Placental Insufficiency Associated with Loss of Cited1 Causes Renal Medullary Dysplasia

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ʈ Duncan B. Sparrow,*† Scott C. Boyle,‡ Rebecca S. Sams,§ Bogdan Mazuruk,§ Li Zhang, ʈ Gilbert W. Moeckel, Sally L. Dunwoodie,*†¶ and Mark P. de Caestecker‡§

*Developmental Biology Division, Victor Chang Cardiac Research Institute, and †St. Vincent’s Clinical School, Faculty of Medicine, and ¶School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, Australia; and ‡Department of Cell and Developmental Biology, §Department of ʈ Medicine, Division of Nephrology and Hypertension, and Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee

ABSTRACT A number of studies have shown that placental insufficiency affects embryonic patterning of the kidney and leads to a decreased number of functioning nephrons in adulthood; however, there is circumstantial evidence that placental insufficiency may also affect renal medullary growth, which could account for cases of unexplained renal medullary dysplasia and for abnormalities in renal function among infants who had experienced intrauterine growth retardation. We observed that mice with late gestational placental insufficiency associated with genetic loss of Cited1 expression in the placenta had renal medullary dysplasia. This was not caused by lower urinary tract obstruction or by defects in branching of the ureteric bud during early nephrogenesis but was associated with decreased tissue oxygenation and increased apoptosis in the expanding renal medulla. Loss of placental Cited1 was required for Cited1 mutants to develop renal dysplasia, and this was not dependent on alterations in embryonic Cited1 expression. Taken together, these findings suggest that renal medullary dysplasia in Cited1 mutant mice is a direct consequence of decreased tissue oxygenation resulting from placental insufficiency.

J Am Soc Nephrol 20: 777–786, 2009. doi: 10.1681/ASN.2008050547

Placental insufficiency is the most common cause support this hypothesis. For example, guinea pigs of intrauterine growth retardation (IUGR) in the with IUGR have reduced renal medullary surface United States and is associated with increased peri- areas,7 whereas human fetuses with IUGR have in- natal mortality and a variety of common diseases creased renal medullary echogenicity that could re- with onset in adult life.1–3 IUGR results from com- sult from decreased tissue oxygenation.8–10 This pensatory changes in the fetal circulation in re- may be of clinical importance because many pa- sponse to placental insufficiency, with shunting of tients with congenital renal malformations have blood toward the brain, away from other nonessen- unexplained renal medullary dysplasia,11 which tial organs.4 The kidney is particularly sensitive to could result from IUGR-dependent effects on the effects of placental insufficiency during late gestation, when it undergoes rapid growth.5,6 This is Received June 3, 2008. Accepted October 28, 2008. thought to explain the epidemiologic and experimental data linking IUGR with reduced nephron Published online ahead of print. Publication date available at www.jasn.org. numbers in adults3; however, the renal medulla undergoes rapid expansion during late gestation,5 sug- Correspondence: Dr. Mark P. de Caestecker, Nephrology Division, Vanderbilt University School of Medicine, S3223 Medical Center North, gesting that intrauterine growth of this structure 21st Avenue South, Nashville, TN 37232. Phone: 615-343-2844; Fax: may also be susceptible to the effects of placental 615-343-2675; E-mail: [email protected] insufficiency. There is circumstantial evidence to Copyright ᮊ 2009 by the American Society of Nephrology

J Am Soc Nephrol 20: 777–786, 2009 ISSN : 1046-6673/2004-777 777 BASIC RESEARCH www.jasn.org growth and patterning of the medulla. Furthermore, abnormal growth of the renal medulla could account for abnormalities in urine-concentrating capacity in infants with IUGR.12 Despite A D this, little attention has been given to the effects of placental insufficiency on embryonic development of the renal medulla. Cited1 is a non-DNA binding transcriptional co-factor that is expressed in the developing heart, liver, and trophectoderm- derived cells of the placenta13,14 and is restricted to the con- densed metanephric mesenchyme in the embryonic kidney.15 A’ D’ Cited1 null mice have abnormalities in mammary gland mat- uration16 but are born without evidence of IUGR or developmental defects on a mixed or 129/SvJ strain background15; however, Cited1 null mice on a C57Bl/6 background have abnormal organization of the placental labyrinth, promoting late gestational placental insufficiency with IUGR.13 A proportion B E of female Cited1 heterozygous mutants also show evidence of IUGR. This occurs because Cited1 is expressed in trophectoderm-derived cells of the placenta and is located on the X chromosome,17 and paternally inherited X chromosomes are inactivated in the placenta18; therefore, female Cited1 heterozygotes with a paternally inherited wild type X chromosome are Cited1 null in the placenta and heterozygous in the C F embryo, whereas female Cited1 heterozygotes with a maternally inherited wild-type X chromosome are Cited1 heterozygous in the placenta and embryo. Comparative analysis of female Cited1 heterozygotes with paternally versus maternally inherited wild type X chromosomes shows that IUGR results from placental insufficiency and not loss of embryonic Cited1 expression. In this article, we show that IUGR associated with loss of G placental Cited1 promotes abnormal patterning of the renal medulla. Comparative analysis of female Cited1 heterozygotes with paternal versus maternally inherited wild type X chromosomes indicates that this effect is independent of changes in embryonic Cited1 expression. These findings provide the first direct evidence that placental insufficiency promotes renal medullary dysplasia.

