Identification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in the urine of healthy and stone forming subjects

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced in␣vitro in whole human urine of healthy individuals and kidney stone patients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein contents. Crystallization in urine was induced by addition of an oxalate or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralization with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacrylamide gel electrophoresis, and identified by western blotting technique. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matrices obtained from the urine of normal and stone forming subjects. Albumin (AB), inter-α-inhibitor (IαI) related proteins, α-1 microglobulin (α-1 m), osteopontin (OPN), prothrombin (PT)-related proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal subjects and stone formers. AB, PT-related proteins and OPN were the main constituents. The other proteins were present in smaller but detectable amounts. However, CaP crystal matrix, contained a large amount of THP. In addition CaP crystals contained significantly more proteins than CaOx crystals. Centrifugation and/or filtration of the urine resulted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine. Received: 24 July 1997 / Accepted: 2 January 1998     

Calcium oxalate crystal deposition in metabolic syndrome model rat kidneys

Objective: Although an epidemiological link between the metabolic syndrome and kidney stone formation has been reported, the mechanism by which metabolic syndrome promotes kidney stone formation has yet to be elucidated. We investigated calcium oxalate (CaOx) kidney stone formation in a rat metabolic syndrome model. Methods: We induced hyperoxaluria in 8‐week‐old male Otsuka Long‐Evans Tokushima fatty (OLETF) rats, and a control strain, Long‐Evans Tokushima Otsuka (LETO) rats, by administering 1.0% ethylene glycol (EG) as their drinking water for 2 weeks. Rats were divided into four groups: LETO‐C (control, n = 7); LETO‐SF (stone forming, n = 8); OLETF‐C (n = 7); and OLETF‐SF (n = 8). Urine and blood samples were collected for biochemistry testing, and the kidneys were harvested for estimation of crystal deposition and determinations of the expression of osteopontin (OPN) and monocyte chemoattractant protein‐1 (MCP‐1). Results: Administration of EG induced hyperoxaluria to the same degree in both strains. The OLETF‐SF group showed a higher grade of renal crystal deposition and significantly higher renal calcium content than the LETO‐SF group. Although the OLETF‐C group excreted significantly higher amounts of uric acid and more acidic urine than the LETO‐C group, similar differences were not observed in rats given EG. Significant upregulation of both OPN and MCP‐1 was seen in the kidneys of hyperoxaluric rats, with higher levels of expression in the OLETF‐SF group than the LETO‐SF group. Conclusions: The present results show for the first time that OLETF rats form more renal CaOx crystal deposits compared with control rats under EG‐induced hyperoxaluric conditions. The model described here should be useful for investigating the mechanisms by which the metabolic syndrome promotes CaOx kidney stone formation.     

Calcium oxalate crystal adherence to hyaluronan-, osteopontin-, and CD44-expressing injured/regenerating tubular epithelial cells in rat kidneys

Retention of crystals in the kidney is an essential early step in renal stone formation. Studies with renal tubular cells in culture indicate that hyaluronan (HA) and osteopontin (OPN) and their mutual cell surface receptor CD44 play an important role in calcium oxalate (CaOx) crystal binding during wound healing. This concept was investigated in vivo by treating rats for 1, 4, and 8 d with ethylene glycol (0.5 and 0.75%) in their drinking water to induce renal tubular cell damage and CaOx crystalluria. Tubular injury was morphologically scored on periodic acid-Schiff-stained renal tissue sections and tissue repair assessed by immunohistochemical staining for proliferating cell nuclear antigen. CaOx crystals were visualized in periodic acid-Schiff-stained sections by polarized light microscopy, and renal calcium deposits were quantified with von Kossa staining. HA was visualized with HA-binding protein and OPN and CD44 immunohistochemically with specific antibodies and quantified with an image analyzer system. Already after 1 d of treatment, both concentrations of ethylene glycol induced hyperoxaluria and CaOx crystalluria. At this point, there was neither tubular injury nor crystal retention in the kidney, and expression of HA, OPN, and CD44 was comparable to untreated controls. After 4 and 8 d of ethylene glycol, however, intratubular crystals were found adhered to injured/regenerating (proliferating cell nuclear antigen positive) tubular epithelial cells, expressing HA, OPN, and CD44 at their luminal membrane. In conclusion, the expression of HA, OPN, and CD44 by injured/regenerating tubular cells seems to play a role in retention of crystals in the rat kidney.     

