Supplementary Material for: Dietary Phosphate Binding and Loading Alter Kidney Angiotensin-Converting Enzyme mRNA and Protein Content in 5/6 Nephrectomized Rats

<i>Background:</i> Vitamin D receptor activation with paricalcitol can modulate the transcription of renin-angiotensin system components in the surgical 5/6 nephrectomy rat model (5/6 NX) of chronic renal insufficiency. We tested the hypothesis whether dietary modification of phosphate influences kidney renin-angiotensin system gene expression at the mRNA level in 5/6 NX rats. <i>Methods:</i> Fifteen weeks after surgery, rats were given control diet (0.3% calcium, 0.5% phosphate), phosphate-lowering diet (3% calcium as carbonate) or high-phosphate diet (1.5%) for 12 weeks. Sham-operated rats were on control diet. <i>Results:</i> Blood pressure, plasma phosphate, parathyroid hormone, glomerulosclerosis, tubulointerstitial damage, and FGF-23 were increased in remnant kidney rats, whereas creatinine clearance was decreased. Phosphate, parathyroid hormone, glomerulosclerosis, tubulointerstitial damage, and FGF-23 were further elevated by the high-phosphate diet, but were reduced by the phosphate-lowering diet. Plasma calcium was increased with the phosphate-lowering diet and decreased with the high-phosphate diet. Remnant kidney rats on control diet showed upregulated kidney angiotensin-converting enzyme (ACE) and angiotensin (Ang) IV receptor (AT₄) transcription, while ACE2, Ang II type 2 receptor and renin receptor transcription were downregulated in comparison with sham rats. Phosphate-lowering diet reduced whereas high-phosphate diet increased kidney ACE, and these effects were observed at both mRNA and protein levels. Dietary phosphate loading also resulted in lower AT_1a gene transcription. <i>Conclusion:</i> Dietary phosphate loading was associated with elevated kidney ACE expression, increased tissue damage and lower AT_1a transcription in 5/6 NX rats. Phosphate binding with 3% calcium carbonate had opposite effects on ACE and kidney damage.

**背景**：使用帕立骨化醇（paricalcitol）激活维生素D受体（vitamin D receptor），可在慢性肾功能不全的5/6肾切除（5/6 nephrectomy, 5/6 NX）大鼠模型中调节肾素-血管紧张素系统（renin-angiotensin system）组分的转录。本研究旨在验证膳食磷酸盐调控是否会在5/6 NX大鼠的mRNA层面影响肾脏肾素-血管紧张素系统基因的表达这一假说。

**方法**：术后15周，将大鼠分为三组，分别给予普通对照饲料（钙含量0.3%、磷酸盐含量0.5%）、降磷酸盐饲料（以碳酸钙形式提供3%钙）或高磷酸盐饲料（磷酸盐含量1.5%），干预时长为12周；假手术组大鼠仅给予普通对照饲料。

**结果**：残余肾大鼠的血压、血浆磷酸盐、甲状旁腺激素、肾小球硬化、肾小管间质损伤以及成纤维细胞生长因子23（fibroblast growth factor 23, FGF-23）水平均升高，而肌酐清除率降低。高磷酸盐饲料进一步升高了上述磷酸盐、甲状旁腺激素、肾小球硬化、肾小管间质损伤及FGF-23指标，而降磷酸盐饲料则可降低这些指标。降磷酸盐饲料会升高血浆钙水平，而高磷酸盐饲料则会降低血浆钙水平。与假手术组大鼠相比，给予普通对照饲料的残余肾大鼠肾脏血管紧张素转换酶（angiotensin-converting enzyme, ACE）与血管紧张素（angiotensin, Ang）Ⅳ受体（AT₄）的转录水平上调，而ACE2、血管紧张素Ⅱ2型受体及肾素受体的转录水平下调。降磷酸盐饲料可降低肾脏ACE的表达，高磷酸盐饲料则升高肾脏ACE的表达，上述效应在mRNA与蛋白层面均有体现。膳食磷酸盐负荷还会降低AT₁a基因的转录水平。

**结论**：在5/6 NX大鼠中，膳食磷酸盐负荷与肾脏ACE表达升高、组织损伤加重及AT₁a转录水平降低相关。以3%碳酸钙结合磷酸盐的膳食干预，对ACE表达与肾脏损伤产生了相反的调控效应。