【作者】 胡波；

【导师】 贺修胜； 吴冠青；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 研究背景常染色体隐性多囊肾（Autosomal recessive polycystic kidney disease, ARPKD）是一种多发于婴幼儿的遗传性疾病,全球发病率约为1:20000,其主要临床症状为双肾集合管和肝胆管扩张伴肝肾纤维化,约50%患者在胎儿期已经发病,出生时伴随因60-90%集合管扩张引起的双肾梭形囊肿,这些患者常在婴儿期即由于呼吸和肾功能障碍而死亡,死亡率约为30%。ARPKD的主要致病基因是Pkhd1,该基因定位于人染色体6p21.1-12,编码一个由4074个氨基酸组成的单跨膜受体样蛋白Fibrocystin（FPC）。FPC定位于肾上皮细胞的初级纤毛和基体内。多囊肾的发生很可能由人类肾脏小管生长发育分子失调所造成,因此揭示多囊肾发病机制,将有助于揭示肾脏小管发生、发育的分子机制。目前,我们已通过基因打靶技术建立Pkhd1基因第15和16号外显子敲除小鼠模型,由于Pkhd1基因缺失,该小鼠模型的肝和肾表现出不同程度的管道扩张、囊肿和纤维化;另外,Pkhd1缺失小鼠的胰腺和脑组织也出现囊肿和小管扩张;这些症状很好地模拟了人类ARPKD。为阐明Pkhd1基因缺失在ARPKD发生发展中的作用及其相关机制,本课题拟利用我们新建立的Pkhd1基因敲除模型和肾集合管上皮细胞株,观察FPC缺失对肾集合管上皮细胞增殖和凋亡的影响,并研究其可能存在的相关机制,为探索ARPKD的发病机制和今后靶向治疗提供新思路。第一部分ARPKD致病基因Pkhd1细胞生物学基本功能的研究目的:利用我们新建立的Pkhd1基因敲除模型和肾集合管上皮细胞株,观察FPC缺失对肾集合管上皮细胞生物学的影响。方法:1.将Pkhd1杂合子小鼠（Pkhd1+/-）与Immorto （Im）小鼠交配产生Im::Pkhd1-/-小鼠和同窝Im::WT野生型小鼠。从8周龄Im::Pkhd1-/-小鼠和同窝Im::WT小鼠中取出肾脏,应用Dolichus biflorus agglutinin （DBA）分离方法,获得永生化肾集合管上皮细胞系。利用免疫荧光染色法,检测永生化肾集合管细胞DBA（集合管细胞标志物）、E-cadherin和Cytokeratin（上皮细胞标准物）的表达情况,鉴定细胞原始来源。施用PCR、实时定量PCR、RT-PCR和Western blot方法,分别从DNA、RNA及蛋白水平鉴定Pkhd1野生型和纯合型细胞Pkhd1表达情况。2.应用胶原/基质胶三维（3D）培养法,观察FPC缺失肾集合管上皮细胞形成管状分支结构状况。3.用ZO-1和E-cadherin抗体进行免疫荧光染色和Western blot方法,检测FPC缺失是否破坏肾集合管上皮细胞紧密连接和间隙连接。4.通过EVOM/STX2电阻测量仪测定细胞跨膜电阻抗（TER）,观测FPC缺失对细胞紧密连接的影响。5.使用罗丹明-鬼笔环肽抗体对细胞F-actin进行免疫荧光染色,观察FPC缺失后肾集合管上皮细胞中F-actin形态和分布的改变。6.应用细胞粘附和细胞穿膜实验,检测FPC缺失是否影响细胞-胞外基质间的相互作用。7.用Acetylated-α-tubulin抗体进行免疫荧光染色,并利用Zeiss LSM成像系统进行多层扫描叠加,以观察FPC缺失对细胞纤毛结构和数量的影响。8.采用3^H（氚）-TdR掺入法,检测肾集合管上皮细胞的增殖效应。9.应用磷酸化组蛋白H3和PCNA抗体分别进行免疫荧光染色、免疫组织化学染色和Western blot,从体内、外检测肾上皮细胞增殖情况。10.利用末端转移酶介导的dUTP缺口末端标记（TUNEL）和Caspase-3免疫荧光染色法（或免疫组织化学）,分别从体内、外观察肾上皮细胞凋亡。结果:1.应用Dolichus biflorus agglutinin （DBA）分离方法,从8周龄Im::Pkhd1^-/-小鼠和同窝Im::WT小鼠肾脏中,获得38个Im::Pkhd1^-/-和Im::WT永生化肾集合管细胞系;随后,用DBA、E-cadherin和Cytokeratin鉴定细胞原始来源,最终获得26个Im::Pkhd1^-/-纯合型和21个Im::WT野生型来源于肾脏集合管的细胞克隆。我们随机选取四株细胞W10B6、W10B2（Im::WT）和M10H2、M10C7(Im::Pkhd1^-/-)用于进一步实验研究;应用PCR鉴定各株细胞基因型,结果显示,野生型为270bp,Pkhd1^-/-为380bp;同时,通过实时定量PCR和RT-PCR证实野生型细胞株表达Pkhd1,而Pkhd1^-/-细胞株不表达Pkhd1;利用我们实验室前期制备的FPC单克隆抗体hAR-C2m4E12进行Western blot检测,结果显示,Pkhd1^-/-细胞株没有FPC的表达,而只有野生型细胞表达FPC。2.在胶原/基质胶三维（3D）培养条件下野生型肾集合管上皮细胞（W10B6、W10B2）约60%的细胞能形成4个及其以上的分支,约40%的细胞能形成1-3个分支,仅极少数（不足5%）细胞不能形成分支结构;而纯合型细胞（M10H2、M10C7）约95%的细胞不能建立管状分支结构,形成肾囊样结构,并具有细胞聚集性,约5%的细胞能形成1-3个分支管状结构,仅不足2%的细胞形成4个以上的分支结构。3.用ZO-1抗体进行免疫荧光染色,发现野生型（W10B6、W10B2）细胞具有整齐而规则的紧密连接,而纯合型（M10H2、M10C7）细胞连接呈不规则弯曲形状,并且许多细胞间紧密连接不完整;用E-cadherin抗体进行免疫荧光染色,结果显示,野生型（W10B6、W10B2）细胞E-cadherin主要分布在细胞-细胞连接处,而纯合型（M10H2、M10C7）细胞E-cadherin在细胞连接处分布不连续,且在胞浆内呈大量弥散分布;然而,Western blot检测两者细胞中ZO-1和E-cadherin蛋白表达均无显著性差异。4.利用EVOM/STX2电阻仪测量细胞跨膜电阻情况,结果表明,野生型（W10B6、W10B2）细胞从第4天开始跨膜电阻明显升高,表明此时细胞已经建立了紧密连接,并能持续至第7天;而纯合型（M10H2、M10C7）细胞跨膜电阻增加缓慢,且在第7天时开始出现下降趋势。5.