John H. Wiessner

Study of a rat model for calcium oxalate crystal formation without severe renal damage in selected conditions

Abstract  Background:  Although nephrotoxic in high doses, ethylene glycol (EG) has been used with ammonium chloride (NH4Cl) or vitamin D3 to study calcium oxalate stone formation in rat models. In the present study we used EG alone or with NH4Cl to study hyperoxaluria, crystaluria, and crystal attachment to renal epithelial cells in rats with minimal renal damage.

Calcium oxalate-crystal membrane interactions: dependence on membrane lipid composition.

PURPOSE Urolithiasis is clearly a multifaceted process, progressing from urine supersaturation to the formation of mature renal calculi. Retention of microcrystals by the urothelium is a critical event in stone maturation. Membrane phospholipids appear to be involved in the attachment of stone crystals to kidney epithelium. MATERIALS AND METHODS The current study quantitates crystal-membrane interactions following selective changes in the red blood cell (RBC) membrane phospholipid composition by using a crystal-induced membranolytic assay. RESULTS Membrane enrichment with anionic phospholipids was found to greatly increase crystal-membrane interactions. Crystal-membrane interaction was associated with an increase in the negative charge on the RBC membrane surface. CONCLUSIONS Specific membrane compositions seem to facilitate the formation of crystal attachment region on the RBC surface that is necessary for effective crystal attachment to the cell membrane.

Membrane Interactions with Calcium Oxalate Crystals: Variation in Hemolytic Potentials with Crystal Morphology

Crystal-induced membranolysis of human red blood cells has been quantitated for calcium oxalate monohydrate and calcium oxalate dihydrate crystals. Calcium oxalate monohydrate crystals are significantly more membranolytic than calcium oxalate dihydrate crystals at constant surface area. If the crystal morphology of calcium oxalate monohydrate is altered by grinding, the lytic potential at constant surface area is markedly reduced. However, altered calcium oxalate dihydrate crystals are as lytic as natural calcium oxalate dihydrate crystals at constant surface area. Differences in the calcium oxalate monohydrate and dihydrate crystal structures, specifically the structural characteristics of the disordered water channel in calcium oxalate dihydrate, can explain these different membranolytic characteristics.

Regulation of pH in rat papillary tubule cells in primary culture.

To investigate the mechanisms responsible for urinary acidification in the terminal nephron, primary cultures of cells isolated from the renal papilla were grown as monolayers in a defined medium. Morphologically, cultured cells were epithelial in type, and similar to collecting duct principal cells. Cell pH measured fluorometrically in monolayers grown on glass slides showed recovery from acid loads in Na+-free media. Recovery was inhibited by cyanide, oligomycin A, and N-ethylmaleimide. Cyanide and oligomycin inhibited recovery less in the presence than in the absence of glucose. When cells were first acid loaded in a Na+-free medium and then exposed to external Na+, pH recovery also took place. This recovery exhibited first-order dependence on Na+ concentration and was inhibited by 5-(N-ethyl-N-isopropyl)amiloride. These studies demonstrate that in culture, collecting duct principal cells possess at least two mechanisms for acid extrusion: a proton ATP-ase and an Na+-H+ exchanger. The former may be responsible for some component of the urinary acidification observed in the papillary collecting duct in vivo; the role of the latter in acid-base transport remains uncertain.

CALCIUM OXALATE CRYSTAL ATTACHMENT TO CULTURED KIDNEY EPITHELIAL CELL LINES

PURPOSE Cultured kidney epithelial cell lines have frequently been used in urolithiasis research, and in particular in studies related to the interactions between stone crystals and cell membranes. There is evidence that when epithelial cell lines are transformed or serially passed to immortalize them, they experience changes in both cell physiology and morphology. Stone research utilizing cell cultures is frequently necessary due to the lack of an animal model for spontaneous stone disease. However, the interpretation of these cell culture research studies might be clouded by any significant differences in cell physiology between primary cells and continuous cell cultures. Therefore, the present study was conducted to compare calcium oxalate monohydrate (COM) crystal attachment to two primary kidney epithelial cell lines and to various continuous cell lines. MATERIALS AND METHODS The cell lines surveyed were primary mouse proximal tubule cells (pMPT), primary inner medullary collecting duct cells (pIMCD), semi-continuous inner medullary collecting duct cells (cIMCD), BSC-1 cells, COS-1 cells, LLC-PK1 cells, MDCK cells, NRK-52E cells, and OK cells. All cell lines were cultured under identical conditions and the amount of COM attachment was measured using radioactive labeled COM crystals. RESULTS COM crystal interaction with continuous kidney epithelial cells varied by a factor of two among the different cell lines. In general, cells that grew as regular, confluent cell monolayers, such as pMPT, pIMCD and cIMCD cells, exhibited the lowest levels of crystal attachment. Neither changes in membrane fluidity nor loss of normal epithelial cell membrane asymmetry seemed to correlate well with crystal attachment. After nine days of continuous cell culture, COM attachment to cIMCD cells dropped by 61 percent while crystal attachment to MDCK cells remained unchanged. It is unclear what makes these cell lines more resistant to crystal attachment compared to continuous cell lines. CONCLUSIONS The significant difference in COM attachment between primary kidney epithelial cells and continuous epithelial cell cultures and the apparent differences in growth morphology between primary and continuous cell cultures must be considered when selecting a cell line for use in kidney stone research. Comparison of cIMCD cells and MDCK cells during extended culture time revealed one possible explanation for the differences in COM attachment: the formation of a mature, end-differentiated, non-dividing cell monolayer could protect the cells from crystal attachment.

