Role of Smads during renal branching morphogenesis

Transforming growth factor-p (TGFp) pathways have been shown to regulate renal branching morphogenesis, one of the key processes determining the final number of nephrons. Although Smads are known to be downstream mediators of these signals, little is known about their role in kidney development. Using RT-PCR and in situ hybridization we investigated the expression of Smads during kidney development. We have found that the receptor regulated Smads (1, 2, 3, 5 and 8) as well as the common partner (Smad4), are expressed in the developing kidney during nephrogenesis. These factors have a specific expression pattern that overlaps with the expression of the signalling molecules BMP-4 and TGFpi. Expression of Smads 1, 3, 4, 5 and 8 is mainly found in undifferentiated mesenchymal cells of the nephrogenic zone as well as ureteric bud tips, but is downregulated in condensing mesenchymal structures. Smad 2 is mostly expressed in the stromal cell compartment. The distinct, yet overlapping, expression of Smads suggests that the specificity of TGFp signals might be determined in part by the combination of Smads expressed in a particular cell type. To better characterize the role of TGFP in kidney development, mouse embryonic kidney explants were treated with soluble TGFP ligands. Addition of BMP-2 and TGFpi inhibited renal branching morphogenesis through their effects on proliferation and survival. Similarly, proliferation and survival appeared to be the critical processes affected by TGFP signalling in the mIMCD-3 model of tubulogenesis. The direct role of Smads was examined by overexpression of a Smad3-GFP fusion protein in mIMCD-3 cells. Apoptosis was most frequently co-localized to Smad-3 transfected cells, which was consistent with the effects observed from the ligand, TGFpi. To test these observations in vivo, we developed a novel method for the manipulation of embryonic kidney explants in culture. GFP-expression vectors were introduced in kidney explants without loss in viability, and the expression of GFP was found to be rapid and sustained for several days in culture. In summary, this study has placed Smads downstream of TGFP signals in the developing kidney, and it opens the door to finding molecules that might be affected by these signals