Midkine Is Involved in Kidney Development and in Its Regulation by Retinoids
José Vilar*,
Claude Lalou*,
Jean-Paul Duong Van Huyen,
Stéphanie Charrin*,
Sylvie Hardouin*,
Daniel Raulais,
Claudie Merlet-Bénichou* and
Martine Leliévre-Pégorier*
*Unité de Recherches, INSERM U356, IFR 58, Université Paris 6; Unité de Recherches, INSERM U430, IFR 58, Hôpital Broussais; and Unité de Recherches, INSERM U440, Institut du Fer à Moulin, Paris, France.
Correspondence to Dr. M. Leliegrave;vre-Pégorier, INSERM U319/U356, Université Paris 7, 2, place Jussieu, 75251 Paris Cedex 05, France. Phone: 1-43-25-04-70; Fax: 1-43-25-67-89; E-mail: pegorier{at}paris7.jussieu.fr
ABSTRACT. In the kidney, in which development depends on epithelial-mesenchymalinteractions, it has been shown that retinoids modulate nephrogenesisin a dose-dependent manner in vivo and in vitro. Midkine (MK)is a retinoic acid responsive gene for a heparin-binding growthfactor. The aim of the present study was therefore to quantifythe expression of MK mRNA during renal development in the rat,to analyze the regulation of MK expression by retinoids in vivoand in vitro, and, finally, to study the role of MK in rat metanephricorgan cultures. The spatiotemporal expression of MK in fetalkidney was studied. In control rats, MK expression is ubiquitousat gestational day 14, i.e., at the onset of nephrogenesis.On day 16, MK is expressed in the condensed mesenchyme and inearly epithelialized mesenchymal derivatives. On gestationalday 21, MK is rather localized in the nonmature glomeruli ofthe renal cortex. In utero exposure to vitamin A deficiencydid not modify the specific spatial and temporal expressionpattern of MK gene in the metanephros, although a decrease inmRNA expression occurred. In metanephroi explanted from 14-d-oldfetuses and cultured in a defined medium, expression of MK mRNAwas found to be stimulated when retinoic acid (100 nM) was addedin the culture medium. Finally, in vitro nephrogenesis was stronglyinhibited in the presence of neutralizing antibodies for MK:the number of nephrons formed in vitro was reduced by 50% withoutchanges in ureteric bud branching morphogenesis. These resultsindicated that MK is implicated in the regulation of kidneydevelopment by retinoids. These results also suggested thatMK plays an important role in the molecular cascade of the epithelialconversion of the metanephric blastema.
Retinoids play a critical role in fetal organogenesis by regulatingthe expression of genes responsible for pattern formation andmorphogenesis (1). Like other embryonic processes, renal organogenesis,which is the result of interactions between the metanephricmesenchyme and the ureteric bud, depends on retinoids (2–4).In utero depletion of vitamin A or inactivation of retinoicacid receptors generates renal malformations (5–8). Usingmetanephric organ culture and a rat model of mild vitamin Adeficiency, we clearly established that vitamin A, via its activemetabolite, retinoic acid, strictly controls the number of nephrons(9,10). We then began to investigate the mechanisms of sucha control, finding that the expression of the proto-oncogenec-ret was modulated in vivo and in vitro according to the retinoidenvironment (10,11). The proto-oncogene c-ret, a receptor tyrosinekinase present at the tips of the ureteric bud branches, isrequired for ureteric bud outgrowth and branching (12). Geneslike Wnt11 and WT1 are also controlled by retinoic acid in themetanephros (13). In this study, we were interested in anotherdevelopmentally regulated gene, midkine (MK), a low-molecular-weightheparin-binding growth/differentiation factor (13 kD) encodedby a retinoic acid-responsive gene (14,15). MK expression increasesat early stages of retinoic acid induced differentiation ofembryonal carcinoma cells (16–18). In adult mice, MK mRNAis significantly expressed only in the kidney (19), but in adultrat, MK is more widely distributed (20). No data exist on thespatiotemporal pattern of MK expression in the rat embryos.In the early stages of mouse embryogenesis, MK is ubiquitouslyexpressed. In the midgestation period, MK expression becomesrestricted to tissues undergoing epithelial-mesenchymal interactions,which suggests a key role in the regulation of organ developmentand cell differentiation (19,21,22). The involvement of MK inepithelial-mesenchymal interactions, which characterize toothmorphogenesis, has been demonstrated (23). In vitro studieshave shown that MK also promotes neurite outgrowth and survivalof various embryonic nerve cells (15,24,25) and is mitogenicto certain fibroblasts, PC12 cells, and neuroectoderm cells(18,25).
