Molecular mechanisms of adaptation to intrauterine and perinatal changes of oxygen partial pressure: The liver-to-kidney switch of Erythropoietin production as model system
Final Report Abstract
In this project, we have characterized the developmental adaptation of the oxygen sensing system by using the liver-to-kidney switch of Erythropoietin (Epo) production as a model system (subprojects A) and studied regulators of Epo expression (subprojects B). General Results Subprojects A: We have studied the developmental expression of the oxygen sensing system genes (Phd1/2/3, Fih), the Hifs(1/2/3) and bona fide hypoxia-regulated genes (HRGs: Vegfa, Epo, Ca9, Glut1) from embryonic (E12) over fetal to perinatal to young and old adult stages in both sexes in different mouse organs (liver, kidney, lung, heart, gonads, brain, gut, placenta, eye). This report focusses on liver, kidney and lung. Very few age- or sex-specific expression effects were observed. Results Subproject A1: In liver, kidney and lung, Hif1α protein was stabilized at all intrauterine stages and destabilized after onset of ventilation with birth. Unexpectedly, this did not generally coincide with a higher HRG expression in utero and a lower HRG expression after birth. This only was the case for Glut1 and Phd3 in liver and Hif2a and Hif3a in both liver and kidney. The detection of (small amounts of) Hif2α protein has proven difficult due to the lack of a mouse-specific antibody. Trying to stabilize Hif proteins in mice in utero for extended periods of time by the use of the prolyl hydroxylase inhibitor Roxadustat, we have achieved transplacental, but only transient (8 h post treatment) Hif stabilization in the embryos’ organs, incapacitating our original experimental idea to study the effects of acute and chronic Hif stabilization during intrauterine development. Instead, we have expanded our expertise on embryonic explant cultures to older (fetal, postnatal and adult) stages of organ development. This way, we could show that the capacity to induce a Hif-driven gene expression profile already existed at all main developmental stages but approaches with systemic hypoxia or Roxadustat treatment revealed only a limited in vivo response of HRGs, indicating potential compensatory mechanisms in vivo. Results Subproject A2: We investigated the oxygen sensing system in developing lung tissue, utilizing single cell RNA-sequencing data and immunohistochemistry across different developmental stages. The study identifies the Hif1a-Phd1 axis as the principal regulator of the HIF-PHD system in mouse lung development, with a supplementary role of the Hif3a-Phd3 axis during gestation. It also highlights the dynamic changes in the composition of the HIF-PHD system during intrauterine and neonatal phases, specifically, the stepwise adaptation of Hif3a isoform expression during saccular and alveolar differentiation. Understanding these dynamics is crucial for addressing prematurity-related conditions like bronchopulmonary dysplasia and preventing associated neurodevelopmental sequelae. Results Subproject A3: To date, our understanding of the physiological and pathological roles of the Hif isoform Hif3α remains limited. In the context of adult systemic hypoxia, the Hif3a isoform exhibited induction in all organs studied, including the liver, kidney, lung, brain, and heart. In contrast, other HRGs displayed highly organ-specific responses to reduced inspiratory pO2 levels. An examination of the inhibitory Hif3a isoform, Ipas, revealed a peak in expression shortly after birth (P0/1) across all organs investigated, suggesting a systemic response to increased pO2 levels or other concurrent physiological adaptation processes. Results Subproject B1: In addition, Hif3a emerged as being important in utero in the Hif3a-Phd3 axis during lung development (subproject A2) and showed a similarly declining expression pattern after birth as hepatic Epo. This implicates that Hif3α could act as a constitutive factor activating hepatic Epo expression in relative hypoxia (low intrauterine pO2). This hypothesis will require further studies. Results Subproject B2: Our original hypothesis that the transcription factor Cxxc5 was involved in the developmental liver-to-kidney switch or the hypoxic regulation of Epo production could not be confirmed. Results Subproject B3: It has long been known that serum Epo shows a diurnal variation in humans, but until now, it was unclear how this was regulated on the molecular level. We have provided evidence for transcriptional control of this circadian oscillation by the core clock regulators Clock/Bmal1 as well as Cryptochromes 1 and 2 via the E-box motif in the minimal EPO promoter. Consideration of the circadian peak levels of Epo in high activity periods of the day may advance concepts of introducing chronotherapy in hematology for adult and pediatric/neonatal use.
Publications
- The circadian clock regulates rhythmic erythropoietin expression in the murine
kidney. Kidney Int.:S0085-2538(21)00729-8.
Sciesielski LK, Felten M*, Michalick L*, Kirschner KM, Lattanzi G+, Jacobi CLJ+, Wallach T, Lang V, Landgraf D, Kramer A, Dame C.
(See online at https://doi.org/10.1016/j.kint.2021.07.012) - Adaptation of the oxygen sensing system during lung development.
Oxid Med Cell Longev.:9714669.
Kirschner KM, Kelterborn S, Stehr H, Penzlin JLT, Jacobi CLJ, Endesfelder S, Sieg M, Kruppa J, Dame C, Sciesielski LK.
(See online at https://doi.org/10.1155/2022/9714669) - Change point detection for clustered expression data. BMC Genomics.;23:491
Sieg M, Sciesielski LK, Kirschner KM, Kruppa J.
(See online at https://doi.org/10.1186/s12864-022-08680-9)
