Patricia A. Glenton

Expression of Osteopontin in Rat Kidneys: Induction During Ethylene Glycol Induced Calcium Oxalate Nephrolithiasis

PURPOSE Osteopontin is a well-known component of stone matrix and a strong inhibitor of the nucleation, growth and aggregation of calcium oxalate crystals in vitro. To understand its involvement in vivo in calcium oxalate nephrolithiasis we investigated the renal expression and urinary excretion of osteopontin in normal rats, and rats with hyperoxaluria and calcium oxalate crystal deposits in the kidneys. MATERIALS AND METHODS Calcium oxalate nephrolithiasis was induced by administering ethylene glycol. Immunohistochemistry and in situ hybridization were done to localize osteopontin and osteopontin messenger RNA in the kidneys, while sensitive reverse transcriptase quantitative competitive template polymerase chain reaction was performed to detect and quantify osteopontin messenger RNA expression. Urinary excretion was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, and then quantified by densitometry of the Western blots. RESULTS Osteopontin expression in the kidneys was significantly increased after hyperoxaluria and it increased further after the deposition of calcium oxalate crystals in the kidneys. Urinary excretion of osteopontin increased concomitantly. The results reveal differences in renal responses after exposure to oxalate and calcium oxalate crystals. In normal kidneys osteopontin expression was limited to a small number of cells of the thin limbs of the loop of Henle and papillary surface epithelium. During hyperoxaluria osteopontin expression in the kidneys was increased but still mostly limited to cells of the thin limb and papillary surface epithelium. However, after calcium oxalate crystal deposition osteopontin expression was observed throughout the kidneys, including segments of the proximal tubules. CONCLUSIONS In response to exposure to oxalate and calcium oxalate crystals renal epithelial cells increase the production of osteopontin, which may have a significant role in calcium oxalate nephrolithiasis.

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

Abstract 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-α-inhibitor (IαI) related proteins, α-1 microglobulin (α-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.

Investigative Urology: 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.

EXPRESSION OF INTER-α INHIBITOR RELATED PROTEINS IN KIDNEYS AND URINE OF HYPEROXALURIC RATS

Purpose: To investigate the involvement of the inter-α 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-α inhibitor family consists of inter-α inhibitor, pre-α 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-α inhibitor family.Results: In normal kidneys staining for inter-α inhibitor ...

Effect of NADPH oxidase inhibition on the expression of kidney injury molecule and calcium oxalate crystal deposition in hydroxy-L-proline-induced hyperoxaluria in the male Sprague-Dawley rats.

BACKGROUND Renal calcium oxalate (CaOx) crystal deposition is associated with epithelial injury and movement of inflammatory cells into the interstitium. We have proposed that oxalate (Ox)- and CaOx crystal-induced injury is most likely caused by reactive oxygen species (ROS) produced by activation of membrane nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. METHODS Present study was undertaken to determine the effect of NADPH oxidase inhibitor apocynin on the expression of kidney injury molecule-1 (KIM-1) and renal CaOx crystal deposition in rats with hyperoxaluria. We also investigated the urinary excretion of KIM-1, osteopontin (OPN) and monocyte chemoattractant protein-1 (MCP-1) and renal expression of OPN and ED-1. Male Sprague-Dawley rats were fed a diet containing 5% hydroxyl-L-proline (HLP) and 4 mmol apocynin to drink for 28 days. Urine was collected on Days 7, 14, 21 and 28. After that, rats were sacrificed and their kidneys processed for various microscopic and molecular investigations. RESULTS HLP consumption produced heavy deposits of CaOx crystals. Renal expression of KIM-1 and OPN and urinary excretion of KIM-1, OPN, H(2)O(2) and MCP-1 was significantly increased. ED-1-positive cells migrated into renal interstitium. Apocynin treatment caused significant reduction of crystal deposits, injured and dilated tubules; renal expression of KIM-1, OPN and ED-1 and urinary excretion of KIM-1, OPN, MCP-1 and H(2)O(2). Apocynin had no effect on the urinary excretion of Ox. CONCLUSIONS This is the first study of urinary excretion and renal expression of KIM-1 in association with renal CaOx crystal deposition, experimental or clinical. The results indicate that NADPH oxidase inhibition leads to reduction in KIM-1 expression and urinary excretion as well as renal CaOx crystal deposition. KIM-1 is an important marker of renal epithelial injury. The results provide further support to our proposal that renal epithelial injury is critical for crystal retention and that injury is in part caused by the production of ROS with the involvement of NADPH oxidase.

