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In our aging society an increasing prevalence of chronic kidney disease (CKD) meets a general shortage of organ donors. As the economic burden of CKD is increasing, kidney research made significant progress in the early detection, repair, or replacement of damaged renal tissue, taking in consideration sex-related differences, and precision medicine principles. Despite recent advancements in renal organoid research, the generation of fully functional nephrons in-vitro is so far not achievable. Here we describe a novel lectin-guided protocol to sort renal tissue into four pure, functionally diverse nephron segments with high yield and intact 3D tubular morphology. We validated tissue-sorted fragments with proximal or distal convoluted tubular identity using bottom-up proteomics and assessed sex-related differences. Compared to males, females showed higher abundance of proteins involved in metabolism and excretion of xenobiotics, as well as in cell cycle progression or proliferation, which together might enhance their ability to better cope with renal injury. The major advantage of our protocol – high yield of sorted nephron segments with preserved 3D structure – will allow us to reconstruct miniature kidneys ex-vivo, by combining it with cutting-edge bio-printing techniques. Besides large-scale drug screening or the identification of segment specific biomarkers in renal disease, they could pave the way to generate functional renal replacement units for regenerative medicine. Furthermore, if applied to human biopsies, our method could improve patient-specific renal disease assessment. In conclusion, our method represents a cost-efficient and broadly applicable tool with the potential to propel personalized regenerative kidney research.

Pflügers Archiv 474(8), 949-962 (2022). doi:10.1007/s00424-022-02688-6 special issue: "Special Issue: Kidney Control of Homeostasis", Published by Springer, Berlin ; Heidelberg

BackgroundCalcineurin inhibitors (CNI) such as Cyclosporine A (CsA) and Tacrolimus (FK506) are commonly used after renal transplantation in order to suppress the immune system. In lymphoid cells, CsA acts via the Calcineurin-NFAT axis, whereas in non-lymphoid cells, such as kidney epithelial cells, CsA induces Calcineurin inhibitor toxicity (CNT). Up to date, it is unknown via which off-targets CsA induces CNT in kidney epithelial cells.MethodsIn vitro experiments using a surrogate marker to measure CNT induction as well as in vivo studies with acute CNT, were used in order to elucidate the underlying molecular mechanism.ResultsInhibition of the NFAT axis does not show any nephrotoxicity. However, inhibition of p38 and PI3K/Akt Kinases showed induction of nephrotoxicity.ConclusionsThese findings show that CsA acts NFAT independent on kidney epithelial cells. Moreover, inhibition of certain protein kinases mimic CsA activity on kidney epithelial cells indicating that p38 and PI3K/Akt kinase pathways might be involved in CNT progression on kidney epithelial cells.

Intrauterine growth restriction (IUGR) is associated with reduced kidney size at birth, accelerated renal function decline, and increased risk for chronic kidney and cardiovascular diseases in adults. Precise mechanisms underlying fetal programming of adult diseases remain largely elusive and warrant extensive investigation. Setting up a mouse model of hypoxia-induced IUGR, fetal adaptations at mRNA, protein and cellular levels, and their long-term functional consequences are characterized, using the kidney as a readout. Here, we identify fetuin-A as an evolutionary conserved HIF target gene, and further investigate its role using fetuin-A KO animals and an adult model of ischemia-reperfusion injury. Beyond its role as systemic calcification inhibitor, fetuin-A emerges as a multifaceted protective factor that locally counteracts calcification, modulates macrophage polarization, and attenuates inflammation and fibrosis, thus preserving kidney function. Our study paves the way to therapeutic approaches mitigating mineral stress-induced inflammation and damage, principally applicable to all soft tissues., Intrauterine growth restriction is associated with increased risk for chronic diseases in adults. Here the authors identify fetuin-A as a HIF target gene and describe its protective role in the kidney, counteracting disease mechanisms such as calcification, macrophage polarization, and fibrosis.

