This grant focuses on one of the most critical mechanistic events in urolithiasis, the nucleation and retention of urinary stone crystals through the molecular attachment of crystals to the epithelium of the kidney papillary tip which then serve as maturation sites for urinary stones. The NIH Consensus Development Conference on the Prevention and Treatment of Kidney Stone Disease in 1988 cited the process of crystal retention as one of the most critical areas for future research. Stone research literature during the past decade is replete with references to both human and animal studies showing the close relationship between tissue injury and stone disease. The proposed studies will use rat inner medullary collecting duct (IMCD) cells in culture and intact papillary tips from rats which have been subjected to established protocols which induce specific forms of cellular injury shown to enhance calcium oxalate stone formation. The working hypothesis in this grant is that the attachment of crystals to cell membranes requires long range order interactions between the crystals and membranes, and that for effective attachment, there must be an alteration in lipid and/or protein domain structures on those cell surfaces which bind crystals.
The Specific Aims and questions to be addressed are:
Specific Aim 1. To study the relationship of membrane composition and domain structure to crystal nucleation on and attachment to IMCD cells. 1. Since there are known differences in the composition of the apical and basolateral membranes, is there enhanced attachment of calcium oxalate monohydrate (WH), calcium oxalate dihydrate (WE), apatite (AP), and uric acid (UA) crystals to monolayer cells whose apical membrane composition has been selectively altered so as to approximate that of the basolateral membrane? 2. What are the specific phospholipid and protein domain structures necessary for enhanced attachment of the various stone crystals? 3. Can the cell surfaces which exhibit enhanced crystal attachment also present the necessary molecular arrays which are supportive of WH, WE, AP, and UA crystal nucleation when the cells are exposed to metastable crystallizing solutions? Specific Aim II. To study the effect of cellular injury on crystal nucleation and attachment. 1. Does injury caused by gentamicin exposure or Mg depletion prime renal papillary tip epithelium to exhibit enhanced nucleation and/or attachment of WH, WE, AP, and UA crystals? 2. Does the preincubation with either nucleating solutions or preformed stone crystals injure the papillary tip epithelium in such a way as to induce enhanced attachment of stone crystals? 3. What changes in membrane composition and/or structure induced by these forms of injury are correlated with changes in the nucleation and/or attachment potential of stone crystals?
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