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.

Operation of long-range substituent effects in rigid opiates: protonated and unprotonated oxymorphone.

The structure of protonated oxymorphone (amine salt) was determined by an X-ray crystallographic study. Significant differences were found with the previously determined structure of unprotonated oxymorphone (free base). Upon protonation on nitrogen, an elongation of the N-C bound occurred, accompanied by subtle changes in bond lengths and angles distant from the site of protonation. These changes in geometry are interpreted as a reflection of long-range substituent effects.

Urinary tract stone disease in the United States veteran population. II. Geographical analysis of variations in composition.

The geographical distribution of crystalline components observed in urinary tract stones in the continental United States has been studied in the United States veteran population. Since the veteran population is at risk for urolithiasis the National Veterans Administration Crystal Identification Center was established in 1983 for the characterization of all crystal-containing veteran patient samples using high resolution x-ray powder diffraction. The geographical distribution of whewellite, weddelite, apatite, brushite, struvite, uric acid and uric acid dihydrate is presented. The percentage occurrence of the crystalline components, percentage occurrence of admixed stones and geographical distribution of the number of components in admixed stones also are presented. The data highlight that although the southeastern United States has the highest patient discharge rate for stones, this high discharge rate appears to be correlated specifically with a high discharge rate for calcium oxalate stones and not with a high discharge rate for any of the other common stone components.

Urinary tract stone disease in the United States veteran population. I. Geographical frequency of occurrence.

The geographical distribution of hospitalization for urinary tract stone disease in the continental United States has been studied in the United States veteran population. The current study has been facilitated by the availability of a centralized computer data base containing the International Classification of Disease Codes for all hospital discharges at all Veterans Administration medical facilities. These data have allowed for an accurate mapping of the hospital discharge rate for urinary tract stone disease (stone discharge rate) in a population at risk for urolithiasis. Stone discharge rate data have been compared to those from the 2 previous studies conducted in general hospitals in 1952 and 1974. The stone discharge rate was 7.9 +/- 3.4 in 1952, 9.97 +/- 2.82 in 1974 and 7.58 +/- 2.01 in our study. These data indicate that the urinary tract stone discharge rate has not markedly varied during the last 34 years and also that the southeastern states still evidence the highest hospital discharge rate for urinary tract stone disease.

A model for human calcium pyrophosphate crystal deposition disease: crystallization kinetics in a gelatin matrix.

A model for the deposition of calcium pyrophosphate dihydrate (CPPD) crystals in cartilage observed in human CPPD crystal deposition disease has been developed using diffusion of calcium and pyrophosphate ions through a denatured collagen matrix environment at physiologic pH. This model system uses biological grade gelatin and has allowed for the study of crystal deposition over a wide range of calcium and pyrophosphate concentrations, including physiologic levels. The model has reproducibly formed the two crystallographic dimorphs observed clinically: triclinic and monoclinic calcium pyrophosphate dihydrate. In addition, amorphous calcium pyrophosphate has been identified, and is the first species to form in the crystallization process and transforms to orthorhombic calcium pyrophosphate tetrahydrate. This in turn dissolves with a very localized increase in available pyrophosphate leading to the formation of triclinic and monoclinic calcium pyrophosphate dihydrate. The denatured collagen matrix has allowed for the formation of the two in vivo crystals at pyrophosphate concentrations lower than previously reported in solution studies.

Calcium pyrophosphate crystal deposition: the effect of soluble iron in a kinetic study using a gelatin matrix model.

The kinetics of calcium pyrophosphate dihydrate (CPPD) crystal growth was studied by allowing calcium and pyrophosphate (PPi-4) ions to diffuse through a denatured collagen matrix (biological grade gelatin) in the presence of either ferric or ferrous ions. Ferric and, to some extent, ferrous ions blocked the migration of the PPi-4 diffusion gradient. This retardation in the [PPi-4] gradient led to numerous changes in the patterns of CPPD crystal formation. At the initial stages of crystal growth, the iron ions induced more crystal growth compared to control. At later incubation times, ferrous and ferric ions enhanced crystal growth at the expense of crystal nucleation. The presence of both ferrous and ferric ions resulted in the more rapid formation of the two crystals observed in vivo, triclinic CPPD and monoclinic CPPD. Further, both ferrous and ferric ions also reduced the solubility of the crystalline material in the broad diffuse band which formed when the Ca+2 and PPi-4 gradients first met. In this system, the presence of either ferrous or ferric ions increased the amount of hydroxyproline included in the crystalline precipitates. Iron was also incorporated into the crystals, particularly into the triclinic CPPD and monoclinic CPPD crystals.

Calcium pyrophosphate crystal deposition: the effect of monosodium urate and apatite crystals in a kinetic study using a gelatin matrix model.

