These data indicate that mTORC1 is required to maintain podocyte

These data indicate that mTORC1 is required to maintain podocyte function and glomerular architecture. Figure 2 Podocyte-specific deletion of the mTORC1 complex results in progressive glomerulosclerosis. Time-dependent deletion of Raptor indicates Vandetanib cancer the importance of mTORC1 activity during glomerular development. Growth of an organ during development can be controlled by alterations in either the number or the size of cells. The 2 mechanisms are fundamentally different and require distinct regulation, whereby mTORC1 is centrally involved in controlling cell size. The convergence of multiple growth factor�Cinitiated pathways on mTORC1 likely allows its participation in multiple developmental processes, underlined by the absolute requirement for mTORC1 during early embryonic development (25, 26).

In agreement with this, morphometric analysis of 8-week-old Raptor��podocyte mice indicated that glomeruli and podocytes appear to be smaller in Raptor��podocyte mice than in WT controls (Supplemental Figure 3). To determine whether podocytes might be particularly sensitive to the loss of mTORC1 activity during glomerular development, we used a conditional expression model (Tet-On system) in which the target gene is deleted only in the presence of a tetracycline derivative. In this system, the reverse tetracycline-controlled transcriptional activator (rtTA) is placed under the control of the NPHS2 promoter (27). A second transgene uses the tetO promoter elements upstream of a minimal CMV promoter to drive expression of Cre recombinase.

We used this strategy to delete the Raptor gene either during glomerular development or in adult mice. As in the Raptor��podocyte mice, all transgenes for this experiment were transferred to a pure C57BL/6 strain (Figure (Figure3A).3A). Doxycycline was added to the drinking water of pregnant female animals and continued throughout gestation and nursing to initiate Cre-mediated excision of Raptor in developing glomeruli. To initiate Cre-mediated excision of Raptor in mature glomeruli, doxycycline was added to the drinking water of 8-week-old mice. Western blot analysis confirmed the reduction of glomerular raptor protein levels in mice induced in utero or after 8 weeks of age (Figure (Figure3,3, B and C). We further assessed the efficiency of doxycycline-regulated Cre expression by crossing the double-transgenic NPHS2.

rtTA;tetO.Cre mice to the mT/mG transgenic reporter strain, which carries a loxP-flanked Tomato cassette (28). Upon Cre-mediated excision of membrane-targeted tandem dimer GSK-3 Tomato (mT), an alternate reporter protein, membrane-targeted GFP (mG), is expressed. Immunofluorescence of kidney sections indicated that almost all podocytes were positive for the excision event in early-induced mice (Figure (Figure3D),3D), whereas most, but not all (ca. 80%) podocytes were positive for the excision event in the late-induced (8 weeks) group.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>