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     

Role of crystal surface adhesion in kidney stone disease

PURPOSE OF REVIEW: Atomic force microscopy has been used recently to characterize the adhesion force between selected calcium oxalate crystal surfaces and biologically relevant chemical groups attached to the atomic force microscopy probe tip. These measurements have permitted comparisons of the adhesion properties of different, well defined crystal faces, as well as determination of the influence of solution-phase macromolecules on adhesion. These studies have produced new insight into the specific chemical interactions that regulate kidney stone formation. RECENT FINDINGS: The adhesion force measurements have demonstrated that the large hexagonal (100) face of calcium oxalate monohydrate is the most adhesive. In contrast, the large (101) face of calcium oxalate dihydrate is the least adhesive. Carboxylate and amidinium groups on the atomic force microscopy tip exhibit equivalently large adhesion at a given crystal face, implicating specific binding to crystal surface lattice ions. Solution-phase macromolecules modulate adhesion in a face-selective manner, dependent on their chemical structures. SUMMARY: The low adhesion force for calcium oxalate dihydrate predicts a decreased ability of these crystals to aggregate or attach to cells, and correlates with the relative absence of calcium oxalate dihydrate in kidney stones. These measurements provide new understanding of the macromolecular regulation of crystal aggregation and attachment to cells in stone formation.     

人肾组织γ-谷氨酰基羧化酶基因启动子区克隆与活性分析

目的 克隆人肾组织γ-谷氨酰基羧化酶(cGCX)基因5'侧翼启动子区,并对其转录活性进行分析.方法 对GGCX基因5'侧翼区进行生物信息学分析,预测其转录调控区域;从人肾组织中提取基因组DNA,以其为模板扩增GGCX基因启动子区,通过酶切鉴定及测序分析无误后,将其构建至萤光报告基因载体pGL3-basic中,瞬时转染至Hela细胞,检测萤光素酶水平,从而分析扩增启动子区的转录活性.结果 成功克隆长度为804 bp(-891～-88)的GGCX基因启动子片段,并构建相应的萤光素酶重组子pGL3.GGCX Promoter,转染Hela细胞48 h后检测萤光素酶活性显示,pGL3-GGCX Promoter与空质粒对照pGL3-basic相对萤光素酶值分别为16.92±4.01与0.02±0.01,pGL3-GGCXPromoter萤光素酶活性显著增强(P<0.05).Matlnspector等分析显示该片段具有多个转录因子结合位点.结论 成功构建含有GGCX基因启动子序列的萤光素酶报告系统,为进一步研究GGCX基因功能及其转录调控奠定了基础.     

Three pathways for human kidney stone formation

No single theory of pathogenesis can properly account for human kidney stones, they are too various and their formation is too complex for simple understanding. Using human tissue biopsies, intraoperative imaging and such physiology data from ten different stone forming groups, we have identified at least three pathways that lead to stones. The first pathway is overgrowth on interstitial apatite plaque as seen in idiopathic calcium oxalate stone formers, as well as stone formers with primary hyperparathyroidism, ileostomy, and small bowel resection, and in brushite stone formers. In the second pathway, there are crystal deposits in renal tubules that were seen in all stone forming groups except the idiopathic calcium oxalate stone formers. The third pathway is free solution crystallization. Clear examples of this pathway are those patient groups with cystinuria or hyperoxaluria associated with bypass surgery for obesity. Although the final products may be very similar, the ways of creation are so different that in attempting to create animal and cell models of the processes one needs to be careful that the details of the human condition are included.     

L-shaped cross-fused kidney with stone

L-shaped crossed renal ectopia is a rare congenital anomaly. A unique stone location on plain film and reconstructive computed tomography is demonstrated in a case of renal and ureteral calculi in a 58-year-old woman with right-to-left cross-fused ectopic kidney.     

