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.

Peptide-carrier interaction: induction of liposome fusion and aggregation by insulin.

As the roles of peptidic agents in therapy expand, the need for gaining the knowledge for formulating peptides and/or polypeptides becomes increasingly urgent. In an attempt to study various approaches to formulating peptidic agents for therapeutic applications, we investigated the interactions between drug carriers and peptides, using liposomes and insulin as a model. The fusion and aggregation properties of dipalmitoylphosphatidylcholine (DPPC) small, unilamellar liposomes, on the binding of insulin was studied by the techniques of resonance energy transfer of fluorescent labeled lipids, electron microscopy, and right-angle scattering. Within 1 h of adding insulin to DPPC liposomes at 25 degrees C, the average size of the liposomes increased from 239 to 361 A in diameter. There was no further increase in the size of the liposomes after the fused liposomes reached this size. However, the aggregation of the fused liposomes continued to increase for several hours after the insulin-induced fusion stopped. Our results suggest that insulin induces the aggregation of newly fused liposomes, when the temperature is below the gel----liquid crystalline phase-transition temperature (Tc) of the liposomes. The aggregation of fused liposomes is markedly affected by not only the zinc content of insulin but also the pH and ionic strength of the solution. The results of the present study demonstrate that an amphyphilic molecule, such as insulin, could induce the fusion of liposomes via hydrophobic interaction and facilitate liposome aggregation via hydrophilic interaction. Thus, when entrapping insulin by small, unilamellar liposomes, care should be taken to avoid fusion and aggregation.

The zinc-reversible antimicrobial activity of neutrophil lysates and abscess fluid supernatants.

There is some evidence to suggest that microbial growth inhibition may occur in chronic abscesses. A substance perhaps responsible for this phenomenon is calprotectin, a neutrophil cytoplasmic protein that inhibits microbial growth and that belongs to a class of proteins often having specific binding sites for zinc. In the present study, the suppressive effects of either human or mouse neutrophil lysates on Candida albicans growth were found to be completely reversed by micromolar quantities of zinc but not by iron or other trace elements. Similarly, supernatants of exudates from experimental abscesses in mice or from clinical specimens of abscesses in humans markedly inhibited the proliferation of C. albicans, and this effect was also completely reversed by zinc. A protein complex characteristic of calprotectin was identified in the abscess fluids. Preparations of the neutrophil growth-inhibiting protein, containing predominantly calprotectin, were shown to have zinc-binding activity by a dialysis technique. These findings suggest that the major mechanism of C. albicans growth inhibition by abscess fluids is through competition for zinc by a cytoplasmic protein apparently released from dying neutrophils.

Antimicrobial activity of an abundant calcium-binding protein in the cytoplasm of human neutrophils.

Studies of experimental infections in animals indicate that phagocytic cells may sometimes control infective foci without actually ingesting or contacting the invading microorganisms. In the present study, an effective inhibitor of Candida albicans growth, previously detected in neutrophils cytoplasm and found to be released only after lysis of the cells, was identified as an abundant calcium-binding protein originally described in neutrophils as the L1 myelomonocytic antigen or the cystic fibrosis antigen. This substance was demonstrated also to have static activity against several other important human pathogens, including Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli. Growth of the various microorganisms was inhibited to considerably different degrees by the neutrophil protein, with the effects on S. aureus (the least responsive organism) being significantly enhance by addition of calcium to the medium. These findings suggest that after its release by the death of neutrophils at sites of tissue infection, this abundant calcium-binding protein could have a host defense function by controlling the growth of pathogenic microorganisms that escape being killed initially and would otherwise be free to proliferate.

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.

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 chemical modification of quartz surfaces on particulate-induced pulmonary inflammation and fibrosis in the mouse.