In this study, we investigated the regulation of MK expressionin vitamin A-deficient (VAD) fetal rat kidneys. We systematicallyanalyzed the distribution and the expression pattern of MK inkidneys of fetuses issued from control and VAD mothers, usingNorthern blot and in situ hybridization techniques. We alsostudied the role of retinoic acid as signal mediating MK expressionin rat metanephric organ culture. Finally, we addressed thequestion of MK implication in nephrogenesis by analyzing theeffects of neutralizing antibodies against MK protein on morphogenesisand cell differentiation in organ culture of embryonic kidney.
Animals
Female Sprague Dawley rats weighing 200 to 300 g were givenfree access to water and standard laboratory pellets (UAR Laboratory,Villemoison sur Orge, France). They were caged overnight witha male, and vaginal smears were taken the following morning.The day a positive smear was obtained was designated day 0 ofgestation.
Two groups of pregnant females were used, control and VAD females.Pregnant females were made vitamin A deficient by administrationof an isocaloric and isoproteic diet deprived of vitamin A 6wk before mating, as described elsewhere (10). Blood sampleswere taken from the cut tip of the tail, and the plasma retinolwas determined by HPLC (Beckman Instruments, Inc., Fullerton,CA). The mean maternal plasma vitamin A concentration of thedeficient group was 50% lower (16.8 ± 0.5 µg/dl)than in the controls (31.3 ± 0.7 µg/dl, n = 65for both groups).
Fetuses were removed from anesthetized pregnant females on days14, 15, 16, 18, and 20 of gestation. Newborns and 20-d-old pupswere also used. Kidneys were surgically removed from embryos,newborns, and pups, immediately frozen in liquid nitrogen, andstored at -80°C. RNA were isolated from kidneys of fetusesfrom one to three litters taken at 14, 15, and 16 d of gestation,from two fetuses at 18 and 20 d of gestation, and from one 20-d-oldpup.
Isolation of RNA and Northern Blot Analysis
Total RNA was isolated from kidneys by one-step liquid-phaseseparation by use of the Trireagent procedure (Life TechnologiesBRL, Grand Island, NY). RNA was subjected to agarose gel electrophoresis,and Northern blot analysis was performed as described elsewhere(26) by use of a mouse MK cDNA probe kindly provided by Prof.M. Vigny (INSERM U440, Paris, France). Signal intensity wasquantified by densitometric analysis of autoradiograms (imageanalysis software, NIH Image) by use of hybridization with an18S ribosomal RNA probe, to allow correction for variationsin RNA loading.
In Situ Hybridization In situ hybridization was performed in fetuses on days 14 and16 and in the kidneys of fetuses on day 21 of gestation, asdescribed elsewhere (26), by use of 4% paraformaldehyde-fixed,paraffin-embedded tissues and the 35S-labeled MK probe. No labelingwas detected in tissue sections that were first treated withribonuclease A and then hybridized with probe.
Western Blot Analysis
Recombinant human MK was obtained from medium of transfectedQ2bn cells and purified by affinity column on heparin-sepharose,followed by an inverse-phase chromatography on C4. Antibodiesagainst MK protein were generated in a chicken. Immunizationwas performed by two injections of 200 ng of MK, in the presenceof complete, then incomplete, Freund adjuvant. Yolks were treatedby 2 vol of phosphate-buffered saline and 3% of polyethyleneglycol (molecular weight 6000, Merck) to precipitate phospholipids.The supernatant, filtered through cotton wool, was precipitatedin presence of 12% polyethylene glycol. The precipitate wasthen dissolved in 1 vol of phosphate-buffered saline and dialysedextensively. Excess polyethylene glycol was removed througha Sephadex G-75 filtration column (Amersham). The protein concentrationof the antibody solution was determined by absorbance at 280nm that used IgG as a standard. Nonimmune IgY was obtained bythe same method from the yolk of a nonimmunized chicken’segg. The specificity of antibodies was checked by Western blot,first by use of recombinant human pleiotrophin (PTN), recombinantrat nerve growth factor, recombinant human MK, and a and b recombinanthuman fibroblast growth factor and second by use of differentfetal tissue from fetuses on gestational day 15.