Pioglitazone inhibits cell growth and reduces matrix production in human kidney fibroblasts

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists are increasingly used in patients with diabetes, and small studies have suggested a beneficial effect on renal function, but their effects on extracellular matrix (ECM) turnover are unknown. The aims of this study were to investigate the effects of the PPAR-gamma agonist pioglitazone on growth and matrix production in human cortical fibroblasts (CF). Cell growth and ECM production and turnover were measured in human CF in the presence and absence of 1 and 3 muM pioglitazone. Exposure of CF to pioglitazone caused an antiproliferative (P < 0.0001) and hypertrophic (P < 0.0001) effect; reduced type IV collagen secretion (P < 0.01), fibronectin secretion (P < 0.0001), and proline incorporation (P < 0.0001); decreased MMP-9 activity (P < 0.05); and reduced tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 secretion (P < 0.001 and P < 0.0001, respectively). These effects were independent of TGF-beta1. A reduction in ECM production was similarly observed when CF were exposed to a selective PPAR-gamma agonist (L-805645) in concentrations that caused no toxicity, confirming the antifibrotic effects of pioglitazone were mediated through a PPAR-gamma-dependent mechanism. Exposure of CF to high glucose conditions induced an increase in the expression of collagen IV (P < 0.05), which was reversed both in the presence of pioglitazone (1 and 3 muM) and by L-805645. In summary, exposure of human CF to pioglitazone causes an antiproliferative effect and reduces ECM production through mechanisms that include reducing TIMP activity, independent of TGF-beta1. These studies suggest that the PPAR-gamma agonists may have a specific role in ameliorating the course of progressive tubulointerstitial fibrosis under both normoglycemic and hyperglycemic states.     

尿草酸钙晶体基质蛋白的分离及理化特征

为了进一步了解晶体基质蛋白（ＣＭＰ）的理化特性和其在结石形成中的作用，利用ＤＥＡＥＳｅｐｈａｄｅｘＡ５０、ＳｅｐｈａｄｅｘＧ２００对草酸钙结晶中的基质蛋白进行了分离，并用ＳＤＳＰＡＧＥ、双向电泳和Ｗｅｓｔｅｒｎｂｌｏｔ进行检测。结果表明：ＣＭＰ是草酸钙过饱和方法制备的晶体基质中的主要成分，其分子量为３１０００，等电点为５．０～５．５；Ｗｅｓｔｅｒｎｂｌｏｔ证明与抗人凝血酶原抗体有交叉反应。结果认为ＣＭＰ是活化的人凝血酶原片段     

Inhibition by sodium-potassium citrate (CG-120) of calcium oxalate crystal growth on to kidney stone fragments obtained from extracorporeal shock wave lithotripsy

Retention of fragments within the kidney after extracorporeal shock wave lithotripsy (ESWL) continues to be a major shortcoming of this form of stone treatment. The aim of this study was to evaluate the ability of sodium-potassium citrate to inhibit calcium oxalate crystal nucleation and growth on to stone fragments remaining after ESWL. The continuous flow crystallisation technique was adapted to induce calcium oxalate crystal nucleation and growth on to the surface of fragmented kidney stones and the inhibitory effect of sodium-potassium citrate was assessed by scanning electron microscopy and by determining the relative increase in crystalline mass at final concentrations of 0, 2, 4, 6, 8, 10 mmol/l. Sodium-potassium citrate significantly inhibited the deposition of new crystalline calcium oxalate in a dose-dependent manner above 2 mmol/l; these findings were confirmed by scanning electron microscopy. It was concluded that sodium-potassium citrate may provide an effective means of preventing the formation of new kidney stones by the deposition of calcium oxalate on to residual stone fragments resulting from ESWL and that the technique used is an efficient means of testing the efficacy of therapeutic agents to prevent stone recurrence in patients treated with ESWL.     