用罗丹明-鬼笔环肽抗体对细胞F-actin进行免疫荧光染色,100倍显微镜下结果显示,野生型（W10B6、W10B2）细胞F-actin具有整齐而均一的应力纤维,主要分布在细胞-细胞连接处,而纯合型（M10H2、M10C7）细胞F-actin分布有纤细而不规则弯曲的应力纤维,并且细胞间连接不完整;激光共聚焦观察野生型（W10B6、W10B2）细胞F-actin主要分布在细胞周边和核周围,分布均匀、排列整齐紧密,细胞间连接紧密,没有间隙形成,在细胞与细胞接触处呈束状相互连接,形成周边肌动蛋白丝带,而纯合型（M10H2、M10C7）细胞F-actin排列紊乱,变短,变细,应力纤维断裂,形成细胞间隙。在胶原/基质胶三维（3D）培养下观察F-actin分布,野生型（W10B6、W10B2）细胞在细胞周边和核周围可形成丰富均一的应力纤维,而纯合型（M10H2、M10C7）细胞在三维状态下仅有少量、单一的应力纤维。6.细胞粘附实验结果发现,当胶原浓度达到0.125μg/ml时,约40%的野生型（W10B6、W10B2）细胞粘附于细胞培养板,而仅15%的纯合型（M10H2、M10C7）细胞粘附于培养板,随着胶原浓度增高,野生型（W10B6、W10B2）细胞粘附进一步增强,纯合型（M10H2、M10C7）细胞并没有显著升高;细胞穿膜实验显示,纯合型（M10H2、M10C7）细胞迁移能力显著低于野生型（W10B6、W10B2）细胞。7.用Acetylated-α-tubulin抗体进行免疫荧光染色,激光共聚焦观察,约80%的野生型（W10B6、W10B2）细胞具有纤毛结构,而约30%的纯合型（M10H2、M10C7）细胞具有纤毛结构。同时,将染色细胞进行多层扫描叠加后,测量纤毛长度发现,野生型（W10B6、W10B2）细胞纤毛长度平均超过2.5μm,而纯合型（M10H2、M10C7）细胞纤毛长度平均不足1.5μm。8. 3^H（氚）-TdR掺入法结果显示,与野生型（W10B6 and W10B2）细胞相比,Pkhd1^-/- （M10H2、M10C7）细胞摄入3H的能力明显下降,提示Pkhd1缺失抑制细胞增殖。9.施用磷酸化组蛋白H3染色法,发现纯合型（M10H2、M10C7）细胞有丝分裂期细胞数明显减少（*P<0.05）。利用Western blot检测磷酸化组蛋白H3和PCNA在野生型（W10B6、W10B2）和纯合型（M10H2、M10C7）细胞中的表达,结果显示,纯合型（M10H2、M10C7）细胞中磷酸化组蛋白H3和PCNA蛋白表达水平显著低于野生型（W10B6、W10B2）细胞。应用免疫组织化学和免疫荧光染色,分别检测了Pkhd1^-/-和野生型小鼠6周,3个月,6个月肾组织中PCNA和磷酸化组蛋白H3蛋白表达情况。结果表明, Pkhd1^-/-小鼠中肾上皮细胞PCNA阳性细胞较野生型小鼠明显减少;磷酸化组蛋白H3染色也得到相似结果。10.在常规培养条件下,细胞经过Ionomycin诱导凋亡后,采用末端转移酶介导的dUTP缺口末端标记（TUNEL）法和Caspase-3活化标记检测Pkhd1野生型和纯合型细胞凋亡情况,发现纯合型细胞凋亡率为14%,显著多于野生型细胞凋亡率（5%）。为了证实体外实验结果,采用免疫组织化学和免疫荧光染色,在Pkhd1^-/-和野生型小鼠3个月,6个月,12个月肾组织中检测Caspase-3蛋白表达情况,发现Pkhd1缺失小鼠中Caspase-3活化细胞明显多于野生型小鼠。TUNEL染色检测结果证实,12个月大的Pkhd1缺失小鼠中阳性染色细胞显著高于野生型小鼠,同时,在Pkhd1缺失小鼠的肾小管中可见许多凋亡碎片。结论:1.成功建立Pkhd1野生型和纯合型小鼠肾脏集合管上皮细胞。2. Pkhd1为肾集合管细胞在3D培养条件下管状分支结构形成所必须。3. FPC缺失可导致肾集合管细胞-细胞连接、细胞-胞外基质黏附异常。4. FPC缺失能导致肾集合管纤毛结构和细胞骨架异常。5. FPC缺失可抑制肾集合管细胞增殖,促进细胞凋亡。第二部分细胞凋亡信号通路在Pkhd1缺失的肾集合管细胞中的作用目的:探讨Pkhd1缺失抑制细胞增殖,促进细胞凋亡导致ARPKD囊肿形成的分子机制。方法:1. Western blot检测磷酸化Akt改变对Pkhd1纯合型细胞凋亡信号通路的影响。2. Western blot检测Pkhd1纯合型细胞中Ras-Raf-MEK-ERK信号通路的改变。3. Western blot检测FPC缺失对细胞增殖和凋亡FAK磷酸化位点的影响。4. Western blot检测Pkhd1纯合型细胞中Akt上游信号通路PI3K-PDK1的改变。结果:1. Western blot结果显示,经CI诱导后,Pkhd1纯合型（M10H2、M10C7）细胞中磷酸化Akt（Ser473）活性较野生型（W10B6、W10B2）细胞明显下降,而Pkhd1纯合型细胞Bax、Caspase-9和Caspase-3表达均明显高于野生型细胞。2. Western blot结果表明,经CI诱导10,30,60分钟后,磷酸化c-Raf、Pan-Ras、磷酸化MEK和磷酸化ERK在Pkhd1纯合型（M10H2、M10C7）细胞中明显下降,而B-Raf表达与野生型细胞相比差异没有显著性意义。3. Western blot结果显示,经CI不同时间点诱导的纯合型（M10H2、M10C7）细胞与野生型（W10B6、W10B2）细胞相比较, FAK pY 861,397, 576和925均明显下降。4.我们首先应用PI3K classⅢ和PI3K p110α抗体进行分析,结果发现PI3K classⅢ在纯合型（M10H2、M10C7）和野生型（W10B6、W10B2）细胞中差异没有显著性意义。相反,PI3K p110α在正常培养和经CI诱导的纯合型中比野生型细胞显著下降。进而,我们分析了PDK1在纯合型和野生型细胞中磷酸化改变情况,与PI3Kp110α结果相似,PDK1p241磷酸化水平在正常培养和CI诱导的纯合型细胞中明显低于野生型细胞。结论:1. FAK-PI3K-Akt-Caspase 3凋亡信号通路可能参与了Pkhd1缺失的肾集合管上皮细胞囊肿形成。2. FPC缺失可能经Ras-c-Raf-MEK-ERK信号通路抑制肾集合管上皮细胞增殖。更多还原