Western blot analysis was performed as described elsewhere (26).Protein were separated by electrophoresis in a sodium dodecylsulfate-polyacrylamide gel (15%) to test antibody specificityregarding other growth factors and in a sodium dodecyl sulfate-polyacrylamidegel gradient (6% to 30%) to detect MK protein in fetal tissueor cultured metanephros. Proteins were transferred to nitrocellulosemembranes (Hybond-C extra, Amersham), and the membranes wereblocked by incubation for 1 h with 3% dried skim milk in Tris-bufferedsaline with Tween (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05%Tween 20 [pH 8]). MK protein was detected by incubating withantibody diluted in 3% dried skim milk in Tris-buffered saline with Tween for 1 h at room temperature.A peroxidase-coupled anti-IgY (Jackson Immunoresearch laboratories,West Grove, PA) was used as the secondary antibody. Antigen-antibodycomplexes were detected by enhanced chemiluminescence as recommendedby the manufacturer (Amersham). Each membrane was labeled witha monoclonal anti-ß-actin antibody (Sigma) to normalizethe amount of protein loaded, as described elsewhere (26). Bandswere quantified by densitometry that used image analysis software(NIH image).
Metanephric Organ Culture
Metanephroi were cultured on a defined serum-free medium asdescribed elsewhere (9). A stock solution of retinoic acid (10mM, Sigma) was prepared in ethanol and stored in the dark at-20°C, and dilutions were made daily in culture medium.The antibody used in culture is a chicken anti-mouse MK polyclonalantibody diluted in culture medium.
One metanephros from each fetus was grown in control medium,and the opposite metanephros was grown in the same medium witheither retinoic acid (100 nM) or MK antibody (100 µg/ml).IgY nonimmune purified fraction was added in control mediumfor experiments with neutralizing antibodies to MK. All mediawere changed daily.
Glomerular and Tubular Structure Assessment and Protein Content in Metanephric Organ Culture
Nephrons formed in vitro were counted after labeling the glomerularstructures in the explanted metanephroi by use of specific lectin-bindingsites located on podocyte membranes (27). Briefly, the explantedmetanephroi were fixed in 2% paraformaldehyde phosphate-bufferedsaline, detached from the filter, permeabilized with saponin,and labeled with rhodamine-coupled peanut agglutinin, whichstains glomeruli. The use of another lectin, Dolichos biflorusagglutinin, fluorescein coupled, allowed us to analyze the uretericbud arborization and to count the end buds. The growth of theexplanted metanephroi was then determined by measurement oftheir protein content. The labeled metanephroi were placed inindividual tubes that contained 0.5 ml distilled water, rinsed,and sonicated for 15 s. The protein content was measured accordingto the procedure of Lowry et al. (28) as modified by Larsonet al. (29), with the use of bovine serum albumin as a standard.
Statistical Analyses
All values are expressed as means ± SEM. Differencesbetween developmental stages were calculated by ANOVA. Controland experimental groups, at the same stage, were compared byStudent’s unpaired t test. Student’s paired t testwas used to compare in vitro data. The threshold of significancewas taken to be P < 0.05.
Specificity of the Purified Anti-MK Antibody
The purified chicken anti-MK antibody reacted strongly withMK, and a unique band of 15 kD was detected on Western blots(Figure 1A, lane 4); however, it did not react with recombinanthuman PTN (lane 1), even though MK and PTN have 50% sequenceidentity (30,31). Anti-MK also did not react with nerve growthfactor (lane 2) and a fibroblast growth factor (lane 4) or bfibroblast growth factor (lane 5), which are other typical heparin-bindinggrowth factors. We conclude that the anti-MK antibody has highspecificity. Figure 1B reported MK expression in a broad rangeof molecular-weight proteins from different fetal tissue homogenatesfrom fetuses on gestational day 15. An unique band of 15 kDwas also detected.
