Kruppel-like factor 5 is Required for Urothelial Maturation

Kruppel-like transcription factor 5 (Klf5) is expressed during late embryogenesis in the forming murine bladder urothelium. Targeted disruption of the Klf5flox alleles by the ShhGfpCre transgene resulted in failure of the bladder urothelium to mature accompanied by hydronephrosis, hydroureter, and vesicoureteric reflux in all E18.5 fetuses. The bladder urothelium did not stratify nor did it express terminal differentiation markers characteristic of basal, intermediate, and umbrella cells including keratins 20, 14, and 5, and uroplakins. At E18.5, an ectopic alpha smooth muscle actin positive layer of cells was identified subjacent to the undifferentiated Klf5-deficient urothelium. The effects of Klf5 deficiency were unique to the urothelium since maturation of the epithelium comprising the bladder neck and urethra were unaffected by the lack of KLF5. mRNA microarray analysis of whole E14.5 control and Klf5 deficient bladders identified Ppar-gamma and Grhl3 as putative downstream intermediary transcription factors that regulate urothelial maturation. Transient transfection assays demonstrated that KLF5 regulated expression of the mGrhl3 promoter. These observations show that alterations in maturation of the bladder urothelium alone are sufficient to induce bladder dysfunction leading to prenatal hydronephrosis. Total RNA from E14.5 whole bladders isolated from Klf5D/D and Klf5+/+ShhGfpCre+ embryos (n=3 samples/genotype, each sample a pool of two bladders) was isolated using the Qiagen MicroRNA Kit. The cDNA was then hybridized according to the manufacturer's protocol to the Affymetrix Mouse Gene 1.0 ST ArrayKruppel样转录因子5（Klf5）在小鼠膀胱尿路上皮形成阶段的胚胎发育晚期表达。通过ShhGfpCre转基因对Klf5 flox等位基因进行靶向敲除后，所有E18.5胎鼠的膀胱尿路上皮均无法正常成熟，并伴随肾积水、输尿管积水以及膀胱输尿管反流。该膀胱尿路上皮既未形成复层结构，也无法表达基底细胞、中间细胞及伞细胞所特有的终末分化标志物，包括角蛋白20、14、5以及尿斑蛋白（uroplakins）。在E18.5胎鼠中，于未分化的Klf5缺陷型尿路上皮下方，可观察到一层异位表达α平滑肌肌动蛋白（alpha smooth muscle actin，α-SMA）的细胞层。Klf5缺陷所产生的表型仅局限于尿路上皮，因为膀胱颈及尿道上皮的成熟过程并未因KLF5缺失而受到影响。对E14.5胎鼠的正常及Klf5缺陷型完整膀胱进行mRNA芯片（mRNA microarray）分析后，发现过氧化物酶体增殖物激活受体γ（Ppar-gamma）和粒状头样转录因子3（Grhl3）可作为调控尿路上皮成熟的潜在下游中介转录因子。瞬时转染实验证实，KLF5可调控mGrhl3启动子的表达。上述研究结果表明，仅膀胱尿路上皮成熟异常即可引发膀胱功能障碍，进而导致产前肾积水。研究人员采用Qiagen MicroRNA试剂盒，从Klf5D/D及Klf5+/+ShhGfpCre+胚胎的E14.5胎鼠完整膀胱中提取总RNA；每组基因型设置3个生物学重复样本，每个样本由2个膀胱混合制备。随后按照试剂盒说明书的操作流程，将制备得到的cDNA与Affymetrix小鼠基因1.0 ST芯片（Affymetrix Mouse Gene 1.0 ST Array）进行杂交。