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Experimental Nephrolithiasis In Rats: The Effect Of Ethylene Glycol And Vitamin D3 On The Induction Of Renal Calcium Oxalate Crystals, R. De Water, E. R. Boevé, P. P. M. C. Van Miert, G. Deng, L. C. Cao, Th. Stijnen, W. C. De Bruijn, F. H. Schröder

Scanning Microscopy

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), 𝝲-glutamyl transpeptidase (𝝲GT), and N-acetyl-𝝱-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 …

Pathological And Immunocytochemical Changes In Chronic Calcium Oxalate Nephrolithiasis In The Rat, R. De Water, E. R. Boeve, P. P. M. C. Van Miert, C. P. Vermaire, P. R. W. A. Van Run, L. C. Cao, W. C. De Bruijn, F. H. Schroder

Scanning Microscopy

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 …

Patterns Of Calcium Oxalate Crystal Production By Three Species Of Wood Decay Fungi, Jon H. Connolly, Howard J. Arnott, Jody Jellison

Scanning Microscopy

Wood decay experiments using red spruce wood resting on moist soil were conducted to discern temporal and spatial patterns of calcium oxalate (CaOx) crystal production by three species of fungi over the course of decay. All three species produced crystals of calcium oxalate dihydrate, but not monohydrate, in and on wood. Over the course of decay, the production of CaOx crystals was shown to be heterogeneous in both space and time. The relative quantity, morphology and longevity of CaOx crystals varied among species. Gloeophyllum (G.) trabeum produced substantial quantities of "free" crystals; Fomitopsis (F.) pinicola produced encrusting crystals; and Trichaptum …

Formation Of Hydrated Calcium Oxalates In The Presence Of Poly-L-Aspartic Acid, Jeffrey A. Wesson, Elaine Worcester

Scanning Microscopy

The effect of poly-L-aspartic acid (PA) on the crystal structure of calcium oxalate crystals grown after spontaneous nucleation was evaluated as a function of relative supersaturation and calcium:oxalate ratio in a buffered salt solution, with pH and ionic strength in the range of normal human urine. PA was used as a model for naturally occurring acidic urine proteins that have been shown to inhibit nucleation and growth of calcium oxalate crystals. The crystals grown were characterized by optical microscopy and X-ray powder diffraction. It was observed that calcium oxalate monohydrate was the preferred crystalline form in the absence of added …

Crystallization And Stone Formation Inside The Nephron, Dirk J. Kok

Scanning Microscopy

A model is presented visualizing the events leading to calcium-salt, crystal- and stone-formation inside the nephron. For each nephron segment, handling of urine components relevant to stone formation is considered and urine composition determined. This information was applied to nucleation experiments simulating passage of urine through a nephron. The model and in vitro experiments suggest that within normal transit times for the respective nephron segments, particles of a hydroxyapatite-like material first form near the bend in the Loop of Henle of juxtamedullary nephrons. From there on, calcium oxalate particles start to appear: first dihydrate, then monohydrate. In the collecting duct …

Studies On The Role Of Urinary Macromolecules In Urolithiasis: Review Of Methodologies And A Proposal For A Standard Reference Crystallization System, A. L. Rodgers, D. Jappie

Scanning Microscopy

In this study, urine from a calcium oxalate kidney stone former was ultrafiltered (10 kD cut-off). Crystallization was induced in the ultrafiltrate and retentate fractions as well as in a sample of the whole urine. The progress of crystallization was monitored by Coulter Counter and flow cytometry techniques. (The latter has not been used in studies of the role of urinary macro-molecules in urolithiasis). Deposited crystals were examined by scanning electron microscopy. Results indicated that urinary macromolecules in this subject are inhibitors of nucleation and aggregation. These results agree with the findings of some workers but disagree with those of …

Inter-Alpha-Inhibitor: A Protein Family Involved In The Inhibition Of Calcium Oxalate Crystallization, Fouad Atmani, Jacques Mizon, Saeed R. Khan

Scanning Microscopy

Inter-α-inhibitor (IαI) is a serine protease inhibitor present in human plasma. It has a molecular weight of about 220 kDa which encompasses 3 chains including two heavy chains and one light chain. The light chain, known as bikunin, is responsible for the antitryptic activity of IαI in the inhibition of various enzymes, such as trypsin and chymotrypsin. Under physiologic or certain pathologic circumstances, several macromolecules related to IαI appear in plasma and urine. However, the physiologic role of IαI remains unclear. As far as urolithiasis is concerned, two urinary macromolecules related to IαI have been isolated and shown to be …

