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、高草酸尿和相关肾结石

• 批准号: 9888079
• 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 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9888079
• 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; Epithelium; 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) 影响约五分之一的男性和约十分之一的女性，非常痛苦且费用高昂（每年 >$10B），并且 与 CKD 和 ESRD 相关。高复发率（5 年内 50%）表明当前的干预措施是 需要不充分的替代疗法。大多数KS由草酸钙组成，少量 尿液中草酸盐的增加会增加结石形成的风险。降低尿草酸钙过饱和度 确实可以减少 KS 的形成，但不幸的是没有药物可以减少尿中草酸盐的排泄。这 肠道细菌 Oxalobacter formigenes (Of) 诱导结肠草酸盐分泌并减少尿草酸盐 通过未知的促分泌剂排泄。考虑到再定植的困难，单独使用 Of 并不能起到治疗作用 可行并强调需要确定促泌剂。我们鉴定了其分泌的衍生因子 刺激（>2.8倍）人肠道Caco2-BBE转运草酸盐的培养条件培养基（CM） (C2) 细胞通过 PKA 激活并刺激草酸转运蛋白 SLC26A6 (A6) 和 SLC26A2 (A2)。 在体内，直肠给药 CM 可减少尿草酸盐排泄 >32.5%，并刺激结肠草酸盐 高草酸尿小鼠的分泌量>42%，反映了这些因素的治疗潜力。我们已经确定 Sel1 蛋白作为主要的 Of 衍生因子，它们同样刺激（1.4-2.4 倍）C2 的草酸盐转运 细胞通过 PKA 和 A2/A6 转运蛋白。我们还在 Sel1 蛋白中鉴定出小肽 (P8+9) 刺激（>2.4倍）C2细胞的草酸盐转运。 P8+9 还刺激人类乙状结肠转运草酸盐 结肠（1.8 倍）、远端结肠（1.7 倍）和回肠（2 倍）类器官（离体肠上皮模型完全 模仿体内反应），证实 P8+9 在人体组织中起作用，并且它们可能会刺激 人体内结肠和回肠上皮细胞的草酸盐分泌。 基于这些发现，我将测试特定 P8 和 P9 子结构域介导结肠草酸盐的假设 可以识别分泌和潜在的治疗基序，并且 P8+9 通过细胞表面受体发挥作用 激活相关草酸转运蛋白（包括 A2、A6 或许还有其他）的特定信号通路。 将追求以下具体目标： 1. 识别刺激草酸盐的 P8 和 P9 肽亚结构域 C2 细胞和人类类器官中的转运：1a 识别最短的功能性 P8 和 P9 肽子结构域 通过删除特定的氨基酸残基。 1b.使用以下方法鉴定 P8 和 P9 肽中的关键氨基酸残基 一种替代方法。 2a.识别 C2 细胞中涉及的细胞表面受体。 2b.定义信令 介导 C2 细胞中草酸转运刺激的途径。 2c.识别草酸转运蛋白和 P8+9 在 C2 细胞中激活它们的机制，并使用 A6 缺失小鼠来确认 A6 的体内作用。 实现该项目的目标将对预防和/或治疗以下疾病产生治疗影响： 高草酸血症和高草酸尿症，影响 CaOx KS 患者的预后 高草酸尿症、原发性高草酸尿症，以及可能的 CKD 和 ESRD。