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Biomedical subjects

P A Glenton

Publications and source records attributed to P A Glenton.

16 recordsLinked to original sources
PubMed2006

Modeling of hyperoxaluric calcium oxalate nephrolithiasis: experimental induction of hyperoxaluria by hydroxy-L-proline.

A number of animal models have been developed to investigate calcium oxalate (CaOx) nephrolithiasis. Ethylene glycol (EG)-induced hyperoxaluria in rats is most common, but is criticized because EG and some of its metabolites are nephrotoxic and EG causes metabolic acidosis. Both oxalate (Ox) and CaOx crystals are also injurious to renal epithelial cells. Thus, it is difficult to distinguish the effects of EG and its metabolites from those induced by Ox and CaOx crystals. This study was performed to investigate hydroxy-L-proline (HLP), a common ingredient of many diets, as a hyperoxaluria-inducing agent. In rats, HLP has been shown to induce CaOx nephrolithiasis in only hypercalciuric conditions. Five percent HLP mixed with chow was given to male Sprague-Dawley rats for 63 days, resulting in hyperoxaluria, CaOx crystalluria, and nephrolithiasis. Crystal deposits were surrounded by ED-1-positive inflammatory cells. Cell injury and death was followed by regeneration, as suggested by an increase in proliferating cell nuclear antigen-positive cells. Both osteopontin (OPN) and CD44 were upregulated. Staining for CD44 and OPN was intense in cells lining the tubules that contained crystals. Along with a rise in urinary Ox and lactate dehydrogenase, there were significant increases in 8-isoprostane and hydrogen peroxide excretion, indicating that the oxidative stress induced cell injury. Thus, HLP-induced hyperoxaluria alone can induce CaOx nephrolithiasis in rats.

Animals↗
PubMed2001

Expression of inter-alpha inhibitor related proteins in kidneys and urine of hyperoxaluric rats.

PURPOSE: To investigate the involvement of the inter-alpha inhibitor family of proteins in calcium oxalate stone formation we determined immunohistochemical distribution in the kidneys and excretion in the urine of these proteins in normal and hyperoxaluric rats. Various members of the family have been shown to inhibit the formation and retention of calcium oxalate crystals in the kidneys. MATERIALS AND METHODS: Hyperoxaluria was induced in male Sprague-Dawley rats by administering 0.75% ethylene glycol. The inter-alpha inhibitor family consists of inter-alpha inhibitor, pre-alpha inhibitor, the so-called heavy chains H1, H2 and H3, and the light chain bikunin. Antibodies against these molecules were used to localize various proteins in rat kidneys by immunohistochemical techniques. Urine was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis to determine the expression of various members of the inter-alpha inhibitor family. RESULTS: In normal kidneys staining for inter-alpha inhibitor and other members of the family was mostly limited to the proximal tubules and generally to their luminal contents. Eight weeks after the induction of hyperoxaluria various sections of renal tubules stained positive for inter-alpha inhibitor, bikunin and H3. Positive staining was observed in the tubular lumina as well as in the cytoplasm of epithelial cells. Crystal associated material was heavily stained. Western blot analysis recognized 7 protein bands in the urine. The urinary expression of H1, H3 and pre-alpha-inhibitor was significantly increased. CONCLUSIONS: Apparently hyperoxaluria and renal calcium oxalate crystal deposition result in the increased expression of crystallization inhibitors, such as inter-alpha-inhibitor related proteins, in the kidneys and urine. Results indicate that kidneys respond to nephrolithic challenges by producing proteins that inhibit crystal formation and retention.

Alpha-Globulins↗
PubMed2001

Characterization of Tamm-Horsfall protein in a rat nephrolithiasis model.

PURPOSE: The role of Tamm-Horsfall protein in calcium oxalate stone formation is controversial. It is unclear whether Tamm-Horsfall protein has a role in crystallization. If it does, does it act as an inhibitor or promoter of crystallization? To elucidate the nature of its involvement we characterized Tamm-Horsfall protein in a rat model of calcium oxalate nephrolithiasis by in vivo and in vitro techniques. MATERIALS AND METHODS: Calcium oxalate nephrolithiasis was induced in male Sprague-Dawley rats. The amino acid and carbohydrate composition of Tamm-Horsfall protein from normal rats and those with nephrolithiasis was determined. The Tamm-Horsfall protein gene and protein expression in the kidneys were examined by in situ hybridization and immunohistochemistry. Furthermore, the interaction of Tamm-Horsfall protein and calcium oxalate crystals was assessed by an in vitro crystal aggregation assay. RESULTS: Tamm-Horsfall protein from rats with nephrolithiasis was biochemically similar to that from normal rats. Although Tamm-Horsfall protein was associated with crystal deposits in the renal papillae of rats with nephrolithiasis, Tamm-Horsfall protein messenger RNA expression in the kidneys remained unchanged. In each group Tamm-Horsfall protein inhibited calcium oxalate crystal aggregation by 47%, indicating no change in functional capabilities. CONCLUSIONS: The results of this study indicate that urinary excretion, and the biochemical nature and functional capabilities of Tamm-Horsfall protein remain unchanged during experimental calcium oxalate nephrolithiasis. Although staining for Tamm-Horsfall protein was evident in the papillae of rats with nephrolithiasis, the site of Tamm-Horsfall protein synthesis remained cells of the thick ascending limbs of the loop of Henle.

