) NPCs (60% knockdown efficiency) and noted a 58% reduction in Neil3 transcript levels. We showed that WT1 directly binds to the Neil3 promoter and that activity of a Neil3 promoter-reporter vector was increased two-fold in WT1(+) vs WT1(-) cells. We propose that Neil3 is a WT1-dependent DNA-repair gene, expressed at high levels in d1(+) NPCs where it repairs mutational injury to the genome during nephrogenesis. NEIL3 is likely just one of many such lineage-specific repair mechanisms that respond to genomic injury during kidney development.

Activation of the hypoxia-signalling pathway induced by deletion of the ubiquitin-ligase von Hippel-Lindau protein causes an endocrine shift of renin-producing cells to erythropoietin (EPO)-expressing cells. However, the underlying mechanisms have not yet been investigated. Since oxygen-regulated stability of hypoxia-inducible transcription factors relevant for EPO expression is dependent on the activity of prolyl-4-hydroxylases (PHD) 2 and 3, this study aimed to determine the relevance of different PHD isoforms for the EPO expression in renin-producing cells in vivo. For this purpose, mice with inducible renin cell-specific deletions of different PHD isoforms were analysed. Our study shows that there are two subgroups of renal renin-expressing cells, juxtaglomerular renin+ cells and platelet-derived growth factor receptor-β+ interstitial renin+ cells. These interstitial renin+ cells belong to the cell pool of native EPO-producing cells and are able to express EPO and renin in parallel. In contrast, co-deletion of PHD2 and PHD3, but not PHD2 deletion alone, induces EPO expression in juxtaglomerular and hyperplastic renin+ cells and downregulates renin expression. A strong basal PHD3 expression in juxtaglomerular renin+ cells seems to prevent the hypoxia-inducible transcription factor-2-dependent phenotype shift into EPO cells. In summary, PHDs seem important for the stabilization of the juxtaglomerular renin cell phenotype. Moreover, these findings reveal tubulointerstitial cells as a novel site of renal renin expression and suggest a high endocrine plasticity of these cells. Our data concerning the distinct expression patterns and functions of PHD2 and PHD3 provide new insights into the regulation of renin-producing cells and highlight the need for selective PHD inhibitors. KEY POINTS: Renal renin-expressing cells can be clearly distinguished into two subgroups, the typical juxtaglomerular renin-producing cells and interstitial renin+ cells. Interstitial renin+ cells belong to the cell pool of native erythropoietin (EPO)-producing cells, show a fast EPO response to acute hypoxia-inducible factor-2 (HIF-2) stabilization and are able to express EPO and renin in parallel. Only co-deletion of the prolyl-4-hydroxylases (PHD) 2 and 3, but not PHD2 deletion alone, induces EPO expression in juxtaglomerular renin+ cells. Chronic HIF-2 stabilization in juxtaglomerular renin-expressing cells leads to their phenotypic shift into EPO-producing cells. A strong basal PHD3 expression in juxtaglomerular renin+ cells seems to prevent a HIF-2-dependent phenotype shift into EPO cells suggesting PHD3 fulfils a stabilizer function for the juxtaglomerular renin cell phenotype.

Ureteric bud induction and branching morphogenesis is fundamental to the establishment of the renal architecture and is a key determinant of nephron number. Defective ureteric bud morphogenesis could give rise to a spectrum of malformations associated with congenital anomalies of the kidney and urinary tract (CAKUT). Signaling involving glial cell line-derived neurotrophic factor and its receptor RET and coreceptor GFRA1 appears to be particularly important in ureteric bud development. Recent epigenome profiling studies have uncovered dynamic changes of histone H3 lysine K4 (H3K4) methylation during metanephros development, and dysregulated H3K4 methylation has been associated with a syndromic human CAKUT.To investigate whether and how inactivation of Ash2l, which encodes a subunit of the COMPASS methyltransferase responsible for genome-wide H3K4 methylation, might contribute to CAKUT, we inactivated Ash2l specifically from the ureteric bud lineage in C57BL/6 mice and examined the effects on genome-wide H3K4 methylation and metanephros development. Genes and epigenome changes potentially involved in these effects were screened using RNA-seq combined with CUT&Tag-seq.Ureteric bud-specific inactivation of Ash2l caused CAKUT-like phenotypes mainly involving renal dysplasia at birth, which were associated with deficient H3K4 trimethylation. Ash2l inactivation slowed proliferation of cells at the ureteric bud tip, delaying budding and impairing ureteric bud branching morphogenesis. These effects were associated with downregulation of Ret, Gfra1, and Wnt11, which participate in RET/GFRA1 signaling.These experiments identify ASH2L-dependent H3K4 methylation in the ureteric bud lineage as an upstream epigenetic regulator of RET/GFRA1 signaling in ureteric bud morphogenesis, which, if deficient, may lead to CAKUT.