Renal Epithelial Cell Differentiation Research Articles

A Simple Bioreactor-Based Method to Generate Kidney Organoids fromPluripotent Stem Cells.

SummaryKidney organoids made from pluripotent stem cells have the potential to revolutionize how kidney development, disease, and injury are studied. Current protocols are technically complex, suffer from poor reproducibility, and have high reagent costs that restrict scalability. To overcome some of these issues, we have established a simple, inexpensive, and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day 8 and show optimal tissue morphology at day 14. A comparison with fetal human kidneys suggests that day-14 organoid tissue most closely resembles late capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient, and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant kidney development.

Calcium ions promote primary renal epithelial cell differentiation into cells with bone-associated phenotypes via transforming growth factor-β1-induced epithelial-mesenchymal transition in idiopathic hypercalciuria patients.

The present study aimed to identify the characteristics and cross‑talk between transforming growth factor β1 (TGF‑β1) and calcium ions in nephrolithiasis patients with idiopathic hypercalciuria (IH) in order to elucidate the potential mechanisms underlying changes in cell phenotype induced by bone‑associated factors and their influence on renal nephrolithiasis formation. Blood samples from a total of 29 nephrolithiasis patients with IH, 29 renal stone patients without IH and 29 healthy age‑matched normal controls were subjected to quantification of peripheral serum TGF‑β1, osteopontin (OPN) and bone morphogenetic protein 2 (BMP2) using ELISA. This was followed by detection of BMP2, OPN and 1,25‑dihydroxyvitamin D3 receptor (VDR) mRNA and protein levels in primary renal epithelial cells (PRECs) of IH and HK‑2 human proximal tubular cell lines (control) using reverse transcription quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses. The mRNA expression levels of BMP2, OPN and VDR in PRECs and HK‑2 were evaluated following stimulation with various concentrations of TGF‑β1 (0.5, 2.0 and 5.0 ng/ml), Ca2+ (0.5, 1.5 and 2.5 mM) or TGF‑β1 and Ca2+ combined using RT‑qPCR, respectively. TGF‑β1, BMP2 and OPN expression levels in patients with IH were all significantly higher than those in the control group. The mRNA and protein expression levels of BMP2 and VDR were significantly higher in PRECs than those in HK‑2 cells. Following incubation with TGF‑β1 and/or Ca2+, the mRNA expression levels of BMP2, OPN and VDR in PRECs increased in a dose‑dependent manner; however, no significant differences were observed in HK‑2 cells with increasing TGF‑β1 dosage. Co‑incubation with TGF‑β1 and Ca2+ in PRECs and HK‑2 cell lines resulted in similar effects and the expression of BMP2, OPN and VDR mRNA increased in a time‑dependent manner. In conclusion, the results of the present study demonstrated that TGF‑β1 regulated the expression of BMP2, OPN and VDR in PRECs, but not in HK‑2 cells. Co‑incubation with TGF‑β1 and Ca2+ significantly increased the expression levels of bone‑associated factors in PRECs and HK‑2 cells, which suggested that this process may be partially responsible for the pathogenesis of calcium stone development, and also associated with bone formation and the TGF‑β1‑induced epithelial to mesenchymal transition.

Xenopus pronephros development—past, present, and future

Kidney development is a multi-step process where undifferentiated mesenchyme is converted into a highly complex organ through several inductive events. The general principles regulating these events have been under intense investigation and despite extensive progress, many open questions remain. While the metanephric kidneys of mouse and rat have served as the primary model, other organisms also significantly contribute to the field. In particular, the more primitive pronephric kidney has emerged as an alternative model due to its simplicity and experimental accessibility. Many aspects of nephron development such as the patterning along its proximo-distal axis are evolutionarily conserved and are therefore directly applicable to higher vertebrates. This review will focus on the current understanding of pronephros development in Xenopus. It summarizes how signaling, transcriptional regulation, as well as post-transcriptional mechanisms contribute to the differentiation of renal epithelial cells. The data show that even in the simple pronephros the mechanisms regulating kidney organogenesis are highly complex. It also illustrates that a multifaceted analysis embracing modern genome-wide approaches combined with single gene analysis will be required to fully understand all the intricacies.