One of the critical steps in the development of crystal-induced lung diseases is thought to be the interaction of crystal surfaces with cell membranes. The effect of chemical modifications of the surface of alpha-quartz on the development of lung disease has been investigated by treating quartz with various organosilanes. The functional groups attached to the quartz surfaces were (-CN), (-CH3), (-NH2), and -(N(CH3)3+). After intratracheal injection of each modified crystal at a constant surface area into mice, pulmonary inflammation and fibrosis were assessed 6 wk postexposure to the crystals by lung wet weight (lung index) and by the level of hydroxyproline in the lung. The crystals showing the highest degree of biologic activity were native quartz, which has a negative charge, -N(CH3)3+ modified quartz, which has a positive charge, and -CN modified quartz, which has no charge. One of the crystals with chemical groups capable of hydrogen bonding, the -NH2 modified quartz, was as unreactive as the crystal preparation modified with a hydrophobic group, -CH3. If the -CH3 and -NH2 modified quartz are compared as a less reactive group with the more reactive native quartz and -N(CH3)3+ modified quartz, these experiments suggest that electrostatic interactions may be more important in determining effective biologic activities than are hydrogen bonding interactions.

Surface exposure of phosphatidylserine increases calcium oxalate crystal attachment to IMCD cells.

The development of urolithiasis is a multifaceted process, starting at urine supersaturation and ending with the formation of mature renal calculi. The retention of microcrystals by the urothelial cell membrane is a critical event in the process. The current study examines calcium oxalate monohydrate (COM) crystal attachment to inner medullary collecting duct (IMCD) cells following selective changes in cell membrane phospholipid composition. Both primary culture of IMCD cells and a continuous IMCD cell line were used for these studies. Cell membrane composition was selectively altered by either exogenous addition of membrane phospholipids or using membrane lipid scrambling agents. Enrichment with anionic phospholipids was found to greatly increase attachment of crystals to the cells. This increased attachment correlated with the exposure of phosphatidylserine (PS) on the exofacial leaflet of the cell membrane as demonstrated by the use of the membrane scrambling agent A-23187. Furthermore, the increased COM attachment following PS exposure could be blocked by incubating the cells with the PS-specific binding protein, annexin V. These results support the hypothesis that exposure of PS head groups on the papillary epithelial cell surface may mediate stone crystal attachment to the kidney tubule cell epithelium in the renal papilla, possibly as an initiating event in urolithiasis.

The dependence on membrane fluidity of calcium oxalate crystal attachment to IMCD membranes.

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.

Comparison of serum and plasma leukotriene B4 levels in normal and asthmatic subjects.

BACKGROUND: Leukotriene B4 (LTB4) serum and plasma concentrations were reported to be higher in some asthmatic patients than in normal subjects; however, reported LTB4 concentrations in normal subjects vary widely. One study suggested that blood clotting causes the increased LTB4 concentration. OBJECTIVE: To determine whether LTB4 concentration is increased in asthmatic patients, and whether it is affected by clotting. METHODS: We studied seven normal subjects and nine clinically stable asthmatic patients. Venous blood was drawn into test tubes without additives; containing heparin; or containing heparin and cyclo- and lipoxygenase inhibitors. Cells were separated after 30 minutes. Leukotriene B4 was measured by radioimmunoassay following its extraction from serum or plasma. In three subjects, plasma was separated also at times 0 through 30 minutes. RESULTS: Serum and plasma concentrations of LTB4 in normal volunteers and asthmatic patients were similar, but the variance of LTB4 concentrations among the asthmatic patients was significantly higher than in the normal subjects. Leukotriene B4 concentrations, measured in plasma only, were significantly reduced in both asthmatic and nonasthmatic subjects in the presence of inhibitors. There was no significant difference in LTB4 concentrations between time 0 and 30 minutes, but there was considerable variability. CONCLUSIONS: We conclude that clotting is unlikely to affect serum LTB4 concentrations. Leukotriene B4 serum and plasma concentrations are not consistently increased in asthmatic patients; however, LTB4 is synthesized during and possibly after blood has been drawn. Proper handling of the specimens and probably the addition of cyclo-oxygenase and lipoxygenase inhibitors is of the utmost importance for accurate LTB4 determination.

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.

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.