【Abstract】 BackgroundAutosomal recessive polycystic kidney disease （ARPKD） is one of the most common hereditary renal cystic diseases in infants and children, with an estimated incidence of ¹ in 20,000 live births and prevalence ¹ in 70 for heterozygosity. ARPKD is characterized by cystic dilatation of collecting ducts of the kidney and hepatic abnormalities consisting of bile duct dysgenesis and periportal fibrosis. Approximately 50% of patients with ARPKD present with their disease as neonates and are born with two very large kidneys with 60 to 90% of the renal tubules being ectatic. These neonates suffer a 30% mortality rate as a result of respiratory and/or renal dysfunction. ARPKD is caused by mutations in Pkhd1, which encodes a 16-kb transcript, contains at least 86 exons, and spans 470 kb on chromosome 6p12. The longest ORF is predicted to have 66 exons and to yield a 4074-amino acid membrane-associated receptor-like protein, fibrocystin/polyductin （FPC）. FPC has been localized to primary cilia and basal body.The cystogenesis of kidney probably results in the molecular dysfunction of growth and development of renal collecting ducts in human. Illustrating the molecular mechanism of the cystogenesis will greatly contribute in the molecular mechanism of the growth and development of renal collecting ducts. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted exon 15 and 16 deletion in the mouse orthologue of human Pkhd1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. Pkhd1?/? mice that escaped embryonic lethality and survived into adulthood exhibited mild to severe tubular dilation or cyst formation in the kidney and liver accompanied by fibrosis and necrosis. Cystic or dilated-duct phenotypes were also seen in the pancreas and brain of Pkhd1?/? mice. To illustrate the role and associated mechanism of Pkhd1 loss in the cystogeneiss and development in ARPKD, we observe the effect of FPC loss on the proliferation and apoptosis of renal collecting duct epithelial cells and explore the related mechanisam using our Pkhd1 knockout mouse model and renal collecting duct cell lines derived from this model. We provided new concept for the pathogenic mechanism of ARPKD cystogenesis which will lead to new therapeutic strategies for human ARPKD.Part 1FPC loss impairs tubulomorphogenesis of collecting duct epithelial cells in Pkhd1 mutant kidneysObjective:Using our Pkhd1 knockout mouse model and renal collecting duct cell lines derived from this model, we observed the biological effect of FPC loss on the renal collecting duct epithilia cells.Methods:1. We mated the Pkhd1+/- mice with Immorto mice （Im） (both with C57Bl/6 congenic background to produce Im:: Pkhd1^-/- mice and their Im::WT littermates. To establish cell lines with or without Pkhd1, kidneys from an 8-week-old Im:Pkhd1^-/- mouse and its wildtype littermate were removed and minced finely with a scalpel. A Dolichus biflorus agglutinin （DBA）-based isolation approach was used to develop immortalized renal collecting duct cell lines from the kidneys. The collecting duct cell lines with and without Pkhd1 were selected from the Im:Pkhd1^-/- and