Hess B, Jordi S, Zipperle L, Ettinger E, Giovanoli R
Department of Medicine and Laboratory of Electron Microscopy, University of Berne, Berne, Switzerland.
Nephrol Dial Transplant. 2000 Mar;15(3):366-74. doi: 10.1093/ndt/15.3.366.
The aim of this study was to measure the effects of normal (nTHP) and abnormal stone former Tamm-Horsfall protein (SF-THP) on calcium oxalate (CaOx) nucleation and aggregation as well as on crystal morphology, in presence or absence of citrate.
Nucleation and aggregation of CaOx crystals from a supersaturated, stirred solution (200 mM NaCl, 10 mM Na-acetate, pH 5.70, 5 mM Ca and 0.5 mM Ox) were studied by spectrophotometric time-course measurements of OD at 620 nm (OD(620)). Measured parameters were induction time t(I) (time to induce formation of detectable particles), S(N), (slope of increase of OD(620), mainly due to crystal nucleation), and S(A), (slope of decrease of OD(620) after equilibrium has been reached, due to crystal aggregation). Effects of citrate, nTHP and SF-THP on these parameters were measured, and scanning electron microscopy (SEM) was performed.
At 1.5, 2.5 and 3.5 mM, citrate increased t(I) and inhibited crystal nucleation (by 78-87%) as well as aggregation (by 63-70%), and smaller CaOx crystals (length/width ratio 1.7+/-0.1) than under standard conditions (length/width 3.9+/-0.5) were visible (P<0.001). Normal THP at 30 and 40 mg/l inhibited crystal nucleation and, more strongly, aggregation (inhibition 76-81%). SEM revealed a decrease in length/width ratio to 2.6+/-0.4 (P=0.051 vs standard conditions) and less aggregation than without nTHP. At all concentrations tested, SF-THP reduced t(I) (P=0.0001 vs standard conditions) and promoted aggregation (inhibition -48 to -33%); crystals were elongated with a length/width ratio of 4.3+/-0.6 (P<0. 05 vs nTHP). When simultaneously present with nTHP, citrate enhanced the inhibitory effects of nTHP, producing the smallest (length/width 1.5+/-0.1) and least aggregated crystals. Finally, 3.5 mM citrate turned promotory SF-THP into a crystallization inhibitor with abundant small and clustered, but not aggregated crystals.
Citrate appears to be the main determinant of CaOx crystallization rates and crystal morphology in the presence of nTHP as well as SF-THP. Its effects appear to predominate over those of THP, since even promotory SF-THP is turned into a crystallization inhibitor in the presence of citrate. This re-emphasizes at a morphological level what has been concluded from functional as well from clinical studies, namely that citrate is needed in urine at equimolar concentrations to calcium in order to prevent the formation of large crystal aggregates in presence of abnormal THP.
本研究旨在测定正常（nTHP）和异常结石形成者的Tamm-Horsfall蛋白（SF-THP）在有或无柠檬酸盐存在的情况下对草酸钙（CaOx）成核、聚集以及晶体形态的影响。
通过在620nm处对光密度（OD）进行分光光度法时间进程测量（OD(620)），研究了来自过饱和搅拌溶液（200mM NaCl、10mM Na-乙酸盐、pH 5.70、5mM Ca和0.5mM草酸）中CaOx晶体的成核和聚集。测量的参数有诱导时间t(I)（诱导形成可检测颗粒的时间）、S(N)（OD(620)增加的斜率，主要由于晶体成核）和S(A)（达到平衡后OD(620)降低的斜率，由于晶体聚集）。测定了柠檬酸盐、nTHP和SF-THP对这些参数的影响，并进行了扫描电子显微镜（SEM）检查。
在1.5、2.5和3.5mM时，柠檬酸盐增加了t(I)，抑制了晶体成核（78 - 87%）以及聚集（63 - 70%），并且可见到比标准条件下更小的CaOx晶体（长宽比1.7±0.1）（标准条件下长宽比为3.9±0.5）（P<0.001）。30和40mg/l的正常THP抑制晶体成核，并且更强烈地抑制聚集（抑制率76 - 81%）。SEM显示长宽比降至2.6±0.4（与标准条件相比P = 0.051），并且聚集比没有nTHP时少。在所有测试浓度下，SF-THP降低了t(I)（与标准条件相比P = 0.0001）并促进了聚集（抑制率 - 48至 - 33%）；晶体拉长，长宽比为4.3±0.6（与nTHP相比P<0.05）。当与nTHP同时存在时，柠檬酸盐增强了nTHP的抑制作用，产生了最小的（长宽比1.5±0.1）且聚集最少的晶体。最后，3.5mM柠檬酸盐将促进结晶的SF-THP转变为具有大量小的、聚集但未聚合的晶体的结晶抑制剂。
在存在nTHP以及SF-THP的情况下，柠檬酸盐似乎是CaOx结晶速率和晶体形态的主要决定因素。其作用似乎比THP的作用更占主导，因为即使是促进结晶的SF-THP在有柠檬酸盐存在时也会转变为结晶抑制剂。这在形态学水平上再次强调了从功能研究以及临床研究中得出的结论，即在尿液中柠檬酸盐需要与钙等摩尔浓度存在，以防止在存在异常THP的情况下形成大的晶体聚集体。
