Improved methodology to induce hyperoxaluria without treatment using hydroxyproline.

The use of hydroxyproline (HP) to generate hyperoxaluria in the rat is a problem because it is impossible to separate the effect of oxalate on renal injury from the effects of HP and the large array of metabolic intermediates formed when HP is converted to oxalate. Previously, the Dahl salt-sensitive (SS) and Brown Norway (BN) rat strains were studied to determine genetic control of resistance or susceptibility to HP-induced renal injury and crystal deposition. To develop a better model to induce hyperoxaluria without causing injury from HP metabolites, animals were fed a diet containing various levels of added oxalate (0, 1, 2, 3, or 5%). After 5 weeks rats were killed and the kidneys were removed for microscopic evaluation of tubule changes and crystal deposition. The 3 and 5% oxalate-fed groups had a substantial increase in urine oxalate, about 50 and 140 µmol/g body weight over controls, respectively. Both the SS and BN 3% oxalate-fed animals showed only slightly elevated tubule area and no crystal deposition. However, BN animals fed 5% oxalate had a dramatic increase in their percent tubule areas compared to control BN rats and treated SS rats. Crystal deposition in the kidneys was only observed in the 5% oxalate-fed groups. The BN kidneys demonstrated a threefold higher crystal deposition compared to oxalate-fed SS rats. We conclude that oxalate-supplemented food is a better method of producing hyperoxaluria in the rat than using HP which may introduce metabolic intermediates injurious to the kidney.

Study of a rat model for calcium oxalate crystal formation without severe renal damage in selected conditions.

BACKGROUND: Although nephrotoxic in high doses, ethylene glycol (EG) has been used with ammonium chloride (NH(4)Cl) or vitamin D(3) to study calcium oxalate stone formation in rat models. In the present study we used EG alone or with NH(4)Cl to study hyperoxaluria, crystaluria, and crystal attachment to renal epithelial cells in rats with minimal renal damage. METHODS: Six-week-old male Sprague-Dawley (SD) rats were given food and special drinking water. In experiment 1 the drinking water contained 1.0% NH(4)Cl plus four different concentrations of EG (0.8%, 0.4%, 0.2%, 0.1%). In experiment 2 the drinking water contained EG alone (0.8%, 0.4%, 0.2%, 0.1%). Urine was collected for 24 h before the rats were sacrificed. In experiment 1 the rats were sacrificed 5-13 days after starting the special water. In experiment 2 the rats were sacrificed 7-21 days after starting the special water. Bladder urine was also obtained. Blood and urine were tested for calcium, phosphorus, and creatinine. In addition, urine was tested for pH, oxalate and N-acetyl-beta-D glucosaminidase (NAG). Kidney sections were stained with hematoxylin-eosin, von Kossa and Pizzolato stain. Crystal morphology was determined using polarizing microscopy, and composition was determined using high-resolution X-ray powder diffraction. RESULTS: Experiment 1: Aggravation of renal function, an increase in urinary oxalate and NAG excretion, and crystals observed in the kidneys all correlated with EG concentration and length of drinking time. In bladder urine, calcium oxalate monohydrate (COM) crystals exceeded calcium oxalate dihydrate (COD) crystals. Experiment 2: Renal function remained unchanged. Oxalate excretion increased and NAG increased slightly. Crystals occurred only in the papillary tip region. Crystals in bladder urine were mostly COD. CONCLUSION: In the current rat model, calcium oxalate crystaluria could be induced without severe renal damage in selected cases. Either and/or both COM and COD might form and interact with kidney epithelium. We propose different experimental conditions to study the various phases of calcium oxalate stone formation in young male SD rats.

A porcine model of calcium oxalate kidney stone disease.

PURPOSE: The pig has been extensively used in biomedical research because of the similarities in organ structure and function to humans. It is desirable to have an animal model of oxaluria and urolithiasis with physiological, anatomical and nutritional characteristics that more closely resemble those of man. In this study we determined if feeding pigs trans-4-hydroxy-l-proline (HP) increased urine oxalate levels and if it would serve as a model for human hyperoxaluria and stone disease. MATERIALS AND METHODS: Male Yorkshire-Durox cross-bred pigs were fed HP for up to 20 days. Urine was periodically collected and analyzed for oxalate levels and the presence of crystalluria. After 20 days of feeding the kidneys were removed and examined grossly and microscopically for indications of injury, crystal deposition and stone formation. RESULTS: Feeding pigs 10% HP (weight per weight HP/food) produced hyperoxaluria, which reached a maximum and leveled off by day 6. Urine oxalate remained near this level until the study ended at 20 days regardless of the further increase in HP to 20% of the weight of the food. When the kidneys were removed and grossly examined, calcium oxalate encrustations were observed on multiple papillary tips. Histopathological observation of the papillary tissue showed tissue injury and crystal deposition. CONCLUSIONS: Pigs fed HP have hyperoxaluria and calcium oxalate crystalluria, and calcium oxalate papillary deposits form that may be precursors of kidney stones. The use of the pig as a model of human hyperoxaluria and stone formation should prove ideal for studies of these human diseases.

Crystal attachment to injured renal collecting duct cells: influence of urine proteins and pH.

BACKGROUND: The attachment of crystals to injured kidney epithelium is thought to be a necessary event in the development of urolithiasis. In vivo, the crystals are coated with urinary macromolecules that define the surface properties of the crystals. The present study examines the influence of coating of calcium oxalate crystals with urinary macromolecules on their attachment to both healthy (polarized) and injured (nonpolarized) primary inner medullary collecting duct (IMCD) cells. METHODS: Calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals were coated with urine macromolecules by incubating the crystals in urine from normal healthy volunteers at pH 5, 6, and 7. The level of attachment of the coated crystals to IMCD cells was also determined at pH 5, 6, and 7. The adsorbed proteins were extracted from the crystal surfaces and separated by gel electrophoresis. RESULTS: The coating of calcium oxalate crystals with urine proteins greatly reduced the attachment of crystals to both control and injured IMCD cells. At pH levels below 6, the crystals readily attached to injured cells. Extraction and separation of the adsorbed proteins showed that both COM and COD crystals adsorbed a similar array of proteins. At pH 5 and 6, several trace proteins were adsorbed to the crystals and were not apparent at pH 7. CONCLUSION: The coating of crystals with urine macromolecules greatly reduces the attachment of the crystals to normal healthy epithelia. The coating and attachment of the crystals below pH 6 enhances the attachment to injured cells. The enhanced crystal attachment could possibly be associated with one or more proteins adsorbed to the crystal surface that are not adsorbed to the crystals at higher pH.