Hydroxycitrate: A Novel Therapy for Calcium Phosphate Urinary Stones

项目总结/摘要 在过去的四十年中，磷酸钙（CaP）结石病的发病率显著增加。 CaP结石形成者的结石复发率高，经常需要重复结石手术， 表明目前的预防性医疗方案是次优的。最常见的代谢异常 在CaP结石形成者中确定的是高钙尿、低柠檬酸尿和尿pH升高，所有这些因素都增加了 钙磷饱和度。碱疗法（柠檬酸钾）是行之有效的，以防止草酸钙结石，部分 通过提高尿柠檬酸盐。然而，当在CaP结石形成者中以使柠檬酸盐正常化的目标剂量进行测试时， 排泄，碱治疗可能会恶化钙磷结石，由于伴随尿pH值上升。羟基柠檬酸是一种 与柠檬酸盐密切相关的分子，已显示其在体外阻止CaP成核。然而，其功效 在体内对抗CaP结石形成的作用以前没有研究过。我们的初步研究表明：1） 羟基柠檬酸盐在低浓度下对CaP晶体生长有较强的抑制作用; 羟基柠檬酸盐摄入耐受性良好，并将尿OHCit排泄量提高至可降低CaP的浓度 尿液中的结晶; 3）口服羟基柠檬酸盐给药增加尿柠檬酸盐，但无显著性差异 因此，在本提案中，我们将测试羟基柠檬酸盐可以作为 作为预防CaP结石病复发的治疗方法。为了验证这个假设，我们将联合收割机 补充方法：1）在对CaP结石形成者进行的人体代谢研究中，我们将检查 A的短期影响。递增剂量的羟基柠檬酸盐和B.羟基柠檬酸盐的比较有效性 与碱疗法（柠檬酸钾）相比，对尿生化和理化参数以及CaP晶体的影响 生长和溶解使用原子力显微镜和散装结晶研究进行的参与者的 尿样2)在CaP沉淀的动物模型中，我们将评估同样的长期影响。 在人类中测试干预措施，测量尿液化学，晶体， 肾脏钙化3)在体外，我们将操作羟基柠檬酸盐、钙、柠檬酸盐和pH值，并评估CaP 晶体生长和溶解度的独特组合的技术跨越多个长度尺度。值得注意的是， 将通过原子力显微镜检查微观表面生长，宏观晶体成核和生长 在微流体装置中，以及在临床相关的羟基柠檬酸盐范围内的本体结晶动力学 浓度的我们将测试羟基柠檬酸盐对CaP晶体生长产生影响的机制 和溶出度，并使用一系列方法评估羟基柠檬酸盐和柠檬酸盐浓度的协同作用 临床相关pH值和离子强度。总的来说，根据我们强大的初步数据，我们的创新，但 严格的技术，和我们的跨学科的互补研究，我们希望这个建议将有助于 确定最佳的治疗方法，越来越多地遇到和难以治疗的CaP结石。

PROJECT SUMMARY / ABSTRACT The incidence of calcium phosphate (CaP) stone disease has markedly increased over the last four decades. CaP stone formers experience high rates of stone recurrence and frequently need repeat stone surgery, indicating that current prophylactic medical regimen are suboptimal. The most common metabolic abnormalities identified in CaP stone formers are hypercalciuria, hypocitraturia, and elevated urine pH, all factors that increase CaP saturation. Alkali therapy (potassium citrate) is well-established to prevent calcium oxalate stones, in part by raising urine citrate. However, when tested in CaP stone formers at doses targeted at normalizing citrate excretion, alkali therapy may worsen CaP stones due to a concomitant rise in urine pH. Hydroxycitrate is a molecule closely related to citrate that has been shown to prevent CaP nucleation in vitro. However, its efficacy against CaP stone formation in vivo has not been previously studied. Our preliminary studies suggest that: 1) Hydroxycitrate can exert a powerful inhibitory effect on CaP crystal growth at low concentrations; 2) Oral hydroxycitrate ingestion is well-tolerated and raises urine OHCit excretion to concentrations that reduce CaP crystallization in urine; 3) Oral hydroxycitrate administration increases urine citrate without significantly increasing urine pH. In this proposal, we will therefore test the overall hypothesis that hydroxycitrate can serve as a treatment to prevent recurrence of CaP stone disease. To test this hypotheses, we will combine three complementary approaches: 1) In human metabolic studies conducted in CaP stone formers, we will examine the short-term impact of a. escalating doses of hydroxycitrate and b. comparative effectiveness of hydroxycitrate vs alkali therapy (potassium citrate) on urinary biochemical and physicochemical parameters, and CaP crystal growth and dissolution using atomic force microscopy and bulk crystallization studies conducted on participants’ urine specimen. 2) In an animal model of CaP precipitation, we will assess the long-term impact of the same interventions to be tested in humans with measurement of urine chemistry, crystalluria, and development of calcification in kidneys. 3) In vitro, we will manipulate hydroxycitrate, calcium, citrate, and pH, and assess CaP crystal growth and solubility by a unique combination of techniques spanning multiple length scales. Notably, we will examine microscopic surface growth by atomic force microscopy, macroscopic crystal nucleation and growth in a microfluidic device, and the kinetics of bulk crystallization over a range of clinically relevant hydroxycitrate concentrations. We will test the mechanisms by which hydroxycitrate exerts its impact on CaP crystal growth and dissolution, and assess the cooperative effects of hydroxycitrate and citrate concentration using a range of clinically relevant pH and ionic strengths. Overall, based on our strong preliminary data, our innovative but rigorous techniques, and our cross-disciplinary complimentary studies, we hope that this proposal will help identify optimal therapeutics for the increasingly encountered and difficult to treat CaP stones.