Heterogeneous nucleation of calcium oxalate crystals in the presence of membrane vesicles

Abstract Membrane-assisted crystallization of calcium oxalate was studied in vitro, using constant composition methodology. Rat renal tubular brush border membrane vesicles were incubated in supersaturated solution of calcium oxalate. Calcium and oxalate depletion started much earlier in the presence of the vesicles than in their absence; within 8, 32, or 258 min of the incubation of vesicles in calcium oxalate solutions of relative supersaturation of 12, 10 or 6 respectively. Thin plate-like crystals with jagged edges formed in association with the membrane vesicles. Since crystal nucleation in the presence of membrane vesicles started within 8 min at a relative supersaturation as low as 12, it will start significantly earlier in the urine of stone formers which is known to have higher relative supersaturation with respect to calcium oxalate. These results demonstrate that cellular membranes can efficiently induce nucleation of calcium oxalate crystals from a metastable solution in an vitro system. Similar membrane induced heterogeneous nucleation of calcium oxalate in vivo within the renal tubules is a distinct possibility.

Lipids and Membranes in the Organic Matrix of Urinary Calcific Crystals and Stones

Abstract. The ultrastructure of the organic matrix of demineralized urinary stones was examined by standard transmission and scanning electron microscopy as well as after malachite green-glutaraldehyde fixation. Crystal ghosts of both calcium oxalate and calcium phosphate were made of amorphous material and were dispersed in a matrix containing amorphous, fibrillar, and membranous substances. Malachite green positive material was seen to be associated with the ghosts, as well as with the membranous and fibrillar components of the organic matrix. Calcium oxalate and calcium-phosphate crystals, induced in human urine in vitro were also found to be associated with an organic matrix containing lipids and proteins. It is suggested that the intimate association between crystals and lipids is a result of the involvement of cellular membranes in the nucleation of these crystals.

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.

Experimental Induction of Calcium Oxalate Nephrolithiasis in Mice

PURPOSE The availability of various transgenic and knockout mice provides an excellent opportunity to better understand the pathophysiology of calcium oxalate stone disease. However, attempts to produce calcium oxalate nephrolithiasis in mice have not been successful. We hypothesized that calcium oxalate nephrolithiasis in mice requires increasing urine calcium and oxalate excretion, and experimentally induced hyperoxaluria alone is not sufficient. To provide evidence we induced hyperoxaluria by administering hyperoxaluria inducing agents in normocalciuric and hypercalciuric mice, and investigating various aspects of nephrolithiasis. MATERIALS AND METHODS We administered ethylene glycol, glyoxylate or hydroxyl proline via diet in male and female normocalciuric B6 mice, and in hypercalciuric sodium phosphate co-transporter type 2 a -/- mice for 4 weeks. We collected 24-hour urine samples on days 0, 3, 7, 14, 21 and 28, and analyzed them for pH, creatinine, lactate dehydrogenase calcium and oxalate. Kidneys were examined using light microscopy. Urine was examined for crystals using light and scanning electron microscopy. RESULTS Hypercalciuric mice on hydroxyl proline did not tolerate treatment and were sacrificed before 28 days. All mice on ethylene glycol, glyoxylate or hydroxyl proline became hyperoxaluric and showed calcium oxalate crystalluria. No female, normocalciuric or hypercalciuric mice showed renal calcium oxalate crystal deposits. Calcium oxalate nephrolithiasis developed in all mice on glyoxylate and in some on ethylene glycol. In all mice the kidneys showed epithelial injury. Male mice particularly on glyoxylate had more renal injury and inflammatory cell migration into the interstitium around the crystal deposits. CONCLUSIONS Results confirm that hyperoxaluria induction alone is not sufficient to create calcium oxalate nephrolithiasis in mice. Hypercalciuria is also required. Kidneys in male mice are more prone to injury than those in female mice and are susceptible to calcium oxalate crystal deposition. Perhaps epithelial injury promotes crystal retention. Thus, calcium oxalate nephrolithiasis in mice is gender dependent, and requires hypercalciuria and hyperoxaluria.

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.