Large-scale growth and characteristics of N-doped carbon nanotubes with ultra-large cavity.

N-doped carbon nanotubes (CNTs) with ultra-large cavity have been synthesized by using a mixture of ZnO and graphite as catalyst in the floating catalyst method. The as-synthesized N-doped CNTs are very pure, and a striking characteristic structure is that the outer diameter is at least 10 times larger than the wall thickness. Moreover, electronic properties analysis reveals that the N-doped CNTs with ultra-large cavity have a reduced room temperature resistance compared with those of the common N-doped CNTs, which give an experimental prove for the previous theoretical predictions.

Oxalate-induced redistribution of phosphatidylserine in renal epithelial cells: implications for kidney stone disease.

AIMS: The present studies assessed the possibility that exposure to oxalate leads to alterations in membrane structure that promote crystal binding to renal epithelial cells. Specifically, we determined whether oxalate exposure produces a redistribution of membrane phosphatidylserine (PS) and an increase in the binding of (14)C-oxalate crystals to renal epithelial cells. METHODS: PS distribution was monitored in MDCK cells and in phospholipid-containing vesicles using NBD-PS, a fluorescent derivative of PS. Superficial PS was also detected by monitoring the binding of annexin V to MDCK cells. RESULTS: Oxalate exposure rapidly increased the abundance of superficial NBD-PS and increased the binding of annexin V to MDCK cells. Oxalate exposure also increased PS at the surface of phospholipid vesicles, suggesting that oxalate may interact directly with PS. The oxalate concentrations that increased superficial PS also increased binding of (14)C-oxalate crystals to MDCK cells, and the increased crystal binding was blocked by annexin V. CONCLUSIONS: These findings provide direct evidence that oxalate exposure promotes both a redistribution of PS and an increase in crystal binding in renal epithelial cells and support the notion that oxalate toxicity may contribute to the development of stone disease by altering the properties of the renal epithelial cell membrane.

Oxalate-induced ceramide accumulation in Madin-Darby canine kidney and LLC-PK1 cells.

BACKGROUND: Oxalate exposure produces oxidant stress in renal epithelial cells leading to death of some cells and adaptation of others. The pathways involved in these diverse actions remain unclear, but appear to involve activation of phospholipase A2 (PLA2) and redistribution of membrane phospholipids. The present studies examined the possibility that oxalate actions may also involve increased accumulation of ceramide, a lipid-signaling molecule implicated in a variety of pathways, including those leading to apoptotic cell death. METHODS: Ceramide accumulation was examined in renal epithelial cells from pig kidney (LLC-PK1 cells) and from dog kidney [Madin-Darby canine kidney (MDCK cells)] using the diacylglycerol kinase assay. Sphingomyelin degradation was assessed by monitoring the disappearance of 3H-sphingomyelin from cells that had been prelabeled with [3H]-choline. The effects of oxalate were compared with those of other oxidants (peroxide, xanthine/xanthine oxidase), other organic acids (formate and citrate), and a known activator of sphingomyelinase in these cells [tumor necrosis factor-alpha (TNF-alpha)]. Separate studies determined whether oxalate-induced accumulation of ceramide could be blocked by pretreatment with antioxidants [Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn TMPyP, a superoxide dismutase mimetic) or N-acetylcysteine (NAC; an antioxidant)], with an inhibitor of ceramide synthase [fumonisin B1 (FB1)] or with an inhibitor of PLA2 [arachidonyl trifluoromethylketone (AACOCF3)]. RESULTS: Oxalate exposure produced a significant time- and concentration-dependent increase in cellular ceramide. A reciprocal decrease in 3H-sphingomyelin was observed under these conditions. Increases in cellular ceramide levels were also observed after treatment with other oxidants (hydrogen peroxide, and xanthine/xanthine oxidase), activators of sphingomyelinase (TNF-alpha), exogenous sphingomyelinase, or arachidonic acid. Formate produced similar (albeit smaller) effects, and citrate did not. The oxidant-induced increases in ceramide were attenuated by pretreatment with NAC (a glutathione precursor) and MnTMPyP (a superoxide dismutase mimetic), suggesting a role for cellular redox states. The oxalate-induced increase in ceramide was also attenuated by pretreatment with AACOCF3, suggesting a role for PLA2. Pretreatment with FB1 produced a small but statistically insignificant attenuation of the response to oxalate. CONCLUSIONS: Oxalate exposure produces a marked accumulation of ceramide in renal epithelial cells by a process that is redox sensitive and mediated in part by activation of PLA2. Since cellular sphingomyelin decreased as ceramide increased, it seems likely that oxalate actions are mediated, at least in part, by an increase in sphingomyelinase activity, although alterations in ceramide synthase are also possible. Further study is required to define the steps involved in oxalate actions and to determine the extent to which ceramide signaling mediates oxalate actions.

