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

项目概要/摘要： 肾结石（KS）影响~1/5的男性和~1/11的女性，非常痛苦和昂贵（每年> 100亿美元）， 与CKD和ESRD相关。高复发率（5年内50%）表明目前的干预措施 需要适当的替代疗法。大多数KS由草酸钙组成， 尿草酸盐的增加增加了结石形成的风险。下尿路草酸钙过饱和 确切地减少KS形成，但不幸的是，没有减少尿草酸排泄的药物。的 肠道细菌产草酸杆菌（Of）诱导结肠草酸分泌并减少尿草酸 通过一种未知的促分泌素排泄。考虑到去乙酰化的困难， 可行并强调需要识别促分泌素。我们鉴定了其分泌的Of-衍生因子， 刺激（>2.8倍）人肠Caco 2-BBE的草酸转运的培养条件培养基（CM） (C2)通过PKA活化和刺激草酸盐转运蛋白SLC 26 A6（A6）和SLC 26 A2（A2）， 在体内，直肠给药CM可减少尿草酸排泄>32.5%，并刺激结肠草酸排泄 在高尿酸小鼠中，分泌>42%，反映了这些因子的治疗潜力。我们已经确定 Sel 1蛋白是主要的Of-derived因子，它们同样刺激C2转运草酸（1.4-2.4倍） 细胞通过PKA和A2/A6转运蛋白。我们还鉴定了Sel 1蛋白中的小肽（P8+9）， 刺激（>2.4倍）C2细胞草酸转运。P8+9还刺激人乙状结肠的草酸转运 结肠（1.8倍）、远端结肠（1.7倍）和回肠（2倍）类器官（离体肠上皮细胞模型完全 模拟体内反应），证实了P8+9在人体组织中起作用，并且它们可能会刺激 体内人结肠和回肠上皮的草酸盐分泌。 基于这些发现，我将测试特定的P8和P9亚结构域介导结肠草酸的假设 分泌和潜在的治疗基序，P8+9通过细胞表面受体起作用， 特定的信号通路，以激活相关的草酸转运蛋白（包括A2，A6，也许其他）。 具体目标如下：1.鉴定刺激草酸盐的P8和P9肽亚结构域 C2细胞和人类类器官中的转运：1a鉴定最短的功能性P8和P9肽亚结构域 通过删除特定的氨基酸残基。1b.鉴定P8和P9肽中的关键氨基酸残基， 替代方法。2a.鉴定C2细胞中涉及的细胞表面受体。2b.定义信令 途径介导的刺激草酸转运在C2细胞。2c.确定草酸转运蛋白和 P8+9在C2细胞中激活它们的机制，并使用A6缺失小鼠来证实A6的体内作用。 实现该项目的目标将对预防和/或治疗 高尿酸血症和高尿酸血症，影响CaOx KS患者的结局，肠 高尿酸血症，原发性高尿酸血症，可能还有CKD和ESRD。

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.