Animals↗
PubMed2000

Membranes and their constituents as promoters of calcium oxalate crystal formation in human urine.

We have proposed that membranes of cellular degradation products are a suitable substrate for the nucleation of calcium oxalate (CaOx) crystals in human urine. Human urine is generally metastable with respect to CaOx. To demonstrate that cellular membranes present in the urine promote nucleation of CaOx we removed these substrates by filtration or centrifugation and induced crystallization by adding sodium oxalate, before and after filtration or centrifugation. In a separate experiment, membrane vesicles isolated from rat renal tubular brush border were added into the filtered or centrifuged urine before crystal induction. Crystals were counted using a particle counter. Urine, the pellet, and retentate were analyzed for the presence of membranes, lipids, and proteins. Lipids were further separated into different classes, identified, and quantified. Both filtration and centrifugation removed lipids, proteins, and membrane vesicles, causing a reduction in lipid and protein contents of the urine. More crystals formed in whole than in filtered or centrifuged urine. The number of crystals significantly increased when filtered urine was supplemented with various urinary components such as the retentate and phospholipids, which are removed during filtration. We also determined the urinary metastable limit with respect to CaOx. Filtration and centrifugation were associated with increased metastable limit which was reduced by the addition of membrane vesicles. These results support our hypothesis that urine normally contains promoters of CaOx crystal formation and that membranes and their constituents are the most likely substrate for crystal nucleation in the urine.

Animals↗
PubMed2000

Changes in urine macromolecular composition during processing.

PURPOSE: To determine the urinary crystallization inhibitory activity, urine is generally centrifuged and/or filtered. These preparative procedures may result in a total or partial removal of many macromolecular constituents implicated in crystallization. The main purpose of this study was to investigate the changes in urinary macromolecular composition following centrifugation and filtration. MATERIALS AND METHODS: Twenty-four hour urine samples were collected from human volunteers. Each was divided into 4 aliquots; one was filtered, the other was centrifuged, another was centrifuged and filtered. The control sample was neither filtered nor centrifuged. Total protein and lipid contents of each sample were determined. Proteins were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis was performed using antibodies against osteopontin (OPN), prothrombin (PT) related proteins, inter-alpha-inhibitor (IalphaI) related proteins, Tamm-Horsfall protein (THP), and albumin (ALB). The effect of processing on incorporation of urinary proteins in crystal matrices was also examined. Calcium oxalate crystals were produced in processed and unprocessed urine samples by the addition of sodium oxalate. Crystals were harvested, de-mineralized and their proteins analyzed by SDS-PAGE and Western blotting. RESULTS: Processing reduced the amounts of both proteins and lipids in the urine. Previously we identified phospholipids in the matrix of calcium oxalate crystals as well as the filtrate and retentate removed during filtration and centrifugation. Phospholipids have a high affinity for calcium-containing crystals. In the case of proteins, those with high molecular weights appeared to be clearly affected by filtration and centrifugation. Processing also appeared to influence the incorporation of proteins in the crystals. The matrix of crystals produced in processed urine contained less THP than those made in unprocessed urine, apparently a result of the loss of this higher molecular weight protein during processing. Incorporation of PT-related proteins, particularly fragment 1, was increased. CONCLUSIONS: We propose that selective inclusion of macromolecules is a result of an increase in available binding sites on crystal surfaces because of the removal of certain calcium binding substances such as phospholipids and proteins. Removal of larger macromolecules from the milieu may also provide a better access to the crystal surfaces.

Centrifugation↗
PubMed1999

Role of inter-alpha-inhibitor and its related proteins in experimentally induced calcium oxalate urolithiasis. Localization of proteins and expression of bikunin gene in the rat kidney.

