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The renin-angiotensin system (RAS) plays a critical role in ureteric bud (UB) and kidney morphogenesis. Mutations in the genes encoding components of the RAS cause a spectrum of congenital abnormalities of the kidney and urinary tract (CAKUT). However, the mechanisms by which aberrations in the RAS result in CAKUT are poorly understood. Given that c-Ret receptor tyrosine kinase (RTK) is a major inducer of UB branching, the present study tested the hypothesis that angiotensin (Ang) II-induced activation of c-Ret plays a critical role in UB branching morphogenesis. E12.5 mice metanephroi were grown for 24 hours in the presence or absence of Ang II, Ang II AT1 receptor (AT1R) antagonist candesartan, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or ERK½ inhibitor PD98059. Ang II increased the number of UB tips (61±2.4 vs. 45±4.3, p<0.05) compared with control. Quantitative RT-PCR analysis demonstrated that Ang II increased c-Ret mRNA levels in the kidney (1.35±0.05 vs. 1.0±0, p<0.01) and in the UB cells (1.28±0.04 vs. 1.0±0, p<0.01) compared to control. This was accompanied by increased Tyr1062Ret phosphorylation by Ang II (5.5±0.9 vs. 1.8±0.4 relative units, p<0.05). In addition, treatment of UB cells with Ang II (10−5 M) increased phosphorylation of Akt compared to control (213±16 vs. 100±20%, p<0.05). In contrast, treatment of metanephroi or UB cells with candesartan decreased c-Ret mRNA levels (0.72±0.06 vs. 1.0±0, p<0.01; 0.68±0.07 vs. 1.0±0, p<0.05, respectively) compared with control. Ang II-induced UB branching was abrogated by LY294002 (24±2.6 vs. 37±3.0, p<0.05) or PD98059 (33±2.0 vs. 48±2.2, p<0.01). These data demonstrate that Ang II-induced UB branching depends on activation of Akt and ERK½. We conclude that cross-talk between the RAS and c-Ret signaling plays an important role in the development of the renal collecting system.
Branching morphogenesis of the ureteric bud (UB) is a key developmental process that controls not only formation of the renal collecting system (collecting ducts, ureter, calyces and renal pelvis), but organogenesis of the entire metanephros (Grobstein, 1953; Ekblom, 1989; Al-Awqati, Goldberg, 1998; Horowitz, Simons, 2008). UB tips induce formation of nephrons (from the glomerulus through the distal tubule) (Ekblom, 1989). Even subtle defects in UB branching result in a significant decrease in nephron endowment (Sakurai, Nigam, 1998). In turn, decreased nephron endowment is linked to renal hypodysplasia, hypertension and eventual progression to chronic renal failure (Brenner et al., 1988; Lisle et al., 2003). In addition, aberrant UB morphogenesis leads to a spectrum of congenital abnormalities of the kidney and urinary tract (CAKUT) (Pope et al., 1999).
Mutations in the genes encoding components of the renin-angiotensin system (RAS) or pharmacological inhibition of RAS in animals or humans cause diverse forms of CAKUT that include papillary and medullary hypodysplasia, hydronephrosis, collapsed collecting ducts, aberrant UB budding, duplicated collecting system, and urinary concentrating defect (Nagata et al., 1996; Niimura et al, 1995; Takahashi et al., 2005; Esther et al., 1996; Oliverio et al., 1998; Tsuchida et al., 1998). Since CAKUT are the major cause of renal failure in childhood (NAPRTCS Annual Report, 2006), identification of the molecular mechanisms that lead to diverse forms of CAKUT under conditions of disrupted RAS is critical.
