Probes for INS

Abstract Background Notch signaling is required during kidney development for nephron formation and principal cell fate selection within the collecting ducts. Whether Notch signaling is required in the adult kidney to maintain epithelial diversity, or whether its loss can trigger principal cell transdifferentiation (which could explain acquired diabetes insipidus in patients receiving lithium) is unclear. Methods To investigate whether loss of Notch signaling can trigger principal cells to lose their identity, we genetically inactivated Notch1 and Notch2, inactivated the Notch signaling target Hes1, or induced expression of a Notch signaling inhibitor in all of the nephron segments and collecting ducts in mice after kidney development. We examined renal function and cell type composition of control littermates and mice with conditional Notch signaling inactivation in adult renal epithelia. In addition, we traced the fate of genetically labeled adult kidney collecting duct principal cells after Hes1 inactivation or lithium treatment. Results Notch signaling was required for maintenance of Aqp2-expressing cells in distal nephron and collecting duct segments in adult kidneys. Fate tracing revealed mature principal cells in the inner stripe of the outer medulla converted to intercalated cells after genetic inactivation of Hes1 and, to a lesser extent, lithium treatment. Hes1 ensured repression of Foxi1 to prevent the intercalated cell program from turning on in mature Aqp2+ cell types. Conclusions Notch signaling via Hes1 regulates maintenance of mature renal epithelial cell states. Loss of Notch signaling or use of lithium can trigger transdifferentiation of mature principal cells to intercalated cells in adult kidneys.

Background: Eluforsen (previously known as QR-010) is a 33-mer antisense oligonucleotide under development for oral inhalation in cystic fibrosis (CF) patients with the delta F508 mutation. Previous work has shown that eluforsen restores CF transmembrane conductance regulator (CFTR) function in vitro and in vivo. To be effective, eluforsen has first to reach its primary target, the lung epithelial cells. Therefore, it has to diffuse through the CF airway surface layer (ASL), which in CF is characterized by the presence of thick and viscous mucus, impaired mucociliary clearance, and persistent infections. The goal of this study was to assess delivery of eluforsen through CF-like ASL. Methods and Results: First, air-liquid interface studies with cultured primary airway epithelial cells revealed that eluforsen rapidly diffuses through CF-like mucus at clinically relevant doses when nebulized once or repeatedly, over a range of testing doses. Furthermore, eluforsen concentrations remained stable in CF patient sputum for at least 48 hours, and eluforsen remained intact in the presence of various inhaled CF medications for at least 24 hours. When testing biodistribution of eluforsen after orotracheal administration in vivo, no differences in lung, liver, trachea, and kidney eluforsen concentration were observed between mice with a CF-like lung phenotype (ENaC-overexpressing mice) and control wild-type (WT) littermates. Also, eluforsen was visualized in the airway epithelial cell layer of CF-like muco-obstructed mice and WT littermates. Finally, studies of eluforsen uptake and binding to bacteria prevalent in CF lungs, and diffusion through bacterial biofilms showed that eluforsen was stable and not absorbed by, or bound to bacteria. In addition, eluforsen was found to be able to penetrate Pseudomonas aeruginosa biofilms. Conclusions: The thickened and concentrated CF ASL does not constitute a significant barrier for delivery of eluforsen, and feasibility of oral inhalation of eluforsen is supported by these data.

Abstract

RATIONALE:

Pulmonary hypertension (PH) complicates the care of patients with chronic lung disease, such as idiopathic pulmonary fibrosis (IPF), resulting in a significant increase in morbidity and mortality. Disease pathogenesis is orchestrated by unidentified myeloid-derived cells.

OBJECTIVE:

We used murine models of PH and pulmonary fibrosis to study the role of circulating myeloid cells in disease pathogenesis and prevention.

METHODS:

We administered clodronate liposomes to bleomycin treated wild-type mice in order to induce pulmonary fibrosis and PH with a resulting increase in circulating bone marrow-derived cells.

MEASUREMENTS AND MAIN RESULTS:

We discovered that a population of CXCR2+ myeloid-derived suppressor cells (MDSCs), granulocytic subset (G-MDSC), is associated with severe PH in mice. Pulmonary pressures worsened despite improvement in bleomycin-induced pulmonary fibrosis. PH was attenuated by CXCR2 inhibition, with antagonist SB 225002, through decreasing G-MDSC recruitment to the lung. Molecular and cellular analysis of clinical patient samples confirmed a role for elevated MDSCs in IPF and IPF with PH.

CONCLUSIONS:

These data show that MDSCs play a key role in PH pathogenesis and that G-MDSC trafficking to the lung, through chemokine receptor CXCR2, increases development of PH in multiple murine models. Furthermore, we demonstrate pathology similar to the preclinical models in IPF with PH patient lung and blood samples, suggesting a potential role for CXCR2 inhibitor use in this patient population. These findings are significant as there are currently no approved disease specific therapies for patients with PH complicating IPF.

