Tamm-Horsfall glycoprotein-inhibitor or promoter of calcium oxalate monohydrate crystallization processes?

Summary The processes of calcium oxalate monohydrate (COM) crystal nucleation, growth and aggregation (agglomeration) generally have been studied using a wide variety of assay systems/conditions. This paper reviews the apparently conflicting data on the effects of Tamm-Horsfall glycoprotein (THP) on COM crystallization processes in vitro, with the main emphasis on crystal aggregation. According to its well-known physico-chemical properties. THP has a dual role in modifying crystal aggregation: at high pH and low ionic strength (IS), THP is a powerful crystal aggregation inhibitor. Upon lowering pH and rasing IS. THP viscosity increases, leading to reduced crystal aggregation inhibition. In the presence of additional calcium ions, some THPs even become strong promoters of crystal aggregation. This phenomenon seems to be more pronounced in THPs isolated from recurrent calcium stone formers whose proteins exhibit an abnormally high tendency of polymerization. Recent studies suggest an inherited molecular abnormality of THP among some severe recurrent calcium stone formers.     

不同浓度TH黏蛋白对草酸钙成核、生长、聚集的影响

目的 观察不同浓度TH黏蛋白(THM)对体外成石系统内一水草酸钙(COM)的成核、生长和聚集的影响.方法 盐析法提取THM,利用原子分光光度计测定0、10、50、100 mg/LTHM人工尿体外成核和种籽晶溶液内Ca~(2+)变化,偏光显微镜计数COM数目.结果 THM为0、10、50、100mg/L时,Ca~(2+)减少量0.81、0.57、0.51、0.96mmol/L;成核数3.20×10~4、2.32×10~4、1.83 × 10~4、2.85×10~4;生长率53.9%、48.7%、34.6%、19.8%;聚集率33.2%、11.7%、9.4%、66.2%,差异均有统计学意义(P<0.01).结论 在生理浓度范围内,THM可以抑制尿结石的形成,且随浓度增加其抑制作用逐渐增强,但超过一定浓度时,其转为结石促进剂.     

Calcium oxalate monohydrate binding protein: a diagnostic biomarker for calcium oxalate kidney stone formers

Urinary oxalate is a biomarker for calcium oxalate kidney stone disease; however, its assay is insensitive and nonspecific. Calcium oxalate monohydrate (COM) binding protein (45 kDa) is a promoter of calcium oxalate kidney disease, which is markedly upregulated by oxalate induced oxidative stress. The current study was carried out to evaluate whether COM binding protein can serve as a diagnostic marker for calcium oxalate kidney stone formers. COM binding protein was isolated, purified and antibody was raised against it in rabbits. Urine samples (24 h) were collected from patients suffering from various kidney diseases such as acute nephritis, chronic nephritis, nephrotic syndrome, calcium oxalate (CaOx) stone formers, uric acid stone formers, struvite stone formers and calcium phosphate stone formers. This COM binding protein was quantified by an in house ELISA method and the excretion was found to lie between 2 and 3 mg in control samples, while in CaOx stone formers it was detected between 11 and 19 mg. Urinary risk factors were assayed. We conclude that COM binding protein can serve as a diagnostic marker for CaOx stone formers.     

Calcium oxalate monohydrate (whewellite) renal lithiasis

Pure calcium oxalate monohydrate lithiasis is rare--about 5 to 10% of total renal lithiases. The proportion is the same in men and women. Pure calcium oxalate stones are a polished dark brown color, and are very hard. They are visualized radiologically as regular and homogeneous. From a biological standpoint, this type of stone is frequently associated with normal calciuria and oxaluria. Pure calcium oxalate monohydrate stones rarely evolve.     