Oxalate toxicity in renal epithelial cells: characteristics of apoptosis and necrosis.

Studies in various tissues, including the kidney, have demonstrated that toxins elicit apoptosis under certain conditions and necrosis under others. The nature of the response has important consequences for the injured tissue in that necrotic cells elicit inflammatory responses, whereas apoptotic cells do not. Thus, there has been considerable interest in defining the mode of cell death elicited by known cytotoxins. The present studies examined the response of renal epithelial cells to oxalate, a metabolite excreted by the kidney that produces oxidant stress and death of renal cells at pathophysiological concentrations. These studies employed LLC-PK1 cells, a renal epithelial cell line from pig kidney and NRK-52E (NRK) cells, a line from normal rat kidney, and compared the effects of oxalate with those of known apoptotic agents. Changes in cellular and nuclear morphology, in nuclear size, in ceramide production, and in DNA integrity were assessed. The ability of bcl-2, an anti-apoptotic gene product, to attenuate oxalate toxicity was also assessed. These studies indicated that oxalate-induced death of renal epithelial cells exhibits several features characteristic of apoptotic cell death, including increased production of ceramide, increased abundance of apoptotic bodies, and marked sensitivity to the level of expression of the anti-apoptotic gene bcl-2. Oxalate-induced cell death also exhibits several characteristics of necrotic cell death in that the majority of the cells exhibited cellular and nuclear swelling after oxalate treatment and showed little evidence of DNA cleavage by TUNEL assay. These results suggest that toxic concentrations of oxalate trigger both forms of cell death in renal epithelial cells.

Oxalate-induced changes in renal epithelial cell function: role in stone disease.

Many studies on the etiology of stone disease have focused on the properties of urine that affect crystal nucleation and growth. More recent studies have focused on the properties of the renal epithelium and the role of injury in crystal retention. The latter studies have shown that oxalate exposure per se can damage renal epithelial cells and enhance crystal binding. This overview summarizes findings of specific biochemical and genetic alterations observed in renal epithelial cells after exposure to oxalate. In LLC-PK1 and MDCK cells, oxalate exposure produces marked effects on membranes, causing a redistribution of phosphatidylserine and activation of two lipid signaling cascades, one involving phospholipase A(2) (PLA(2)) and one involving ceramide. Longer exposure to oxalate leads to membrane damage and cell death. Adaptive responses are also observed, including proliferation (for replacement of damaged cells) and induction of various genes (for cellular replacement and repair). Many or all of these responses are blocked by antioxidants, and many can be mimicked by PLA(2) agonists/products. This finding suggests links between oxalate-induced increases in oxidant stress, lipid signaling pathways, and subsequent molecular responses that may eventuate in renal cell damage or death. Whether such changes play a role in stone disease in vivo, and whether strategies to inhibit these changes would be beneficial therapeutically, is unknown.

Does urinary oxalate interfere with the inhibitory role of glycosaminoglycans and semisynthetic sulfated polysaccharides in calcium oxalate crystallization?