Our earlier studies indicated that members of the inter-alpha-inhibitor (IalphaI) family of glycoproteins may play an important role in urolithiasis. Indeed bikunin, the light chain of IalphaI is a potent inhibitor of calcium oxalate crystallization. In order to understand this role, the distribution of IalphaI and its related proteins, as well as the expression of bikunin, were studied in normal and nephrolithic rats. In normal rats, IalphaI immunoreactivity was located mainly in proximal tubules. However, in nephrolithic rats, in addition to proximal tubules, the staining was intensively extended to tubules in the corticomedullary junction. Furthermore, by using polymerase chain reaction technique, we demonstrated that gene encoding for bikunin was activated in kidneys of nephrolithic rats. We have previously demonstrated increased staining for osteopontin in association with calcium oxalate crystal deposition in rat kidneys. Others have shown an increase in osteopontin production by renal epithelial cells on exposure to calcium oxalate crystals. Based on these observations we conclude that kidney cells possess an auto-defense system against calcium oxalate crystallization and stone formation in which members of the IalphaI family may be closely involved.

Alpha-Globulins↗
PubMed1999

Temporal changes in mRNA expression for bikunin in the kidneys of rats during calcium oxalate nephrolithiasis.

Inter-alpha-inhibitor and other bikunin-containing proteins are synthesized in relatively large quantities by the liver. These proteins function as Kunitz-type serine protease inhibitors and appear capable of inhibiting calcium oxalate (CaOx) crystallization in vitro. Preliminary studies have shown that renal tubular epithelial cells synthesize bikunin in response to CaOx challenge. To examine this response in vivo, a sensitive reverse transcription-quantitative competitive template-PCR was developed to detect and quantify poly(A)+ -tailed bikunin mRNA expression in kidney tissue from normal rats and rats developing CaOx nephrolithiasis after challenge with ethylene glycol. Bikunin mRNA expression in rat liver tissue was assessed as a positive control. The expression of bikunin mRNA in liver did not differ significantly between normal control rats and experimental rats with induced hyperoxaluria and renal CaOx crystallization. In contrast, there were significant temporal increases in the levels of bikunin mRNA expression in rat kidneys during CaOx nephrolithiasis after challenge with ethylene glycol. Urinary excretion of bikunin-containing proteins seemed to increase concomitantly. These findings indicate an association between the induction of hyperoxaluria/CaOx nephrolithiasis and the expression of the bikunin gene in rat kidneys.

Alpha-Globulins↗
PubMed1998

Identification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in the urine of healthy and stone forming subjects.

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced in vitro in whole human urine of healthy individuals and kidney stone patients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein contents. Crystallization in urine was induced by addition of an oxalate or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralization with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacrylamide gel electrophoresis, and identified by western blotting technique. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matrices obtained from the urine of normal and stone forming subjects. Albumin (AB), inter-alpha-inhibitor (IalphaI) related proteins, alpha-1 microglobulin (alpha-1 m), osteopontin (OPN), prothrombin (PT)-related proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal subjects and stone formers. AB, PT-related proteins and OPN were the main constituents. The other proteins were present in smaller but detectable amounts. However, CaP crystal matrix, contained a large amount of THP. In addition CaP crystals contained significantly more proteins than CaOx crystals. Centrifugation and/or filtration of the urine resulted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine.

Albumins↗
PubMed1997

Biochemical and quantitative analysis of Tamm Horsfall protein in rats.

The involvement of Tamm Horsfall protein (THP) in nephrolithiasis is currently under investigation in several laboratories. Although rat is a commonly used species as an in vivo model for such studies, there is only limited information available about the biochemical properties and excretion profile of THP in normal rats. In order to characterize rat THP, we purified and analyzed normal male rat THP, and compared it with normal human male urinary THP by gel electrophoresis. Both THPs migrated at approximately 90 KDa, and stained similarly for protein (Coomassie blue) as well as carbohydrates (periodic acid Schiff reagent). Compositional analysis revealed that rat THP was largely similar to human THP in amino acid and carbohydrate contents but showed differences in the individual sugar components from other mammals. There was considerable variation in the day-to-day urinary excretion of THP in normal rats, with values ranging from 552.96 micrograms to 2865.60 micrograms and a mean value of 1679.54 micrograms per 24 h. It was concluded from this study that rat THP did not contain any unusual biochemical components and was primarily similar to human THP in composition and mean urinary concentration.

Amino Acids↗
PubMed1996

Increased urinary excretion of lipids by patients with kidney stones.

OBJECTIVE: To test the hypothesis that patients with calcific kidney stones (stone formers) excrete more lipids and cell membranes in their urine than do normal individuals. PATIENTS, SUBJECTS AND METHODS: Lipids were isolated from the urine of patients with calcific stones (four women and six men, age range 28-69 years) and from normal subjects (three men and three women, age range 31-54 years). Different phospholipids, neutral lipids and glycolipids were identified using thin layer chromatography and individual lipids were characterized by several assays after scraping the spots from the plates, or by densitometry. RESULTS: Stone formers excreted more lipids and acidic phospholipids than did normal individuals; the urinary excretion of glycolipid, cholesterol and cholesterol esters was also increased. CONCLUSIONS: The greater excretion of lipids may reflect the increased turnover of cells in the tubular epithelium and sloughing of cells into the urine in response to a challenge by oxalate and calcium oxalate crystals. Acidic phospholipids from cellular membranes of the sloughed epithelial cells may be involved in crystal nucleation and retention within the kidneys and thus the initial development of stone nidus and the continued growth thereafter.

