Podocytes play a critical role in glomerular barrier function, both in health and disease. However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. ...Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. The plating densities of iPSC stocks as well as the seeding densities for differentiation cultures turned out to be a crucial parameter for differentiation results. Conditionally immortalized human podocytes served as reference controls. iPSC-derived podocyte-like cells showed a typical podocyte-specific morphology and distinct expression of podocyte markers synaptopodin, podocin, nephrin and WT-1 after 10 days of differentiation as assessed by immunofluorescence staining or Western blot analysis. qPCR results showed a downregulation of pluripotency markers Oct4 and Sox-2 and a 9-fold upregulation of the podocyte marker synaptopodin during the time course of differentiation. Cultured podocytes exhibited endocytotic uptake of albumin. In toxicological assays, matured podocytes clearly responded to doxorubicin (Adriamycin™) with morphological alterations and a reduction in cell viability after 48 h of incubation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases ...and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Growing evidence suggests that a proportion of interstitial myofibroblasts detected during renal tubulointerstitial fibrosis originates from tubular epithelial cells by a process called ...epithelial-mesenchymal transition (EMT). The IL-6-type cytokine oncostatin M (OSM) has been recently implicated in the induction of EMT. We investigated OSM effects on the expression of both cell-cell contact proteins and mesenchymal markers and studied OSM-induced intracellular signaling mechanisms associated with these events in human proximal tubular cells. Human recombinant OSM attenuated the expression of N-cadherin, E-cadherin, and claudin-2 in human kidney-2 (HK-2) cells associated with the induction of HK-2 cell scattering in 3D collagen matrices. Conversely, expression of collagen type I, vimentin, and S100A4 was induced by OSM. OSM-stimulated cell scattering was inhibited by antibodies against gp130. Besides inducing phosphorylation of Stat1 and Stat3, OSM led to a strong concentration- and time-dependent phosphorylation of the mitogen-activated protein kinases ERK1, ERK2, and ERK5. MEK1/2 inhibitor U0126 (10 muM) blocked basal and OSM-induced ERK1/2 phosphorylation but not phosphorylation of either ERK5 or Stat1/3. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at concentrations which inhibit ERK1/2 phosphorylation but not ERK5 phosphorylation, restored N-cadherin expression in the presence of OSM, inhibited basal claudin-2 expression, but did not affect either basal or OSM-inhibited E-cadherin expression or OSM-induced expression of collagen type I and vimentin. These results suggest that in human proximal tubular cells ERK1/2 signaling represents an important component of OSM's inhibitory effect on N-cadherin expression. Furthermore, functional ERK1/2 signaling is necessary for basal claudin-2 expression.
Within the glomerulus, podocytes are highly specialized visceral epithelial cells that are part of the glomerular filtration barrier. Human podocyte cell culture is rather challenging for primary or ...immortalized cells, due to the nonproliferative state of the cells. In addition, rapid dedifferentiation is often observed. Hence, iPSC-derived podocytes offer an exciting alternative to culture podocyte-like cells from different donors over prolonged time. Here we report a simple and rapid one-step protocol that drives iPSC into podocyte-like cells in 10 days.
1 Department of Physiology, University of
Innsbruck, A-6010 Innsbruck, Austria; and 2 Department
of Morphology, University Medical Center, CH-1211 Geneva 4, Switzerland
Submitted 3 October 2002
; ...accepted in final form 18 May 2003
Constitutive activation of the MAPK/ERK kinase (MEK)1-ERK2 signaling module
in Madin-Darby canine kidney (MDCK)-C7 cells disrupts their ability to form
cystlike structures in collagen gels and induces an invasive,
myofibroblastlike phenotype. However, the reversibility of these cellular
events, as well as the relative role of both MEK isoforms (MEK1 and MEK2) and
both ERK isoforms (ERK1 and ERK2) during these processes, has not yet been
investigated. We now report that loss of constitutively active MEK1 (caMEK1)
and, thus, loss of active ERK1/2 in C7caMEK1 cells is associated with
increased MEK2 protein expression, reexpression of ERK1 protein, and
epithelial redifferentiation of these cells. The morphological changes toward
an epithelial phenotype in these revertant cell lines (C7rev4, C7rev5, C7rev7)
are reflected by the upregulation of epithelial marker proteins, such as
E-cadherin, -catenin, and cytokeratin, by the loss of -smooth
muscle actin expression, and by the ability of these epithelial revertants to
form well-organized spherical cysts when grown in three-dimensional collagen
gels. Further evidence for a role of the MEK1-ERK1/2 module in
epithelial-mesenchymal transition was obtained from the analysis of two novel,
spontaneously transdifferentiated MDCK-C7 cell clones (C7e1 and C7e2 cells).
In these clones, increased MEK1/2-ERK1/2 phosphorylation, reduced MEK2 protein
expression, and loss of ERK1 protein expression is associated with phenotypic
alterations similar to those observed in transdifferentiated C7caMEK1 cells.
