Elisabeth Feifel

ERK1/2-driven and MKP-mediated inhibition of EGF-induced ERK5 signaling in human proximal tubular cells.

The MEK1‐ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial‐to‐mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5‐ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal‐regulated kinases in epidermal growth factor (EGF)‐stimulated human kidney‐2 (HK‐2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK‐2 cell proliferation, which was largely U0126‐sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl‐1040, when used at 10 and 1 µM, respectively, inhibited basal and EGF‐induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long‐term inhibition of MEK1/2‐ERK1/2 signaling and/or vanadate‐sensitive protein phosphatases enhanced and prolonged EGF‐induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad‐caMEK1) construct completely blocked EGF‐induced ERK5 phosphorylation. Expression of Ad‐caMEK1 in HK‐2 cells resulted in the upregulation of the dual‐specificity phosphatases MKP‐3/DUSP6, MKP‐1/DUSP1, and DUSP5. The EGF‐mediated time‐dependent induction of MKP‐3, MKP‐1 and DUSP5 mRNA levels was U0126‐sensitive at a concentration, which blocked EGF‐mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF‐induced MKP‐3 and MKP‐1 protein expression. Both MKP‐3 and MKP‐1 co‐immunoprecipitated with ERK5 in unstimulated as well as in EGF‐stimulated HK‐2 cells. These results suggest the existence of an ERK1/2‐driven negative feed‐back regulation of ERK5 signaling in EGF‐stimulated HK‐2 cells, which is mediated by MKP‐3, DUSP5 and/or MKP‐1. J. Cell. Physiol. 211: 88–100, 2007.

Differential activities of H+extrusion systems in MDCK cells due to extracellular osmolality and pH

The aim of the present study was to obtain detailed information on MDCK cell proton secretion characteristics under various growth conditions. Confluent monolayers cultured on glass coverslips were adapted over 48 h to media with different osmolality and pH (200 mosmol/kgH2O, pH 7.4; 300 mosmol/kgH2O, pH 7.4; and 600 mosmol/kgH2O, pH 6.8) corresponding to the luminal fluid composition of the collecting duct segments found in the in renal cortex, the outer stripe of outer medulla and inner medulla. Proton fluxes were determined from the recovery of intracellular pH following an acid load induced by an NH4Cl pulse times the corresponding intrinsic buffering power (βi). The intracellular buffering power was found to change only with culture medium osmolality but not with culture medium pH. In addition to an amiloride and Hoe-694-sensitive Na+/H+exchange, Madin-Darby canine kidney (MDCK) cells possess a Sch-28080-sensitive, K+-dependent H+ extrusion mechanism that is increased upon adaptation of monolayers to hyperosmotic-acidic culture conditions. A significant contribution of the bafilomycin A1-sensitive vacuolar H+-ATPase could be found only in cells adapted to hyposmotic culture conditions. Exposure of MDCK cells to 10-5 or 10-7 M aldosterone for either 1 or 18 h did not alter the H+ extrusion characteristics significantly. The results obtained show that different extracellular osmolality and pH induce different MDCK phenotypes with respect to their H+-secreting systems.

Effects of constitutively active and dominant negative MAPK kinase (MKK) 3 and MKK6 on the pH-responsive increase in phosphoenolpyruvate carboxykinase mRNA.

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 mm \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document}). 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.

Protection of human podocytes from Shiga toxin 2-induced phosphorylation of mitogen-activated protein kinases and apoptosis by human serum amyloid P component

ABSTRACT Hemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occur in the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce proteinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP can protect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-associated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immortalized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-binding globotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38α MAPK and c-Jun N-terminal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in an increased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregulation of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option.