RESULTS

Figure 1. Renal medullary dysplasia in E18.5 Cited1C57Bl/6 null Cited1C57Bl/6 Null Mice Have Renal Medullary mice. (A through C) Cited1C57Bl/6 null kidneys have renal medul- Dysplasia C57Bl/6 lary dysplasia associated with disorganized expansion of the me- Cited1 null mice on a C57Bl/6 background (Cited1 null dulla into the renal pelvis (arrows). (D through F) Wild-type kid- mice) have renal medullary dysplasia that is apparent from neys are shown for comparison. Boxes in A and D indicate embryonic day postcoitus 17.5 (E17.5). Renal dysplasia is bi- enlarged insets. (AЈ and DЈ) Insets showing normal cortical thick- lateral and associated with disorganized growth and patterning ness and nephronic patterning in Cited1C57Bl/6 null and wild-type of the renal medulla during late gestation when compared with kidneys. (G) Frequency of renal medullary dysplasia in E18.5 wild type littermate controls (E18.5; Figure 1, A through F). kidneys from five Cited1C57Bl/6 null and eight wild-type male Ten of 10 kidneys from five Cited1C57Bl/6 null mice had vari- littermates (kidney numbers). Magnification, ϫ100. able, disorganized expansion of the renal medulla, whereas only two of 16 kidneys (both from the same mouse) from eight evidence of abnormal ureteric bud (UB) branching in wild-type littermates had mild medullary dysplasia (Figure Cited1C57Bl/6 null mice earlier in development. There was no 1G). Cortical thickness and glomerular numbers, indicators of reduction in UB branching in metanephroi isolated from nephron patterning, were normal for this stage of development E12.5 Cited1C57Bl/6 null mice and grown in culture for 3 d (wild (Figure 1, AЈ and DЈ). To evaluate this further, we looked for type [n ϭ 5], UB tips (mean Ϯ SEM) 63.2 Ϯ 5.6, Cited1C57Bl/6

778 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 777–786, 2009 www.jasn.org BASIC RESEARCH null [n ϭ 4], UB tips 73.5 Ϯ 9.9; t test, P Ͼ 0.05 versus wild- type and four of four Cited1C57Bl/6 null mice evaluated (Figure type; Figure 2, A and B). Furthermore, no reduction in UB tips 2, G and H). Taken together, these studies indicate that was detected by Calbindin D28K staining in the outer nephro- Cited1C57Bl/6 null mice have renal medullary dysplasia that genic zone of E15.5 kidneys from Cited1C57Bl/6 null embryos does not result from defects in UB branching or lower urinary (wild type [n ϭ 6], UB tips 22.1 Ϯ 0.82, Cited1C57Bl/6 null [n ϭ tract obstruction. 3], 24.7 Ϯ 0.82; t test, P Ͼ 0.05 versus wild-type; Figure 2, C and D). These findings indicate that renal medullary dysplasia in Renal Function in Adult Cited1C57Bl/6 Null Mice Cited1C57Bl/6 null mice does not result from a defect in UB To determine whether this embryonic defect affects renal func- branching. We therefore looked for evidence of ureteric ob- tion, we evaluated renal histology, glomerular number, and struction in Cited1C57Bl/6 null mice, because this could account GFR in adult male Cited1C57Bl/6 null mice (age 53.6 Ϯ 3.3 wk) for renal dysplasia later in gestation. There was no pelvicaliceal and wild-type littermates (age 50.3 Ϯ 3.0 wk). There were no dilation or abnormal ureteric muscularization (␣-smooth obvious differences in renal structure (M.P.d.C., data not muscle actin expression) in E16.5 kidneys before renal medul- shown) or GFR between wild-type and Cited1C57Bl/6 null mice lary dysplasia is apparent (Figure 2, E and F). Furthermore, (Figure 3A). There was a minor reduction in glomerular num- analysis of E18.5 kidneys showed no evidence of ureteric ob- bers in Cited1C57Bl/6 null mice, but this difference failed to struction: There was free flow of methylene blue down the reach statistical significance (Figure 3B). There was, however, a ureter after injection into the renal pelvis in four of four wild- urine-concentrating defect in Cited1C57Bl/6 null mice: Whereas baseline urinary osmolality was similar to wild-type mice, there was a reduction in urinary osmolality in response to A B water deprivation or treatment with desmopressin in Cited1C57Bl/6 null mice (Figure 3C). This is consistent with a defect in renal medullary function in Cited1C57Bl/6 null mice.