The glial-derived neurotrophic factor (GDNF)/c-Ret/Wnt11 signaling pathway is a major inducer of UB branching in the metanephros (Majumdar et al., 2003). Metanephric mesenchymal cells secrete GDNF which signals via the c-Ret receptor tyrosine kinase (RTK) and GFR 1 co-receptor expressed in the UB tip cells to induce UB branching (Arighi et al., 2005; Sariola, Saarma, 1999). Genetic inactivation of GDNF, c-Ret or GFR 1 in mice leads to kidney agenesis (Sanchez et al., 1996; Schuchardt et al., 1996; Cacalano et al., 1998). Using in situ hybridization, we have recently reported that angiotensin (Ang) II, the principal effector peptide of the RAS, induces GDNF and c-Ret gene expression in the metanephros during active UB branching (Yosypiv et al., 2008). In this work, we examined the cross-talk between Ang II and c-Ret in Ang II-induced UB branching morphogenesis. We report here that the stimulatory effects of Ang II on metanephric UB branching are mediated via activation of c-Ret/Akt and ERK½ signaling pathways.
The GDNF/c-Ret/Wnt11 signaling pathway is a major positive regulator of UB branching morphogenesis program (Majumdar et al., 2003). Using in situ hybridization, we previously demonstrated that Ang II-induced UB branching is accompanied by increased c-Ret gene expression in the UB tip cells (Yosypiv et al., 2008). To confirm the observed effect of Ang II on c-Ret and to allow a more quantitative analysis of changes in c-Ret gene expression, in the present study we examined the effect of Ang II on c-Ret mRNA levels in whole metanephroi grown ex vivo by quantitative real-time RT-PCR. Treatment of E12.5 metanephroi with Ang II (10−5 M) for 24 h resulted in an increase of c-Ret mRNA levels compared to control (1.35±0.05 vs. 1.0±0, p<0.01) (Fig. 1B). To examine the role of endogenous Ang II in the regulation of c-Ret, we utilized the AT1R antagonist candesartan. Treatment of E12.5 metanephroi with candesartan (10−6 M) for 24 h decreased c-Ret mRNA levels compared to control (0.72±0.06 vs. 1.0±0, p<0.01) (Fig. 1B). To test the hypothesis that Ang II and c-Ret may interact directly, we used UB cells derived from isolated intact ureteric buds (Barasch et al., 1996). We previously demonstrated that cultured UB cells express Ang II AT1R mRNA (Iosipiv, Schroeder, 2003). Here, we demonstrate that cultured UB cells maintain expression of c-Ret mRNA (Fig. 1A). Treatment of UB cells with Ang II (10−5 M) for 24 h resulted in an increase of c-Ret mRNA levels compared to control (1.28±0.04 vs. 1.0±0, p<0.01) (Fig. 1C). In contrast, treatment of UB cells with candesartan for 24 h decreased c-Ret mRNA levels compared to control (0.68±0.07 vs. 1.0±0, p<0.05) (Fig. 1C). Our present findings that Ang II upregulates c-Ret mRNA expression in the metanephros as well as in UB cells indicate that Ang II-induced increase in c-Ret gene expression may be involved in Ang II-induced UB branching. Using in situ hybridization, we recently reported that Ang II induces GDNF gene expression in the developing metanephros (Yosypiv et al., 2008). Our present findings that Ang II increases c-Ret mRNA levels in UB cells indicate that Ang II induces c-Ret gene expression directly via a mechanism independent, in part, of GDNF. Since candesartan downregulates c-Ret mRNA levels in whole intact metanephroi and in UB cells, the stimulatory effects of endogenous Ang II on c-Ret gene expression are therefore mediated by the AT1R. As candesartan treatment inhibits cell process formation in UB cells grown in collagen matrix gels (Iosipiv, Schroeder, 2003) and UB branching in the whole intact metanephroi grown ex vivo (Yosypiv et al., 2006), AT1R-mediated effects on c-Ret are physiologically important.