Human Platelet Lysates Successfully Replace Fetal Bovine Serum in Adipose-Derived Adult Stem Cell Culture

Fetal bovine serum (FBS) is still the gold standard as a cell culture medium additive due to its high level of growth stimulatory factors. Although supplementation of growth media with FBS is common practice in cell and tissue culture, FBS bears a number of disadvantages and its use has been questioned recently: (1) an ill-defined medium supplement, (2) qualitative and quantitative batch-to-batch variations, and (3) animal welfare concerns regarding the harvest of bovine fetal blood. Recently, we were able to show the capacity of human platelet � -granule lysates to replace FBS in a variety of human and animal cell culture systems. Thus, lysates of human donor platelets may become a valuable non animal-derived substitute for FBS in cultures of mammalian cells and in human and animal stem cell technology. Stem cells may become the future for human-based alternative to animal testing, in vitro toxicology, and drug safety assessment. New stem cell-based test systems are continuously established, and their performance under animal- derived component free culture conditions has to be defined in prevalidation and validation studies. In order to accomplish these tasks, adipose-derived mesenchymal stem cells (ADSC) were expanded in media supplemented with platelet lysates. Proliferation assays by resazurin and WST-8 compared with direct cell counting confirmed the growth promoting effect of platelet lysate, comparable to high FBS. Furthermore, we established culture conditions that ADSC kept their undifferentiated state as determined by CD73, CD90 and CD105 expression and the lack of negative marker CD45. Preliminary tests whether ADSC can be differentiated towards adipogenic, osteogenic, or chondrogenic phenotypes under platelet lysate supplemented growth conditions were also successful.

PMA and staurosporine affect expression of the PCK gene in LLC-PK1-F+ cells.

The addition of phorbol 12-myristate 13-acetate (PMA) to renal LLC-PK1-F+cells caused a rapid decrease in the level of phospho enolpyruvate carboxykinase (PCK) mRNA and reversed the stimulatory effects of exposure to acidic medium (pH 6.9, 10 mM [Formula: see text]) or cAMP. In contrast, prolonged treatment with PMA increased the levels of PCK mRNA. The two effects correlated with the membrane translocation and downregulation of the α-isozyme of protein kinase C and were blocked by pretreatment with specific inhibitors of protein kinase C. The rapid decrease in PCK mRNA caused by PMA occurred with a half-life ( t ½ = 1 h) that is significantly faster than that measured during recovery from acid medium or following inhibition of transcription ( t ½ = 4 h). The effect of PMA was reversed by staurosporine, which apparently acts by inhibiting a signaling pathway other than protein kinase C. Staurosporine had no effect on the half-life of the PCK mRNA, but it stimulated the activity of a chloramphenicol acetyltransferase gene that was driven by the initial 490 base pairs of the PCK promoter and transiently transfected into LLC-PK1-F+cells. This effect was additive to that of cAMP, and neither stimulation was reversed by PMA. The stimulatory effect of staurosporine was mapped to the cAMP response element (CRE-1) and P3(II) element of the PCK promoter. The data indicate that, in LLC-PK1-F+cells, activation of protein kinase C decreases the stability of the PCK mRNA, whereas transcription of the PCK gene may be suppressed by a kinase that is inhibited by staurosporine.The addition of phorbol 12-myristate 13-acetate (PMA) to renal LLC-PK1-F+ cells caused a rapid decrease in the level of phosphoenolpyruvate carboxykinase (PCK) mRNA and reversed the stimulatory effects of exposure to acidic medium (pH 6.9, 10 mM HCO-3) or cAMP. In contrast, prolonged treatment with PMA increased the levels of PCK mRNA. The two effects correlated with the membrane translocation and downregulation of the alpha-isozyme of protein kinase C and were blocked by pretreatment with specific inhibitors of protein kinase C. The rapid decrease in PCK mRNA caused by PMA occurred with a half-life (t1/2 = 1 h) that is significantly faster than that measured during recovery from acid medium or following inhibition of transcription (t1/2 = 4 h). The effect of PMA was reversed by staurosporine, which apparently acts by inhibiting a signaling pathway other than protein kinase C. Staurosporine had no effect on the half-life of the PCK mRNA, but it stimulated the activity of a chloramphenicol acetyltransferase gene that was driven by the initial 490 base pairs of the PCK promoter and transiently transfected into LLC-PK1-F+ cells. This effect was additive to that of cAMP, and neither stimulation was reversed by PMA. The stimulatory effect of staurosporine was mapped to the cAMP response element (CRE-1) and P3(II) element of the PCK promoter. The data indicate that, in LLC-PK1-F+ cells, activation of protein kinase C decreases the stability of the PCK mRNA, whereas transcription of the PCK gene may be suppressed by a kinase that is inhibited by staurosporine.