its wildtype littermate cell pool. After using E-cadherin and cytokeratin as epithelial markers and DBA as the collecting duct marker to identify their origin, we detect the Pkhd1 expression in null-Pkhd1and their wildtype control at DNA, RNA and protein levels using PCR, RT-PCR, real time PCR and Western blot methods.2. We characterized the cell lines by performing experiments in 3-D Matrigel culture to examine whether the loss of FPC induced abnormal tubulomorphogenesis in vitro.3. We also analyzed the cell-cell contacts in these cell lines. E-cadherin and ZO-1 which are putative cell-cell junctional markers were detected using immunofluorescence staining and Western blot.4. To confirm the altered cell-cell interactions by loss of FPC, we measured the cell transepithelial resistance （TER） to determine if the integrity of the cell-cell contacts was impaired.5. We performed rhodamine-phalloidin staining to label cellular cytoplasmic actin to observe the locoliazation and shape changes of F-actin by loss of FPC.6. We investigated the effect of null-Pkhd1 on the integrin-dependent adhesion to CI. We also tested the transwell migration capability upon CI between the cells with and without Pkhd1.7. To determine whether the lack of FPC also disrupts ciliogenesis in Pkhd1-deficient mice. We used IF with an anti-acetylatedα-tubulin antibody to examine the number and morphology of renal primary cilia in Pkhd1^-/- cells M10H2 and M10C7 and their wildtype littermate cells W10B6 and W10B2.8. Performing a tritiated thymidine proliferation assay, to determine whether the loss of FPC caused a decrease in renal epithelial proliferation.9. Pkhd1^-/- cells M10H2 and M10C7 and their wildtype littermate cells W10B6 and W10B2 were subjected to Phospho-Histone H3 staining to evaluate cell proliferation.10. At last, we used DeadEndTMflurometric TUNEL system or Caspase-3 Active Apoptosis Kit to examine whether apoptosis also occurred in vitro and in vivo.Results:1. To establish null-Pkhd1 cell lines, the kidneys from an 8-week-old Im::Pkhd1^-/- mouse and its Im::WT littermate were removed, and a Dolichus biflorus agglutinin （DBA）-based isolation approach was used to develop immortalized renal collecting duct cell lines of both genotypes. After a limiting dilution, 38 immortalized renal collecting duct cell colonies were isolated from each of the Im::Pkhd1^-/- and Im::WT cell pools. To identify the origin of the cell lines, we used E-cadherin and cytokeratin as epithelial markers and DBA as a collecting duct marker. By these biomarkers, 26 collecting duct cell lines with the Im::Pkhd1^-/- genotype were selected from the Im::Pkhd1^-/- cell pool, and 21 Im::WT collecting duct cell lines were selected from the Im::WT cell pool. Of these, two randomly selected lines from each pool (W10B6 and W10B2 for wildtype