Hypoxia-induced intrauterine growth restriction increases the risk for cardiovascular, renal, and other chronic diseases in adults, representing thus a major public health problem. Still, not much is known about the fetal mechanisms that predispose these individuals to disease. Using a previously validated mouse model of fetal hypoxia and bottom-up proteomics, we characterize the response of the fetal kidney to chronic hypoxic stress. Fetal kidneys exhibit a dichotomous response to chronic hypoxia, comprising on the one hand cellular adaptations that promote survival (glycolysis, autophagy, and reduced DNA and protein synthesis), but on the other processes that induce a senescence-like phenotype (infiltration of inflammatory cells, DNA damage, and reduced proliferation). Importantly, chronic hypoxia also reduces the expression of the antiaging proteins klotho and Sirt6, a mechanism that is evolutionary conserved between mice and humans. Taken together, we uncover that predetermined aging during fetal development is a key event in chronic hypoxia, establishing a solid foundation for Barker's hypothesis of fetal programming of adult diseases. This phenotype is associated with a characteristic biomarker profile in tissue and serum samples, exploitable for detecting and targeting accelerated aging in chronic hypoxic human diseases.

Background In pregnancy, a high plasma volume maintains uteroplacental perfusion and prevents placental ischemia, a condition linked to elevated maternal blood pressure ( BP ). Reducing BP by increasing Na + intake via plasma volume expansion appears contra‐intuitive. We hypothesize that an appropriate Na + intake in pregnancy reduces maternal BP and adapts the renin‐angiotensin system in a pregnancy‐specific manner. Methods and Results BP was measured by implanted telemetry in Sprague‐Dawley rats before and throughout pregnancy. Pregnant and nonpregnant animals received either a normal‐salt (0.4%; NS ), high‐salt (8%; HS ), or low‐salt (0.01%; LS ) diet, or HS (days 1–14) followed by LS (days 14–20) diet ( HS / LS ). Before delivery (day 20), animals were euthanized and organs collected. Food, water, and Na + intake were monitored in metabolic cages, and urinary creatinine and Na + were analyzed. Na + intake and retention increased in pregnancy ( NS , LS ), leading to a positive Na + balance ( NS , LS ). BP was stable during LS , but reduced in HS conditions in pregnancy. The renin‐angiotensin system was adapted as expected. Activating cleavage of α‐ and γ‐subunits of the renal epithelial Na + channel and expression of‐full length medullary β‐subunits, accentuated further in all LS conditions, were upregulated in pregnancy. Conclusions Pregnancy led to Na + retention adapted to dietary changes. HS exposure paradoxically reduced BP . Na + uptake while only modestly linked to the renin‐angiotensin system is enhanced in the presence of posttranslational renal epithelial Na + channel modifications. This suggests (1) storage of Na + in pregnancy upon HS exposure, bridging periods of LS availability; and (2) that potentially non–renin‐angiotensin–related mechanisms participate in EN aC activation and consecutive Na + retention.

Calcineurin inhibitor toxicity (CNT) is a frequent occurrence in transplanted renal grafts and autochthone kidneys from patients undergoing long-term treatment with calcineurin inhibitors, notably cyclosporin A (CsA) and tacrolimus. Here, we show an indispensable role of the tumor necrosis factor superfamily (TNFS) molecule TNF-related weak inducer of apoptosis (TWEAK) (TNFSF12) in the pathogenesis of acute CNT lesions in mice. A deficiency in TWEAK resulted in limited tubulotoxicity after CsA exposure, which correlated with diminished expression of inflammatory cytokines and reduced intraparenchymal infiltration with immune cells. We further identified tubular epithelial cells of the kidney as major targets of CsA activity and found that Fn14 (tumor necrosis factor receptor superfamily 12A), the receptor for TWEAK, is a highly CsA-inducible gene in these cells. Correlating with this, CsA pretreatment sensitized tubular epithelial cells specifically to the pro-inflammatory activities of recombinant TWEAK in vitro. Moreover, injection of rTWEAK alone into mice induced moderate disease similar to CsA, and rTWEAK combined with CsA resulted in synergistic nephrotoxicity. These findings support the importance of tubular epithelial cells as cellular targets of CsA toxicity and introduce TWEAK as a critical contributor to CNT pathogenesis.