OBJECTIVES: Previously it was shown that the polysaccharide G872 in vitro strongly inhibits calcium oxalate monohydrate crystallization processes. However, when rats on a stone-inducing diet of ethylene glycol plus vitamin D3 are given this polysaccharide, no changes in the urine capacity for crystallization inhibition were found. We investigated here how the inhibitory action of polysaccharides changes under high oxalate conditions, as they exist in the stone inducing diet. METHODS: Calcium oxalate monohydrate (COM) crystals were incubated in a series of 0.05 M PBS buffers containing polysaccharides with increasing oxalate concentrations (0-0.4 mmol/l). The coated crystals were collected, washed and resuspended in an artificial urine. We then measured the zeta potential of the crystals, using a Coulter DELSA 440, and the initial rates for crystal growth and agglomeration, using the Coulter Multisizer II. RESULTS: Addition of oxalate to the medium shifts the negative zeta potential distribution of COM crystals coated by polysaccharides in positive direction. Particle size analysis demonstrated that the initial rates of COM crystal growth and agglomeration responding to oxalate concentration changes (0.1-->0.4 mmol/l) in the presence of G872 (0.2 mg/l) are approximately 2.5 times faster than that in the absence of G872. CONCLUSIONS: Oxalate interferes with the binding of polysaccharides to crystals. This can be envisioned to occur through changes in the crystal surface properties or by induction of functional and secondary structural changes of urinary macromolecular inhibitors such as GAGs, resulting in a decrease of their inhibitory activity against COM crystallization. Thus, in urine, a high oxalate may increase the rate of crystallization both by increasing the supersaturation and by decreasing the inhibitory potential of the urine.

Ultrastructural osteopontin localization in papillary stones induced in rats.

OBJECTIVES: To detect in situ the precise osteopontin (OPN) localization in papillary stones. METHODS: Immunocytochemical labelling procedures are applied to detect OPN localizations in crystalline material of renal papillary stones. The tissue-processing procedure for electron microscopy, which includes OsO4 postfixation, preserves both immunocytochemical OPN reactivity and cellular membrane contrast up to the ultrathin section. Reflection-contrast light microscopical images are correlated with high resolution transmission-electron microscopical observations from consecutive ultrathin epon sections. RESULTS: Preserved crystalline material in interstitial and peripheral papillary stones is recognized as calcium oxalate monohydrate. After section incubation with markers conjugated to an antibody against OPN (alpha OPN) the crystals are converted into ghosts. In the ghosts, alpha OPN markers are present around microcrystals. The size of these microcrystals ranges from several nanometers to micrometers. It is observed (due to the OsO4-preserved membranes) that interstitial cells are separated from the stone surfaces by unidentified extracellular material, also present in the center as a stone matrix. CONCLUSION: The microcrystal-growth inhibitor OPN is detected in situ in interstitial stones induced in the rat's papilla and at the surface of the papilla.

Crystal-cell interaction inhibition by polysaccharides.

PURPOSE: We studied the effect of polysaccharides on interactions between calcium oxalate monohydrate (COM) crystals and cultured renal cells. MATERIALS AND METHODS: Monolayers of Madin-Darby canine kidney (MDCK) cells were incubated with radiolabeled crystals in the presence of various concentrations of natural glycosaminoglycans (GAGs) and semisynthetic polysaccharides (SSPs). RESULTS: While most GAGs were found to have relatively little effect, SSPs (SP54, G871 and G872) were potent inhibitors of crystal-cell association. Pretreatment of crystals, but not of cells, was similarly effective, suggesting polysaccharide-induced modification of crystal surface properties. CONCLUSIONS: This result further supports the idea that SSPs, and especially G872, are of potential interest for treatment of recurrent stone disease.

Effect of two new polysaccharides on growth, agglomeration and zeta potential of calcium phosphate crystals.

PURPOSE: To study the effect of semisynthetic sulphated polysaccharides in the different calcium phosphate crystallization processes in vitro. MATERIALS AND METHODS: Crystallization of hydroxyapatite (HAP) and brushite (DCPD) in the presence and absence of 2 new semisynthetic sulphated polysaccharides (G871, G872) were defined by a constant composition technique, particle size analysis and zeta potential measurement. RESULTS: These polysaccharides demonstrated strong inhibitory effect on HAP and DCPD crystal growth and agglomeration. The increase of negative zeta potential values after addition of polysaccharides suggests the binding of these polysaccharides to HAP and DCPD crystals. CONCLUSION: We conclude that both G871 and G872 could be of potential use for calcium phosphate urolithiasis prevention in addition to their use for calcium oxalate.

Zeta potential measurement and particle size analysis for a better understanding of urinary inhibitors of calcium oxalate crystallization.

