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Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia
Submitted 4 April 2007 ; accepted in final form 23 May 2007
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ABSTRACT |
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 TOP ABSTRACT METHODS AND MATERIALSRESULTSDISCUSSIONGRANTSREFERENCES |
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1 (TGF-1). Similar gene expression changes were found in E16.5 metanephroi in which the dam had been exposed to 2 days of DEX (0.2 mg·kg–1·day–1) at E14.5/15.5 in vivo. However, in kidneys collected at E20.5 after in vivo exposure for 2 days, GDNF expression was increased and BMP-4 and TGF-1 expression decreased suggesting a biphasic response in gene expression to DEX exposure. These results show for the first time that inhibition of ureteric branching morphogenesis may be a key mechanism through which DEX exposure results in a reduced nephron endowment. hypertension; reduced nephron endowment
An interesting commonality in all three species mentioned above is that timing of DEX exposure is synonymous for the stage of kidney development. In sheep at E26–28, the rat at 
E14–16, and the Spiny mouse at E23–26, the ureteric bud has invaded the metanephric mesenchyme and begun branching in all three species (9, 18, 28). This suggests that this very early stage of renal development is particularly susceptible to DEX exposure. Despite this evidence, the exact mechanisms through which maternal DEX administration results in a nephron deficit in the offspring are not known.
Mammalian kidney development involves complex molecular reciprocal interactions between the ureteric bud (arising from the Wolffian duct) and the surrounding metanephric mesenchyme (7, 15). Branching of the ureteric tree is thought to be a critical process that determines the final number of nephrons, as new nephrons only form adjacent to ureteric tips. In the present study, we hypothesized that the decrease in nephron number previously reported in the rat following in vivo exposure to DEX (20, 21) is a result of an inhibition in ureteric branching morphogenesis. To test this hypothesis, we utilized a whole metanephric organ culture to determine the effect of exogenous DEX on ureteric branching morphogenesis and nephron number.
We also assessed the effects of DEX on the expression levels of key regulatory genes involved in branching morphogenesis. Glial cell line-derived neurotrophic factor (GDNF) is an important promoter of ureteric branching as studies have revealed that GDNF heterozygous mice have less ureteric branches (22) and reduced nephron number (8). In contrast, bone morphogenetic protein-4 (BMP-4), another member of the TGF- superfamily, inhibits ureteric branching. Culture of mouse metanephroi in the presence of exogenous BMP-4 results in asymmetric ureteric branching and reduced ureteric growth (3). In vitro studies with exogenous transforming growth factor-1 (TGF-1) have also reported inhibition of ureteric branching and abnormal tubular development (25) as well as reported inhibition of ureteric growth and a reduction in nephron number (5). These last two genes are of great interest as recently, Dickinson et al. (10) reported that 60 h of DEX exposure to pregnant Spiny mice increased expression of both BMP-4 and TGF-1 in the fetal metanephroi. The renal renin-angiotensin system (RRAS) has also been shown to be important in kidney development with the major receptor the angiotensin II type 1 receptor (AT1R) able to regulate ureteric branching morphogenesis as well as renal growth (16). This is of great importance as maternal DEX exposure alters the expression of fetal angiotensin II receptors in other models of DEX exposure (17). Hence, we investigated the effect of exogenous DEX exposure on the metanephric expression of GDNF, BMP-4, TGF-1, and the angiotensin II type 1 (AT1R) and 2 (AT2R) receptors. Finally, to test the relevance of the gene changes found in vitro, we examined the effects of in vivo DEX exposure during pregnancy in the rat on gene expression in fetal metanephroi.
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METHODS AND MATERIALS |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONGRANTSREFERENCES |
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All animal experiments were approved in advance by an Animal Ethics Committee at Monash University and were carried out in accordance with the guidelines of the National Health and Medical Research Council of Australia.
