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: 10614325
• 负责人: HATIM A HASSAN
• 金额: $ 34.98万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10614325
• 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~1/5男性和~1/11女性，非常痛苦和昂贵(每年100亿美元)，而且 与慢性肾脏病和终末期肾病相关。高复发率(5年内为50%)表明目前的干预措施 不充分的替代疗法是必要的。大多数KS是由草酸钙组成的，而且很小 尿草酸增加会增加结石形成的风险。下尿草酸钙过饱和度 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的排泄。这个 肠道细菌草酸杆菌(OF)诱导结肠草酸分泌和减少尿草酸 通过未知的促分泌器排泄。考虑到重新殖民化的困难，单独的不是治疗上的 这是可行的，并强调了确定分泌者的必要性。我们鉴定了其分泌的衍生因子 人肠道Caco 2-BBE促进(2.8倍)草酸转运的条件培养液(CM) (C2)通过激活PKA和刺激草酸转运蛋白SLC26A6(A6)和SLC26A2(A2)来刺激细胞。 在活体内，直肠给药可减少尿草酸排泄32.5%，并刺激结肠草酸。 高草酸尿小鼠的分泌物&gt；42%，反映了这些因素的治疗潜力。我们已经确定了 Sel1蛋白是主要的衍生因子，它们类似地刺激C2(1.4-2.4倍)草酸转运 细胞通过PKA和A2/A6转运蛋白。我们还在Sel1蛋白中发现了小肽(P8+9)， 通过C2细胞刺激(2.4倍)草酸转运。P8+9也刺激了人乙状结肠的草酸运输 结肠(1.8倍)、远端结肠(1.7倍)和回肠(2倍)器质(完全体外肠道上皮模型 模拟体内反应)，证实P8+9在人体组织中起作用，它们可能会刺激 活体人结肠和回肠上皮细胞草酸分泌。 基于这些发现，我将测试特定的P8和P9亚区介导结肠草酸的假设 可以确定P8+9的分泌和潜在的治疗基序，并且P8+9通过细胞表面受体和 激活相关草酸转运体(包括A2、A6等)的特定信号通路。 将追求以下具体目标：1.确定刺激草酸的P8和P9肽亚域 C2细胞和人类类器官的转运：1识别最短的功能性P8和P9多肽亚域 通过删除特定的氨基酸残基。1B.P8和P9多肽中关键氨基酸残基的鉴定 一种替代的方法。2A。鉴定C2细胞中涉及的细胞表面受体(S)。2B。定义信令 刺激C2细胞草酸转运的途径。2C。确定草酸转运体和 P8+9在C2细胞中激活它们的机制，并使用A6缺失的小鼠来确认A6在体内的作用。 实现项目目标将对预防和/或治疗红斑狼疮产生治疗影响 高草酸血症和高草酸尿对肠源性CaOx KS患者预后的影响 高草酸尿，原发性高草酸尿，可能还有慢性肾脏病和终末期肾病。