and M10H2 and M10C7 for Pkhd1^-/-) were used as the genotype-representative cell lines for further analysis. PCR genotyping was performed to identify cell lines with Pkhd1^-/- cells （M10H2 and M10C7） and wildtype cells （W10B6 and W10B2）. A 380bp PCR band was observed for the Pkhd1^-/- allele whereas a 270bp PCRband was observed for the wild type. Quantitative PCR verified that the wildtype cell lines expressed Pkhd1 and the Pkhd1^-/- cell lines did not. To further confirm the genotypes of these cell lines, an anti-FPC monoclonal antibody hAR-C2m4E12, which is a subclone from hAR-C2m3C10, was used to detect the FPC expression levels in the cell lines by western blot. Consistent with the quantitative PCR results, the Pkhd1^-/- cell lines did not express any detectable FPC, while the wildtype cell lines expressed it.2. Most of the wildtype cells formed normal tubular structures in the 3-D culture, and only 5-10% of them failed to exhibit tubulogenesis. In sharp contrast, ⁹5% of the 3-D cultured Pkhd1^-/- cells failed to undergo tubulogenesis （Suppl. Fig. 1A-D）. In addition, less than 5% of the colonies in the Pkhd1^-/- cell cultures had 3 or more branches, whereas ⁶0% of the wildtype tubular structures did.3. Although ZO-1 staining was observed on the cell-cell junctions for wildtype W10B6 and W10B2 cells, a more diffuse and discontinuous pattern of junctional staining was seen in Pkhd1^-/- cells （M10H2 and M10C7）. In the wildtype cell lines, E-cadherin was predominantly seen at the cell-cell junctions; while in the Pkhd1^-/- cells junctional staining of E-cadherin was nearly indistinguishable from cytosolic. Given the IF staining showed different distribution patterns for E-cadherin and ZO-1 in wildtype and Pkhd1^-/- cells, we performed western blot analysis to determine whether there were variations in E-cadherin or ZO-1 expression levels. We found there was no detectable immunoreactive change in either protein among the cell lines. 4. The TER was significantly lower in the null-Pkhd1 cells than in the wildtype ones after 3 days of transwell culture （*P<0.05）, and this difference persisted to day 7.5. Under 100×microscope, the Pkhd1^-/- and wildtype cells were stained with a rhodamine-phalloidin （F-actin） antibody. We found that wildtype cells exhibit nominal cortical actin distribution and epithelial shape with fine and even stress fibers in sub-confluent cultures. Pkhd1^-/- cells cells lose their normal cortical actin distribution and exhibit an irregular shape with thick and enriched stress fibers. The cell-cell junction gaps and irregular cell-cell borders were seen in Pkhd1^-/- cells. Confocal microscope images showed that F-actin in cultured wildtype cells （W10B6 and W10B2） localized around the nucleas and cells. Fine, stress fiber distributed at cell-cell junctions. The cell-cell junction