Renal Organogenesis Research Articles

Comprehensive analysis of FRAS1/FREM family as potential biomarkers and therapeutic targets in renal clear cell carcinoma.

Background: FRAS1 (Fraser syndrome protein 1), together with FREM1 (the Fras1-related extracellular matrix proteins 1) and FREM2, belonging to the FRAS1/FREM extracellular matrix protein family, are considered to play essential roles in renal organogenesis and cancer progression. However, their roles in kidney renal clear cell carcinoma (KIRC) remain to be elucidated. Methods: FRAS1/FREM RNA expression analysis was performed using TCGA/GTEx databases, and valided using GEO databases and real-time PCR. Protein expression was peformed using CPTAC databases. Herein, we employed an array of bioinformatics methods and online databases to explore the potential oncogenic roles of FRAS1/FREM in KIRC. Results: We found that FRAS1, FREM1 and FREM2 genes and proteins expression levels were significantly decreased in KIRC tissues than in normal tissues. Decreased FRAS1/FREM expression levels were significantly associated with advanced clinicopathological parameters (pathological stage, grade and tumor metastasis status). Notably, the patients with decreased FRAS1/FREM2 expression showed a high propensity for metastasis and poor prognosis. FRAS1/FREM were correlated with various immune infiltrating cells, especially CD4+ T cells and its corresponding subsets (Th1, Th2, Tfh and Tregs). FRAS1 and FREM2 had association with DNA methylation and their single CpG methylation levels were associated with prognosis. Moreover, FRAS1/FREM might exert antitumor effects by functioning in key oncogenic signalling pathways and metabolic pathways. Drug sensitivity analysis indicated that high FRAS1 and FREM2 expression can be a reliable predictor of targeted therapeutic drug response, highlighting the potential as anticancer drug targets. Conclusion: Together, our results indicated that FRAS1/FREM family members could be potential therapeutic targets and valuable prognostic biomarkers of KIRC.

Sildenafil Citrate Enhances Renal Organogenesis Following Metanephroi Allotransplantation into Non-Immunosuppressed Hosts.

In order to harness the potential of metanephroi allotransplantation to the generation of a functional kidney graft on demand, we must achieve further growth post-transplantation. Sildenafil citrate (SC) is widely known as a useful inductor of angiogenesis, offering renoprotective properties due to its anti-inflammatory, antifibrotic, and antiapoptotic effects. Here, we performed a laparoscopic metanephroi allotransplantation after embedding sildenafil citrate into the retroperitoneal fat of non-immunosuppressed adult rabbit hosts. Histology and histomorphometry were used to examine the morphofunctional changes in new kidneys 21 days post-transplantation. Immunofluorescence of E-cadherin and renin and erythropoietin gene expression were used to assess the tubule integrity and endocrine functionality. After the metanephroi were embedded in a 10 µM SC solution, the new kidneys’ weights become increased significantly. The E-cadherin expression together with the renin and erythropoietin gene expression revealed its functionality, while histological mature glomeruli and hydronephrosis proved the new kidneys’ excretory function. Thus, we have described a procedure through the use of SC that improves the outcomes after a metanephroi transplantation. This study gives hope to a pathway that could offer a handsome opportunity to overcome the kidney shortage.

Unravelling the Role of PAX2 Mutation in Human Focal Segmental Glomerulosclerosis.

No effective treatments are available for familial steroid-resistant Focal Segmental Glomerulosclerosis (FSGS), characterized by proteinuria due to ultrastructural abnormalities in glomerular podocytes. Here, we studied a private PAX2 mutation identified in a patient who developed FSGS in adulthood. By generating adult podocytes using patient-specific induced pluripotent stem cells (iPSC), we developed an in vitro model to dissect the role of this mutation in the onset of FSGS. Despite the PAX2 mutation, patient iPSC properly differentiated into podocytes that exhibited a normal structure and function when compared to control podocytes. However, when exposed to an environmental trigger, patient podocytes were less viable and more susceptible to cell injury. Fixing the mutation improved their phenotype and functionality. Using a branching morphogenesis assay, we documented developmental defects in patient-derived ureteric bud-like tubules that were totally rescued by fixing the mutation. These data strongly support the hypothesis that the PAX2 mutation has a dual effect, first in renal organogenesis, which could account for a suboptimal nephron number at birth, and second in adult podocytes, which are more susceptible to cell death caused by environmental triggers. These abnormalities might translate into the development of proteinuria in vivo, with a progressive decline in renal function, leading to FSGS.