Tamm-Horsfall蛋白对草酸钙结晶成核和聚集的影响

目的 在人工尿液中，观察不同浓度及不同性质的Tamm-Horsfall蛋白对草酸钙的成核，生长，聚集的影响。方法 通过分光光度计在620μm时的A值的测定可在过饱和溶液中观察草酸钙晶体的成核和积聚。观察参数包括诱导时间（inducetime），简写为，TI，即形成可观察微粒所需的时间；成核曲线（slope of nucleation）的速率，简写为SN，即分光光度计A620所观察的曲线上升支；聚积曲线（slopeofaggregation）的速率，简写为SA，即下降支，以及加入不同浓度的NTHP（正常人尿液中所提取的TH蛋白），SF-THP（结石患者尿液中所提取的TH蛋白）后对参数的影响。结果 草酸钙结晶实验的诱导时间为（60．0±2．0）s，成核速率（SN）（1．058±0．036）×10^-3／s，聚集速率（SA）为（0．074±0．006）×10^-3／s．NTHP浓度20mg／L时，诱导时间（59±8）s；NTHP30mg／L时，诱导时间（63±11）s；NTHP40mg／L时，诱导时间（57±6）s，NTHP既可抑制晶体成核，又可抑制晶体的聚集。SF-THP20mg／L时，诱导时间（37±2）s；SF-THP30mg／L时，诱导时间（34±3）s；SF-THP40mg／L时，诱导时间（31±5）s，ST-THP降低TI并且促进聚集。结论 NTHP和ST-THP对草酸钙晶体的结晶动力学的影响是不同的。     

Effects of Tamm-Horsfall protein on the protection of MCDK cells from oxalate induced free radical injury

Renal cell injury and fixed particle formation is one of the theories of urinary stone formation. The exposure of renal epithelial cells to oxalate ions and calcium oxalate monohydrate crystals can cause free radical generation and increase lipid peroxidation. Tamm-Horsfall protein (THP) has a protective effect on the production of free radicals in vitro. We aimed to show that THP (and its deglycosylated products, D-THP) could protect culture cells from free radical injury in vivo as well as the possible mechanism by which this is done. Exposure of Madin-Darby canine kidney (MDCK) cells to Ox resulted in a significant increase in the release LDH, NBT and MDA, as well as an increase in caspase 3 activity, all of which were further elevated when COM crystals were added. With the addition of THP at 500 nM, there was a significant decrease in the release of LDH and the production of MDA and NBT. A decrease in capase 3 activity was observed when 500 nM THP was added to the culture medium that reached 32.7% and 40.4% of inhibition in CaOx+THP and CaOx+COM+THP, respectively. THP decreased the adhesion of COM crystals to the MDCK cells but lost its effect when THP was deglycosylated. The results indicate that both Ox and COM crystals cause the release of LDH, MDA, NBT and increase the activity of capase 3 in MDCK cells. As a free radical scavenger, THP reduces the amount of free radicals and provides significant protection at a critical concentration of 500 nM. The deglycosylated THP decreased the effect of the protection of the MDCK cells from oxalate-induced injury and an increase of adhesion of the COM crystals to the MDCK cells. Therefore, the effects of THP on the protection of oxalate induced radical injury may be partly due to its intact glycosylation and its adhesion to the cell membrane.     

Effect of urinary material and glycosaminoglycans on calcium oxalate monohydrate crystal aggregation

We have examined by the coulter counter method whether some substances promote or inhibit calcium oxalate monohydrate crystal aggregation. The substances tested were hyaluronic acid, chondroitin sulfate and urinary lyophilized material of less than 10 k dalton fractions having aggregate activity. As a result, hyaluronic acid promoted aggregation at low concentrations but inhibited it at higher concentrations, and chondroitin sulfate only inhibited it. They seem, therefore, to have quite different effects, depending on their urinary concentrations, on calcium oxalate crystal aggregation process. However, urinary fractions only promoted aggregation in a dose-response manner. This promoting effect might be caused by not only hyaluronic acid that was contained in the fractions but also by some other promoters.     