gaps and irregular cell-cell borders were seen in Pkhd1^-/- cells. Confocal microscope images of the Pkhd1^-/- cells （M10H2 and M10C7） and their wildtype littermate cells （W10B6 and W10B2） cultured for 7 days in 3-D CI gels using rhodamine-phalloidin. Extensive tubulomorphogenesis was seen in the wildtype cells, but it was rare in the Pkhd1^-/- cells.6. The Pkhd1^-/- cells adhered less well than that of wildtype cells at concentrations of CI from 0.125 to 2μg/ml. At 2μg/ml CI, the null-Pkhd1 cells showed only 40% cell adhesion compared to over 90% for the control cell lines. Transwell migration assay results indicated the absolute number of cells that migrated to the underside of the transwell. There was a significant decrease in the number of migrated Pkhd1^-/- cells compared to wildtype cells （W10B6 and W10B2）.7. Compared with the cultured WT cells, a shortened ciliary structure and decreased ciliary staining were seen in the Pkhd1^-/- cells. The cilia stained in approximately 80% of WT cells and in fewer than 30% of Pkhd1^-/- cells. The mean length of primary cilia was 2.5μm in cultured WT cells and was <1.5μm in Pkhd1?/? littermate cells.8. Pkhd1^-/- cells M10H2 and M10C7 and their wildtype littermate cells W10B6 and W10B2 were incubated with 3H-thymidine, then the rate of 3H-thymidine incorporation was determined. The 3H-thymidine values were significantly decreased in the Pkhd1^-/- cells than that in the wildtype cells.9. Pkhd1^-/- cells M10H2 and M10C7 and their wildtype littermate cells W10B6 and W10B2 were subjected to Phospho-Histone H3 staining to evaluate cell proliferation. The percentage of cells positive for Phospho-Histone H3 was significantly greater in the cells with Pkhd1 than in those without. Western analyses with Phospho-Histone H3 and PCNA antibodies showed that both these proliferation markers were significantly downregulated in the null-Pkhd1 cells （M10H2 and M10C7）, compared with their wildtype littermate cells W10B6 and W10B2. Furthermore, we tested immunohistochemical （IHC） and immunofluorescent （IF） staining of Phospho-Histone H3 and PCNA in the kidney of 6-week-old, 3-month-old and 6-months old Pkhd1^-/- mice and its wildtype littermate. Many positive-stained cells were seen in the wildtype kidneys, but only a few appeared in the corresponding region of Pkhd1^-/- kidneys.10. We examined apoptosis rates for the Pkhd1^-/- and wildtype cell lines using TUNEL （Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling） assays. Under routine culture conditions, approximately 5% of the wildtype cells and around 14% of the null-Pkhd1 cells were apoptotic. To confirm this result, we assayed active caspase-3 as an indictor of apoptosis in the same cell lines. In agreement with the TUNEL assay results, the percentage of apoptotic Pkhd1^-/- cells was significantly higher