Supplementary Material for: Chronic Kidney Disease Results in Deficiency of ABCC6, the Novel Inhibitor of Vascular Calcification

<b><i>Background:</i></b> Chronic kidney disease (CKD) is associated with arterial medial calcification which plays a major role in the pathogenesis of cardiovascular disease in this population. Several factors are known to promote soft tissue and accelerated arterial calcification in CKD including systemic inflammation, altered calcium and phosphate homeostasis, hypertension, and deficiency of endogenous calcification inhibitors. The ABCC6 transporter (ATP-binding cassette subfamily C number 6), also known as multidrug resistance-associated protein 6 (MRP6), is highly expressed in the liver and kidney. Mutation of <i>ABCC6</i> results in pseudoxanthoma elasticum, an inherited disorder characterized by arterial and soft tissue calcification. Given the prevalence of arterial medial calcification in CKD, the present study was undertaken to test the hypothesis that CKD may lead to acquired ABCC6 deficiency. <b><i>Methods:</i></b> CKD was induced via 5/6 nephrectomy in male Sprague-Dawley rats and by adenine-containing diet to cause chronic interstitial nephropathy in female DBA/2J mice. Sham-operated rats and mice fed regular diet served as controls. Liver and kidney tissues were harvested and processed for ABCC6 protein and mRNA analysis. <b><i>Results:</i></b> ABCC6 protein levels were significantly reduced in the liver and kidney tissues from CKD rats and mice. However, ABCC6 mRNA levels were unchanged, pointing to post-transcriptional or post-translational mechanisms for the observed ABCC6 deficiency. Additionally, plasma levels of the calcification inhibitor fetuin-A were significantly decreased in CKD animals compared to controls. <b><i>Conclusions:</i></b> CKD results in acquired ABCC6 transporter deficiency. To our knowledge this abnormality has not been previously reported and may contribute to CKD-associated vascular and soft tissue calcification.

<b><i>研究背景：</i></b> 慢性肾脏病(Chronic kidney disease, CKD)与动脉中层钙化相关，而动脉中层钙化在该人群心血管疾病的发病机制中发挥核心作用。已知多种因素可促进慢性肾脏病患者的软组织钙化与动脉钙化加速，包括全身性炎症、钙磷稳态失衡、高血压以及内源性钙化抑制因子缺乏。ATP结合盒转运蛋白C亚家族成员6(ATP-binding cassette subfamily C number 6, ABCC6)，亦称多药耐药相关蛋白6(multidrug resistance-associated protein 6, MRP6)，在肝脏与肾脏中呈高表达。ABCC6基因突变可导致弹性假黄瘤(pseudoxanthoma elasticum)，这是一种以动脉及软组织钙化为特征的遗传性疾病。鉴于慢性肾脏病患者动脉中层钙化的高患病率，本研究旨在验证“慢性肾脏病可导致获得性ABCC6缺乏”这一假说。<b><i>研究方法：</i></b> 本研究通过5/6肾切除术构建雄性Sprague-Dawley大鼠慢性肾脏病模型，通过含腺嘌呤的饮食构建雌性DBA/2J小鼠慢性间质性肾病模型。假手术组大鼠及喂食普通饲料的小鼠作为对照。采集肝脏与肾脏组织，用于ABCC6蛋白与mRNA水平分析。<b><i>研究结果：</i></b> 慢性肾脏病模型大鼠与小鼠的肝脏、肾脏组织中ABCC6蛋白水平均显著降低。但ABCC6的mRNA水平未发生明显变化，提示上述ABCC6缺乏现象可能由转录后或翻译后机制介导。此外，与对照组相比，慢性肾脏病模型动物的血浆钙化抑制因子胎球蛋白-A(fetuin-A)水平显著降低。<b><i>研究结论：</i></b> 慢性肾脏病可导致获得性ABCC6转运蛋白缺乏。据我们所知，这一异常此前尚未见报道，其可能参与慢性肾脏病相关的血管与软组织钙化过程。