Renal failure causes early death of bcl-2 deficient mice

Mechanisms of Ageing and Development 127 (2006) 600–609 www.elsevier.com/locate/mechagedev

Renal failure causes early death of bcl-2 deficient mice Lev M. Fedorov a,c,*, Carolin Schmittwolf b, Kerstin Amann d, Wolf-Hans Thomas b, Albrecht M. Mu¨ller b, Harald Schubert c, Jos Domen e, Burkhard Kneitz f a

Theodor-Boveri-Institut fu¨r Biowissenschaften, Biozentrum, Bayerische Julius-Maximilians-Universita¨t, Am Hubland, 97074 Wu¨rzburg, Germany b Institut fu¨r Medizinische Strahlenkunde und Zellforschung, Bayerische Julius-Maximilians-Universita¨t, Versbacher-Str. 5, 97078 Wu¨rzburg, Germany c Core Unit ‘‘ Transgene Tiere’’, Institut fu¨r Versuchstierkunde, Friedrich-Schiller-Universita¨t, Erlanger Allee 101, 07740 Jena, Germany d Pathologisches Institut, Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Krankenhausstr. 8-10, 91054 Erlangen, Germany e Cellerant Therapeutics, 1531 Industrial Road, San Carlos, CA 94070, United States f Urologische Klinik und Poliklinik, Julius-Maximilians-Universita¨t Wu¨rzburg, Oberdu¨rrbacherstr. 6, 97080 Wu¨rzburg, Germany Received 12 October 2005; received in revised form 20 February 2006; accepted 23 February 2006 Available online 18 April 2006

Abstract BCL-2 functions as a death repressor molecule in an evolutionary conserved cell death pathway. Inactivation of bcl-2 in mice results in pleiotropic effects including postnatal growth retardation, massive apoptosis in lymphoid tissues, polycystic kidney disease (PKD) and shortened lifespan. To evaluate the influence of the affected bcl-2 deficient kidneys on the postnatal development and lifespan of bcl-2 knockout mice we used ‘‘the rescue of (n 1) affected tissues’’ strategy. According to this strategy bcl-2 heterozygous animals were crossed with H2K-hbcl-2 transgenic mice expressing human BCL-2 in most tissues and organs excluding the kidney. Overexpression of hBCL-2 in bcl-2 / mice rescues growth retardation, normalizes and protects the hematolymphoid system from g-radiation. However, the hbcl-2 transgene is not expressed in kidneys and the rescued mice have PKD and a shortened lifespan. Thus, our results indicated that PKD is the main reason of early mortality in bcl-2 deficient mice. Moreover, we have created mouse model, similar to the kidney specific knockout of bcl-2. Such models can be useful to study the influence of bcl-2 or other gene deficiency in individual organs (or tissues) on development and ageing of whole organism. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: BCL-2; Polycystic kidney; Hematolymphoid system; Lifespan

1. Introduction The Bcl-2 proto-oncogene was originally discovered in follicular B-cell lymphoma with t(14,18) chromosomal translocation (Tsujimoto et al., 1984; Bakhshi et al., 1985; Cleary and Sklar, 1985). The product of the bcl-2 gene, a 25 kDa protein inhibits programmed cell death and thereby promotes tumorigenesis (Hockenbery et al., 1990; Allsopp

* Corresponding author at: Core Unit for Transgenic animals/IVTK, Forschungszentrum Lobeda, Uni-Klinikum, Erlanger Allee 101, 07740 Jena, Germany. Tel.: +49 3641 932 6776; fax: +49 3641 932 5952. E-mail address: [email protected] (L.M. Fedorov). 0047-6374/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2006.02.009

et al., 1993). One of the well-investigated mechanisms to suppress apoptosis is in the ability of BCL-2 to close pores in external mitochondrial membranes, which prevents the release of cytochrome C into the cytosol, and prevents initiation of caspase-9 dependent apoptosis cascades (Kroemer et al., 1997; Shimizu et al., 1999). Other mechanism by which BCL-2 suppresses apoptosis consists of its ability to accumulate Ca2+ in mitochondria, to prevent their dysfunction and as a consequence, cellular death (Murphy and Fiskum, 1999). BCL-2 is widely expressed in the embryonic tissues and plays a significant role in the embryogenesis (LeBrun et al., 1993; Novack and Korsmeyer, 1994). In an adult however the protein is mainly expressed in early hematopoietic progenitors and B and T cell lineages. A limited number of non-lymphoid

L.M. Fedorov et al. / Mechanisms of Ageing and Development 127 (2006) 600–609

tissues expressing BCL-2 is normally restricted to stem cell zones of the intestine, epidermis, hormonally regulated epithelium and long-lived post-mitotic neurons (Hockenbery et al., 1991). Overexpression of BCL-2 in different tissues of transgenic mice defends the cells against apoptosis induced by different stimuli in vivo and in vitro (McDonnell et al., 1989; Strasser et al., 1991a,b; Sentman et al., 1991; Martinou et al., 1994; Lacronique et al., 1996; Domen et al., 1998; Chen et al., 2001). Knockout of bcl-2 leads to pleiotropic effects including postnatal growth retardation, round snout, small external ears, lymphoid apoptosis, polycystic kidneys and hypopigmented hair (Veis et al., 1993; Kamada et al., 1995; for review Sorenson, 2004). Immune system without bcl-2 is subjected to significant pathological changes. The lymphocyte differentiation is initially normal, but thymus and spleen undergo massive apoptotic involution and the amount of mature B and T cells significantly decreases and almost completely disappears by 6–8 weeks postnatally (Veis et al., 1993; Nakayama et al., 1993). Another organ which is subjected to significant pathological changes in bcl-2 / mice is the kidney. Already at E17-E19 bcl-2 deficient embryos have a narrow nephrogenic zone containing five times less glomeruli compared to wild type or heterozygous embryos (Nagata et al., 1996). Soon after birth the kidneys of bcl-2 knockout mice develop structural damage, progressing to glomerulosclerosis and cystic renal degeneration (Gassler et al., 1998). Adult bcl-2 knockout mice demonstrated various degrees of PKD, but there was no significant correlation between the age of mice and the volume of cysts (Veis et al., 1993). The lifespan of bcl-2 deficient mice ranges from 2 weeks to 1 year on C57Bl/6x 129/Sv mixed background (Veis et al., 1993; Michaelidis et al., 1996). On the C57Bl/6 background phenotype of bcl-2 negative mice was more uniformly severe. All animals had fragile lymphoid system, strong PKD, other pathological changes and died between third and eighth weeks of life (Bouillet et al., 2001). Moreover, the authors created the mice lacking bcl-2 and its proapoptotic antagonist bim (Bouillet et al., 2001, 2005). Removal of bim in bcl-2 deficient mice restored normal development of the kidney, hematolymphoid system, growth and coat pigmentation. These studies provided strong genetic evidence that Bcl-2 and Bim function as the main opposing players in the control of cell death in organs and tissues, which are affected in bcl-2 deficient mice. The authors concluded that bcl-2 / mice succumbed to PKD. However, since bcl-2 negative mice have multiple abnormalities it is not possible to exclude the influence of failure of other organs (particularly immunodeficiency) on the health status and lifespan of these animals. Thus, inactivation of bcl-2 by conventional gene targeting leads to multiple physiological changes in the different organs and their pleiotropic effects result in a complicated cumulative phenotype and the main causes of death are presumed to be severe kidney disease or/and failure of the immune system. To answer this question it is necessary to perform a conditional, tissue or organ specific, knock out of bcl-2 in immune system or kidneys. An alternative strategy is the rescue of all affected tissues (excluding immune system or kidneys) in the bcl-2 deficient mice by introduction of a bcl-2