Renal Medullary Dysplasia in Cited1C57Bl/6 C D Heterozygotes with Placental Insufficiency Late gestational renal medullary dysplasia is difficult to explain on the basis of the expression of Cited1 during nephrogenesis, because Cited1 expression is restricted to the outer nephrogenic zone in the developing kidney.15 An E F alternative explanation is that it results from the placental insufficiency described in Cited1C57Bl/6 null mice.13 Mice characterized in this article include a subset of the total Cited1C57Bl/6 mutant embryos reported by Rodriguez et al.13 Fetal weights in E18.5 Cited1C57Bl/6 null embryos with renal GH medullary dysplasia were lower than those of their wild-type littermates (wild-type [n ϭ 8; mean Ϯ SEM] 1.1850 Ϯ 0.0350 g, Cited1C57Bl/6 null [n ϭ 5] 1.0010 Ϯ 0.0149 g; t test, P Ͻ 0.05 versus wild-type). This indicates that Cited1C57Bl/6 null mice used for our studies have IUGR and suggests that renal medullary dysplasia could result from placental insufficiency. To address this, we compared E18.5 kidneys from Pϩ/Ϫ Figure 2. UB branching and patency in Cited1C57Bl/6 null mice. female Cited1 heterozygotes with a paternally inherited (A and B) Whole-mount preparations of cultured metanephroi wild-type X chromosome (they do not express Cited1 in the ϩ Ϫ isolated from two E12.5 wild-type (A) and two Cited1C57Bl/6 null placenta and have IUGR) with female Cited1M / heterozy- (B) embryos, cultured for 3 d and stained using anti–Calbindin gotes with a maternally inherited wild-type X chromosome D28K antibodies (a UB marker). (C and D) Calbindin D28K stain- (they express Cited1 in the placenta and do not have C57Bl/6 ϩ Ϫ ing of E15.5 kidneys from two wild-type (C) and two Cited1 IUGR13). Fetal weights in Cited1P / mutants were lower null (D) embryos. Dotted blue lines in C illustrate outer nephro- than those of wild-type mice (Cited1Pϩ/Ϫ mutants [n ϭ 4] genic zone areas used to count Calbindin D28K–positive UB tips. 0.9160 Ϯ 0.0364 g; t test, P Ͻ 0.05 versus wild-type mice (E and F) Sections through the renal pelvis of E16.5 kidneys from Pϩ/Ϫ C57Bl/6 ␣ above), indicating that these Cited1 mice have IUGR. wild-type (E) and Cited1 null (F) mice stained for -smooth Mϩ/Ϫ Pϩ/Ϫ muscle actin. (G and H) Representative images illustrating meth- Unlike Cited1 mice, E18.5 Cited1 kidneys have ylene blue dye passage down the ureters of E18.5 wild-type (G) renal medullary dysplasia (Figure 4). Because Cited1 is de- C57Bl/6 and Cited1C57Bl/6 (H) null mice after injection into the renal pelvis. tected equally in E18.5 kidneys from female Cited1 Images obtained using a dissecting microscope. Magnification, heterozygotes with or without placental insufficiency (Sup- ϫ100 in A through F; ϫ10 in G and H. plemental Figure 1), this suggests that placental insuffi-

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A B were indistinguishable from wild-type controls (Fig- ure 5, C and D); therefore, placental insufficiency is associated with increased medullary apoptosis whether the embryos are heterozygous or null for Cited1, indicating that this apoptotic response is un- related to the level of Cited1 expression in Cited1C57Bl/6 mutant embryos

Tissue Hypoxia in Cited1C57Bl/6 Mutants with Placental Insufficiency Apoptosis may be triggered by decreased tissue oxygenation associated with placental insufficiency.19,20 We therefore compared tissue oxygenation in embryonic kidneys of wild-type and Cited1C57Bl/6 mice C with placental insufficiency. Hypoxia (oxygen tensions Ͻ10 mmHg) can be detected by analyzing bioreductive protein adducts of pimonidazole in embryonic tissues after intraperitoneal injection of pregnant dams.21 Using this approach, we were able to detect pimonidazole adducts in renal tubules at the corticomedullary junction, the tip of the renal medulla, and ureteric epithelium by immunohistochemical staining of E17.5 wild-type embryonic kidneys after maternal injection of pimonidazole but not in uninjected controls (Supplemental Figure 2). Comparison of E17.5 kidneys from wild-type and Figure 3. Renal function in adult Cited1C57Bl/6 mutant mice. (A through C) Cited1C57Bl/6 mutants showed increased pimonida- GFR (measured by FITC-inulin clearance in ␮l/min per mouse; A), glomerular zole-adduct formation at the corticomedullary junc- counts (direct counting of glomeruli after acid digestion of the kidneys; B), tion and within the renal medulla of mutants with and urine-concentrating capacity (spot urine osmolality in mOsm/kg; C) were C57Bl/6 placental insufficiency (Figure 6, A through C). determined in adult male wild-type (WT) and Cited1 null mice. Sam- Analysis of pimonidazole adducts in E17.5 kidneys ples were obtained from controls with free access to water, after 23 h of by Western blot revealed multiple bands only detect- water deprivation and after intraperitoneal injection with 1 ng/g desmopressin intraperitoneally. Individual data points with mean vales shown. Kruskal- able after maternal injection of pimonidazole (Fig- Wallis ANOVA, *P Ͻ 0.05 versus WT controls. No significant differences were ure 6D). Intensity of these bands was increased in C57Bl/6 detected between glomerular counts or GFR between genotypes. Cited1 null kidneys (Figure 6E). For semiquantitative analysis, we performed densitometry on ciency and not the loss of renal Cited1 causes renal medul- a defined 80-kD band that is distinct from other bands and lary dysplasia in Cited1C57Bl/6 mutant mice. detected in all of the lysates (Figure 6, D and E, arrows). The 80-kD pimonidazole band intensity was increased ϩ Ϫ Cited1C57Bl/6 Mutants Have Increased Renal Medullary Cited1C57Bl/6 null and Cited1P / mice compared with wild- ϩ Ϫ Apoptosis type and Cited1M / littermates (Figure 6F), indicating that To explore the mechanisms mediating this effect, we evaluated there is decreased oxygenation of the embryonic kidneys from apoptosis in the kidneys of Cited1C57Bl/6 mutant mice. Termi- Cited1C57Bl/6 mutants with placental insufficiency. nal deoxynucleotidyl transferase–mediated digoxigenin-de- oxyuridine nick-end labeling (TUNEL)-positive, mainly non- tubular interstitial cells were detected in the renal medulla of DISCUSSION wild-type mice at E17.5 and increased in Cited1C57Bl/6 null mice at E17.5 and E18.5 (Figure 5, A and B). This was not Mice with placental insufficiency associated with genetic loss associated with alterations in cell proliferation, because prolif- of Cited1 in the placenta have renal medullary dysplasia that is erating (PCNA and phospho-histone H3 positive) cells are vir- first apparent during late gestation. These dysplastic changes tually undetectable in the medulla during this period in wild- are not caused by abnormal UB branching or lower urinary type and Cited1C57Bl/6 null mice (S.C.B., data not shown). tract obstruction but are associated with decreased oxygen- Renal medullary apoptosis was increased in Cited1Pϩ/Ϫ mu- ation and increased apoptosis in the renal medulla, which are tants with placental insufficiency, whereas embryonic kidneys detected only in late gestation (from E17.5). Furthermore, re- from Cited1Mϩ/Ϫ mutants (with normal placental function) nal dysplasia occurs only in Cited1C57Bl/6 mutants with placen-