The kinetics of calcium pyrophosphate dihydrate (CPPD) crystal growth was studied by allowing calcium and pyrophosphate (PPi-4) ions to diffuse through a denatured collagen matrix (biological grade gelatin) in the presence of either monosodium urate monohydrate (MSU) or hydroxyapatite (HA) crystals. In this in vitro model system, MSU crystals significantly altered the kinetics of PPi-4 ionic diffusion through the gelatin matrix by allowing the [PPi-4] gradient to fall off much more rapidly, suggesting an increased level of scavenging of PPi-4 ions into crystalline materials. Even more significantly, the presence of MSU crystals markedly influenced the crystal growth morphology of triclinic CPPD, producing that observed in vivo. A large number of epitaxially dimensional matches between MSU and triclinic (t) and monoclinic (m) CPPD were identified, suggesting that MSU crystals can epitaxially induce CPPD crystal growth. This finding supports the hypothesis that the association of urate gout and CPPD crystal deposition disease is based on the nucleating potential of MSU crystals for CPPD crystal growth. In contrast, the HA crystal structure did not appear to serve as a nucleating agent for CPPD crystals. However, HA crystals did serve as effective traps for PPi-4 ions and their presence led to more stable CPPD crystal growth.

The effect of some urinary stone inhibitors on membrane interaction potentials of stone crystals.

The effect of stone growth inhibitors (citrate, pyrophosphate, ethane diphosphonate, methane diphosphonate, chondroitin sulfate A, chondroitin sulfate C, heparin and ribonucleic acid) on crystal-membrane interactions of whewellite, weddellite, apatite, brushite, struvite, uric acid, monosodium urate and quartz (control) stones was quantitated. As a model for the initial retention of microcrystals by kidney epithelial membranes, crystal-induced membranolysis of red blood cells served as a measure of crystal-membrane interactions. The inhibitors induced changes in hemolytic potential from approximately 320 per cent enhancement to 80 per cent inhibition. No inhibitor behaved the same way for all crystals studied. However, some crystals showed consistent trends in altered hemolytic potential in the presence of inhibitors. These crystals included weddellite and sodium urate, which were inhibited consistently, and apatite and quartz, which were enhanced consistently. Whewellite, uric acid, brushite and struvite exhibited mixed patterns in the altered hemolytic potentials owing to the inhibitors.

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.

Calcium pyrophosphate crystal deposition: a kinetic study using a type I collagen gel model.

Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is characterized by deposits of triclinic (t) and monoclinic (m) CPPD crystals in articular and fibrocartilage. Many investigators have attempted to model CPPD crystal growth using both solution and a variety of gel systems. We have investigated the effect of type I collagen fibrils on CPPD crystal nucleation and growth using an ionic diffusion model. Collagen was isolated from porcine menisci using a pepsin solubilization procedure and gelled in three layers, with one containing 10 mM pyrophosphate (PPi) plus physiologic ions, the middle containing only the ions, while the third contained 25 mM Ca plus physiologic ions. Initially, amorphorous calcium pyrophosphate formed at the Ca-PPi interface. Monoclinic CPPD crystallized in 6 weeks when the [Ca] was between 2 and 3 mM and the [PPi] was between 50 and 75 microM. At 13 weeks, t-CPPD formed when the [Ca] was also between 2 and 3 mM, but the PPi was less than 25 microM. One of the most striking differences between this system and all previous solution and gel model systems is the total absence of orthorhombic calcium pyrophosphate tetrahydrate (o-CPPT) from the gels made of collagen fibrils in near native conformation. Further, crystals of t-CPPD appear as large single crystals with the classic prismatic growth habit observed in vivo, and crystals of m-CPPD also evidence the in vivo rod habit. In contrast, the crystal growth habits of t-CPPD, m-CPPD, and o-CPPT grown in all of the other model systems never matched that observed in vivo. When compared to the previous studies, these results, particularly the crystal growth habit data, suggest that the native collagen fibrils themselves can nucleate CPPD crystal formation.

The role of complement in experimental silicosis.

The role of the complement system in the pathogenesis of crystal-induced pulmonary inflammation and fibrosis was evaluated using a mouse model of silicosis and congenitally complement-deficient mice. Mice lacking the fifth component of complement (B10.D2/o) were compared to C5-sufficient animals (B10.D2/n) for pulmonary changes following intratracheal instillation of silica crystals. Complement-deficient mice demonstrated a significant reduction compared to complement-sufficient mice in both cell number and protein content of lung lavage fluid throughout the 12 weeks following silica exposure. Lung hydroxyproline content (indicative of collagen deposition) was equivalent for both strains and significantly higher than controls at all time points following silica instillation. Moreover, studies in vitro have shown that silica crystals are capable of activating complement via the alternative pathway. These studies indicate that the complement system may be responsible for some of the pulmonary inflammation, but not fibrosis elicited by silica exposure.

Kinetics of inflammatory and fibrotic pulmonary changes in a murine model of silicosis.