To better understand urinary inhibitors of calcium oxalate crystallization, both zeta potential measurement and particle size analysis were chosen to illustrate: (1) the potential therapeutic efficacy of G872, a semi-synthetic sulfated polysaccharide, in stone prevention; and (2) the relative contribution of various urinary fractions ¿e.g., ultrafiltered urine (UFU), Tamm-Horsfall protein (THP), urinary polyanions precipitated with cetylpyridinium chloride (CPC), urinary macromolecular substances with different concentration ratios (UMS10,50,90 and UMS'10,50,90) and THP-free urine (THPFU)¿ to total urinary inhibitory activity. The results showed: (1) addition of G872 significantly enhances urinary inhibitory activity and negative zeta potential values; (2) re-addition of the CPC to UFU completely restores urinary inhibitory activity; and (3) artificial urines prepared by mixing UMS'10,50,90 from THPFU with UFU differed in inhibitory activity from that prepared by mixing UMS10,50,90 from a pooled normal urine with UFU. Based on these experimental results, the following speculations can be made: (1) normal human urines are considered to be a protective colloidal system; (2) urinary inhibitory activity originates mainly from CPC and/or UMS; (3) normal THP is a protective material to maintain urinary inhibitory activity; and (4) mutual interaction between urinary inhibitors may change the total urinary inhibitory activity.

Lectin-cytochemistry of experimental rat nephrolithiasis.

Lectin reactivity in epithelial apical cell coats of normal rat kidneys was compared to that from animals subjected to crystal inducing diets (CID). The aim was to see whether the absence of lectin reactivity in cell coats is related to intratubular calcium oxalate crystal retention. In normal rat kidneys, after a pre-embedding procedure, it was observed that at the ultrastructural level, reactivity was present but that the lectin specificity for the various parts of the nephron might have to be reconsidered. There was heterogeneity between the epithelial cells with respect to the presence of coat material in the tubular cell apices. Tubular epithelial cell apices from CID rats showed no obvious changes in lectin reactivity pattern. Lectin reactivity was present at the periphery of intratubular crystals but undetectable at true crystal attachment sites or reduced at cell apices in the vicinity of recently attached crystals or agglomerates. After a post-embedding reaction procedure, wheat-germ agglutinin (WGA)-lectin reactivity confirmed the presence of coat material in the cleft between cell apex and retained crystal at crystal-attachment sites. The WGA/Au-10 nm reaction products were also seen inside epithelial cells. WGA/Au-10 nm reaction products mark a crystal matrix component inside intratubular and retained crystals. A similar matrix was also marked by an alpha-osteopontin (alpha OPN/Au-10 nm) reaction product.

Pathological and immunocytochemical changes in chronic calcium oxalate nephrolithiasis in the rat.

In the present study, we exposed rats to a crystal-inducing diet (CID) consisting of vitamin D3 and 0.5% ethylene glycol (EG), and we investigated histologically the kidney damage induced by the deposition of calcium oxalate (CaOx) crystals. After 28 days, 50% of the animals had renal CaOx crystals, of which 60% also had small papillary stones. Most crystals were present in the cortex. The occurrence of these crystals coincided with morphological and cytochemical changes: glomerular damage, tubular dilatation and necrosis, and an enlargement of the interstitium. The number of epithelial and interstitial cells positive for the proliferating cell nuclear antigen (PCNA) was increased. Tamm-Horsfall protein (THP) was not only demonstrable in the thick ascending limb of the loop of Henle (TAL), but also frequently in glomeruli, in the proximal tubular epithelium, and in the papilla. In the lumen of the tubular system, it was associated with urinary casts. Reflection contrast microscopy (RCM) showed that the crystals were coated with a thin layer of THP. In spite of the high urinary oxalate concentrations, the above described cellular changes were not observed in CID-fed rats without renal crystals. We conclude, therefore, that in the kidney, the retained CaOx crystals rather than the urinary oxalate ions are responsible for the observed morphological and immunocytochemical changes.

Experimental nephrolithiasis in rats: the effect of ethylene glycol and vitamin D3 on the induction of renal calcium oxalate crystals.