Identification of a foetal epigenetic compartment in adult human kidney

ABSTRACT The mammalian kidney has extensive repair capacity; however, identifying adult renal stem cells has proven elusive. We applied an epigenetic marker of foetal cell origin (FCO) in diverse human tissues as a probe for developmental cell persistence, finding a 5.4-fold greater FCO proportion in kidney. Normal kidney FCO proportions averaged 49% with extensive interindividual variation. FCO proportions were significantly negatively correlated with immune-related gene expression and positively correlated with genes expressed in the renal medulla, including those involved in renal organogenesis (e.g., FGF2, PAX8, and HOXB7). FCO associated genes also mapped to medullary nephron segments in mouse and rat, suggesting evolutionary conservation of this cellular compartment. Renal cancer patients whose tumours contained non-zero FCO scores survived longer. The kidney appears unique in possessing substantial foetal epigenetic features. Further study of FCO-related gene methylation may elucidate regenerative regulatory programmes in tissues without apparent discrete stem cell compartments.

Nephronectin as a Matrix Effector in Cancer.

Simple SummaryThe extracellular matrix provides an important scaffold for cells and tissues of multicellular organisms. The scaffold not only provides a secure anchorage point, but also functions as a reservoir for signalling molecules, sequestered and released when necessary. A dysregulated extracellular matrix may therefore modulate cellular behaviour, as seen during cancer progression. The extracellular matrix protein nephronectin was discovered two decades ago and found to regulate important embryonic developmental processes. Loss of either nephronectin or its receptor, integrin α8β1, leads to underdeveloped kidneys. Recent findings show that nephronectin is also dysregulated in breast cancer and plays a role in promoting metastasis. To enable therapeutic intervention, it is important to fully understand the role of nephronectin and its receptors in cancer progression. In this review, we summarise the literature on nephronectin, analyse the structure and domain-related functions of nephronectin and link these functions to potential roles in cancer progression.The extracellular matrix protein nephronectin plays an important regulatory role during embryonic development, controlling renal organogenesis through integrin α8β1 association. Nephronectin has three main domains: five N-terminal epidermal growth factor-like domains, a linker region harbouring two integrin-binding motifs (RGD and LFEIFEIER), and a C-terminal MAM domain. In this review, we look into the domain-related functions of nephronectin, and tissue distribution and expression. During the last two decades it has become evident that nephronectin also plays a role during cancer progression and in particular metastasis. Nephronectin is overexpressed in both human and mouse breast cancer compared to normal breast tissue where the protein is absent. Cancer cells expressing elevated levels of nephronectin acquire increased ability to colonise distant organs. In particular, the enhancer-motif (LFEIFEIER) which is specific to the integrin α8β1 association induces viability via p38 MAPK and plays a role in colonization. Integrins have long been desired as therapeutic targets, where low efficiency and receptor redundancy have been major issues. Based on the summarised publications, the enhancer-motif of nephronectin could present a novel therapeutic target.

Novel Aspects of Extracellular Vesicles in the Regulation of Renal Physiological and Pathophysiological Processes.

Extracellular vesicles (EV) are nanosized particles released by a large variety of cells. They carry molecules such as proteins, RNA and lipids. While urinary EVs have been longer studied as a source of biomarkers for renal and non-renal disorders, research on EVs as regulatory players of renal physiological and pathological processes has experienced an outbreak recently in the past decade. In general, the microenvironment and (patho)physiological state of the donor cells affect the cargo of the EVs released, which then determines the effect of these EVs once they reach a target cell. For instance, EVs released by renal epithelial cells modulate the expression and function of water and solute transporting proteins in other cells. Also, EVs have been demonstrated to regulate renal organogenesis and blood flow. Furthermore, a dual role of EVs promoting, but also counteracting, disease has also been reported. EVs released by renal tubular cells can reach fibroblasts, monocytes, macrophages, T cells and natural killer cells, thus influencing the pathogenesis and progression of renal disorders like acute kidney injury and fibrosis, nephrolithiasis, renal transplant rejection and renal cancer, among others. On the contrary, EVs may also exert a cytoprotective role upon renal damage and promote recovery of renal function. In the current review, a systematic summary of the key studies from the past 5 years addressing the role of EVs in the modulation of renal physiological and pathophysiological processes is provided, highlighting open questions and discussing the potential of future research.

