Calcium signaling induces partial EMT and renal fibrosis in a Wnt4mCherry knock-in mouse model.

The renal tubular epithelial cells (TEC) have a strong capacity for repair after acute injury, but when this mechanism becomes uncontrollable, it leads to chronic kidney diseases (CKD). Indeed, in progress toward CKDs, the TECs may dedifferentiate, undergo epithelial-to-mesenchyme transition (EMT), and promote inflammation and fibrosis. Given the critical role of Wnt4 signaling in kidney ontogenesis, we addressed whether changes in this signaling are connected to renal inflammation and fibrosis by taking advantage of a knock-in Wnt4mCh/mCh mouse. While the Wnt4mCh/mCh embryos appeared normal, the corresponding mice, within one month, developed CKD-related phenotypes, such as pro-inflammatory responses including T-cell/macrophage influx, expression of fibrotic markers, and epithelial cell damage with a partial EMT. The Wnt signal transduction component ß-catenin remained unchanged, while calcium signaling is induced in the injured TECs involving Nfat and Tfeb transcription factors. We propose that the Wnt4 signaling pathway is involved in repairing the renal injury, and when the signal is overdriven, CKD is established.

Deciphering the minimal quantity of mouse primary cells to undergo nephrogenesis ex vivo.

BACKGROUND: Tissue organoids derived from primary cells have high potential for studying organ development and diseases in numerous organs. They recreate the morphological structure and mimic the functions of given organ while being compact in size, easy to produce, and suitable for use in various experimental setups. RESULTS: In this study we established the number of cells that form mouse kidney rudiments at E11.5, and generated renal organoids of various sizes from the mouse primary cells of the metanephric mesenchyme (MM). We investigated the ability of renal organoids to undergo nephrogenesis upon Wnt/ ß-catenin pathway-mediated tubule induction with a GSK-3 inhibitor (BIO) or by initiation through the ureteric bud (UB). We found that 5000 cells of MM cells are necessary to successfully form renal organoids with well-structured nephrons as judged by fluorescent microscopy, transmission electron microscopy (TEM), and quantitative Polymerase Chain Reaction (qPCR). These mouse organoids also recapitulated renal secretion function in the proximal tubules. CONCLUSIONS: We show that a significant decrease of cells used to generate renal mouse organoids in a dissociation/re-aggregation assay, does not interfere with development, and goes toward 3Rs. This enables generation of more experimental samples with one mouse litter, limiting the number of animals used for studies.

Mouse Embryonic Stem Cell-Derived Ureteric Bud Progenitors Induce Nephrogenesis.

Generation of kidney organoids from pluripotent stem cells (PSCs) is regarded as a potentially powerful way to study kidney development, disease, and regeneration. Direct differentiation of PSCs towards renal lineages is well studied; however, most of the studies relate to generation of nephron progenitor population from PSCs. Until now, differentiation of PSCs into ureteric bud (UB) progenitor cells has had limited success. Here, we describe a simple, efficient, and reproducible protocol to direct differentiation of mouse embryonic stem cells (mESCs) into UB progenitor cells. The mESC-derived UB cells were able to induce nephrogenesis when co-cultured with primary metanephric mesenchyme (pMM). In generated kidney organoids, the embryonic pMM developed nephron structures, and the mESC-derived UB cells formed numerous collecting ducts connected with the nephron tubules. Altogether, our study established an uncomplicated and reproducible platform to generate ureteric bud progenitors from mouse embryonic stem cells.

Experimental Tubulogenesis Induction Model in the Mouse.

Kidney development and induction of tubulogenesis have been studied for almost seven decades. The experimental setup of metanephric mesenchyme induction ex vivo allows to control the environment, to perform cellular manipulations, and to learn about renal development. Since the establishment of the ex vivo kidney culture technique in 1953, the method was modified to suit well the progress in biological and medical fields and still today present many advantages over the traditional in vivo studies.

Embryonic Stem Cells Derived Kidney Organoids as Faithful Models to Target Programmed Nephrogenesis.

The kidney is a complex organ that is comprised of thousands of nephrons developing through reciprocal inductive interactions between metanephric mesenchyme (MM) and ureteric bud (UB). The MM undergoes mesenchymal to epithelial transition (MET) in response to the signaling from the UB. The secreted protein Wnt4, one of the Wnt family members, is critical for nephrogenesis as mouse Wnt4-/- mutants fail to form pretubular aggregates (PTA) and therefore lack functional nephrons. Here, we generated mouse embryonic stem cell (mESC) line lacking Wnt4 by applying the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems 9 (Cas9). We describe here, differentiation of the wild type and Wnt4 knockout mESCs into kidney progenitors, and such cells induced to undergo nephrogenesis by the mouse E11.5 UB mediated induction. The wild type three-dimensional (3D) self-organized organoids depict appropriately segmented nephron structures, while the Wnt4-deficient organoids fail to undergo the MET, as is the case in the phenotype of the Wnt4 knockout mouse model in vivo. In summary, we have established a platform that combine CRISPR/Cas9 and kidney organoid technologies to model kidney development in vitro and confirmed that mutant organoids are able to present similar actions as in the in vivo studies.

