Studies on calcium oxalate binding proteins: effect of lipid peroxidation

OBJECTIVE: Urolithiasis and free radicals have long been associated. In this study, we have isolated calcium oxalate monohydrate (COM) binding proteins from rat kidney before and after lipid peroxidation (LPO) and studied its properties on calcium oxalate crystal growth. MATERIALS AND METHODS: LPO was carried out using t-butyl hydroperoxide, cumene hydroperoxide and an ascorbate system. The COM binding proteins from control and peroxidised tissues were isolated using a modified procedure. Protein was extracted using 25 mM EDTA, and the extract was loaded onto a DEAE cellulose column and eluted with Tris-HCl buffer (pH 6.5), 0.05 M NaCl in the above buffer and 0.3 M NaCl in the same buffer. Three major protein fractions were obtained, and they were designated as fractions I, II and III according to their order of elution. The proteins were subjected to calcium oxalate crystal nucleation and aggregation. RESULTS: A positive correlation was observed between LPO and COM adsorption, while a negative correlation was observed between reduced glutathione and COM adsorption. Peroxidised protein did not show any alteration in the elution profile on the DEAE cellulose column. The -SH content of the peroxidised fractions were lower than that of the control fractions, but their oxalate binding activities were increased. Peroxidised fraction I promoted crystal growth to a greater extent than the control fraction I. Peroxidised fractions II and III were less inhibitory in nature compared to their control fractions. Light-microscopic examination of the crystals formed in the presence of the peroxidised fractions showed the formation of large aggregates of COM. CONCLUSION: Peroxidation of the renal proteins favoured their adsorption to COM crystals. -SH depletion increased the oxalate binding activity and also their affinity to the COM crystals. The peroxidised fraction I was found to favour the formation of large aggregates, suggesting that peroxidation may be one of the mechanisms altering the crystal inhibitory activity of the proteins in hyperoxaluria.     

Effect of gamma-glutamyl carboxylation of renal microsomes on calcium oxalate monohydrate crystal binding in hyperoxaluria

AIM: The gamma-carboxy glutamic acid (gla) containing proteins have been shown recently to have a role in calcium oxalate urolithiasis. Therefore, the effect of carboxylation on calcium oxalate monohydrate (COM) binding was studied in experimental hyperoxaluria. METHODS: Urolithiasis was induced in experimental rats by oral feeding 1% ethylene glycol (EG). The microsomes were carboxylated in presence of reduced vitamin K and COM binding activity was determined. RESULTS: Microsomal proteins adsorbed maximally with COM crystals. On carboxylation of the renal microsomal proteins, a significant increase in the COM crystal adsorption by 2.5-fold (p < 0.001) was observed in the hyperoxaluric condition. Further cyboxylated microsomes of EG-treated rats showed significant binding with calcium oxalate. CONCLUSION: This study presents an increased COM adsorption after the carboxylation of renal microsomes of hyperoxaluric rats.     

Uric acid-binding proteins in calcium oxalate stone formers and their effect on calcium oxalate crystallization.

OBJECTIVES: To study the effect of urinary uric acid-binding proteins of controls and stone formers on calcium oxalate crystal nucleation and aggregation. MATERIALS AND METHODS: Urine samples were collected over 24 h from 20 stone formers and from 20 age-matched normal controls. Uric acid crystallization was induced by adding equal volumes of 2.5 mmol/L uric acid. The bound proteins were separated on a cellulose column, and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The effect of the separated fractions on calcium oxalate crystal nucleation and aggregation was assessed. RESULTS: The protein bound to unit mass of uric acid crystals was higher in hyperoxaluric urine than in control urine. On cellulose-column separation, the uric acid-crystal binding proteins produced three major protein peaks, i.e. fraction I (buffer), fraction II (0.05 mol/L sodium chloride in Tris-HCl buffer) and fraction III (0.3 mol/L sodium chloride in buffer), with a minor peak obtained on elution with increasing concentrations of sodium chloride in Tris-HCl buffer (pH 7.0). Fraction I derived from either stone formers or controls promoted calcium oxalate crystallization. Fraction II from the control samples was a strong inhibitor, whereas hyperoxaluric fraction II was less inhibitory. CONCLUSION: Uric acid-binding proteins isolated either from the urine of stone formers or controls modulated calcium oxalate crystal growth. Proteins isolated from stone formers were less inhibitory of crystal nucleation and aggregation. These proteins may act as a bridge, leading to the epitaxial deposition of calcium oxalate over a urate core.     