Variability of protein content in calcium oxalate monohydrate stones

BACKGROUND AND PURPOSE: Urinary stones are heterogeneous in their fragility to lithotripter shockwaves. As a first step in gaining a better understanding of the role of matrix in stone fragility, we measured extractible protein in calcium oxalate monohydrate (COM) stones that were extensively characterized by micro-computed tomography (micro CT). MATERIALS AND METHODS: Stones were scanned using micro CT (Scanco mCT20, 34 microm). They were ground, and the protein extracted using four methods: 0.25M EDTA, 2% SDS reducing buffer, 9M urea buffer, and 10% acetic acid. Protein was measured using NanoOrange. The SDS extracts were also examined using polyacrylamide electrophoresis (PAGE). RESULTs: Extracted protein was highest with the SDS or urea methods (0.28% +/- 0.13% and 0.24% +/- 0.11%, respectively) and lower using the EDTA method (0.17% +/- 0.05%; P < 0.02). Acetic acid extracted little protein (0.006 +/- 0.002%; P < 0.001). Individual stones were significantly different in extractability of protein by the different methods, and SDS-PAGE revealed different protein patterns for individual stones. Extracted protein did not correlate with X-ray-lucent void percentage, which ranged from 0.06% to 2.8% of stone volume, or with apatite content. CONCLUSIONS: Extractible stone-matrix protein differs for individual COM stones, and yield is dependent on the extraction method. The presence of X-ray-lucent voids or minor amounts of apatite in stones did not correlate with protein content. The amounts of protein recovered were much lower than reported by Boyce, showing that these methods extracted only a fraction of the protein bound up in the stones. The results suggest that none of the methods tested will be useful for helping to answer the question of whether matrix content differs among stones of differing fragility to lithotripter shockwaves.     

Calcium oxalate monohydrate crystal binding substance produced from Madin-Darby canine kidney cells

BACKGROUND: The interaction between kidney urothelium and crystals is a critical event in the growth of renal calculi. When studying calcium oxalate monohydrate (COM) crystal binding to Madin-Darby canine kidney (MDCK) cells in culture, we observed that crystals also attached to areas on the coverslips devoid of cells. This phenomenon could be the result of substances produced by the cells that adhere to the glass and subsequently bind COM crystals. We investigated the characteristics of this COM binding substance. METHODS: Media was collected from cultures of MDCK cells (conditioned media) and proteins were separated by high performance liquid chromatography. The molecular weights and purity of isolated proteins were determined by polyacrylamide gel electrophoresis. The conditioned media and each separated fraction were applied to glass and to MDCK cells and COM-binding ability determined using 14C-labeled crystals. The binding of radio-labelled calcium oxalate dihydrate, brushite, uric acid, and apatite to coverslips were also studied. RESULTS: Fourteen times more COM bound to coverslips incubated with conditioned media than those with control media. The molecular weight of the protein bound to the glass was determined to be 200 kDa. The COM crystals binding to this protein was 1.5 micro g/ng. Other crystals bound to a lesser extent. The incubation of cells with this protein inhibited COM binding by 39%. CONCLUSION: The MDCK cells produce a 200-kDa protein that has a high binding affinity for COM crystals. This protein binds to glass and is responsible for crystal binding to areas devoid of cells. This protein also has an inhibitory effect on COM binding to MDCK cells in culture.     

Estimation by infrared spectrophotometer of the calcium oxalate dihydrate to calcium oxalate monohydrate ratio

According to the theoretical expression for calibration curve as a function of the optical absorption ratio of two peaks and with the analysis of the infrared spectra of the mixture samples of commercial calcium oxalate monohydrate and synthesized calcium oxalate dihydrate, the following quadratic equation was obtained; Y = 1.79 X2 - 30.90 X + 107.04 in which Y is the percentage of the purity of calcium oxalate dihydrate and X is the ratio of the relative optical absorption at 660 cm.-1 (the wave number at a characteristic absorption peak of calcium oxalate monohydrate) to that at 610 cm.-1 (that of calcium oxalate dihydrate) by regarding the line as a base-line that links the absorption valley at around 700 cm.-1 with that at 550 cm.-1 The linear correlation coefficient of the actual purity to the estimated purity obtained from this formula of calcium oxalate dihydrate is 0.995. When this formula is applied to the results derived from the infrared spectra of the mixture samples of commercial calcium oxalate monohydrate and calcium oxalate dihydrate obtained from urinary stones in duplicate in each percentage, the linear correlation coefficient is 0.991. This estimation method by infrared spectrophotometer of the calcium oxalate dihydrate to calcium oxalate monohydrate ratio gave a very close correlation between actual and estimated purity of calcium oxalate dihydrate and seems useful in the study of calcium oxalate urolithiasis.     