Wnt/β-catenin signaling is involved in patterning of bone primordia, but also plays an important role in the differentiation of chondrocytes and osteoblasts. During these processes the level of β-catenin must be tightly regulated. Excess β-catenin leads to conditions with increased bone mass, whereas loss of β-catenin is associated with osteoporosis or, in extreme cases, the absence of limbs. In this study, we examined skeletogenesis in mice, which retain only 25% of β-catenin. These embryos showed severe morphological abnormalities of which the lack of hindlimbs and misshaped front paws were the most striking. Surprisingly however, calcification of bone primordia occurred normally. Moreover, the Wnt-dependent regulatory network of transcription factors driving the differentiation of cartilage and bone, as well as the expression of extracellular matrix components, were preserved. These findings show that 25% β-catenin is insufficient for the correct patterning of bone primordia, but sufficient for their mineralization. Our approach helps to identify bone morphogenetic processes that can proceed normally even at low β-catenin levels, in contrast to those that require high β-catenin dosages. This information could be exploited to improve the treatment of bone diseases by fine-tuning the individual β-catenin dosage requirements., Summary: The correct allocation and patterning of skeletal elements during development requires a higher dosage of β-catenin than the maturation and mineralization of these primordia.

International audience; Acute kidney injury (AKI) is common in hospitalized patients and has a poor prognosis, the severity of AKI being linked to progression to chronic kidney disease. This stresses the need to search for protective mechanisms during the acute phase. We investigated kidney repair after hypoxic injury using a rat model of renal artery branch ligation, which led to an oxygen gradient vertical to the corticomedullary axis. Three distinct zones were observed: tubular necrosis, infarction border zone and preserved normal tissue. EphA2 is a receptor tyrosine kinase with pivotal roles in cell architecture, migration and survival, upon juxtacrine contact with its membrane-bound ligand EphrinA1. Following hypoxia, EphA2 was up-regulated in cortical and medullary tubular cells, while EphrinA1 was up-regulated in interstitial cells adjacent to peritubular capillaries. Moreover, erythropoietin (EPO) messenger RNA (mRNA) was strongly expressed in the border zone of infarcted kidney within the first 6 h. To gain more insight into the biological impact of EphA2 and EphrinA1 up-regulation, we activated the signalling pathways in vitro using recombinant EphrinA1/Fc or EphA2/Fc proteins. Stimulation of EphA2 forward signalling in the proximal tubular cell line HK2 increased cell attachment and laminin secretion at the baso-lateral side. Conversely, activation of reverse signalling through EphrinA1 expressed by Hep3B cells promoted EPO production at both the transcriptional and protein level. Strikingly, in co-culture experiments, juxtacrine contact between EphA2 expressing MDCK and EphrinA1 expressing Hep3B was sufficient to induce a significant up-regulation of EPO mRNA production in the latter cells, even in the absence of hypoxic conditions. The synergistic effects of EphA2 and hypoxia led to a 15–20-fold increase of EPO expression. Collectively, our results suggest an important role of EphA2/EphrinA1 signalling in kidney repair after hypoxic injury through stimulation of (i) tubular cell attachment, (ii) secretion of basal membrane proteins and (iii) EPO production. These findings could thus pave the way to new therapeutic approaches. © 2016, Springer-Verlag Berlin Heidelberg.

One of the most visually striking patterns in the early developing embryo is somite segmentation. Somites form as repeated, periodic structures in pairs along nearly the entire caudal vertebrate axis. The morphological process involves short- and long-range signals that drive cell rearrangements and cell shaping to create discrete, epithelialized segments. Key to developing novel strategies to prevent somite birth defects that involve axial bone and skeletal muscle development is understanding how the molecular choreography is coordinated across multiple spatial scales and in a repeating temporal manner. Mathematical models have emerged as useful tools to integrate spatiotemporal data and simulate model mechanisms to provide unique insights into somite pattern formation. In this short review, we present two quantitative frameworks that address the morphogenesis from segment to somite and discuss recent data of segmentation and epithelialization.

