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【摘要】 In response to gestational high salt intake, BdkrB2 -/- embryos acquire an aberrant renal phenotype mimicking renal dysplasia in humans. Genetic analysis identified p53 as a mediator of the renal dysplasia in salt-stressed BdkrB2 -/- mice, acting partly via repression of terminal epithelial differentiation genes. The present study tested the hypothesis that inactivation of BdkrB2 predisposes the salt-stressed embryo to p53-mediated metanephric apoptosis. Newborn BdkrB2 -/- pups exhibited hyperphosphorylation of metanephric p53 on serine 20 (mouse serine 23), a modification known to increase p53 stability and apoptotic activity. As a result, there was widespread, ectopic expression of p53 in the BdkrB2 -/- kidney. However, no differences were found in the apoptosis index or gene expression in BdkrB2 -/- and +/+ kidneys, indicating that p53 stabilization as a result of BdkrB2 inactivation is not sufficient to induce metanephric apoptosis. On gestational salt stress, fulminant metanephric apoptosis and enhanced Bax gene expression occurred in BdkrB2 -/- but not their +/- or +/+ littermates. Germline deletion of p53 from BdkrB2 -/- mice prevented Bax activation and normalized the apoptosis index. Rescue of metanephric apoptosis in BdkrB2 -/- mice was similarly achieved by Bax gene deletion. Aberrant apoptosis in salt-stressed BdkrB2 -/- mice was triggered on embryonic day E15.5 and involved both ureteric bud (UB) and metanephric mesenchyme-derived nephron elements. Cultured E12.5 salt-stressed BdkrB2 -/- metanephroi manifested stunted UB branching compared with +/- and +/+ littermates; the abnormal UB branching was corrected by p53 deletion. Our results suggest a model whereby a seemingly silent genetic mutation of BdkrB2 predisposes mice to renal dysplasia by creating a "preapoptotic" state through p53 activation.
【关键词】 kidney development ureteric bud branching kallikreinkinin system
GENE TARGETING IN MICE HAS elucidated a complex network of genes encoding transcription factors, soluble factors, receptors, cell cycle, cell-cell, and cell-matrix molecules, which regulate ureteric bud (UB) outgrowth and branching, mesenchymal-to-epithelial transition, nephrogenesis, and terminal differentiation ( 3, 7, 42 ). Abnormal kidney development, the leading cause of neonatal renal failure in humans, results from disruption of one or more of these processes ( 44 ). In general, defects occurring at the early steps of nephron development lead to either complete lack of kidneys or rudimentary severely malformed kidneys, such as mice lacking GDNF/c-Ret, Pax-2, Wnt-4, FoxD1, Eya1, and WT-1. On the other hand, mutations of other genes, such as polycystins, preferentially affect terminal nephron differentiation ( 21 ). In such cases, the phenotype consists of focal or diffuse cystic tubules and/or glomeruli, epithelial-mesenchymal transition, deregulated cell cycle progression, and excessive cell death. In other cases, like Brn1 or Foxi1 deficiency ( 4, 32 ), there is loss of cell-type specification and differentiation, leading to functional renal impairment.
The bradykinin B2 receptor (gene: BdkrB2; protein: B2R) is a G protein-coupled receptor that is highly expressed in differentiating nephrons ( 10, 13 ). Activation of collecting duct B2R by endogenous kinins elicits sodium and water excretion. Hence, BdkrB2 -deficient mice are prone to salt-sensitive hypertension ( 1, 8, 25 ). Interestingly, salt sensitivity in BdkrB2 -/- mice is established during embryogenesis, because gestational salt loading induces abnormal renal development in homozygous BdkrB2 null but not heterozygous or wild-type progeny ( 14 ). Salt-stressed BdkrB2 -/- kidneys exhibit repression of terminal differentiation genes such as E-cadherin, Dlg, HNF-1, AQP-2, and renin ( 14, 15, 18 ). Repression of E-cadherin gene expression is mediated by p53, because it is normalized by deletion of p53 in BdkrB2 -/- mice ( 15 ). Moreover, cell culture experiments revealed that p53-mediated repression of E-cadherin is associated with hypoacetylation of promoter-associated histone H4, which was reversed by treatment with the histone deacetylase inhibitor trichostatin A ( 15 ). These data suggested an important role for p53 in mediating renal dysgenesis in gestational salt-stressed BdkrB2 -/- mice. However, the final pathway(s) and downstream events leading to renal dysgenesis have not been elucidated.
Previous work from our laboratory has shown that the tumor suppressor protein p53 is a transcriptional activator of the BdkrB2 gene ( 27, 35 - 37 ). During collecting duct differentiation, p53 recruits the histone acetylase CREB-binding protein (CBP) to the promoter region of the BdkrB2 gene in a developmentally regulated manner ( 36 ). Genetic deletion of p53 in mice impairs CBP recruitment and BdkrB2 gene expression, supporting the notion that BdkrB2 is a physiological target of p53 ( 35, 36 ). The present study provides evidence for the presence of a functional loop between p53 and BdkrB2, which is required for metanephric survival. Thus whereas p53 activates BdkrB2 transcription, B2R is required to prevent uncontrolled p53 activation and cell death in response to embryonic salt stress. BdkrB2 disruption creates a "preapoptotic" state as a result of NH 2 -terminal phosphorylation and stabilization of the p53 protein. Together, BdkrB2 disruption and gestational salt stress induce fulminant metanephric apoptosis via a p53-Bax-dependent pathway.
MATERIALS AND METHODS
Animals. BdkrB2 -/- mice on a C57BL/6 background have been described ( 6, 14 ). p53 +/- and Bax +/- mice on a C57BL/6 background were obtained from Jackson Laboratories. Crossing of BdkrB2 -/- with p53 +/- or Bax +/- mice and genotyping were carried out according to protocols established by Jackson Laboratories. Following overnight mating, pregnant mice were placed on either a normal (0.3% NaCl) or high-salt (5% NaCl) isocaloric chow (TD no. 92102; Harlan Teklad, Madison, WI) for the duration of gestation. The animal study protocol was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Tulane University.