Integrins, Extracellular Matrix, and Terminal Differentiation of Renal Epithelial Cells

The mechanism whereby epithelial cells terminally differentiate is an active area of investigation. One potential interface is the spatial and temporal expression of the transmembrane receptors known as integrins and the extracellular matrix (ECM) proteins to which they bind. In this issue of JASN

Transcriptional control of terminal nephron differentiation

Terminal differentiation of epithelial cells into more specialized cell types is a critical step in organogenesis. Throughout the process of terminal differentiation, epithelial progenitors acquire or upregulate expression of renal function genes and cease to proliferate, while expression of embryonic genes is repressed. This exquisite coordination of gene expression is accomplished by signaling networks and transcription factors which couple the external environment with the new functional demands of the cell. While there has been much progress in understanding the early steps involved in renal epithelial cell differentiation, a major gap remains in our knowledge of the factors that control the steps of terminal differentiation. A number of signaling molecules and transcription factors have been recently implicated in determining segmental nephron identity and functional differentiation. While some of these factors (the p53 gene family, hepatocyte nuclear factor-1beta) promote the terminal epithelial differentiation fate, others (Notch, Brn-1, IRX, KLF4, and Foxi1) tend to regulate differentiation of specific nephron segments and individual cell types. This review summarizes current knowledge related to these transcription factors and discusses how diverse cellular signals are integrated to generate a transcriptional output during the process of terminal differentiation. Since these transcriptional processes are accompanied by profound changes in nuclear chromatin structure involving the genes responsible for creating and maintaining the differentiated cell phenotype, future studies should focus on identifying the nature of these epigenetic events and factors, how they are regulated temporally and spatially, and the chromatin environment they eventually reside in.

Hypertonic-induced lamin A/C synthesis and distribution to nucleoplasmic speckles is mediated by TonEBP/NFAT5 transcriptional activator

Lamin A/C is the most studied nucleoskeletal constituent. Lamin A/C expression indicates cell differentiation and is also a structural component of nuclear speckles, which are involved in gene expression regulation. Hypertonicity has been reported to induce renal epithelial cell differentiation and expression of TonEBP (NFAT5), a transcriptional activator of hypertonicity-induced gene transcription. In this paper, we investigate the effect of hypertonicity on lamin A/C expression in MDCK cells and the involvement of TonEBP. Hypertonicity increased lamin A/C expression and its distribution to nucleoplasm with speckled pattern. Microscopy showed codistribution of TonEBP and lamin A/C in nucleoplasmic speckles, and immunoprecipitation demonstrated their interaction. TonEBP silencing caused lamin A/C redistribution from nucleoplasmic speckles to the nuclear rim, followed by lamin decrease, thus showing that hypertonicity induces lamin A/C speckles through a TonEBP-dependent mechanism. We suggest that lamin A/C speckles could serve TonEBP as scaffold thus favoring its role in hypertonicity.

Xenopus Bicaudal-C is required for the differentiation of the amphibian pronephros

The RNA-binding molecule Bicaudal-C regulates embryonic development in Drosophila and Xenopus. Interestingly, mouse mutants of Bicaudal-C do not show early patterning defects, but instead develop polycystic kidney disease (PKD). To further investigate the molecular mechanism of Bicaudal-C in kidney development, we analyzed its function in the developing amphibian pronephros. Bicaudal-C mRNA was present in the epithelial structures of the Xenopus pronephros, the tubules and the duct, but not the glomus. Inhibition of the translation of endogenous Bicaudal-C with antisense morpholino oligomers ( xBic-C-MO) led to a PKD-like phenotype in Xenopus. Embryos lacking Bicaudal-C developed generalized edemas and dilated pronephric tubules and ducts. This phenotype was caused by impaired differentiation of the pronephros. Molecular markers specifically expressed in the late distal tubule were absent in xBic-C-MO-injected embryos. Furthermore, Bicaudal-C was not required for primary cilia formation, an important organelle affected in PKD. These data support the idea that Bicaudal-C functions downstream or parallel of a cilia-regulated signaling pathway. This pathway is required for terminal differentiation of the late distal tubule of the Xenopus pronephros and regulates renal epithelial cell differentiation, which – when disrupted – results in PKD.

Alterations in cell-adhesive and migratory properties of proximal tubule and collecting duct cells from bcl-2 -/- mice.