601

transgene and evaluation of the disease development and lifespan dependant on pathological changes in a single tissue or organ. To rescue the hematolymphoid system and other organs of bcl-2 negative animals we chose H2K-hbcl-2 transgenic mice which express the transgene-derived human BCL-2 protein in different organs including hematopoietic system, spleen, thymus, lung and liver (Domen et al., 1998). H2K-hbcl-2 mice develop normally, have normal body- and organ-weight except for a several fold increase in size of the lymphoid organs (thymus, spleen, lymph nodes), in the number of hematopoietic stem cells and in the number of circulating white blood cells. Moreover, overexpression of BCL-2 increased survival of populations of splenocytes, B and T cells in vitro in medium without growth factors and protected these cell populations from the death induced by g-irradiation in vivo (Domen et al., 1998). Our previous analysis of BCL-2 expression by Western blotting in different organs of H2K-hbcl-2 transgenic mice showed similar abundant BCL-2 protein in different organs. However, the total level of BCL-2 protein in kidneys of young transgenic animals was very low and comparable with that in wild type mice (unpublished results). We suggested that BCL-2 protein detected in the kidney is of endogenous origin and that the promoter/enhancer combination used in the transgene is not active in the mouse kidney. Thus, the H2K-hbcl-2 transgene is a potentially useful tool to rescue all affected tissues (excluding kidneys) of bcl-2 negative mice. To clarify the role of bcl-2 in the development of pathological changes in different organs and their connection with the lifespan of bcl-2 knockout mice we rescued the phenotype of bcl-2 negative mice by introducing the H2K-hbcl2 transgene on the bcl-2 / background. This strategy allowed us to rescue the growth retardation, anatomical defects, immunodeficiency but not the kidney phenotype observed in bcl-2 deficient mice. Thereby we identified the affected kidneys as the main reason of early mortality of bcl-2 negative mice. 2. Materials and methods 2.1. Animals and genotyping All mice were housed under conventional conditions, handled and sacrificed in accordance with institutional guidelines. bcl-2 deficient mice on the 129/Sv background were a gift from M. Sendtner (Michaelidis et al., 1996). H2K-hbcl2 transgenic mice (C57Bl/6 background) expressing human BCL-2 under control of the H2K-promoter/enhancer were described early (Domen et al., 1998). To produce mice hemizygous for the transgene and homozygous for bcl2 null allele hemizygous H2K-hbcl-2 were mated with bcl-2+/ mice. Resultant compound mice were subsequently backcrossed with bcl-2+/ mice to yield H2K-hbcl-2/bcl-2 / and H2K-hbcl-2/bcl-2+/ animals with (C57Bl/6x129/ Sv) F2 background. Genotyping of animals was performed by PCR using tail DNA as described earlier (Fedorov et al., 2002).

2.2. Histopathological evaluation and immunohistochemistry Mice were sacrificed by cervical dislocation, subjected to complete autopsy and both gross and microscopic examinations were conducted. Organs were fixed in 3.7% formaldehyde in PBS, embedded in paraffin, sectioned at 6 mm and stained with haematoxylin/eosin. For the immunohistochemical detection of mouse or/and human BCL-2 protein, paraffin-embedded 6 mm thick sections

602

L.M. Fedorov et al. / Mechanisms of Ageing and Development 127 (2006) 600–609

were deparaffinized, rehydrated and microwaved for 6 min in 10 mM sodium citrate buffer pH 5.5. Subsequently the slices were incubated for 6 min in peroxidase blocking solution (3% H2O2 in PBS). After antigen retrieval, slides were rinsed in distilled water, incubated in 20% sucrose in PBS at +4 8C for 30 min, washed in PBS and placed in blocking buffer (2.5% goat serum in PBS) for 40 min. Slides were then incubated with a rabbit antiserum, which is crossreactive with mouse and human BCL-2 (sc-492, Santa Cruz, CA) diluted 1:150 in blocking buffer at +4 8C overnight. Antigen–antibody complexes were detected with the immunoperoxidase system (Vectastain ABC Kit, Vector) and DAB. The sections were then counterstained with haematoxylin. All immunohistochemical reactions were carried out in parallel with reactions lacking primary antibodies to ensure the specificity of the observed staining.

