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.     

Dietary oxalate and calcium oxalate nephrolithiasis

PURPOSE: Patients with calcium oxalate kidney stones are advised to decrease the consumption of foods that contain oxalate. We hypothesized that a cutback in dietary oxalate would lead to a decrease in the urinary excretion of oxalate and decreased stone recurrence. We tested the hypothesis in an animal model of calcium oxalate nephrolithiasis. MATERIALS AND METHODS: Hydroxy-L-proline (5%), a precursor of oxalate found in collagenous foods, was given with rat chow to male Sprague-Dawley rats. After 42 days rats in group 1 continued on hydroxy-L-proline, while those in group 2 were given chow without added hydroxy-L-proline for the next 21 days. Food and water consumption as well as weight were monitored regularly. Once weekly urine was collected and analyzed for creatinine, calcium, oxalate, lactate dehydrogenase, 8-isoprostane and H(2)O(2). Urinary pH and crystalluria were monitored. Rats were sacrificed at 28, 42 and 63 days, respectively. Renal tissue was examined for crystal deposition by light microscopy. RESULTS: Rats receiving hydroxy-L-proline showed hyperoxaluria, calcium oxalate crystalluria and nephrolithiasis, and by day 42 all contained renal calcium oxalate crystal deposits. Urinary excretion of lactate dehydrogenase, 8-isoprostane and H(2)O(2) increased significantly. After hydroxy-L-proline was discontinued in group 2 there was a significant decrease in urinary oxalate, 8-isoprostane and H(2)O(2). Half of the group 2 rats appeared to be crystal-free. CONCLUSIONS: Dietary sources of oxalate can induce hyperoxaluria and crystal deposition in the kidneys with associated degradation in renal biology. Eliminating oxalate from the diet decreases not only urinary oxalate, but also calcium oxalate crystal deposits in the kidneys and improves their function.     

Decreased renal expression of the putative calcium oxalate inhibitor Tamm-Horsfall protein in the ethylene glycol rat model of calcium oxalate urolithiasis

PURPOSE: Tamm-Horsfall protein is believed to inhibit calcium oxalate crystallization, aggregation or adhesion to the renal epithelium. We determined whether ethylene glycol induced urolithiasis changes the expression of renal and urinary Tamm-Horsfall protein. For comparison the expression of another calcium oxalate inhibitor, osteopontin, was also analyzed. MATERIALS AND METHODS: Male rats were treated with 0.75% ethylene glycol plus an AIN-76 diet (Dyets, Bethlehem Pennsylvania) (ethylene glycol group) or standard rat chow and water (control group) for up to 8 weeks (6 per group for 8 weeks and 3 per group for 3 days to 6 weeks). Kidneys and urine (8 weeks only) were harvested and analyzed by Northern and Western blot analysis, and immunohistochemistry. RESULTS: Tamm-Horsfall protein message and protein (membrane bound form) were decreased, while those of osteopontin were increased in the kidneys of rats treated with ethylene glycol for 8 weeks. As judged by immunochemistry Tamm-Horsfall protein and osteopontin were consistently present in a few tubules in rats in the ethylene glycol and control groups, respectively. In urine expression of the free form of Tamm-Horsfall protein (approximately 75 kDa.) was decreased but detectable in ethylene glycol treated rats. Although readily detected in tissue, osteopontin was not detected in the urine of control or ethylene glycol treated rats. In the time course experiment Tamm-Horsfall protein did not decrease until 4 weeks, when calcium oxalate crystals were detectable in the kidneys of treated rats. In contrast, osteopontin was increased, although inconsistently, beginning at 3 days. CONCLUSIONS: Unlike other calcium oxalate inhibitors, such as osteopontin, renal message and protein for Tamm-Horsfall protein was decreased in ethylene glycol treated rats. Tamm-Horsfall protein expression did not decrease until aggregates of crystals had been deposited in the kidneys, while osteopontin expression began to increase almost immediately. Comparisons of the data on kidneys and urine obtained by RNA or protein blot analysis and immunochemistry underscore the need to examine tissue and urine by multiple techniques to obtain the most accurate assessment of how protein expression is changed by a given treatment.     

