The circadian clock regulates rhythmic erythropoietin expression in the murine kidney.

Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1 and 2 (Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietin mRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5' erythropoietin promoter. RNAscope® in situ hybridization confirmed the presence of Bmal1 in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy.

Amine oxidase copper-containing 1 (AOC1) is a downstream target gene of the Wilms tumor protein, WT1, during kidney development.

Amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) is a secreted glycoprotein that catalyzes the degradation of putrescine and histamine. Polyamines and their diamine precursor putrescine are ubiquitous to all organisms and fulfill pivotal functions in cell growth and proliferation. Despite the importance of AOC1 in regulating polyamine breakdown, very little is known about the molecular mechanisms that control its expression. We report here that the Wilms tumor protein, WT1, which is necessary for normal kidney development, activates transcription of the AOC1 gene. Expression of a firefly luciferase reporter under control of the proximal AOC1 promoter was significantly enhanced by co-transfection of a WT1 expression construct. Binding of WT1 protein to a cis-regulatory element in the AOC1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Antisense inhibition of WT1 protein translation strongly reduced Aoc1 transcripts in cultured murine embryonic kidneys and gonads. Aoc1 mRNA levels correlated with WT1 protein in several cell lines. Double immunofluorescent staining revealed a co-expression of WT1 and AOC1 proteins in the developing genitourinary system of mice and rats. Strikingly, induced changes in polyamine homeostasis affected branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-specific manner. These findings suggest that WT1-dependent control of polyamine breakdown, which is mediated by changes in AOC1 expression, has a role in kidney organogenesis.

Transcriptional regulation by the Wilms tumor protein, Wt1, suggests a role of the metalloproteinase Adamts16 in murine genitourinary development.

ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs) is a secreted mammalian metalloproteinase with unknown function. We report here that murine Adamts16 is co-expressed with the Wilms tumor protein, Wt1, in the developing glomeruli of embryonic kidneys. Adamts16 mRNA levels were significantly reduced upon transfection of embryonic murine kidney explants with Wt1 antisense vivo-morpholinos. Antisense knockdown of Adamts16 inhibited branching morphogenesis in kidney organ cultures. Adamts16 was detected by in situ mRNA hybridization and/or immunohistochemistry also in embryonic gonads and in spermatids and granulosa cells of adult testes and ovaries, respectively. Silencing of Wt1 by transfection with antisense vivo-morpholinos significantly increased Adamts16 mRNA in cultured embryonic XY gonads (11.5 and 12.5 days postconception), and reduced Adamts16 transcripts in XX gonads (12.5 and 13.5 days postconception). Three predicted Wt1 consensus motifs could be identified in the promoter and the 5'-untranslated region of the murine Adamts16 gene. Binding of Wt1 protein to these elements was verified by EMSA and ChIP. A firefly luciferase reporter gene under control of the Adamts16 promoter was activated ∼8-fold by transient co-transfection of human granulosa cells with a Wt1 expression construct. Gradual shortening of the 5'-flanking sequence successively reduced and eventually abrogated Adamts16 promoter activation by Wt1. These findings demonstrate that Wt1 differentially regulates the Adamts16 gene in XX and XY embryonic gonads. It is suggested that Adamts16 acts immediately downstream of Wt1 during murine urogenital development. We propose that Adamts16 is involved in branching morphogenesis of the kidneys in mice.

Adaptation of the Oxygen Sensing System during Lung Development.

During gestation, the most drastic change in oxygen supply occurs with the onset of ventilation after birth. As the too early exposure of premature infants to high arterial oxygen pressure leads to characteristic diseases, we studied the adaptation of the oxygen sensing system and its targets, the hypoxia-inducible factor- (HIF-) regulated genes (HRGs) in the developing lung. We draw a detailed picture of the oxygen sensing system by integrating information from qPCR, immunoblotting, in situ hybridization, and single-cell RNA sequencing data in ex vivo and in vivo models. HIF1α protein was completely destabilized with the onset of pulmonary ventilation, but did not coincide with expression changes in bona fide HRGs. We observed a modified composition of the HIF-PHD system from intrauterine to neonatal phases: Phd3 was significantly decreased, while Hif2a showed a strong increase and the Hif3a isoform Ipas exclusively peaked at P0. Colocalization studies point to the Hif1a-Phd1 axis as the main regulator of the HIF-PHD system in mouse lung development, complemented by the Hif3a-Phd3 axis during gestation. Hif3a isoform expression showed a stepwise adaptation during the periods of saccular and alveolar differentiation. With a strong hypoxic stimulus, lung ex vivo organ cultures displayed a functioning HIF system at every developmental stage. Approaches with systemic hypoxia or roxadustat treatment revealed only a limited in vivo response of HRGs. Understanding the interplay of the oxygen sensing system components during the transition from saccular to alveolar phases of lung development might help to counteract prematurity-associated diseases like bronchopulmonary dysplasia.