Figure 1. Specificity of the purified anti-midkine (MK) antibody by Western blotting. (A) Cross-reaction with other growth factors in 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Lane 1, Recombinant human pleiotrophin (PTN); lane 2, recombinant rat nerve growth factor (NGF); lane 3, recombinant human MK; lane 4, recombinant human a fibroblast growth factor (FGF); and lane 5, recombinant human bFGF. Of each protein factor, 0.5 µg was loaded. (B) MK detection in fetal tissues on gestational day 15 in a 6% to 30% SDS-PAGE gradient. Lane 1, kidney; lane 2, liver; lane 3, lung; lane 4, testis; and lane 5, spinal cord. Of each tissue homogenate, 30 µg of protein was loaded. The positions of protein markers are shown on the left (as kD).
Vitamin A Controls MK Expression in Fetal Kidney
MK expression was ubiquitous in 14-d-old control fetuses. Inkidney, it was equally displayed between the mesenchymal andepithelial tissue (Figure 2A). From day 16 onward, MK expressionbecomes restricted to brain (neopallial cortex), stomach, intestine,lung, and kidney. In the latter, on day 16 of gestation, MKwas mainly found in the metanephrogenic zone, i.e., in the condensedmesenchyme, in the ureteric bud branches, and in nephron anlagen.The interstitium was not labeled, and the expression in thecollecting ducts in the profound zone of the kidney was verylow (Figure 2B). On day 21, MK was still strongly expressedin the immature glomeruli but was decreased in the mesenchyme.Its expression was also weak in the mature glomeruli (Figure 2C).MK mRNA was detected as a unique 0.9-kb band by Northernblot hybridization analysis. Densitometric analysis was madetaking control fetuses of 14 d as 100%. MK expression was maximalon the early stages of nephrogenesis and then declined to reacha very low level after birth. The decrease was 65% on fetalday 16 and reached 90% on postnatal day 20 (Figure 3). In VADfetuses, a similar level of MK expression was observed at gestationalday 14. From day 16, a decrease in MK mRNA occurred that isof 50% for the whole period studied. Exposure to vitamin A deficiencyin utero did not modify the specific spatial and temporal expressionpatterns of MK. However, consistent with Northern blot results,the MK expression decreases from day 16 in the metanephrogeniczone and in the S-shaped bodies of VAD fetal kidneys, comparedwith that in controls (Figure 4, A through D). On day 21, MKexpression was weak in the mature glomeruli of both controland VAD fetuses (Figure 4, E and F).
Figure 2. Expression of MK mRNA in the kidney during embryonic development as detected by in situ hybridization. Sections of kidneys from (A) 14-, (B) 16-, and (C) 21-d-old fetuses were hybridized with an 35S-labeled MK probe and counterstained with hematoxylin-eosin. (A) Section of metanephros showing a labeling of both ureteric bud () and mesenchyme (). (B) Section of kidney showing a strong MK expression in the nephrogenic zone, in the mesenchyme () and in the nonmature glomeruli (). In contrast, low staining was observed in the mature tubule in the profound zone of the kidney (). (C) Section of kidney disclosing a decreased expression of MK in the mesenchyme (). The expression was restricted to the nonmature glomeruli (). In the mature glomeruli, the MK expression was weak (). Magnification, x400.
Figure 3. Patterns of MK mRNA in the kidney of fetuses (14–20 d of gestation) and of postnatal rats (age 1 and 20 d) from control () and vitamin A-deficient (VAD; ) rats. Values are means ± SE of five to six experiments. Results are expressed as percentage of the gestational day 14 control values after normalization for RNA loading on the basis of hybridization of 18S rRNA. *P < 0.05 and **P < 0.001 compared with aged-matched controls.
Figure 4. Expression of MK mRNA in the kidney during embryonic development as detected by in situ hybridization. Sections of kidneys 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 VAD than in control fetuses. (C and D) S-shaped body disclosing a staining that appears to be lower in VAD than in control fetuses. (E and F) Weak labeling was observed in the mature glomeruli of both control and VAD kidneys. Magnification, x1000.
Retinoic Acid Stimulates MK Expression in Metanephric Organ Culture
We quantified the changes in MK expression induced by retinoicacid in the metanephros explanted from 14-d-old fetuses. Retinoicacid was added to the culture medium at the final concentrationof 100 nM, which increased by 40% the number of end buds after2 d of culture and led to a threefold increase in the numberof glomeruli after 6 d of culture. Densitometric analysis ofNorthern blots shows that, under these conditions, MK mRNA expressionwas increased by 70% and 100% after 24 and 48 h of culture,respectively (Figure 5A). The amount of protein increased significantly,by 30%, after 2 d of culture (Figure 5B).
