J Am Soc Nephrol 13: 668–676, 2002 Midkine Is Involved in Kidney Development and in Its Regulation by Retinoids

JOSE´ VILAR,* CLAUDE LALOU,* JEAN-PAUL DUONG VAN HUYEN,† STE´ PHANIE CHARRIN,* SYLVIE HARDOUIN,* DANIEL RAULAIS,‡ CLAUDIE MERLET-BE´ NICHOU,* and MARTINE LELIE` VRE-PE´ GORIER* *Unite´de Recherches, INSERM U356, IFR 58, Universite´Paris 6; †Unite´de Recherches, INSERM U430, IFR 58, Hoˆpital Broussais; and ‡Unite´de Recherches, INSERM U440, Institut du Fer a`Moulin, Paris, France.

Abstract. In the kidney, in which development depends on the renal cortex. In utero exposure to vitamin A deficiency did epithelial-mesenchymal interactions, it has been shown that not modify the specific spatial and temporal expression pattern retinoids modulate nephrogenesis in a dose-dependent manner of MK in the metanephros, although a decrease in mRNA in vivo and in vitro. Midkine (MK) is a responsive expression occurred. In metanephroi explanted from 14-d-old gene for a -binding . The aim of the fetuses and cultured in a defined medium, expression of MK present study was therefore to quantify the expression of MK mRNA was found to be stimulated when retinoic acid (100 mRNA during renal development in the rat, to analyze the nM) was added in the culture medium. Finally, in vitro nephro- regulation of MK expression by retinoids in vivo and in vitro, genesis was strongly inhibited in the presence of neutralizing and, finally, to study the role of MK in rat metanephric organ antibodies for MK: the number of nephrons formed in vitro cultures. The spatiotemporal expression of MK in fetal kidney was reduced by ϳ50% without changes in ureteric bud branch- was studied. In control rats, MK expression is ubiquitous at ing morphogenesis. These results indicated that MK is impli- gestational day 14, i.e., at the onset of nephrogenesis. On day cated in the regulation of kidney development by retinoids. 16, MK is expressed in the condensed mesenchyme and in These results also suggested that MK plays an important role in early epithelialized mesenchymal derivatives. On gestational the molecular cascade of the epithelial conversion of the met- day 21, MK is rather localized in the nonmature glomeruli of anephric blastema.

Retinoids play a critical role in fetal organogenesis by regu- gene, midkine (MK), a low-molecular-weight heparin-binding lating the expression of responsible for pattern formation growth/differentiation factor (13 kD) encoded by a retinoic and morphogenesis (1). Like other embryonic processes, renal acid–responsive gene (14,15). MK expression increases at organogenesis, which is the result of interactions between the early stages of retinoic acid induced differentiation of embry- metanephric mesenchyme and the ureteric bud, depends on onal carcinoma cells (16–18). In adult mice, MK mRNA is retinoids (2–4). In utero depletion of vitamin A or inactivation significantly expressed only in the kidney (19), but in adult rat, of retinoic acid receptors generates renal malformations (5–8). MK is more widely distributed (20). No data exist on the Using metanephric organ culture and a rat model of mild spatiotemporal pattern of MK expression in the rat embryos. In vitamin A deficiency, we clearly established that vitamin A, the early stages of mouse embryogenesis, MK is ubiquitously via its active metabolite, retinoic acid, strictly controls the expressed. In the midgestation period, MK expression becomes number of nephrons (9,10). We then began to investigate the restricted to tissues undergoing epithelial-mesenchymal inter- mechanisms of such a control, finding that the expression of actions, which suggests a key role in the regulation of organ the proto-oncogene c-ret was modulated in vivo and in vitro development and cell differentiation (19,21,22). The involve- according to the retinoid environment (10,11). The proto- ment of MK in epithelial-mesenchymal interactions, which oncogene c-ret, a receptor present at the tips of characterize tooth morphogenesis, has been demonstrated (23). the ureteric bud branches, is required for ureteric bud out- In vitro studies have shown that MK also promotes neurite growth and branching (12). Genes like Wnt11 and WT1 are also outgrowth and survival of various embryonic nerve cells controlled by retinoic acid in the metanephros (13). In this (15,24,25) and is mitogenic to certain fibroblasts, PC12 cells, study, we were interested in another developmentally regulated and neuroectoderm