CaOx Crystal Deposition Research Articles

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 deposition in metabolic syndrome model rat kidneys

Although an epidemiological link between the metabolic syndrome and kidney stone formation has been reported, the mechanism by which metabolic syndrome promotes kidney stone formation has yet to be elucidated. We investigated calcium oxalate (CaOx) kidney stone formation in a rat metabolic syndrome model. We induced hyperoxaluria in 8-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) rats, and a control strain, Long-Evans Tokushima Otsuka (LETO) rats, by administering 1.0% ethylene glycol (EG) as their drinking water for 2 weeks. Rats were divided into four groups: LETO-C (control, n = 7); LETO-SF (stone forming, n = 8); OLETF-C (n = 7); and OLETF-SF (n = 8). Urine and blood samples were collected for biochemistry testing, and the kidneys were harvested for estimation of crystal deposition and determinations of the expression of osteopontin (OPN) and monocyte chemoattractant protein-1 (MCP-1). Administration of EG induced hyperoxaluria to the same degree in both strains. The OLETF-SF group showed a higher grade of renal crystal deposition and significantly higher renal calcium content than the LETO-SF group. Although the OLETF-C group excreted significantly higher amounts of uric acid and more acidic urine than the LETO-C group, similar differences were not observed in rats given EG. Significant upregulation of both OPN and MCP-1 was seen in the kidneys of hyperoxaluric rats, with higher levels of expression in the OLETF-SF group than the LETO-SF group. The present results show for the first time that OLETF rats form more renal CaOx crystal deposits compared with control rats under EG-induced hyperoxaluric conditions. The model described here should be useful for investigating the mechanisms by which the metabolic syndrome promotes CaOx kidney stone formation.

Effects of Orthosiphon grandiflorus, Hibiscus sabdariffa and Phyllanthus amarus Extracts on Risk Factors for Urinary Calcium Oxalate Stones in Rats

We evaluated the antilithic effect of Orthosiphon grandiflorus, Hibiscus sabdariffa and Phyllanthus amarus extracts on known risk factors for calcium oxalate stones in rats. We divided 30 male Wistar rats into 5 equal groups. Controls were fed a standard diet and the remaining groups received a 3% glycolate diet for 4 weeks to induce hyperoxaluria. One glycolate fed group served as the untreated group and the others were given oral extracts of Orthosiphon grandiflorus, Hibiscus sabdariffa or Phyllanthus amarus at a dose of 3.5 mg daily. We collected 24-hour urine and blood samples. Kidneys were harvested for histological examination. We measured the renal tissue content of calcium and oxalate. The Hibiscus sabdariffa group showed significantly decreased serum oxalate and glycolate, and higher oxalate urinary excretion. The Phyllanthus amarus group showed significantly increased urinary citrate vs the untreated group. Histological examination revealed less CaOx crystal deposition in the kidneys of Hibiscus sabdariffa and Phyllanthus amarus treated rats than in untreated rats. Those rats also had significantly lower renal tissue calcium content than untreated rats. All parameters in the Orthosiphon grandiflorus treated group were comparable to those in the untreated group. Hibiscus sabdariffa and Phyllanthus amarus decreased calcium crystal deposition in the kidneys. The antilithic effect of Hibiscus sabdariffa may be related to decreased oxalate retention in the kidney and more excretion into urine while that of Phyllanthus amarus may depend on increased urinary citrate. In contrast, administering Orthosiphon grandiflorus had no antilithic effect.

