CaP, crystal responsible for idiopathic nephrolithasis

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DESCRIPTION (provided by applicant): Nephrolithksis is a result of crystal formation and retention within the kidneys. Several studies have shown that conditions are favorable for the formation of calcium phosphate (CaP) in early segments of the nephron namely proximal tubules and the loops of Henle. It is proposed that CaP formed in early segments promote nucleation of calcium oxalate (CaOx) in the collecting ducts. Renal epithelial cells exposed to non-physiologically high levels of oxakte and/or CaOx crystals experience both structural and functional changes, which may eventually lead to cell injury and death. These changes also promote crystal attachment to cell surfaces and retention within the renal tubules. As a protective response, CaOx crystals are often endocytosed at the luminal side and exocytosed into die interstitium where they are processed by the inflammatory defense indicated by the presence of monocytes, macrophages and giant cells. Interestingly CaP crystals seen in the renal interstitium do not induce an inflammatory response. Even though CaP is considered the primary crystal and crystal induced changes in epithelial cells are essential for crystal retention, studies have yet to investigate crystal cell interactions between CaP and renal epithelial cells. CaP has also been shown to dissolve in the acidic environment of the collecting ducts. It is our hypothesis that crystals formed in the urine contain a surface coat, which inhibits crystal dissolution. We also hypothesize that similar to CaOx crystals, an exposure to crystals of CaP also challenges the cells and leads to alterations in renal epithelial cells, which promote crystal retention. We propose to test our hypotheses by exposing proximal tubular origin HK-2 and collecting duct origin MDCK cells to CaP crystals in culture. Two forms of CaP, inorganic apatite and brushite as well as apatite and brushite coated with urinary macromolecules will be used. In addition we propose to investigate the interaction between CaP crystals and renal interstitial fibroblasts to find out why renal interstitial CaP deposits do not cause inflammation. We have the following Specific Aims. 1. To confirm that CaP crystals coated with urinary macromolecules, are resistant to dissolution and good nucleators of CaOx in acidic environment of collecting ducts. 2. To probe that exposure to CaP crystals is associated with renal epithelial cellular injury and that exposure of renal epithelial cells to CaP crystals causes free radical production and lipid peroxidation. 3. To show that production by renal epithelial cells of crystallization modulators such as osteopontin (OPN) and bikunin and a-1 microglobulin is upregulated on exposure to CaP crystals. 4. To demonstrate that CaOx crystal adherence to renal epithelial cells is increased by their previous exposure to CaP crystals and nucleation of CaOx crystals is promoted by surface membranes of the cells pre-exposed to CaP crystals. 5. To determine the molecular mechanism for the inability of renal interstitial CaP crystals to provoke an inflammatory reaction.