Oxalate-induced exposure of phosphatidylserine on the surface of renal epithelial cells in culture.

A molecular mechanism of crystal attachment to renal cells after injury has been proposed in which the exposure of phosphatidylserine (PS) on the cell membrane surface following injury provides attachment sites for calcium-containing crystals. Annexin V was used to determine whether injury to kidney cells by oxalate in culture resulted in PS exposure on the cell surface. When continuous cultures of intermedullary collecting duct cells were exposed to various levels of oxalate, a dose-dependent increase in PS exposure was observed on the cell surfaces. Initially, only scattered cells expressed PS on the surface. However, as the level of oxalate increased, groups of cells began to express PS, suggesting that the injured cells may have an influence on neighboring cells. Exposure of PS on the cell membrane surface correlated with a corresponding increase in calcium oxalate monohydrate crystal attachment to the cells. This indicates that damage to kidney epithelial cells by elevated concentrations of urinary components, in this case oxalate, could result in exposure of PS on cells, which could provide a point of fixation or nucleation for calcium-containing crystals.

Uric acid crystal binding to renal inner medullary collecting duct cells in primary culture.

Attachment of microcrystals to cellular membranes may be an important component in the pathophysiology of urolithiasis. This study characterizes the concentration-dependent binding of uric acid crystals to rat renal inner medullary collecting duct cells in primary culture. Collecting duct cell cultures grew as monolayers with interspersed aggregates of rounded cells. Cultures were incubated with 14C-uric acid crystals, and the crystals that bound were quantitated by adherent radioactivity. Uric acid crystal adherence demonstrated concentration dependent saturation with a 1/alpha value (maximum micrograms of crystals adhering to 1 cm2 of binding area) of 645 micrograms/cm2. The beta values (fraction of cross-sectional area which bound crystals) of uric acid (mean = 0.15) and calcium oxalate monohydrate (mean = 0.13) crystals did not differ significantly. Uric acid crystal binding was inhibited by pre-bound calcium oxalate monohydrate crystals in a concentration dependent manner. These data suggest that uric acid and calcium oxalate crystals exhibit similar binding patterns to rat renal inner medullary collecting duct cells in primary culture.

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 effect of crystal structure on mouse lung inflammation and fibrosis.

In order to identify the physical and structural parameters that relate best to the membranolytic, inflammatory, and fibrotic potentials of different silicon dioxide (SiO2) and titanium dioxide (TiO2) crystals, we have studied the potential of four different SiO2 and two different TiO2 crystal structures to lyse human red blood cells and to induce pulmonary inflammation and fibrosis in mice. The crystals studied were quartz, tridymite, cristobalite, coesite, anatase, and rutile. Mice were injected intratracheally with each crystal at constant surface area. Inflammation and fibrosis were assessed 6 wk after crystal instillation by wet lung weight (lung index), protein concentration of lung lavage fluid, the level of hydroxyproline in the lung, and histologic examination. In vitro red blood cell (RBC) lysis was evaluated by incubating the crystals with 51Cr-labeled RBC and measuring the release of 51Cr into the medium. Known crystallographic data for each of the minerals were used to calculate the percent occupied volume. Biologic activity seemed to correlate with percent occupied volume, suggesting that surface molecular topology may be important in crystal-cell interactions. The crystals with more irregular surfaces and protruding oxygen atoms, which form surface pockets (quartz, tridymite, and cristobalite), showed a dramatic increase over saline controls for lung index (greater than 2 x), cell number and lavage protein concentration (greater than 4 x), and hydroxyproline level (greater than 2 x). The other more boxlike crystals (coesite, anatase, and rutile) displayed little change in these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

L-arginine uptake and metabolism by lung macrophages and neutrophils following intratracheal instillation of silica in vivo.