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that renal dysplasia does not depend on alteration in embryonic Cited1 expression and suggests that placental insufficiency plays a direct role in promoting renal patterning defects in these mice. Renal medullary dysplasia has been described in a number of other mutant mouse lines. Unlike Cited1C57Bl/6 mutant mice, renal medullary dysplasia is often associated with overt defects in UB branching (Igf-1, Fgf-7, and Fgf-10 null mice23–25) or results from defects in postnatal growth and maturation of the renal medulla (angiotensinogen, Ang-1a/1b receptor, Nfat-5, and Aqp-2 null mice26–29). Glipican-3 mutant mice develop late gestational cystic dysplasia of the renal medulla.30 Like Cited1, Glipican-3 is an X-linked gene, and the renal phenotype of Glipican-3 null mice becomes progressively more severe as the mutation is backcrossed onto the C57Bl/6 background. Furthermore, these mice do not have overt UB branching defects, but, unlike Cited1C57Bl/6 mutants, they have a generalized increase in prolifera- Figure 4. Renal patterning defects in female Cited1C57Bl/6 heterozygotes. tion and apoptosis of UB epithelium that can be de- Hematoxylin- and eosin-stained sections through the renal pelvis of E18.5 tected from E12.5 and is thought to account for the 30,31 embryonic kidneys from female Cited1C57Bl/6 mutants. Female Cited1C57Bl/6 cystic dysplastic phenotype in the mice. Mice heterozygotes with Pϩ/Ϫ (with placental insufficiency) but not Mϩ/Ϫ (with- with germ-line deletion of the cyclin-dependent ki- out placental insufficiency) wild-type X chromosomes have abnormal orga- nase inhibitor p57KIP2 also have a defect in renal nization and expansion of the renal medulla into the pelvis (arrows). Wild- medullary expansion and patterning that is first de- type female littermate kidneys are shown from comparison. Magnification, tectable in late gestation.32 There are no data to indi- ϫ100. cate whether this is associated with abnormalities in UB branching or proliferation; however, like Cited1 tal insufficiency and is not dependent on alterations in embry- and Glipican-3, p57KIP2 is a paternally imprinted gene,33 and onic Cited1 expression. Taken together, these studies suggest loss of placental p57KIP2 expression in p57KIP2 null mice and in that renal dysplasia is a direct consequence of decreased tissue female p57KIP2 heterozygotes with a paternally inherited wild- oxygenation resulting from late gestational placental insuffi- type allele is associated with placental insufficiency and late ciency in Cited1C57Bl/6 mutant mice. These findings provide gestational IUGR.32,33 Furthermore, perinatal mortality asso- the first direct evidence that placental insufficiency promotes ciated with IUGR in p57KIP2 mutants is higher on a C57Bl/6 renal medullary dysplasia and suggest a novel genetic model than on a mixed genetic background.32 This suggests that pla- that could be used to explore the fetal mechanisms regulating cental insufficiency could be the common underlying mecha- renal patterning defects associated with placental insufficiency. nism mediating renal medullary dysplasia in both Cited1 and Our studies were performed with Cited1 mutant mice on a p57KIP2 mutants on a C57Bl/6 background; however, p57KIP2 C57Bl/6 strain background and contrast with earlier observa- null mice have placental overgrowth and develop hypertension tions indicating that renal development is normal in Cited1 with a preeclampsia-like syndrome during pregnancy.34 This null mice on a 129/Svj background.15 This is notable because contrasts with Cited1C57Bl/6 mutants in which placental size is Cited1 null mice on the 129/Svj background do not have pla- normal.13 Furthermore, we found no evidence that cental insufficiency or IUGR.13 The explanation for this is un- Cited1C57Bl/6 null mice developed hypertension during preg- clear but presumably results from strain-dependent genetic nancy (Supplemental Figure 3); therefore, although renal pat- modifiers.22 Important, however, is that these findings demon- terning defects may be similar, the mechanism of placental strate an association between renal dysplasia and placental in- insufficiency and its impact on maternal physiology and fetal sufficiency in Cited1C57Bl/6 mutant mice. The time course over growth in the two mouse lines is likely to be different. which this occurs (from E17.5) parallels the time course of Our findings contrast with observations that have linked pla- IUGR in Cited1C57Bl/6 null mice.13 Furthermore, as Cited1 is an cental insufficiency with reduced nephron numbers in adults.3 X-linked, paternally imprinted gene,17 comparative analysis of This is thought to occur as decreased nutrition and/or oxygen- female Cited1C57Bl/6 heterozygotes with paternally versus ma- ation of the developing kidney interferes with the burst of ternally inherited wild-type X chromosomes indicates that re- nephron growth that occurs during the latter part of gestation. nal dysplasia is associated with placental insufficiency whether Our studies show that Cited1C57Bl/6 null mice have a minor de- the embryos are heterozygous or null for Cited1. This indicates crease in glomerular numbers compared with their wild-type lit-