To determine the role of c-Ret in Ang II signal transduction, we examined the effect of Ang II on tyrosine phosphorylation of c-Ret. c-Ret activation by GDNF results in phosphorylation of key docking tyrosines that bind to specific adaptor proteins. Phospho-Tyr1062Ret serves as a docking site for protein complexes that activate phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase ½ (ERK½) signaling cascades (Besset et al., 2000). Given that mutations in phospho-Tyr1062Ret result in defective UB branching (Wong et al., 2005; Jain et al., 2005), we investigated the ability of Ang II to induce phosphorylation of Tyr1062Ret. Treatment of E12.5 metanephroi with Ang II (10−5 M) for 24 h resulted in an increase of Tyr1062Ret phosphorylation compared to control (301±16 vs. 100±11%, p<0.05) (Fig. 2A, B). The results indicate that Ang II activates Tyr1062 phosphorylation of c-Ret in whole intact metanephroi grown ex vivo.
In our previous study, we demonstrated that Ang II downregulates expression of Sprouty (Spry) 1, a physiological inhibitor of c-Ret tyrosine kinase activity (Basson et al., 2005), in cultured embryonic kidneys and UB cells (Yosypiv et al., 2008). These findings indicate that inductive effects of Ang II on Tyr1062Ret phosphorylation observed in the present study are indirect (via repression of an repressor). Given that total c-Ret protein levels are not affected by Ang II, the significance of c-Ret mRNA induction by Ang II is not clear. The mechanisms by which Ang II induces c-Ret gene expression remain to be determined. Interestingly, Ang II causes nuclear accumulation of the AT1R in COS-7 cells accompanied by activation of cAMP response element-binding protein (CREB) and enhanced cell proliferation (Cook et al., 2006, 2007). Additional potential mechanisms may involve induction of Gata3, beta-catenin, retinoic acid or other positive transcriptional regulators of c-Ret (Grote et al., 2008, Bridgewater et al., 2008, Batourina et al., 2001). Regardless, dependence of Ang II-induced UB branching on Tyr1062Ret phosphorylation indicates that activation of c-Ret function is a key mediator of Ang II-induced UB morphogenesis. One mechanism by which Ang II may induce c-Ret phosphorylation may involve transphosphorylation by the epidermal growth factor (EGF) receptor. In this regard, we have previously shown that activation of AT1R by Ang II induces tyrosine phosphorylation of EGF receptor in UB cells (Yosypiv et al., 2006). Given that EGF receptor kinase inhibitors inhibit Ret-induced signaling in kidney Cos-7 cells and cell growth in NIH3T3 fibroblasts (Croyle et al., 2008), Ang II-induced activation of Ret may be mediated, in part, by an increase in EGF receptor tyrosine kinase activity. In addition, Ang II may cross-talk with c-Ret RTK via facilitation of physical interaction between Ang II AT1R/AT2R and c-Ret. In this regard, GDNF has been shown to recruit Ret into the lipid rafts and prevent its proteosomal degradation (Pierchala et al., 2006).
Inhibition of PI3K/Akt blocks GDNF/c-Ret-dependent UB branching in the metanephric kidney (Tang et al., 2002; Kim, Dressler, 2007). Thus, one of the possible mechanisms leading to Ang II-stimulated c-Ret signaling may involve stimulation of PI3K/Akt pathway. Given that phosphorylation of Tyr1062Ret activates signaling via the PI3K/Akt cascade (Besset et al., 2000), we next examined the ability of Ang II to induce phosphorylation of Akt. Treatment of E12.5 metanephroi with Ang II (10−5 M) for 24 h increased phosphorylation of Akt compared to control (194±23 vs. 100±11%, p<0.05), whereas the total levels of Akt did not change (Fig. 2C, D). We also tested the ability of Ang II to induce Akt phosphorylation in UB cells. Ang II (10−5 M) increased Akt phosphorylation compared with control when factored per total AKT protein levels (213±16 vs. 100±20%, p<0.05) (Fig. 2E, F). These findings suggest that activation of PI3K/Akt signaling in response to Ang II-induced phosphorylation of Tyr1062Ret may link Ang II receptors to UB branching morphogenesis.
