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.

Whether genetics may play a role in the pathophysiologic response of kidney tubules to oxalate exposure remains unexplored despite that as many as 15% of the U.S. population annually will experience a kidney stone composed of calcium oxalate. To explore this issue, we utilized a panel of chromosome substitution strains in which one chromosome at a time was transferred from the Brown Norway (BN) rat onto the Dahl salt-sensitive (SS) genetic background. Hyperoxaluria was induced by adding hydroxyproline (HP) to the drinking water. A dose-response (0-2% HP) study found that both SS and BN exhibited the same level of oxalate excretion as HP concentration increased, but only the BN exhibited changes in urothelial pathology and demonstrated crystal deposition at sites of urothelial injury as a function of dose (at 1.5-2.0%). The consomic panel was treated with 2.0% HP and evaluated for hyperoxaluria, renal injury, and crystal deposition. Tubular injury (% Area) and crystal deposition (% Area) were similar between the resistant SS and SS-4, -6, -7, -8, -9, -11, -16, and -20(BN) consomic rats. However, tubular injury was significantly increased in SS-2(BN) compared with the SS parental (9.8 +/- 1.56 and 4.2 +/- 1.09%, respectively). Crystal deposition was observed in SS-2(BN) and SS-18(BN) (4.7 +/- 0.70 and 3.5 +/- 1.3%, respectively) to the same extent as seen in the susceptible BN (3.2 +/- 0.44%). The fact that crystal deposition was observed in SS-18(BN) without extensive overall tubule injury, compared with the more severe widespread tubular injury seen in SS-2(BN), suggests that the underlying mechanism of each locus is different. In conclusion, these studies establish that BN rats demonstrate oxalate-associated pathology and they retain calcium oxalate crystals coincident with urothelial injury but SS rats do not. These observations establish that BN rat chromosome 2 and 18 harbor genes that contribute to these processes.

PURPOSEThe 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 METHODSMale 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.RESULTSFeeding 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.CONCLUSIONSPigs 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.

Background: The interaction between kidney urothelium and crystals is a critical event in the growth of renal calculi. When studying calcium oxalate monohydrate (COM) crystal binding to Madin-Darby canine kidney (MDCK) cells in culture, we observed that crystals also attached to areas on the coverslips devoid of cells. This phenomenon could be the result of substances produced by the cells that adhere to the glass and subsequently bind COM crystals. We investigated the characteristics of this COM binding substance.Methods: Media was collected from cultures of MDCK cells (conditioned media) and proteins were separated by high performance liquid chromatography. The molecular weights and purity of isolated proteins were determined by polyacrylamide gel electrophoresis. The conditioned media and each separated fraction were applied to glass and to MDCK cells and COM-binding ability determined using C-14-labeled crystals. The binding of radio-labelled calcium oxalate dihydrate, brushite, uric acid, and apatite to coverslips were also studied.Results: Fourteen times more COM bound to coverslips incubated with conditioned media than those with control media. The molecular weight of the protein bound to the glass was determined to be 200 kDa. The COM crystals binding to this protein was 1.5 mug/ng. Other crystals bound to a lesser extent. The incubation of cells with this protein inhibited COM binding by 39%.Conclusion: The MDCK cells produce a 200-kDa protein that has a high binding affinity for COM crystals. This protein binds to glass and is responsible for crystal binding to areas devoid of cells. This protein also has an inhibitory effect on COM binding to MDCK cells in culture.

Crystalline hydroxyapatite is a component of bone, teeth, and numerous pathological calcifications. The apatite crystal structure can accommodate a wide variety of atomic substitutions which gives apatite crystals an unusually high degree of variability in biochemical and physical properties. Apatite crystallites interact with numerous cellular systemsin vivo, and some of these interactions may lead to altered cellular function. One measure of crystal-membrane interactions is crystal-induced membranolysis of human red blood cells. Hemolytic potentials at constant crystal surface areas were measured at 1, 2, and 4 hours for 29 different preparations of apatite. Each apatite sample was characterized by its morphology, particle size, % CO3, zeta potential, and broadening of the (211), (112), (300), (202), and (002) diffraction maxima. Only the surface area/g and the X-ray powder diffraction line broadening showed a significant inverse correlation with hemolytic potential. These parameters were related to each other, and are indications of the degree of crystallinity.