Hydroxycitrate: A Novel Therapy for Calcium Phosphate Urinary Stones

项目摘要/摘要 在过去的40年里，磷酸钙结石(CaP)的发病率显著增加。 冠状结石成形者结石复发率高，经常需要重复结石手术， 说明目前的预防医疗方案不是最优的。最常见的代谢异常 在帽状结石形成中发现的是高钙尿、低柠檬酸尿和尿液pH升高，所有这些因素都会增加。 封口饱和度。碱疗法(柠檬酸钾)在一定程度上可以预防草酸钙结石。 通过增加尿液中的柠檬酸。然而，当以使柠檬酸盐正常化为目标的剂量在盖石成型剂中进行测试时 排泄，碱疗法可能会恶化帽结石，因为伴随而来的尿液pH值上升。羟基柠檬酸是一种 与柠檬酸盐密切相关的分子，已被证明在体外可以防止帽核。然而，它的功效 在体内抗帽状结石的形成以前还没有研究过。我们的初步研究表明：1) 羟基柠檬酸在低浓度下对帽晶生长有很强的抑制作用；2)口服 摄入羟基柠檬酸的耐受性很好，并会增加尿液羟色胺的排泄量，使其浓度降低 3)口服羟基柠檬酸可增加尿液中柠檬酸的含量，但无显著差异 尿液pH值升高。因此，在这项提案中，我们将检验羟基柠檬酸盐可以起作用的总体假设。 作为预防帽状石疾病复发的一种治疗方法。为了检验这一假设，我们将结合三个 互补方法：1)在盖石症患者进行的人体代谢研究中，我们将检查 A.增加羟基柠檬酸盐剂量的短期影响和B.羟基柠檬酸盐的比较效果 比较碱疗法(柠檬酸钾)对尿液生化指标和尿帽结晶的影响 使用原子力显微镜和整体结晶研究参与者的生长和溶解 尿液样本。2)在盖帽降水的动物模型中，我们将评估同样的长期影响 对人体进行的干预措施，包括尿液化学、结晶性尿和尿毒症的发展 肾脏内的钙化。3)在体外，我们将操纵羟基柠檬酸、钙、柠檬酸盐和pH，并评估CAP 通过跨越多个长度尺度的独特技术组合进行晶体生长和溶解。值得注意的是，我们 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 在微流控装置中，以及在一系列临床相关的羟基柠檬酸盐上的本体结晶动力学 浓度。我们将测试羟基柠檬酸对帽子晶体生长的影响机制。 和溶解，并使用一系列范围评估羟基柠檬酸盐和柠檬酸盐浓度的协同效应 临床相关的pH和离子强度。总体而言，基于我们强劲的初步数据，我们的创新但 严谨的技术和我们跨学科的互补性研究，我们希望这项建议将有所帮助 为越来越多遇到的和难以治疗的帽状结石确定最佳治疗方法。

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.