BSA. Where appropriate, erythrocytes were lysed by incubation in Gey´s solution for 5 min at RT. Filtered single-cell suspensions were rinsed with FACS buffer (PBS, pH 7.4, supplemented with 0.4% BSA and 0.02% NaN3), and, after blocking of Fc receptors (FcgRIII/II) with 2.4G2 antibody, 5  105 cells were added to 50 ml of FACS buffer supplemented with a combination of anti-CD4 fluorescein isothiocyanate (FITC) and anti-CD8 phycoerythrin (PE) or anti-B220-PE and anti-IgM-FITC antibodies and incubated at 4 8C for 30 min. The following antibodies were used: CD4-FITC (RM4-5, BD PharMingen), CD8-PE (53-6.7, BD PharMingen), IgMb-FITC (AF6-78, BD PharMingen), B220-PE (RA3-6B2, BD PharMingen). FACS analysis was performed on a Becton Dickinson four-colour FACSCalibur.

3. Results 2.3. Western blot analysis Tissues obtained from euthanized mice were frozen in liquid nitrogen. Frozen tissue samples were homogenised in PBS buffer containing a protease inhibitor cocktail (Roche cat # 1873 580) using an Ultra Turrax blender. After centrifugation at 13,000  g for 10 min the protein content of the supernatant was determined using the Dc Protein Assay kit (BioRad). The protein lysates were separated by electrophoresis in 12% SDS polyacrylamide gel and blotted onto nitrocellulose membranes. Mouse and human BCL-2 proteins were detected by rabbit polyclonal antiserum (sc-492, Santa Cruz Biotech) diluted 1:200 and ERK 2 rabbit anti mouse (sc-154, Santa Cruz Biotech) polyclonal antiserum diluted 1:5000 for loading control. Donkey anti-rabbit immunoglobulins linked to horse radish peroxidase (Amersham) diluted 1:1000 were used as secondary antibodies. Bound antibodies were detected by chemiluminescence (ECL, Amersham).

2.4. Irradiation Healthy 8–12-week-old mice received sublethal irradiation with 9.5 Gy (split dose, 137Cs-Gammatron, 0.511 MeV, dose rate = 0.322 Gy/min). Mice were given acid water before irradiation and antibiotic water (1.1 g/L neomycin sulfate) during 1 week after irradiation.

2.5. Flow cytometry analysis For FACS analysis single-cell suspensions were prepared from spleen, thymus, bone marrow, and mesenteric lymph nodes in ice-cold PBS/0.3%

3.1. Phenotype of H2K-hbcl-2/bcl-2

/

mice

H2K-hbcl-2 mice expressing human BCL-2 were crossed with bcl-2 hemizygous mice to produce H2K-hbcl-2/bcl-2 / animals (Figs. 1A and 2A). Expression of the H2K-hbcl-2 transgene was estimated by Western blotting in different organs of H2K-hbcl-2 and H2K-hbcl-2/bcl-2 / transgenic mice. BCL-2 specific antibodies successfully recognized the endogenous (mouse) and human transgenic BCL-2 proteins. Since growth retardation of bcl-2 negative mice is already visible at 7–9 days, we initially estimated expression of BCL-2 in the mouse tissues of this age. As expected, the expression of transgenic BCL-2 was detected in a variety of organs and tissues of young (9-day old) and adult animals and was significantly stronger than endogenous BCL-2 (Fig. 1B). Analysis of BCL-2 expression in H2K-hbcl-2/bcl-2 / mice of different ages shows the raising of transgenic BCL-2 expression level three to five-fold above the level of endogenous BCL-2 (Fig. 1C). To test the physiological effects of the hbcl-2 transgene the mice of different genotypes from the same litters were observed during 1.5 years. Monitoring of the growth of these mice revealed that 9-day-old bcl-2 deficient mice of both

Fig. 1. Western blot analysis of endogenous and transgenic BCL-2 expression in the tissues of H2K-hbcl-2/bcl-2 / mice. (A) PCR genotyping of transgenic mice. KO and WT, knockout and wild type, respectively. (B) BCL-2 expression in different tissues of 9-day-old mice. Liv, liver; Lng, lung; Msl, muscle; Spl, spleen; *, tissues from 1.5-month-old mice. As a loading control, the amount of ERK-2 protein was determined. (C) BCL-2 expression in different tissues of adult mice; * and **, tissues from 1.5 and 6-month-old mice, respectively. (D) BCL-2 expression in renal tissues of different age mice; *, tissues from 1.5-month-old mice.

L.M. Fedorov et al. / Mechanisms of Ageing and Development 127 (2006) 600–609

603

Fig. 2. Rescue of growth retardation of bcl-2 deficient mice by human BCL-2. (A) Three-month-old mice from the same litter. (B, D) Weight increase of rescued bcl2 / females and males, respectively. Columns correspond to 9 days, 1.5, 2.5, 4, 6 and 8.5-month-old mice (left to right). Each data point represents the average of five to six mice. The bcl-2 negative, H2K-bcl-2, H2K-bcl-2/bcl-2 / and age-matched wild type animals were compared by ANOVA test using a p < 0.05. ANOVA shows in global testing that bcl-2 / mice have significant difference compared to mice of other genotypes ( p < 0.001). No difference was detected between H2K-bcl-2, H2K-bcl-2/bcl-2 / and wild type animals. (C) Survival curves of transgenic and wild type mice were created by Kaplan-Meier program. The bcl-2 negative and H2K-bcl-2/bcl-2 / mice have significant difference vs. H2K-bcl-2 and wild type animals ( p < 0.001). The bcl-2 negative have significant difference vs. H2K-bcl-2/ bcl-2 / mice ( p < 0.03). No difference was detected between H2K-bcl-2 and wild type animals.