Whole Metanephric Organ Culture
Sprague-Dawley female rats were time-mated overnight where presence of a vaginal plug indicated time of mating. This time of mating was designated as E0.5. Whole metanephroi were isolated from E14.5 Sprague-Dawley embryos within a weight range of 0.115–0.125 g and placed on 3.0-µm pore polycarbonate transfilter membranes (Transwell, Corning Star, Cambridge, MA) in 24-well tissue culture plates with wells containing 350 µl of serum-free culture medium at 37°C and 5% CO2. The culture medium consisted of DMEM:Ham's F12 liquid medium (Trace Biosciences, Castle Hill, New South Wales, Australia) supplemented with 5 µg/ml transferrin (Sigma, Castle Hill, Australia), 12.9 µl/ml L-glutamine (Trace Biosciences), penicillin (100 µg/ml), and streptomycin (100 U/ml). Metanephroi were randomly assigned to one of the four following groups: control (media alone), media containing DEX (10–5 M), media containing DEX (10–7 M), or vehicle (VEH). The VEH used was ethanol (DEX was only soluble in ethanol). The VEH control had an ethanol concentration of 0.04% vol/vol, equivalent to the ethanol concentration used with the highest concentration of DEX, i.e., (10–5 M). To determine the effect of DEX on ureteric branching morphogenesis, metanephroi were cultured in treatment media for 2 days. To determine the effect of DEX on glomerular number, metanephroi were cultured for 2 days in treatment media and then for a further 3 days in control media. Metanephroi were cultured in treatment media for 2 days only, to mimic the previous in vivo studies in sheep and rat that have reported a nephron deficit in offspring following 2 days glucocorticoid exposure in utero (20, 21, 30). At the conclusion of the culture period, metanephroi were fixed for examination with whole mount immunofluorescence microscopy.
Immunofluorescence Staining
To visualize ureteric branching morphogenesis, metanephroi were wholemount immunostained with a monoclonal anti-pan cytokeratin mixture (Sigma-Aldrich). At the conclusion of the culture period, metanephroi were fixed whole in methanol at –20°C for a minimum of 15 min. After fixation, metanephroi were washed briefly in PBS containing 1% Tween 20 (PBST) and incubated with the primary antibody; monoclonal mouse anti-pan cytokeratin (Sigma-Aldrich) at a dilution of 1:100 at 37°C for 2 h. Metanephroi were then washed in PBST before addition of the secondary antibody; Alexa 588 goat antimouse IgG (Molecular Probes, Eugene, OR) at a dilution of 1:100 at 37°C for 2 h. Metanephroi were then washed briefly in PBST before mounting in PBS/glycerol mounting media (Sigma-Aldrich).
Quantification of Ureteric Branching Morphogenesis
Following immunostaining, metanephroi were visualized under an epifluorescence microscope (Olympus). By focusing through the whole metanephros, the ureteric "tree" was sketched as a skeletonized image. The number of branch points and terminal tips was determined. Branch points were defined as the intersection of three or more branches (lines on the skeletonized image).
Lectin Histochemistry
To determine glomerular number, metanephroi were stained with peanut agglutinin (PNA). At the end of the culture period, metanephroi were fixed as above. Following a brief wash in PBS, metanephroi were incubated in 50 mM NH4Cl at room temperature for 1 h following which the tissue was permeabolized with 0.1% saponin in PBS for 1 h at room temperature. After being digested with 2% H2O2 in methanol at room temperature for 30 min, metanephroi were washed in 0.1% saponin in PBS for 30 min. Metanephroi were then incubated in 0.1 U/ml of neuraminidase (Sigma-Aldrich) in 1% CaCl2 in PBS for 2 h at 37°C, before being washed twice for 30 min in 0.2% saponin in PBS. To stain glomeruli, metanephroi were incubated in PNA (50 µg/ml; Sigma Aldrich) in 0.3% Triton in PBS with 1:100 dilution of ions overnight at 4°C. Metanephroi were then extensively washed in 0.1% saponin to remove excess PNA. This washing period averaged 3 days with regular changes of 0.1% saponin. Metanephroi were then mounted on cavity slides with fluorescence preserving mounting media (Sigma-Aldrich).