Possible role of Tamm-Horsfall glycoprotein in calcium oxalate crystallisation

The role of Tamm-Horsfall glycoprotein (THGP) in the crystallisation of calcium oxalate was investigated. The results showed THGP to have a weak inhibitory effect on crystal growth. In contrast, urinary macromolecules showed a strong inhibitory effect. THGP should, therefore, not be considered the main component of urinary macromolecules which have a strong inhibitory effect on crystal growth. However, THGP did enhance crystal production in the early phase of crystallisation. It appears that THGP should be viewed as a promoter rather than an inhibitor of calcium oxalate crystallisation at physiological concentrations.     

Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation

BACKGROUND: The tubular fluid of the mammalian kidney is often supersaturated with mineral salts, but crystallization rarely occurs under normal conditions. The unique ability of the kidney to avoid harmful crystal formation has long been attributed to the inhibitory activity of the urinary macromolecules, although few in vivo studies have been carried out to examine this hypothesis. Here we examined the role of Tamm-Horsfall protein (THP), the principal urinary protein, in urinary defense against renal calcium crystal formation, using a THP knockout model that we recently developed. METHODS: Wild-type and THP knockout mice were examined for the spontaneous formation of renal calcium crystals using von Kossa staining. The susceptibility of these mice to experimentally induced renal crystal formation was evaluated by administering mice with ethylene glycol, a precursor of oxalate, and vitamin D(3), which increases calcium absorption. Renal calcium crystals were visualized by von Kossa stain, dark field microscopy with polarized light and scanning electron microscopy. RESULTS: Inactivating the THP gene in mouse embryonic stem cells results in spontaneous formation of calcium crystals in adult kidneys. Excessive intake of calcium and oxalate, precursors of the most common type of human renal stones, dramatically increases both the frequency and the severity of renal calcium crystal formation in THP-deficient, but not in wild-type mice. Under high calcium/oxalate conditions, the absence of THP triggers a marked, adaptive induction in renal epithelial cells of osteopontin (OPN), a potent inhibitor of bone mineralization and vascular calcification. Thus, OPN may serve as an inducible inhibitor of calcium crystallization, whereas THP can serve as a constitutive and apparently more effective inhibitor. CONCLUSION: These results provide the first in vivo evidence that THP is a critical urinary defense factor and suggest that its deficiency could be an important contributing factor in human nephrolithiasis, a condition afflicting tens of millions of people in the world annually.     

Decreased renal expression of the putative calcium oxalate inhibitor Tamm-Horsfall protein in the ethylene glycol rat model of calcium oxalate urolithiasis

PURPOSE: Tamm-Horsfall protein is believed to inhibit calcium oxalate crystallization, aggregation or adhesion to the renal epithelium. We determined whether ethylene glycol induced urolithiasis changes the expression of renal and urinary Tamm-Horsfall protein. For comparison the expression of another calcium oxalate inhibitor, osteopontin, was also analyzed. MATERIALS AND METHODS: Male rats were treated with 0.75% ethylene glycol plus an AIN-76 diet (Dyets, Bethlehem Pennsylvania) (ethylene glycol group) or standard rat chow and water (control group) for up to 8 weeks (6 per group for 8 weeks and 3 per group for 3 days to 6 weeks). Kidneys and urine (8 weeks only) were harvested and analyzed by Northern and Western blot analysis, and immunohistochemistry. RESULTS: Tamm-Horsfall protein message and protein (membrane bound form) were decreased, while those of osteopontin were increased in the kidneys of rats treated with ethylene glycol for 8 weeks. As judged by immunochemistry Tamm-Horsfall protein and osteopontin were consistently present in a few tubules in rats in the ethylene glycol and control groups, respectively. In urine expression of the free form of Tamm-Horsfall protein (approximately 75 kDa.) was decreased but detectable in ethylene glycol treated rats. Although readily detected in tissue, osteopontin was not detected in the urine of control or ethylene glycol treated rats. In the time course experiment Tamm-Horsfall protein did not decrease until 4 weeks, when calcium oxalate crystals were detectable in the kidneys of treated rats. In contrast, osteopontin was increased, although inconsistently, beginning at 3 days. CONCLUSIONS: Unlike other calcium oxalate inhibitors, such as osteopontin, renal message and protein for Tamm-Horsfall protein was decreased in ethylene glycol treated rats. Tamm-Horsfall protein expression did not decrease until aggregates of crystals had been deposited in the kidneys, while osteopontin expression began to increase almost immediately. Comparisons of the data on kidneys and urine obtained by RNA or protein blot analysis and immunochemistry underscore the need to examine tissue and urine by multiple techniques to obtain the most accurate assessment of how protein expression is changed by a given treatment.     

