BASIC RESEARCH www.jasn.org Placental Insufficiency Associated with Loss of Cited1 Causes Renal Medullary Dysplasia 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 ges- tation, when it undergoes rapid growth.5,6 This is Received June 3, 2008. Accepted October 28, 2008. thought to explain the epidemiologic and experi- mental 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 un- dergoes 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 develop- mental defects on a mixed or 129/SvJ strain background15; however, Cited1 null mice on a C57Bl/6 background have ab- normal 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 trophecto- derm-derived cells of the placenta and is located on the X chro- mosome,17 and paternally inherited X chromosomes are inactivated in the placenta18; therefore, female Cited1 het- erozygotes with a paternally inherited wild type X chromo- some 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 het- erozygotes with paternally versus maternally inherited wild type X chromosomes shows that IUGR results from placental insuffi- ciency 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 chromo- somes 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).