PI3K/Akt pathway has been shown to mediate the effects of Ang II on cell proliferation and hypertrophy in renal mesangial and inner medullary collecting duct cells (Huwiler et al., 1995; Karihaloo et al., 2001). Given that inhibition of PI3K/Akt blocks GDNF/c-Ret-dependent UB branching in the metanephric kidney (Tang et al., 2002), we next tested the hypothesis that Ang II stimulates UB morphogenesis via PI3K/Akt pathway. Treatment with the specific PI3K inhibitor LY294002 (20 μM) decreased the number of UB tips compared with media (21±0.8 vs. 27±2.0, p<0.01) (Fig. 3). These data are consistent with the findings reported by Tang et al. (2002) and demonstrate that PI3K is essential for UB morphogenesis. Ang II (10−5 M) increased the number of UB tips compared with control (37±2.8 vs. 27±2.0, p<0.05) (Fig. 3). Ang II-induced increase in UB branching was abrogated by pretreatment with LY294002 (24±2.5 vs. 37±2.8, p<0.05). Coupled with Ang II-induced phosphorylation of Tyr1062Ret and Akt, these data suggest that Ang II-induced activation of c-Ret signals via PI3K to stimulate UB branching.
Previously, we showed that Ang II-induced upregulation of c-Ret is accompanied by a decrease in Spry1 gene expression (Yosypiv et al., 2008). Thus, it is conceivable that inductive effects of Ang II on Tyr1062Ret phosphorylation and UB branching observed in the present study are mediated via repression of Spry1. One mechanism by which Ang II-induced decrease in Spry1 may enhance c-Ret signaling to stimulate UB branching may involve activation of extracellular signal-regulated kinase ½ (ERK½). In this regard, Spry1 inhibits ERK½ signaling downstream of c-Ret Tyr1062 (Basson et al., 2006, Jain et al., 2006). Notably, ERK½ inhibition decreases UB branching (Fisher et al., 2001). To test the hypothesis that Ang II stimulates UB morphogenesis via ERK½, we examined the effect of ERK½ inhibition on UB branching in mouse metanephroi grown ex vivo. Ang II-induced increase in UB branching was prevented by pretreatment with the specific ERK½ inhibitor PD98059 (33±2.0 vs. 48±2.2, p<0.01) (Fig. 4). Interestingly, PD98059 alone did not alter the number of UB tips compared with control (37±1.6 vs. 38±2.5, p=0.6) (Fig. 4). Our findings that PD98059 alone did not inhibit UB branching are not consistent with the findings by Fisher et al. (2001), but are in concert with data reported by Tang et al. (2002). Together, these findings indicate that ERK½ is essential for Ang II-induced UB branching and that the mechanism of ERK½ activation may involve Spry1-dependent de-repression of c-Ret. Spry1 can antagonize ERK1/2 signaling by inhibiting Ras and Raf directly or by a decrease in phospholipase C (PLC) -dependent activation of Raf (Mason et al., 2006). An important role for PLC -Raf pathway is supported by the findings of reduced ERK1/2 phosphorylation in neuronal cells that lack c-Ret Tyr1015, the binding site for PLC (Encinas et al., 2008).
The mechanisms of Ang II-induced UB branching may involve c-Ret/PI3K- and ERK½-dependent UB cell proliferation/survival and migration. In this regard, the stimulatory effects of Ang II on UB branching are accompanied by preferential proliferation and survival of UB tip cells (Yosypiv et al., 2008). Notably, ERK½ antagonism inhibits proliferation of the UB tip cells (Fisher et al., 2001). In addition, inhibition of PI3K blocks GDNF-dependent migration of c-Ret-transfected MDCK cells (Tang et al., 2002). Whether Ang II can induce UB cell migration remains to be determined.
In summary, the present study demonstrates that Ang II induces phosphorylation of Tyr1062Ret and Akt in the intact metanephros. The stimulatory effects of Ang II on UB branching depend on serine phosphorylation of Akt and activation of ERK½. Collectively, these findings support the hypothesis that cooperation of Ang II with c-Ret/Akt and ERK½ signaling performs essential functions during renal collecting system development via control of UB branching morphogenesis.