Embryogenesis relies on the precise interplay of signaling cascades to activate tissue-specific differentiation programs. An important player in these morphogenetic processes is β-catenin, which is a central component of adherens junctions and canonical Wnt signaling. Lack of β-catenin is lethal before gastrulation, but mice heterozygous for β-catenin (Ctnnb1) develop as wild type. Here, we confine β-catenin amounts below the heterozygous expression level to study the functional consequences for development. We generate embryonic stem (ES) cells and embryos expressing β-catenin only from the ubiquitously active ROSA26 promoter and thereby limit β-catenin expression to ~12.5% (ROSA26β/+) or ~25% (ROSA26β/β) of wild-type levels. ROSA26β/+ is sufficient to maintain ES cell morphology and pluripotent characteristics, but is insufficient to activate canonical target genes upon Wnt stimulation. This Wnt signaling deficiency is incompletely restored in ROSA26β/β ES cells. We conclude that even very low β-catenin levels are able to sustain cell adhesion, but not Wnt signaling. During development, ROSA26β/β as well as ROSA26β/+ partially rescues the knockout phenotype, yet proper gastrulation is absent. These embryos differentiate according to the neural default hypothesis, indicating that gastrulation depends on high β-catenin levels. Strikingly, if ROSA26β/+ or ROSA26β/β is first activated after gastrulation, subsequent development correlates with the dosage of β-catenin. Moreover, molecular evidence indicates that the amount of β-catenin controls the induction of specific Wnt target genes. In conclusion, by restricting its expression we determine the level of β-catenin required for adhesion or pluripotency and during different morphogenetic events.

Background In pregnancy, plasma volume expansion by high aldosterone levels is crucial to prevent placental ischaemia, a condition linked to maternal arterial hypertension. Objective We hypothesise that an adapted salt intake will further lead to a favourable maternal blood pressure response. Methods Sprague–Dawley rats were followed by blood pressure telemetry and metabolic measurements before mating and throughout pregnancy in four experimental groups receiving either normal (NS; 0.4%), high (HS; 8%), low (LS; 0.01%), or high (8%) for the first 14 d of pregnancy followed by a switch to low Na + diet (0.01%) until day 20 (HS/LS). On day 20 the animals were sacrificed. Food, water and Na + balance was recorded. Results In pregnancy, Na + intake ( p = 0.02) and retention ( p = 0.004) increased already in NS conditions. The ratio of Na + intake vs. excretion was high in pregnant rats on NS ( p = 0.015) and LS ( p = 0.008 vs. non-pregnant) diet and low ( p p = 0.004–0.044 vs. non-pregnant) as compared to NS diet ( p Conclusions Pregnancy determines the response to dietary changes in salt intake aiming to preserve salt thus overcoming periods of low salt availability as shown in our HS/LS model. Increased salt intake reduces maternal blood pressure in pregnancy. Though the exact dose and temporal relationship needs to be elucidated, these results highlight the important role of environmental factors, such as salt, for a successful pregnancy.

Beta-thymosins constitute a group of small actin-sequestering peptides. These highly conserved peptides are involved in cytoskeleton dynamics and can influence different cell properties such as motility, substrate adhesion, shape and chemotaxis. As a marker for tumour metastasis, the mammalian thymosin beta15 is believed to have an important diagnostic relevance in cancer prognosis, although little is known about its physiological function. In order to study the role of thymosin beta15 avian in embryogenesis, we cloned the chicken and quail orthologues of thymosin beta15 and used the chicken as a model for vertebrate development. Avian thymosin beta15, the first known non-mammalian thymosin beta15-like gene, encodes a peptide that possesses a cysteine at position one after the methionine which is a significant difference compared to its mammalian counterparts. Thymosin beta15 avian expression starts at an early stage of development. The expression pattern changes rapidly with development and differs from that of the related thymosin beta4 gene. The most prominent expression domain is seen in developing muscles of limbs and trunk. Gain-of-function experiments revealed that thymosin beta15avian has a function in normal myotome development. Ectopic over-expression of thymosin beta15 avian leads to premature elongation of myotome cells trespassing segment borders. We conclude that thymosin beta15 avian has a still undescribed function in promoting myocyte elongation.