Zeta Potential Measurement And Particle Size Analysis For A Better Understanding Of Urinary Inhibitors Of Calcium Oxalate Crystallization, L. C. Cao, G. Deng, E. R. Boeve, W. C. De Bruijn, R. De Water, C. F. Verkoelen, J. C. Romijn, F. H. Schroder

Scanning Microscopy

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 polyanionsprecipitated with cetylpyridinium chloride (CPC), urinary macromolecular substances with different concentration ratios (UMSl0,50,90 and UMS'l0,50,90) 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 …

Induction Of Crystallization Of Calcium Oxalate Dihydrate In Micellar Solutions Of Anionic Surfactants, H. Füredi-Milhofer, L. Tunik, N. Filipovic-Vincekovic, D. Skrtic, V. Babic-Ivancic, N. Garti

Scanning Microscopy

Calcium oxalate dihydrate (CaC₂O₄.(2+x)H₂O; COD; x ≤ 0.5) does not readily crystallize from electrolytic solutions but appears as a component in crystalluria. In this paper, we review in vitro studies on the factors responsible for its nucleation and growth with special attention given to the role of surfactants. The following surfactants were tested: dodecyl ammonium chloride (cationic), octaethylene monohexadecylether (non-ionic), sodium dodecyl sulfate (SOS, anionic), dioctyl sulphosuccinate (AOT, anionic), and sodium cholate (NaC, anionic). The cationic and some of the anionic surfactants (SOS, AOT) induced different habit modifications of growing calcium oxalate crystals by …

Heterogeneous Nucleation Of Calcium Oxalate Crystals In Mammalian Urine, Saeed R. Khan

Scanning Microscopy

It is generally recognized that calcium oxalate crystal formation in urine is induced by heterogeneous nucleation. However, there is no consensus as to the nature of the nucleation substrate. Evidence is provided in this paper that membranous cellular degradation products are the most likely candidates because they: (1) are ubiquitous in urine and urinary stones; (2) are found in close association with crystal deposits in the kidneys; and (3) can induce nucleation of crystals from a meta-stable solution of calcium oxalate in vitro and metastable urine in vivo.

Alterations In Mdck And Llc-Pk1 Cells Exposed To Oxalate And Calcium Oxalate Monohydrate Crystals, R. L. Hackett, P. N. Shevock, S. R. Khan

Scanning Microscopy

Structural analysis of human kidney stones reveals the presence of cellular membranes and other cell fragments. Experimentally, calcium oxalate crystallization is facilitated when an exogenous nephrotoxin is given with ethylene glycol, thus providing cellular degradation products to act as heterogenous nuclei. In this report, we tested whether oxalate alone could act as a cell toxin capable of producing damaged cells without the presence of an exogenous agent. Cultured LLC-PK₁ and MDCK cells, when exposed to 1.0 mmol KOx, a concentration at the limit of metastability for calcium oxalate nucleation, were severely damaged as measured by specific lactate dehydrogenase (LDH) …

Etiology Of Calcium Oxalate Nephrolithiasis In Rats. I. Can This Be A Model For Human Stone Formation?, W. C. De Bruijn, E. R. Boevé, P. R. W. A. Van Run, P. P. M. C. Van Miert, R. De Water, J. C. Romijn, C. F. Verkoelen, L. C. Cao, F. H. Schröder

Scanning Microscopy

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 …

Experimental Calcium Oxalate Nephrolithiasis And The Formation Of Human Urinary Stones, Saeed R. Khan

Scanning Microscopy

Calcium oxalate nephrolithiasis in rats requires induction of hyperoxaluria which results in increased urinary calcium oxalate supersaturation. As a result of low to mild chronic hyperoxaluria, calcium oxalate crystals deposit first in the papillary collecting ducts. Crystal deposition in the kidneys is preceded by calcium oxalate crystalluria and starts with the retention of aggregated calcium oxalate crystals in the renal tubules. Retained crystals move from the tubules to the interstitium, and in the process, become anchored to the tubular basement membrane. Crystal aggregates present in the superficial peripheral collecting ducts of the renal papillae ulcerate through to the papillary surface …

Urinary Glycosaminoglycans Are Selectively Included Into Calcium Oxalate Crystals Precipitated From Whole Human Urine, Koji Suzuki, Keiko Mayne, Ian R. Doyle, Rosemary L. Ryall