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A B ferences in severity and/or timing of placental insufficiency could have distinct effects on fetal growth and renal patterning. The impact of this patterning defect on the structure and function of the adult kidney in Cited1C57Bl/6 null mice was less pronounced than we expected. There was no significant alteration in renal structure, glomerular numbers, or GFR, and although urine- concentrating capacity was diminished, Cited1 mutants maintained urinary osmolalities that are suffi- cient to sustain life. This suggests that decreased urine-concentrating capacity results from subtle abnormalities in organization of the renal tubules/interstitium and vasa recta that affect medullary function without disrupting overall structure. This C D discrepancy between medullary defects in fetal and adult kidneys may result from selection bias if mice that die postnatally have more severe renal dysplasia than the survivors. This is consistent with the variable penetrance of renal dysplasia seen in Cited1C57Bl/6 null embryos; however, we were unable to test this hypothesis because the majority of Cited1C57Bl/6 mutants with placental insufficiency that die cannot be analyzed because death occurs within a few hours of birth.13 An alternative explanation for our findings is that much of the structural defect that is apparent in utero is lost postnatally. This would occur if postnatal Figure 5. Apoptosis in renal medullas of Cited1C57Bl/6 mutant mice with expansion of the medulla progresses normally in placental insufficiency. (A) Representative images showing TUNEL staining Cited1C57Bl/6 mutants despite an initial delay in pat- for apoptotic nuclei in E17.5 embryonic kidneys from wild type (ϩ/ϩ) and terning of the medulla in utero. On this basis, persis- Cited1 null (Ϫ/Ϫ) embryonic kidneys. (B) Graph indicates mean Ϯ SEM tent abnormalities in organization of the renal me- apoptotic indices in embryonic kidneys isolated at E16.5, E17.5, and E18.5 dulla might result from a developmental delay in from wild type and Cited1 null embryos (mouse numbers). Kruskal-Wallis renal medullary growth, whereas gross defects in Ͻ ANOVA, *P 0.05 versus wild-type controls. (C) Representative images medullary patterning are lost by the time the surviv- showing TUNEL staining of E17.5 embryonic kidneys from wild-type, ing mice reach adulthood. Cited1C57Bl/6 null, and Cited1C57Bl/6 heterozygotes with Mϩ/Ϫ (no placental insufficiency) and Pϩ/Ϫ (with placental insufficiency) wild-type X chromo- Finally, our studies show that there is increased somes. (D) Graph illustrates mean Ϯ SEM apoptotic indices in the renal apoptosis and decreased oxygenation of the renal C57Bl/6 medullas of E17.5 kidneys from the same genotypes (mouse numbers). medulla in Cited1 mutants with placental in- ANOVA, *P Ͻ 0.05 versus WT controls. Magnification, ϫ400. sufficiency. A number of other models of IUGR showed increased apoptosis in growth restricted organs.19,20,36–39 This is thought to result from nutri- termates, but this difference does not reach the level of statistical tional and/or oxygen deficiency promoting stress-induced significance. We used a direct maceration/counting method to proapoptotic responses. Previous studies also showed in- evaluate glomerular numbers. This is an established technique creased apoptosis in the renal medullary interstitium of human that produced reproducible results in our hands15 but lacked sen- embryonic kidneys with medullary dysplasia40,41; therefore, sitivity to detect minor differences in glomerular numbers35; our studies suggest that placental insufficiency causes renal therefore, it is possible we may have failed to detect a minor, medullary dysplasia by promoting hypoxia-induced apoptosis although significant, reduction in nephron numbers in at a critical stage of renal medullary development. Cited1C57Bl/6 null mice. Nevertheless, unlike other models of placental insufficiency, the dominant abnormality seen in Cited1C57Bl/6 mutant mice is renal medullary dysplasia. The expla- CONCISE METHODS nation for this dominant effect on renal medullary patterning is unclear but is likely to reflect differences in severity and/or timing Mouse Line and Breeding Strategies of placental insufficiency compared with other models. This We maintain a colony of Cited1 mutants (Cited1tm1Dunw)13 on a pure could be relevant to human disease, because it suggests that dif- C57Bl/6 background (after backcrossing seven generations). These

