Calcium Oxalate Kidney Stones: Pathogenesis of Obesity-associated Hyperoxaluria

描述（由申请人提供）：70-80%的肾结石（KS）由草酸钙组成，尿草酸盐的微小变化会影响结石风险。肥胖是一个危险因素 对于KS和肥胖的结石形成者通常有轻度的高尿酸血症。据报告，体型增大与尿草酸排泄量升高呈正相关;然而，其潜在机制仍未知。因此，我们在肥胖ob/ob（ob）小鼠中开展研究，以确定肥胖相关性高尿酸（OAH）的发病机制。发现ob小鼠与对照相比具有显著更高的尿草酸盐（针对肌酸酐调整）（>2.9倍），这不是由于使用成对喂养的过量进食。在肥胖db/db小鼠中也观察到显著的高尿症（> 2.3倍）。与对照组相比，我们观察到ob小鼠空肠（> 46%）和回肠（>30%）14 C-草酸盐和3 H-甘露醇（细胞旁标记物）的吸收通量显著更高。重要的是，与对照组相比，在口服管饲13 C-草酸盐后，在ob小鼠中观察到显著更高（>35%）的尿13 C-草酸盐。促炎细胞因子（PC）IFN-？[IFN]和TNF-α [TNF]在肥胖症中升高，导致人肠Caco 2-BBE（C2）细胞中顶侧至基底侧14 C-草酸盐和3 H-甘露醇通量增加> 3倍。PC诱导的吸收通量增加被AMP-18和GLP-2（已知可改善肠屏障功能的肽）预处理完全阻断。BALB/c小鼠在分离空肠段并将其固定在Ussing室中之前用TNF预处理，导致14 C-草酸盐和3 H-甘露醇吸收通量增加> 1.9倍，AMP或GLP预处理可完全阻断该效应。IFN、TNF和IL-6还通过涉及降低SLC 26 A6（A6）的机制显著抑制C2细胞对顶端14 C-草酸盐的摄取，SLC 26 A6（A6）是一种阴离子交换剂，在肠草酸盐分泌、mRNA/总蛋白表达中起重要作用。此外，ob小鼠具有显著降低的空肠A6 mRNA（>65%）和总蛋白表达。在对照空肠组织中观察到小的净草酸分泌通量，而在OB组织中观察到大的净吸收通量，这是由于显著减少的分泌通量和增加的吸收通量。基于这些发现，我将检验这一假设，即PC在OAH中起重要作用，并且抗TNF、抗IFN和/或抗IL-6将正常化和/或改善所观察到的高尿酸血症。将努力实现以下具体目标：评价抗TNF、抗IFN和抗IL-6对观察到的高尿酸血症的治疗效果。1b.评价ob小鼠与TNF、IFN和/或IL-6缺失小鼠杂交对观察到的高尿症的影响，以确认它们在OAH中的作用。2a.评估ob小鼠和对照小鼠体内小肠和大肠的细胞旁通透性。2b.检查AMP和GLP对观察到的高血压尿的治疗作用。2c.阐明AMP、GLP、抗TNF、抗IFN和/或抗IL-6使观察到的高尿酸正常化和/或改善的体内机制。3.评价ob小鼠与A6缺失小鼠杂交对观察到的高尿症的影响，以证实A6在OAH中的作用。

DESCRIPTION (provided by applicant): 70-80% of kidney stones (KS) are composed of calcium oxalate, and minor changes in urine oxalate affect the stone risk. Obesity is a risk factor for KS and obese stone formers often have mild hyperoxaluria. A positive correlation between increased body size and elevated urinary oxalate excretion was reported; however, the underlying mechanism(s) remain(s) unknown. Therefore, we initiated studies in the obese ob/ob (ob) mice to define the pathogenesis of the obesity-associated hyperoxaluria (OAH). ob mice were found to have significantly higher urine oxalate (adjusted for creatinine) compared to controls (>2.9-fold), which is not due to overeating using pair-feeding. Significant hyperoxaluria was also seen in the obese db/db mice (> 2.3-fold). We observed significantly higher jejunal (> 46%) and ileal (>30%) 14C-oxalate and 3H-mannitol (a paracellular marker) absorptive fluxes in ob mice compared to controls ex vivo. Importantly, a significantly higher (>35%) urine 13C-oxalate was observed in ob mice compared to controls following an oral gavage with 13C-oxalate. The proinflammatory cytokines (PCs) IFN-? [IFN] and TNF-¿ [TNF], which are elevated in obesity, caused > 3- fold increase in apical to basolateral 14C-oxalate and 3H-mannitol fluxes in human intestinal Caco2-BBE (C2) cells. The PC-induced increased absorptive fluxes were completed blocked by pretreatment with AMP-18 and GLP-2, peptides known to improve intestinal barrier function. Pretreatment of BALB/c mice with TNF before isolating and mounting jejunal segments in Ussing chambers led to > 1.9-fold increase in 14C -oxalate and 3H- mannitol absorptive fluxes, an effect completely blocked by pretreatment with AMP or GLP. IFN, TNF, and IL-6 also significantly inhibited apical 14C-oxalate uptake by C2 cells through mechanisms involving reduced SLC26A6 (A6), an anion exchanger with essential role in intestinal oxalate secretion, mRNA/total protein expression. In addition, ob mice have significantly reduced jejunal A6 mRNA (>65%) and total protein expression. while a small net oxalate secretory flux was observed in control jejunal tissues, a large net absorptive flux was seen in ob tissues, due to significantly reduced secretory flux and an increased absorptive flux . Based on these findings, I will test the hypothesis that PCs play an important role in the OAH, and that anti-TNF, anti-IFN, and/or anti-IL-6 will normalize and/or ameliorate the observed hyperoxaluria. The following specific aims will be pursued: 1a. Evaluate the therapeutic effects of anti-TNF, anti-IFN, and anti-IL-6 on the observed hyperoxaluria.1b. Evaluate the effects of crossing the ob mice with the TNF, IFN, and/or IL-6 null mice on the observed hyperoxaluria to confirm their roles in the OAH. 2a. Assess small and large intestinal paracellular permeability in vivo in ob mice and their controls. 2b. Examine the therapeutic effects of AMP and GLP on the observed hyperoxaluria. 2c. Elucidate the in vivo mechanisms by which AMP, GLP, anti-TNF, anti- IFN, and/or anti-IL-6 normalize and/or ameliorate the observed hyperoxaluria. 3. Evaluate the effects of crossing the ob mice with the A6 null mice on the observed hyperoxaluria to confirm the role of A6 in the OAH.