Adhesion, internalization and metabolism of calcium oxalate monohydrate crystals by renal epithelial cells (original) (raw)
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Renal epithelial cells rapidly bind and internalize calcium oxalate monohydrate crystals
Proceedings of the National Academy of Sciences of the United States of America, 1994
Renal tubular fluid is supersaturated with calcium and oxalate ions, which can nucleate to form crystals of calcium oxalate monohydrate (COM), the most abundant constituent of kidney stones. However, the mechanisms by which nascent crystals are retained in the nephron and then grow into kidney stones are unclear. An interaction of COM crystals with the surface of renal epithelial cells could be a critical initiating event in nephrolithiasis. To investigate this possibility we used cultures of monkey kidney epithelial cells (BSC-1 line) as a model system and found that [14C]COM crystals bound to the cell surface within seconds. Scanning electron microscopy revealed that crystals bind first to apical microvilli, which subsequently migrate over the crystalline surface. When visualized by transmission electron microscopy, intracellular crystals were located within vesicles. Cytoskeletal responses to crystal uptake were sought by immunofluorescence microscopy, which revealed concentratio...
Modulation of Proliferating Renal Epithelial Cell Affinity for Calcium Oxalate Monohydrate Crystals
Journal of the American Society of Nephrology, 2004
Adhesion of urinary crystals to distal tubular cells could be a critical event that triggers a cascade of responses ending in kidney stone formation. Monolayer cultures of distal nephronderived MDCKI cells were used as a model to study crystal-cell interactions. COM crystal adhesion reached a peak 2 d after plating and progressively fell thereafter. The decline in crystal binding was accelerated by prostaglandin E 2 (PGE 2) supplementation and delayed by blockade of PG production. Crystals avidly adhered to cells that migrated in to repair a scrape wound made in the monolayer and after a transient hypoglycemic insult. Exposure of MDCKI cells to uric acid crystals and soluble uric acid was also associated with increased crystal adhesion. Treatment of physically or hypoglycemically injured cells with trypsin or neuraminidase reduced crystal binding to baseline levels, suggesting that increased exposure of cell surface glycoproteins mediated the Materials and Methods Cell Culture Renal epithelial cells of the MDCK line, type I, were a gift of Carl
Calcium Oxalate Crystal Attachment to Cultured Rat Kidney Epithelial Cell, NRK-52E
Urologia Internationalis, 2001
Madin-Darby canine kidney (MDCK) cells have been used in research on crystal adhesion to epithelial cells. Recently, matrix proteins were identified, and studies of the genes and proteins expressed in renal epithelial cells have become active. The present study confirms the usefulness of the NRK-52E cell line, derived from the rat, in the study of attachment with calcium oxalate crystals. The calcium oxalate crystal suspension was distributed on top of the cells. After incubation, the monolayers were rinsed to remove non-associated crystals. After fixation, the association of crystals and NRK-52E cells was visualized using scanning electron microscopy. Calcium oxalate crystals were attached to the surface of NRK-52E cells. Under high magnification, many of the microvilli of the cells had elongated towards the crystals, and microvilli projections appeared to catch the crystals. The NRK-52E cell line is useful in the study of attachment between crystals and urinary epithelial cells in the kidney, especially for the regulation and analysis of genes and proteins.
Journal of the American Society of Nephrology, 2005
Renal tubular fluid in the distal nephron of the kidney is supersaturated with calcium oxalate (CaOx), which crystallizes in the tubules as either calcium oxalate monohydrate (COM) or calcium oxalate dihydrate (COD). Kidney stones are aggregates, most commonly containing microcrystals of COM as the primary inorganic constituent. Stones also contain small amounts of embedded proteins, which are thought to play an adhesive role in these aggregates, and they often are found attached to the tip of renal papilla, presumably through adhesive contacts. Voided urine, however, often contains COD in the form of single micron-sized crystals. This suggests that COD formation protects against stone disease because of its reduced capacity to form stable aggregates and strong adhesion contacts to renal epithelial cells. Using atomic force microscopy configured with tips modified with biologically relevant functional groups, we have compared the adhesion strengths of the morphologically important faces of COM and COD. These measurements provide direct experimental evidence, at the near molecular level, for poorer adhesion at COD crystal faces, which explains the benign character of COD and has implications for resolving one of the mysteries of kidney stone formation.
Morphology of crystals in calcium oxalate monohydrate kidney stones
Urological Research, 2007
Both scanning electron microscopy and atomic force microscopy (AFM) have shown that calcium oxalate monohydrate kidney stones are made up from arrangements of sub micron crystals. The purpose of this investigation was to determine the morphology of these crystals which was obscured by the presence of organic matrix in our earlier study. Sections of stones were treated to remove the protein component of the matrix and then imaged using AFM. Images obtained after proteolysis show that the crystals are in the form of plates stacked on (100) surfaces. These results were confirmed by scanning electron microscopy observations from selected regions of calcium oxalate kidney stone surfaces. The observed crystal sizes are consistent with both the known matrix mass fraction and crystallite growth in the passage through the collecting duct.