Architecture of calcium oxalate monohydrate urinary calculi

We have developed a partial dissolution method which enabled us to observe the internal architecture of urinary calculi. Using this method with improved organic matrix fixative, we studied the architecture of small calcium oxalate monohydrate urinary calculi with special attention to crystal-matrix interrelations. Calculi, we have observed, are invariably composed of 3 distinct zones. A core area is composed of randomly aggregated plate-like crystals with tendency of rosette formation. This core area is surrounded by an intermediate layer which shows prominent radial striations. This layer is composed of radially arranged piles of sheet-like crystals which extend beyond many layers of lamination. This layer gradually shifts to a peripheral layer where concentric laminations are prominent and each layer of lamination is composed of minute crystals which have lost radial arrangement. The organic matrix is observed only at the outside of the crystals, and matrix-rich crystal-poor bands separate each layer of concentric lamination. The concentric lamination seems to be a manifestation of the altered density of organic matrix which encrusted on the developing calculi and offered a milieu for crystal growth within.     

Crystal-matrix relationships in experimentally induced urinary calcium oxalate monohydrate crystals,an ultrastructural study

Summary Calcium oxalate monohydrate crystalluria was experimentally induced in male rats by administration of ethylene glycol. The crystalluria particles were separated by filtration and studied by scanning and transmission electron microscopy. They were associated with cellular degradation products. Organic material was present both on the surfaces of crystalluria particles as well as within them and was organized in radial striations and concentric laminations. It is proposed that crystal nuclei were formed by heterogeneous nucleation. These nuclei then adsorbed organic material and aggregated. The organic material polymerized resulting in the formation of a crystal-matrix unit which then grew and incorporated more organic material during the crystal growth.     

一水草酸钙与二水草酸钙结石形成机理的研究

目的　探讨一水草酸钙（ＣＯＭ） 与二水草酸钙（ＣＯＤ） 结石形成的机制。　方法　应用红外光谱仪对２５８ 块尿结石成分进行检测,同时检测３０ 例患者２４ｈ 尿液生化指标,对测定结果利用ＳＰＳＳ软件进行ｔ 检验。　结果　（１） 尿钙：ＣＯＭ 组（４．８３ ±１ ．９８）ｍ ｍｏｌ／２４ｈ,ＣＯＤ 组（９．８８ ±４ ．２８）ｍｍｏｌ／２４ｈ,Ｐ＜ ０ ．０１ ;（２） 尿磷：ＣＯＭ 组（１９ ．４０ ±９．６９）ｍ ｍｏｌ／２４ｈ,ＣＯＤ 组（２９．２０ ±１２．００）ｍ ｍｏｌ／２４ｈ,Ｐ＜ ０．０５,两组尿钙、尿磷差异有显著性。　结论　二水草酸钙结石患者尿钙、尿磷高于一水草酸钙结石患者,表明二水草酸钙的形成与高钙尿及磷酸盐异质成核有关,而一水草酸钙的形成可能与尿中抑制物缺乏有关。