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. Author manuscript; available in PMC: 2009 Sep 4.
Published in final edited form as: Kidney Int. 2008 Jul 23;74(10):1287–1293. doi: 10.1038/ki.2008.378

Downregulation of Spry-1, an inhibitor of GDNF/Ret, as a mechanism for angiotensin II-induced ureteric bud branching

Ihor V Yosypiv 1
PMCID: PMC2738599  NIHMSID: NIHMS124638  PMID: 18650792

Abstract

Mutations of the renin-angiotensin system (RAS) genes are associated with congenital abnormalities of the kidney and urinary tract. We have shown that angiotensin (Ang) II stimulates ureteric bud (UB) branching in vitro. The present study tested the hypothesis that Ang II stimulates the GDNF/c-Ret/Wnt11 pathway. E12.5 mice metanephroi were grown for 24 hours in the presence or absence of Ang II or AT1R receptor antagonist candesartan and subjected to whole-mount ISH. c-Ret, a receptor tyrosine kinase for GDNF, and its downstream target Wnt11 were induced by Ang II in the UB tip cells. GDNF, a Wnt11-regulated gene expressed in the mesenchyme, was also upregulated by Ang II. In contrast, Ang II treatment downregulated Spry1, an endogenous inhibitor of Ret tyrosine kinase activity, in an AT1R-dependent manner. Quantitative RT-PCR analysis confirmed that Ang II decreases Spry1 mRNA levels in cultured UB cells. In vivo BrdU incorporation indicated that exogenous Ang II preferentially stimulates UB tip cell proliferation, while AT1R blockade increases TUNEL-positive apoptotic cells. These findings suggest a model in which AT1R-mediated inhibition of Spry1 gene expression releases c-Ret tyrosine kinase activity leading to upregulation of c-Ret and its downstream target gene, Wnt11. Enhanced Wnt11 expression induces GDNF in the adjacent mesenchyme. This causes focal bursts of UB tip cell proliferation, a decrease in UB tip cell apoptosis and branching.

Keywords: kidney development, ureteric bud, renin-angiotensin, GDNF, c-Ret, Wnt11, Spry1, proliferation, apoptosis, branching morphogenesis

INTRODUCTION

The metanephros develops via reciprocal inductive interactions among the ureteric bud (UB), the metanephric mesenchyme (MM) and the stroma (1, 2, 3). Branching morphogenesis involves UB outgrowth from the nephric duct followed by repetitive branching to form the renal collecting system (ureters, pelvis, calyces and collecting ducts). In turn, emerging UB tips induce surrounding mesenchymal cells to form nephrons (from the glomerulus to the distal tubule). Even subtle decreases in the efficiency of UB branching result in a profound decrease in nephron endowment (4). Decreased nephron endowment is linked to hypertension and eventual progression to chronic renal failure (5, 6). In addition, aberrant UB branching morphogenesis causes renal dysplasia, a leading cause of chronic renal failure in infants (7).

The GDNF/c-Ret/Wnt11 signaling pathway is a major positive regulator of UB branching in the metanephros (8). Glial-derived neurotrophic factor (GDNF) is released from the MM and interacts with the c-Ret tyrosine kinase receptor expressed in the UB tip cells to induce branching (9). GDNF/c-Ret and Wnt11 cooperate genetically to induce branching morphogenesis (10). Uncontrolled activation of the GDNF/c-Ret/Wnt11 pathway is prevented by Sprouty (Spry) proteins which function as intracellular inhibitors of receptor tyrosine kinase signaling (11). Genetic inactivation of Spry1 in mice results in ectopic UB outgrowth from the Wolffian duct, increased number of UB branches, and expanded GDNF, c-Ret and Wnt-11 expression domains (11, 12), indicating that Spry1 is a negative regulator of the GDNF-c-Ret-Wnt11 pathway.

Exposure to angiotensin converting enzyme (ACE) inhibitors or AT1R antagonists during fetal life, as well as mutations in the genes encoding angiotensinogen (AGT), renin, ACE or AT1R in humans are associated with renal tubular dysgenesis (13). Inactivation of the renin-angiotensin system (RAS) genes in mice causes abnormalities in the development of the ureter, renal pelvis and papilla (14–18). AGT, renin, ACE or AT1R-deficient mice exhibit pelvic dilation (hydronephrosis) and a small papilla. Mutations in the AT2R gene are associated with increased incidence of lower urinary tract anomalies including double ureters and vesicoureteral reflux (19). These findings indicate that UB growth and development is a major target for angiotensin (Ang) II actions.