Analysis of renal phenotype. Two-micrometer frontal corticomedullary sections taken through the hilus were stained with periodic acid-Schiff to delineate the proximal tubular brush border and glomerular mesangial matrix. On examination of the stained sections, particular attention was focused on the integrity of the nephrogenic zone, the organization of the medullary rays, the presence of tubular cysts/dysplasia and glomerular cysts, and the thickness and integrity of the renal microvessels ( 14, 18 ). "Hypoplasia" was defined as a reduction in the number of mature glomeruli and generations of nephrons. Four microscopic fields (800 x 530 µm) were counted in the cortex and medulla of each kidney. "Dysplasia" referred to abnormal spatial organization of nephron segments within the cortex or medulla, cystic dilatation or "ectasia" of the tubules, mesenchymal expansion, metaplasia, and primitive glomeruli or ducts. Histological evaluations of kidney tissue sections were performed in a blinded fashion in 6-10 mice/genotype.
Cell proliferation and apoptosis assays. Cell proliferation was determined by in vivo incorporation of 5-bromo-2-deoxyuridine (BrdU). Newborn mice were subcutaneously injected with 10 µl concentrated BrdU (Zymed)/g body wt. Two hours following the injection, kidneys were harvested and 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% H 2 O 2 and trypsin digestion, the sections were treated with blocking solution and sequentially incubated with biotinylated mouse anti-BrdU, streptavidin-peroxidase substrate, and stained with diaminobenzidine (Zymed). The slides were counterstained with hematoxylin, mounted, and coverslipped. The BrdU incorporation index was assessed by light microscopy. The proliferation index (percentage of BrdU-positive cells) was determined in four microscopic fields (800 x 530 µm) in the cortex and medulla of each kidney section.
Terminal deoxynucleotide transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assays were performed using an in situ cell death detection kit (Chemicon). Following digestion with 20 µg/ml proteinase K for 15 min at room temperature, the sections were peroxidase quenched with 30% H 2 O 2, and the TUNEL reaction mixture was added to cover each section. The slides were then incubated in a humidified chamber for 60 min at 37°C with a coverslip to prevent drying of sections. The reaction was stopped by a stop/wash buffer. Sections were incubated with anti-digoxigenin conjugate, and color was developed in a peroxidase substrate. The slides were counterstained with 0.5% methyl green and examined by light microscopy. The apoptosis index (percentage of TUNEL-positive cells) was determined in four microscopic fields (800 x 530 µm) in the cortex and medulla of each kidney section.
Immunostaining. Kidneys were fixed in 10% buffered formalin, dehydrated in graded solutions of alcohol, and embedded in paraffin blocks, and 3- to 5-µm sections were cut and mounted on Vectabond-coated slides (Vector Laboratories, Burlingame, CA). Immunostaining was performed by the immunoperoxidase technique using a Vectastain Elite kit (Vector Laboratories) as described ( 18, 19 ). Antigen retrieval was performed by immersing slides in 10 mM citric acid and boiled in a microwave for 20 min. Primary antibodies used were as follows: polyclonal anti-B2R antibody (1:500; from W. Müller-Esterl) and anti-phospho (P)-serine 20-p53 (1:1,000; Cell Signaling). Slides were photographed using an Olympus model SC35 camera mounted to an Olympus model BH-2 microscope, and digital images were captured using Adobe Photoshop software.
Metanephric organ culture. Mouse embryos were staged by considering noon of the day of the vaginal plug as embryonic day 0.5. E12.5 metanephroi were collected from females under anesthesia and cultured in serum-free medium until fixed with methanol at 48 h. After washes in 0.1% Tween/PBS, metanephroi were incubated with monoclonal anti-pan-cytokeratin (Sigma) antibody in PBS overnight at 4°C. Following extensive washes, metanephroi were incubated in FITC-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology), washed, and mounted in Vectashield Mounting Medium.
In situ hybridization. Preparation of RNA probes and whole-mount in situ hybridization procedures were performed according to protocols ( http://www.hhmi.ucla.edu/derobertis/protocol_page/mouse.PDF ) established in the De Robertis laboratory.
RT-PCR. RNA was isolated from each pair of newborn mouse kidneys using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using 3-5 µg of total RNA as a template, 0.1 µg of random hexamers (Invitrogen), 4 µl of 5 x first-strand buffer (Invitrogen), 2 µl of 10 mM dithiothreitol, 0.5 µl of 20 mM dNTPs, and 1 µl of Superscript II (Invitrogen) in a volume of 20 µl. Two microliters of cDNA template, 5 µl of 10 x PCR buffer, 1.5 µl of 50 mM MgCl 2, 0.5 µl of 20 mM dNTPs, 1 µl each of 25 pmol forward and reverse primers, and 1 µl (5 U) of Taq polymerase (Invitrogen) were applied to the following PCR program: 4 min at 95°C, 40 s at 95°C, 30 s at 53°C, 1 min at 72°C, and a final extension for 7 min at 72°C for 30 cycles. After amplification, 10 µl of each PCR reaction mixture were electrophoresed through a 1% agarose gel with ethidium bromide (0.5 µg/ml). The gel was scanned with ultraviolet illumination using digital imaging and analysis (Alpha Innotech). The GAPDH primers were forward 5'-AATGCATCCTGCACCACCAA and reverse 5'-GTAGCCATATTCATTGTCATA. Apoptosis genes were assayed using a mouse p53 Apoptosis Effectors I MultiGene RT-PCR profiling kit (SuperArray Bioscience).
Western blot analysis. Immunoblotting was performed as described ( 18 ). Antibodies, dilutions, and commercial sources were as follows: monoclonal p53 (PAb 240, 1:500, Novacastra); polyclonal anti-phospho serine p53 (1:1,000, Cell Signaling); polyclonal anti-acetyl p53 directed to lysines 373, 382 (1:500, Upstate Biotechnology); and a polyclonal Bax antibody (1:500, Santa Cruz Biotechnology). Membranes were reprobed with monoclonal -actin antibody (1:4,000, Sigma) as a loading control. Signals were detected using enhanced chemiluminescence (Amersham), and protein expression was analyzed densitometrically using the Digital Imaging and Analysis Systems (Apha Innotech).