Nitric oxide (NO) has been associated with lung inflammation following exposure to silica. L-arginine can be converted to NO and L-citrulline by nitric oxide synthase (NOS), or into urea and L-ornithine by arginase. We tested the hypothesis that after instillation of silica into rat lungs in vivo, lung inflammatory cells increase L-arginine metabolism by both NOS and arginase, which is associated with an increase in L-arginine uptake. We isolated lung inflammatory cells 3 d after silica or saline (control) exposure. The uptake of [3H]L-arginine at 24 h by cells from silica-exposed lungs (73.9 +/- 4.8%) was significantly greater than uptake by control cells (24.7 +/- 2.2%; P < 0.05) and was a saturable process. The greater [3H]L-arginine uptake by cells from silica-exposed lungs was associated with greater NO and urea production than by control cells. The uptake of [3H]L-arginine by cells from control or silica-exposed lungs was blocked in a dose-dependent manner by L-ornithine (an inhibitor of L-arginine transport) and by Nomega-nitro-L-arginine methyl ester (L-NAME) (an NOS inhibitor), but not by L-valine (an arginase inhibitor). The production of NO by cells from silica-exposed lungs was completely blocked by L-NAME. The addition of L-arginine to media resulted in dose-dependent production of NO and urea. The results show that lung inflammatory cells increase L-arginine uptake and metabolism by both NOS and arginase following in vivo silica exposure. The increase in L-arginine uptake may represent a mechanism to maintain an intracellular supply of this amino acid. NO can react to generate peroxynitrite, a potential mediator of lung injury following silica exposure.

Mechanisms of calcium oxalate crystal attachment to injured renal collecting duct cells.

BACKGROUND: Renal cell or tissue injury results in a loss of membrane lipid asymmetry and/or loss of cell polarity, and both events lead to changes on the surface of the cell membranes that enhance crystal attachment. We have proposed two distinct mechanisms of crystal attachment following membrane changes induced by various modes of injury. METHODS: Annexin V was used to determine whether phosphatidylserine (PS) exposure on the cell membrane surface plays a role in calcium oxalate monohydrate (COM) crystal attachment to cells that have lost their polarity as well as to cells that have lost their lipid asymmetry. We utilized two different experimental models of injury to renal epithelial cells in culture. The first model used calcium ionophore A23187 to induce a loss of lipid asymmetry, and the second model used EGTA to break down tight junctions and lose cell polarity. RESULTS: Inner medullary collecting duct cells that have lost lipid asymmetry demonstrated an increase in the number of cells that bound annexin V. However, when cells lost their polarity, they did not bind annexin V. In addition, the attachment of crystals to cells following a loss of cell polarity was not inhibited by annexin V. CONCLUSIONS: This study indicates that both individual cell injury (loss of lipid asymmetry) and generalized cell monolayer injury (loss of cell polarity) result in the presentation of different cell surfaces and that both forms of injury result in an increased affinity for crystal attachment. Both mechanisms could be important independently or collectively in the retention of microcrystals to renal collecting duct cells in urolithiasis.

Specificity in calcium oxalate adherence to papillary epithelial cells in cultures.

Attachment of microcrystallites to cellular membranes may be an important component of the pathophysiology of many diseases including urolithiasis. This study attempts to characterize the interaction of calcium oxalate (CaOx) crystals and apatite (AP) crystals with renal papillary collecting tubule (RPCT) cells in primary culture. Primary cultures of RPCT cells showed the characteristic monolayer growth with sporadically interspersed clumped cells. Cultures were incubated with [14C]CaOx crystals, and the crystals that bound were quantified by microscopy and adherent radioactivity. Per unit of cross-sectional area, 32 times more CaOx crystals were bound to the clumps than to the monolayer. CaOx adherence demonstrated concentration-dependent saturation with a beta value (fraction of cell culture area binding CaOx crystals) of 0.179 and a 1/alpha ox value (maximum micrograms of crystallites adhering to 1 cm2 of binding area) of 287 micrograms/cm2. On coincubation with AP crystals, CaOx binding demonstrated concentration-dependent inhibition with a 1/alpha AP value of 93 micrograms/cm2. Microcrystallite adherence to RPCT cells demonstrates selectivity for cellular clumps, saturation, and inhibition. These features suggest specific binding.