We have recently reported that Ang II, acting via the AT1R, stimulates UB branching morphogenesis in the intact metanephric kidney cultured in vitro, and that activation of the epidermal growth factor receptor tyrosine kinase activity is a critical step in the signal transduction pathway downstream of the AT1R leading to UB branching (20). We report here that Ang II stimulates the GDNF/Ret/Wnt11 pathway indirectly via repression of Spry1. This effect is accompanied by increased proliferation of UB tip cells. Furthermore, inhibition of AT1R signaling induces apoptosis preferentially in UB tip cells.

RESULTS

Effect of Ang II on c-Ret, Wnt11 and GDNF gene expression in the ex vivo cultured metanephric kidney

The GDNF/c-Ret/Wnt11 signaling pathway is a major positive regulator of UB branching morphogenesis program (8, 21, 22, 23). In a previous study, we demonstrated that Ang II stimulates UB branching morphogenesis in E12.5 metanephric kidneys grown ex vivo (20). In the present study, we examined whether Ang II-induced UB branching is accompanied by activation of the GDNF/c-Ret/Wnt11 pathway. Treatment with Ang II (10−5 M) for 24 hours increased c-Ret and Wnt11 mRNA expression in the UB tip cells compared to control as determined by ISH (Fig. 1). GDNF expression in the mesenchyme was also upregulated by Ang II. These data suggest that activation of this signaling pathway is critical in Ang II-mediated UB branching (20).

Figure 1.

Figure 1

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Angiotensin (Ang) II upregulates c-RetWnt11 and GDNF mRNA expression in E12.5 mouse metanephroi that were grown ex vivo for 24 hours. After 24 hours in culture, kidney explants were processed for whole mount in situ hybridization. Representative images demonstrate that treatment with Ang II increased c-Ret and Wnt11 expression in the UB tips and GDNF expression in the mesenchyme.

Effect of Ang II on Spry1 gene expression in the ex vivo cultured metanephric kidney and UB cells

Sprouty proteins function as intracellular inhibitors of receptor tyrosine kinase signaling. Genetic inactivation of Spry1 in mice results in increased number of UB branches, and expanded GDNF, c-Ret and Wnt-11 expression domains, indicating that Spry1 is a negative regulator of the GDNF/c-Ret/Wnt11 pathway (11, 12). We therefore examined whether ANG II stimulates the GDNF/c-Ret/Wnt11 pathway indirectly via repression of Spry1. E12.5 wild-type metanephroi were treated with media or Ang II (10−5 M) for 24 hours and processed for whole-mount in situ hybridization. As previously reported (11, 12), Spry1 mRNA was expressed in UB branches and to a lesser extent in condensing mesenchyme (Fig. 2). Ang II treatment downregulated Spry1 expression in the UB and surrounding mesenchyme (Fig. 2). These findings indicate that Ang II is an important regulator of Spry1 in the intact metanephros.

Figure 2.

Figure 2

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Angiotensin (Ang) II downregulates Spry1 mRNA expression in E12.5 mouse metanephroi that were grown ex vivo for 24 hours. After 24 hours in culture, kidney explants were processed for whole mount in situ hybridization. Representative images demonstrate that treatment with Ang II (10−5 M, B) decreases Spry1 mRNA expression in the UB and in the mesenchyme compared to control (Media, A). AT1R antagonist candesartan (D) abrogates Ang II-induced decrease in Spry1 gene expression (C, D). C- Ang II alone (10−5 M); D- Ang II (10−5 M) + candesartan (10−6 M).

To confirm the observed effect of Ang II on Spry1 and to allow a more quantitative analysis of changes in Spry1 gene expression, we examined the effect of Ang II on Spry1 mRNA levels in cultured UB cells by quantitative real-time RT-PCR. Treatment of UB cells with Ang II (10−5 M) for 24 hours resulted in a decrease of Spry1 mRNA levels compared to control (0.66±0.03 vs 1.0±0.01, p<0.01; n=3 per treatment group). We recently demonstrated that cultured UB cells maintain expression of Ang II AT1R mRNA (24). Our present findings that Ang II downregulates Spry1 mRNA expression in the UB cell lineage indicates that Ang II-mediated inhibition of Spry1 gene expression may play a crucial role in Ang II-induced UB branching.