Bcl-2 protects cells from apoptosis initiated by a variety of stimuli including loss of cell adhesion. Bcl-2 -/- mice develop renal hypoplastic/cystic dysplasia with renal cyst formation coinciding with renal maturation in normal mice. To gain a better understanding of the role cell-adhesive mechanisms play during renal maturation, we generated proximal tubule and collecting duct cell lines from postnatal day 10 (P10) and P20 bcl-2 +/+ and bcl-2 -/- mice. Very little is known about the role cell-adhesive and migratory mechanisms play during renal maturation. We observed that modulation of cell-adhesive properties, which normally occur in a nephron segment-specific manner during renal maturation, and cell migration were altered in cells from bcl-2 -/- mice. Enhanced migration of bcl-2 -/- proximal tubule cells in a scratch wound assay was completely inhibited by incubation with PP1 (Src inhibitor) and moderately affected by incubation with SB-203580 (p38 inhibitor). These cells expressed increased levels of fibronectin and had numerous central focal adhesions. P20 bcl-2 -/- proximal tubule cells adhered to fibronectin but adhered poorly to collagen, vitronectin, or laminin. Collecting duct cells, similar to proximal tubule cells from bcl-2 -/- mice, demonstrated enhanced migration in a scratch wound assay that was inhibited by incubation with PP1. Migration of these cells was moderately affected by incubation with PD-98059 (MEK inhibitor) or LY-294002 (PI3 kinase inhibitor), whereas incubation with SB-203580 had no effect. P10 bcl-2 -/- collecting duct cells also expressed increased levels of fibronectin but decreased levels of thrombospondin-1 and demonstrated precocious binding to fibronectin and vitronectin compared with bcl-2 +/+ cells. The ability of P20 bcl-2 +/+ collecting duct cells to adhere to fibronectin and vitronectin corresponded with a decline in thrombospondin-1 expression. Therefore, alterations in cell-adhesive and migratory characteristics may be an early indicator of aberrant renal epithelial cell differentiation.

Spatial repression of PCNA by p53 during kidney development.

Transcriptional repression is a key mechanism for the spatial specification of gene expression and cell fate determination. During kidney development, proliferating cell nuclear antigen (PCNA) is expressed in the nephrogenic zone and is downregulated rapidly as renal epithelial cells enter terminal differentiation and acquire functional characteristics. Our laboratory reported that the transcription factor p53 stimulates the terminal differentiation of renal epithelial cells by means of transcriptional activation of renal function genes (Saifudeen Z, Dipp S, and El-Dahr SS. J Clin Invest 109: 1021-1030, 2002). Because p53-induced growth arrest correlates with downregulation of PCNA gene expression, we examined the impact of p53 inactivation on PCNA expression in mice and evaluated the effect of p53 on PCNA transcription. Immunohistochemistry revealed that the transition from nephrogenesis to terminal epithelial cell differentiation correlates with accumulation of the transcription factor p53. Importantly, the spatially restricted pattern of PCNA expression is disrupted in kidneys of p53-deficient pups, in which there was a redistribution of PCNA expression into the differentiation zone (without a change in total kidney PCNA content) and distortion of the tubular architecture. Electrophoretic mobility shift assays revealed that the binding of kidney nuclear extracts to the p53 response elements in human and rat PCNA promoters is developmentally regulated. Transient transfection assays performed in p53-deficient HeLa cells revealed that exogenous p53 strongly represses transcription from human PCNA promoter-reporter constructs. Interestingly, deletion of the p53-binding site confers enhanced responsiveness to p53-mediated repression, suggesting that transcriptional repression of PCNA by p53 is achieved by a mechanism other than direct DNA binding. On the basis of these results, we propose the hypothesis that p53-mediated transcriptional repression plays a role in the spatial restriction of PCNA gene expression during normal renal development.

Characterisation, chromosomal localisation and expression of the mouse Kid3 gene

Kid1 encodes a transcriptional repressor implicated in the differentiation of renal epithelial cells. Here we report the characterisation of Kid3, a novel mouse gene related to Kid1. Kid3 encodes a C 2H 2 zinc finger protein with an N-terminal KRAB transcriptional repression domain. It maps to chromosome 11, adjacent to Kid1 and another related gene Kid2. Northern analysis shows that Kid3 is highly expressed in embryonic and adult brain, with lower levels in adult and embryonic (E16.5) kidney, gut, lung and heart. Expression of Kid3 in the kidney is developmentally regulated and suggests a role for Kid3 in the early stages of nephrogenesis.