Prevention of renal crystal deposition by an extract of Ammi visnaga L. and its constituents khellin and visnagin in hyperoxaluric rats

In Egypt, teas prepared from the fruits of Ammi visnaga L. (syn. "Khella") are traditionally used by patients with urolithiasis. The aim of this study was to evaluate whether oral administration of an aqueous extract prepared from the fruits of A. visnaga as well as two major constituents khellin and visnagin could prevent crystal deposition in stone-forming rats. Hyperoxaluria was induced in male Sprague-Dawley rats by giving 0.75% ethylene glycol and 1% ammonium chloride via the drinking water. The Khella extract (KE; 125, 250 or 500mg/kg) was orally administered for 14days. The histopathological examination of the kidneys revealed that KE significantly reduced the incidence of calcium oxalate (CaOx) crystal deposition. In addition, KE significantly increased urinary excretion of citrate along with a decrease of oxalate excretion. Comparable to the extract, khellin and visnagin significantly reduced the incidence of CaOx deposition in the kidneys. However, both compounds did not affect urinary citrate or oxalate excretion indicating a mechanism of action that differs from that of the extract. For KE, a reasonably good correlation was observed between the incidence of crystal deposition, the increase in citrate excretion and urine pH suggesting a mechanisms that may interfere with citrate reabsorption. In conclusion, our data suggest that KE and its compounds, khellin and visnagin, may be beneficial in the management of kidney stone disease caused by hyperoxaluria but that it is likely that different mechanism of action are involved in mediating these effects.

Berberis vulgaris root bark extract prevents hyperoxaluria induced urolithiasis in rats

Berberis vulgaris is a widely used plant for the treatment of urolithiasis. To evaluate its antiurolithic potential, the crude aqueous-methanol extract of Berberis vulgaris root bark (Bv.Cr) was tested in an animal model of urolithiasis, developed in male Wistar rats by adding 0.75% ethylene glycol in drinking water. Bv.Cr (50 mg/kg) inhibited CaOx crystal deposition in renal tubules and protected against associated changes including polyuria, weight loss, impaired renal function and the development of oxidative stress in kidneys. Activity-guided fractionation revealed the concentration of antiurolithic constituent(s) mainly in the aqueous fraction. These data, indicating the presence of antiurolithic activity in Berberis vulgaris root bark, rationalize its medicinal use for the treatment of urolithiasis.

Proteome Changes in Human Monocytes upon Interaction with Calcium Oxalate Monohydrate Crystals

Monocytic infiltration in renal interstitium is commonly found surrounding the site of calcium oxalate (CaOx) crystal deposition in the kidney. Monocytes are supposed to eliminate the deposited crystals. However, effects of CaOx crystals on the infiltrating monocytes remain unknown. Therefore, this study investigated the altered cellular proteome of human monocytes in response to interaction with CaOx monohydrate (COM) crystals. After 24-h culture with or without 100 microg/mL COM crystals, U937 cells were harvested and subjected to 2-DE analysis with Deep Purple fluorescence staining (n = 5 gels/group; each was derived from independent culture). Spot matching, quantitative intensity analysis, and statistics revealed 22 differentially expressed proteins (9 up-regulated and 13 down-regulated proteins), which were successfully identified by Q-TOF MS and MS/MS analyses, including those involved in cell cycle, cellular structure, carbohydrate metabolism, lipid metabolism, mRNA processing, and protein synthesis, stabilization, and degradation. Randomly selected changes [up-regulated ALG-2 interacting protein 1 (Alix), elongation factor-2 (EF-2), and down-regulated beta-actin] were confirmed by Western blot analysis. Our data may help to understand how monocytes interact with COM crystals. These processes are proposed to cause subsequent inflammatory response in kidney stone disease through oxidative stress pathway(s).