To examine the role of endogenous Ang II in the regulation of Spry1, we utilized the AT1R antagonist, candesartan. Treatment of E12.5 metanephroi with candesartan (10−6 M) for 24 hours abrogated Ang II-induced downregulation of Spry1 gene expression (Fig. 2). The inhibitory effects of endogenous Ang II on Spry1 gene expression are therefore mediated via AT1R. Since candesartan treatment decreases UB branching (20), AT1R- mediated effect on Spry1 is physiologically important.

Effect of Ang II on UB cell proliferation in the ex vivo cultured embryonic kidney

To begin to understand the cellular events leading to stimulation of UB branching by Ang II, we examined the direct effects of Ang II on proliferation of the UB epithelium utilizing BrdU incorporation as an index of DNA synthesis in vivo. Treatment with Ang II (10−5 M) for 48 hours increased cell proliferation index in the UB tip cells (28.5±2.4 vs. 9.7±1.2; p<0.001) but not in UB stalks (11.3±1.9 vs. 9.3±1.2; p=0.4) as compared to control (Fig. 3). These results demonstrate a preferential stimulatory effect of Ang II on UB cell proliferation in the tip cells and are consistent with the notion that growth factor-induced stimulation of UB branching is initiated by focal bursts of tip cell proliferation.

Figure 3.

Figure 3

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Angiotensin (Ang) II stimulates proliferation of the ureteric bud (UB) tip cells. Ang II-treated metanephroi (B, D) have more BrdU-positive cells (brown staining, arrows) in UB tips compared to control (Media, A, C). E: Ang II increases cell proliferation index in UB tip but not in stalk cells.

AT1R blockade promotes apoptosis in metanephric kidneys

Aberrant apoptosis is a cardinal feature of renal dysplasia and hypoplasia (25). Genetic inactivation of angiotensinogen, renin, ACE or AT1R in mice causes hypoplasia of the medulla and papilla (14–18), which may be a result of excessive cell death in the UB derivatives. Accordingly, we examined the effect of AT1R antagonism on UB cell apoptosis in metanephroi grown ex vivo. Treatment of E13.5 metanephroi with candesartan (10−6 M) for 24 hours significantly increased the number of TUNEL-positive cells in the UB tips but not in the stalks (tips: 0.12±0.08 vs. 0.65±0.2, p<0.05; stalks: 0.88±0.19 vs. 1.0±0.25; p=0.72) (Fig. 4). These findings indicate a preferential inhibitory role of endogenous Ang II and its AT1R on apoptosis in UB tip cells, suggesting a role for endogenous Ang II in epithelial cell survival during UB branching morphogenesis.

Figure 4.

Figure 4

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The AT1R antagonist, candesartan, stimulates apoptosis in UB tip cells. Apoptotic cells were identified by TUNEL (brown staining). A- Media, B- Candesartan (10−6 M), C- Kidney treated with TACS-nuclease to generate DNA breaks in every cell (positive control); D- Bar graph shows the effect of media or candesartan on the number of TUNEL-positive cells in the UB tip and stalk cells. G- glomerulus, UB- ureteric bud, ND- not different. B- TUNEL-positive cells in UB are indicated by arrows.

DISCUSSION

The present study demonstrates that Ang II stimulates GDNF, c-Ret, Wnt-11 gene expression, while inhibiting expression of Spry1 in the metanephric kidney cultured in vitro. This effect of Ang II on Spry1 is mediated by the AT1R. In addition, Ang II induces preferential proliferation and provides a survival signal to UB tip cells.