EMSA. Kidney nuclear extracts were prepared as described ( 37 ). Double-stranded oligonucleotides (Operon Biotechnologies) were 5'-end labeled with [ - 32 P]dATP with T4 kinase for use as probes in EMSA. The labeled probe was incubated for 20 min at room temperature with nuclear extracts and the binding buffer [20 mM HEPES, pH 7.9, 10% glycerol, 50 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1 mM spermidine, and 1.0 µg of poly(dI-dC)]. Specific competitor oligonucleotides were added in 100- to 200-fold molar excess 15 min before addition of the radioactive probe. The sequences of the oligonucleotides (double-stranded) used in EMSA as probes or competitors were as follows: high-affinity p53-binding site (P1) from rat BdkrB2 gene, 5'-AGGGGGGAGGTGCCCAGGAGAGTGATGACA-3'; p53-consensus sequence binding site, 5'-AGGCATGTCTAGGCATGTCT-3'; and p53-binding site from human p21, 5'-ATCAGGAACATGTCCCAACATGTTGGAACATGTCCCAACATGTTGA-GCTC-3'.
RESULTS
Contribution of impaired cell proliferation and excessive apoptosis to renal dysgenesis. Previous studies have shown that BdkrB2 -/- mice have normal embryonic development and postnatal survival compared with their BdkrB2 +/+ littermates and that gestational high salt in the maternal diet induces renal dysgenesis in the BdkrB2 -/- but not +/- or +/+ progeny ( 14, 15, 18 ). The present study further shows that the extent of dysgenesis ranged from frank dysplasia (primitive ducts, expanded mesenchyme, and metaplasia) ( Fig. 1, A and B ) to focal tubular ectasia ( Fig. 1 E ). Evaluation of cellular proliferation by in vivo BrdU incorporation revealed that more severely affected kidneys exhibited reduced BrdU-positive but increased TUNEL-positive cells within and around the dysplastic foci ( Fig. 1, C and D ). Kidneys affected with tubular dysgenesis had a reduced thickness of the BrdU-positive nephrogenic zone, accompanied by ectopic BrdU-positive cells in proliferating cystic tubules ( Fig. 1, F and G ). Quantitative analysis confirmed that salt-stressed BdkrB2 -/- mice had a significant thinning of the nephrogenic zone and a reduced number of glomerular generations and total glomeruli compared with Bdrk2 +/+ mice ( Tables 1 and 2 ). Moreover, the percentage of BrdU-positive cells (proliferation index) was lower in salt-stressed BdkrB2 -/- than in BdkrB2 +/+ kidneys ( Tables 1 and 2 ). In contrast, salt-stressed BdkrB2 +/+ and nonstressed BdkrB2 -/- kidneys had a normal proliferation index and nephron number compared with wild-type mice (Supplemental Fig. 1; the online version of this article contains supplemental data). Thus the combination of gestational salt stress and BdkrB2 disruption compromises cellular proliferation and nephrogenesis.
Fig. 1. Gestational salt-stressed bradykinin B2 receptor null ( BdkrB2 -/-) mice exhibit abnormalities in renal development and cell proliferation. A - D : renal dysplasia: primitive epithelial ducts, mesenchymal expansion, metaplasia ( A and B ), terminal deoxynucleotide transferase-mediated dUTP-biotin nick end-labeling (TUNEL)-positive apoptotic cells ( C ), and paucity of 5-bromo-2-deoxyuridine (BrdU)-positive proliferating cells ( D ). HS, high salt. A - G : renal tubular dysgenesis: distorted renal cortical architecture, poorly developed medullay rays, and tubular ectasia mostly involving periodic-acid-Schiff (PAS)-negative distal nephron segments (arrows, E ). The nephrogenic zone is thin (F), and ectopic BrdU uptake is seen in cystic tubular segments (arrowheads, G ) and hyperproliferative tubular epithelium (arrow, G ).
Table 1. General characteristics and renal growth and morphology in BdkrB2-/- mice on gestational normal salt
Table 2. General characteristics and renal growth and morphology in salt-stressed BdkrB2-/- mice
Aberrant apoptosis is a cardinal feature of renal dysplasia ( 44 ). Our previous studies have shown that the earliest histological evidence of tubular dysgenesis in salt-stressed BdkrB2 -/- mice appears on E16 ( 14 ). We therefore examined whether excessive apoptosis precedes the onset of dysgenesis and thus contributes to the renal phenotype. On E15.5, metanephroi of BdkrB2 -/- on gestational normal salt had minimal apoptosis in both the cortex and medulla, mainly confined to the stroma and involving <1% of the cells ( Fig. 2, A and B ). In contrast, E15.5 salt-stressed BdkrB2 -/- mice showed a dramatic induction of the apoptosis index (up to 6% of total cells) in both the nephrogenic zone and medullary region ( Fig. 2, C and D ). At higher magnification, the apoptotic cells were observed in early tubular elements, S-shaped nephrons, and stroma ( Fig. 2 E ). On E14.5, salt-stressed BdkrB2 -/- mice showed minimal evidence of apoptosis ( Fig. 2, F and G ), mostly in the peripheral mesenchyme. Therefore, excess epithelial apoptosis in salt-stressed BdkrB2 -/- mice is not detectable until E15.5 and precedes the development of renal dysgenesis.
Fig. 2. A - F : onset of metanephric apoptosis in gestational salt-stressed BdkrB2 -/- embryos. In situ TUNEL assay shows minimal apoptosis in the renal cortex ( A ) and medulla ( B ) of embryonic day E15.5 nonstressed BdkrB2 -/- embryos. NS, normal salt. In contrast, E15.5 salt-stressed BdkrB2 -/- embryos have abundant apoptosis in both cortex ( C ) and medulla ( D ), preceding the appearance of the abnormal renal phenotype on E16 ( 14 ). Apoptotic cells are seen in the tubular precursors, S-shaped nephrons, and stroma ( E ). There is minimal apoptosis in the cortex ( F ) and medulla ( G ) in E14.5 salt-stressed BdkrB2 -/- embryos. H - O : in vivo early ureteric bud (UB) branching morphogenesis is unaffected by gestational salt stress in either BdkrB2 +/+ or -/- mice. Freshly isolated metanephroi ( H - M ) or urogenital blocks ( N and O ) were harvested on E12.5 - E15.5 and processed for whole-mount cytokeratin immunofluorescence staining.