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.     

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.     

The hole truth: intracrystalline proteins and calcium oxalate kidney stones

The ultimate aim of our research is to understand the role of macromolecules in the formation of human kidney stones, particularly their interactions with calcium oxalate (CaOx) crystals. The invariable association of stones with proteins raises the possibility that proteins play a role in their formation, similar to the role of proteins in healthy biomineralization. Do these proteins induce mineralization? Are they merely a response to the disease process? Or are they protective molecules that were overwhelmed by mineral supersaturation? A protein of particular interest is fragment 1 (F1) of prothrombin. We have shown that mRNA for prothrombin is present in the kidney. Because the F1 fragment of prothrombin present in urine is slightly different from that found in the blood, we refer to this protein as "urinary prothrombin fragment 1" (UPTF1). Available evidence suggests that the kidney manufactures the protein for protection against stone disease and that the protein has a directive role in stone formation. We now have evidence that proteins are interred within CaOx crystals precipitated from human urine, where it is distributed in continuous channels. These proteins could facilitate crystal deconstruction and removal after attachment to the renal epithelium and endocytosis. We suspect that the formation of CaOx crystals in the urine is a normal process designed to permit harmless disposal of an excess of calcium, oxalate, or both. The incorporation of proteins provides a second line of defense against stone formation by enabling the destruction and removal of retained crystals. Understanding the basic molecular strategies by which plants produce protein-containing CaOx crystals may provide insight into human CaOx stone formation.     

Increased expression of monocyte chemoattractant protein-1 (MCP-1) by renal epithelial cells in culture on exposure to calcium oxalate, phosphate and uric acid crystals.

BACKGROUND: During the development of non-infectious kidney stones, crystals form and deposit in the kidneys and become surrounded by monocytes/macrophages (M/M). We have proposed that in response to crystal exposure renal epithelial cells produce chemokines, which attract the M/M to the sites of crystal deposition. We investigated the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein by NRK52E rat renal tubular epithelial cells exposed to calcium oxalate (CaOx), brushite (Br, a calcium phosphate) and uric acid (UA) crystals. METHODS: Confluent cultures of NRK52E cells were exposed to CaOx, Br or UA at a concentration of 250 micro g/ml (66.7 micro g/cm(2)). They were exposed for 1, 3, 6, 12, 24 and 48 h for isolation of mRNA and 24 h for ELISA to determine the secretion of protein into the culture medium. Since cells are known to produce free radicals on exposure to CaOx crystals we also investigated the effect of free radical scavenger catalase on the crystal induced expression of MCP-1 mRNA and protein. RESULTS: Exposure of NRK52E cells to the crystals resulted in increased expression of MCP-1 mRNA and production of the chemoattractant. CaOx crystals were most provocative while UA the least. Treatment with catalase had a negative effect on the increased expression of both MCP-1 mRNA and protein, which indicates the involvement of free radicals in up-regulation of MCP-1 production. CONCLUSION: Exposure to both CaOx and calcium phosphate crystals stimulates increased production of MCP-1. Free radicals appear to be involved in this up-regulation. Results indicate that MCP-1, which is often associated with localized inflammation, may be one of the chemokine mediators associated with the deposition of various urinary crystals in the kidneys during kidney stone formation. Because of the small number of experiments performed here, results must be confirmed by more extensive studies with larger sample size.     