We recently reported that Ang II, acting via the AT1R, stimulates UB branching morphogenesis in the metanephric kidney cultured in vitro (20). Furthermore, we found that activation of the epidermal growth factor (EGF) receptor tyrosine kinase (RTK) activity is a critical step in the signal transduction pathway downstream of the AT1R leading to UB branching (20). These results indicated that Ang II can directly stimulate UB branching, and that cross-talk between AT1R and RTK signaling plays an important role in the development of the renal collecting system.

c-Ret is a RTK that is activated by GDNF and plays a critical role in UB morphogenesis in the developing kidney. GDNF is expressed in the metanephric mesenchyme (26), whereas c-Ret is expressed along the nephric duct and subsequently- in the UB tip cells (9). Genetic inactivation of c-Ret or GDNF in mice leads to a complete absense of the UB or significant impairment of UB morphogenesis (21, 22). Like c-Ret, Wnt11 is expressed in the UB tip cells and interacts genetically with GDNF/c-Ret pathway to induce UB branching (10). Wnt11 expression is reduced in c-Ret−/− metanephroi, indicating that Wnt11 is a downstream target gene of c-Ret. UB branching and GDNF expression is decreased in Wnt11−/− metanephroi, indicating that both mesenchymal GDNF expression and UB tree morphogenesis are dependent on Wnt11 signal from UB tip cells (10). Thus, the GDNF/c-Ret/Wnt11 pathway is a positive feedback loop that acts to stimulate proliferation of UB tip cells and thus promote further UB growth and branching. Our present findings that Ang II enhances GDNF, c-Ret and Wnt11 expression indicate that activation of this pathway by Ang II plays a critical role in Ang II-mediated signaling to stimulate UB tree morphogenesis.

RTK signaling is tightly controlled by positive and negative regulators. Sprouty (Spry) proteins function as intracellular inhibitors of RTK signaling (27). Genetic inactivation of Spry1 in mice results in increased number of UB branches, and expansion of GDNF, c-Ret and Wnt-11 expression domains (11, 12). Therefore, Spry1 is a physiological negative regulator of the GDNF/c-Ret/Wnt11 pathway. In the present study, we found that exogenous Ang II suppresses Spry1 gene expression in cultured embryonic kidneys. Thus, Ang II may stimulate the GDNF/c-Ret/Wnt11 pathway indirectly via repression of Spry1. Moreover, Ang II-induced downregulation of Spry1 expression is abrogated by AT1R antagonism. Based on these findings, we propose that AT1R signaling negatively regulates Spry1 gene expression. This in turn facilitates c-Ret RTK signaling leading to activation of the GDNF/Ret/Wnt11 positive feedback loop (Fig. 5). The mechanism of AT1R-Spry1 interactions may involve clustering in lipid rafts. Caveolae/lipid rafts are essential for Ang II–induced transactivation of EGF receptor (28). We have demonstrated that activation of AT1R by Ang II induces tyrosine phosphorylation of EGF receptor in UB cells (20). Since Spry proteins rapidly translocate to lipid rafts following stimulation with EGF (29), AT1R activation by Ang II may hinder association of Spry1 with RTK (EGF receptor, c-Ret) to prevent inhibition and stimulate UB branching.

Figure 5.

Figure 5

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A proposed model for Ang II-induced UB branching morphogenesis. Ang II AT1R-mediated inhibition of Spry1 gene expression releases c-Ret tyrosine kinase activity leading to upregulation of c-Ret and its downstream target gene, Wnt-11. Enhanced Wnt-11 expression, in turn, induces GDNF expression in the adjacent mesenchyme. This causes focal bursts of UB tip cell proliferation and branching. Decreased UB tip cell apoptosis may also contribute to Ang II-induced UB branching. The mechanisms of Spry1 inhibition by Ang II remain to be determined.

The balance of cell proliferation and apoptosis plays a critical role in UB branching and nephron endowment (30, 31, 32, 33, 34, 35). Derangements of the regulatory mechanisms that control these events are implicated in the pathogenesis of renal hypodysplasia (25, 34), a leading cause of pediatric ESRD (7). The present study demonstrates that Ang II causes preferential proliferation of UB tip cells, whereas inhibition of endogenous AT1R signaling inhibits UB tip cell apoptosis. Since Ang II stimulates UB tip cell proliferation and both c-Ret and Wnt11 are expressed in the UB tip cells, it is likely that observed increase in Ret and Wnt11 expression by Ang II is due in part to enhanced UB tip cell proliferation. We speculate that Ang II induces focal bursts of proliferation of UB tip cells, and together with decreased apoptosis, plays an important role in the expansion of the ampulla, subsequent branching and directional bud elongation.