cells (18,25). In this study, we investigated the regulation of MK expres- sion in vitamin A–deficient (VAD) fetal rat kidneys. We Received June 18, 2001. Accepted October 1, 2001. systematically analyzed the distribution and the expression Correspondence to Dr. M. Lelie`vre-Pe´gorier, INSERM U319/U356, Universite´ pattern of MK in kidneys of fetuses issued from control and Paris 7, 2, place Jussieu, 75251 Paris Cedex 05, France. Phone: 1-43-25-04-70; Fax: 1-43-25-67-89; E-mail: [email protected] VAD mothers, using Northern blot and in situ hybridization 1046-6673/1303-0668 techniques. We also studied the role of retinoic acid as signal Journal of the American Society of Nephrology mediating MK expression in rat metanephric organ culture. Copyright © 2002 by the American Society of Nephrology Finally, we addressed the question of MK implication in J Am Soc Nephrol 13: 668–676, 2002 Midkine Involvement in Nephrogenesis 669 nephrogenesis by analyzing the effects of neutralizing antibod- ence of 12% polyethylene glycol. The precipitate was then ies against MK on morphogenesis and cell differenti- dissolved in 1 vol of phosphate-buffered saline and dialysed ation in organ culture of embryonic kidney. extensively. Excess polyethylene glycol was removed through a Sephadex G-75 filtration column (Amersham). The protein Materials and Methods concentration of the antibody solution was determined by Animals absorbance at 280 nm that used IgG as a standard. Nonimmune Female Sprague Dawley rats weighing 200 to 300 g were given IgY was obtained by the same method from the yolk of a free access to water and standard laboratory pellets (UAR Laboratory, nonimmunized chicken’s egg. The specificity of antibodies Villemoison sur Orge, France). They were caged overnight with a was checked by Western blot, first by use of recombinant male, and vaginal smears were taken the following morning. The day human (PTN), recombinant rat nerve growth fac- a positive smear was obtained was designated day 0 of gestation. tor, recombinant human MK, and a and b recombinant human Two groups of pregnant females were used, control and VAD and second by use of different fetal females. Pregnant females were made vitamin A deficient by admin- tissue from fetuses on gestational day 15. istration of an isocaloric and isoproteic diet deprived of vitamin A 6 Western blot analysis was performed as described elsewhere (26). wk before mating, as described elsewhere (10). Blood samples were Protein were separated by electrophoresis in a sodium dodecyl sul- taken from the cut tip of the tail, and the plasma retinol was deter- fate–polyacrylamide gel (15%) to test antibody specificity regarding mined by HPLC (Beckman Instruments, Inc., Fullerton, CA). The other growth factors and in a sodium dodecyl sulfate–polyacrylamide mean maternal plasma vitamin A concentration of the deficient group gel gradient (6% to 30%) to detect MK protein in fetal tissue or ϳ Ϯ ␮ Ϯ was 50% lower (16.8 0.5 g/dl) than in the controls (31.3 0.7 cultured metanephros. were transferred to nitrocellulose ␮ ϭ g/dl, n 65 for both groups). membranes (Hybond-C extra, Amersham), and the membranes were Fetuses were removed from anesthetized pregnant females on days blocked by incubation for 1 h with 3% dried skim milk in Tris- 14, 15, 16, 18, and 20 of gestation. Newborns and 20-d-old pups were buffered saline with Tween (50 mmol/L Tris-HCl, 150 mmol/L NaCl, also used. Kidneys were surgically removed from embryos, newborns, 0.05% Tween 20 [pH 8]). MK protein was detected by incubating Ϫ and pups, immediately frozen in liquid nitrogen, and stored at 80°C. with antibody 1/5000 diluted in 3% dried skim milk in Tris-buffered RNA were isolated from kidneys of fetuses from one to three litters saline with Tween for1hatroom temperature. A peroxidase-coupled taken at 14, 15, and 16 d of gestation, from two fetuses at 18 and 20 d anti-IgY (Jackson Immunoresearch laboratories, West Grove, PA) of gestation, and from one 20-d-old pup. was used as the secondary antibody. Antigen-antibody complexes were detected by enhanced chemiluminescence as recommended by Isolation of RNA and Northern Blot Analysis the manufacturer (Amersham). Each membrane was labeled with a Total RNA was isolated from kidneys by one-step liquid-phase monoclonal anti–␤-actin antibody (Sigma) to normalize the amount of separation by use of the Trireagent procedure (Life Technologies protein loaded, as described elsewhere (26). Bands were quantified by BRL, Grand Island, NY). RNA was subjected to agarose gel electro- densitometry that used image analysis software (NIH image). phoresis, and Northern blot analysis was performed as described elsewhere (26) by use of a mouse MK cDNA probe kindly provided Metanephric Organ Culture by Prof. M. Vigny (INSERM U440, Paris, France). Signal intensity Metanephroi were cultured on a defined serum-free medium as was quantified by densitometric analysis of autoradiograms (image described elsewhere (9). A stock solution of retinoic acid (10 mM, analysis software, NIH Image) by use of hybridization with an 18S Sigma) was prepared in ethanol and stored in the dark at Ϫ20°C, and ribosomal RNA probe, to allow correction for variations in RNA dilutions were made daily in culture medium. The antibody used in loading. culture is a chicken anti-mouse MK polyclonal antibody 1/100 diluted in culture medium. In Situ Hybridization One metanephros from each fetus was grown in control medium, In situ hybridization was performed in fetuses on days 14 and 16 and the opposite metanephros was grown in the same medium with and in the kidneys of fetuses on day 21 of gestation, as described either retinoic acid (100 nM) or MK antibody (100 ␮g/ml). IgY elsewhere (26), by use of 4% paraformaldehyde-fixed, paraffin-em- nonimmune purified fraction was added in control medium for exper- bedded tissues and the 35S-labeled MK probe. No labeling was iments with neutralizing antibodies to MK. All media were changed detected in tissue sections that were first treated with ribonuclease A daily. and then hybridized with probe. Glomerular and Tubular Structure Assessment and Western Blot Analysis Protein Content in Metanephric Organ Culture Recombinant human MK was obtained from medium of Nephrons formed in vitro were counted after labeling the glomer- transfected Q2bn cells and purified by affinity column on ular structures in the explanted metanephroi by use of specific lectin- heparin-sepharose, followed by an inverse-phase chromatogra- binding sites located on podocyte membranes (27). Briefly, the ex- phy on C4. Antibodies against MK protein were generated in a planted metanephroi were fixed in 2% paraformaldehyde phosphate- buffered saline, detached from the filter, permeabilized with saponin, chicken. Immunization was performed by two injections of 200 and labeled with rhodamine-coupled peanut agglutinin, which stains ng of MK, in the presence of complete, then incomplete, glomeruli. The use of another lectin, Dolichos biflorus agglutinin, Freund adjuvant. Yolks were treated by 2 vol of phosphate- fluorescein coupled, allowed us to analyze the ureteric bud arboriza- buffered saline and 3% of polyethylene glycol (molecular tion and to count the end buds. The growth of the explanted meta- weight 6000, Merck) to precipitate phospholipids. The super- nephroi was then determined by measurement of their protein content. natant, filtered through cotton wool, was precipitated in pres- The labeled metanephroi were placed in individual tubes that con- 670 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 668–676, 2002 tained 0.5 ml distilled water, rinsed, and sonicated for 15 s. The protein content was measured according to the procedure of Lowry et al. (28) as modified by Larson et al. (29), with the use of bovine serum albumin as a standard.

Statistical Analyses All values are expressed as means Ϯ SEM. Differences between developmental stages were calculated by ANOVA. Control and ex- perimental groups, at the same stage, were compared by Student’s unpaired t test. Student’s paired t test was used to compare in vitro data. The threshold of significance was taken to be P Ͻ 0.05.

Results Specificity of the Purified Anti-MK Antibody The purified chicken anti-MK antibody reacted strongly with MK, and a unique band of 15 kD was detected on Western blots (Figure 1A, lane 4); however, it did not react with recombinant human PTN (lane 1), even though MK and PTN have ϳ50% sequence identity (30,31). Anti-MK also did not react with (lane 2) and a fibroblast growth factor (lane 4) or b fibroblast growth factor (lane 5), which are other typical heparin-binding growth factors. We conclude that the anti-MK antibody has high specificity. Figure 1B reported MK expression in a broad range of molecular-weight proteins from different fetal tissue homogenates from fetuses on gesta- tional day 15. An unique band of 15 kD was also detected.

