| [Objective] TRPS1is the gene responsible for the Tricho-rhino-phalangeal syndromes, which are characterized by sparse and slow-growing scalp hair as well as craniofacial and skeletal abnormalities. To investigate the role of Trpsl during kidney development, we generated Trps1KO mice. In a previous study, we demonstrated that Trps1-deficient (KO) mice show an expanded renal interstitium compared to wild-type (WT) mice, because the loss of Trpsl affects the mesenchymal-epithelial transition (MET) in the cap mesenchyme and ureteric bud (UB) branching. Wnt9b and Wnt4revealed decreased branching of the UBs and sparse distribution of the cap mesenchyme in E14.5kidneys from Trpsl KO mice. These findings indicated that Trpsl is essential for normal nephron formation during early renal development. However, how Trpsl regulates UB branching is still unknown. In this study, we unveil the molecular mechanisms by which the loss of Trps1suppresses UB branching.[Methods] Firstly, UBs were isolated from the E11.5WT and Trpsl KO embryonic kidneys and cultured in Matrigel, while Whole kidneys isolated from E12.5WT and Trps1KO embryos were cultured on Transwell filters. The UBs were removed from the Matrigel on day4of culture, and total RNA was isolated from batches of35-pooled UBs using RNeasy Mini Kit. We compared gene expression patterns by a DNA GeneChip Array (Mouse Gene1.0ST Array, Affymetrix) and statistical analyses were conducted with a GeneChip3000Scanner using the Comparison Analysis feature to identify genes that were differentially expressed in the two groups. Levels of positive signaling for kidney development in Trpsl E12.5,14.5and16.5WT and KO embryonic kidney were verified by whole-mount in situ hybridization (ISH), real-time PCR (qPCR), and immunofluorescence, including RET and GDNF. Aberrant expression of genes associated with the transforming growth factor (TGF)-β/Smad3signaling pathway in the E14.5,16.5and18.5KO UBs compared with WT UBs were tested by real-time PCR (qPCR), western blot and double staining immunofluorescence, including Rb1ccl, Arkadia1, phosphorylated Smad3(psmad3), Smurf2, Smad7, c-Ski and phosphorylated p38(pp38). Total activated TGF-β1protein was measured with commercially available ELISA kits and immunofluorescence. In addition, TUNEL staining and immunohistochemistry for PCNA showed the apoptosis and proliferation of UB cells, respectively. Finally, we applied exogenous addition of Smad3inhibitor SIS3for the restoration of UB branching in the KO UBs, whereas the addition of TGF-β1for the suppression of UB branching in the organ culture of both the isolated UBs from E11.5embryos and the whole embryonic kidneys from E12.5embryos. To evaluate and quantify branching in the cultured UBs, photomicrographs were taken using a camera attached to a stereomicroscope and were analyzed using Image-Pro software. All data were analyzed by the SPSS13.0software.[Results]1. UB branching is suppressed in embryonic kidneys and cultured UBs from Trpsl KO mice. The numbers of UB tips in the KO kidneys were decreased to46%and59%of the numbers observed in WT kidneys on E14.5and E16.5, respectively. T-shaped UBs from E11.5KO kidneys showed no differences in size and shape compared to the WT UBs, however, on day4of culture, quantification of the cultured UBs revealed that the KO UBs had decreased numbers of tips and branching compared to the WT UBs. In addition, the length of the stalk in the KO UBs was longer than that in the WT UBs. These were no clear difference in mRNA expression and protein levels of Ret and GDNF between E12.5kidneys from the WT and KO mice, however, on E14.5and E16.5, mRNA and protein expression was decreased in the KO kidneys.2. Gene expression measured through DNA array is altered in Trpsl KO UBs. To identify genes whose expression is altered by the loss of Trpsl, we conducted a DNA microarray analysis using isolated UBs from WT and Trps1KO kidneys. In Trpsl KO UBs, the expression levels of Smad3and Arkadial were increased2.67-fold and2.26-fold, respectively, compare to WT UBs, whereas the levels of Smad7and Smurf2, which are inhibitors of TGF-P signaling, were decreased by at least50%. Although the Cpal, Kdm5d, Hoxal3, and Croxos1expression levels were also altered, after further analysis, there were no significant differences in these measures. Genes regulated by GDNF, such as those for a secreted factor (Wnt11), a receptor (Ret), a