Using ethylene glycol (EG) and vitamin D3 as crystal-inducing diet (CID) in rats, we investigated the effect of the dosage of EG on the generation of chronic calcium oxalate (CaOx) nephrolithiasis. We collected weekly 24 hour urines and measured herein the amount of oxalate, calcium, glycosaminoglycans (GAG's), creatinine, protein, alkaline phosphatase (AP), gamma-glutamyl transpeptidase (gamma-GT), and N-acetyl-beta-glucosaminidase (NAG). The potential of these urines to inhibit crystal growth and agglomeration was also evaluated. After four weeks, the kidneys were screened by histology and radiography for the presence of CaOx crystals and the amount of kidney-associated oxalate was biochemically measured. Using 0.5 vol.% EG, only a part of the rats showed CaOx deposition in the renal cortex and/or medulla, without obvious differences between Wistar and Sprague-Dawley (SD) rats. If a dietary EG concentration of 0.75, 1.0, or 1.5 vol.% was used, the amount of kidney-associated oxalate was proportionally higher and CaOx crystal formation was consistently found in all rats. Most crystals were encountered in the cortex, whereas in the medulla and the papillary region, crystals were only occasionally detected. From these data, we conclude that in the chronic rat model, based on EG and vitamin D3, a consistent deposition of CaOx crystals is obtained using a EG concentration of at least 0.75%.

Association of calcium oxalate monohydrate crystals with MDCK cells.

Many factors are presently known which determine the risk of calcium oxalate (CaOx) stone formation in the kidney, although the early events in the pathogenesis of this disease are still to be elucidated. One of these early events is the interaction of intraluminal crystals with the epithelial cells lining the renal tubules. In this study we determined the interaction of approximately 2 microns calcium oxalate monohydrate (COM) crystal with monolayers of Madin-Darby canine kidney (MDCK) cells grown on porous supports in a two-compartment culture system. Crystal-cell interaction studies were performed after the monolayers reached their highest level of gamma-glutamyltranspeptidase (gamma GT) enzyme activity, a marker for brush border development. Technical aspects were evaluated, such as the size and morphology of the crystals and the influence of incubation time, temperature and pH on crystal-cell interaction. Kinetic data demonstrated that an equilibrium between free and associated particles was reached within 30 minutes. Crystal-cell interaction was often associated with cell damage. However, evidence is provided that in an environment that was saturated with calcium oxalate, MDCK cells in an environment that was a certain amount of COM crystals without sustaining measurable injury. After initial attachment to the cell surface, crystals were taken up and subsequently eliminated again from the monolayers. The model system described in this paper provides a tool for detailed studies of processes that are involved in renal cellular handling of luminal COM crystals.

Etiology of calcium oxalate nephrolithiasis in rats. I. Can this be a model for human stone formation?

Crystal retention is studied in a rat-model system as a possible mechanism for the etiology of human nephrolithiasis. A crystal-inducing diet (CID) of ethylene glycol plus NH4Cl in their drinking-water is offered to healthy rats to generate intratubular crystals. Subsequently, the fate of retained crystals is investigated by allowing the rats a tissue recovery/crystalluria phase for three, five and ten days, respectively, on normal drinking water. The process of exotubulosis is observed in cortex and medulla of aldehyde-fixed kidneys after three days recovery. After five days, crystals are predominantly seen there in the interstitium. After ten days, cortex and medulla are virtually free of crystals. However, in the papillary regions after five and ten days recovery, three types of calcium oxalate monohydrate (COM) crystals are present: (1) free in the calycine space, (2) sub-epithelially located surrounded by interstitial cells within, and (3) covered by macrophage-like cells, outside the original papillary surface. After a CID plus three days recovery, a further thirty-seven days extra oxalate challenge with solely 0.3 vol% ethylene glycol induced intratubular and interstitial oxalate crystals. In the papillary region, large sub-epithelial crystals are seen. However, no crystals are seen in kidneys from rats given solely (0.5 or 0.8 vol.%) ethylene glycol for thirty days. An oxalate re-challenge retards crystal removal.

Etiology of calcium oxalate nephrolithiasis in rats. II. The role of the papilla in stone formation.