C7e1 cells at least partially regained some of their epithelial
characteristics at higher passages. In contrast, C7e2 cells maintained a
transdifferentiated phenotype at high passage, were unable to generate
cystlike epithelial structures, and retained invasive properties when grown on
a three-dimensional collagen matrix. We conclude that in renal epithelial
MDCK-C7 cells, stable epithelial-to-mesenchymal transition (EMT) is associated
with loss of ERK1 protein expression, reduced MEK2 protein expression, and
increased basal ERK2 phosphorylation. In contrast, loss of active MEK1-ERK1/2
results in increased MEK2 protein expression and reexpression of ERK1 protein,
concomitant with the restoration of epithelial phenotype and the ability to
form cystic structures.
mitogen-activated protein kinase; extracellular signal-regulated kinase; epithelial differentiation; epithelial-to-mesenchymal transition; invasion; Madin-Darby canine kidney cells
Address for reprint requests and other correspondence: H. Schramek, Dept. of
Physiology, Fritz-Pregl-Strasse 3, A-6010 Innsbruck, Austria (E-mail:
herbert.schramek{at}uibk.ac.at ).
Metabolic acidosis is partially compensated by a pronounced increase in renal catabolism of glutamine. This adaptive response is sustained, in part, through increased expression of ...phosphoenolpyruvate carboxykinase (PEPCK). Previous inhibitor studies suggested that the pH-responsive increase in PEPCK mRNA in LLC-PK1-FBPase+ cells is mediated by a p38 mitogen-activated protein kinase (MAPK). These cells express high levels of the upstream kinase MAPK kinase (MKK) 3 but relatively low levels of the alternative upstream kinase MKK6. To firmly establish the role of the p38 MAPK signaling pathway, clonal lines of LLC-PK1-FBPase+ cells that express constitutively active (ca) and dominant negative (dn) forms of MKK3 and MKK6 from a tetracycline-responsive promoter were developed. Western blot analyses confirmed that 0.5 μg/ml doxycycline was sufficient to block transcription and that removal of doxycycline led to pronounced and sustained expression of the caMKKs and dnMKKs. Expression of caMKK6 (but not caMKK3) caused an increase in phosphorylation of p38 MAPK and an increase in the level of PEPCK mRNA that closely mimicked the effect of treatment with acidic medium (pH 6.9, 10 mmHCO3−). Only caMKK6 activated transcription of a PEPCK-luciferase reporter construct. Co-expression of both dnMKKs blocked the increases in phosphorylation of p38 MAPK and PEPCK mRNA. The latter effect closely mimicked that of the p38 MAPK inhibitor SB203580. The expression of either dnMKK3 or dnMKK6 was less effective than co-expression of both dnMKKs. Thus, the pH-responsive increase in PEPCK mRNA in the kidney is mediated by the p38 MAPK signaling pathway and involves activation of MKK3 and/or MKK6.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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/cm 2 after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm 2 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 G 0 /G 1 to G 2 /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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The search for alternatives to the use of fetal bovine serum (FBS) in cell and tissue culture media has become a major goal in terms of the 3R principles in order to reduce or to avoid harvesting of ...FBS from bovine fetuses, and, in terms of Good Manufacturing Practice (GMP), to ensure safe and animal product-free conditions for biomedical tissue engineering and (adult) stem cell therapy, respectively. In the present study, we investigated the feasibility of using platelet lysates (PL) as a substitute for FBS, based on the fact that most of the potent mitogenic factors present in serum are derived from activated thrombocytes. Platelet lysates were obtained from outdated human donor platelet concentrates. Methods were established to activate human donor platelets in order to achieve a maximum yield of platelet a-granule growth factors. Platelet lysates were successfully introduced to grow and maintain anchorage-dependent and -independent human and animal cell lines. For cell culture experiments, cells were either grown in culture media supplemented with 10% FBS, 5% PL, or under serum-free conditions. Growth experiments, viability assays, and platelet lysate-induced activation of ERK1/2 mitogen-activated protein kinase revealed platelet lysates as a valuable alternative to FBS in cell culture media.
Abstract only
Cultured renal epithelia form monolayers of differentiated, polarized cells. On permeable supports, an apical and a basolateral compartment are separated by the cultured epithelium to ...study epithelial transport in vitro. However, culture conditions and culture media have a significant impact. In this respect, the quality of fetal bovine serum (FBS) seemed to be of major importance. The high lot‐to‐lot variablity of FBS was found to substantially influence the differentiation of epithelial cultures with regard to the generation of a transepithelial electrical resistance (TEER). Thus, batch‐testing of FBS was required. We recently reported on the use of human platelet lysates (PL) as a replacement for FBS. The growth promoting capacity of PL was tested on renal epithelial cell lines, for which differentiation end points are well established. PL support growth, proliferation and differentiation of LLC‐PK1, HK‐ 2 and MDCK cells. In addition, TEER was monitored in filter‐grown LLC‐PK1 and in MDCK II and MDCK I epithelia. Low‐resistance epithelia generated a TEER of 150–250 Ω•cm2 in PL‐media, as seen with FBS, and high‐resistance MDCK I retained their TEER of 8,000 Ω•cm2. PL are a valuable, animal‐derived component‐free substitute for FBS in renal epithelial cell culture with a proven high batch‐to batch uniformity.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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