Wild-type CD1 mice embryos (Charles River Laboratories, New York, NY) were dissected aseptically from the surrounding tissues on E12.5 and the metanephroi were isolated. The day when the vaginal plug was observed was considered to be E0.5. Paired metanephroi were grown on air-fluid interface on polycarbonate transwell filters (Corning Costar, 0.5 m) inserted into 6-well plates containing DMEM/F12 medium (Gibco BRL) alone (n=4) or in the presence of angiotensin (Ang) II (10−5 M, n=4), PI3 kinase (PI3K) inhibitor LY294002 (20 mcM, Calbiochem, n=4) or ERK½ inhibitor PD98059 (10 mcM, Cell Signaling, n=4) for 24 hours at 37° C and 5% CO2 as previously described (Yosypiv et al., 2006). After 24 hours in culture, kidney explants were stained with anti-pancytokeratin antibody (Sigma, C2562, 1:200) to label the ureteric bud (UB) and the number of UB tips was compared between the treatment groups. Images of branching UBs were obtained at 24 hours of incubation directly from the inserts via an Olympus IX70 inverted phase-contrast microscope and Olympus MagnaFire FW camera, and processed with Adobe PhotoShop 7.0.
Quantitative real-time RT-PCR was utilized to determine whether Ang II or Ang II AT1R antagonist candesartan alters c-Ret mRNA expression in the whole metanephroi grown ex vivo and in UB cells in vitro. E12.5 CD1 mice metanephroi or UB cells were grown on air-fluid interface in the presence of media (control), Ang II (10−5 M) or AT1R antagonist candesartan (10−6 M; Sigma) for 24 hours at 37° C and 5% CO2. UB cells were initially obtained from microdissected ureteric buds of an embryonic day 11.5 mouse transgenic for simian virus 40 (SV40) large T antigen (Immorto-mouse, Charles River) (Barasch et al., 1996). SYBR Green quantitative real-time RT-PCR was conducted in the Mx3000P equipment (Stratagene, La Jolla, CA) using MxPro QPCR software (Stratagene) as described previously (Yosypiv et al., 2008). The quantity of each target mRNA expression was normalized by that of GAPDH mRNA expression. Three RNA samples per treatment group were analyzed in triplicates in each run. PCR reaction was performed three times.
Wild-type CD1 mice E12.5 kidneys were treated with DMEM/F12 medium (n=4, Gibco BRL) alone or in the presence of Ang II (10−5 M, n=4/group) for 24 hours. Quiescent UB cells (generously provided by Dr. Jonathan Barasch, Columbia University) were treated with DMEM/F12 medium alone or combined with Ang II (10−5 M) for 1 hour. Kidneys or UB cells were homogenized in cold lysis buffer containing a cocktail of enzyme inhibitors (Yosypiv et al., 2006). The samples were centrifuged and the supernatants containing proteins (40 μg/lane) were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking nonspecific binding, the membranes were incubated with the phosphospecific anti-Tyr1062Ret (Santa Cruz, sc-20252-R) or anti-phospho-Ser473Akt (Cell Signaling) antibodies. After stripping, membranes were reprobed with anti-total Ret C-19 (sc-167) or anti-total Akt (Cell Signaling) antibodies to document equal protein loading. Immunoreactive bands were visualized using the enhanced chemiluminescence detection system (ECL, Amersham) as previously described (Yosypiv et al, 2006).
Data are presented as means±SEM. Differences among the treatment groups in the number of UB tips, c-Ret mRNA, Tyr1062Ret, Ser473AKT protein levels were analyzed by Student's t test. A p value of <0.05 was considered statistically significant.
This work was supported by NIH Grants P20 RR17659 and DK-071699 to Ihor Yosypiv. We thank Dr. Jonathan Barasch, Columbia University, for providing UB cells.
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