Vitamin A Controls MK Expression in Fetal Kidney MK expression was ubiquitous in 14-d-old control fetuses. In kidney, it was equally displayed between the mesenchymal and epithelial tissue (Figure 2A). From day 16 onward, MK expression becomes restricted to brain (neopallial cortex), stomach, intestine, lung, and kidney. In the latter, on day 16 of

Figure 2. Expression of MK mRNA in the kidney during embryonic Figure 1. Specificity of the purified anti–midkine (MK) antibody by development as detected by in situ hybridization. Sections of kidneys Western blotting. (A) Cross-reaction with other growth factors in 15% from (A) 14-, (B) 16-, and (C) 21-d-old fetuses were hybridized with sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis an 35S-labeled MK probe and counterstained with hematoxylin-eosin. (PAGE). Lane 1, Recombinant human pleiotrophin (PTN); lane 2, (A) Section of metanephros showing a labeling of both ureteric bud recombinant rat nerve growth factor (NGF); lane 3, recombinant (4) and mesenchyme (‹). (B) Section of kidney showing a strong human MK; lane 4, recombinant human a fibroblast growth factor MK expression in the nephrogenic zone, in the mesenchyme (€) and (FGF); and lane 5, recombinant human bFGF. Of each protein factor, in the nonmature glomeruli (‹). In contrast, low staining was ob- 0.5 ␮g was loaded. (B) MK detection in fetal tissues on gestational served in the mature tubule in the profound zone of the kidney (4). day 15 in a 6% to 30% SDS-PAGE gradient. Lane 1, kidney; lane 2, (C) Section of kidney disclosing a decreased expression of MK in the liver; lane 3, lung; lane 4, testis; and lane 5, spinal cord. Of each tissue mesenchyme (€). The expression was restricted to the nonmature homogenate, 30 ␮g of protein was loaded. The positions of protein glomeruli (4). In the mature glomeruli, the MK expression was weak markers are shown on the left (as kD). (4). Magnification, ϫ400. J Am Soc Nephrol 13: 668–676, 2002 Midkine Involvement in Nephrogenesis 671

Figure 3. Patterns of MK mRNA in the kidney of fetuses (14–20dof gestation) and of postnatal rats (age 1 and 20 d) from control (᭛) and vitamin A–deficient (VAD; }) rats. Values are means Ϯ SE of five to six experiments. Results are expressed as percentage of the gesta- tional day 14 control values after normalization for RNA loading on Figure 4. Expression of MK mRNA in the kidney during embryonic the basis of hybridization of 18S rRNA. *P Ͻ 0.05 and **P Ͻ 0.001 development as detected by in situ hybridization. Sections of kidneys compared with aged-matched controls. from (A through D) 16- and (E and F) 21-d-old fetuses issued from (A, C, and E) control and (B, D, and F) VAD mothers were hybridized with an 35S-labeled MK probe and counterstained with hematoxylin- eosin. (A and B) MK staining in the nephrogenic zone was lower in gestation, MK was mainly found in the metanephrogenic zone, VAD than in control fetuses. (C and D) S-shaped body disclosing a i.e., in the condensed mesenchyme, in the ureteric bud staining that appears to be lower in VAD than in control fetuses. (E branches, and in nephron anlagen. The interstitium was not and F) Weak labeling was observed in the mature glomeruli of both ϫ labeled, and the expression in the collecting ducts in the control and VAD kidneys. Magnification, 1000. profound zone of the kidney was very low (Figure 2B). On day 21, MK was still strongly expressed in the immature glomeruli Retinoic Acid Stimulates MK Expression in but was decreased in the mesenchyme. Its expression was also Metanephric Organ Culture weak in the mature glomeruli (Figure 2C). MK mRNA was We quantified the changes in MK expression induced by detected as a unique 0.9-kb band by Northern blot hybridiza- retinoic acid in the metanephros explanted from 14-d-old fe- tion analysis. Densitometric analysis was made taking control tuses. Retinoic acid was added to the culture medium at the fetuses of 14 d as 100%. MK expression was maximal on the final concentration of 100 nM, which increased by ϳ40% the early stages of nephrogenesis and then declined to reach a very number of end buds after2dofculture and led to a threefold low level after birth. The decrease was 65% on fetal day 16 and increase in the number of glomeruli after6dofculture. reached 90% on postnatal day 20 (Figure 3). In VAD fetuses, Densitometric analysis of Northern blots shows that, under a similar level of MK expression was observed at gestational these conditions, MK mRNA expression was increased by day 14. From day 16, a decrease in MK mRNA occurred that ϳ70% and 100% after 24 and 48 h of culture, respectively is of ϳ50% for the whole period studied. Exposure to vitamin (Figure 5A). The amount of protein increased significantly, by A deficiency in utero did not modify the specific spatial and ϳ30%, after2dofculture (Figure 5B). temporal expression patterns of MK. However, consistent with Northern blot results, the MK expression decreases from day Neutralizing Antibodies to MK Impaired In 16 in the metanephrogenic zone and in the S-shaped bodies of Vitro Nephrogenesis VAD fetal kidneys, compared with that in controls (Figure 4, The addition of neutralizing antibodies to MK alters nephro- A through D). On day 21, MK expression was weak in the genesis in E14 metanephros compared with the opposite met- mature glomeruli of both control and VAD fetuses (Figure 4, E anephros cultured in the presence of the IgY nonimmune- and F). purified fraction as control conditions (Figure 6). The number 672 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 668–676, 2002

2 d and subsequently labeled with Dolichos biflorus agglutinin was not changed by the presence of the antibody (Figure 6, C and D). This was confirmed by the lack of difference in the number of end buds (Figure 7B). No effect of MK antibody was observed on the surface area (Figure 6) or total protein content per metanephros (Figure 7C) in E14 metanephric kid- neys cultured for 4 d.