Oxalate binding proteins in calcium oxalate nephrolithiasis

The existence of several oxalate specific binding proteins have been demonstrated in human and rat kidney. These occur in both cortical and medullary cells and are distributed mostly in the subcellular organelles. About 1/3 of the total cellular oxalate binding was localised in the inner mitochondrial membrane while the rest was in the nucleus. The purified mitochondrial oxalate binding protein (62 kDa) was composed, with a higher molar proportion, of basic amino acids, and could accumulate oxalate on incorporation into liposomes. In the nucleus, histone H_1B (27.5 kDa), nuclear membrane protein (68 kDa) and nuclear pore complex protein (205 kDa) were present with oxalate binding activities. In addition, a 45 kDa calcium oxalate binding protein was identified in most of the subcellular organelles. Both mitochondrial and nuclear oxalate binding proteins and calcium oxalate binding protein have shown the kinetic properties of specificity, saturability, pH and temperature dependency, energy of activation and inhibition by substrate analogues. All oxalate binding proteins were sensitive to the transport inhibitor 4-4 diisothiocyano stilbene-2–2 disulphonic acid (DIDS), which is known to interact with the lysine moiety of the proteins. Calcium oxalate monohydrate (COM) crystals adsorbed oxalate binding proteins from human and rat kidney, and oxalate binding proteins isolated from human kidney stone matrix also exhibited the above kinetic properties. In experimental hyperoxaluria, all of the renal oxalate binding proteins showed enhanced oxalate binding activity with increased protein concentration. This enhanced oxalate binding activity was also attributed to increased lipid peroxidation, which correlated positively, and to decreased thiol status, which correlated negatively. A positive correlation was observed between the lipid peroxidation and both the oxalate binding activity of the in vitro peroxidised subcellular organelles and the purified protein. Similarly, in an in vivo hyperoxaluric condition, a negative correlation was observed between thiol content and both the oxalate binding activity of the peroxidised subcellular organelles and the purified protein. In the calcium oxalate crystal growth system, the oxalate binding proteins behaved either as promoters or inhibitors of the nucleation and aggregation of crystals. Following the peroxidation of the proteins, the degree of effect of the promoter protein was further stimulated while the degree of inhibition caused by the inhibitor protein further declined. Similar observations were duplicated with the proteins derived from hyperoxaluric rat kidney or kidney homogenate subjected to in vitro lipid peroxidation. The oxalate binding proteins were thought to modulate the crystallisation process in an hyperoxaluric condition similar to calcium specific binding protein modulators.     

Effect of hyperoxaluria on the inhibitory activity of a 45-kD urinary protein

Proteins are thought to play a major role in stone formation and structurally abnormal proteins have been reported to be present in the urine of stone formers. This study was aimed to determine whether hyperoxaluria modifies the kinetic properties of urinary inhibitory proteins. Hyperoxaluria was induced by feeding 1% ethylene glycol to rats. Oxalate, uric acid and calcium excretion were increased progressively during hyperoxaluria, while magnesium level was decreased. Urinary proteins were separated on a DEAE-cellulose column by eluting with stepwise increasing salt concentration in 0.05 M Tris-HCl buffer (pH 7.0). Each protein fraction was studied for its crystallization inhibitory potential by the spectrophotometric method. The protein eluted in 0.3 M NaCl containing buffer had the maximal nucleation as well as inhibitory activity. The protein had a molecular weight of 45 kD. In hyperoxaluria, the urinary excretion of this protein significantly increased. In the crystal growth assay, the control rat 45-kD protein inhibited nucleation by 75% and aggregation by 100%. In contrast, it is very interesting to note that the protein derived from 28th day hyperoxaluric urine, behaved as a promoter of nucleation (-113%, percentage inhibition) and weak inhibitor of aggregation (28%). A significantly high negative correlation (r = -0.97) between oxalate excretion and the inhibitory activity of the 45-kD protein was observed suggesting a modification of the protein by oxalate.     

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.     

