Nie Mingzhu, Bal Manjot S, Yang Zhufeng, Liu Jie, Rivera Carolina, Wenzel Andrea, Beck Bodo B, Sakhaee Khashayar, Marciano Denise K, Wolf Matthias T F
Pediatric Nephrology.
Nephrology, and.
J Am Soc Nephrol. 2016 Nov;27(11):3447-3458. doi: 10.1681/ASN.2015101100. Epub 2016 Apr 1.
Hypercalciuria is a major risk factor for nephrolithiasis. We previously reported that Uromodulin (UMOD) protects against nephrolithiasis by upregulating the renal calcium channel TRPV5. This channel is crucial for calcium reabsorption in the distal convoluted tubule (DCT). Recently, mutations in the gene encoding Mucin-1 (MUC1) were found to cause autosomal dominant tubulointerstitial kidney disease, the same disease caused by UMOD mutations. Because of the similarities between UMOD and MUC1 regarding associated disease phenotype, protein structure, and function as a cellular barrier, we examined whether urinary MUC1 also enhances TRPV5 channel activity and protects against nephrolithiasis. We established a semiquantitative assay for detecting MUC1 in human urine and found that, compared with controls (n=12), patients (n=12) with hypercalciuric nephrolithiasis had significantly decreased levels of urinary MUC1. Immunofluorescence showed MUC1 in the thick ascending limb, DCT, and collecting duct. Applying whole-cell patch-clamp recording of HEK cells, we found that wild-type but not disease mutant MUC1 increased TRPV5 activity by impairing dynamin-2- and caveolin-1-mediated endocytosis of TRPV5. Coimmunoprecipitation confirmed a physical interaction between TRPV5 and MUC1. However, MUC1 did not increase the activity of N-glycan-deficient TRPV5. MUC1 is characterized by variable number tandem repeats (VNTRs) that bind the lectin galectin-3; galectin-3 siRNA but not galectin-1 siRNA prevented MUC1-induced upregulation of TRPV5 activity. Additionally, MUC1 lacking VNTRs did not increase TRPV5 activity. Our results suggest that MUC1 forms a lattice with the N-glycan of TRPV5 via galectin-3, which impairs TRPV5 endocytosis and increases urinary calcium reabsorption.
高钙尿症是肾结石的主要危险因素。我们之前报道过，尿调节蛋白（UMOD）通过上调肾钙通道瞬时受体电位香草酸亚型5（TRPV5）来预防肾结石。该通道对远端小管（DCT）中的钙重吸收至关重要。最近，发现编码粘蛋白-1（MUC1）的基因突变会导致常染色体显性遗传性肾小管间质性肾病，这与UMOD突变导致的疾病相同。由于UMOD和MUC1在相关疾病表型、蛋白质结构以及作为细胞屏障的功能方面存在相似性，我们研究了尿MUC1是否也能增强TRPV5通道活性并预防肾结石。我们建立了一种用于检测人尿中MUC1的半定量检测方法，发现与对照组（n = 12）相比，高钙尿性肾结石患者（n = 12）的尿MUC1水平显著降低。免疫荧光显示MUC1存在于髓袢升支粗段、DCT和集合管中。应用全细胞膜片钳记录HEK细胞，我们发现野生型而非疾病突变型MUC1通过损害发动蛋白2和小窝蛋白1介导的TRPV5内吞作用来增加TRPV5活性。免疫共沉淀证实了TRPV5与MUC1之间存在物理相互作用。然而，MUC1并未增加N-聚糖缺陷型TRPV5的活性。MUC1的特征是具有可变数量串联重复序列（VNTRs），其可结合凝集素半乳凝素-3；半乳凝素-3小干扰RNA而非半乳凝素-1小干扰RNA可阻止MUC1诱导的TRPV5活性上调。此外，缺乏VNTRs的MUC1并未增加TRPV5活性。我们的结果表明，MUC1通过半乳凝素-3与TRPV5的N-聚糖形成晶格，从而损害TRPV5内吞作用并增加尿钙重吸收。
