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.
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