Quantification of Glomerular Number
Following lectin PNA histochemistry, metanephroi were observed under an epifluorescence microscope where by focusing through the whole metanephros the number of glomeruli were counted and recorded. Since background staining is observed with PNA staining and glomeruli are observed at different stages of development, glomeruli were counted on three separate occasions and the average was taken. Glomeruli were counted if the podocytes (specific site of PNA binding) were observed through any level of the metanephros.
In Vitro Gene Expression Studies
Gene expression was determined in metanephroi that had been cultured in control media or media containing VEH or DEX (10–5 M) for 48 h only. At the end of the culture period, metanephroi were placed in RNA later (Qiagen) for later extraction of RNA. Total RNA was extracted using RNeasy extraction kits (Qiagen). One microgram of each RNA sample was reverse transcribed in a 30.75-µl reaction containing 5 µl of 10x Taqman reverse transcriptase buffer, 11 µl of 25 mM MgCl2, 10 µl of 100 µM 2'-deoxynucleoside 5'-triphosphate, 2.5 µl of 50 mM random hexamers, 1 µl of RNase inhibitor, and 1.25 µl of Multiscribe reverse transcriptase (PE Applied Biosystems, Foster City, CA). To control for genomic DNA contamination, negative reactions were prepared, where the 1.25 µl of Multiscribe was replaced with sterile water. Reverse transcription reactions were run in a Bio-Rad i-cycler at 25°C for 10 min, 48°C for 30 min, 95°C for 5 min, and then held at 4°C. These samples were stored at –20°C.
Gene expression levels were determined via real-time PCR using an ABI-PRISM 7700 real-time machine as described previously (11, 26). We previously reported the sequences and optimal concentrations of primer/probes for AT1Ra, AT1Rb, and AT2R (26). GDNF was analyzed using an Assay-on-Demand from Applied Biosystems while the primer/probe sequences for BMP-4 and TGF-1 were as follows: BMP-4: forward primer: 5'-CGAGCCATGCTAGTTTGATACCT-3', reverse primer: 5'-CCGCGTGGCCCTGAA-3'; probe: 5'-TCGGCGATTTTTTCTTCCCGGTCT-3'; TGF-1: forward primer: 5'-TCGACATGGAGCTGGTGAAA-3', reverse primer: 5'-GAGCCTTAGTTTGGACAGGATACTG-3', probe: 5'-AAGCGCATCGAAGCCTCCGTG-3'.
A comparative cycle of threshold fluorescence (CT) method was used with 18S as an internal control. GDNF, BMP-4, TGF-1, AT1Ra, and AT1Rb receptor primer/probe sets were multiplexed with 18S. AT2R was run in a separate tube to 18S after optimization experiments revealed the CT was altered if run in a multiplex reaction.
Calculations of Relative Gene Expression
The CT value for 18S was subtracted from the CT value for the gene of interest to give a 
CT for each sample. The CT of the calibrator [in this case the mean CT of control media group for in vitro and the mean of the saline (SAL) group for in vivo] was then subtracted from each sample to give a CT value. This was then inserted into the equation 2 – CT to give a final relative expression relative to the calibrator.
In Vivo Model for Gene Expression Studies
To investigate whether DEX alters metanephric gene expression similarly in an in vivo model, pregnant Sprague-Dawley rats were injected intraperitoneally twice daily with DEX (0.2 mg·kg–1·day–1 split over 2 doses) or an equivalent dose of SAL. Rats were injected on E14.5/E15.5 and three pregnant animals from each group (SAL and DEX) were culled with an overdose of pentobarbital sodium (Nembutal Sodium, Abbott Australia) on either E16.5 or E20.5. The abdomen was opened and the uterus was located. Embryos were removed, cleaned, and weighed before decapitation. Kidneys were then removed, weighed, and frozen for later extraction of RNA. At E16.5, kidneys from two or three embryos were pooled while kidneys from individual animals were collected at E20.5. RNA extraction was performed as described in the previous section.