1964 OF MICE AND MEN: MALE GENDER, HYPEROXALURIA AND HYPERCALCIURIA PROMOTE CALCIUM OXALATE STONE FORMATION IN MICE

You have accessJournal of UrologyStone Disease: Basic Research1 Apr 20101964 OF MICE AND MEN: MALE GENDER, HYPEROXALURIA AND HYPERCALCIURIA PROMOTE CALCIUM OXALATE STONE FORMATION IN MICE Saeed Khan and Benjamin Canales Saeed KhanSaeed Khan More articles by this author and Benjamin CanalesBenjamin Canales More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1974AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Despite years of sustained research, the pathophysiology of idiopathic CaOx stone disease is still poorly understood. The availability of various genetically engineered transgenic and knock out (KO) mice provides an opportunity to advance our knowledge in this regard. It has however proven difficult to produce CaOx stones in mice by inducing hyperoxaluria alone. We hypothesized that CaOx nephrolithiasis in mice requires an increase in the urinary excretion of both calcium and oxalate. To investigate it further and develop a dependable mouse model of CaOx stone formation, we tested whether hyperoxaluria inducing agents that have proven successful in rats could induce CaOx stones in the normocalciuric and hypercalciuric mice. METHODS Four to six month old male and female normocalciuric B6 wild type (WT) and hypercalciuric Npt2a KO mice weighing 18-30 gm were given 1.25% ethylene glycol (EG), or 1.5% glyoxylate (Gox). 24-hour urine samples were collected on days 0, 3, 7, 14, 21 and 28 and their pH measured. Urine was analyzed for crystalluria and calcium and oxalate. Mice were sacrificed on day 28, using carbon dioxide. RESULTS Npt2a KO mice are hypercalciuric and produce calcium phosphate deposits in their kidneys. By day 7 of receiving hyperoxaluria inducing diets, all mice, normocalciuric B6 and hypercalciuric KO mice, male and female became hyperoxaluric and showed CaOx crystalluria. There were no identifiable crystal deposits in the kidneys of any B6 mice, on hyperoxaluric or regular diet. On the other hand, only the male hypercalciuric Npt2a KO mice, receiving Gox or EG, developed CaOx crystal deposits, and only after 28 days of treatment. Kidneys of all mice on hyperoxaluria inducing diets, with or without renal CaOx crystals showed signs of injury and inflammation. Vacuolar degeneration of the tubules was common and inflammatory cells were visible in the cortical interstitium. Male mice showed more injury than the female mice. CONCLUSIONS Results indicate that CaOx stone formation can be induced in the mice. As is the case in the humans, male gender, hyperoxaluria, and hypercalciuria are risk factors for CaOx stone formation in mice. High oxalate even in the absence of renal crystal deposits is injurious to the renal epithelium. Gainesville, FL© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e763 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Saeed Khan More articles by this author Benjamin Canales More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Role of Lithium Carbonate and Lithium Citrate in Regulating Urinary Citrate Level and Preventing Nephrolithiasis

Background and purpose: Urinary Citrate is an inhibitor of Calcium oxalate stone formation. It is reabsorbed in the proximal kidney through sodium dicarboxylate co-transporters (NaDC-1, NaDC-3) present in the renal tubular epithelium. Lithium (Li) is a known potent inhibitor of these transporters. We investigated the effect of lithium carbonate (LiC) and lithium citrate (LiCit) in regulating urinary citrate levels and preventing nephrolithiasis (NL) in the rat model. Experimental approach: We took 220 Wistar rats and induced nephrolithiasis in 130 of them by administering high doses of 5% ammonium oxalate (AmOx) for seven days and labeled them as Group B. Rest were labeled as Group A. Each group was then divided into 3 subgroups. First sub-group acted as control while other two were treated with either lithium carbonate (LiC) or lithium citrate (LiCit) for 21 days. Ten rats from each of the six sub-groups were randomly selected for sacrifice on 3rd, 7th and 14th day and additional 10th and 21st day from Li treated groups. Blood and urine samples were collected and analyzed on these days. The kidneys of the sacrificed rats were dissected and studied under light microscopy for crystal deposition (left kidney) and histological changes (right kidney). Key results: Urinary citrate levels were significantly increased in response to either LiC (p<0.001) or LiCit (p<0.001). Increased urinary citrate levels resulted in the reduction of calcium oxalate (CaOx) crystal deposition, kidney tubular dilatation and infiltration of inflammatory cell in the tubulo-interstitium. Conclusions and implications: Use of lithium salts might be a potentially useful approach in the prevention of recurrent NL.