Increased calcium oxalate crystal nucleation and aggregation by peroxidized protein of human kidney stone matrix and renal cells

Kidney stone matrix protein fractions eluted from DEAE cellulose column showed increased oxalate binding activity and had negative correlation with reduced thiol content. Fraction 1 (eluted in Tris-HCl, pH 7.4) and fraction 3 (0.3 M NaCl in buffer) showed nucleation and aggregation-promoting properties while fraction 2 (0.05 M NaCl in buffer) showed an inhibitory effect in an in vitro crystallization system. On peroxidation, fractions 1 and 3 showed a further increase in the promoting effect whereas fraction 2 showed a reduction in the inhibitory effect of nucleation and aggregation of calcium oxalate crystals. Protein peroxidation was negatively correlated with the inhibitory activity of the protein on calcium oxalate nucleation and aggregation. A similar promoting effect of nucleation and aggregation was seen with mitochondria and nucleus after peroxidation. These studies suggested that peroxidation of protein or tissue had greater influence on the nucleation and aggregation property of calcium oxalate crystal growth. Received: 25 January 2000 / Accepted: 1 February 2001     

An oxalate-binding protein with crystal growth promoter activity from human kidney stone matrix

OBJECTIVE: To fractionate renal-stone matrix proteins, identify the presence of oxalate-binding protein and assess its effect in a calcium oxalate (CaOx) crystal growth system. MATERIALS AND METHODS: Proteins were isolated from the matrix of kidney stones containing CaOx as the major constituent, using EDTA as a demineralizing agent. The solubilized proteins were subjected to cellulose-column chromatography by eluting with increasing sodium chloride concentrations in Tris-HCl buffer. Three protein fraction peaks were eluted, i.e. fraction I in buffer, fraction II in 0.05 mol/L NaCl in buffer and fraction III in 0.3 mol/L NaCl in buffer. The protein fractions were tested for their effects on CaOx crystal growth. RESULTS: All three fractions had maximum CaOx binding activity at pH 7.4 but fraction II also had activity at pH 4.5. Fraction I promoted in vitro CaOx crystal growth, while fractions II and III were inhibitory. When fraction I was further separated on a Sephadex G-200 column, two protein fractions (Ia and Ib) were obtained. Fraction Ia protein had high and fraction Ib low CaOx-binding activity. Fraction Ia had a molecular weight of 48 kDa on gel electrophoresis and Western blotting. The 48 kDa protein did not cross-react with crystal matrix protein antibody, band-3 protein antibody, or albumin. The protein promoted CaOx crystal growth, with an optimum temperature of 37 degrees C and pH 6.5. The inhibitory effect of citrate on crystal growth was significantly lower in the presence of the 48 kDa protein. The protein promoted nucleation and aggregation of CaOx crystals in the in vitro crystallization system at pH 6.5, whereas fraction Ib (29 kDa) inhibited both nucleation and aggregation. Using the 48 kDa antibody, the yield of the protein from the stone matrix was 32% by EDTA extraction and only 3% with other methods. The protein was also detected in the nucleus and mitochondria, and in other matrix fractions of calcium phosphate and uric acid stones. CONCLUSION: The 48 kDa protein isolated from stone matrix is a potent promoter of CaOx crystal growth with high oxalate-binding activity; it is enriched in the nucleus and mitochondria.     

Multifunctional character of osteopontin and strategy for clinical applications

Osteopontin (OPN) is the major constituent of calcium-containing urinary stones and is involved in the inhibition of nucleation and aggregation of calcium oxalate (CaOx) crystals, promotion of the adherence of CaOx crystals to cultured renal epithelial cells, and regulation of inflammatory cells as chemokine. OPN has different effects (inhibitor and promoter) at each stage of stone formation in vitro and these multifunctional actions of OPN have not been fully elucidated. We developed a modified crystal method using collagen granules (CG) and immobilized OPN. OPN had strong inhibitory activity on the aggregation/growth of CaOx crystals, but the inhibitory activity decreased by use of OPN-immobilized CG. OPN is also a critical promoter of adherence for CaOx crystals to cultured renal epithelial cells in an in vitro experimental system. We examined the effect of OPN in vivo, by OPN siRNA transfection in rats. Hydrodynamic intravenous and renal subcapsular injections with lipofection were performed on days 1 and 8. The calcium concentration in the kidney was significantly lower and the frequency of CaOx crystal deposits in the tubules was lower in the OPN siRNA transfection group (drinking 1.5% ethylene glycol (EG)), than in the EG drinking group (sham operation) at day 15. We examined the effect of candesartan, an angiotensin II (Ang II) type 1 receptor blockers (ARB) in hyperoxaluric rats. ARB reduced crystal formation and calcium concentrations in the whole kidney. Hyperoxaluria leads to CaOx crystallization and the development of tubulointerstitial lesions in the kidney. AngII mediates OPN synthesis, which is involved in both macrophage recruitment and CaOx crystallization. OPN synthesis and production increased with hyperoxaluria but to a lesser extent in ARB-treated hyperoxaluric rats. These results show that oxalate can activate the renal renin-angiotensin system and that oxalate-induced upregulation of OPN is in part mediated via the renal renin-angiotensin system.     

Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals

OBJECTIVE: To compare the ultrastructure and protein content, particularly prothrombin fragment 1 and osteopontin, of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals precipitated from human urine, and their susceptibility to proteolysis, to try to clarify the role of intracrystalline proteins in urolithiasis, as differences between these types of crystal may determine whether calcium oxalate crystals nucleated in urine progress to stone formation. MATERIALS AND METHODS: Sodium dodecyl sulphate gel electrophoresis and Western blotting were used to analyse demineralized extracts of COM and/or COD crystals deposited from the same centrifuged and filtered urine (which contains abundant urinary proteins) by adjusting the calcium concentration to 2 and 7 mmol/L, respectively. Similar analyses were performed on COM and COD crystals deposited from ultrafiltered urine (which contains only proteins of < 10 kDa) and then incubated in centrifuged and filtered urine, as well as crystals generated in the presence of increasing concentrations of proteins derived from the organic matrix of urinary calcium oxalate crystals. Field-emission scanning electron microscopy was used to assess effects of proteinase K and cathepsin D on internal and superficial crystal structure. RESULTS: Osteopontin was undetectable in COM extracts, but clearly visible in COD. Prothrombin fragment 1 was abundant in COM, but present in COD in lesser amounts than osteopontin. The selectivity was also the same with crystals from ultrafiltered urine that were incubated in centrifuged and filtered urine: prothrombin fragment 1 binding was favoured by low calcium concentration, while osteopontin bound at higher levels. Scanning electron microscopy of COM and COD digested with proteinase K and cathepsin D revealed superficial and internal texture, as wells as surface erosion, in crystals from centrifuged and filtered urine, thus confirming the presence of intracrystalline proteins. Such features were absent from crystals precipitated from ultrafiltered urine. CONCLUSION: Binding of osteopontin and prothrombin fragment 1 to calcium oxalate is dictated primarily by ambient calcium concentration. Each protein may inhibit urolithiasis by inhibiting crystallization of its preferred crystal habit, and by facilitating the intracellular disintegration and dissolution of crystals attached to and internalized by renal epithelial cells.     

Pyridoxamine lowers kidney crystals in experimental hyperoxaluria: a potential therapy for primary hyperoxaluria

BACKGROUND: Primary hyperoxaluria is a rare genetic disorder of glyoxylate metabolism that results in overproduction of oxalate. The disease is characterized by severe calcium oxalate nephrolithiasis and nephrocalcinosis, resulting in end-stage renal disease (ESRD) early in life. Most patients eventually require dialysis and kidney transplantation, usually in combination with the replacement of the liver. Reduction of urinary oxalate levels can efficiently decrease calcium oxalate depositions; yet, no treatment is available that targets oxalate biosynthesis. In previous in vitro studies, we demonstrated that pyridoxamine can trap reactive carbonyl compounds, including intermediates of oxalate biosynthesis. METHODS: The effect of PM on urinary oxalate excretion and kidney crystal formation was determined using the ethylene glycol rat model of hyperoxaluria. Animals were given 0.75% to 0.8% ethylene glycol in drinking water to establish and maintain hyperoxaluria. After 2 weeks, pyridoxamine treatment (180 mg/day/kg body weight) started and continued for an additional 2 weeks. Urinary creatinine, glycolate, oxalate, and calcium were measured along with the microscopic analysis of kidney tissues for the presence of calcium oxalate crystals. RESULTS: Pyridoxamine treatment resulted in significantly lower (by approximately 50%) levels of urinary glycolate and oxalate excretion compared to untreated hyperoxaluric animals. This was accompanied by a significant reduction in calcium oxalate crystal formation in papillary and medullary areas of the kidney. CONCLUSION: These results, coupled with favorable toxicity profiles of pyridoxamine in humans, show promise for therapeutic use of pyridoxamine in primary hyperoxaluria and other kidney stone diseases.     

