Response Of Renal Cells To Osmotic Stress

由于与渗透保护转录因子TONEBP/OREBP/NFAT5(TONEBP)物理上相关的蛋白质可能调节或支持其活性，我们使用蛋白质组学来鉴定它们。我们在HEK 293细胞中稳定表达了TONEBP的1-547个氨基酸，并将其与暴露在高盐下的细胞核中的相关蛋白进行了免疫沉淀，从而鉴定了14种相关蛋白。相关蛋白质分为几类：1)DNA依赖的蛋白激酶，包括其催化亚基和调节亚基Ku86；2)RNA解旋酶，即RNA解旋酶A、核仁RNA解旋酶II/Gu和DEAD-box RNA解旋酶P72；3)小的或异质核核糖核蛋白(SnRNP或hnRNP)，即U5 snRNP特异的116 kDa蛋白质、U5 SnRNP特异的200 kDa蛋白质、hnRNP U、hnRNP M、hnRNP K和hnRNP F；4)热休克蛋白，即Hsp90beta和Hsc70；以及5)聚(ADP-RNP)聚合酶-1(PARP-1)。我们通过Western分析证实了大多数蛋白质的鉴定，并通过电泳迁移率改变分析证明它们存在于与TONEBP/OREBP特异结合的大复合体中。此外，我们还发现PARP-1和Hsp90对TONEBP/OREBP活性有调节作用。PARP-1的表达降低了TONEBP/OREBP的转录活性及其反式激活结构域的活性。HSP90增强了这些活性，并维持了暴露于高盐的细胞中TONEBP/OREBP蛋白的丰度增加。我们目前正在扩展这些蛋白质组学研究，以确定与TONEBP-1-1531相关的其他蛋白质，并确定TONEBP中被高盐磷酸化的氨基酸。到目前为止，我们已经鉴定了几个磷酸化的氨基酸，并用磷酸特异性抗体证实了其中一些的磷酸化是受渗透调节的。此外，这些氨基酸的突变会影响TONEBP的渗透调节。 甘油磷酸胆碱(GPC)是一种具有渗透保护作用的亲和性和中和性的有机渗透压物质，在肾脏内髓细胞中积聚，以响应高盐和尿素的反应。我们先前发现，高浓度的氯化钠和/或尿素增加了肾脏(Madin-Darby Canine Renal，MDCK)细胞的GPC，GPC是由磷脂酰胆碱衍生的，由当时尚未发现的磷脂酶催化。神经病靶标酯酶(NTE)是一种催化磷脂酰胆碱合成GPC的磷脂酶B。因此，我们测试了NTE是否在高盐诱导的肾细胞GPC合成增加中起作用，发现确实是这样。在小鼠内髓内集合管(MIMCD3)细胞中，高盐使NTE基因和蛋白表达增加。抑制NTE酯酶活性的二异丙基氟磷酸盐减少GPC的积累，就像siRNA特异性地降低NTE蛋白丰度一样。NTE mRNA的20h半衰期不受高盐的影响，但用特定的siRNA敲除TONEBP可抑制高盐诱导的NTE mRNA的增加。此外，长期服用速尿的ClCK1-/-小鼠和服用速尿的正常小鼠肾脏髓内间质氯化钠浓度降低与NTE mRNA和蛋白的降低有关。因此，在组织培养和体内实验中，高浓度的氯化钠促进了由TONEBP介导的NTE的转录，导致NTE表达的增加，从而促进了哺乳动物肾脏细胞GPC的产生和积累。 我们先前发现，高浓度的尿素和/或氯化钠抑制了磷酸二酯酶(GPC-PDE)的活性，该酶催化GPC分解为胆碱和甘油磷酸，这有助于GPC的渗透诱导。在目前未发表的研究中，我们已经确定了磷酸二酯酶为Gdpd5。从mIMCD3细胞获得的重组Gdpd5免疫共沉淀物具有GPC-PDE活性，当细胞暴露于高盐或尿素时，其比活性较低。此外，高盐降低了Gdpd5的mRNA丰度，而针对Gdpd5的特异性siRNA增加了GPC。因此，Gdpd5是一种GPC-PDE，其活性有助于GPC的渗透调节。 在其他未发表的研究中，我们发现了更多调节TONEBP活性的信号分子。在许多受TONEBP调控的基因中，c-jun和c-Fos与矿石相邻的AP-1位点结合，该位点的存在以及c-fos和c-jun活性的存在是高盐完全激活TONEBP所必需的。磷脂酶C伽马结合到TONEBP的特定位置，也有助于TONEBP的活性。磷脂酰肌醇3激酶类IB(PI3K-IB)被高盐激活，负向调节TONEBP，在许多方面与PI3K-IA相反，我们之前已经证明PI3K-IA也被高盐激活，但正向调节TONEBP。 最后，我们先前发现，在细胞培养和体内肾脏髓质中，高渗透压都会导致DNA断裂和氧化应激。DNA损伤和氧化应激与细胞衰老有关，尤其是在衰老和癌症中。我们现在发现，在组织培养中，高盐会导致细胞衰老，在正常小鼠的肾脏髓质以及暴露在高盐下的线虫中，与年龄相关的衰老细胞的积累会加速。因此，高渗透压不仅会造成DNA损伤和氧化应激，还会导致细胞衰老。

Reasoning that proteins that physically associate with the osmoprotective transcription factor TonEBP/OREBP/NFAT5 (TonEBP) are likely to regulate or support its activity we used proteomics to identify them. We stably expressed amino acids 1-547 of TonEBP in HEK 293 cells and immunoprecipitated it plus associated proteins from the nuclei of cells exposed to high NaCl, thereby identifying 14 associated proteins. The associated proteins fall into several classes: 1) DNA-dependent protein kinase, both its catalytic subunit and regulatory subunit, Ku86; 2) RNA helicases, namely RNA helicase A, nucleolar RNA helicase II/Gu, and DEAD-box RNA helicase p72; 3) small or heterogeneous nuclear ribonucleoproteins (snRNPs or hnRNPs), namely U5 snRNP-specific 116 kDa protein, U5 snRNP-specific 200 kDa protein, hnRNP U, hnRNP M, hnRNP K, and hnRNP F; 4) heat shock proteins, namely Hsp90beta and Hsc70; and 5) poly(ADP-ribose) polymerase-1 (PARP-1). We confirmed identification of most of the proteins by Western analysis and also demonstrated by electrophoretic mobility-shift assay that they are present in the large complex that binds specifically along with TonEBP/OREBP to its cognate DNA element. In addition, we found that PARP-1 and Hsp90 modulate TonEBP/OREBP activity. PARP-1 expression reduces TonEBP/OREBP transcriptional activity and the activity of its transactivating domain. Hsp90 enhances those activities and sustains the increased abundance of TonEBP/OREBP protein in cells exposed to high NaCl. We are currently extending these proteomic studies to identify additional proteins that associate with TonEBP-1-1531 and also to identify amino acids in TonEBP that are