Exosomes as secondary inductive signals involved in kidney organogenesis.

The subfraction of extracellular vesicles, called exosomes, transfers biological molecular information not only between cells but also between tissues and organs as nanolevel signals. Owing to their unique properties such that they contain several RNA species and proteins implicated in kidney development, exosomes are putative candidates to serve as developmental programming units in embryonic induction and tissue interactions. We used the mammalian metanephric kidney and its nephron-forming mesenchyme containing the nephron progenitor/stem cells as a model to investigate if secreted exosomes could serve as a novel type of inductive signal in a process defined as embryonic induction that controls organogenesis. As judged by several characteristic criteria, exosomes were enriched and purified from a cell line derived from embryonic kidney ureteric bud (UB) and from primary embryonic kidney UB cells, respectively. The cargo of the UB-derived exosomes was analysed by qPCR and proteomics. Several miRNA species that play a role in Wnt pathways and enrichment of proteins involved in pathways regulating the organization of the extracellular matrix as well as tissue homeostasis were identified. When labelled with fluorescent dyes, the uptake of the exosomes by metanephric mesenchyme (MM) cells and the transfer of their cargo to the cells can be observed. Closer inspection revealed that besides entering the cytoplasm, the exosomes were competent to also reach the nucleus. Furthermore, fluorescently labelled exosomal RNA enters into the cytoplasm of the MM cells. Exposure of the embryonic kidney-derived exosomes to the whole MM in an ex vivo organ culture setting did not lead to an induction of nephrogenesis but had an impact on the overall organization of the tissue. We conclude that the exosomes provide a novel signalling system with an apparent role in secondary embryonic induction regulating organogenesis.

Kidney development and perspectives for organ engineering.

Organ transplantation is currently the best strategy for treating end stage renal disease (ESRD) but the numbers of donor kidneys available are not sufficient to meet the needs of the ever-increasing ESRD population. Therefore, developments in the field of tissue engineering are necessary to provide alternative treatments. Decellularization and three-dimensional (3D) bioprinting strategies may serve as attractive novel options. Since successful tissue engineering requires an in -depth understanding of organ development and regulatory pathways, we discuss signaling in renal development and the composition of the renal extracellular matrix before presenting progress in the decellularization and 3D bioprinting fields.

Novel fixed z-direction (FiZD) kidney primordia and an organoid culture system for time-lapse confocal imaging.

Tissue, organ and organoid cultures provide suitable models for developmental studies, but our understanding of how the organs are assembled at the single-cell level still remains unclear. We describe here a novel fixed z-direction (FiZD) culture setup that permits high-resolution confocal imaging of organoids and embryonic tissues. In a FiZD culture a permeable membrane compresses the tissues onto a glass coverslip and the spacers adjust the thickness, enabling the tissue to grow for up to 12 days. Thus, the kidney rudiment and the organoids can adjust to the limited z-directional space and yet advance the process of kidney morphogenesis, enabling long-term time-lapse and high-resolution confocal imaging. As the data quality achieved was sufficient for computer-assisted cell segmentation and analysis, the method can be used for studying morphogenesis ex vivo at the level of the single constituent cells of a complex mammalian organogenesis model system.

Nephrogenesis in organoids to develop novel drugs and progenitor cell based therapies.

New therapies that are derived from small molecules and stem/progenitor cells should be developed to face the increasing occurrence of end stage renal disease where treatments are currently limited. Over the last decade a significant progress in the knowledge of how the organs are assembled have been made and led to development of novel three-dimensional organoid assays, also for the kidney. Indeed, such organoids provide novel tool to study aspects of drugs nephrotoxicity, openings for renal disease modeling and cell therapy development and may offer solutions for end stage renal disease.

The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3.

Adrenal glands are zonated endocrine organs that are essential in controlling body homeostasis. How zonation is induced and maintained and how renewal of the adrenal cortex is ensured remain a mystery. Here we show that capsular RSPO3 signals to the underlying steroidogenic compartment to induce ß-catenin signaling and imprint glomerulosa cell fate. Deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth. Importantly, Rspo3 function remains essential in adult life to ensure replenishment of lost cells and maintain the properties of the zona glomerulosa. Thus, the adrenal capsule acts as a central signaling center that ensures replacement of damaged cells and is required to maintain zonation throughout life.