Although the onset of apoptosis in salt-stressed BdkrB2 -/- mice is not detected until E15.5, it is conceivable that abnormalities in UB growth and branching could occur earlier. To examine this possibility, we harvested metanephroi from E12.5 - E15.5 salt-stressed BdkrB2 +/+ and -/- mice and evaluated their UB branching patterns by whole-mount cytokeratin staining to label the epithelium of the UB and its branches. As shown in Fig. 2, H - O, the pattern of UB branching was remarkably similar in both groups of mice. Quantitative analysis of E12.5 and E13.5 metanephroi revealed no significant differences in the numbers of UB branches and tips between the two groups (data not shown). Thus the aberrant apoptosis in BdkrB2 -/- mice was not preceded by a period of impaired UB growth and branching.
Salt-stressed BdkrB2-/- kidneys undergo abnormal UB branching in vitro. During the branching morphogenesis stage of kidney development, B2R is expressed in UB branches, and at later stages is localized to mature collecting ducts ( Fig. 3 A' and Ref. 13 ). Although the above results indicated that UB branching morphogenesis proceeds similarly in salt-stressed BdkrB2 -/- and BdkrB2 +/+ mice, it is possible that the presence of compensatory mechanisms masks the susceptibility to abnormal branching in vivo. To test this hypothesis, E12.5 metanephroi from salt-stressed BdkrB2 -/-, +/-, and BdkrB2 +/+ littermates were cultured in serum-free medium for 48 h ( n = 34). The organ cultures were subjected to whole-mount cytokeratin immunostaining to evaluate the branching and tip patterns of the UB tree. Figure 3 depicts a representative experiment, which shows that salt-stressed BdkrB2 -/- metanephroi cultured for 2 days in the absence of serum had, on average, 40% fewer tips than similarly treated BdkrB2 +/+ or +/- metanephroi (25 ± 4 vs. 35 ± 3 and 38 ± 6; P < 0.05; Fig. 3, A - F ). In addition, the UB tips in BdkrB2 -/- metanephroi appeared flattened and thickened ( Fig. 3, E and F ).
Fig. 3. Ex vivo UB branching morphogenesis is compromised in salt-stressed BdkrB2 -/- embryos. A ': at E13.5, the bradykinin B2 receptor (B2R) is expressed in UB branches and tips. A - H : left and right metanephroi from littermates with various genotypes were isolated on E12.5 and cultured in serum-free medium for 48 h. Salt-stressed BdkrB2 -/- metanephroi ( E and F ) exhibited stunted UB branching and tip formation compared with BdkrB2 +/+ ( A and B ) or +/- metanephroi ( C and D ). In addition, the UB stalks were disorganized and tips were thick and flat in appearance (arrows and arrowheads, E and F ). p53 deletion normalized UB branching pattern in salt-stressed BdkrB2 -/- metanephroi ( G and H ). I - L : in situ hybridization for c-Ret, confirming the defects in UB branching. Salt-stressed Bdkrb2 -/- metanephroi displayed reduced numbers of c-Ret- positive tips compared with BdkrB2 +/+ metanephroi ( K and L ).
To further characterize this UB branching defect, we examined the expression of c-Ret by in situ hybridization. c-Ret is a tyrosine kinase receptor for the glial-derived neurotrophic factor (GDNF) and is essential for UB branching morphogenesis ( 39 ). In the developing kidney, c-Ret is highly expressed in the UB tips ( 33 ). This expression pattern is maintained in nonstressed BdkrB2 -/- metanephroi cultured for 48 h in serum-free medium ( Fig. 3, I and J ). In contrast, salt-stressed BdkrB2 -/- metanephroi displayed fewer c-Ret -positive tips ( Fig. 3, K and L ), which is consistent with the cytokeratin staining results ( Fig. 3, A - F ). Collectively, these results suggest that BdkrB2 loss of function predisposes the embryonic kidney to abnormal development in a stressful external environment.
BdkrB2 disruption results in p53 phosphorylation and stabilization. In response to cellular stress, p53 is phosphorylated on serine residues 15 (serine 18 of mouse p53) and/or 20 (mouse serine 23) as well as other serine residues ( 5 ). These posttranslational modifications stabilize the p53 protein by preventing the association of the E3 ubiquitin ligase MDM-2 with the NH 2 terminus of p53. In addition, several lysine residues (e.g., K320, K373, and K382) in the COOH terminus of p53 are acetylated by acetyltransferases, such as PCAF and CBP, increasing DNA-binding affinity and transactivation ( 34 ) ( Fig. 4 A ).