sexes have already significant growth retardation. Body weight of H2K-hbcl-2/bcl-2 / animals, in contrast to bcl-2 / mice, was undistinguishable from the wild type or H2K-hbcl-2 transgenic mice during 8 months of observation (Fig. 2A, B and D). Furthermore, examinations of internal organs revealed that the size and appearance of the liver, lung, heart, intestine and reproductive organs of H2K-hbcl-2/bcl-2 / mice were also similar to these organs of wild type or H2K-hbcl-2 transgenic animals. Moreover, the round snout and small external ears, the typical anatomical defects of bcl-2 deficient mice, were also corrected in H2K-hbcl-2/bcl-2 / animals. In spite that hbcl-2 transgene rescued many tissues in H2Khbcl-2/bcl-2 / mice observation demonstrated that these animals have some features of bcl-2 deficient mice. The initially wild type coat of the rescued mice underwent hypopigmentation, similar to age-matched bcl-2 / mice (Fig. 2A). The change of original coat colour reflects apparently the absence of H2K-bcl-2 transgene expression in the melanocyte cell lineage and supports conclusion that bcl-2 deficiency leads to apoptosis of melanocyte stem cells (Nishimura et al., 2005). Furthermore, the monitoring of animal health revealed that some H2K-hbcl-2/bcl-2 / mice showed disease symptoms similar to bcl-2 negative mice (Veis et al., 1993). Sick animals were often lethargic, anorexic, humpbacked and lost approximately 20% of body weight. In addition, sick H2K-hbcl-2/bcl-2 / mice had a reduced lifespan. The life expectancy of wild type or H2K-hbcl-2 transgenic littermates was normal, as expected. The survival curve of H2K-hbcl-2/bcl-2 / mice is comparable to that of

bcl-2 deficient mice, but 2–3 weeks delayed (Fig. 2C, solid triangles versus solid squares). The lifespan of mice of both genotypes was variable. The first bcl-2 / mice died or were moribund at 3–5 weeks. The same was seen in H2K-hbcl-2/bcl2 / mice, but 2–3 weeks later. However, many mice of both genotypes were healthy for at least several months and some survive for more than 1 year. In contrast with bcl-2 negative or bcl-2 negative rescued mice, animals that carried one or two alleles of endogenous bcl-2 or the H2K-hbcl-2 transgene on a bcl-2 wild type background showed no phenotype. 3.2. H2K-hbcl-2 transgene rescues immune system of bcl-2 knockout mice To identify whether abnormalities in the immune system or kidneys are the main reason for this severe disease and concomitant mortality of H2K-hbcl-2/bcl-2 / mice comparative analysis of these organs was performed on wild type, bcl-2 / , H2K-hbcl-2 and H2K-hbcl-2/bcl-2 / mice. Morphometric analysis of lympho-hematopoietic organs showed marked decrease of weight and cell numbers of the thymus, spleen and bone marrow of bcl-2 deficient mice (Table 1). In contrast, rescued mice as well as H2K-hbcl-2 animals demonstrated a two to three-fold increase of these parameters compared to wild type counterparts. These results are consistent with the abundant human BCL-2 expression in spleen, thymus and lymph nodes and other organs of the rescued mice (Fig. 1C and data not shown). To determine whether transgene-derived bcl-2 was important for lymphocyte maturation and differentiation, we

604

L.M. Fedorov et al. / Mechanisms of Ageing and Development 127 (2006) 600–609

Table 1 Organ weights and cell numbers in lymphomyeloid organs in bcl-2 KO rescued mice Organs

Wild type Nonirradiated

bcl-2

/

H2K-hbcl-2

H2K-hbcl-2/bcl-2

/

Irradiateda

Nonirradiated

Irradiateda

Nonirradiated

Irradiateda

Nonirradiated

Irradiateda

37  15, p = 0.013b 26  11, p < 0.01b 14  4, p = 0.015b 24  9, p < 0.01b 1.5  0.3, p = 0.018b

6  3, p < 0.01b 0.17  0.03, p < 0.001b 2  1, p < 0.01b 0.2  0.02, p < 0.01b 0.005  0.002, p < 0.01b

247  90, p < 0.01b 259  115, p < 0.001b 86  8, p = 0.033b 285  49, p = 0.012b 16.6  3.5, p = 0.81

134  65, p < 0.01b 17.6  8.3, p < 0.01b 31  3, p < 0.01b 17.2 + 5, p < 0.01b 1.1  0.2, p < 0.01b

221  86, p = 0.014b 247  88, p < 0.01b 81  8, p = 0.037b 275 + 54, p < 0.01b 16.4  2.8, p = 0.87

138  71, p < 0.01b 16.4  7.5, p < 0.01b 27  4, p < 0.01b 18.2 + 3, p < 0.01b 1.1  0.4, p < 0.01b

Spleen (mg)

85  9

31  4

Spleen (106)

85  32

2.2  0.5

Thymus (mg)

59  6

10  4

Thymus (106)

144  42

1.4  1

Bone marrow (106)

15.4  2

0.1  0.01

Organ weight and cell numbers of 8–12-week-old animals. Each data point represents the average of five to six mice per genotype. Non-irradiated bcl-2 / , H2K-bcl2 and H2K-bcl-2/bcl-2 / mice had statistically significant difference vs. wild type animals (t-test; p < 0.05). No difference was detected between H2K-bcl-2 and H2K-bcl-2/bcl-2 / animals. a Animals were irradiated by the lethal dose (9.5 Gy) of g-irradiation. Organ weights and cell numbers were measured 7 days after irradiation. The irradiated bcl-2 / , H2K-bcl-2 and H2K-bcl-2/bcl-2 / mice showed significant difference vs. wild type animals. No difference was detected between irradiated H2K-bcl-2 and H2K-bcl-2/bcl-2 / mice. b Values considered to show a significant difference (t-test).

Fig. 3. Flow cytometric analysis of B-lymphocytes from 2 to 3-month-old transgenic mice and wild type littermates. Genotypes are indicated on the tops of the columns. Flow cytometric analysis was done for three to four animals of each genotype in three separate experiments. (A) IgM-B220 staining of lymphocytes from bone marrow (BM) and spleen (Spl) of non-irradiated mice. (B) IgM-B220 staining of lymphocytes from mice 7 days after total body irradiation by the 9.5 Gy dose. To have sufficient amount of cells for the FACS staining from irradiated bcl-2 deficient mice the cells were pooled from two to three animals.