regulator of signal transduction (Spry1), and for transcription factors (Etv4and Etv5), did not display significant differences in mRNA expression levels between the WT and KO UBs. Additionally, the mRNA levels of fibroblast growth factors (FGFs), which regulate Etv4and Etv5, showed no significant differences between the WT and KO UBs3. Expression levels of genes in the TGF-β/Smad3signaling pathway are altered in Trpsl KO embryonic kidneys. In agreement with our DNA array results showing that the levels of several regulators of the TGF-β signaling pathway were altered, we assumed that TGF-β signaling might affect UB branching. Both the Arkadial and Rblccl mRNA and protein levels rose in Trps1KO kidneys to levels much higher than those found in the WT kidneys. There were no significant differences in Smad7and c-Ski mRNA levels at E14.5and E16.5between WT and Trpsl KO kidneys. The protein levels, however, were dramatically reduced in the Trpsl KO kidneys compared to the WT kidneys, as determined by Western blot and immunofluorescence. Western blot analysis also showed that the levels of Smurf2protein were lower in the Trpsl KO kidneys than in the WT kidneys. Given the critical roles of TGF-β signaling in kidney development, phosphorylated Smad3(p-Smad3) is predicted to be central to the inhibition of UB branching. P-Smad3was readily detected in the nuclei of KO UB cells, especially in the tips, whereas only a few p-Smad3expressing cells were observed in the WT UB tips.4. The expression of TGF-β1and phosphorylated-p38(p-p38) is upregulated in Trpsl KO embryonic kidneys. The total amount of TGF-βmeasured by Elisa was significantly increased in the KO kidneys compared to the WT kidneys. In addition, immunofluorescence revealed that intense staining of TGF-β1was largely observed in the cap mesenchyme, UB tips, and renal vesicles in the Trpsl KO kidneys. However, only slight positive staining was observed in the cap mesenchyme on E14.5, and virtually no positive staining was observed in the stromal cells of E16.5WT kidneys. We also found intense p-p38staining in the UB tips, renal vesicles, and cap mesenchyme of Trpsl KO kidneys (Fig.6F). Conversely, virtually no staining was observed in the WT UBs. Total p38mRNA levels were not significantly different between the WT and KO kidneys, whereas the level of p-p38protein was increased in the KO kidneys compared to the WT kidneys.5. Trpsl regulates cell proliferation and apoptosis in UB tips in normal UB branching. A balance between cell proliferation and apoptosis is important to UB branching and nephron formation. Immunohistochemistry for PCNA clearly showed that the number of PCNA-positive cells was significantly decreased in the KO UB tips and in the whole KO kidney compared to WT. In regards to apoptosis, the numbers of TUNEL-positive cells in the whole kidney or in the UBs were increased in the Trpsl KO kidneys as compared to the WT kidneys. The changes in both TUNEL and PCNA staining were mainly observed in the UB tips.6. TGF-p/Smad3signaling regulates UB branching in the WT and KO embryonic kidneys. To further examine whether TGF-β/Smad3signaling plays crucial roles in UB branching, the isolated UBs and the whole embryonic kidneys were cultured in the presence of TGF-β or Smad3inhibitor SIS3. In the isolated UB culture, the growth and branching were dramatically inhibited by the exogenous addition of TGF-β1in both the WT and KO UBs. By contrast, the exogenous addition of SIS3to the isolated UBs restored the growth and branching of the KO UBs to the same levels of the WT UBs.[Conclusions]1. Ureteric bud morphogenesis is abnormal in Trps1KO mice and in vitro culturing UB, including branching decrease, stalks elongation and thicker tips.2. Trpsl KO embryonic kidney and UB have aberrant apoptosis and decreased proliferation.3. Most positive regulators for UB branching show similar levels in Trpsl KO mice, however GDNF and RET decreased in the late stage of Trpsl KO kidney development.4. The TGF-β/Smad3pathway members are altered in Trpsl KO embryonic kidney5. TGF-β1and pp38are upregulated in Trpsl KO kidney; and they may be reciprocal causation6. Smad3inhibitor SIS3rescued the suppressed UB branching in the KO UBs and kidneys.7. Taken together, we concluded that the loss of Trps1promotes TGF-β/Smad3signaling, which directly regulates the morphogenesis of UB branching. |
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