Fig. 4. Phosphorylation, accumulation, and ectopic expression of p53 in kidneys of newborn BdkrB2 -/- mice. A : schematic of p53 protein depicting the transactivation domain (AD), DNA-binding domain (DBD), and oligomerization domain (OD). Amino acid residues, serine (S) and lysine (K), which are modified by phosphorylation and acetylation, are also shown. B : Western blot analysis of kidney nuclear extracts from nonstressed BdkrB2 +/+ and -/- mice probed with anti-p53 (PAb 240) antibody. Right : densitometric analysis revealed 2-fold higher levels of nuclear p53 in BdkrB2 -/- than in +/+ kidneys. The same blot was reprobed with an antibody for actin as a loading control. C : EMSA using kidney nuclear extracts from BdkrB2 -/- ( lanes 1, 3, and 5 ) or +/+ ( lanes 2, 4, and 6 ) mice and a 32 P-labeled p53-consensus oligoduplex. Consistent with immunoblotting results, p53-DNA binding activity is higher in BdkrB2 -/- than +/+ kidneys. The presence of several protein-DNA complexes is probably related to the extent of p53 multitetramer formation and/or presence of other proteins in the complexes. Increasing concentrations (100- to 200-fold) of cold competitors were used representing the p53-binding site in the human p21 promoter ( lanes 3 and 4 ) or the P1-p53 binding site in the rat/mouse BdkrB2 promoter ( lanes 5 and 6 ). D : Western blot analysis of kidney nuclear extracts probed with anti-phospho-p53 and anti-acetylated p53 antibodies. BdkrB2 -/- express 3-fold higher levels of phospho-Ser23-p53 than +/+ kidneys, and a modest increase in phospho-Ser6 and Ser18-p53 (1.4- to 1.8-fold). Phosphorylation of COOH-terminal Ser392 and acetylation of K373 and K382 were not altered. E : immunostaining utilizing a phospho-Ser23-p53 antibody. In nonstressed or salt-stressed BdkrB2 +/+ kidneys, phospho-Ser23-p53 is predominantly localized in nephron precursors (bracket; top and bottom left ). In comparison, phospho-Ser23-p53 is ectopically expressed throughout the renal cortex in BdkrB2 -/- kidneys ( top right ). Control kidney section from p53 -/- mouse incubated with the p53 antibody shows absence of specific immunostaining ( bottom right ).
We initially determined the effects of BdkrB2 disruption on metanephric p53 abundance. Western blotting using a monoclonal antibody to p53 (Pab 240) revealed that kidney nuclear p53 levels were twofold higher in BdkrB2 -/- than BdkrB2 +/+ mice ( Fig. 4 B ). Furthermore, EMSA utilizing a radiolabeled consensus p53 probe and kidney nuclear extracts showed higher DNA binding activity in BdkrB2 -/- than BdkrB2 +/+ mice ( Fig. 4 C, lanes 1 and 2 ). Formation of the high-and low-molecular-size complexes could be competitively inhibited by preincubation with cold oligoduplexes representing the p53-binding site in the human p21 gene ( Fig. 4 C, lanes 3 and 4 ) or the P1-p53 binding site of the rat BdkrB2 gene ( Fig. 4 C, lanes 5 and 6 ). Next, we examined the effects of BdkrB2 -/- and gestational salt stress on metanephric p53 activation by assessing the phosphorylation and acetylation status of p53. Western blot analysis of newborn kidney nuclear extracts using phospho-serine-specific p53 antibodies revealed threefold higher levels of P-serine 23-p53 in nonstressed BdkrB2 -/- compared with BdkrB2 +/+ mice ( Fig. 4 D ). In addition, there was a modest increase in serine 6 and serine 18 phosphorylation (1.4- to 1.8-fold). p53-serine 392 phosphorylation and K373 and K382 acetylation were not consistently altered by BdkrB2 disruption ( Fig. 4 D ). Gestational salt stress alone had no detectable effect on p53 phosphorylation or acetylation in either BdkrB2 +/+ or -/- mice ( Fig. 4 D ). Together, these findings indicate that BdkrB2 disruption stabilizes the p53 protein via phosphorylation of NH 2 -terminus serine residues, whereas phosphorylation and acetylation of the COOH terminus remain unaltered.
BdkrB2 disruption induces ectopic expression of p53. Considering the focal nature of renal dysgenesis, the extent of p53 modification and accumulation in the dysplastic kidneys is probably underestimated in whole kidney extracts. We therefore determined whether accumulation of P-serine 23-p53 in BdkrB2 -/- kidneys is accompanied by alterations in its spatial distribution ( Fig. 4 E ). Immunostaining of BdkrB2 +/+ kidneys demonstrated that P-serine 23-p53 was predominantly expressed at low levels in the metanephric mesenchyme and early nephron precursors ( top left ). This limited intrarenal distribution was also observed in salt-stressed BdkrB2 +/+ kidneys ( bottom left ). In comparison, nonstressed BdkrB2 -/- kidneys exhibited enhanced and widespread distribution of P-serine 23-p53 in both the nephrogenic and differentiation zones ( top right ). Thus BdkrB2 disruption not only increases metanephric P-serine 23-p53 levels but also results in its intrarenal redistribution. In salt-stressed BdkrB2 -/- mice, P-serine 23-p53 was expressed in both nephrogenic and differentiating epithelial elements in a pattern similar to nonstressed BdkrB2 -/- mice (Supplemental Fig. 2 ). These findings confirm that gestational salt stress did not change the abundance or distribution of P-serine 23-p53. Control p53 -/- kidneys lacked specific p53 immunostaining ( Fig. 4 E, bottom right ).
p53 mediates aberrant metanephric apoptosis and growth impairment. Because p53 activation is known to induce apoptosis, we determined whether phosphorylation and accumulation of p53 in BdkrB2 -/- kidneys contribute to metanephric cell death. Salt-stressed BdkrB2 -/- metanephroi exhibited profound apoptosis as well as anoikis (detachment of tubular epithelial cells from the underlying basement membrane; Fig. 5, A and B ). Quantitative analysis of TUNEL-positive cells revealed that the apoptosis index was 10-fold higher in salt-stressed BdkrB2 -/- mice than in their BdkrB2 +/- and BdkrB2 +/+ littermates ( Tables 1 and 2 ). To address the role of p53, we generated compound mutants of p53 and BdkrB2. p53 gene dosage reduction ( p53 +/-; BdkrB2 -/- or p53 -/-; BdkrB2 -/-) attenuated the apoptotic index by fourfold ( Tables 1 and 2 ) and rescued the morphological development of the kidney ( Fig. 5, C and D ). Salt stressed p53 +/+; BdkrB2 +/+ or nonstressed p53 +/+; BdkrB2 -/- mice had no significant increase in metanephric apoptosis, compared with wild-type nonstressed mice ( Tables 1 and 2 ). p53 deletion normalized the number of nephrons, thickness of the nephrogenic zone, and number of BrdU-positive proliferating cells ( Fig. 5, E and F, and Tables 1 and 2 ). In addition, p53 deletion corrected the UB branching defects in cultured metanephroi of salt-stressed BdkrB2 -/- embryos ( Fig. 3, G and H ). Thus the induction of apoptosis and growth impairment in salt-stressed BdkrB2 -/- mice is a p53-mediated response.