L.M. Fedorov et al. / Mechanisms of Ageing and Development 127 (2006) 600–609

compared the development of B- and T-cell lineages in animals with different genotypes. Development of B cells was assessed by measuring IgM and B220 expression in bone marrow, spleen and lymph nodes (Fig. 3A and data not shown). In spite of the dramatically decreased weight and absolute cell numbers of hematopoietic organs the IgM/B220 profiles of B-lymphocytes in bcl-2 negative mice was not different from wild type mice. The percentage of B220 positive cells in bone marrow, spleen and lymph nodes was similar in both genotypes. However, after introduction of the H2K-hbcl-2 transgene into the genome of bcl2 deficient mice the profile of IgM and B220 expression in all organs studied changed and became very similar to that of H2Khbcl-2 transgenic mice. T cell development was assessed by measuring CD4 and CD8 expression in thymus, spleen and lymph nodes (Fig. 4A and data not shown). As expected, the bcl-2 / mice have a reduced percentage of double positive thymocytes compared with wild type mice. Moreover, the profile of mature Tlymphocytes in thymus was shifted toward the CD4+ CD8 cells. A strong shift in the same direction was also observed in spleen and lymph nodes of these mice. Thymi of H2K-hbcl-2 and rescued mice have near normal percentages of CD4+ CD8+ cells, in contrast to bcl-2 deficient animals. Furthermore, the

605

ratio of CD4+ CD8 and CD4 CD8+ mature T lymphocytes was also corrected in the rescued mice. To test whether the H2K-hbcl-2 transgene increases resistance of immune cells in H2K-hbcl-2/bcl-2 / mice to apoptosis, the animals were exposed to a lethal dose (9.5 Gy) of g-irradiation. Seven days after irradiation animals with different genotypes had reduced numbers of cells and sizes of the haematopoietic organs (Table 1). bcl-2 deficient mice had more dramatic changes after irradiation compared to mice of other genotypes. Weight of the hematopoietic organs in bcl-2 / mice was reduced six to seven-fold whereas the total cellularity of the organs was reduced by two to three orders of magnitude. The reduction was less severe in wild type and far less severe in H2K-hbcl-2 or H2K-hbcl-2/bcl-2 / mice. The compositions of B- and T-cell populations were also significantly changed in irradiated mice. The flow cytometry analysis of B-cells in irradiated mice demonstrated significant reduction of mature (IgM+/B220+) B-cells in bone marrow and spleen of wild type mice (Fig. 3B). Moreover, wild type B and pre-B lymphocytes were more radiosensitive than T cells and the lymphocyte population in the spleens of irradiated mice contained more than 60% T lymphocytes (Figs. 3B and 4B). bcl-2 negative mice lost nearly all their lymphocytes after

Fig. 4. Flow cytometric analysis of T-lymphocytes from 2 to 3-month-old transgenic mice and wild type littermates. Flow cytometric analysis was done for three to four animals of each genotype in three separate experiments. Genotypes are indicated on the tops of the columns. (A) CD4–CD8 staining of lymphocytes from thymus (Th) and spleen (Spl) of non-irradiated mice. (B) CD4–CD8 staining of lymphocytes from mice 7 days after total body irradiation by split dose 9.5 Gy. To have sufficient amount of cells for the FACS staining from irradiated Bcl-2 deficient mice the cells were pooled from two to three animals.

606

L.M. Fedorov et al. / Mechanisms of Ageing and Development 127 (2006) 600–609

Fig. 5. Histology and immunohistochemistry of tissues from wild type and transgenic mice. Renal tissues from 3-month-old wild type (A), bcl-2 / (C) and H2Khbcl-2/bcl-2 / (E) mice at the low magnification. Several tubules in transgenic kidneys appear hypertrophied. Kidney from 9-month-old bcl-2 / (B) and 10-monthold H2K-bcl-2/bcl-2 / (D) mouse at the high magnification. Kidneys of mice of both genotypes have dilated renal tubules and hypertrophic epithelium. (F) Infiltration of the mononuclear cells in kidney of H2K-hbcl-2/bcl-2 / mouse. Arrows indicate accumulation of the cells. Haematoxylin–eosin staining. (G–J) Immunostaining for BCL-2. Absence of staining in the kidney of wild type (G) and bcl-2 / (H) mouse. Expression of human BCL-2 protein in white blood cells infiltrated in kidney (I) and splenocytes in spleen (J) of 4-month-old H2K-hbcl-2/bcl-2 / mouse. Immunoperoxidase staining (DAB brown); in addition sections were counterstained with haematoxylin (blue). Scale bars = 400 mm (A, C, E, F), 800 mm (B, D), 50 mm (G), 100 mm (H, I, J).

L.M. Fedorov et al. / Mechanisms of Ageing and Development 127 (2006) 600–609

irradiation (Table 1), cells pooled from two to three animals were necessary to perform one FACS analysis. Irradiation resulted in the strong reduction of mature B cells in spleen and lymph nodes of bcl-2 negative mice (Fig. 3B and data not shown). In excess of 80% of the few surviving splenocytes consisted of B220+ cells, indicating that a small subpopulation of B cells may be BCL-2 independent with respect to radioresistance. The mice containing transgenic bcl-2 were less sensitive to irradiation. The percentage and absolute numbers of immature (IgM /B220+) and mature (IgM+/B220+) B-cells was significantly larger in H2K-hbcl-2 or H2K-hbcl-2/ bcl-2 / mice compared with wild type animals. The T cell lineage composition was also significantly changed in irradiated mice (Fig. 4B). Thymi of bcl-2 deficient mice lost nearly all double positive CD4+/CD8+ immature T cells. In addition, mature T lymphocytes were also more sensitive and their populations were significantly reduced or absent in bcl-2 negative mice. Furthermore, bcl-2 deficient or wild type CD8+ mature cells were more radiosensitive compared to CD4+ cells. Human BCL-2 protein restored both immature and mature T cells in H2K-hbcl-2/bcl-2 / mice and in addition corrected ratio of CD4+/CD8+ mature lymphocytes. Thus, transgenederived BCL-2 rescued the immunodeficiency of bcl-2 negative mice and increased the resistance of lymphocytes against girradiation induced apoptosis. 3.3. BCL-2 expression and renal morphology of H2K-hbcl2/bcl-2 / mice Extensive histological analysis showed that all 44 ill bcl-2 / mice at different ages developed polycystic kidneys in the terminal stage of life (Figs. 2C and 5A–C). Surprisingly, all 37 sick H2K-hbcl-2/bcl-2 / mice at different ages showed also a variety of renal abnormalities (Figs. 2C and 5D and E). Sick mice had yellow-coloured kidneys, thin renal cortex and similar glomerular rows in bcl-2 / and H2K-hbcl-2/bcl-2 / mice. In both cases the structural damages progressed to glomerulosclerosis and cystic renal degeneration (Fig. 5D and E). In addition, glomeruli and the corresponding tubules were hypertrophied like in bcl-2 negative kidneys (Fig. 5B and D). There was no linear relationship between the degree of cystic disease and the age of the mice; cystic disease was observed in mice as young as 1 month or as old as 12 months. Ill mice always had severe affected kidneys. Thus, kidney abnormality is the main reason for mortality of H2K-hbcl-2/bcl-2 / as well as bcl-2 deficient mice. To identify the relationship between BCL-2 expression and pathological changes in the kidneys in H2K-hbcl-2/bcl-2 / mice at first we evaluated the level of BCL-2 protein in kidneys by Western blotting. Expression of endogenous BCL-2 in the kidneys of wild type mice was very low (Fig. 1D). To our surprise, we found BCL-2 expression in the kidneys of H2Khbcl-2/bcl-2 / mice. In general, they expressed low levels of transgenic BCL-2. The kidneys of some animals, however, (one out of every eight to nine mice) had elevated expression of the transgene. The level of transgene expression was not connected with kidney pathology and health status of the mice. High BCL-2