Enhancement of HGF-induced tubulogenesis by endothelial cell-derived GDNF.

Tubulogenesis, the organization of epithelial cells into tubular structures, is an essential step during renal organogenesis as well as during the regeneration process of renal tubules after injury. In the present study, endothelial cell-derived factors that modulate tubule formation were examined using an in vitro human tubulogenesis system. When human renal proximal tubular epithelial cells (RPTECs) were cultured in gels, tubular structures with lumens were induced in the presence of hepatocyte growth factor (HGF). Aquaporin 1 was localized in the apical membrane of these tubular structures, suggesting that these structures are morphologically equivalent to renal tubules in vivo. HGF-induced tubule formation was significantly enhanced when co-cultured with human umbilical vein endothelial cells (HUVECs) or in the presence of HUVEC-conditioned medium (HUVEC-CM). Co-culture with HUVECs did not induce tubular structures in the absence of HGF. A phospho-receptor tyrosine kinase array revealed that HUVEC-CM markedly enhanced phosphorylation of Ret, glial cell-derived neurotrophic factor (GDNF) receptor, in HGF-induced tubular structures compared to those without HUVEC-CM. HUVECs produced GDNF, and RPTECs expressed both Ret and GDNF family receptor alpha1 (co-receptor). HGF-induced tubule formation was significantly enhanced by addition of GDNF. Interestingly, not only HGF but also GDNF significantly induced phosphorylation of the HGF receptor, Met. These data indicate that endothelial cell-derived GDNF potentiates the tubulogenic properties of HGF and may play a critical role in the epithelial-endothelial crosstalk during renal tubulogenesis as well as tubular regeneration after injury.

Macrophages restrict the nephrogenic field and promote endothelial connections during kidney development.

The origins and functions of kidney macrophages in the adult have been explored, but their roles during development remain largely unknown. Here we characterise macrophage arrival, localisation, heterogeneity, and functions during kidney organogenesis. Using genetic approaches to ablate macrophages, we identify a role for macrophages in nephron progenitor cell clearance as mouse kidney development begins. Throughout renal organogenesis, most kidney macrophages are perivascular and express F4/80 and CD206. These macrophages are enriched for mRNAs linked to developmental processes, such as blood vessel morphogenesis. Using antibody-mediated macrophage-depletion, we show macrophages support vascular anastomoses in cultured kidney explants. We also characterise a subpopulation of galectin-3+ (Gal3+) myeloid cells within the developing kidney. Our findings may stimulate research into macrophage-based therapies for renal developmental abnormalities and have implications for the generation of bioengineered kidney tissues.

The Origins and Functions of Tissue-Resident Macrophages in Kidney Development

The adult kidney hosts tissue-resident macrophages that can cause, prevent, and/or repair renal damage. Most of these macrophages derive from embryonic progenitors that colonize the kidney during its development and proliferate in situ throughout adulthood. Although the precise origins of kidney macrophages remain controversial, recent studies have revealed that embryonic macrophage progenitors initially migrate from the yolk sac, and later from the fetal liver, into the developing kidney. Once in the kidney, tissue-specific transcriptional regulators specify macrophage progenitors into dedicated kidney macrophages. Studies suggest that kidney macrophages facilitate many processes during renal organogenesis, such as branching morphogenesis and the clearance of cellular debris; however, little is known about how the origins and specification of kidney macrophages dictate their function. Here, we review significant new findings about the origins, specification, and developmental functions of kidney macrophages.