Strain differences in urinary factors that promote calcium oxalate crystal formation in the kidneys of ethylene glycol-treated rats

Ethylene glycol (EG)-induced hyperoxaluria is the most commonly employed experimental regimen as an animal model of calcium oxalate (CaOx) stone formation. The variant sensitivity to CaOx among different rat strains has not been fully explored, although the Wistar rat is known to accumulate more CaOx in kidney tissue after low-dose EG exposure than in the Fischer 344 (F344) rats. Supersaturation of CaOx in tubular fluid contributes to the amount of CaOx crystal formation in the kidney. We hypothesized that the urinary supersaturation of CaOx in Wistar rats is higher than that of F344 rats, thereby allowing for greater CaOx crystal deposition in the Wistar rat. Age-matched male Wistar and F344 rats were treated with 0.75% EG or drinking water for 8 wk. Twenty-four-hour urine was collected at 0, 2, 4, 6, and 8 wk for analysis of key electrolytes to calculate the CaOx supersaturation. Plasma oxalate level was also measured. Our data confirmed the different sensitivity to renal toxicity from EG between the two rat strains (Wistar > F344). After EG treatment, the plasma oxalate level and urine oxalate excretion were markedly greater in the Wistar rats than in the F344 rats, while urine calcium was slightly decreased in Wistars. Thus, the CaOx supersaturation in urine of Wistar rats was higher, which led to a greater crystal deposition in kidney in Wistar rats. These studies suggest that during EG treatment, changes in urine electrolytes and in CaOx supersaturation occur to a greater extent in the Wistar rat, in agreement with its greater sensitivity to EG toxicity.

Chronic l-arginine administration increases oxidative and nitrosative stress in rat hyperoxaluric kidneys and excessive crystal deposition

Hyperoxaluric kidneys show an impaired diuretic response to acute infusion of L-arginine. In this study, we examined the chronic effect of l-arginine supplementation on CaOx crystal formation in hyperoxaluric rat kidneys. Eight groups were tested: control (received drinking water), L group (received L-arginine, 0.6%), LN group [received NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg)], L + LN group (received L-arginine + l-NAME), HP group [received hydroxyl-L-proline (HP, 5%) mixed with chow to induce hyperoxaluria], L + HP group (received HP + L-arginine), HP + LN group, and L + HP + LN group. The duration was 42 days, and each group had eight animals. Urinary biochemistry and renal CaOx amounts were measured, as well as renal expressions of nitric oxide synthase (NOS) isoforms and NAD(P)H oxidase. The distribution of inducible NOS (iNOS), NAD(P)H oxidase, ED1-positive cells, and nitrotyrosine was examined by immunohistochemical and immunofluorescence studies, whereas superoxide production from the kidneys was examined by fluorescence spectrometric assay. Compared with the HP group, the L + HP group had excessive CaOx crystal accumulation and enhanced endothelial NOS (eNOS), iNOS, and NAD(P)H oxidase protein expression in the kidney. Urinary excretion of nitrotyrosine was markedly increased. Increased superoxide formation in the L + HP kidney was derived from NAD(P)H oxidase and uncoupled eNOS, and increased nitrotyrosine formation might derive from iNOS and ED1-positive cells that gathered around the CaOx crystals. L-NAME cotreatment (L + HP + LN group) reduced renal oxidative nitrosative stress and tubular damage, which were induced by L + HP. The results showed that chronic l-arginine treatment to the hyperoxaluric kidney with massive CaOx crystal deposition may have a toxic effect by enhancing intrarenal oxidative and nitrosative stress.

