Jill M. Ricono

Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells

Angiotensin II (Ang II) stimulates hypertrophy of glomerular mesangial cells. The signalling mechanism by which Ang II exerts this effect is not precisely known. Downstream potential targets of Ang II are the extracellular-signal-regulated kinases 1 and 2 (ERK1/ERK2). We demonstrate that Ang II activates ERK1/ERK2 via the AT1 receptor. Arachidonic acid (AA) mimics the action of Ang II on ERK1/ERK2 and phospholipase A2 inhibitors blocked Ang II-induced ERK1/ERK2 activation. The antioxidant N-acetylcysteine as well as the NAD(P)H oxidase inhibitors diphenylene iodonium and phenylarsine oxide abolished both Ang II- and AA-induced ERK1/ERK2 activation. Moreover, dominant-negative Rac1 (N17Rac1) blocks activation of ERK1/ERK2 in response to Ang II and AA, whereas constitutively active Rac1 resulted in an increase in ERK1/ERK2 activity. Antisense oligonucleotides for Nox4 NAD(P)H oxidase significantly reduce activation of ERK1/ERK2 by Ang II and AA. We also show that protein synthesis in response to Ang II and AA is inhibited by N17Rac1 or MEK (mitogen-activated protein kinase/ERK kinase) inhibitor. These results demonstrate that Ang II stimulates ERK1/ERK2 by AA and Nox4-derived reactive oxygen species, suggesting that these molecules act as downstream signal transducers of Ang II in the signalling pathway linking the Ang II receptor AT1 to ERK1/ERK2 activation. This pathway involving AA, Rac1, Nox4, reactive oxygen species and ERK1/ERK2 may play an important role in Ang II-induced mesangial cell hypertrophy.

Morphological Insights into the Origin of Glomerular Endothelial and Mesangial Cells and Their Precursors

Glomerular endothelial and mesangial cells may originate from the metanephric mesenchyme. We used the MAb Thy1.1, a mesangial cell marker in the adult rat kidney, and rat endothelial cell markers MAb RECA-1, MAb PECAM-1 (CD31), and MAb Flk-1 as potential markers to characterize the spatial and temporal distribution of mesangial and endothelial cell precursors during nephrogenesis in the rat. At early stages of glomerulogenesis, RECA-1- and Thy1.1-positive cells were detected in the metanephric blastema at 14 days post conception (dpc) embryos and 15 dpc, respectively, with Thy1.1 expression in cells surrounding the ureteric bud. At 17 and 18 dpc, both RECA-1- and Thy1.1-positive cells were found in the cleft of the S-shaped bodies and in the capillary loops of maturing glomeruli. Double staining for BrdU, a marker of proliferation, and for RECA-1 or BrdU and Thy1.1 also localize in the cleft of S-shaped bodies and in glomerular capillary loops at later stages of development. PDGFRβ co-localizes in cells expressing endothelial or mesangial markers. The data suggest that endothelial and mesangial cell precursors share common markers during the course of glomerulogenesis and that full differentiation of these cells occurs at late stages of glomerular maturation. Thy1.1- and RECA-1-positive cells may be derived from the metanephric blastemal cells at early stages of kidney development. A sub-population of these Thy1.1- or RECA-1-positive cells may be precursors that can migrate into the cleft of comma and S-shaped bodies and proliferate in situ to form glomerular capillary tufts.

PDGF receptor-β modulates metanephric mesenchyme chemotaxis induced by PDGF AA

PDGF B chain or PDGF receptor (PDGFR)-beta-deficient (-/-) mice lack mesangial cells. To study responses of alpha- and beta-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and -/- mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for -/- MMCs. The mechanism by which PDGFR-beta inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular Ca(2+) and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of -/- MMCs with the wild-type beta-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of Ca(2+) signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-l-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular Ca(2+) concentration, suggesting that ROS act as upstream mediators of Ca(2+) in suppressing PDGF AA-induced migration. These data indicate that ROS and Ca(2+) generated by active PDGFR-beta play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs. During kidney development, PDGFR beta-mediated ROS generation and Ca(2+) influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme.

Reciprocal Induction of Simple Organogenesis by Mouse Kidney Progenitor Cells in Three-Dimensional Co-Culture

Kidney development is regulated by a coordinated reciprocal induction of metanephric mesenchymal (MM) and ureteric bud (UB) cells. Here, established MM and UB progenitor cell lines were recombined in three-dimensional Matrigel implants in SCID mice. Differentiation potential was examined for changes in phenotype, organization, and the presence of specialized proteins using immunofluorescence and bright-field and electron microscopy. Both cell types, when grown alone, did not develop into specialized structures. When combined, the cells organized into simple organoid structures of polarized epithelia with lumens surrounded by capillary-like structures. Tracker experiments indicated the UB cells formed the tubuloid structures, and the MM cells were the source of the capillary-like cells. The epithelial cells stained positive for pancytokeratin, the junctional complex protein ZO-1, collagen type IV, as well as UB and collecting duct markers, rearranged during transfection (RET), Dolichos biflorus lectin, EndoA cytokeratin, and aquaporin 2. The surrounding cells expressed α-smooth muscle actin, vimentin, platelet endothelial cell adhesion molecule 1 (PECAM), and aquaporin 1, a marker of vasculogenesis. The epithelium exhibited apical vacuoles, microvilli, junctional complexes, and linear basement membranes. Capillary-like structures showed endothelial features with occasional pericytes. UB cell epithelialization was augmented in the presence of MM cell-derived conditioned medium, glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), or fibronectin. MM cells grown in the presence of UB-derived conditioned medium failed to undergo differentiation. However, UB cell-derived conditioned medium induced MM cell migration. These studies indicate that tubulogenesis and vasculogenesis can be partially recapitulated by recombining individual MM and UB cell lineages, providing a new model system to study organogenesis ex vivo.