Mushtaq Shamim, Siddiqui Anwar Ali, Naqvi Zulfiqar Ali, Rattani Ahmed, Talati Jamsheer, Palmberg Carina, Shafqat Jawed
Department of Biological and Biomedical Sciences, Research Laboratory, Juma Building, Pakistan.
Clin Chim Acta. 2007 Sep;384(1-2):41-7. doi: 10.1016/j.cca.2007.05.015. Epub 2007 May 26.
In order to understand the mechanism of stone genesis, it is essential to determine the characteristics of macromolecules constituting the urinary stones. We characterized proteins from the inner core and outer matrix of calcium oxalate (CaOx) renal stones.
Inner core and outer matrix of CaOx renal stones were separated and proteins were extracted with a buffer containing SDS and beta-mercaptoethanol. Proteins were analyzed and purified by SDS-PAGE and RP-HPLC respectively. The protein bands from gel and protein fractions were sequenced by MALDI TOF mass spectrometry. ELISA, western and slot blot immunoassays were performed to confirm the identity of the proteins in stones and urine of the stone formers. The potential of the identified protein as an effective promoter or inhibitor was assessed by observing their effects on CaOx crystallization using aggregometer.
The inner core extract predominantly exhibited protein species in the molecular weight range of 12-14 kDa. However, a 66 kDa band, identified as osteopontin was also detected in the inner core along with outer matrix and in the urine of stone formers and non stone formers. Purification of low molecular weight proteins was carried out by reversed phase HPLC. Tandem mass spectrometry analysis identified them as myeloperoxidase chain A (MPO-A), alpha-defensin, and calgranulin. ELISA, western blot and slot-blot immuno-assays further confirmed their presence restricted to the inner core and not in the outer matrix. Turbidity assays showed that low molecular weight renal stone proteins promoted the aggregation of CaOx crystals.
Persistent hyperoxaluria leads to tubular epithelial injury, resulting in the release of these anti-inflammatory proteins. These proteins could have been first adsorbed on CaOx crystals thereby become a part of nucleation process leading to inner matrix formation.
为了了解结石形成的机制,确定构成尿路结石的大分子特征至关重要。我们对草酸钙(CaOx)肾结石的内核和外基质中的蛋白质进行了表征。
分离CaOx肾结石的内核和外基质,并用含有SDS和β-巯基乙醇的缓冲液提取蛋白质。分别通过SDS-PAGE和RP-HPLC对蛋白质进行分析和纯化。通过MALDI TOF质谱对凝胶中的蛋白条带和蛋白质组分进行测序。进行ELISA、western和斑点印迹免疫分析以确认结石形成者结石和尿液中蛋白质的身份。通过使用凝集仪观察它们对CaOx结晶的影响,评估鉴定出的蛋白质作为有效促进剂或抑制剂的潜力。
内核提取物主要显示分子量在12 - 14 kDa范围内的蛋白质种类。然而,在结石形成者和非结石形成者的内核、外基质以及尿液中也检测到一条66 kDa的条带,鉴定为骨桥蛋白。通过反相HPLC对低分子量蛋白质进行纯化。串联质谱分析将它们鉴定为髓过氧化物酶链A(MPO-A)、α-防御素和钙粒蛋白。ELISA、western印迹和斑点印迹免疫分析进一步证实它们仅存在于内核中,而不存在于外基质中。比浊法表明低分子量肾结石蛋白促进了CaOx晶体的聚集。
持续性高草酸尿症导致肾小管上皮损伤,从而导致这些抗炎蛋白的释放。这些蛋白质可能首先吸附在CaOx晶体上,从而成为导致内核基质形成的成核过程的一部分。