Statistics
Values are means ± SE except where otherwise indicated. A one-way ANOVA was used to analyze the effects of DEX on ureteric branching morphogenesis and glomerular number. A post hoc analysis using the all pairwise multiple comparison Tukey's test was used to determine the differences within treatment. Gene expression data were analyzed using unpaired two-tailed Student's t-tests. Significance was accepted at P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONGRANTSREFERENCES |
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Culture in the presence of DEX did not appear to affect either the gross structure or the pattern of ureteric branching in metanephroi. However, metanephroi cultured in the presence of DEX (10–5 M) appeared smaller than metanephroi cultured under the other conditions (Fig. 1). This observation was confirmed in the quantitative analysis which showed that metanephroi cultured in DEX (10–5 M) had significantly less ureteric branch points (P < 0.05; Fig. 2A) and terminal tips (P < 0.05; Fig. 2B) than those cultured in control or VEH media. The number of branch points and terminal tips in metanephroi cultured in DEX (10–7 M) was not significantly different to the control or VEH groups. Although not statistically significant, metanephroi cultured in DEX (10–5 M) had 29% fewer branch points (P = 0.06; Fig. 2A) and 30% fewer terminal tips (P = 0.06; Fig. 2B) than those cultured in the DEX (10–7 M).
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No differences were observed in total glomerular number between the VEH and control culture groups. Metanephroi cultured in DEX (10–5 M) had 45% fewer glomeruli than the VEH cultures and 39% fewer glomeruli than the control cultures (P < 0.001 for both; Fig. 2C). Interestingly, metanephroi cultured in the presence of DEX (10–7 M) also had significantly fewer glomeruli than VEH (26% less) and control (22%) cultures (P < 0.001 for VEH and P < 0.05 for control; Fig. 2C). No significant difference in glomerular number was observed between the DEX (10–5 M) and DEX (10–7 M) groups.
Expression of GDNF, BMP-4, and TGF-1 In Vitro
In vitro gene expression at E16.5.
Metanephroi cultured in the presence of DEX (10–5 M) for 2 days had significantly reduced mRNA expression for GDNF (Fig. 3A; P < 0.001), whereas expression of both BMP-4 and TGF-1 was significantly increased (Fig. 3, B and C; P < 0.001 and P < 0.05, respectively).
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1 In Vivo
Kidneys of embryos of mothers administered DEX on E14.5/E15.5 demonstrated a significantly reduced expression of GDNF at E16.5 followed by an upregulation in the expression at E20.5 (Fig. 3D; P < 0.001 for both ages). In contrast, expression of BMP-4 was increased at E16.5 followed by a decrease in expression at E20.5 (Fig. 3E; P < 0.001, P < 0.01). Like BMP-4 expression, TGF-1 expression was increased at E16.5 (Fig. 3F; P = 0.06) and significantly decreased at E20.5 (Fig. 3F; P < 0.01).
Gene Expression of Components of RRAS
Effect of exogenous DEX on expression of angiotensin receptors in vitro. AT1Ra, AT1Rb, and AT2R expression was not different between the control and VEH groups. However, metanephroi cultured in DEX (10–5 M) for 2 days had a significant upregulation in the expression of AT1Ra (Fig. 4A; P < 0.05) and AT2R (Fig. 4C; P < 0.05) compared with both the control and VEH groups. In contrast, mRNA expression of AT1Rb was markedly reduced in the DEX (10–5 M) group compared with both the control and the VEH groups (Fig. 4B; P < 0.001).