Measures of kidney function by minimally invasive techniques correlate with histological glomerular damage in SCID mice with adriamycin-induced nephropathy.

Maximising the use of preclinical murine models of progressive kidney disease as test beds for therapies ideally requires kidney function to be measured repeatedly in a safe, minimally invasive manner. To date, most studies of murine nephropathy depend on unreliable markers of renal physiological function, exemplified by measuring blood levels of creatinine and urea, and on various end points necessitating sacrifice of experimental animals to assess histological damage, thus counteracting the principles of Replacement, Refinement and Reduction. Here, we applied two novel minimally invasive techniques to measure kidney function in SCID mice with adriamycin-induced nephropathy. We employed i) a transcutaneous device that measures the half-life of intravenously administered FITC-sinistrin, a molecule cleared by glomerular filtration; and ii) multispectral optoacoustic tomography, a photoacoustic imaging device that directly visualises the clearance of the near infrared dye, IRDye 800CW carboxylate. Measurements with either technique showed a significant impairment of renal function in experimental animals versus controls, with significant correlations with the proportion of scarred glomeruli five weeks after induction of injury. These technologies provide clinically relevant functional data and should be widely adopted for testing the efficacies of novel therapies. Moreover, their use will also lead to a reduction in experimental animal numbers.

Organ In Vitro Culture: What Have We Learned about Early Kidney Development?

When Clifford Grobstein set out to study the inductive interaction between tissues in the developing embryo, he developed a method that remained important for the study of renal development until now. From the late 1950s on, in vitro cultivation of the metanephric kidney became a standard method. It provided an artificial environment that served as an open platform to study organogenesis. This review provides an introduction to the technique of organ culture, describes how the Grobstein assay and its variants have been used to study aspects of mesenchymal induction, and describes the search for natural and chemical inducers of the metanephric mesenchyme. The review also focuses on renal development, starting with ectopic budding of the ureteric bud, ureteric bud branching, and the generation of the nephron and presents the search for stem cells and renal progenitor cells that contribute to specific structures and tissues during renal development. It also presents the current use of Grobstein assay and its modifications in regenerative medicine and tissue engineering today. Together, this review highlights the importance of ex vivo kidney studies as a way to acquire new knowledge, which in the future can and will be implemented for developmental biology and regenerative medicine applications.

Signaling during Kidney Development.

The kidney plays an essential role during excretion of metabolic waste products, maintenance of key homeostasis components such as ion concentrations and hormone levels. It influences the blood pressure, composition and volume. The kidney tubule system is composed of two distinct cell populations: the nephrons forming the filtering units and the collecting duct system derived from the ureteric bud. Nephrons are composed of glomeruli that filter the blood to the Bowman's capsule and tubular structures that reabsorb and concentrate primary urine. The collecting duct is a Wolffian duct-derived epithelial tube that concentrates and collects urine and transfers it via the renal pelvis into the bladder. The mammalian kidney function depends on the coordinated development of specific cell types within a precise architectural framework. Due to the availability of modern analysis techniques, the kidney has become a model organ defining the paradigm to study organogenesis. As kidney diseases are a problem worldwide, the understanding of mammalian kidney cells is of crucial importance to develop diagnostic tools and novel therapies. This review focuses on how the pattern of renal development is generated, how the inductive signals are regulated and what are their effects on proliferation, differentiation and morphogenesis.

Development of embryonic stem cells in recombinant kidneys.

Embryonic stem cells (ESC) are self-renewing and can generate all cell types during normal development. Previous studies have begun to explore fates of ESCs and their mesodermal derivatives after injection into explanted intact metanephric kidneys and neonatal kidneys maturing in vivo. Here, we exploited a recently described recombinant organ culture model, mixing fluorescent quantum dot labeled mouse exogenous cells with host metanephric cells. We compared abilities of undifferentiated ESCs with ESC-derived mesodermal or non-mesodermal cells to contribute to tissue compartments within recombinant, chimeric metanephroi. ESC-derived mesodermal cells downregulated Oct4, a marker of undifferentiated cells, and, as assessed by locations of quantum dots, contributed to Wilms' tumor 1-expressing forming nephrons, synaptopodin-expressing glomeruli, and organic ion-transporting tubular epithelia. Similar results were observed when labeled native metanephric cells were recombined with host cells. In striking contrast, non-mesodermal ESC-derived cells strongly inhibited growth of embryonic kidneys, while undifferentiated ESCs predominantly formed Oct4 expressing colonies between forming nephrons and glomeruli. These findings clarify the conclusion that ESC-derived mesodermal cells have functional nephrogenic potential, supporting the idea that they could potentially replace damaged epithelia in diseased kidneys. On the other hand, undifferentiated ESCs and non-mesodermal precursors derived from ESCs would appear to be less suitable materials for use in kidney cell therapies.