Discussion The effect of retinoids on the expression of several genes known to play a key role in fetal organogenesis has been demonstrated elsewhere. Particularly in VAD animals, genes involved in the retinoic acid signaling pathway and other genes encoding for transcription factors, growth factors, and extra- cellular matrix components have been reported to be regulated by vitamin A status (32–37). As already mentioned, the pro- tooncogene c-ret is down-regulated in the metanephros of the VAD fetus (10). In this study, both Northern blot and in situ hybridization analysis of kidneys of VAD fetuses clearly show that vitamin A status also regulates MK expression during nephrogenesis. In addition, a significant increase in MK mRNA expression occurred within 24 h of retinoic acid stim- ulation in metanephric organ culture, which suggests that MK regulation is directly mediated by retinoic acid. This hypothe- sis is supported by the mapping and characterization of a retinoic acid–responsive enhancer ϳ900 nt upstream of the MK mRNA transcription start site reported for the mouse gene (38). The core element was mapped to positions Ϫ976 to Ϫ951. The binding of a retinoic acid receptor heterodimer DR5 type RARE was verified by use of gel-shift analysis. Trans- fection of a MK promoter/CAT reporter construct in EC, F9, and HM-1 cells showed a fivefold to tenfold induction of CAT activity by retinoic acid at the same concentration as that used in this study (100 nM). Spatiotemporal localization of MK mRNA has been reported in the whole mouse embryo, but only partial data exist about the developing kidney (19,22). No data exist in the rat embryo. The present data, which show that the in vivo pattern of MK expression is developmentally regulated in the metanephros, support the hypothesis of a role of MK in nephrogenesis. The ubiquitous expression of MK determined on day 14 of gesta- tion, i.e., at the onset of kidney organogenesis, becomes re- stricted to the metanephrogenic mesenchyme as nephrogenesis proceeds. At the end of fetal nephrogenesis, expression reached very low level. This suggests that MK may play a role as a Figure 5. MK expression (A, mRNA and B, protein) in metanephros survival factor for mesenchymal cell compartment, as pro- from embryonic day 14 embryos cultured in a defined medium during posed by Burrow and colleagues (39). We also found MK 24 and 48 h in the presence (dotted and plain bars) or absence (open transcript accumulation in early epithelialized mesenchymal bars) of 100 nM retinoic acid (RA). Values are means Ϯ SE of three or four experiments. Results are expressed as percentage of the control derivatives, which suggests that MK promotes the cell prolif- value after normalization for RNA loading on the basis of hybridiza- eration and/or differentiation during the first stage of the epi- tion of 18S rRNA and for protein loading by labeling with anti–␤- thelial conversion of the metanephric blastema. The role of actin antibody. *P Ͻ 0.05 and **P Ͻ 0.01 compared with controls. MK in epithelialization of the mesenchyme also emerged from anti-MK antibody in vitro experiments that have shown an important alteration of nephrogenesis leading to a reduction in of glomeruli counted after labeling (Figure 6, A and B) was the number of nephrons. However, no modification of the reduced by ϳ50% (Figure 7A), whereas ureteric bud branching growth of the explant and of the ureteric bud branching mor- morphogenesis assessed on explanted metanephroi grown for phogenesis has been observed. Misiadis et al. (40), using J Am Soc Nephrol 13: 668–676, 2002 Midkine Involvement in Nephrogenesis 673

Figure 6. Metanephroi development in vitro assessed by lectin histochemistry. Explanted metanephroi from 14-d-old embryos were grown for 2or6dinadefined medium in the (A and C) absence or (B and D) presence of neutralizing antibody to MK. Peanut agglutinin labeling allowed us to visualize and quantify nephron formation (A and B) in metanephroi after4dofculture. Dolichos biflorus agglutinin labeling (C and D) was used in metanephroi after2dofculture to analyze ureteric bud branching morphogenesis and determine the number of end buds. polyclonal rabbit antibody against MK, were unable to show suppressor gene WT1 plays a key role in nephrogenesis (45). any role for MK in nephrogenesis. This difference could be During normal kidney development, WT1 is