Scanning Microscopy

Urinary glycosaminoglycans are selectively included into calcium oxalate (CaOx) crystals precipitated from whole human urine: The presence of glycosaminoglycans (GAGs) in the organic matrix of urinary stones, and their known effects on CaOx crystallization have prompted speculation regarding their role in CaOx urolithiasis. The aim of this study was to examine the involvement of GAGs in the early stages of CaOx crystallization in human urine. Urine samples were collected from healthy men and CaOx crystallization was induced by the addition of a sodium oxalate load. The crystals were harvested and demineralized, and the GAG content of the resulting extract analysed …

Etiology Of Experimental Calcium Oxalate Monohydrate Nephrolithiasis In Rats, W. C. De Bruijn, E. R. Boevé, P. R. W. A. Van Run, P. P. M. C. Van Miert, J. C. Romijn, C. F. Verkoelen, L. C. Cao, F. H. Schröder

Scanning Microscopy

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 …

Effect Of Urinary Macromolecules And Chondroitin Sulphate On Calcium Oxalate Crystallization In Urine, A. L. Rodgers, D. Ball, W. Harper

Scanning Microscopy

After filtration and ultrafiltration (10 kD) of 24 hour urine specimens from 12 healthy male subjects, calcium oxalate crystallization was induced in the filtered (FILTD) and ultrafiltered (UF) fractions by administration of a sodium oxalate load. In addition crystallization was also induced in UF fractions to which physiological quantities of chondroitin sulphate (CHON) had been added (UF + CHON). The rate of calcium oxalate crystallization was determined by measuring the rate at which turbidity increased. Crystal numbers and sizes were measured with a Malvern particle size analyzer and by scanning electron microscopy.

Crystallization rates, crystal numbers and crystal sizes were …

Crystallization Of Calcium Oxalate And Calcium Phosphate At Supersaturation Levels Corresponding To Those In Different Parts Of The Nephron, Ján Lupták, Hanne Bek-Jensen, Anne-Marie Fornander, Inge Höjgaard, Mari-Anne Nilsson, Hans-Göran Tiselius

Scanning Microscopy

The risk of crystallization in solutions, with a composition corresponding to that of urine in various parts of the nephron, was assessed by studying urine samples containing standardized increments of calcium and oxalate concentrations. The experiments were carried out in salt solutions with and without dialysed urine and the appearance of crystals was detected and measured with a Coulter counter. With increased concentrations of calcium, crystals of calcium phosphate were predominantly formed in solutions with a composition corresponding to that in the proximal and distal tubuli, whereas, calcium oxalate was the primary crystallization product in solutions with a composition corresponding …

Effects Of Acute Acid Loading On The Risk Of Calcium Phosphate And Calcium Oxalate Crystallization In Urine, P. J. Osther, J. Bollerslev, J. R. Nørgård, K. Engel, P. Kildeberg

Scanning Microscopy

The aim of this study was to examine the risk of calcium phosphate and calcium oxalate crystallization during acute acid loading under controlled conditions.

The effects of acute acid loading on rates of renal excretion of calcium, magnesium, phosphate, citrate, oxalate and urine pH were studied in healthy subjects. The risk of calcium phosphate and calcium oxalate crystallization were evaluated by estimates of the ion activity products of calcium phosphate [AP(CaP)-index] and calcium oxalate [AP(CaOx)-index] according to Tiselius. In addition, the risk of brushite [AP(Bru)-index] crystallization was estimated.

An acute acid load administered as ammonium chloride (NH₄Cl) produced …

Urinary Calculi: Review Of Classification Methods And Correlations With Etiology, M. Daudon, C. A. Bader, P. Jungers, O. Beaugendre, M. P. Hoarau

Scanning Microscopy

Current physical and chemical methods available for urinary stones analysis are critically reviewed. No one method is sufficient to provide all the clinically useful information on the structure and composition of the stones. We show that a combination of refined morphological and structural examination of stone with optical microscopy, complemented by compositional analysis using infrared spectroscopy of the core, cross-section and surface of calculi, provides a precise and reliable method for identifying the structure and crystalline composition, and permits quantification of stone components while being highly cost effective. Using such morphoconstitutional studies leads to a classification of urinary stones in …

The Nucleation And Growth Of Calcium Oxalate Monohydrate On Self-Assembled Monolayers (Sams), Allison A. Campbell, Glen E. Fryxell, Gordon L. Graff, Peter C. Rieke, Barbara J. Tarasevich

Scanning Microscopy

A physical chemical approach was used to study calcium oxalate monohydrate (COM) nucleation and growth on various organic interfaces. Self-assembling monolayers (SAMs), containing derivatized organic functional groups, were designed to mimic various amino acid residues present in both urine and stone matrix macromolecules. Derivatized surfaces include SAMs with terminal methyl, bromo, imidazole, and thiazolidine-carboxylic acid functional groups. Pronounced differences in COM deposition were observed for the various interfaces with the imidazole and thiazolidine surfaces having the greatest effect and the methyl and bromo groups having little or no nucleating potential.

