Neurogenic and pericytic plasticity of conditionally immortalized cells derived from renal erythropoietin‐producing cells

In adult mammals, the kidney is the main source of circulating erythropoietin (Epo), the master regulator of erythropoiesis. In vivo data in mice demonstrated multiple subtypes of interstitial renal Epo-producing (REP) cells. To analyze the differentiation plasticity of fibroblastoid REP cells, we used a transgenic REP cell reporter mouse model to generate conditionally immortalized REP-derived (REPD) cell lines. Under nonpermissive conditions, REPD cells ceased from proliferation and acquired a stem cell-like state, with strongly enhanced hypoxia-inducible factor 2 (HIF-2α), stem cell antigen 1 (SCA-1), and CD133 expression, but also enhanced alpha-smooth muscle actin (αSMA) expression, indicating myofibroblastic signaling. These cells maintained the “on-off” nature of Epo expression observed in REP cells in vivo, whereas other HIF target genes showed a more permanent regulation. Like REP cells in vivo, REPD cells cultured in vitro generated long tunneling nanotubes (TNTs) that aligned with endothelial vascular structures, were densely packed with mitochondria and became more numerous under hypoxic conditions. Although inhibition of mitochondrial oxygen consumption blunted HIF signaling, removal of the TNTs did not affect or even enhance the expression of HIF target genes. Apart from pericytes, REPD cells readily differentiated into neuroglia but not adipogenic, chondrogenic, or osteogenic lineages, consistent with a neuronal origin of at least a subpopulation of REP cells. In summary, these results suggest an unprecedented combination of differentiation features of this unique cell type

Knee Extensors Muscle Plasticity Over a 5-Years Rehabilitation Process After Open Knee Surgery

We investigated molecular and cellular parameters which set metabolic and mechanical functioning of knee extensor muscles in the operated and contralateral control leg of 9 patients with a chronically insufficient anterior cruciate ligament (ACL; 26.6 ± 8.3 years, 8 males, 1 female) after open reconstructive surgery (week 0), after ambulant physiotherapy under cast immobilization (week 9), succeeding rehabilitation training (up to week 26), and subsequent voluntary physical activity (week 260). Clinical indices of knee function in the operated leg were improved at 52 weeks and remained at a comparable level at week 260. CSA of the quadriceps (-18%), MCSA of muscle fibers (-24%), and capillary-to-fiber ratio (-24%) in m. vastus lateralis from the ACL insufficient leg were lower at week 0 than reference values in the contralateral leg at week 260. Slow type fiber percentage (-35%) and mitochondrial volume density (-39%) were reduced in m. vastus lateralis from the operated leg at weeks 9 and 26. Composition alterations in the operated leg exceeded those in the contralateral leg and, with the exception of the volume density of subsarcolemmal mitochondria, returned to the reference levels at week 260. Leg-specific deterioration of metabolic characteristics in the vasti from the operated leg was reflected by the down-regulation of mitochondrial respiration complex I-III markers (-41–57%) at week 9. After rehabilitation training at week 26, the specific Y397 phosphorylation of focal adhesion kinase (FAK), which is a proxy for mechano-regulation, was elevated by 71% in the operated leg but not in the contralateral leg, which had performed strengthening type exercise during ambulant physiotherapy. Total FAK protein and Y397 phosphorylation levels were lowered in both legs at week 26 resulting in positive correlations with mitochondrial volume densities and mitochondrial protein levels. The findings emphasize that a loss of mechanical and metabolic characteristics in knee extensor muscle remains detectable years after untreated ACL rupture, which may be aggravated in the post-operative phase by the deterioration of slow-oxidative characteristics after reconstruction due to insufficient load-bearing muscle activity. The reestablishment of muscle composition subsequent to years of voluntary physical activity reinforces that slow-to-fast fiber transformation is reversible in humans

Establishment of Novel Cell Lines Derived from Renal Erythropietin-Producing Cells

