Trps1通过TGF-β/Smad3信号通路控制肾脏以及输尿管芽分化和发育机制的研究
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摘要
[目的]毛发-鼻-指(趾)综合征(tricho rhino phalangeal syndrome, TRPSs)是一种以头皮毛发、颅面和骨骼发育异常为特征的遗传病,主要致病基因为Trps1。 Trpsl作为转录因子调控多种基因表达,以往研究表明其在软骨以及毛囊发育中起重要作用。我们制造并培育Trps1基因敲除(KO)小鼠并对Trps1在肾脏发育中的作用机制进行研究。本实验室以往研究证实Trps1KO小鼠胚胎肾脏间质相较野生型(WT)扩大,并且发现这主要是由于敲除Trps1基因后,控制间充质细胞分化的间质上皮转化(MET)受到影响。Wnt9b和Wnt4原位杂交显示在Trps1KO小鼠14.5天的胚胎肾中,输尿管芽分叉以及与之相连的帽间充质分布减少。这些发现均表明,Trps1基因对于肾脏发育中的肾单位形成具有必要作用。由于胚胎期输尿管芽分支的减少将导致出生后wilms瘤,多囊肾,并增加高血压,慢性肾病的风险,因此对于Trps1基因如何调控输尿管芽以及集合管发育进行研究具有临床意义。此项研究主要探讨与揭示Trps1敲除后输尿管芽及集合管减少的生物分子学机制。
[方法]T型输尿管自胚胎期11.5天Trsp1WT和KO小鼠肾脏中分离,放置于matrigel中培养(生物胶用于器官3D培养)。同时将胚胎期12.5天Trsp1WT和KO小鼠肾脏放置于过滤膜上培养。输尿管芽培养4天后使用RNeasy Mini Kit提取总RNA,并利用DNA基因芯片以及基因芯片3000扫描仪以分析统计基因在WT和KO两组间的差异表达。之后分析Trps1WT和KO胚胎期小鼠肾脏的体内基因表达。首先通过整体原位杂交,实时定量PCR和免疫荧光染色确定肾脏发育正性调控因子RET和GDNF的表达水平。由于众多位于TGF-β Smad3通路中的基因在DNA芯片结果中有差异,我们利用实时定量PCR, western blot和免疫荧光双重染色重点分析了通路中的调控因子Rblccl, Arkadial,磷酸化Smad3(psmad3), Smurf2, Smad7, c-Ski和磷酸化p38(pp38)在Trps1WT和KO胚胎肾脏中的表达位置及表达量的差异。同时使用ELISA和免疫荧光双重染色定位量化确定分泌型TGF-β1蛋白。凋亡与增殖对于输尿管芽发育起主要作用,因此我们还通过TUNEL染色及PCNA表达分析胚胎肾脏中输尿管芽凋亡与增殖情况。最后,于输尿管芽和肾脏培养时添加Smad3抑制剂及TGF-β1,观察输尿管芽和肾脏发育情况。此研究项目中所有统计分析均通过SPSS13.0进行。
[结果]
1. Trps1KO小鼠胚胎肾脏中输尿管芽分叉和体外培养输尿管芽相较野生型均受到抑制。于胚胎期14.5天和16.5天输尿管芽分别减少46%和59%。分离自胚胎期11.5天T型输尿管芽在WT和KO中无差别,3D培养4天后KO芽尖数量减少,分支宽度增加。我们首先分析肾脏发育正性调控因子RET和GDNF,于12.5天胚胎发育期中两者无差别,然而两者在胚胎期14.5天Trpsl KO肾脏中mRNA和蛋白水平开始下降。
2.DNA芯片分析基因在Trpsl WT和KO输尿管芽表达差异明显。为了确定敲除Trpsl对于其他肾脏发育基因表达的影响,利用DNA微阵列分析培养4天的WT和KO输尿管芽基因表达差异。在Trpsl KO中,Smad3和Arkadial cDNA分别升高2.67倍和2.26倍,同时Smad7以及Smurf2作为TGF-β通路的抑制分子降低至少50%。尽管Cpal, Kdm5d, Hoxa13,和Croxosl作为在胰脏导管发育中重要的基因在DNA芯片结果中发现有表达变化,然而经过进一步分析其在WT和KO肾脏中无统计学差异。GDNF/Ret通路正常情况下正性调控肾脏发育,我们分析上下游分子包括分泌因子Wntll,受体Ret,转录调控因子Spryl,和转录因子Etv4and Etv5。各个基因mRNA水平在两种基因型小鼠肾脏中均无差异。同时我们还分析另一重要正性调控系统成纤维细胞生长因子FGF通路,其在WT和KO肾脏中也无变化。