than that of the wildtype cells. To examine apoptosis, the same kidneys were subjected to caspase-3 staining. More positive staining was seen in the Pkhd1^-/- than in the wildtype kidneys 3-month-old, 6-months old and 12-months old. Since older Pkhd1^-/- mice exhibit severer cystic kidney, the kidneys of 12-month-old Pkhd1^-/- and wildtype mice were also examined by TUNEL staining. Increased positive staining was seen in the Pkhd1^-/- than that in wildtype kidneys. Notably, much apoptotic debris was observed in the lumen of the Pkhd1^-/- renal tubules.Conclusion:1. Establishment of renal collecting duct cell lines bearing null-Pkhd1 alleles from Pkhd1^-/- mutant kidneys.2. FPC expression is required for normal tubulogenesis in 3-D culture of renal collecting duct cells.3. Loss of FPC impairs normal renal collecting duct cell-cell contacts and decreases integrin-dependent cell adhesion.4. Loss of FPC exhibits aberrant ciliogenesis and disturbs normal actin cytoskeleton distribution in collecting duct epithelial cells5. Loss of FPC reduces cell proliferation and promotes apoptosis in vitro and in vivo.Part 2The role of apoptosis singnaling Pathway in collecting duct epithelial cells of Pkhd1 mutant kidneysObjective:To explore the molecular mechanism of Pkhd1 deletion in cystogenesis in ARPKD through reducing proliferation and increasing apoptosis.Methods:1. Western blot was use to detect the expression of phosphorylated Akt （pS473） and test its effects on the apoptosis cell singnaling Pathway Bax-Caspase9-Caspase 3 in collecting duct epithelial cells of Pkhd1 mutant kidneys.2. We used western blot analyses to examine the Akt-downstream singnaling Ras-Raf-MEK-ERK.3. Four major FAK phosphorylation sites which regulate multiple cellular processes from cell migration and polarity to proliferation and apoptosis were examined by Western blot.4. Lysates from our null-Pkhd1 and wildtype cell lines were examined for changes in the phosphorylation of PI3K and PDK1.Results:1. Western blot analysis showed that phosphorylated Akt （pS473） was significantly reduced in the null-Pkhd1 cells compared to the wildtype-littermate cells after collagen I （CI） induction. Because Bax and Bcl2 are putative downstream factors of Akt, and their dysfunction can also induce abnormal apoptosis, we next compared their factors between the null-Pkhd1 and wildtype cells. We found that no significant difference in Bcl2 expression between the cells with and without Pkhd1, at all time points examined after CI induction （data not shown）. In contrast, Bax expression was significantly higher in the null-Pkhd1 cells compared to the wildtype cells. The basal and collagen I （CI）-inducible levels of Bax were both much lower in the null-Pkhd1 cells than that in the wildtype cells. Since both caspase-9 and caspase-3 are putative downstream factors of Bax, we next examined their expression levels in the same cell lines. Western analyses showed that the caspase-9 and -3 were significantly elevated in the null-Pkhd1 cells compared to the wildtype cells. 