Quantum dots do not affect the behaviour of mouse embryonic stem cells and kidney stem cells and are suitable for short-term tracking.

Quantum dots (QDs) are small nanocrystals widely used for labelling cells in order to enable cell tracking in complex environments in vitro, ex vivo and in vivo. They present many advantages over traditional fluorescent markers as they are resistant to photobleaching and have narrow emission spectra. Although QDs have been used effectively in cell tracking applications, their suitability has been questioned by reports showing they can affect stem cell behaviour and can be transferred to neighbouring cells. Using a variety of cellular and molecular biology techniques, we have investigated the effect of QDs on the proliferation and differentiation potential of two stem cell types: mouse embryonic stem cells and tissue-specific stem cells derived from mouse kidney. We have also tested if QDs released from living or dead cells can be taken up by neighbouring cells, and we have determined if QDs affect the degree of cell-cell fusion; this information is critical in order to assess the suitability of QDs for stem cell tracking. We show here that QDs have no effect on the viability, proliferation or differentiation potential of the two stem cell types. Furthermore, we show that the extent of transfer of QDs to neighbouring cells is <4%, and that QDs do not increase the degree of cell-cell fusion. However, although the QDs have a high labelling efficiency (>85%), they are rapidly depleted from both stem cell populations. Taken together, our results suggest that QDs are effective cell labelling probes that are suitable for short-term stem cell tracking.

Integration potential of mouse and human bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a multipotent cell population which has been described to exert renoprotective and regenerative effects in experimental models of kidney injury. Several lines of evidence indicate that MSCs also have the ability to contribute to nephrogenesis, suggesting that the cells can be employed in stem cell-based applications aimed at de novo renal tissue generation. In this study we re-evaluate the capacity of mouse and human bone marrow-derived MSCs to contribute to the development of renal tissue using a novel method of embryonic kidney culture. Although MSCs show expression of some genes involved in renal development, their contribution to nephrogenesis is very limited in comparison to other stem cell types tested. Furthermore, we found that both mouse and human MSCs have a detrimental effect on the ex vivo development of mouse embryonic kidney, this effect being mediated through a paracrine action. Stimulation with conditioned medium from a mouse renal progenitor population increases the ability of mouse MSCs to integrate into developing renal tissue and prevents the negative effects on kidney development, but does not appear to enhance their ability to undergo nephrogenesis.

The potential of small chemical functional groups for directing the differentiation of kidney stem cells.

In the future, stem-cell-based therapies could offer new approaches to treat kidney disease and reduce the incidence of ESRD (end-stage renal disease), but, as yet, research in this area is only being conducted in rodents and it is not clear whether or when it could be applied to human patients. Drug therapies, on the other hand, have been very effective at delaying the progression of kidney disease, but, for various reasons, current drug regimes are not suitable for all patients. A greater understanding of the molecular mechanisms that underlie disease progression in chronic kidney disease could help to identify novel drug targets. However, progress in this area is currently hindered due to the lack of appropriate in vitro culture systems for important renal cell types, such as proximal tubule cells and podocytes. This problem could be overcome if it were possible to direct the differentiation of kidney stem cells to renal cell types in vitro. In the present review, we highlight the potential of surface gradients of small chemical functional groups to direct the differentiation of kidney stem cells.

The expression pattern of the 70-kDa heat shock protein Hspa2 in mouse tissues.

The highest expression level of a 70-kDa heat shock protein family member Hspa2 is detected specifically in meiotic and post-meiotic male germ cells, which is reflected by original name of this protein, i.e., testis-specific Hsp70. However, this chaperon protein could be also detected in certain somatic tissues. Here, the extra-testicular expression pattern of mouse Hspa2 was analyzed. We found expression of Hspa2 in various epithelial cells including lining of bronchioles and oviduct, columnar epithelium of endometrium, epithelial reticular cells of thymus, transitional epithelium of the urinary bladder, or ependymal cells covering walls of the ventricular system of the brain. Surprisingly, Hspa2 was a putative secretory protein in intestine, endometrial glands and subcommissural organ. Hspa2 was detected in central and peripheral nervous system: in neuron's bodies and fiber tracts, in the subventricular zone of the lateral ventricles, in the dentate gyrus of the hippocampus, in enteric ganglia of the gastrointestinal tract. Hspa2 was also expressed in smooth muscles and at low level in immune system (in germinal centers associated with B-lymphocyte production). In addition to somatic tissues listed above, Hspa2 was detected in oocytes arrested at diplotene of the first meiotic division.