Journal of Structural …, 2001
The external appearance of urinary calcium oxalate (CaOx) crystals suggests that they are solid, homogeneous structures, despite their known association with proteins. Our aim was to determine whether proteins comprising the organic matrix of CaOx crystals are superficial or intracrystalline in order to clarify the role of urinary proteins in the formation of kidney stones. CaOx crystals were precipitated from centrifuged and filtered, or ultrafiltered, healthy human urine. They were then treated with dilute NaOH to remove bound proteins, partially demineralized with EDTA, or fractured and subjected to limited proteolysis before examination by low-resolution scanning electron microscopy or field emission scanning electron microscopy. Crystals precipitated from centrifuged and filtered urine had a complex interior network of protein distributed throughout the mineral phase, which appeared to comprise closely packed subcrystalline particles stacked in an orderly array among an amorphous organic matrix. This ultrastructure was not evident in crystals deposited in the absence of macromolecules, which were completely solid. This is the first direct evidence that crystals generated from cell-free systems contain significant amounts of protein distributed throughout a complex internal cribriform ultrastructure. Combined with mineral erosion in the acidic lysosomal environment, proteins inside CaOx crystals would render them susceptible to attack by urinary and intracellular renal proteases and facilitate their further dissolution or disruption into small particles and ions for removal by exocytosis. The findings also have broader ramifications for industry and the materials sciences, as well as the development and resorp-tion of crystals in biomineralization systems throughout nature.
Kidney International, 2001
Intratubular crystallization of calcium oxalate in the presence Exactly where and how urinary stones originate are of membrane vesicles: An in vitro study. still unclear. Most urinary stones are located in the Background. Since urine spends only a few minutes in the kidneys. Some are seen attached to the renal papillae. renal tubules and has a low supersaturation with respect to Others demonstrate signs of earlier attachment to the calcium oxalate (CaOx), nucleation of CaOx crystals in the kidneys, such as remains of renal tubules in small depreskidneys is most probably heterogeneous. We have proposed sions on stone surface [1]. Kidneys of many stone patients that membranes of cellular degradation products are the main substrate for crystal nucleation. The purpose of our study was contain subepithelial plaques on their papillae [2]. These to determine the site of membrane-mediated crystal nucleation plaques are suggested to be the sites of stone developwithin the renal tubules and the required lag time, factors ment. Obviously, for a stone to form, crystallization must that determine whether crystallization results in crystalluria or occur, and crystals must be retained in the kidneys. Since nephrolithiasis. urine spends only three to five minutes in the renal tu-Methods. Nucleation of CaOx was allowed to occur in five different artificial urine solutions with ionic concentrations sim-bules and is generally undersaturated for CaOx before ulating urine in proximal tubules (PTs), descending (DLH) reaching the collecting ducts (CDs), it is suggested that and ascending (ALH) limbs of the loop of Henle, distal tubules nucleation of CaOx crystals within the renal tubules is (DTs), and collecting ducts (CDs). A constant composition most probably heterogeneous [3]. Investigations of the crystallization system was used. Experiments were run for two ionic conditions within different segments of the nephron hours with or without the renal tubular brush border membrane and application of the data to in vitro studies have shown (BBM) vesicles. Results. The addition of BBM significantly reduced the nuthat urine of the loop of Henle can support calcium cleation lag time and increased the rate of crystallization. The phosphate (CaP) nucleation [4-10]. It was proposed that average nucleation lag time decreased from 84.6 Ϯ 43.4 minutes CaP crystals formed in the loops could promote nucleto 24.5 Ϯ 19 minutes in PTs, from 143.6 Ϯ 29 to 70.2 Ϯ 53.4 ation of CaOx further along the nephron in the CDs. minutes in DLH, from 17.6 Ϯ 8.6 minutes to 0.625 Ϯ 0.65 min-The results of most in vitro crystallization studies showed utes in DTs and from 9.54 Ϯ 3.03 minutes to 0.625 Ϯ 0.65 that it took hours for the precipitation of CaP in solutions minutes in CDs. There was no nucleation in the ALH solution without BBM for two hours. CaOx dihydrate (COD) was comsimulating urine in the loop; however, urine spends only mon in most solutions. Calcium phosphate (CaP) also nucleminutes in the tubules and seconds in various segments ated in the DLH and CD solutions. [7, 8]. Since crystallization must occur in moving urine, Conclusions. In the absence of membrane vesicles, there studies in one laboratory utilized a dynamic crystallizawas no crystallization in any of the solutions within the time tion system [7, 8]. The solution composition simulated urine spends in the renal tubules. As a result, homogeneous changing conditions existing in the proximal tubule (PT), nucleation of crystals anywhere within the nephron appears unlikely. However, BBM-supported nucleation is possible in the descending limb of the loop of Henle (DLH), the the DTs as well as CDs. A high crystallization rate in CDs ascending limb of the loop of Henle (ALH), the distal would promote rapid crystal growth and aggregation, resulting tubule (DT), and finally to the conditions existing in the in crystal retention within the kidneys and development of CD. When the solution conditions became similar to nephrolithiasis. those in the DLH, precipitation of CaP required less than three seconds. The CaP precipitate, however, began to dissolve in the ascending limb conditions (ALH) and