Expression of inter-alpha inhibitor related proteins in kidneys and urine of hyperoxaluric rats

PURPOSE: To investigate the involvement of the inter-alpha inhibitor family of proteins in calcium oxalate stone formation we determined immunohistochemical distribution in the kidneys and excretion in the urine of these proteins in normal and hyperoxaluric rats. Various members of the family have been shown to inhibit the formation and retention of calcium oxalate crystals in the kidneys. MATERIALS AND METHODS: Hyperoxaluria was induced in male Sprague-Dawley rats by administering 0.75% ethylene glycol. The inter-alpha inhibitor family consists of inter-alpha inhibitor, pre-alpha inhibitor, the so-called heavy chains H1, H2 and H3, and the light chain bikunin. Antibodies against these molecules were used to localize various proteins in rat kidneys by immunohistochemical techniques. Urine was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis to determine the expression of various members of the inter-alpha inhibitor family. RESULTS: In normal kidneys staining for inter-alpha inhibitor and other members of the family was mostly limited to the proximal tubules and generally to their luminal contents. Eight weeks after the induction of hyperoxaluria various sections of renal tubules stained positive for inter-alpha inhibitor, bikunin and H3. Positive staining was observed in the tubular lumina as well as in the cytoplasm of epithelial cells. Crystal associated material was heavily stained. Western blot analysis recognized 7 protein bands in the urine. The urinary expression of H1, H3 and pre-alpha-inhibitor was significantly increased. CONCLUSIONS: Apparently hyperoxaluria and renal calcium oxalate crystal deposition result in the increased expression of crystallization inhibitors, such as inter-alpha-inhibitor related proteins, in the kidneys and urine. Results indicate that kidneys respond to nephrolithic challenges by producing proteins that inhibit crystal formation and retention.     

Reduced crystallization inhibition by urine from women with nephrolithiasis

BACKGROUND: Human urine is known to inhibit growth, aggregation, nucleation, and cell adhesion of calcium oxalate monohydrate (COM) crystals, the main solid phase of human kidney stones. This study tested the hypothesis that low levels of inhibition are present in women with calcium oxalate stones and, therefore, could promote stone production. METHODS: In 17 stone-forming women and 17 normal women matched in age within five years, inhibition by dialyzed urine proteins of COM growth and aggregation was examined, as well as whole urine upper limits of metastability (ULM) for COM and calcium phosphate (CaP) in relation to the corresponding supersaturation (SS). RESULTS: Compared to normals, patient urine showed a reduced ULM in relation to SS. In contrast to men, there was no difference in growth inhibition. CONCLUSIONS: Reduced CaP and CaOx ULM values in relation to SS are a characteristic of female stone formers. This defect could promote stones by facilitating crystal nucleation. Abnormal inhibition may well be a very important cause of human nephrolithiasis.     

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.     

Stone prevention: why so little progress?*

Despite intensive research the knowledge of stone pathogenesis, which is the basis of every rational stone metaphylaxis, has remained rather scanty. Epidemiology shows that stone formation in most patients is only a sporadic event, probably resulting from a coincidence of different factors. The hypercalciuria, hypocitraturia, hyperuricosuria and hyperoxaluria frequently found in calcium stone formers can be influenced therapeutically and, in affluent societies, seem to be the result of protein over-consumption. These four factors favour crystallization processes in urine. However, urine is normally protected from nucleation, growth and aggregation of calcium minerals by crystallization inhibitors. In urine, crystallization of calcium oxalate can only be induced by an extreme supersaturation, a deficient inhibitor activity and promoters of crystallization. To form a stone, crystals have to be retained in the urinary collecting system. Two mechanisms of retention are discussed: large crystal aggregates trapped in collecting ducts of renal papillae, or a pre-existing calcification of the papilla (mainly calcium phosphate) that may be responsible for growth of an initially fixed particle to a concretion large enough to become symptomatic. An excessive oxalate intake combined with a low calcium consumption can produce marked hyperoxaluria. In the animal model, hyperoxaluria induces not only calcium oxalate crystallization but also papillary damage and incrustrations. Hypercalciuria at a low pH favours the aggregation of calcium oxalate, and at a high pH the crystallization of calcium phosphate, a promoter of heterogeneous nucleation of calcium oxalate. All these factors and further complex phenomena mentioned in this paper have to be taken in account to perform rational stone metaphylaxis. Received: 5 June 1997 / Accepted: 13 August 1997     

Urinary material affecting calcium oxalate monohydrate stone formation

We previously reported that the low molecular and uronic acid-rich fractions in the urine from calcium oxalate stone formers promoted aggregation of calcium oxalate monohydrate seed crystals. In this study, we have demonstrated that lyophilized material of the fractions contains hyaluronic acid as a sole glycosaminoglycans, as well as acidic amino acid-rich proteins and urinary pigment which is supposed to combine with protein. It is known that hyaluronic acid is present in stone matrix, that calcium containing stones contain proteins rich in acidic amino acids, and that the external color of calcium oxalate monohydrate calculi is usually brownish. These facts correspond with our present results. Therefore, it is suggested that the urinary material promotes the calcium oxalate crystal aggregation, sticks the crystals together and is incorporated into the stone.     