Human Platelet Lysates Promote the Differentiation Potential of Adipose-Derived Adult Stem Cell Cultures

Adipose tissue from liposuction is a rich source for human mesenchymal stem cells. This type of adult stem cell is ethically acceptable, that paved the way for research on their potential use in regenerative medicine. However, any clinical application of adult stem cells is impeded by the use of FBS as an animal-derived growth supplement. In addition, stem cell cultures gained importance as innovative human-based alternative to animal testing, in vitro toxicology, drug testing and safety assessment. Thus, animal-derived component-free culture protocols are mandatory for a successful application of human stem cell-based testing systems under humanized conditions. Recently, we succeeded in using human platelet lysates (PL) as a serum alternative in the cell culture of a number of human and animal cell lines, and human mesenchymal stem cells. PL were prepared as cell-free extracts from activated donor thrombocytes. The minimal criteria defining multipotent mesenchymal cells are (1) the capacity to adhere to plastic, (2) the expression of specific surface antigens (e.g. CD73, CD90, CD105) for undifferentiated state, and (3) the potential of the cells to differentiate into the adipogenic, chondrogenic and osteogenic lineage. In the present study, adipose-derived stem cells (ADSC) were used as cell model. ADSC were maintained under PL or FBS and then switched to the respective media to induce mesodermal differentiation. Differentiation endpoints were assessed by phase-contrast microscopy and by histochemical staining: (1) lipid droplets in adipocytes were stained by Oil red O, (2) proteoglycans in chondrogenic spheroids were detected by toluidineblue, and fine structure of spheroids was monitored by scanning electron microscopy, and (3) calcium deposits in differentiated osteoblasts were stained with silver nitrate (von Kossa staining). Adipogenic differentiation was further confirmed by quantitative real-time PCR of selected marker genes (PREF1 vs. FABP4). There were no differences between FBS- and PL-grown ADSC, indicative for retention of the differentiation potential of ADSC under animal-derived component-free culture conditions in PL-supplemented culture media. The degree of adipogenic and osteogenic differentiation was even more pronounced under PL compared to FBS.

Human Platelet Lysates as a Serum Substitute in Cell Culture Media

The search for alternatives to fetal bovine serum (FBS) has become a major goal in the field of cell and tissue culture research. Although the supplementation of culture media with FBS is routine practice, FBS bears a number of disadvantages: unknown composition, high lot-to-lot variablity, ethical concerns about the harvest from bovine fetuses, and possible shortage in global supply. Several strategies have been developed to reduce or replace FBS in cell culture media (Bjare 1992; Even et al. 2006; Gstraunthaler 2003; van der Valk et al. 2004, 2010). Here we report on the use of human platelet lysates (PL) as a serum replacement (Alden et al. 2007; Bernardo et al. 2007; Bieback et al. 2009; Doucet et al. 2005; Johansson et al. 2003; Kocaoemer et al. 2007; Muller et al. 2009; Schallmoser et al. 2009). PL in DMEM support growth, proliferation and differentiation, as assessed by dome formation, of proximal tubule-like LLC-PK1 (porcine kidney) and HK-2 (human kidney) cells, as well as PL-supplemented DMEM/Ham F-12 for distal tubule-like MDCK (dog kidney) cells. In addition to adherent epithelial cell lines, anchorage-independent Raji human lymphoma cells were investigated. PL fully supported growth and proliferation of Raji cells in RPMI-1640 medium in suspension. In order to biochemically determine the proliferative potential of PL, the stimulation of extracellular signal-regulated MAP kinase (ERK1/2) was determined. Addition of PL to quiescent LLC-PK1 cultures resulted in specific phosphorylation, and thus activation, of ERK1/2 within minutes. The time course is identical with ERK1/2 activation upon addition of FBS. The data show the high potential of PL as a valuable substitute for FBS in mammalian cell and tissue culture.

Differentiation of human iPSCs into functional podocytes

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.