maximally ex- attributed to the different antibody affinities for MK or/and to pressed in the mature glomeruli (46–48). In this study, we species differences. It is worthwhile to note that addition of have shown that the MK gene is expressed in condensing MK to embryonic mouse lung explants stimulated mesenchy- metanephric mesenchyme and in early epithelial structures mal tissue with no effect on branching morphogenesis (41). derived from the mesenchyme; then, it is down-regulated as the Mice lacking the MK gene present no abnormalities in the nephron matures. It was not detected in mature glomeruli, as organs, including lung and kidney. In fact, genetic redundancy described elsewhere, in the adult kidney (49). The down- due to genes of the same family can severely limit the power regulation of MK is coincident with the up-regulation of WT1 of this gene knockout study (42). Indeed, PTN, another hepa- in the developing nephron, which suggests that MK could be a rin-binding growth factor, is found in epithelio-mesenchymal target gene for WT1. Using Wilms’s tumor cells, Adachi et al. organs such as kidney and lung (21). Finally, the fact that the (43) provided evidence that a WT1 gene product indeed sup- presence of anti-MK antibodies in the culture media did not presses MK through binding to its promoter change the ureteric bud branching morphogenesis, together with region. Similarly, WT1 suppresses pax2 expression during late the fact that MK is regulated by retinoic acid, suggest that this stages of nephron differentiation (50). latter may have an effect on the epithelial conversion of the Taken together, the in vivo and in vitro data provide evi- metanephric blastema. Until now, only an effect of retinoic acid dence for the implication of MK in molecular process of on the ureteric bud branching capacity had been reported (9). nephrogenesis. They indicate that MK is a target gene for Other factors that regulate MK expression have been re- retinoic acid and is involved in the stimulating effect of retin- ported elsewhere (43,44). Among them, the Wilms’s tumor oids on nephrogenesis. 674 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 668–676, 2002

Acknowledgments We acknowledge the technical assistance of M.F. Belair for in situ hybridization studies and M. Vigny for kindly providing MK probe. This study was supported by a grant from the Fondation pour la Recherche Medical. References 1. Means A, Gudas L: The role of retinoids in vertebrate develop- ment. Annu Rev Biochem 64: 201–233, 1995 2. Merlet-Be´nichou C, Vilar J, Lelie`vre-Pe´gorier M, Gilbert T: Role of retinoids in renal development: Pathophysiological implica- tion. Curr Opin Nephrol Hypertens 8: 39–43, 1999 3. Burrow CR: Retinoids and renal development. Exp Nephrol 8: 219–225, 2000 4. Gilbert T, Merlet-Be´nichou C: Retinoids and nephron mass con- trol. Pediatr Nephrol 14: 1137–1144, 2000 5. Wilson JG, Warkany J: Malformations in the genito-urinary tract induced by maternal vitamin A deficiency in the rat. Am J Anat 83: 357–407, 1948 6. Mendelsohn C, Lohnes D, De´cimo D, Lufkin T, LeMeur M, Chambon P, Mark M: Function of the retinoic acid receptors (RARs) during development. (II) Multiple abnormalities at var- ious stages of organogenesis in RAR double mutants. Develop- ment 120: 2749–2771, 1994 7. Luo J, Sucov HM, Bader JA, Evans RM, Giguere V: Compound mutants for retinoic acid receptor (RAR) beta and RAR alpha 1 reveal developmental functions for multiple RAR beta isoforms. Mech Dev 55: 33–44, 1996 8. Kastner P, Mark M, Ghyselinck N, Krezel W, Dupe V, Grondona JM, Chambon P: Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development. Development 124: 313–326, 1997 9. Vilar J, Gilbert T, Moreau E, Merlet-Be´nichou C: Metanephros organogenesis is highly stimulated by vitamin A derivatives in organ culture. Kidney Int 49: 1478–1487, 1996 10. Lelie`vre-Pe´gorier M, Vilar J, Ferrier M, Moreau E, Freund N, Gilbert T, Merlet-Be´nichou C: Mild vitamin A deficiency leads to inborn nephron deficit in the rat. Kidney Int 54: 1455–1462, 1998 11. Moreau E, Vilar J, Lelie`vre-Pe´gorier M, Merlet-Be´nichou C, Gilbert T: Regulation of c-ret expression by retinoic acid in rat metanephros: Implication in nephron mass control. Am J Physiol 275: F938–F945, 1998 12. Sariola H, Sainio K: The tip-top branching ureter. Curr Opin Cell Biol 9: 877–884, 1997 13. Moreau E, Vilar J, Marlier