Calcium oxalate crystal deposition in kidneys of hypercalciuric mice with disrupted type IIa sodium-phosphate cotransporter

The most common theories about the pathogenesis of idiopathic kidney stones consider precipitation of calcium phosphate (CaP) within the kidneys critical for the development of the disease. We decided to test the hypothesis that a CaP substrate can promote the deposition of calcium oxalate (CaOx) in the kidneys. Experimental hyperoxaluria was induced by feeding glyoxylate to male mice with knockout (KO) of NaP(i) IIa (Npt2a), a sodium-phosphate cotransporter. Npt2a KO mice are hypercalciuric and produce CaP deposits in their renal tubules. Experimental hyperoxaluria led to CaOx crystalluria in both the hypercalciuric KO mice and the normocalciuric control B6 mice. Only the KO mice produced CaOx crystal deposits in their kidneys, but the CaOx crystals deposited separately from the CaP deposits. Perhaps CaP deposits were not available for a CaOx overgrowth. These results also validate earlier animal model observations that showed that CaP substrate is not required for renal deposition of CaOx and that other factors, such as local supersaturation, may be involved. The absence of CaOx deposition in the B6 mice despite extreme hyperoxaluria also signifies the importance of both calcium and oxalate in the development of CaOx nephrolithiasis.

Effect of Ethanolic Extract of Leaves of Cocculus hirsutus (L.) Diels on Experimentally Induced Urothiliasis in Rats

In the present study, we report anti-urolithiasis effect of ethanolic extract of leaves of Cocculus hirsutus (L.) Diels (CH-eth) on the biochemical and histopathological parameters against sodium oxalate (NaOx) induced urolithiasis model in rat. The increased severity of microscopic calcium oxalate (CaOx) crystal deposition along with increased CaOx concentration in kidney was seen after seven days of NaOx (70 mg/kg, i.p) pretreatment. CH-eth (100 and 200 mg/kg, p.o.) and standard marketed formulation, Cystone ® (500 mg/kg, p.o.) caused significant reversal of NaOx induced changes in ion excretion and urinary CaOx concentration in seven day treatement (prophylactic and therapeutic schedule). In conclusion, CH-eth showed beneficial effect against urolithiasis by decreasing CaOx excretion and preventing crystal deposition in kidney tubules.

Increased Expression of Heparan Sulfate Proteoglycan on the Cultured Renal Epithelial Cells during Oxalate Exposure

We have previously reported that heparan sulfate (HS) / heparan sulfate proteoglycan (HSPG, syndecan-1) expression significantly increased in the rat kidney during calcium oxalate (CaOx) nephrolithiasis. Although the exact mechanism of the increased syndecan expression still remains unclear, HS/syndecan is thought to have some important roles in CaOx crystal formation. The present study examined the role of HS during oxalate exposure by using a newly developed cell line (KIC-synd-1) that expresses human heparan sulfate proteoglycan (syndecan-1). Quantitative competitive (QC)-RT-PCR was used to examine change of syndecan-1 mRNA expression in KIC-synd-1 cells. Production of syndecan-1 core protein and glycosaminoglycans (GAGs) were also confirmed by Western blot, immunohistochemistry and HPLC, respectively. Wild type Mardin-Darby canine kidney (MDCK) cells were also examined in the same manner. The stable expression of syndecan-1 gene and production of both core protein and HS chains were confirmed in the newly developed KIC-synd-1 cell line. Increased syndecan-1 mRNA expression and production of core proteins were confirmed in KIC-synd-1 cells during oxalate exposure. MTT assay revealed that the cell viability decreased significantly in the MDCK cells after 1 mM oxalate exposure (p<0.05). On the other hand, there was no significant difference in the oxalate exposed KIC-synd-1 cells. However, the cell viability in KIC-synd-1 cells pretreated with heparitinase digestion decreased significantly before oxalate exposure (p<0.05). The present data suggests that both exogenous and endogenous HS exerts protective effect against oxalate-induced cell injuries. Previous studies in our laboratory have indicated that hyperoxaluria and deposition of CaOx crystals resulted in renal tubular cellular injury inducing the synthesis of HSPG to protect and repair the damaged epithelial cell surface. The present data offers strong support for this hypothesis. Finally, HS could be potent inhibitor of CaOx nephrolithiasis and the absence of this substance on the tubular surface may increase the risk of CaOx crystal formation and retention.