近端肾小管上皮细胞损伤后对草酸钙结晶的促进作用

目的 研究过氧化氢(H2O2)对人肾小管上皮细胞(HKC)的氧化损伤作用,探讨结晶时间对损伤HKC调控草酸钙(CaOxa)晶体生长的影响.方法 通过检测细胞成活率和细胞中丙二醛释放量的变化评价HKC的损伤程度;利用扫描电镜研究HKC损伤对CaOxa结晶的影响.结果 0.3 mmol/L H2O2作用HKC 1 h后,细胞活性降为79.0%,作用2 h后,细胞活性仅37.8%(P＜0.05).正常HKC形态饱满,细胞连接成片,鞭毛、突触等均完好.0.3 mmol/L H2O2作用1 h后,HKC发生明显皱缩,细胞表面粗糙,周围出现细胞碎片;作用2 h后,细胞皱缩更明显,部分细胞脱落.对照组细胞只诱导少量二水草酸钙形成,损伤细胞不仅诱导一水草酸钙(COM)形成,而且增加CaOxa晶体的数量和聚集程度;用CaOxa过饱和溶液长时间孵育对照组细胞后亦可以产生损伤.结论 H2O2能使HKC产生氧化性损伤,促进COM晶体成核和聚集;晶体在尿路中长时间滞留是肾结石形成的危险因素.

Abstract：

Objective To investigate the injury caused by hydrogen peroxide (H2O2) on human renal tubular epithelial cell (HKC) and its effect on calcium oxalate (CaOxa) crystal crystallization time before and after the injury. Methods The injury degree of HKC by H2O2 was measured by detecting the cell survival rate and the concentration change of malonaldehyde (MDA). CaOxa crystallization was investigated by scanning electron microscopy (SEM). Results Control cells induced only a small amount of calcium oxalate dihydrate (COD) crystals, while the injured cells not only induced calcium oxalate monohydrate (COM) crystals, but also increased the number and aggregation of CaOxa crystals. After incubating with CaOxa supersaturated solution, the control group HKC cells could be injured as well. Conclusions H2O2 can cause oxidative damage on HKC. The injured HKC promotes the nucleation and aggregation of COM crystals. In the body environment, the long-term presence of crystals in urinary tract is a risk factor for stone formation.     

A study of crystallization on urolithiasis in vitro]

The formation, growth and aggregation of calcium oxalate crystals were evaluated with a Coulter counter. CG-120 and sodium pentosan polysulfate (SPP) has inhibitory activity on calcium oxalate crystal formation, growth and aggregation in the seeded crystal system and whole urine system. In continuous crystallizer system, they inhibit the nucleation rate. A new system of observing the crystal formation and growth of fragmented stones after extracorporeal shock wave lithotripsy has been developed. CG-120 has an inhibitory effect on their growth. The deposition of calcium oxalate crystals in the rat kidney was induced, and the, the volume and number of crystals were estimated using a Coulter counter. SPP has an inhibitory effect on calcium oxalate crystal growth in vivo.     

Reduced crystallization inhibition by urine from men with nephrolithiasis.

BACKGROUND: Human urine is known to inhibit growth, aggregation, nucleation, and cell adhesion of calcium oxalate monohydrate (COM) crystals, the main solid phase of human kidney stones. This study tests the hypothesis that low levels of inhibition are present in men with calcium oxalate stones and could therefore promote stone production. METHODS: In 17 stone-forming men and 17 normal men that were matched in age to within five years, we studied the inhibition by dialyzed urine proteins of COM growth, aggregation, and binding to cultured BSC-1 renal cells, as well as whole urine upper limits of metastability (ULM) for COM and calcium phosphate (CaP) in relationship to the corresponding supersaturation (SS). RESULTS: Compared with normals, patient urine showed reduced COM growth inhibition and reduced ULM in relationship to SS. When individual defects were considered, 15 of the 17 patients were abnormal in one or more inhibition measurements. ULM and growth inhibition defects frequently coexisted. CONCLUSIONS: Reduced COM growth and CaP and CaOx ULM values in relationship to SS are a characteristic of male stone formers. Both defects could promote stones by facilitating crystal nucleation and growth. Abnormal inhibition may be a very important cause of human nephrolithiasis.