Pyridoxamine lowers kidney crystals in experimental hyperoxaluria: a potential therapy for primary hyperoxaluria

BACKGROUND: Primary hyperoxaluria is a rare genetic disorder of glyoxylate metabolism that results in overproduction of oxalate. The disease is characterized by severe calcium oxalate nephrolithiasis and nephrocalcinosis, resulting in end-stage renal disease (ESRD) early in life. Most patients eventually require dialysis and kidney transplantation, usually in combination with the replacement of the liver. Reduction of urinary oxalate levels can efficiently decrease calcium oxalate depositions; yet, no treatment is available that targets oxalate biosynthesis. In previous in vitro studies, we demonstrated that pyridoxamine can trap reactive carbonyl compounds, including intermediates of oxalate biosynthesis. METHODS: The effect of PM on urinary oxalate excretion and kidney crystal formation was determined using the ethylene glycol rat model of hyperoxaluria. Animals were given 0.75% to 0.8% ethylene glycol in drinking water to establish and maintain hyperoxaluria. After 2 weeks, pyridoxamine treatment (180 mg/day/kg body weight) started and continued for an additional 2 weeks. Urinary creatinine, glycolate, oxalate, and calcium were measured along with the microscopic analysis of kidney tissues for the presence of calcium oxalate crystals. RESULTS: Pyridoxamine treatment resulted in significantly lower (by approximately 50%) levels of urinary glycolate and oxalate excretion compared to untreated hyperoxaluric animals. This was accompanied by a significant reduction in calcium oxalate crystal formation in papillary and medullary areas of the kidney. CONCLUSION: These results, coupled with favorable toxicity profiles of pyridoxamine in humans, show promise for therapeutic use of pyridoxamine in primary hyperoxaluria and other kidney stone diseases.     

Effect of angiotensin II receptor blockage on osteopontin expression and calcium oxalate crystal deposition in rat kidneys

Hyperoxaluria leads to calcium oxalate (CaOx) crystallization and development of tubulointerstitial lesions in the kidneys. Treatment of hyperoxaluric rats with angiotensin II (Ang II) type I receptor blocker (ARB) reduces lesion formation. Because Ang II mediates osteopontin (OPN) synthesis, which is involved in both macrophage recruitment and CaOx crystallization, it was hypothesized that ARB acts via OPN. Hyperoxaluria was induced in 10-wk-old male Sprague-Dawley rats, and they were treated with ARB candesartan. At the end of 4 wk, kidneys were examined for crystal deposits, ED-1-positive cells, and expression of OPN mRNA. PCR was used to quantify OPN, renin, and angiotensin-converting enzyme (ACE) mRNA in kidneys. RIA was used to determine renal, plasma, and urinary OPN; plasma renin; Ang II and ACE; and renal Ang II. For evaluating oxidative stress, malondialdehyde was measured. Urinary calcium, oxalate, creatinine, and albumin were also determined. Despite similar urinary calcium and oxalate levels, kidneys of hyperoxaluric rats on candesartan had fewer CaOx crystals, fewer ED-1-positive cells, reduced OPN expression, and reduced malondialdehyde than hyperoxaluric rats. Urinary albumin excretion and serum creatinine levels improved significantly on candesartan treatment. mRNA for OPN, renin, and ACE were significantly elevated in hyperoxaluric rats. OPN synthesis and production increased with hyperoxaluria but to a lesser extent in candesartan-treated hyperoxaluric rats. These results show for the first time that oxalate can activate the renal renin-angiotensin system and that oxalate-induced upregulation of OPN is in part mediated via renal renin-angiotensin system.     

Enteric hyperoxaluria, nephrolithiasis, and oxalate nephropathy: potentially serious and unappreciated complications of Roux-en-Y gastric bypass.