Vitamin E attenuates crystal formation in rat kidneys: Roles of renal tubular cell death and crystallization inhibitors

We previously reported that oxidative stress and renal tubular damage occur in chronic hyperoxaluric rats. However, the in vivo responses of renal epithelial cells after vitamin E administration and their correlations with calcium oxalate (CaOx) crystal formation have not been evaluated. Male Wistar rats received 0.75% ethylene glycol (EG) for 7, 21, or 42 days to induce CaOx deposition (EG group). Another group of EG-treated rats received 200 mg kg(-1) of vitamin E intraperitoneally (EG+E group) to evaluate its effect on hyperoxaluria. Urinary electrolytes and biochemistry and levels of lipid peroxides and enzymes were examined, together with serum vitamin E levels. Levels of the tubular markers, alpha and mu glutathione S-transferase, proliferating cell nuclear antigen (PCNA), osteopontinin (OPN), and Tamm-Horsfall protein (THP) were also measured, and TUNEL staining was performed to examine the viability of the tubular epithelium. There were no significant differences between the two age-matched controls either untreated or given vitamin E. Compared to untreated controls, tubular cell death was increased at all time points in EG rats with a gradual increase in CaOx crystals, whereas the number of PCNA-positive cells was only significantly increased on day 21. In EG+E rats, tubular cell death was decreased compared to the EG group, and cell proliferation was seen at all time points, while CaOx crystal deposition was decreased, but hyperoxaluria, urinary lipid peroxides, and enzymuria were unaffected. Vitamin E supplement prevented the loss of OPN and THP in renal tissues by EG and the reduction in their levels in the urine. The beneficial effect of vitamin E in reducing CaOx accumulation is due to attenuation of tubular cell death and enhancement of the defensive roles of OPN and THP.

Reactive oxygen species mediated calcium oxalate crystal-induced expression of MCP-1 in HK-2 cells

Under severe hyperoxaluric conditions calcium oxalate crystals often deposit in the renal interstitium and produce localized inflammation. We have proposed that renal epithelial cells exposed to CaOx crystals produce chemoattractants such as monocyte chemoattractant protein-1 (MCP-1). MCP-1 synthesis is mediated by reactive oxygen species (ROS). HK-2 cells of human renal epithelial line were exposed to CaOx crystals for different lengths of time. The culture media was tested for cell injury marker LDH, and subjected to enzyme-linked immunosorbent assay to determine the secretion of MCP-1 protein. Cell expression of MCP-1 was assessed by Western blot analysis. Gene expression was determined by reverse transcriptase-polymerase chain reaction. The data clearly showed that the HK-2 cells express MCP-1 gene and protein. The MCP-1 mRNA expression was increased following exposure to CaOx crystals, which was reduced upon treatment with free radical scavengers, catalase and superoxide dismutase. Results indicate that CaOx crystals strongly induce MCP-1 synthesis and secretion by the HK-2 cells and production is mediated by intracellular ROS production. Based on these and other data, antioxidant therapy and blockade of rennin-angiotensin system may prove beneficial for the prevention of end stage renal disease caused by hyperoxaluria and CaOx crystal deposition.

Hyperoxaluria-induced oxidative stress and antioxidants for renal protection

Renal cellular exposure to oxalate (Ox) and/or CaOx crystals leads to the production of reactive oxygen species (ROS), development of oxidative stress followed by injury and inflammation. Renal injury and inflammation appear to play a significant role in stone formation. ROS are produced from many sources and involve a variety of signaling pathways. Tissue culture and animal model studies show that treatments with anti-oxidants and free radical scavengers reduce Ox/CaOx crystal induced injuries. In addition, CaOx crystal deposition in kidneys is significantly reduced by treatments with antioxidants and free radical scavengers, indicating their efficacy. These results point towards a great potential for the therapeutic application of antioxidants and free radical scavengers to reduce stone recurrence particularly after shock wave lithotripsy, which is itself known to generate ROS and cause renal damage.