3. TGF-β/Smad3信号通路中的基因在Trpsl KO胚胎肾脏中发生改变。与DNA array结果相契合,我们进一步分析发现TGF-β/Smad3信号通路及其调控因子在不同时期的Trps1KO胚胎肾脏中均有变化,此结果进一步表明Trps1通过TGF-β/Smad3信号通路控制肾脏和输尿管芽发育。Arkadia和Rblccl通过分解Smad7和c-Ski蛋白正性调控TGF-β/Smad3信号通路,前两者mRNA和蛋白水平在Trps1KO中明显升高.后两者Smad7和c-Ski解离psmad3和smad4的结合并抑制psmad3向核内转运,其mRNA在Trpsl KO胚胎期14.5和16.5天无变化,然而Western blot和immunofluorescence显示蛋白质于输尿管芽中明显降低。Smurf2可解离TGF-β受体与smad3的结合,蛋白质水平在Trps1KO'肾脏中也降低。磷酸化smad位于TGF-β通路的中心位置,其往核内的转运调控下游多种转录因子表达。我们发现pSmad3稳定表达与Trps1KO输尿管芽的细胞核中,特别位于输尿管芽尖的细胞核中,然而在野生型肾脏中只有少量psamd3表达于输尿管芽细胞.
4.进一步检测发现TGF-β1和磷酸化p38(p-p38)在Trps1KO胚胎肾脏中表达量增加。首先利用Elisa试剂盒发现分泌型TGF-β1总量在KO肾脏中显著升高。免疫荧光双染显示TGF-β1强染色主要位于Trps1KO胚胎肾脏的帽间充质细胞,输尿管芽尖,以及肾囊胞。而胚胎期14.5天的野生型肾脏仅有少量阳性染色位于间充质细胞,16.5天的肾脏几乎无阳性表达。由于磷酸化p38位于TGF-β下游,并且pp38也可反向正调控TGF-β的升高,我们检测pp38在输尿管芽的表达量。其表达位置以及表达量在Trps1KO肾脏中与TGF-β相同。Western blot证明在Trps1KO肾脏中pp38蛋白量升高,尽管p38mRNA于WT和KO肾脏中无统计学差异。
5. Trps1调节输尿管芽细胞凋亡与增殖。细胞凋亡与增殖间的平衡对于输尿管芽分支和肾单位形成起到重要的调节作用。PCNA阳性免疫组化染色在Trps1KO肾脏和输尿管芽中显著减少,表明Trps1KO'肾脏的细胞增殖降低。TUNEL阳性细胞在Trps1KO肾脏和输尿管芽中相比野生型肾脏升高,说明Trps1敲除后,肾脏细胞凋亡提升。
6. TGF-β1/Smad3通路直接调控输尿管分叉与分化。前面结果已显示TGF-β1/Smad3通路中的成员在Trps1KO中表达变化差异明显,为了进一步证明TGF-β1/Smad3通路直接调控输尿管芽的分化,11.5天的输尿管芽与12.5天的胚胎肾脏在添加了TGF-β1或者Smad3抑制剂SIS3的培养基中培养。大量TGF-β1可以明显抑制输尿管芽与肾脏的分化和发育。与之相反,Smad3抑制剂SIS3增加Trps1KO输尿管芽的分支,逆转了Trps1基因敲除对于肾脏发育的抑制作用。
[结论]
1. Trps1基因敲除小鼠的胚胎肾以及体外培养输尿管芽形态表型异常,包括分支数量减少,长度延长以及宽度增加。
2. Trps1KO胚胎肾脏及输尿管芽细胞凋亡增加,增殖减少。
3.多数肾脏发育正性调控因子在WT和KO之间无统计学差异,然而GDNF和RET在Trps1KO'肾脏发育中表达降低。
4. TGF-β/smad3通路过度激活,其组成因子以及调控因子在Trps1KO胚胎肾脏中表达改变
5. TGF-β1和pp38于Trps1KO肾脏中上调,并且两者互为因果。
6. Smad3抑制剂SIS3可逆转敲除Trps1基因后输尿管芽的分化抑制
7.通过体外及体内实验证明,Trps1敲除后TGF-β/smad3通路在胚胎肾脏中过度激活,并直接调控输尿管芽分化及表型。
[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 UBs
3. 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 kidney
5. TGF-β1and pp38are upregulated in Trpsl KO kidney; and they may be reciprocal causation
6. 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.