Resolution of proteins in the kidney stone matrix using high-performance liquid chromatography

The organic matrix accounts for 2-3% of the total stone weight and has been considered to play a role in stone formation and growth. Thus far, fractionation of the matrix proteins has been insufficient due to low resolution and reproducibility. In this report the matrix proteins of 22 stones were resolved by means of high-performance liquid chromatography. Following pulverization, the organic matrix was obtained by dialysis against EDTA. The average content of nondialyzable extractable proteins was 1.6% of the total stone weight. Analysis of the matrix proteins with high-performance gel permeation liquid chromatography and high-performance ion-exchange liquid chromatography has indicated that the protein composition of the stone matrix is identical regardless of the mineral composition. The major component of the matrix proteins was identified as glycoprotein and/or proteoglycan from their absorption to a concanavalin A Sepharose column. Higher molecular weight matrix proteins seem to be polymers or condensation products, since they have been degraded into lower molecular weight subfractions by sodium dodecyl sulfate treatment.     

Distribution of osteopontin and calprotectin as matrix protein in calcium-containing stone

We recently reported that osteopontin (OPN) and calprotectin (CPT) are present in the matrix of urinary calcium stones, and that OPN mRNA is expressed in the renal distal tubular cells. In the present study, we examined the immunohistochemical distributions of OPN and CPT in urinary stones. The stones used in this study were passed spontaneously from the upper urinary tract. One half of each of the stones was analyzed with an infrared spectrophotometer, and were shown to be comprised of calcium oxalate, calcium phosphate, uric acid and cystine. The other half of each stone was immersed in tetrasodium ethylenediamine-tetraacetate (EDTA) solution. The half-stones were embedded in paraffin and cut into 5-μm sections. The avidin-biotin-peroxidase complex technique was employed. A monoclonal antibody to human milk-derived OPN and a monoclonal antibody to human granulocyte-derived CPT were used as primary antibodies. The immunochemical study using the OPN and CPT antibodies showed positive staining of the matrix of the urinary calcium stones. The stones showed staining in two distinct zones: a core area was stained with randomly aggregated OPN and CPT, and peripheral layers were stained in concentric circles. On the basis of our observations, it is reasonable to presume that OPN and CPT play roles as the matrix in the structure of urinary calcium stones. Received: 24 February 1998 / Accepted: 28 January 1999     

A polymorphism of matrix Gla protein gene is associated with kidney stones

PURPOSE: Matrix Gla protein, a potent calcification inhibitor in arterial vessels, is also expressed in the kidney and is up-regulated following the administration of ethylene glycol, a precursor of oxalate. Considering the analogous characteristics between arterial calcification and kidney stones, we identified variants of the human matrix Gla protein gene and investigated whether there is an association between MGP genetic polymorphisms and kidney stones. MATERIALS AND METHODS: We studied single nucleotide polymorphisms in matrix Gla protein in 122 kidney stone cases and 125 controls. We resequenced the human genomic MGP gene, including the 1,000 bp promoter 5'-untranslated region, 4 exons and 3'-untranslated regions, and we performed systematic genetic analysis. A single nucleotide polymorphism was genotyped using a fluorescent 5'endonuclease assay and its association with kidney stones was analyzed. RESULTS: We observed 19 polymorphisms. A single nucleotide polymorphism was associated with kidney stones (OR 0.51, 95% CI 0.30-0.87; p = 0.012). The G allele carrier had a 2-fold decreased kidney stone risk compared with A allele carriers in single nucleotide polymorphism 11 (OR 0.55, 95% CI 0.31-1.00, p = 0.047). We found no association between the polymorphism and kidney stone clinical characteristics. CONCLUSIONS: Our findings show that an MGP gene polymorphism is associated with kidney stones and influences genetic susceptibility to kidney stones. In the future functional assays of the polymorphism should permit better understanding of the role of matrix Gla protein genetic variants and kidney stones.     

An unidentified macromolecular inhibitory constituent of calcium oxalate crystal growth in human urine

Summary We have detected and isolated a macromolecular constituent in normal human urine possessing calcium crystal growth inhibitory activity. The purification procedure consisted of two anion exchange chromatographies and one affinity chromatography. The crystal growth inhibitor was found to be heterogeneous in net charge as well as in size. It has not been identified. It is not an uronic acid-containing glycosaminoglycan, hitherto presumed to be responsible for the inhibitory activity. Whether an urinary fragment of inter--trypsin inhibitor is responsible has yet to be resolved.     