The leukemia inhibitory factor (Lif) signaling pathway is a crucial determinant for mouse embryonic stem (mES) cell self-renewal and pluripotency. One of the hallmarks of mES cells, their compact growth morphology, results from tight cell adhesion mediated through E-cadherin, β-catenin (Ctnnb1) and α-catenin with the actin cytoskeleton. β-catenin is also involved in canonical Wnt signaling, which has also been suggested to control mES cell stemness. Here, we analyze Ctnnb1-/- mES cells in which cell adhesion is preserved by an E-cadherin-α-catenin (Eα) fusion protein (Ctnnb1-/-Eα mES cells), and show that mimicking only the adhesive function of β-catenin is necessary and sufficient to maintain the mES cell state, making β-catenin/Wnt signaling obsolete in this process. Furthermore, we propose a role for E-cadherin in promoting the Lif signaling cascade, showing an association of E-cadherin with the Lifr-Gp130 receptor complex, which is most likely facilitated by the extracellular domain of E-cadherin. Without Eα, and thus without maintained cell adhesion, Ctnnb1-/- mES cells downregulate components of the Lif signaling pathway, such as Lifr, Gp130 and activated Stat3, as well as pluripotency-associated markers. From these observations, we hypothesize that the changes in gene expression accompanying the loss of pluripotency are a direct consequence of dysfunctional cell adhesion. Supporting this view, we find that the requirement for intact adhesion can be circumvented by the forced expression of constitutively active Stat3. In summary, we put forward a model in which mES cells can be propagated in culture in the absence of Ctnnb1, as long as E-cadherin-mediated cell adhesion is preserved.

From Wnt Signals to Telomerase Activity Telomerase activity is associated with stem cell renewal and cancers, whereas a decrease in telomerase activity is seen during cell differentiation and senescence. Wnt/β-catenin signaling is also a critical regulator of stem cells, and deregulation of the pathway is associated with cancer. Now, Hoffmeyer et al. (p. 1549 ; see the Perspective by Greider ) have found a link between these two pathways. In embryonic stem cells, β-catenin was able to regulate telomerase expression and activity directly. Similar observations were obtained in adult stem cells, a model of intestinal tumors, and human cancer cells.

Vertebrates have a metameric bodyplan that is based on the presence of paired somites. Somites develop from the segmental plate in a cranio-caudal sequence. At the same time, new material is added from Hensen's node, the primitive streak and the tailbud. In this way, the material residing in the segmental plate remains constant and comprises 12 prospective somites on each side. Prospective segment borders are not yet determined in the caudal segmental plate. Prior to segmentation, the cranial segmental plate undergoes epithelialization, which is controlled by signals from the neural tube and ectoderm. The bHLH transcription factor Paraxis is critically involved in this process. Formation of a new somite from the cranial end of the segmental plate is a highly controlled process involving complex cell movements in relation to each other. Hox genes specify regional identity of the somites and their derivatives. In the chicken a transposition of thoracic into cervical vertebrae has occurred as compared to the mouse. Transcription factors of the bHLH and homeodomain type also specify the cranio-caudal polarity and that of particular cell groups within the somites. According to segmentation models, somitogenesis is under the control of a "segmentation clock" in combination with a morphogen gradient. This hypothesis has recently found support from molecular data, especially the cycling expression of genes such as cHairy1 and Lunatic Fringe, which depend on the Notch/Delta pathway of signal transduction. FGF8 has been described to be distributed along a cranio-caudal gradient. The first oscillating gene described shown to be independent of Notch is Axin2, encoding a negative regulator of the canonical Wnt pathway and a target of Wnt3a. Wnt3a and Axin2 show a similar distribution as FGF8 with high levels in the tailbud. The chick embryo has recently become accessible to molecular approaches such as overexpression by electroporation and RNA interference which can be expected to help elucidating some of the still open questions concerning somitogenesis.