BACKGROUND: Neither the presence nor prevalence of enteric hyperoxaluria has been recognized after Roux-en-Y gastric bypass (RYGBP). We have noted a high rate of oxalate nephrolithiasis and even 2 patients with oxalate nephropathy in this patient population postoperatively. Our aim was to determine the frequency of the occurrence and effects of enteric hyperoxaluria after RYGBP. METHODS: Retrospective review of all patients at our institution diagnosed with calcium oxalate nephrolithiasis or oxalate nephropathy after standard (n = 14) or distal (n = 9) RYGBP. The mean postoperative follow-up was 55 months. RESULTS: A total of 23 patients (14 men and 9 women; mean age 45 years; mean preoperative body mass index 55 kg/m(2)) developed enteric hyperoxaluria after RYGBP, defined by the presence of oxalate nephropathy (n = 2) or calcium oxalate nephrolithiasis (n = 21) and increased 24-hour excretion of urinary oxalate and/or calcium oxalate supersaturation. Enteric hyperoxaluria was recognized after a mean weight loss of 46 kg at 29 months (range 2-85) after RYGBP. Two patients developed renal failure and required chronic hemodialysis. Of the 21 patients with nephrolithiasis, 14 had no history of nephrolithiasis preoperatively, and 19 of 21 required lithotripsy or other intervention. Of the 23 patients, 20 tested had increased oxalate excretion, and 14 of 15 tested had high urine calcium oxalate supersaturation. CONCLUSION: Enteric hyperoxaluria, nephrolithiasis, and oxalate nephropathy must be considered with the other risks of RYGBP. Efforts should be made to identify factors that predispose patients to developing hyperoxaluria.     

Experimentally induced hyperoxaluria in MCP-1 null mice

Experimental animal model studies suggest that calcium oxalate (CaOx) crystal deposition in the kidneys is associated with the development of oxidative stress, epithelial injury and inflammation. There is increased production of inflammatory molecules including osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1) and various subunits of inter-alpha-inhibitor such as bikunin. What does the increased production of such molecules suggest? Is it a cause or consequence of crystal deposition? We hypothesized that over-expression and increased production of MCP-1 is a result of the interaction between renal epithelial cells and CaOx crystals after their deposition in the renal tubules. We induced hyperoxaluria in MCP-1 null as well as wild type mice and examined pathological changes in their kidneys and urine. Both wild type and MCP-1 null male mice became hyperoxaluric and demonstrated CaOx crystalluria. Neither of them developed crystal deposits in their kidneys. Both showed some morphological changes in their renal proximal tubules. Significant pathological changes such as cell death and increased urinary excretion of LDH were not seen. Results suggest that at least in mice (1) Increase in oxalate and decrease in citrate excretion can lead to CaOx crystalluria but not CaOx nephrolithiasis; (2) MCP-1 does not play a role in crystal retention within the kidneys; (3) Expression of OPN and MCP-1 is not increased in the kidneys in the absence of crystal deposition; (4) Crystal deposition is necessary for significant pathological changes and movement of monocytes and macrophages into the interstitium.     

Calcium oxalate crystal interaction with renal tubular epithelium,mechanism of crystal adhesion and its impact on stone development

The interaction between renal epithelial cells and calcium oxalate (CaOx) crystals and/or oxalate ions plays a critical role in the formation of urinary stones. Epithelial cells respond to hyperoxaluria and the presence of CaOx crystals in the kidneys by increased enzymuria and internalization of the crystals. Crystal cell interaction results in movement of crystals from the luminal to the basolateral side between the cells and the basement membrane. Once beneath the epithelium, crystals adhere to the basement membrane and become anchored inside the kidneys. Crystals anchored to basement membrane of the peripheral collecting duct aggregate with other crystals and move through an eroding epithelium to the papillary surface, furnishing an encrustation platform or a nidus for future development of a kidney stone. Thus interaction between renal epithelial cells and CaOx crystals and/or oxalate ions is an essential element in the development of urinary stone disease.     

Oxalate ions and calcium oxalate crystal-induced up-regulation of osteopontin and monocyte chemoattractant protein-1 in renal fibroblasts