607

expression levels were detected in healthy as well as polycystic kidneys. Extensive histological examination revealed that kidneys sometimes contained infiltrates of lymphocytes and other mono-nucleated white blood cells (Fig. 5F). In order to identify the BCL-2 expressing cells kidney sections were stained with antibodies specific for mouse and human BCL-2. In immunohistochemistry as well as in Western blotting experiments we used the organs and tissues from wild type or H2Khbcl-2/bcl-2 / mice expressing only one type of BCL-2 protein. Therefore wild type and transgenic BCL-2 did not interfere and we were able to evaluate the expression of every BCL-2 separately. Low expression of BCL-2 was found in few infiltrated white blood cells and endothelium of renal arteries of wild type animals (data not shown) but was not detectable in epithelial cells of the renal tissues of wild type as well as negative control bcl-2 / mice (Fig. 5G and H). The epithelial cells of nephrons, tubules and cysts in the kidneys of sick H2K-hbcl-2/bcl-2 / mice were also negative (Fig. 5I and data not shown). However, infiltrated white blood cells expressed high levels of BCL-2 comparable to the BCL-2 positive cells in spleen of these mice (Fig. 5J). Thus, only infiltrated white blood cells express transgenic BCL-2 protein in the kidney of H2K-hbcl-2/bcl-2 / mice and this protein was detectable by Western blotting and immunohistochemistry. Hence, the H2K-hbcl-2 transgene does not express in epithelial cells of the kidney. In summary our results show that the lack of BCL-2 expression in the kidneys of H2K-hbcl-2/bcl-2 / as well as bcl-2 negative animals causes early death by severe renal abnormalities. 4. Discussion To avoid pleiotropic effects of mutated gene and study the role of this gene in different physiological processes in the cells of certain tissue the best strategy is the production of mice by tissue specific gene targeting or conditional/tissue specific gene targeting technology. However, these methods are very expensive and time consuming. In many cases the answer to questions of interest can be obtained by crossing heterozygous knockout mice with transgenic mice expressing the same gene in definite tissues. In the present study we used ‘‘the rescue of (n 1) affected tissues’’ strategy to avoid a complicated cumulative phenotype in homozygous adult mice, which develops after conventional gene targeting. According to this strategy all affected tissues or organs (excluding one) of adult homozygous mutant mouse can be rescued by introduction in its genome of a transgene containing the same gene expressed under the regulation of a promoter/enhancer combination active in multiple tissues. This method is limited by the number of transgenic lines with suitable expression patterns. In addition, the transgene expression profile and level is often not identical to the endogenous gene. Nevertheless, the method allows the evaluation of physiological changes caused by inactivation of a gene of interest in a single tissue or organ. Moreover, this strategy can be modified in ‘‘the rescue of (1 n) affected tissues’’ version to save homozygous embryos when severe malformation of a single tissue (organ) leads to embryonic lethality. For this purpose homozygous mutant embryos can be

608

L.M. Fedorov et al. / Mechanisms of Ageing and Development 127 (2006) 600–609

rescued by introduction in its genome of a transgene containing the same gene expressed under the regulation of a tissue specific promoter, which expressed in affected tissue of knockout embryos. This approach allows the evaluation of the influence of a mutated gene on the other tissues or organs in postnatal development. The expression of the BCL-2 oncoprotein is essential for the embryonic and postnatal development of the mammalian organism and it is in consistent with a role played by bcl-2 in the control of cellular survival. Inactivation of endogenous bcl-2 by conventional gene targeting results in multiple physiological changes in the different tissues and organs. Especially, bcl-2 deficient mice are characterized by dramatic apoptosis in the immune system. Intrinsic fragility of the lymphoid compartment of bcl-2 / mice was demonstrated by elegant experiments on reconstituted animals where the stress from renal insufficiency was precluded (Matsuzaki et al., 1997; Bouillet et al., 2001). In fact abnormality of immune system influences often the health of animals. For example severe combined immunodeficient (scid) mice are very sensitive to different pathogens what resulted in an infection and correspondingly shortened lifespan in conventional conditions (Percy and Barta, 1993). Rag-2 / negative mice have no gross abnormality except B and T cell deficiency, but in non-barrier facility these animals are smaller in size and routinely develop infections (Shinkai et al., 1992). Therefore it was speculated that immunodeficiency could potentially be responsible for the lethality in bcl2 deficient mice. We were able to rescue the immunodeficiency of bcl-2 negative mice totally by introducing the H2Khbcl-2 transgene. Our findings showed that transgene-derived human BCL-2 protein restores T cell and B cell lineage development in bcl-2 negative mice. Organ sizes, cell numbers and expression profiles in B and T cell lineages of rescued animals were very similar to those of H2K-hbcl-2 transgenic mice. Furthermore, transgene-derived BCL-2 increased the resistance of lymphocytes of bcl-2 negative mice against g-irradiation induced apoptosis. Moreover, we rescued the growth retardation and other anatomical defects of bcl-2 deficient mice. However, the rescue of multiple abnormalities (except the renal failure and coat pigmentation) increases the lifespan of these animals only by 2–3 weeks in general. Hence, the bcl-2 deficiency in kidneys of bcl-2 / and H2K-hbcl-2/bcl-2 / mice results in early death of these animals and the variability of their lifespan is depended on the degree of kidney malformation. During embryonic and postnatal life the expression of BCL2 in the kidney varies considerably. Development of the kidney anlage (metanephros) depends on the inductive events, which occur between the ureteric bud and the metanephric mesoderm. At E12.5 of mouse embryonic development these structures express high levels of BCL-2 (Novack and Korsmeyer, 1994), however during consecutive development the expression of BCL-2 is significantly reduced. The BCL-2 expression in adult kidneys is very low. Activation of its expression, however, prevents apoptosis in kidney tissue with renal failure. For example, BCL-2 expression was detected in kidneys of