Calcium oxalate monohydrate aggregation induced by aggregation of desialylated Tamm-Horsfall protein

Tamm-Horsfall protein (THP) is thought to protect against calcium oxalate monohydrate (COM) stone formation by inhibiting COM aggregation. Several studies reported that stone formers produce THP with reduced levels of glycosylation, particularly sialic acid levels, which leads to reduced negative charge. In this study, normal THP was treated with neuraminidase to remove sialic acid residues, confirmed by an isoelectric point shift to higher pH. COM aggregation assays revealed that desialylated THP (ds-THP) promoted COM aggregation, while normal THP inhibited aggregation. The appearance of protein aggregates in solutions at ds-THP concentrations ≥1 μg/mL in 150 mM NaCl correlated with COM aggregation promotion, implying that ds-THP aggregation induced COM aggregation. The aggregation-promoting effect of the ds-THP was independent of pH above its isoelectric point, but was substantially reduced at low ionic strength, where protein aggregation was much reduced. COM aggregation promotion was maximized at a ds-THP to COM mass ratio of ~0.025, which can be explained by a model wherein partial COM surface coverage by ds-THP aggregates promotes crystal aggregation by bridging opposing COM surfaces, whereas higher surface coverage leads to repulsion between adsorbed ds-THP aggregates. Thus, desialylation of THP apparently abrogates a normal defensive action of THP by inducing protein aggregation, and subsequently COM aggregation, a condition that favors kidney stone formation.     

Hyperoxaluria-induced oxidative stress and antioxidants for renal protection

Renal cellular exposure to oxalate (Ox) and/or CaOx crystals leads to the production of reactive oxygen species (ROS), development of oxidative stress followed by injury and inflammation. Renal injury and inflammation appear to play a significant role in stone formation. ROS are produced from many sources and involve a variety of signaling pathways. Tissue culture and animal model studies show that treatments with anti-oxidants and free radical scavengers reduce Ox/CaOx crystal induced injuries. In addition, CaOx crystal deposition in kidneys is significantly reduced by treatments with antioxidants and free radical scavengers, indicating their efficacy. These results point towards a great potential for the therapeutic application of antioxidants and free radical scavengers to reduce stone recurrence particularly after shock wave lithotripsy, which is itself known to generate ROS and cause renal damage.     

Calcium oxalate crystal interaction with renal tubular epithelium,mechanism of crystal adhesion and its impact on stone development

The interaction between renal epithelial cells and calcium oxalate (CaOx) crystals and/or oxalate ions plays a critical role in the formation of urinary stones. Epithelial cells respond to hyperoxaluria and the presence of CaOx crystals in the kidneys by increased enzymuria and internalization of the crystals. Crystal cell interaction results in movement of crystals from the luminal to the basolateral side between the cells and the basement membrane. Once beneath the epithelium, crystals adhere to the basement membrane and become anchored inside the kidneys. Crystals anchored to basement membrane of the peripheral collecting duct aggregate with other crystals and move through an eroding epithelium to the papillary surface, furnishing an encrustation platform or a nidus for future development of a kidney stone. Thus interaction between renal epithelial cells and CaOx crystals and/or oxalate ions is an essential element in the development of urinary stone disease.     

Citrate determines calcium oxalate crystallization kinetics and crystal morphology-studies in the presence of Tamm-Horsfall protein of a healthy subject and a severely recurrent calcium stone former.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.     

Effects of an aqueous extract from <Emphasis Type="Italic">Phyllantus niruri</Emphasis> on calcium oxalate crystallization in vitro

Phyllanthus niruri is a plant used in Brazilian folk medicine for the treatment of urolithiasis. It was previously observed that P. niruri shows no toxicity, potentially increases calculus voiding by stone forming patients and inhibits the endocytosis of calcium oxalate (CaOx) crystals by MDCK cells. In addition, in a rat model of urolithiasis it reduced calculus growth. In the present study, we evaluated the effect of an aqueous extract of P. niruri on CaOx crystallization in vitro. CaOx precipitation was induced by the addition of 0.1 M sodium oxalate to unfiltered urine samples from Wistar rats (n=14) and normal humans (n=18) in the presence or absence of P. niruri extract (0.25 mg/ml of urine). The presence of CaOx crystals was evaluated immediately and 24 h later. In vitro crystallization of human urine produced typical mono- and dihydrated CaOx crystals, but only a few typical CaOx crystals were found in rat urine. The presence of P. niruri extract did not inhibit CaOx precipitation and even more crystals were obtained, although they were significantly smaller than those in the control urine. Crystal aggregation observed 24 h after crystallization was also inhibited by P. niruri extract. The results showed an inhibitory effect of P. niruri extract on CaOx crystal growth and aggregation in human urine, suggesting that it may interfere with the early stages of stone formation and may represent an alternative form of treatment and/or prevention of urolithiasis     