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are referred to as Cited1C57Bl/6 mice in the text. Because AB +/+ -/- both Cited1C57Bl/6 null and Pϩ/Ϫ female heterozygotes have reduced viability,13 we maintain this colony by breeding wild-type C57Bl/6 females with Cited1C57Bl/6 null males. Comparison between Cited1C57Bl/6 null and wild- type mice (both adult and embryonic studies) was performed on offspring derived from crossing Cited1C57Bl/6 heterozygous females with wild-type males. Comparison between female Cited1C57Bl/6 heterozygotes with paternal (Cited1Pϩ/Ϫ with placental insufficiency) and maternal Pimonidazole Mϩ/Ϫ C P+/- D - ++ (Cited1 with normal placental function) inherited wild-type X chromosomes was performed on offspring kDa Mϩ/Ϫ 93 from female Cited1 heterozygotes to limit potentially confounding effects of maternal genotype on embryonic growth. These were mated either with wild-type males (ex- 52 pected genotypes 25% female Cited1Pϩ/Ϫ, 25% wild type female, 25% wild-type male, and 25% null male) or 37 Cited1C57Bl/6 null males (expected genotypes 25% female ϩ Ϫ 28 Cited1M / without placental insufficiency, 25% null fe- 123 male, 25% null male, and 25% wild-type male). The main E Cited1 F Wild type null analysis involved side-by-side comparison between female Pϩ/Ϫ Mϩ/Ϫ -actin (3)* 3.5 (5) 170 β * Cited1 and Cited1 heterozygotes, but other ex- 130 Anti-HP 3.0 pected genotypes provided internal controls for each litter.

95 2.5 Timed pregnancies were performed with the morning of 72 2.0 the vaginal plug counted as 0.5 d post coitus (referred to as 55 1.5 E0.5). Embryonic heads and adult tails were used for geno- (13) (4) 43 1.0 typing using primers to detect Cited1 mutant alleles and 34 0.5 the Y and X chromosome–specific forms of smooth mus- 0 cle cells for sex determination in embryos.13,42 43 β-actin WT M+/- P+/- Null 80kDa pimonidazole adduct band/ 162345 Cited1 genotypes Preparation of Renal Tissue and C57Bl/6 Figure 6. Renal hypoxia in Cited1 mutant mice with placental Quantification of UB Tips and Glomerular insufficiency. (A through C) Representative immunohistochemical staining Numbers for pimonidazole adducts in transverse sections through E17.5 kidneys ϩ Ϫ Kidneys isolated from E15.5 to E18.5 embryos were fixed from Cited1C57Bl/6 null mice (Ϫ/Ϫ) and Cited1P / female heterozygotes for1to2hin10%formalin at 4°C before processing and with placental insufficiency (Pϩ/Ϫ), compared with wild-type female ␮ (ϩ/ϩ) mice derived from the same litter. Tissue preparation and immu- mounting in paraffin. Serial sagittal sections (5 M) were nohistochemical staining were performed simultaneously under identical cut on Superfrost Plus slides (Fisher, PA) and evaluated conditions on all three sections. Arrows indicate staining for pimonidazole under an inverted microscope: Only sections cut through adducts in inner and outer medullas. (D) Immunoblot for pimonidazole the medulla (assessed from appearance of the renal pelvis adducts in E17.5 kidney lysates obtained from wild-type embryos with or and/or evidence of longitudinal sectioning through prox- without maternal pimonidazole 2 h before killing, as indicated. Lanes 1 imal collecting ducts) were used for subsequent analysis. and 3, 10 mg of protein; lane 2, 5 mg of protein. (E) Representative Sections were stained with hematoxylin and eosin, and im- Western blot illustrating changes in density of pimonidazole adduct pro- munohistochemical and TUNEL assays were performed as tein bands in E17.5 kidney lysates obtained from wild-type and outlined in TUNEL Staining and Assessment of Apoptotic Cited1C57Bl/6 null embryos derived from the same litter. Arrows indicate Indices. Staining for pimonidazole protein adducts was the distinctive approximately 80-kD band that is used for quantitative densitometry. ␤-Actin control shown. (F) Semiquantitative analysis of performed on transverse sections through the renal me- E17.5 kidney pimonidazole adduct immunoblots from wild type (WT), dullas of E17.5 embryos. For quantification of UB tips in Cited1C57Bl/6 null, and Cited1C57Bl/6 heterozygotes with Mϩ/Ϫ and Pϩ/Ϫ E15.5 kidneys, Calbindin D28K–positive UB tips were wild-type X chromosomes. Densitometry of conserved 80-kD pimonida- counted in the outer nephrogenic zone by a blinded ob- zole adduct band corrected for ␤-actin loading. Each litter (n ϭ 5) was server (M.P.d.C.). Sections (5 ␮M) were evaluated every evaluated on individual blots, and results were normalized to WT controls 10th section around the future medulla (as determined by Ϯ to correct for between-litter variations. Data are means SEM (mouse the presence of proximal ureteric stalks), and results are Ͻ Mϩ/Ϫ numbers). Kruskal-Wallis ANOVA, *P 0.05 versus WT and Cited1 expressed as the mean of at least three values (three to six ϫ controls. Magnification, 200. sections counted per kidney). Glomeruli were counted in adult kidneys using a direct maceration/counting method, as

