高钙激活Nox4/ROS/p38-MAPK介导的BMP2信号通路在肾脏Randall钙斑形成中的作用

Randall’s plaque, located in the renal interstitium, is an ectopic calcification and is considered as the initial nidi of kidney stones according to the Randall’s plaque theory. In our previous studies, we demonstrated that BMP2 signaling pathway in renal tubular epithelial cells could be abnormally activated by high-calcium. And the abnormal activation of BMP2 would result in the transdifferention of renal tubular epithelial cells to osteoblast-like (chondrocyte-like) cells which have important roles in the formation of Randall’s plaque. Recent studies have shown that the ROS/MAPK signaling pathway which is mediated by Nox4 isoform is closely associated with many ectopic calcification diseases. In addition, Nox4/ROS is also involved in the cellular osteogenesis differentiation which was regulated by BMP2. Our preliminary studies have found that BMP2 expression in the renal papillae of hypercalciuric stone-forming rats was significantly increased and the Nox4/ROS/p38-MAPK pathway was abnormally activated. Furthermore, renal tubular epithelial cells showed similar changes after receiving high-calcium treatment. Based on the above evidence we could make this assumption: the activation of Nox4/ROS/p38-MAPK pathway is the key points for the high-calcium induced over expression of BMP2 in renal tubular epithelial cells and the formation of Randall’s plaque. This research program is intended to explore the effects and the underlying mechanism of Nox4/ROS/p38-MAPK on BMP2 signaling pathway and Randall’s plaque formation at different levels including humans, animals and cells. This study is expected to provide new theoretical basis for the pathogenesis of Randall’s plaque as well as the prevention and treatment of nephrolithiasis.

Randall钙斑是肾间质的异位钙化，钙斑学说认为其为肾结石形成的起始病变。我们研究发现，高钙可激活肾小管上皮细胞BMP2通路并导致细胞发生成（软）骨样转分化而参与钙斑形成。最新研究表明，Nox4介导的ROS/p38-MAPK通路与多种异位钙化疾病密切相关，此外Nox4/ROS亦可调控BMP2通路参与的细胞成骨分化。我们初步研究发现高钙尿大鼠肾乳头内BMP2表达升高，同时伴随Nox4/ROS/p38-MAPK通路异常活化；肾小管上皮细胞经高钙处理后亦发生类似变化。藉此我们推测：Nox4/ROS/p38-MAPK通路活化是高钙促进肾小管上皮细胞内BMP2高表达及钙斑形成的关键环节。本研究拟在人体、动物、细胞水平，探索Nox4/ROS/p38-MAPK对BMP2通路和钙斑形成的调控作用及分子机制。本研究将为Randall钙斑的形成和肾结石的防治提供新的理论依据。

活性氧（ROS）介导的氧化应激在草酸钙结石的形成中发挥着关键作用。其中NADPH氧化酶Nox4亚型是肾脏ROS的主要来源。但Nox4在肾结石 发生发展中的作用机制尚不清楚。我们首先通过检测草酸钙结石患者肾乳头Randall钙斑芯片发现Nox4基因在草酸钙结石患者肾乳头组织的表达明显升高。随后我们通过体内外实验发现高钙可激活NRK-52E细胞中p-PKC和Nox4的表达，而PKC抑制剂能抑制Nox4的表达，提示高钙可能通过PKC来激活Nox4的表达。高钙可增加肾脏和细胞中ROS/H2O2的产生，调节Nox4的表达和活性可改变这种现象。高钙调控的肾脏和细胞氧化应激水平依赖于Nox4的表达。高钙尿大鼠肾脏损伤和凋亡明显增加，而抑制Nox4的表达或活性可明显改善这些损害。同样高钙处理下NRK-52E细胞会发生损伤和凋亡，而调节Nox4的表达会加重或减轻上述变化。高钙诱导NRK-52细胞和大鼠肾脏内Keap1蛋白表达增加，Nrf2蛋白表达下降。此外，高钙可诱导大鼠肾脏和NRK-52E细胞的钙盐沉积，抑制Nox4表达或活性时，钙盐沉积现象明显减弱，上调Nox4表达时，钙盐沉积则显著增加。高钙激活大鼠肾脏和NRK-52E细胞中的Nox4和MAPK信号通路蛋白的表达，抑制Nox4表达或活性明显减弱了高钙诱导的MAPK磷酸化蛋白表达，而上调Nox4表达显著增强了高钙诱导的MAPK磷酸化蛋白表达。此外，Nox4参与调控高钙诱导下肾小管上皮细胞中成骨相关蛋白BMP2和OPN的表达。ERK和JNK抑制剂会显著降低高钙诱导的NRK-52E细胞中BMP2和OPN表达。此外，MAPK抑制剂能显著减弱高钙诱导的NRK-52E细胞中Nox4的增加，MAPK可通过正反馈回路调控Nox4的表达。我们通过体内外实验初步发现并证实了Nox4在Randall钙斑和草酸钙结石病理过程中的作用。一方面，高钙通过PKC来激活肾小管上皮细胞内Nox4的表达，Nox4衍生的ROS/H2O2导致氧化应激的发生和氧化还原稳态的失调，介导了肾小管上皮细胞的损伤和凋亡，进而导致草酸钙结石的形成。另一方面高钙诱导的Nox4通过ERK/JNKMAPK信号通路介导成骨相关蛋白BMP2和OPN的异常激活，诱导肾小管上皮细胞转分化为成骨样细胞，导致Randall钙斑和草酸钙结石的形成。Nox4抑制剂有望成为防治草酸钙结石的候选药物。

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