Calcium oxalate crystal localization and osteopontin immunostaining in genetic hypercalciuric stone-forming rats

BACKGROUND: The inbred genetic hypercalciuric stone-forming (GHS) rats develop calcium phosphate (apatite) stones when fed a normal 1.2% calcium diet. The addition of 1% hydroxyproline to this diet does not alter the type of stone formed, while rats fed this diet with 3% hydroxyproline form mixed apatite and calcium oxalate stones and those with 5% hydroxyproline added form only calcium oxalate stones. The present study was designed to determine the localization of stone formation and if this solid phase resulted in pathologic changes to the kidneys. METHODS: GHS rats were fed 15 g of the standard diet or the diet supplemented with 1%, 3%, or 5% hydroxyproline for 18 weeks. A separate group of Sprague-Dawley rats (the parental strain of the GHS rats), fed the standard diet for a similar duration, served as an additional control. At 18 weeks, all kidneys were perfusion-fixed for structural analysis, detection of crystal deposits using the Yasue silver substitution method, and osteopontin immunostaining. RESULTS: There were no crystal deposits found in the kidneys of Sprague-Dawley rats. Crystal deposits were found in the kidneys of all GHS rats and this Yasue-stained material was detected only in the urinary space. No crystal deposits were noted within the cortical or medullary segments of the nephron and there was no evidence for tubular damage in any group. The only pathologic changes occurred in 3% and 5% hydroxyproline groups with the 5% group showing the most severe changes. In these rats, which form only calcium oxalate stones, focal sites along the urothelial lining of the papilla and fornix of the urinary space demonstrated a proliferative response characterized by increased density of urothelial cells that surrounded the crystal deposits. At the fornix, some crystals were lodged within the interstitium, deep to the proliferative urothelium. There was increased osteopontin immunostaining in the proliferating urothelium. CONCLUSION: Thus in the GHS rat, the initial stone formation occurred solely in the urinary space. Tubular damage was not observed with either apatite or calcium oxalate stones. The apatite stones do not appear to cause any pathological change while those rats forming calcium oxalate stones have a proliferative response of the urothelium, with increased osteopontin immunostaining, around the crystal deposits in the fornix.     

Hydroxyapatite induction and secondary aggregation of calcium oxalate, two important processes in calcium stone formation

Stone formation has often been ascribed to crystal aggregation and fixed particle growth on kidney calcifications. In this paper, the influence of hydroxyapatite (HAP) and of preformed calcium oxalate (CaOx) aggregates on CaOx crystallization was studied in freshly voided urine. Crystallization was induced by different oxalate loads and precipitates were analyzed by the spectrophotometric measurement of sedimentation time (ST), which decreases with increasing particle size. The fact that the ST of aggregates (STA) is significantly lower than the ST of other particles demonstrates that STA is a useful indicator for aggregation. At relatively low oxalate loads the addition of HAP to urine increased STA by a factor of 4.3 (P < 0.001). After a second oxalate load, STA decreased by 56% (P < 0.001), indicating secondary growth of the preexisting aggregates. HAP induced and primary CaOx aggregation occurred at low pH at which a high ionic calcium concentration (Ca2+) was measured. In urine, crystals are coated by macromolecules creating a negative surface potential with a consecutive accumulation of cations such as Ca2+. This Ca2+ accumulation could be responsible for the enhancement of aggregation by preexisting particles, which seems to be important for stone formation and which can otherwise hardly be explained in the presence of coated crystals.     

