CALCIUM OXALATE CRYSTAL INTERACTIONS WITH RENAL CELLS

个别尿结晶变成肾结石的过程 在很大程度上是不确定的。肾小管内离子核的简单生长 从动力学的角度来看，流体不太可能产生足够大的颗粒， 堵塞管腔并因此被冲洗掉。一个替代 假设是新形成的晶核粘附在肾小管细胞上 表面并进行内吞作用，允许这些锚定的 核进入肾结石。该项目的目标是定义 介导结石中发现的主要尿晶体结合的因子， 草酸钙一水合物（COM），对培养的肾上皮细胞， 并识别随之而来的生物反应。 在未转化猴肾上皮细胞的初步实验中， 细胞（BSC-1系），COM晶体迅速粘附到细胞上， 内化，启动DNA合成，并刺激细胞增殖。 这种反应是特异性的，在透钙磷石中，另一种含钙 尿结晶，不内化，不刺激DNA合成。 COM晶体的内吞作用被纤连蛋白和四- 肽RGDS，表明晶体可能与纤连蛋白结合 受体的我们还发现了其他生物活性分子， 抑制COM晶体摄取（TGF-β 2、肝素）和三种有效的促分裂剂 增强这一过程（表皮生长因子，血清， 核苷酸ADP）。具有潜在临床意义的最常见蛋白质 在人尿中，Tamm-Horsfall糖蛋白（THP）抑制COM晶体 通过BSC-1细胞摄取。此外，4例患者中有4例的THP 加速型肾结石不能抑制COM晶体摄取。 功能失调的THP可能在某些患者中起致病作用， 肾结石，可能与其他尚未认识到的 肾细胞-晶体相互作用调节器的异常。 该项目的具体目标是：1）定义特定的表面 肾上皮细胞上COM晶体的受体。2)表征 THP与肾上皮细胞的相互作用。3)调查THP 肾结石及结石形成患者肾组织结构和功能 特发性高钙尿症大鼠。4)定义调节因素 COM晶体的粘附和随后的内吞作用。5)识别 质膜的结构和功能改变， 细胞骨架和细胞核 和COM晶体的内吞作用。6)确定是否具有粘附性和/或 COM晶体内吞作用诱导自分泌/旁分泌因子释放 肾脏细胞。 实现这些具体目标将增加对肾脏如何 上皮细胞对尿结晶有反应阐明这些 细胞和分子水平的过程可以帮助我们实现长期的， 制定合理的新的治疗策略， 肾结晶滞留和结石形成。

The processes by which individual urinary crystals become kidney stones remain largely undefined. Simple growth of ionic nuclei within tubular fluid is unlikely, on kinetic grounds, to create particles large enough to occlude the lumen and would therefore be washed away. An alternative hypothesis is that newly formed crystal nuclei adhere to the tubular cell surface and undergo endocytosis, permitting growth of these anchored nuclei into renal calculi. The objective of this project is to define factors which mediate binding of the main urinary crystal found in stones, calcium oxalate monohydrate (COM), to renal epithelial cells in culture, and identity the biological responses that ensue. In preliminary experiments with nontransformed monkey kidney epithelial cells (BSC-1 line), COM crystals rapidly adhered to the cells, were internalized, initiated DNA synthesis, and stimulated cell multiplication. The response was specific, in that brushite, another calcium-containing urinary crystal, was not internalized and did not stimulate DNA synthesis. Endocytosis of COM crystals is inhibited by fibronectin and the tetra- peptide RGDS, suggesting that the crystal may bind to the fibronectin receptor. We also identified other biologically active molecules which inhibit COM crystal uptake (TGF-beta2, heparin), and three potent mitogens that enhance the process (epidermal growth factor, serum, and the nucleotide ADP). Of potential clinical importance the most common protein in human urine, Tamm-Horsfall Glycoprotein (THP), inhibited COM crystal uptake by BSC-1 cells. Furthermore, THP from 4 of 4 patients with an accelerated form of nephrolithiasis failed to inhibit COM crystal uptake. Dysfunctional THP may play a pathogenic role in certain patients with nephrolithiasis, perhaps in concert with other as yet unrecognized abnormalities in regulators of renal cell-crystal interactions. Specific aims of the project are to: 1) Define a specific surface receptor(s) for COM crystals on renal epithelial cells. 2) Characterize the interaction of THP with renal epithelial cells. 3) Investigate THP structure and function in patients with nephrolithiasis and stone-forming rats with Idiopathic Hypercalciuria. 4) Define factors that regulate adhesion and subsequent endocytosis of COM crystals. 5) Identify structural and functional alterations in the plasma membrane, cytoskeleton, and nucleus of renal epithelial cells following attachment and endocytosis of COM crystals. 6) Determine if adhesion and/or endocytosis of COM crystals induces release of autocrine/paracrine factors from renal cells. Achieving these specific aims will increase understanding of how kidney epithelial cells respond to urinary crystals. Elucidation of these processes at the cellular and molecular level could help attain our long- term goal of formulating rational new therapeutic strategies to prevent renal crystal retention and the formation of calculi.