Purification and characterization of a growth factor from human bone matrix

A growth factor was purified from human bone matrix by extraction with EDTA, acetone treatment, gel filtration column chromatography, ion exchange column chromatography, and reversed-phase HPLC. Purified protein migrated as a single band to an area with a molecular weight of about 6,000 on SDS-polyacrylamide gel. The purified protein stimulated dose dependent osteoblast proliferation. The sequence of the first 30 N-terminal amino acids of the protein was identical to that of the human insulin-like growth factor-II (IGF-II). MC3T3-E1 cells reacted immunocytochemically to the monoclonal antibody against IGF-II. The culture medium of MC3T3-E1 cells contained immunoreactive IGF-II, which was measured by an enzyme immunoassay. These results indicate that IGF-II is contained in bone matrix, is effective in osteoblast proliferation, produced by the osteoblasts, and secreted from the osteoblasts. Taken together, these results show that IGF-II is present as a local growth factor in the bone system, playing an important role in bone formation following bone resorption.     

Purification and properties of a growth factor from human bone matrix

A growth factor was isolated from demineralized human bone-matrix and purified approximately 2,300-fold with 5.6% yield by the procedures of acetone treatment, Blue-A matrix gel chromatography, Sephadex G-75 gel filtration and Mono-Q fast protein liquid chromatography. The final preparation was homogeneous and a single polypeptide of 18,000 daltons as judged by SDS-polyacrylamide gel electrophoresis. The purified growth factor stimulated DNA synthesis in embryonic chick osteoblast in a dose-dependent manner and was saturated at the concentration of 4 ng/ml. By comparing various properties of the final preparation with those of other growth factors derived from bone, it appears to be a novel growth factor, and may be one of the important local regulators of bone remodeling.     

The riddle of kidney stone disease: lessons from Africa

Urolithiasis has not been extensively researched in the African continent due to a general lack of facilities and resources. Consideration of the few published papers indicates that there are some regions where the occurrence of stones is extremely rare. South Africa is unique in two respects. Firstly, it has both stone-prone and stone-free population groups and secondly, it is an African country in which a fair amount of research has been conducted in this field. These studies have shown that routine urine parameters cannot explain stone rarity, but that structural differences of inhibitory urinary proteins appear to be important. Similarly, the studies have demonstrated that common dietary components cannot necessarily be correlated with urine composition, particularly oxaluria, nor can they necessarily explain stone rarity, but that the role of oxalate-degrading bacteria has the potential to offer explanatory insights. By investigating the factors influencing stone rarity, those affecting stone formation have been concomitantly scrutinized. As a result, it is suggested that a paradigm shift from a focus on pathology to one on physiology is needed in urolithiasis research in general.     

The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin.

Urine contains proteins that inhibit the growth of calcium oxalate (CaOx) crystals and may prevent the formation of kidney stones. We have identified a potent crystal growth inhibitor in the conditioned media from primary cultures of mouse kidney cortical cells. Conditioned media, incubated with the kidney cells for 6-72 h, was assayed for crystal growth inhibition; inhibitory activity increased 15-fold by 24 h. Inhibitory activity was purified from serum-free media containing proteinase inhibitors using anion-exchange and gel-filtration chromatography. A single band of molecular weight 80,000 daltons was seen after SDS-polyacrylamide gel electrophoresis. The sequence of the N-terminal 21 amino acids of this protein matched that of osteopontin (OP), a phosphoprotein initially isolated from bone matrix. Antisera raised to fusion proteins produced by plasmids containing the N-terminal or C-terminal portions of OP cDNA also cross-reacted with the protein purified from cell culture media on western blots. The effect of the purified protein on the growth of CaOx crystals was measured using a constant composition assay. A 50% inhibition of growth occurred at a protein concentration of 0.85 micrograms/ml, and the dissociation constant of the protein with respect to CaOx crystal was 3.7 x 10(-8) M. The concentration of OP in mouse urine, measured using antibodies raised to the purified protein, was approximately 8 micrograms/ml. We conclude that OP is synthesized by kidney cortical tubule cells and functions as a crystal growth inhibitory protein in urine.