Oxalobacter formigenes-derived Factors Impact Hyperoxalemia, Hyperoxaluria, and Related Kidney Stones

产草酸杆菌衍生因素影响高草酸血症、高草酸尿症和相关肾结石

• 批准号: 10080732
• 负责人: HATIM A HASSAN
• 金额: $ 35.64万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-06 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10080732
• 关键词: Affect; Alternative Therapies; Amino Acids; Caco-2 Cells; Calcium Oxalate; Cell Surface Receptors; Cells; Colon; Conditioned Culture Media; Crystallization; Cyclic AMP-Dependent Protein Kinases; Distal; End stage renal failure; Enteral; Epithelial; Event; Excretory function; FDA approved; Human; Hyperoxaluria; Individual; Intervention; Intestines; Kidney Calculi; Knockout Mice; Lead; Mediating; Modeling; Mus; Organoids; Oxalates; Oxalobacter; Oxalobacter formigenes; Pain; Patient-Focused Outcomes; Patients; Peptides; Pharmaceutical Preparations; Physiological; Plasma; Prevention; Primary Hyperoxaluria; Proteins; Rectal Administration; Recurrence; Risk; Risk Factors; Role; Sigmoid colon; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Structure; Testing; Therapeutic; Translating; Urine; Woman; Work; base; cost; gut bacteria; human tissue; ileum; in vivo; intestinal epithelium; men; new therapeutic target; novel therapeutics; protein protein interaction; response; urinary

Project Summary/Abstract: Kidney stones (KS) affect ~1 in 5 men and ~1 in 11 women, are very painful & costly (>$10B annually), and are associated with CKD and ESRD. High recurrence rates (50% in 5 years) indicate that current interventions are inadequate and alternative therapies are needed. Most KS are composed of calcium oxalate, and small increases in urine oxalate enhance the risk for stone formation. Lower urinary calcium oxalate supersaturation definitively reduces KS formation, but unfortunately there are no drugs that reduce urinary oxalate excretion. The gut bacterium Oxalobacter formigenes (Of) induces colonic oxalate secretion and reduces urinary oxalate excretion via an unknown secretagogue. Given the difficulties with recolonization, Of alone is not therapeutically feasible and underscores the need to identify the secretagogue. We identified Of-derived factors secreted in its culture conditioned medium (CM) that stimulate (>2.8-fold) oxalate transport by human intestinal Caco2-BBE (C2) cells through PKA activation and stimulation of the oxalate transporters SLC26A6 (A6) and SLC26A2 (A2). In vivo, rectal administration of CM reduced urinary oxalate excretion >32.5% and stimulated colonic oxalate secretion >42% in hyperoxaluric mice, reflecting the therapeutic potential of these factors. We have identified Sel1 proteins as the major Of-derived factors and they similarly stimulate (1.4-2.4-fold) oxalate transport by C2 cells via PKA and the A2/A6 transporters. We also identified small peptides (P8+9) within a Sel1 protein that stimulate (>2.4-fold) oxalate transport by C2 cells. P8+9 also stimulated oxalate transport by human sigmoid colon (1.8-fold), distal colon (1.7-fold), and ileum (2-fold) organoids (ex vivo intestinal epithelia models fully mimicking the in vivo responses), confirming that P8+9 work in human tissues and that they will likely stimulate oxalate secretion in human colonic and ileal epithelia in vivo. Based on these findings I will test the hypotheses that specific P8 and P9 subdomains mediate colonic oxalate secretion and potential therapeutic motifs can be identified, and that P8+9 act via cell surface receptors and specific signaling pathways to activate the involved oxalate transporters (including A2, A6, and perhaps others). The following specific aims will be pursued: 1. Identify the P8 and P9 peptide subdomains that stimulate oxalate transport in C2 cells and human organoids: 1a Identify the shortest functional P8 and P9 peptides subdomains by deleting specific amino acid residues. 1b. Identify the critical amino acid residues in P8 and P9 peptides using a substitution approach. 2a. Identify the involved cell surface receptor(s) in C2 cells. 2b. Define the signaling pathways mediating stimulation of oxalate transport in C2 cells. 2c. Identify the oxalate transporters and the mechanisms by which P8+9 activate them in C2 cells and use A6 null mice to confirm the in vivo role of A6. Achieving the project’s objectives will have therapeutic implications for the prevention and/or treatment of hyperoxalemia and hyperoxaluria, impacting the outcomes of patients suffering from CaOx KS, enteric hyperoxaluria, primary hyperoxaluria, and possibly CKD and ESRD.

项目摘要/摘要： 肾结石（KS）影响〜1分之一的男性和11个女性中的约1个，非常痛苦和昂贵（每年> $ 10B），并且是 与CKD和ESRD相关联。高复发率（5年内为50％）表明当前干预措施为 需要不足和替代疗法。大多数KS由草酸钙组成，小 草酸盐的尿液增加增强了石材形成的风险。草酸钙过饱和尿钙 明确地减少了KS的形成，但不幸的是，没有药物可以减少草酸盐排泄。 肠道细菌草杆菌形式（Of）诱导草酸盐分泌并减少草酸盐 通过未知的秘密排泄。考虑到重新殖民的困难，单独的是没有治疗的 可行并强调了识别秘密的需求。我们确定了其分泌的衍生因素 培养培养基（CM），刺激人类肠道CACO2-BBE刺激（> 2.8倍）草酸盐转运（> 2.8倍） （C2）细胞通过PKA激活和草酸转运蛋白转运蛋白SLC26A6（A6）和SLC26A2（A2）的刺激。 在体内，直肠施用CM降低了草酸盐排泄> 32.5％，并刺激了草酸盐 分泌> 42％的高氧小鼠，反映了这些因素的治疗潜力。我们已经确定了 SEL1蛋白作为衍生的主要因素，它们类似地刺激了C2的草酸盐转运（1.4-2.4倍） 细胞通过PKA和A2/A6转运蛋白。我们还确定了SEL1蛋白中的小肽（P8+9） 通过C2细胞刺激（> 2.4倍）草酸盐转运。 P8+9还刺激了人类乙状结 结肠（1.8倍），远端结肠（1.7倍）和回肠（2倍）类器官（Ex Vivo肠上皮模型完全 模仿体内反应），确认P8+9在人体组织中起作用，并且它们可能会刺激 体内人类结肠和回肠上皮的草酸盐分泌。 根据这些发现，我将测试特定的P8和P9子域培养基结肠的假设 可以鉴定分泌和潜在的治疗基序，并且P8+9通过细胞表面受体和 特定的信号通路可以激活所涉及的草酸盐转运蛋白转运蛋白（包括A2，A6，也许还有其他）。 将追求以下特定目标：1。识别刺激草酸盐的P8和P9胡椒子域 C2细胞和人体类器官中的运输：1A确定最短的功能性P8和P9肽子域 通过删除特定的氨基酸保留。 1B。使用使用P8和P9肽保留的关键氨基酸保留 一种替代方法。 2a。识别C2细胞中涉及的细胞表面受体。 2b。定义信号 C2细胞中草酸盐转运刺激的途径。 2C。确定草酸盐转运蛋白和 P8+9在C2细胞中激活它们并使用A6 NULL小鼠确认A6的体内作用的机制。 实现该项目的目标将对预防和/或治疗的治疗意义 高氧血症和高黄油尿症，影响患有CAOX KS的患者的结局，进入 高黄油，原发性高氧化尿症以及可能的CKD和ESRD。