2. Western blot results indicated that there was no significant difference in the B-Raf phosphorylation in the M10H2 and M10C7 (Pkhd1^-/-) cell lines versus the wildtype-littermate W10B6 and W10B2 cell lines. We therefore shifted our focus to c-Raf, which is another important regulator of cell proliferation. Compared to the wildtype cell lines, western results showed that the null-Pkhd1 cells exhibited a significant downregulation of phosphorylated c-Raf, in the M10H2 and M10C7 (Pkhd1^-/-) cell lines versus the wildtype-littermate W10B6 and W10B2 cell lines.we examined the Ras/c-Raf downstream factors MEK and ERK, both of which are key markers for cell proliferation, using lysates from the Pkhd1^-/- and wildtype cell lines. The results showed that the phosphorylated MEK and ERK were significantly decreased in the null-Pkhd1 cells at all time points after CI induction. Given that Raf is the first effector that is positioned downstream of Ras, we next investigated if the aberrant c-Raf phosphorylation was caused by Ras dysregulation. Western analyses of lysates from the Pkhd1^-/- and wildtype cell lines showed that the level of Ras was significantly downregulated in the basal condition and after 30 minutes of CI induction in the null-Pkhd1 cells.3. Besides FAKpY861, which we reported previously, the phosphorylations of FAKpY397, 576, and 925 were also significantly decreased in the M10H2 and M10C7 (Pkhd1^-/-) cells compared to the wildtype-littermate W10B6 and W10B2 lines after CI induction.4. We analyzed the PI3K phosphorylation using anti-PI3K class III and PI3Kp110αantibodies. We observed no difference in PI3K class III phosphorylation between the cells with and without Pkhd1. In contrast, the basal and induced levels of PI3Kp110αwere much lower in the null-Pkhd1 M10H2 and M10C7 cells than that in wildtype-littermate W10B6 and W10B2 cells. Notably, in the null-Pkhd1 cells, a low level of PI3Kp110αwas observed in the basal and CI-induced condition. Since PDK1 is a downstream factor of PI3K that is activated by PI3K’s phosphorylation on p110, we analyzed the phosphorylation of PDK1 between the null-Pkhd1 and wildtype cells. Similar to PI3Kp110, both the basal and induced pPDK1 levels were significantly lower in the null-Pkhd1 cells than that in wildtype cells.Conclusion:1. FAK phosphorylation promotes Bax and provoke apoptosis in null-Pkhd1 cells via the PI3Kp110α/PDK1 inactivation.2. Downregulation of the Ras/c-Raf/MEK/ERK cascade may be the molecular mechanism underlying the decreased renal epithelial proliferation in collecting duct epithelial cells of Pkhd1 mutant kidneys.更多还原