Figure 5. MK expression (A, mRNA and B, protein) in metanephros from embryonic day 14 embryos cultured in a defined medium during 24 and 48 h in the presence (dotted and plain bars) or absence (open bars) of 100 nM retinoic acid (RA). Values are means ± SE of three or four experiments. Results are expressed as percentage of the control value after normalization for RNA loading on the basis of hybridization of 18S rRNA and for protein loading by labeling with anti-ß-actin antibody. *P < 0.05 and **P < 0.01 compared with controls.
Neutralizing Antibodies to MK Impaired In Vitro Nephrogenesis
The addition of neutralizing antibodies to MK alters nephrogenesisin E14 metanephros compared with the opposite metanephros culturedin the presence of the IgY nonimmune-purified fraction as controlconditions (Figure 6). The number of glome6ruli counted afterlabeling (Figure 6, A and B) was reduced by 50% (Figure 7A),whereas ureteric bud branching morphogenesis assessed on explantedmetanephroi grown for 2 d and subsequently labeled with Dolichosbiflorus agglutinin was not changed by the presence of the antibody(Figure 6, C and D). This was confirmed by the lack of differencein the number of end buds (Figure 7B). No effect of MK antibodywas observed on the surface area (Figure 6) or total proteincontent per metanephros (Figure 7C) in E14 metanephric kidneyscultured for 4 d.
Figure 6. Metanephroi development in vitro assessed by lectin histochemistry. Explanted metanephroi from 14-d-old embryos were grown for 2 or 6 d in a defined 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 after 4 d of culture. Dolichos biflorus agglutinin labeling (C and D) was used in metanephroi after 2 d of culture to analyze ureteric bud branching morphogenesis and determine the number of end buds.
Figure 7. Quantitative analysis of in vitro development of kidneys from 14-d-old embryos cultured in absence (open bars) or in the presence (hatched bars) of neutralizing antibody to MK (MK Ab). (A) Differentiation was analyzed by counting the total number of glomeruli present within the metanephroi. (B) Branching morphogenesis of the ureteric bud was analyzed by counting the number of end buds. (C) Growth was assayed by protein content determination. **P < 0.01 compared with controls.
The effect of retinoids on the expression of several genes knownto play a key role in fetal organogenesis has been demonstratedelsewhere. Particularly in VAD animals, genes involved in theretinoic acid signaling pathway and other genes encoding fortranscription factors, growth factors, and extracellular matrixcomponents have been reported to be regulated by vitamin A status(32–37). As already mentioned, the protooncogene c-retis down-regulated in the metanephros of the VAD fetus (10).In this study, both Northern blot and in situ hybridizationanalysis of kidneys of VAD fetuses clearly show that vitaminA status also regulates MK expression during nephrogenesis.In addition, a significant increase in MK mRNA expression occurredwithin 24 h of retinoic acid stimulation in metanephric organculture, which suggests that MK regulation is directly mediatedby retinoic acid. This hypothesis is supported by the mappingand characterization of a retinoic acid-responsive enhancer900 nt upstream of the MK mRNA transcription start site reportedfor the mouse gene (38). The core element was mapped to positions-976 to -951. The binding of a retinoic acid receptor heterodimerDR5 type RARE was verified by use of gel-shift analysis. Transfectionof a MK promoter/CAT reporter construct in EC, F9, and HM-1cells showed a fivefold to tenfold induction of CAT activityby retinoic acid at the same concentration as that used in thisstudy (100 nM).