[方法]T型输尿管自胚胎期11.5天Trsp1WT和KO小鼠肾脏中分离,放置于matrigel中培养(生物胶用于器官3D培养)。同时将胚胎期12.5天Trsp1WT和KO小鼠肾脏放置于过滤膜上培养。输尿管芽培养4天后使用RNeasy Mini Kit提取总RNA,并利用DNA基因芯片以及基因芯片3000扫描仪以分析统计基因在WT和KO两组间的差异表达。之后分析Trps1WT和KO胚胎期小鼠肾脏的体内基因表达。首先通过整体原位杂交,实时定量PCR和免疫荧光染色确定肾脏发育正性调控因子RET和GDNF的表达水平。由于众多位于TGF-β Smad3通路中的基因在DNA芯片结果中有差异,我们利用实时定量PCR, western blot和免疫荧光双重染色重点分析了通路中的调控因子Rblccl, Arkadial,磷酸化Smad3(psmad3), Smurf2, Smad7, c-Ski和磷酸化p38(pp38)在Trps1WT和KO胚胎肾脏中的表达位置及表达量的差异。同时使用ELISA和免疫荧光双重染色定位量化确定分泌型TGF-β1蛋白。凋亡与增殖对于输尿管芽发育起主要作用,因此我们还通过TUNEL染色及PCNA表达分析胚胎肾脏中输尿管芽凋亡与增殖情况。最后,于输尿管芽和肾脏培养时添加Smad3抑制剂及TGF-β1,观察输尿管芽和肾脏发育情况。此研究项目中所有统计分析均通过SPSS13.0进行。
[结果]
1. Trps1KO小鼠胚胎肾脏中输尿管芽分叉和体外培养输尿管芽相较野生型均受到抑制。于胚胎期14.5天和16.5天输尿管芽分别减少46%和59%。分离自胚胎期11.5天T型输尿管芽在WT和KO中无差别,3D培养4天后KO芽尖数量减少,分支宽度增加。我们首先分析肾脏发育正性调控因子RET和GDNF,于12.5天胚胎发育期中两者无差别,然而两者在胚胎期14.5天Trpsl KO肾脏中mRNA和蛋白水平开始下降。
2.DNA芯片分析基因在Trpsl WT和KO输尿管芽表达差异明显。为了确定敲除Trpsl对于其他肾脏发育基因表达的影响,利用DNA微阵列分析培养4天的WT和KO输尿管芽基因表达差异。在Trpsl KO中,Smad3和Arkadial cDNA分别升高2.67倍和2.26倍,同时Smad7以及Smurf2作为TGF-β通路的抑制分子降低至少50%。尽管Cpal, Kdm5d, Hoxa13,和Croxosl作为在胰脏导管发育中重要的基因在DNA芯片结果中发现有表达变化,然而经过进一步分析其在WT和KO肾脏中无统计学差异。GDNF/Ret通路正常情况下正性调控肾脏发育,我们分析上下游分子包括分泌因子Wntll,受体Ret,转录调控因子Spryl,和转录因子Etv4and Etv5。各个基因mRNA水平在两种基因型小鼠肾脏中均无差异。同时我们还分析另一重要正性调控系统成纤维细胞生长因子FGF通路,其在WT和KO肾脏中也无变化。
3. TGF-β/Smad3信号通路中的基因在Trpsl KO胚胎肾脏中发生改变。与DNA array结果相契合,我们进一步分析发现TGF-β/Smad3信号通路及其调控因子在不同时期的Trps1KO胚胎肾脏中均有变化,此结果进一步表明Trps1通过TGF-β/Smad3信号通路控制肾脏和输尿管芽发育。Arkadia和Rblccl通过分解Smad7和c-Ski蛋白正性调控TGF-β/Smad3信号通路,前两者mRNA和蛋白水平在Trps1KO中明显升高.后两者Smad7和c-Ski解离psmad3和smad4的结合并抑制psmad3向核内转运,其mRNA在Trpsl KO胚胎期14.5和16.5天无变化,然而Western blot和immunofluorescence显示蛋白质于输尿管芽中明显降低。Smurf2可解离TGF-β受体与smad3的结合,蛋白质水平在Trps1KO'肾脏中也降低。磷酸化smad位于TGF-β通路的中心位置,其往核内的转运调控下游多种转录因子表达。我们发现pSmad3稳定表达与Trps1KO输尿管芽的细胞核中,特别位于输尿管芽尖的细胞核中,然而在野生型肾脏中只有少量psamd3表达于输尿管芽细胞.