Wnt8a expands the pool of embryonic kidney progenitors in zebrafish

During zebrafish embryogenesis the pronephric kidney arises from a small population of posterior mesoderm cells that then undergo expansion during early stages of renal organogenesis. While wnt8 is required for posterior mesoderm formation during gastrulation, it is also transiently expressed in the post-gastrula embryo in the intermediate mesoderm, the precursor to the pronephros and some blood/vascular lineages. Here, we show that knockdown of wnt8a, using a low dose of morpholino that does not disrupt early mesoderm patterning, reduces the number of kidney and blood cells. For the kidney, wnt8a deficiency decreases renal progenitor growth during early somitogenesis, as detected by EdU incorporation, but has no effect on apoptosis. The depletion of the renal progenitor pool in wnt8a knockdown embryos leads to cellular deficits in the pronephros at 24 hpf that are characterised by a shortened distal-most segment and stretched proximal tubule cells. A pulse of the canonical Wnt pathway agonist BIO during early somitogenesis is sufficient to rescue the size of the renal progenitor pool while longer treatment expands the number of kidney cells. Taken together, these observations indicate that Wnt8, in addition to its well-established role in posterior mesoderm patterning, also plays a later role as a factor that expands the renal progenitor pool prior to kidney morphogenesis.

Chronic kidney disease in children and the role of epigenetics: Future therapeutic trajectories.

Global differences in the observed causes of chronic kidney disease (CKD) in children are well documented and are attributed to dissimilarities in clime, race, hereditary, and ancestry. Thus, familial clustering and disparities in CKD prevalence rates across ethnic and racial groups indicate that the progression of renal disease has a strong genetic component. Mammalian studies have demonstrated a feasible nexus between nutrition and non-genetic exposure (around the time of conception and in epigenetic changes) in the expression of major genes identified in renal organogenesis. The major consequence is a reduction in the number of nephrons, with subsequent predisposition to hypertension and CKD. Identifying these epigenetic changes is crucial (due to their potentially reversible nature), as they may serve as future therapeutic targets to prevent kidney fibrosis and CKD. Despite progress in the field of epigenetics in oncology, research in other subspecialties of medicine is largely experimental with few existing studies regarding the clinical implication of epigenetics in renal disease. Therapeutic trajectories for CKD in children based on the influence of epigenetics may eventually revolutionize the management of this disease. The aim of the current narrative review is to appraise the role of epigenetics in CKD, and highlight the potential future therapeutic pathways.

Ablation of the epithelial-specific splicing factor Esrp1 results in ureteric branching defects and reduced nephron number.

Background:Abnormalities in ureteric bud (UB) branching morphogenesis lead to congenital anomalies of the kidney and reduced nephron numbers associated with chronic kidney disease (CKD) and hypertension. Previous studies showed that the epithelial fibroblast growth factor receptor 2 (Fgfr2) IIIb splice variant supports ureteric morphogenesis in response to ligands from the metanephric mesenchyme during renal organogenesis. The epithelial‐specific splicing regulator Esrp1 is required for expression of Fgfr2‐IIIb and other epithelial‐specific splice variants. Our objective was to determine whether Esrp1 is required for normal kidney development.Results:Ablation of Esrp1 in mice, alone or together with its paralog Esrp2, was associated with reduced kidney size and increased incidence of renal aplasia. Three‐dimensional imaging showed that embryonic Esrp1 knockout (KO) kidneys had fewer ureteric tips and reduced nephron numbers. Analysis of alternative splicing in Esrp‐null ureteric epithelial cells by RNA‐Seq confirmed a splicing switch in Fgfr2 as well as numerous other transcripts.Conclusions:Our findings reveal that Esrp1‐regulated splicing in ureteric epithelial cells plays an important role in renal development. Defects in Esrp1 KO kidneys likely reflect reduced and/or absent ureteric branching, leading to decreased nephron induction secondary to incorrect Fgfr2 splicing and other splicing alterations. Developmental Dynamics 245:991–1000, 2016. © 2016 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

The potential role of regucalcin in kidney cell regulation: Involvement in renal failure (Review).