Factors produced by activated leukocytes alter renal epithelial cell differentiation

The development of tubulointerstitial fibrosis in inflammatory renal diseases has been linked to disease progression to end-stage renal failure. Understanding the interactions of the factors influencing inflammation and activating the fibrotic process, that is, the inflammatory infiltrate and the resident proximal tubular cells, may lead to a determination of the mechanisms that regulate tubulointerstitial fibrosis. We used an in vitro model of human proximal tubule cells that were stimulated with supernatant from activated peripheral blood mononuclear cells (leukocytes) to study the alterations in cellular phenotype, and examined the signaling pathways mediating epithelial-fibroblast like transdifferentiation. Our hypothesis of the proposed sequence of events leading to tubulointerstitial fibrosis is explained.

Autocrine Insulin-like Growth Factor Binding Protein-2 (IGFBP-2) Modulates the Effects of Insulin-like Growth Factor-II (IGF-II) on Differentiating Human Fetal Glomerular Epithelial Cells † 1823

We have previously implicated the IGF system in the process of normal mammalian nephrogenesis. IGF-II is an important fetal mitogen and is abundantly expressed in the undifferentiated metanephric blastema, while the IGF binding proteins, which modulate IGF biological effects, display specific spatial and temporal expression in the developing human fetal kidney. To study the role of IGF-II and IGFBP-2 in renal epithelial cell differentiation, we developed and characterized a primary fetal glomerular epithelial cell line and a kidney explant model from human fetal kidneys obtained at 12-18 weeks gestation. Stimulation of the cell monolayers with IGF-II resulted in a dose-dependent increase in 3H-thymidine incorporation to 126 +/- 4.6% control at a concentration of 200 ng/ml for 24 hours. This stimulation was abrogated by addition of a Type I IGF receptor (IGF-IR) antibody. Stimulation with IGFBP-2, the major endogenous IGFBP of this cell type, did not affect mitogenesis, however co-incubation of the monolayers with IGFBP-2 inhibited IGF-II-stimulated mitogenesis in a dose-dependent fashion at all concentrations of IGF-II tested (1-200 ng/ml). Furthermore, incubation of the unstimulated monolayers with a neutralizing IGFBP-2 antibody (1:4000) resulted in an increase in mitogenesis to 121 +/- 2.5% of control. By Northern blot analysis, IGF-II upregulated IGFBP-2 and IGFBP-4 mRNAs, markers of glomerular epithelial cell differentiation (119% and 137% respectively vs control), while IGFBP-2 also upregulated IGFBP-4 mRNA (141% vs control) in the cell monolayers. Finally, incubation of the kidney explants with a neutralizing antibody to IGFBP-2 resulted in an inhibition of explant differentiation at 24 hours, with an expanded nephrogenic zone and abundant metanephric blastema. These data suggest that IGF-II stimulated IGFBP-2 production in the developing fetal glomerular epithelial cell plays a role in further differentiation of this cell type.

Constitutively Active Mutant of the Mitogen-activated Protein Kinase Kinase MEK1 Induces Epithelial Dedifferentiation and Growth Inhibition in Madin-Darby Canine Kidney-C7 Cells