phosphorylated by high salt. Until now we have identified several phosphorylated amino acids and have confirmed with phospho-specific antibodies that phosphorylation of some of them is osmotically regulated. Also, mutation of those amino acids affects osmotic regulation of TonEBP. Glycerophosphocholine (GPC) is an osmoprotective compatible and counteracting organic osmolyte that accumulates in renal inner medullary cells in response to high NaCl and urea. We previously found that high NaCl and/or urea increases GPC in renal (Madin-Darby canine kidney, MDCK) cells and that the GPC is derived from phosphatidylcholine, catalyzed by a phospholipase that was not identified at that time. Neuropathy target esterase (NTE) was recently shown to be a phospholipase B that catalyzes production of GPC from phosphatidylcholine. Therefore, we tested whether NTE contributes to the high NaCl-induced increase of GPC synthesis in renal cells, finding that it does. In mouse inner medullary collecting duct (mIMCD3) cells, high NaCl increases NTE mRNA and protein. Diisopropyl fluorophosphate, which inhibits NTE esterase activity, reduces GPC accumulation, as does an siRNA that specifically reduces NTE protein abundance. The 20-h half-life of NTE mRNA is unaffected by high NaCl, but knockdown of TonEBP by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA. Further, the lower renal inner medullary interstitial NaCl concentration that occurs chronically in ClCK1-/- mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. Thus, high NaCl increases transcription of NTE, mediated by TonEBP, and the resultant increase of NTE expression contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo. We previously found that high urea and/or NaCl inhibit the activity of a phosphodiesterase (GPC-PDE) that catalyzes breakdown of GPC to choline and glycerol phosphate, and that this contributes to osmotic induction of GPC. In current unpublished studies we have identified the phosphodiesterase as Gdpd5. Recombinant Gdpd5 immunoprecipitated from mIMCD3 cells has GPC-PDE activity and the specific activity is lower if the cells have been exposed to high NaCl or urea. Further, high salt reduces Gdpd5 mRNA abundance and a specific siRNA against Gdpd5 increases GPC. Thus, Gdpd5 is a GPC-PDE, whose activity contributes to osmotic regulation of GPC. In additional unpublished studies we have identified several more signaling molecules that regulate TonEBP activity. c-Jun and c-Fos bind to an AP-1 site adjacent to the OREs in many genes that are regulated by TonEBP, and presence of the site and of c-fos and c-Jun activity are necessary for full activation of TonEBP by high salt. Phospholipase C gamma binds to a specific site in TonEBP, also contributing to TonEBP activity. Phosphatidylinositol 3 kinase Class IB (PI3K-IB) is activated by high salt and negatively regulates TonEBP, having in many respects an effect opposite to PI3K-IA, which we previously showed is also activated by high salt, but positively regulates TonEBP. Finally, we previously found that hyperosmolality causes DNA breaks and oxidative stress both in cell culture and in kidney medullas in vivo. DNA damage and oxidative stress are associated with cellular senescence, most striking in aging and in cancer. We are now finding that high salt causes cellular senescence in tissue culture and that age-associated accumulation of a senescent cells is accelerated in kidney medullas of normal mice, as well as in C. Elegans exposed to high salt. Thus, hyperosmolality not only causes DNA damage and oxidative stress, but also causes cellular senescence.