4.进一步检测发现TGF-β1和磷酸化p38(p-p38)在Trps1KO胚胎肾脏中表达量增加。首先利用Elisa试剂盒发现分泌型TGF-β1总量在KO肾脏中显著升高。免疫荧光双染显示TGF-β1强染色主要位于Trps1KO胚胎肾脏的帽间充质细胞,输尿管芽尖,以及肾囊胞。而胚胎期14.5天的野生型肾脏仅有少量阳性染色位于间充质细胞,16.5天的肾脏几乎无阳性表达。由于磷酸化p38位于TGF-β下游,并且pp38也可反向正调控TGF-β的升高,我们检测pp38在输尿管芽的表达量。其表达位置以及表达量在Trps1KO肾脏中与TGF-β相同。Western blot证明在Trps1KO肾脏中pp38蛋白量升高,尽管p38mRNA于WT和KO肾脏中无统计学差异。
5. Trps1调节输尿管芽细胞凋亡与增殖。细胞凋亡与增殖间的平衡对于输尿管芽分支和肾单位形成起到重要的调节作用。PCNA阳性免疫组化染色在Trps1KO肾脏和输尿管芽中显著减少,表明Trps1KO'肾脏的细胞增殖降低。TUNEL阳性细胞在Trps1KO肾脏和输尿管芽中相比野生型肾脏升高,说明Trps1敲除后,肾脏细胞凋亡提升。
6. TGF-β1/Smad3通路直接调控输尿管分叉与分化。前面结果已显示TGF-β1/Smad3通路中的成员在Trps1KO中表达变化差异明显,为了进一步证明TGF-β1/Smad3通路直接调控输尿管芽的分化,11.5天的输尿管芽与12.5天的胚胎肾脏在添加了TGF-β1或者Smad3抑制剂SIS3的培养基中培养。大量TGF-β1可以明显抑制输尿管芽与肾脏的分化和发育。与之相反,Smad3抑制剂SIS3增加Trps1KO输尿管芽的分支,逆转了Trps1基因敲除对于肾脏发育的抑制作用。
[结论]
1. Trps1基因敲除小鼠的胚胎肾以及体外培养输尿管芽形态表型异常,包括分支数量减少,长度延长以及宽度增加。
2. Trps1KO胚胎肾脏及输尿管芽细胞凋亡增加,增殖减少。
3.多数肾脏发育正性调控因子在WT和KO之间无统计学差异,然而GDNF和RET在Trps1KO'肾脏发育中表达降低。
4. TGF-β/smad3通路过度激活,其组成因子以及调控因子在Trps1KO胚胎肾脏中表达改变
5. TGF-β1和pp38于Trps1KO肾脏中上调,并且两者互为因果。
6. Smad3抑制剂SIS3可逆转敲除Trps1基因后输尿管芽的分化抑制
7.通过体外及体内实验证明,Trps1敲除后TGF-β/smad3通路在胚胎肾脏中过度激活,并直接调控输尿管芽分化及表型。
[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 UBs
3. 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 kidney
5. TGF-β1and pp38are upregulated in Trpsl KO kidney; and they may be reciprocal causation
6. 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.
引文
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