Functional genetic targeting of embryonic kidney progenitor cells ex vivo.

The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor-treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting.

Fibrotic response in fibroblasts from congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are characterized by a generalized underglycosylation of proteins. CDG is associated with multiple symptoms such as psychomotor retardation, hypotonia, hormonal disturbances, liver fibrosis and coagulopathies. The molecular basis of these symptoms is poorly understood considering the large extent of affected glycoproteins. To better understand the cellular responses to protein underglycosylation in CDG, we have investigated the differences in gene expression between healthy control and CDG fibroblasts by transcriptome comparison. This analysis revealed a strong induction of several genes encoding components of the extracellular matrix, such as collagens, COMP, IGFBP5 and biglycan. The extent of this response was confirmed at the protein level by showing increased production of collagen type-I for example. This fibrotic response of CDG fibroblasts was not paralleled by a differentiation to myofibroblasts and by increased TGF-ß signalling. We could show that the addition of recombinant IGFBP5, one of the induced proteins in CDG, to healthy control fibroblasts increased the production of collagen type-I to levels similar to those found in CDG fibroblasts. The fibrotic response identified in CDG fibroblasts may account for the elevated tissue fibrosis, which is often encountered in CDG patients.

Genome-wide gene expression profiling reveals renal genes regulated during metabolic acidosis.

Production and excretion of acids are balanced to maintain systemic acid-base homeostasis. During metabolic acidosis (MA) excess acid accumulates and is removed from the body, a process achieved, at least in part, by increasing renal acid excretion. This acid-secretory process requires the concerted regulation of metabolic and transport pathways, which are only partially understood. Chronic MA causes also morphological remodeling of the kidney. Therefore, we characterized transcriptional changes in mammalian kidney during MA to gain insights into adaptive pathways. Total kidney RNA from control and 2- and 7-days NH(4)Cl treated mice was subjected to microarray gene profiling. We identified 4,075 transcripts significantly (P < 0.05) regulated after 2 and/or 7 days of treatment. Microarray results were confirmed by qRT-PCR. Analysis of candidate genes revealed that a large group of regulated transcripts was represented by different solute carrier transporters, genes involved in cell growth, proliferation, apoptosis, water homeostasis, and ammoniagenesis. Pathway analysis revealed that oxidative phosphorylation was the most affected pathway. Interestingly, the majority of acutely regulated genes after 2 days, returned to normal values after 7 days suggesting that adaptation had occurred. Besides these temporal changes, we detected also differential regulation of selected genes (SNAT3, PEPCK, PDG) between early and late proximal tubule. In conclusion, the mammalian kidney responds to MA by temporally and spatially altering the expression of a large number of genes. Our analysis suggests that many of these genes may participate in various processes leading to adaptation and restoration of normal systemic acid-base and electrolyte homeostasis.

Genome-wide analysis of the unfolded protein response in fibroblasts from congenital disorders of glycosylation type-I patients.

Congenital disorders of glycosylation (CDG) are a family of diseases characterized by defects of N-linked glycosylation. In CDG-I, several genetic defects cause a shortage of dolichol-linked oligosaccharides, which leads to underglycosylation of nascent glycoproteins. N-linked glycosylation is important for proper folding and trafficking of glycoproteins. Inhibition of glycosylation results in the buildup of misfolded proteins in the endoplasmic reticulum, which induces a protective reaction known as the unfolded protein response (UPR). To investigate whether UPR components are induced in CDG, we have performed a transcriptome analysis of primary fibroblasts from unaffected control subjects and from CDG-I patients using oligonucleotide gene expression arrays. The stress imposed by CDG was also compared with the stress induced by tunicamycin and glucose deprivation. Whereas tunicamycin elicited a strong transcriptional response typical for the UPR, CDG fibroblasts displayed a qualitatively similar yet moderate induction of genes encoding components of the UPR. Among these genes, the PERK kinase inhibitor DNAJC3/P58(IPK) gene showed the highest induction throughout all CDG-I types tested. This was paralleled by elevated expression of genes involved in amino acid biosynthesis and transport, which defined a new component of the cellular response to glycosylation stress.

WT1 controls antagonistic FGF and BMP-pSMAD pathways in early renal progenitors.

Kidney organogenesis requires the tight control of proliferation, differentiation and apoptosis of renal progenitor cells. How the balance between these cellular decisions is achieved remains elusive. The Wilms' tumour suppressor Wt1 is required for progenitor survival, but the molecular cause for renal agenesis in mutants is poorly understood. Here we demonstrate that lack of Wt1 abolishes fibroblast growth factor (FGF) and induces BMP/pSMAD signalling within the metanephric mesenchyme. Addition of recombinant FGFs or inhibition of pSMAD signalling rescues progenitor cell apoptosis induced by the loss of Wt1. We further show that recombinant BMP4, but not BMP7, induces an apoptotic response within the early kidney that can be suppressed by simultaneous addition of FGFs. These data reveal a hitherto unknown sensitivity of early renal progenitors to pSMAD signalling, establishes FGF and pSMAD signalling as antagonistic forces in early kidney development and places WT1 as a key regulator of pro-survival FGF signalling pathway genes.