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

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.

Generation and characterization of iPSC-derived renal proximal tubule-like cells with extended stability.

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.

A Protocol for One-Step Differentiation of Human Induced Pluripotent Stem Cells into Mature Podocytes.

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.

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.

Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media.

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.

TGF-beta signaling and its effect on glutaminase expression in LLC-PK1-FBPase+ cells.

During systemic acidosis, renal proximal tubular cells exhibit enhanced rates of bicarbonate and ammonium ion synthesis and undergo extensive hypertrophy. The former adaptations are accomplished, in part, by increased expression of glutaminase (GA). LLC-PK(1)-FBPase+ cells, a gluconeogenic line of porcine kidney cells, exhibit a rapid activation of the ERK1/2 and p38 MAPK pathways and a two- to threefold increase in GA mRNA when transferred to acidic medium (pH 6.9). Transforming growth factor-beta (TGF-beta), a potent activator of MAPK and Smad signaling cascades, also causes extensive renal hypertrophy. Thus the potential role of TGF-beta in the renal response to metabolic acidosis was investigated. Western blot analyses established that in LLC-PK(1)-FBPase+ cells, TGF-beta activated the ERK1/2, p38 MAPK, and Smad1/5/8 pathways, but not the JNK and Smad2/3 pathways. Addition of TGF-beta to cells cultured in normal medium (pH 7.4) produced a steady increase in GA mRNA, resulting in a twofold induction after 18 h. Western blot analysis indicated that treatment with either TGF-beta or acidic medium resulted in an increased level of fibronectin. However, the effects of the two treatments on both GA mRNA and fibronectin levels occurred with different time courses and were additive. In addition, the rates of ammonia production were decreased slightly by addition of TGF-beta. Finally, a GA-luciferase reporter construct, which is activated 3.5-fold by treatment with acidic medium, is not affected by TGF-beta. Therefore, TGF-beta and metabolic acidosis activate some of the same signaling pathways in LLC-PK(1)-FBPase+ cells, but produce separate effects on GA expression.

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 microM, 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.

Oncostatin M-induced effects on EMT in human proximal tubular cells: differential role of ERK signaling.

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.

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 microg/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 HCO3-). 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.

Loss of active MEK1-ERK1/2 restores epithelial phenotype and morphogenesis in transdifferentiated MDCK cells.

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 cyst-like structures in collagen gels and induces an invasive, myofibroblast-like 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, beta-catenin, and cytokeratin, by the loss of alpha-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 cyst-like 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.

p38 MAPK mediates acid-induced transcription of PEPCK in LLC-PK(1)-FBPase(+) cells.

LLC-PK(1)-FBPase(+) cells are a gluconeogenic and pH-responsive renal proximal tubule-like cell line. On incubation with acidic medium (pH 6.9), LLC-PK(1)-FBPase(+) cells exhibit an increased rate of ammonia production as well as increases in glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels and enzyme activities. The increase in PEPCK mRNA is due to an enhanced rate of transcription that is initiated in response to intracellular acidosis. The involvement of known MAPK activities (ERK1/2, SAPK/JNK, p38) in the associated signal transduction pathway was examined by determining the effects of specific MAPK activators and inhibitors on basal and acid-induced PEPCK mRNA levels. Transfer of LLC-PK(1)-FBPase(+) cultures to acidic medium resulted in specific phosphorylation, and thus activation, of p38 and of activating transcription factor-2 (ATF-2), respectively. Anisomycin (AI), a strong p38 activator, increased PEPCK mRNA to levels comparable to those observed with acid stimulation. AI also induced a time-dependent phosphorylation of p38 and ATF-2. SB-203580, a specific p38 inhibitor, blocked both acid- and AI-induced PEPCK mRNA levels. Western blot analyses revealed that the SB-203580-sensitive p38alpha isoform is strongly expressed. The octanucleotide sequence of the cAMP-response element-1 site of the PEPCK promotor is a perfect match to the consensus element for binding ATF-2. The specificity of ATF-2 binding was proven by ELISA. We conclude that the SB-203580-sensitive p38alpha-ATF-2 signaling pathway is a likely mediator of the pH-responsive induction of PEPCK mRNA levels in renal LLC-PK(1)-FBPase(+) cells.