The kidneys play a physiologic role in the regulation of urine formation and nutrient reabsorption in the proximal tubule epithelial cells. Kidney development has been shown to be regulated through calcium(Ca2+) signaling processes that are present through numerous steps of tubulogenesis and nephron induction during embryonic development of the kidneys. Ca2+-binding proteins, such as calbindin-D28k and regucalcin are important proteins that are commonly used as biomarkers in pronephric tubules, and the ureteric bud and metanephric mesenchyme. Previous research on regucalcin focused on Ca2+ sensors that are involved in renal organogenesis and the link between Ca2+-dependent signals and polycystins. Moreover, regucalcin has been highlighted to play a multifunctional role in kidney cell regulation. The regucalcin gene, which is localized on the Xchromosome, is regulated through various transcription factors. Regucalcin has been found to regulate intracellular Ca2+ homeostasis in kidney proximal tubule epithelial cells. Regucalcin has been demonstrated to regulate the activity of various enzymes that are involved in intracellular signaling pathways. It has been noted that regucalcin suppresses DNA synthesis and regulates the gene expression of various proteins related to mineral transport, transcription factors, cell proliferation and apoptosis. The overexpression of regucalcin has been shown to exert suppressive effects on cell proliferation and apoptotic cell death, which are stimulated by various stimulatory factors. Moreover, regucalcin gene expression was found to to be involved in various pathophysiological states, including renal failure. This review discusses recent findings concerning the potential role of regucalcin as a regulatory protein in the kidney proximal tubule epithelial cells.

DICER1 mutations in childhood cystic nephroma and its relationship to DICER1-renal sarcoma

The pathogenesis of cystic nephroma of the kidney has interested pathologists for over 50 years. Emerging from its initial designation as a type of unilateral multilocular cyst, cystic nephroma has been considered as either a developmental abnormality or a neoplasm or both. Many have viewed cystic nephroma as the benign end of the pathologic spectrum with cystic partially differentiated nephroblastoma and Wilms tumor, whereas others have considered it a mixed epithelial and stromal tumor. We hypothesize that cystic nephroma, like the pleuropulmonary blastoma in the lung, represents a spectrum of abnormal renal organogenesis with risk for malignant transformation. Here we studied DICER1 mutations in a cohort of 20 cystic nephromas and 6 cystic partially differentiated nephroblastomas, selected independently of a familial association with pleuropulmonary blastoma and describe four cases of sarcoma arising in cystic nephroma, which have a similarity to the solid areas of type II or III pleuropulmonary blastoma. The genetic analyses presented here confirm that DICER1 mutations are the major genetic event in the development of cystic nephroma. Further, cystic nephroma and pleuropulmonary blastoma have similar DICER1 loss of function and ‘hotspot' missense mutation rates, which involve specific amino acids in the RNase IIIb domain. We propose an alternative pathway with the genetic pathogenesis of cystic nephroma and DICER1-renal sarcoma paralleling that of type I to type II/III malignant progression of pleuropulmonary blastoma.

MiRNA let-7e targeting MMP9 is involved in adipose-derived stem cell differentiation toward epithelia

miRNA let-7e is involved in stem cell differentiation, and metalloproteinases are among its potential target genes. We hypothesized that the inhibitory action of let-7e on regulation of MMP9 expression could represent a crucial mechanism during differentiation of adipose-derived stem cells (ASCs). ASCs were differentiated with all-trans retinoic acid (ATRA) to promote differentiation, and the effect of let-7 silencing during differentiation was tested. Results indicate that ASCs cultured with ATRA differentiated into cells of the epithelial lineage. We found that ASCs cultured with ATRA or transfected with miRNA let-7e expressed epithelial markers such as cytokeratin-18 and early renal organogenesis markers such as Pax2, Wt1, Wnt4 and megalin. Conversely, the specific knockdown of miRNA let-7e in ASCs significantly decreased the expression of these genes, indicating its vital role during the differentiation process. Using luciferase reporter assays, we also showed that MMP9 is a direct target of miRNA let-7e. Thus, our results suggest that miRNA let-7e acts as a matrix metalloproteinase-9 (MMP9) inhibitor and differentiation inducer in ASCs.

