Membranes and Their Constituents as Promoters of Calcium Oxalate Crystal Formation in Human Urine S. R. Khan, S. A. Maslamani, F. Atmani, P. A. Glenton, F. J. Opalko, S. Thamilselvan, C. Hammett-Stabler Department of Pathology, Box 100275, College of Medicine, University of Florida, Gainesville, Florida 32610, USA Received: 11 August 1998 / Accepted: 26 July 1999 Abstract. We have proposed that membranes of cellular degradation products are a suitable substrate for the nucle- ation 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 cen- trifugation. 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 mem- branes, lipids, and proteins. Lipids were further separated into different classes, identified, and quantified. Both filtra- tion and centrifugation removed lipids, proteins, and mem- brane vesicles, causing a reduction in lipid and protein con- tents of the urine. More crystals formed in whole than in filtered or centrifuged urine. The number of crystals signifi- cantly increased when filtered urine was supplemented with various urinary components such as the retentate and phos- pholipids, which are removed during filtration. We also de- termined the urinary metastable limit with respect to CaOx. Filtration and centrifugation were associated with increased metastable limit which was reduced by the addition of mem- brane vesicles. These results support our hypothesis that urine normally contains promoters of CaOx crystal forma- tion and that membranes and their constituents are the most likely substrate for crystal nucleation in the urine. Key words: Calcium oxalate — Nephrolithiasis — Cell membrane — Matrix Vesicles — Phospholipids. Human urine is generally metastable with respect to calcium oxalate (CaOx) [1, 2], the most common constituent of uri- nary stones. As a result, crystal nucleation, the first step towards stone formation, requires a substrate. We have pro- posed that in the human urine, membranes of cellular deg- radation products act as nucleators of CaOx crystals [3]. Membranes and their lipids have also been implicated in the nucleation of calcium phosphate (CaP) crystals in biological fluids [4–15]. In vitro studies have shown that acidic phos- pholipids, lipid extracts from various calcified tissues, membranes of so-called matrix vesicles, and liposomes all can initiate the formation of CaP crystals in metastable so- lutions. Lipids can also induce CaP crystallization in vivo. We have demonstrated that membranes of renal tubular ori- gin and lipids of CaOx stones promote the precipitation of CaOx crystals in vitro in metastable solutions [16–18]. In addition we have shown that hyperoxaluric rats develop CaOx crystalluria and nephrolithiasis only in the presence of sloughed cellular membranes [19]. The studies described here were carried out to determine the role of membranes and their constituents in the forma- tion of CaOx crystals in the urine. Urine samples were filtered and/or centrifuged and analyzed for lipids and mem- branes. Metastable limit with respect to CaOx was deter- mined for various samples with or without the addition of membrane vesicles. Nucleation of CaOx was induced by addition of sodium oxalate. Results indicate that human urine contains promoters of CaOx crystal nucleation and, that cellular membranes are the most likely nucleators. Re- moval of these nucleators from the urine through filtration and centrifugation changes the CaOx crystallization activity in the urine, and reintroduction of membranous substances into the processed urine boosts their crystallization poten- tial. Materials and Methods Urine Collection for Crystallization Studies Twenty-four-hour urine samples were collected from normal healthy human males. During collection, the specimens were maintained at room temperature (approximately 24°C). One mil- liliter of 20% sodium azide, an antibacterial agent, was placed in the collection bottles prior to collection. The pH and total urinary volume were recorded. The total urinary protein was determined using dipsticks and/or a Bio-Rad (Bio-Rad Laboratories, Hercules, CA) protein assay kit. Urine was examined microscopically. Ab- sence of proteinuria, overt crystalluria, and blood cells in the urine were considered to indicate the absence of kidney diseases. Following a generally accepted protocol of urine processing [2, 20–22] before performing crystallization studies, aliquots were filtered through 5 m or 0.22 m filters and/or centrifuged at 10,000 g at 20°C for 20 minutes using the J-14 rotor of the J2-21 centrifuge. Both the pellets and the retentate on the filters were processed for and examined by transmission electron microscopy (TEM) as described in our earlier publications [18]. Calcium and oxalate contents of sample urine were determined both before and after various manipulations to verify that filtration and centrifuga- tion did not alter such urinary constituents. Crystal Induction and Particle Counting Crystallization of CaOx was induced by the addition of 30 l of 0.1 M sodium oxalate to 2 ml of native or filtered urine. The mixture was incubated for 30 minutes in a shaking water bath after Correspondence to: S. R. Khan Calcif Tissue Int (2000) 66:90–96 © 2000 Springer-Verlag New York Inc.