A, Merlet-Be´nichou C, Gilbert T: Retinoic acid act on both ureteric bud and metanephric blastema to promote renal organogenesis. J Am Soc Nephrol 11: 380A, 2000 14. Tomomura M, Kadomatsu K, Nakamoto M, Muramatsu H, Kon- doh H, Imagawa K, Muramatsu T: A retinoic acid responsive gene, MK, produces a secreted protein with heparin binding activity. Biochem Biophys Res Commun 171: 603–609, 1990 15. Muramatsu H, Muramatsu T: Purification of recombinant mid- Figure 7. Quantitative analysis of in vitro development of kidneys kine and examination of its biological activities: Functional from 14-d-old embryos cultured in absence (open bars) or in the comparison of new heparin binding factors. Biochem Biophys presence (hatched bars) of neutralizing antibody to MK (MK Ab). (A) Res Commun 177: 652–658, 1991 Differentiation was analyzed by counting the total number of glomer- 16. Kadomatsu K, Tomomura M, Muramatsu T: cDNA cloning and uli present within the metanephroi. (B) Branching morphogenesis of sequencing of a new gene intensely expressed in early differen- the ureteric bud was analyzed by counting the number of end buds. tiation stages of embryonal carcinoma cells and in mid-gestation (C) Growth was assayed by protein content determination. **P Ͻ period of mouse embryogenesis. Biochem Biophys Res Commun 0.01 compared with controls. 151: 1312–1318, 1988 J Am Soc Nephrol 13: 668–676, 2002 Midkine Involvement in Nephrogenesis 675

17. Tomomura M, Kadomatsu K, Matsubara S, Muramatsu T: A 32. Zhai Y, Sperkova Z, Napoli JL: Cellular expression of retinal retinoic acid-responsive gene, MK, found in the teratocarcinoma dehydrogenase types 1 and 2: Effects of vitamin A status on system. Heterogeneity of transcript and the nature of translation testis mRNA. J Cell Physiol 186: 220–232, 2001 product. J Biol Chem 265: 10765–10770, 1990 33. McMenamy KR, Zachman RD: Effect of gestational age and 18. Michikawa M, Xu RY, Muramatsu H, Muramatsu T, Kim SU: retinol (vitamin A) deficiency on fetal rat lung nuclear retinoic Midkine is a mediator of retinoic acid induced neuronal differ- acid receptors. Pediatr Res 33: 251–255, 1993 entiation of embryonal carcinoma cells. Biochem Biophys Res 34. Chen Y, Kostetskii I, Zile MH, Solursh M: Comparative study of Commun 192: 1312–1318, 1993 Msx-1 expression in early normal and vitamin A-deficient avian 19. Kadomatsu K, Huang RP, Suganuma T, Murata F, Muramatsu T: embryos. J Exp Zool 272: 299–310, 1995 A retinoic acid responsive gene MK found in the teratocarcinoma 35. Mahmood R, Flanders KC, Morriss-Kay GM: The effects of system is expressed in spatially and temporally controlled man- retinoid status on TGF beta expression during mouse embryo- ner during mouse embryogenesis. J Cell Biol 110: 607–616, genesis. Anat Embryol (Berl) 192: 21–33, 1995 1990 36. Fu Z, Noguchi T, Kato H: Vitamin A deficiency reduces - 20. Kretschmer PJ, Fairhurst JL, Decker MM, Chan CP, Gluzman Y, like growth factor (IGF)-I gene expression and increases IGF-I Bohlen P, Kovesdi I: Cloning, characterization and developmen- receptor and insulin receptor gene expression in tissues of Jap- tal regulation of two members of a novel human gene family of anese quail (Coturnix coturnix japonica). J Nutr 131: 1189– neurite outgrowth-promoting proteins. Growth Factors 5: 99– 1194, 2001 114, 1991 37. Chailley-Heu B, Chelly N, Lelie`vre-Pe´gorier M, Barlier-Mur 21. Mitsiadis TA, Salmivirta M, Muramatsu T, Muramatsu H, Rau- vala H, Lehtonen E, Jalkanen M, Thesleff I: Expression of the AM, Merlet-Benichou C, Bourbon JR: Mild vitamin A defi- heparin-binding cytokines, midkine (MK) and HB-GAM ciency delays fetal lung maturation in the rat. Am J Respir Cell (pleiotrophin) is associated with epithelial-mesenchymal interac- Mol Biol 21: 89–96, 1999 tions during fetal development and organogenesis. Development 38. Matsubara S, Take M, Pedraza C, Muramatsu T: Mapping and 121: 37–51, 1995 characterization of a retinoic acid-responsive enhancer of 22. Nakamoto M, Matsubara S, Miyauchi T, Obama H, Ozawa M, midkine, a novel heparin-binding growth/differentiation fac- Muramatsu T: A new family of heparin binding growth/differ- tor with neurotrophic activity. J Biochem (Tokyo) 115: 1088– entiation factors: Differential expression of the midkine (MK) 1096, 1994 and HB-GAM genes during mouse development. J Biochem 39. Qui L, Hyink D, Amsler K, Wilson P, Burrow C: Midkine (Tokyo) 112: 346–349, 