The effects of intracrystalline and surface-bound proteins on the attachment of calcium oxalate monohydrate crystals to renal cells in undiluted human urine.
The effects of intracrystalline and surface-bound proteins on the attachment of calcium oxalate monohydrate crystals to renal cells in undiluted human urine.
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DOI：
10.1111/j.1464-410x.2009.08816.x
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发表时间：
2010-03
影响因子：
4.5
通讯作者：
Ryall RL
中科院分区：
文献类型：
--
作者：
Grover PK;Thurgood LA;Wang T;Ryall RL
比较以下物质与马丁 - 达比犬肾（MDCK）- II细胞的结合情况：（i）无机草酸钙一水合物（iCOM）晶体以及从含不同蛋白质浓度的尿液中沉淀出的草酸钙（COM）晶体；（ii）含有晶内蛋白以及晶内 + 表面结合蛋白的尿液COM晶体。
在混合人尿液经筛分（sCOM）、离心和过滤（cfCOM）以及超滤（ufCOM）的部分中生成尿液COM晶体，并使用六种不同的超滤尿液样本作为结合介质来比较它们与MDCK - II细胞的黏附情况。通过对从人尿液中沉淀出的草酸钙晶体进行脱矿质处理来制备晶体基质提取物（CME），并用于制备在蛋白质浓度为0、0.05、0.1、0.5和5.0 mg/L时含有晶内以及晶内 + 表面结合CME的COM晶体。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和蛋白质印迹法，以凝血酶原片段1（PTF1）作为标记物，对与晶体相关的蛋白质含量进行定性评估。测定另外10份尿液样本经筛分、离心和过滤以及超滤后的蛋白质浓度。
六种尿液类型中的晶体附着中位数按iCOM > ufCOM > cfCOM = sCOM的顺序降低，与它们所沉淀的溶液或尿液中的蛋白质浓度成反比。sCOM和cfCOM晶体的结合比iCOM晶体少约□23%。在CME蛋白质浓度增加的情况下生成的COM晶体的附着在浓度达到5 mg/L之前不受影响，但含有相同浓度晶内 + 表面结合蛋白的晶体的结合在蛋白质浓度从0到5.0 mg/L时成比例下降。
无机COM晶体与MDCK - II细胞的结合明显比从经筛分、离心和过滤以及超滤的尿液中沉淀出的尿液晶体更强，并且结合亲和力与形成它们的尿液中的蛋白质浓度成反比。虽然晶内和表面的CME蛋白质都会降低COM晶体与MDCK - II细胞的附着，但位于晶体表面的蛋白质比嵌在矿物质内部的蛋白质影响更大。未来的细胞 - 晶体相互作用研究应使用尿液晶体并在人尿液中进行。
To compare the binding to Madin-Darby canine kidney (MDCK)-II cells of: (i) inorganic calcium oxalate monohydrate (iCOM) crystals and COM crystals precipitated from urine containing different concentrations of protein; and (ii) urinary COM crystals containing intracrystalline and intracrystalline + surface-bound protein. Urinary COM crystals were generated in sieved (sCOM), centrifuged and filtered (cfCOM), and ultrafiltered (ufCOM) portions of a pooled human urine and their adhesion to MDCK-II cells was compared using six different ultrafiltered urine samples as the binding medium. Crystal matrix extract (CME) was prepared by demineralizing calcium oxalate crystals precipitated from human urine and used to prepare COM crystals with intracrystalline, and intracrystalline + surface-bound CME at protein concentrations of 0, 0.05, 0.1, 0.5 and 5.0 mg/L. The amount of protein associated with the crystals was qualitatively assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting, using prothrombin fragment 1 (PTF1) as a marker. Protein concentration was determined in sieved, centrifuged and filtered, and ultrafiltered fractions of 10 additional urine samples. The median crystal attachment in the six urine types decreased in the order iCOM > ufCOM > cfCOM = sCOM, in inverse proportion to the concentration of protein in the solution or urine from which they were precipitated. sCOM and cfCOM crystals bound ≈□ 23% less than iCOM crystals. The attachment of COM crystals generated in the presence of increasing concentrations of CME proteins was unaffected up to a concentration of 5 mg/L, but binding of crystals containing the same concentrations of intracrystalline + surface-bound proteins decreased proportionally at protein concentrations from 0 to 5.0 mg/L. Inorganic COM crystals bind significantly more strongly to MDCK-II cells than urinary crystals precipitated from sieved, centrifuged and filtered, and ultrafiltered urine, and binding affinity is inversely related to the concentration of protein in the urine in which they are formed. While both intracrystalline and superficial CME proteins reduce the attachment of COM crystals to MDCK-II cells, those located on the crystal surface have a greater influence than those incarcerated within the mineral bulk. Future cell–crystal interaction studies should use urinary crystals and be performed in human urine.