OBJECTIVE: To examine the responses of renal fibroblasts to high oxalate (Ox) and calcium Ox (CaOx) crystals, as the latter are found in the renal interstitium of patients with primary or enteric hyperoxaluria, and in animals with experimental CaOx nephrolithiasis, and are associated with tubulointerstitial inflammation (TI). TI might begin with the production of chemoattractants by the renal epithelial cells exposed to high Ox and/or CaOx crystals; as Ox levels are also high in the renal interstitium and crystal deposition in nephrolithiasis might start in the interstitium, we hypothesized that renal fibroblasts might also be involved in the development of TI. MATERIALS AND METHODS: We exposed renal fibroblast cells of line NRK 49F in vitro to Ox ions (500 micromol/L) or CaOx monohydrate crystals (67 microg/cm(2)). We assessed the production of osteopontin and monocyte chemoattractant protein-1 (MCP-1), and expression of their mRNA, in the cells. We also determined the cellular malondialdehyde content as a marker of reactive oxygen species (ROS)-induced lipid peroxidation, and Trypan blue staining and the release of lactate dehydrogenase as markers of injury. RESULTS: Similar to renal epithelial cells, renal fibroblasts were stimulated by exposure to Ox and CaOx crystals. They showed signs of injury and ROS-induced lipid peroxidation. The mRNA expression and production of osteopontin and MCP-1 increased significantly. CONCLUSIONS: These results indicate that fibroblasts respond to high Ox and CaOx crystals by up-regulating specific pathways producing pro-inflammatory conditions. Migration of monocytes/macrophages to sites of interstitial crystal deposits can lead to localized interstitial inflammation and fibrosis.     

The influence of sex hormones on renal osteopontin expression and urinary constituents in experimental urolithiasis

PURPOSE: To our knowledge the influence of sex hormones on urinary stone formation remains undetermined. We investigated the effect of castration on urinary lithogenic factors and renal osteopontin expression in rats previously treated with ethylene glycol. MATERIALS AND METHODS: Sprague-Dawley rats were divided normal males, castrated males, males with 2 weeks of 0.75% ethylene glycol treatment, castrated males with 2 weeks of 0.75% ethylene glycol treatment, normal females, castrated females, females with 2 weeks of 0.75% ethylene glycol treatment and castrated females with 2 weeks of 0.75% ethylene glycol treatment. We analyzed 24-hour urine samples for urinary constituents, such as calcium, oxalate, citrate, uric acid, phosphate, magnesium, sodium, potassium and creatinine. The kidneys were examined for osteopontin expression by Northern blot analysis and for crystal deposition by histological examination. RESULTS: In intact male rats calcium and citrate excretion decreased and oxalate excretion increased significantly after ethylene glycol treatment. Castrated male rats with ethylene glycol had greater calcium and less oxalate excretion than male intact rats with ethylene glycol. In intact female rats uric acid excretion decreased and only calcium excretion increased significantly after ethylene glycol treatment. Castrated female rats with ethylene glycol excreted significantly more oxalate and less calcium than intact female rats with ethylene glycol. Renal osteopontin expression was the same in male intact and castrated rats, and in female intact and castrated rats. In males with ethylene glycol expression was stronger in castrated than in intact rats. In females with ethylene glycol expression was weaker in castrated than in intact rats. No crystal deposits were found in the kidneys in any group. CONCLUSIONS: Testosterone appears to promote stone formation by suppressing osteopontin expression in the kidneys and increasing urinary oxalate excretion. Estrogen appears to inhibit stone formation by increasing osteopontin expression in the kidneys and decreasing urinary oxalate excretion.     

Osteopontin knockdown in the kidneys of hyperoxaluric rats leads to reduction in renal calcium oxalate crystal deposition

Osteopontin (OPN) expression is increased in kidneys of rats with ethylene glycol (EG) induced hyperoxaluria and calcium oxalate (CaOx) nephrolithiasis. The aim of this study is to clarify the effect of OPN knockdown by in vivo transfection of OPN siRNA on deposition of CaOx crystals in the kidneys. Hyperoxaluria was induced in 6-week-old male Sprague–Dawley rats by administering 1.5 % EG in drinking water for 2 weeks. Four groups of six rats each were studied: Group A, untreated animals (tap water); Group B, administering 1.5 % EG; Group C, 1.5 % EG with in vivo transfection of OPN siRNA; Group D, 1.5 % EG with in vivo transfection of negative control siRNA. OPN siRNA transfections were performed on day 1 and 8 by renal sub-capsular injection. Rats were killed at day 15 and kidneys were removed. Extent of crystal deposition was determined by measuring renal calcium concentrations and counting renal crystal deposits. OPN siRNA transfection resulted in significant reduction in expression of OPN mRNA as well as protein in group C compared to group B. Reduction in OPN expression was associated with significant decrease in crystal deposition in group C compared to group B. Specific suppression of OPN mRNA expression in kidneys of hyperoxaluric rats leads to a decrease in OPN production and simultaneously inhibits renal crystal deposition.     