Fig. 5. Fulminant metanephric apoptosis in salt-stressed BdkrB2 -/- mice and rescue by germline p53 gene deletion. A - D : in situ TUNEL stain depicting widespread metanephric apoptosis in the left (LK) and right (RK) kidneys of a gestational salt-stressed BdkrB2 -/- pup ( A and B ). Note the detachment of the tubular epithelium from the underlying basement membrane ( inset, A ) and the dysplastic tubules ( inset, B ). p53 gene dosage reduction abrogates metanephric apoptosis in gestational salt-stressed BdkrB2 -/- mouse ( C and D ). A few remaining tubules with mild ectasia are occasionally observed. E and F : BrdU uptake and thickness of the nephrogenic zone (bracket) are normalized by p53 deletion in salt-stressed BdkrB2 -/- mouse (see Tables 1 and 2 for quantitative analysis of apoptosis and proliferation index).
p53 is a tumor suppressor that maintains genomic integrity by acting as a sequence-specific transcription factor for cell cycle, apoptosis, and DNA repair genes ( 17, 29, 40 ). In response to cellular stress, such as DNA damage, osmotic stress, and hypoxia, p53 induces cell cycle arrest largely via stimulation of p21 Cip1/Waf-1 gene expression. On the other hand, an expanding list of proapoptotic genes is induced by p53, including Bax, PUMA, Bad, Perp, Noxa, Fas/Apo-1, and caspases. Because the increased apoptosis in salt-stressed BdkrB2 -/- mice can be rescued by p53 gene deletion, we performed RT-PCR screens to identify p53 downstream genes differentially induced by gestational salt stress in BdkrB2 -/- mice compared with their +/- and +/+ littermates. Figure 6 A shows that metanephroi of salt-stressed BdkrB2 -/- pups expressed higher levels of Bax (2.3-fold) and Fas/Apo-1 (2.7-fold) mRNA than their BdkrB2 +/+and +/- littermates. Densitometric analysis of Western blots performed on kidney tissue lysates confirmed that Bax protein levels were higher in salt-stressed BdkrB2 -/- than +/+ pups (4.7 ± 0.5 vs. 2.2 ± 0.4 units; P < 0.05, n = 3/group; Fig. 6 B ). Importantly, germline deletion of one or both alleles of p53 in BdkrB2 -/- mice returned Bax and Fas/Apo-1 gene expression to control levels ( Fig. 6 A ). The expression of other proapoptotic genes, such as caspase 1, 2, 3, 9, and 11, Bad, c-Myc, Noxa, Perp, pmp22/gas3, TNF, DR5/KILLER, and Trp53BP2 was not altered in salt-stressed BdkrB2 -/- mice (Supplemental Fig. 3 ). There was a tendency for decreased expression of the antiapoptotic Bcl-2 gene in salt-stressed BdkrB2 -/- kidneys, whereas Bcl-x L expression was not altered ( Fig. 6 A and Supplemental Fig. 3 ). Thus salt-stressed BdkrB2 -/- mice exhibit alterations in a selected subset of p53-target genes, which include Bax, Fas/Apo-1, and Bcl-2.
Fig. 6. Activation of Bax and Fas/APO-1 gene expression in kidneys of gestational salt-stressed BdkrB2 -/- mice and rescue of the renal phenotype by germline Bax gene deletion. A : analysis of proapoptosis gene expression by semiquantitative RT-PCR. The example shown represents RNA isolated from kidneys of a litter of gestational salt-stressed pups. The genotypes are shown on top of the figure. Bax and Fas/APO-1 gene expression is higher (2.3- and 2.7-fold, respectively) in BdkrB2 -/- than +/+ or +/- kidneys (asterisk). p53 gene dosage reduction returns Bax and Fas expression to wild-type levels. Casp, caspase. B : Western blot showing increased Bax protein expression in kidneys of salt-stressed BdkrB2 -/- compared with +/+ littermates. C - J : partial rescue of apoptosis in salt-stressed double Bax -/-; BdkrB2 -/- mice. C and D : low- and high-power views of PAS-stained sections showing focal dysplasia affecting the lower pole of the kidney in a salt-stressed Bax +/+; BdkrB2 -/- pup. E and F : in situ TUNEL assay performed on consecutive sections showing aberrant cortical and medullary apoptosis in and around the dysplastic tubular epithelium. G and H : Bax deletion improved the renal phenotype in salt-stressed BdkrB2 -/- pups. I and J : scattered tubular cysts were commonly observed, and TUNEL-positive cells in the medulla were decreased but not ameliorated, indicating that Bax deletion provides partial rescue of the renal dysgenesis.
Germline Bax deletion rescues renal dysgenesis. Because Bax mRNA and protein were upregulated in salt-stressed BdkrB2 -/- kidneys, we evaluated its contribution to the phenotype. Bax is a proapoptotic member of the Bcl-2 family and is regulated transcriptionally by p53 through an intronic p53-binding site in both human and mouse genes ( 41 ). Bax plays a key role in p53-mediated apoptosis in vivo, because Bax deletion provides partial rescue for mdm-2 null mice ( 9 ). To assess the contribution of Bax to p53-mediated apoptosis in salt-stressed BdkrB2 -/- mice, we crossed BdkrB2 -/- to Bax +/- mice. Both strains of mice were on a C57BL/6 genetic background. The F1 double-heterozygous progeny were intercrossed and subjected to gestational salt stress. Examination of the F2 progeny ( n = 62) revealed that salt-stressed Bax +/+; BdkrB2 -/- pups exhibited the typical histological characteristics of renal dysgenesis ( Fig. 6, C - F ). In comparison, salt-stressed Bax -/-; BdkrB2 -/- mice had a well-developed nephrogenic zone, four generations of glomeruli, and lacked macrocysts ( Fig. 6, G - J ). However, the renal abnormalities in salt-stressed BdkrB2 -/- mice were not completely ameliorated by homozygous Bax deletion, because they continued to exhibit microcysts and medullary apoptosis ( Fig. 6, H and I ). In addition, there was a significant attenuation of the apoptosis index in the renal cortex of Bax -/-; BdkrB2 -/- (2.4 ± 0.7) compared with Bax +/+; BdkrB2 -/- mice (4.9 ± 0.9; P < 0.01; Fig. 6, I and J ). However, the extent of reduction in the apoptotic index as a result of Bax deletion was significantly less than that resulting from p53 deletion (1.3 ± 0.3; P = 0.02), particularly in the medulla ( Fig. 6 J ). Accordingly, Bax deletion provided a partial rescue of apoptosis. Unlike the case of p53 crosses, where p53 haploinsufficiency was sufficient to rescue apoptosis, the rescue provided by Bax deletion required the loss of both alleles of the gene (data not shown). Collectively, these findings underscore the direct involvement of p53 and its target gene, Bax, in the pathogenesis of uncontrolled apoptosis and renal dysgenesis. The partial rescue of apoptosis by Bax deletion is not surprising given that p53 mediates apoptosis through activation of multiple downstream pathways.