The mechanisms by which Ang II regulates UB tip cell proliferation and apoptosis are not known. Potential mechanisms include upregulation of antiapoptotic (Bcl-2) and downregulation of proapoptotic (bax, p53) factors. In this regard, decreased UB branching is observed in Bcl-2−/− mice (35). Moreover, Bcl-2 overexpression in the UB suppresses UB cell apoptosis, stimulates branching of the UB tree, and increases nephron endowment (34). The finding that p53 or bax inactivation rescues both aberrant apoptosis and UB branching in salt-stressed bradykinin B2 receptor-null mice (36) provides further evidence that G protein-coupled receptor signaling is intimately linked to cell survival in the metanephric kidney. Stimulation of AT1R by Ang II increases intracellular calcium and activates PKC (37). Activation of the ERK-MAPK pathway by PKC stimulates transcription of cell cycle progression genes, such as cyclin D1, through activation of the transcription factor AP-1 (38). Ang II may regulate these pathways directly or may favor the release of a mesenchymal factor, such as GDNF, which, in turn, stimulates UB tip cell proliferation (30, 31) and migration (39, 40). Recent data indicate that GDNF-induced migration of Ret-transfected MDCK cells is critically dependent on Ret and its downstream signaling via the PI3 kinase pathway (39, 40). We propose a model in which stimulation of GDNF and c-Ret by Ang II induces preferential proliferation and survival of UB tip cells leading to UB growth and branching (Fig. 5).

In summary, the present study demonstrates that Ang II, acting via the AT1R, downregulates Spry1 and upregulates GDNF/Ret/Wnt11 gene expression in the metanephros. The stimulatory effects of Ang II on the GDNF/Ret/Wnt11 pathway are accompanied by preferential proliferation and survival of UB tip cells. These results support the hypothesis that abnormal collecting system development in angiotensinogen, renin, ACE or AT1R-deficient mice is at least partly due to dysregulation of the UB branching morphogenesis program as well as aberrant UB cell proliferation and apoptosis.

MATERIALS AND METHODS

Metanephric organ culture

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. Metanephroi were grown on air-fluid interface on polycarbonate transwell filters (Corning Costar, 0.5 µm) inserted into 6-well plates containing DMEM/F12 medium (Gibco BRL) alone or in the presence of ANG II (10−5 M) alone or combined with the AT1 receptor (AT1R) antagonist candesartan (10−6 M; Sigma) for 24 hours at 37° C and 5% CO2 (20) and then processed for the whole mount in situ hybridization. The effect of drug treatment was studied in paired kidneys obtained from the same fetus (i.e., left kidney was incubated with media and right kidney- with Ang II or left kidney- with media and right kidney- with candesartan).

In situ hybridization

The effect of Ang II on the GDNF/c-Ret/Wnt11 pathway during UB branching was examined by whole-mount in situ hybridization. c-Ret, GDNF, Wnt11 and Spry1 cDNAs were kind gifts from Drs. F. Costantini, A. McMahon and J. D. Licht, respectively. Preparation of RNA probes and whole-mount in situ hybridization were performed according to protocols (http://www.hhmi.ucla.edu/derobertis/protocol_page/mouse.PDF) established in the De Robertis laboratory. 5 embryonic kidneys per treatment group per probe were examined. All experiments were done at least twice. The metanephroi were photographed using an Olympus model SC35 camera mounted on an Olympus model BH-2 microscope, and digital images were captured using Adobe Photoshop software.

Quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR)

Quantitative real-time RT-PCR was utilized to determine whether Ang II alters c-Ret, Wnt11 and Spry1 mRNA expression in ureteric bud (UB) cells (generously provided by Dr. Jonathan Barasch, Columbia University). We have previously demonstrated that these cells express AT1R mRNA (Iosipiv, 2003). UB cells were grown in MEM medium (Gibco BRL) that contained 10% FBS at 37° C in an incubator with 5% CO2. Cells were starved overnight and treated with media (control, n=3) or Ang II (10−6 M, n=3) for 24 hours at 37° C and 5% CO2. The cells were used at passages 5–8. Total RNA was extracted using the TRIzol reagent (Invitrogen). 3 µg RNA was reverse-transcribed in the presence of 100 ng random hexamers, 0.001 ml of 10 mM dNTP, 0.002 ml of 10x RT buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2), and 200 U of Superscript II reverse transcriptase (Invitrogen) as previously described (24).

