Urinary Calcium Oxalate-Binding Proteins Are a Critical Component in the Pathogenesis of Stone Disease
Jonathan A. Gerber, M.D., Nicolette K. Janzen, M.D., Paul F. Austin, M.D., Scott R. Manson, Ph.D..
Texas Children's Hospital, Houston, TX, USA.
BACKGROUND: Clinical approaches for urinary stone disease (USD) focus almost exclusively on managing urine chemistry but this is often ineffective at preventing recurrence (>50%). About 40% of USD has a genetic basis and defining its underlying causes will improve patient phenotyping and guide the development of novel therapies. This project tests the hypothesis that urinary proteins are critical regulators of calcium oxalate monohydrate (COM) crystallization in supersaturated urine with important implications for the evaluation and treatment of patients with USD. METHODS: Urine samples were collected from patients without a history of USD and the protein was purified in a centrifugal fractionator with a 3-kDa cutoff. COM crystallization was assessed in synthetic urine by adding the specified treatment, initiating crystallization with 1 mM CaCl2 and 2 mM Na2Ox, and then measuring the decrease in free calcium that occurs during crystal formation with the calcium-sensitive dye Arsenazo-III at λ=650 nm. COM-binding proteins (COM-BPs) were isolated by passing urine samples through affinity chromatography columns containing COM crystals. Proteomic identification of COM-BPs was then conducted by high-pressure liquid chromatography and mass spectrometry. RESULTS: Human urine contains potent inhibitors of crystallization, as demonstrated by our finding that the addition of just 1% urine to supersaturated solutions inhibits COM crystal formation by 74.1%. Biochemical fractionation revealed that the urinary protein fraction is required for this inhibitory activity (urinary protein - 79.7% inhibition; protein-free urine - 14.0% inhibition). Affinity chromatography showed that only 11.9% of urinary proteins have COM-binding activity. Selective depletion of these COM-BPs diminishes the inhibitory effects of urinary protein by 87.4%. Proteomic analysis identified 47 high-affinity COM-BPs in human urine. These results were validated by two approaches: (1) Immunoblotting analysis measured COM-binding affinities ranging from 68.7-78.1% for COM-BPs compared to 0.8-2.8% for other urinary proteins. (2) Treatment with recombinant COM-BPs inhibits COM crystallization with IC50 values ranging from 0.06-1.38 μg/mL while other urinary proteins have no measurable effects. Deficits in urinary COM-BPs have the potential to contribute to USD since the variation in their levels spans a 3.5-fold range between individuals (41.3-145.1 μg/dL). Intrinsic differences in COM-BP activity can be rapidly assessed in 24-hour urine samples by purifying urinary protein and then assessing its effects on COM crystallization. In a pilot study, this assay measured an average patient COM IC50 value of 6.5 μg/dL with an intra-sample variance of only 7.7%. Defining the mechanisms of COM-BPs may reveal novel therapeutic strategies for USD. Bioinformatic analysis showed that COM-BPs have a 32.6% increase in negatively charged Glu/Asp residues and 57.4% increase in Ca2+-binding capacity compared to other urinary proteins. The effects of COM-BPs can be partially mimicked by treatment with poly-Asp peptides which inhibit COM crystallization with an IC50 value of 10 μg/mL [all results: n=7, p<0.05]. CONCLUSIONS: This study demonstrates that urinary COM-BPs are critical regulators of COM crystallization. Their analysis in patients is likely to reveal genetic factors underlying susceptibility to USD. Furthermore, COMBP-mimetic peptides have a therapeutic potential to overcome these deficiencies, inhibit stone growth, and prevent recurrence in patients with USD.
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