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.

Guaifenesin stone matrix proteomics: a protocol for identifying proteins critical to stone formation.

Drug-related kidney stones are a diagnostic problem, since they contain a large matrix (protein) fraction and are frequently incorrectly identified as matrix stones. A urine proteomics study patient produced a guaifenesin stone during her participation, allowing us to both correctly diagnose her disease and identify proteins critical to this drug stone-forming process. The patient provided three random midday urine samples for proteomics studies; one of which contained stone-like sediment with two distinct fractions. These solids were characterized with optical microscopy and Fourier transform infrared spectroscopy. Immunoblotting and quantitative mass spectrometry were used to quantitatively identify the proteins in urine and stone matrix. Infrared spectroscopy showed that the sediment was 60 % protein and 40 % guaifenesin and its metabolite guaiacol. Of the 156 distinct proteins identified in the proteomic studies, 49 were identified in the two stone-components with approximately 50 % of those proteins also found in this patient's urine. Many proteins observed in this drug-related stone have also been reported in proteomic matrix studies of uric acid and calcium containing stones. More importantly, nine proteins were highly enriched and highly abundant in the stone matrix and 8 were reciprocally depleted in urine, suggesting a critical role for these proteins in guaifenesin stone formation. Accurate stone analysis is critical to proper diagnosis and treatment of kidney stones. Many matrix proteins were common to all stone types, but likely not related to disease mechanism. This protocol defined a small set of proteins that were likely critical to guaifenesin stone formation based on their high enrichment and high abundance in stone matrix, and it should be applied to all stone types.

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.

Guaifenesin- and ephedrine-induced stones.

PURPOSE: We report a new type of drug-induced stone that is caused by overconsumption of preparations containing guaifenesin and ephedrine. MATERIALS AND METHODS: Clinical and stone analysis data from the Molecular Structure Laboratory at the Veterans Affairs Medical Center in Milwaukee, Wisconsin, were reviewed. Stone analysis was performed by Fourier transform infrared spectroscopy, high-resolution X-ray crystallographic powder diffraction, or both. The urine and stone material from one of the subjects were analyzed with high-performance liquid chromatography. RESULTS: Stone analysis from seven patients demonstrated metabolites of guaifenesin. High-performance liquid chromatography revealed that the stone and urine from one subject had a high content of guaifenesin metabolites and a small amount of ephedrine. Demographic data were available on five patients. Three had a history of alcohol or drug dependency. All were consuming over-the-counter preparations containing ephedrine and guaifenesin. Four admitted to taking excessive quantities of these agents, mainly as a stimulant. Hypocitraturia was identified in two individuals subjected to urinary metabolic testing. These stones are radiolucent on standard X-ray imaging but can be demonstrated on unenhanced CT. Shockwave lithotripsy was performed in two patients, and the calculi fragmented easily. CONCLUSIONS: Individuals consuming large quantities of preparations containing ephedrine and guaifenesin may be at risk to develop stones derived mainly from metabolites of guaifenesin and small quantities of ephedrine. These patients may be prone to drug or alcohol dependency.

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.

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.

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.

Evidence of calcium phosphate supersaturation in the loop of Henle.

We have used published rat micropuncture data to construct a matrix of ion concentrations along the rat nephron. With an iterative computer model of known ion interactions, we calculated relative supersaturation ratios in all nephron segments. The collecting ducts and urine showed expected supersaturation with stone-forming salts. Fluid in the thin segment of the loop of Henle may be supersaturated with calcium carbonate and calcium phosphate under certain conditions. Because calculations cannot predict the actual course of crystallization, we made solutions to mimic, in vitro, presumed conditions in the loop of Henle. The solid phases that formed were analyzed by X-ray powder diffraction, electron microprobe, and infrared spectroscopy. All samples were identified as poorly crystallized or immature apatite. The descending limb of Henle's loop creates a unique condition as it extracts water but not sodium, bicarbonate, calcium, or phosphate, giving a calcium concentration at the bend of 3 mM, pH 7.4, and a phosphate concentration that varies from 0.8 to 48 mM, depending on parathyroid hormone and dietary phosphate. We conclude that conditions in the thin segment potentially could create a solid calcium phosphate phase, which may initiate nucleation of calcium oxalate salts in the collecting ducts, potentiating nephrolithiasis and nephrocalcinosis.

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.

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.

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.

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.

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.

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.