DISCUSSION
The present study demonstrates novel genetic interactions between the BdkrB2 and p53 genes during kidney development. Morphological, biochemical, and genetic analyses indicate that loss of BdkrB2 function induces a preapoptotic state characterized by p53 activation and its intrarenal redistribution. Together with gestational salt stress, BdkrB2 disruption induces transcription of Bax, provoking dramatic metanephric apoptosis. Deletion of either p53 or Bax rescues the renal dysgenesis in salt-stressed BdkrB2 -/- mice. Collectively, these findings illustrate an example of how the developmental consequences of genetic mutations can be unmasked by environmental stress.
The kinin B1 and B2 receptors belong to the seven-transmembrane, G protein-coupled receptor family. The BdkrB1 gene is expressed at very low levels in healthy tissues but is induced in response to injury and inflammation. In contrast, BdkrB2 is constitutively expressed in most tissues ( 24 ). However, BdkrB2 expression is stimulated by inflammatory cytokines, growth factors, and during organogenesis ( 10 ). The onset of terminal nephron differentiation correlates temporally and spatially with activation of BdkrB2 gene expression and kinin synthesis ( 12, 13 ). These findings suggest that kinin-BdkrB2 participates in terminal differentiation and in the physiological adaptation of the kidney to perinatal life.
Recent studies have provided genetic evidence supporting the hypothesis that kinins are physiologically important protective factors. For example, in the adult, kinins protect the cardiac muscle against ischemia-reperfusion injury and prevent the development of hypertension in response to a high-salt diet ( 1 ). The same stressor, i.e., salt, can induce renal dysgenesis in the progeny of BdkrB2 knockout mice ( 14, 15 ). Because the Western diet has a high salt content and mutations/polymorphisms in the BdkrB2 gene are known to occur in humans with renal disease, it is conceivable these gene-environment interactions may represent an unrecognized cause of renal dysplasia.
Kidneys of salt-stressed BdkrB2 -/- mice exhibited thinning of the nephrogenic zone, decreased cellular proliferation, and reduced nephron number. However, wet kidney weight and kidney weight/body weight ratio were not different from those of wild-type mice. This finding can be explained by increased proportion of mesenchymal tissue in the dysplastic kidneys ( Fig. 1 ). In addition, some dysplastic kidneys had hydronephrosis and thus increased water content. Importantly, p53 deletion normalized these abnormalities, including nephron number and cellular proliferation. p53 induces cell cycle arrest in either the G 1 or G 2/M phases through transcriptional activation of the CDK inhibitor p21 or inhibition of CDC2, respectively ( 17 ). In addition, high levels of p53 interfere with S-phase progression via transcriptional repression of the proliferating cell nuclear antigen ( PCNA ) ( 38, 46 ). However, p21 and PCNA gene expression is not altered in salt-stressed BdkrB2 -/- mice. Furthermore, because UB branching morphogenesis proceeds normally up to E15.5, it is unlikely that the reduced nephron number is secondary to interference with early inductive events. We previously reported downregulation of the transcription factor hepatocyte nuclear factor 1 (HNF1- ) in salt-stressed BdkrB2 -/- kidneys ( 15 ). HNF-1 mutations in humans and mice cause renal dysplasia, characterized by deregulated growth and cyst formation ( 20 ), raising the possibility that dysregulation of HNF-1 may contribute to distorted growth and cystogenesis in salt-stressed BdkrB2 -/- mice.
A uniform finding of this study is the enhanced apoptosis in salt-stressed BdkrB2 -/- metanephroi. Quantitatively, wild-type newborn mouse kidneys exhibit minimal apoptosis in the nephrogenic cortex in <1% of cells. Salt-stressed BdkrB2 +/+ and +/- pups had a similarly low apoptotic index. In comparison, salt-stressed BdkrB2 -/- mice had 10-fold higher levels of the apoptosis index than controls (4-6% of cells). Apoptosis involved epithelial cells of nephrogenic precursors, collecting ducts and tips, interstitial stroma cells, and more differentiated tubules. In addition, the dysplastic kidneys exhibited detachment of tubular epithelial cells from the underlying basement membrane (anoikis) and expanded mesenchymal tissue around the tubules. Apoptosis, anoikis, and myofibroblastic transformation (a feature of epithelial-mesenchymal transition) are characteristic features of human renal dysplasia ( 44 ).
The present study demonstrates that derangements of the regulatory mechanisms that control metanephric apoptosis are established on or after E15.5 of gestation in salt-stressed BdkrB2 -/- mice. Several lines of evidence support the hypothesis that p53 mediates the uncontrolled metanephric apoptosis in salt-stressed BdkrB2 -/- mice. The most direct evidence is the rescue of apoptosis and nephrogenesis by germline deletion of p53. Surprisingly, p53 haploinsufficiency was as efficient as homozygous deletion in restoring apoptosis. We interpret this finding that the metanephric kidney is extremely sensitive to even modest increases in p53 levels, as previously demonstrated in mice expressing the MMTV-p53 transgene in the kidney ( 16 ). In the latter study, transgenic expression p53 in the kidney caused accelerated apoptosis during late fetal development and altered the differentiation of the UB and its derivatives.