Publisher Summary This chapter presents an overview of the present knowledge regarding Wnt signaling, focusing primarily on the Wnt/β-catenin pathway and its role in invertebrate and vertebrate development. Further, it briefly discusses recent progress concerning the non-canonical Wnt/PCP pathway. In general, the canonical Wnt pathway involves the fine-tuned interaction of activators, inhibitors, and many co-factors at the cell membrane, in the cytoplasm, and in the nucleus. This complex molecular machinery is put into place to control cytoplasmic turnover of β-catenin, thus regulating the transcription of Wnt target genes. Various processes during chordate embryogenesis depend on canonical Wnt signaling. To explain this, this chapter uses the example of the mouse and Xenopus laevis to discuss some major developmental processes in Wnt signaling. Under this, it states that Wnt signaling controls early patterning of the body plan, when the dorsal–ventral (DV) and anterior–posterior (AP) body axes are laid down. Finally, it states that Wnt signaling is involved in many morphogenetic events leading to organogenesis. Over recent years, stem cell research has evolved into one of the most prominent topics in biological science. Given the importance of Wnt signaling during development, many reports have connected β-catenin and other components of the Wnt signaling machinery to embryonic and somatic stem cell renewal and cancer.

Vertebrates have a metameric bodyplan that is based on the presence of paired somites. Somites develop from the segmental plate in a cranio-caudal sequence. At the same time, new material is added from Hensen’s node, the primitive streak and the tail bud. In this way, the material residing in the segmental plate remains constant and comprises 12 prospective somites on each side. Prospective segment borders are not yet determined in the caudal segmental plate. Prior to segmentation, the cranial segmental plate undergoes epithelialization, which is controlled by signals from the neural tube and ectoderm. The bHLH transcription factor Paraxis is critically involved in this process. Formation of a new somite from the cranial end of the segmental plate is a highly controlled process involving complex cell movements in relation to each other. Hox genes specify regional identity of the somites and their derivatives. In the chicken a transposition of thoracic into cervical vertebrae has occurred as compared to the mouse. Transcription factors of the bHLH and homeodomain type also specify the cranio-caudal polarity and that of particular cell groups within the somites. According to segmentation models, somitogenesis is under the control of a “segmentation clock” in combination with a morphogen gradient. This hypothesis has recently found support from molecular data, especially the cycling expression of genes such as chairy1 and Lunatic Fringe, which depend on the Notch/Delta pathway of signal transduction. FGF8 has been described to be distributed along a cranio-caudal gradient. The first oscillating gene described shown to be independent of Notch is Axin2, encoding a negative regulator of the canonical Wnt pathway and a target of Wnt3a. Wnt3a and Axin2 show a similar distribution as FGF8 with high levels in the tailbud. The chick embryo has recently become accessible to molecular approaches such as overexpression by electroporation and RNA interference which can be expected to help elucidating some of the still open questions concerning somitogenesis.

Development of vertebrate embryos is regulated by a number of different signaling pathways. These pathways are frequently not independent of each other but are connected by crosstalk between cells and tissues. Furthermore, different signaling pathways have been found to interact at the cellular level. Development of cranial and limb structures is an example, in which FGF, BMP, and SHH signaling interact. Mutations in the different signaling pathways may therefore result in complex but similar phenotypes. This indicates the existence of integrator molecules, which depend in their expression or activity on the combination of different signaling pathways. Here we show that expression of the bHLH transcription factor Twist in the paraxial mesoderm requires an induction from the notochord. This induction can only be substituted by a combination of FGF and SHH signaling, but not by individual application of FGF8 or SHH alone. Furthermore, the expression of Twist can be modified by BMP2 in a complex, age-dependent manner. We propose that Twist is one of the integrating parts of the three signaling pathways and mediates some of the common effects.