Effects Of Citrate On The Different Phases Of Calcium Oxalate Crystallization, H. -G. Tiselius, C. Berg, A. -M. Fornander, M. -A. Nilsson

Scanning Microscopy

Urinary citrate appears to be an important factor in the crystallization process of calcium oxalate and calcium phosphate. The urinary excretion of citrate was found to be significantly lower in patients with calcium oxalate stone disease as compared with normal subjects, and about 30 per cent of the calcium stone formers can be considered as hypocitraturic. The lowest excretion of citrate was recorded in urine collected during the night. Citrate has significant effects on supersaturation with respect to both calcium oxalate and calcium phosphate, it also inhibits the growth of these crystals. In addition, citrate appears to be capable of …

Evaluation And Management Of Dietary Habits In Japanese Renal Stone Formers, Haruo Ito, Tadashi Kotake, Naoto Miura

Scanning Microscopy

To elucidate the relationship between the formation of kidney stones and diet, we carried out a dietary investigation in patients with urinary tract stones. Dietary intakes were estimated for 36 patients (24 men, 12 women) with calcium stones, and compared with the official dietary requirements for the Japanese.

Total protein intake, animal protein intake and animal protein ratio were significantly higher for patients with stones in both men and women. Dietary salt intake was significantly higher for male patients and the total group. Dietary calcium and carbohydrate intakes were significantly lower for patients with stones in men and the total …

Methods For The Study Of Calcium Oxalate Crystallisation And Their Application To Urolithiasis Research, J. P. Kavanagh

Scanning Microscopy

Many methods have been used to study calcium oxalate crystallisation. Most can be characterised by changes in supersaturation during the experiment, which may increase, remain constant or decay. Their ability to quantify various aspects of crystallisation often reflects the extent to which nucleation, growth and aggregation can be measured independently, when two or three of these processes may be occurring simultaneously.

The mixed suspension, mixed product removal technique reaches a steady state supersaturation, is a good model for intrarenal crystallisation and allows both growth and nucleation rates to be measured. Using 92% urine and comparing control urines with samples from …

Influence Of Ageing, Ph And Various Additives On Crystal Formation In Artificial Urine, A. L. Rodgers, M. A. E. Wandt

Scanning Microscopy

In order to investigate the effect of various factors on urinary crystallization processes, a series of five experiments was carried out using an artificial urine (AU) in a rotary evaporator. The influence of ageing, pH and organic, inorganic and potential inhibitory additives formed the basis of the study. Precipitates were characterized by X-ray powder diffraction, scanning electron microscopy and energy dispersive X-ray analysis. In the ageing experiment, AU aliquots, adjusted to various pH values, were allowed to stand for several days and were not evaporated. Calcium oxalate monohydrate (COM) was formed at low pH, while whitlockite, apatite and struvite occurred …

The Role Of Tamm-Horsfall Glycoprotein And Nephrocalcin In Calcium Oxalate Monohydrate Crystallization Processes, B. Hess

Scanning Microscopy

Theoretical considerations as well as clinical observations suggest that the aggregation of nucleated crystals is the most dangerous step in the formation of calcium oxalate (CaOx) renal stones. The effects of 2 major urinary glycoproteins, Tamm-Horsfall glycoprotein (THP) and Nephrocalcin (NC), on calcium oxalate monohydrate (COM) crystal aggregation in vitro are studied. At low ionic strength (IS) and high pH (within urinary limits), THP is a powerful crystal aggregation inhibitor (90% inhibition at 40 mg/l). Decreasing pH to 5.7 and raising IS to 0.21 increases THP viscosity, thereby lowering THP crystal aggregation inhibition. Upon addition of calcium …

Retention Of Calcium Oxalate Crystals In Renal Tubules, Saeed R. Khan, Raymond L. Hackett

Scanning Microscopy

Crystal retention within the renal tubules is essential for nephrolithiasis and the development of urinary stone disease. We studied the mechanisms involved in this process by inducing calcium oxalate crystal deposition within the rat renal tubules and examining them using various microscopic techniques. Crystals appeared to be retained either by attachment to the tubular epithelium or by aggregating with other crystals thus becoming large enough to be retained by their collective size.