patients with glomerulonephritis (Goumenos et al., 2004). BCL-2 was found to be ectopically expressed in dysplastic epithelia of tubules in children with chronic renal failure (Winyard et al., 1996) and renal cell carcinomas (Chandler et al., 1994; Paraf et al., 1995). In our experiments we identified low levels of BCL-2 protein by Western blotting in kidney lysates of adult wild type mice, but immunohistochemistry failed to show BCL-2 in epithelial cells of tubules and nephrons. Low expression was only identified in renal arteries and infiltrates of white blood cells. Nevertheless, we cannot exclude that the epithelial cells of wild type kidneys express small amount of BCL-2 (undetectable by immunohistochemistry). Low BCL-2 expression does not equate dispensable function. For example, the epithelia of the small intestine in mice are not immunohistochemically positive for BCL-2 protein yet there was a modest but significant increase in apoptosis in bcl-2 null mice (Pritchard et al., 1999). Moreover, type II pneumocytes in lung of adult animals also express very low levels of BCL-2. Despite the fact that this is not detectable by immunohistochemistry, BCL-2 plays an important role in development of Raf oncogene-induced lung adenomas (Fedorov et al., 2002). As in other organs, the BCL-2 protein in kidney is probably important not only for embryonic development but also for postnatal development and functionality. Knockout of bcl-2 results in extensive apoptosis and dramatic reduction of glomeruli in kidneys of bcl-2 deficient embryos at E17-19 (Nagata et al., 1996). Moreover, no new nephrons develop after birth in bcl-2 / mice (Gassler et al., 1998). Hence, oligonephronia is already established at the birth. It seems that BCL-2 inhibits apoptosis in renal stem cells during the induction of nephrons and a minimal level of BCL-2 is probably necessary to support the pool of these stem cells in postnatal kidneys. Future experiments that utilise a bcl-2 conditional knockout in the kidney should help to settle the question about role of bcl-2 for the postnatal kidney development. In conclusion, we have shown that PKD is the main reason of early mortality in bcl-2 deficient mice. Moreover, we have created a mouse model similar to the kidney specific knockout of bcl-2. This model can be useful to study the role of bcl-2 in renal pathology. Several inherited human diseases (including autosomal dominant PKD) are characterized by the development of renal tubular cysts and other kidney defects similar to these mice. In addition our model could be useful to study the influence of bcl-2 deficiency in the individual organs (tissues) on development and ageing of whole organism. Acknowledgements We thank G. Fedorova, S. Kirste and M. Klewer for expert technical assistance and K. Hoffmann for help with the statistical analysis. We gratefully acknowledge M. Sendtner and I.L. Weissman for bcl-2 deficient and H2K-hbcl-2 transgenic mice, respectively. We are grateful to M. Schartl, D. Weih and F. Weih for the critical reading of manuscript and stimulation of helpful discussion. This work was partly supported by Wilhelm-Sander-Stiftung (2002.091.2), German