Adult↗
PubMed1996

Localization of tamm-horsfall protein and osteopontin in a rat nephrolithiasis model.

The possibility of more than one urinary protein being simultaneously associated with calcium oxalate (CaOx) crystallization in vivo was investigated by examining the localization of Tamm-Horsfall protein (THP) and osteopontin (Opn) in a rat model of nephrolithiasis. CaOx crystal deposits were induced in male Sprague-Dawley rats by feeding 0.75% ethylene glycol in drinking water. THP and Opn were localized on kidney sections by immunoperoxidase technique, using specific polyclonal antibodies. When only occasional crystal deposits were seen in the kidney, THP showed a similar to normal pattern of distribution, with positive staining in the thick ascending limbs of the loop of Henle. Opn was localized in some nephrons in the thin limb of loop of Henle and on the papillary surface in the calyceal fornix. In contrast, in samples with a significantly increased number of deposits in the kidneys, the staining for both THP and Opn was strikingly enhanced and altered, with positive staining around the crystals as well as abnormal localization in the papilla. Interestingly, the occurrence of Opn was, however, more consistent than that of THP. This is a first study showing that in this nephrolithiasis model, normal localization of THP and Opn is altered and they are closely and concurrently associated with crystal deposits in vivo.

Animals↗
PubMed1995

Deposition of calcium phosphate and calcium oxalate crystals in the kidneys.

Calcium phosphate (CaP) and calcium oxalate (CaOx) are the two most common crystalline constituents of human urinary stones. Calcium phosphate is often recognized as the nucleator of CaOx crystals, but the relationship between the two is not yet clearly understood. Using rat models of nephrolithiasis, we studied the role of CaP in renal deposition of CaOx. Calcium oxalate nephrolithiasis was brought about by inducing hyperoxaluria, while CaP CaOx nephrolithiasis was produced by dietary manipulation. Under similar urinary CaOx or CaP supersaturations, male rats were prone to form CaOx deposits while female rats were susceptible to produce CaP deposits in their kidneys. Crystal deposition in females was generally localized to the corticomedullary junction and in males to the renal papillae. The results indicate that gender plays an important role in the type and location of crystal deposition in the kidneys. In addition, deposition of CaP does not appear to influence the deposition of CaOx.

Animals↗
PubMed1978

Characterization of receptors mediating 5-hydroxytryptamine- and catecholamine-induced platelet aggregation, assessed by the actions of alpha- and beta-blockers, butyrophenones, 5-HT antagonists and chlorpromazine.

1 Blood platelets from normal human volunteers were isolated and aggregated in vitro with 5-hydroxytryptamine (5-HT), noradrenaline (NA), dopamine and N-dimethyldopamine (DMDA). 2 The effects of 5-HT antagonists, alpha- and beta-adrenoceptor blocking agents, butyrophenones and chlorpromazine upon aggregation induced by the four amines were investigated. 3 Only the the 5-HT antagonists, chlorpromazine and spiroperidol were potent inhibitors of 5-HT-induced aggregation, and only phentolamine was a potent inhibitor of the catecholamine-induced aggregation. 4 Evidence was obtained for two populations of receptors, one for 5-HT and one for the three catecholamines.

Adrenergic alpha-Antagonists↗
PubMed1976

A comparison of affinity constants for muscarine-sensitive acetylcholine receptors in guinea-pig atrial pacemaker cells at 29 degrees C and in ileum at 29 degrees C and 37 degrees C.

1 The affinity of 17 compounds for muscarine-sensitive acetylcholine receptors in atrial pacemaker cells and ileum of the guinea-pig has been measured at 29 degrees C in Ringer-Locke solution. Measurements were also made at 37 degrees C with 7 of them. 2 Some of the compounds had much higher affinity for the receptors in the ileum than for those in the atria. For the most selective compound, 4-diphenylacetoxy-N-methylpiperidine methiodide, the difference was approximately 20-fold. The receptors in the atria are therefore different the structure from those in the ileum. 3 The effect of temperature on affinity are not the same for all the compounds, tested indicating different enthalpies and entropies of adsorption and accounting for some of the difficulty experienced in predicting the affinity of new compounds.

Acetylcholine↗