Crystal-induced inflammation of the kidneys: results from human studies, animal models, and tissue-culture studies.

Calcium oxalate (CaOx), calcium phosphate (CaP), and uric acid or urate are the most common crystals seen in the kidneys. Most of the crystals evoke an inflammatory response leading to fibrosis, loss of nephrons, and eventually to chronic renal failure. Of the three, CaOx monohydrate is the most reactive, whereas some forms of CaP do not evoke any discernible response. Reactive oxygen species are produced during the interactions between the crystals and renal cells and are responsible for the various cellular responses. CaOx crystals generally form in the renal tubules. Exposure of renal epithelial cells to CaOx crystals results in the increased synthesis of osteopontin, bikunin, heparan sulfate, monocyte chemoattractant protein 1 (MCP-1), and prostaglandin (PG) E2, which are known to participate in inflammatory processes and in extracellular matrix production. CaOx crystal deposition in rat kidneys also activates the renin-angiotensin system. Both Ox and CaOx crystals selectively activate p38 mitogen-activated protein kinase (MAPK) in exposed tubular cells. CaP crystals can form in the tubular lumen, tubular cells, or tubular basement membrane. Renal epithelial cells exposed to brushite crystals produce MCP-1. Basic CaP and calcium pyrophosphate dihydrate induce mitogenesis in fibroblasts, stimulate production of PGE2, and up-regulate the synthesis of metalloproteinases (MMP) while down-regulating the production of inhibitors of MMPs through activation of p42/44 MAPK. Deposition of urate crystals in the kidneys becomes associated with renal tubular atrophy, interstitial fibrosis, and development of inflammatory infiltrate. Renal epithelial cells exposed to uric acid crystals synthesize MCP-1 as well as PGE2. Monocytes or neutrophils exposed to urate crystals produce tumor necrosis factor alpha, interleukin-1 (IL-1), IL-6, and IL-8. Expression of IL-8 is mediated through extracellular signal-regulated kinase 1 (ERK-1)/ERK-2 and nuclear transcription factors activated protein 1 and nuclear factor kappabeta. Urate crystals also stimulate the macrophages to produce MMPs.

Minipump Induced Hyperoxaluria and Crystal Deposition in Rats: A Model for Calcium Oxalate Urolithiasis

Unraveling the mechanisms leading to clinically active calcium oxalate (CaOx) stone disease and the development of effective medical therapies to treat it have been hampered by the lack of appropriate animal models. To address this problem we developed a model of hyperoxaluria and calcium oxalate crystal deposition by implanting osmotic minipumps subcutaneously into male rats, that is minipump induced hyperoxaluria and crystal deposition in rats. Male Harlan-Sprague Dawley rats (225 to 290 gm) were implanted subcutaneously with 1-week 2 ml osmotic minipumps containing 1.5 M potassium oxalate (360 microM KOx/24 hours, [KOx-trt], 11) or phosphate buffered saline (PBS-trt, 9) on days 1 and 7. The 24-hour urine collections were performed on days 0, 4, 7, 11 and 14. Data were analyzed by ANOVA and Tukey's HSD. Urinary crystals were analyzed by light microscopy. Kidneys were harvested on day 14 and processed for light and polarizing microscopy, and RNA analysis.RESULTS Mean overall creatinine excretion +/- SEM (PBS-trt 107 +/- 7 and KOx-trt 123 +/- 6 microM/24 hours, p >0.07) and day 14 serum creatinine (PBS-trt 83 +/- 4 and KOx-trt 83 +/- 5 microM, p >or=0.9) were similar in the 2 treatment groups. Overall urinary volume (PBS-trt 11.3 +/- 0.8 and KOx-trt 18.0 +/- 1.5 ml/24 hours, p <or=0.001) and oxalate (OX) excretion (PBS-trt 9.2 +/- 0.6 and KOx-trt 44 +/- 4.2 microM/24 hours, p <or=0.001) were higher in KOx-trt vs PBS-trt rats. In KOx-trt rats OX excretion on day 0 was significantly less than on any day after implantation (p <or=0.01). All KOx-trt rats excreted calcium oxalate dihydrate crystals by day 4 and had intrarenal deposits of birefringent crystals by day 14. Overall the morphology of kidneys of OX rats was normal, although localized regions of inflammation and tubular debris were occasionally observed. Reverse transcriptase-polymerase chain reaction and Northern blot analysis revealed that the expression of 3 distress molecules tumor necrosis factor receptor, osteopontin and kidney injury molecule were up-regulated in KOx-trt kidneys. The model of minipump induced hyperoxaluria and crystal deposition in rats reliably induces hyperoxaluria, CaOx crystalluria and CaOx crystal deposition. These characteristics make it an appropriate model for investigations of the effects of OX on renal physiology as well as investigating the efficacy of new therapeutics.