J Am Soc Nephrol 20: 777–786, 2009 Renal Dysplasia and the Placenta 783 BASIC RESEARCH www.jasn.org described previously.15 For this, kidneys were minced into 2-mm cubes, mouse (MOM) blocking reagent (Vector Laboratories, Burlingame, CA) and the fragments were incubated in 5 ml of 6 M HCl at 37°C for 90 min. or 10% goat serum in PBS for rabbit primary antibodies. In addition, Tissue was homogenized through repeated pipetting, and 25 ml of water 0.5% hydrogen peroxide was added to inhibit endogenous peroxidases. was added. After overnight incubation at 4°C, glomeruli in 5 ϫ 1mlof Primary antibodies were incubated in the respective blocking reagents this solution were counted in a 35-mm counting dish (cat. no. 174926; and detected using species specific biotinylated antibodies and the ABC Nalgen, RI). Total glomerular number per kidney was extrapolated system (Vector Laboratories) or HRP-conjugated secondary antibodies. mathematically from the mean of these five counts. For metanephric ␣-Smooth muscle actin was detected using mouse anti–␣-smooth mus- organ cultures, E12.5 kidneys were isolated and grown for3donTrans- cle actin (Dako M0851; Dako, Carpinteria, CA) incubated at 1:50 in well filters, as described previously.15 Filters were fixed in cold methanol MOM diluent for1hatroom temperature. Pimonidazole adducts were for 10 min before immunostaining, as outlined in Immunohistochemis- detected using FITC-conjugated Hypoxyprobe-1 mouse mAb (Chemi- try. Individual Calbindin D28K UB tips were counted directly from pho- con 90531) in MOM diluent for 30 min at room temperature and de- tomicrographs by a blinded observer (M.P.d.C.). tected using HRP-conjugated mouse anti-FITC mAb (Chemicon 90532). Calbindin D28K was detected using rabbit ant[an]Calbindin Assessment of Ureter Patency D28K (Calbiochem PC-2532; Calbiochem, San Diego, CA) at 1:100 over- E18.5 kidneys, ureters, and bladder were isolated en block, and the night at 4°C. For immunofluorescence analysis of Calbindin D28K ex- renal pelvises were punctured with a 30-G needle and injected man- pression in cultured metanephroi, methanol fixed explants were incu- ually with 10 mg/ml methylene blue at approximately 100 ␮l/min, as bated with the same primary antibody overnight in Tris-buffered saline described previously.43 Images of whole-mount preparations illus- (25 mM Tris base with 100 mM NaCl) with 0.1% Tween 20 (TBST) with trate dye passage along the ureter. 10% goat serum, washed three times for1hinTBST the following day, and incubated with Cy2-conjugated donkey anti-rabbit secondary anti- Assessment of Tissue Oxygenation body (Jackson Immunological Research 711225152; JAX, West Grove, Renal tissue oxygenation (oxygen tensions Ͻ10 mmHg) was evalu- PA) at 1:50 in TBST without serum for an additional hour before washing ated by detecting bioreductive protein adducts of pimonidazole hy- and mounting with Vectashield (Vector Laboratories). drochloride (Hypoxyprobe; Millipore, Billerica, MA) in embryonic tissues after intraperitoneal injection of pregnant dams, as described TUNEL Staining and Assessment of Apoptotic Indices previously.21 For this, E17.5 pregnant dams were injected with 60 We used an FITC-labeling in situ TUNEL detection kit (Roche mg/kg pimonidazole 2 h before dissection. Mothers were killed by 11684795910 Roche Laboratories, Ontario, CA) to detect apoptotic cells cervical dislocation after isofluorane, the uterus was isolated, and em- in embryonic renal medullas. Briefly, paraffin-embedded tissue sections bryos were dissected while maintained in ice-cold PBS to minimize were deparaffinized and treated with 20 ␮g/ml Proteinase K for 15 min at postmortem formation of new pimonidazole adducts. For prevention room temperature, and apoptosis-associated 3Ј hydroxyl-DNA terminal of variability in postmortem effects between littermates, embryonic overhangs were labeled by terminal deoxynucleotidyl transferase–de- kidneys isolated from each litter were kept in ice-cold PBS until all of pendent incorporation of FITC-conjugated nucleotides. Slides were the embryos had been dissected and then were simultaneously snap- mounted in medium containing 4Ј-6Ј diamino-2-phenyllindole (DAPI) frozen for Western blot analysis or fixed in ice-cold 10% formalin for to label nuclei (Vector Laboratories). Apoptotic indices were evaluated by 2 h for immunohistochemical analysis, as outlined in Immunohisto- epifluorescence microscopy. For this, a blinded observer (S.C.B.) deter- chemistry. For Western blot analysis, snap-frozen kidneys were lysed mined the total number of FITC-positive nuclei out of approximately in RIPA buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 800 DAPI-positive nuclei in the medulla and inner cortex of sections cut 1% NP40, 1% sodium deoxycholate, and 0.1% SDS) and 10 ␮gof through the renal pelvis. Results were expressed as the percentage of lysate protein separated by SDS-PAGE, transferred to polyvinylidene FITC/TUNEL positive to total DAPI staining nuclei. difluoride membranes and probed with FITC-conjugated mouse anti–Hypoxyprobe-1 followed by a secondary horseradish peroxidase Renal Function Tests (HRP)-conjugated mouse anti-FITC (both antibodies from Chemi- GFR was determined by plasma clearance kinetics of FITC after a con). After development of the films, membranes were stripped and single bolus intravenous injection of FITC-inulin (Sigma), as de- reprobed with mouse anti–␤-actin (Sigma, St. Louis, MO) as a control scribed previously.44 Urine-concentrating capacity was determined for protein loading. Quantification of pimonidazole adducts was per- by collecting spot urine samples for osmolality before (10:00 am), formed by densitometry of the constant 80-kD protein band, ex- after 23 h of water deprivation, or 4 h after water deprivation and pressed as the ratio of band density to the ␤-actin control. Results intraperitoneal injection of 1 ng/g desmopressin (Sigma). Urine sam- were normalized to the wild-type littermate controls for each of the ples were prepared and osmolalities were measured using the Ad- litters to allow for between-litter variations. vanced Micro Osmometer Model 3300 (Advanced Instruments, Nor- wood, MA) as described previously.45 Immunohistochemistry Tissue sections were deparaffinized, and antigen retrieval was performed Statistical Analysis by heating slides in sodium citrate buffer (Biogenex, San Roman, CA). Statistical analysis was performed by t test for paired group compar- Sections were blocked with avidin/biotin blocking reagents (Vectastain isons or one-way ANOVA for comparison between multiple groups kit) followed by block for nonspecific antibody binding using mouse-on- using Bonferroni correction or post hoc, pair-wise, between-group