Erythropoietin (Epo) is the principal humoral factor regulating red blood cell homeostasis by directly influencing the survival, proliferation and differentiation of erythrocyte progenitors in the bone marrow. Epo is almost exclusively regulated on the transcriptional level, mainly by the transcription factor hypoxia-inducible factor (HIF)-2α. HIF-2α stands at the centre of the cellular oxygen sensing machinery: under conditions of sufficient oxygen, it is constantly hydroxylated by the prolyl hydroxylase enzymes PHD1, PHD2, and PHD3 as well as by the asparagine hydroxylase factor inhibiting HIF (FIH), ultimately resulting in its proteasomal degradation. PHD2 and PHD3 both are induced in hypoxia, which serves as a negative feedback regulation of HIF activity and therefore also Epo expression. The main source of Epo in the human adult are peritubular specialized renal Epo-producing (REP) cells located at border of the kidney cortex and medulla. REP cells are of mesenchymal origin with fibroblast properties, and have a neuron-like phenotype with long, thin, mitochondria-containing protrusions, which is reminiscent of the recently described telocyte cell type. Active REP cells are rare in number, present with a telocyte-like phenotype, and do not possess a known marker expression pattern clearly distinguishing them from other kidney cells. Upon a reduction in tissue oxygenation resulting in local hypoxia, HIF-2α in REP cells is stabilized, resulting in the de novo transcription, synthesis, and secretion of Epo. In diseases such as chronic kidney disease (CKD), production of Epo is impaired, resulting in a pathological reduction of erythrocyte production. This reduction in blood oxygen carrying capacity, also called anaemia, results in an undersupply of oxygen and thus to complications in organs throughout the body. It is suggested that in this condition, REP cells either differentiate into myofibroblasts, thereby losing their Epo-producing capabilities, or that their ability to sense changes in blood oxygenation is hampered e.g. due to changes in tissue composition and structure. The mechanisms by which REP cells lose their capability to produce Epo in response to hypoxia are not well understood. This is owed to the fact that up to date, the generation of a REP cell-derived cell line still capable of hypoxic Epo expression has not been achieved. While several groups have attempted to, all of the published cell lines mostly seized Epo production briefly after the start of in vitro culture. As a tool to investigate the fate of REP cells, we have generated a novel mouse model, expressing a tamoxifen-inducible Cre recombinase under the control of Epo-regulatory elements. We then crossed this Epo-CreERT2tg/tg mice with both, a reporter mouse expressing tdTomato downstream of a floxed transcriptional STOP cassette, as well as further crossing it with a mouse expressing a floxed diphtheria toxin receptor to facilitate enrichment. In this manner, we are able to efficiently and reproducibly label actively Epo-producing cells, extract them from the kidney and to take them in culture. We have generated a number of REP cell derived (REPD) cell lines, either with constitutive or conditional SV40 large T immortalization. While these cells present with sporadic hypoxic Epo expression, its degree and reliability are not sufficient for downstream applications, such as investigating the genetic regulation of Epo by HIF-2α, or elucidating the intricacies of its negative feedback regulation, which might be one of the reasons for the transient nature of Epo expression. It has been suggested that cellular quiescence is required for REP cells to produce Epo in response to hypoxia. Indeed, AB-REPD2-22 cells, which were immortalized using a temperature-labile mutant form of the SV40 large T antigen, stop proliferation when cultivated at 37°C and show marked changes in marker expression. However, also long-term culture at 37°C does not restore hypoxic Epo induction. Still, we managed to obtain several interesting insights from studying REPD cells. First, interfering with the negative feedback loop of HIF signalling is not sufficient to restore hypoxic Epo induction in REPD cell lines in vitro. Second, when cultivated at non-permissive temperatures, REPD cells have an increased expression of mesenchymal stem cell (MSC)-associated markers, such as Sca1 or Cd81. This is in line with recent findings suggesting Sca1 as a marker for a REP cell population. REPD cells also present a certain potential for neurogenic differentiation. This is in support of their mesenchymal origin, as well as their neuron-like phenotype and their plasticity observed in vivo as shown e.g. by their myofibroblast differentiation potential during CKD. Third, we realized that some of the generated REPD cells underwent only incomplete Cre-mediated recombination of the floxed STOP cassette in vivo, which resulted only in partial tdTomato fluorescence. This was reproducible in vitro and demonstrates that experiments involving the recombination of this widely used reporter construct need to be evaluated and controlled carefully. Fourth, REP cells as well as REPD cells have characteristic long, thin, mitochondria-containing cellular protrusions which appear to increase in number upon hypoxic exposure. There also appears to be a shedding and perhaps even an uptake of mitochondria-containing membrane compartments. However, at least in vitro, these cellular protrusions or the mitochondria contained within do not seem to be relevant for oxygen sensing. To summarize, while not suitable to answer open questions regarding the hypoxic regulation of REP cells, REPD cells still present a useful and interesting tool to investigate various aspects of this enigmatic cell type. This includes, among others, the mechanisms behind the transient nature of Epo expression, their surprising plasticity, and the role of cellular projections in oxygen sensing

Cre-mediated, loxP independent sequential recombination of a tripartite transcriptional stop cassette allows for partial read-through transcription