Glucagon-like peptide-1 receptor (GLP-1R) agonists, used to treat type 2 diabetes and obesity, reduce rates of myocardial infarction and cardiovascular death. The GLP-1R has been localized to the human sinoatrial node; however, its expression in ventricular tissue remains uncertain. Here we studied GLP-1R expression in the human heart using GLP-1R-directed antisera, quantitative PCR, reverse transcription PCR to detect full length mRNA transcripts, and in situ hybridization. GLP1R mRNA transcripts, encompassing the entire open reading frame, were detected in all four cardiac chambers from 15 hearts at levels approximating those detected in human pancreas. In contrast, cardiac GLP2R expression was relatively lower, whereas cardiac GCGR expression was sporadic and not detected in the left ventricle. GLP1R mRNA transcripts were not detected in RNA from human cardiac fibroblasts, coronary artery endothelial, or vascular smooth muscle cells. Human Brunner's glands and pancreatic islets exhibited GLP-1R-immunopositivity and abundant expression of GLP1R mRNA transcripts by in situ hybridization. GLP1R transcripts were also detected by in situ hybridization in human cardiac sinoatrial node tissue. However definitive cellular localization of GLP1R mRNA transcripts or immunoreactive GLP-1R protein within human cardiomyocytes (CMs) or cardiac blood vessels remained elusive. Moreover, validated GLP-1R antisera lacked sufficient sensitivity to detect expression of the endogenous islet or cardiac GLP-1R by Western blotting. Hence, although human cardiac ventricles express the GLP1R, the identity of one or more ventricular cell type(s) that express a translated GLP1R protein requires further clarification with highly sensitive methods of detection.

Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblastdifferentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease.

Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway.

Abstract

Study question

Can novel meiotic RNA targets of DAZL (deleted in azoospermia-like) be identified in the human foetal ovary?

Summary answer

SYCP1 (synaptonemal complex protein-1), TEX11 (testis expressed 11) and SMC1B (structural maintenance of chromosomes 1B) are novel DAZL targets in the human foetal ovary, thus DAZL may have previously unrecognised roles in the translational regulation of RNAs involved in chromosome cohesion and DNA recombination in the oocyte from the time of initiation of meiosis.

What is known already

The phenotype of Dazl deficiency in mouse is infertility in both sexes and DAZL has also been linked to infertility in humans. Few studies have explored targets of this RNA-binding protein. The majority of these investigations have been carried out in mouse, and have focussed on the male thus the basis for its central function in regulating female fertility is largely unknown.

Study design size, duration

We carried out RNA sequencing after immunoprecipitation of endogenous DAZL from human foetal ovarian tissue (17 weeks of gestation, obtained after elective termination of pregnancy) to identify novel DAZL targets involved in meiosis (n = 3 biological replicates).

Participants/materials, setting, methods

Using quantitative RT-PCR, we examined the expression of selected RNAs identified by our immunoprecipitation across gestation, and visualised the expression of potential target SMC1B in relation to DAZL, with a combination of in situ hybridisation and immunohistochemistry. 3′ untranslated region (3′UTR)-luciferase reporter assays and polysome profile analysis were used to investigate the regulation of three RNA targets by DAZL, representing key functionalities: SYCP1, TEX11 and SMC1B.

Main results and the role of chance

We identified 764 potential RNA targets of DAZL in the human foetal ovary (false discovery rate 0.05 and log-fold change ≥ 2), with functions in synaptonemal complex formation (SYCP1, SYCP3), cohesin formation (SMC1B, RAD21), spindle assembly checkpoint (MAD2L1, TRIP13) and recombination and DNA repair (HORMAD1, TRIP13, TEX11, RAD18, RAD51). We demonstrated that the translation of novel targets SYCP1 (P = 0.004), TEX11 (P = 0.004) and SMC1B (P = 0.002) is stimulated by the presence of DAZL but not by a mutant DAZL with impaired RNA-binding activity.

Large scale data

The raw data are available at GEO using the study ID: GSE81524.

Limitations, reasons for caution

This analysis is based on identification of DAZL targets at the time when meiosis starts in the ovary: it may have other targets at other stages of oocyte development, and in the testis. Representative targets were validated, but detailed analysis was not performed on the majority of putative targets.

Wider implications of the findings

These data indicate roles for DAZL in the regulation of several key functions in human oocytes. Through the translational regulation of novel RNA targets SMC1B and TEX11, DAZL may have a key role in regulating chromosome cohesion and DNA recombination; two processes fundamental in determining oocyte quality and whose establishment in foetal life may support lifelong fertility.

Abstract