Distribution of calcium oxalate crystals in the secondary phloem of conifers: a constitutive defense mechanism?

• Calcium oxalate (CaOx) crystals were studied in the secondary phloem of 46 conifer species from six families to characterize their distribution relative to defense. • Scanning electron microscopy, and polarized light microscopy of stem tissue allowed analysis of the quantity, location, morphology and size of the crystals. • Vastly different patterns of CaOx crystal deposition were observed in stems of Pinaceae and nonPinaceae lineages. The CaOx crystals were present in all species but the highest density occurred along the nonPinaceae lineage. In Pinaceae, all species accumulated crystals intracellularly in crystalliferous parenchyma, whereas all nonPinaceae species had only extracellular crystals. A possible relationship between the number of aggressive bark beetles species and the amount of CaOx accumulation was noted where increased crystal accumulation appears to be antagonistic to beetle attack. Mapping along with this trend was the presence of phloem fibers. • We conclude that in conifer stems the patterns and frequency of CaOx crystals function as a constitutive defense and in combination with fiber rows, provides an effective barrier against small bark-boring insects.

Effect of prothrombin and its activation fragments on calcium oxalate crystal growth and aggregation in undiluted human urine in vitro: relationship between protein structure and inhibitory activity

In recent years there has been great interest in the putative role of prothrombin and its activation peptides, especially the urinary form of prothrombin fragment 1, in the pathogenesis of calcium oxalate (CaOx) urolithiasis. Previously, we showed that prothrombin and its activation peptides inhibit CaOx crystallization in inorganic conditions in vitro. The aim of the present study was to determine if this inhibitory activity is retained in undiluted human urine and, therefore, whether it is likely to have any influence under physiological conditions. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. Prothrombin was purified from Prothrombinex-HT, cleaved with thrombin and the resulting fragment 1 (F1) and fragment 2 (F2) were purified. The purity of each protein was confirmed by SDS/PAGE, and their effects on CaOx crystallization in undiluted ultrafiltered human urine were determined at a final concentration 80.65 nmol/l using Coulter Counter and [(14)C]oxalate analysis. The precipitated crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that, whereas prothrombin and its activation peptides did not affect the urinary metastable limit and the size of the precipitated particles, F1 did significantly reduce the latter. These findings were corroborated with scanning electron microscopy which also revealed that the reduction in particle size caused by F1 resulted from a decrease in the degree of crystal aggregation, rather than in the size of the individual crystals. The [(14)C]oxalate data showed that none of the proteins added significantly inhibited the mineral deposition. It was concluded that with the exception of F1, which does inhibit CaOx crystal aggregation, prothrombin and its activation peptides do not alter the deposition and aggregation of CaOx crystals in ultrafiltered human urine in vitro. Also, the gamma-carboxyglutamic acid domain of prothrombin and F1, which is absent from thrombin and F2, is the region of the molecules that determines their potent inhibitory effects. The superior potency of F1, compared with prothrombin, probably results from the molecule's greater charge-to-mass ratio.