SYBR Green quantitative real-time RT-PCR was conducted in the Mx3000P equipment (Stratagene, La Jolla, CA) using MxPro QPCR software (Stratagene). Mouse Spry1 gene-specific primers obtained from SuperArray (Frederick, MD). Each PCR reaction was run in 25 µl with the 12.5 mcl SYBR Green ER qPCR SuperMix (Invitrogen), 1 µl first strand cDNA template and 1 µl primer set (10 µM each). The program conditions were: 95°C, 10 min followed by 40 cycles of 95°C, 15 sec and 60°C, 1 min. The quantity of each target mRNA expression was normalized by that of GAPDH mRNA expression. Three UB cell RNA samples per treatment group were analyzed in duplicates in each run. PCR reaction was performed twice.

Cell proliferation and apoptosis assays

To examine the role of cell proliferation in Ang II-induced UB branching, we examined the effect of exogenous Ang II on in vivo incorporation of 5-bromo-2-deoxyuridine (BrdU). CD1 mice metanephroi isolated on E11.5 were grown on filters in the presence of ANG II (10−5 M, n=4) or DMEM/F12 medium alone (control, n=4) for 48 hours at 37° C. BrdU (10−4 M, Sigma) was added to the media during the last 6 hours of incubation. The kidneys were fixed in 10% neutral buffered formalin overnight at 4°C, processed for paraffin embedding, and 4-µm-thick sections were cut. Slides were deparaffinized in two exchanges of xylene and rehydrated in a series of graded ethanol. After quenching of endogenous peroxidase with 30% H2O2 and trypsin digestion, the sections were treated with blocking solution and sequentially incubated with biotinylated mouse anti-BrdU antibody (Sigma, 1:50), streptavidin-peroxidase substrate, and stained with diaminobenzidine (Zymed, San Francisco, CA). The slides were counterstained with hematoxylin, mounted, and coverslipped. The number of BrdU-positive (brown) and -negative (blue) cells was determined in four randomly selected UBs of each kidney section by light microscopy. Cell proliferation index (percentage of BrdU-positive cells) was calculated from the ratio of BrdU-positive to total nuclei.

To investigate the role of endogenous Ang II and AT1R in UB cell apoptosis, E12.5 CD1 mice metanephroi were grown on filters in the presence of DMEM/F12 medium alone (n=10) or with AT1R antagonist candesartan (10−6 M, n=10) for 24 hours at 37° C. Apoptosis was assessed by terminal uridine triphosphate (UTP) end-labeling (TUNEL) (TACS TdT Kit, R&D Systems, Minneapolis, MN). Following digestion with 20 µg/ml proteinase K for 15 min at room temperature, the sections were peroxidase quenched with 30% H2O2, and the TUNEL labeling reaction mixture was added to cover each section. The slides were then incubated in a humidified chamber for 60 min at 37°C. The reaction was stopped by a stop buffer. The slides were counterstained with 0.5% methyl green and examined by light microscopy. The number of TUNEL-positive cells per UB tip or stalk was determined in each kidney section (n=10 kidneys per group; 3 sections per kidney) and the mean number of TUNEL-positive cells per UB tip or stalk was calculated.

Statistical analysis

Differences among the treatment groups in Spry1 mRNA levels and the number of BrdU- and TUNEL-positive cells in media vs. Ang II or candesartan vs. Ang II were analyzed by Student’s t test. A p value of <0.05 was considered statistically significant.

AKNOWLEDGMENTS

This work was supported by NIH Grants P20 RR17659 and DK-71699 to I.V.Y. and DK-56264 and DK-62250 to S.E.D. We thank Dr. Frank Costantini (Columbia University Medical Center), Andrew P. McMahon (Harvard University) and Jonathan D. Licht (Northwestern University) for providing the probes for in situ hybridization, and Dr. Renfang Song for help with qPCR.

Footnotes

DISCLOSURE

Nothing to disclose.

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