An interesting finding of this study is the induction of abnormal branching in salt-stressed BdkrB2 -/- metanephroi cultured in vitro. This abnormality did not occur in salt-stressed BdkrB2 +/+ or BdkrB2 +/- metanephroi. These results clearly demonstrate the direct requirement of the BdkrB2 gene in metanephric development. Furthermore, the rescue of UB branching by p53 deletion further highlights the genetic and functional interactions between B2R- and p53-mediated pathways. The developmental pathways that are affected by BdkrB2 disruption and salt stress are currently unknown and were not addressed in the present study. Because the p53 gene is expressed in both UB and metanephric mesenchyme, it would be important to examine developmentally regulated genes in both cell lineages. In this regard, several genes having crucial roles in renal development (e.g., Wnt-4, BMP-4, BMP-7, GDNF ) harbor p53-binding sites in their promoter regions and are bound to p53 in vivo ( 43 ).
A targeted RT-PCR screen identified Bax among genes that were activated in salt-stressed BdkrB2 -/- compared with their wild-type littermates. Deletion of p53 returned metanephric Bax expression to normal levels, demonstrating that p53 activation is responsible for its activation. Furthermore, deletion of Bax attenuated metanephric apoptosis in salt-stressed BdkrB2 -/- mice, strongly implicating Bax in p53-mediated apoptotic signaling in this model of renal dysgenesis. Previous studies have shown that tumor growth is accelerated and apoptosis drops by 50% in Bax -deficient mice, indicating that it is required for a full p53-mediated response ( 47 ). Bax is a proapoptotic member of the Bcl-2 gene family. In the intrinsic pathway of apoptosis, Bax translocates to the mitochondria causing mitochondrial dysfunction and release of cytochrome c ( 22 ). Also, Bax amplifies the extrinsic apoptotic pathway initiated by Fas/APO-1 ( 31 ). Interestingly, we found that Fas was upregulated in salt-stressed BdkrB2 -/- kidneys. Accordingly, these findings suggest that the rescue of p53-mediated metanephric apoptosis by Bax deletion may have resulted from partial inhibition of the intrinsic and extrinsic pathways of apoptosis.
The present study demonstrates that loss of BdkrB2 function promotes NH 2 -terminal phosphorylation and widespread distribution of metanephric p53. NH 2 -terminal phosphorylation of p53 prevents its interactions with mdm-2 and results in p53 stabilization ( 28 ). The pathways linking BdkrB2 disruption with p53 phosphorylation are not known but may involve activation of the checkpoint kinase Chk1 ( 2 ), recently identified in a differential screen as an upregulated protein in salt-stressed BdkrB2 -/- kidneys ( 15 ). Although Chk1 activation suggests the involvement of a genotoxic stress-response pathway, we failed to detect direct evidence of DNA damage in BdkrB2 -/- metanephroi irrespective of the gestational salt stress and p53 genotype (data not shown). We therefore believe that p53 phosphorylation in this model of renal dysgenesis is due to alternative mechanisms.
In addition to p53 stabilization (shown in this study), BdkrB2 disruption can negatively regulate cell survival via other mechanisms. First, B2R is linked to activation of the mitogen-activated protein kinase/ERK and PI3K-Akt pathways ( 11, 30, 45 ), both of which are known to promote survival and proliferation. Second, stimulation of B2R by kinins activates nitric oxide (NO) synthase and NO production. Interestingly, salt-stressed BdkrB2 -/- mice have lower kidney endothelial NO synthase levels than wild-type controls ( 15 ). The NO-cGMP pathway inhibits apoptosis through activation of the serine-threonine kinase, Akt and Bad phosphorylation, increased Bcl-2, and decreased caspase 3 activity ( 48 ). Moreover, NO inhibits Fas-mediated apoptosis via a cGMP-independent mechanism ( 23, 26 ). Last, NO causes thiol nitrosylation of caspases? active site, leading to their inactivation ( 23 ).
In summary, the present study demonstrates that BdkrB2 and p53 regulate each other during kidney development and suggests the following model ( Fig. 7 ): BdkrB2 disruption creates a preapoptotic state by stabilizing p53 (via NH 2 -terminal phosphorylation) and results in ectopic p53 expression. BdkrB2 loss of function also interferes with intracellular survival pathways. Gestational salt stress is required to trigger p53 apoptotic signaling and impaired cell proliferation and differentiation. The mechanisms by which gestational salt stress activates p53 apoptotic gene expression are currently unknown; we speculate that they may involve epigenetic modulation of chromatin in p53-target genes.
Fig. 7. Working hypothesis of gene ( BdkrB2, p53 ) and environment (gestational salt stress) interactions in kidney development. The tumor suppressor protein p53 acts as a transcriptional activator upstream of BdkrB2 during kidney development through direct binding to the promoter and recruitment of coactivators. Conversely, as shown in the present study, B2R signaling provides inhibitory inputs, preventing metanephric p53 activation. BdkrB2 disruption activates p53 kinases (e.g., Chk1 and Junk), leading to NH 2 -terminal phosphorylation and stabilization of the p53 protein. We postulate that these changes render apoptotic genes poised for activation on superimposed embryonic salt stress. The aberrant apoptosis is p53/Bax-dependent. AQP2, aquaporin-2.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56264 and DK-62250 and grants from the American Heart Association and the Tulane Renal and Hypertension Center of Excellence.
ACKNOWLEDGMENTS
We thank Drs. Cathy Mendelsohn for the c-Ret cDNA plasmid, Werner Muller-Esterl for the B2R antibody, and Oliver Wessely for assistance in the in situ hybridization procedures and a critical review of the manuscript.
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作者单位：Section of Pediatric Nephrology, Department of Pediatrics, Tulane University Health Sciences Center, New Orleans, Louisiana