Vitamin E attenuates crystal formation in rat kidneys: roles of renal tubular cell death and crystallization inhibitors

We previously reported that oxidative stress and renal tubular damage occur in chronic hyperoxaluric rats. However, the in vivo responses of renal epithelial cells after vitamin E administration and their correlations with calcium oxalate (CaOx) crystal formation have not been evaluated. Male Wistar rats received 0.75% ethylene glycol (EG) for 7, 21, or 42 days to induce CaOx deposition (EG group). Another group of EG-treated rats received 200 mg kg(-1) of vitamin E intraperitoneally (EG+E group) to evaluate its effect on hyperoxaluria. Urinary electrolytes and biochemistry and levels of lipid peroxides and enzymes were examined, together with serum vitamin E levels. Levels of the tubular markers, alpha and mu glutathione S-transferase, proliferating cell nuclear antigen (PCNA), osteopontinin (OPN), and Tamm-Horsfall protein (THP) were also measured, and TUNEL staining was performed to examine the viability of the tubular epithelium. There were no significant differences between the two age-matched controls either untreated or given vitamin E. Compared to untreated controls, tubular cell death was increased at all time points in EG rats with a gradual increase in CaOx crystals, whereas the number of PCNA-positive cells was only significantly increased on day 21. In EG+E rats, tubular cell death was decreased compared to the EG group, and cell proliferation was seen at all time points, while CaOx crystal deposition was decreased, but hyperoxaluria, urinary lipid peroxides, and enzymuria were unaffected. Vitamin E supplement prevented the loss of OPN and THP in renal tissues by EG and the reduction in their levels in the urine. The beneficial effect of vitamin E in reducing CaOx accumulation is due to attenuation of tubular cell death and enhancement of the defensive roles of OPN and THP.     

Randall's plaque: pathogenesis and role in calcium oxalate nephrolithiasis

The purpose of these studies was to test the hypothesis that Randall's plaque develops in unique anatomical sites of the kidney and their formation is conditioned by specific stone-forming pathophysiologies. We performed intraoperative papillary biopsies from kidneys of idiopathic-calcium oxalate (CaOx), intestinal bypass for obesity, brushite (BR) and cystine stone formers (SF) during percutaneous nephrolithotomy. Tissues were examined by infrared analysis and light and electron microscopy. Our analysis revealed a distinct pattern of mineral deposition and papillary pathology for each type of SF. CaOx SF had interstitial apatite crystals beginning at thin loops of Henle. These deposits termed Randall's plaque are thought to serve as sites for stone attachment. No tubular injury was noted. Intestinal bypass patients possessed intraluminal apatite deposits in inner medullary collecting ducts (IMCD) with associated cell injury. BR SF showed the most severe form of cortical and medullary changes with sites of Randall's plaque, and yellowish intraluminal deposits of apatite in IMCD. Cystine SF had plugging of ducts of Bellini with cystine crystals and apatite deposits in IMCD and loops of Henle. Intratubular sites of crystalline deposits were always associated to adjacent regions of interstitial fibrosis. The metabolic, anatomic, and surgical pathologic findings in four distinct groups of SF clearly show that 'the histology of the renal papilla from a stone former, is particular to the clinical setting'. We believe our approach to studying stone disease will provide insights into the pathogenesis of stone formation for each type of SF that will lead to improved clinical treatment.     

Urate and calcium stones--picking up a drop of mercury with one's fingers?

The evidence invariably cited to support the suspicion that urinary urate is a predisposing factor in calcium oxalate (CaOx) stone formation is critically reviewed. Analysis of the relevant literature shows that speculation is based on the clinical impression that CaOx stone-formers appear to excrete more urate than do normal subjects, and that allopurinol reduces the rate of CaOx stone recurrences. On balance, this is sufficient to suggest that a high urinary excretion of urate promotes CaOx stone formation. However, in the past, evidence to disclose the mechanism by which urate could exert this effect has been largely shrouded in confusion and controversy. The evidence for two theories that have dominated thinking in this area are reviewed and new findings are reported that indicate that neither can account for the purported effect of urate. It is concluded that dissolved urate in urine, at normal physiological pH values, directly provokes CaOx crystal nucleation by the phenomenon of salting-out. The possibility that urate promotes CaOx stone formation is further strengthened by its ability to increase significantly the amount of CaOx precipitated from solution and to cause the aggregation of individual crystals into large clusters. Future avenues of investigation that should assist in the formulation of diagnostic and therapeutic guidelines are presented.     