Overexpression of a constitutively active mitogen-activated protein kinase kinase (MAPKK or MEK) induces neuronal differentiation in adrenal pheochromocytoma 12 cells but transformation in fibroblasts. In the present study, we used a constitutively active MAPK/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) mutant to investigate the function of the highly conserved MEK1-ERK2 signaling module in renal epithelial cell differentiation and proliferation. Stable expression of constitutively active MEK1 (CA-MEK1) in epithelial MDCK-C7 cells led to an increased basal and serum-stimulated ERK1 and ERK2 phosphorylation as well as ERK2 activation when compared with mock-transfected cells. In both mock-transfected and CA-MEK1-transfected MDCK-C7 cells, basal and serum-stimulated ERK1 and ERK2 phosphorylation was almost abolished by the synthetic MEK inhibitor PD098059. Increased ERK2 activation due to stable expression of CA-MEK1 in MDCK-C7 cells was associated with epithelial dedifferentiation as shown by both a dramatic alteration in cell morphology and an abolished cytokeratin expression but increased vimentin expression. In addition, we obtained a delayed and reduced serum-stimulated cell proliferation in CA-MEK1-transfected cells (4.6-fold increase in cell number/cm2 after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm2 after 5 days). This result was confirmed by flow cytometric DNA analysis showing that stable expression of CA-MEK1 decreased the proportion of MDCK-C7 cells moving from G0/G1 to G2/M as compared with both untransfected and mock-transfected cells. Taken together, our data demonstrate an association of increased basal and serum-stimulated activity of the MEK1-ERK2 signaling module with epithelial dedifferentiation and growth inhibition in MDCK-C7 cells. Thus, the MEK1-ERK2 signaling pathway could act as a negative regulator of epithelial differentiation thereby leading to an attenuation of MDCK-C7 cell proliferation.

Effects of attachment substrates on the growth and differentiation of LLC-PK1 cells

The growth and differentiation of an established renal epithelial cell line, LLC-PK1, on membrane bound mussel adhesive protein (MAP), collagen, and extracellular matrix (ECM) in serum-containing medium was studied. Cell attachment and growth on uncoated- vs. protein-coated cellulose nitrate and acetate membranes did not differ significantly, and confluence was achieved on all membranes. However, cells remained in a single monolayer only when plated on collagen or ECM. LLC-PK1 monolayers grown on ECM-coated membranes displayed the highest transepithelial D-glucose transport (333 +/- 22 ng.cm-2.min-1) whereas cells plated on collagen-coated membranes displayed the lowest (94 +/- 23 ng.cm-2.min-1). Glucose flux values increased with age of the culture, reaching a plateau at 28 d postseeding. These results indicate that the underlying substratum and cell age can affect differentiation of renal epithelial cells in vitro.

Cyclic adenosine monophosphate modulates cell morphology and behavior of a cultured renal epithelial

The role of cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) in modulating functions of differentiated renal cells is well established. Its importance in controlling their growth and differentiation is less clear. We have used somatic cell genetic techniques to probe the role of PKA in controlling morphology and behavior of a renal epithelial cell line, LLC-PK1, which acquires many properties characteristic of the renal proximal tubular cell. Mutants of this line altered in PKA activity have been isolated and their behavior compared to that of the parent line. The results indicate that PKA is involved, either directly or indirectly, in maintenance of cell morphology, cell-cell and cell-substratum interactions, density-dependent growth regulation, and expression of one function characteristic of the renal proximal tubular cell, Na-hexose symport. The relevance of these results to the role of PKA in controlling growth and differentiation of renal epithelial cells in vivo is discussed.

Proximal Renal Tubular Surface Membrane Antigens Identified in Primary and Metastatic Renal Cell Carcinomas

Polyclonal antibodies to the brush border membrane of the human renal proximal tubule were found to identify two proteins with apparent molecular weights of 35,000 and 46,000 daltons. The antigens are distinct in their molecular weights from other antigens that have been localized to the human proximal tubular brush border. The antibodies were extensively studied for their immunoreactivity to fixed, embedded tissue sections by indirect immunoperoxidase staining. These sections were from 36 primary and 37 metastatic renal cell carcinomas, three renal oncocytomas, 87 cases of 28 different types of neoplasms, and 33 different types of normal tissues. Ninety-four percent of primary and 78% of metastatic renal cell carcinomas demonstrated expression of the antigens. Eight cases in which both primary tumor and its metastases were available revealed a general quantitative decrease in expression of the antigens in the metastases. Specificity analysis revealed immunoreactivity to the luminal surfaces of cells lining the epididymis, breast lobule, and bile ductules; the cytoplasm of the submandibular gland ductules; and to some adenocarcinomas of lung, breast, and small intestine, astrocytomas of brain, and squamous carcinomas of skin. The antibodies may be of some utility in the surgical pathology laboratory to help determine the primary site of metastatic clear cell tumors and may also be of use in the in vitro differentiation of renal epithelial cells. Development of monoclonal antibodies to these antigens may improve specificity and clinical utility.