1992 stimulates expansion of the renal progenitor cell compartment 23. Thesleff I, Partanen AM, Vainio S: Epithelial-mesenchymal in- and inhibits nephrogenesis in metanephric organ culture. JAm teractions in tooth morphogenesis: The roles of extracellular Soc Nephrol 11: 381A, 2000 matrix, growth factors, and cell surface receptors. J Craniofac 40. Mitsiadis TA, Muramatsu T, Muramatsu H, Thesleff I: Midkine Genet Dev Biol 11: 229–237, 1991 (MK), a heparin-binding growth/differentiation factor, is regu- 24. Maruta H, Bartlett PF, Nurcombe V, Nur EKMS, Chomienne C, lated by retinoic acid and epithelial-mesenchymal interactions in Muramatsu T, Muramatsu H, Fabri L, Nice E, Burgess AW: the developing mouse tooth, and affects cell proliferation and Midkine (MK), a retinoic acid (RA)-inducible gene product, morphogenesis. J Cell Biol 129: 267–281, 1995 produced in E. coli acts on neuronal and HL60 leukemia cells. 41. Toriyama K, Muramatsu H, Hoshino T, Torii S, Muramatsu T: Growth Factors 8: 119–134, 1993 Evaluation of heparin-binding growth factors in rescuing mor- 25. Nurcombe V, Fraser N, Herlaar E, Heath JK: MK: A pluripo- phogenesis of heparitinase-treated mouse embryonic lung ex- tential embryonic stem-cell-derived neuroregulatory factor. De- plants. Differentiation 61: 161–167, 1997 velopment 116: 1175–1183, 1992 42. Nakamura E, Kadomatsu K, Yuasa S, Muramatsu H, Mamiya T, 26. Amri K, Freund N, Duong Van Huyen J, Merlet-Be´nichou C, Nabeshima T, Fan QW, Ishiguro K, Igakura T, Matsubara S, Lelie`vre-Pe´gorier M: Altered nephrogenesis due to maternal Kaname T, Horiba M, Saito H, Muramatsu T: Disruption of the diabetes is associated with increased expression of IGF-II/man- midkine gene (Mdk) resulted in altered expression of a calcium nose-6-phosphate receptor in fetal kidney. Diabetes 50: 1069– binding protein in the hippocampus of infant mice and their 1075, 2001 abnormal behaviour. Genes Cells 3: 811–822, 1998 27. Gilbert T, Gaonach S, Moreau E, Merlet-Be´nichou C: Defect of 43. Adachi Y, Matsubara S, Pedraza C, Ozawa M, Tsutsui J, Taka- nephrogenesis induced by gentamicin in rat metanephric organ matsu H, Noguchi H, Akiyama T, Muramatsu T: Midkine as a culture. Lab Invest 70: 656–666, 1994 novel target gene for the Wilms’ tumor suppressor gene (WT1). 28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein mea- surement with the Folin phenol reagent. J Biol Chem 193: 265– Oncogene 13: 2197–2203, 1996 275, 1951 44. Minegishi T, Karino S, Tano M, Ibuki Y, Miyamoto K: Regu- 29. Larson E, Howlett B, Jagendorf A: Artificial reductant enhance- lation of midkine messenger ribonucleic acid levels in cultured ment of the Lowry method for protein determination. Anal Bio- rat granulosa cells. Biochem Biophys Res Commun 229: 799– chem 155: 243–248, 1986 805, 1996 30. Li YS, Milner PG, Chauhan AK, Watson MA, Hoffman RM, 45. Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Kodner CM, Milbrandt J, Deuel TF: Cloning and expression of Housman D, Jaenisch R: WT-1 is required for early kidney a developmentally regulated protein that induces mitogenic and development. Cell 74: 679–691, 1993 neurite outgrowth activity. Science 250: 1690–1694, 1990 46. Pritchard-Jones K, Fleming S, Davidson D, Bickmore W, Por- 31. Merenmies J, Rauvala H: Molecular cloning of the 18-kDa teous D, Gosden C, Bard J, Buckler A, Pelletier J, Housman D: growth-associated protein of developing brain. J Biol Chem 265: The candidate Wilms’ tumour gene is involved in genitourinary 16721–16724, 1990 development. Nature 346: 194–197, 1990 676 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 668–676, 2002

47. Armstrong JF, Pritchard-Jones K, Bickmore WA, Hastie ND, 49. Kitamura M, Shirasawa T, Mitarai T, Muramatsu T, Maruyama Bard JB: The expression of the Wilms’ tumour gene. WT1,inthe N: A retinoid responsive cytokine gene, MK, is preferentially developing mammalian embryo. Mech Dev 40: 85–97, 1993 expressed in the proximal tubules of the kidney and human tumor 48. Mundlos S, Pelletier J, Darveau A, Bachmann M, Winterpacht A, cell lines. Am J Pathol 142: 425–431, 1993 Zabel B: Nuclear localization of the protein encoded by the 50. Ryan G, Steele-Perkins V, Morris JF, Rauscher FJ III, Dressler Wilms’ tumor gene WT1 in embryonic and adult tissues. Devel- GR: Repression of Pax-2 by WT1 during normal kidney devel- opment 119: 1329–1341, 1993 opment. Development 121: 867–875, 1995