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The observation that a significant percentage of GDNF and Ret knockouts develop tuclazepam rudimentary kidneys (Costantini and Shakya, 2006?and?Pichel et al., 1996) has led to the search for a GDNF�CRet independent bypass pathway for budding. Such a pathway has been identified in vitro (Maeshima et al., 2007) and has recently been strongly supported by in vivo evidence (Michos et al., 2010). To ascertain whether the role of PKA is on the GDNF�CRet dependent pathway only or includes the bypass pathway as well, the effect of PKA activity was compared in Ret-null WDs cultured in the standard Ret-dependent culture media (containing GDNF), versus a previously-established media condition that obviates Tenofovir the needs for Ret to induce WD budding (and which does not contain GDNF) (Maeshima et al., 2007). This latter condition bypasses the requirement for GDNF and ensures that there is no Ret activation when used with Ret knockout tissue. We demonstrated that an increase in PKA activity did not suppress the outgrowth of the UB from the WD in this latter condition, suggesting that the PKA-mediated suppression of WD budding is only associated with Ret-dependent budding rather than affecting the Ret-independent bypass process for in-vitro budding (Fig. 7A�CD). Given that activation of PKA appears to inhibit budding of the WD via downregulation of Ret, we then sought to determine what possible growth factors may stimulate the PKA pathway in the regulation of ureteric bud formation. Members of the TGF�� family have long been considered candidate molecules for suppression of WD budding due to their inhibitory effect on UB and epithelial cell branching (Bush et al., 2004, Sakurai and Nigam, 1997?and?Santos et al., 1993). This is supported by in vivo data where deletion of Grem1, a BMP antagonist that preferentially antagonizes BMP2 and BMP4 (Avsian-Kretchmer and Hsueh, 2004), leads to renal agenesis due to failure of UB invasion into the adjacent MM (Michos et al., 2004). BMP4 is localized to the MM surrounding the ureteric stalk while BMP2 is expressed XAV 939 in the mesenchyme adjacent to the site of UB formation (Dudley and Robertson, 1997). This suggests that BMP2 may be involved in limiting further UB formation around the initial UB. To determine if BMP2 may be mediating this process, we added BMP2 (5?nM) to the isolated WD in the presence of GDNF and FGF1, conditions which normally lead to the formation of one or more buds. As expected, BMP2 completely suppressed budding (Fig.?8A). We then measured PKA activity and found it to be increased 3-fold in the BMP2 treated WD compared to the WD treated with GDNF/FGF1 alone which underwent budding (338 RFU/mM and 110 RFU/mM respectively; p?