Calcium Oxalate Crystal Interaction with Rat Renal Inner Papillary Collecting Tubule Cells

Rat renal inner papillary collecting tubule cells (RPCT) have been isolated and maintained in primary culture. The cells have been found to be of only one type and they have maintained the characteristics of RPCT cells. The RPCT cells in culture appear as a monolayer with intermittent clumps of rounded cells. When small calcium oxalate monohydrate crystals (COM) or calcium oxalate dihydrate crystals (COD) are added to the monolayer of RPCT cells, the crystals bind on or about these clumps of rounded-up cells. The use of this system as a model for the study of crystal membrane interactions in crystalluria and urolithiasis is discussed.

The Dependence on Membrane Fluidity of Calcium Oxalate Crystal Attachment to IMCD Membranes

Abstract. The development of urolithiasis is a multifaceted process, starting with urine supersaturation and ending with the formation of mature renal calculi. The retention of microcrystals by kidney tubule epithelium cell membranes has been proposed as a critical event in the process. To date, attachment of kidney stone constituent crystals to urothelial cells has been demonstrated both in vitro and in vivo yet the mechanism of crystal attachment remains unknown. We hypothesize that for effective stone crystal attachment to the epithelium there must be cell membrane rearrangement that would allow for long-range bonding between the stone crystal and the cell membrane. This rearrangement may be influenced by the physical state of the membrane. The current study examines calcium oxalate monohydrate (COM) crystal attachment to inner medullary collecting duct (IMCD) cells following changes in cell membrane fluidity. Radioactively labeled COM crystals were used to quantitate crystal attachment. Membrane fluidity was altered by changing temperature, cell membrane cholesterol content, or extended length of cell culture. Crystal attachment to IMCD cells was directly correlated to changes in membrane fluidity. This finding was consistently observed regardless of the method used to alter membrane fluidity. The results are consistent with the theory that the ability to form a crystal attachment region on the cell surface may be related to the ease of rearrangement of membrane components at the cell surface. Variations in the urothelial cell environment during certain pathological conditions in the kidney could induce these physical perturbations and prime kidney epithelial cells at or near the papillary tip to bind COM crystals.

Effect of Particle Size on Quartz-Induced Hemolysis and on Lung Inflammation and Fibrosis

To assess the role of crystal size in biologic responses, we quantitated red blood cell lysis and lung inflammation and fibrosis in the mouse using 4 alpha-quartz preparations with average diameters of 1, 5, 7.8, and 11.2 microns. When compared on the basis of identical crystal surface areas, the 1-micron fraction was more hemolytic than the other 3 fractions. The three larger fractions had equivalent membranolytic activities. After 6 weeks of postintratracheal instillation of the crystals into mice, the 1-micron-diameter crystal fraction increased wet lung weights by 1.25 x that of saline controls, while a 1.75 x increase was found for the three larger crystal fractions. A similar response was found when evaluating fibrosis development by determining lung hydroxyproline levels. Measurement of the percentage of the crystal dose remaining in the lungs revealed that the biologic differences observed were not due to a difference in the clearance of the smaller crystal fraction. Thus, larger crystals of alpha-quartz produce a greater degree of inflammation and fibrosis when instilled into the lung than those of 1 micron diameter, even though the smaller crystals are more membranolytic in vitro and appear to be cleared from the lung at the same rate as the larger crystals.

The effect of hydroxyapatite crystallinity on hemolysis.

SummaryCrystalline hydroxyapatite is a component of bone, teeth, and numerous pathological calcifications. The apatite crystal structure can accommodate a wide variety of atomic substitutions which gives apatite crystals an unusually high degree of variability in biochemical and physical properties. Apatite crystallites interact with numerous cellular systemsin vivo, and some of these interactions may lead to altered cellular function. One measure of crystal-membrane interactions is crystal-induced membranolysis of human red blood cells. Hemolytic potentials at constant crystal surface areas were measured at 1, 2, and 4 hours for 29 different preparations of apatite. Each apatite sample was characterized by its morphology, particle size, % CO3, zeta potential, and broadening of the (211), (112), (300), (202), and (002) diffraction maxima. Only the surface area/g and the X-ray powder diffraction line broadening showed a significant inverse correlation with hemolytic potential. These parameters were related to each other, and are indications of the degree of crystallinity.

Calcium oxalate monohydrate crystal binding substance produced from Madin-Darby canine kidney cells

Abstract Background: The interaction between kidney urothelium and crystals is a critical event in the growth of renal calculi. When studying calcium oxalate monohydrate (COM) crystal binding to Madin‐Darby canine kidney (MDCK) cells in culture, we observed that crystals also attached to areas on the coverslips devoid of cells. This phenomenon could be the result of substances produced by the cells that adhere to the glass and subsequently bind COM crystals. We investigated the characteristics of this COM binding substance.