Spatiotemporal localization of MK mRNA has been reported inthe whole mouse embryo, but only partial data exist about thedeveloping kidney (19,22). No data exist in the rat embryo.The present data, which show that the in vivo pattern of MKexpression is developmentally regulated in the metanephros,support the hypothesis of a role of MK in nephrogenesis. Theubiquitous expression of MK determined on day 14 of gestation,i.e., at the onset of kidney organogenesis, becomes restrictedto the metanephrogenic mesenchyme as nephrogenesis proceeds.At the end of fetal nephrogenesis, expression reached very lowlevel. This suggests that MK may play a role as a survival factorfor mesenchymal cell compartment, as proposed by Burrow andcolleagues (39). We also found MK transcript accumulation inearly epithelialized mesenchymal derivatives, which suggeststhat MK promotes the cell proliferation and/or differentiationduring the first stage of the epithelial conversion of the metanephricblastema. The role of MK in epithelialization of the mesenchymealso emerged from anti-MK antibody in vitro experiments thathave shown an important alteration of nephrogenesis leadingto a reduction in the number of nephrons. However, no modificationof the growth of the explant and of the ureteric bud branchingmorphogenesis has been observed. Misiadis et al. (40), usingpolyclonal rabbit antibody against MK, were unable to show anyrole for MK in nephrogenesis. This difference could be attributedto the different antibody affinities for MK or/and to speciesdifferences. It is worthwhile to note that addition of MK toembryonic mouse lung explants stimulated mesenchymal tissuewith no effect on branching morphogenesis (41). Mice lackingthe MK gene present no abnormalities in the organs, includinglung and kidney. In fact, genetic redundancy due to genes ofthe same family can severely limit the power of this gene knockoutstudy (42). Indeed, PTN, another heparin-binding growth factor,is found in epithelio-mesenchymal organs such as kidney andlung (21). Finally, the fact that the presence of anti-MK antibodiesin the culture media did not change the ureteric bud branchingmorphogenesis, together with the fact that MK is regulated byretinoic acid, suggest that this latter may have an effect onthe epithelial conversion of the metanephric blastema. Untilnow, only an effect of retinoic acid on the ureteric bud branchingcapacity had been reported (9).
Other factors that regulate MK expression have been reportedelsewhere (43,44). Among them, the Wilms’s tumor suppressorgene WT1 plays a key role in nephrogenesis (45). During normalkidney development, WT1 is maximally expressed in the matureglomeruli (46–48). In this study, we have shown that theMK gene is expressed in condensing metanephric mesenchyme andin early epithelial structures derived from the mesenchyme;then, it is down-regulated as the nephron matures. It was notdetected in mature glomeruli, as described elsewhere, in theadult kidney (49). The down-regulation of MK is coincident withthe up-regulation of WT1 in the developing nephron, which suggeststhat MK could be a target gene for WT1. Using Wilms’stumor cells, Adachi et al. (43) provided evidence that a WT1gene product indeed suppresses MK gene expression through bindingto its promoter region. Similarly, WT1 suppresses pax2 expressionduring late stages of nephron differentiation (50).
Taken together, the in vivo and in vitro data provide evidencefor the implication of MK in molecular process of nephrogenesis.They indicate that MK is a target gene for retinoic acid andis involved in the stimulating effect of retinoids on nephrogenesis.
Acknowledgments
We acknowledge the technical assistance of M.F. Belair for insitu hybridization studies and M. Vigny for kindly providingMK probe. This study was supported by a grant from the Fondationpour la Recherche Medical.
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Received for publication June 18, 2001.
Accepted for publication October 1, 2001.
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Top
Abstract
Introduction
50% without changes in ureteric bud branching morphogenesis. These results indicated that MK is implicated in the regulation of kidney development by retinoids. These results also suggested that MK plays an important role in the molecular cascade of the epithelial conversion of the metanephric blastema. 
diluted in 3% dried skim milk in Tris-buffered saline with Tween for 1 h at room temperature. A peroxidase-coupled anti-IgY (Jackson Immunoresearch laboratories, West Grove, PA) was used as the secondary antibody. Antigen-antibody complexes were detected by enhanced chemiluminescence as recommended by the manufacturer (Amersham). Each membrane was labeled with a monoclonal anti-ß-actin antibody (Sigma) to normalize the amount of protein loaded, as described elsewhere (26). Bands were quantified by densitometry that used image analysis software (NIH image). 
diluted in culture medium. 
) and mesenchyme (
). (B) Section of kidney showing a strong MK expression in the nephrogenic zone, in the mesenchyme (
) and in the nonmature glomeruli (
) and vitamin A-deficient (VAD; 
) rats. Values are means ± SE of five to six experiments. Results are expressed as percentage of the gestational day 14 control values after normalization for RNA loading on the basis of hybridization of 18S rRNA. *P < 0.05 and **P < 0.001 compared with aged-matched controls.
