Ma Lijun, Shelness Gregory S, Snipes James A, Murea Mariana, Antinozzi Peter A, Cheng Dongmei, Saleem Moin A, Satchell Simon C, Banas Bernhard, Mathieson Peter W, Kretzler Matthias, Hemal Ashok K, Rudel Lawrence L, Petrovic Snezana, Weckerle Allison, Pollak Martin R, Ross Michael D, Parks John S, Freedman Barry I
Internal Medicine-Nephrology,
Pathology-Lipid Sciences, and.
J Am Soc Nephrol. 2015 Feb;26(2):339-48. doi: 10.1681/ASN.2013091017. Epub 2014 Jul 10.
Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms' tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytes observed in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.
虽然载脂蛋白L1（APOL1）基因变异与非裔美国人的肾病相关，但对于APOL1蛋白在肾细胞中的合成、摄取及定位却知之甚少。为解决这些问题，我们检测了APOL1蛋白及mRNA在人肾组织和人肾源性细胞系中的定位。对肾功能正常者的非病变肾切除冷冻切片进行间接免疫荧光显微镜检查发现，APOL1蛋白在足细胞中显著富集（与突触素和肾母细胞瘤抑制因子共定位），而在肾小管细胞中的含量较低。荧光原位杂交检测到肾小球（足细胞和内皮细胞）及肾小管中有APOL1 mRNA，这与这些细胞类型中的内源性合成一致。当将这些分析扩展到肾源性细胞系时，定量逆转录聚合酶链反应（qRT-PCR）未在人系膜细胞中检测到APOL1 mRNA；然而，在近端肾小管细胞和肾小球内皮细胞中观察到大量的APOL1 mRNA，在足细胞中的表达较低。蛋白质印迹分析显示这些细胞系中APOL1蛋白水平与之相对应。为解释在冷冻切片中观察到的肾足细胞中APOL1蛋白含量显著丰富与足细胞系中含量较低之间的明显差异，我们研究了APOL1的细胞摄取情况。APOL1蛋白在体外易被人足细胞摄取，但系膜细胞、肾小球内皮细胞或近端肾小管细胞对其摄取效率不高。我们推测，人冷冻切片足细胞中较高水平的APOL1蛋白可能反映了内源性蛋白合成以及从循环或肾小球滤液中摄取的APOL1。