ECM modulated early kidney development in embryonic organ culture

The use of exogenous signals is gaining importance in renal regenerative therapies. We wanted to explore the role of extracellular matrix (ECM) constituents on renal structure formation during renal organogenesis. We used a recently established organ culture setup to expose embryonic kidney rudiments directly to a large set of surface-immobilized or dissolved ECM molecules and growth factors. Organ culture was also performed on immobilized adult kidney ECM extracts and on reactive polymer films without any biomolecular components. The applied conditions resulted in distinct differences of organ phenotypes, underlining the multifaceted role of exogenous signals during kidney development. Specific ECM components, including collagen I and laminin, supported nephronal and tubular structure formation of the developing organ. ECM biopolymers, e.g. hyaluronic acid, were found to determine the fate of developing explants in a concentration- and molecular weight-dependent manner. The organ culture system used was an effective and robust means to identify exogenous signals that direct kidney development. This system can provide valuable insight for future regenerative therapies of kidney diseases.

Three calcium-sensitive genes, fus, brd3 and wdr5, are highly expressed in neural and renal territories during amphibian development

Numerous Ca2+ signaling events have been associated with early development of vertebrate embryo, from fertilization to organogenesis. In Xenopus laevis, Ca2+ signals are key regulators in the earliest steps of the nervous system development. If neural determination is one of the best-characterized examples of the role of Ca2+ during embryogenesis, increasing literature supports a determining role of organogenesis and differentiation. In blastula the cells of the presumptive ectoderm (animal caps) are pluripotent and can be induced toward neural fate with an intracellular increase of free Ca2+ triggered by caffeine. To identify genes that are transcribed early upon Ca2+ stimuli and involved in neural determination, we have constructed a subtractive cDNA library between neuralized and non-neuralized animal caps. Here we present the expression pattern of three new Ca2+-sensitive genes: fus (fused in sarcoma), brd3 (bromodomain containing 3) and wdr5 (WD repeat domain 5) as they all represent potential regulators of the transcriptional machinery. Using in situ hybridization we illustrated the spatial expression pattern of fus, brd3 and wdr5 during early developmental stages of Xenopus embryos. Strikingly, their domains of expression are not restricted to neural territories. They all share a specific expression throughout renal organogenesis which has been found to rely also on Ca2+ signaling. This therefore highlights the key function of Ca2+ target genes in specific territories during early development. We propose that Ca2+ signaling through modulation of fus, brd3 and wdr5 expressions can control the transcription machinery to achieve proper embryogenesis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Generation of Kidney from Pluripotent Stem Cells via Blastocyst Complementation

Because a shortage of donor organs has been a major obstacle to the expansion of organ transplantation programs, the generation of transplantable organs is among the ultimate goals of regenerative medicine. However, the complex cellular interactions among and within tissues that are required for organogenesis are difficult to recapitulate in vitro. As an alternative, we used blastocyst complementation to generate pluripotent stem cell (PSC)-derived donor organs in vivo. We hypothesized that if we injected PSCs into blastocysts obtained from mutant mice in which the development of a certain organ was precluded by genetic manipulation, thereby leaving a niche for organ development, the PSC-derived cells would developmentally compensate for the defect and form the missing organ. In our previous work, we showed proof-of-principle findings of pancreas generation by injection of PSCs into pancreas-deficient Pdx1(-/-) mouse blastocysts. In this study, we have extended this technique to kidney generation using Sall1(-/-) mouse blastocysts. As a result, the defective cells were totally replaced, and the kidneys were entirely formed by the injected mouse PSC-derived cells, except for structures not under the influence of Sall1 expression (ie, collecting ducts and microvasculature). These findings indicate that blastocyst complementation can be extended to generate PSC-derived kidneys. This system may therefore provide novel insights into renal organogenesis.

Antenatal detection of fetuses with renal pelvic dilatation and their 6 months outcomes at surrey and Sussex healthcare NHS Trust

Antenatally diagnosed urinary tract anomalies form approximately 20% of fetal malformations. Renal pelvic dilatation forms 2-3% cases.Embryologically, the renal organogenesis is complex involving multitude of pathologic factors. Obstruction to urinary...

Renal organogenesis

The increasing prevalence of chronic kidney disease in the absence of new treatment modalities has become a strong driver for innovation in nephrology. An increasing understanding of stem cell biology has kindled the prospects of regenerative options for kidney disease. However, the kidney itself is not a regenerative organ, as all the nephrons are formed during embryonic development. Here, we will investigate advances in the molecular genetics of renal organogenesis, including what this can tell us about lineage relationships, and discuss how this may serve to inform us about both the normal processes of renal repair and options for regenerative therapies.