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Embryonic kidneys from the maternal DEX-treated group demonstrated significantly higher expression of AT1Ra at E16.5 (Fig. 4D; P < 0.001) and significantly decreased expression at E20.5 (Fig. 4D; P < 0.01) compared with the SAL group. The expression of AT1Rb in embryonic kidneys from the DEX group was significantly downregulated at E16.5 (Fig. 4E; P < 0.001) and remained downregulated at E20.5 compared with the SAL group (Fig. 4E; P < 0.01). AT2R expression was reduced at E16.5 (Fig. 4F; P < 0.05) in embryonic kidneys from the DEX group and remained reduced at E20.5 (Fig. 4F; P < 0.001).
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DISCUSSION |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONGRANTSREFERENCES |
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Administration of DEX for just 2–3 days during pregnancy in rats (E15–16 out of a 22-day pregnancy) (20, 21), sheep (26–28 out of a 150-day pregnancy) (30), and most recently in Spiny mouse (E20–23 out of a 39-day pregnancy) (10) results in a significant nephron deficit in the offspring. In many cases, the nephron deficit is thought to be a contributing factor to the development of hypertension. In the rat, sheep, and Spiny mouse, the timing of DEX administration coincided with early ureteric branching morphogenesis. This suggests that there is a critical window early in kidney development that is highly susceptible to DEX exposure. However, no study has ever explored the mechanisms through which DEX may alter nephron endowment. The data from the present study reveal that metanephroi cultured with DEX for 2 days, thus mimicking the timing of exposure of the in vivo models, have reduced ureteric branching and glomerular number. This effect appeared to be dose dependent as the lower concentration of DEX did not directly inhibit ureteric branching. Interestingly, glomerular number was reduced after a 5-day culture period, even though DEX was present for only the first 2 days. This decrease in glomerular number was observed in metanephroi cultured at the lower dose of DEX despite no significant inhibition in branching morphogenesis. This strongly suggests DEX exposure for just 2 days during the period of ureteric branching can permanently alter kidney development and even if conditions are restored to normal (i.e., 3-day culture in control media), the developing kidney is unable to "catch up" to restore a normal nephron complement.
Furthermore, it also suggests that branching morphogenesis is not the only developmental mechanism affected by DEX. The fact that nephron number was reduced with no observable effect on ureteric branching morphogenesis at DEX (10–7 M) suggests that other mechanisms may be involved. Increased apoptosis has been reported in kidneys from embryonic rats where the mothers were exposed to a low-protein diet (29). DEX has been shown to induce apoptosis of eosinophils (34). Further studies in our DEX model are required to investigate if apoptosis plays a role.
Results from this study suggest DEX has the potential to act directly on the kidney to influence renal development. DEX acts primarily on the glucocorticoid receptor (GR) which is widely expressed in the vascular tufts of developing glomeruli, in collecting duct, epithelial cells of renal tubules, and Bowman's capsule suggesting a direct action is possible. However, glucocorticoids are also transcriptional regulators and glucocorticoid response elements (GREs) are found in the promoter regions of many genes including TGF-1 and genes of renal RAS. To see whether glucocorticoids may exert their action in part by modulation of genes involved in branching morphogenesis, we examined the expression of key genes involved in this process. This study reveals that DEX exposure for 2 days alters the expression levels of some key members of the TGF- superfamily and RRAS suggesting that DEX may, in part, exert its actions indirectly through alterations in gene expression patterns.
GDNF has a direct role in ureteric branching as its binding to its receptor, c-ret, induces bifurcation of the ureteric bud (2). Our laboratory showed that GDNF heterozygous mice have reduced ureteric epithelial volume and nephron endowment compared with wild-type controls (8). These observations complement our data, where we report a decrease in expression of GDNF and a reduction in ureteric branching and subsequent glomerular number. This suggests by reducing expression of GDNF, DEX may decrease nephron number by inhibiting early stages of ureteric branching morphogenesis. DEX exposure for 2 days also significantly increased the expression of BMP-4 and TGF-1, two known inhibitors of ureteric branching morphogenesis. BMP-4 has been shown to be expressed in the main branches of the ureteric tree (23) and metanephroi cultured in exogenous BMP-4 have reduced nephron number (3). TGF-1 mRNA is expressed in the nephrogenic zone mesenchyme, branching ureteric bud epithelium, and early nephron structures (6). Ritvos et al. (24) reported branching defects in metanephroi cultured with TGF-1.