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.     

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.     

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.     

Animal models of kidney stone formation: an analysis

Calcific kidney stones in both humans and mildly hyperoxaluric rats are located on renal papillary surfaces and consist of an organic matrix and crystals of calcium oxalate and/or calcium phosphate. The matrix is intimately associated with the crystals and contains substances that can promote as well as inhibit calcification. Osteopontin, Tamm-Horsfall protein, bikunin, and prothrombin fragment 1 have been identified in matrices of both human and rat stones. Hyperoxaluria can provoke calcium oxalate nephrolithiasis in both humans and rats. Kidney-stone-forming rats are hypomagnesuric and hypocitraturic during nephrolithiasis. Human stone formers may have the same disorders. Males of both species are prone to develop calcium oxalate nephrolithiasis, whereas females tend to form calcium phosphate stones. Oxalate metabolism is considered to be almost identical between rats and humans. Thus, there are many similarities between experimental nephrolithiasis induced in rats and human kidney-stone formation, and a rat model of calcium oxalate nephrolithiasis can be used to investigate the mechanisms involved in human kidney stone formation.     

Preventive effects of green tea on renal stone formation and the role of oxidative stress in nephrolithiasis

PURPOSE: Urinary stones are similar to arteriosclerosis in epidemiology, mechanism, calcification composition and age at frequent occurrence. The calcification that occurs in arteriosclerosis is inhibited by antioxidants. Green tea leaves contain approximately 13% catechins, which have been shown to have antioxidant effects. We investigated the inhibitory, antioxidative effects of green tea on calcium urinary stone formation. MATERIALS AND METHODS: A total of 120 Wistar rats were divided into 4 groups, namely group 1-control rats receiving saline, group 2-stone group rats administered ethylene glycol (EG) and vitamin D3, group 3-drink group rats administered EG, vitamin D3 and green tea given as drinking water, and group 4-powder group rats administered EG, vitamin D3 and 2.5% powdered green tea leaves mixed in a powder diet. Pooled 24-hour urine samples and blood samples were collected and the 2 kidneys were excised 7, 14 and 21 days after administration, respectively. One kidney was used for immunohistological examination of osteopontin, superoxide dismutase (SOD), p65, p53 and bcl-2 expression, in situ hybridization of osteopontin and detection of apoptosis, while the other was used for quantitative analysis of SOD activity. RESULTS: Green tea treatment decreased urinary oxalate excretion and calcium oxalate deposit formation. Green tea treatment increased SOD activity compared with the stone group. The degree of apoptosis in the stone group was significantly increased compared with the drink and powder groups. CONCLUSIONS: The inhibitory effect of green tea on calcium oxalate urolithiasis is most likely due to antioxidative effects.     

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.     

An animal model of calcium oxalate urolithiasis based on a cyclooxygenase 2 selective inhibitor

Our aim was to develop a stone-forming animal model involving renal tubular injury using a cyclooxygenase 2 selective inhibitor. Male Sprague-Dawley rats fed chow containing 3% sodium oxalate with or without 100 mg/kg celecoxib were compared to animals fed normal chow. Rats were killed after 2 or 4 weeks and the kidneys were harvested for morphological examination. Collections of 24-h urine were made before kidney harvest. After 2 weeks only a few crystals were observed in rats that received oxalate and celecoxib, but after 4 weeks more crystals were observed at the renal papilla than in rats that received only oxalate. Few crystals were found in rats fed normal chow with or without celecoxib. The urinary activities of gamma-glutamyl transpeptidase (GGT) were increased by celecoxib administration whereas creatinine clearance rates were unchanged. In rats fed oxalate, urinary oxalate excretion increased, but calcium excretion decreased. This model using a cyclooxygenase 2 selective inhibitor is a useful stone forming animal model involving mild renal tubular injury together with mild hyperoxaluria.