One of the widely used applications of the popular Cre-loxP method for targeted recombination is the permanent activation of marker genes, such as reporter genes or antibiotic resistance genes, by excision of a preceding transcriptional stop signal. The STOP cassette consists of three identical SV40-derived poly(A) signal repeats and is flanked by two loxP sites. We found that in addition to complete loxP-mediated recombination, limiting levels of the Cre recombinase also cause incomplete recombination of the STOP cassette. Partial recombination leads to the loss of only one or two of the three identical poly(A) repeats with recombination breakpoints always precisely matching the end/start of each poly(A) signal repeat without any relevant similarity to the canonical or known cryptic loxP sequences, suggesting that this type of Cre-mediated recombination is loxP-independent. Incomplete deletion of the STOP cassette results in partial read-through transcription, explaining at least some of the variability often observed in marker gene expression from an otherwise identical locus

Fount, fate, features, and function of renal erythropoietin-producing cells

Renal erythropoietin (Epo)-producing (REP) cells represent a rare and incompletely understood cell type. REP cells are fibroblast-like cells located in close proximity to blood vessels and tubules of the corticomedullary border region. Epo mRNA in REP cells is produced in a pronounced “on–off” mode, showing transient transcriptional bursts upon exposure to hypoxia. In contrast to “ordinary” fibroblasts, REP cells do not proliferate ex vivo, cease to produce Epo, and lose their identity following immortalization and prolonged in vitro culture, consistent with the loss of Epo production following REP cell proliferation during tissue remodelling in chronic kidney disease. Because Epo protein is usually not detectable in kidney tissue, and Epo mRNA is only transiently induced under hypoxic conditions, transgenic mouse models have been developed to permanently label REP cell precursors, active Epo producers, and inactive descendants. Future single-cell analyses of the renal stromal compartment will identify novel characteristic markers of tagged REP cells, which will provide novel insights into the regulation of Epo expression in this unique cell type

Genetic activation of Nrf2 reduces cutaneous symptoms in a murine model of Netherton syndrome

Netherton syndrome is a monogenic autosomal recessive disorder primarily characterized by the detachment of the uppermost layer of the epidermis, the stratum corneum. It results from mutations in the SPINK5 gene, which codes for a kallikrein inhibitor. Uncontrolled kallikrein activity leads to premature desquamation, resulting in a severe epidermal barrier defect and subsequent life-threatening systemic infections and chronic cutaneous inflammation. Here, we show that genetic activation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nfe2l2/Nrf2) in keratinocytes of Spink5 knockout mice, a model for Netherton syndrome, significantly alleviates their cutaneous phenotype. Nrf2 activation promoted attachment of the stratum corneum and concomitant epidermal barrier function, and reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor α and thymic stromal lymphopoietin. Mechanistically, we show that Nrf2 activation induces overexpression of secretory leukocyte protease inhibitor (Slpi), a known inhibitor of kallikrein 7 and elastase 2, in mouse and human keratinocytes in vivo and in vitro, respectively. In the Spink5-deficient epidermis, the upregulation of Slpi is likely to promote stabilization of corneodesmosomes, thereby preventing premature desquamation. Our results suggest pharmacological NRF2 activation as a promising treatment modality for Netherton syndrome patients

The Deubiquitinase OTUB1 Is a Key Regulator of Energy Metabolism

Dysregulated energy metabolism is a major contributor to a multitude of pathologies, including obesity and diabetes. Understanding the regulation of metabolic homeostasis is of utmost importance for the identification of therapeutic targets for the treatment of metabolically driven diseases. We previously identified the deubiquitinase OTUB1 as substrate for the cellular oxygen sensor factor-inhibiting HIF (FIH) with regulatory effects on cellular energy metabolism, but the physiological relevance of OTUB1 is unclear. Here, we report that the induced global deletion of OTUB1 in adult mice (Otub1 iKO) elevated energy expenditure, reduced age-dependent body weight gain, facilitated blood glucose clearance and lowered basal plasma insulin levels. The respiratory exchange ratio was maintained, indicating an unaltered nutrient oxidation. In addition, Otub1 deletion in cells enhanced AKT activity, leading to a larger cell size, higher ATP levels and reduced AMPK phosphorylation. AKT is an integral part of insulin-mediated signaling and Otub1 iKO mice presented with increased AKT phosphorylation following acute insulin administration combined with insulin hypersensitivity. We conclude that OTUB1 is an important regulator of metabolic homeostasis

Immediate effect of Acacia mearnsii tannins on methane emissions and milk fatty acid profiles of dairy cows

acceptedVersio

Immediate effect of Acacia mearnsii tannins on methane emissions and milk fatty acid profiles of dairy cows

acceptedVersio