The role of osteopontin on calcium oxalate crystal formation

OBJECTIVE: We evaluated whether osteopontin (OPN) and other proteins with the RGD sequence as in OPN (RGD family proteins) that are present in renal tubular cells (fibronectin [FN], Tamm-Horsfall glycoprotein [THP], vitronectin [VN], and laminin [LN]) inhibit the aggregation and growth of calcium oxalate (CaOx) crystals by a novel seed crystal method using collagen granules (CG) with and without OPN adhered on the surface. We also evaluated the effect of solid phase OPN, FN and THP in which the relationship between their proteins and CaOx crystallization was reported. Moreover, the state and time-course changes in CaOx crystals adhered to CG were observed under scanning electron microscopy (SEM). METHODS: The inhibitory activity (IA) on the aggregation and growth of CaOx crystals was measured in vitro by the conventional seed crystal method using isotopes. In this study, the following nine samples were used: OPN alone; FN alone; THP alone; VN alone; LN alone; CG alone; and CG with OPN, FN, or THP adhered on the surface (OPN/FN/THP-immobilized CG). In addition, the state and time-course changes in CaOx crystals adhered to CG were evaluated by SEM. RESULTS: Using the conventional seed crystal method, the following values of IA were obtained: 91.7% (37.5 micro g/ml) for OPN, 5.0% (100 micro g/ml) for FN, 2.0% (100 micro g/ml) for THP, 3.0% (100 micro g/ml) for VN, and 1.0% (100 micro g/ml) for LN. However, the value of IA obtained by our seed crystal method using CG was 92.1% (180cm(2)/5ml PBS) when CG alone was used. Although the value of IA was decreased by 33.6% when OPN-immobilized CG was used, it did not significantly change when FN/THP-immobilized CG was used. When CG alone was used, the evaluation of CaOx crystallization by SEM demonstrated mild adherence and aggregation of CaOx crystal suspension (seed crystals) on the CG surface, although newly formed crystals only slightly adhered to the CG surface. When OPN-immobilized CG was used, marked adherence and aggregation of seed crystals were observed, in addition to the relatively increased adherence of newly formed crystals. When FN/THP-immobilized CG was used, newly formed crystals only slightly adhered to the CG surface, although the degree of seed crystal adherence and aggregation did not significantly change. CONCLUSIONS: These findings suggest that the immobilization of OPN to the CG surface enhances the adherence and aggregation of seed crystals, as well as enhancing the adherence of newly formed crystals, resulting in decreased IA of CG (overall promotion of crystal deposition). Therefore, the results of this study clarified that OPN enhances the formation and aggregation of CaOx crystals in this experimental system.     

Thiazides reduce brushite, but not calcium oxalate, supersaturation, and stone formation in genetic hypercalciuric stone-forming rats

Over 59 generations, a strain of rats has been inbred to maximize urine calcium excretion. The rats now excrete eight to 10 times as much calcium as controls. These rats uniformly form calcium phosphate (apatite) kidney stones and have been termed genetic hypercalciuric stone-forming (GHS) rats. The addition of a common amino acid and oxalate precursor, hydroxyproline, to the diet of the GHS rats leads to formation of calcium oxalate (CaOx) kidney stones. Hydroxyproline-supplemented GHS rats were used to test the hypothesis that the thiazide diuretic chlorthalidone would decrease urine calcium excretion, supersaturation, and perhaps stone formation. All GHS rats received a fixed amount of a standard 1.2% calcium diet with 5% trans-4-hydroxy-l-proline (hydroxyproline) so that the rats would exclusively form CaOx stones. Half of the rats had chlorthalidone (Thz; 4 to 5 mg/kg per d) added to their diets. Urine was collected weekly, and at the conclusion of the study, the kidneys, ureters, and bladders were radiographed for the presence of stones. Compared with control, the addition of Thz led to a significant reduction of urine calcium and phosphorus excretion, whereas urine oxalate excretion increased. Supersaturation with respect to the calcium hydrogen phosphate fell, whereas supersaturation with respect to CaOx was unchanged. Rats that were fed Thz had fewer stones. As calcium phosphate seems to be the preferred initial solid phase in patients with CaOx kidney stones, the reduction in supersaturation with respect to the calcium phosphate solid phase may be the mechanism by which thiazides reduce CaOx stone formation.