L.M. Fedorov et al. / Mechanisms of Ageing and Development 127 (2006) 600–609

Programm Neue Bundesland (NBL-3), Deutsche Forschungsgemeinschaft (SFB 423) and the ‘‘Interdisziplina¨ren Zentrum fu¨r Klinische Forschung’’ of the University of Wu¨rzburg. References Allsopp, T.E., Wyatt, S., Paterson, H.F., Davies, A.M., 1993. The protooncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis. Cell 73, 295–307. Bakhshi, A., Jensen, J.P., Goldman, P., Wright, J.J., McBride, O.W., Epstein, A.L., Korsmeyer, S.J., 1985. Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 41, 899–906. Bouillet, P., Cory, S., Zhang, L.C., Strasser, A., Adams, J.M., 2001. Degenerative disorders caused by Bcl-2 deficiency prevented by loss of its BH3only antagonist Bim. Dev. Cell 1, 645–653. Bouillet, P., Robati, M., Bath, M., Strasser, A., 2005. Polycystic kidney disease prevented by transgenic RNA interference. Cell Death Differ. 12, 831– 833. Chandler, D., el-Naggar, A.K., Brisbay, S., Redline, R.W., McDonnell, T.J., 1994. Apoptosis and expression of the bcl-2 proto-oncogene in the fetal and adult human kidney: evidence for the contribution of bcl-2 expression to renal carcinogenesis. Hum. Pathol. 25, 789–796. Chen, Z., Chua, C.C., Ho, Y.S., Hamdy, R.C., Chua, B.H., 2001. Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice. Am. J. Physiol. Heart Circ. Physiol. 280, 2313–2320. Cleary, M.L., Sklar, J., 1985. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpointcluster region near a transcriptionally active locus on chromosome 18. Proc. Natl. Acad. Sci. USA 82, 7439–7443. Domen, J., Gandy, K.L., Weissman, I.L., 1998. Systemic overexpression of BCL-2 in the hematopoietic system protects transgenic mice from the consequences of lethal irradiation. Blood 91, 2272–2282. Fedorov, L.M., Tyrsin, O.Y., Papadopoulos, T., Camarero, G., Gotz, R., Rapp, U.R., 2002. Bcl-2 determines susceptibility to induction of lung cancer by oncogenic CRaf. Cancer Res. 62, 6297–6303. Gassler, N., Elger, M., Inoue, D., Kriz, W., Amling, M., 1998. Oligonephronia, not exuberant apoptosis, accounts for the development of glomerulosclerosis in the bcl-2 knockout mouse. Nephrol. Dial. Transplant. 13, 2509–2518. Goumenos, D.S., Tsamandas, A.C., Kalliakmani, P., Tsakas, S., Sotsiou, F., Bonikos, D.S., Vlachojannis, J.G., 2004. Expression of apoptosis-related proteins bcl-2 and bax along with transforming growth factor (TGF-beta1) in the kidney of patients with glomerulonephritides. Renal Failure 26, 361– 367. Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R.D., Korsmeyer, S.J., 1990. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334–336. Hockenbery, D.M., Zutter, M., Hickey, W., Nahm, M., Korsmeyer, S.J., 1991. BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc. Natl. Acad. Sci. USA 88, 6961–6965. Kamada, S., Shimono, A., Shinto, Y., Tsujimura, T., Takahashi, T., Noda, T., Kitamura, Y., Kondoh, H., Tsujimoto, Y., 1995. bcl-2 deficiency in mice leads to pleiotropic abnormalities: accelerated lymphoid cell death in thymus and spleen, polycystic kidney, hair hypopigmentation, and distorted small intestine. Cancer Res. 55, 354–359. Kroemer, G., Zamzami, N., Susin, S.A., 1997. Mitochondrial control of apoptosis. Immunol. Today 18, 44–51. Lacronique, V., Mignon, A., Fabre, M., Viollet, B., Rouquet, N., Molina, T., Porteu, A., Henrion, A., Bouscary, D., Varlet, P., Joulin, V., Kahn, A., 1996. Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice. Nat. Med. 2, 80–86. LeBrun, D.P., Warnke, R.A., Cleary, M.L., 1993. Expression of bcl-2 in fetal tissues suggests a role in morphogenesis. Am. J. Pathol. 142, 743–753.

609

Martinou, J.C., Dubois-Dauphin, M., Staple, J.K., Rodriguez, I., Frankowski, H., Missotten, M., Albertini, P., Talabot, D., Catsicas, S., Pietra, C., et al., 1994. Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. Neuron 13, 1017– 1030. Matsuzaki, Y., Nakayama, K., Nakayama, K., Tomita, T., Isoda, M., Loh, D.Y., Nakauchi, H., 1997. Role of bcl-2 in the development of lymphoid cells from the hematopoietic stem cell. Blood 89, 853–862. McDonnell, T.J., Deane, N., Platt, F.M., Nunez, G., Jaeger, U., McKearn, J.P., Korsmeyer, S.J., 1989. bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell 57, 79–88. Michaelidis, T.M., Sendtner, M., Cooper, J.D., Airaksinen, M.S., Holtmann, B., Meyer, M., Thoenen, H., 1996. Inactivation of bcl-2 results in progressive degeneration of motoneurons, sympathetic and sensory neurons during early postnatal development. Neuron 17, 75–89. Murphy, A.N., Fiskum, G., 1999. Bcl-2 and Ca(2+)-mediated mitochondrial dysfunction in neural cell death. Biochem. Soc. Symp. 66, 33–41. Nagata, M., Nakauchi, H., Nakayama, K., Loh, D., Watanabe, T., 1996. Apoptosis during an early stage of nephrogenesis induces renal hypoplasia in bcl-2-deficient mice. Am. J. Pathol. 148, 1601–1611. Nakayama, K., Negishi, I., Kuida, K., Shinkai, Y., Louie, M.C., Fields, L.E., Lucas, P.J., Stewart, V., Alt, F.W., et al., 1993. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science 261, 1584–1588. Nishimura, E.K., Granter, S.R., Fisher, D.E., 2005. Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche. Science 307, 720–724. Novack, D.V., Korsmeyer, S.J., 1994. Bcl-2 protein expression during murine development. Am. J. Pathol. 145, 61–73. Paraf, F., Gogusev, J., Chretien, Y., Droz, D., 1995. Expression of bcl-2 oncoprotein in renal cell tumours. J. Pathol. 177, 247–252. Percy, D.H., Barta, J.R., 1993. Spontaneous and experimental infections in scid and scid/beige mice. Lab. Anim. Sci. 43, 127–132. Pritchard, D.M., Potten, C.S., Korsmeyer, S.J., Roberts, S., Hickman, J.A., 1999. Damage-induced apoptosis in intestinal epithelia from bcl-2-null and bax-null mice: investigations of the mechanistic determinants of epithelial apoptosis in vivo. Oncogene 18, 7287–7293. Sentman, C.L., Shutter, J.R., Hockenbery, D., Kanagawa, O., Korsmeyer, S.J., 1991. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 67, 879–888. Shimizu, S., Narita, M., Tsujimoto, Y., 1999. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399, 483–487. Shinkai, Y., Rathbun, G., Lam, K.P., Oltz, E.M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A.M., et al., 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855–867. Sorenson, C.M., 2004. Bcl-2 family members and disease. Biochim. Biophys. Acta 1644, 169–177. Strasser, A., Harris, A.W., Cory, S., 1991a. bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell 67, 889–899. Strasser, A., Whittingham, S., Vaux, D.L., Bath, M.L., Adams, J.M., Cory, S., Harris, A.W., 1991b. Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and elicits autoimmune disease. Proc. Natl. Acad. Sci. USA 88, 8661–8665. Tsujimoto, Y., Finger, L.R., Yunis, J., Nowell, P.C., Croce, C.M., 1984. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science 226, 1097–1099. Veis, D.J., Sorenson, C.M., Shutter, J.R., Korsmeyer, S.J., 1993. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75, 229–240. Winyard, P.J., Risdon, R.A., Sams, V.R., Dressler, G.R., Woolf, A.S., 1996. The PAX2 transcription factor is expressed in cystic and hyperproliferative dysplastic epithelia in human kidney malformations. J. Clin. Invest. 98, 451–459.