Although the decrease in nephron number may be associated with a decrease in ureteric branching, GDNF, BMP-4, and TGF-1 are also expressed in the mesenchyme. The reduction in nephron number could be due to DEX causing a reduction in the mesenchymal cell population or an increase in inhibitory signaling from the mesenchyme delaying ureteric branching and thus nephron number. It is probable that both compartments (ureteric bud epithelium and the mesenchyme) contribute to the observed nephron deficit but further studies need to be undertaken to investigate this.
The present study also shows altered expression of the receptors of the RRAS following 2-day DEX exposure. All the components of the RRAS are present within the fetal kidney from early in development where ANG II has been reported to cause apoptosis and inhibition of growth through AT2R and to mediate growth and proliferation via the AT1R receptor (31, 32). The upregulated expression of AT2R observed in the present study may be mediating apoptosis. As noted above, increased apoptosis has been hypothesized as a mechanism through which a prenatal perturbation could result in decreased nephron endowment. Blockade of the AT1R has previously been reported to inhibit ureteric branching morphogenesis (16). Our data show differential effect of DEX on the AT1R subtypes, with an increase in AT1Ra and a decrease in AT1Rb. Most studies that have investigated the effect of glucocorticoids on AT1R regulation in rodents do not distinguish between the receptor subtypes (19). However, Chansel et al. (4) reported that rat mesangial cells cultured in the presence of DEX have a reduced expression of AT1Rb with no alteration in the expression of AT1Ra, whereas Uno et al. (27) reported increased expression of AT1Ra with no change in expression of AT1Rb in smooth muscle cells cultured in DEX. These conflicting results may reflect relative expression levels of the receptor subtypes in specific cell populations. Importantly, in our studies where the overall effect on whole metanephroi was examined, it would appear that DEX causes a downregulation of AT1a expression which is associated with decreased branching morphogenesis.
The second part of this study investigated whether the same dose and duration of DEX treatment previously reported to decrease nephron number in the rat result in similar alterations in gene expression in vivo to those we observed in vitro. This would give an insight into whether the changes observed in vitro are likely to be responsible for the observed in vivo changes. Metanephroi collected at E16.5 after maternal DEX administration for 2 days complements our in vitro data in there being reduced GDNF, AT1Rb, and AT2R expression and increased gene expression of BMP-4, TGF-1, and AT1Ra. Indeed, in most cases the changes were quantitatively similar. AT1Ra expression was upregulated, while AT1Rb and AT2R were reduced. However, at E20.5, these changes in gene expression were reversed. The reasons for this are unclear but it may represent a compensatory adaptation to reverse the detrimental effects on branching morphogenesis caused by the DEX exposure earlier in gestation. However, results from our in vitro data and the overall reduction in nephron endowment observed in the in vivo model suggest that this is insufficient to prevent a nephron deficit. Studies of maternal nutrient restriction or maternal glucocorticoid exposure have reported suppression of RRAS during the time of insult, followed by compensatory upregulation in RRAS later in development (19, 33). The differential expression patterns at two developmental time points highlight the importance of studying the long-term effects of a perturbation at more than one time point.
Conclusion
This study shows that DEX exposure in vitro for 2 days inhibits ureteric branching morphogenesis and nephrogenesis, thus suggesting that a reduction in ureteric branching morphogenesis may be a key mechanism through which DEX reduces nephron endowment. DEX may act in part through indirectly altering mRNA expression of key genes regulating branching morphogenesis. The changes in gene expression observed in vitro were reflected in vivo at the same dose and duration of DEX exposure previously reported to decrease nephron number. This strongly suggests that the reduction in nephron number seen in vivo may be a consequence of DEX-induced inhibition of ureteric branching morphogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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