784 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 777–786, 2009 www.jasn.org BASIC RESEARCH comparisons for sample sizes of five or more per group. Smaller sam- 13. Rodriguez TA, Sparrow DB, Scott AN, Withington SL, Preis JI, Michal- ple sizes (four or fewer per group) were compared using the nonpara- icek J, Clements M, Tsang TE, Shioda T, Beddington RS, Dunwoodie metric Kruskal-Wallis ANOVA test with Siegel (Bonferroni) correc- SL: Cited1 is required in trophoblasts for placental development and for embryo growth and survival. Mol Cell Biol 24: 228–244, 2004 tion for post hoc pair-wise contrasts. The minimal level of significance 14. Dunwoodie SL, Rodriguez TA, Beddington RS: Msg1 and Mrg1, was set at P Ͻ 0.05, and statistical analyses were performed using founding members of a gene family, show distinct patterns of gene Analyze-It 1.73 for Microsoft Excel. expression during mouse embryogenesis. Mech Dev 72: 27–40, 1998 15. Boyle S, Shioda T, Perantoni AO, de Caestecker M: Cited1 and Cited2 are differentially expressed in the developing kidney but are not required for nephrogenesis. Dev Dyn 236: 2321–2330, 2007 ACKNOWLEDGMENTS 16. Howlin J, McBryan J, Napoletano S, Lambe T, McArdle E, Shioda T, Martin F: CITED1 homozygous null mice display aberrant pubertal This work was supported by National Institutes of Health grants R01 mammary ductal morphogenesis. Oncogene 25: 1532–1542, 2006 17. Fenner MH, Parrish JE, Boyd Y, Reed V, MacDonald M, Nelson DL, DK61558 and P50 DK39261 (M.P.d.C.) and T32 HD007502 (S.C.B.), Isselbacher KJ, Shioda T: MSG1 (melanocyte-specific gene 1): Map- a Westfield-Belconnen Fellowship (D.B.S.), a Pfizer Foundation Aus- ping to chromosome Xq13.1, genomic organization, and promoter tralia Senior Research Fellowship (S.L.D.), and a NHMRC Senior Re- analysis. Genomics 51: 401–407, 1998 search Fellowship (S.L.D.). 18. Papaioannou VE, West JD: Relationship between the parental origin We thank Robert Lane for helpful advice and comments, Zhong- of the X chromosomes, embryonic cell lineage and X chromosome expression in mice. Genet Res 37: 183–197, 1981 hua Qi from the Mouse Metabolic Phenotyping Core at Vanderbilt 19. Lane RH, Ramirez RJ, Tsirka AE, Kloesz JL, McLaughlin MK, Gruetz- for performing the FITC-inulin clearance studies, and Annabelle macher EM, Devaskar SU: Uteroplacental insufficiency lowers the Scott for technical assistance. threshold towards hypoxia-induced cerebral apoptosis in growth-retarded fetal rats. Brain Res 895: 186–193, 2001 20. Burke C, Sinclair K, Cowin G, Rose S, Pat B, Gobe G, Colditz P: Intrauterine growth restriction due to uteroplacental vascular insuffi- DISCLOSURES ciency leads to increased hypoxia-induced cerebral apoptosis in new- None. born piglets. Brain Res 1098: 19–25, 2006 21. 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