In kidneys of healthy rats submitted to a crystal-inducing diet (CID) with ethylene glycol (EG) and NH4Cl, the fate of retained crystals in the papillar region is studied during a recovery period of one, five or ten days, as model system for human nephrolithiasis. Scanning electron microscopy (SEM) shows, at papillary tips bulging into the calycine space, crystal masses covered either by the epithelium or a thin fibrous veil, or by unidentified mobile cuboidal cells. After CID plus one or five days recovery, small sub-epithelial swellings are seen of large sub-epithelial crystals at or around the papillary tip. After CID plus ten days, massive sub-surface crystal-containing micrometer-sized stones are seen in which the presence of calcium is confirmed by X-ray microanalysis. The papillary tip of rats after a re-challenge with an oxalate load from 0.1 vol% EG for twelve or forty-two days shows minor lesions. But a re-challenge with 0.3 vol% EG for thirty-seven days induces large sub-epithelial papillary millimeter-sized stones. The Von Kossa section staining converts the crystals into a black precipitate, but large peri-tubular or peri-vascular calcium deposits are absent. A new hypothesis about the etiology of an inductive calcium oxalate monohydrate nephrolithiasis is formulated which differs from the one proposed by Randall based on his deductive human kidney studies.

Zeta potential distribution on calcium oxalate crystal and Tamm-Horsfall protein surface analyzed with Doppler electrophoretic light scattering.

The zeta potential distribution (ZPD) and particle size of Tamm-Horsfall protein (THP) and of calcium oxalate monohydrate (COM) crystals were measured using a Doppler Electrophoretic Light Scattering Analysis Instrument. The studies showed differences in the ZPD pattern between THP derived from normal subjects (nTHP) and from stone patients (pTHP). Both nTHP and pTHP can shift the zeta potential of calcium oxalate crystals towards more negative values; nTHP is significantly more potent than pTHP. The zeta potential of both nTHP and pTHP becomes less negative with decreasing pH and with increasing calcium concentration or ionic strength. Tamm-Horsfall protein particle size measurements showed that nTHP particles are significantly smaller than pTHP particles. The size of both nTHP and pTHP increases with increasing calcium concentration or increasing ionic strength and with decreasing pH. The differences between nTHP and pTHP in surface charge and particle size may be based on differences in molecular structure and may cause functional differences in their ability to inhibit calcium oxalate crystal aggregation.

Glycosaminoglycans and other sulphated polysaccharides in calculogenesis of urinary stones.

Naturally occurring glycosaminoglycans (GAGs) and other, semisynthetic, sulphated polysaccharides are thought to play an important role in urolithiasis. Processes involved in urinary stone formation are crystallization and crystal retention. Oxalate transport and renal tubular cell injury are determining factors in these processes. In this article experimental results concerning the possible mechanisms of action of GAGs and other sulphated polysaccharides are reviewed. GAGs are inhibitors of crystal growth and agglomeration and possibly also of nucleation. They can prevent crystal adherence, correct an abnormal oxalate flux and prevent renal tubular cell damage.

Etiology of experimental calcium oxalate monohydrate nephrolithiasis in rats.

In a rat-model system, tubular crystal retention as a possible mechanism for the etiology of nephrolithiasis in man, was studied by conventional transmission electron microscopy. The animals were supplied for nine days with a crystal-inducing diet, with ethylene glycol plus NH4Cl in their drinking-water. After this induction period, a two day regime with fresh drinking-water was included, to allow crystals to be removed by spontaneous crystalluria. After aldehyde fixation of the rat kidneys, large crystals were seen inside the tubular lumen. The crystals were attached to cell surfaces and covered by neighboring epithelial cells. Some crystals were overgrown by several epithelial cells and underwent a process of so-called exotubulosis, resulting in free or cell-surrounded crystals in the interstitium, and possibly in crystals in Giant cells. To investigate the fate of the retained crystals, some animals were additionally exposed to a low-oxalate challenge from drinking water containing 0.1 volume per cent of ethylene glycol for 12 or 30 days, respectively. It was assumed that this would interfere with the retained intratubular or interstitial crystals, and allow the crystals to grow into mini-stones. This was not observed. After the oxalate challenge, no crystals were found to be retained in the tubules (free or covered by cells). Interstitial crystals were observed, but it remains to be demonstrated whether such crystals actually grow into mini-stones or that they are removed by the sterile inflammation process observed.