CALCIUM OXALATE CRYSTAL INTERACTIONS WITH RENAL CELLS

个体尿晶变成肾结石的过程 在很大程度上仍然是不确定的。管内离子核的简单生长 从动力学角度看，流体不太可能产生大到足以 堵塞管腔，因此会被冲走。另一种选择 假说是新形成的晶核附着在肾小管细胞上。 表面并经历内吞作用，允许锚定的这些细胞生长 核变成肾结石。这个项目的目标是定义 调节结石中发现的主要尿晶结合的因素， 一水草酸钙(COM)诱导培养的肾上皮细胞， 并确定随之而来的生物反应。 在未转化的猴肾上皮细胞的初步实验中 细胞(BSC-1系)，COM晶体迅速附着在细胞上， 内化，启动DNA合成，并刺激细胞增殖。 这种反应是特殊的，在那个刷子石中，另一种含钙的 尿液结晶，没有内化，也不刺激DNA合成。 纤维连接蛋白和四环素抑制COM晶体的内吞作用 多肽RGDS，提示晶体可能与纤维连接蛋白结合 受体。我们还鉴定了其他具有生物活性的分子 抑制COM晶体摄取(转化生长因子-β2、肝素)和三种有效的有丝分裂原 促进这一过程(表皮生长因子、血清和 核苷酸ADP)。最常见的蛋白质具有潜在的临床重要性 人尿中Tamm-Horsfall糖蛋白(THP)抑制COM晶体 BSC-1细胞摄取。此外，4例AND患者中有4例的THP 加速形成的肾结石未能抑制COM晶体的摄取。 THP功能障碍可能在某些患者中起致病作用 肾结石，可能与其他尚未被认识的疾病合并 肾细胞-晶体相互作用调节因子的异常。 该项目的具体目标是：1)定义特定的表面 肾上皮细胞COM晶体受体(S)。2)描述 THP与肾上皮细胞的相互作用。3)调查THP 肾结石和结石患者的结构和功能 特发性高钙尿症大鼠。4)定义监管因素 COM晶体的黏附和随后的内吞作用。5)识别 质膜的结构和功能改变， 附着后肾上皮细胞的细胞骨架和细胞核 和COM晶体的内吞作用。6)确定粘附性和/或 COM晶体的内吞作用诱导自分泌/旁分泌因子释放 来自肾细胞。 实现这些特定的目标将增加对肾脏如何 上皮细胞对尿液晶体有反应。对这些问题的解释 细胞和分子水平的过程可以帮助实现我们的长期- 制定合理的新治疗策略以预防 肾结晶滞留和结石的形成。

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.