A murine model of experimental silicosis has been developed after the intratracheal injection of alpha-quartz crystals. Pulmonary inflammation was monitored by increases in wet lung weight and cell number and protein content of the lung lavage fluid; fibrosis was assessed by measuring increases in hydroxyproline content of the lungs. Acute pulmonary cellular inflammation occurred between weeks 1 and 2, followed by a chronic inflammatory response at week 12. Lung hydroxyproline content, an indication of collagen deposition, was initiated as early as 1 week after silica injection and continued to increase steadily over time. The inflammatory and fibrotic changes induced by silica appeared to be a specific effect of the injection of this toxic particulate and not the result of the introduction of a foreign body, because mice injected with silica crystals were found to have significantly greater increases in acute cellular inflammation and chronic collagen deposition than did mice injected with latex beads. A possible role for the immune system in modulating silica-induced damage was suggested by the variability in response of six different strains of mice (C3H/He, CBA/J, Balb/c, DBA/2, C57BL/6, C57BL/10), which differed at specific genetic loci. Both strains with high (DBA/2) and low (C3H/He) response demonstrated similar patterns of inflammation and fibrosis over a period of 12 weeks. This model demonstrates great potential in future studies for elucidating the role of the immune system in the development of pulmonary inflammation and fibrosis induced by toxic inorganic particulates.

Calcium pyrophosphate crystal deposition. An in vitro study using a gelatin matrix model.

Deposition of crystalline triclinic (t) and monoclinic (m) calcium pyrophosphate dihydrate (CPPD) in fibrocartilage and articular cartilage is the hallmark of chondrocalcinosis. Using biologic grade gelatin to model this crystal growth process, t-CPPD, m-CPPD, amorphous calcium pyrophosphate, orthorhombic calcium pyrophosphate tetrahydrate (o-CPPT), and 3 mixed calcium/sodium pyrophosphate salts were grown at physiologic pH. Amorphous and o-CPPT appeared to be kinetic precursor crystals in the formation of t-CPPD and m-CPPD. Optimal concentration ranges for the different crystals were determined.

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.

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.

Effect of pH on growth of mouse renal cortical tubule cells in primary culture.

We examined the effect of the medium pH on growth of primary cultures of mouse cortical tubule cells grown in defined medium. A significantly higher DNA content was observed within 24 h of lowering medium pH from 7.4 to 6.8 or 7.1 and persisted for the duration of the study. Further studies revealed that either medium acidification or insulin plus prostaglandin E1 nearly doubled uptake of [3H]thymidine in cells deprived of other growth factors for the previous 72-110 h. Moreover, the effects of insulin, prostaglandin E1, and medium acidification on [3H]thymidine uptake of quiescent cells were additive. An alkaline medium pH appeared to have a small but significant effect on cell hypertrophy, since cells exposed to pH 7.4 and 7.7 had a higher protein-to-DNA ratio than cells incubated at a lower pH. Cell pH of monolayers grown on glass slides determined from fluorescence of the carboxyfluorescein analogue 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was linearly correlated with medium pH, and changes in medium pH resulted in changes in steady-state cell pH of a similar magnitude. Four hours after medium acidification, relative increases in cell Na+ and water content occurred, whereas medium alkalinization led to decreases in cell Na+ and water content. The increases in cell Na+ and cell water content at pH 6.8 could be inhibited by amiloride. We conclude that decreasing the cell pH can be a mitogenic stimulus for renal tubule cells. Medium acidification is accompanied by changes in cell Na+ transport, which may be mediated in part by altered Na+-H+ antiporter activity.

Pneumoconiosis in workers exposed to silicon carbide.

Two men who had been exposed only to silicon carbide for many years in a factory manufacturing refractory bricks developed bilateral reticulonodular densities, as shown on chest radiograms, and complained of dyspnea. An open lung biopsy of one of them showed a large amount of black material in the fibrosed alveolar septums. Studies by X-ray diffraction revealed that the silicon carbide to which they were exposed did not contain quartz; X-ray powder diffraction analysis of the lung tissue revealed at least 6 different silicon carbides, traces of tungsten carbide, and an insignificant amount of quartz. Line-width analysis of the pattern suggested that the lung contained foreign material, with a significant number of particles smaller than 0.1 micron. The etiologic role of silicon carbide for tissue fibrosis is discussed.

Cell polarity and calcium oxalate crystal adherence to cultured collecting duct cells.

The relationship between cell membrane polarity and calcium oxalate (CaOx) crystal binding was studied in rat renal inner medullary collecting duct (IMCD) cells in primary culture. Cultures grew as simple monolayers (M) with interspersed cellular aggregates (A), and CaOx bound preferentially to A. An antibody that recognizes an exclusively basolateral epitope in intact IMCD binds to some of the cells in A but not to cells in M. Lysing of intercellular junctions with 3 mM EGTA (monitored by transepithelial resistance, R) resulted in basolateral antibody binding to the previously negative cells in M and a 21-fold increase in CaOx adherence to M over control (P less than 0.01). Enhanced CaOx attachment appeared to lag behind the fall in R by 5-10 min. Crystal attachment returned to control between 30 and 120 min after removal of EGTA and readdition of Ca. These data suggest that loss of epithelial membrane polarity may result in enhanced capacity to bind CaOx. Such loss of cell membrane polarity may occur in IMCD with some forms of epithelial injury and repair and may provide a site of crystal fixation to initiate nephrolithiasis.